8th Annual Meeting of the Oligonucleotide Therapeutics Society The Science of RNAi Therapeutics. October 28, 2012

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1 8th Annual Meeting of the Oligonucleotide Therapeutics Society The Science of RNAi Therapeutics October 28, 212

RNAi Therapeutics Harness natural pathway» Catalytic mechanism» Mediated by small interfering RNAs or sirnas Treat disease with therapeutic gene

2 RNAi Therapeutics Harness natural pathway» Catalytic mechanism» Mediated by small interfering RNAs or sirnas Treat disease with therapeutic gene silencing» Any gene in genome» Creates unique opportunities for innovative medicines RNA Interference (RNAi) A New Class of Innovative Medicines 2

3 Making sirna into Drugs? Not Easy! Remember sirna are highly negatively charged, hydrophilic and large, so they don t cross cell membranes They are also readily prone to exonuclease and/or endonuclease degradation And, to make matters worse, they can have off target effects including effects on innate immunity pathways Therefore Need to impart drug-like properties» Potency, selectivity, stability, PK/PD, biodistribution, cellular uptake 3

4 Lead Selection & Optimization Alnylam RNAi Product Platform Turning sirnas into Drugs Delivery Selection Direct sirna sequence sirna» Specificity Systemic» Crossreactivity» Potency conjugation Optimization Introduce chemical modifications for drug-like properties sirna chemistry» Stability» Immuno-silence» Potency formulation PK/PD Biodistribution Cellular uptake Small Scale Gene walks In vitro assays Chemistry, Manufacturing and Controls Medium Scale In vivo biology Large Scale GMP Production Clinical trials 4

5 Lead Selection & Optimization Alnylam RNAi Product Platform Turning sirnas into Drugs Delivery Selection Direct sirna sequence» Specificity» Crossreactivity» Potency Systemic sirna conjugation Optimization Introduce chemical modifications for drug-like properties sirna chemistry» Stability» Immuno-silence» Potency formulation PK/PD Biodistribution Cellular uptake Small Scale Gene walks In vitro assays Chemistry, Manufacturing and Controls Medium Scale In vivo biology Large Scale GMP Production Clinical trials 5

6 The Delivery Challenge PK/Tissue Distribution Cellular Uptake and Trafficking RNA Release and RISC Loading anti-ago2 6

7 Lipid Nanoparticles (LNPs) for Systemic RNAi Multi-component lipid formulation» Amino lipid» Structural lipid» PEG lipid» Cholesterol Highly efficient for liver delivery» Hepatocyte-specific gene silencing achieved N O O DLin-KC2-DMA mpeg 2 -C14 glyceride Cholesterol Low surface charge Small uniform size particle <1 nm DSPC 7

% Residual Factor VII New Generation LNPs Major Potency Improvements with Novel Lipids Novel LNPs set new benchmark for systemic RNAi with ~1-fold improved potency

digit mg/kg ED 5 12 1 Dlin-MC3-DMA-b C12-2 2 nd Generation LNPs ALN-PCS (Phase I Completed) ALN-TTR2 (Phase II Ongoing) 1st Generation LNPs ALN-VSP (Phase I Completed)

8 % Residual Factor VII New Generation LNPs Major Potency Improvements with Novel Lipids Novel LNPs set new benchmark for systemic RNAi with ~1-fold improved potency Efficacy in pre-clinical models following single IV injection Each LNP comprised of distinct cationic lipid component Improvements in potency have resulted in single digit mg/kg ED Dlin-MC3-DMA-b C nd Generation LNPs ALN-PCS (Phase I Completed) ALN-TTR2 (Phase II Ongoing) 1st Generation LNPs ALN-VSP (Phase I Completed) ALN-TTR1 (Phase I Completed) 8 DLin-KC2-DMA-b DLinDMA DLinDAP 6 MD1 DLin-KC2-DMA-a 4 2 ED 5 DLin-K-DMA 98N12-5(I) FVII sirna Dose (mg/kg) 8 Oligo Ther Soc., Sep 211

9 % Serum TTR Knockdown Second Generation LNPs Translation of Improved Potency to NHP Control ALN-TTR1 (1. mg/kg) ALN-TTR2 (.1 mg/kg) >1-fold improved efficacy in animal models 2 nd generation MC3 LNP formulation ~9% Knockdown at.3 mg/kg Rapid knockdown after single dose Durability supportive of q3-q6 day dosing sirna Dose Day Post Dose ALN-TTR2 (.3 mg/kg) 9 Oligo Ther Soc., Sep 211

10 Mechanism of ApoE Mediated ilnp Delivery LNPs Endogenous internalized traffic through ApoEand bind fenestrated disrupt to LNPs endosome; endothelium release of liver; sirna bindintocytoplasm LDL receptor ph ~ 5 As endosome acidifies, cationic charge on vesicle increases Θ Θ Θ Cationic lipid combines with anionic membrane lipids to disrupt endosomal membrane Lipoprotein particle ApoE Exchange of ApoE Fenestration ApoE-binding cell surface receptor sirna cargo is released into cytoplasm where it can enter RISC ph 7.4 Blood Compartment Space of Disse 1 Molecular Therapy, 18: , July 21 Hepatocyte

FVII mrna (% of Baseline) FVII mrna (% of baseline) In Vivo Potency of sirna Relationship of sirna Liver Concentration to FVII mrna Silencing EC 5 ~1 ng/g Corresponds to roughly 4-5

11 FVII mrna (% of Baseline) FVII mrna (% of baseline) In Vivo Potency of sirna Relationship of sirna Liver Concentration to FVII mrna Silencing EC 5 ~1 ng/g Corresponds to roughly 4-5 molecules per cell Demonstrates power of natural RNAi pathway in vivo FVII sirna (ng/g Liver) Factor VII sirna, ng/g Liver 11 Oligo. Ther. Soc., Nov 29 Confidential

% Gene expression sirna Conjugates Subcutaneous Administration

, Cholesterol Promotes cellular uptake Improves plasma PK O O 3

12 % Gene expression sirna Conjugates Subcutaneous Administration Lipophilic molecules Vitamins Carbohydrates Peptides Antibodies sirna Ligand e.g., Cholesterol Promotes cellular uptake Improves plasma PK O O 3 ' O O N H O P O N O H RNAi activity in vitro without transfection reagent 14 Unmodified Cholesterol conjugated O sirna dose (nm) 12 Nature;432:173-8(24)

13 ASGPR Highly expressed in hepatocytes».5-1 million copies/cell Clears serum glycoproteins via clathrin-mediated endocytosis High rate of uptake Recycling time ~15 minutes Conserved across species GalNAc-siRNA Trivalent GalNAc carbohydrate cluster has high affinity (nm) for ASGPR GalNAc ligand conjugated to chemically-modified sirna to mediate targeted delivery Administered subcutaneously (SC) sirna Conjugate Approach Targeted Delivery to Hepatocytes and SC Dosing Glycosylated ligand ASGPR (ph>5) Lysosome Transport vesicle Clathrin-coated pit Recycling ASGPR Clathrin-coated vesicle Early endosome 13 GalNAc ligand Late endosome (ph <5) Adapted from Essentials of Glycobiology (29)

14 MFI Design of GalNAc Ligand for sirna Conjugation Alnylam Design Site of Conjugation Spacing: 2 Å Spacing: 2 Å 4 Kd = 2.48 nm 3 Ligand Mouse Hepatocyte Ki 2 1 Kd = 28.8 nm (GalNAc)3 (GalNAc) [(GalNAc)n] nm GalNAc 2 GalNAc 3 ~24 nm 2.7 nm van Berkel, JBC, 21, 276, 37577; Bioconjugate Chem., 22, 13 (2), 295; Sliedregt, L. A. et. al. J. Med Chem. 1999, 42, AASLD Annual Meeting, Oct 21

15 ng sirna/gm tissue TTR-GalNAc sirna Biodistribution GalNAc-siRNA conjugates efficiently target liver Achieve liver levels at ~5% delivered dose hr post dose Testes Jejunum Heart Lung Thymus liver spleen kidney 15

16 TTR mrna (%Control) Robust Silencing with TTR-GalNAc Conjugate SC Single Dose, 72h, Mouse Major advance in RNAi delivery: In vivo efficacy with subcutaneous dosing Single SC dose; Transthyretin target ED 5 ~5 mg/kg Control TTR-GalNAc (mg/kg) 16 AAAS, Feb 21 Confidential

17 % Serum TTR Knockdown (Normalized to Pre-dose) Sustained TTR Suppression with SC Dosing of ALN-TTRsc Non-Human Primate SC administration of ALN-TTRsc results in robust and sustained TTR suppression ~8% suppression at doses as low as 2.5 mg/kg Nadir knockdown at ~day 14; sustained suppression with weekly maintenance dose Recovery to baseline levels >4 days from last dose mg/kg 5 mg/kg PBS 1 mg/kg sirna Doses: 17 IRT, Aug. 212 Study Day

18 mangptl3/mgapdh Relative to PBS=1 hpcsk9 Protein, Group Average Relative to Individual Pre-Dose GalNAc-siRNA Conjugates Increasing Applications for Other Molecular Targets 1.2 GalNAc-siRNA Targeting ANGPTL3 Single Dose ED5 <3 mg/kg 1.2 GalNAc-siRNA Targeting PCSK9 Single Dose ED5 <.3 mg/kg PBS Lead Lead Opt ANGPTL3-GalNAc (mg/kg) PBS PCS-GalNAc (mg/kg) 18 OTS, Aug. 212

19 Antithrombin (AT) and ALN-AT3 Program A hepatocyte-expressed serpin» Encoded by SERPINC1 on Ch1» Abundant plasma glycoprotein (58 kda)» Heterozygous AT deficiency associated with increased risk for venous thromboembolism (VTE) Anticoagulant function» Major stoichiometric inhibitor of thrombin and FXa» Inhibits other activated factors VIIa, IXa, XIa, and XIIa to lesser degree» Forms inhibitory stochiometric complex with serine protease Human AT deficiency associated with increased thrombin generation Expressed in liver; circulates in plasma Intrinsic system Hemophilia B FIX FIXa FX Extrinsic system FVIIa TFPI FVII Hemophilia A FVIII FVIIIa FXa AT FVa FV Prothrombin Thrombin Fibrinogen Fibrin Blood clot 19

20 % Antithrombin Knockdown (Normalized to Pre-Dose and PBS) ALN-AT3 Maintenance of AT Levels in Wild Type Mice Weekly SC administration of ALN-AT3 results in potent and consistent AT suppression Repeat dose ED 5 for AT knockdown <.75 mg/kg Nadir knockdown at ~day mg/kg qw 1.5 mg/kg qw.75 mg/kg qw sirna Doses: Day 2 IRT, Aug. 212

21 Thrombin (nm) Thrombin Generation AUC (nm min) ALN-AT3 Activity in Hemophilia B Mice Hypothesis: AT knock-down should increase thrombin generation in hemophilic mice N = 3, hemophilia B (FIX -/- ) mice or wild-type mice Single subcutaneous injection at 3 mg/kg 72 hour time point Blood collected in sodium citrate with CTI TGA using Calibrated Automated Thrombinoscope (CAT) 5 Thrombin Generation in HB Mice WT Control 6 Normalization of ETP in HB Mice 4 HB Control ALN-AT3 treated #1 5 3 ALN-AT3 treated #2 ALN-AT3 treated # Time (min) WT Control HB Control ALN-AT3 Treated 21 WFH, July 212; In collaboration with Dr. Claude Negrier

22 GalNAc-siRNA Conjugate Tolerability ALN-TTRsc Results Cytokine/Complement Assessment No evidence of cytokine induction» Ex vivo whole blood assay» Up to 125mg/kg SC in CD1 mice» Up to 3mg/kg SC in NHP No evidence of complement activation (NHP)» No increases in plasma Bb or C3a Non-GLP Toxicology Assessment Rat: 5 daily SC doses of 3, 1, or 3mg/kg» No adverse in-life findings or clinical pathology changes» No injection site reactions» No adverse histology findings (liver, spleen, kidney, heart, lung, injection site) NHP: single SC dose of 3mg/kg» No adverse in-life findings or clinical pathology changes» No injection site reactions 22

23 23 RNAi Human Translation

24 % Serum TTR Knockdown Relative to Baseline at Nadir (Days 1-28) % Subjects Achieving LDL-c Target 1. Human Safety and PK >575 Subjects/patients enrolled overall Systemic delivery in human trials» ~1 Patients dosed» >325 Doses administered 2» >24 months of dosing RNAi therapeutics generally well tolerated Pharmacologically relevant human tissue levels achieved p<.1 RNAi Human Translation Achieved Multiple Alnylam Therapeutic Programs 2. Human Proof of Mechanism 3. Human Proof of Concept 4. Human Clinical Efficacy ALN-TTR Pre- Post- Positive, p<.1 5 location along VEGF transcript RNAi Cleavage ALN-PCS p=.3 ALN-VSP Control sirna *** *** *** Placebo ALN-TTR2 (mg/kg) 2 <.15 mg/kg.15 mg/kg (n=9) (n=15) ALN-PCS Dose Groups

Severe Hypercholesterolemia Program Unmet Need and Product Opportunity Cardiovascular disease (CVD) resulting from elevated low-density lipoprotein cholesterol (LDL-C) is leading cause of death

25 Severe Hypercholesterolemia Program Unmet Need and Product Opportunity Cardiovascular disease (CVD) resulting from elevated low-density lipoprotein cholesterol (LDL-C) is leading cause of death worldwide 1 Unmet need: >5, patients with severe hypercholesterolemia despite current therapies Recent clinical trials suggest further health benefits with even lower LDL-C levels in patients with CVD 2 (<7 mg/dl) Multiple studies indicate many patients (up to 8%) do not meet LDL-C goals even on statins 3,4» Poor adherence» Incomplete response to statins» Intolerance to statins 25 1 World Health Organization (24) 2 Ridker et al., NEJM; 359: (28) 3 Jacobson et al., NHANES II, Arch Intern Med; 16: (2) 4 Pearson et al., L-TAP Study, Arch Intern Med; 16: (25)

26 ALN-PCS Phase I Study Study Design Randomized, single-blind, placebo-controlled, single-dose escalation study» 3:1 Randomization» 4 Subjects/cohort (except.25 and.4 mg/kg cohorts with 8 subjects, 6:2)» 6 Dose levels ( mg/kg) Healthy volunteers with elevated baseline LDL-C (>116mg/dL), not on statin or lipid therapy Dose levels and dosing schedule.15,.45,.9,.15,.25 (n=6), and.4 (n=6) mg/kg Single 6-min IV infusion Premedicate night before and day of dosing with dexamethasone, H1 and H2 blockers, and acetaminophen Primary objective Safety and tolerability Secondary objectives Assess pharmacodynamics and clinical activity» Plasma PCSK9 protein and serum LDL-C levels Characterization of pharmacokinetics Study Status Two study centers in the UK participating Dosing complete; follow-up ongoing 26

27 ALN-PCS Phase I Study Safety and Tolerability - Overall TEAEs 1%.15 (n=3).45 (n=3).9 (n=3).15 (n=3).25 (n=6).4 (n=6) Overall ALN-PCS (N=24) Placebo (N=8) Preferred Term n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%) Rash 1 (33.3) 1 (33.3) (.) (.) 4 (66.7) 6 (1) 12 (5.) 4 (5.) Headache 1 (33.3) 1 (33.3) 2 (66.7) (.) (.) 1 (16.7) 5 (2.8) 2 (25.) Hiccups 2 (66.7) (.) (.) (.) (.) 2 (33.3) 4 (16.7) 2 (25.) Cold Symptoms (.) 1 (33.3) (.) 2 (66.7) 1 (16.7) (.) 4 (16.7) 1 (12.5) Paraesthesia 1 (33.3) (.) (.) (.) (.) 2 (33.3) 3 (12.5) 2 (25.) Polyuria/Dysuria 1 (33.3) (.) (.) (.) (.) 1 (16.7) 2 (8.4) 1 (12.5) All TEAEs mild or moderate in severity No SAEs related to study drug administration» One subject at.45 mg/kg dose level was diagnosed with bilateral PEs and DVTs; determined by PI to be unrelated to study drug given subject s prior history and symptoms prior to study drug administration No significant laboratory abnormalities (LFTs, CRP and cytokines) 27 ATVB, April 212

28 % Knockdown PCSK9 Plasma Levels Mean Relative to Baseline and Placebo % Knockdown LDL-C Serum Levels Mean Relative to Baseline and Placebo ALN-PCS Phase I Study Results Pharmacodynamics and Clinical Efficacy PCSK9 knockdown and LDL-C reduction after single dose without statins Randomized, placebo-controlled, single dose escalation study in healthy volunteers with elevated LDL (n=32) Rapid, dose-dependent, and durable knockdown of PCSK9 of up to 84% with mean lowering of 68% at.4 mg/kg group (p<.1) Major reductions in LDL-C of up to 5% with mean lowering of 41% at.4 mg/kg group (p<.1) No significant decreases in HDL-C -4 PCSK9 * Data for n=6 from the.4 mg/kg group through Day 14 only LDL-C * Data for n=6 from the.4 mg/kg group through Day 14 only sirna Dose ALN-PCS dose group.15 mg/kg.45 mg/kg.9 mg/kg Day.15 mg/kg.25 mg/kg.4 mg/kg 4 6 sirna Dose ALN-PCS dose group.15 mg/kg.45 mg/kg.9 mg/kg mg/kg.25 mg/kg.4 mg/kg 28 ATVB, April 212

Transthyretin (TTR)-Mediated Amyloidosis (ATTR) Program Unmet Need and Product Opportunity ATTR is significant orphan disease» ~5, Patients worldwide Clinical pathology» Onset ~4 to >6 yr» Two

29 Transthyretin (TTR)-Mediated Amyloidosis (ATTR) Program Unmet Need and Product Opportunity ATTR is significant orphan disease» ~5, Patients worldwide Clinical pathology» Onset ~4 to >6 yr» Two predominant forms Familial amyloidotic polyneuropathy (FAP) Familial amyloidotic cardiomyopathy (FAC)» Peripheral sensorimotor neuropathy, autonomic neuropathy, and/or cardiomyopathy» Fatal within 5-15 years Current treatments limited» Liver transplant current standard of care <3, Patients eligible» EU approval of Pfizer s Vyndaqel TM (tafamidis) for FAP in Nov 211» CRL from FDA in Jun

30 ALN-TTR2 Phase I Study Study Design Randomized, placebo-controlled, single-blind, single-dose escalation study in normal healthy volunteers» 3:1 randomization» 4 subjects/cohort Dose Levels and Dosing Schedule.1,.5,.15,.3 and.5 mg/kg ALN-TTR2 Single 6-min IV infusion Premedicate the night before and day of dosing with dexamethasone, H1 and H2 blockers, and acetaminophen Primary Objective Evaluate safety and tolerability of ALN-TTR2 Secondary Objectives Assess preliminary pharmacokinetics and pharmacodynamics» Serum TTR, RBP, Vitamin A levels Status Study enrollment complete; follow-up ongoing 3

31 Mean % Serum TTR Knockdown Relative to Baseline ALN-TTR2 Phase I Study Results Duration of Response by Dose Group sirna Dose Day Treatment (mg/kg) Placebo (n=4).15 (n=3).1 (n=3).3 (n=3).5 (n=3).5 (n=1) Control sirna.4 mg/kg (n=6) 31 Serum TTR levels were measured in separate Phase I study of ALN-PCS, an RNAi therapeutic targeting PCSK9, which uses identical LNP formulation as ALN-TTR2 B.U. Med. Center, July 212

32 Blinded pre- and post-treatment serum samples from ALN-TTR2 subjects at.3 mpk.4um filtration/ 1,g spin 5 RACE Using Circulating RNA Method RNA adaptor cdna synthesis Cleaved mrna Incubate at 4 C with 1M LiCl Spin at 16,g, for 2 hours Pellet Extract RNA using Trizol:ChCl3 Pellet RNA with isopropanol +LPA PCR amplify with GSP Clone Sequence 96 colonies per sample Aligns with human TTR? Yes Predicted cleavage product? Yes Positive 32

33 ALN-TTR2 Phase I Study Results Detection of Circulating Cleavage Product Using 5 RACE Subject # Dose (mg/kg) # of TTR-aligned Clones With Predicted Cleavage Pre Post 13 PBO h post-dose p < 2.2e-16 by Mantel-Haenszel chi-squared test Predicted cleavage product detected in blood 24h post-treatment in all 3 subjects at.3 mg/kg dose 33

34 Mean % Serum TTR Knockdown Relative to Baseline ALN-TTR2 Phase I Study Results Correlation of TTR Knockdown in NHP and Human at.3 mg/kg 2 ALN-TTR2 Human (n=3) NHP (n=3) sirna Dose Day 34 B.U. Med. Center, July 212

35 ALN-TTR2 Phase I Trial Safety and Tolerability - Overall TEAEs 1%.1 (n=3).5 (n=3).15 (n=3).3 (n=3).5 (n=1) Overall ALN-TTR2 (N=13) Placebo (N=4) Preferred Term n (%) n (%) n (%) n (%) n (%) n (%) n (%) Skin erythema 1 (33.3%) 2 (66.7%) (%) 2 (66.7%) 1 (1%) 6 (46%) 2 (5%) Infusion reaction (%) (%) (%) (%) 1 (1%) 1 (7.7%) (%) Headache (%) 1 (33.3%) (%) (%) (%) 1 (7.7%) (%) Backache (%) (%) (%) (%) (%) (%) 1 (25%) Nausea (%) (%) (%) (%) (%) (%) 1 (25%) Sore throat (%) (%) 1 (33.3%) (%) (%) 1 (7.7%) (%) Chest pain (%) (%) (%) (%) (%) (%) 1 (25%) Lightheadedness 1 (33.3%) (%) (%) (%) (%) 1 (7.7%) (%) Sleepiness 1 (33.3%) (%) (%) (%) (%) 1 (7.7%) (%) All TEAEs mild or moderate in severity» Infusion reaction at.5 mg/kg; subject able to complete dose with slowing of infusion rate No SAEs, no discontinuations due to study drug LFTs remained within normal limits; no elevation of cytokines or CRP 35 B.U. Med. Center, July 212

36 The Science of RNAi Therapeutics Summary Alnylam has industrialized process for discovery of potent, stable and selective sirnas Proprietary algorithm developed for sirna design that maximizes efficacy and selectivity High throughput screening assays for sirna potency Proprietary chemistry tool kit for stabilization and immuno-silencing Screening assays for measuring potential off-target effects and immune stimulation Alnylam has robust, mechanism-based delivery platform for silencing hepatic target genes with IV and SC dosing LNPs» Clinically validated for IV dosing» 2nd generation LNPs w/ ~1-fold potency improvement over 1st generation Conjugates» Efficacy in liver via SC route in rodents and NHP» Simple formulation» Very broad therapeutic index» Plan to enter clinical testing in Q4 12 Alnylam has demonstrated translation of RNAi therapeutics in man, enabling advancement of innovative medicines to patients 36

37 Thank You

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

TIDES 2014 GalNAc-siRNA with Enhanced Stabilization Chemistry: ESC-GalNAc-siRNA May 14, 2014 Muthiah Manoharan Agenda sirna-galnac Conjugates: Background Standard Template Chemistry (STC)» Human POC of