Slide 1: Building a Therapeutic A-to-I RNA Editing Platform Through Oligonucleotide Chemistry Optimization
Building a Therapeutic A-to-I RNA Editing Platform Through Oligonucleotide Chemistry Optimization Ian Harding, Senior Scientist January 19, 2024 Deaminet 2024, San Diego, CA
Slide 2: Forward-looking statements
Forward-looking statements This document contains forward-looking statements. All statements other than statements of historical facts contained in this document, including statements regarding possible or assumed future results of operations, preclinical and clinical studies, business strategies, r
Slide 3: Disclosures
Disclosures • Ian Harding is an employee of Wave Life Sciences 3
Slide 4: Wave's ability to rationally design oligonucleotides enables access to unique disease targets
Wave’s ability to rationally design oligonucleotides enables access to unique disease targets O 5’ B B B Base O - 3’ 2’ XX O R R R 2’-Ribose P O O 5’ B B X Stereochemistry and O backbone modification 3’ R 2’ O R Phosphoryl guanidine O: Phosphodiester S: Phosphorothioate N: Phosphoryl guanidine 4
Slide 5: Unlocking RNA editing with PRISM to develop AIMers: A-to-I editing oligonucleotides
Unlocking RNA editing with PRISMTM to develop AIMers: A-to-I editing oligonucleotides Substantial Substantial and and durable editing in durable editing GalNAc-mediated uptake PRISMTM-driven gains inNHP NHP liver in vivo liver in vivo Primary human GalNAc AIMers hepatocytes 100 ACTB-1 80 ACTB-2 ASGR
Slide 6: Overview
Overview 1 Optimizing editing activity with PRISMTM 2 Achieving RNA editing in multiple tissues 3 Applications 6
Slide 7: Overview: Optimizing editing activity with PRISM
Overview 1 Optimizing editing activity with PRISMTM 2 3 7
Slide 8: Enhancing editing efficiency across nearest neighbors
PRISMTM Enhancing editing efficiency across nearest neighbors 3’ AIMer NCN 5’ 5’ Target RNA XAX 3’ Approach: Structure-activity relationship analysis of AIMer backbone, sugar and orphan base 1 Optimizing orphan site base 5’ 3’ N-3-Uracil Cytosine N3U Optimizing sugar and backbone 2 modification patt
Slide 9: N3U and AIMer-D chemistry mask increase editing across nearest neighbor sequences
PRISMTM N3U and AIMer-D chemistry mask increase editing across nearest neighbor sequences AIMer-S AIMer-D Orphan base: C N3U C N3U Orphan site base Sugar and backbone 5’ AIMer 3’ 5’ 3’ C N3U AIMer-S AIMer-D 5’ G, 3’A %Ugp2 mRNA editing 5’ C, 3’C %Ugp2 mRNA editing Nearest Neighbors %Ugp2 mRNA editin
Slide 10: N3U supports enhanced editing efficiency in vivo
PRISMTM N3U supports enhanced editing efficiency in vivo Orphan site base, sugar Liver hADAR-p110 mice 5’ 3’ 100 Primary mouse hepatocytes ✱✱✱✱ %Ugp2 mRNA editing (GalNAc-mediated delivery) 80 ✱✱✱✱ D0 D2 D4 D7 100 N3U, Sugar Y 60 %Ugp2 mRNA editing Dose 10 Necropsy 80 N3U, Sugar X mg/kg 60 40 40 C,
Slide 11: Overview: Achieving RNA editing in multiple tissues
Overview 1 2 Achieving RNA editing in multiple tissues 3 11
Slide 12: Systemic in vivo editing without delivery vehicles
Extrahepatic Systemic in vivo editing without delivery vehicles Substantial RNA editing across multiple mouse tissues following single subcutaneous dose of Ugp2 AIMer Control Specific liver associated cells Ugp2 AIMer (unconjugated) T cell Cholan- Macro- NK cell LSEC giocyte phage subset %Ugp2 mRNA
Slide 13: Durable editing observed out to 4 months post-single dose
Extrahepatic Durable editing observed out to 4 months post-single dose Peak editing observed 4-weeks post-single ICV dose across tissues hADAR mice Cortex Hippocampus Striatum Brain stem Cerebellum Spinal cord Peak editing 30% >40% 25% >40% 50% >65% 13 Transgenic human ADAR mice were administered 10
Slide 14: AIMer directs widespread RNA editing in CNS of NHP
Extrahepatic AIMer directs widespread RNA editing in CNS of NHP Distribution to Frontal Cortex In vivo CNS editing in NHP (ACTB, 1 week) ACTB AIMer ACTB AIMer 80 AIMer (single, 10 mg IT dose) % ACTB Editing PBS 60 10X 40X 40 ACTB AIMer aCSF 20 0 40X 10X 14 Cynomolgus monkeys (NHPs) were administered
Slide 15: Overview: Applications
Overview 1 2 3 Applications 15
Slide 16: ADAR editing for therapeutic applications
ADAR editing for therapeutic applications Applications ADAR editing of mRNA Downstream Applications Post- Mutation translational Correction1 modification I(G) Protein Upregulation activity (e.g. kinase activity) Protein- Processing protein interaction 16 1Monian et al., 2022. Nat Biotechnol DOI: 10.
Slide 17: AIMers can restore protein expression
AIMers can restore protein expression Applications MECP2 protein expression Mecp2R168X WT MECP2 AIMer Mecp2 RNA editing Primary cortical Ladder (Primary cortical neurons, PBS PBS Mecp2R168X KI mouse) neurons (mouse) 90- MECP2 100 70- HDAC2 % Mecp2 RNA Editing 75 NTC AIMer 50- * 50 MECP2 AIMer 25 DAP
Slide 18: AIMers disrupt protein-protein interaction in vivo
AIMers disrupt protein-protein interaction in vivo Applications NRF2 downstream gene upregulation following GalNAc AIMer mRNA KEAP1 editing in vivo in liver of mice Nrf2 liver editing in vivo Nqo1 Gstm1 NRF2 8 *** **** (Normalized to HPRT) 80 Relative Expression NRF2 is **** RNA editing efficiency *
Slide 19: Summary
Summary • Optimized AIMer design enhances editing in vitro and in vivo • Design improvements include N3U orphan base modification and optimization of sugar modification and backbone modification patterns • Editing efficiency improved across nearest neighbor combinations • AIMers support RNA editing
Slide 20: Acknowledgements
Acknowledgements 2022-2023 Publications • Thanks to all colleagues and contributors from Wave Life Sciences and our collaborators. Silencing Splicing RNA Editing • Nicole Neuman, Wave Life Sciences, assisted in preparing this presentation. 20