10-K 1 mgx-10k-20231231.htm 10-K 10-K

 

 

UNITED STATES

SECURITIES AND EXCHANGE COMMISSION

Washington, D.C. 20549

 

FORM 10-K

 

(Mark One)

ANNUAL REPORT PURSUANT TO SECTION 13 OR 15(d) OF THE SECURITIES EXCHANGE ACT OF 1934

For the fiscal year ended December 31, 2023

OR

TRANSITION REPORT PURSUANT TO SECTION 13 OR 15(d) OF THE SECURITIES EXCHANGE ACT OF 1934 FOR THE TRANSITION PERIOD FROM TO

Commission File Number 001-41949

 

Metagenomi, Inc.

(Exact name of Registrant as specified in its Charter)

 

Delaware

81- 3909017

(State or other jurisdiction of

incorporation or organization)

(I.R.S. Employer

Identification No.)

5959 Horton Street, 7th Floor

Emeryville, California

94608

(Address of principal executive offices)

(Zip Code)

Registrant’s telephone number, including area code: (510) 871-4880

 

Securities registered pursuant to Section 12(b) of the Act:

 

Title of each class

 

Trading

Symbol(s)

 

Name of each exchange on which registered

Common stock, par value $0.0001 per share

 

MGX

 

The Nasdaq Global Select Market

 

Securities registered pursuant to Section 12(g) of the Act: None

Indicate by check mark if the Registrant is a well-known seasoned issuer, as defined in Rule 405 of the Securities Act. YES ☐ NO ☒

Indicate by check mark if the Registrant is not required to file reports pursuant to Section 13 or 15(d) of the Act. YES ☐ NO ☒

Indicate by check mark whether the Registrant: (1) has filed all reports required to be filed by Section 13 or 15(d) of the Securities Exchange Act of 1934 during the preceding 12 months (or for such shorter period that the Registrant was required to file such reports), and (2) has been subject to such filing requirements for the past 90 days. YES ☒ NO ☐

Indicate by check mark whether the Registrant has submitted electronically every Interactive Data File required to be submitted pursuant to Rule 405 of Regulation S-T (§232.405 of this chapter) during the preceding 12 months (or for such shorter period that the Registrant was required to submit such files). YES ☒ NO ☐

Indicate by check mark whether the registrant is a large accelerated filer, an accelerated filer, a non-accelerated filer, smaller reporting company, or an emerging growth company. See the definitions of “large accelerated filer,” “accelerated filer,” “smaller reporting company,” and “emerging growth company” in Rule 12b-2 of the Exchange Act.

 

Large accelerated filer

Accelerated filer

 

 

 

 

Non-accelerated filer

Smaller reporting company

 

 

 

 

 

 

 

Emerging growth company

 

 

 

 

 

 

If an emerging growth company, indicate by check mark if the registrant has elected not to use the extended transition period for complying with any new or revised financial accounting standards provided pursuant to Section 13(a) of the Exchange Act. ☒

Indicate by check mark whether the registrant has filed a report on and attestation to its management’s assessment of the effectiveness of its internal control over financial reporting under Section 404(b) of the Sarbanes-Oxley Act (15 U.S.C. 7262(b)) by the registered public accounting firm that prepared or issued its audit report. ☐

If securities are registered pursuant to Section 12(b) of the Act, indicate by check mark whether the financial statements of the registrant included in the filing reflect the correction of an error to previously issued financial statements. ☐

Indicate by check mark whether any of those error corrections are restatements that required a recovery analysis of incentive-based compensation received by any of the registrant’s executive officers during the relevant recovery period pursuant to §240.10D-1(b). ☐

Indicate by check mark whether the Registrant is a shell company (as defined in Rule 12b-2 of the Exchange Act). YES ☐ NO ☒

As of June 30, 2023, the last business day of the Registrant’s most recently completed second quarter, there was no established public trading market for the Registrant’s equity securities as the Registrant was not a public company and therefore cannot calculate the aggregate market value of its voting and non-voting equity held by non-affiliates as of such date. The Registrant’s common stock began trading on the Nasdaq Global Select Market on February 9, 2024.

 

The number of shares of Registrant’s Common Stock outstanding as of March 15, 2024 was 37,472,351.

 

DOCUMENTS INCORPORATED BY REFERENCE

None

 

 

A

 


 

Table of Contents

 

 

 

Page

PART I

 

Item 1.

Business

4

Item 1A.

Risk Factors

83

Item 1B.

Unresolved Staff Comments

135

Item 1C.

Cybersecurity

135

Item 2.

Properties

136

Item 3.

Legal Proceedings

136

Item 4.

Mine Safety Disclosures

136

 

PART II

 

Item 5.

Market for Registrant’s Common Equity, Related Stockholder Matters and Issuer Purchases of Equity Securities

137

Item 6.

[Reserved]

138

Item 7.

Management’s Discussion and Analysis of Financial Condition and Results of Operations

139

Item 7A.

Quantitative and Qualitative Disclosures About Market Risk

155

Item 8.

Financial Statements and Supplementary Data

155

Item 9.

Changes in and Disagreements With Accountants on Accounting and Financial Disclosure

155

Item 9A.

Controls and Procedures

155

Item 9B.

Other Information

156

Item 9C.

Disclosure Regarding Foreign Jurisdictions that Prevent Inspections

156

 

PART III

 

Item 10.

Directors, Executive Officers and Corporate Governance

157

Item 11.

Executive Compensation

160

Item 12.

Security Ownership of Certain Beneficial Owners and Management and Related Stockholder Matters

167

Item 13.

Certain Relationships and Related Transactions, and Director Independence

169

Item 14.

Principal Accounting Fees and Services

170

 

PART IV

 

Item 15.

Exhibits, Financial Statement Schedules

172

Item 16.

Form 10-K Summary

173

 

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SPECIAL NOTE REGARDING FORWARD-LOOKING STATEMENTS

This Annual Report on Form 10-K contains express or implied forward-looking statements that are based on our management’s belief and assumptions and on information currently available to our management and which are made pursuant to the safe harbor provisions of Section 27A of the Securities Act of 1933, as amended, or the or the Securities Act, and Section 21E of the Securities Exchange Act of 1934, as amended, or the Exchange Act. Although we believe that the expectations reflected in these forward-looking statements are reasonable, these statements relate to future events or our future operational or financial performance, and involve known and unknown risks, uncertainties and other factors that may cause our actual results, performance or achievements to be materially different from any future results, performance or achievements expressed or implied by these forward-looking statements. Forward-looking statements in this Annual Report on Form 10-K include, but are not limited to, statements about:

the initiation, timing, progress and results of our research and development programs, preclinical studies and future clinical trials;
our ability to demonstrate, and the timing of, preclinical proof-of-concept in vivo and ex vivo for multiple programs;
our ability to advance any product candidates that we may identify and successfully complete any clinical studies, including the manufacture of any such product candidates;
our ability to quickly leverage programs within our initial target indications and to progress additional programs to further develop our pipeline;
the timing of our Investigational New Drug (“IND”) applications submissions;
the implementation of our strategic plans for our business, programs and technology;
the scope of protection we are able to establish and maintain for intellectual property rights covering our genome editing technology and platform;
developments related to our competitors and our industry;
our ability to leverage the clinical, regulatory, and manufacturing advancements made by genome editing programs to accelerate our clinical trials and approval of product candidates;
our ability to identify and enter into future license agreements and collaborations;
developments related to our genome editing technology and platform;
regulatory developments in the United States and foreign countries;
our ability to attract and retain key scientific and management personnel; and
estimates of our expenses, capital requirements, and needs for additional financing.

In some cases, you can identify forward-looking statements by terminology such as “may,” “will,” “should,” “expects,” “intends,” “plans,” “anticipates,” “believes,” “estimates,” “predicts,” “potential,” “continue” or the negative of these terms or other comparable terminology. These statements are only predictions and are subject to change. You should not place undue reliance on forward-looking statements because they involve known and unknown risks, uncertainties, and other factors, which are, in some cases, beyond our control and which could materially affect results. Moreover, we operate in an evolving environment. New risk factors and uncertainties may emerge from time to time, and it is not possible for management to predict all risk factors and uncertainties.

Factors that may cause actual results to differ materially from current expectations include, among other things, those listed under the section entitled “Risk Factors” and elsewhere in this Annual Report on Form 10-K. If one or more of these risks or uncertainties occur, or if our underlying assumptions prove to be incorrect, actual events or results may vary significantly from those implied or projected by the forward-looking statements. No forward- looking statement is a guarantee of future performance. You should read this Annual Report on Form 10-K and the documents that we reference in this Annual Report on Form 10-K and have filed with the SEC as exhibits to this Annual Report on Form 10-K completely and with the understanding that our actual future results may be materially different from any future results expressed or implied by these forward-looking statements.

The forward-looking statements in this Annual Report on Form 10-K represent our views as of the date of this Annual Report on Form 10-K. We anticipate that subsequent events and developments will cause our views to change. However, while we may elect to update these forward-looking statements at some point in the future, we have no current intention of doing so except to the extent required by applicable law. You should therefore not rely on these forward- looking statements as representing our views as of any date subsequent to the date of this Annual Report on Form 10-K.

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In addition, statements that “we believe” and similar statements reflect our beliefs and opinions on the relevant subject. These statements are based upon information available to us as of the date of this 10-K, and while we believe such information forms a reasonable basis for such statements, such information may be limited or incomplete, and our statements should not be read to indicate that we have conducted an exhaustive inquiry into, or review of, all potentially available relevant information. These statements are inherently uncertain.

This Annual Report on Form 10-K also contains estimates, projections and other information concerning our industry, our business and the markets for our programs and product candidates. Information that is based on estimates, forecasts, projections, market research or similar methodologies is inherently subject to uncertainties and actual events or circumstances may differ materially from events and circumstances that are assumed in this information. Unless otherwise expressly stated, we obtained this industry, business, market, and other data from our own internal estimates and research as well as from reports, research surveys, studies, and similar data prepared by market research firms and other third parties, industry, medical and general publications, government data and similar sources. While we are not aware of any misstatements regarding any third-party information presented in this Annual Report on Form 10-K, their estimates, in particular, as they relate to projections, involve numerous assumptions, are subject to risks and uncertainties and are subject to change based on various factors, including those discussed under the section entitled “Risk Factors” and elsewhere in this Annual Report on Form 10-K.

Trademarks and Service Marks

This Annual Report on Form 10-K contains references to our trademarks and service marks and to those belonging to other entities. Solely for convenience, trademarks and trade names referred to in this Annual Report on Form 10-K, including logos, artwork and other visual displays, may appear without the ® or TM symbols, but such references are not intended to indicate in any way that we will not assert, to the fullest extent under applicable law, our rights or the rights of the applicable licensor to these trademarks and trade names. We do not intend our use or display of other entities’ trade names, trademarks or service marks to imply a relationship with, or endorsement or sponsorship of us by, any other entity.

Market, Industry and Other Data

Unless otherwise indicated, information contained in this Annual Report on Form 10-K concerning our industry and the markets in which we operate, including our general expectations about our product candidates, market position, market opportunity, market size, competitive position and the incidence of certain medical conditions, is based on or derived from publicly available information released by industry analysts and third-party sources, independent market research, industry and general publications and surveys, governmental agencies, our internal research and our industry experience. Our estimates of the potential market opportunities for our product candidates include a number of key assumptions based on our industry knowledge and industry publications, the latter of which may be based on small sample sizes and fail to accurately reflect such information, and you are cautioned not to give undue weight to such estimates. While we believe that our internal assumptions are reasonable, no independent source has verified such assumptions. Industry publications and third-party research often indicate that their information has been obtained from sources believed to be reliable, although they do not guarantee the accuracy or completeness of such information and such information is inherently imprecise. In some cases, we do not expressly refer to the sources from which this data is derived. In that regard, when we refer to one or more sources of this type of data in any paragraph, you should assume that other data of this type appearing in the same paragraph is derived from the same sources, unless otherwise expressly stated or the context otherwise requires. In addition, projections, assumptions and estimates of our future performance and the future performance of the industry in which we operate is necessarily subject to a high degree of uncertainty and risk due to a variety of factors, including those described in Part I, Item 1A of this Annual Report on Form 10-K titled “Risk Factors” and elsewhere in this Annual Report on Form 10-K. These and other factors could cause results to differ materially from those expressed in the estimates made by independent third parties and by us.

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SUMMARY OF MATERIAL RISKS ASSOCIATED WITH OUR BUSINESS

Our business is subject to numerous risks and uncertainties that you should be aware of in evaluating our business. These risks include, but are not limited to, the following:

We have incurred significant losses since inception. We expect to incur losses for the foreseeable future and may never achieve or maintain profitability.
We will need substantial additional funding in addition to the net proceeds we received from our initial public offering. If we are unable to raise additional capital when needed on acceptable terms, or at all, we may be forced to delay, reduce, or terminate certain of our research and product development programs, future commercialization efforts or other operations.
We are very early in our development efforts, and we have not yet initiated IND-enabling studies or clinical development of any product candidate. As a result, we expect it will be many years before we commercialize any product candidate, if ever. If we are unable to advance our future product candidates into and through clinical trials, obtain regulatory approval and ultimately commercialize our product candidates or experience significant delays in doing so, our business will be materially harmed.
We are subject to additional development challenges and risks due to the novel nature of our genome editing technology.
The genome editing field is relatively new and is evolving rapidly. We are focusing our research and development efforts on genome editing using programmable nucleases, base editing, and RNA and DNA-mediated integration systems (including prime editors and CRISPR-associated (“Cas”) transposases), but other genome editing technologies may be discovered that provide significant advantages over such technologies, which could materially harm our business.
While we intend to seek designations for our potential product candidates with the FDA and comparable foreign regulatory authorities that are intended to confer benefits such as a faster development process or an accelerated regulatory pathway, there can be no assurance that we will successfully obtain such designations. In addition, even if one or more of our potential product candidates are granted such designations, we may not be able to realize the intended benefits of such designations.
Because we are developing product candidates in the field of genetic medicines in which there is little clinical experience, there is increased risk that the FDA, the EMA or other regulatory authorities may not consider the endpoints of our clinical trials to provide clinically meaningful results and that these results may be difficult to analyze.
If conflicts arise between us and our collaborators or strategic partners, these parties may act in a manner adverse to us and could limit our ability to implement our strategies.
We have entered into collaborations, and may enter into additional collaborations, with third parties for the research, development, manufacture and commercialization of programs or product candidates. If these collaborations are not successful, our business could be adversely affected.
Our commercial success depends on our ability to obtain, maintain, enforce, and otherwise protect our intellectual property and proprietary technology, and if the scope of the intellectual property protection obtained is not sufficiently broad, our competitors or other third parties could develop and commercialize products and product candidates similar to ours and our ability to successfully develop and commercialize our genome editing systems may be adversely affected.
It is difficult and costly to protect our intellectual property and our proprietary technologies, and we may not be able to ensure their protection.
The impacts of cyber attacks and data privacy breaches on our business.

The summary risk factors described above should be read together with the text of the full risk factors below in the section titled “Risk Factors” in Part I, Item 1.A. and the other information set forth in this Annual Report on Form 10-K, as well as in other documents that we file with the U.S. Securities and Exchange Commission (SEC). The risks summarized above or described in full below are not the only risks that we face. Additional risks and uncertainties not precisely known to us, or that we currently deem to be immaterial, may also materially adversely affect our business, financial condition, results of operations and future growth prospects.

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PART I

Item 1. Business.

Overview

We are a precision genetic medicines company committed to developing curative therapeutics for patients using our proprietary, comprehensive metagenomics-derived genome editing toolbox. Genetic diseases are caused by a diverse set of mutations that have been largely inaccessible by genome engineering approaches to date. Genetic mutations are seen in a variety of forms, including deletions, insertions, single-base-pair changes and sequence repeats, and are found throughout the genome and across a variety of different cell types, tissues, and organ systems. Additionally, many diseases lack a genetic origin but have the potential to be effectively and permanently addressed through genome editing. We are harnessing the power of metagenomics, the study of genetic material recovered from the natural environment, to unlock four billion years of microbial evolution to discover and develop a suite of novel editing tools capable of correcting any type of genetic mutation found anywhere in the genome. Our comprehensive genome editing toolbox includes programmable nucleases, base editors, and RNA and DNA-mediated integration systems (including prime editing systems and clustered regularly interspaced short palindromic repeat (“CRISPR”)-associated transposases (“CASTs”)). We believe our diverse and modular toolbox positions us to access the entire genome and select the optimal tool to unlock the full potential of genome editing for patients.

The company was founded by pioneers in the field of metagenomics, a powerful science that allows us to tap into the diversity of microbial life on this planet. The metagenomics process starts by collecting samples from microbe-rich ecosystems ranging from simple home gardens to extreme locations such as hydrothermal vents below the ocean. We then extract the DNA from these environmental samples and deeply sequence them to fully reconstruct the genomes of the resident microbes. Each sample may include thousands of distinct genomes from previously unknown organisms revealing novel cellular machinery that we utilize as building blocks for our editing systems. Using high-throughput screening, artificial intelligence (“AI”), and proprietary algorithms, we rapidly mine through billions of novel proteins from our genome-resolved metagenomics database to create genome editing tools. To date, we have analyzed over 460 trillion base pairs of DNA sequencing data, predicted over 7.4 billion proteins, including over 322 million CRISPR-associated (“Cas”) proteins, and identified over 1.75 million CRISPRs, which we estimate has resulted in the identification of over 20,000 novel genome editing systems. Simultaneously, we have assembled extensive libraries of millions of nucleases, deaminases, reverse transcriptase (“RTs”) and over one thousand CASTs. Our platform is designed to enable us to rapidly and effectively find, screen, and select tools with the highest potential targetability, specificity, and efficiency in order to develop them into genetic medicines. The iterative nature of our process, underpinned by AI, allows us to continuously push the boundaries of innovation.

Our proprietary toolbox of editing systems

We have developed an expansive and modular toolbox of next-generation genome editing systems that will allow us to interact with the human genome in a site-specific manner, where each tool can be matched to specific disease targets. Figure 1 summarizes our diverse and versatile toolbox of different editing capabilities with the potential to address the full spectrum of genetic diseases.

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Figure 1. Our Toolbox.

 

img190561166_0.jpg 

 

Our programmable nucleases are the backbone of our broad set of genome editing tools. These novel nucleases including type II and type V Cas nucleases, of which some are ultra-small systems that we call SMall Arginine- Rich sysTems (“SMART”) nucleases, have unique targeting abilities and can be programmed by guide RNAs (“gRNAs”) to target and cut at specific locations in any genome sequence. Targeted genomic breaks trigger DNA repair pathways that can be used for genome editing, for example, to integrate a gene at a target site (knock-in) or for gene inactivation (knock-down).

Our toolbox contains thousands of CRISPR nucleases with diverse abilities to target different parts of the genome, allowing us to potentially select the ideal nuclease for targeting any given gene in a site-specific manner and overcome a major limitation of first-generation CRISPR/Cas9 systems.

We also modify our nucleases to either nick the genome (i.e., a nickase that cuts one strand of the DNA) or to simply bind to target sites (i.e., a nuclease dead variant). These capabilities (searching, cutting, nicking, and binding) can be leveraged as a chassis by adding on additional effector enzymes to create base editors for single nucleotide changes, RNA-mediated integration systems (“RIGS”) for both small and large genomic integrations using “Little RIGS” for prime editing and “Big RIGS” for large integrations. Using modular engineering, we match nickases with deaminases and RTs for base editing and RIGS, respectively. Furthermore, nucleases can be engineered by swapping the search modules of the enzyme to expand the targetability of the chassis, which is critical for site-specific genomic modifications. Given the measured targeting density of our toolbox, we believe that essentially any codon in the human genome could be addressed with our gene editing systems.

Our highly active nucleases have gone through extensive preclinical evaluation for both in vivo and ex vivo applications, with demonstration of broad potency of these systems across human primary cells, mouse, and nonhuman primate (“NHP”) models. Our base editors, RIGS, and CAST systems have demonstrated activity across various cell-based models. In addition to evaluating system activity, we have undertaken detailed characterization of guide-specific on-and off-target effects. We routinely identify guides that have no or minimal verifiable off-target editing, thus overcoming another limitation of first-generation CRISPR/Cas9 systems.

In addition to overcoming the activity, targetability, and specificity limitations of first-generation systems, our nuclease toolbox was designed to have broad compatibility with viral and nonviral delivery technologies. This compatibility is accomplished by having a variety of nuclease and gRNA structures, which range in terms of their size and biochemistry. For example, small guides for some type V Cas systems streamline manufacturing for delivery by lipid nanoparticle (“LNP”) approaches, and SMART nickases can be

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used to construct base editors that are small enough to fit within the packaging limitations of adeno-associated viruses (“AAV”). SpCas9, which is currently used in most base editing applications, is roughly three times the size of some of the smallest SMART nickases and cannot be efficiently packaged into a single AAV. Combined, we believe these features will facilitate delivery of our genome editing tools to previously inaccessible tissue types and organ systems.

While nucleases, base editors, and prime editors can precisely address a wide variety of genomic modifications required to treat disease, the fact that many diseases are caused by a multitude of mutations across a gene means that a diverse set of editing tools are required to fully address these patient populations. The integration of a complete and functional gene through targeted genome editing may provide a way in which every patient with a given disease could potentially be treated by a single genetic medicine. Big RIGS and CASTs are novel genome editing systems that are under development to achieve what has been a major challenge for the genome editing field — large, targeted genomic integrations. Initial preclinical readouts conducted in mammalian cells indicate that these systems could potentially have a major impact on how diseases caused by loss-of-function mutations, the most common cause of genetic diseases, can be addressed through genome editing.

Therapeutic translation roadmap and initial programs

We are taking a stepwise approach deploying our genome editing toolbox to develop potentially curative therapies for patients. Our lead programs are selected to both address important diseases and to establish new standards in targetability, precision, efficiency, and scope of editing capabilities. Figure 2 summarizes the portfolio of programs that we and our partners are advancing, as we aim to match the optimal genome editing tools for each indication. Each of these indications were chosen based on our conviction in the underlying biology, existence of validating preclinical and clinical data, availability of pharmacodynamic and translational tools to assess early proof-of-concept, relevant value supporting outcome measures, and ongoing clinical unmet need. While we do not currently have any approved products and all of our product candidates are preclinical, our lead programs capture an ever-growing set of translational learnings and insights that will inform and accelerate future programs.

Figure 2. Therapeutic Translation.

 

img190561166_1.jpg 

 

Hemophilia A—novel, durable, knock-in approach for expression of Factor VIII

Hemophilia A is the most common X-linked inherited bleeding disorder and is caused by mutations in the Factor VIII (“FVIII”) gene leading to loss of functional FVIII protein that impacts the body’s ability to form normal clots in response to injury. FVIII is normally produced in the liver within sinusoidal endothelial cells and is then secreted into the bloodstream where it acts as a cofactor for the catalytic activation of Factor X in the clotting pathway. The lack of functional FVIII disrupts the normal clotting cascade and predisposes patients to increased risk of bleeding, either spontaneously or in response to injury or surgery. Repeated bleeding episodes in joints or soft tissues can lead to progressive joint damage, inflammation, pain, and mobility impairment. Intracranial bleeding is of greatest concern as this can be rapidly fatal or lead to major morbidity.

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The standard of care for patients with severe hemophilia A, involves lifelong repeated intravenous (“IV”) infusions of recombinant FVIII preparations prophylactically and in response to bleeding events. The major limitation of this approach is fluctuating FVIII activity levels, with trough values that can still result in breakthrough microscopic and macroscopic bleeding events, particularly within sensitive and previously damaged joints. Additionally, frequent FVIII infusions are inconvenient, which can be associated with suboptimal compliance, and in some patients result in inhibitor formation (antibodies against FVIII) that compromise efficacy. More recently, emicizumab, a bispecific antibody, has been approved for hemophilia A in the United States. Valoctocogene roxaparvovec, the first hemophilia A gene therapy, was conditionally approved for use in Europe in August 2022 and was approved in the United States in June 2023. This genetic medicine delivers a FVIII gene construct to the liver using an AAV vector; however, longitudinal clinical data has demonstrated that FVIII levels drop over time. Importantly, AAV gene therapy is also not a feasible treatment approach for infants or children due to the high degree of liver growth during pre-adulthood that would dilute out the episomal FVIII levels during progressive rounds of liver cell division.

Rather than provide the FVIII gene in an episomal location, which risks dilution from cell division or cell death as well as episomal transcriptional silencing, our approach is to insert a FVIII DNA cassette into a “safe harbor location,” within an intron of the albumin gene that is not expected to have deleterious effects. FVIII expression is then driven off the strength of the native albumin promoter. This approach has previously been demonstrated in preclinical studies to lead to therapeutically relevant expression of a different clotting factor (Factor IX) with negligible impact to systemic circulating albumin levels. Our FVIII knock-in approach is designed to provide stable expression and clinically relevant circulating levels of FVIII, even at low integration rates because of the strength of the albumin promoter.

We have demonstrated the feasibility of the FVIII gene knock-in approach in mice with several mouse specific guides and different FVIII DNA donor cassettes, with integration of the FVIII gene leading to FVIII mRNA expression and therapeutically relevant levels of FVIII protein in the blood. In an ongoing NHP study we demonstrated integration of a surrogate cynomolgus-FVIII cassette (used to avoid immune response that would occur with a foreign human FVIII protein) and observed therapeutically relevant levels of the cyno- FVIII protein encoded by the integrated cassette in all 3 treated animals that has extended for 4.5 months following a single dose of the AAV-cFVIII virus followed five weeks later by a liver trophic LNP encapsulating the mRNA encoding MG29-1 and guide 2 at a dose of 1mg/kg body weight. We intend to continue measuring FVIII levels in these monkeys up to the 12 month time point to generate a robust data set on durability.

Evaluation of different human FVIII donor DNA cassettes has been completed in mice resulting in the selection of 2 lead cassettes that will be compared in another NHP study, potentially leading to a development candidate selection anticipated in mid-2024.

In parallel, we are manufacturing mRNA, gRNA, AAV and LNP to support future investigational new drug (“IND”) enabling studies.

Primary Hyperoxaluria, Type 1 (“PH1”)—a durable knock-down of HAO1 for substrate reduction therapy

PH1 is a rare autosomal recessive metabolic disease arising from loss of function mutations in the alanine- glyoxylate aminotransferase (“AGXT”) gene that encodes alanine glyoxylate aminotransferase. This enzyme is found in peroxisomes of the liver where it catalyzes the conversion of glyoxylate to glycine and pyruvate. Lack of functional AGXT leads to an accumulation of glyoxylate substrate, which is then converted to oxalate and excreted in the kidney. The excess urinary oxalate forms an insoluble complex with urinary calcium that leads to the production of calcium oxalate crystal precipitates. This pathologic process results in the formation of repeated calcium oxalate urolithiasis and nephrolithiasis, which in turn leads to obstructive uropathy, inflammation, fibrosis, tubular toxicity, and progressive loss of kidney function. PH1 is a serious disease that causes kidney failure. More than 70% of individuals with PH1 mutations will develop end-stage renal disease, with a median age in young adulthood.

Until recently, the standard of care for treating PH1 was primarily supportive in nature, with hydration and diuretics used to reduce urinary oxalate concentration, pyridoxine (vitamin B6) to enhance residual function of alanine glyoxylate aminotransferase catalytic activity, and hemodialysis once renal function progressed to end stage. More recently, the standard of care has been updated to include treatment with lumasiran, a small interfering RNA (“siRNA”) therapeutic approved in adults and children with PH1 that acts to reduce the levels of urinary oxalate. Using a therapeutic approach known as substrate reduction therapy, lumasiran targets mRNA from a separate gene, HAO1, that encodes glycolate oxidase (“GO”). Lumasiran has been generally well tolerated in clinical studies of adults and children with PH1 but as a siRNA therapy, it requires repeat subcutaneous administration indefinitely in order to maintain its effect. An additional RNAi drug, Nedosiran, which targets LDH, a different enzyme in the same pathway as HAO1, was also given FDA approval for adults and children with PH1 in October 2023.

The goal of our genome editing approach is to durably knock down HAO1 resulting in stable and permanent reduction of oxalate levels to effect a lifelong benefit. We have performed nuclease and guide screening to select an optimal nuclease and gRNA combination. Along with our partner ModernaTX, Inc. (“Moderna”), we have achieved preclinical proof-of-concept in an AGXT

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knock-out mouse which is an accepted disease model of PH1. We are in the final stages of confirming the candidate to take into NHP studies and expect to have NHP data in 2024 to support final development candidate selection.

Transthyretin Amyloidosis—a single treatment to knock-down TTR gene expression

Transthyretin amyloidosis is a disease of misfolded and aggregated transthyretin (“TTR”) protein that can deposit in tissues causing organ dysfunction, primarily in the heart and/or peripheral nerves. The TTR protein is normally produced in the liver and circulates in a homotetramer (four copies of the same TTR protein bound together) where it serves as a carrier protein for vitamin A and thyroxine. Certain mutations have been identified that can cause TTR homotetramers to fall apart, misfold, and aggregate into insoluble fibrils that deposit in cardiac tissue and peripheral nerves. However, more commonly, the normal aging process is associated with an increased propensity for TTR misfolding and aggregation in the heart without any known genetic sequence variation. These distinctions lead to TTR amyloidosis being characterized as either hereditary transthyretin amyloidosis (“ATTRv”) caused by mutations in TTR, or wild-type ATTR amyloidosis (“ATTRwt”). It is estimated that globally there are approximately 50,000 patients with ATTRv and between 300,000 and 500,000 patients with ATTRwt. Among the larger ATTRwt patient population, the most common presentation is a rapidly progressive, restrictive, and hypertrophic cardiomyopathy due to progressive deposition of insoluble TTR fibrils, which result in thickening of the myocardium and stiffening of the ventricles. These pathologic processes lead to impaired diastolic function and progressive cardiomyopathy that typically leads to progressive heart failure and often death within three to five years from disease onset. Although cardiac manifestations are more common and severe, patients with neurologic manifestations also experience significant morbidity, loss of functionality, and impaired quality of life.

Using our novel nucleases, we aim to provide efficient TTR knock-down and halt further deposition of amyloid fibrils. Previous experience suggests a clinical correlation between the degree of TTR knock-down and potential for benefit in familial forms of the disease, which are expected to translate similarly to wild type forms. The high degree of in vivo editing efficiency and specificity of our nuclease platform suggest the potential for a single treatment to knock-down TTR gene expression and remove the requirement for life-long therapy. Along with our partner Ionis Pharmaceuticals, Inc. (“Ionis”), we are currently in advanced stages of nuclease and guide selection, having achieved more than 90% knock-down of human TTR protein in a humanized TTR mouse model, and expect to move into NHP studies in 2024.

Further areas of therapeutic activity and interest

In parallel with our translation efforts in our lead programs using our novel programmable nucleases to knock-in or knock-down gene expression in liver-associated targets, we are developing more complex editing systems for liver associated targets as well as moving beyond the liver. Given that our genome editing toolbox contains small editing systems designed to be amenable to viral vector delivery, and given the progress established in targeting the central nervous system and muscle with established AAV capsids, our first extrahepatic indications will be neurodegenerative and neuromuscular diseases.

Building on our experience delivering our nucleases to the liver via LNP systems, we are extending that experience delivering novel RIGS to the liver to potentially correct ATP7B mutations in Wilson’s disease and PiZ mutations in alpha-1-antitrypsin deficiency (“A1AT deficiency”). We are also exploring addressing A1AT deficiency via a base editor approach given the predominant mutation involves a single base pair. Both of these liver diseases have well-defined biology, readily available translational biomarkers for early proof-of-concept, established development pathways based on prior drug approvals, and important unmet medical needs.

Building on our experience with our novel type II and type V programmable nucleases, we are extending that experience by working to deliver these nucleases via AAV to the central nervous system to potentially knock down genetic targets important for both spontaneous and familial amyotrophic lateral sclerosis (SOD1, ATXN2) and Charcot-Marie-Tooth Type 1a (PMP22). In addition, we are working to address a series of mutations common in Duchenne Muscular Dystrophy with our programmable nucleases through exon skipping approaches. In diseases outside of the liver, we intend to initially leverage known biology and clinical validation achieved with RNA-targeted approaches like antisense and siRNA to advance more potent and definitive one-time genome editing treatments.

Building on our experience with both knock-in gene expression and smaller gene corrections with RIGS, we are progressing our larger RNA- and DNA-mediated integration systems to potentially provide a single curative approach to cystic fibrosis. As opposed to currently-available therapies limited to subsets of patients with individual mutations, we intend to deliver a full copy of a functional cystic fibrosis transmembrane conductance regulator (“CFTR”) gene. This approach can similarly be pursued across many other diseases characterized by loss of function mutations.

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Our Team

We have assembled a world-class team that is driven by a passion to create potentially curative genetic medicines through the discovery of novel genome editing technologies by harnessing the power of metagenomics. Key members of our executive and leadership team include:

Brian C. Thomas, Ph.D., Chief Executive Officer and Founder, prior to co-founding the company, Dr. Thomas spent more than 20 years in academic research at UC Berkeley helping to pioneer the field of metagenomics. Dr. Thomas has been cited over 16,000 times and listed as an inventor in 28 patent families.
Jian Irish, Ph.D., MBA, President and Chief Operating Officer, has held biopharma executive leadership roles for nearly 20 years at Kite Pharma / Gilead, Sanofi, and Amgen in drug development and global operations, and has helped launch several breakthrough medicines.
Pamela Wapnick, MBA, Chief Financial Officer, has over 20 years of diversified financial leadership experience spanning strategic and operational finance roles at public and private companies including life sciences and biotechnology companies, Diality Inc, Capsida Biotherapeutics, True North Therapeutics and Amgen.
Sarah Noonberg, M.D., Ph.D., Chief Medical Officer, has spent more than 20 years in translational and clinical development leadership roles with a track record of advancing therapeutic programs from discovery to commercialization, including at Medivation and BioMarin.
Simon Harnest, M.Sc, Chief Investment Officer and SVP of Strategy, has held leadership roles in corporate finance and strategy in the life sciences sector, having raised over $1 billion in public and private capital, including leading Cellectis’ U.S. IPO and subsequent spin-out and IPO of Calyxt.
Luis G. Borges, Ph.D., Chief Scientific Officer, has over 27 years of experience in the biotechnology industry, including Amgen, Five Prime Therapeutics, Cell Medica, and Century Therapeutics, where he held leadership roles in the research and development of multiple therapeutic candidates.
Simren Delaney, Ph.D., LLM, VP of Intellectual Property and Legal Operations, is specialized in Intellectual Property and Patent law, having previously worked at Wilson Sonsini Goodrich & Rosati, and plays an instrumental role in driving the development of the company’s growing IP portfolio.
Matthew L. Wein, J.D., VP of Corporate Legal and Compliance, and Corporate Secretary, has spent more than 20 years in legal leadership positions at biotechnology companies, including Mustang Bio and Amgen.
Christopher T. Brown, Ph.D., VP of Discovery, is a former scientist at the Jillian F. Banfield laboratory at UC Berkeley and an expert in using metagenomics to discover novel microbial systems for use in genome editing. Dr. Brown’s research has resulted in over 35 publications and over 20 patent family filings.
Michael Conway, MBA, CPA, VP of Finance, has spent nearly 20 years in finance leadership positions at life science and technology companies, including Adamas Pharmaceuticals, InterMune, and Intel.
Alan Brooks, Ph.D., SVP of Preclinical, has worked on genetic medicines providing scientific leadership in translational research for more than 25 years, including at Casebia Therapeutics and Bayer Healthcare. Dr. Brooks’ research has led to 20 publications and numerous patent filings.

Our company is supported by our board of directors, Scientific Advisory Board, and a leading syndicate of investors, with more than 30 funds supporting our Series B preferred unit and Series B-1 preferred unit financings (collectively, the “Series B preferred unit financing”).

Our Strategy

Our goal is to harness the power of our proprietary metagenomics platform to create curative genetic medicines for patients. Key components of our strategy to achieve this goal include:

Leverage our leadership position in metagenomics to continually advance and expand innovative genome editing tools. We expect to build on the diversity and versatility of our toolbox through continuous interrogation of novel microbial genomic information, identification of highly active natural enzymes, design and optimization of genome editing systems, and continuous integration of learnings to accelerate development. In connection with these discoveries, we will continue to strenuously file and protect our intellectual property. Coupled with our trade secret protection around our discovery platform, our broad intellectual property estate creates a significant barrier to entry.

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Develop and deliver products that make precise modifications to the human genome to cure disease. We focus on disease areas with well understood disease biology, readily available translational biomarkers for early proof-of-concept, clear development pathways, and important unmet medical need. We are taking a stepwise approach deploying our genome editing toolbox to develop potentially curative therapies for patients. Along with our development efforts using our novel programmable nucleases to knock-in or knock-down gene expression in liver-associated targets, we are leveraging our toolbox to deliver more complex editing systems to targets in and outside the liver. Our approach allows us to systematically incorporate knowledge and insights from our initial development programs, thereby accelerating therapeutic translation across our genome editing technologies.
Build a fully integrated genome editing company. Our team includes experts in discovery, preclinical and clinical development, encompassing all major functions necessary to take a molecule from target identification through registrational clinical trials. To rapidly translate editing technologies into genetic medicines, we strategically invest in automation, characterization, and manufacturing capabilities. This applies not only to process development and manufacturing for clinical trial materials, but also high throughput automated screening and genome sequencing, and state-of-the-art characterization assays. We believe our ability to develop and characterize complex human genome editing components is essential to pursue a successful regulatory pathway for genetic medicine development.
Expand therapeutic impact to patients through continued investment in business development and enabling partnerships. We carefully consider opportunities for business development such as collaborations and partnerships with industry leaders that have unique strengths and we may pursue additional partnership opportunities which complement our technologies, with the objective of accelerating our programs and pushing forward our therapeutic translation efforts. Our existing partnerships with Moderna, Ionis, and Affini-T Therapeutics, Inc. (“Affini-T”) demonstrate our thoughtful approach to collaborating with industry pioneers to accelerate and optimize the development of our genetic therapeutic candidates.
Maintain our entrepreneurial outlook, scientifically rigorous approach, and culture of tireless commitment to patients. We are a team of experienced drug discoverers, developers, and company builders who are united by our mission and passion to unlock the full potential of genome editing for patients with high unmet needs. We are dedicated to attracting and retaining top talent and partnerships at the intersection of academia and industry. We are unwavering in our commitment to deliver cutting-edge technology and unlock the long-awaited, transformative potential of genome editing.

Introduction to Genome Editing and Limitations with Current Approaches

Genome editing is a new treatment modality that has the potential to revolutionize healthcare by creating permanent, one-time treatments that address disease at the genomic level. Genome editing involves the alteration of genetic material of a living organism by inserting, replacing, converting, or deleting nucleotides within the DNA. Several approaches and technologies are being studied and developed in order to perform these edits, including:

Nuclease-based genome editing: Several genome editing methods rely on a class of enzymes called nucleases to create double-stranded breaks in DNA at a targeted location to cause gene inactivation, gene insertion, or alter gene splicing. Examples of nucleases include CRISPR associated nucleases, zinc finger nucleases (“ZFNs”), engineered meganucleases, and transcription-activator like effector nucleases (“TALENs”). The discovery and characterization of a particular nuclease, Cas protein 9 from Streptococcus pyogenes (“SpCas9”), has been leveraged to develop a number of different therapeutic approaches. Importantly, additional novel and distinct Cas nucleases exist in nature and have the potential to be developed into tools for genome editing. When introduced at target sites in a genome sequence, genomic breaks trigger DNA repair pathways that can be used for genome editing. If a DNA template is provided, the DNA repair machinery may incorporate the sequence at the site of the genomic break, resulting in a site-specific knock-in. If not, the cut will lead to the disruption of a gene sequence and subsequent knock- down of the encoded protein.

Base editing: Base editing is a genome editing approach that relies on using deaminases to chemically convert specific nucleotides in a genome. Deaminases are enzymes that catalyze chemical reactions to remove an amino group. Multiple programmable nuclease platforms, such as CRISPR nucleases, have been harnessed for base editing by using the programmable nature of these enzymes to direct deaminases to specific genomic target sites. In these cases, the nuclease activity is deactivated, thus creating a nicking or nuclease-dead version that does not disrupt the ability of the enzyme to be programmed to target specific genomic sites for editing. There are two primary types of base editors: adenine base editors (“ABEs”) and cytosine base editors (“CBEs”). ABEs convert adenine-thymine base pairs to guanine-cytosine base pairs. CBEs target cytosine-guanine and convert them to thymine-adenine.

RNA-mediated integration, including prime editing: RNA-mediated integration systems (“RIGS”) are genome editing systems that make programmable genomic modifications that are encoded in RNA templates. Because the modifications are encoded in RNA, these systems have the ability to repair diverse mutations, including insertions, deletions, and all types of point mutations. These systems rely on RTs to convert messages encoded as RNA into DNA. CRISPR systems are used to direct RTs to genomic target sites. Some systems use a nickase to create a target-specific site that primes the activity of the RT and results in the corrected genomic sequence encoded in the RNA to be incorporated into the genome. Prime editing can be accomplished with RIGS, as can large, targeted genomic integrations.

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DNA-mediated integration, including CAST: CASTs are a class of genome editing systems that provide directed and programmable genomic integration of large DNA templates. CASTs are naturally occurring systems that have been engineered to accomplish large integrations for genome editing in various cell types and for therapeutic applications. The systems consist of a catalytically dead Cas effector that can be programmed by gRNAs to target a transposase to integrate large DNA cargos into specific genomic target sites. DNA-templated integrations can be accomplished with other transposase and recombinase systems; however, these systems typically require extensive protein engineering in order to alter their targetability, or need to be used in concert with other genome editing tools such as prime editing systems in order to incorporate targeting motifs into specific genomic sites.

There have been significant advancements in genome editing since the seminal research that led to the discovery of CRISPR SpCas9 and its application in humans. However, there remain key limitations that must be addressed to unlock the full potential of genome editing. We believe the key limitations facing current genome editing platforms are:

1)
First-generation technology lacks the ability and flexibility for accomplishing complex genome editing. The majority of genome editing platforms are limited to a single genome editing approach, such as gene insertions/deletions, single nucleotide changes, or small gene corrections. As a result, they are faced with inherent limitations including the diversity of edits in which they can employ and, as a result, an inability to address a range of diseases. In addition, they lack the flexibility to tailor their genome editing system to a broad range of genomic targets of interest.
2)
Lack specificity and control over resulting edits. Current genome editing platforms have a narrow armamentarium of genome editing systems and therefore limited access to systems capable of high activity and specificity at desired target sites. This lack of control and specificity is often measured by “off-target” edits which can pose a risk for undesirable side effects or unexpected safety findings.
3)
Size of current genome editing technologies limits in vivo delivery methods and target organs. First- generation SpCas9 systems are about 1,300 amino acids (“aa”) in length and as such are not feasible to package into many delivery vectors such as AAV. As such, their delivery is largely limited to LNP systems, which precludes delivery to many tissues outside of the liver.
4)
Inability to access certain sequences in the genome. SpCas9 is only able to target DNA sequences which contain a flanking sequence of “NGG”, restricting the range in genetic targets it can be programmed to locate, and subsequently limiting the ability to address certain underlying mutations that drive disease.
5)
Substantial engineering requirements. Limited access to highly active natural nucleases and effectors drives the need for substantial modifications to make a system operate at therapeutically-relevant levels, resulting in long lead times from discovery to candidate nomination.
6)
Narrow terms of license agreement from academic institutions. The majority of genome editing platforms have been formed as a result of a licensing agreement for specific genome editing systems or technology from academic institutions and are therefore limited to the confines of that technology arrangement. Alternatively, genome editing tools developed by us are built from highly novel components derived from our metagenomics database, and thus are not subject to these constraints.

In order to address these broad challenges with current genome editing approaches, we have leveraged our deep expertise with metagenomics to develop a proprietary discovery platform that is designed to continuously identify novel editing systems and optimize our expansive editing toolbox. Starting at the microbial level, our multifaceted platform enables discovery beyond nucleases, translating highly active natural enzymes into powerful genome editing systems optimized for efficiency and specificity.

Our Metagenomics Platform

We believe genome editing tools with capabilities that go beyond the current technology landscape will be required in order to treat the vast majority of genetic diseases. Our goal is to unlock the full potential of genome editing by developing tools with new capabilities using novel cellular machinery discovered from the natural environment. Our company was founded by Brian C. Thomas and Jillian F. Banfield, pioneers in the field of metagenomics. They recognized the power that naturally evolved microbial systems could have in revolutionizing access to enhanced genome editing technologies to create potentially highly efficacious and curative genetic medicines. Our metagenomics discovery platform is foundational to our business and therapeutic pipeline. This platform enables us to rapidly and effectively find and engineer highly active natural enzymes sourced from nature into genome editing tools that are highly specific, efficient, and have enhanced targetability.

CRISPR systems, having been studied for decades, are known to be ubiquitous in the microbial world. However, it was not until the recent discovery and characterization of CRISPR-SpCas9 that it became possible to use these systems as tools for genome editing. SpCas9 is only a single representative of the CRISPR systems that exist in nature, leaving open the potential to identify and develop new genome editing tools through continued and systematic discovery efforts. The vast majority of microbes, including bacteria, archaea, viruses, fungi, and single-celled eukaryotes, are extremely difficult, and potentially impossible, to study using traditional laboratory methods. We aim to address the limitations of traditional laboratory methods through a process whereby microbes are

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recovered from the natural environment and studied based on their genetic blueprint- their genome sequences. This approach has supported the ability to characterize the extent of CRISPR biology on the planet, and to expand beyond CRISPR to identify a vast collection of novel enzymes and other cellular machinery.

Samples from diverse climates and geographies have been used to build a metagenomic database that is continuously analyzed via high-throughput screening that utilizes AI and proprietary algorithms to direct our discovery efforts. This continuous genome mining process generates expansive libraries of novel systems, including nucleases, deaminases, RTs, and CAST systems (together, “effector enzymes”), that make up the foundational building blocks necessary to assemble a modular, novel genome editing toolbox that can be harnessed to make precise changes to the human genome to address a variety of important diseases with curative intent. We have simultaneously developed a modular approach to engineering that involves interchanging key components in a high-speed process to translate the discovery of novel nucleases and other effector enzymes into optimized genome editing systems with substantially reduced translation time compared with previously described systems. We estimate that our metagenomics platform and modular engineering process has resulted in the discovery of over 20,000 novel genome editing systems, to which we are seeking coverage through our pending patent portfolio. These systems span hundreds of novel nuclease families and fuel a growing genome editing toolbox.

Mining Our Natural Environment to Create an Expansive Genomics Database

We aim to harness the power of the metagenome by using our continuous genome mining process to assemble a broad, diverse library of novel genome editing nucleases and other effector enzymes. This is accomplished by studying all the DNA from microbial communities at the same time. Our mining process begins with proprietary sampling in which our scientists collect samples from diverse climates and geographies to build a database that spans broad biodiversity including, but not limited to our local natural environments and extreme environments such as from high-altitudes, high-temperatures, and hydrothermal vents below the ocean. Samples collected in a natural environment may contain billions of cells representing tens of thousands of distinct species. Every sample collected from a natural environment is subjected to deep DNA sequencing and bioinformatics analysis to identify and functionally analyze recovered microbial genomes for the discovery of genetic elements of interest.

Through our metagenomics process, DNA is extracted from these environmental samples and sequenced in order to reconstruct the genomes of the resident microbes. The process of DNA extraction and sequencing results in the fragmentation of each individual microbial genome into small sections which are blended into a backdrop of all other sequences present. Due to the clonal replication of the organisms in microbial communities, multiple copies of nearly-identical genome sequences are recovered, and the overlap between these sequences provides the information needed to reconstruct the original genomes. This complex process is only the beginning. Once reconstructed, it is necessary to predict the function of each section of the newly reconstructed genomes. Our platform has facilitated the discovery of vast sequences and functional components that, to our knowledge, have never been published before. These novel components have resulted from the unique selective pressures that microbes face from different environments and that drive immense genomic diversification. The evolutionary process of natural selection provides highly optimized enzymes that require little, if any, protein engineering. The novelty of the genomes recovered from metagenomics requires the de novo prediction of genes, proteins, non-coding RNAs, and other essential features. This is challenging and necessary due to the fact that previously studied ‘reference’ sequences do not provide enough information to sufficiently guide this process. Considering that these evolutionary processes have been at work for billions of years, there is considerable genetic diversity to mine for the development of highly-optimized genome editing tools. Our industry-leading database of novel microbial genetic sequences provides the basis for our discovery process. To date, our continuous genome mining process has analyzed over 460 trillion base pairs of DNA sequence, an amount of data roughly equivalent to what would be required to sequence hundreds of thousands of human genomes, and resulted in the prediction of over 7.4 billion proteins and over 1.75 million CRISPRs – including over 322 million Cas proteins.

Leveraging AI and High-Throughput Screening to Identify Novel Editing Systems

To interrogate our metagenomics database, our platform combines AI, including machine learning, and proprietary algorithms run on expansive cloud computing infrastructure to screen for novel CRISPR nucleases and other effector enzymes at high speed. The screening process consists of high-throughput sequencing that allows us to quickly analyze large amounts of genomic data and subsequently synthesize the identified components, building blocks, into functioning CRISPR nucleases and other effector enzymes that can be interrogated biochemically and in mammalian cells. Once extensively characterized, novel nucleases and other effector enzymes are categorized into our ever-expanding library. Through the metagenomics-driven discovery process we have characterized one of the largest known libraries of novel nucleases, which we estimate includes 20,000 novel genome editing systems from hundreds of nuclease families. We have also assembled a robust library of other effector enzymes to design and engineer the next generation of genome editing systems that are fit for essentially any therapeutic purpose. Our effector enzyme library includes over three million deaminases, over five million RTs, and thousands of CAST systems, as shown in Figure 3 below. The continuous interrogation of an ever-expanding database of programmable nucleases and other enzyme effectors accelerates the pace of learning and insights to feedback into proprietary machine learning algorithms and further separates us from peer companies. As we continue

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to build upon our metagenomic library, we expect to make additional discoveries of novel genome editing technology and expand our toolbox.

Figure 3. Our Effector Enzyme Library.

 

img190561166_2.jpg 

 

From this library, we select our lead nucleases and other effector enzymes through a funnel of stringent performance and safety criteria that involves testing in a series of cell-free, cell-based, and in vivo experiments. Ultimately, we prioritize editing systems that exhibit high editing efficiency and precision, and have a compact size that will optimize their delivery. Because most systems added to our genome editing toolbox have different genomic targeting specifications, we believe that our expanding toolbox could enable us to target every base pair in the human genome. This process, highlighted in Figure 4, helps us to identify highly active natural enzymes that require minimal engineering and optimization to translate into potentially curative genetic medicine.

Figure 4. Overview of High-Throughput Screening Process.

 

img190561166_3.jpg 

 

Modular Engineering Translates Metagenomic Discoveries into Genetic Medicines

Our metagenomics platform and modular engineering process has supported the discovery and development of our broad genome editing toolbox at a rapid pace since our company’s founding in 2018. By selecting highly active nucleases from our library, our process requires minimal optimization to develop genome editing systems. We utilize a modular engineering approach to match lead nuclease candidates with an optimal gRNA and targeting domain in order to optimize targeting, specificity, and editing efficiency. Furthermore, additional effector enzymes can be included to modify the function of the system, for example by adding a deaminase to a nickase variant for base editing.

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In order to achieve these modifications, we leverage our vast library of editing systems to perform targeting domain swaps between enzymes, substituting domains from less-active enzymes into the backbone of highly- active nucleases. This results in chimeric enzymes that can provide high efficiency editing at gene loci.

The modular engineering process is accelerated using in silico screening algorithms to predict the optimal chimera. This predicative, high-speed engineering process allows us to continually iterate across each component of the genome editing system to quickly develop an engineered system that is optimized for various therapeutic applications.

Modular engineering can be used to adjust the targeting of high-performing systems by leveraging the diverse targeting capabilities of diverse Metagenomi nucleases. Given the measured targeting density of our toolbox, we believe that essentially any codon and ultimately every base pair in the human genome could be addressed with our gene editing systems.

Genome editing remains in the early stages of development and our platform allows us to continuously learn, iterate, and optimize our genome editing toolbox in pursuit of curative genome editing medicines. The increasing insights from our modular engineering and target domain swapping are captured and further interrogated and organized by our proprietary AI platform to accelerate the pace of future development and further separate us from our peers. As the genetic medicine field continues to rapidly evolve, our platform positions us to be at the forefront of unlocking the full potential of genome editing through the continuous discovery of new editing systems and the development of the next wave of genetic medicines.

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Our Solution: Proprietary Toolbox Derived from Our Metagenomic Approach

Our Platform of Genome Editing Technologies

 

Gene Edit

Metagenomi (“MG”) Tools

Key Attributes

 

Type II Nucleases

Extensive targetability (Alexander et al 2023, Lamothe et al 2023)
Can be converted to nickases for base editing and RIGS

Double-strand DNA break (e.g for knock-down, knock-in, gene activation, and exon skipping)

Type V Nucleases

Systems with small gRNAs (Goltsman et al 2020)
Includes ultra-small systems that expand delivery approaches (e.g., AAV)

 

SMART Nucleases

Ultra–small systems expand delivery approaches (e.g., AAV) (Goltsman et al 2022)
Can be converted to nickases for base editing and RIGS

Nucleotide changes

(i.e. base editing)

ABE and CBE

Engineered from MG nickase and MG deaminase
CBE also include MG uracil glycosylase inhibitor (“UGI”)
Base editors engineered from type II have extensive genomic targetability
SMART base editors are smallest nickase-based systems characterized to date

Small changes (1-100 base

pair replacement, insertion,

or deletion,

i.e. prime editing)

Little RIGS

Engineered from MG nickase and MG RT
Ultra-small RTs are highly active and accurate for prime editing
Enabled by extensive targetability and deliverability of MG nickases

 

RNA-templated: Big RIGS

Engineered from MG nickase and MG RT
MG RT are accurate, and can convert >4,000 bp RNA templates into DNA
Programmable integration of large transgenes delivered as RNA

Large insertions

(>100 base

pair integrations)

DNA-templated: CAST and other systems

CASTs are naturally occurring programmable transposase systems
Ability to site-specifically integrate large transgenes delivered as DNA, possibly including templates much larger than 4,000 bp
Potential to addresses any genetic disease driven by loss of function

 

All components of our toolbox have been discovered and derived from our proprietary metagenomics library. We have assembled a full suite of differentiated tools that, together, can potentially effectuate any desired gene modification – gene knock-down, gene knock-in and replacements. We believe there is no other single platform enabling the breadth and differentiation of genome editing technologies as our toolbox. All elements of our toolbox are wholly owned, and we have constructed a broad patent estate that protects our intellectual property, and it will continue to expand as we discover, interrogate, and optimize our novel editing systems. As we continue to expand our metagenomic library, we expect to make additional discoveries of novel genome editing technology. The core technologies in our toolbox to date are outlined in the above table.

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Key Attributes of Our Proprietary Toolbox

Key advantages of our platform and technologies are:

1)
Potential to create a full spectrum of genetic medicines – Our broad suite of genome editing technologies include: programmable nucleases, base editors, RIGS and CASTs, that, together, can potentially effectuate any desired modification to the genome – gene knock-down, gene knock-in, and replacements. This allows us to address a diverse set of mutations by matching the right tool to a specific target, with limited unintended effects such as off-target editing. As such, we intend to prosecute a genetic medicine therapeutic development strategy across a broad array of diseases and target organs including liver, central nervous system, muscle, kidney, and lung.
2)
Potential next generation genome editing systems – Our scientific underpinnings based in metagenomics provide a continuous engine for discovering and developing potential next generation genome editing systems. For example, RIGS and CAST. As we continue to build upon our metagenomic library we expect to expand our toolbox as we make more discoveries. We have constructed a broad patent estate that protects our intellectual property, and it will continue to expand as we discover, interrogate, and optimize our novel editing systems.
3)
Ultra-small nuclease platform to expand in vivo delivery of multiple genome editing systems – Compact systems create potential advantages for delivery, manufacturing, and dosing. For example, at 429 aa in length, one of our SMART nucleases is a fraction of the size of the industry-standard SpCas9 system, which is 1,300 aa and exceeds the delivery capacity of standard AAV vectors. The ability to package our systems into a single AAV will enable more efficient targeting of organs and diseases beyond what is currently possible with LNP delivery.
4)
Designed to edit any target in the human genome – Our metagenomics library contains hundreds of nucleases with diverse targeting abilities that allow us to address a diverse set of mutations that cause disease, including those found at sites that often cannot be targeted by first-generation nucleases. This allows us to select the ideal nuclease for any target site of interest. Given the measured targeting density of our toolbox, we believe that essentially any codon in the human genome could be addressed with our gene editing systems.
5)
Shortened optimization period – We benefit from a diverse set of highly-active nucleases and effectors which have required little -if any- protein engineering to optimize. These highly active natural enzymes allow us to quickly identify systems for therapeutic development. As a result of not having to heavily engineer systems to be active in human genome editing applications, we are able to move quickly from discovery to candidate nomination for particular genetic disease applications.
6)
Ability to target large gene integrations into the genome using our RIGS and CAST systems – Our novel RIGS and CAST systems allow for programmable, large gene insertions, an outcome which has been a major challenge for the genome editing field. RIGS are a proprietary genome editing system engineered from nickases and RTs, while CAST are systems that exist in nature but have required engineering to allow for mammalian genome editing. We believe we are the first to demonstrate targeted genomic integration in human cells using compact CAST systems. While CAST have the theoretical capability to integrate very large DNA templates into the genome, RIGS are also being developed in order to achieve targeted, large genomic integrations when all components need to be delivered as RNA, for example when using standard LNP delivery technology.

Specific Components of Our Toolbox

Programmable Nucleases

Overview

Figure 5. Schematic Showing Programmable Nucleases and Their Use for Genome Editing.

 

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We are building a toolbox that includes programmable nucleases that are selected to target any site in the human genome with high precision. Most therapeutic genome editing applications to date use CRISPR/SpCas9 as the programmable nuclease. However, this system has several limitations that prevent its broad use across the thousands of genetic diseases that impact patients. Most importantly, SpCas9 is limited by where it can be targeted in the human genome and in some cases has a lack of specificity that leads to frequent off-target editing. In addition, the size of the SpCas9 enzyme complicates options for delivery using industry-standard

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methods. We have explored diverse programmable nucleases found in nature in order to identify novel systems that overcome all of these limitations (Figure 5).

The discovery of new CRISPR enzymes with unique functionality and structure may offer the potential to further disrupt genome editing technologies, improving speed, specificity, functionality, and ease of use. CRISPR systems are commonly organized into two classes, six types and an expanding number of subtypes based on functional characteristics and evolutionary relatedness. We focus our attention on CRISPR type II and type V systems, owing to the simplicity of these programmable nucleases. Type II and type V nucleases are RNA-guided enzymes that can be programmed to target specific sequences of DNA, and sometimes RNA. Unlike type II nucleases, type V nucleases are more likely to generate staggered versus blunt-end cuts in double stranded DNA. Nucleases from both of these systems are extensively used in biotechnology, despite limitations that prevent their more widespread use in therapeutic applications. Our type II and type V CRISPR systems are distinct from previously studied CRISPR enzymes (based on their protein sequence, size, and biochemistry), while maintaining the core functionality of being programmable nucleases. Our lead systems have undergone extensive study across multiple mammalian cell types and animal models, demonstrating their utility as genome editing tools in both in vivo and ex vivo applications.

Throughout our search for novel programmable nucleases, we have identified several new types of ultra-small nucleases that range in size from approximately 450 aa to 1,000 aa, compared with type II and type V nucleases which are typically between 1,000 aa and 1,500 aa in length (for comparison, the most studied SpCas9 enzyme is 1,368 aa). Our newly-discovered, ultra-small nucleases include new types that we collectively refer to as SMART, as well as those that come from novel type V sub-groups. Given their small size, these new systems have the potential to be delivered to additional therapeutic target sites that expand beyond what is possible with first-generation systems. In adapting these novel systems into potential precision genetic medicines, our programmable nucleases are designed to have the capability to target essentially any therapeutically relevant genomic site with a high level of specificity (i.e. with limited off-target editing), while expanding compatibility with available delivery technologies.

Our Approach

To date, we have identified thousands of novel CRISPR type II, type V and SMART nucleases, including ultra- small systems, expanding the collection of known programmable nucleases by mining our proprietary metagenomics database. Using high-throughput in vitro testing, we have validated the activity of hundreds of novel nucleases. This has led to the identification of highly-active natural nucleases while also enabling us to catalog the unique targeting capabilities of each system. We select for natural, un-engineered systems with high activity and specificity. Our best-characterized nucleases have demonstrated activity levels meeting and often greatly exceeding SpCas9 while exhibiting low levels of off-target editing in mammalian cells. Given the measured targeting density of our toolbox, we believe that essentially any codon in the human genome could be addressed with our gene editing systems. The targeting density (frequency of targetable sites in the human genome) of the toolbox greatly increases the likelihood of identifying a highly active and specific nuclease and gRNA combination for any therapeutically relevant genomic target. We can use these programmable nucleases for genome editing, and also as a chassis for developing base editors and RIGS. The diverse biochemistry of these novel systems, including their size and DNA cutting profiles, makes it possible to link editing systems to the particular genomic target, edit type, and delivery technology we believe are required to develop genetic medicines. Additional discovery and characterization of programmable nucleases will further expand the targeting density of our toolbox, improving the overall activity and specificity of lead systems identified for therapeutic development.

Our best-characterized type II and type V nucleases are suitable for a wide variety of genome editing applications

Nucleases with the activity and specificity required for potential therapeutic genome editing applications are identified through a series of high-throughput screening and characterization steps. Novel protein and gRNAs are first predicted bioinformatically and then validated in both cell-free and mammalian cell screens. These steps validate the activity of a system and provide an accurate measure of targeting and specificity profiles. This approach has been applied to both type II and type V CRISPR nucleases in order to establish an expanding collection of well-characterized systems. In addition to discovery of novel nucleases with distinct targeting capabilities, a modular protein engineering approach is also used in order to create chimeric systems. The chimera approach leverages the high activity of top-performing systems but changes where they can edit by incorporating distinct targeting domains from related systems. Through continuous discovery and refinement, this collection of systems will expand, with the potential to target essentially every site in the human genome, while also creating a chassis to support the accelerated development of base editing, prime editing, and targeted integration systems.

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Given the measured targeting density of our toolbox, we believe that essentially any codon in the human genome could be addressed with our gene editing systems.

Figure 6. Our Expanding Nuclease Toolbox is Designed to Enable Extensive Targeting throughout the Human Genome.

 

Figure 6a. Our nucleases have shown high editing efficiency in mammalian cells.

 

Figure 6b. Given the measured targeting density of our toolbox, we believe that essentially any codon in the human genome could be addressed with our gene editing systems.

 

 

 

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* Editing efficiency was determined based on the frequency of InDels detected by next generation sequencing (“NGS”) at genomic sites targeted by each nuclease.

 

* Graph depicts cumulative targetability.

 

 

* Graph depicts editing efficiency for the top five guides for each of six nucleases.

 

* Targeting density (targetability) is the average distance between nuclease target sites in the human genome.

 

 

 

Our nucleases are capable of highly efficient genome editing in mammalian cells. Systems are tested across a collection of gRNAs designed based on the unique requirements and targeting preferences determined for each system. If a nuclease is routinely capable of editing at near-saturating levels, sometimes up to 99% editing efficiency, the system will go through further characterization and therapeutic development. Figure 6a shows the high editing efficiency of seven systems observed in mammalian cells, indicating that they have the potential for broad use in therapeutic development given an ability to rapidly identify high performing guides that require minimal optimization.

Given that each nuclease has distinct genomic targeting capabilities, we are able to determine which sites in the human genome can be addressed by each system. Figure 6b shows how the targeting density of our toolbox increases with each new system. Given the measured targeting density of our toolbox, we believe that essentially any codon in the human genome could be addressed with our gene editing systems. This collection of systems includes both natural enzymes as well as chimeric forms that have been engineered to alter the targetability of the nuclease. For reference, SpCas9 is able to target roughly every ten base pairs in the human genome. The targeting density of the toolbox increases the likelihood of identifying a highly-potent guide for any desired genome edit.

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Our nuclease toolbox is on track to target anywhere in the human genome

Figure 7. Our Nucleases are Compact with Diverse Targeting Sequence Motifs, Selected to Enable Broad Genome Editing Applications.

 

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* Graph depicts a selection of our nucleases, including their size in aa on the horizontal axis and their corresponding targeting motifs shown as sequence logos (PAM and TAM sequences), compared to SpCas9.

Multiple parameters are considered when screening and promoting nucleases for further development, including the size of the system, activity across various assays and cell types, and targeting. For CRISPR systems, nuclease targeting is limited by a PAM sequence, or protospacer adjacent motif. The PAM is a short DNA sequence motif that must be present next to a target sequence in order for the nuclease to cut at the target site. Other systems have similar requirements that go by different names. For example, some SMART nucleases recognize functionally similar sequences called target-adjacent motifs (“TAMs”). Figure 7 shows the diversity of protein sizes and targeting motifs recognized by our nucleases. The vast diversity of the protein size and targeting motif requirements of our nucleases continues to fuel toolbox development and will enable the identification of additional systems that could make it possible to target nearly every base pair in the human genome.

Our nucleases are highly active across many human cell types which may indicate broad potential utility for human therapeutic applications

Beyond initial nuclease benchmarking conducted in immortalized mammalian cell lines, we have conducted extensive surveys to show that these systems can be used in a variety of primary cells important for preclinical studies (i.e., T cells, natural killer (“NK”) cells, B cells, hematopoietic stem cells (“HSCs”) and induced pluripotent stem cells (“iPSCs”)), as illustrated by Figure 8 below. The versatility of these exemplary systems indicates their potential use in broad therapeutic applications. These benchmarks against SpCas9 also indicate that our systems may have potency advantages in which fewer genome editing reagents will need to be delivered in order to achieve high levels of genome editing.

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Figure 8. Our Nucleases Show High Editing Efficiency in Human T Cells, NK Cells, B Cells, HSCs, and iPSCs.

 

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* Two example nucleases are shown, type II MG3-6 and type V MG29-1.

* Editing efficiency was determined based on the frequency of InDels detected by NGS at genomic sites targeted by each nuclease.

Our nucleases exhibit high specificity when benchmarked in multiple mammalian cell types

Our therapeutic development of genome editing systems require that they exhibit high activity and specificity across multiple cell-based models. We employ a broad set of specificity and off-target assessment methodologies, including in silico, biochemical, and cell-based approaches. Figure 9 shows peer-reviewed and published off-target assessments conducted using an unbiased, industry standard, cell-based, oligonucleotide capture method. In Figure 9a we show that two of our nucleases are more specific than SpCas9 when compared across multiple guides, and in Figure 9b we show that we can identify guides with no to minimal detectable off-targets with two lead nucleases tested in both immortalized and primary human cells. This trend has continued, and we are able to identify guides that have minimal or no detectable off-target activity for therapeutic targets. It is expected that the higher specificity of these systems will translate into better safety profiles across various therapeutic genome editing applications.

Figure 9. Our Nucleases are Highly Specific.

Figure 9a. Our nucleases are highly specific in multiple mammalian cell types.

 

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Figure 9b. Our best-characterized nucleases have guides that showed no to minimal detectable off-target edits when assayed in immortalized and primary human cells.

 

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* Figure 9a: Off-target analysis showed that our type II MG3-6 and MG3-8 nucleases have high specificity in multiple mammalian cell types as measured by the number of off-target sites and the fraction of on-target reads. The target sites are the same between the three nucleases, including SpCas9. Off-target experiments were conducted at N = 2, and reads for both replicates were summed for analysis. Double strand break (“DSB”) discovery via capture of a double-stranded oligonucleotide in primary T cells using the same MG3-6 and MG29-1 guides from (A; averaged across three biological replicates). Source: Alexander et al 2023 CRISPR Journal

* Figure 9b: DSB discovery via capture of a double-stranded oligonucleotide in HEK293T cells with SpCas9 (on target read in gray), guides MG3-6 TRAC B2, TRAC D2, TRAC 6, and GR 3 (on target in blue), and MG29-1 guides TRAC 9, TRAC 19, TRAC 35, and GR 13 (on target in yellow) across three biological replicates. Source: Lamothe et al 2023 CRISPR Journal

Our nucleases enable highly efficient and specific in vivo genome editing in preclinical evaluations

Type II MG3-6/8 is our chimeric nuclease that has been extensively characterized in vitro and in vivo. This system is engineered from the MG3-6 chassis, which was discovered from the genome of a commensal, non-pathogenic representative of the human microbiome that showed high editing efficiencies when tested across multiple mammalian cell types (see Figures 6, 8 and 9 above). The engineering involved changing the PAM interacting domain of the chassis, which enables the enzyme to be targeted to new sites in the human genome. Figure 10 shows that this system is suitable for in vivo applications, based on saturating levels of editing achieved in a mouse study with a single administration of nuclease mRNA and guide encapsulated in a LNP with tropism to the liver.

Figure 10. In Vivo Editing in Mice with MG3-6/8 Nuclease.

 

Editing efficiency was determined based on the frequency of InDels detected by NGS at genomic sites targeted by each nuclease.

 

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* Delivery by mRNA and LNP, dosing at 1 mg/kg.

* Mean of 79% genome editing efficiency as measured by the frequency of InDels detected by NGS at the targeted genomic site.

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In addition, one of our most highly characterized type V nucleases, MG29-1, demonstrated high activity and specificity during multiple preclinical studies spanning from in vitro to NHP. Originating from a bacterial genome found in a deep sea hydrothermal vent, MG29-1 has a smaller protein (1,280 aa) and gRNA (~70 nt) compared with MG3-6 (1,135 aa and ~110 nt) and SpCas9 (1,368 aa and ~100 nt). MG29-1 has demonstrated up to 97% editing in primary mouse hepatocytes in culture (Figure 11a), superior liver editing to an exemplary SpCas9 guide targeting an overlapping genomic site when delivered to mice using a LNP that delivers primarily to the hepatocytes in the liver (Figure 11b), and on average 50% editing in the whole liver of NHP when delivered in a LNP that delivers primarily to the hepatocytes in the liver (Figure 11c, guide 2). Approximately 76% of the genomes in mouse liver are from hepatocytes based upon a published analysis of gene editing in whole liver compared to hepatocytes isolated from the same edited mice. Using this conversion factor of 76% (1/0.76), we achieved 100% editing of hepatocytes with MG3-6/3-8 (Figure 10) and 94% editing of hepatocytes with MG29-1 (Figure 11b).

In cynomolgus monkeys, it is estimated that hepatocytes make up 60% of the cells in the liver. Therefore, 50% editing in the whole liver achieved with MG29-1 in NHP (Figure 11c, guide 2) translates to editing of approximately 80% of the hepatocyte genomes which are the therapeutically relevant target cell.

Figure 11. Our Type V MG29-1 Nuclease is a Highly Active Natural Enzyme for In Vivo Genome Editing.

 

Figure 11a. MG29-1 achieved saturating levels of editing in primary mouse hepatocytes.

 

Figure 11b. MG29-1 has higher potency than SpCas9 when editing mouse liver cell genomes in vivo.

 

Figure 11c. MG29-1 demonstrates successful editing in NHPs.

 

 

 

 

 

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* 50% whole liver editing = 80% hepatocytes editing.

 

* MG29-1 and SpCas9 were delivered by LNP with co-formulated mRNA and gRNA.

* gRNAs for both MG29-1 and SpCas9 have overlapping target sites.

* mRNAs for both nucleases use the same overall design and were codon optimized with the same algorithm.

* The SpCas9 gRNA incorporated extensive optimizations published in the literature.

* InDels were analyzed by NGS four days after IV infusion.

* L, M, H, refer to low, medium and high doses.

We use multiple unbiased, industry standard methods to identify putative off-target edits, which are then investigated using sensitive targeted sequencing. To date no detectable off-target editing has been observed for the MG29-1 lead guide for two of our therapeutic programs at therapeutically relevant doses, which is consistent with MG29-1 being a highly efficient, specific and programmable nuclease suitable for broad in vivo genome editing applications. Beyond MG29-1, the multitude of nucleases in our toolbox enables us to screen an extensive guide library for any genomic locus of interest. For exemplary lead guides that have been examined for off-target editing we observe minimal or no detectable off-target editing.

Our ultra-small nucleases are efficient genome editors that unlock additional delivery modalities

Delivery of genome editing payloads is often restricted by the size limitations of the delivery vehicle. Therefore, we leverage our metagenomics database to find new nucleases that are significantly smaller than current systems. After searching through billions of predicted proteins from bioinformatically reconstructed microbial genomes, we uncovered several distinct types of ultra-small nucleases. Our lead ultra-small nucleases are significantly smaller than CRISPR SpCas9, enabling new and improved in vivo delivery methods and new possibilities for building base editing systems and prime editors that can be packaged in size-constrained delivery vehicles. Typical CRISPR Cas9 systems are about 1,300 aa in length whereas some of these new systems are just over 450 aa. Compact systems create potential advantages for delivery, manufacturing, and dosing. Furthermore, they can be delivered to organs and tissues currently only accessible by AAV, given that the nucleases are well-within the packaging limitations of these delivery vectors. The smaller size of these nucleases compared to previous systems potentially unlock target indications beyond applications in

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the liver. Because of their novel biochemistry and divergence from typical type II, we refer to some of these ultra-small nucleases as SMART. In addition, we identified several novel families of ultra-small type V nucleases. Unlike compact type V, and all other previously studied type V, SMART have a dual catalytic domain structure that enables engineering of nickase variants that can be used for base and prime editing.

Figure 12 shows that genome editing efficiencies achieved for SMART and compact type V nucleases are comparable to other lead systems. These results show that our ultra-small nucleases are highly efficient genome editing systems that have the potential for extensive therapeutic applications, including for those in which delivery is a limiting factor.

Figure 12. Our Ultra-Small Nucleases are Designed to be Highly Efficient Genome Editing Systems that Are Well- Within the Packaging Limits of AAV Vectors.

 

Figure 12a. Schematic representation of how our ultra- small nucleases are a fraction of the size of a typical CRISPR SpCas9 nuclease.

 

Figure 12b. SMART nuclease showed high editing efficiency in mammalian cells, prior to optimization.

 

Figure 12c. Compact type V nuclease showed high editing efficiency in mammalian cells, prior to optimization.

 

 

 

 

 

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* InDels are measured by NGS.

* Each bar represents a distinct guide.

* MG3-6 used as internal control.

 

* InDels are measured by NGS.

* Each bar represents a distinct guide.

 

Base Editors

Overview

Figure 13. Schematic Showing the Two Primary Types of Base Editors under Development.

 

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We leverage our toolbox of programmable nucleases to develop a highly targetable and flexible base editing platform compatible with various delivery technologies. Base editing modifies individual bases in the genome without making double-stranded breaks in the DNA. This approach uses a chemical reaction designed to create precise, predictable, and efficient genetic outcomes at the targeted sequence. These precise changes to individual base pairs in the genome can be used to correct or change genomic sequence in order to address disease. Notably, the most common class of genetic mutations are errors of a single base, known as point mutations. These point mutations, many of which could be addressed with a base editor, represent approximately 58% of all the known genetic errors associated with disease. Furthermore, base editors can be used to precisely knock-down genes by introducing premature stop codons or interrupting gene splice sites.

There are two types of base editors: ABEs and CBEs. ABEs convert adenine-thymine base pairs to guanine- cytosine base pairs. CBEs target cytosine-guanine and convert them to thymine-adenine. We are developing both ABEs and CBEs, which have been validated across multiple mammalian cell types and in vivo, to enable broad use of the base editing approach for addressing disease (Figure 13).

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Base editors are composed of a targeting enzyme, typically a programmable nuclease that has been engineered to localize to a specific site in the genome but not cause a dsDNA break, and a deaminase, which is responsible for the chemical conversion of targeted genomic bases. Base editors can be engineered using modified nickase programmable nucleases that have been engineered to nick rather than cut genomic targets (i.e. a nickase variant). The nickase recognizes and binds to specific DNA sequences, determined by a gRNA, enabling deaminases that are fused to the nickase to modify the targeted bases. Typically, the modified bases are found on the single-stranded DNA that is exposed at a target site when the gRNA is bound to one of the strands of DNA. The efficiency of cytosine base editors can be improved by the addition of another enzyme called a UGI. The UGI protects edited bases against DNA repair machinery that would otherwise remove them and revert the edited sequence back to its original form.

Base editing results in one or more mismatched bases in the dsDNA, which are resolved by DNA repair mechanisms in the cell. Nickases are typically used such that the DNA strand opposite to the modified bases is nicked, thus biasing DNA repair pathways to favor the modified strand over the original sequence. The result is a precise change in one or more targeted bases without creating double-stranded breaks or requiring a donor template.

Our Approach

We are leveraging our toolbox of programmable nucleases and our metagenomics discovery platform to develop next generation base editing tools. Our programmable nucleases provide the ability to efficiently and precisely target locations in the genome required in order to potentially create a wide-variety of genetic medicines. In addition, the availability of ultra-small SMART effectors enables us to develop base editors that could be delivered to various target tissues and organs. While established base editing systems using SpCas9 require splitting the system into two AAV vectors for delivery, those developed from SMART are designed to be within the packaging limitations of a single AAV. We have discovered and engineered what we believe to be two of the smallest base editors developed to date, one SMART ABE that is 623 aa in length and another SMART ABE that is 969 aa. These systems provide substantial opportunities for vector optimization compared with SpCas9 ABEs that are 1,588 aa. Furthermore, the flexibility of the modular, chimeric nuclease platform, enables rapid optimization of base editors capable of editing at a multitude of target sites, since engineering and optimization applied to one base editor can rapidly be applied to other systems with the capability of targeting to new genomic sites.

Given that base editors require deaminases, and a UGI in the case of cytosine base editors, substantial enzyme discovery beyond programmable nucleases has been required in order to develop highly efficient base editors that have the potential to overcome the limitations of the current technology. We have mined over three million deaminases in order to identify enzymes with the capability to function as part of base editing systems. In many cases these novel deaminases have required minimal protein engineering to achieve therapeutically-relevant editing efficiencies in multiple studies. The ability of our metagenomics discovery platform to identify novel enzymes with high activity makes it possible to rapidly develop differentiated technology that surpasses first- generation systems.

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Our base editors are highly active in mammalian cells across various genomic targets

Figure 14. ABE and CBE Developed from Type II MG3-6 System Have Been Shown to be Highly Active in Mammalian Cells, with Extensive Genome Targetability to Enable Potential In Vivo and Ex Vivo Therapeutic Development.

 

Figure 14a. PAM interacting domain engineering was used to develop a suite of chimeric MG3-6 base editors with extensive genome targetability.

 

Figure 14b. ABE screening identified guides capable of achieving saturating levels of A-to-G genome editing in mammalian cells.

 

 

 

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* ABE: SpCas9 = 18% and MG3-6 = 95% targetability.

* CBE (not shown): SpCas9 = 5% and MG3-6 = 53%.

 

* Optimized ABE construct tested in Hepa 1-6 cells with mRNA delivery.

 

 

 

 

 

 

Figure 14c. CBE screening identified highly active deaminases for C-to-T editing, compared to published control.

 

Figure 14d. MG3-6 PAM chimera ABE and CBE achieved efficient editing at splice-sites for enacting therapeutically-relevant knock-out of genes in human cells.

 

 

 

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* Unoptimized CBE construct tested in HEK293T cells with plasmid delivery.

* CBE control uses hyperactive CDA A0A2K5RDN7.

 

* Top three guides shown for each target and system.

* Constructs tested in HEK293T cells with mRNA delivery for the BE along with chemically synthesized guides.

 

 

 

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Figure 14e. MG3-6 CBEs are efficient for multiplexed knock-outs in primary human T cells with no impact on cell viability.

 

 

 

 

 

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* Top panel shows two guides that were used to knock-out two different proteins, either when edited independently or when multiplexed.

 

* Experiments were conducted in primary human T cells using mRNA delivery for the BE along with chemically synthesized guides.

 

 

 

Our base editors are highly active and progressing towards in vivo therapeutic applications. Both of our ABE and CBE systems have been tested in mammalian cells in order to determine editing efficiency, where a large collection of guides spanning various genomic loci were tested for each system. Figure 14 shows the development of a highly efficient and targetable base editing platform established from the MG3-6 nuclease chassis. Using PAM interacting domain engineering, the theoretical targetability of the ABE and CBE was shown to be significantly greater than that of typical base editors developed from SpCas9. Based on PAM availability the ABE was able to target over 95% of the adenine bases in the human genome, and the CBE was able to target over 50% of the cytosine bases, compared with approximately 18% and 5%, respectively, for SpCas9.

Furthermore, over 95% editing efficiency was achieved with both an optimized ABE and CBE construct delivered by mRNA. Initial CBE testing without construct optimization using a less-efficient plasmid delivery approach achieved up to 58% editing efficiency, which was notable because it outperformed an industry-standard control CBE by several fold in the benchmarking experiment shown in Figure 14. We demonstrated that these efficient ABE and CBE systems could be used for creating edits that have the potential to result in protein knock-down, which could be applied for therapeutically relevant editing in vivo. Furthermore, we believe the observed CBE multiplexing in primary T cells shows that this platform has the potential to advance ex vivo cell therapy development by making it possible to efficiently knock-out multiple proteins without impacting cell viability.

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Our base editing platform has broad in vivo therapeutic potential

Figure 15. Lead ABE System Developed from Type II MG3-6 System Demonstrated High Activity in Primary Cells and in an in vivo Mouse Model.

 

Figure 15a. Our lead ABE system efficiently edited primary mouse hepatocytes at example target sites.

Figure 15b. Our lead ABE system demonstrated activity in vivo when delivered to mouse livers by mRNA and LNP.

 

 

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In order to demonstrate the utility of the MG3-6 ABE platform for in vivo base editing, a lead construct design was tested both in primary cells and in mice. Multiple guides were tested in each study, designed to target different genomic loci. Figure 15 shows the results of these evaluations of the ABE platform, where up to 60% editing efficiency was demonstrated in primary hepatocytes and 35% efficiency was achieved across all liver cells in an initial in vivo study using mRNA and LNP delivery. Given that approximately 70% of liver cells are hepatocytes, and therefore targetable by the LNP platform, this editing experiment suggests that approximately 50% of all targetable cells were successfully edited. We believe this level of editing, which is comparable to other initial in vivo base editing studies, supports the potential to rapidly progress towards therapeutic applications. Given that both the ABE and CBE data shown in Figures 14 and 15 are based on a chimeric MG3-6 nickase, we anticipate that the broad targetability of this system will enable rapid development of multiple potential new therapeutic applications of base editing technology.

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Our SMART have been engineered to create ultra-small base editors, expanding the potential deliverability of base editing technology

Figure 16. SMART Can be Engineered into Ultra-Small Base Editors with Naturally High Editing Efficiency and AAV Compatibility.

 

Figure 16a. Ultra-small SMART base editors are a fraction of the size of a typical Cas9 system, enabling AAV delivery.

 

Figure 16b. SMART ABE exhibited a similar editing profile in vitro compared to SpCas9.

 

Figure 16c. SMART ABE were active in mammalian cells.

 

 

 

 

 

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* SMART ABE-MG34-1 base editing at four genomic targets loci in E. coli. vs. reference SpCas9 system.

 

* Heatmap values represent the mean of two independent experiments.

 

* SMART ABE achieved up to 22% base editing efficiency in human cells.

 

* Base editing efficiency shown at three genomic targets tested in human HEK293T cells.

 

* Heatmap values represent the mean of three independent experiments.

 

 

 

 

 

 

 

 

* The target sequence for each locus is shown above the heatmap.

 

* Heatmaps represent the percentage of NGS reads supporting an edit at each position.

 

 

 

Source: Goltsman et al 2022 Nature Communications

Figure 16d. SMART ABE optimization improved editing efficiency in human cells.

 

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* Top guide shown for each SMART ABE system.

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Figure 16 illustrates how our ultra-small SMART nucleases provide an opportunity to develop compact base editors with the potential to improve delivery using existing AAV technology. Our engineered, but unoptimized SMART ABE systems were observed to edit target loci at levels comparable to reference SpCas9 systems, and with up to 22% editing efficiency in mammalian cells. Achieving this level of efficiency with an unoptimized system, comparable to initial base editing readouts achieved with SpCas9, indicates the potential of this novel CRISPR platform. Additional optimization enabled editing in human cells at over 80% efficiency. The SMART ABE highlighted in Figure 16 is one of the smallest nickase-based systems with activity in mammalian cells (969 aa), and at 623 aa another SMART ABE under development is even smaller. Together, these systems provide us with unique opportunities to optimize base editors using the naturally compact, precise, and programmable SMART platform.

RNA-Mediated Integration Systems: RIGS for prime editing and large genomic integrations

Overview

Figure 17. Schematic Showing Application of RIGS for Small Replacements and Large Integrations.

 

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Programmable nucleases, which use dsDNA breaks to trigger DNA repair machinery to enact specific genomic changes, and base editors, which use chemical modifications to convert specific base pairs in the human genome, cannot address all mutations that cause disease. RIGS are being developed in order to encode any type of genomic modification in an RNA template, and thus create any type of genome modification necessary to address a disease (Figure 17). RIGS involve using RTs to convert genomic corrections encoded in RNA into DNA, and are engineered with programmable nucleases or nickases to incorporate newly synthesized DNA messages into the genome at specified target sites. Importantly, any genomic modification can be encoded in the RNA template. Furthermore, the potential for having an all-RNA format for the system, including delivery of the protein components as mRNA, could simplify delivery for some applications. For example, the all-RNA format could enable use of LNPs for delivery of systems to the liver for large, targeted genomic integrations. The enzymatic nature of the genome integration, combined with the ability to deliver the entire system as RNA is of particular importance when considering large integrations, since the delivery of DNA templates at concentrations required for integration mediated by DNA repair machinery can be toxic.

One implementation of RIGS is to use our programmable nickases and RTs for prime editing. Prime editing can repair diverse mutations, including all types of point mutations, deletion mutations, insertion and duplication mutations and insertion-deletion mutations. As with other genome editing approaches, prime editing systems, and RIGS more broadly, create permanent modifications at natural genomic locations, resulting in durable edits that are passed on through cell divisions and that are expressed under natural mechanisms specific to the gene or target of interest. One key aspect of prime editing is the modification of a CRISPR gRNA to create a pegRNA (“prime editing gRNA”). In this design, the typical backbone and targeting components of the gRNA are maintained; however, additional sequence is added to the guide in order to code for a desired genomic correction and to prime a RT to begin synthesizing new DNA sequence. During prime editing a portion of the pegRNA containing the genomic modification is copied into DNA at a specific site in the genome sequence. Once the RT has incorporated the new DNA sequence into the target site, DNA repair

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machinery will finalize the genome edit by removing the corresponding section of the original sequence, synthesizing any required complementary DNA, and ligating the ends of the nicked DNA strands. We have discovered a collection of novel RTs that can be combined with our nickases to perform prime editing in mammalian cells at levels and with accuracy that surpasses industry-standard systems.

Current prime editing and RNA-templated editing approaches are limited by the size of the RNA template that can be incorporated into a genomic site. This limitation is based on multiple aspects of the system design, including 1) the need to encode gRNA and RT template sequences in a single pegRNA, and 2) the processivity and fidelity of the RT. The first constraint can be addressed by creating new system designs in which the gRNA and repair template are encoded in separate RNA molecules, where additional engineering ensures that the templates and RT are able to co-localize to the nucleus, and more specifically, to targeted sites in the genome. Regarding the second point, processivity and fidelity are biochemical characteristics that relate to the ability of an RT to traverse through large and structurally complex RNA templates (processivity) and to do so without introducing errors (fidelity). We have identified and developed novel RTs that we believe surpass the processivity and fidelity requirements for therapeutic delivery of transgenes as RNA templates, for example in order to treat diseases by introducing a complete and correct copy of a gene in order to overcome a loss-of-function mutation. Furthermore, we are engineering these RTs with programmable nucleases and nickases to achieve large, targeted genomic integrations.

Our Approach

Similar to base editing, development of RIGS leverages our expansive platform of programmable nucleases that can be converted into nickases, including ultra-small SMART, as well as a metagenomics discovery approach that enables the rapid identification of novel RTs that have the specific characteristics necessary for different genome editing applications. The targetability of our toolbox of programmable nucleases will enable essentially any target sites of interest to be addressed, and compatibility with ultra-small SMART effectors could expand the deliverability of RIGS to therapeutically relevant tissues and organs.

Little RIGS describes systems used for prime editing (e.g., for small genomic replacements such as transversions, transitions, insertions, and deletions), while Big RIGS describes systems capable of making large targeted genomic integrations. The mechanisms driving these systems differ from one another, but both are based on using a reverse transcriptase to incorporate genomic corrections encoded in RNA into target sites in the genome identified by a programmable nickase or nuclease. We identified five million RT candidates from novel families in order to find systems for Little and Big RIGS development. RTs found in nature are highly diverse, but typically do not have all the characteristics suited towards being useful in a therapeutic context. Based on measured enzyme characteristics such as fidelity and processivity, RTs are nominated and combined with our programmable nucleases/nickases for either Little and/or Big RIGS engineering. Our Little RIGS are benchmarked against industry-standard prime editing systems in order to rapidly identify top-performing systems. Currently, we believe no genome editing system has been described that is comparable to Big RIGS. Our Big and Little RIGS are undergoing indication-specific optimization. We believe that this combination of discovery and system optimization will enable the rapid development of best genome editing systems that, together, can enact any type of genomic edit.

Our RIGS have shown complex prime editing in mammalian cells

Figure 18. Our RTs Outperformed Benchmarks for G-to-T Prime Editing in Mammalian Cells. Graph Depicts a Broad Range of RTs that Exhibited Higher Natural Activity than Unengineered Prime Editing RT.

 

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* Unengineered prime editing control RT is from Moloney murine leukemia virus (“MMLV”).

* RTs were cloned into a plasmid backbone that was co-transfected with chemically synthesized pegRNAs designed to edit the genome of human HEK293T cells.

* Genomic DNA was isolated 72 hours post transfection and target loci were amplified for NGS to evaluate editing outcomes.

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Our RTs are benchmarked against natural and engineered variants of an RT widely used for prime editing in order to identify highly active natural enzymes for further optimization along with our programmable nickases. Figure 18 shows that our natural RTs are more active than a control RT when used for prime editing. In addition, our minimally engineered RTs demonstrated up to 6 times higher activity than the unengineered control RT for a G-to-T change. This indicates that we can potentially optimize gene editing activity quicker than comparable prime editing RTs. Figure 19 shows that a minimally engineered Metagenomi RT has editing efficiencies equivalent to or better than engineered, industry-standard prime editing RT. Notably, this has been achieved with an ultra-small RT that is only 251 aa in length, compared with the 671 aa of the standard prime editing RT. For therapeutic applications, complex corrections must be precise, and the precision of RIGS depends on the unique characteristics of the reverse transcriptase used. Figure 19 also shows that one of our RTs has demonstrated significantly higher editing accuracy compared to the engineered, industry-standard control. In order to measure optimal activity for new systems, multiple primer binding sequence (“PBS”) lengths must be tested. In some case, Metagenomi RT are more active across a broader range and with smaller PBS lengths compared with the control, providing more flexibility around pegRNA design. We believe our RIGS are distinguished from typical systems used for prime editing by the high efficiency and accuracy of the RTs, as well as the broad targetability of our programmable nickases.

Figure 19. Our Ultra-Small RTs are Efficient and Accurate Systems for Conducting Complex Genomic Corrections in Human Cells.

 

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* The engineered prime editing benchmark RT is the MMLV variant used in PE2 prime editing systems.

* RT were tested with an unoptimized plasmid delivery system that enables high-throughput benchmarking of novel RT to identify leads.

* Plasmids encoding RT and Cas were co-transfected with chemically synthesized pegRNAs designed to edit the genome of human HEK293T cells.

* Genomic DNA was isolated 72 hours post transfection and target loci were amplified for NGS to evaluate editing outcomes.

Figure 19d. 5 bp Replacement

 

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* The engineered prime editing benchmark RT is the MMLV variant used in PE2 prime editing systems.

* Our ultra-small MG RT was fused to MG Cas and tested with mRNA delivery and chemically synthesized pegRNAs designed to edit the genome of human HEK293T cells.

* Co-transfection with chemically synthesized guides designed to program the Cas to create a nick in the vicinity of the edit were included to bias DNA repair towards the edited strand.

* Genomic DNA was isolated 72 hours post transfection and target loci were amplified for NGS to evaluate editing outcomes.

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Our novel RTs are selected to enable the development of Big RIGS for RNA-templated, large, targeted therapeutic transgene integration

Figure 20. Our RTs Have Been Observed to be Active, Processive, and High-Fidelity.

 

Figure 20a. Our RTs were more processive than RT typically used for prime editing, enabling transcriptions of large gene templates in mammalian cells.

 

Figure 20b. Our RTs were more accurate than industry-standard RT.

 

 

 

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* Prime editing benchmark RT is MMLV.

 

* Processivity was measured by quantitative polymerase chain reaction (“qPCR”) quantification of cDNA produced in mammalian cells from 4 kb templates.

 

* Typically, less than 1% of 4 kb templates are fully transcribed by MMLV.

 

* Template modifications are N1-methylpseudouridine.

 

* Fidelity was measured by NGS.

 

Figure 20c. Our RT can be combined with our Cas to achieve targeted integration of >900 bp in human cells.

 

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* Targeted integration confirmed by Sanger sequencing.

* Our MG RT was combined with MG Cas and tested using mRNA delivery along with chemically synthesized gRNA and the RNA integration template.

* Integration was targeted to an engineered landing pad located in the genome of HEK293T cells.

Our RTs were screened based on their activity and processivity in mammalian cells in order to identify highly active systems capable of reverse transcribing RNA templates over 4,000 base pairs in length (>4 kbp). We chose the 4 kb benchmark as many potential therapeutic targets could be addressed with a genomic integration of this size (current prime editing systems are limited to RNA-templated integration around 100 bp). In Figure 20, our best-characterized RTs were compared with industry standard systems in order to benchmark their activity, processivity, and fidelity. When tested in mammalian cells, our RTs were routinely able to fully transcribe a 4 kbp template into DNA. In Figure 20a, we show that our RTs demonstrated orders of magnitude more processivity than control RT when tested on these large templates, which we believe is a strong indication of being able to convert therapeutically relevant templates delivered as RNA into DNA for genomic integration. Importantly, Figure 20b also shows that on average our RTs transcribed over 4 kbp without introducing errors. Furthermore, template modifications (N1-methylpseudouridine), which could enable delivery of RNA templates less likely to trigger an immune response, improved fidelity. We are engineering our RTs along with our programmable nucleases and nickases for targeted genomic integration of a donor template delivered as RNA. Initial readouts show that when combined with a CRISPR effector and targeting gRNA, Metagenomi RT are able to incorporate newly synthesized DNA from an RNA template into a target site. In Figure 20c, we report what we believe to be the first-ever targeted integration of >900 bp in human cells with all-RNA delivery. In principle, these systems, which represent a major step forward in the genome editing space, could be delivered entirely as RNA and could enable large, targeted exogenous gene integrations.

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DNA-Mediated Integration Systems: CAST and other approaches to achieving large genomic integrations

Overview

Figure 21. Schematic Showing Application of CAST for Large, Targeted Genomic Integrations.

 

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Genome editing approaches based on nucleases, base editing, and prime editing are capable of precisely modifying the genome to address disease. However, for therapeutic approaches that necessitate expression of an exogenous gene or complete gene correction, large integration approaches are needed. Many individual diseases are associated with a wide variety of genetic mutations and thus may require an entire healthy gene to counteract each of the many different underlying causes. For example, there are over 1,800 mutations in the CFTR gene associated with cystic fibrosis. While in theory most of these mutations could be addressed using distinct and mutation-specific base or prime editing systems, this would require the optimization and translation of a large number of genome editing therapies. Alternatively, integration of a complete and correct copy of the CFTR gene could potentially cure patients with varying mutations in a one-and-done treatment. Directed DNA integration has largely been considered the ultimate goal of corrective genome editing, where enzymatic systems with this capability could provide safe and sustained therapeutic protein expression. By developing DNA-mediated integration systems and RIGS, we are at the forefront of creating a new class of genome editing therapeutics.

Many efforts over the past decades have sought to achieve direct DNA integration in order to develop treatments that work across diverse tissues, cell types, and genetic variants. Established transposase and lentiviral systems are efficient at inserting large DNA cargos into the human genome but result in non-specific and sometimes hyperactive integrations that have led to severe adverse events during clinical trials. Furthermore, recombinases have gained attention recently as a possible solution, given that they are able to incorporate large (>10 kbp) genomic cargos in a non-random, site-specific manner in mammalian cells. However, these systems typically require additional genome editing tools, such as prime editing systems, in order to first install recognition sites into the genome. These recognition sites are required by the recombinase and thus control where the enzyme can incorporate new genomic material.

CASTs are a new technology that is exciting because of their programmable, site-specific, and enzymatic integration capabilities. While these systems have been challenging to translate for mammalian cell and human therapeutic applications, we have had recent breakthrough success by demonstrating that the most compact type of CAST, based on catalytically dead nucleases, are capable of programmable and targeted DNA integration into the genome of human cells. Our CASTs are being developed in order to enable large (>10 kbp), targeted genomic integrations for therapeutic applications. This technology has the potential to address a large collection of complex genetic diseases driven by a loss of function mutation, such as cystic fibrosis.

Our Approach

We are pursuing multiple approaches to achieve targeted, large genomic integrations, including both RNA and DNA templated systems. Unlike RNA-templated systems that undergo a copying mechanism in order to integrate into the genome, DNA templated systems orchestrate the direct mobilization of the template into the genome. This direct integration avoids copying mechanisms that may be inhibited by certain template features, and thus allows for the incorporation of much larger templates compared with RNA-mediated systems. Given that RNA-templated systems may have delivery advantages for some applications, we are developing both technologies to have the broadest potential to address diseases through targeted integration of large transgenes.

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The development of our novel CAST systems was made possible by the discovery and engineering of active natural variants from our metagenomic library. CASTs are being developed to achieve accurate and efficient integration of large DNA cargos at a target locus, without depending on dsDNA breaks. CASTs are unique in that they combine the programmability of a CRISPR effector with the enzymatic integration capability of a transposase. We hypothesized that activity in human cells could be accomplished by combining novel system discovery with systematic development designed to tune the systems to the mammalian nuclear environment. We conducted a detailed survey of newly-discovered CAST systems and demonstrated the ability of novel systems for programmable integration of a transgene in vitro and in E. coli. The high efficiency of our systems enabled us to demonstrate directed transposase activity into single copy, safe harbor loci in the genome of human cells, as shown in Figure 21. Our results will enable the rapid development and optimization of CASTs to address unmet therapeutic and biotechnological needs. We believe we are the first to achieve this milestone using the most compact variety of CASTs.

We are also developing systems based on recombinases and other mobile elements that can be used in combination with programmable nucleases, including catalytically dead variants, as well as with Little RIGS in order to affect large, targeted genomic integrations under circumstances where CASTs are not the ideal genome editing technology. Beyond therapeutic use, novel systems with these capabilities could enable synthetic biology, antibody discovery, functional genomics, animal model development, and various other unmet needs in the biotechnology space.

Our novel and engineered CASTs are capable of integrating large DNA cargo at a safe-harbor locus in the human genome

We have shown preclinical proof-of-capability for our CASTs by demonstrating targeted integration of a large DNA template in the genome of mammalian cells. Translation from bacteria to human cell editing required protein engineering to mitigate the complex coordination between multiple protein and nucleotide components. Figure 22 shows polymerase chain reaction (“PCR”)-based confirmation achieved by detecting the junction between the donor DNA template and the target sequence in the genome of HEK293T cells. Targeted integration was achieved by delivering the CAST system as an all-in-one plasmid, while the DNA donor is delivered on a second plasmid. In addition, NGS was used to both confirm and quantify integration efficiency, which showed that about 1.5% of sequencing reads had evidence of target-specific integration. We believe the ability to target payloads to a single copy, safe-harbor locus now allows for further therapeutic-driven optimization and development.

Figure 22. Our CASTs Demonstrated Targeted Integration of a DNA Template at a Safe-harbor Locus in the Genome of Human Cells.

Figure 22a. Targeted integration confirmed by PCR.

 

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* PCR of junction between template and target confirms integration at target loci in HEK293T cells.

* Successful transposition is indicated by a band of the correct size in the gel in treated samples, but not in the null target (control) sample.

* Sequencing of PCR products from the junction between the template and target confirmed integration.

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Figure 22b. Integration efficiency measured by NGS.

 

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* On-target integration efficiency measured by NGS sequencing.

* Quantified integration efficiencies are shown as the mean (bar) of six AAVS1 or three “No Target” biological replicates (dots) with one standard deviation (whisker). “No Target” represents the non-targeting spacer (negative control), AAVS1 is the targeted safe-harbor locus.

Future Novel Editing System Discoveries

Our scientific underpinnings based in metagenomics provides a continuous engine for discovering and developing potentially next generation genome editing systems. As we continue to build upon our metagenomic library, we expect to make additional discoveries of novel technology, and expand our toolbox to further unlock the field of genome editing.

Our Pipeline

We are taking a stepwise approach deploying our genome editing toolbox to develop potentially curative therapies for patients. Our lead programs are selected to both address important diseases and to establish new standards in targetability, precision, efficiency, and scope of editing capabilities. Figure 23 summarizes the portfolio of programs that we and our partners are advancing, as we aim to match the optimal genome editing tools for each indication. Each of these indications were chosen based on our conviction in the underlying biology, existence of validating preclinical and clinical data, availability of pharmacodynamic and translational tools to assess early proof-of-concept, relevant value-supporting outcome measures, and ongoing clinical unmet need. Our lead programs capture an ever-growing set of translational learnings and insights that will inform and accelerate future programs.

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Figure 23. Therapeutic Translation.

 

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Hemophilia A—novel, durable, knock-in approach for expression of Factor VIII

The Disease

Hemophilia A is the most common X-linked inherited bleeding disorder and is caused by mutations in the FVIII gene leading to loss of functional FVIII protein that impacts the body’s ability to form normal clots in response to injury. FVIII is normally produced in the liver within sinusoidal endothelial cells and is then secreted into the bloodstream where it acts as a cofactor for the catalytic activation of Factor X in the clotting pathway. The lack of functional FVIII disrupts the normal clotting cascade and predisposes patients to increased risk of bleeding, either spontaneously or in response to injury or surgery. Repeated bleeding episodes in joints or soft tissues can lead to progressive joint damage, inflammation, pain, and mobility impairment. Intracranial bleeding is of greatest concern as this can be rapidly fatal or lead to major morbidity.

The severity of hemophilia A is directly correlated to the amount of residual FVIII activity. Severe hemophilia is defined as less than 1% of normal FVIII activity, moderate hemophilia defined as 1-5% of normal FVIII activity and mild hemophilia defined as 5 to 40% of normal FVIII activity. There are estimated to be nearly 30,000 patients with hemophilia A in the United States and more than 500,000 patients with hemophilia A globally. Of these, approximately 60% have severe disease and are at the greatest risk of spontaneous life-threatening bleeding events. In these patients, diagnosis typically occurs in infancy due to exaggerated bleeding in response to minor injury or routine medical procedures. As the inheritance of hemophilia A is X-linked, the vast majority of patients are male.

Limitations of Current Approaches

The standard of care for patients with severe hemophilia A involves life-long repeated IV infusions of recombinant FVIII preparations prophylactically and in response to bleeding events. The major limitation of this approach is fluctuating FVIII activity levels, with trough values that can still result in breakthrough microscopic and macroscopic bleeding events, particularly within sensitive and previously damaged joints. Additionally, frequent FVIII infusions are inconvenient, which can be associated with suboptimal compliance, and in some patients result in inhibitor formation (antibodies against FVIII) that compromise efficacy. More recently, emicizumab, a bispecific antibody, has been approved for hemophilia A in the United States that acts as functional FVIII mimetic in binding Factors IXa and X to support catalytic activation. This antibody approach has the benefit of a longer half-life than typical recombinant FVIII protein infusions that allows for less frequent administration but has the drawback of not being a true FVIII protein replacement therapy and breakthrough bleeding has been reported. Both the bispecific antibody and FVIII protein replacement approaches have a high economic burden (estimated lifetime cost of $15 to 18 million per patient).

Valoctocogene roxaparvovec, the first hemophilia A gene therapy, was conditionally approved for use in Europe in August 2022 and was approved in the United States in June 2023. This genetic medicine delivers a FVIII gene construct to the liver using an AAV

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vector. Once transduction of liver cells occurs, the FVIII gene resides in an episomal state (meaning not integrated in the genome) where it is transcribed from an artificial engineered exogenous promoter to produce FVIII mRNA which is translated into FVIII protein. This gene therapy approach has the potential benefit of constant production of FVIII protein by the liver; however, longitudinal clinical data has demonstrated that FVIII levels drop over time. To date, repeat dosing of a gene therapy has not been possible due to the production of high titers of neutralizing antibodies to the AAV vector. Importantly, AAV gene therapy is also not a feasible treatment approach for infants or children due to the high degree of liver growth during pre-adulthood that would dilute out the episomal FVIII gene during progressive rounds of liver cell division. Thus, there continues to be a significant unmet need in hemophilia A for a curative therapy that can provide life-long protection from bleeding events and joint damage in adults and children.

Our Approach and Results

Experience with early hemophilia A gene therapy approaches suggest that morbidity and mortality from the disease can be markedly reduced by achieving only moderate amounts of stable FVIII expression. For instance, achieving stable FVIII activity above 5-10% of normal activity level has the potential to convert a patient with severe hemophilia at risk of catastrophic bleeding into a patient only at risk for bleeding in the setting of major trauma or surgery. Achieving stable levels of FVIII above 15% activity level could provide complete joint protection and may allow patients to have a functional cure from their disease.

Rather than provide the FVIII gene in an episomal location, which risks dilution from cell division or cell death as well as episomal transcriptional silencing, our approach is to insert a FVIII DNA cassette into a “safe harbor location,” within an intron of the albumin gene that is not expected to have deleterious effects. FVIII expression is then driven by the strong native albumin promoter. This approach has previously been demonstrated in preclinical studies to lead to therapeutically relevant expression of a different clotting factor (Factor IX) with negligible impact to systemic circulating albumin levels. Our FVIII knock-in approach is designed to provide stable expression and clinically relevant circulating levels of FVIII, even at low integration rates because of the strength of the albumin promoter.

Our approach is fundamentally different from the AAV gene therapy approaches. AAV gene therapy approaches use a viral vector to deliver a replacement FVIII gene driven off a non-natural promoter that exists in an episomal state (not integrated in the genome). This AAV FVIII gene therapy approach has been associated with the loss of FVIII expression over time in patients, a phenomenon hypothesized to be due to a combination of loss of the DNA encoding the FVIII gene (due to liver cell replication) and silencing of the episomal FVIII expression. Because our approach is designed to permanently integrate the FVIII gene into the genome of the patient, the FVIII gene should not be lost from the liver when the liver cells divide (because it is integrated in the genome and therefore transferred to the daughter cells during cell division), which may allow for a therapeutic option for children with hemophilia. Silencing of expression may be less likely to occur with our approach because we are using an endogenous albumin promoter rather than a synthetically designed promoter in a non-natural episomal state.

As shown in Figure 24, our hemophilia A genome editing program has two components: a LNP component that is designed to deliver mRNA along with a gRNA to the liver to produce a highly efficient and specific nuclease to create a precise cut at the albumin safe harbor gene locus; and an AAV vector that is designed to deliver the donor template FVIII DNA that becomes inserted into the nuclease cut site by the naturally occurring DNA repair process called non-homologous end joining. The DNA template encodes a FVIII protein, and the sequence has been optimized to improve expression. In both preclinical mouse and NHP models we have demonstrated that this FVIII knock-in approach leads to stable integration and clinically relevant circulating levels of FVIII.

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Figure 24. Therapeutic Approach to Hemophilia A Genome Editing.

 

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We have performed gRNA screening in human and mouse cells to identify both candidate clinical guides and mouse surrogate guides. In the case of the human guide screen, a total of 77 guides for three of our nucleases were screened against albumin intron 1 in liver cell lines, and two guides (called guide 1 and guide 2) were selected as leads. Primary human hepatocytes (“PHH”) isolated from the livers of deceased individuals are the most appropriate preclinical model to evaluate human liver genome editing. Guide 1 and guide 2 displayed dose dependent editing in PHH with guide 2 exhibiting the highest potency (Figure 25).

Figure 25. Editing at the Human Albumin Locus by Lead Nuclease and Guides in Primary Human Hepatocytes. Each Set of Four Bars is a Dose Response (from High Dose to Low Dose Going from Left to Right) Performed at Three Different Molar Ratios of mRNA to gRNA.

 

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* Each set of four bars is a dose response (from high dose to low dose going from left to right) performed at three different molar ratios of mRNA to gRNA.

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Genome editing nucleases can edit at sites other than the intended target site, an activity that is dependent on the specific nuclease and guide. Editing at non-target sites (called off-target editing) is undesirable due to the potential to cause damage to the host cell DNA (sometimes called “genotoxicity”). In accordance with recent FDA guidelines and insights from the FDA workshop (Assessing Genetic Heterogeneity in the Context of Genome Editing Off-Targets in Gene Therapy Products, December 2022) we have applied a combination of 3 methods; in cell oligo integration in PHH, a biochemical assay, and in silico prediction using the variant aware software CRISPRme referencing gnomad v4 variants to identify potential off-target sites for our two lead guides. We generated two panels to query these potential off-target sites. In Panel 1 we combined cell based and biochemical based discovery to query potential off-target sites. Panel 2 combined Panel 1 and variant aware in silico methods. There were a total of 372 potential off-target sites for guide 1 and 113 potential off-target sites for guide 2 in Panel 1. Editing at these potential off-target sites was then evaluated in PHH by amplicon sequencing in which a pair of PCR primers flanking each predicted off-target site is used to PCR amplify that site followed by next generation sequencing. To efficiently analyze all of the potential off-target sites, sets of barcoded PCR primers to multiple sites are carefully designed and combined in a single PCR amplification (a methodology called rhAMP-Seq). Any sites that failed to be detected from the rhAMP-Seq panels were re-tested as single amplicons. The sequence data was analyzed using the CRISPResso software to quantify InDels in each of the amplicons representing the potential off-target sites. The sensitivity of this method is high because of the depth of sequencing reads that can be readily achieved, typically enabling detection of InDels at frequencies as low as 0.1% (i.e. 1 InDel in 1,000 sequencing reads). Because double strand breaks can occur naturally in cells and because of sequence specific background signals arising from PCR primer slippage we compared the InDel rates in PHH edited at a dose that results in saturating editing (1x dose) and at a 27-fold higher dose (27x dose) to the InDel rate in un-edited PHH. We used LNP to deliver the MG29-1 mRNA and gRNA to mimic in vivo delivery and because it provided high editing efficiency and low toxicity. We believe the cells edited with the dose 27-fold higher than that which results in saturating editing provides an additional safety margin for the detection of off-target editing that may occur at undetectable frequencies at saturating doses but is detectable at a higher dose. The InDel frequencies of the potential off-target sites from Panel 1 were plotted as a scatter plot (Figure 26). InDel frequencies that are the same in the edited and unedited cells lie on the diagonal axis of Figure 26. Sites that have a higher InDel frequency in the edited cells than in the un-edited cells and thus represent a true off-target edit will appear as a point significantly above the diagonal axis of Figure 26. In addition, real off-target sites will have InDel frequencies higher than the lower limit of detection of the assay of 0.1%. Only the on-target site lies above the diagonal axis of the scatter plot in Figure 26 having an InDel rate in both 1x and 27x treated cells of 60% to 90% and an InDel rate in untreated cells of 0.01 to 0.05%. No editing significantly above the signal in the control cells was detected at any of the off-target sites nominated in Panel 1 for either guide 1 or guide 2. No signal was detected above the limit of detection after correcting for InDel frequencies in unedited control samples.

In summary, no editing was detectable at any of the potential off-target sites in Panel 1 for the lead albumin targeting guides in the most relevant human cell, primary human hepatocytes, including at a super saturating dose that is 27-fold higher than that required for saturating editing.

We are expanding the number of potential off-target sites to be evaluated by amplicon sequencing by including sites from variant aware in silico prediction using CRISPRme software referencing genomic variants from gnomAD v4 (Panel 2) that evaluates the impact of naturally occurring sequence variation within the human population on potential off-target activity. Additional tests have been conducted using the same methodology but with different PHH donors and these reached 2X target dose with similar on-target editing. Higher doses demonstrated cellular toxicity and reduced on-target editing in the new donors. Further studies using expanded methodologies to identify potential risks of off-target editing and expand our experience with supersaturating doses are ongoing.

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Figure 26. Measurement of editing at potential off-target sites from Panel 1 for the two lead guides for Hemophilia A in PHH using amplicon sequencing.

 

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* 372 and 113 sites were nominated and evaluated for guide 1 and guide 2, respectively at both a 1x and a 27x saturating dose. For each potential off-target site as well as the on-target site, the InDel frequency was plotted for 1x compared to untreated (black points) and for 27x compared to untreated (blue points). For Guide 2 the on-target site for 1x vs untreated (black dot) and 27x vs untreated (blue dot) overlap and so appear as only a blue dot.

We have demonstrated the feasibility of the human FVIII gene knock-in approach in mice with a mouse specific guide and 11 different FVIII DNA donor cassettes as shown in Figure 27, which shows the human FVIII protein levels achieved in the blood at day 10 post LNP dosing. A FVIII level of 1 IU/ml is equivalent to 100% of the normal level of FVIII in humans. FVIII levels ranged from 0.02 IU/ml (2% of normal) to 0.75 IU/ml (75% of normal). Seven of the 11 FVIII donor designs achieved human FVIII levels at or above 15% of normal, a level that would be sufficient to prevent the majority of bleeding events in hemophilia patients. We quantified integration of the FVIII gene in the liver in the correct (forward) orientation at the target site in the albumin locus in selected groups of mice at the end of the study (day 14 post LNP). The mean forward integration frequency in these groups was between approximately 0.5% and 2.5% (meaning approximately 0.5 to 2.5 copies per 100 mouse genomes, Figure 28). The finding that different human FVIII donor designs resulted in a range of FVIII levels in the blood of mice despite similar levels of integration implicate the donor DNA design as a critical component to maximize FVIII protein expression and these learnings are being incorporated into FVIII donor designs that will be evaluated in NHP studies to enable final development candidate selection.

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Figure 27. Expression of FVIII in Mice after Genome Editing.

 

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Figure 28. Integration of FVIII in Mice after genome editing.

 

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* Results are from a subset of the groups of Mice in Figure 27 (Integration was only measured for selected donors for technical reasons).

The editing efficiency of the two lead gRNA sequences (guide 1 and guide 2) were evaluated in non-human primates (“NHPs”). Guide 1 is a perfect match to the target sequence in the NHP genome while guide 2 required a single nucleotide change in the spacer to match perfectly to the target sequence in NHPs. The same nuclease mRNA sequence was used with both guides and the guide and mRNA were co-delivered with the same LNP formulation by IV infusion of three cynomolgus macaques per group at a dose of 1.25 mg RNA per kg body weight. The editing efficiency in five different regions of the liver (representing the different liver lobes) were measured by determining the InDels at the target site using next generation sequencing in each of the three animals per treatment group as shown in Figure 29. Editing levels were similar across the five liver regions (represented by the five dots in each bar) demonstrating that editing was homogenous across the entire liver. Guide 2 resulted in a mean editing efficiency of 50% amongst the three animals. This represents editing in approximately 80% of hepatocytes because hepatocytes make up about 60% of the cells in the liver of NHPs and the LNP delivers primarily to hepatocytes.

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Figure 29. Efficient Editing in NHPs at the Safe Harbor Locus for the Hemophilia A Program.

 

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* Each bar is an animal and each datapoint represents a different lobe of the liver.

To evaluate our FVIII gene integration approach in a more relevant preclinical species we performed combined AAV-FVIII and LNP dosing in three NHP (cynomolgus macaques). The monkeys were pre-screened for neutralizing antibodies (“NaB”) against AAV and only animals with a titre of less than 1:5 (meaning that no Nab were detected at a 1:5 fold dilution of the monkey plasma) were selected for the study. The target site in albumin intron 1 was sequenced from each animal to confirm the absence of sequence differences that might otherwise impact recognition by the gRNA. Plasma was collected prior to dosing to provide a baseline for the FVIII activity assay. A small amount of liver tissue was collected by biopsy two weeks prior to dosing to provide a baseline for the measurement of FVIII mRNA in the liver. The three animals were given a single dose of the anti-inflammatory drug dexamethasone (2 mg/kg) prior to AAV dosing and again prior to LNP dosing. It is well known that human FVIII is immunogenic in monkeys resulting in generation of anti-human-FVIII antibodies (typically within weeks to months of exposure) which prevents the detection of the human FVIII protein. A cellular immune response against human FVIII is also likely to occur in monkeys which may result in the destruction of liver cells expressing the human FVIII. We therefore used the cynomolgus FVIII gene in the AAV construct to ensure that the monkeys would not have an immune response against the FVIII protein expressed from the integrated FVIII gene and thereby enable a long term evaluation of the durability of FVIII expression. The AAV construct was designed with a codon optimized B-domain deleted cynomolgus FVIII (cFVIII) coding sequence with a single amino acid change that had no observed impact on the function of the cFVIII protein but enables the specific detection of the AAV encoded cFVIII protein in the background of the endogenous cFVIII protein. All three monkeys received a single dose of the AAV-cFVIII virus followed five weeks later by a liver trophic LNP encapsulating the mRNA encoding MG29-1 and guide 2 at a dose of 1 mg/kg body weight. Plasma was collected either weekly or bi-weekly starting at 14 days post LNP dosing and assayed for the vector derived cFVIII using a commercial activity assay (chromogenic assay). As of the November 11, 2023 data cutoff date, we have collected and assayed cFVIII activity up to day 126 post LNP (18 weeks, 4.5 months) and results are shown in Table 1. The plasma for each post-LNP time point was assayed multiple times (between 5 and 7 times) and the mean value and standard deviation were calculated. Plasma collected prior to dosing (day -14, n=3 replicates) had no detectable cFVIII using this assay, demonstrating that the assay method does not detect endogenous cFVIII. The mean FVIII activity derived from the administered c-FVIII gene over the time period from day 14 to 126 was 0.75 IU/ml, 0.13 IU/ml and 0.29 IU/ml in the 3 animals which corresponds to 75%, 13%, and 29% of normal FVIII activity in humans. These average levels of FVIII are within the desirable therapeutic range (approximately 10% to 150% of normal).

Samples of liver tissue were collected on day 7 and day 70 post LNP dosing by biopsy of all three NHP. The day 7 liver biopsy was analyzed for editing at the target site in albumin intron by NGS which revealed that the cleavage by MG29-1 was efficient and consistent, with InDel frequencies of 45%, 50%, and 55% in the 3 animals, similar to what was observed in our earlier editing only study in NHP with the same guide 2 (Figure 29). Integration of the cFVIII gene encoded in the AAV at the target site in albumin intron 1 was quantified by a digital droplet PCR assay that specifically detects the junction between the albumin intron 1 sequence at the target site and the 5’ end of the AAV encoded cFVIII gene. Integration was detected in all 3 animals and the integration frequency ranged from 0.7 to 2.9 copies per 100 genomes, similar to the frequency observed in mice.

The specific albumin-cFVIII hybrid mRNA that is generated by the precise splicing between albumin exon 1 and the 5’ end of the integrated cFVIII gene derived from the AAV was quantified in liver tissue from the day 7 and day 70 biopsies as well as the pre-dose biopsy using a digital droplet PCR assay. No signal was detected in the liver tissue from the pre-dose biopsy as expected, demonstrating that this assay does not detect endogenous cFVIII mRNA. The specific albumin-cFVIII hybrid mRNA was clearly detected in all three animals at both the day 7 and day 70 timepoints with no significant change in the mRNA level between day 7 and

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day 70. The mean of the albumin-cFVIII hybrid mRNA levels at day 7 and day 70 post LNP dose were 177, 79, and 131 in the 3 animals (the units are percentage of an endogenous control mRNA that was quantified in the same samples). The integration frequency and the hybrid albumin-cFVIII mRNA levels correlated with the plasma FVIII activity. The data in Table 1 provides important proof of concept for our gene editing approach in a relevant NHP model. We intend to continue this study up to 12 months with continued measurement of FVIII activity to evaluate the durability of FVIII expression from the integrated cFVIII gene.

Table 1. cFVIII donor integration frequency, FVIII mRNA level and Mean FVIII activity level in NHPs.

 

Animal ID

 

Editing in
the liver
(InDel
%)
1

 

 

FVIII gene integration
frequency
(copies per 100 genomes)
2

 

 

FVIII mRNA
(% of endogenous control mRNA)
3

 

 

Mean FVIII activity
% of normal (d14
to d126)
4

1001

 

 

45

%

 

 

2.9

 

 

 

177

 

 

75 +/- 9

1002

 

 

50

%

 

 

0.7

 

 

 

79

 

 

13 +/- 4

1003

 

 

55

%

 

 

1.4

 

 

 

131

 

 

29 +/- 5

 

Table footnotes: (1) InDelS at the on-target site in albumin intron 1 were measured by NGS in a liver biopsy taken at day 7 post LNP dosing. (2) The FVIII donor gene integration frequency in the forward orientation was quantified by droplet digital PCR analysis of the liver biopsy at day 7 post LNP and is normalized to an endogenous single copy gene. (3) FVIII mRNA was quantified with a digital droplet PCR assay specific for the hybrid mRNA created by correct splicing between albumin exon 1 and the 5’ end of the FVIII gene and is the average of the levels in the liver biopsy at day 7 and day 70 post LNP dosing and is normalized to the levels of an endogenous mRNA. (4) The mean of the FVIII activity in IU/ml measured at each time point between day 14 and day 126 was calculated and converted to the percentage of normal by multiplying by 100 (1 IU/ml is approximately 100% FVIII activity in a healthy human being).

No safety signals of concern were observed during this NHP study as of the November 11, 2023 cut-off date. A comprehensive set of safety markers were measured in the blood of the monkeys after both AAV and LNP dosing. These included assays for coagulation, serum chemistry and hematology as well as liver enzymes (transaminases and bilirubin levels) and a panel of 10 cytokines. We observed mild and transient elevations of alanine aminotransferases (“ALT”) and aspartate aminotransferase (“AST”) and no significant change in total bilirubin post AAV and post LNP (Figure 30). Importantly we observed no changes in serum albumin levels over the course of the study demonstrating that the high level of editing in albumin intron 1 (mean 48% InDels which converts to approximately 78% of hepatocytes) did not alter expression of the albumin protein. Three cytokines (IL-6, MCP-1 and IL-12/IL-23) exhibited mild and transient increases in the blood post AAV and post LNP. The data for IL-6 is shown in Figure 31. As of the data cut-off date, all three animals in the study remained healthy, without notable clinical observations, and continued to gain weight as expected.

Figure 30a. ALT, AST, and total bilirubin levels in the blood of NHP up to day 8 after AAV dosing in the integration study.

 

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Figure 30b. ALT and AST levels in the blood of NHP up to day 10 after LNP dosing in the integration study.

 

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Figure 30c. ALT, AST and total bilirubin levels in the blood of NHP throughout the study to the data cut-off date.

 

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Figure 30. Albumin levels in the blood of the 3 NHP after AAV and LNP dosing in the integration study.

 

img190561166_50.jpg 

 

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Figure 31. Levels of the cytokine IL-6 in the blood of the 3 NHP in the integration study post AAV dosing and post LNP dosing.

Figure 31a. Levels of the cytokine IL-6 in the blood of the 3 NHP in the integration study post AAV dosing.

 

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Figure 31b. Levels of the cytokine IL-6 in the blood of the 3 NHP in the integration study post LNP dosing.

 

img190561166_52.jpg 

 

Next Steps

We have selected two human FVIII donor DNA cassette designs, and are in the process of producing the AAV, mRNA and gRNA to perform a second NHP integration study to select a single donor and guide and expect to nominate a development candidate by mid-2024. In parallel, we are initiating GxP manufacturing of mRNA, gRNA, AAV and LNP to support future IND-enabling studies. The progress we have made on this program not only validates the efficiency and specificity of our novel nucleases in rodent and NHP models, but also supports our ongoing efforts with other large gene integration approaches.

Primary Hyperoxaluria, Type 1—a durable knock-down of HAO1 for substrate reduction therapy

The Disease

PH1 is a rare autosomal recessive metabolic disease arising from loss of function mutations in the AGXT gene that encodes alanine glyoxylate aminotransferase. This enzyme is found in peroxisomes of the liver where it catalyzes the conversion of glyoxylate to glycine and pyruvate. Lack of functional AGXT leads to an accumulation of glyoxylate substrate, which is then converted to oxalate and excreted in the kidney. The excess urinary oxalate forms an insoluble complex with urinary calcium that leads to the production of calcium oxalate crystal precipitates. This pathologic process results in the formation of repeated calcium oxalate urolithiasis and nephrolithiasis, which in turn leads to obstructive uropathy, inflammation, fibrosis, tubular toxicity, and progressive loss of kidney function.

PH1 is a serious disease that causes kidney failure. More than 70% of individuals with PH1 mutations will develop end-stage renal disease, with a median age in young adulthood. Patients with PH1 continue to experience morbidity and mortality even after the development of end-stage renal disease due to progressive systemic calcium oxalate precipitation in various organs (systemic oxalosis). Despite renal replacement therapy or kidney transplantation, patients with PH1 have an overall shorter lifespan than patients with other causes of renal failure, highlighting the progressive and severe nature of this metabolic disease.

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PH1 is the most common of the primary hyperoxalurias but is a rare disease with an estimated prevalence of approximately one to three in 1,000,000 individuals. While epidemiologic data on PH1 is limited, these estimates suggest there are approximately 1,000 to 3,000 patients in both the United States and Europe, and possibly up to 20,000 patients globally.

Limitations of Current Approaches

Until recently, the standard of care for treating PH1 was primarily supportive in nature, with hydration and diuretics used to reduce urinary oxalate concentration, pyridoxine (vitamin B6) to enhance residual function of alanine glyoxylate aminotransferase catalytic activity, and hemodialysis once renal function progressed to end stage. Liver transplantation has also been explored as a means of providing patients with a normal copy of AGXT and has been used alone in early-stage patients or as a combined liver-kidney transplant in more advanced patients. Transplantation approaches have limitations due to donor availability, morbidity associated with the surgical procedure, and lifelong immunosuppression required to inhibit graft rejection.

More recently, the standard of care has been updated to include treatment with lumasiran, a siRNA therapeutic approved in adults and children with PH1 that acts to reduce the levels of urinary oxalate. Using a therapeutic approach known as substrate reduction therapy, lumasiran targets mRNA from a separate gene, HAO1, that encodes GO. By inhibiting GO, levels of glyoxylate are reduced, which results in reduced downstream levels of oxalate. As a result, lower urinary oxalate results in decreased urinary calcium oxalate stone formation. It is anticipated that inhibition of renal oxalate accumulation and stone formation can slow or prevent continued loss of renal function. Lumasiran has been generally well tolerated in clinical studies of adults and children with PH1 but it requires repeat subcutaneous administration indefinitely in order to maintain its effect. In addition, injection site reactions are common among patients taking lumasiran and the degree of urinary oxalate reduction has been observed to not reach normal levels in many patients. An additional RNAi drug, Nedosiran, which targets LDH, a different enzyme in the same pathway as HAO1, was also granted marketing approval by the FDA for adults and children with PH1 in October 2023. Thus, there is a potential to improve clinical outcomes for PH1 patients with a one-time administration of a therapy that inhibits urinary oxalate accumulation, prevents calcium oxalate stone formation, and protects renal function.

Our Approach and Results

The goal of our genome editing approach is to durably knock down HAO1 resulting in stable and permanent reduction of oxalate levels to effect a lifelong benefit. We plan to deliver mRNA for one of our lead nucleases and a guide encapsulated within a single LNP as shown in Figure 32 below. We expect the mRNA and HAO1 gRNA will be released in hepatocytes (the cell type in the liver that expresses the HAO1 gene) where the mRNA will be expressed into the nuclease that forms a complex with the guide and create a double stranded break in the HAO1 gene and inhibits gene expression.

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Figure 32. Genome Editing Strategy for Targeting HAO1.

 

img190561166_53.jpg 

 

The safety of this durable knock-down of HAO1 is supported by human genetic observations of an individual lacking a functional copy of the HAO1 gene with no evidence of a pathologic phenotype. The safety of this approach is also supported by up to three years of clinical data using a siRNA approach to silence this gene.

We have performed nuclease and guide screening to select an optimal nuclease and gRNA combination. The double strand break created by the selected nuclease has been observed to be efficiently and rapidly repaired by the cell via a process that introduces small InDels to the sequence at the target site. When these InDels alter the reading frame of the gene (so called “out of frame” InDels) this results in degradation of the HAO1 mRNA. Because different nucleases and guides generate distinct InDel profiles, we included analysis of the InDel profile as a selection criterion during our nuclease /gRNA screen. In addition to the InDel profile, we also screened for the efficiency of HAO1 mRNA reduction and impact on the HAO1 mRNA sequences using whole transcriptome RNA sequencing (“RNAseq”). This screening process resulted in selection of a lead guide that targets an identical sequence in humans and mice which enables the evaluation of the lead human guide in mouse models. The potency of this lead nuclease and guide was tested in PHH in vitro as shown in Figure 29, demonstrating dose-dependent editing when delivered by a LNP with tropism to the liver.

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Figure 33. Editing Dose Response in Primary Human Hepatocytes with the Lead Nuclease mRNA and Guide Targeting HAO1 Delivered in a LNP.

 

img190561166_54.jpg 

 

The off-target profile of the lead human guide targeting HAO1 is being evaluated in accordance with FDA guidelines published in March 2022. Potential off-target sites were identified using a combination of in silico prediction, biochemical discovery assays, and a cell-based discovery assay. To predict potential off-target sites in silico we used the variant aware CRISPRme software using a permissive PAM for MG29-1 (YYN) and permitting up to 5 changes (4MM + 1 bulge) from the HAO1 reference protospacer to identify potential off-target sites referencing both the GRCh38 human genome reference and the Genome Aggregation Database gnomAD v4 (Minor Allele Frequency of ≥1% in subpopulations).

We applied two biochemical off-target discovery methods, Digenome-seq and SITE-Seq both of which digest purified human genomic DNA with the editing nuclease/guide complex in vitro.

To identify potential off-target sites with a cell-based discovery method we transfected primary human hepatocytes with MG29-1 and the lead guide together with a short double stranded oligonucleotide that integrates into the genome of cells at double strand breaks. Using a PCR based method we selectively amplified the DNA junction between the integrated oligonucleotide and the human genome and evaluated the profile of potential off-target sites by NGS.

A total of 318 potential sites were identified by the combination of the three empirical discovery methods; the overlap of potential off-target sites identified from each of these methods is shown in Figure 34.

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Figure 34. Venn diagram demonstrating the overlap of potential off-target sites identified through cell-based and biochemical discovery methods for the lead HA01 targeting guide (n=318) (left panel). Venn diagram demonstrating the overlap of the potential off-target sites identified through cell-based and biochemical discovery methods with in silico predicted off-target sites (right panel).

 

img190561166_55.jpg 

img190561166_56.jpg 

 

These 318 potential off-target sites were tested in primary human hepatocytes that had been edited by MG29-1 and the lead HAO1 targeting gRNA using LNP delivery.

Off-target editing at all 317 amplifiable potential off-target sites was measured in the genomic DNA from primary human hepatocytes edited by MG29-1 and the lead HAO1 targeting gRNA. No editing was detectable at any of the 318 potential off-target sites (one centromere-proximal site identified in one SITE-seq replicate and by CRISPRme could not be amplified by standard PCR methods) for the lead HAO1 targeting guide in the most relevant human cell, primary human hepatocytes, indicating that MG29-1 in combination with the lead HAO1 targeting gRNA is highly specific.

In normal mice we have demonstrated dose dependent saturating levels of hepatocyte genome editing of HAO1 and up to 90% reduction of target GO protein, providing strong preclinical proof-of-concept as shown in Figure 35.

Figure 35. Dose Dependent Editing, mRNA Knock-down and Protein Knock-down of HAO1 in Normal Mice after a Single Administration of the Lead Nuclease mRNA and Lead Guide Encapsulated in a LNP with Tropism to the Liver.

 

img190561166_57.jpg 

 

Additionally, along with our partner Moderna, we have achieved preclinical proof-of-concept in an AGXT knock- out mouse which is an accepted disease model of PH1 (Figure 36). AGXT knock-out mice have elevated oxalate in the urine (measured with a mass spec assay) of about 500 mg per gram of creatinine compared to about 190 mg per gram of creatinine in mice of the same strain (BL/6)

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with a wild type AGXT genotype. Cohorts of male and female mice were given a single administration of the lead nuclease mRNA and gRNA encapsulated in a LNP with tropism to the liver. Dose dependent editing at the target site in HAO1 in the liver was measured at the end of this study (when the mice were sacrificed). The editing of HAO1 had the expected effect of dose dependently reducing urinary oxalate, with the highest dose achieving oxalate levels comparable to that of wild type mice.

Figure 36. Preclinical Proof of Concept in PH1 Disease Model (AGXT -/- mice).

 

Figure 36a. Dose dependent editing of the HAO1 gene in AGXT -/- mice.

 

Figure 36b. Dose-dependent reduction in urinary oxalate reaching normalization at highest dose.

 

 

 

img190561166_58.jpg 

 

img190561166_59.jpg 

 

 

 

Source: Moderna

 

Source: Moderna

 

The durability of this approach was demonstrated by continued knock-down of GO protein even after partial hepatectomy (removal of about two thirds of the liver) and rapid liver re-growth (Figure 37). In this study wild type mice were given a single injection of either buffer or a liver tropic LNP encapsulating the MG nuclease mRNA and the lead gRNA targeting HAO1. As a control a Cas9 mRNA and a potent Cas9 guide targeting HAO1 from a published source were also packaged in the LNP with tropism to the liver and tested at a single dose. Eight days after dosing a liver hepatectomy was performed in which about two thirds of the liver was removed. This stimulates the majority of the remaining liver cells to enter cell division and results in rapid re-growth of the liver that restores the normal liver size within 7 to 10 days. Eight days after partial hepatectomy the levels of GO protein (the product of the HAO1 gene) in the liver were reduced by more than 90%, the same as the level of knock-down in edited mice that did not undergo hepatectomy. This result demonstrates that the knock-down of HAO1 expression was not compromised by extensive liver growth and that the fitness of edited hepatocytes was not impacted by editing.

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Figure 37. Knock-down of the Protein Encoded by HAO1 after HAO1 Gene Editing was Maintained Following Partial Hepatectomy. Control Mice were Injected with Buffer as a Control.

 

img190561166_60.jpg 

 

Source: Moderna

Next Steps

We are in the final stages of confirming the candidate to take into NHP studies and expect to have NHP data in 2024 to support final development candidate selection.

This program is partnered with Moderna for both development and commercialization. The partnership enables us to leverage Moderna’s expertise in mRNA and LNP technology to ensure efficient delivery of our nuclease to hepatocytes. In turn, we provide the novel programmable nuclease and guide chemistry to support precise targeting of the HAO1 gene. In addition to further validating our therapeutic platform, to the best of our knowledge this program represents the first time a type V nuclease is being developed for a therapeutic in vivo genome editing approach.

Transthyretin Amyloidosis—a single treatment to knock-down TTR gene expression

The Disease

Transthyretin amyloidosis is a disease of misfolded and aggregated TTR protein that can deposit in tissues causing organ dysfunction, primarily in the heart and/or peripheral nerves. The TTR protein is normally produced in the liver and circulates in a homotetramer (four copies of the same TTR protein bound together) where it serves as a carrier protein for vitamin A and thyroxine. Certain mutations have been identified that can cause TTR homotetramers to fall apart, misfold, and aggregate into insoluble fibrils that deposit in cardiac tissue and peripheral nerves. However, more commonly, the normal aging process is associated with an increased propensity for TTR misfolding and aggregation in the heart without any known genetic sequence variation. These distinctions lead to TTR amyloidosis being characterized as either ATTRv caused by mutations in TTR, or ATTRwt. It is estimated that globally there are approximately 50,000 patients with ATTRv and between 300,000 and 500,000 patients with ATTRwt. Among the larger ATTRwt patient population, the most common presentation is a rapidly progressive, restrictive, and hypertrophic cardiomyopathy due to progressive deposition of insoluble TTR fibrils, which result in thickening of the myocardium and stiffening of the ventricles.

These pathologic processes lead to impaired diastolic function and progressive cardiomyopathy that typically leads to progressive heart failure and often death within three to five years from disease onset. Although cardiac manifestations are more common and severe, patients with neurologic manifestations also experience significant morbidity, loss of functionality, and impaired quality of life.

Limitations of Current Approaches

To date, treatment options for patients with TTR amyloidosis, including those with either cardiomyopathy or polyneuropathy manifestations, consist of efforts to stabilize the TTR tetramer with a small molecule (tafamadis) or knock down TTR levels through

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antisense oligonucleotides (i.e., inotersen and eplontersen which is currently under regulatory review in the U.S.) or siRNA strategies (i.e., patisiran and vutrisiran).

Tafamadis was studied in a randomized trial of patients with either ATTRv and ATTRwt cardiomyopathy and demonstrated reductions in all-cause mortality and cardiovascular-related hospitalizations and reduced the decline in quality of life and functional capacity over 30 months compared to placebo. Tafamadis is currently approved to treat patients with ATTR cardiomyopathy in the U.S. and patients with ATTRv and ATTR cardiomyopathy in the EU and in other parts of the world. Inotersen has demonstrated benefit in patients with ATTRv and polyneuropathy and is approved in the U.S., EU, and in other parts of the world. Eplontersen has demonstrated benefit in patients with ATTRv and polyneuropathy and is currently under regulatory review in the U.S. Eplontersen is also being studied in a Phase 3 trial for patients with ATTR cardiomyopathy. Patisiran is approved for the treatment of polyneuropathy of ATTRv in adults in the U.S., EU, and other parts of the world. Patisiran is currently under regulatory review for the treatment of patients with ATTR cardiomyopathy based on a randomized study demonstrating improvement in the 6-Minute Walk Test and quality of life over 12 months compared to placebo. Vutrisiran has demonstrated benefit in patients with ATTRv and polyneuropathy and is approved for use in ATTRv in the U.S., EU, and other parts of the world. Although these approaches have improved clinical outcomes for patients with TTR amyloidosis, the disease is still associated with significant morbidity and mortality and requires lifelong therapy to maintain therapeutic benefit.

Our Approach/Next Steps

More recently, early-stage third-party studies have demonstrated the feasibility of knocking down TTR (wild type or mutated versions) using a CRISPR based genome editing approach in a small number of patients. Clinical validation of this TTR knock-down approach is provided by antisense and siRNA clinical experience and further suggests the potential longer-term safety and tolerability of this approach.

Using our novel nucleases, we aim to provide efficient TTR knock-down and halt further deposition of amyloid fibrils. Previous experience suggests a clinical correlation between the degree of TTR knock-down and potential for benefit in familial forms of the disease, which are expected to translate similarly to wild type forms. The high degree of in vivo editing efficiency and specificity of our nuclease platform suggest the potential for a single treatment to knock-down TTR gene expression and remove the requirement for life-long therapy.

Along with our partner Ionis, we are currently in advanced stages of nuclease and guide selection and expect to move into NHP studies in 2024. We believe one of the strengths of our technology platform is applying our multiple nucleases that have distinct non-overlapping PAMs to create a larger number of guides for a given target. Starting with a larger number of guides should increase the chance of finding highly active and specific guides/nuclease combinations. Leveraging multiple nucleases with a diversity of PAMs enabled a high targeting density for the TTR gene as illustrated in Figure 38a. By comparison significantly fewer (3.8-fold fewer) guides are available when using SpCas9 (Figure 38b). We have screened more than 500 guides against the human TTR gene using six of our nucleases. With one additional nuclease in progress we will ultimately have screened 535 guides. Our high throughput screening platform has enabled us to screen approximately 500 guides in a four- month time span.

Figure 38a. High density of gRNA targeting the coding and regulatory regions of the TTR gene enabled by our platform of nucleases with diverse PAMs.

 

img190561166_61.jpg 

 

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Figure 38b. Density of SpCas9 gRNA targeting the coding and regulatory regions of the TTR gene.

 

img190561166_62.jpg 

 

We are in the process of selecting lead guides and nucleases for evaluation in mouse models that carry the human TTR gene. Promising early leads have shown potent TTR mRNA knock-down in primary human hepatocytes. We plan to deliver our programmable nuclease and its associated gRNA to the liver using LNP technology, which has been shown to be highly selective for hepatocytes. This program not only provides a novel nuclease approach to knocking down TTR levels, but also leverages our partner’s long-standing clinical development expertise in the TTR amyloidosis field to accelerate our efforts.

A preliminary evaluation of the in vivo potency of lead human guides was performed in a humanized mouse model in which multiple copies of the human TTR gene were integrated at random sites in the mouse genome. These mice express human TTR protein which can be measured in the blood using an assay specific for the human TTR protein. In this study, Lead 1 reduced the human TTR mRNA and protein by 75% and 88%, respectively. Leads 2 and 3 both reduced the human TTR mRNA and protein by 97% and 98%, respectively. Leads 2 and 3 are particularly attractive because these guides and mRNA have undergone only minimal sequence and chemistry optimization to date. Additional leads are currently being evaluated in this mouse model.

Figure 39. Proof of concept for knock-down of human TTR protein by lead nucleases/guides in human TTR transgenic Mice.

 

img190561166_63.jpg 

 

Cardiovascular Disease – a gene editing solution to eliminate angiotensinogen gene expression

The Disease

Cardiovascular disease is the leading cause of death worldwide and implicated in the deaths of approximately 17.9 million individuals each year. Although cardiovascular diseases are not genetically defined diseases, there are well validated gene targets and signaling pathways that address potent sources of vasoconstriction allowing for the creation of an important genetic medicine for these common

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diseases. Among the most important of these signaling pathways that have demonstrated marked clinical benefit in both hypertension and heart failure is the renin-angiotensin-aldosterone system (RAS) pathway, that has been successfully targeted by a number of important medications including renin inhibitors, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and aldosterone antagonists. Beyond its effect on blood pressure control, inhibitors of the RAS pathway have been shown to provide meaningful clinical benefit in ischemic heart disease and chronic heart failure as well as in diabetic nephropathy, and other forms of chronic renal insufficiency. However, continuous 24-hour inhibition of the RAS pathway with oral agents is not always successful, especially if compliance is suboptimal. Importantly, despite use of RAS inhibitors, diurnal variability in blood pressure and nocturnal blood pressure elevations can still occur. Recent early stage clinical studies using antisense and siRNA approaches to durably suppress the RAS pathway by targeting liver derived angiotensinogen provides important clinical validation of this approach as a means of safely reducing blood pressure without incurring hypotension, hypokalemia, or acute renal injury.

Limitations of Current Therapy

Although there are numerous approved classes of drugs that have demonstrated clinical benefit in patients with cardiovascular disease and refractory hypertension patients, many patients do not reach their blood pressure goals or continue to have cardiovascular disease progression. In addition to contributions from underlying stiffened arteries and/or increased sympathetic tone, patients may have suboptimal adherence in taking a large number of daily oral pills. Importantly, current treatments often do not fully provide reliable and consistent 24-hour control of blood pressure which can leave patients exposed to diurnal variation and early morning blood pressure surges associated with cardiovascular events. Adverse effects of polypharmacy approaches and complicated food effects or drug-drug interactions can further negatively affect compliance and lead to poor outcomes. Therefore, despite the availability of oral medications, there remains a significant unmet need for a well-tolerated, durable approach to blood pressure control and cardiovascular protection with a clinically validated target.

Our Approach/Next Steps

Our goal is to knock-down the expression of angiotensinogen in the liver using one of our programmable nucleases to generate a durable reduction in blood pressure from a single treatment. Angiotensinogen, encoded by the gene AGT, is at the top of the RAS pathway and the precursor to highly vasoconstrictive peptides angiotensin I and II and is thus an attractive and novel target for reduction of blood pressure. Inhibiting angiotensinogen protein production from the liver has the advantage of minimizing inhibition of the RAS pathway in the kidney which will provide a better safety profile by keeping the small amount of kidney production of AGT intact and thus avoiding renal dysfunction that is common with current medications and ensuring adequate vasoactive homeostasis in stress situations. A second potential advantage of targeting AGT is that it may limit escape mechanisms that restore angiotensin II levels or angiotensin II signaling. Clinical proof of concept for reduction of AGT protein levels comes from ongoing clinical trials of antisense (IONIS-AGT-LRx) and siRNA (ALN-AGT) drug candidates that have shown reduced mRNA levels of AGT in the liver and consequently reduced levels of AGT protein in the blood. We plan to deliver a programmable nuclease and its associated gRNA to the liver using LNP technology.

Along with our partner Ionis Pharmaceuticals we are currently in advanced stages of nuclease and guide screening and expect to move into NHP studies in late 2024 or early 2025. We believe one of the strengths of our technology platform is being able to access a larger proportion of the human genome by applying our multiple nucleases that have distinct non-overlapping PAMs. We have the potential to screen up to 1,490 guides using six MG nucleases against the AGT gene. To date we have screened 441 guides and are planning to screen an additional 420 guides. Leveraging the multiple MG nucleases with a diversity of PAMs enables a high targeting density as illustrated for the seven MG nucleases that are being screened against AGT. By comparison significantly fewer guides (2.5-fold fewer) are available when using spCas9. Multiple guides with potent editing of the AGT gene in human cells were identified in our initial guide screen against the coding sequence. We are in the process of completing the guide screen and selecting lead guides/nucleases for evaluation in mouse models that carry the human AGT gene.

We believe that this program is expected to be one of the first co-development efforts to develop a gene editing therapy for more common, non-genetically defined cardiovascular indications.

A preliminary evaluation of the in vivo potency of lead human guides was performed in a humanized mouse model in which multiple copies of the human AGT gene were integrated at random sites in the mouse genome. These mice express human AGT protein which can be measured in the blood using an assay specific for the human AGT protein. In this study, human AGT protein in the blood and the human AGT mRNA in the livers were measured 7 days post LNP dosing at both 1 and 0.3 mg/kg (Figure 40). At a dose of 1 mg/kg, lead 1 reduced the human AGT mRNA and protein by 85% and 93%, respectively. Lead 2 reduced the human AGT mRNA and protein by 91% and 92%, respectively. Additional leads are currently being evaluated in this mouse model.

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Figure 40. Proof of concept for knock-down of human AGT protein by lead nucleases/guides in transgenic human AGT mice.

 

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A1AT Deficiency

The Disease

A1AT deficiency is an autosomal recessive disease arising from loss of the normal A1AT protein encoded by the gene SERPINA1. Clinical manifestations of A1AT deficiency are primarily in the lung due to the toxic loss of normal function of A1AT, and occasionally in the liver due to the toxic effects of accumulation of abnormal A1AT.

AIAT is a highly abundant plasma protein that acts as an inhibitor of the potent proteolytic enzyme neutrophil elastase. Neutrophil elastase is involved in the host response to infection, but when unchecked can degrade connective tissue. In the lung, clinical manifestations of A1AT deficiency include emphysema and bronchiectasis from destruction of normal alveolar connective tissue. In patients with liver disease, the primary pathophysiology is liver inflammation and cirrhosis due to buildup of abnormal A1AT protein. Liver disease is more often found in children with A1AT deficiency, and lung disease is more often found in adults with A1AT.

A1AT deficiency is a clinically underrecognized disease, especially those with pulmonary manifestations as it is often mistaken for other forms of chronic obstructive pulmonary disease. Recent estimates from genetic screening suggest that 80,000 to 100,000 individuals in the United States have severe deficiencies of A1AT and approximately 40,000 to 60,000 have clinically manifest emphysema caused by A1AT deficiency. As not all individuals with A1AT deficiency present similarly, it is believed that environmental factors such as exposure to smoke, allergens, chemicals, and other environmental factors likely impact the severity and clinical manifestations.

Limitations of Current Therapy

Patients with A1AT deficiency are often treated with protein augmentation therapy. The goal of augmentation therapy is to increase circulating levels of A1AT sufficiently to balance the adverse effects of unchecked neutrophil elastase and slow the progression of emphysema. Augmentation therapy consists of an IV infusion of purified pooled donor plasma enriched for A1AT protein levels given weekly. While studies have demonstrated the ability to achieve higher circulating levels of A1AT with augmentation therapy, longer term impacts on protecting lung function are limited. This approach is also costly and not available for many patients. Infusions have also been associated with adverse events including flu-like reactions, fever, and rarely anaphylaxis. In severe cases of lung or liver disease, organ transplantation may be required to preserve life but are associated with post-transplant risks and long-term immunosuppression requirements.

Our Approach/Next Steps

The most frequent mutation responsible for A1AT deficiency is the PiZ mutation that changes the normal amino acid at position 342, which is glutamic acid, to lysine (E342K). Patients that are homozygous for the PiZ allele (called ZZ allele) make up more than 90% of the patients globally. We propose to correct this mutation back to normal using either an adenine base editor or Little RIGS. Because individuals that are heterozygous for the PiZ mutation (so called ZM allele) have minimal pathology, it is expected that editing of 50% of the alleles would be therapeutic. The most common DNA sequence change that creates the E342K mutation is a G

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to A change at the first position of the codon; GAG (normal) to AAG (PiZ). Converting the PiZ sequence (AAG) to the normal sequence (GAG) requires changing the underlined A to G which can be achieved using an adenine base editor.

For this approach to be successful, a potent gRNA needs to be identified that targets the editing window to the ABE at the precise location of the codon for amino acid 342. There are five additional A bases within 5 bp on either side of the target A base in the 342 codon which could be subject to bystander edits (defined as edits at non-target bases within the site targeted by the guide), several of which would change additional aa with unpredictable impacts on the structure and function of the protein. Given the complexity of this edit, we will evaluate multiple genome editing systems, including base editors and RIGS, in order to advance the most effective genome editing approach.

Wilson’s Disease

The Disease

Wilson’s disease is an autosomal recessive disease of copper metabolism resulting from impaired function of the intracellular copper transporter encoded by the gene ATP7B. Mutations in ATP7B lead to impaired biliary excretion of copper, leading to copper accumulation in multiple organs including liver, brain, and eye. Given the large size of the gene and allelic heterogeneity, the disease has been difficult to target with any genetic medicine to date. While impaired copper excretion begins at birth, the effect is typically not observed clinically until later in childhood or early adulthood and the majority of patients are diagnosed between ages five and 35. The most common manifestation is chronic active hepatitis that can progress to liver cirrhosis; however, a variety of neurologic and psychiatric manifestations may also be present and undiagnosed. Such symptoms may include dysarthria, gait impairment, dystonia, depression, irritability, and personality changes. Diagnosis is typically suggested by abnormalities in blood ceruloplasmin and/or 24-hour urine copper as well as the presence of Kayser-Fleisher rings on ocular examination. Additional diagnostic certainty can come from liver biopsy and ATP7B sequencing.

Wilson’s disease has an estimated global prevalence of one patient per 10,000 to 30,000 individuals although recent genotyping studies suggest that the actual genetic prevalence may be substantially higher. This estimate suggests that there are more than 30,000 patients with Wilson’s disease in the United States alone.

Limitations of Current Therapy

Wilson’s disease is a serious disease that is fatal if left untreated, typically from cirrhosis and liver failure. Once diagnosed, the standard of care for Wilson’s disease involves chronic treatments to try and remove copper from the body using copper chelators and efforts to minimize copper absorption from the gastrointestinal tract. Treatment must be lifelong to prevent copper reaccumulation and side effects from chelator therapy (e.g. hypersensitivity reactions, fever, changes to blood counts) are common and can lead to treatment discontinuation. If treatment is discontinued, patients are at risk for hepatic decompensation or the development of new neurologic symptoms. In patients with delayed diagnosis or rapidly progressive disease, liver transplant may be required with associated post-transplant risks and need for immunosuppression.

There are no genetic medicines approved for Wilson’s disease. A genetic medicine has the potential to eliminate the need for life-long therapy and copper monitoring as well as reduce the risk for adverse events.

Our Approach/Next Steps

Wilson’s disease is inherited in a recessive manner meaning that heterozygotes (people having one mutant allele and one wild type allele) are not affected. This means that correction of only one of the two mutant alleles should be sufficient to confer a normal phenotype on the hepatocyte, the cell type in the liver in which the ATP7B gene performs its function. Patients with Wilson’s disease have a diverse spectrum of different mutation types (point mutations, deletions, splice site mutations) that are located throughout the ATP7B gene although different ethnic groups bear different predominant mutations. Common mutations in specific population groups include H1069Q (30-70% of Wilson’s disease patients in Germany, 30-70% in US Caucasians), M645R (approximately 30% of Wilson’s disease patients in Spain), R778L (40% of Wilson’s disease patients in Korea, approximately 30% in China).

Our approach would be to treat Wilson’s disease using a Big RIGS insertion, potentially the first known instance of a targeted large gene insertion with this approach. We believe this represents the ideal approach for genome editing for Wilson’s disease because we expect that it could enable the majority of patients to be treated with the same therapy irrespective of their specific mutation(s) in ATP7B. The ATP7B gene spans 76,000 bp of DNA in the human genome, and the protein coding sequence of ATP7B is 4,398 bp in length which encodes a protein of 1,465 aa. The majority of the pathogenic mutations identified in ATP7B are located in the C-terminal half of the protein between amino acid positions 645 and 1373 which are encoded in exons 6 to 21 (Figure 41). This region includes the three most frequent mutations (H1069Q, R778L, M645R, I1148T) as well as many other less common mutations. We are evaluating a genome editing approach in which a donor template that encodes exons 6 to 21 of the ATP7B gene (2460 bp) is inserted

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in to the ATP7B locus at intron 6 (downstream of exon 5) such that exons 1 through 5 of the endogenous gene are functionally fused to the inserted wild type exons 6 to 21. We believe this will generate a wild type ATP7B protein driven from the endogenous ATP7B promoter thereby preserving the normal levels of ATP7B and normal regulation of expression. This approach should be applicable to all Wilson’s disease patients except for those with mutations in exons 1 to 5 which represent only a small fraction of the patient population. This approach leverages an all RNA based editing system such that the editing components could theoretically be delivered in a single LNP that is taken up by the hepatocytes of the liver.

Figure 41. Genome Editing Strategy for Wilson’s Disease.

 

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Familial Amyotrophic Lateral Sclerosis (“ALS”)

The Disease

ALS is a rapidly progressive neurodegenerative disorder of upper and lower motor neurons leading to weakness, disability, and death. The prevalence of ALS is approximately four to six patients per 100,000 individuals and it is estimated that over 5,000 patients are diagnosed each year in the United States. While the majority of ALS cases have no known family history, it is estimated that approximately 10% of cases are due to inherited causes. Of these, among the more prominent causes is from a mutation in the gene superoxide dismutase 1 (“SOD1”). SOD1 ALS (occasionally referred to as ALS1), is an autosomal dominant condition associated with toxic gain of function of SOD1 leading to protein misfolding and intraneuronal cytotoxicity.

While the mechanism of action of SOD1 pathophysiology is poorly understood, it is unique in that histopathologically it does not contain typical cytoplasmic inclusions of the nuclear binding protein TDP-43 seen in other spontaneous and familial forms of ALS. The median age of diagnosis of SOD1 ALS is mid-to-late 40s, and similar to other forms of ALS, progressive weakness and loss of voluntary function is rapid with a median survival after diagnosis of approximately three years.

Limitations of Current Therapy

Despite significant investment in research over recent decades, the standard of care for treating all forms of ALS is suboptimal and there remains no cure. There are currently three drugs approved in the United States to treat all forms of ALS (riluzole, sodium phenylbutyrate and taurursodiol, edaravone), and one recent accelerated drug approval for SOD1 ALS (tofersen). To date, only modest benefit in slowing disease progression or improving survival has been observed, and all of these medications require repeat administration or dosing and are associated with adverse effects. In a Phase 3 clinical trial, tofersen, which is an antisense olignonucleotide targeting SOD1 delivered directly to the cerebrospinal fluid and that requires repeated lumbar puncture monthly, did not result in a statistically significant change on clinical measures after 28 weeks of treatment, but did show a nominally statistically significant benefit on a biomarker, neurofilament light chain, and longer term administration was associated with slowing of disease. Clinically significant adverse reactions with tofersen include myositis and/or radiculitis, papilledema and elevated intracranial pressure, and aseptic meningitis, and additionally adverse reactions can occur from repeated lumbar puncture procedures. Thus, despite this precision medicine approach, there remains significant unmet need for effective therapies that can halt disease progression and improve overall survival from SOD1 ALS.

Our Approach/Next Steps

Our approach is to build upon the data generated by tofersen and use one of our programmable nucleases to durably knock down SOD1 levels with a single administration, thus capturing durable benefit with a clinically validated disease target without requiring the patient burden of repeat intrathecal administrations of an oligonucleotide. We plan to deliver the nuclease and associated guide using an AAV vector with sufficient tropism for lower motor neurons. We have performed guide screening with 8 of our small MG nucleases that can fit in a single AAV. A total of 99 lead guides were identified for SOD1 and 60 leads for ATXN2 which are being evaluated in neuronal cells in culture. We are in the process of optimizing AAV vector designs to package and efficiently express our small nucleases and gRNA from a single virus to enable evaluation of lead SOD1 and ATXN2 guides in mouse models.

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Spontaneous ALS

The Disease and Limitations of Current Therapy

As noted above, ALS is a relentless and ultimately fatal disease of motor neurons with no known cure. Approximately 90% of ALS does not have a known family history or clear genetic cause. While the underlying cause of ALS in most cases is unknown, a common histopathologic finding involves misfolded cytoplasmic protein aggregates that include TDP-43. As TDP-43 is a highly conserved nuclear RNA and DNA binding protein involved in RNA processing, the clinical manifestations of ALS may arise both from the toxic cytoplasmic TDP-43 aggregates as well as RNA processing abnormalities from the loss of normal nuclear functions of TDP-43. The initial symptom of ALS is a gradual onset of muscle weakness that is typically painless. These symptoms are often followed by muscle twitching, loss of coordination, falls, and fatigue that impair functionality. As the disease progresses, loss of ambulation, spasticity, and diaphragmatic weakness progresses with the most common cause of death being respiratory failure. Despite the approval of several classes of oral compounds for spontaneous ALS, effects on preserving clinical functionality and survival are modest. There remains significant unmet need for safe and effective therapies that can halt disease progression and improve overall survival from spontaneous ALS.

Our Approach/Next Steps

TDP-43 intracytoplasmic inclusions and proteinopathy are hallmarks of the vast majority of ALS histopathology; however, targeting TDP-43 directly has not been feasible due to its critical role in RNA processing and other cellular functions. We intend to develop an ALS therapy that targets the ATXN2 gene, which we believe is an attractive target based on strong third-party preclinical data targeting the gene ATXN2, which encodes the protein Ataxin 2, and has been shown to be a powerful genetic modifier of TDP-43 in yeast and flies, and importantly, knock-down of Ataxin 2, either by antisense oligonucleotides or by genetic manipulation has improved survival and motor function in a mouse model of ALS. A Phase 1/2 clinical trial of an investigational antisense oligonucleotide targeting Ataxin 2 in adults with ALS that is administered intrathecally is in progress, which will provide further information about the safety and efficacy of this approach.

Our plan is to deliver a nuclease and associated guide using an AAV vector with sufficient tropism for lower motor neurons. We are currently initiating guide screening efforts to potently and selectively knock down Ataxin 2 levels and determining the appropriate nuclease/guide system for AAV packaging.

Charcot-Marie-Tooth Type 1a (“CMT1a”)

The Disease

CMT1a is part of a larger classification of hereditary peripheral motor and sensory neuropathies caused by pathogenic mutations in proteins associated with myelin formation and axonal signal propagation. CMT1a is an autosomal dominant disease arising from a gene duplication of PMP22 (peripheral myelin protein-22) and overexpression of PMP22 protein. PMP22 is tightly regulated and expressed in Schwann cells that control the production of myelin sheaths around axons. Patients with CMT1a have altered myelination that impairs nerve conduction and neuromuscular function. Patients typically present in the first or second decade of life with lower extremity weakness, atrophy, falls, and sensory deficits. The disease is slowly progressive and impairs mobility, and can also involve changes to distal upper extremities and limb deformities. Although life expectancy is typically preserved, CMT1a leads to significant disability and impaired quality of life.

Although exact prevalence estimates vary, CMT is believed to affect one patient per 2,500 individuals, with approximately 126,000 patients in the United States alone. Of those approximately half are believed to have CMT1a, the most common form of the disease.

Limitations of Current Therapy

The treatment of CMT1a and other forms of CMT are largely supportive and there are no approved treatments. Existing standard of care involves physical therapy, stretching, orthotics, and occasional foot surgery to improve deformities that impair ambulation. Patients should be screened for conditions that can exacerbate neuropathies such as vitamin deficiencies or diabetes in an attempt to mitigate against more rapid progression. There are investigational efforts for RNA targeted therapeutics to reduce PMP22 overexpression with strong preclinical data to support the approach, but clinical studies are very early and therapies would need to be given lifelong.

Our Approach/Next Steps

Our approach to CMT1A is a permanent reduction of PMP22 protein levels to the normal range by knock-down of PMP22 expression using our nuclease platform. There is preclinical proof-of-concept for this approach in a mouse model of CMT1A using AAV9 to express a siRNA against PMP22. We intend to use our novel nucleases to introduce InDels in the promoter region of PMP22 that lead to a reproducible reduction of PMP22 expression to the normal range. We anticipate that this editing approach will be delivered via an

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AAV with the goal of achieving uniformly high levels of genome editing while minimizing the risk of toxicity resulting from reducing PMP22 levels to below normal at a cellular level.

We believe the diversity of our nuclease platforms including type V systems such as MG29-1 that create larger deletions and are more likely to inactivate promoters by eliminating transcription factor binding sites, lends itself to identification of nuclease guide combinations that are efficient and result in partial reduction in the expression of the PMP22 gene. We plan to perform a guide screen targeting the promoter region first in HEK293 cells (a cell line that expresses PMP22 mRNA) and active guides with promising InDel profiles and editing potency will be further evaluated for PMP22 mRNA knock-down. Lead systems will be vectorized into AAV9 viruses and evaluated in the CMT1a mouse model. Once reduction of PMP22 expression is confirmed, the therapeutic benefit can be evaluated via various endpoints including tissue morphology, nerve conduction velocity, circulating biomarkers and behavioral tests to support candidate selection.

Duchenne Muscular Dystrophy (“DMD”)

The Disease

DMD is an X-linked recessive myopathic disease involving loss of function mutations of dystrophin, a large protein critical for the stabilization and protection of muscle fibers encoded by the DMD gene. In the setting of absent or abnormal dystrophin, muscle fibers are prone to injury, degeneration, fibrosis, and ultimately fatty infiltration and replacement. DMD typically presents in early childhood with initial symptoms of muscle weakness and progresses rapidly to loss of ambulation as well as respiratory muscle fatigue and cardiomyopathy. In general, large proximal muscles are affected earlier than smaller distal muscles, and lower extremity muscles are affected earlier than upper extremity muscles. Patients are often wheelchair bound before teen years and often die in their late teens and early twenties from cardiopulmonary complications.

Prior to clinically overt findings, patients typically have marked elevations in the muscle enzyme creatine kinase that can serve as the earliest indicator of muscle inflammation and degeneration. DMD is estimated to occur in one of every 3,500 live births, and as an x linked recessive disease is observed almost entirely in males.

DMD is the largest gene identified in humans to date spanning approximately 2.3 Mbp, and the severity of clinical manifestations of the muscular dystrophy is in part related to the location and type of dystrophin mutation and resulting residual amount of dystrophin present. Accordingly, in-frame mutations with residual dystrophin of 5-50% are associated with a less severe clinical course than early truncating mutations with residual dystrophin levels of 0-5%. These findings have led to drug development efforts to restore out of frame mutations into in-frame mutations that result in an abnormal but partially effective dystrophin protein. Additionally, it has been observed that in-frame deletions of large portions of the central part of the dystrophin protein coding region are well tolerated, which has provided the basis for gene transfer approaches of a “microdystrophin” protein.

Limitations of Current Approaches

Despite significant efforts to develop precision medicines for DMD, results to date have not led to meaningful improvements in the standard of care for patients. The mainstay of treatment for DMD is glucocorticoids to address muscle inflammation and improve proximal muscle strength and respiratory function. However, the benefits of glucocorticoids have to be weighed against their long-term risks that include excessive weight gain, impact on growth, cataract formation, bone loss/fracture risk, and behavioral changes. More recently several genetic therapies that induce exon skipping have been approved in the United States to address specific mutations (e.g., exon 45 (casimersen), exon 51 (etiplirsen), exon 53 (golodirsen)) or induce readthrough of stop mutations (ataluren). These therapies have shown modest improvement in muscle dystrophin levels but their overall impact on clinical outcomes has not been established.

Our Approach/Next Steps

DMD patients often have deletions of entire exons (frequently in the hotspot region of exons 45-55) which results in the flanking exons being spliced out of frame at the mRNA level. Approved therapies have aimed to correct these mutations by restoring the open reading frame through “exon skipping”, thus avoiding degradation of the mRNA and/or premature protein termination.

Our proposed genome editing approach is to more effectively facilitate exon skipping by making permanent changes at the DNA level using genome editing that inactivate the splice acceptor (5’ exon splice junction sequence) of the exon that follows the deleted exon such that after transcription the RNA splicing machinery “skips” over that exon, splices to the following exon, and restores the proper reading frame. The resulting proteins will lack the domains encoded by the mutated and skipped exons but these are known to function nearly as well as normal dystrophin.

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For example, one common mutation is deletion of exon 44, which results in exon 43 being aberrantly spliced to exon 45 and an out of frame mRNA (designated as “STOP” in Figure 42) which is then degraded or translated into a truncated protein. As shown in the Figure 42 below, skipping of exon 45 results in splicing from exon 43 to 46 and restores protein expression.

Figure 42. Example of Exon Skipping Approach for Dystrophin Exon 45 that Results in Splicing of Exon 43 to 46 Thereby Restoring the Correct Translational Reading Frame and Thus Dystrophin Function.

 

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We believe a similar strategy can be employed for deletions of exons 50, 51, and 53, creating a franchise of development programs for DMD. We are in the process of initiating a guide screen using our toolbox of DSB nucleases and base editors that are small enough to package in a single AAV vector to inactivate the splice acceptors of the various exons within this hotspot region. The splice acceptor sequence is short (composed of a poly-pyrimidine tract of about 10 to 15 bases followed a short distance away by the three base consensus sequence C/T-A-G) which limits the number of guides that can be designed to inactivate the splice acceptor when using a single nuclease. We are leveraging our collection of small nucleases that have a variety of different PAMs to enable more guides to be designed against each splice acceptor. Hits from this guide screen will be assayed in cardiac and/or skeletal muscle cells in vitro. The nuclease/guide combinations that show potent activity in muscle cells will then be evaluated in human cells in which the DMD gene contains the relevant pathogenic mutations to evaluate exon skipping activity. In vivo evaluation of leads will be performed in mouse models of DMD.

Because DMD affects skeletal and cardiac muscles throughout the body of DMD patients, effective therapy will require delivery to a large proportion of these muscles with the diaphragm and heart muscle being of particular importance. To date the only delivery system that is able to deliver to these tissues is AAV, with AAV serotype 9 showing the most promising delivery profile. Because a maximum of up to 5 kb of DNA can be packaged inside the AAV virus it is not possible to deliver SpCas9 and a guide or current SpCas9 derived base editors and a guide in a single AAV. While these larger systems can be split between two AAVs, this reduces potency due to the dose limiting toxicity of AAV and the need to administer two viruses (effectively doubling the dose). A dual AAV approach also increases manufacturing complexity and costs. We are leveraging our collection of smaller editing systems that can be packaged in a single AAV (currently three nucleases).

The potential therapeutic benefit of editing the DMD gene in mouse models will be assayed at the mRNA and protein level, and by functional endpoints such as maximum force output.

Cystic fibrosis

Cystic fibrosis is an autosomal recessive lung disease caused by mutations in the CFTR gene. Mutations impact electrolyte transport in cells that produce mucus, sweat, and digestive fluids causing these secretions to be thicker and more viscous than normal. As a result, these secretions become sticky and can clog passageways and ducts, particularly in the lung and pancreas. Over time, these thick mucus secretions in the lung cause chronic lung infections, inflammation, fibrosis and ultimately destructive bronchoalveolar lung disease resulting in progressive pulmonary failure. The pancreas is similarly affected and blockage of ducts in the pancreas lead to loss of exocrine function and pancreatic insufficiency.

The overall incidence of cystic fibrosis is estimated at one patient in 3,000 to 6,000 live births in the US and Europe, but rates depend strongly on geographic location and ethnicity with higher rates in Caucasians of northern European descent and much lower rates in Asians. It is estimated that there are approximately 30,000 individuals living with cystic fibrosis in the US and 70,000 individuals worldwide. Over the past few decades, improvements in antibiotic treatments, supportive measures, multidisciplinary care centers, and newer targeted medications have increased the overall life expectancy from late childhood to the fourth decade.

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Limitations in Current Approaches

One of the challenges in developing precision medicines for cystic fibrosis is the large nature of the gene and the varied mutations along the gene that result in loss of protein expression, loss of function, misfolding and mislocalization within the cell. Accordingly, therapeutic efforts have required individualized approaches tailored to certain mutations to improve CFTR function. Although such medications have improved rates of lung function decline, not all patients have mutations amenable to CFTR targeted therapies. Further, none of these therapies offer a true cure to the underlying gene mutation, and patients continue to experience morbidity and mortality from cystic fibrosis disease progression.

Our Approach/Next Steps

Our goal for a genome editing based treatment for cystic fibrosis is two-fold: (1) a permanent curative therapy from a single treatment, and (2) a therapy that is applicable to the majority of cystic fibrosis patients. We believe this could be achieved by integrating a functional version of the CFTR gene into the genome of the lung basal stem cells (alternatively called bronchioalveolar stem cells). The basal stem cells are believed to give rise to the lung epithelial cells that are the site of CFTR expression that is defective in cystic fibrosis patients. Stem cells are a specialized population of cells that are maintained for a person’s lifetime and are the source for renewal of differentiated cells. Editing the stem cells should ensure that the introduced functional CFTR gene is not lost over time due to the shedding of differentiated epithelial cells. By integrating a functional CFTR gene rather than correcting individual cystic fibrosis causing mutations, a single therapy could treat the majority of cystic fibrosis patients.

We intend to explore two of our genome editing modalities in cystic fibrosis. The Big RIGS technology and CAST systems both have the potential to integrate large pieces of DNA (in this case encoding a CFTR gene) into a specific site in the genome. For the Big RIGS approach, the CFTR gene would be delivered as an RNA that is reverse transcribed into DNA to provide the DNA template for integration. In the case of CAST, the CFTR gene would be delivered as double stranded DNA that is recognized by the CAST system and integrated at the desired site by the transposase. Delivery to the basal stem cells of the lungs can theoretically be achieved by IV dosing. We may also evaluate a non-viral delivery system for lung delivery.

We believe the Big RIGS system has the advantage that it can be delivered using only RNA for which non-viral RNA delivery technologies such as LNP are well established. In contrast, delivery of DNA (that would be required for CAST) by non-viral delivery vectors is not well established with the main barrier being transit of the DNA into the nucleus. However, in a preliminary study we have recently demonstrated delivery of a 4.6 kb double stranded DNA to the nuclei of cells in the liver of mice by IV administered LNP.

Cell Therapy Applications Using Our Platform

Identifying and optimizing novel nucleases from our metagenomics platform has involved a series of stringent efficiency, activity, and specificity testing in a variety of immune cell types which yields a comprehensive dataset that demonstrates our genome editing capabilities for cell therapy applications. Many cell therapies require multiple simultaneous genome edits to enhance efficacy, safety, and/or durability of these products. In the allogeneic cell therapy setting, additional gene edits are required to decrease the immunogenicity of the cell therapies to prevent rejection by the host immune system. Our toolbox provides an important advantage compared to the current cell therapy landscape as we are able to use either single or multiple genome editing enzymes to implement multiple gene edits (“multiplex editing”) with high efficiency and specificity. These gene edits could be either knock-outs or knock-ins.

We are currently working on various discovery stage projects in-house and with collaborators to evaluate the efficacy and specificity of our enzymes for the engineering CAR-T and TCR-T cell product candidates.

Our initial lead nucleases can be used to efficiently engineer primary human T cells, demonstrating utility for cell therapy applications.

Our programmable nucleases have achieved key requirements for T cell engineering, including genome editing dsDNA-break induced knock-in of a chimeric antigen receptor (CAR, Figure 43).

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Figure 43. Example of Two Lead Nucleases, Type II MG3-6 and Type V MG29-1, Capable of Engineering Primary Human T Cells.

 

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* Nucleases were delivered as RNP and CAR donor template by AAV.

* Nucleases were programmed to target the TRAC locus.

* CAR integration into T cells from 10 T cell donors with MG3-6 and MG29-1 and either 5E4 or 1E5 MOI. Data generated by Metagenomi.

Our MG29-1 nuclease is a highly active and specific nuclease being used for ex vivo T cell engineering in collaboration with Affini-T Therapeutics. Affini-T Therapeutics is developing a cell therapy based on engineering the TCR of T cells to recognize mutant KRAS for the treatment of patients with solid tumors. Affini-T scientists showed that the knock-out of the endogenous TCR improved expression of a transgenic TCR, and that MG29-1 can be used to engineer functional TCR T cells by knock-in of a transgene into the TRAC locus.

 

Figure 44a. MG29-1 tested in primary human CD4/CD8 T cells showed specificity in two loci.

Figure 44b. Oligonucleotide capture in primary T cells identified putative MG29-1 off-targets. More sensitive methods did not verify such results.

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*
InDel formation was evaluated for 590 computationally predicted off- targets across TRAC and TRBC, allowing for up to six mismatches.
*
Off-targets had 100-fold fewer barcodes compared with target sites.
*
Off-target sites could not be validated with amplicon-based NGS sequencing.
*
The presence of >9 mismatches in the off-target sites may indicate that they are not likely to be true positives.

 

Data generated by Affini-T Therapeutics.

We plan to use our gene editing enzymes to engineer different T cell subsets with either chimeric antigen receptors (CARs) or engineered TCRs targeting antigens expressed in various tumor indications, including hematological and solid tumor malignancies. We are currently investigating different tumor targets and tumor indications to select future T cell therapy product candidates. In addition, we plan to use CAR-T cells in auto- immune diseases driven by the production of autoantibodies, including conditions such as systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, autoimmune hemolytic anemia, immune-mediated

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thrombotic thrombocytopenic purpura, and possibly others. We will use T cells engineered with chimeric antigen receptors against B cell markers to deplete the B cell compartment and eliminate the production of autoantibodies.

Our License and Collaboration Agreements

Moderna Strategic Collaboration and License Agreement

On October 29, 2021, the effective date, we entered into a Strategic Collaboration and License Agreement (the “Moderna Agreement”) with Moderna. We will collaborate with Moderna on the research and development of in vivo genome editing therapies directed at certain targets and the commercialization of such genome editing therapies. The collaboration provides Moderna with exclusive access to our technology platform during the research period in (1) the field of in vivo gene editing technology for a therapeutic, ameliorative or prophylactic application by way of knock-out through InDel formation or base editing or insertion of an exogenous DNA template (such field, “DT Field”) and (2) the field of in vivo gene editing technology for a therapeutic, ameliorative or prophylactic application outside the use of (a) DNA donor templates and (b) no exogenous template at all but including (c) correction by base editing (such field, “RT Field”). The use of RIGS with mRNA and base editing correction with mRNA is within the RT Field exclusive to Moderna within the Term. We formed a joint steering committee, a joint research subcommittee and a joint patent subcommittee to oversee the collaboration activities. Each of us and Moderna must use commercially reasonable efforts to perform and complete our respective activities under research plans and commercialization plans approved by the joint steering committee.

Under the terms of the Moderna Agreement, we and Moderna will collaborate on one or more programs in the RT Field (the “Moderna RT program”) and two programs in the DT Field (the “Moderna DT program” and the “DT Co- Co program”). We and Moderna have each granted the other party a non-exclusive license in such party’s (i) background technology, including intellectual property rights controlled by each party related to each respective program, and (ii) the know-how and patents that come into control of each party relating to the program and during the respective program term, in each case to carry out activities in the applicable research programs. We shall own certain intellectual property that relates to our technology platform (“Metagenomi Program Technology”). Moderna shall own certain intellectual property that relates to Moderna’s technology platform (“Moderna Program Technology”). Any intellectual property discovered, invented, conceived or created during an applicable research term that is not Metagenomi Program Technology or Moderna Program Technology shall be jointly owned by us and Moderna. Further, we granted Moderna a perpetual, irrevocable, royalty-free, nonexclusive license under Metagenomi Program Technology to the extent pertaining to the exploitation of donor templates or guides to which Moderna has any inventive contribution.

With respect to the Moderna RT and Moderna DT programs, we will collaborate on the research and development of product candidates under the approved research plans. The initial research term of the Moderna RT program is four years, which may be extended by Moderna for an additional three years upon written notice and a payment of extension fees. The initial research term of the Moderna DT program is four years. We granted to Moderna an option to obtain an exclusive license to develop, manufacture and commercialize up to ten Moderna RT program candidates and up to two Moderna DT program candidates at any time during the research term and prior to filing of an investigational new drug (“IND”) application with the Food and Drug Administration (“FDA”) or any similar application filed with a regulatory authority in a country other than the United States (“U.S.”), subject to Moderna’s payment of an option exercise fee of $10.0 million per target. If we or any of our affiliates wish to grant any third party rights in certain targets in the DT Program prior to the earlier of the (a) second anniversary of the agreement or (b) 90 days after achievement of certain readiness standards, we shall provide Moderna with written notice thereof and Moderna shall have a right of first negotiation to negotiate an agreement on the terms of a collaboration and license agreement for such targets.

With respect to the DT Co-Co program, we will work together with Moderna on the co-development and commercialization of products and share costs and profits equally. We granted Moderna a co-exclusive (with us and our affiliates) license under our patents and know-how related to PH1, the DT Co-Co target, to exploit all applications of such target in the DT Co-Co program. We maintain commercialization rights in the U.S. (subject to Moderna’s right to appoint up to 50% of the U.S. sales force for the DT Co-Co program), while Moderna maintains these rights in countries other than the U.S. The initial research term for the DT Co-Co program is four years, and each party has a right to opt-out of the DT Co-Co program at any time, at which point the other party has the right to solely continue the development and commercialization activities, provided that the party which has opted-out shall have a right of first offer in the event that the other party wishes to grant a license or sublicense to a third party with respect to the DT Co-Co program. If there is no development candidate nomination by the end of the initial research term, the DT Co-Co program will expire, unless we have mutually agreed to continue the program.

During the year ended December 31, 2021, we received a non-refundable upfront payment of $40.0 million and a $5.0 million payment for the first year of research costs. Concurrent with the Moderna Collaboration Agreement, Moderna also provided $30.0 million in cash in the form of a convertible promissory note pursuant to a convertible promissory note agreement dated October 29, 2021 (the “Moderna Convertible Promissory Note Agreement”). The convertible promissory note was converted into shares of Series

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B redeemable convertible preferred units in January 2022. Moderna will reimburse us up to $5.0 million in annual research and development costs related to the Moderna DT and Moderna RT programs, or up to the agreed amount of expenses per the budget. As of December 31, 2023, we have received a total of $49.6 million under the Moderna Collaboration Agreement, not including cost-sharing payments under the DT Co-Co program.

For the Moderna RT and Moderna DT programs, we are eligible to receive (i) technology milestone fees related to the achievement of certain preclinical research objectives of up to $75.0 million, (ii) development and regulatory milestones of up to $100.0 million per target, (iii) sales milestones of up to $200.0 million per target, and (iv) royalties ranging from a mid-single digit to a low-teens percentage of annual net sales of a licensed product. Any profits and losses from the co-development and commercialization of the DT Co-Co program are shared equally between us and Moderna. With respect to the DT Co-Co program for which the opt-out party has exercised its opt-out right, the continuing party will pay to the opt-out party, certain development, regulatory and sales milestone payments that will not exceed an aggregate $239.0 million per DT Co-Co target, and opt-out royalties ranging from a high-single digit to a low-teens percentage of annual net sales of a licensed product.

The term of the Moderna Agreement will continue on a licensed product-by-licensed product and country-by-country basis, until the expiration of the applicable royalty term. The royalty term commences on the first commercial sale of a licensed product and terminates on the latest of: (a) the expiration or abandonment of the last valid claim of a patent within the licensed Moderna DT or RT technology; (b) 10 years after the first commercial sale of a licensed product; and (c) expiration of the regulatory exclusivity. Upon the expiration of the term of a licensed product in the Moderna DT or Moderna RT program, the licenses granted to Moderna will survive and become perpetual, fully paid and royalty-free. Each party may terminate the Moderna Agreement on a program-by-program basis upon written notice to the other party for an uncured material breach or insolvency. In lieu of termination for our material breach, Moderna may, upon written notice, continue the agreement with respect to the relevant collaboration target at Moderna’s amounts payable reduced by 50%, or in the case of a DT Co-Co-Target, Moderna may propose, subject to arbitration, to adjust the profit and loss share for DT Co-Co Products to provide Moderna with an additional share of the net profits (not to exceed 75% of the total net profits). We may terminate the Moderna Agreement upon written notice to Moderna for a patent challenge. Additionally, Moderna may terminate the agreement at its convenience with respect to Moderna DT or Moderna RT programs for any reason upon at least: (a) 60 days’ prior written notice if a first commercial sale has not occurred for the products in such program, or (b) 180 days’ prior written notice if a first commercial sale of a product in such program has occurred. Upon termination, all licenses granted under the agreement with respect to the applicable products under the agreement shall terminate, subject to an orderly wind- down period, provided that any permitted sublicense granted to a third party shall survive (provided such third party did not cause the termination through uncured material breach).

Affini-T Development, Option and License Agreement

On June 14, 2022, the effective date, we entered into a Development, Option and License Agreement (the “Affini-T Agreement”) with Affini-T. Pursuant to the Affini-T Agreement, we and Affini-T have agreed to identify, develop or optimize certain reagents using our proprietary technology for Affini-T to use such reagents to develop and commercialize gene edited TCR-based therapeutic products exclusively in the field of treatment, prevention or diagnosis of any human cancer using products with any engineered primary TCR alpha/beta T cells and non-exclusively in the field of treatment, prevention or diagnosis of any human cancer using products with certain other engineered immune cells worldwide. A joint steering committee was established by both parties to assign alliance managers and project leaders to oversee the collaboration activities. We must use commercially reasonable efforts to perform and complete our obligations under research plans approved by the joint steering committee.

Pursuant to the Affini-T Agreement, we granted Affini-T options to receive, on a pre-specified target-by-pre-specified target basis, for up to six pre-specified targets, either (i) an exclusive, royalty-bearing, sublicensable worldwide license under all of our applicable intellectual property to research, develop, manufacture, use, commercialize and otherwise exploit any TCR-based therapy, preventative treatment, or diagnostic for humans that is directed to such pre-specified target, contains or comprises Primary TCR alpha/beta T Cells and is derived from ex vivo application of our reagent (the “Exclusive Option”) or (ii) a non-exclusive, royalty-bearing, sublicensable worldwide license under all our applicable intellectual property to research, develop, manufacture, use commercialize and otherwise exploit any TCR-based therapy, preventative treatment, or diagnostic for humans that is directed to such pre-specified target, contains or comprises TCR natural killer (“NK”) cells derived from iPSC immune cells or TCR T cells derived from donor-derived or iPSC immune cells. Affini-T can exercise its options for either an exclusive license or a non-exclusive license, or both, for each pre-specified target by providing written notice prior to the earlier of (x) the end of the Affini-T Agreement term or (y) 90 days following the filing of an IND for a licensed product directed to a pre-specified target, subject to the payment of certain fees per each option exercised. After the option exercise, Affini-T has agreed to use commercially reasonable efforts to conduct all development and commercialization activities for a licensed product, and development and commercialization of all licensed products will be at Affini-T’s sole cost and expense.

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On a target-by-target basis, (1) until the earlier of Affini-T’s (a) exercise of an Exclusive Option, (b) written notice not to exercise an Exclusive Option or (c) expiration of an applicable Exclusive Option or (2) upon exercise of an Exclusive Option, we and our affiliates shall not exploit, or work with any third party to exploit, any ex vivo gene edited products directed to the applicable target covered by such Exclusive Option.

In connection with the Affini-T Agreement, we received upfront equity consideration of 719,920 shares of Affini- T’s common stock with an estimated fair value of $1.3 million in June 2022. The fair value of Affini-T’s shares of common stock was estimated by our management, considering the most recent third-party valuation. Affini-T has also agreed to reimburse us for expenses incurred while performing research activities under the research plans. As of December 31, 2023, we received a total of $4.3 million from Affini-T related to reimbursable expenses. Additionally, we are eligible to receive (i) 933,650 shares of Affini-T’s common stock upon the achievement of a regulatory milestone, which is the earlier of a submission of a drug master file to the FDA or an acceptance of an IND filing for a licensed product by the FDA, (ii) up to $18.8 million in future developmental milestone payments depending on the completion of or the number of patients dosed in, the relevant human clinical trial, or the initiation of a pivotal trial, and $40.6 million in future regulatory approval milestone payments, which include regulatory approvals in the U.S. and other markets for licensed products directed to a pre-specified target if options for both exclusive and non-exclusive licenses are exercised with respect to such target, (iii) up to $250.0 million in sales-based milestones for aggregate sales of all licensed products directed to a given pre-specified target and (iv) royalties ranging from a low-single digit to high-single digit percentage of worldwide annual net sales of licensed products.

The initial term of the Affini-T Agreement is five years from the effective date. If Affini-T exercises an Exclusive Option with respect to any pre-specified target during the initial term, the initial term will be extended by an additional five years. Following the expiration of the extended term, if any, the agreement will continue on a target-by-target basis and expire with respect to such target upon the expiration of the royalty term for all licensed products directed to such target. The Affini-T Agreement may be terminated during the term by either party for an uncured material breach by, or bankruptcy of, the other party. Additionally, Affini-T may terminate the Affini-T Agreement for convenience, in its entirety, on a research plan-by-research plan basis, on a target-by- target basis or on a licensed product-by-licensed product basis, by providing prior written notice. Upon a material breach and with written notice to us, in lieu of termination, Affini-T shall have the right to continue the agreement at payments payable at a certain percentage reduction.

Ionis Collaboration and License Agreement

On November 10, 2022, the effective date, we entered into a Collaboration and License Agreement (the “Ionis Agreement”) with Ionis to collaborate on drug discovery and exploratory research activities to advance new medicines using gene editing strategies, with the goal of discovering novel medicines. Pursuant to the terms of the Ionis Agreement, we granted Ionis and its affiliates a worldwide exclusive, royalty-bearing license, with the right to grant sublicenses, to use all licensed systems and licensed products in the field of in vivo gene editing for all therapeutic, prophylactic, palliative, and analgesic uses in humans. In connection with the Ionis Agreement, we also have the right to exercise an exclusive option to co-develop and co-commercialize certain products under a drug discovery program. A joint steering committee was established by both parties to coordinate, oversee, and monitor the research and drug discovery activities under the Ionis Agreement. Each party must use commercially reasonable efforts to perform and complete its respective activities under the applicable program plans approved by the joint steering committee.

We will collaborate to discover therapeutic products under a drug discovery program and develop a drug discovery plan for each target, selected by Ionis. The target selection is divided into two waves: up to four targets in Wave 1 and up to four targets in Wave 2. For each drug discovery program, once the parties identify a development candidate that is suitable for further development, Ionis will be responsible for the development and commercialization of products resulting from such program. Per the terms of the Ionis Agreement, at any time prior to the designation of a development candidate for a drug discovery program and for any reason, Ionis may replace the collaboration target, provided such target has not previously been substituted out. Ionis may substitute (i) up to two Wave 1 targets and (ii) up to two Wave 2 targets.

The drug discovery activities for a program commence on the selection of a target and expire upon the earlier of (a) completion of all drug discovery activities for such program, (b) the fifth anniversary of the effective date and (c) selection of a development candidate for such drug discovery program. If one or more Wave 2 targets become collaboration targets as a result of the parties achieving enabled delivery and less than two years are remaining in the drug discovery term, then the term will be extended to the earlier of (i) the time that we complete all of our activities under the applicable drug discovery plan and (ii) the seventh anniversary of the effective date, subject to our consent.

We will also conduct an exploratory research program, and will jointly optimize gRNA and select delivery technologies and other activities. The exploratory research activities commence on the effective date and expire upon the earlier of (a) completion of all exploratory research activities established in the exploratory research plan, and (b) the fifth anniversary of the effective date.

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We have the exclusive option to co-develop and co-commercialize the licensed products under a drug discovery program (the “Co-Co Option”) with Ionis. The Co-Co Option may be exercised for (a) the initial Wave 1 target (“Target 1”), (b) no more than one of the other three discovery programs for the Wave 1 targets, and (c) no more than two drug discovery programs for the Wave 2 targets that become collaboration targets. If we exercise the Co-Co Option for a particular drug discovery program, that drug discovery program will automatically be deemed a “Co-Co Program”, all corresponding licensed products be deemed “Co-Co Products,” we will be obligated to pay Ionis an option exercise fee, and we and Ionis will enter into a separate co- development and co-commercialization agreement. The Co-Co Option exercise fee will equal 50% of Ionis’ internal costs and out-of-pocket costs incurred in the conduct of the drug discovery activities prior to the exercise of the Co-Co Option and be reduced by 50% of our corresponding costs incurred. Future development and commercialization costs will be shared equally. We may elect to reduce our cost-share percentage anywhere between 50% and 25% on a go-forward basis, provided we will continue to bear 50% of the costs of any clinical trials ongoing at the time of the election through the completion of the clinical trials.

We will manufacture all licensed systems and certain components of the applicable licensed products that are needed by Ionis for use in its development activities and all of our manufactured components needed by Ionis for use in its commercialization activities. We will provide the manufactured components at a price that represents the cost of goods plus 15%.

Pursuant to the terms of the Ionis Agreement, we have also been granted an option to obtain a non-exclusive, royalty-bearing license, with the right to grant sublicenses, for certain Ionis’ background technology to use in up to eight therapeutic products discovered by us in the field of in vivo gene editing and directed to a Collaboration Target (each such product, a “Metagenomi Product” and each such option an “Ionis IP Option”), but subject to encumbrance checks with respect to particular targets. A Collaboration Target is a target that is selected by Ionis, and, with respect to us, is not the subject of discussions with a third party, is not the subject of a contractual grant of rights to a third party nor the subject to an internal research and development program. If we exercise our Ionis IP Option, we will pay to Ionis up to several million dollars per Metagenomi Product upon achievement of certain clinical and regulatory milestones. We are also obligated to pay Ionis royalties in an amount equal to a low single-digit royalty on the net sales of the applicable Metagenomi Product on product-by-product and country-by-country basis.

In November 2022, we received an $80.0 million upfront payment from Ionis for the Wave 1 drug discovery research collaboration and selected Target 1. Ionis selected its second target (“Target 2”) in Wave 1 in December 2022, its third target (“Target 3”) in Wave 1 in November 2023, and its fourth target (“Target 4”) in Wave 1 in February 2024. Ionis has an option to select up to four Wave 2 targets at any time during the drug discovery term, if (a) an IND for any licensed product directed to a Wave 1 target is filed with the applicable regulatory authority or (b) the parties achieve enabled delivery for a non-liver target under the exploratory research activities, by providing written notice and by paying a Wave 2 target selection fee of $15.0 million or $30.0 million, depending on and per the selected target.

Ionis is obligated to reimburse us for all internal costs and out-of-pocket costs incurred in the performance of the exploratory research activities, up to an aggregate of $10.0 million, which is payable in quarterly installments of $0.5 million during the exploratory research term. As of December 31, 2023, we received a total of $2.0 million related to the reimbursable expenses. We are also eligible to receive (a) up to $29.0 million in future development milestone payments for each licensed product; (b) up to $60.0 million in future regulatory milestone payments for each licensed product; (c) up to $250.0 million in sales-based milestones for each licensed product; and (d) royalties on annual net sales of licensed products from a mid-single-digit to low-teens percentage, subject to customary reductions.

The term of the Ionis Agreement will continue (i) with respect to the drug discovery programs, until the expiration of all applicable royalty terms for a licensed product, (ii) with respect to the Co-Co Programs, until the parties cease all exploitation for the Co-Co Products that are the subject to such Co-Co Program, and (iii) with respect to the Metagenomi Products, until the expiration of the royalty term for a Metagenomi Product.

The royalty term ends on the latest of the following two dates: (i) the expiration of (A) the last claim of any issued and unexpired patent, or (B) a claim within a patent application that has not been pending for more than seven years from the earliest date to which the claim or applicable patent application is entitled to claim priority and which claim has not been revoked, cancelled, withdrawn, held invalid, or abandoned, or (ii) 12 years following the first commercial sale of a licensed product.

The Ionis Agreement may be terminated during the term by either party for an uncured material breach or bankruptcy by the other party. Additionally, Ionis may terminate the Ionis Agreement for convenience and without penalty, in its entirety or on a licensed product-by-licensed product basis, by providing 90 days’ written notice. Upon termination, Ionis will transfer to us ownership of all regulatory approvals, and all licenses granted under the agreement with respect to the applicable products under the agreement shall terminate, subject to an orderly wind-down period and a right for Ionis to sell or otherwise dispose of applicable products on hand at the time of such termination. Upon our written request within 30 days following termination, Ionis will grant us an exclusive, royalty-bearing (as agreed by the parties at such time), right and license, with the right to grant sublicenses through multiple tiers, to patent

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rights and know-how controlled by Ionis and used in the development, commercialization, or exploitation of terminated products, solely for the exploitation of such terminated products in the terminated countries.

Competition

The pharmaceutical and biotechnology industries, including the gene therapy and genome editing fields, are characterized by rapidly advancing technologies, intense competition, and a reliance on strong intellectual property. We believe our metagenomics powered discovery platform along with our expertise in genome editing, drug discovery, clinical development, manufacturing and our ever-increasing IP portfolio, provide us with several key competitive advantages over our peers. Despite our competitive advantages, we face competition from several companies. There are numerous publicly traded companies utilizing CRISPR/Cas nuclease technology, including Caribou Biosciences, Inc., Editas Medicine, Inc., CRISPR Therapeutics AG, Intellia Therapeutics, Inc., and Graphite Bio, Inc., among others. Beam Therapeutics Inc. and Verve Therapeutics, Inc. utilize base editing technology and Prime Medicine utilizes prime editing technology. Several other companies such as Sangamo Therapeutics, Inc., Precision BioSciences, Inc., Cellectis S.A., and bluebird bio, Inc. utilize first- generation nuclease-based genome editing technologies, including ZFNs, engineered meganucleases and TALENs. We also face competition from companies utilizing gene therapy, oligonucleotides, and CAR-T therapeutic approaches.

There are several other private companies such as Arbor Biotech, Chroma Medicine, Inc., Mammoth Biosciences, Scribe Therapeutics, Tessera Therapeutics, Tome Biosciences, and Tune Therapeutics, Inc. that have announced they are working on genome- and epigenome-editing therapies.

Any product candidates that we successfully develop and commercialize will compete with existing therapies and new therapies that may become available in the future that are approved to treat the same diseases for which we may obtain approval for our product candidates. This may include other genome editing companies using antiquated or next generation genome editing approaches or other types of therapies, such as small molecule, antibody, and/or protein therapies.

In addition, many of our current or potential competitors, either alone or with their collaboration partners, have significantly greater financial resources and expertise in research and development, manufacturing, preclinical testing, conducting clinical trials and approved products than we do today. Mergers and acquisitions in the pharmaceutical, biotechnology and gene therapy industries may result in even more resources being concentrated among a smaller number of our competitors. Smaller or early-stage companies may also prove to be significant competitors, particularly through collaborative arrangements with large and established companies. We also compete with these companies in recruiting, hiring, and retaining qualified scientific and management talent, establishing clinical trial sites and patient registration for clinical trials, obtaining manufacturing slots at contract manufacturing organizations. Our commercial opportunity could be reduced or eliminated if our competitors develop and commercialize products that are safer, more effective, particularly if they represent cures, have fewer or less severe side effects, are more convenient, or are less expensive than any products that we may develop. Our competitors also may obtain FDA or other regulatory approval for their products more rapidly than we may obtain approval for ours, which could result in our competitors establishing a strong market position before we are able to enter the market. The key competitive factors affecting the success of all of our programs are likely to be their efficacy, safety, convenience, and availability of reimbursement.

Manufacturing

Our genome editing technology is composed of multiple genome editing components including the nuclease, mRNA, gRNA, and in some instances may include a donor DNA or RNA template for insertions. We have extensively characterized each of these components and have made significant investment in scalable manufacturing and process automation to meet stringent current good manufacturing practices (“cGMP”). Our in-house cGMP facility is capable of manufacturing clinical grade nucleases and mRNA to supply both wholly- owned and collaboration programs. We partner with contact manufacturing organizations (“CMOs”) for gRNA and DNA template development and supply and continue to invest in both viral and non-viral delivery technologies internally and with partners. We believe our ability to develop, characterize, and manufacture complex human genome editing components is essential to maintaining a competitive edge while pursuing a successful regulatory pathway for genetic medicine.

Intellectual Property

Our success depends in large part upon our ability to obtain and maintain our technology and intellectual property. To protect our intellectual property rights, we primarily rely on patents and trade secret laws, confidentiality procedures, and employee disclosure and invention assignment agreements. Our intellectual property is critical to our business and we strive to protect it through a variety of approaches, including by obtaining and maintaining patent protection in various countries for our genome editing technology and other inventions that are important to our business.

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Patents have a limited lifespan. In the United States, the natural expiration of a patent is generally 20 years from its earliest U.S. non-provisional filing date. The time required for development, testing, and regulatory review of our genome editing systems limits the commercially useful lifespan of our patents.

The patent positions of companies like ours are generally uncertain and involve complex legal and factual questions. No consistent policy regarding the scope of patentable claims in the field of genome editing has emerged, for example, in the United States and in Europe. Changes in the patent laws and rules, either by legislation, judicial decisions, or regulatory interpretation may diminish our ability to protect our inventions and enforce our intellectual property rights. These changes could affect the scope and value of our intellectual property.

Filing, prosecuting, enforcing, and defending patents protecting our genome editing systems in all countries throughout the world would be prohibitively expensive. We cannot seek patent protection for our genome editing systems throughout the world. Furthermore, the intellectual property rights we obtain in some countries outside the United States can be less extensive than those obtained in the United States. The requirements for patentability may differ in certain countries, particularly in developing countries; thus, even in countries where we do pursue patent protection, there can be no assurance that any patents will issue with claims that cover our products.

Our ability to stop third parties from infringing any of our patented inventions, either directly or indirectly, will depend in part on our success in obtaining, defending, and enforcing patent claims that cover our genome editing systems. We cannot be sure that any patents will be granted with respect to any of our pending patent applications or with respect to any patent applications filed by us in the future. We cannot be sure that any of our existing patents or any patents that may be granted to us in the future will be found by a court to be enforceable. Protecting our competitive position around our genome editing systems may involve lawsuits to enforce our patents or other intellectual property, which is expensive and time consuming, and may ultimately be unsuccessful. Furthermore, our issued patents and those that may issue in the future may be challenged, narrowed, circumvented, or invalidated, which could limit our ability to stop competitors from marketing related genome editing systems or limit the length of the term of patent protection that we may have for our genome editing systems and future gene therapies. We cannot be sure that any of our existing patents or any patents that may be granted to us in the future will be useful in protecting our commercialized genome editing systems. The rights granted under any issued patents may not provide us with complete protection or competitive advantages against competitors with similar but not identical technology or technologies that achieve similar outcomes but with different approaches. For these reasons, we may have competition for our genome editing systems.

Our issued patents and those that may issue in the future do not guarantee us the right to practice our genome editing systems. Third parties may have issued patents or be granted patents in the future that could block our ability to commercialize our genome editing systems.

We and third parties rely on trade secrets to protect certain aspects of our genome editing systems. If we are unable to protect the confidentiality of our trade secrets, our competitive position could be harmed. Furthermore, reliance on trade secrets does not prevent third parties from independently inventing those aspects of our genome editing systems. While we take commercially reasonable steps to ensure that our employees do not use the trade secrets of third parties, third parties may file claims asserting that we or our employees have misappropriated their trade secret.

For this and other risks related to our technology, inventions, improvements, platforms, and genome editing technology, please see the section entitled “Risk Factors—Risks Related to Our Intellectual Property.”

Patent Portfolio

As of December 31, 2023, we own three issued U.S. patents, 21 pending U.S. non-provisional patent applications, 38 pending U.S. provisional patent applications, nine issued foreign patents in Great Britain, Hong Kong, Mexico and Australia, 129 pending foreign patent applications, including in Australia, Canada, China, Europe, Great Britain, Hong Kong, India, Japan, Korea, Mexico and Brazil, and 23 Patent Cooperation Treaty (“PCT”) patent applications.

The patent portfolios for our genome editing systems as of December 31, 2023 are summarized below.

Our type II CRISPR systems are protected by two issued U.S. patents with composition of matter claims covering genome editing systems using Type II nucleases, six pending U.S. non-provisional patent applications with composition of matter claims covering genome editing systems using Type II nucleases and methods of using them, and one pending U.S. provisional patent application with composition of matter claims covering genome editing systems using Type II nucleases and methods of using them. Our type II CRISPR systems are also protected by three issued foreign patents with composition of matter claims covering genome editing systems using Type II nucleases, including in South Korea, Great Britain, Australia and Mexico, 25 pending foreign patent applications with composition of matter claims covering genome editing systems using Type II nucleases and methods of using them, including in Australia, Canada, China, Europe, Great Britain, Hong Kong, India, Japan, Korea, Mexico, and Brazil, and one PCT

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patent application with composition of matter claims covering genome editing systems using Type II nucleases and methods of using them. The aforementioned issued US patents will expire on February 14, 2040, and the issued foreign patents will expire on February 14, 2040. If issued, the aforementioned patent applications are expected to expire between February 14, 2040 and May 6, 2041.

Our type V CRISPR systems are protected by one issued U.S. patent and four pending U.S. non-provisional patent applications with composition of matter claims covering genome editing systems using Type V nucleases with composition of matter claims covering genome editing systems using Type V nucleases and methods of using them. Our type V CRISPR systems are also protected by two issued foreign patents with composition of matter claims covering genome editing systems using Type V nucleases, including in Great Britain and Hong Kong, 19 pending foreign patent applications with composition of matter claims covering genome editing systems using Type V nucleases and methods of using them, including in Australia, Brazil, Canada, China, Europe, Great Britain, Hong Kong, India, Japan, South Korea, and Mexico, and one PCT patent applications with composition of matter claims covering genome editing systems using Type V nucleases and methods of using them. If issued, the aforementioned patent applications are expected to expire between March 6, 2041 and July 29, 2043. The aforementioned issued foreign patents will expire on March 6, 2041.

Our base editor systems are protected by seven pending U.S. non-provisional patent applications with composition of matter claims covering genome editing systems using nucleases and base editors and methods of using them, and three pending U.S. provisional patent applications with composition of matter claims covering genome editing systems using nucleases and base editors and methods of using them. Our base editor systems are also protected by 12 pending foreign patent applications with composition of matter claims covering genome editing systems using nucleases and base editors and methods of using them, including in Australia, Canada, China, Europe, Great Britain, Hong Kong, India, Japan, Korea, Mexico, and Brazil, and one PCT patent applications with composition of matter claims covering genome editing systems using nucleases and base editors and methods of using them. If issued, the aforementioned patent applications are expected to expire on September 10, 2041.

Our CAST systems are protected by two pending U.S. non-provisional patent applications with composition of matter claims covering genome editing systems using nucleases in combination with either recombinases or transposases and methods of using them, and two pending U.S. provisional patent application with composition of matter claims covering genome editing systems using nucleases in combination with either recombinases or transposases and methods of using them. Our CAST systems are also protected by 21 pending foreign patent applications with composition of matter claims covering genome editing systems using nucleases in combination with either recombinases or transposases and methods of using them, including in Australia, Brazil, Canada, China, Europe, Great Britain, Hong Kong, India, Japan, South Korea, and Mexico, and two PCT patent applications with composition of matter claims covering genome editing systems using nucleases in combination with either recombinases or transposases and methods of using them. If issued, the aforementioned patent applications are expired to expire between August 23, 2041 and March 23, 2043.

Our Cas chimera systems are protected by one pending U.S. non-provisional patent application with composition of matter claims covering genome editing systems using chimeric nucleases and methods of using them and one pending U.S. provisional patent application with composition of matter claims covering genome editing systems using chimeric nucleases and methods of using them. Our Cas chimera systems are also protected by one issued foreign patent in Great Britain and eleven pending foreign patent applications, including in Australia, Brazil, Canada, China, Europe, India, Japan, South Korea, Mexico and Hong Kong with composition of matter claims covering genome editing systems using chimeric nucleases, and one PCT patent applications with composition of matter claims covering genome editing systems using chimeric nucleases and methods of using them. If issued, the aforementioned patent applications are expected to expire on January 21, 2042.

Our SMART nuclease systems are protected by one pending U.S. non-provisional patent applications with composition of matter claims covering genome editing systems using small nucleases, and two pending U.S. provisional patent applications with composition of matter covering genome editing systems using small nucleases and methods of using them. Our SMART nuclease systems are also protected by 10 pending foreign patent applications with composition of matter covering genome editing systems using small nucleases and methods of using them, including in Australia, Canada, China, Europe, Great Britain, Hong Kong, India, Japan, Korea, and Mexico, and two PCT patent applications with composition of matter claims covering genome editing systems using small nucleases and methods of using them. If issued, the aforementioned patent applications are expected to expire on March 31, 2040.

We cannot predict whether the patent applications we pursue or may license in the future will issue as patents in any particular jurisdiction or whether the claims of any issued patents will provide any protection from competitors. Even if our pending patent applications are granted as issued patents, those patents, as well as any patents we may license in the future from third parties now or in the future, may be challenged, circumvented or invalidated by third parties. Consequently, we may not obtain or maintain adequate patent protection for any of our programs and genome editing systems.

The term of individual patents depends upon the legal term of the patents in the countries in which they are obtained. In most countries in which we file, the patent term is 20 years from the earliest date of filing of a non-provisional patent application. In the United

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States, the patent term of a patent may be extended by patent term adjustment, which compensates the patent owner for patent office delays. Additionally, in the United States, patents that cover an FDA-approved drug or biologic may also be eligible for patent term extension, which permits patent term restoration as compensation for the patent term lost during FDA regulatory review process. The Hatch-Waxman Act permits a patent term extension of up to five years beyond the expiration of the patent. The length of the patent term extension is related to the length of time the drug or biologic is under regulatory review. Patent term extension cannot extend the remaining term of a patent beyond a total of 14 years from the date of product approval, only one patent applicable to an approved drug or biologic may be extended and only those claims covering the approved drug or biologic, a method for using it, or a method for manufacturing it may be extended. Similar provisions are available in European Member States and other foreign jurisdictions to extend the term of a patent that covers an approved drug or biologic. In the future, if our investigational gene therapies receive FDA approval, we expect to apply for patent term extensions where applicable on patents covering those products. We plan to seek patent term extensions to any of our issued patents in any jurisdiction where these are available, however there is no guarantee that the applicable authorities, including the U.S. Patent and Trademark Office (“USPTO”) in the United States, will agree with our assessment of whether these extensions should be granted, and if granted, the length of these extensions.

Our intellectual property is critical to our business and we strive to protect it through a variety of approaches, including by obtaining and maintaining patent protection in various countries for our genome editing technology and other inventions that are important to our business.

Trademarks

As of December 31, 2023, we own the trademark registrations for Metagenomi in the United States and we have trademark applications for Metagenomi SMART in the United States.

Trade Secrets and Proprietary Information

In addition to our reliance on patent protection for our inventions, investigational gene therapies and research programs, we also rely on trade secrets, know-how, confidentiality agreements and continuing technological innovation to develop and maintain our competitive position. Although we take steps to protect our proprietary information and trade secrets, including through contractual means with our employees, advisors and consultants, these agreements may be breached and we may not have adequate remedies for any breach. In addition, third parties may independently develop substantially equivalent proprietary information and techniques or otherwise gain access to our trade secrets or disclose our technology. As a result, we may not be able to meaningfully protect our trade secrets. It is our policy to require our employees, consultants, outside scientific collaborators, sponsored researchers and other advisors to execute confidentiality agreements upon the commencement of employment or consulting relationships with us. These agreements provide that all confidential information concerning our business or financial affairs developed or made known to the individual or entity during the course of the party’s relationship with us is to be kept confidential and not disclosed to third parties except in specific circumstances. In the case of employees, the agreements provide that all inventions conceived of by the individual during the course of employment, and which relate to or are reasonably capable or being used in our current or planned business or research and development are our exclusive property. In addition, we take other appropriate precautions, such as physical and technological security measures, to guard against misappropriation of our technology by third parties. However, such agreements and policies may be breached and we may not have adequate remedies for such breaches. For more information regarding the risks related to our intellectual property, see “Risk Factors—Risks Related to Our Intellectual Property.”

Government Regulation

In the United States, biological products, including gene therapy products, are subject to regulation under the Federal Food, Drug, and Cosmetic Act (“FD&C Act”), and the Public Health Service Act (“PHS Act”), and other federal, state, local and foreign statutes and regulations. Both the FD&C Act and the PHS Act and their corresponding regulations govern, among other things, the research, development, clinical trial, testing, manufacturing, safety, efficacy, labeling, packaging, storage, record keeping, distribution, reporting, advertising, and other promotional practices involving biological products. Each clinical trial protocol for a gene therapy product must be reviewed by the FDA. FDA approval must be obtained before the marketing of biological products. The process of obtaining regulatory approvals and the subsequent compliance with appropriate federal, state, local and foreign statutes and regulations require the expenditure of substantial time and financial resources and we may not be able to obtain the required regulatory approvals.

U.S. Biological Product Development Process

The process required by the FDA before a biological product candidate may be marketed in the United States generally involves the following:

completion of nonclinical laboratory tests and animal studies according to good laboratory practices (“GLPs”), unless justified and applicable requirements for the humane use of laboratory animals or other applicable regulations;

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submission to the FDA of an application for an IND, which must become effective before human clinical trials may begin;
approval of the protocol and related documentation by an independent institutional review board (“IRB”), or ethics committee at each clinical trial site before each study may be initiated;
performance of adequate and well-controlled human clinical trials according to the FDA’s regulations commonly referred to as good clinical practices (“GCPs”), and any additional requirements for the protection of human research subjects and their health information, to establish the safety and efficacy of the proposed biological product for its intended use;
submission to the FDA of a biologics license application (“BLA”), for regulatory approval that includes sufficient evidence of establishing the safety, purity and potency of the proposed biological product for its intended indication, including from results of nonclinical testing and clinical trials;
satisfactory completion of an FDA inspection of the manufacturing facility or facilities where the biological product is produced to assess compliance with cGMP, to assure that the facilities, methods and controls are adequate to preserve the biological product’s identity, strength, quality and purity and, if applicable, the FDA’s current good tissue practices (“CGTPs”), for the use of human cellular and tissue products;
potential FDA audit of the nonclinical study and clinical trial sites that generated the data in support of the BLA in accordance with any applicable expedited programs or designations;
review of the product candidate by an FDA advisory committee, where appropriate or if applicable;
payment of user fees for FDA review of the BLA (unless a fee waiver applies); and
FDA review and approval, or licensure, of the BLA.

Before testing any biological product candidate, including a gene therapy product, in humans, the product candidate enters the preclinical testing stage. Preclinical tests, also referred to as nonclinical studies, include laboratory evaluations of product biological characteristics, chemistry, toxicity, and formulation, as well as animal studies to assess the potential safety and activity of the product candidate. The conduct of the preclinical tests must comply with federal regulations and requirements including GLPs.

An IND is an exemption from the FD&C Act that allows an investigational product candidate to be shipped in interstate commerce for use in a clinical trial and a request for FDA authorization to administer such investigational product candidate to humans. Such authorization must be secured prior to interstate shipment and administration of any product candidate that is not the subject of an approved BLA. In support of a request for an IND, applicants must submit a protocol for each clinical trial and any subsequent protocol amendments must be submitted to the FDA as part of the IND. In addition, the results of the preclinical tests, together with manufacturing information, analytical data, any available clinical data or literature and plans for clinical trials, among other things, must be submitted to the FDA as part of an IND. The FDA requires a 30-day waiting period after the filing of each IND before clinical trials may begin. This waiting period is designed to allow the FDA to review the IND to determine whether human research subjects will be exposed to unreasonable health risks. At any time during this 30-day period the FDA may raise concerns or questions about the conduct of the trials as outlined in the IND and impose a clinical hold or partial clinical hold. In this case, the IND sponsor and the FDA must resolve any outstanding concerns before clinical trials can begin.

Following commencement of a clinical trial, the FDA may also place a clinical hold or partial clinical hold on that trial. A clinical hold is an order issued by the FDA to the sponsor to delay a proposed clinical investigation or to suspend an ongoing investigation. A partial clinical hold is a delay or suspension of only part of the clinical work requested under the IND. No more than 30 days after imposition of a clinical hold or partial clinical hold, the FDA will provide the sponsor a written explanation of the basis for the hold. Following issuance of a clinical hold or partial clinical hold, an investigation may only resume after the FDA has notified the sponsor that the investigation may proceed. There also are requirements governing the reporting of ongoing clinical trials and completed clinical trial results to public registries. Information about certain clinical trials, including clinical trial results, must be submitted within specific timeframes for publication on the www.clinicaltrials.gov website.

A sponsor may choose, but is not required, to conduct a foreign clinical trial under an IND. When a foreign clinical trial is conducted under an IND, all FDA IND requirements must be met unless waived. When a foreign clinical trial is not conducted under an IND, the sponsor must ensure that the study complies with certain regulatory requirements of the FDA in order to use the study as support for an IND or application for regulatory approval or licensing. In particular, such studies must be conducted in accordance with GCP, including review and approval by an independent ethics committee (“IEC”), and informed consent from subjects. The FDA must be able to validate the data through an onsite inspection, if deemed necessary by the FDA.

An IRB representing each institution participating in the clinical trial must review and approve the plan for any clinical trial before it commences at that institution, and the IRB must conduct continuing review and reapprove the study at least annually. The IRB must

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review and approve, among other things, the study protocol and informed consent information to be provided to study subjects. An IRB must operate in compliance with FDA regulations. An IRB can suspend or terminate approval of a clinical trial at its institution, or an institution it represents, if the clinical trial is not being conducted in accordance with the IRB’s requirements or if the product candidate has been associated with unexpected serious harm to patients. Some trials are overseen by an independent group of qualified experts organized by the trial sponsor, known as a data safety monitoring board or committee (“DSMB”). This group provides authorization as to whether or not a trial may move forward at designated check points based on access that only the group maintains to available data from the study.

In addition to the submission of an IND to the FDA before initiation of a clinical trial in the United States, certain human clinical trials involving recombinant or synthetic nucleic acid molecules may be subject to oversight of institutional biosafety committees (“IBCs”), as set forth in the National Institutes of Health (“NIH”), Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (“NIH Guidelines”). Under the NIH Guidelines, recombinant and synthetic nucleic acids are defined as: (i) molecules that are constructed by joining nucleic acid molecules that can replicate in a living cell (i.e., recombinant nucleic acids); (ii) nucleic acid molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules (i.e., synthetic nucleic acids); or (iii) molecules that result from the replication of those described in (i) or (ii). Specifically, under the NIH Guidelines, supervision of human gene transfer trials includes evaluation and assessment by an IBC, a local institutional committee that reviews and oversees research utilizing recombinant or synthetic nucleic acid molecules at that institution. The IBC assesses the safety of the research and identifies any potential risk to public health or the environment, and such review may result in some delay before initiation of a clinical trial. While the NIH Guidelines are not mandatory unless the research in question is being conducted at or sponsored by institutions receiving NIH funding for recombinant or synthetic nucleic acid molecule research, many companies and other institutions not otherwise subject to the NIH Guidelines voluntarily follow them.

Clinical trials typically are conducted in three sequential phases that may overlap or be combined:

Phase 1. The biological product candidate is initially introduced into healthy human subjects and tested for safety. In the case of some biological product candidates for severe or life-threatening diseases, especially when the product candidate may be too inherently toxic to ethically administer to healthy volunteers, the initial human testing is often conducted in patients.
Phase 2. The biological product candidate is evaluated in a limited patient population to identify possible adverse effects and safety risks, to preliminarily evaluate the efficacy of the product candidate for specific targeted diseases and to determine dosage tolerance, optimal dosage and dosing schedule.
Phase 3. Clinical trials are undertaken to further evaluate dosage, clinical efficacy, potency and safety in an expanded patient population at geographically dispersed clinical trial sites. These clinical trials are intended to establish the overall risk/benefit ratio of the product candidate and provide an adequate basis for approval and product labeling.

Post-approval clinical trials, sometimes referred to as Phase 4 clinical trials, may be conducted after initial regulatory approval. These clinical trials are used to gain additional experience from the treatment of patients in the intended therapeutic indication, particularly for long-term safety follow-up. The FDA generally recommends that sponsors of human gene therapy product candidates and genome editing product candidates observe subjects for potential gene therapy-related delayed adverse events for up to a 15-year period, including five years of annual examinations followed by ten years of annual queries, either by telephone or by questionnaire, of study subjects.

During all phases of clinical development, the FDA requires extensive monitoring and auditing of all clinical activities, clinical data, and clinical trial investigators. Annual progress reports detailing the results of the clinical trials must be submitted to the FDA. Written IND safety reports must be promptly submitted to the FDA and the investigators for serious and unexpected suspected adverse events, any findings from other studies, tests in laboratory animals or in vitro testing that suggest a significant risk for human subjects, or any clinically important increase in the rate of a serious suspected adverse reaction over that listed in the protocol or investigator brochure. The sponsor must submit an IND safety report within 15 calendar days after the sponsor determines that the information qualifies for reporting. The sponsor also must notify the FDA of any unexpected fatal or life-threatening suspected adverse reaction within seven calendar days after the sponsor’s initial receipt of the information. The FDA or the sponsor, acting on its own or based on a recommendation from the sponsor’s data safety monitoring board may suspend a clinical trial at any time on various grounds, including a finding that the research subjects are being exposed to an unacceptable health risk. Similarly, an IRB can suspend or terminate approval of a clinical trial at its institution if the clinical trial is not being conducted in accordance with the IRB’s requirements or if the biological product candidate has been associated with unexpected serious harm to patients.

Concurrent with clinical trials, companies usually complete additional animal studies and also must develop additional information about the physical characteristics of the biological product candidate as well as finalize a process for manufacturing the product candidate in commercial quantities in accordance with cGMP. To help reduce the risk of the introduction of adventitious agents with

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use of biological product candidates, the PHS Act emphasizes the importance of manufacturing control for product candidates whose attributes cannot be precisely defined. The manufacturing process must be capable of consistently producing quality batches of the product candidate and, among other things, the sponsor must develop methods for testing the identity, strength, quality, potency, and purity of the final biological product. Additionally, appropriate packaging must be selected and tested and stability studies must be conducted to demonstrate that the biological product candidate does not undergo unacceptable deterioration over its shelf life.

U.S. Review and Approval Processes

After the completion of clinical trials of a biological product candidate, FDA approval of a BLA must be obtained before commercial marketing of the biological product. The BLA must include results of product development, laboratory and animal studies, human studies, information on the manufacture and composition of the product, proposed labeling and other relevant information. The testing and approval processes require substantial time and effort and there can be no assurance that the FDA will accept the BLA for filing and, even if filed, that any approval will be granted on a timely basis, if at all.

Within 60 days following submission of the application, the FDA reviews a BLA to determine if it is substantially complete before the FDA accepts it for filing. The FDA may refuse to file any BLA that it deems incomplete or not properly reviewable at the time of submission and may request additional information. In this event, the BLA must be resubmitted with the additional information. The resubmitted application also is subject to review before the FDA accepts it for filing. In most cases, the submission of a BLA is subject to a substantial application user fee, although the fee may be waived under certain circumstances. Under the performance goals and policies implemented by the FDA under the Prescription Drug User Fee Act (“PDUFA”), for original BLAs, the FDA targets ten months from the filing date in which to complete its initial review of a standard application and respond to the applicant, and six months from the filing date for an application with priority review. The FDA does not always meet its PDUFA goal dates, and the review process is often significantly extended by FDA requests for additional information or clarification. This review typically takes 12 months from the date the BLA is submitted to the FDA because the FDA has approximately two months to make a “filing” decision. The review process and the PDUFA goal date may be extended by three months if the FDA requests or the BLA sponsor otherwise provides additional information or clarification regarding information already provided in the submission within the last three months before the PDUFA goal date.

Once the submission is accepted for filing, the FDA begins an in-depth substantive review of the BLA. The FDA reviews the BLA to determine, among other things, whether the proposed product is safe, pure, and potent for its intended use, and whether the product is being manufactured in accordance with cGMP to ensure the continued safety, purity, and potency of such product. The FDA may refer applications for novel biological products or biological products that present difficult or novel questions of safety or efficacy to an advisory committee, typically a panel that includes clinicians and other experts, for review, evaluation, and a recommendation as to whether the application should be approved and under what conditions. The FDA is not bound by the recommendations of an advisory committee, but it considers such recommendations carefully when making decisions. During the biological product approval process, the FDA also will determine whether a Risk Evaluation and Mitigation Strategy (“REMS”), is necessary to assure the safe use of the biological product. If the FDA concludes a REMS is needed, the sponsor of the BLA must submit a proposed REMS; the FDA will not approve the BLA without a REMS, if required.

Before approving a BLA, the FDA typically will inspect the facilities at which the product is manufactured. The FDA will not approve the product unless it determines that the manufacturing processes and facilities are in compliance with cGMP and adequate to assure consistent production of the product within required specifications. For a gene therapy product, the FDA also will not approve the product if the manufacturer is not in compliance with the CGTPs. These are FDA regulations that govern the methods used in, and the facilities and controls used for, the manufacture of human cells, tissues, and cellular and tissue-based products (“HCT/Ps”), which are human cells or tissue intended for implantation, transplant, infusion, or transfer into a human recipient. The primary intent of the CGTP requirements is to ensure that cell and tissue-based products are manufactured in a manner designed to prevent the introduction, transmission and spread of communicable disease. FDA regulations also require tissue establishments to register and list their HCT/Ps with the FDA and, when applicable, to evaluate donors through appropriate screening and testing. Additionally, before approving a BLA, the FDA will typically inspect one or more clinical sites to assure that the clinical trials were conducted in compliance with IND study requirements and GCP requirements. To assure cGMP, CGTP and GCP compliance, an applicant must incur significant expenditure of time, money, and effort in the areas of training, record keeping, production and quality control.

Under the Pediatric Research Equity Act (“PREA”), a BLA or supplement to a BLA for a novel product (e.g., new active ingredient, new indication, etc.) must contain data to assess the safety and effectiveness of the biological product for the claimed indications in all relevant pediatric subpopulations and to support dosing and administration for each pediatric subpopulation for which the product is safe and effective. The FDA may grant deferrals for submission of data or full or partial waivers. Unless otherwise required by regulation, PREA does not apply to any biological product for an indication for which orphan designation has been granted.

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After the FDA evaluates a BLA and conducts inspections of manufacturing facilities where the biological product will be manufactured, the FDA may issue an approval letter or a Complete Response Letter. An approval letter authorizes commercial marketing of the product with specific prescribing information for specific indications. A Complete Response Letter indicates that the review cycle of the application is complete, and the application will not be approved in its present form. A Complete Response Letter will usually describe all of the deficiencies that the FDA has identified in the BLA, except that where the FDA determines that the data supporting the application are inadequate to support approval, the FDA may issue the Complete Response Letter without first conducting required inspections, testing submitted product lots and/or reviewing proposed labeling. In issuing the Complete Response Letter, the FDA may recommend actions that the applicant might take to place the BLA in condition for approval, including requests for additional information or clarification, which may include the potential requirement for additional preclinical studies or clinical trials or additional manufacturing activities. If a Complete Response Letter is issued, the applicant may either resubmit the BLA, addressing all of the deficiencies identified in the letter, or withdraw the application or request an opportunity for a hearing. The FDA may delay or refuse approval of a BLA if applicable regulatory criteria are not satisfied, require additional testing or information and/or require post-marketing testing and surveillance to monitor safety or efficacy of a product.

If a product receives regulatory approval, the approval may be significantly limited to specific diseases and dosages or the indications for use may otherwise be limited, including to subpopulations of patients, which could restrict the commercial value of the product. Further, the FDA may require that certain contraindications, warnings precautions or interactions be included in the product labeling. The FDA may impose restrictions and conditions on product distribution, prescribing or dispensing in the form of a REMS, or otherwise limit the scope of any approval. In addition, the FDA may require post-marketing clinical trials, sometimes referred to as Phase 4 clinical trials, designed to further assess a biological product’s safety and effectiveness, and testing and surveillance programs to monitor the safety of approved products that have been commercialized.

Orphan Drug Designation

Under the Orphan Drug Act, the FDA may grant orphan designation to a drug or biological product candidate intended to treat a rare disease or condition, which is generally a disease or condition that affects fewer than 200,000 individuals in the United States, or more than 200,000 individuals in the United States and for which there is no reasonable expectation that the cost of developing and making a drug or biological product candidate available in the United States for this type of disease or condition will be recovered from sales of the product. Orphan product designation must be requested before submitting a BLA. After the FDA grants orphan product designation, the identity of the therapeutic agent and its potential orphan use are disclosed publicly by the FDA.

If a product candidate that has orphan drug designation subsequently receives the first FDA approval for the disease or condition for which it has such designation, the product is entitled to orphan drug exclusive approval (or exclusivity), which means that the FDA may not approve any other applications, including a full BLA, to market the same product for the same indication for seven years, except in limited circumstances, such as a showing of clinical superiority to the product with orphan drug exclusivity by means of greater effectiveness, greater safety or providing a major contribution to patient care or if the holder of the orphan drug exclusivity cannot assure the availability of sufficient quantities of the orphan drug to meet the needs of patients with the disease or condition for which the product was designated. Orphan drug exclusivity does not prevent the FDA from approving a different drug or biologic for the same disease or condition, or the same drug or biologic for a different disease or condition. Among the other benefits of orphan drug designation are tax credits for certain research and a waiver of the BLA application fee. A designated orphan drug may not receive orphan drug exclusivity if it is approved for a use that is broader than the indication for which it received orphan drug designation. In addition, exclusive marketing rights in the United States may be lost if the FDA later determines that the request for designation was materially defective or if the manufacturer is unable to assure sufficient quantities of the product to meet the needs of patients with the rare disease or condition.

Expedited Development and Review Programs

The FDA has various programs, including fast track designation, breakthrough therapy designation, priority review and accelerated approval, that are intended to expedite or simplify the process for the development and FDA review of drugs and biologics that are intended for the treatment of serious or life-threatening diseases or conditions. These programs do not change the standards for approval but may help expedite the development or approval process. To be eligible for fast track designation, new drugs and biological products must be intended to treat a serious or life-threatening condition and demonstrate the potential to address unmet medical needs for the condition. Fast track designation applies to the combination of the product and the specific indication for which it is being studied. The sponsor of a new drug or biologic may request the FDA to designate the drug or biologic as a fast track product at any time during the clinical development of the product. One benefit of fast track designation, for example, is that the FDA may consider for review sections of the marketing application for a product that has received fast track designation on a rolling basis before the complete application is submitted.

Under the FDA’s breakthrough therapy program, products intended to treat a serious or life-threatening disease or condition may be eligible for the benefits of the fast track program when preliminary clinical evidence demonstrates that such product may have

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substantial improvement on one or more clinically significant endpoints over existing therapies. Additionally, the FDA will seek to ensure the sponsor of a breakthrough therapy product receives timely advice and interactive communications to help the sponsor design and conduct a development program as efficiently as possible.

Any product is eligible for priority review if it has the potential to provide safe and effective therapy where no satisfactory alternative therapy exists or a significant improvement in the treatment, diagnosis or prevention of a disease compared to marketed products. The FDA will attempt to direct additional resources to the evaluation of an application for a new drug or biological product designated for priority review in an effort to facilitate the review. Under priority review, the FDA’s goal is to review an application in six months once it is filed, compared to ten months for a standard review.

Additionally, a product may be eligible for accelerated approval. Drug or biological products studied for their safety and effectiveness in treating serious or life-threatening illnesses and that provide meaningful therapeutic benefit over existing treatments may receive accelerated approval, which means that they may be approved on the basis of adequate and well-controlled clinical trials establishing that the product has an effect on a surrogate endpoint that is reasonably likely to predict a clinical benefit, or on the basis of an effect on an intermediate clinical endpoint other than survival or irreversible morbidity. As a condition of approval, the FDA may require that a sponsor of a drug or biological product receiving accelerated approval perform adequate and well-controlled post-marketing clinical trials with due diligence, and, under the Food and Drug Omnibus Reform Act of 2022 (“FDORA”), the FDA is now permitted to require, as appropriate, that such trials be underway prior to approval or within a specific time period after the date of approval for a product granted accelerated approval. Under FDORA, the FDA has increased authority for expedited procedures to withdraw approval of a product or indication approved under accelerated approval if, for example, the confirmatory trial fails to verify the predicted clinical benefit of the product. In addition, for products being considered for accelerated approval, the FDA generally requires, unless otherwise informed by the agency, that all advertising and promotional materials intended for dissemination or publication be submitted to the agency for review, which could adversely affect the timing of the commercial launch of the product.

RMAT Designation

As part of the 21st Century Cures Act, Congress amended the FD&C Act to facilitate an efficient development program for and expedite review of regenerative medicine advanced therapies (“RMAT”), which include cell and gene therapies, therapeutic tissue engineering products, human cell and tissue products and combination products using any such therapies or products. RMAT do not include those HCT/Ps regulated solely under section 361 of the PHS Act and 21 CFR Part 1271. This program is intended to facilitate efficient development and expedite review of regenerative medicine therapies, which are intended to treat, modify, reverse, or cure a serious or life-threatening disease or condition and qualify for RMAT designation. A drug sponsor may request that FDA designate a drug as a RMAT concurrently with or at any time after submission of an IND. FDA has 60 calendar days to determine whether the drug meets the criteria, including whether there is preliminary clinical evidence indicating that the drug has the potential to address unmet medical needs for a serious or life-threatening disease or condition. A BLA for a regenerative medicine therapy that has received RMAT designation may be eligible for priority review or accelerated approval through use of surrogate or intermediate endpoints reasonably likely to predict long-term clinical benefit, or reliance upon data obtained from a meaningful number of sites. Benefits of RMAT designation also include early interactions with FDA to discuss any potential surrogate or intermediate endpoint to be used to support accelerated approval. A regenerative medicine therapy with RMAT designation that is granted accelerated approval and is subject to post-approval requirements may fulfill such requirements through the submission of clinical evidence from clinical trials, patient registries, or other sources of real world evidence, such as electronic health records; the collection of larger confirmatory data sets; or post-approval monitoring of all patients treated with such therapy prior to its approval. Like some of the FDA’s other expedited development programs, RMAT designation does not change the standards for approval but may help expedite the development or approval process.

Rare Pediatric Disease Designation and Priority Review Vouchers

Under the or FD&C Act, as amended, the FDA incentivizes the development of product candidates that meet the definition of a “rare pediatric disease,” defined to mean a serious or life-threatening disease in which the serious of life-threatening manifestations primarily affect individuals aged from birth to 18 years and the disease affects fewer than 200,000 individuals in the United States or affects 200,000 or more in the United States and for which there is no reasonable expectation that the cost of developing and making in the United States a drug or biologic for such disease or condition will be received from sales in the United States of such drug or biologic. The sponsor of a product candidate for a rare pediatric disease may be eligible for a voucher that can be used to obtain a priority review for a subsequent marketing application after the date of approval of the rare pediatric disease drug product. A sponsor may request rare pediatric disease designation from the FDA prior to the submission of its BLA. A rare pediatric disease designation does not guarantee that a sponsor will receive a priority review voucher (“PRV”) upon approval of its BLA. Moreover, a sponsor who chooses not to submit a rare pediatric disease designation request may nonetheless receive a PRV upon approval of their marketing application if they request such a voucher in their original marketing application and meet all of the eligibility criteria. If a PRV is received, it may be sold or transferred an unlimited number of times. Congress has extended the PRV program until September 30, 2024, with the potential for PRVs to be granted until September 30, 2026.

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Post-Approval Requirements

Maintaining substantial compliance with applicable federal, state and local statutes and regulations requires the expenditure of substantial time and financial resources. Products manufactured or distributed pursuant to FDA approvals are subject to pervasive and continuing regulation by the FDA, including, among other things, requirements relating to recordkeeping, periodic reporting, product sampling and distribution, advertising and promotion and reporting of adverse experiences with the product. If there are any modifications to the product, including changes in indications, labeling or manufacturing processes or facilities, the applicant may be required to submit and obtain FDA approval of a new BLA or BLA supplement, which may require the development of additional data or preclinical studies and clinical trials. Such regulatory reviews can result in denial or modification of the planned changes, or requirements to conduct additional tests or evaluations that can substantially delay or increase the cost of the planned changes. The FDA may also impose a number of post-approval requirements as a condition of approval of a BLA. For example, the FDA may require post-marketing testing, including Phase 4 clinical trials, and surveillance to further assess and monitor the product’s safety and effectiveness after commercialization.

Manufacturers of biological products are required to comply with applicable requirements in the cGMP, including quality control and quality assurance and maintenance of records and documentation. Other post- approval requirements applicable to biological products include reporting of cGMP deviations that may affect the identity, potency, purity and overall safety of a distributed product, record-keeping requirements, reporting of adverse effects, reporting updated safety and efficacy information, and complying with electronic record and signature requirements. After a BLA is approved, the product also may be subject to official lot release. As part of the manufacturing process, the manufacturer is required to perform certain tests on each lot of the product before it is released for distribution. If the product is subject to official release by the FDA, the manufacturer submits samples of each lot of product to the FDA together with a release protocol showing a summary of the history of manufacture of the lot and the results of all of the manufacturer’s tests performed on the lot. The FDA also may perform certain confirmatory tests on lots of some products, such as viral vaccines, before releasing the lots for distribution by the manufacturer. In addition, the FDA conducts laboratory research related to the regulatory standards on the safety, purity, potency, and effectiveness of biological products.

We also must comply with the FDA’s advertising and promotion requirements, such as those related to direct-to-consumer advertising, the prohibition on promoting products for uses or in patient populations that are not described in the product’s approved labeling (known as “off-label use”), industry-sponsored scientific and educational activities, and promotional activities involving the internet. Discovery of previously unknown problems or the failure to comply with the applicable regulatory requirements may result in restrictions on the marketing of a product or withdrawal of the product from the market as well as possible civil or criminal sanctions. Failure to comply with the applicable U.S. requirements at any time during the product development process, approval process or after approval, may subject an applicant or manufacturer to administrative or judicial civil or criminal sanctions and adverse publicity. FDA sanctions could include refusal to approve pending applications, withdrawal of an approval, clinical holds, warning or untitled letters, product recalls, product seizures, total or partial suspension of production or distribution, injunctions, fines, refusals of government contracts, mandated corrective advertising or communications with doctors or other stakeholders, debarment, restitution, disgorgement of profits, or civil or criminal penalties.

Biological product manufacturers and other entities involved in the manufacture and distribution of approved biological products, and those supplying products, ingredients, and components of them, are required to register their establishments with the FDA and certain state agencies and are subject to periodic unannounced inspections by the FDA and certain state agencies for compliance with cGMP and other laws. Accordingly, manufacturers must continue to expend time, money, and effort in the area of production and quality control to maintain cGMP compliance. Discovery of problems with a product after approval may result in restrictions on a product, manufacturer, or holder of an approved BLA, including withdrawal of the product from the market. In addition, changes to the manufacturing process or facility generally require prior FDA approval before being implemented and other types of changes to the approved product, such as adding new indications and additional labeling claims, are also subject to further FDA review and approval.

U.S. Patent Term Restoration and Marketing Exclusivity

Depending upon the timing, duration, and specifics of the FDA approval of the use of our product candidates, some U.S. patents that may issue from our pending patent applications may be eligible for limited patent term extension under the Hatch-Waxman Amendments. The Hatch-Waxman Amendments permit a patent restoration term of up to five years as compensation for patent term lost during product development and the FDA regulatory review process. However, patent term restoration cannot extend the remaining term of a patent beyond a total of 14 years from the product’s approval date. The patent term restoration period is generally one-half the time between the effective date of an IND and the submission date of a BLA plus the time between the submission date of a BLA and the approval of that application, except that the review period is reduced by any time during which the applicant failed to exercise due diligence. Only one patent applicable to an approved biological product is eligible for the extension and the application for the extension must be submitted prior to the expiration of the patent. In addition, only those claims covering the approved product, a method for using it, or a method for manufacturing it may be extended, and a patent can only be extended once and only for a single product. The USPTO, in consultation with the FDA, reviews and approves the application for any patent term extension or restoration.

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In the future, we may apply for restoration of patent term for one of the patents that may issue from our pending patent applications, if and as applicable, to add patent life beyond its current expiration date, depending on the expected length of the clinical trials and other factors involved in the filing of the relevant BLA. However, there can be no assurance that our pending patent applications will issue or that we will benefit from any patent term extension or favorable adjustments to the terms of any patents we may own or in-license in the future.

A biological product can obtain pediatric market exclusivity in the United States. Pediatric exclusivity, if granted, adds six months to existing exclusivity periods and may be granted based on the voluntary completion of a pediatric study in accordance with an FDA-issued “Written Request” for such a study.

The Biologics Price Competition and Innovation Act of 2009 created an abbreviated approval pathway for biological products shown to be similar to, or interchangeable with, an FDA-licensed reference biological product. This amendment to the PHS Act attempts to minimize duplicative testing. Biosimilarity, which requires that there be no clinically meaningful differences between the biological product and the reference product in terms of safety, purity, and potency, can be shown through analytical studies, animal studies and a clinical trial or trials. Interchangeability requires that a product is biosimilar to the reference product and the product must demonstrate that it can be expected to produce the same clinical results as the reference product and, for products administered multiple times, the biologic and the reference biologic may be switched after one has been previously administered without increasing safety risks or risks of diminished efficacy relative to exclusive use of the reference biologic.

A reference biological product is granted four- and 12-year exclusivity periods from the time of first licensure of the product. FDA will not accept an application for a biosimilar or interchangeable product based on the reference biological product until four years after the date of first licensure of the reference product, and FDA will not approve an application for a biosimilar or interchangeable product based on the reference biological product until twelve years after the date of first licensure of the reference product. “First licensure” typically means the initial date the particular product at issue was licensed in the United States. Date of first licensure does not include the date of licensure of (and a new period of exclusivity is not available for) a biological product if the licensure is for a supplement for the biological product or for a subsequent application by the same sponsor or manufacturer of the biological product (or licensor, predecessor in interest, or other related entity) for a change (not including a modification to the structure of the biological product) that results in a new indication, route of administration, dosing schedule, dosage form, delivery system, delivery device or strength, or for a modification to the structure of the biological product that does not result in a change in safety, purity, or potency. Therefore, one must determine whether a new product includes a modification to the structure of a previously licensed product that results in a change in safety, purity, or potency to assess whether the licensure of the new product is a first licensure that triggers its own period of exclusivity. Whether a subsequent application, if approved, warrants exclusivity as the “first licensure” of a biological product is determined on a case-by-case basis with data submitted by the sponsor.

Regulation Outside of the United States

In addition to regulations in the United States, we are subject to a variety of regulations in other jurisdictions governing clinical studies, commercial sales, and distribution of our products. Most countries outside of the United States require that clinical trial applications be submitted to and approved by the local regulatory authority for each clinical study. In the European Union, for example, an application must be submitted to the national competent authority and an independent ethics committee in each country in which we intend to conduct clinical trials, much like the FDA and IRB, respectively. Under the new Clinical Trials Regulation (EU) No 536/2014, which replaced the Clinical Trials Directive 2001/20/EC on January 31, 2022, a single application is now made through the Clinical Trials Information System (“CTIS”) for clinical trial authorization in up to 30 EU/ EEA countries at the same time and with a single set of documentation.

The assessment of applications for clinical trials is divided into two parts (Part I contains scientific and medicinal product documentation and Part II contains the national and patient-level documentation). Part I is assessed by a coordinated review by the competent authorities of all European Union Member States in which an application for authorization of a clinical trial has been submitted (Member States concerned) of a draft report prepared by a Reference Member State. Part II is assessed separately by each Member State concerned. The role of the relevant ethics committees in the assessment procedure will continue to be governed by the national law of the Member State concerned, however overall related timelines are defined by the Clinical Trials Regulation. The new Clinical Trials Regulation also provides for simplified reporting procedures for clinical trial sponsors.

In addition, whether or not we obtain FDA approval for a product, we must obtain approval of a product by the comparable regulatory authorities of countries outside the United States before we can commence marketing of the product in those countries. The approval process and requirements vary from country to country, so the number and type of nonclinical, clinical, and manufacturing studies needed may differ, and the time may be longer or shorter than that required for FDA approval.

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To obtain regulatory approval of our medicinal products under the European Union regulatory system, we are required to submit a marketing authorization application (“MAA”), to be assessed in the centralized procedure. The centralized procedure allows applicants to obtain a marketing authorization (“MA”) that is valid throughout the European Union, and the additional Member States of the European Economic Area (Iceland, Liechtenstein and Norway) (“EEA”). It is compulsory for medicinal products manufactured using biotechnological processes, orphan medicinal products, advanced therapy medicinal products (gene-therapy, somatic cell-therapy or tissue- engineered medicines) and human products containing a new active substance which is not authorized in the European Union and which is intended for the treatment of HIV, AIDS, cancer, neurodegenerative disorders, auto-immune and other immune dysfunctions, viral diseases or diabetes. The centralized procedure is optional for any other products containing new active substances not authorized in the European Union or for products which constitute a significant therapeutic, scientific, or technical innovation or for which a centralized authorization is in the interests of patients at European Union level. When a company wishes to place on the market a medicinal product that is eligible for the centralized procedure, it sends an application directly to the European Medicines Agency (“EMA”), to be assessed by the Committee for Medicinal Products for Human Use (“CHMP”). The CHMP is responsible for conducting the assessment of whether a medicine meets the required quality, safety, and efficacy requirements, and whether the product has a positive risk/benefit profile. The procedure results in a European Commission decision, which is valid in all European Union Member States. The centralized procedure is as follows: full copies of the MAA are sent to a rapporteur and a co-rapporteur designated by the competent EMA scientific committee. They coordinate the EMA’s scientific assessment of the medicinal product and prepare draft reports. Once the draft reports are prepared (other experts might be called upon for this purpose), they are sent to the CHMP, whose comments or objections are communicated to the applicant. The rapporteur is therefore the privileged interlocutor of the applicant and continues to play this role, even after the MA has been granted.

The rapporteur and co-rapporteur then assess the applicant’s replies, submit them for discussion to the CHMP, and taking into account the conclusions of this debate, prepare a final assessment report. Once the evaluation is completed, the CHMP gives a favorable or unfavorable opinion as to whether to grant the authorization.

When the opinion is favorable, it shall include the draft summary of product characteristics (“SmPC”), the package leaflet, and the texts proposed for the various packaging materials. The time limit for the evaluation procedure is 210 days (excluding clock stops, when additional written or oral information is to be provided by the applicant in response to questions asked by the CHMP). The EMA then has fifteen days to forward its opinion to the European Commission, which will make a binding decision on the grant of an MA within 67 days of the receipt of the CHMP opinion.

There are two other procedures in the European Union for the grant of an MA in multiple European Union Member States. The decentralized procedure provides for approval by one or more other, or Concerned Member States, of an assessment of an application performed by one Member State, known as the Reference Member State. Under this procedure, an applicant submits an application, or dossier, and related materials including a draft SmPC, and draft labeling and package leaflet, to the Reference Member State and Concerned Member States. The Reference Member State prepares a draft assessment and drafts of the related materials within 120 days after receipt of a valid application. Within 90 days of receiving the Reference Member State’s assessment report, each Concerned Member State must decide whether to approve the assessment report and related materials. If a Member State cannot approve the assessment report and related materials on the grounds of potential serious risk to the public health, the disputed points may eventually be referred to the European Commission, whose decision is binding on all Member States. Where a product has already been authorized for marketing in a European Union Member State, this national MA can be recognized in other Member States through the mutual recognition procedure.

The criteria for designating an “orphan medicinal product” in the European Union are similar in principle to those in the United States. Under Article 3 of Regulation (EC) 141/2000, a medicinal product may be designated as an orphan medicinal product if it is intended for the diagnosis, prevention, or treatment of a life-threatening or chronically debilitating condition that affects no more than five in 10,000 persons in the European Union when the application is made. In addition, orphan designation can be granted if the product is intended for a life threatening, seriously debilitating, or serious and chronic condition in the European Union and, without incentives, it is unlikely that sales of the product in the European Union would be sufficient to justify the necessary investment in its development. Orphan designation is only available if there is no other satisfactory method approved in the European Union of diagnosing, preventing, or treating the applicable orphan condition, or if such a method exists, the proposed orphan medicinal product will be of significant benefit to patients affected by such condition, as defined in Regulation (EC) 847/2000.

Orphan designation provides opportunities for fee reductions, protocol assistance, and access to the centralized procedure. Fee reductions are limited to the first year after an MA, except for small and medium enterprises. In addition, if a product which has an orphan designation subsequently receives a centralized MA for the indication for which it has such designation, the product is entitled to orphan market exclusivity, which means the EMA may not approve any other application to market a similar medicinal product for the same indication for a period of ten years. A “similar medicinal product” is defined as a medicinal product containing a similar active substance or substances as contained in an authorized orphan medicinal product, and which is intended for the same therapeutic indication. The exclusivity period may be reduced to six years if, at the end of the fifth year, it is shown that the designation criteria

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are no longer met, including where it is shown that the product is sufficiently profitable not to justify maintenance of market exclusivity. Additionally, an MA may be granted to a similar medicinal product for the same indication at any time if:

the second applicant can establish that its product, although similar to the authorized product, is safer, more effective or otherwise clinically superior;
the MA holder of the authorized product consents to a second orphan medicinal product application; or
the MA holder of the authorized product cannot supply enough orphan medicinal product.

A pediatric investigation plan (“PIP”), in the European Union is aimed at ensuring that the necessary data are obtained to support the authorization of a medicine for children, through studies in children. All applications for Mas for new medicines have to include the results of studies as described in an agreed PIP, unless the medicine is exempt because of a deferral or waiver. This requirement also applies when an MA holder wants to add a new indication, pharmaceutical form, or route of administration for a medicine that is already authorized and covered by intellectual property rights. Several rewards and incentives for the development of pediatric medicines for children are available in the European Union. Medicines authorized across the European Union with the results of studies from a PIP included in the product information are eligible for an extension of their supplementary protection certificate (“SPC”) by six months (provided an application for such extension is made at the same time as filing the SPC application for the product, or at any point up to two years before the SPC expires). This is the case even when the studies’ results are negative. For orphan medicinal products, the incentive is an additional two years of market exclusivity. Scientific advice and protocol assistance at the EMA are free of charge for questions relating to the development of pediatric medicines. Medicines developed specifically for children that are already authorized but are not protected by a patent or supplementary protection certificate are eligible for a pediatric-use MA (“PUMA”). If a PUMA is granted, the product will benefit from ten years of market protection as an incentive.

In March 2016, the EMA launched an initiative, the PRIority Medicines (“PRIME”) scheme, to facilitate development of product candidates in indications, often rare, for which few or no therapies currently exist. The PRIME scheme is intended to encourage development of products in areas of unmet medical need and provides accelerated assessment of products representing substantial innovation reviewed under the centralized procedure. Products from small- and medium-sized enterprises may qualify for earlier entry into the PRIME scheme than larger companies on the basis of compelling non-clinical data and tolerability data from initial clinical trials. Many benefits accrue to sponsors of product candidates with PRIME designation, including but not limited to, early and proactive regulatory dialogue with the EMA, frequent discussions on clinical trial designs and other development program elements, and potentially accelerated MAA assessment once a dossier has been submitted. Importantly, once a candidate medicine has been selected for the PRIME scheme, a dedicated contact and rapporteur from the CHMP or from the Committee for Advanced Therapies (“CAT”) are appointed early in the PRIME scheme facilitating increased understanding of the product at EMA’s committee level. An initial meeting with the CHMP/CAT rapporteur initiates these relationships and includes a team of multidisciplinary experts at the EMA to provide guidance on the overall development and regulatory strategies. PRIME eligibility does not change the standards for product approval, and there is no assurance that any such designation or eligibility will result in expedited review or approval.

The aforementioned European Union rules are generally applicable in the EEA.

The United Kingdom left the European Union on January 31, 2020, and the United Kingdom and the European Union have concluded a trade and cooperation agreement (“TCA”) which was provisionally applicable since January 1, 2021 and has been formally applicable since May 1, 2021.

The TCA includes specific provisions concerning pharmaceuticals, which include the mutual recognition of GMP, inspections of manufacturing facilities for medicinal products and GMP documents issued, but does not provide for wholesale mutual recognition of United Kingdom and European Union pharmaceutical regulations. At present, Great Britain has implemented European Union legislation on the marketing, promotion and sale of medicinal products through the Human Medicines Regulations 2012 (as amended). Except in respect of the new European Union Clinical Trials Regulation, the regulatory regime in Great Britain therefore largely aligns with current European Union medicines regulations, however it is possible that these regimes will diverge more significantly in future now that Great Britain’s regulatory system is independent from the European Union and the TCA does not provide for mutual recognition of United Kingdom and European Union pharmaceutical legislation. However, notwithstanding that there is no wholesale recognition of European Union pharmaceutical legislation under the TCA, under a new framework mentioned below which will be put in place by the Medicines and Healthcare products Regulatory Agency (“MHRA”), the United Kingdom’s medicines regulator, from January 1, 2024, the MHRA has stated that it will take into account decisions on the approval of Mas from the EMA (and certain other regulators) when considering an application for a Great Britain MA.

On February 27, 2023, the United Kingdom government and the European Commission announced a political agreement in principle to replace the Northern Ireland Protocol with a new set of arrangements, known as the “Windsor Framework”. This new framework fundamentally changes the existing system under the Northern Ireland Protocol, including with respect to the regulation of medicinal

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products in the United Kingdom. In particular, the MHRA will be responsible for approving all medicinal products destined for the United Kingdom market (i.e., Great Britain and Northern Ireland), and the EMA will no longer have any role in approving medicinal products destined for Northern Ireland. A single United Kingdom-wide MA will be granted by the MHRA for all medicinal products to be sold in the United Kingdom, enabling products to be sold in a single pack and under a single authorization throughout the United Kingdom. The Windsor Framework was approved by the European Union-United Kingdom Joint Committee on March 24, 2023, so the United Kingdom government and the European Union will enact legislative measures to bring it into law.

The MHRA has introduced changes to national licensing procedures, including procedures to prioritize access to new medicines that will benefit patients, an accelerated assessment procedure and new routes of evaluation for novel products and biotechnological products. All existing European Union Mas for centrally authorized products were automatically converted (grandfathered) into United Kingdom Mas free of charge on January 1, 2021. For a period of three years from January 1, 2021, the MHRA may rely on a decision taken by the European Commission on the approval of a new MA in the centralized procedure, in order to more quickly grant a new Great Britain MA. A separate application will, however, still be required. On January 24, 2023, the MHRA announced that a new international recognition framework will be put in place from January 1, 2024, which will have regard to decisions on the approval of Mas made by the EMA and certain other regulators when determining an application for a new Great Britain MA. There is now no pre-MA orphan designation in Great Britain. Instead, the MHRA reviews applications for orphan designation in parallel to the corresponding MAA. The criteria are essentially the same, but have been tailored for the Great Britain market, i.e., the prevalence of the condition in Great Britain (rather than the European Union) must not be more than five in 10,000. Should an orphan designation be granted, the period of market exclusivity will be set from the date of first approval of the product in Great Britain.

Other Healthcare Laws and Compliance Requirements

Other Healthcare Laws

Biotechnology companies are subject to additional healthcare laws in the jurisdictions in which they conduct their business that may constrain the financial arrangements and relationships through which we research, as well as, in the future sell, market and distribute any products for which we obtain regulatory approval. Such laws include, without limitation, state and federal patient data privacy and security laws, federal and state anti- kickback laws, physician-self referral laws, false claims and transparency laws and regulations with respect to drug pricing and payments and other transfers of value made to physicians and other health care providers, and similar healthcare laws and regulations in the EU and other jurisdictions. Violations of any of such laws or any other governmental regulations that apply may result in significant penalties, including, without limitation, administrative, civil and criminal penalties, damages, fines, disgorgement, the curtailment or restructuring of operations, integrity oversight and reporting obligations to resolve allegations of noncompliance, and exclusion from participation in federal and state healthcare programs and imprisonment.

Coverage and Reimbursement

Sales of any product depend, in part, on the extent to which such product will be covered by third-party payors, such as federal, state, and foreign government healthcare programs, commercial insurance and managed healthcare organizations, and the level of reimbursement for such product by third-party payors. Decisions regarding the extent of coverage and amount of reimbursement to be provided are made on a plan-by-plan basis. These third-party payors are increasingly reducing coverage and reimbursement for medical products, drugs and services. For products administered under the supervision of a physician, obtaining coverage and adequate reimbursement may be particularly difficult because of the higher prices often associated with such drugs. Additionally, separate reimbursement for the product itself or the treatment or procedure in which the product is used may not be available, which may impact physician utilization.

The U.S. government, state legislatures and foreign governments have also continued implementing cost- containment programs, including price controls, restrictions on coverage and reimbursement and requirements for substitution of generic (or biosimilar) products. Adoption of price controls and cost-containment measures, and adoption of more restrictive policies in jurisdictions with existing controls and measures, could further limit sales of any product. Decreases in third-party reimbursement for any product or a decision by a third-party payor not to cover a product could reduce physician usage and patient demand for the product and also have a material adverse effect on sales.

Healthcare Reform

In the United States, in March 2010, the Patient Protection and Affordable Care Act, as amended by the Health Care and Education Reconciliation Act, each as amended, collectively known as the ACA, was enacted, which substantially changed the way healthcare is financed by both governmental and private insurers, and significantly affected the pharmaceutical industry. The ACA contained a number of provisions, including those governing enrollment in federal healthcare programs, reimbursement adjustments and changes to fraud and abuse laws. Since its enactment, there have been judicial and Congressional challenges to certain aspects of the ACA. Other legislative changes have been proposed and adopted since the ACA was enacted, including aggregate reductions of Medicare payments to providers of 2% per fiscal year.

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Additionally, President Biden has issued multiple executive orders that have sought to reduce prescription drug costs. There has also been increasing legislative and enforcement interest in the United States with respect to drug pricing practices. Specifically, there has been heightened governmental scrutiny over the manner in which manufacturers set prices for their marketed products, which has resulted in several U.S. Congressional inquiries and proposed and enacted federal and state legislation. The Inflation Reduction Act of 2022 (“IRA”), includes several provisions that impact the pharmaceutical industry, including provisions that reduce the out-of-pocket spending cap for Medicare Part D beneficiaries to $2,000 starting in 2025; impose new manufacturer financial liability on certain drugs under Medicare Part D, allow the U.S. government to negotiate Medicare Part B and Part D price caps for certain high-cost drugs and biologics without generic or biosimilar competition; require companies to pay rebates to Medicare for drug prices that increase faster than inflation; and delay until January 1, 2032 the implementation of the HHS rebate rule that would have limited the fees that pharmacy benefit managers can charge. Further, under the IRA, orphan drugs are exempted from the Medicare drug price negotiation program, but only if they have one orphan designation and for which the only approved indication is for that disease or condition. If a product receives multiple orphan designations or has multiple approved indications, it may not qualify for the orphan drug exemption. The effects of the IRA on our business and the healthcare industry in general is not yet known.

We expect that additional state and federal healthcare reform measures will be adopted in the future, any of which could impact the amounts that federal and state governments and other third-party payors will pay for healthcare products and services.

Data Privacy & Security

Numerous state, federal and foreign laws govern the collection, dissemination, use, access to, confidentiality and security of personal information, including health-related information. As our operations and business grow, we may become subject to or affected by U.S. federal and state laws and regulations, including the Health Information Portability and Accountability Act of 1996, and its implementing regulations, as amended (“HIPAA”), that govern the collection, use, disclosure, and protection of health-related and other personal information. In California the California Consumer Protection Act (“CCPA”), which went into effect on January 1, 2020 and was amended effective January 1, 2023, establishes a new privacy framework for covered businesses by creating an expanded definition of personal information, establishing new data privacy rights for consumers in the State of California, imposing special rules on the collection of consumer data from minors, and creating a new and potentially severe statutory damages framework for violations of the CCPA and for businesses that fail to implement reasonable security procedures and practices to prevent data breaches. While clinical trial data and information governed by HIPAA are currently exempt from the current version of the CCPA, other personal information may be applicable and possible changes to the CCPA may broaden its scope. Other states, including Virginia (effective January 1, 2023), Colorado (effective July 1, 2023), Connecticut (effective July 1, 2023), and Utah (effective December 31, 2023) have passed privacy legislation and more states may do so in the future, including Iowa, where the Iowa state legislature passed a comprehensive privacy legislation on March 15, 2023. State and non-U.S. laws, including for example the EU General Data Protection Regulation, also govern the privacy and security of health information in some circumstances, many of which differ from each other in significant ways and often are not preempted by HIPAA, thus complicating compliance efforts. Failure to comply with these laws, where applicable, can result in the imposition of significant civil and/or criminal penalties and private litigation. Privacy and security laws, regulations, and other obligations are constantly evolving, may conflict with each other to complicate compliance efforts, and can result in investigations, proceedings, or actions that lead to significant civil and/or criminal penalties and restrictions on data processing.

Employees and Human Capital Resources

As of December 31, 2023, we had 236 full-time employees, of which 80 have M.D. or Ph.D. degrees. Within our workforce, 199 employees are engaged in research and development and 37 are engaged in business development, finance, legal, and general management and administration. None of our employees are represented by labor unions or covered by collective bargaining agreements. We consider our relationship with our employees to be good.

Our human capital resources objectives include, as applicable, identifying, recruiting, retaining, incentivizing and integrating our existing and new employees, advisors and consultants. The principal purposes of our equity incentive plans are to attract, retain and reward personnel through the granting of equity-based compensation awards in order to increase shareholder value and the success of our company by motivating such individuals to perform to the best of their abilities and achieve our objectives.

Information Available on the Internet

Our Internet website address is https://metagenomi.co. The information contained on, or that can be accessed through, our website is not a part of or incorporated by reference in this Annual Report on Form 10-K. We have included our website address in this in this Annual Report on Form 10-K solely as an inactive textual reference. We will make available, free of charge, through our website, our Annual Report on Form 10-K, Quarterly Reports on Form 10-Q, Current Reports on Form 8-K and amendments to those reports filed or furnished pursuant to Sections 13(a) and 15(d) of the Exchange Act. We will make these reports available through the “Investors” section of our website as soon as reasonably practicable after we electronically file such reports with, or furnish such reports to, the Securities and Exchange Commission, or SEC. We will also make available, free of charge on our website, the reports filed with the

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SEC by our executive officers, directors and 10% stockholders pursuant to Section 16 under the Exchange Act as soon as reasonably practicable after copies of those filings are provided to us by those persons. You can review our electronically filed reports and other information that we file with the SEC on the SEC’s website at http://www.sec.gov.

Investors and others should note that we announce material information to our investors using one or more of the following: SEC filings, press releases, public conference calls and webcasts and our corporate website, including without limitation the “Investors-News & Events” sections of our website. We use these channels, as well as social media channels such as LinkedIn and X (formerly Twitter), to communicate with the public about our company, our business, our approved drugs and drug candidates and other matters. It is possible that the information we post on our corporate website or other social media could be deemed to be material information. Therefore, we encourage investors, the media, and others interested in our company to review the information we post on the “Investors-News & Events” section of our corporate website and on our social media channels. The contents of our corporate website and social media channels are not, however, a part of this Annual Report on Form 10-K.

Facilities

Our corporate headquarters is located in Emeryville, California, where we sublease and occupy approximately 75,662 square feet of combined office, research and laboratory space at 5959 Horton Street, 7th Floor, Emeryville, California 94608. The current term of our sublease expires in March 2031. The company also leases approximately 23,155 square feet of office space at 1485 Park Avenue, Emeryville, California 94608 and approximately 23,851 square feet of laboratory and office space at 1545 Park Avenue, Emeryville, California 94608.

We believe that our facilities are adequate for our current needs and for the foreseeable future. To meet the future needs of our business, we may lease additional or alternate space. We believe that suitable additional or substitute space at commercially reasonable terms will be available as needed to accommodate any future expansion of our operations.

Legal proceedings

From time to time, we may become involved in legal proceedings arising from the ordinary course of business. We record a liability for such matters when it is probable that future losses will be incurred and that such losses can be reasonably estimated. Significant judgment by us is required to determine both probability and the estimated amount. Our management is currently not aware of any legal matters that could have a material adverse effect on our financial position, results of operations or cash flows.

Corporate Information

We commenced our current operations and converted to a Delaware limited liability company in September 2018. We were originally founded as Metagenomi.co, a Delaware corporation, in September 2016. On January 24, 2024, we completed a series of transactions, which we refer to collectively as the Reorganization. As a result of the Reorganization, Metagenomi Technologies, LLC merged with and into its wholly-owned subsidiary, Metagenomi, Inc., a Delaware corporation, with Metagenomi, Inc. continuing as the surviving corporation. In connection with the Reorganization, (i) all of the outstanding common unitholders of Metagenomi Technologies, LLC received shares of common stock of Metagenomi, Inc., (ii) all of the outstanding preferred unitholders of Metagenomi Technologies, LLC received shares of preferred stock of Metagenomi, Inc. and (iii) certain holders of profits interest units in Metagenomi Technologies, LLC received shares of common stock and restricted common stock in Metagenomi, Inc. as determined by the applicable provisions of the Metagenomi Technologies, LLC operating agreement in effect immediately prior to the Reorganization. Immediately prior to the completion of the IPO, all outstanding shares of preferred stock of Metagenomi, Inc. were converted into shares of common stock.

Metagenomi, Inc. is the registrant for purposes of Annual Report on form 10-K. Our consolidated financial statements for reporting periods prior to the Reorganization were reported from Metagenomi Technologies, LLC.

Our principal executive offices are located at 5959 Horton Street, 7th Floor, Emeryville, California 94608, and our telephone number is (510) 871-4880.

Our website address is https://www.metagenomi.co. The information contained in or accessible from our website is not incorporated into this prospectus, and you should not consider it part of this prospectus. We have included our website address in this prospectus solely as an inactive textual reference.

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Item 1A. Risk Factors.

Investing in our common stock involves a high degree of risk. You should carefully consider the risks and uncertainties described below together with all of the other information contained in this Annual Report on Form 10-K, including our consolidated financial statements and related notes appearing at the end of this Annual Report on Form 10-K, before deciding to invest in our common stock. If any of the events or developments described below were to occur, our business, prospects, operating results and financial condition could suffer materially, the trading price of our common stock could decline and you could lose all or part of your investment. The risks and uncertainties described below are not the only ones we face. Additional risks and uncertainties not presently known to us or that we currently believe to be immaterial may also adversely affect our business.

Risks Related to Financial Position and Need for Capital

We have incurred significant losses since inception. We expect to incur losses for the foreseeable future and may never achieve or maintain profitability.

Since inception, we have incurred significant operating losses. Our net loss was $68.3 million and $43.6 million for the years ended December 31, 2023 and 2022, respectively. As of December 31, 2023, we had an accumulated deficit of $144.9 million. We have financed our operations primarily through issuing redeemable convertible preferred units and convertible promissory notes, entering into collaboration agreements, and through the IPO proceeds. Substantially all of our losses have resulted from expenses incurred in connection with our research and development and from general and administrative costs associated with our operations. We expect to continue to incur significant expenses and increasing operating losses for the foreseeable future. The net losses we incur may fluctuate significantly from quarter to quarter. We anticipate that our expenses will increase substantially if and as we:

advance our current research activities and further develop our platform;
continue preclinical development and initiate clinical trials for any product candidates we may identify;
seek regulatory approval for any product candidates for which we successfully complete clinical trials;
establish our manufacturing capabilities, including internal manufacturing facilities and contracting with other vendors;
ultimately, commercialize our future product candidates requiring significant marketing, sales, and distribution infrastructure expenses;
hire additional research and development, clinical, commercial, general and administration personnel;
develop, maintain, expand, protect, and enforce our intellectual property portfolio;
acquire or in-license product candidates, intellectual property and technologies;
confirm, maintain or obtain freedom to operate for any of our owned or licensed technologies and product candidates;
establish and maintain collaborations;
add operational, financial and management information systems and personnel; or
incur additional legal, audit, accounting, compliance, insurance, investor relations and other expenses to operate as a public company that we did not incur as a private company.

As a result, we will need substantial additional funding to support our continuing operations and pursue our growth strategy. Until such time as we can generate significant revenue from product sales, if ever, we expect to finance our operations through the sale of equity, debt financings, or other capital sources, which may include collaborations with other companies or other strategic transactions. We may be unable to raise additional funds or enter into such other agreements or arrangements when needed on favorable terms, or at all. If we fail to raise capital or enter into such agreements as and when needed, we may have to significantly delay, reduce or eliminate the development and commercialization of our platform or delay our pursuit of potential in-licenses or acquisitions.

We have not initiated clinical development of any potential product candidate and expect that it will be many years, if ever, before we have a product candidate ready for commercialization. To become and remain profitable, we must develop and, either directly or through collaborators, eventually commercialize a therapy or therapies with market potential. This will require us to be successful in a range of challenging activities, including identifying product candidates, completing preclinical studies and clinical trials of product candidates, obtaining regulatory approval for these product candidates, manufacturing, marketing and selling those therapies for which we may obtain regulatory approval and satisfying any post-marketing requirements. We may never succeed in these activities and, even if we do, may never generate revenues that are significant or large enough to achieve profitability.

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Because of the numerous risks and uncertainties associated with developing our technology and any potential product candidates, we are unable to predict the extent of any future losses or when we will become profitable, if at all. If we do achieve profitability, we may not be able to sustain or increase profitability on a quarterly or annual basis. Our failure to become and remain profitable would decrease the value of our company and could impair our ability to raise capital, maintain our research and development efforts, expand our business or continue our operations. A decline in the value of our company could also cause you to lose all or part of your investment.

We have never generated revenue from product sales and may never become profitable.

Our ability to generate revenue from product sales and achieve profitability depends on our ability, alone or with collaborative partners, to successfully complete the development of, and obtain the regulatory approvals necessary to commercialize, product candidates we may identify for development. We may not generate revenues from product sales for many years, if ever. Our ability to generate future revenues from product sales depends heavily on our or our collaborators’ ability to successfully:

identify product candidates and successfully complete research development of any product candidates we may identify;
seek and obtain regulatory approvals for any product candidates for which we successfully complete clinical trials;
launch and commercialize any product candidates for which we may obtain regulatory approval by establishing a sales force, marketing and distribution infrastructure, or alternatively, collaborating with a commercialization partner;
qualify for adequate coverage and reimbursement by government and third-party payors for any product candidates for which we may obtain regulatory approval;
establish and maintain supply and manufacturing relationships with third parties that can provide adequate, in both amount and quality, products and services to support clinical development and the market demand for any product candidates for which we obtain regulatory approval;
develop, maintain and enhance a sustainable, scalable, reproducible and transferable manufacturing process for the product candidates we may develop;
address competing technological and market developments;
negotiate favorable terms in any collaboration, licensing or other arrangements into which we may enter and performing our obligations in such collaborations;
receive market acceptance by physicians, patients, healthcare payors, and others in the medical community;
maintain, protect, enforce, defend and expand our portfolio of intellectual property and other proprietary rights, including patents, trade secrets and know-how;
defend against third-party intellectual property claims of infringement, misappropriation or other violation; and
attract, hire and retain qualified personnel.

Our expenses could increase beyond expectations if we are required by the U.S. Food and Drug Administration (the “FDA”) or other regulatory authorities to perform preclinical studies or clinical trials in addition to those that we currently anticipate. Even if one or more of the product candidates we may develop are approved for commercial sale, we anticipate incurring significant costs associated with commercializing any approved product candidate. Additionally, such products may become subject to unfavorable pricing regulations, third- party reimbursement practices or healthcare reform initiatives. Even if we are able to generate revenues from the sale of any approved product candidates, we may not become profitable and may need to obtain additional funding to continue operations.

We will need substantial additional funding in addition to the net proceeds we received from our initial public offering (the “IPO”). If we are unable to raise additional capital when needed on acceptable terms, or at all, we may be forced to delay, reduce, or terminate certain of our research and product development programs, future commercialization efforts or other operations.

Developing gene editing products, including conducting preclinical studies and clinical trials, is a very time- consuming, expensive and uncertain process that takes years to complete. Our operations have consumed substantial amounts of cash since inception, and we expect our expenses to increase in connection with our ongoing activities, particularly as we identify, continue the research and development of, initiate and conduct clinical trials of, and seek regulatory approval for, any product candidates we may identify. In addition, if we obtain regulatory approval for any product candidates we may identify, we expect to incur significant commercialization expenses related to product sales, marketing, manufacturing, and distribution to the extent that such sales, marketing, manufacturing, and distribution are not the responsibility of a collaborator. Other unanticipated costs may also arise. Furthermore, we expect to continue incurring additional costs associated with operating as a public company. Accordingly, we will

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need to obtain substantial additional funding in connection with our continuing operations. If we are unable to raise capital when needed or on acceptable terms, we would be forced to delay, reduce, or eliminate our research and product development programs, future commercialization efforts or other operations.

As of December 31, 2023, our cash, cash equivalents and available-for-sale marketable securities were $271.2 million. We expect that the net proceeds from the IPO, approximately $81.1 million, together with our existing cash, cash equivalents, and available-for-sale marketable securities, will enable us to fund our operating expenses and capital expenditure requirements into 2027, and provide cash runway to support 2 INDs and 2 additional Development Candidate nominations. However, our operating plan may change as a result of factors currently unknown to us, and we may need to seek funding sooner than planned. Our future capital requirements will depend on many factors, including:

the timing and progress of research and development, preclinical and clinical development activities;
the number, scope and duration of clinical trials required for regulatory approval of our future product candidates;
the costs, timing, and outcome of regulatory review of any of our future product candidates;
the costs of manufacturing clinical and commercial supplies of our future product candidates;
the costs and timing of future commercialization activities, including product manufacturing, marketing, sales and distribution, for any of our future product candidates for which we receive regulatory approval;
the costs of preparing, filing and prosecuting our patent applications, maintaining and enforcing our patents and other intellectual property rights and defending intellectual property-related claims;
our ability to maintain existing, and establish new, strategic collaborations, licensing or other arrangements, and the financial terms of any such agreements, including the timing and amount of any future milestone, royalty or other payments due under any such agreement;
our ability to establish and maintain collaboration and license agreements on favorable terms, if at all;
the extent to which we acquire or in-license other product candidates and technologies;
any product liability or other lawsuits related to our future product candidates;
our implementation of various computerized informational systems and efforts to enhance operational systems;
expenses incurred to attract, hire and retain skilled personnel;
the costs of operating as a public company;
our ability to establish a commercially viable pricing structure and obtain approval for coverage and adequate reimbursement from third-party and government payers;
the extent to which we acquire or invest in businesses, products, and technologies;
the effect of competing technological and market developments; and
the impact of the COVID-19 pandemic, as well as other factors, including economic uncertainty and geopolitical tensions, which may exacerbate the magnitude of the factors discussed above.

Identifying potential product candidates and conducting preclinical testing and clinical trials is a time- consuming, expensive, and uncertain process that takes years to complete, and we may never generate the necessary data or results required to obtain regulatory approval and achieve product sales. In addition, our future product candidates, if approved, may not achieve commercial success. Our commercial revenues, if any, will be derived from sales of products that we do not expect to be commercially available for many years, if at all. Accordingly, we will need to continue to rely on additional financing to achieve our business objectives.

Adequate additional financing may not be available to us on acceptable terms, or at all. In addition, we may seek additional capital due to favorable market conditions or strategic considerations even if we believe we have sufficient funds for our current or future operating plans. To the extent that we raise additional capital through the sale of equity or convertible debt securities, your ownership interest will be diluted, and the terms of these securities may include liquidation or other preferences that adversely affect your rights as a common stockholder. Debt financing, if available, may involve agreements that include covenants limiting or restricting our ability to take specific actions, such as incurring additional debt, making capital expenditures, declaring dividends, and possibly other restrictions.

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Any additional fundraising efforts may divert our management from their day-to-day activities, which may adversely affect our ability to develop and commercialize our product candidates. We have no committed sources of additional capital and, if we are unable to raise additional capital in sufficient amounts or on terms acceptable to us, we may have to significantly delay, scale back or discontinue the development or commercialization of our future product candidates or other research and development initiatives. Without sufficient funding, our license agreements and any future collaboration agreements may also be terminated if we are unable to meet the payment or other obligations under such agreements.

If we are unable to raise additional funds through equity or debt financings when needed, we may be required to delay, limit, reduce, or terminate our product development or future commercialization efforts or grant rights to develop and market product candidates that we would otherwise prefer to develop and market ourselves. Additionally, if we raise funds through additional collaborations, strategic alliances, or licensing arrangements with third parties, we may have to relinquish valuable rights to our technologies, future revenue streams, research programs, or product candidates we develop, or we may have to grant licenses on terms that may not be favorable to us and/or that may reduce the value of our common stock.

Our short operating history may make it difficult for you to evaluate the success of our business to date and to assess our future viability.

We are an early-stage company. We commenced our operations in September 2018. Our operations to date have been limited to organizing and staffing our company, business planning, raising capital, and research and development activities such as acquiring and developing our platform and technology and identifying and beginning to advance preclinical testing of potential product candidates. All of our programs are still in the research or lead optimization stage of development and their risk of failure is high. We have not yet demonstrated an ability to initiate or successfully complete any clinical trials, including large-scale, pivotal clinical trials, obtain regulatory approvals, manufacture a commercial-scale therapy, arrange for a third party to do so on our behalf or conduct sales and marketing activities necessary for successful commercialization.

Our limited operating history, particularly in light of the rapidly evolving genome editing field, may make it difficult to evaluate our technology and industry and predict our future performance. Our short history as an operating company makes any assessment of our future success or viability subject to significant uncertainty. We will encounter risks and difficulties frequently experienced by very early stage companies in rapidly evolving fields. If we do not address these risks successfully, our business will suffer.

In addition, as a new business that is rapidly growing, we may encounter other unforeseen expenses, difficulties, complications, and delays in our product development. We will need to transition from a company with a research focus to a company capable of conducting clinical trials and ultimately supporting commercial activities if any of our future product candidates are approved. We may not be successful in such a transition.

Our future ability to utilize our net operating loss carryforwards and certain other tax attributes may be limited.

Since our inception, we have incurred losses and we may never achieve profitability. As of December 31, 2023, we had U.S. federal net operating loss carryforwards of less than $0.1 million (which are not subject to expiration) and state net operating loss carryforwards of $17.5 million (which begin to expire in various amounts in 2037), and $4.7 million of research credit carryforwards for state income tax purposes (which do not expire and can be carried forward indefinitely). To the extent that we continue to generate taxable losses, under current law, our unused U.S. federal net operating losses (“NOLs”) may be carried forward to offset a portion of future taxable income, if any. Additionally, we continue to generate business tax credits, including research and development tax credits, which generally may be carried forward to offset a portion of future taxable income, if any, subject to expiration of such credit carryforwards. Under Sections 382 and 383 of the Internal Revenue Code of 1986, as amended (the “Code”), if a corporation undergoes an “ownership change,” generally defined as one or more shareholders or groups of shareholders who own at least 5 percent of the corporation’s equity increasing their equity ownership in the aggregate by more than 50 percentage points (by value) over a three-year period, the corporation’s ability to use its pre-change NOLs and other pre-change tax attributes (such as research and development tax credits) to offset its post-change income or taxes may be limited. Similar rules may apply under state tax laws. As of December 31, 2023, the Company has completed an IRC Section 382 analysis from inception through the year ended December 31, 2023. The Company experienced two ownership changes in August 2019 and January 2022. Net operating losses generated prior to December 31, 2017, of $0.3 million are permanently limited for federal tax purposes. Net federal operating losses generated after December 31, 2017 are not limited as they can be carried forward indefinitely, subject to an 80% income limitation. Net operating losses of $0.1 million are permanently limited for California tax purposes. In addition, we may experience ownership changes in the future, some of which are outside of our control. As a result, if we earn net taxable income, our ability to use our pre-change NOLs or other pre-change tax attributes to offset U.S. federal taxable income may be subject to limitations, which could potentially result in increased future tax liability to us. Additional limitations on our ability to utilize our NOLs to offset future taxable income may arise as a result of our corporate structure whereby NOLs generated by our subsidiary may not be available to offset taxable income earned by our subsidiary. There is a risk that due to changes under the tax law, regulatory changes or other unforeseen reasons, our existing NOLs or business tax credits could expire or otherwise be unavailable to offset future income tax liabilities. At the state level, there

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may also be periods during which the use of NOLs or business tax credits is suspended or otherwise limited, which could accelerate or permanently increase state taxes owed. For these reasons, we may not be able to realize a tax benefit from the use of our NOLs or tax credits, even if we attain profitability.

Changes in tax laws or in their implementation or interpretation may adversely affect our business and financial condition.

The rules dealing with U.S. federal, state and local income taxation are constantly under review by persons involved in the legislative process and by the Internal Revenue Service and the U.S. Treasury Department. Changes to tax laws (which changes may have retroactive application) could adversely affect our business and our financial condition. In recent years, many such changes have been made and changes are likely to continue to occur in the future. We cannot predict whether, when, in what form or with what effective dates, tax laws, regulations and rulings may be enacted, promulgated or decided or whether they could increase our tax liability or require changes in the manner in which we operate in order to minimize increases in our tax liability.

Risks Related to Business, Technology, and Industry

We are very early in our development efforts, and we have not yet initiated IND-enabling studies or clinical development of any product candidate. As a result, we expect it will be many years before we commercialize any product candidate, if ever. If we are unable to advance our future product candidates into and through clinical trials, obtain regulatory approval and ultimately commercialize our product candidates or experience significant delays in doing so, our business will be materially harmed.

We are very early in our development efforts and have focused our research and development efforts to date on research efforts including preclinical studies. Currently, all of our programs are still in the research or lead optimization stage of development. Our ability to generate product revenues, which we do not expect will occur for many years, if ever, will depend heavily on the successful development, regulatory approval and eventual commercialization of our future product candidates, which may never occur. We have not yet generated revenue from product sales, and we may never be able to develop or commercialize a marketable product.

Commencing clinical trials in the United States is subject to acceptance by the FDA of an investigational new drug (“IND”) application and finalizing the trial design based on discussions with the FDA. In the event that the FDA requires us to complete additional preclinical studies or we are required to satisfy other FDA requests prior to commencing clinical trials, the start of our first clinical trials may be delayed or we may be unsuccessful obtaining clearance to proceed into clinical development. Even after we receive and incorporate guidance from the FDA, the FDA could disagree that we have satisfied their requirements to commence any clinical trial or change their position on the acceptability of our trial design or the clinical endpoints selected, which may require us to complete additional preclinical studies or clinical trials, delay the enrollment of our clinical trials, abandon our clinical development plans or meet stricter approval conditions than we currently expect. There are equivalent processes and risks applicable to clinical trial applications in other countries, including countries in the European Union.

In addition, clinical trials conducted in one country may not be accepted by regulatory authorities in other countries, and regulatory approval in one country does not guarantee regulatory approval in any other country. We may conduct one or more of our clinical trials with one or more trial sites that are located outside the United States. Although the FDA may accept data from clinical trials conducted outside the United States, acceptance of these data is subject to conditions imposed by the FDA, and there can be no assurance that the FDA will accept data from trials conducted outside of the United States. If the FDA does not accept the data from any trial that we conduct outside the United States, it would likely result in the need for additional trials, which would be costly and time-consuming and could delay or permanently halt our development of the applicable product candidates.

Commercialization of any product candidates we may develop will require preclinical and clinical development; regulatory approval in multiple jurisdictions; manufacturing supply, capacity and expertise; a commercial organization; and significant marketing efforts. The success of product candidates we may identify and develop will depend on many factors, including the following:

timely and successful completion of preclinical studies, including toxicology studies, biodistribution studies and minimally efficacious dose studies in animals, where applicable;
effective INDs or comparable foreign applications that allow commencement of our planned clinical trials or future clinical trials for any product candidates we may develop;
successful enrollment and completion of clinical trials, including under the FDA’s current Good Clinical Practices (“GCPs”), current Good Laboratory Practices (“GLPs”), and any additional regulatory requirements from foreign regulatory authorities;
positive results from our future clinical trials that support a finding of safety and effectiveness and an acceptable risk-benefit profile in the intended populations;
receipt of regulatory approvals from applicable regulatory authorities;

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establishment of arrangements through our own facilities or with third-party manufacturers for clinical supply and, where applicable, commercial manufacturing capabilities;
establishment, maintenance, defense and enforcement of patent, trademark, trade secret and other intellectual property protection or regulatory exclusivity for any product candidates we may develop;
commercial launch of any product candidates we may develop, if approved, whether alone or in collaboration with others;
acceptance of the benefits and use of our product candidates we may develop, including method of administration, if and when approved, by patients, the medical community and third-party payors;
effective competition with other therapies;
maintenance of a continued acceptable safety, tolerability and efficacy profile of any product candidates we may develop following approval; and
establishment and maintenance of healthcare coverage and adequate reimbursement by payors.

If we do not succeed in one or more of these factors in a timely manner or at all, we could experience significant delays or an inability to successfully commercialize any product candidates we may develop, which would materially harm our business. If we are unable to advance our product candidates to clinical development, obtain regulatory approval and ultimately commercialize our product candidates, or experience significant delays in doing so, our business will be materially harmed.

We are subject to additional development challenges and risks due to the novel nature of our genome editing technology.

Because our in vivo technology potentially involves genome editing across multiple cell and tissue types, we are subject to many of the challenges and risks that other genome editing therapeutics and gene therapies face, including:

regulatory guidance regarding the requirements governing gene and genome editing therapy product candidates have changed and may continue to change in the future;
to date, only a limited number of products that involve in vivo gene transfer have been approved globally;
improper modulation of a gene sequence, including unintended editing events or insertion of a sequence into certain locations in a patient’s chromosome, could lead to cancer, other aberrantly functioning cells or other diseases, including death;
corrective expression of a missing protein in patients’ cells could result in the protein being recognized as foreign, and lead to a sustained immunological reaction against the expressed protein or expressing cells, which could be severe or life-threatening; and
regulatory agencies may require extended follow-up observation periods of patients who receive treatment using genome editing product candidates including, for example, the FDA’s recommended 15-year follow-up observation period for these patients, and we will need to adopt such observation periods for product candidates we develop if required by the relevant regulatory agency, which could vary by country or region.

Furthermore, our technology has potential application for ex vivo immune cell editing strategies. Because ex vivo application of our technology potentially involves editing human cells and then delivering modified cells to patients, we may be subject to many of the challenges and risks that engineered cell therapies face. For example, clinical trials using engineered cell-based gene therapies may require unique products to be created for each patient and such individualistic manufacturing may be both inefficient and cost-prohibitive.

Clinical drug development involves a lengthy and expensive process, with an uncertain outcome. Because genome editing is relatively novel and the regulatory landscape that will govern our potential product candidates is uncertain and may change, we cannot predict the time and cost of obtaining regulatory approval, if we receive it at all, for our potential product candidates.

The time required to obtain approval for any of our potential product candidates from the FDA, the European Medicines Agency (“EMA”) or other comparable foreign regulatory authorities is unpredictable but typically takes many years following the commencement of clinical trials and depends upon numerous factors, including the substantial discretion of regulatory authorities. For more information on the regulatory approval process, see “Business—Government Regulation.” Clinical trials may fail to demonstrate that our product candidates are safe for humans and effective for indicated uses. Even if initial clinical trials in any of any product candidates we may develop are successful, such product candidates may fail to show the desired safety and efficacy in later stages of clinical development despite having successfully advanced through preclinical studies and initial clinical trials. There is a high failure rate for drugs and biologics proceeding through clinical trials. A number of companies in the pharmaceutical and biotechnology

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industries have suffered significant setbacks in later stage clinical trials even after achieving promising results in earlier stage clinical trials.

Because genome editing is relatively novel, the regulatory requirements that will govern any novel genome editing product candidates we develop may continue to evolve. Only one genome editing therapy, CASGEVY, has received marketing authorization from the FDA and EMA to date and, within the broader genetic therapy field, a limited number of gene therapy products have received marketing authorization from the FDA and the EMA. Even with respect to more established products that fit into the categories of gene therapies or cell therapies, the regulatory landscape is still developing. Regulatory requirements governing gene therapy products and cell therapy products have changed frequently and may continue to change in the future. For example, in January 2020, the FDA issued several new guidance documents on gene therapy products, and in January 2024, the FDA published a final guidance document providing recommendations for human genome editing gene therapy products. Moreover, there is substantial, and sometimes uncoordinated, overlap in those responsible for regulation of existing gene therapy products and cell therapy products. For example, in the United States, the FDA has established the Office of Therapeutic Products (“OTP”) within its Center for Biologics Evaluation and Research (“CBER”) to consolidate the review of gene therapy and related products, and the Cellular, Tissue and Gene Therapies Advisory Committee to advise CBER on its review. Gene therapy clinical trials may also be subject to review and oversight by an institutional biosafety committee (“IBC”), a local institutional committee that reviews and oversees certain basic and clinical research conducted at the institution participating in the clinical trial. Although the FDA decides whether individual gene therapy protocols may proceed, the review process and determinations of other reviewing bodies, such as an IBC or institutional review board (“IRB”), can impede or delay the initiation of a clinical trial, even if the FDA has reviewed the trial and approved its initiation. For example, more recently, some genome editing companies have seen significant delays in receiving FDA authorization to allow the initiation of their clinical trials, and has suspended ongoing trials, due to the FDA’s placement of clinical holds on their INDs.

The same applies in the European Union. The EMA’s Committee for Advanced Therapies (“CAT”) is responsible for assessing the quality, safety and efficacy of advanced-therapy medicinal products (i.e. gene therapy, somatic-cell therapy or tissue-engineered medicines). The role of the CAT is to prepare a draft opinion on an application for marketing authorization for a gene therapy medicinal candidate that is submitted to the Committee for Medicinal Products for Human Use (“CHMP”) before CHMP adopts its opinion which is submitted to the European Commission for the final decision on whether to grant a marketing authorization or not. In the European Union, the EMA publishes guidelines for the development and evaluation of gene therapy medicinal products to assist in preparing marketing authorization applications, however these are continually under review. The EMA may issue new guidelines concerning the development and marketing authorization for gene therapy medicinal products and require that we comply with these new guidelines.

Adverse developments in post-marketing experience or in clinical trials conducted by others of gene therapy products, cell therapy products or products developed through the application of genome editing technology may cause the FDA, the EMA and other regulatory bodies to revise the requirements for development or approval of our potential product candidates or limit the use of products utilizing genome editing technologies, either of which could materially harm our business. In addition, the clinical trial requirements of the FDA, the EMA and other regulatory authorities and the criteria these regulators use to determine the safety and efficacy of a product candidate vary substantially according to the type, complexity, novelty and intended use and market of the potential products. The regulatory approval process for novel product candidates can be more expensive and take longer than for other, better known or more extensively studied pharmaceutical or other product candidates. Regulatory agencies administering existing or future regulations or legislation may not allow production and marketing of products utilizing genome editing technology in a timely manner or under technically or commercially feasible conditions. In addition, regulatory action or private litigation could result in expenses, delays or other impediments to our research programs or the commercialization of resulting products.

The regulatory review committees and advisory groups described above and any new guidelines they promulgate may lengthen the regulatory review process, require us to perform additional studies or trials, increase our development costs, lead to changes in regulatory positions and interpretations, delay or prevent approval and commercialization of these treatment candidates or lead to significant post-approval limitations or restrictions. As we advance our research programs and develop future product candidates, we will be required to consult with these regulatory and advisory groups and to comply with applicable guidelines. If we fail to do so, we may be required to delay or discontinue development of any product candidates we identify and develop.

The genome editing field is relatively new and is evolving rapidly. We are focusing our research and development efforts on genome editing using programmable nucleases, base editing, and RNA and DNA-mediated integration systems (including prime editors and CRISPR-associated (“Cas”) transposases), but other genome editing technologies may be discovered that provide significant advantages over such technologies, which could materially harm our business.

To date, we have focused our efforts on genome editing technologies using programmable nucleases, base editing, and RNA and DNA-mediated integration systems (including prime editors and Cas transposases backed by our metagenomics database. Other companies have previously undertaken research and development of genome editing technologies using zinc finger nucleases,

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engineered meganucleases and transcription activator-like effector nucleases, but to date none have obtained regulatory approval for a product candidate. There can be no certainty that genome editing technology will lead to the development of genetic medicines or that other genome editing technologies will not be considered better or more attractive for the development of medicines. A number of alternative approaches are being developed by others. Our investments may not be consistent with the expectations of our stockholders and may not produce the benefits that we expect, in which case our growth, business, financial condition, and results of operations could be adversely affected. See “Risk Factors—Risks Related to Business, Technology and Industry—We face significant competition in an environment of rapid technological change, and there is a possibility that our competitors may achieve regulatory approval before us or develop therapies that are safer or more advanced or effective than ours, which may harm our financial condition and our ability to successfully market or commercialize any product candidates we may develop.” Similarly, another new genome editing technology that has not been discovered yet may be more attractive than programmable nucleases, base editing, and RNA and DNA-mediated integration systems.

Moreover, if we decide to develop genome editing technologies other than those involving such technologies, we cannot be certain we will be able to obtain rights to such technologies. Any of these factors could reduce or eliminate our commercial opportunity, and could have a material adverse effect on our business, financial condition, results of operations and prospects.

If any of the product candidates we may develop or the delivery modes we rely on cause undesirable side effects, it could delay or prevent their development or potential regulatory approval, limit the commercial potential or result in significant negative consequences following any potential regulatory approval.

To date, we have not evaluated any product candidates in human clinical trials. It is impossible to predict when or if any product candidates we may develop will ultimately prove safe in humans. In the genomic medicine field, there have been several significant adverse events from gene therapy treatments in the past, including reported cases of leukemia and death. Product candidates we may develop may be associated with undesirable side effects, unexpected characteristics or other serious adverse events, including off-target cuts of DNA, or the introduction of cuts in DNA at locations other than the target sequence. These off-target cuts could lead to disruption of a gene or a genetic regulatory sequence at an unintended site in the DNA, or, in those instances where we also provide a segment of DNA to serve as a repair template, it is possible that following off-target cut events, DNA from such repair template could be integrated into the genome at an unintended site, potentially disrupting another important gene or genomic element. There is also the potential risk of delayed adverse events following exposure to genome editing therapy due to persistent biologic activity of the genetic material or other components of products used to carry the genetic material. Possible adverse side effects that could occur with treatment with genome editing products include an immunologic reaction after administration which could substantially limit the effectiveness of the treatment If any of our genome editing technologies demonstrate a similar effect, we may decide or be required to halt or delay preclinical development or clinical development of any product candidates we may develop. In addition to serious adverse events or side effects caused by any product candidate we may develop, the administration process or related procedures also can cause undesirable side effects. If any such events occur, our preclinical studies or clinical trials could be delayed, suspended or terminated. There can be no assurance that our genome editing technologies will not cause severe or undesirable side effects.

If in the future we are unable to demonstrate that such adverse events were caused by factors other than our product candidate, the FDA, the EMA or other comparable foreign regulatory authorities could order us to cease further clinical studies of, or deny approval of, any product candidates we develop for any or all targeted indications. Even if we are able to demonstrate that all future serious adverse events are not product-related, such occurrences could affect patient recruitment or the ability of enrolled patients to complete the trial.

Moreover, if we elect, or are required, to delay, suspend or terminate any clinical trial of any product candidate we may develop, the commercial prospects of such product candidates may be harmed and our ability to generate product revenues from any of these product candidates may be delayed or eliminated. Any of these occurrences may harm our ability to identify and develop product candidates, and may harm our business, financial condition, result of operations and prospects significantly.

Viral vectors, including the adeno-associated virus (“AAV”), which are relatively new approaches used for disease treatment, also have known side effects, and for which additional risks could develop in the future. In past clinical trials that were conducted by others with non-AAV vectors, significant side effects were caused by gene therapy treatments, including reported cases of myelodysplasia, leukemia and death. Other potential side effects could include an immunologic reaction and insertional oncogenesis, which is the process whereby the insertion of a functional gene near a gene that is important in cell growth or division results in uncontrolled cell division, which could potentially enhance the risk of cancer. If the vectors we use demonstrate a similar side effect, or other adverse events, we may be required to halt or delay further clinical development of any potential product candidates. Such delayed adverse events may also occur in other viral vectors, including AAV vectors.

In addition to side effects and adverse events caused by our product candidates, the conditioning, administration process or related procedures which may be used to condition a patient for gene therapy treatment also can cause adverse side effects and adverse events.

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A gene therapy patient is generally administered cytotoxic drugs to remove stem cells from the bone marrow to create sufficient space in the bone marrow for the modified stem cells to engraft and produce new cells. This procedure compromises the patient’s immune system, and conditioning regimens have been associated with adverse events in clinical trial participants.

Additionally, if we successfully develop a product candidate and it receives regulatory approval, the FDA could require us to adopt a Risk Evaluation and Mitigation Strategy (“REMS”) to ensure that the benefits of treatment with such product candidate outweighs the risks for each potential patient, which may include, among other things, a medication guide outlining the risks of the product for distribution to patients, a communication plan to healthcare practitioners, extensive patient monitoring, or distribution systems and processes that are highly controlled, restrictive, and more costly than what is typical for the industry. Furthermore, if we or others later identify undesirable side effects caused by any product candidate that we may develop that receives regulatory approval, several potentially significant negative consequences could result, including:

regulatory authorities may revoke licenses or suspend, vary or withdraw approvals of such product candidate;
regulatory authorities may require additional warnings on the label;
we may be required to change the way a product candidate is administered or conduct additional clinical trials;
we could be sued and held liable for harm caused to patients; and
our reputation may suffer.

Any of these events could prevent us from achieving or maintaining market acceptance of our genome editing technology and any product candidates we may identify and develop and could have a material adverse effect on our business, financial condition, results of operations and prospects.

Positive results from early preclinical studies of any product candidates we may develop may not necessarily be predictive of the results of later preclinical studies and any future clinical trials of such product candidates. If we cannot replicate the positive results from our earlier preclinical studies of any product candidates we may develop in our later preclinical studies and future clinical trials, we may be unable to successfully develop, obtain regulatory approval for and commercialize such product candidates.

Any positive results from our preclinical studies of any product candidates we may develop may not necessarily be predictive of the results from later preclinical studies and clinical trials of such product candidates. Similarly, even if we are able to complete our planned preclinical studies or any future clinical trials of any product candidates we may develop according to our current development timeline, the positive results from such preclinical studies and clinical trials of our product candidates may not be replicated in subsequent preclinical studies or clinical trial results.

Many companies in the pharmaceutical and biotechnology industries have suffered significant setbacks in late- stage clinical trials after achieving positive results in early-stage development and we cannot be certain that we will not face similar setbacks. These setbacks have been caused by, among other things, preclinical findings made while clinical trials were underway or safety or efficacy observations made in preclinical studies and clinical trials, including previously unreported adverse events. Moreover, preclinical and clinical data are often susceptible to varying interpretations and analyses and many companies that believed their product candidates performed satisfactorily in preclinical studies and clinical trials nonetheless failed to obtain regulatory approval.

We may also consider additional delivery modes, which may carry additional known and unknown risks.

We may also consider additional delivery modes, which may carry additional known and unknown risks. For example, we intend to use lipid nanoparticles (“LNPs”) to deliver our nucleases. While LNPs have been used to deliver smaller molecules, such as small interfering RNA (“siRNA”), they have not been clinically proven to deliver large RNA molecules. Furthermore, as with many AAV-mediated gene therapy approaches, certain patients’ immune systems might prohibit the successful delivery, thereby potentially limiting treatment outcomes of these patients. Even if initial clinical trials in any of our potential product candidates we may develop are successful, these product candidates we may develop may fail to show the desired safety and efficacy in later stages of clinical development despite having successfully advanced through preclinical studies and initial clinical trials.

We may find it difficult to enroll patients in our future clinical trials given the limited number of patients who have the diseases any product candidates we identify or develop are intended to target. If we experience delays or difficulties in the enrollment of patients in clinical trials, our clinical development activities and our receipt of necessary regulatory approvals could be delayed or prevented.

As we progress our programs, we may not be able to initiate or continue clinical trials for any product candidates we identify or develop if we are unable to locate and enroll a sufficient number of eligible patients to participate in these trials as required by the

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FDA, the EMA or other comparable regulatory authorities outside the United States, or as needed to provide appropriate statistical power for a given trial. Enrollment may be particularly challenging for some of the rare genetically defined diseases we are targeting in some of our discovery programs. In addition, if patients are unwilling to participate in our genome editing trials because of negative publicity from adverse events related to the biotechnology, gene therapy or genome editing fields, competitive clinical trials for similar patient populations, clinical trials in competing product candidates or for other reasons, the timeline for recruiting patients, conducting studies and obtaining regulatory approval of our potential product candidates may be delayed. Moreover, some of our competitors may have ongoing clinical trials for product candidates that would treat the same indications as our potential product candidates, and patients who would otherwise be eligible for our future clinical trials may instead enroll in clinical trials of our competitors’ product candidates.

Patient enrollment is also affected by other factors, some of which may include:

severity of the disease under investigation;
size of the patient population and process for identifying patients, including proximity and availability of clinical trial sites for prospective patients with conditions that have small patient pools;
design of the trial protocol, including efforts to facilitate timely enrollment in clinical trials;
availability and efficacy of approved medications for the disease under investigation;
availability of genetic testing for potential patients and ability to monitor patients adequately during and after treatment;
ability to obtain and maintain patient informed consent;
risk that enrolled patients will drop out before completion of the trial;
eligibility and exclusion criteria for the trial in question;
perceived risks and benefits of the product candidate under trial and genome editing as a therapeutic approach; and
patient referral practices of physicians.

In addition, our ability to successfully initiate, enroll and complete a clinical trial in any foreign country is subject to numerous risks unique to conducting business in foreign countries, some of which may include:

difficulty in establishing or managing relationships with clinical research organizations (“CROs”) and physicians;
different standards for the conduct of clinical trials;
different standard-of-care for patients with a particular disease;
difficulty in locating qualified local consultants, physicians and partners; and
potential burden of complying with a variety of foreign laws, medical standards and regulatory requirements, including the regulation of pharmaceutical and biotechnology products and treatment and of genome editing technologies.

Enrollment delays in our future clinical trials may result in increased development costs for our potential product candidates, which would cause the value of our company to decline and limit our ability to obtain additional financing. If we or our collaborators have difficulty enrolling a sufficient number of patients to conduct our future clinical trials as planned, we may need to delay, limit or terminate ongoing or planned clinical trials or entire clinical programs, any of which would have an adverse effect on our business, financial condition, results of operations and prospects.

Genetic therapies are novel, and any product candidates we develop may be complex and difficult to manufacture. We could experience delays in satisfying regulatory authorities or production problems that result in delays in our development programs, limit the supply of the product candidates we may develop or otherwise harm our business.

Any product candidates we may develop will likely require processing steps that are more complex than those required for most chemical pharmaceuticals. Moreover, unlike chemical pharmaceuticals, the physical and chemical properties of a biologic such as the product candidates we intend to develop generally cannot be fully characterized. As a result, assays of the finished product candidate may not be sufficient to ensure that the product candidate will perform in the intended manner. Problems with the manufacturing process, even minor deviations from the normal process, could result in product defects or manufacturing failures that result in lot failures, product recalls, product liability claims, insufficient inventory or potentially delay progression of our potential IND filings. If we successfully develop product candidates, we may encounter problems achieving adequate quantities and quality of clinical-grade materials that meet the FDA, the EMA or other comparable applicable foreign standards or specifications with consistent and acceptable production yields and costs. For example, the current approach of manufacturing AAV vectors may fall short of supplying

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required number of doses needed for advanced stages of preclinical studies or clinical trials, and the FDA may ask us to demonstrate that we have the appropriate manufacturing processes in place to support the higher-dose group in our preclinical studies or clinical trials. In addition, any product candidates we may develop will require complicated delivery methods, each of which will introduce additional complexities in the manufacturing process.

In addition, the FDA, the EMA and other regulatory authorities may require us to submit samples of any lot of any approved product together with the protocols showing the results of applicable tests at any time. Under some circumstances, the FDA, the EMA or other regulatory authorities may require that we not distribute a lot until the agency authorizes its release. Slight deviations in the manufacturing process, including those affecting quality attributes and stability, may result in unacceptable changes in the product that could result in lot failures or product recalls. Lot failures or product recalls could cause us to delay clinical trials or product launches, which could be costly to us and otherwise harm our business, financial condition, results of operations and prospects.

Given the nature of biologics manufacturing there is a risk of contamination during manufacturing. Any contamination could materially harm our ability to produce product candidates on schedule and could harm our results of operations and cause reputational damage. Some of the raw materials that we anticipate will be required in our manufacturing process are derived from biologic sources. Such raw materials are difficult to procure and may be subject to contamination or recall. A material shortage, contamination, recall or restriction on the use of biologically derived substances in the manufacture of any product candidates we may develop could adversely impact or disrupt the commercial manufacturing or the production of clinical material, which could materially harm our development timelines and our business, financial condition, results of operations and prospects.

Any problems in our manufacturing process or the facilities with which we contract could make us a less attractive collaborator for potential partners, including larger pharmaceutical companies and academic research institutions, which could limit our access to additional attractive development programs. Problems in third-party manufacturing process or facilities also could restrict our ability to ensure sufficient clinical material for any clinical trials we may be conducting or are planning to conduct and meet market demand for any product candidates we develop and commercialize.

We face significant competition in an environment of rapid technological change, and there is a possibility that our competitors may achieve regulatory approval before us or develop therapies that are safer or more advanced or effective than ours, which may harm our financial condition and our ability to successfully market or commercialize any product candidates we may develop.

The development and commercialization of new drug products is highly competitive. Moreover, the genome editing field is characterized by rapidly changing technologies, significant competition and a strong emphasis on intellectual property. We will face competition with respect to any product candidates that we may seek to develop or commercialize in the future from major pharmaceutical companies, specialty pharmaceutical companies and biotechnology companies worldwide. Potential competitors also include academic institutions, government agencies and other public and private research organizations that conduct research, seek patent or other intellectual property protection and establish collaborative arrangements for research, development, manufacturing and commercialization.

There are a number of large pharmaceutical and biotechnology companies that currently market and sell products or are pursuing the development of products for the treatment of the disease indications for which we have research programs. Some of these competitive products and therapies are based on scientific approaches that are the same as or similar to our approach, while others are based on entirely different approaches.

Amongst publicly traded peers, there are several companies utilizing CRISPR/Cas technology, including Caribou Biosciences, Inc., Editas Medicine, Inc., CRISPR Therapeutics AG, Intellia Therapeutics, Inc. and Graphite Bio, Inc. Several additional companies such as Sangamo Therapeutics, Inc., Precision BioSciences, Inc., bluebird bio, Inc., and Cellectis Inc. utilize alternative nuclease-based genome editing technologies, including zinc finger nucleases (“ZFNs”), engineered meganucleases and transcription-activator like effector nucleases (“TALENs”). Beam Therapeutics utilizes base editing technology. Prime Medicine utilizes prime editing technology.

In addition, other private companies such as Tessera Therapeutics, Inc. and Tome Biosciences, Inc. have announced their work in recombinase DNA and RNA gene writers, although little is known publicly about their science or portfolio. Other companies have announced intentions to enter the genome editing field, such as Moderna, Inc. and Pfizer Inc. Most recently, new epigenetic editing companies have emerged, such as Chroma Medicine, Inc. and Tune Therapeutics, Inc. In addition, we face competition from companies utilizing gene therapy, oligonucleotides and cell therapy therapeutic approaches.

Several private companies such as Arbor Biotechnologies, Inc., Scribe Therapeutics Inc., and Mammoth Biosciences, Inc. are actively searching for novel genome editing components and have reported the discovery of new DNA-cutting enzymes. Other companies are

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active in LNP delivery technologies and advancing those into therapeutics using genetic therapies, including Recode Therapeutics, Inc., Verve Therapeutics, Inc., Generation Bio Co. and Beam Therapeutics, among others.

Any product candidates that we successfully develop and commercialize will compete with existing therapies and new therapies that may become available in the future that are approved to treat the same diseases for which we may obtain approval for any product candidates we may develop. This may include other types of therapies, such as small molecule, antibody and/or protein therapies.

Many of our current or potential competitors, either alone or with their collaboration partners, may have significantly greater financial resources and expertise in research and development, manufacturing, conducting preclinical studies and clinical trials, obtaining regulatory approvals and marketing approved products than we do. Mergers and acquisitions in the pharmaceutical, biotechnology and gene therapy industries may result in even more resources being concentrated among a smaller number of our competitors. Smaller or early-stage companies may also prove to be significant competitors, particularly through collaborative arrangements with large and established companies. These competitors also compete with us in recruiting and retaining qualified scientific and management personnel and establishing clinical trial sites and patient registration for clinical trials, as well as in acquiring technologies complementary to, or necessary for, our programs. Our commercial opportunity could be reduced or eliminated if our competitors develop and commercialize product candidates that are safer, more effective, have fewer or less severe side effects, are more convenient, or are less expensive than any product candidates that we may develop or that would render any product candidates that we may develop obsolete or non-competitive. Our competitors also may obtain FDA or other regulatory approval for their product candidates more rapidly than we may obtain approval for ours, which could result in our competitors establishing a strong market position before we are able to enter the market. Additionally, technologies developed by our competitors may render our potential product candidates uneconomical or obsolete, and we may not be successful in marketing any product candidates we may develop against competitors.

In addition, as a result of the expiration or successful challenge of our patent or other intellectual property rights, we could face risks relating to our ability to successfully prevent or delay launch of competitors’ products. The availability of our competitors’ products could limit the demand and the price we are able to charge for any product candidates that we may develop and commercialize.

Adverse public perception of genome editing and cellular therapy products may negatively impact demand for, or regulatory approval of, any product candidates we may develop.

The product candidates we may develop will involve editing the human genome. The clinical and commercial success of any product candidates we may develop will depend in part on public acceptance of the use of genome editing therapies for the prevention or treatment of human diseases. Public attitudes may be influenced by claims that genome editing is unsafe, unethical, or immoral, and, consequently, our products may not gain the acceptance of the public or the medical community. Negative public reaction to gene therapy in general could result in greater government regulation and stricter labeling requirements of genome editing products, including any of our product candidates, and could cause a decrease in the demand for any products we may develop. Adverse public attitudes may adversely impact our ability to enroll clinical trials. Additionally, ethical, social and legal concerns about genome editing and gene therapy could result in additional regulations restricting or prohibiting any product candidates we may develop.

The commercial success of any of the product candidates we may develop will depend upon the degree of market acceptance by physicians, patients, third-party payors and others in the medical community.

Even if we obtain the requisite approvals from the FDA in the United States, the EMA in the European Union and other regulatory authorities internationally, the commercial success of any product candidates we may develop will depend, in part, on the acceptance of physicians, patients and health care payors of genome editing and gene therapy products in general, and our product candidates in particular, as medically necessary, cost- effective and safe. Any product that we commercialize may not gain acceptance by physicians, patients, health care payors and others in the medical community who may opt for existing treatments with which they are already familiar and for which greater clinical data may be available. The degree of market acceptance of genome editing and gene therapy products and, in particular, any product candidates we may develop, if approved for commercial sale, will depend on several factors, including:

the efficacy, durability and safety of such product candidates as demonstrated in any future clinical trials;
the potential and perceived advantages of such product candidates over alternative treatments;
the cost of treatment relative to alternative treatments;
the clinical indications for which the product candidate is approved by the FDA, the EMA or other regulatory authorities;
patient awareness of, and willingness to seek, genotyping;
the willingness of physicians to prescribe new therapies;

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the willingness of the target patient population to try new therapies;
the prevalence and severity of any side effects;
product labeling or product insert requirements of the FDA, the EMA or other regulatory authorities, including any limitations or warnings contained in a product’s approved labeling;
relative convenience and ease of administration;
the strength of marketing and distribution support;
the timing of market introduction of competitive products;
publicity concerning our products or competing products and treatments; and
sufficient third-party payor coverage and reimbursement.

Even if a potential product displays a favorable efficacy and safety profile in preclinical studies and future clinical trials, market acceptance of the product will not be fully known until after it is launched. If any product candidates we may develop do not achieve an adequate level of acceptance following regulatory approval, if ever, we may not generate significant product revenue and may not become profitable.

We may expend our limited resources to pursue a particular product candidate or indication and fail to capitalize on product candidates or indications that may be more profitable or for which there is a greater likelihood of success.

We have limited financial and managerial resources. As a result, we may forego or delay pursuit of opportunities with other product candidates or for other indications that later prove to have greater commercial potential. Our resource allocation decisions may cause us to fail to timely capitalize on viable commercial products or profitable market opportunities. Our spending on current and future research and development programs and product candidates for specific indications may not yield any commercially viable products. If we do not accurately evaluate the commercial potential or target market for a particular product candidate, we may relinquish valuable rights to that product candidate through collaboration, licensing or other royalty arrangements in cases in which it would have been more advantageous for us to retain sole development and commercialization rights to such product candidate.

If, in the future, we are unable to establish sales and marketing capabilities or enter into agreements with third parties to sell and market products based on our technologies, we may not be successful in commercializing our future product candidates if and when any such product candidates are approved and we may not be able to generate any revenue.

We do not currently have a sales or marketing infrastructure and, as a company, have no experience in the sale, marketing or distribution of therapeutic products. To achieve commercial success for any potential approved product candidate for which we retain sales and marketing responsibilities, we must build our sales, marketing, managerial and other non-technical capabilities or make arrangements with third parties to perform these services. In the future, we may choose to build a focused sales and marketing infrastructure to sell, or participate in sales activities with our collaborators for, some of our product candidates if and when they are approved.

There are risks involved with both establishing our own sales and marketing capabilities and entering into arrangements with third parties to perform these services. For example, recruiting and training a sales force is expensive and time consuming and could delay any product launch. If the commercial launch of a product candidate for which we recruit a sales force and establish marketing capabilities is delayed or does not occur for any reason, we would have prematurely or unnecessarily incurred these commercialization expenses. This may be costly and our investment would be lost if we cannot retain or reposition our sales and marketing personnel.

Factors that may inhibit our efforts to commercialize our product candidates on our own include:

our inability to recruit, train and retain adequate numbers of effective sales and marketing personnel;
the inability of sales personnel to obtain access to physicians or persuade adequate numbers of physicians to prescribe any future product that we may develop;
the lack of complementary treatments to be offered by sales personnel, which may put us at a competitive disadvantage relative to companies with more extensive product lines; and
unforeseen costs and expenses associated with creating an independent sales and marketing organization.

If we enter into arrangements with third parties to perform sales, marketing and distribution services, our product revenue or the profitability to us from these revenue streams is likely to be lower than if we were to market and sell any product candidates that we develop ourselves. In addition, we may not be successful in entering into arrangements with third parties to sell and market our product candidates or may be unable to do so on terms that are favorable to us. We likely will have little control over such third parties

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and any of them may fail to devote the necessary resources and attention to sell and market our product candidates effectively. If we do not establish sales and marketing capabilities successfully, either on our own or in collaboration with third parties, we may not be successful in commercializing our product candidates. Further, our business, results of operations, financial condition and prospects will be materially adversely affected.

Due to the novel nature of our technology and the potential for any product candidates we may develop to offer therapeutic benefit in a single administration or limited number of administrations, we face uncertainty related to pricing and reimbursement for such product candidates.

Our initial target patient populations are relatively small, as a result of which the pricing and reimbursement of any product candidates we may develop, if approved, must be adequate to support the necessary commercial infrastructure. If we are unable to obtain adequate levels of reimbursement, our ability to successfully market and sell any such product candidates will be adversely affected. The manner and level at which reimbursement is provided for services related to any product candidates we may develop (e.g., for administration of our product candidate to patients) is also important. Inadequate reimbursement for such services may lead to physician and payor resistance and adversely affect our ability to market or sell our product candidates. In addition, we may need to develop new reimbursement models in order to realize adequate value. Payors may not be able or willing to adopt such new models, and patients may be unable to afford that portion of the cost that such models may require them to bear. If we determine such new models are necessary but we are unsuccessful in developing them, or if such models are not adopted by payors, our business, financial condition, results of operations, and prospects could be adversely affected.

We expect the cost of a single administration of a genome editing therapy, such as those we are seeking to develop, to be substantial, when and if they achieve regulatory approval. We expect that coverage and reimbursement by government and private payors will be essential for most patients to be able to afford these treatments. Accordingly, sales of any such product candidates will depend substantially, both domestically and abroad, on the extent to which the costs of any of our product candidates will be paid by government authorities, private health plans, and other third-party payors. Payors may not be willing to pay high prices for a single administration. Coverage and reimbursement by a third-party payor may depend upon several factors, including the third-party payor’s determination that use of a product is:

a covered benefit under its health plan;
safe, effective, and medically necessary;
appropriate for the specific patient;
cost-effective; and
neither experimental nor investigational.

Obtaining coverage and reimbursement for a product from third-party payors is a time-consuming and costly process that could require us to provide to the payor supporting scientific, clinical, and cost-effectiveness data. There is significant uncertainty related to third-party coverage and reimbursement of newly approved products. We may not be able to provide data sufficient to gain acceptance with respect to coverage and reimbursement. If coverage and reimbursement are not available, or are available only at limited levels, we may not be able to successfully commercialize any of our product candidates. Even if coverage is provided, the approved reimbursement amount may not be adequate to realize a sufficient return on our investment.

In the United States, no uniform policy exists for coverage and reimbursement for products among third-party payors. Therefore, decisions regarding the extent of coverage and amount of reimbursement to be provided can differ significantly from payor to payor. The process for determining whether a payor will provide coverage for a product may be separate from the process for setting the reimbursement rate a payor will pay for the product. One third-party payor’s decision to cover a particular product or service does not ensure that other payors will also provide coverage for the medical product or service. Third-party payors may limit coverage to specific products on an approved list or formulary, which may not include all FDA-approved products for a particular indication.

Further, third-party payors are increasingly challenging the price and examining the medical necessity and cost-effectiveness of medical products and services, in addition to their safety and efficacy. In order to secure coverage and reimbursement for any product that might be approved for sale, we may need to conduct expensive pharmacoeconomic studies in order to demonstrate the medical necessity and cost-effectiveness of any product candidates we may develop, in addition to the costs required to obtain FDA or comparable regulatory approvals. Additionally, we may also need to provide discounts to purchasers, private health plans or government healthcare programs. Despite our best efforts, any product candidates we may develop may not be considered medically necessary or cost-effective. If third-party payors do not consider a product to be cost- effective compared to other available therapies, they may not cover an approved product as a benefit under their plans or, if they do, the level of payment may not be sufficient to allow us to sell our products at a profit. A decision by a third-party payor not to cover a product could reduce physician utilization once the product is approved and have a material adverse effect on sales, our operations and financial condition. Finally, in some

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foreign countries, the proposed pricing for a product candidate must be approved before it may be lawfully marketed. The requirements governing product pricing vary widely from country to country. For example, in the EU, pricing and reimbursement of pharmaceutical products are regulated at a national level under the individual EU Member States’ social security systems. Some foreign countries provide options to restrict the range of medicinal products for which their national health insurance systems provide reimbursement and can control the prices of medicinal products for human use. To obtain reimbursement or pricing approval, some of these countries may require the completion of clinical trials that compare the cost effectiveness of a particular product candidate to currently available therapies. A country may approve a specific price for the medicinal product or it may instead adopt a system of direct or indirect controls on the profitability of the company placing the medicinal product on the market. There can be no assurance that any country that has price controls or reimbursement limitations for products will allow favorable reimbursement and pricing arrangements for any of our product candidates. Even if approved for reimbursement, historically, product candidates launched in some foreign countries, such as some countries in the EU, do not follow price structures of the United States and prices generally tend to be significantly lower.

If we are not able to establish collaborations on a timely basis, on commercially reasonable terms, or at all, we may have to alter, reduce or delay our development and commercialization plans, or increase our expenditures to fund development or commercialization activities at our own expense.

For some of the product candidates we may develop, we may decide to collaborate with other pharmaceutical and biotechnology companies for the development and potential commercialization of those product candidates. We face significant competition in seeking appropriate collaborations and collaborations are complex and time-consuming to negotiate and document. Whether we reach a definitive agreement for a collaboration will depend, among other things, upon our assessment of the collaborator’s resources and expertise, the terms and conditions of the proposed collaboration, and the proposed collaborator’s evaluation of a number of factors. Those factors may include the design or results of clinical trials, the likelihood of approval by the FDA, the EMA or similar regulatory authorities outside the United States, the potential market for the subject product candidate, the costs and complexities of manufacturing and delivering such product candidate to patients, the potential of competing products, the existence of uncertainty with respect to our ownership of technology, which can exist if there is a challenge to such ownership without regard to the merits of the challenge, and industry and market conditions generally. The collaborator may also consider alternative product candidates or technologies for similar indications that may be available to collaborate on and whether such a collaboration could be more attractive than the one with us.

We may also be restricted under existing collaboration agreements from entering into future collaboration agreements on certain terms with potential collaborators. In addition, there have been a significant number of recent business combinations among large pharmaceutical companies that have resulted in a reduced number of potential future collaborators, which further increases competition we face in seeking potential collaborations.

We may not be able to negotiate collaborations on a timely basis, on acceptable terms, or at all. If we are unable to do so, we may have to curtail the development of the product candidate for which we are seeking to collaborate, reduce or delay its development program or one or more of our other development programs, delay its potential commercialization or reduce the scope of any sales or marketing activities, or increase our expenditures and undertake development or commercialization activities at our own expense. If we elect to increase our expenditures to fund development or commercialization activities on our own, we may need to obtain additional capital, which may not be available to us on acceptable terms or at all. If we do not have sufficient funds, we may not be able to develop product candidates or bring them to market and generate product revenue.

Unfavorable global economic conditions could adversely affect our business, financial condition or results of operations.

Our results of operations could be adversely affected by general conditions in the global economy, geopolitical tensions and in the global financial markets. A severe or prolonged economic downturn or additional global financial and political crises could result in a variety of risks to our business, including weakened demand for any product candidates we develop or our ability to raise additional capital when needed on acceptable terms, if at all. A weak or declining economy could also strain our suppliers or other third parties and create import and export issues, possibly resulting in supply disruption. Any of the foregoing could harm our business and we cannot anticipate all of the ways in which the current economic climate and financial market conditions could adversely impact our business.

We face risks related to health epidemics, pandemics and other widespread outbreaks of contagious disease, including the COVID-19 pandemic, which could significantly disrupt our operations, impact our financial results or otherwise adversely impact our business.

Significant outbreaks of contagious diseases and other adverse public health developments could have a material impact on our business operations and operating results. For example, the spread of COVID-19 has affected segments of the global economy and our operations. As a result of the COVID-19 pandemic or similar public health crises that may arise, we may experience disruptions that could adversely impact our operations, research and development, and as we continue developing, any preclinical studies, clinical trials and manufacturing activities we may conduct, some of which may include:

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delays or disruptions in research programs, preclinical studies, clinical trials or IND-enabling studies that we or our collaborators may conduct;
interruption or delays in the operations of the FDA, the EMA and comparable foreign regulatory agencies;
interruption of, or delays in receiving and distributing, supplies of drug substance and drug product from our contract manufacturing organizations (“CMOs”), to preclinical or clinical research sites or delays or disruptions in any preclinical studies or clinical trials performed by CROs;
limitations imposed on our business operations by local, state or federal authorities to address a pandemic or similar public health crises; and
business disruptions caused by potential workplace, laboratory and office closures and an increased reliance on employees working from home, disruptions to or delays in ongoing laboratory experiments and operations, staffing shortages, travel limitations, and cybersecurity and data accessibility or security issues.

In addition, the trading prices for biopharmaceutical companies have been highly volatile as a result of the COVID- 19 pandemic and we may face similar volatility in our stock price after we complete this public offering. We cannot predict the scope and severity of any economic recovery after the COVID-19 pandemic abates, including following any additional “waves” or other intensifying of the pandemic. If we or any of the third parties with whom we engage were to experience additional shutdowns or other business disruptions, our ability to conduct our business in the manner and on the timelines presently planned could be materially and negatively affected, which could have a material adverse impact on our business, financial condition, our results of operations and prospects. Furthermore, the COVID-19 pandemic could exacerbate the other risks described in this section.

Our operations are vulnerable to interruption by disasters, terrorist activity, pandemics and other events beyond our control, which could harm our business.

Our facilities are located in California. We have not undertaken a systematic analysis of the potential consequences to our business and financial results from a major flood, power loss, terrorist activity, pandemics or other regional or global disasters and generally do not have a recovery plan for such events. In addition, we do not carry sufficient insurance to compensate us for actual losses from interruption of our business that may occur, and any losses or damages incurred by us could harm our business. The occurrence of any of these business disruptions could seriously harm our operations and financial condition and increase our costs and expenses.

We may use artificial intelligence in our business, and challenges with properly managing its use, as well as uncertainty regarding the legal landscape surrounding the use of AI could result in reputational harm, competitive harm, and legal liability, and adversely affect our results of operations.

We incorporate artificial intelligence (“AI”) solutions into our platform, and these applications may become important in our operations over time. There are significant risks involved in utilizing AI and no assurance can be provided that the usage of such AI will enhance our business or assist our business in being more efficient or profitable. Known risks of AI currently include inaccuracy, bias, toxicity, intellectual property infringement or misappropriation, data privacy and cybersecurity and data provenance. In addition, AI may have errors or inadequacies that are not easily detectable. AI may also be subject to data herding and interconnectedness (i.e., multiple market participants utilizing the same data), which may adversely impact our business. If the data used to train AI or the content, analyses, or recommendations that AI applications assist in producing are or are alleged to be deficient, inaccurate, incomplete, overbroad or biased, our business, financial condition, and results of operations may be adversely affected. The legal landscape and subsequent legal protection for the use of AI remains uncertain, and development of the law in this area could impact our ability to enforce our proprietary rights or protect against infringing uses. If we do not have sufficient rights to use the data on which AI relies or to the outputs produced by AI applications, we may incur liability through the violation of certain laws, third-party privacy or other rights or contracts to which we are a party. Our use of AI applications may also, in the future, result in cybersecurity incidents that implicate the personal data of customers or patients. Any such cybersecurity incidents related to our use of AI applications could adversely affect our reputation and results of operations.

Risks Related to Regulatory, Legal, and Clinical Trials

While we intend to seek designations for our potential product candidates with the FDA and comparable foreign regulatory authorities that are intended to confer benefits such as a faster development process or an accelerated regulatory pathway, there can be no assurance that we will successfully obtain such designations. In addition, even if one or more of our potential product candidates are granted such designations, we may not be able to realize the intended benefits of such designations.

The FDA and comparable foreign regulatory authorities offer certain designations for product candidates that are designed to encourage the research and development of product candidates that are intended to address conditions with significant unmet medical

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need. These designations may confer benefits such as additional interaction with regulatory authorities, a potentially accelerated regulatory pathway and priority review.

However, there can be no assurance that we will successfully obtain such designations for any potential product candidates. In addition, while such designations could expedite the development or approval process, they generally do not change the standards for approval. Even if we obtain such designations for one or more of our potential product candidates, there can be no assurance that we will realize their intended benefits. For example, we may seek fast track designation for some of our potential product candidates. If a therapy is intended for the treatment of a serious or life threatening condition and the therapy nonclinical or clinical data demonstrates the potential to address unmet medical needs for this condition, the therapy sponsor may apply for fast track designation. The FDA has broad discretion whether or not to grant this designation, so even if we believe a particular product candidate is eligible for this designation, there can be no assurance that the FDA would decide to grant it. Even if we do receive fast track designation, we may not experience a faster development process, review or approval compared to conventional FDA procedures, and receiving a fast track designation does not provide assurance of ultimate FDA approval. In addition, the FDA may withdraw fast track designation if it believes that the designation is no longer supported by data from our clinical development program. Additionally, we may seek a breakthrough therapy designation for some of our potential product candidates. A breakthrough therapy is defined as a therapy that is intended, alone or in combination with one or more other therapies, to treat a serious or life-threatening disease or condition, and preliminary clinical evidence indicates that the therapy may demonstrate substantial improvement over existing therapies on one or more clinically significant endpoints, such as substantial treatment effects observed early in clinical development. For therapies that have been designated as breakthrough therapies, interaction and communication between the FDA and the sponsor of the trial can help to identify the most efficient path for clinical development while minimizing the number of patients placed in ineffective control regimens. Therapies designated as breakthrough therapies by the FDA may also be eligible for accelerated approval. Designation as a breakthrough therapy is within the discretion of the FDA. Accordingly, even if we believe one of our potential product candidates meets the criteria for designation as a breakthrough therapy, the FDA may disagree and instead determine not to make such designation. In any event, the receipt of a breakthrough therapy designation for a product candidate may not result in a faster development process, review or approval compared to therapies considered for approval under conventional FDA procedures and does not assure ultimate approval by the FDA. In addition, even if one or more of our potential product candidates qualify as breakthrough therapies, the FDA may later decide that such product candidates no longer meet the conditions for qualification. In addition, we may seek a regenerative medicine advanced therapy (“RMAT”) designation for some of our potential product candidates. An RMAT is defined as cell therapies, therapeutic tissue engineering products, human cell and tissue products and combination products using any such therapies or products. Gene therapies, including genetically modified cells that lead to a durable modification of cells or tissues may meet the definition of a regenerative medicine therapy. The RMAT program is intended to facilitate efficient development and expedite review of RMATs, which are intended to treat, modify, reverse or cure a serious or life-threatening disease or condition. A new drug application or a biologics license application (“BLA”) for an RMAT may be eligible for priority review or accelerated approval through (1) surrogate or intermediate endpoints reasonably likely to predict long-term clinical benefit or (2) reliance upon data obtained from a meaningful number of sites. Benefits of such designation also include early interactions with the FDA to discuss any potential surrogate or intermediate endpoint to be used to support accelerated approval. A regenerative medicine therapy that is granted accelerated approval and is subject to post-approval requirements may fulfill such requirements through the submission of clinical evidence, clinical trials, patient registries or other sources of real world evidence, such as electronic health records; the collection of larger confirmatory data sets; or post-approval monitoring of all patients treated with such therapy prior to its approval. RMAT designation is within the discretion of the FDA. Accordingly, even if we believe one of our potential product candidates meets the criteria for designation as a regenerative medicine advanced therapy, the FDA may disagree and instead determine not to make such designation. In any event, the receipt of RMAT designation for a product candidate may not result in a faster development process, review or approval compared to drugs considered for approval under conventional FDA procedures and does not assure ultimate approval by the FDA. In addition, even if one or more of our potential product candidates qualify as for RMAT designation, the FDA may later decide that the biological products no longer meet the conditions for qualification. We may also seek rare pediatric disease designation for some of our potential product candidates. The FDA defines “rare pediatric disease” as a (i) serious or life-threatening disease in which the serious or life-threatening manifestations primarily affect individuals aged from birth to 18 years, including age groups often called neonates, infants, children, and adolescents; and (ii) a rare disease or condition within the meaning of the Orphan Drug Act. Designation of a product candidate as a product for a rare pediatric disease does not guarantee that a marketing application for such product candidate will meet the eligibility criteria for a rare pediatric disease priority review voucher (“PRV”) at the time the application is approved. Under the U.S. Federal Food, Drug, and Cosmetic Act (“FDCA”), we will need to request a rare pediatric disease PRV in our original marketing application for any potential product candidates for which we have received rare pediatric disease designation. The FDA may determine that a marketing application for any such product candidates, if approved, does not meet the eligibility criteria for a PRV. Under the current statutory sunset provisions, after September 30, 2024, the FDA may only award a PRV for an approved rare pediatric disease product application if the sponsor has rare pediatric disease designation for the drug or biologic that is the subject of such application, and that designation was granted by September 30, 2024. After September 30, 2026, the FDA may not award any rare pediatric disease PRVs. However, it is possible the authority for FDA to award rare pediatric disease PRV will be further extended by Congress. As such, if we do not obtain approval of a marketing

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application for any of our potential product candidates on or before September 30, 2026, and if the PRV program is not extended by Congressional action, we may not receive a PRV.

In the future, we may also seek approval of product candidates under the FDA’s accelerated approval pathway. A product may be eligible for accelerated approval if it is designed to treat a serious or life-threatening disease or condition and generally provides a meaningful advantage over available therapies upon a determination that the product candidate has an effect on a surrogate endpoint or intermediate clinical endpoint that is reasonably likely to predict clinical benefit or on a clinical endpoint that can be measured earlier than irreversible morbidity or mortality (“IMM”) that is reasonably likely to predict an effect on IMM or other clinical benefit. The FDA considers a clinical benefit to be a positive therapeutic effect that is clinically meaningful in the context of a given disease, such as IMM. For the purposes of accelerated approval, a surrogate endpoint is a marker, such as a laboratory measurement, radiographic image, physical sign or other measure that is thought to predict clinical benefit, but is not itself a measure of clinical benefit. An intermediate clinical endpoint is a clinical endpoint that can be measured earlier than an effect on irreversible morbidity or mortality that is reasonably likely to predict an effect on irreversible morbidity or mortality or other clinical benefit. The accelerated approval pathway may be used in cases in which the advantage of a new drug over available therapy may not be a direct therapeutic advantage, but is a clinically important improvement from a patient and public health perspective. If granted, accelerated approval is usually contingent on the sponsor’s agreement to conduct, in a diligent manner, additional post-approval confirmatory studies to verity and describe the drug’s clinical benefit. Under the Food and Drug Omnibus Reform Act of 2022 (“FDORA”), the FDA is permitted to require, as appropriate, that a post-approval confirmatory study or studies be underway prior to approval or within a specified time period after the date of approval for a product granted accelerated approval.

FDORA also requires sponsors to send updates to the FDA every 180 days on the status of such studies, including progress toward enrollment targets, and the FDA must promptly post this information publicly. FDORA also gives the FDA increased authority to withdraw approval of a drug or biologic granted accelerated approval on an expedited basis if the sponsor fails to conduct such studies in a timely manner, send the necessary updates to the FDA, or if such post-approval studies fail to verify the drug’s predicted clinical benefit. Under FDORA, the FDA is empowered to take action, such as issuing fines, against companies that fail to conduct with due diligence any post-approval confirmatory study or submit timely reports to the agency on their progress. There can be no assurance that the FDA would allow any of the product candidates we may develop to proceed on an accelerated approval pathway, and even if the FDA did allow such pathway, there can be no assurance that such submission or application will be accepted or that any expedited development, review or approval will be granted on a timely basis, or at all. Moreover, even if we received accelerated approval, any post-approval studies required to confirm and verify clinical benefit may not show such benefit, which could lead to withdrawal of any approvals we have obtained. Receiving accelerated approval does not assure that the product’s accelerated approval will eventually be converted to a traditional approval.

If the FDA determines that a product candidate offers a treatment for a serious condition and, if approved, the product would provide a significant improvement in safety or effectiveness, the FDA may designate the product candidate for priority review. A priority review designation means that the goal for the FDA to review an application is six months, rather than the standard review period of ten months. We may request priority review for the product candidates that we may develop. The FDA has broad discretion with respect to whether or not to grant priority review status to a product candidate, so even if we believe a particular product candidate is eligible for such designation or status, the FDA may decide not to grant it. Moreover, a priority review designation does not necessarily result in an expedited regulatory review or approval process or necessarily confer any advantage with respect to approval compared to conventional FDA procedures. Receiving priority review from the FDA does not guarantee approval within the six-month review cycle or at all.

In addition, in the European Union, we may seek to participate in the PRIority Medicines (“PRIME”) scheme for our potential product candidates. The PRIME scheme is intended to encourage development of products in areas of unmet medical need and provides accelerated assessment of products representing substantial innovation, where the marketing authorization application will be made through the centralized procedure in the European Union. Products from small-and medium-sized enterprises may qualify for earlier entry into the PRIME scheme than larger companies on the basis of compelling non-clinical data and tolerability data from initial clinical trials. Eligible products must target conditions for which there is an unmet medical need (no treatment option exists in the European Union or, they can offer a major therapeutic advantage over existing treatments). Many benefits accrue to sponsors of product candidates with PRIME designation, including but not limited to, early and proactive regulatory dialogue with the EMA, frequent discussions on clinical trial designs and other development program elements, and accelerated marketing authorization application assessment once a dossier has been submitted. There is no guarantee, however, that our potential product candidates would be deemed eligible for the PRIME scheme and even if we do participate in the PRIME scheme, where during the course of development a product no longer meets the eligibility criteria, support under the PRIME scheme may be withdrawn. PRIME eligibility does not change the standards for product approval, and there is no assurance that any such designation or eligibility will result in expedited review or approval.

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Healthcare and other reform legislation may increase the difficulty and cost for us and any collaborators we may have to obtain regulatory approval of and commercialize any product candidates we may develop and affect the prices we, or they, may obtain.

In the United States and some foreign jurisdictions, there have been and continue to be ongoing efforts to implement legislative and regulatory changes regarding the healthcare system. Such changes could prevent or delay regulatory approval of any product candidates that we may develop, restrict or regulate post-approval activities and affect our ability to profitably sell any product candidates for which we obtain regulatory approval. Although we cannot predict what healthcare or other reform efforts will be successful, such efforts may result in more rigorous coverage criteria, in additional downward pressure on the price that we, or our future collaborators, may receive for any approved products or in other consequences that may adversely affect our ability to achieve or maintain profitability.

Within the United States, the federal government and individual states have aggressively pursued healthcare reform, as evidenced by the passing of the Affordable Care Act, as amended by the Health Care and Education Reconciliation Act of 2010 (collectively, the “ACA”), and the ongoing efforts to modify or repeal that legislation. The ACA significantly changed the way healthcare is financed by both governmental and private insurers and contains a number of provisions that affect coverage and reimbursement of drug products and/or that could potentially reduce the demand for pharmaceutical products such as increasing drug rebates under state Medicaid programs for brand name prescription drugs and extending those rebates to Medicaid managed care and assessing a fee on manufacturers and importers of brand name prescription drugs reimbursed under certain government programs, including Medicare and Medicaid. Other aspects of healthcare reform, such as expanded government enforcement authority and heightened standards that could increase compliance-related costs, could also affect our business. Modifications have been implemented under the former Trump administration and additional modifications or repeal may occur.

There have also been executive, judicial, and congressional challenges to certain aspects of the ACA. It is unclear how other healthcare reform measures of the Biden administration or other efforts, if any, to challenge, repeal or replace the ACA will impact our business. There is no assurance that federal or state healthcare reform will not adversely affect our future business and financial results, and we cannot predict how future federal or state legislative, judicial or administrative changes relating to healthcare reform will affect our business.

In addition, other legislative changes have been proposed and adopted in the United States since the ACA was enacted. For example, the American Rescue Plan Act of 2021 eliminates the statutory Medicaid drug rebate cap, currently set at 100 percent of a drug’s average manufacturer price, for single source and innovator multiple source drugs, beginning January 1, 2024. The U.S. Budget Control Act of 2011 and subsequent legislation, among other things, included aggregate reductions of Medicare payments to providers of 2% per fiscal year, which remain in effect through 2032. The American Taxpayer Relief Act of 2012 further reduced Medicare payments to several providers, including hospitals, imaging centers and cancer treatment centers, and increased the statute of limitations period for the government to recover overpayments to providers from three to five years.

Additionally, there has been increasing legislative and enforcement interest in the United States with respect to drug pricing practices, which has resulted in several U.S. Congressional inquiries and federal and state legislation designed to, among other things, bring more transparency to drug pricing, reduce the cost of prescription drugs, and review the relationship between pricing and manufacturer patient programs. The Inflation Reduction Act of 2022 (the “IRA”), for example, includes several provisions that may impact our business to varying degrees, including provisions that reduce the out-of-pocket spending cap for Medicare Part D beneficiaries to $2,000 starting in 2025, eliminating the prescription drug coverage gap; impose new manufacturer financial liability on certain drugs under Medicare Part D, allow the U.S. government to negotiate Medicare Part B and Part D price caps for certain high-cost drugs and biologics without generic or biosimilar competition; require companies to pay rebates to Medicare for certain drug prices that increase faster than inflation; and delay until January 1, 2032 the implementation of an HHS rebate rule that would have limited the fees that pharmacy benefit managers can charge. Further, under the IRA, orphan drugs are exempted from the Medicare drug price negotiation program, but only if they have one orphan designation and for which the only approved indication is for that disease or condition. If a product receives multiple orphan designations or has multiple approved indications, it may not qualify for the orphan drug exemption. The effects of the IRA on our business and the healthcare industry in general is not yet known.

In addition, President Biden has issued multiple executive orders that have sought to reduce prescription drug costs. In February 2023, HHS issued a proposal in response to an October 2022 Biden executive order that proposes a Medicare drug pricing model that will test whether targeted Medicare payment adjustments will sufficiently incentivize manufacturers to complete confirmatory trials for drugs approved through the FDA’s accelerated approval pathway. Although a number of these and other proposed measures may require authorization through additional legislation to become effective, and the Biden administration may reverse or otherwise change these measures, both the Biden administration and Congress have indicated that they will continue to seek new legislative measures to control drug costs.

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We cannot predict the initiatives that may be adopted in the future. The continuing efforts of the government, insurance companies, managed care organizations and other payors of healthcare services to contain or reduce costs of healthcare and/or impose price controls may adversely affect:

the demand for our product candidates, if we obtain regulatory approval;
our ability to set a price that we believe is fair for our approved products;
our ability to generate revenue and achieve or maintain profitability;
the level of taxes that we are required to pay; and
the availability of capital.

Any reduction in reimbursement from Medicare or other government programs may result in a similar reduction in payments from private payors, which may adversely affect our future profitability.

Individual states in the United States have also become increasingly active in passing legislation and implementing regulations designed to control pharmaceutical and biological product pricing, including price or patient reimbursement constraints, discounts, restrictions on certain drug access and marketing cost disclosure and transparency measures, and designed to encourage importation from other countries and bulk purchasing. Legally mandated price controls on payment amounts by third-party payors or other restrictions could harm our business, financial condition, results of operations and prospects. In addition, regional healthcare authorities and individual hospitals are increasingly using bidding procedures to determine what pharmaceutical products and which suppliers will be included in their prescription drug and other healthcare programs. This could reduce the ultimate demand for our drugs or put pressure on our drug pricing, which could negatively affect our business, financial condition, results of operations and prospects.

Because we are developing product candidates in the field of genetic medicines in which there is little clinical experience, there is increased risk that the FDA, the EMA or other regulatory authorities may not consider the endpoints of our clinical trials to provide clinically meaningful results and that these results may be difficult to analyze.

In order to proceed into clinical development of any product candidates we identify, we will need to submit INDs or clinical trial applications to regulatory authorities and obtain regulatory clearance to commence clinical development. Because the product candidates we identify are based on novel gene-editing technology, we may be unsuccessful in obtaining clearance from regulatory authorities to proceed into clinical development. In order to commence clinical development, we will need to identify success criteria and endpoints such that the FDA, the EMA or other regulatory authorities will be able to determine the clinical efficacy and safety profile of any product candidates we may develop. As we are initially seeking to identify and develop product candidates to treat diseases in which there is little clinical experience using new technologies, and while we may have opportunities to discuss our clinical development plans with regulatory authorities prior to commencing clinical development, there is heightened risk that the FDA, the EMA or other regulatory authorities may not consider the clinical trial endpoints that we propose to provide clinically meaningful results (reflecting a tangible benefit to patients). In addition, the resulting clinical data and results may be difficult to analyze. Even if the FDA does find our success criteria to be sufficiently validated and clinically meaningful, we may not achieve the pre-specified endpoints to a degree of statistical significance. This may be a particularly significant risk for many of the genetically defined diseases for which we plan to develop product candidates because many of these diseases such as PH1 have small patient populations, and designing and executing a rigorous clinical trial with appropriate statistical power is more difficult than with diseases that have larger patient populations.

Furthermore, even if we do achieve the pre-specified criteria, we may produce results that are unpredictable or inconsistent with the results of the non-primary endpoints or other relevant data. The FDA also weighs the benefits of a product against its risks, and the FDA may view the efficacy results in the context of safety as not being supportive of regulatory approval. Other regulatory authorities in the European Union and other countries may make similar comments with respect to these endpoints and data. Any product candidates we may develop will be based on a novel technology that makes it difficult to predict the time and cost of development and of subsequently obtaining regulatory approval. No genome editing therapeutic product has been approved in the United States or in Europe. Within the broader genome product field, only a limited number of gene therapy products, such as uniQure N.V.’s Glybera and Abecma from Bristol Myers Squibb and bluebird bio, have received marketing authorization or regulatory approval from the European Commission or the FDA. Some of these products have taken years to register and have had to deal with significant issues in their post-marketing experience.

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If preclinical studies or clinical trials of any product candidates we may identify and develop fail to demonstrate safety and efficacy to the satisfaction of regulatory authorities or do not otherwise produce positive results, we may incur additional costs or experience delays in completing, or ultimately be unable to complete, the development and commercialization of such product candidates.

Before obtaining regulatory approval from regulatory authorities for the sale of any product candidates we may identify and develop, we must complete preclinical development and then conduct extensive clinical trials to demonstrate the safety and efficacy in humans. Clinical testing is expensive, difficult to design and implement, can take many years to complete, and is uncertain as to outcome. A failure of one or more clinical trials can occur at any stage of testing. The outcome of preclinical testing and early clinical trials may not be predictive of the success of later clinical trials, and interim results of a clinical trial do not necessarily predict final results.

Moreover, preclinical and clinical data are often susceptible to varying interpretations and analyses. Many companies that have believed their product candidates performed satisfactorily in preclinical studies and clinical trials have nonetheless failed to obtain regulatory approval of their product candidates.

We and our collaborators, if any, may experience numerous unforeseen events during, or as a result of, clinical trials that could delay or prevent our ability to receive regulatory approval or commercialize any product candidates we may identify and develop, including:

delays in reaching a consensus with regulators on trial design;
regulators, IRBs, or independent ethics committees may not authorize us or our investigators to commence a clinical trial or conduct a clinical trial at a prospective trial site;
delays in reaching or failing to reach agreement on acceptable clinical trial contracts or clinical trial protocols with prospective CROs and clinical trial sites;
clinical trials of any product candidates we may develop may produce negative or inconclusive results, and we may decide, or regulators may require us, to conduct additional clinical trials or abandon product development or research programs;
difficulty in designing well-controlled clinical trials due to ethical considerations which may render it inappropriate to conduct a trial with a control arm that can be effectively compared to a treatment arm;
difficulty in designing clinical trials and selecting endpoints for diseases that have not been well-studied and for which the natural history and course of the disease is poorly understood;
the number of patients required for clinical trials of any product candidates we may develop may be larger than we anticipate; enrollment of suitable participants in these clinical trials, which may be particularly challenging for some of the rare genetically defined diseases we are targeting in our most advanced programs, may be delayed or slower than we anticipate; or patients may drop out of these clinical trials at a higher rate than we anticipate;
our third-party contractors may fail to comply with regulatory requirements or meet their contractual obligations to us in a timely manner, or at all;
regulators, IRBs, or independent ethics committees may require that we or our investigators suspend or terminate clinical research or clinical trials of any product candidates we may develop for various reasons, including noncompliance with regulatory requirements, a finding of undesirable side effects or other unexpected characteristics, or that the participants are being exposed to unacceptable health risks or after an inspection of our clinical trial operations or trial sites;
the cost of clinical trials of any product candidates we may develop may be greater than we anticipate;
the supply or quality of any product candidates we may develop or other materials necessary to conduct preclinical studies or clinical trials of any product candidates we may develop may be insufficient or inadequate, including as a result of delays in the testing, validation, manufacturing, and delivery of any product candidates we may develop to the preclinical study sites or clinical sites by us or by third parties with whom we have contracted to perform certain of those functions;
delays in having patients complete participation in a trial or return for post-treatment follow-up;
clinical trial sites dropping out of a trial;
selection of clinical endpoints that require prolonged periods of clinical observation or analysis of the resulting data;
occurrence of serious adverse events associated with any product candidates we may develop that are viewed to outweigh their potential benefits;
occurrence of serious adverse events in trials of the same class of agents conducted by other sponsors; and
changes in regulatory requirements and guidance that require amending or submitting new clinical protocols.

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If we or our collaborators are required to conduct additional clinical trials or other testing of any product candidates we may develop beyond those that we currently contemplate, if we or our collaborators are unable to successfully complete clinical trials or other testing of any product candidates we may develop, or if the results of these trials or tests are not positive or are only modestly positive or if there are safety concerns, we or our collaborators may:

be delayed in obtaining regulatory approval for any such product candidates we may develop or not obtain regulatory approval at all;
obtain approval for indications or patient populations that are not as broad as intended or desired;
obtain approval with labeling that includes significant use or distribution restrictions or safety warnings, including boxed warnings;
be subject to changes in the way the product is administered;
be required to perform additional clinical trials to support approval or be subject to additional post- marketing testing requirements;
have regulatory authorities withdraw, or suspend, their approval of the product or impose restrictions on its distribution in the form of a REMS, or through modification to an existing REMS;
be sued; or
experience damage to our reputation.

Product development costs will also increase if we or our collaborators experience delays in clinical trials or other testing or in obtaining regulatory approvals. We do not know whether any clinical trials will begin as planned, will need to be restructured, or will be completed on schedule, or at all. Significant clinical trial delays also could shorten any periods during which we may have the exclusive right to commercialize any product candidates we may develop, could allow our competitors to bring products to market before we do, and could impair our ability to successfully commercialize any product candidates we may develop, any of which may harm our business, financial condition, results of operations, and prospects.

Failure to access or a significant delay in accessing animal research models may materially adversely affect our ability to advance our preclinical programs and successfully develop any product candidates we may identify, which could result in significant harm to our business.

Consistent with various rules, regulations and current good manufacturing practices (“cGMP”), our ability to advance our preclinical programs and successfully develop any product candidates we may identify requires access to animal research models sufficient to assess safety and in some cases to establish the rationale for therapeutic use. Failure to access or a significant delay in accessing animal research models that meet our needs or that fulfil regulatory requirements may materially adversely affect our ability to advance our preclinical programs and successfully develop any product candidates we may identify and this could result in significant harm to our business. During the COVID-19 pandemic, researchers and CROs (including those engaged by us) experienced significant limitations in their access to animal research models, specifically including a sharp reduction in the availability of non-human primates (“NHPs”) originating from breeding farms in Southeast Asia and limited access to the generation of genetically-modified rodent models used in efficacy evaluations. Prior to the pandemic, China was the leading exporter of NHPs employed in basic and applied research; however, early in 2020, China ceased exportation of cynomolgus monkeys, the species most commonly involved in pharmaceutical product development. This change in the world supply of a critical research model has resulted in increased demand from breeding farms principally located in Cambodia, Vietnam, and Mauritius Island, with a resultant marked increase in unit pricing. Consequently, this has further exacerbated an already constrained NHP supply for research purposes. If we are unable to obtain NHPs in sufficient quantities and in a timely manner to meet the needs of our preclinical research programs, if the price of NHPs that are available increases significantly, or if our suppliers are unable to ship the NHPs in their possession that are reserved for us, our ability to advance our preclinical programs and successfully develop any preclinical candidates we may identify may be materially adversely affected or significantly delayed.

Even if we complete the necessary clinical trials, we cannot predict when, or if, we will obtain regulatory approval to commercialize a product candidate we may develop in the United States or any other jurisdiction, and any such approval may be for a more narrow indication than we seek.

We cannot commercialize a product candidate until the appropriate regulatory authorities have reviewed and approved the product candidate. Even if our product candidates meet their safety and efficacy endpoints in clinical trials, the regulatory authorities may not complete their review processes in a timely manner, or we may not be able to obtain regulatory approval. Additional delays may result if an FDA advisory committee or other regulatory authority recommends non-approval or restrictions on approval. In addition, we

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may experience delays or rejections based upon additional government regulation from future legislation or administrative action, or changes in regulatory authority policy during the period of product development, clinical trials, and the review process.

Regulatory authorities also may approve a product candidate for more limited indications than requested or they may impose significant limitations in the form of narrow indications, warnings or a REMS. These regulatory authorities may require labeling that includes precautions or contraindications with respect to conditions of use, or may grant approval subject to the performance of costly post-marketing clinical trials. In addition, regulatory authorities may not approve the labeling claims that are necessary or desirable for the successful commercialization of our product candidates. Any of the foregoing scenarios could materially harm the commercial prospects for our product candidates and materially adversely affect our business, financial condition, results of operations, and prospects.

Regulatory approval by the FDA in the United States, if obtained, does not ensure approval by regulatory authorities in other countries or jurisdictions. In addition, clinical trials conducted in one country may not be accepted by regulatory authorities in other countries, and regulatory approval in one country does not guarantee regulatory approval in any other country. Approval processes vary among countries and can involve additional product candidate testing and validation and additional administrative review periods. Seeking regulatory approval outside the United States could result in difficulties and costs for us and require additional preclinical studies or clinical trials which could be costly and time-consuming. Regulatory requirements can vary widely from country to country and could delay or prevent the introduction of our product candidates in those countries. The foreign regulatory approval process involves all of the risks associated with FDA approval. We do not have any product candidates approved for sale in any jurisdiction, including international markets, and we do not have experience in obtaining regulatory approval in international markets. If we fail to comply with regulatory requirements in international markets or to obtain and maintain required approvals, or if regulatory approvals in international markets are delayed, our target market will be reduced and our ability to realize the full market potential of our product candidates will be unrealized.

Even if we, or any of our collaborators or strategic partners, obtain regulatory approvals for any product candidates we may develop, the terms of approvals and ongoing regulation of such product candidates could require the substantial expenditure of resources and may limit how we, or they, manufacture and market such product candidates, which could materially impair our ability to generate revenue.

Any product candidate for which we obtain regulatory approval, along with the manufacturing processes, post- approval clinical data, labeling, advertising and promotional activities for such product, will be subject to continual requirements of and review by the FDA, the EMA and other regulatory authorities. These requirements include submissions of safety and other post-marketing information and reports, facility registration and drug listing requirements, cGMP relating to quality control, quality assurance and corresponding maintenance of records and documents, applicable product tracking and tracing requirements and requirements regarding the distribution of samples to physicians and recordkeeping. In addition, our manufacturing and testing facilities will be required to undergo pre-license inspections and pre-approval inspections. Even if regulatory approval of a product candidate is granted, the approval may be subject to limitations on the indicated uses for which the products may be marketed or to the conditions of approval, or contain requirements for costly post-marketing testing and surveillance to monitor the safety or efficacy of the products.