Stereochemical Control of Antisense Oligonucleotides Enhances Target Efficacy Chandra Vargeese, PhD SVP, Drug Discovery Wave Life Sciences October 3, 2018 Exhibit 99.1
Acknowledgements & Disclosures All Wave Life Sciences employees Prof. Gregory Verdine, co-founder & Director Wave Life Sciences Prof. Takeshi Wada, co-founder Wave Life Sciences Prof. Matthew Wood, Department of Physiology, Anatomy and Genetics, University of Oxford Chandra Vargeese is an employee of Wave Life Sciences
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Architects of transformation Wave Life Sciences is a clinical-stage, genetic medicines company unlocking the potential of a proprietary chemistry platform that enables the precise design, optimization and production of stereopure nucleic acid therapies. Wave has reinvented the design, synthesis and manufacture of nucleic acid therapies to potentially optimize potency, durability and safety PRECISION Ability to design nucleic acid compounds that have one defined and consistent profile SCALE Platform potential across multiple modalities and tissues Internal expertise and capacity for large-scale GMP manufacturing Wave’s chemistry platform is built on a foundation of two core capabilities
History of oligonucleotide therapeutics Backbone modifications • Introduce chiral centers • Generate mixtures Sugar modifications Drug approvals (FDA) Chiral Phosphorothioate Chiral Phosphorodiamidite Morpholino (PMO) Mixtures of 2n molecules (n=No. of chiral centers) SEQUENCE STEREOCHEMISTRY CHEMISTRY Fomiversen Pegaptanib Mipomerse Eteplirsen Nusinersen Patisiran Stereopure ASOs enter clinic Wave Stereopure ASOs Oka N, Wada T, Saigo K. JACS. 2002 Stec WJ, et al. J Am Chem Soc. 1989 ~500,000 different molecules per dose
Advances in stereopure oligonucleotide synthesis and manufacturing Versatility in Chemistry Versatility in Scale High Crude Purity • Improved synthetic capabilities • Custom building blocks – Tunable ‘R’ groups – Various 2’-modifications Candidate Optimization and Selection GMP Quality High-throughput scale Manufacturing scale
Wave’s chemistry platform INDICATION, TARGET TRANSCRIPT, PRODUCT PROFILE SPLICING RNAi ANTISENSE DEFINE MODALITY DESIGN & OPTIMIZE VALIDATE SEQUENCE STEREOCHEMISTRY CHEMISTRY Free uptake in cellular models Animal models POTENCY STABILITY SPECIFICITY IMMUNE POTENCY DURABILITY TOXICOLOGY Candidates
INDICATION, TARGET TRANSCRIPT, PRODUCT PROFILE SPLICING RNAi ANTISENSE DEFINE MODALITY DESIGN & OPTIMIZE VALIDATE SEQUENCE STEREOCHEMISTRY Free uptake in cellular models Animal models POTENCY STABILITY SPECIFICITY IMMUNE POTENCY DURABILITY TOXICOLOGY Candidates CHEMISTRY Wave’s chemistry platform: Antisense
Precision RNase H-mediated RNA degradation Antisense In RNase H1 assay, ASOs were pre-annealed to surrogate MALAT1 mRNA (1:1, Cf=5 mM). RNase H (250:1, E:S) was added and quenched with EDTA at the indicated times. Products were quantified, and V0 was calculated from the best-fit line (n=3 per time point). RNA-Stereorandom ASO RNA-Stereopure ASO Rp Sp Rp or Sp Linker Nucleotide Initial Velocity (V0) Cleavage Activity
Potency of stereopure oligonucleotides under in vitro free-uptake conditions translates in vivo In vitro: In iCell neurons, 10, 30, 100, 300, 1,000 or 3,000 nM ASO was added to iCell neurons under free-uptake conditions. 4-days post-treatment, RNA was harvested and processed. MALAT1 mRNA expression was determined by qPCR (n=2 per concentration). In vivo: Mice received a single IVT injection. 1 week post-injection, tissues were frozen and processed for RNA. MALAT1 mRNA expression was determined by qPCR (n=7). Antisense MALAT1 Knockdown in Posterior Mouse Eye at 1 Week MALAT1 Knockown in iCell Neurons In Vitro In Vivo
Stereopure oligonucleotides enhance potency across tissues in vivo Muscle Single SC injection in Mice (25 mg/kg) Eye Single IVT injection in Mice (50 µg) CNS Single ICV injection in Mice (50 µg) Antisense Tissues were harvested 1 week post-dose and processed for RNA. mRNAs were quantified by qPCR. Plots show relative fold change or percentage mRNA remaining with respect to control mRNA. Each symbol represents one animal. SR = Stereorandom
Stereopure oligonucleotides induce potent and durable activity in the eye Tissues were harvested at the indicated time points, post-dose and processed for RNA. mRNAs were quantified by qPCR. Plots show percentage mRNA remaining with respect to control mRNA. Each symbol represents one animal. 9-fold greater eye volume Antisense (OTS poster #030)
Stereopurity improves potency and durability of GalNAc-conjugated oligonucleotides Antisense Stereopure ASO yields potency comparable to state-of-the-art GalNAc-dsRNAi Stereopure ASO yields durable effect in transgenic mice
INDICATION, TARGET TRANSCRIPT, PRODUCT PROFILE SPLICING RNAi ANTISENSE DEFINE MODALITY DESIGN & OPTIMIZE VALIDATE Free uptake in cellular models Animal models POTENCY STABILITY SPECIFICITY IMMUNE POTENCY DURABILITY TOXICOLOGY SEQUENCE STEREOCHEMISTRY CHEMISTRY Candidates Wave’s chemistry platform: Splicing
Stereopure oligonucleotides induce exon 23 skipped transcript (OTS poster #119) Splicing Stereorandom (SR) Stereopure
Stereopure oligonucleotide induces dystrophin protein restoration and reduces elevated serum enzymes Splicing Multiple Doses (in vivo mdx23 mice) 70-90% of natural dystrophin restoration 87% reduction in creatine kinase levels ALT=alanine aminotransferase; AST=aspartate aminotransferase; CK=creatine kinase; GLDH=glutamate dehydrogenase. Serum and plasma clinical chemistry were measured with an Olympus AU640 (Olympus America) and the manufacturer’s reagents and procedures. Dystrophin Protein Restoration Serum Enzyme Levels
Stereopure surrogate restores dystrophin in muscle fibers after single dose Neuro DMD PBS DMD-1742 Immunohistochemistry of dystrophin in gastrocnemius in mdx23 mice at 4 weeks 10X Experimental conditions: mdx23 mice received a single IV injection of PBS or DMD-1742 (150 mg/kg). Immunohistochemistry: Blue: Nuclei, Hoechest; Yellow: Rabbit anti-Dystrophin(#ab15277) 1:400 diluent, 555/Cy3, Yellow is a fake color for Cy3. 10X magnification.
Stereopure surrogate restores dystrophin in muscle fibers after multiple doses Experimental conditions: mdx23 mice received 4 weekly IV injections of PBS or DMD-1742 (150 mg/kg). Immunohistochemistry: Blue: Nuclei, Hoechest; Yellow: Rabbit anti-Dystrophin(#ab15277) 1:400 diluent, 555/Cy3, Yellow is a fake color for Cy3. 10X magnification. Neuro DMD PBS DMD-1742 Immunohistochemistry of dystrophin in gastrocnemius in mdx23 mice at 4 weeks 10X 0X
Stereopure oligonucleotide traffics to nuclei in myoblasts Cultured myoblasts were treated with 10 mM of the indicated ASO under free-uptake conditions. ASO was detected with ViewRNA; nuclei are stained with DAPI. Exon skipping efficiency was quantified by Taqman assay. Nuclear ASO was quantified with ImageJ software (https://imagej.nih.gov/ij/). Stereorandom (SR) Stereopure Stereopure (opposite) Splicing Stereopure ASO enters the nuclei of cultured myoblasts and promotes efficient exon 51 skipping WV-3 SR WV-5
Mdx23 mice were treated with a single 30 mg/kg dose of optimized, stereopure ASO (IV). Tissues were collected 24-hours post-dose. ASO was detected using ViewRNA, and nuclei were stained with Hoechst33342 or hematoxylin. Nucleus: Hematoxylin (blue) ASO: ViewRNA (red) Nucleus: Hoechst33342 (blue) ASO: Fast Red (pink) Stereopure oligonucleotides access myofiber nuclei in mice Splicing Stereopure ASO targeting exon 53 rapidly enters myofibers in mdx23 mice 30 mg/kg, 24 hours Bright-field view Fluorescence-field view (z stack)
We have developed a scalable process for generating stereopure ASOs Compared with stereorandom, stereopure ASOs are: Taken up more readily by cells under gymnotic conditions in multiple cell lines More potent in multiple tissues More durable in vivo Optimized, stereopure ASOs exhibit improvements in multiple properties: Precision and activity of RNase H Potency correlation between in vitro and in vivo Exon skipping efficiency Rapid and broad tissue distribution Nuclear uptake Summary
Future: Improving nucleic acid therapeutics through greater understanding of protein-nucleic acid interactions Understanding innate immune receptor and broader DNA/RNA-protein interactions TLR9 bound to stereopure, CpG-containing oligonucleotide Stereochemistry of CpG-containing oligonucleotides impacts TLR9 activity Fold-increase TLR9 signaling Concentration ASO (mM)
Future: More potent and durable CNS targeting with new chemistries PBS = phosphate buffered saline; Ctx = cortex; Str = striatum; Cb = cerebellum; Hp = hippocampus; SC = spinal cord. PBS Stereopure Cortex Spinal cord 15.9 nM 150 nM 2,900 nM Stereopure Stereopure Stereorandom IC50 MALAT1 Knockdown in Mice 10 weeks after single 100 µg injection MALAT1 Knockdown in Mice 1 week after single ICV injection MALAT1 Knockdown in Human iCell Neurons Under Free-Uptake Conditions Antisense In vitro potency In vivo potency In vivo durability