AsiaTIDES 2012: Formulation and Delivery of Peptides and Oligonucleotides Strategies for Delivery of RNAi Therapeutics March 1, 2012 Akin Akinc, PhD
Lead Selection Alnylam RNAi Product Platform Turning sirnas into Drugs Lead Optimization Delivery conjugation Introduce chemical modifications for drug-like properties formulation sirna design Selectivity screen» Off target effects Stabilization Potency Selectivity PK/PD Biodistribution Cellular uptake RISC loading Small Scale Gene walks In vitro assays Chemistry, Manufacturing and Controls Medium Scale In vivo biology Large Scale GMP Production Clinical trials 2
Lead Selection Alnylam RNAi Product Platform Turning sirnas into Drugs Lead Optimization Delivery conjugation Introduce chemical modifications for drug-like properties formulation sirna design Selectivity screen» Off target effects Stabilization Potency Selectivity PK/PD Biodistribution Cellular uptake RISC loading Small Scale Gene walks In vitro assays Chemistry, Manufacturing and Controls Medium Scale In vivo biology Large Scale GMP Production Clinical trials 3
% Remaining Message sirna Conjugates: An Emerging Approach to Delivery Lipophilic molecules Vitamins Carbohydrates Peptides Antibodies 3 5 sirna Ligand Free Uptake and Silencing by GalNAc-siRNA 100 80 TTR-GalNAc 1 TTR-GalNAc 2 TTR-GalNAc 3 60 40 20 GalNAc-siRNA 0 0.001 0.01 0.1 1 um sirna 4 Oligo Ther Soc., Sep 2011
Asialoglycoprotein Receptor (ASGPR) ASGPR Highly expressed in hepatocytes» 0.5-1 million copies/cell Clears serum glycoproteins via clathrinmediated endocytosis High rate of uptake Recycling time ~15 minutes Conserved across species 5 Ashwell and Morell, Adv Enzymol Relat Areas Mol Biol. 1974, 41, 99 Lee, JBC, 1982, 257, 939 Cummings et al Essentials of Glycobiology 2008, Park et al PNAS 2005
% of injected dose % Labeled Dose % Labeled Dose Features of ASGPR Support Dose Optimization 125 I-Tyr-GalNAc 3 -sirna Liver Uptake AD26115 125 I labeled 3 GalNAc 3, IV Liver Uptake 125 I labeled 3 GalNAc 3, IV 100 liveruptake 100 75 50 0.5 mg/kg 1.5mg/kg liver control liver+10 mg/kg liver+100 mg/kg liver+1 mg/kg (30') liver+5 mg/kg liver+1 mg/kg (10') liver control+nacgal 75 50 0.5mg/kg 1.5mg/kg 2 nd dose 30 1.5mg/kg 2 nd dose 10 25 5mg/kg 10mg/kg 25 100mg/kg 0 0 10 20 30 2 nd dose 0 0 10 20 30 40 50 60 time (min) time (min) Fast uptake, saturation and recycling point towards optimizing dosing regimen» Delayed release via SC dosing may help by engaging multiple rounds of receptor uptake» Multi-dose SC administration as one approach to optimize receptor utilization 6 Collaboration with Dr Theo van Berkel
Relative TTR mrna (%PBS Treated) SC Administration of TTR-GalNAc Leads to Robust Gene Silencing 120 100 30mg/kg TTR-GalNAc, IV 30mg/kg TTR-GalNAc, SC 80 60 40 20 0 0 50 100 150 200 250 300 350 400 Time Post-Dose (h) Efficacy via SC greater than via IV administration ~55% bioavailability from SC space Greater liver exposure after SC dosing» C max and AUC ~3-fold higher compared to IV dosing 7
% Serum TTR Knockdown (Relative to Control) Subcutaneous ALN-TTR Program ALN-TTRsc ALN-TTRsc achieves sustained silencing in pre-clinical animal model Multi-dose subq regimen; once weekly Sustained TTR silencing achieved over multi-week period Product differentiation across ATTR patient populations; planned IND in H2 12 0 20 ALN-TTRsc (5.0 mg/kg) 40 60 80 ALN-TTRsc (25.0 mg/kg) 100 0 2 4 6 8 10 12 14 16 18 sirna Doses Q Weekly Dosing 8 Keystone: Nuc. Acid Ther., Jan 2012 Confidential
Subcutaneous Dose Optimization Summary Receptor is available for multiple rounds of uptake» As early as 10 minutes post-initial dose (IV) SC administration enhances liver exposure of GalNAc-siRNA SC split dosing at lower doses demonstrates equivalent silencing to single high dose» Multiple schedules possible for maintenance of silencing at clinically feasible dose levels 9
ALT (U/L) sirna-galnac Conjugates are Well Tolerated In Vivo Rat Tolerability Non-GLP Single Dose Tolerability Animals: S-D rats Dose levels: 100, 250, 500 & 750mg/kg SC Clinical chemistry at 24 and 72 h Necropsy at 72 hours, histopathology of kidneys, spleen, liver, heart, testes, thymus, and injection site Results No test article-related clinical signs of toxicity or alterations in clinical chemistry No histopathology in kidneys, spleen, liver heart, testes, and thymus Non-adverse, test article-related ( 250 mg/kg) minimal histiocytic inflammation at injection site Single-dose of up to 750 mg/kg is well tolerated in rats ~150X safety margin based on ED50 ~ 5mg/kg 120 100 80 60 40 20 0 24h 72h PBS 100 ALT 250 500 750 TTR-GalNAc (mg/kg) 10
sirna-galnac Conjugates are Well Tolerated Cytokine Assessment Whole Blood Assay 2 fold change in cytokines in whole blood assay (13 cytokine panel) In vivo No IL-1b, IFN-γ, TNF-α, IL1-RA or IL6 induction at 4 or 24hrs in NHP dosed at 100mg/kg SC No cytokine induction in CD-1 mice dosed at 125mg/kg SC at multiple time points (22 cytokine panel) No erythema or edema observed at injection sites in rats or NHP 11
Normalized Serum TTR (Fraction Pre-dose) 5X 5mg/kg TTR Protein Suppression in NHP SC Single vs. Multi-Dose Regimen Study Day: 0 1 2 3 4 PD Draw Day: 5 7 9 11 14 18 22 26 32 39 25mg/kg 46 53 Study Day: 0 PD Draw Day: 1 3 5 7 10 14 18 22 28 35 42 49 5X 5mg/kg vs 25mg/kg sirna 1.4 PBS 5x5 mg/kg Control 5x5 mg/kg sirna 25mg/kg sirna 1.2 1 0.8 0.6 0.4 0.2 0-15 -10-5 0 5 10 15 20 25 30 Study Day 12
Subcutaneous sirna Conjugate Program Summary Technology Liver specific silencing via ligand-mediated uptake Simple formulation sirna-galnac in buffer Dosing, Efficacy Single dose ED50 5 mg/kg Low dose multi-dosing enables ED80 silencing Tolerability Non-GLP tolerability assessment in rats consistent with large safety margin Well tolerated with single dose NOAEL > 250mg/kg in rats Overall Goal: Human ALN-TTRsc IND submission in H2 12 13
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 2000 -C14 glyceride Cholesterol Low surface charge Small uniform size particle <100 nm DSPC 14
% Residual Factor VII Second Generation LNPs Remarkable Potency Improvements with Novel Lipids Novel LNPs set new benchmark for systemic RNAi with ~100-fold improved potency Efficacy in rodent models following single IV injection Each LNP comprised of distinct cationic lipid component Improved potency has resulted in single digit mg/kg ED 50 120 100 Dlin-MC3-DMA-b C12-200 2 nd Generation LNPs ALN-PCS (Phase I Data) ALN-TTR02 (CTA ~YE 11) 1st Generation LNPs ALN-VSP (Phase I Completed) ALN-TTR01 (Phase I Data) 80 DLin-KC2-DMA-b DLinDMA DLinDAP 60 MD1 DLin-KC2-DMA-a 40 20 ED 50 DLin-K-DMA 98N12-5(I) 0 0.0001 0.001 0.01 0.1 1 10 FVII sirna Dose (mg/kg) 15 Oligo Ther Soc., Sep 2011
LNP Intracellular Delivery Still Relatively Inefficient Despite Significant Potency Advances Formulation of gold-sirna EM Analysis of Intracellular Localization of Gold-siRNA R-S S Formulation R-S S in vitro: HeLa cells transfected at 20 nm sirna in vivo: Hepatocytes of treated mice dosed at 0.2 mg/kg sirna EM analysis of intracellular localization of gold-sirna both in vitro and in vivo indicates that only 1-2% of total cellular gold is in cytoplasm 16 Collaboration with Dr Marino Zerial, Max-Planck
Majority of LNPs Accumulate in Lysosomes AF647- sirna LAMP1- GFP 20nM-24h Significant co-localization with LAMP1, a marker of late endosomes/lysosomes Similar results obtained in vivo via imaging of hepatocytes 17 Collaboration with Dr Marino Zerial, Max-Planck
DLinDMA (ng/ml) DLinDMA (ng/g) Stable Ionizable Lipids Are Persistent In Vivo Formulation: SNALP (DLinDMA) Dose: 1 mg/kg (sirna) via 15 min IV infusion Animals: SD rats DLinDMA (1, 2-Dilinoleyloxy-3-dimethylaminopropane) Plasma Tissues 10000 1000000 100000 1000 10000 100 1000 100 10 10 Liver Spleen 1 0 50 100 150 200 1 0 50 100 150 200 Time (h) Time (h) Stable ionizable lipids have long elimination half-lives in plasma and tissues 18
Higher Doses of Persistent LNPs Result in Cytotoxicity in Target Tissues LNP GLP Toxicology Summary 4-Dose rat and NHP GLP studies Major clinically-significant target organs: liver and spleen» Liver primary and spleen secondary in rat; spleen primary and liver secondary in NHP» Liver findings: necrosis, incidence and severity increasing with dose; correlates with ALT/AST increases» Spleen findings: vacuolation, histocytic infiltration, lymphoid depletion, incidence and severity increasing with dose NOAEL rat = 1 mg/kg (based on liver findings), NHP = 1-3 mg/kg (based on spleen findings) MTD rat = 3 mg/kg, NHP = 6 mg/kg Safety pharmacology generally unremarkable and mutagenicity studies negative 19
A New Class of Lipids For Rapid Eliminated LNPs (relnps) Rationale Stable ionizable lipid components of LNPs are persistent in cells and tissues may result in accumulation over time and may be responsible for cytotoxicity Novel, rapidly eliminated lipid components are less likely to accumulate with chronic dosing and should be better tolerated Approach Design functional ionizable lipids that are biodegradable in vivo» Ideally enzymatically degradable for rapid degradation in vivo, but chemically stable for long shelf life» Lipid degradation products should be easily metabolized or eliminated 20
Design of Degradable Linkages Within Lipid Chains Expected Initial Metabolic Products Internal Chain Ester Lipid Terminal Chain Ester Lipid Mono-acid (mono-ester) Mono-acid (mono-ester) Di-acid Di-acid 21
Design of Degradable Linkages Within Lipid Chains Metabolites as Potential Substrates for b-oxidation Pathway of Fatty Acids Lipid Chain Modifications Additional enzymes 2,4-dienoyl CoA reductase and cis-δ 3 -enoyl isomerase used to oxidize unsaturated fatty acids 22
Lipid (ng/ml) Novel Biodegradable Lipids Are Rapidly Eliminated From Plasma In Vivo relipid 1 and relipid 2 undetectable in plasma after 8 hours 100000 10000 DLinDMA relipid 1 relipid 2 1000 100 10 1 0 100 200 300 400 Time (h) 0.3 mg/kg (sirna) dose via IV in mice 23
Lipid (ng/g) Lipid (ng/g) Novel Biodegradable Lipids Are Rapidly Eliminated From Tissues In Vivo relipid 1 undetectable in liver and spleen after 8 h and 24 h, respectively relipid 2 undetectable in liver and spleen after 2 h and 8 h, respectively 100000 Liver 10000 Spleen 10000 1000 1000 100 100 10 relipid 1 relipid 2 1 0 100 200 300 400 Time (h) DLinDMA 10 1 0 100 200 300 400 Time (h) 0.3 mg/kg (sirna) dose via IV in mice 24
Rapidly Eliminated LNPs (relnps) Are Well-Tolerated In Vivo Single Dose Toxicology Animals: S-D rats Dose levels: 1, 3, 5, and 10 mg/kg Serum chemistry at 24 and 72 h Necropsy at 72 hours, histopathology of liver and spleen Results No adverse clinical signs noted No significant alterations in serum chemistry at 24 or 72 h No significant histopathology findings (liver and spleen only) NOAEL > 10 mg/kg ALT (24 hours) 25
Relative FVII Protein Level Relative Liver TTR mrna relnps Are Efficacious in Rodents and NHPs relnps maintain potency gains of 2 nd Generation LNPs 1.4 Efficacy in Mice (FVII) 1.2 Efficacy in NHPs (TTR) 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.2 0.4 0.2. 0 PBS 0.1 0.03 0.01 0.003 relnp (mg/kg) 0 Naive relnp sittr 0.3 mg/kg 26
relnps Represent 3 rd Generation LNPs With Significantly Improved Therapeutic Index Derived from LNP research efforts focusing on both mechanistic understanding and lipid chemistry Equivalent potency and substantially improved tolerability relative to second generation LNPs Formulation optimization and in vivo testing on-going Improvement of Therapeutic Index from 1 st to 3 rd Generation LNP 0.01 mg/kg 0.1 mg/kg 1.0 mg/kg 10 mg/kg Rat 2 nd Gen. 1 st Gen. 1 st Gen. ~ 30x ~ 5x 2 nd Gen. 3 rd Gen. ~ 300x 3 rd Gen. Key Efficacy NOAEL Efficacy based on FVII protein reduction NOAEL based on single dose rat tox. 27
Systemic RNAi Delivery Summary Conjugates Efficacy in liver via SC route in rodents and NHP Simple formulation Promising preliminary toxicology Plan to enter clinical testing in H1 2013 Lipid nanoparticles (LNPs) Clinically validated approach for delivering RNAi therapeutics to liver (POC established for 1st and 2nd generation LNPs) Platform continues to advance rapidly» ~100-fold potency improvement over 1st generation» 3rd generation LNPs emerging, suggest substantially improved therapeutic index 28
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