Current Status and Future Directions

Transcription

1 Delivery Roundtable Current Status and Future Directions June 25, 2009

2 Agenda RAi and the Role for Delivery Direct RAi Systemic RAi Future Strategies 5

3 RA Interference Synthetic sira dsra Cleavage dicer Strand separation atural Process of RAi Targeted Gene Silencing mra degradation RISC Complementary pairing mra (A) n Cleavage (A) n 6

4 Structure adapted from Klosterman,P. S.; Shah, S. A.; Steitz, T. A Biochemistry (1999), 38, Sense strand Making Drugs ut of siras The Challenge Characteristics M.W 12,000-14,000 Size: 2 turns of helix 40 negative charges ydrophilic ydrated heavily ca. 5.5 nm X 2 nm Biostability base pairs long 2 bp (double) 3 overhangs dsra Seed sequence (residues 2-8) Anti-sense or Guide strand 7

5 Achieving RAi as Therapy Introducing drug-like properties into siras» Potency» Selectivity» Stability Achieving delivery to target tissues/cells» PK/PD/Biodistribution» Cellular uptake 8

6 Delivery of RAi Therapeutics Key driver of success Delivery» PK/PD/Biodistribution» Cellular uptake Major progress achieved Robust in vivo efficacy Delivery Approaches» >25 Targets Conjugates Includes many un-druggable Liposomal Ps» >5 rgans Peptides Includes lung, liver, and CS Antibodies» 6 Species Includes humans 9

7 RAi Delivery Strategies Direct RAi Systemic RAi Respiratory» RSV/Influenza Multiple major diseases» Cystic fibrosis» Metabolic» Asthma/CPD» Viral disease cular» AMD/Retinopathy» Glaucoma CS» europathic pain» Spinal cord injury» untington s disease» Parkinson s disease Topical/Mucosal» Infectious disease» Cancer» Inflammation» Cardiovascular 10

8 RAi Therapeutics pportunity Large umber of Undruggable Targets ew Drug pportunities World of targets Accessible targets for small molecules/antibodies ew targets and disease ral drug space Mab/Protein Drug space on-oral oral SM RAi accessible targets» Undruggable targetst» Potent, selective lead» Rapid; 3-6 month to lead» Cross species active» Multi-targeting t» Allelic specificity» Mab-like PD 11

9 Alnylam Development Pipeline Key Programs Discovery Development Phase I Phase II Phase III RSV Infection Liver Cancers PCSK9/ypercholesterolemia TTR Amyloidosis ID 2009 candidate untington s Disease Alnylam Proprietary Programs Co-development Programs 3 programs in clinical trials in

10 Agenda RAi and the Role for Delivery Direct RAi Systemic RAi Future Strategies 13

11 Direct Delivery in CS Example: Cy3-tt sira Distribution tion After Intrastriatal Infusion Cortex Striatum Thalamus Substantia igra 14 DSA (2008)

12 orm malized CP/MBP (% PBS) Silencing of Rat CPase Infusion Duration: ~3 d Infusion rate: 10 µl/hr *** ** PBS AD (mg/ml) Direct Delivery in CS Example: An ligodendrocyte Gene Piece 0 Piece 1 *** p< ** p<0.01 vs PBS Two way AVA RA adaptor sira cleavage site GR5 1118/1119 RAi-Mediated Cleavage of CPase in Rats GSP RTGSP 3 ~299bp Predicted product Step I. Ligation of adaptor Step II. cda Synthesis Step III. PCR PBS sicp PBS sicp Relative CPase/MB BP mra (%) Silencing of Primate CPase 500 Injection Site Adjacent Site Infusion Duration: 7 days Infusion Rate: 20 µl/hr Control Animal #1 Animal # nt Regular PCR TD PCR Verified by cloning and sequencing of amplified products (18/20 clones) Querbes et al., ligonucleotides (2008) 15

13 Direct Delivery in CS Therapeutic Silencing of untingtin Strong pre-clinical data support advancement toward clinic RAi therapeutic targeting huntingtin corrects disease in animal model» Silencing of huntingtin gene» Decreased disease pathology and behavioral changes Mean # of foot slips Behavior Improvement in Disease Model 8 ormal mice 7 Control sira TT sira * p 0.01 * Mean number neuropil aggregates/ μm Improved istopathology Luc TT sira * * 0 ormal Pre-Disease Induction 7 Days Post-Disease Induction 0 Group 1 Group 2 16 PAS (2007) 104,

14 FIS Staining in Lung 4 ours after Single 10 mg/kg Dry Powder or Liquid sira Installation Luc sira DP, 4 rs, x10 o Treatment, x10 eg Ctrl sira DP, 4 rs, x10 Luc sira D5W, 4 rs, x10 17

15 Direct Delivery in Lung Example: Anti-viral Activity of RSV siras U/g lung Log10 PF mg/kg 2 mg/kg 4 mg/kg 1 LLD 0 P1 P2 P3 AL-RSV01 2 L1 L2 L3 P4 P4-MM Pgene gene Lgene sira i.n. (25-100ug) 4 hrs Virus (RSV/A 2 ) i.n. (10 6 pfu) 4 days Viral Titer in Lung RSV Symposium, Aug

16 AL-RSV01 Phase II GEMII Study uman PC for RAi Therapeutics AL-RSV01 shows statistically significant reduction in RSV infection Randomized, double-blind, placebo-controlled study (n=88) ~40% Relative reduction in infection rate (P<0.01) 01) ~95% Increase in number of uninfected subjects (P<0.01) % Infec cted Placebo Plaque Assay AL-RSV01 ~40% Reduction o. of Unin nfected P<0.01 Plaque Assay ~95% Increase P= Study Day 0 Placebo AL-RSV01 Int l Symp Res Vir Infect, Feb

17 sira Conjugates Enhance Delivery 3 5 P E.g., Cholesterol Promotes cellular uptake» Direct cell permeation ature (2004) 432, % Gen ne expression RAi Activity in vitro without transfection reagent Unmodified Cholesterol conjugated sira dose (nm) 20

18 Direct Delivery to Mucosal Tissue Intravaginal Application of Chol-siRA against ectin 1 21 Cell ost Microbe (2009) 5,84-94

19 Agenda RAi and the Role for Delivery Direct RAi Systemic RAi Future Strategies 22

20 Systemic RAi Parenteral administration increases access to multiple major diseases» Metabolic» Viral disease» Cancer» Inflammation» Cardiovascular Validated Systemic RAi approaches» Conjugates» Liposomal nanoparticles Multiple approaches» Polymers» Small molecules» Peptides» Antibodies 23

21 Chol-siRAs Silence apob in Liver and Jejunum Saline treatme ent group Liver Jejunum % ApoB mra conte ent relative to saline Chol-luc- Chol-MM unconjugated apob-1 * * Chol-apoB-1 * * Chol-apoB-2 * P< compared to saline control animals ature (2004) 432,

22 Lipophilic Conjugates SAR for Gene Silencing In Vivo 5' 3' P Y Y = or S L Cholesterol (C 27 ) * P<0.05 ** P<0.005 Lithocholic-oleyl (C 43 ) Rel. apob mra (%) * * 60 ** ** 40 Docosanoyl (C 22 ) Stearoyl (C 18 ) Lauroyl (C 12 ) 20 Myristoyl (C 14 ) 0 PBS C27 MM C27 C18 C22 C43 C12 C14 C16 Palmitoyl (C 16 ) Linoleoyl (C 18, two C=C bond) Wolfrum et al., at. Biotech. Sep

23 Biodistribution of Chol-siRAs is Lipoprotein-Mediated Chol-siRAs bind to circulating lipoproteins and traffic to tissue in a receptor-mediated fashion ature Biotech (2007). 25:

24 Lipoprotein-mediated uptake of chol-siras into tissues of Ldlr / and wildtype mice Stoffel, Keystone March

25 Liposomal anoparticles for Systemic RAi Multi-component lipid formulation» Cationic lipid» Fusogenic lipid» PEG lipid» Cholesterol ighly efficient for liver delivery» epatocyte-specific gene silencing achieved Low surface charge Small uniform size particle < 100 nm 28

26 Liposomal sira Delivery to Liver Mouse 100 Clearance from Plasma Detection of Cy3-siRA in Liver % Injected D ose % Injecte ed Dose Time 100 Detection in Tissues mins Composite Control o 10 mg/kg g 3 mg/kg g 1 mg/kg g Liver Lungs Kidney eart Femur Thymus Small intestine Muscle Fat 29

27 Relative Live er FVII mra Dose-Dependent Silencing of Factor VII Liver Factor VII mra Relative FV VII Protein Plasma Factor VII Protein 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 Prothro ombin Time (s) Relative FVII protein *** * 5 0 PBS sicont 10mg/kg sifvii 10mg/kg sifvii 5mg/kg sifvii 2.5mg/kg sifvii 1.25mg/kg *** *** ** *** sicont sifvii Time (d) ature Biotechnology (2008) 26,

28 Repeat dosing over 3 months highly effective Repeat Silencing of Factor VII Comparable potency and durability of silencing with repeat dosing o evidence for tachyphylaxis y a or immunogenicity sicont sifvii Rat n FVII protei Relative Time (days) Dose 1 Dose 2 Dose 3 Molecular Therapy (2009) 17,

29 % Co ontrol 32 Silencing apob on-uman Primate Efficacy in monkeys with Systemic RAi after single IV injection Effects are rapid, potent, dose-dependent and durable RAi effects are specific and lead to measurable therapeutic benefit RAi mechanism proven in vivo * P <.05 ** 31.7 ** ** P < ** mra Protein 1mg/kg 2.5 mg/kg 1 mg/kg 2.5 mg/kg ature (2006) 441, ** day 11 day * % Control * P <.05 ** P <.005 >65% Inhibition * 34.1 >85% Inhibition ** Cholesterol LDL DL Day 11 Post-Dose (2.5 mg/kg)

30 Alnylam Systemic Programs AL-PCS, AL-TTR, AL-VSP AL-PCS to treat hypercholesterolemia Targets PCSK9 ID candidate for 2009 AL-TTR to treat TTR amyloidosis Targets liver expressed TTR ID candidate for 2009 AL-VSP to treat liver cancer Target two key genes» KSP critical for cell division» VEGF critical for angiogenesis Phase I KSP/ VEGF PCS TTR 33

31 RAi to treat liver cancers Prevalent solid tumor and common site of metastatic disease» ~700,000/yr Incidence of CC worldwide» ~500,000/yr Patients with liver metastasis AL-VSP is dual-target product» Targeting 2 pathways increases potential therapeutic impact Proliferation: Kinesin Spindle Protein (KSP) Angiogenesis: VEGF» Liposomal nanoparticle formulation With Tekmira Pharmaceuticals AL-VSP in clinical development» Phase I liver cancer patient study Liver Cancer Program AL-VSP 34

32 RAi-Mediated Cell Cycle Arrest Murine Liver Cancer Model rthotopic tumor model with intrahepatic ep3b seeding in SCID mice Single IV bolus injection of AL-VSP or control sira Mitotic arrest (monoasters) clearly detected in VSP-treated animals KSP and VEGF target mras cleaved in tumors confirming RAi mechanism Control sira hksp mra hvegf mra AL-VSP RA adapter GR5 hksp mra 3 cleavage product hksp mra 3 cleavage product Rev3 Rev2 cda RA adapter GR5 hvegf mra 3 cleavage product hvegf mra 3 cleavage product Rev4 Rev3 cda PCR product 380 nt VSP mg/kg PCR product 210 nt VSP mg/kg Keystone: RAi, Feb 2009

33 AL-VSP Anti-Tumor Activity Murine Liver Cancer Model rthotopic tumor model with intrahepatic ep3b seeding in SCID mice AL-VSP demonstrates clear anti-tumor activity compared with controls Control sira, n=6 AL-VSP, n=7 36 Keystone: RAi, Feb 2009

34 AL-VSP Anti-Tumor Activity Comparative Efficacy vs. Sorafenib rthotopic tumor model with intrahepatic ep3b seeding in SCID mice Significant survival benefit of AL-VSP treatment vs. controls Superior survival advantage of AL-VSP vs. sorafenib treatment Control Sorafenib Soraf/Control Soraf+AL-VSP AL-VSP (%) Survival log-rank Control 20 Soraf S Soraf Soraf/Cntrl S S Soraf/Cntrl VSP p=0.002 p=0.012 p=0.020 VSP Soraf/VSP p= p=0.003 p=0.025 S Keystone: RAi, Feb 2009

35 PCSK9/ypercholesterolemia Program AL-PCS RAi to treat hypercholesterolemia Significant unmet medical need» >500,000 Patients with severe hypercholesterolemia Inadequately managed by statins ti and other drugs PCSK9 is regulator of LDL metabolism» Controls expression of LDL receptor» Validated in human genetics Attractive opportunity for Systemic RAi» PCSK9 expressed in liver» Early clinical markers of activity possible Collaboration with UT Southwestern» orton, obbs, Brown, and Goldstein 38

36 lative to pre-d dose rel RAi Silencing of PCSK9 Protein and LDLc Efficacy of PCSK9 silencing in non-human primates on-uman Primates PCSK9 plasma levels reduced by up to 70% of pre-dose levels Rapid reductions in LDL cholesterol l levels l by 40-60% Durable effects after single injection PCSK9 protein Day 4 Day 7 Day 14 Day 21 * p 0.05 PBS Control PCSK9 sira sira * rel ative to Pre-d dose LDL Cholesterol Day 4 Day 7 Day 14 Day 21 * p 0.05 * * * PBS Control PCSK9 sira sira 39 PAS (2008) 105,

37 RAi to treat significant orphan disease Transthyretin (TTR) Amyloidosis» Caused by mutation in TTR gene» Amyloid deposits in nerves and heart Familial Amyloid Polyneuropathy (FAP) Familial Amyloid Cardiomyopathy (FAC)» ~10, patients WW with FAP Clinical pathology» Typical onset ~30-50 yr» Fatal within 5-15 years» Severe pain/loss of autonomic nervous function TTR is well-validated target» uman and mouse genetics o pathology in knock-out mouse» Produced d almost exclusively l in liver (95%) Liver transplant current standard of care TTR Amyloidosis Program AL-TTR 40

38 RAi Silencing of Mutant and Wild-Type TTR Mouse and on-uman Primate Efficacy in transgenic mouse model and non-human primates Reduced mutant t V30M-TTR plasma a levels e and liver mra >90% in transgenic mice Reduced liver TTR mra levels ~80% in non-human primates httr transgenic mouse* 1mg/kg 3mg/kg 6mg/kg on-human primate** 0.3 mg/kg g 1 mg/kg g 3 mg/kg g TTR/GAPD D TTR/GAP PBS Control TTR sira sira 0.0 Control TTR sira sira 41 Keystone: RAi, Feb 2009 *Lipidoid formulation, w/ MIT; **SALP formulation, Tekmira

39 Agenda RAi and the Role for Delivery Direct RAi Systemic RAi Future Strategies 42

40 Future Strategies ptimizing LPs» ew formulations of lipids» ew lipids» Tailoring PK/PD and biodistribution Targeting ligands» ew conjugates» Targeted liposomes ssrai» ew physical properties 43

41 Liposome Formulation Development Progress Efficacy Improvement ver Time r VII % Residual Facto LP D LP C LP B LP01 LP A ED FVII sira Dose (mg/kg) Int l Symp Athero, June

42 Lipidoid Library for ovel anoparticles Library Components Alnylam-MIT lam Collaboration In Vitro Screen Q10 Q12 Q13 Q14 Q TEST Synthetic Scheme R 1 R ΔT MeI Me R 2 R 2 R 1 R2 91 R 1 R R R 1 ΔT R 2 1 R 1 R 1 ature Biotechnology (2008) 26,

43 Factors That Impact LP Pharmacology Stability of PEGylation Particle Size Molecular Therapy (2009) 17,

44 PK/Biodistribution of LPs Can Be Modified JCI (2009) 119,

45 PBS=1 relative to Initial Dosing and Lower Maintenance Dose PCSK9/GAPD Cholesterol Multiple Injections LP formulated PCS A2 (mg/kg) 0.6 otal Cholesterol elative to PBS=1 To Re PBS 3mg/kg bolus+ PBS once a week 3mg/kg bolus mg/kg per week days post initial bolus Initial 3mg/kg bolus Maintenance: 1 x wk Rats were bled one day prior to repeated dosing Int l Sym of Athero, June

46 Targeted Delivery The use of targeting ligands may substantially develop delivery solutions to increase» Efficiency» Access to other tissues Applicable to both conjugate and LP strategies Targeting approaches» Small molecules» Peptides» Monoclonal antibodies 49

47 Alnylam Delivery Strategy Two key systemic delivery strategies at Alnylam» Lipid nanoparticles Including targeting with ligands» Conjugates Including targeting with ligands A third delivery strategy» Single stranded siras An early an alternate approach to delivery of siras Agreement with Isis 50

48 Single Stranded and Duplex si-ras Physiochemical Properties Single-stranded sira MW ~ formal negative charges Flexible with ~ 1 nm width ydrophobic surfaces accessible for protein interactions» Aromatic bases (green) are exposed Double-stranded sira MW ~13,000 Two molecules (one strand pink) 40 formal negative charges Rigid with ~ 2 nm diameter Very little exposed hydrophobic surface» Aromatic bases (green) are paired and buried in duplex These different physical/chemical properties will likely result in marked differences in pharmacokinetic profiles 51

49 Importance of Mechanistic Insights RISC 52

50 Multiple Ways into the Cell Collaboration with Merino Zerial 53 Keystone: RAi, Feb 2009

51 RAi Delivery Alnylam has broad and long-term commitment to improving and expanding delivery technology Continue development of direct and systemic approaches» Many technologies under evaluation Direct RAi Systemic RAi» ew Liposomes and lipidoids» ew conjugates including peptides and antibodies» Single strand sira Basic research» Marino Zerial Alnylam approached pro-actively by academics and companies» >25 evaluations ongoing with external groups at any one time 54

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