Selection of Well-Tolerated GalNAc-siRNAs by Screening for RNAi-Mediated Off-Target Effects in Rodent Toxicity Studies Maja Janas, PhD, DABT

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1 Selection of Well-Tolerated GalNAc-siRNAs by Screening for RNAi-Mediated Off-Target Effects in Rodent Toxicity Studies Maja Janas, PhD, DABT Senior Scientist, Investigative Toxicology TIDES, May 2 nd 2017

2 Agenda Nonclinical safety profile of GalNAc-siRNAs Mechanisms of GalNAc-siRNA hepatotoxicity in rodents 2

3 GalNAc-siRNA Conjugates SC-Administered Platform for Targeted Delivery to Hepatocytes sirna Metabolic stability Duration of effect Intrinsic potency Safety CMC Ligand Receptor affinity & specificity Metabolic stability Safety CMC Asialoglycoprotein Receptor (ASGPR) Highly expressed in hepatocytes High turnover (recycling time ~15 min) Conserved across species 3

4 GalNAc-siRNA Conjugates Nonclinical Considerations Highly specific distribution to liver via ASGPR Limited uptake/distribution in other organs Liver to kidney ratio >30 at PD doses Short plasma half-life; long tissue half-life; long PD effects Metabolized largely by intracellular exoand endo-nucleases Maximum liver drug concentration limits relevant toxicological doses Loss of dose-proportionality, approaching saturation of liver uptake via ASGPR Cannot achieve MTDs Excess circulating sirna levels not taken up by liver; excreted through kidneys 4

5 GalNAc-siRNA Conjugates Platform-Wide Responses at Toxicological Doses Rat Liver Hepatocellular vacuolation: increased number and size of normal rat hepatocellular vacuoles, most contain lipid Increased single cell necrosis Increased mitosis and regeneration Kidney Basophilic granules in proximal tubular epithelium; represents drug accumulation NHP Liver Basophilic granules in Kupffer cells and hepatocytes; represents drug accumulation Lymph nodes Vacuolated macrophages (with basophilic stippling); represents phagocytosis of drug Primarily dose-dependent and the result of drug accumulation in tissue 5

6 Path Towards a Development Candidate (DC) In silico prediction & In vitro efficacy In vitro screen for predicted off-targets Rodent Knockdown Rat >100x PD dose NHP Knockdown DC Good Actors (60%): No hepatotoxicity Bad Actors (40%): Show hepatotoxicity Single cell necrosis and/or hepatocellular degeneration with LFT 2x upper limit of normal 6

7 Potential Causes of Hepatotoxicity in Rodent Toxicity Studies RISC loading Illustrative design 1. Non-RNAi drug effects e.g. protein binding On-target binding Full sequence match mrna cleavage RISC mrna 3 -UTR 2. Competition for Ago binding with mirnas Off-target binding Partial sequence match 3. RNAi-mediated off-target activity 7 Desired on-target activity

8 n g A S / g liv e r u g A S / g liv e r 5 Modifications on the Antisense Strand Can Block RISC Loading With No Impact on Liver Exposure Dose: 30 mg/kg Regimen: q2d x 6 Necropsy: Day 15 RISC loading Sense strand removal Target RNA cleavage mrna R a t R IS C L o a d in g R a t L iv e r E x p o s u r e R IS C lo a d in g b lo c k R IS C lo a d in g b lo c k s ir N A -1 s ir N A -2 s ir N A -1 s ir N A -2 8

9 U /L Blocking Antisense RISC Loading Without Altering the 2 F/2 OMe/PS Content Mitigates Hepatotoxicity R a t A L T Dose: 30 mg/kg Regimen: q2d x 6 Necropsy: Day R e fe re n c e ra n g e 0 R IS C lo a d in g b lo c k S a lin e s ir N A -1 s ir N A -2 RISC loading block - + * ^ * # 9

10 u g A S / g liv e r n g A S / g liv e r Changing sirna Chemical Modification Pattern Does Not Reduce Liver Exposure or RISC Loading Dose: 100 mg/kg Regimen: qw x 9 Necropsy: Day 58 ESC Advanced ESC Illustrative design Altered modification pattern for improved metabolic stability R a t L iv e r E x p o s u r e R a t R IS C L o a d in g E S C Ad v a n c e d E S C 0 E S C Ad v a n c e d E S C 10

11 U /L Changing sirna Chemical Modification Pattern Does Not Mitigate Hepatotoxicity R a t A L T Dose: 100 mg/kg Regimen: qw x 9 Necropsy: Day R e fe re n c e ra n g e 0 ESC S a lin e E S C Ad v a n c e d E S C Advanced ESC # * # * 11

12 Potential Causes of Hepatotoxicity in Rodent Toxicity Studies RISC loading Illustrative design 1. Non-RNAi drug effects e.g. protein binding On-target binding Full sequence match mrna cleavage RISC mrna 3 -UTR 2. Competition for Ago binding with mirnas Off-target binding Partial sequence match 3. RNAi-mediated off-target activity 12 Desired on-target activity

13 Relative Target Protein Level Reversir Platform Achieves Tailored Control of RNAi Pharmacology ASGPR 120% 100% Reversir Functional RISC 80% mrna target 60% 40% 20% No Reversir mrna cleavage Inactive RISC 0% mg/kg GalNAc-siRNA 0.1 mg/kg GalNAc-Reversir Time (Days) 13

14 u g A S / g liv e r n g A S / g liv e r Blocking RISC-Loaded Antisense Strand with Reversir Does Not Reduce Liver Exposure or RISC Loading Prevention Reversir sirna ( ) 3-10 mg/kg 30 mg/kg Treatment Reversir sirna 3-10 mg/kg 30 mg/kg R a t L iv e r E x p o s u r e R a t R IS C L o a d in g Targeting Ctr - Targeting Ctr Targeting Ctr RVR sirna 1 - Targeting Ctr - Targeting Ctr Targeting Ctr RVR sirna 14

15 U /L Treatment U /L Prevention U /L Reversing Activity of the RISC-Loaded Antisense Strand Mitigates Hepatotoxicity R a t G L D H + Ctr RVR + Targeting RVR 5 0 R eference range Targeting - Targeting Ctr RVR - 1 sirna R a t G L D H * * * ^ * # * * # R eference range 0 - Targeting - Targeting Ctr RVR - 2 sirna # R a t G L D H R eference range Targeting - Targeting Ctr RVR - 3 sirna

16 u g A S / g liv e r u g A S / g liv e r Swapping Seed Regions Does Not Reduce Liver Exposure or RISC Loading Dose: 30 mg/kg Regimen: q2d x 6 Necropsy: Day 15 Illustrative design R a t L iv e r E x p o s u r e R a t R IS C L o a d in g Toxic Non-toxic Non-toxic Toxic Seed Toxic Non-toxic Toxic Non-toxic Non-seed Parent Seed swap 0 Toxic Non-toxic Non-toxic Toxic Seed Toxic Non-toxic Toxic Non-toxic Non-seed Parent Seed swap 16

17 Non-toxic U /L NON-SEED Toxic Swapping Seed Regions Mitigates Hepatotoxicity Dose: 30 mg/kg Regimen: q2d x 6 Necropsy: Day 15 Toxic SEED Non-toxic R a t A L T # * * R e fe re n c e ra n g e 0 S a lin e btoxic a d C 5Non-toxic g o o d Cg 5oNon-toxic o d s e eb da d stoxic e e d Seed Toxic Non-toxic Toxic Non-toxic Non-seed Parent Seed swap * * # 17

18 GalNAc-siRNA Conjugates Can Cause Seed-Mediated Off-Targets at High Doses RNAseq in Rat Hepatocytes (24 hrs, 10 nm) sirna-1 65 sirna Downregulated genes Antisense seed enrichment Sense seed enrichment Upregulated genes Antisense seed enrichment Sense seed enrichment sirna-1 p < 2.2e-16 p = p = sirna-2 p < 2.2e-16 p = p = p =

19 sirna-2 sirna-1 RNAi Activity is Driving Gene Dysregulation In Vivo at High Doses RNAseq in Rat Liver (24 hrs, 50 mg/kg) - RISC loading block % % % % 19 sirna-1 sirna-2 RISC Loading Block Downregulated genes Antisense seed enrichment Sense seed enrichment Upregulated genes Antisense seed enrichment Sense seed enrichment - p = 1.25e-03 p = p = 1 p = p = 1 (NA) p = 1 (NA) p = 1 p = p = 2.06e-10 p = p = 1 p = p = 1 p = 1 p = 1 p = 1

20 Conclusions 3 -UTR Off-target binding Partial sequence match RNAi-mediated offtarget activity Antisense strand-driven RNAi-mediated hybridization-based off-target effects, not chemical modifications, are a major driver of hepatotoxicity of a subset of GalNAc-siRNA conjugates that fail in rodent toxicity studies at >100x PD dose Careful selection of "good actor" sirnas is an important part of identifying optimal sirna molecules for clinical development 20

21 Thank You! 21 Nonclinical Safety and Bioanalysis Carole Harbison Yongfeng Jiang Onur Yilmaz Michael Placke Natalie Keirstead Kirk Brown Brenda Carito Samantha Chigas Sean Dennin Kristin Fong Paul Gedman Toni Hayes Emma Henchy Lauren Moran Elena Ooms Rachel Peters Kristina Perry Kellie Sawyer Catrina Wong Research Adam Castoreno Rubina Parmar Mark Schlegel Svetlana Shulga-Morskaya Huilei Xu Ivan Zlatev Martin Maier Vasant Jadhav Terence Cawley Klaus Charisse Anna Bisbe Rajeev Kallanthottathil Ryan Malone Mano Manoharan Jonathan O'Shea Nate Taneja Christopher Theile Jeff Rollins Stacy Seide