Slide 1: Exploring new oligonucleotide backbone chemistries and their deployment to improve stereopure oligonucleotides
Exploring new oligonucleotide backbone chemistries and their deployment to improve the properties of stereopure oligonucleotides Chandra Vargeese, PhD Chief Technology Officer Sept 22, 2021 TIDES USA
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: PRISM platform enables rational drug design
PRISM platform enables rational drug design Sequence Chemistry O 5’ B B B: bases R: 2’ modifications O A, T, C, mC, G, U, OMe, MOE, F, - 3’ 2’ other modified bases R other modifications XX O Sequence Chemistry P O O X: backbone chemistry 5’ B Stereochemistry Phosphodiester (PO), O Stereochemistry ph
Slide 4: Expanding repertoire of backbone modifications with novel PN backbone chemistry
Expanding repertoire of backbone modifications with novel PN backbone chemistry Backbone linkages PO PS PN Backbone Phosphodiester Phosphorothioate Phosphoramidate diester modification (X) Stereorandom PN backbone Stereorandom Stereochemistry Not chiral Chiral PS backbone Rp Chiral PN backbone Rp PS
Slide 5: New building blocks and chemistry support synthesis of chimeric stereopure backbones
New building blocks and chemistry support synthesis of chimeric stereopure backbones Chiral backbone chemistries Versatility in chemistry Growing library of amidites DMTrO B DMTrO B O O O R O R P P O N O N O O S S O O (L)-PSM Amidite (D)-PSM Amidite (L)-PSM (D)-PSM B Nucleotide base Introducing ster
Slide 6: Chemical and structural validation of dimer stereochemistry
Chemical and structural validation of dimer stereochemistry NH2 NH2 N N HO N O HO N O O NH2 O NH2 N N N N O F N O O F N P P O O N O N O N O O N O OH F OH F PN-Rp-fCfC dimer PN-Sp-fCfC dimer For all dimers evaluated, diastereoselectivity >99% RP-UPLC, reversed-phase ultra performance liquid chromatog
Slide 7: We are exploring multiple types of PN chemistry
We are exploring multiple types of PN chemistry PN-1 PN-5 (1,3-dimethylimidazolidin- (di)pyrrolidine-1-yl)methylene) 2-ylidene)phosphoramidate phosphoramidate PN-2 PN-4 ((4-acetamidophenyl)sulfonyl) (1,3-dimethyl-1,3-diazepan- phosphoramidate 2-ylidene)phosphoramidate PN-3 (1,3-dimethyltetrahydropyr
Slide 8: PN chemistry increases potency in vitro 10-fold
PN chemistry increases potency in vitro 10-fold Silencing RNase H Potency S- Relative percentage expression Relative percentage expression PS 150 150 (MALAT1/SRSF9) (MALAT1/SRSF9) 100 100 10-fold 50 50 0 0 10 -6 10 -4 10 -2 10 0 10 2 10 -6 10 -4 10 -2 10 0 10 2 Log concentration Log concentration EC
Slide 9: Across many modalities, PN-1 chemistry enhances potency, exposure, and durability
Across many modalities, PN-1 chemistry enhances potency, exposure, and durability Modality Pharmacology • Efficient engagement • Target knockdown, Silencing Potency of RNase H or AGO2 splicing or editing • In the right tissues, • Efficient uptake in cells and cellular Splicing Exposure the cell nucl
Slide 10: PN-1 chemistry increases potency in silencing, splicing, and editing preclinical studies
PN-1 chemistry increases potency in silencing, splicing, and editing preclinical studies Potency Silencing Splicing Editing Target knockdown (% remaining) % Skipping % Editing 100 Improved knockdown 80 Improved skipping Improved editing % Editing PS / PO / PN 60 40 PS / PO (Stereopure 20 PS / PO (St
Slide 11: PN-1 chemistry increases potency and durability in mouse CNS for multiple targets
PN-1 chemistry increases potency and durability in mouse CNS for multiple targets Silencing RNase H Low dose leads to Malat1 and C9orf72 knockdown in CNS PBS PS/PO Durability Hippocampus Cerebellum PS/PN 150 150 * Spinal cord Cortex 100 ** 100 Relative percentage expression C9orf72 MALAT1 50 50 (Mal
Slide 12: RNAi-dependent silencing modality
RNAi-dependent silencing modality ESC Advanced ESC G G P P • 2’-OMe & 2’-F ribose • Decreased 2’-F ribose in both strands • Terminal PS modifications • Terminal PS modifications • GalNAc-modified passenger strand • GalNAc-modified passenger strand Application of PRISM to RNAi Control backbone ster
Slide 13: Application of PN-1 chemistry to siRNA: Improving another class of silencers
Application of PN-1 chemistry to siRNA: Improving another class of silencers Silencing RNAi PN-1 chemistry improves potency and durability of ESC format Potency Durability Ago2-loading Ttr mRNA (liver) Ttr protein (serum) Guide strand-Ago2 IP (liver) PBS ESC Ref PO/PS/PN ~30-fold Dose 125 PBS ESC Re
Slide 14: Application of PN-1 chemistry to siRNA: Improving on the state-of-the-art
Application of PN-1 chemistry to siRNA: Improving on the state-of-the-art Silencing RNAi PN-1 chemistry extends duration of Advanced ESC format Durability Durability Dose Ttr protein (serum) • PN-1 increases potency and durability (1 mpk) PBS Adv ESC Ref PO/PS/PN • PN-1 extends 50% knockdown period
Slide 15: PN chemistry improves exon-skipping potency and increases cellular uptake in myoblasts
PN chemistry improves exon-skipping potency and increases cellular uptake in myoblasts Splicing Potency Exposure PS/PO backbone PS/PO/PN backbone DMD mRNA skipping Cellular uptake (Exon 23, H2K mouse myoblasts) (H2K mouse myoblasts) Cytoplasmic Nuclear Concentration (ng/ml) Concentration (ng/ml) 100
Slide 16: PN-based exon skipping molecule led to overall survival benefit in Utr/Dmd mouse model
PN-based exon skipping molecule led to overall survival benefit in Utr/Dmd mouse model Splicing Durability PN-containing molecules led to 100% dKO survival at time of study termination 100 75 Survival probability (%) PS/PO/PN, 150 mg/kg weekly PS/PO/PN, 75 mg/kg bi-weekly 50 PS/PO, 150 mg/kg weekly
Slide 17: PRISM enables practical approach to RNA editing without need for viruses or exogenous protein
PRISM enables practical approach to RNA editing without need for viruses or exogenous protein Wave ADAR-editing oligonucleotides Extracellular space GalNAc Intracellular Endogenous ADAR Unedited Edited RNA RNA 17 No delivery vehicle required No exogenous proteins necessary Potential for reduced o
Slide 18: GalNAc-conjugated oligonucleotides support efficient and durable ADAR editing in NHPs
GalNAc-conjugated oligonucleotides support ADAR efficient and durable ADAR editing in NHPs Systemic Liver In vitro dose-response Substantial and durable in NHP hepatocytes editing in vivo in NHP liver (multiple 5 mpk, SC doses) RNA editing out to at least day 50, or 45 days post-last dose TAG TGG Ba
Slide 19: Leading RNA editing program provides optimal approach for treatment of AATD
Leading RNA editing program provides optimal approach for treatment of AATD Editing 1) Restore circulating, 2) Reduce Z-AAT protein 3) Retain M-AAT physiological functional wild-type M-AAT aggregation in liver regulation Risk of disease Null Highest (no AAT) risk (lung) Z-AAT High (lung + PI*ZZ live
Slide 20: Focused on restoring wild-type M-AAT in vivo
Focused on restoring wild-type M-AAT in vivo Editing In vitro proof of concept In vivo proof of concept SERPINA1 Z allele mRNA editing 100 % Z allele mRNA editing % S e r p in A 1 - P IZ m R N A E d i t in g 80 60 40 20 0 NT SA1-1 SA1-2 -1 -2 T N 1 1 A A AAT protein concentration in media S S 6000 *
Slide 21: Achieving 40% editing of Z allele mRNA at initial timepoint
Achieving 40% editing of Z allele mRNA at Editing initial timepoint SERPINA1 Z allele mRNA editing levels nearing correction to heterozygote (MZ) In vivo Z allele mRNA editing • GalNAc-conjugated compounds 50 • Up to 40% editing of Z allele mRNA in **** liver of transgenic human ADAR mice at 40 ****
Slide 22: Achieving biologically meaningful increases in circulating human AAT protein
Achieving biologically meaningful increases Editing in circulating human AAT protein 3-fold increase in circulating human AAT as compared to PBS at initial timepoint Human AAT concentration in serum 800 AAT serum levels by genotype 3-fold increase at day 7 Serum AAT (µg/ml) 600 **** PI*MZ ~3 to 5-fo
Slide 23: ADAR editing restores circulating, functional M-AAT
ADAR editing restores circulating, functional M-AAT Editing Wild-type M-AAT detected with ADAR editing Significant increase in neutrophil elastase inhibition with ADAR editing % Relative Elastase Inhibition 600 3-fold increase in total AAT 100 ~2.5-fold increase Pre-dose Serum AAT protein µg/mL) 80
Slide 24: RNA editing throughout CNS of huADAR mouse
RNA editing throughout CNS of huADAR mouse Editing PN-containing molecules direct editing of UGP2 in vivo In vitro dose-response curves In vivo CNS editing in proprietary hADAR transgenic mouse (1 week) ACTB UGP2 100 100 80 AIMer (single, 100 µg ICV dose) 80 80 60 % ACTB editing PBS % UGP2 editing %
Slide 25: Summary
Summary Sustained exploration has yielded a novel modality and pharmacologic advances in preclinical studies • Backbone chemistry and stereochemistry profoundly impact pharmacology – Introducing PN backbone chemistry with stereopure synthesis • Improves potency and durability across modalities • Inc