Progress in the Development of LNP Delivery Systems for sirna Advancing LNPs to the Clinic

Transcription

1 Progress in the Development of LNP Delivery Systems for sirna Advancing LNPs to the Clinic Mark A. Tracy, Ph.D. August 4, 2010

2 Alnylam RNAi Product Platform Alnylam has built industry-leading RNAi platform capabilities Approaches to sirna discovery industrialized» Selection of potent sirnas» Stabilization vs. nuclease degradation» Minimizing off-target effects and immune stimulation» Alnylam has integrated all these key steps into a product platform Significant progress in delivery and delivery platform» Direct RNAi and Systemic RNAi» Greatest systemic delivery progress with liposomes/lipidoid nanoparticles (LNPs) Advanced to clinic» Direct delivery approaches offer the potential to address significant unmet medical needs» Conjugates show potential Efficacy observed in multiple animal models for a variety of targets» Access to many additional technologies and IP 2

3 RNA Interference Synthetic sirna dsrna Cleavage dicer Strand separation Natural Process of RNAi Targeted Gene Silencing mrna degradation RISC Complementary pairing mrna (A) n Cleavage (A) n 3

4 Delivery Technologies for RNAi Cholesterol Conjugated sirna sirna Dynamic PolyConjugates Naked sirna LNP sirna Antibody-Protamine Complex sirna Aptamer Conjugated sirna Simple saline solutions or lipophilic conjugates effective in local (direct) administration paradigms» Respiratory» CNS» Epithelial cells Major advances made in systemic/parenteral delivery» Lipid nanoparticles (LNP)» Conjugates» Polymers Other delivery technologies also being investigated 4

5 Alnylam Delivery Funnel Other Non-Lipid Particle Based Delivery Approaches Other Device-based Approaches Other Conjugates Liver Tumor Kidney CNS Immune Cells Targeting Ligand- Containing Lipid Particles (LNPs) Liver Tumors Immune Cells Polymeric Conjugates Liver Lipid Particles (LNPs) Liver Extra-Hepatic Tumors Immune Cells Device-Based CNS RtoD Lipid Particles (LNPs) Liver (ALN-TTR02, ALN-PCS) Clinic Device-Based Nebulized Solution Lung (ALN-RSV01) Lipid Particles (LNPs) Liver Tumors TTR Amyloidosis (ALN-VSP02, ALN-TTR01) Delivery Research Pre-Clinical Development (pre-ind) Clinical Development (post-ind) 5

6 Key Delivery Attributes of LNPs Fully Enabled for Delivery to Cytoplasm Liver directed» Key variable: size» Targeting ligand optional Access to multiple cell types in liver» Including hepatocytes, tumor cells Effective at delivery to cytoplasm» Key variables: lipid chemistry and formulation/process Versatile» Capable of extra-hepatic delivery 6

7 Lipid Nanoparticle Particle (LNP) R&D Approach Principles 1. Advance current generation formulations rapidly to clinic in areas of significant unmet medical need 2. Maintain ongoing formulation and delivery research to identify new formulations 3. Translate next generation formulations to clinic 7

8 Alnylam Development Pipeline Key Programs Discovery Development Phase I Phase II Phase III RSV Infection ALN-RSV02 (Pediatric) ALN-RSV01 (Adult) Liver Cancers ALN-VSP TTR Amyloidosis ALN-TTR02 ALN-TTR01 PCSK9/Hypercholesterolemia ALN-PCS Huntington s Disease ALN-HTT Alnylam Proprietary Programs Co-development Programs Availability of delivery systems translatable to clinic is a key driver for pipeline development 8

9 LNPs for Systemic RNAi Multi-component lipid formulation» Cationic/Ionizeable lipid» Fusogenic lipid» PEG lipid» Cholesterol Highly efficient for liver delivery» Hepatocyte-specific gene silencing achieved Low surface charge Small uniform size particle < 100 nm 9

10 Liver Lungs Kidney Heart Femur Thymus Small intestine Muscle Fat % Injected Dose % Injected Dose Liposomal sirna Delivery to Liver Mouse 100 Clearance from Plasma Detection of Cy3-siRNA in Liver Time 100 Detection in Tissues 30 mins Composite Control 10 mg/kg 3 mg/kg 1 mg/kg 10

11 ALN-VSP for the treatment of solid tumors with liver involvement Liver is common site of primary and metastatic cancer» ~650,000/yr incidence of HCC WW» ~500,000/yr incidence of liver metastasis WW ALN-VSP is a dual-target product targeting KSP for proliferation and VEGF for angiogenesis ALN-VSP is a sterile lipid nanoparticle formulation of sirna with lipid excipients in aqueous isotonic buffer» Tekmira Pharmaceuticals collaboration» Particle diameter ~80 nm» 1:1 molar ratio of VEGF and KSP sirnas» Prolongs half-life vs. unformulated sirna» Distribution predominantly to liver and spleen Organs with fenestrated vascular endothelium» Distributes to hepatic and extrahepatic tumors with porous microvasculature Phase I trial initiated March 2009» Patients enrolled across multiple dose levels Liver Cancer Program ALN-VSP KSP sirna VEGF sirna ALN-VSP 11

12 DLinDMA:DPPC:mPEG2000-C-DMA:Cholesterol Liposome Formulation Composition and Chemistry 1,2-Dilinoleyloxy-N,N-dimethylpropylamine (DLinDMA) 1,2-Di palmitoyl-sn-glycero-3-phosphocholine (DPPC) Cholesterol (Cholest-5-en-3 -ol) O O N H O 3-N-[( -Methoxy poly(ethylene glycol) 2000 )carbamoyl]-1,2-dimyristyloxypropylamine (mpeg2000-c-dma) O O n O Nature, 441, ,

13 A1R A1G A1W A2R A2G A2W B1R B1G B1W B2R B2G B2W C1R C1G C1B C1W C2R C2G C2W Rel. hgapdh mrna ug/ml AFP Rel. hksp mrna (%) Rel. hvegf mrna (%) Efficacy in Target Cell Type: Anti-Tumor Activity of ALN-VSP Murine Liver Cancer Model Tumor KSP PBS LUC* ALN-VSP (mg/kg) Tumor GAPDH; Individual Animals Tumor VEGF PBS LUC* ALN-VSP (mg/kg) Serum AFP Levels 500 LUC ALN-VSP PreRx EndRx*** ALN-VSP reduces tumor burden and expression of tumor KSP and VEGF Human GAPDH mrna levels reflect tumor burden Human KSP or VEGF mrna levels derive from Hep3B tumor Inhibition of tumor growth (AFP levels) and significant survival benefit ISBTC, Oct 2008 PBS LUC** ALN-VSP** *4 mg/kg dose **3 mg/kg dose ***ALN-VSP: d32(n=8) Control: d28 (n=1), d32 (n=3), d33 (n=3) 13

14 ALN-VSP02 Phase I Study Design Screening Cycle 1 Cycle 2 Cycle 3 Cycle 4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 DCE-MRI Tumor biopsy Plasma samples for VEGF/PLGF CT scan for tumor measurement Rx Rx Rx Rx Rx Rx Rx Rx d3-5 d8-10 d3-6 Adverse events 14 Dose levels and dosing schedule 0.1, 0.2, 0.4, 0.7, 1.0, 1.25, 1.5, 1.7 mg/kg cohort design, expansion phase of 20 pts at MTD (10 in US, 10 in Spain) 15-min IV infusion q2 wks; premed with steroids, H1 and H2 blockers, acetaminophen Cycle = 2 doses (1 month), tumor measurements after every 2 cycles, treat until disease progression» ALN-VSP extension study for pts remaining on study beyond 4 cycles ASCO, June 2010

15 ASCO, June ALN-VSP02 Phase I Study Summary Interim results from Phase 1 trial of ALN-VSP02 in advanced cancer patients with liver involvement» Accrual of patients in first 4 of 8 planned dose levels completed (0.1, 0.2, 0.4, and 0.7 mg/kg) Total of 19 patients to date and 62 doses administered» Dose escalation ongoing MTD not yet achieved Accrual continuing beyond 0.7 mg/kg ALN-VSP02 well-tolerated in most patients» In majority, only mild drug-related AEs at doses of mg/kg» Two mild infusion reactions (one each at 0.4 and 0.7 mg/kg) that responded to slowing of infusion» Hepatic grade 5 SAE of unclear etiology in 1 pt (0.7 mg/kg) with liver extensively replaced by tumor Occurred 5 days after 2nd dose, resulting in hepatic failure and death; deemed possibly related to drug As precaution, protocol amended to limit degree of liver involvement to <50% No clinically significant changes in liver function tests (LFTs) seen in any other patients, including 6 additional patients at 0.7 mg/kg Dose escalation beyond 0.7 mg/kg is ongoing ALN-VSP02 human plasma PK showed dose-proportional Cmax and AUC with no evidence of drug accumulation While preliminary, some encouraging PD data» Serial DCE-MRI results suggestive of anti-vegf effect in majority of treated patients» Some patients with DCE-MRI effects also had associated reduction in plasma VEGF Tumor biopsy data: pending

16 Ktrans and IAUGC Change from Baseline (%) Baseline MRI, coronal view Patient DCE-MRI Results: Patient mg/kg T1 T2 T T1 T2 T3 Ktrans2 IAUGC2 Ktrans3 IAUGC3 T: liver tumor number Ktrans2/IAUGC2: Δ DCE-MRI #1 (BL) to DCE-MRI #2 (Day 4) Ktrans3/IAUGC3: Δ DCE-MRI #1 (BL) to DCE-MRI #3 (Day 7) KTrans Baseline (BL) Pre-Dose Day 4 Post-Dose 1 Day 7 Post-Dose 1 T1 T1 T1 T2 T2 T2 T3 T3 T3 ASCO, June

17 Average Ktrans Change From Baseline (%) DCE-MRI Results Summary of Cohorts 1-4 # of evaluable liver tumors: %» 21 evaluable liver tumors in 12 patients» 19/21 tumors (90%) showed decline in Ktrans» 13 of 21 tumors (62%) had Ktrans of 40%» 8 of 12 patients (67%) had average Ktrans of 40% Patient Number Dose level: 0.1 mg/kg 0.2 mg/kg 0.4 mg/kg 0.7 mg/kg ASCO, June

18 TTR/GAPDH mrna (Relative to Control) ALN-TTR Reduces TTR mrna Non-Human Primates ALN-TTR shows dose dependent silencing of TTR mrna Single i.v. infusion of ALN-TTR or control sirna Liver mrna levels measured 48hr post-dose *** *** 47% 62% ED50 ~ 0.3 mg/kg *** p< (one-way ANOVA, Dunn s post-hoc test) 0.4 *** 82% 0.2 Keystone RNAi, Feb Control sirna ALN-TTR (mg/kg) (mg/kg) 18

19 purity [%] Z-average [nm] label claim [mg/ml] (mg/ml) DLinDMA content label claim by denaturing AX-HPLC excluded stability spec(s) release spec(s).% conf. slope(s) DLinDMA confidence expiry desired expiry AP content IC097 by IC098 IC100 IC100 inv RP-HPLC/ IC102 IC102 inv IC103 ELSDL00056 L00058 L00075 Drug Product Shelf-Life Example Data at 2-8 C ex sta sp re sp.% slo co de AP 34 IC IC IC IC IC IC IC L0 L0 L C C month month purity by denaturing AX-HPLC excluded stability spec(s) release spec(s).% conf. slope(s) confidence expiry desired expiry particle AP IC097 size IC098 IC100 IC100 inv Z-average IC102 IC102 inv IC103 L00056 L00058 L C month C month Shelf life stability studies at 2-8 C support a 24 months re-test (expiry) date for drug product. Additional supportive data includes: duplex ratio, % encapsulation, PEG-c-DMA content, cholesterol content, DPPC content, ph, osmolality, and recent 24 month data.

20 Key Attributes of sirna-containing LNPs as Drug Products High potency in vivo (multiple species including NHPs)» ED50 ~ mg/kg in rodents and NHPs achieved Delivery to organs/cells of clinical interest» Liver: Hepatocytes and cancer cells for liver cancer, TTR amyloidosis, hyperlipidemia targets Reproducible manufacturing demonstrated at clinical scale Product stability demonstrated at 2-8 C for 2 years Prior clinical and regulatory experience with LNP formulations (e.g., quality and testing requirements, packaging, administration) 20

21 Next Steps Dosing continues with ALN-VSP02 Dosing begun with ALN-TTR01 Next generation formulations show enhanced potency and ability to silence targets outside liver 21

22 % Residual Factor VII LNP Research Remarkable Potency Improvements with Novel Lipids Novel LNPs set new benchmark for systemic RNAi with ~100 fold improved potency Efficacy in rodents following single IV injection Each LNP comprised of distinct cationic/ionizeable lipid component Improvement in potency has resulted in ED 50 <0.03 mg/kg C DLinDMA DLinDAP 60 DLin-KC2-DMA-a DLin-KC2-DMA-b DLin-K-DMA 98N12-5(I) ED FVII sirna Dose (mg/kg) 22 Keystone: Advance in Biopharm., Jan 2010

23 Relative Serum FVII Levels Relative CD45 Levels Mitotic Figures Delivery Advances Expand RNAi Therapeutic Opportunities ASGPR Targeting to Liver Targeted delivery to liver via asialoglycoprotein receptor (ASGPR) Immune Cell Silencing Gene silencing in leukocytes, including macrophages and dendritic cells Control sirna CD45 sirna Efficacy in Extra-Hepatic Tumors Efficient delivery of sirnas to extra-hepatic tumors, e.g., lymph nodes Monoasters Simple mitotic figures 0.0 PBS ilnp 0.15% GalNAciLNP 0 Macrophages Dendritic Cells 0 Control sirna ALN-VSP Keystone: Advance in Biopharm., Jan

24 Delivery of RNAi Summary: LNP Advances Significant Recent Progress in Systemic Delivery LNPs are a leading approach for systemic delivery of sirna Highly potent Current generation (DLinDMA) LNPs are progressing in the clinic (ALN- VSP02, ALN-TTR01)» Early data is promising 2 year shelf life and reproducible manufacturing at scale demonstrated Versatile» Demonstrated tissue distribution to multiple cell types of therapeutic interest (liver/extra-hepatic, immune cells)» Improved potency demonstrated through chemistry and formulation R&D Dramatic ongoing improvements in potency of LNPs» In past two years, ~100x improvement in potency resulting in expected increase in therapeutic index» Single digit g/kg ED50 achieved» Next generation LNP formulations advancing toward clinic LNPs are promising sirna delivery systems with the potential to create novel products addressing significant unmet medical needs 24

25 Acknowledgments Alnylam Research Pre-clinical CMC Clinical Regulatory Program Management BD AlCana/UBC Pieter Cullis Tom Madden Mick Hope Max Planck Institute Marino Zerial MIT Dan Anderson Bob Langer Tekmira Ian MacLachlan Pete Lutwyche 25

26 Progress on LNP-Mediated Delivery of RNAi Therapeutics Martin Maier, PhD August 4, 2010

27 Lipid Nanoparticles for Systemic RNAi Multi-component lipid formulation» Cationic (ionizable) lipid» Structural lipid» PEG lipid» Cholesterol Highly efficient for liver delivery» Hepatocyte-specific gene silencing achieved Low surface charge Small uniform size particle (60-80 nm) 27

28 LNP Delivery Evolves Around two Distinct Classes of Lipids DLinDMA Ionizable LNP (ilnp) AlCana/UBC/Tekmira In vivo silencing in hepatocytes TETA-5-LAP Cationic LNP (clnp) MIT DLin-KC2-DMA (XTC2) ED50 C MC3 Structure similar to Dlin-KC2-DMA Neutral surface charge at physiological ph Uptake via clathrin-mediated endocytosis ApoE-dependent Higher surface charge at physiological ph Uptake via macropinocytosis ApoE-independent 28

29 Prothrombin Time (s) Relative FVII protein Relative Liver FVII mrna Relative FVII Protein clnp-mediated Dose-Dependent Silencing of Factor VII Liver Factor VII mrna Plasma Factor VII Protein in Rat PBS sicont 10mg/kg sifvii 10mg/kg sifvii 5mg/kg sifvii 2.5mg/kg sifvii 1.25mg/kg 0.0 PBS sicont 10mg/kg sifvii 10mg/kg sifvii 5mg/kg sifvii 2.5mg/kg sifvii 1.25mg/kg Prothrombin Time Durability *** * 5 0 PBS sicont 10mg/kg sifvii 10mg/kg sifvii 5mg/kg sifvii 2.5mg/kg sifvii 1.25mg/kg *** *** ** *** sicont sifvii Time (d) Nature Biotechnology (2008) 26,

30 % Control % Control ilnp-mediated Silencing of ApoB Non-Human Primate Efficacy in monkeys with Systemic RNAi after single IV injection Effects are rapid, potent, dose-dependent and durable RNAi effects are specific and lead to measurable therapeutic benefit RNAi mechanism proven in vivo * P <.05 ** 31.7 ** ** P < ** ** mrna Protein mg/kg 2.5 mg/kg 1 mg/kg 2.5 mg/kg 2 day 11 day * * P <.05 ** P <.005 >65% Inhibition * 34.1 >85% Inhibition ** Cholesterol LDL HDL Day 11 Post-Dose (2.5 mg/kg) 30 Nature (2006) 441,

31 % Residual Factor VII Improved Potency with Novel Lipids Novel LNPs set new benchmark for systemic delivery Efficacy in rodents following single IV injection Each LNP comprised of distinct cationic/ionizeable lipid component Improvement in potency has resulted in ED 50 <0.03 mg/kg C DLinDMA DLinDAP 60 DLin-KC2-DMA-a DLin-KC2-DMA-b DLin-K-DMA 98N12-5(I) ED FVII sirna Dose (mg/kg) 31 Keystone: Advance in Biopharm., Jan 2010

32 Prothrombin Time (s) Relative FVII Protein Level FVII/GAPDH relative mrna Level Relative FVII Protein Level Efficacy in mouse with systemic RNAi after single IV injection Effects are rapid, potent, dose-dependent and durable ED 50 ~ 0.01 mg/kg Next Generation LNPs In Vivo Silencing of Factor VII in Rodent mrna Silencing Protein Suppression PBS sicont sifvii (mg/kg) (mg/kg) Pharmacological Effect PBS sicont sifvii (mg/kg) (mg/kg) Durability PBS sicont sifvii (mg/kg) (mg/kg) Keystone: Advance in Biopharm., Jan PBS sifvii 1.0 mg/kg sifvii 0.1mg/kg Day 32

33 Cellular Uptake ApoE Dependence in Cultured Primary Hepatocytes Cellular uptake of sirna requires ApoE for ionizable LNPs but not cationic LNPs Primary mouse hepatocytes exposed to 20 nm clnp- or ilnp- AF647-siRNA Cultures fixed and stained with DAPI after 4 hours Control + ApoE clnp ilnp 40X Keystone: Advance in Biopharm., Jan

34 Relative to control Relative to control ilnp-mediated Silencing in Liver Is ApoE-Dependent ilnp silencing of FVII is absent in ApoE KO mice but restored by premixing with ApoE Single IV bolus of ilnp-sifvii at 0.2 mg/kg, with or without rhapoe at mg/kg FVII liver mrna and serum protein levels measured 48 hours post-dose WildType FVII Liver mrna FVII Serum Protein rhapoe ApoE -/- Mice PBS - ApoE 0.1 mg/kg LNP-siFVII (0.2 mg/kg) ApoE (mg/kg) LNP-siFVII (0.2 mg/kg) Keystone: Advance in Biopharm., Jan

35 Relative Serum FVII Protein Level Relative Serum FVII Protein Level...While Activity of clnp Appears to Be ApoE Independent ApoE -/- Mice 1.4 ilnp 1.8 clnp PBS ilnp (mg/kg) ilnp + ApoE (mg/kg) 0.0 PBS clnp clnp + ApoE 35

36 WT Internalization of ilnp is Impaired in LDLR KO Primary Hepatocytes WT and LDLR -/- mouse primary hepatocytes incubated with ilnps for 4h Imaging by automated confocal microscopy ilnp-af647sirna ilnp-af647sirna LDLR KO ilnp-af647sirna Zerial collaboration Keystone: RNAi., Jan

37 Relative Serum FVII Protein Level In Vivo Silencing Activity of ilnps Is Diminished in LDLR -/- Mice WT LDLR -/ PBS 0.1 mg/kg 0.03 mg/kg 0.01 mg/kg ilnp Mol. Therapy (2010) 18,

38 Proposed Mechanism of ApoE-Mediated Delivery of ilnps LNPs Endogenous internalized traffic through ApoEand bind fenestrated disrupt to LNPs endosome; endothelium release of liver; sirna bindintocytoplasm LDL receptor As endosome acidifies, cationic charge on vesicle increases ph ~ 5 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 Keystone: Advance in Biopharm., Jan Space of Disse Hepatocyte

39 Relative Serum FVII Protein Most Active LNPs Display Single Digit mg/kg ED DLin-K-C2 DMA MC3 C DLin-K-C2-DMA Structure similar to DLinK-C2-DMA MC Log (μg/kg) LNP ED50 mg/kg (mouse) DLin-K-C2 DMA MC C OH N N N N OH OH C HO N HO 39

40 TTR/GAPDH mrna Level Relative to PBS Relative Serum TTR Protein Next-Generation LNPs Demonstrate Highly Potent and Durable Silencing in NHPs Single i.v. infusion into NHP and measurement of liver TTR mrna 48 hrs later Serum Protein DLin-K-C2 DMA 0.03 mg/kg 0.1 mg/kg 0.3 mg/kg Day ED50 <0.03 mg/kg PBS DLin-K-C2 DMA (mg/kg) MC3 (mg/kg) C (mg/kg) 40

41 Monitoring Cellular Uptake and Silencing Automated Confocal Microscopy Evaluation of novel lipids Mechanism of uptake (e.g. role of apoe) In vitro-in vivo correlation 41

42 Content per cell (Fluoresccent Units) Dual Labeling Demonstrates ApoE-Dependent Uptake of Entire Particle BODIPY BODIPY+ApoE AF-647 AF-647+ApoE m 30m 1h 2h 4h AF647-siRNA BODIPY-XTC BODIPY Labeled lipid AF-647 tag 42 Mol. Therapy (2010) 18, h

43 GFP (RFU) % UTC sirna content per cell (RFU) Effect of Lipid pk a on Cellular Uptake and Silencing Uptake 250 w/o apoe with apoe sirna (nm) UTC pk a = 5.64 pk a = 6.44 pk a = 7.16 pk a = 10.0 Silencing w/o apoe with apoe pk a sirna (nm) UTC pk a = 5.64 pk a = 6.44 pk a = 7.16 pk a =

44 Targeted Delivery The use of targeting ligands may substantially increase» Efficiency of delivery to the target cells» Access to other tissues Applicable to both conjugate and LNP strategies Targeting approaches» Small molecules» Peptides» Monoclonal antibodies 44

45 Asialoglycoprotein Receptor (ASGPR) Targeting Tissue specific» High receptor expression on liver hepatocytes Readily endocytosed (high turnover) Well-studied design requirements for high affinity ligands Well conserved across species» Mouse and human are 89% and 80% identical to rat (HL-1) Proven delivery of cargo to liver» E.g., recombinant proteins with incomplete sialylation Asialoglycoprotein receptor (ASGR) Impact on rate of clearance 45

46 Polarization Polarization Receptor Binding of GalNAc-Containing ilnps (GalNAc) 3 -PEG DSG Cell-free receptor competition assay with ilnps containing mol% GalNAc-PEG-DSG % GalNAc 0.005% GalNAc 0.015% GalNAc 0.05% GalNAc 0.15% GalNAc 0.5% GalNAc 1.5% GalNAc % GalNAc 0.005% GalNAc 0.015% GalNAc 0.05% GalNAc 0.15% GalNAc 0.5% GalNAc 1.5% GalNAc [sirna] ng/ml [GalNAc] M Mol. Therapy (2010) 18,

47 Relative Serum FVII Level Hepatocyte Targeting by GalNAc LNPs ilnp silencing of FVII in ApoE KO mice can be rescued by incorporation of GalNAc3 Single IV bolus of ilnp-sifvii at 0.2 mg/kg, with or without % GalNAc3 incorporated FVII serum protein levels measured 48 hours post-dose 1.2 (GalNAc) 3 -PEG DSG PBS Mol. Therapy (2010) 18, ilnp FVII sirna 0.005% 0.015% 0.05% 0.15% 0.5% ilnp FVII-siRNA + %GalNAc

48 FVII mrna (% of Baseline) FVII mrna (% of baseline) PK/PD Correlation for LNP-Formulated sirna Relationship of sirna Liver Concentration to FVII mrna Silencing in Rat Tissue sirna levels for 50% silencing ~1 ng/g Corresponds to roughly molecules per cell FVII sirna (ng/g Liver) Oligo. Ther. Soc., Nov Factor VII sirna, ng/g Liver

49 Improving Potency in Animal Models: Have We Reached the Limit? Efficiency of RISC loading EC50 = 1 ng sirna/g liver sirnas/cell 1% of sirnas delivered per cell (0.5% of inj. dose) ~ 1% Efficiency LNP-FVII-siRNA ED50: 0.01 mg/kg = nmol/inj. 1 x sirnas Efficiency of cytoplasmic delivery Fraction of sirnas escapes from non-productive compartments 10 8 hepatocytes/g liver 50,000 sirnas/cell Assumption: 50% of inj. dose reaches hepatocytes 5 x sirnas 49

50 Delivery of sirna with LNPs Summary of Research Progress Systemic delivery to liver has advanced» Improved potency with LNPs by ~ 100-fold over prototype» Mechanistic understanding of LNPs Developed two distinct classes of LNPs (ilnps and clnps) Demonstrated role of apoe for delivery of ilnps Proof-of-concept for ASGR-targeted LNPs with GalNAc-based ligand Established PK/PD relationship for sirna delivered by LNP Mechanistic understanding of systemic delivery with LNPs provides basis for design and development of next generation formulations 50

51 Alnylam A. Akinc R. Alvarez S. Barros D. Butler J. Butler W. Cantley E. Carroll K. Charisse T. de Fougerolles S. De H. Epstein-Barash L. Eltepu K. Fitzgerald M. Frank-Kamenetsky R. Hutabarat R. Kallanthottathil A. Kelin V. Kotelianski Y. Landesman R. Li J. Liu M. Manoharan S. Matsuda J. Nair B. Pang W. Querbes J. Qin T. Racie J. Sapiro D. Sah M. Severgnini M. Solomon L. Speciner A. Sprague N. Svrzikapa M. Tracy A. Vaishnaw Q. Wang M. Weinstein X. Zhang C. Zurenko Acknowledgments AlCana Technologies S. Ansell J. Chen M. Hope T. Madden B. Mui Y. Tam UBC P. Cullis MPI-CBG Dresden Eugenio Fava Anja Zeigerer M. Zerial MIT D. Anderson B. Langer Tekmira I. MacLachlan P. Lutwyche 51

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