TIDES 2014 GalNAc-siRNA with Enhanced Stabilization Chemistry: ESC-GalNAc-siRNA. May 14, 2014 Muthiah Manoharan

Transcription

1 TIDES 2014 GalNAc-siRNA with Enhanced Stabilization Chemistry: ESC-GalNAc-siRNA May 14, 2014 Muthiah Manoharan

2 Agenda sirna-galnac Conjugates: Background Standard Template Chemistry (STC)» Human POC of ALN-TTRsc Enhanced Stabilization Chemistry (ESC)» mttrsc» ALN-AT3» ALN-PCSsc Summary 2

3 GalNAcsiRNA conjugate ASGPR (ph>5) GalNAc-siRNA Conjugates for Systemic RNAi Receptor and the Ligand Clathrin-coated pit protein Recycling ASGPR Clathrincoated vesicle RISC mrna Endosome Nucleus Asialoglycoprotein Receptor (ASGPR) Highly expressed in hepatocytes» million copies/cell Clears serum glycoproteins via clathrin-mediated endocytosis High rate of uptake Recycling time ~15 minutes Conserved across species Valency and Affinity Valency Gal GalNAc Mono- ~4 mm ~50 μm Bi- ~3 μm ~24 nm Tri- ~100 nm ~3 nm 3

4 Making Drugs Out of sirna Conjugates Role of Chemical Modifications 5 -sense = PS sirna = 2 OH = 2 -O-methyl Ligand = 2 -F (GalNAc) 3 Ligand GalNAc mediates binding and internalization of conjugate by hepatocytes sirna Chemistry Stabilize conjugate in subcutaneous space, lymphatics, plasma, and hepatocytes Increase specificity Reduce immune stimulation Enhance cellular delivery Improve RISC activity 4

5 sirna Conjugates Encounter Various Metabolic Enzymes En Route to Site of Action GalNAc-siRNA conjugate SC Injection site Excretion Lymphatics Capillary Uptake Nucleases (5 exo, 3 exo, endo) Peptidases D/M Central Compartment (systemic circulation) D/M P-bodies and associated exonucleases Kinases - Clp1 Phosphatases Effect Site Liver Cytosol RISC Lysosome 1 Hepatocyte Endosome Kidney Schröder et. al., The Proteome of the Lysosome, Proteomics.10: (2010) (adapted from Mark Cancilla, Merck) Biliary excretion Phosphatases Nucleases Peptidases N-acetylgalactosidase many others 5

6 GalNAc-siRNA Conjugates for Systemic RNAi Molecular Targets Discussed Today ALN-TTRsc, ALN-AT3, and ALN-PCSsc Potent, rapid, dose-dependent, and durable target knockdown with SC administration Hepatocyte target genes» ALN-TTRsc targeting TTR for treatment of transthyretinmediated amyloidosis» ALN-AT3 targeting AT to restore thrombin generation in hemophilia and rare bleeding disorders» ALN-PCSsc targeting PCSK9 to lower cholesterol for treatment of hypercholesterolemia ED80 levels for target knockdown from mg/kg» Generally achieved with qw dosing regimen More potent conjugates reflect advances in Enhanced Stabilization Chemistry (ESC) Used with ALN-AT3 and ALN-PCSsc Significantly improved potency and durability Maintains wide therapeutic index and safety 6

7 Agenda sirna-galnac Conjugates: Background Standard Template Chemistry (STC)» Human POC of ALN-TTRsc Enhanced Stabilization Chemistry (ESC)» mttrsc» ALN-AT3» ALN-PCSsc Summary 7

8 % Mean TTR Knockdown Relative to Baseline (± SEM) % Mean TTR Knockdown Relative to Baseline ALN-TTRsc Phase 1 Study Results Human POC for GalNAc-siRNA Conjugates Randomized, double-blind, placebo-controlled SAD and MAD study in healthy volunteers Rapid, dose-dependent, consistent, and durable knockdown of serum TTR» Significant knockdown of serum TTR (p<0.01) up to 94% TTR knockdown; mean knockdown up to 92.4% Generally well tolerated» Only AEs associated with drug were generally mild ISRs resolved within ~2 hours of onset Duration of effect longer in human vs. NHP ALN-TTRsc dose groups Placebo (n=3) 2.5 (n=3) 5.0 (n=3) 10.0 (n=3) Human NHP ALN-TTRsc (mg/kg), qd x5; qw x5 Days ALN-TTRsc Single 10.0 mg/kg Injection Days Zimmermann, Heart Failure Society of America, Sept

9 5 -sense Conjugates More stable in Human than NHP = 2 -F = 2 -O-methyl (GalNAc) 3 Standard Template Chemistry (ALN-TTRsc) NHP 24 hour (5µM) Peak Intensity In vitro Metabolic Profile at 24 hr Human 24 hour (5µM) Peak intensity Sense Strand (5-3 ) Metabolic Profile Metabolite -1 Metabolite -2 Metabolite -3 Metabolite -4 Metabolite -5 Metabolite -6 Metabolite -7 Metabolite -8 Metabolite -9 Metabolite -10 Metabolite -11 Metabolite -12 Full Length Sense Full Length AS Antisense Strand (5-3 ) Metabolite -1 Metabolite -2 Metabolite -3 Metabolite -4 Metabolite -5 Metabolite -6 Metabolite -7 Metabolite -8 Metabolite -9 Metabolite -10 Metabolite -11 Metabolite -12 Human vs. NHP metabolic profile explains extended duration of action in human for TTRsc 9

10 Agenda sirna-galnac Conjugates: Background Standard Template Chemistry (STC)» Human POC of ALN-TTRsc Enhanced Stabilization Chemistry (ESC)» mttrsc» ALN-AT3» ALN-PCSsc Summary 10

11 Conjugate Platform Transitioning Standard Template Chemistry (STC) to Enhanced Stabilization Chemistry (ESC) 5 -sense = 2 -F = 2 -O-methyl (GalNAc) 3 Lead Optimization by Further Stabilization Chemistries 5 -sense Standard Template Chemistry, STC (ALN-TTRsc) 5 -antisense = 2 -F = 2 -O-methyl (GalNAc) 3 Enhanced Stabilization Chemistry, ESC (mttr, ALN-AT3, ALN-PCS) 5 -antisense 11

12 TTR mrna levels Depicted as % of PBS Control (mttr/mgapdh) ESC Improves Efficacy of mttrsc ESC design improves in vivo efficacy by 5-fold over STC design in mttrsc 120 Subcutaneous Single Dose PBS 25 mg/kg 5 mg/kg 1 mg/kg 5 mg/kg 1 mg/kg 0.2 mg/kg STC ESC 12

13 % Knockdown Serum TTR (Fraction Pre-dose) GalNAc Conjugates Are a Repeat-Dose Platform Chronic Dosing in Mice with ESC-mTTRsc Steady knockdown maintained with chronic dosing Sustained knockdown at both ED 50 (1 mg/kg) and ED 80 (2.5 mg/kg) dose levels Absence of tachyphylaxis or sensitization No changes in serum TTR levels in PBS control group GalNAc Conjugates are a multi-dose platform, showing dose additive effect -20 Long-Term Dosing: QW Dosing with ESC-mTTRsc PBS 1.0 mg/kg 2.5mg/kg Day 13

14 sirna-galnac Lead Optimization Tuning STC-siRNA-GalNAc to ESC-siRNA-GalNAc STC-siRNA-GalNAc Design chemically modified sirnas in vivo exposure/ PD in rodent or htg Xn sirna synthesis Select 3-6 conjugates for high dose rat toxicity screen In vitro screening NHP PK/PD ESC-siRNA-GalNAc 14

15 Liver Concentration (ng/g) ESC Leads to Higher Liver Exposure Liver Exposure and Metabolic Stability, Single SC Dose, 25 mg/kg in Mice Metabolic profiling in liver 8h post dose S 5 Standard Template Chemistry (STC) (GalNAc) Liver Exposure AS 5 = 2 -F = 2 -O-methyl SC STC ESC Enhanced Stabilization Chemistry (ESC) Time (h) S 5 Liver Exposure AS 5 Target Compound Tmax (h) Cmax (µg/g) AUC 0-t (h µg/g) AUC 0-48 (h µg/g) AT3 STC = Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed) AT3 ESC

16 % Knockdown Serum AT (Relative to Pre-dose) ESC Significantly Enhances Efficacy and Duration Reduction of AT Protein After Single SC Dose in NHP Potent and durable silencing achieved after single SC dose >10-fold improvement in efficacy over standard template chemistry Substantially extended duration of effect STC-AT3 (10 mg/kg) ESC-AT3 (10 mg/kg) ESC-AT3 (1 mg/kg) Day 16

17 % Antithrombin Knockdown Normalized to Pre-Dose and PBS Maintenance of AT Knockdown with Repeat Dosing ALN-AT3 Pre-Clinical Studies Weekly SC administration of ALN-AT3 in WT mice results in potent and consistent AT suppression Repeat dose ED 50 for AT knockdown <0.75 mg/kg Nadir reached at ~day ALN-AT3 (mg/kg) ALN-AT3, qw x5 Day Akinc, WFH, July

18 Liver sirna (ng/g) Drug Levels in Tissue During Chronic Dosing (ALN-AT3) No Evidence for Accumulation in Tissue (Mouse Liver) Day ALN-AT3, dosing (qw x 14) Actual Data with Interpolated Data Added 1.0 mg/kg qw 0.5 mg/kg qw 0.2 mg/kg qw Tissue collection Post-dose (4 h) Pre- and postdose (4 h) Pre- and postdose (4 h, 3 days) Pre- and postdose (4 h) Pre- and postdose (4 h) 18

19 Liver sirna (ng/g) Drug Levels in Tissue During Chronic Dosing (ALN-AT3) No Evidence for Accumulation in Tissue (Mouse Liver) ng/g ng/g 400 ng/g Day ALN-AT3, dosing (qw x 14) Actual Data with Interpolated Data Added 1.0 mg/kg qw 0.5 mg/kg qw 0.2 mg/kg qw Tissue collection Post-dose (4 h) Pre- and postdose (4 h) Pre- and postdose (4 h, 3 days) Pre- and postdose (4 h) Pre- and postdose (4 h) Additional peak and trough concentration data added by interpolation; additional 3-day post-dose data added by assuming fixed ratio relative to peak concentration Liver levels are approximately dose proportional Mean steady state liver drug load of ~3 g/g per mg/kg cumulative weekly dosing 19

20 % Mean AT Knockdown (Pre-dose = 1) Relative Thrombin Generation (Peak Thrombin) ALN-AT3 Pre-Clinical Efficacy Potent AT Knockdown and Normalization of Thrombin Generation ALN-AT3 achieves potent AT knockdown and fully corrects thrombin generation in non-human primates (NHP) Weekly SC doses for >5 months result in potent, dose-dependent, and durable AT knockdown In NHP hemophilia inhibitor model, ALN-AT3 fully restores thrombin generation to normal levels p< ~60% AT KD ~80% AT KD Day 0.5 mg/kg qw x 8 Recovery mg/kg qw x Pre-dose Normal Saline ALN-AT3 (mg/kg) qw Hemophilia A (Induced) Akinc, ISTH, July mg/kg q2w x mg/kg qw x 5 Recovery 0.25 mg/kg qw x 12 Recovery 20

21 Relative AT Level What About Species-Related Differences? ALN-AT3 Single Dose Efficacy and Recovery Day Mouse 2.5 mg/kg Rat 3.0 mg/kg NHP 3.0 mg/kg Single-dose efficacy: mouse rat > NHP Time to nadir and recovery kinetics: NHP > mouse > rat 21

22 hpcsk9 protein (Relative to pre-dose) Fraction PCS message remaining Screen of ca. 200 PCSK9 sirnas identified initial lead 1.4 ALN-PCSsc Lead Selection Use What You Know, Then Iterate 0.1 nm, average activity across 3 human cell lines PCSsc Lead sirna rank order +/- SD Tested Lead as STC and ESC versions in vivo SD 1.2 STC-PCS vs. ESC-PCS hpcsk9 tg, mice 72h, 3 mg/kg PBS STC-PCS ESC-PCS 22

23 % Knockdown PCSK9 (relative to pre-bleed) Multi-Dose ESC-PCS Results in >90% Lowering of PCSK9 But Duration can be Improved (NHP) ESC-PCS q2w 1 mg/kg q1w Dosing Schedule BIW Day ED90 of ESC-PCS ~1 mg/kg BIW (12 doses) Reduction in PCSK9 results in >60% lowering of LDL-C (data not shown) Duration of nadir PCSK9 post-last dose (BIW) is ~20 days The compound potency was sufficient but stability/dose frequency not sufficient to meet TPP Goal of once-monthly dosing Another round of ESC lead optimization was used! 23

24 sirna-galnac Lead Optimization Tuning ESC-siRNA-GalNAc to Development Candidate for PCSK9 ESC-siRNA-GalNAc Design chemically modified sirnas in vivo exposure/ PD in rodent or htg X2 sirna synthesis Select 3-6 conjugates for high dose rat toxicity screen In vitro screening NHP PK/PD ALN-PCSsc Development Candidate 24

25 % PCSK9 Knockdown (relative to pre-bleed) % LDL-C Lowering (relative to pre-bleed) ALN-PCSsc Achieves Potent, Highly Durable PD Pre-Clinical Efficacy in NHP with Single Dose ALN-PCSsc achieves highly durable PCSK9 knockdown and LDL-C reduction with single dose Single SC dose 1-10 mg/kg Up to 96% PCSK9 knockdown Up to 77% LDL-C lowering in absence of statins Highly durable effects, supports once-monthly and possibly once-quarterly dosing» >50% LDL-C lowering maintained for over 3 months in 10 mg/kg group -20 PCSK LDL-C ALN-PCSsc (mg/kg) Days ALN-PCSsc (mg/kg) Days Fitzgerald, ATVB, May

26 Comparing STC- to ESC-GalNAc-siRNA Conjugates Key Data in NHP STC (ALN-TTRsc) ESC (ALN-PCSsc) Potency ED 50 SD 10.0 mg/kg 1.0 mg/kg MD (qw) ca. 2.0 mg/kg 0.2 mg/kg Stability t 1/2 Liver ~24 h ~120 h Duration ~40 days ~120 days Cytokine/Complement Activation None None Safety Adverse ISRs None None NOAEL (MD) >300 mg/kg >300 mg/kg 26

27 Agenda sirna-galnac Conjugates: Background Standard Template Chemistry (STC)» Human POC of ALN-TTRsc Enhanced Stabilization Chemistry (ESC)» mttrsc» ALN-AT3» ALN-PCSsc Summary 27

28 Summary Alnylam has developed an effective, well-tolerated, SC-administered GalNAc-conjugate platform with proven human clinical translation Enhanced Stabilization Chemistry (ESC) of sirna-galnac conjugates has led to increased metabolic stability resulting in:» Increased liver exposure» Improved potency: Up to 10-fold SD potency improvement and less injection volume» Increased duration of effect, which further increases from rodents to NHP» No compromise in safety ESC-conjugate design translates well across multiple sirnas and targets Expect favorable translation of potency and duration in humans for ESC-GalNAc-conjugates 28