WFH 2014 World Congress Medical Free Paper 8 ALN AT3: An Investigational RNAi Therapeutic Targeting Antithrombin for the Treatment of Hemophilia

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1 WFH 214 World Congress Medical Free Paper 8 ALN AT3: An Investigational RNAi Therapeutic Targeting Antithrombin for the Treatment of Hemophilia May 15, 214 Akin Akinc, PhD

2 Antithrombin and ALN-AT3 Program Intrinsic system Extrinsic system Antithrombin (AT) is genetically defined target AT is key natural anticoagulant» Inactivates factors Xa and thrombin» Attenuates thrombin generation Human AT deficiency associated with increased thrombin generation Expressed in liver; circulates in plasma Hemophilia B FIX Hemophilia A FVIII FX FIXa FVIIIa FXa Prothrombin FVIIa AT FVa Thrombin FVII FV Co-inheritance of thrombophilic traits in hemophilia Associated with milder bleeding, reduced factor requirements, fewer complications Includes heterozygous» Antithrombin deficiency» Factor V Leiden» Protein C deficiency» Protein S deficiency Fibrinogen Fibrin Blood clot ALN-AT3 in clinical development GalNAc-siRNA for SC dosing Efficacy in pre-clinical animal models Orphan drug status in US (HA/HB) Phase 1 study ongoing Kurnik et al., Haematologica; 92:982-5 (27); Ettingshausen et al., Thromb Haemost; 85:218-2 (21); Negrier et al., Blood; 81:69-5 (1993); Shetty et al., Br J Haematol; 138:541-4 (27) 2

3 RNAi Therapeutics Harness natural pathway» Catalytic mechanism» Mediated by small interfering RNA or sirna Therapeutic gene silencing» Any gene in genome» Distinct mechanism of action vs. other drug classes» Unique opportunities for innovative medicines Clinically validated platform RNA Interference (RNAi) A New Class of Innovative Medicines 3

4 Relative Antithrombin Level (Normalized to pre-dose and PBS) ALN-AT3 SC-Administered GalNAc-Conjugated sirna Targeting Antithrombin ALN-AT3 (sirna GalNAc conjugate) mg/kg 1.5 mg/kg.75 mg/kg ASGPR (ph>5) Clathrin-coated pit 1. Wild-type mice, N = 5 Weekly (qw) dosing.8 AT protein Recycling ASGPR Clathrin-coated vesicle.6.4 RISC.2 mrna Endosome Nucleus qw x5 Day Enhanced stabilization chemistry (ESC) utilized for potency and durability of effect Weekly SC dosing results in potent and sustained suppression of antithrombin levels 4

5 % Mean AT Knockdown (Pre-dose = 1) ALN-AT3 Pre-Clinical Efficacy Potent AT Knockdown and Normalization of Thrombin Generation ALN-AT3 achieved potent AT knockdown and fully corrected 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 normalizes thrombin generation Relative Thrombin Generation (Peak Thrombin) ~6% AT KD p<.1 ~8% AT KD Day.5 mg/kg qw x 8 1 mg/kg q2w x 4 Recovery Recovery.125 mg/kg qw x mg/kg qw x 12. Pre-dose Saline.25.5 ALN-AT3 (mg/kg) qw Normal Hemophilia A (Induced) similar results obtained by ETP (p<.1 at.5 mg/kg) Akinc, ISTH, July mg/kg qw x 5 Recovery 5

6 Platelets Area (microns 2 ) Fibrin Area (microns 2 ) ALN-AT3 Improved Hemostasis Microvessel Laser Injury Model (HA Mice) WT HA + PBS Deposition of Platelets HA + 1 mg/kg HA + 3 mg/kg HA + 1U/kg Advate 3 2 WT HA + PBS HA + 1 mg/kg HA + 3 mg/kg HA + 1U/kg Advate Deposition of Fibrin Time (sec) Time (sec) Group Animals (N) Injuries (N) Stable Thrombus (N) Percent AT mrna in liver WT % HA + PBS % HA + 1 mg/kg ALN-AT % HA + 3 mg/kg ALN-AT % Akinc, ISTH, July 213; In collaboration with Dr. Lacra Ivanciu and Dr. Rodney Camire 6

7 Number of Hemostatic Events ALN-AT3 Improved Hemostasis Saphenous Vein Bleeding Model (HA Mice) Saphenous vein bleeding model adapted from Whinna 1 Saphenous vein exposed and transected to initiate bleeding Thirty seconds after cessation of blood loss, clot dislodged to re-initiate bleeding Number of hemostatic events in 3 min observation period recorded Pilot study details 2 Treated animals received single SC dose of ALN-AT3 to yield ~7% AT knockdown at time of model initiation Control animals received 25 IU/kg Advate via IV administration 15 min prior to model initiation N = 8 N = 15 N = 6 N = 9 p <.1 -- Saline ALN-AT3 Advate 25 IU/kg WT ~7% AT KD HA 1 Buyue et al., Blood; 112: (28); 2 In collaboration with Dr. Brian Cooley 7

8 Thrombin (nm) Thrombin (nm) Antithrombin Depletion Increased Thrombin Generation in Hemophilia Plasma HA Donor Plasma FVIII<1%, AT=% FVIII<1%, AT=1% FVIII<1%, AT=2% FVIII<1%, AT=4% FVIII<1%, AT=6% FVIII<1%, AT=1% FVIII=1%, AT=1% HB Donor Plasma FIX<1%, AT=% FIX<1%, AT=1% FIX<1%, AT=2% FIX<1%, AT=4% FIX<1%, AT=6% FIX<1%, AT=1% FIX=1%, AT=1% Time (min) Time (min) Antithrombin depletion increases peak height and delays inhibition of thrombin 8

9 FVIII Equivalence of Peak Thrombin (%) FIX Equivalence of Peak Thrombin (%) Antithrombin Depletion Increased Thrombin Generation in Hemophilia Plasma 1 HA Donor Plasma 1 HB Donor Plasma AT Reduction (%) AT Reduction (%) 6% AT reduction results peak thrombin equivalent to ~1% FVIII 4% AT reduction results peak thrombin equivalent to ~15% FIX 9

10 mod APTT (s) Modified APTT Simple Method for Assessing Activity of ALN-AT3 APTT Simple, standard clot time based screening assay that interrogates the intrinsic and common pathways Hemophilia is associated with prolonged APTT, and treatment with replacement factor corrects APTT However, APTT is NOT sensitive to AT levels Modified (heparin) APTT Plasma clotting tests do not contain endothelial cells, the source of heparan sulfate proteoglycans, key cofactor for AT» Heparin enhances activity of AT by ~1-fold Standard plasma clotting tests likely utilize AT in its inactive form Test HA plasmas generated by adding human AT protein to AT-depleted HA plasma (1% AT = 125 µg/ml) Unfractionated heparin (UFH) at.5 and.1 U/mL tested UFH.1 U/mL UFH.5 U/mL Addition of heparin to test plasma should activate AT and increase sensitivity of plasma-based tests like APTT to AT levels AT (%) 1

11 ALN-AT3 Phase 1 Study Dose-escalation Study in Two Parts Part A Study Design Randomized, single-blinded, placebo-controlled SAD study in healthy volunteers Primary Objective Safety and tolerability of single doses with AT knockdown <4% Secondary Objectives Assess clinical activity» AT knockdown Part B Study Design Open-label, MAD study in subjects with moderate to severe hemophilia A or B (N=up to 18) Primary Objective Safety and tolerability of multidose in hemophilia subjects Secondary Objectives Assess clinical activity» AT knockdown» Increase in thrombin generation 11

12 Positive top-line Phase 1 Part A study results ALN-AT3 Phase 1 Study Status Update Single.3 mg/kg dose was safe and well-tolerated in healthy volunteer subjects At.3 mg/kg dose, achieved statistically significant AT knockdown, placebo vs. treated, p<.1 by ANOVA Central Assay Local Assay Mean Nadir Knockdown 19% 28% Maximum Knockdown 28% 32% In addition, achieved statistically significant (p<.1) increase in thrombin generation, consistent with degree of AT knockdown Phase 1 SAD objectives were met, transition to MAD phase in hemophilia patients with no further dosing of healthy volunteers» Per protocol, maximum allowable AT knockdown of 4% (local assay) Initial Phase 1 data to be presented in late 214» To include initial experience in hemophilia patients 12

13 Key Translational Data for GalNAc-Conjugates Standard Template Chemistry vs. Enhanced Stabilization Chemistry Standard Template Chemistry: ALN-TTRsc in Human Dose (mg/kg) Dose (mg/kg) Single-dose Nadir KD (%) Single-dose Nadir KD (%) Multi-dose (qdx5, q weekly) Nadir KD (%) ± 1 NT* ± ± ± 1 88 ± 7 A ± 8 92 ± 2 Enhanced Stabilization Chemistry: ALN-AT3 in NHP Multi-dose (q weekly) Nadir KD (%).1 19 ± 2 27 ± ± ± ± ± NT > ± 13 NT 1 82 ± 7 NT B A. 5-1x Increased potency in NHP with AT3 vs. TTRsc B. 1x Increased potency with SD to MD C. 1x Increased potency in human vs. NHP D. ~5x Increased potency in humans with AT3 vs. TTRsc D Enhanced Stabilization Chemistry: ALN-AT3 in Human Dose (mg/kg) Single-dose Nadir KD (%) % C 13

14 Implications for ESC-GalNAc Conjugate Platform First human POC for ESC-GalNAc-siRNA conjugates» Extends and broadens initial human POC achieved with standard template chemistry conjugates with ALN-TTRsc Unprecedented RNAi activity observed at very low single dose of.3 mg/kg» Represents 5-1x improved efficacy in humans compared to NHP» If confirmed in additional studies, could represent ~5x improved potency for ESC-GalNAc conjugates as compared with standard template chemistry GalNAc-conjugates 14

15 Acknowledgements Quintiles Tim Mant, BSc, FRCP, FFPM Children s Hospital of Philadelphia, USA Lacramioara Ivanciu, PhD Rodney Camire, PhD University of North Carolina, Chapel Hill, USA Brian Cooley, PhD Edouard Herriot University Hospital, Université Claude Bernard, Lyon, France Yesim Dargaud, MD, PhD Claude Negrier, MD, PhD 15

16 Thank You