12th US-Japan Symposium on Drug Delivery Systems Advances in Systemic Delivery of RNAi Therapeutics. Martin Maier

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1 12th US-Japan Symposium on Drug Delivery Systems Advances in Systemic Delivery of RNAi Therapeutics Martin Maier December 18, 213

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

3 Outline GalNAc-siRNA Conjugate Platform» Ligand design for targeted delivery to hepatocytes» GalNAc-siRNA conjugates for subcutaneous administration PK and PD of GalNAc-siRNA Conjugates Translation Across Species 3

4 GalNAc-siRNA Conjugates Targeted Delivery to Hepatocytes via ASGPR-Mediated Uptake ASGPR Clears desialylated serum glycoproteins via clathrin-mediated endocytosis Highly expressed in hepatocytes».5-1 million copies/cell High rate of uptake Recycling time ~15 minutes Conserved across species GalNAc-siRNA GalNAc ligand conjugated to chemically modified sirna for targeted delivery to hepatocytes GalNAc carbohydrate cluster with high affinity for ASGPR (nm) Administered subcutaneously (SC) GalNAc-siRNA conjugate ASGPR Target protein mrna cleavage RISC loading Clathrin-coated pit Recycling ASGPR Clathrin-coated vesicle Endosome Adapted from Essentials of Glycobiology (29) 4

5 Carbohydrate Recognition Domain of ASGPR Subunit H1 Carbohydrate Recognition by ASGPR Structure and Carbohydrate Binding of H1-CRD* 5 *Meier et al., J. Mol. Biol., 3: (2)

6 MFI Design of High-Affinity Ligand for ASGPR Spacing of sugar residues Branching point Attachment to sirna Valency Spacer length C2-substituent Spacer length Y-shaped linker 4 Kd = 2.48 nm 3 2 Kd = 28.8 nm Ligand Mouse Hepatocyte Ki Adapted from Lee et al., Carbohydrates in Chemistry and Biology; 4:549 (2) 1 (GalNAc)3 (GalNAc) [(GalNAc)n] nm GalNAc 2 GalNAc 3 ~24 nm 2.7 nm 6 Biessen et al., J. Med. Chem., 38:1538 (1995); Rensen et al., J. Med. Chem., 47:5798 (24); Biessen et al., Bioconjugate Chem., 13:295 (22)

7 Binding affinity or efficacy Ligand Design Impact of Carbohydrate Structure and Valency Receptor Binding Affinity and In Vivo Efficacy Rel. to Trivalent (GalNAc) Rel. Binding Affinity (KiGalNAc3/Ki) Est. rel. in vivo efficay (Gal) 3 or (GalNAc) 3 rel. to (GalNAc) (Gal)3 (GalNAc)2 ~5-Fold decrease in binding affinity for sirnas containing (GalNAc)2 or (Gal)3 (prim. mouse hepatocytes) ~2-Fold loss of in vivo efficacy (single dose efficacy screen in mouse) (GalNAc) 2 7

8 Relative Uptake (MFI) 18 Uptake of GalNAc-siRNA Conjugates 1 o Mouse Hepatocytes sirna Glu 2 -sirna GalNAc 2 - sirna GalNAc 3 - sirna +EGTA +G3BB (38) ASGR2 KO Cells GalNAc 3 -sirna + Glucose conjugate does not mediate uptake GalNAc 3 BB & EGTA block uptake Substantially decreased uptake in ASGR2 KO cells G3BB 8

9 % Labeled Dose % Labeled Dose IV Administration of 125 I-Labeled sirna-galnac Conjugate % of Injected Dose in Liver Repeat Dose & Re-uptake.5 mg/kg 1.5mg/kg 5mg/kg 1mg/kg 1mg/kg Rapid uptake, receptor saturation and recycling Extended plasma exposure may help by engaging multiple rounds of receptor uptake Slow release after SC administration may improve receptor utilization 9 Collaboration with Dr Theo van Berkel

10 % mrna Remaining Relative to PBS Drug level in Liver g/g Mode of Administration Impacts Pharmacology IV vs. SC Comparison of Efficacy and Drug Level in Mouse Liver 12 TTR mrna KD in Liver 12 sirna in Liver SC 24 h IV SC 24 h IV 1 Oligo Ther Soc., Sept. 212

11 sirna/ tissue (ng/g) TTR-GalNAc sirna Biodistribution After Single SC Administration GalNAc-siRNA conjugates efficiently target liver Achieve liver levels of >5% delivered dose 12 4 hours Post-Dose Liver Spleen Kidney Heart Lung 11 Oligo Ther Soc., Sept. 212

12 Synthesis of GalNAc Support and Conjugate 1 GalNAc 2 Tris 3 trans-4-oh-pro-oh 4 Solid Support Multi-step synthesis GalNAc Solid Support 1 Synthesis/Process Developed at Alnylam Compatible with multiple solid supports Compatible with solid phase RNA synthesis and deprotection conditions GalNAc Solid Support Convergent multi-step synthesis Scalable to multi-kilo scale sirna-galnac Synthesis Solid phase phosphoramidite chemistry Scalable to multi-kilo scale GalNAc-siRNA Conjugate 12

13 % hpcsk9 Knockdown % Antithrombin Knockdown % TTR Knockdown Potent, rapid, dose-dependent and durable target knockdown with SC administration Any hepatocyte target gene ED 8 levels for target knockdown ranging from.5 5 mg/kg» Generally achieved with qw dosing regimen» More potent conjugates reflect benefit of continued platform improvements Doses of 2.5 mg/kg enable SC injection at volumes 1 ml ALN-PCSsc targeting PCSK9 to lower cholesterol for the treatment of hypercholesterolemia GalNAc-siRNA Conjugates Broad Platform for SC Delivery of RNAi Therapeutics PCSK9 2. mg/kg 1. mg/kg.5 mg/kg ALN-TTRsc targeting TTR for the treatment of transthyretin-mediated amyloidosis Control 25 mg/kg 5. mg/kg 2.5 mg/kg sittrsc, qw x12 Transthyretin Day ALN-AT3 targeting AT to restore thrombin generation in in hemophilia and rare bleeding disorders -2 2 Antithrombin 3. mg/kg 1.5 mg/kg.75 mg/kg ALN-PCSsc qd x5; biw x5 Day ALN-AT3, qw x5 Day

14 Outline GalNAc-siRNA Conjugate Platform» Ligand design for targeted delivery to hepatocytes» GalNAc-siRNA conjugates for subcutaneous administration PK and PD of GalNAc-siRNA Conjugates Translation Across Species 14

15 PK/PD Study Design ALN-AT3 Pre-clinical Studies in Wild Type Mice Group sirna Dose (mg/kg) Conc. (mg/ml) Route No. of Males 1 AT3 1.1 SC 44 2 AT SC 44 3 AT3 5.5 SC 44 Blood and Tissues Collection Time Points.83,.25,.5, 1, 2, 4, 8, 24, 48, 96, 12, 168, 24, 336, 48, 54, 576, 672, 744, 84, 912 and 18 h post-dose Male C57BL/6 mice (2-3 grams) Organs: Liver, spleen, kidney, blood (n=2 /time point/group) at specified time points. qpcr assay - LLOQ Plasma =.4 ng/ml & Liver =.4 ng/g RISC-loaded sirna levels were determined by Ago2 IP followed by RT-qPCR Plasma and liver samples at each time point were analyzed for AT protein in Plasma and AT mrna in liver 15

16 sirna conc. in plasma (ng/ml) sirna conc. in liver (ng/g) Time-Concentration Profiles in Plasma and Liver ALN-AT3 Pre-clinical Studies Plasma 1 mg/kg 2.5 mg/kg 5 mg/kg Liver Time (h) ALN-AT Time (h) ALN-AT3 AT3 Conjugate in Plasma Dose (mg/kg) Apparent t1/2β (h) Tmax (h) Cmax (µg/g) AUC-t (h µg/g) mg/kg mg/kg 5 mg/kg Time (h) ALN-AT3 AT3 Conjugate in Liver Dose (mg/kg) Apparent t 1/2β (h) Tmax (h) Cmax (µg/g) AUC -t (h µg/g)

17 % Knockdown AT mrna (Relative to Control) % Knockdown AT protein in serum (Relative to Control) Pharmacodynamic Profiles Single S.C. Dose of ALN-AT3 Potent and durable silencing achieved after single s.c. dose of ALN-AT3 Full recovery to baseline 4 days post-dose Comparable PD profiles for AT mrna and plasma protein Silencing of AT mrna Reduction of AT Protein mg/kg mg/kg 5 mg/kg Time (h) ALN-AT3 4 1 mg/kg mg/kg 5 mg/kg Time (h) ALN-AT3 17

18 % Knockdown AT mrna Relative to control RISC-loaded sirna (ng/g) Total sirna (ng/g) RISC-loaded sirna (ng/g) % Knockdown AT mrna Relative to control Total sirna in liver (ng/g) 1 Total and RISC-loaded sirna Total vs. RISC-loaded sirna in Liver Silencing Correlates with RISC-Loaded sirna Total sirna vs. mrna Silencing Time (h) ALN-AT3 2.5 mg/kg Time (h) ALN-AT3 2.5 mg/kg 18 Tmax of RISC-loaded sirna shifted relative to total sirna Rate of depletion of RISC-loaded sirna slower than total sirna Amount of RISC-loaded sirna correlates well with silencing activity RISC-loaded sirna vs. mrna Silencing ALN-AT3 2.5 mg/kg Time post - dose (h)

19 % AT mrna Silencing % AT mrna Silencing PK/PD Relationship Total ALN-AT3 in Liver Target gene silencing achieved at low liver tissue exposure EC 5 at ~.1 g/g tissue (>12 h post-dose)» Compares very favorably with other oligonucleotide platforms requiring >1 g/g tissue levels Total sirna in liver (µg/g) 6 R 2 = Total sirna in liver (µg/g) 19 1 Mipomersen FDA Advisory Committee Briefing Document, October 212

20 % AT mrna Silencing % AT mrna Silencing PK/PD Relationship RISC-Loaded ALN-AT3 Target gene silencing achieved at very low RISC-loaded sirna concentrations EC 5 at ~1.5 ng/g tissue (~8 molecules/cell)» ~1x lower than EC 5 values obtained for total sirna R² = RISC-loaded sirna in liver (ng/g) RISC - loaded sirna in liver (ng/g) 2

21 % Antithrombin Knockdown Normalized to Pre-Dose and PBS Maintenance of AT Knockdown with Repeat Dosing ALN-AT3 Pre-clinical Studies Weekly SC administration of ALN-AT3 in WT mice results in potent and consistent AT suppression Repeat dose ED 5 for AT knockdown <.75 mg/kg Nadir reached at ~day 14-2 ALN-AT3 (mg/kg) ALN-AT3, qw x5 Day 21 WFH, July 212

22 sirna in liver (ng/g) Drug Levels in Tissue During Chronic Dosing ALN-AT3 Pre-clinical Studies 1 Drug Levels in Mouse Liver Actual Data ALN-AT3, dosing (qw x 14) Day.2 mg/kg qw.5 mg/kg qw Tissue collection post-dose (4 h) Pre- and postdose (4 h) Pre- and postdose (4 h, 3 days) Pre- and postdose (4 h) Pre- and postdose (4 h) 22

23 sirna in liver (ng/g) Drug Levels in Tissue During Chronic Dosing ALN-AT3 Pre-clinical Studies 1 Drug Levels in Mouse Liver Actual data with interpolated data added 1 11 ng/g 4 ng/g 1.2 mg/kg qw 1.5 mg/kg qw ALN-AT3, qw x 14 Day No evidence for drug accumulation during chronic dosing (after 3 rd weekly dose) Mean steady state liver drug load of 4 and 11 ng/g at.2 and.5 mg/kg qw, respectively Mean liver drug load proportional to dose 23

24 % Knockdown Normalized Serum TTR (Fraction Pre-dose) Long Term Conjugate Pharmacology Example: Ongoing Chronic Dosing Study in Mice w/ TTR GalNAc-siRNA Steady knockdown maintained with chronic dosing Sustained knockdown at both ED5 (1 mg/kg) and ED8 (2.5 mg/kg) dose levels» Rodent orthologue with improved potency compared with ALN-TTRsc Demonstrated absence of tachyphylaxis or sensitization No changes in serum TTR levels in PBS control group -2 PBS 1. mg/kg 2.5mg/kg mttr sirna, qw dosing Time (days) 24

25 Outline GalNAc-siRNA Conjugate Platform» Ligand design for targeted delivery to hepatocytes» GalNAc-siRNA conjugates for subcutaneous administration PK and PD of GalNAc-siRNA Conjugates Translation Across Species 25

26 % Mean AT Knockdown (Pre-dose = 1) Peak Thrombin (nm) ALN-AT3 Pre-Clinical Efficacy Potent and Durable Pharmacologic Effect in NHP ALN-AT3 achieves potent AT knockdown and fully corrects thrombin generation in NHP Weekly SC doses result in potent, dose-dependent, and durable AT knockdown over >5 mos In NHP hemophilia inhibitor model, ALN-AT3 fully restores thrombin generation to normal levels % AT reduction 2 1 ** (p<.1) % AT reduction Day.5 mg/kg qw x 8 Recovery.125 mg/kg qw x 12 Pre-dose Saline.25.5 ALN-AT3 (mg/kg) qw Normal Induced HA 1 mg/kg q2w x mg/kg qw x 5 26 ISTH, July 213 Recovery.25 mg/kg qw x 12 Recovery

27 % PCSK9 Knockdown or LDL-C Lowering ALN-PCSsc Pre-Clinical Efficacy Potent PCSK9 Knockdown and LDL-C Lowering in NHP ALN-PCSsc achieves potent PCSK9 knockdown and LDL-C lowering with SC dosing Up to 95% PCSK9 knockdown and up to 67% lowering of LDL-C in absence of statins in NHP Durability >5 days after last dose; supports q2w dosing, possibly q4w Development Candidate selected; IND in late 14 LDL-C PCSK ALN-PCSsc, 2 mg/kg Day qd x5; qw x3 27 AHA, Nov. 213

28 % Mean TTR Knockdown Relative to Baseline (± SEM) Mean (SEM) % TTR KD in human ALN-TTRsc Phase I Study Results TTR Knockdown in Multi-Dose Cohorts Randomized, double-blind, placebo-controlled SAD and MAD study in healthy volunteers Rapid, dose-dependent, consistent, and durable knockdown of serum TTR» Statistically significant knockdown of serum TTR at all doses evaluated (p<.1)» Up to 94% TTR knockdown; Mean TTR knockdown of 87.5% and 92.4% at 5. and 1. mg/kg, respectively Generally safe and well tolerated» Only AEs associated with drug were generally mild ISRs, resolving within ~2 hours of onset Excellent correlation of human to non-human primate TTR knockdown on mg/kg basis» Confirmation of human translation of GalNAc-siRNA conjugate platform ALN-TTRsc (mg/kg) ALN-TTRsc dose groups Placebo (n=3) 2.5 (n=3) 5. (n=3) 1. (n=3) 5 25 R 2 =.83 p <.1 ALN-TTRsc (mg/kg), qd x5; qw x5 Time (days) Mean (SEM) % TTR KD in NHP 28 Heart Failure Society of America, Sept. 213

29 GalNAc-siRNA Conjugates Summary Broad therapeutic platform for any hepatocyte target gene utilizing high capacity receptor-ligand system enabling convenient multi-dosing paradigm» Sub-cutaneous administration» Potent and durable target knockdown demonstrated for multiple targets» No evidence for drug accumulation in target tissue during chronic dosing» Sustained target knockdown without tachyphylaxis or sensitization during long term chronic dosing Rational design and efficient synthesis of trivalent GalNAc ligand» High receptor binding affinity enabling efficient in vivo delivery to hepatocytes» Successful large scale manufacture of sirna-conjugates PK/PD evaluation confirms potency of RNAi» EC 5 for RISC-loaded conjugate: ~1.6 ng/g (~8 molecules/cell)» EC 5 for total conjugate in liver: ~.1 ug/g Wide therapeutic index with favorable safety profile Excellent translation to higher species with potent and durable target knockdown in non-human primates and in human 29

30 Alnylam Akin Akinc Scott Barros Brian Bettencourt Anna Borodovsky Ben Brigham Jim Butler Amy Chan Klaus Charisse Valerie Clausen Rick Duncan Shannon Fishman Don Foster Kevin Fitzgerald Minggeng Gao Greg Hinkle Renta Hutabarat M Jayaraman Rajeev Kallanthottathil Pachamuthu Kandasamy Pia Kasperkovitz Alex Kelin Varun Kumar Matthias Kretschmer Satya Kuchimanchi Abigail Liebow Ju Liu Mano Manoharan Shigeo Matsuda Rachel Meyers Stu Milstein Jay Nair Lubo Nechev Sara Nochur Sue Panesar Bo Pang William Querbes June Qin Alfica Sehgal Acknowledgements Tanya Sengupta Sarfraz Sheik Jon O Shea Svetlana Shulga Morskaya Nate Taneja Jessica Sutherland Akshay Vaishnav Garvin Warner Jennifer Willoughby Kristina Yucius Bill Zedalis Xuemei Zhang Ligang Zhang Tracy Zimmermann Collaborators Theo van Berkel, Leiden/Amsterdam Center for Drug Research, University of Leiden, Netherland Maria Saraiva, Amyloid Unit. Institute of Molecular and Cellular Biology, Portugal Claude Negrier, Hemophilia Treatment Center, University Claude Bernard, Lyon, France 3

31 Thank You