Drug Metabolism and Pharmacokinetic (DMPK) Properties of sirna-galnac Conjugates

Drug Metabolism and Pharmacokinetic (DMPK) Properties of sirna-galnac Conjugates Anshul Gupta, Lead Scientist DSM, Alnylam Pharmaceuticals, Cambridge, MA DIA/FDA Oligonucleotide Based Therapeutic Conference, September 09-11, 2015 1

Presentation Topics RNAi Therapeutics Mechanism of RNAi Introduction to sirna-galnac conjugates Pharmacokinetic Behavior and ADME Properties of sirna-galnac Conjugates Absorption and PK profile- plasma and liver kinetics Distribution in tissues and Elimination Metabolism: Improved stability with ESC vs. STC Exposure vs. efficacy relationship Clinical translation Enhanced potency of second generation conjugates in the clinic 2

RNA Interference (RNAi) Mechanism of Action Synthetic sirna dsrna Cleavage dicer Strand separation Natural Process of RNAi Targeted Gene Silencing mrna degradation RISC Complementary pairing mrna Cleavage 4 Confidential

sirna-galnac Conjugates Subcutaneous Delivery of RNAi Therapeutics Asialoglycoprotein Receptor (ASGPR). Binds to GalNAc Ligand Highly expressed in hepatocytes (0.5-1 million copies per cell) Low to no expression in other tissues High rate of uptake Recycling time ~15 minutes Conserved across species GalNAc-siRNA conjugate ASGPR (ph>5) protein Clathrin-coated pit Recycling ASGPR GalNAc 3 Clathrin-coated vesicle RISC Endosome mrna Nucleus 5

c o n c e n t r a t i o n ( n g / m l ) c o n c e n t r a t i o n ( n g / m l ) SC Dosing Enhances Targeted Liver Exposure Comparison between SC and IV dosing- Rat 3 mg/kg P l a s m a L i v e r 1 0 0 0 0 1 0 0 0 0 0 I n t r a v e n o u s 1 0 0 0 S u b c u t a n e o u s 1 0 0 0 0 I n t r a v e n o u s S u b c u t a n e o u s 1 0 0 1 0 0 0 1 0 1 0 0 1 1 0 0 1 2 3 4 5 0 1 0 0 2 0 0 3 0 0 4 0 0 T i m e ( h ) T i m e ( h ) Plasma Parameter (IV) (SC) T max (h) 0.083 1 C max (µg/ml) 5.86 0.3 AUC 0-t (h µg/ml) 2.01 0.67 t 1/2β (h) 0.27 1 Liver Parameter (IV) (SC) T max (h) 1 4 C max (µg/g) 18.5 24.4 AUC 0-t (h µg/g) 622 1246 t 1/2β (h) 54.8 59.7 Route-dependent PK Profile differences for typical ALNY molecule 8

% o f i n j e c t e d d o s e % Administered Dose Liver Uptake Efficiency of sirna-galnac Conjugates Decreases with Increasing Dose 1 2 5 I - T y r - G a l N A c 3 - s i R N A 1 0 0 7 5 5 0 2 5 A D 2 6 1 1 5 Liver Uptake 125 li i vlabeled e r u p t a k e 3 GalNAc 3 0.5 mg/kg 1.5mg/kg 5mg/kg 10mg/kg Percent administered dose measured in NHP liver after single SC dose of ALN-TTRsc l i v e r c o n t r o l l i v e r + 1 0 m g / k g Time (hr) l i v e r + 1 0 0 m g / k g l i v e r + 1 m g / k g ( 3 0 ' ) l i v e r + 5 m g / k g l i v e r + 1 m g / k g ( 1 0 ' ) l i v e r c o n t r o l + N A c G a l 2.5 mg/kg Dose Group 5.0 mg/kg 7.5 mg/kg 10.0 mg/kg 8 19.8% 20.6% 12.4% 12.8% Theo van Berkel Collaboration 0 0 1 0 2 0 3 0 t i m e ( m i n ) Mouse (IV dose) 100mg/kg Lower dose provides higher % uptake in liver indicative of ASGPR saturation, prior to receptor turnover, at higher doses 9

C o n c e n t r a t i o n ( g / g ) L i v e r ( g / g ) Loss of Dose-Proportionality in Drug Liver Concentration at 30mg/kg sirna-galnac Rat Liver concentrations after multiple SC doses 2 0 0 0 1 5 0 0 1 0 0 0 1 0 0 0 5 0 0 0 1 0 0 2. 5 5. 0 3 0. 0 1 0 0. 0 D o s e ( m g / k g ) 3 0 0. 0 *Group dosed at 5 mg/kg is single dose 0 1 0 0 2 0 0 3 0 0 4 0 0 D o s e ( m g / k g ) Representative Pharmacological Doses: 0.1 3.0mg/kg Toxicity Doses: 30-300 mg/kg- QW x 5 Dose multiples 30x or greater, Liver tissue exposure multiples up to 100X 10

C o n c e n t r a t i o n ( g / g ) a t t = 2 4 h Rat Liver to Kidney Ratio of sirna-galnac Decreases with Increasing Doses 1 0 0 0 0 0 0 1 0 0 0 0 0 K i d n e y L i v e r 1 0 0 0 0 1 0 0 0 1 0 0 1 0 1 1 5 1 0 3 0 1 0 0 3 0 0 C max (mg/g) D o s e ( m g / k g ) Doses 1 5 10 30 100 300 Liver 0.051 0.14 0.09 0.36 0.6 1.6 Kidney 0.002 0.01 0.02 0.07 0.4 1.1 Ratio 25 14 4.5 5 1.5 1.5 AUC 0-t (h*mg/g) Doses 1 5 10 30 100 300 Liver 2.3 8.6 8.8 37.0 77.1 211 Kidney 0.28 1.1 2.8 11.1 59.0 248 Ratio 8.2 7.8 3.1 3.3 1.3 0.85 Kidney concentrations approach liver concentrations at higher doses, reflecting shift to increased % renal clearance vs. liver uptake 11

Tissue Distribution of sirna-galnac Conjugates Near Pharmacological Doses Typically <1-5 % Delivered dose Exposure is the highest in the target organ (liver), followed by kidney; Rest of the tissues examined typically have cumulative levels <1-5% of delivered dose deposited 12

13 Metabolism

Nuclease-Mediated Metabolism 3 & 5 -Exonuclease End products are mononucleotides Endonuclease Cleaves internally Localization 5 3 5 Exo- 3 Exo- Endo- Exo- and endonucleases are ubiquitously distributed in both plasma and tissues Metabolic hot spots on sirna-galnac Standard Template Chemistry (STC) S 5 AS 5 (GalNAc) 3 Metabolite profiling in liver 8h post dose Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed) 14 Manoharan, TIDES, May 2014

Metabolic Profile Time Course: In vivo NHP liver (10mg/kg SC) 8 hr - Peak Intensity 24 hr Peak Intensity Sense Strand (5-3 ) Metabolite 1 Full Length Sense Antisense Strand (5-3 ) Metabolite 35 Metabolite 1 Full Length AS 15 Metabolite 29

Liver Concentration (ng/g) ESC Leads to Higher Liver Exposure Liver Exposure and Metabolic Stability, Single SC Dose, 25 mg/kg in Mice Metabolic profiling in liver 8h post dose 1000000 Liver Exposure Standard Template Chemistry (STC) 100000 10000 S 5 (GalNAc) 3 1000 100 STC SC ESC AS 5 Enhanced Stabilization Chemistry (ESC) 10 0 50 100 150 200 Time (h) Liver t ½ : 1-2 days for STC 1-2 weeks for ESC S 5 Target Compound Tmax (h) Liver Exposure Cmax (µg/g) AUC 0-t (h µg/g) AUC 0-48 (h µg/g) AS 5 AT3 STC 2 59.5 735 735 AT3 ESC 8 285 21546 9697 = Enzymatic cleavage site (thickness reflects frequency of corresponding cleavage products observed) 16 Manoharan, TIDES, May 2014

TTR mrna levels Depicted as % of PBS Control (mttr/mgapdh) Enhanced Stabilization Chemistry (ESC) Improves Efficacy ESC design improves in vivo efficacy by 5-fold over STC design in mttrsc 120 Subcutaneous Single Dose 100 80 60 40 20 0 PBS 25 mg/kg 5 mg/kg 1 mg/kg 5 mg/kg 1 mg/kg 0.2 mg/kg mttrsc-stc mttrsc-esc 17

18 Exposure versus Efficacy

C o n c e n t r a t i o n ( g / g ) Rat PK/PD: Liver Concentration vs. PD Single & Multiple SC Dose 4 0 3 0 2 0 1 0 L i v e r c o n c e n t r a t i o n * t a r g e t % m R N A s i l e n c i n g 1 0 0 5 0 t a r g e t % m R N A s i l e n c i n g 0 0 0 2 0 4 0 6 0 8 0 Single Dose (SD)- 10 mg/kg T i m e ( D a y s ) Multi Dose (MD)- 5mg/kg (2xW; 6 doses) * Sparse sampling scheme (concentration measured at trough only following 1 st through 5 th dose; full concentration-time profile post last dose) 10 mg/kg SD and 5 mg/kg MD schedule achieved similar maximum KD (~ 92%) Minimum threshold liver concentrations to maintain maximum KD- ~ 4 µg/g achieved at a dose of 5 mg/kg; no gain in PD at doses > 5 mg/kg 19

C o n c e n t r a t i o n ( n g / g ) Low Proportion of Effective Drug Concentration in Liver Driving Efficacy Measured by quantifying Ago2 associated protein GalNAc-siRNA Application 1 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 1 0 0 0 R I S C l o a d e d / A g o 2 a s s o c i a t e d T o t a l l i v e r 1 0 Grinding Tissue 1 Liver Lysate In Lysis /IP buffer 0 1 0 0 2 0 0 3 0 0 4 0 0 T i m e ( h ) Initial data suggests: Total tissue sirna levels are generally ~100-1,000 fold higher than RISC-loaded sirna. Limited gain in efficacy with increased dose or dosing frequency. IP Incubation with Ago2 Ab + Beads Detection 20

Relative AAT levels (predose =1) % Silencing ALAS-1 mrna Pharmacodynamic Effect in Non-Human Primate ALN-CC5-20 0 20 40 ALN-AS1 60 PBS 80 1.25 mg/kg 2.5 mg/kg 100 5.0 mg/kg -10 0 10 20 30 40 ALAS-1 GalNAc-siRNAqd x5; q2d x12 Days 1.20 1.00 0.80 0.60 ALN-AAT 1mg/kg q1w x12 1mg/kg q4w X3 3mg/kg q4w x3 3mg/kg x1, 1mg/kg q4w x2 ALN-AT3 0.40 0.20 21 0.00-5 45 95 145 Days

22 Clinical Translation: NHP to Man

% Mean TTR Knockdown Relative to Baseline (± SEM) % Mean TTR Knockdown Relative to Baseline Revusiran Phase 1 Study Results Human POC for GalNAc-siRNA Conjugates Rapid (up to 94%), dose-dependent, consistent, and durable knockdown of serum TTR Generally well tolerated Excellent correlation of human to non-human primate TTR knockdown Duration of effect longer in human vs. NHP Human Human vs. NHP -20 0 20 40 0 20 40 60 80 100 Revusiran dose groups Placebo (n=3) 2.5 (n=3) 5.0 (n=3) 10.0 (n=3) Revusiran (mg/kg), qd x5; qw x5 Days 60 80 Human NHP 100 0 10 20 30 40 50 60 Revusiran Days Single 10.0 mg/kg Injection 23 Zimmermann, HFSA, Sep. 2013; Manoharan, TIDES, May 2014

% AT Knockdown ALN-AT3 Phase 1 Study (MAD) Pharmacodynamics and Clinical Activity: AT Knockdown Potent, dose-dependent, and durable AT knockdown at low microgram/kilogram (mcg/kg) SC doses Mean maximum AT knockdown of 59 ± 7% (p<0.05); durable, lasting ~60 days ALN-AT3 generally well tolerated in both healthy volunteers and hemophilia subjects -20 0 20 40 60 80 15 mcg/kg (N=3) 45 mcg/kg (N=6) 75 mcg/kg (N=3) *p < 0.05, relative to baseline 100 0 10 20 30 40 50 60 70 24 *Data as of 2 June 2015 Time (Days)

% AT Knockdown ALN-AT3 Phase 1 Study (MAD) Pharmacodynamics and Clinical Activity: AT Knockdown Good correlation between NHP and human knockdown Potency in humans significantly enhanced over NHP: 5-10x improved potency in humans with same slope of KD -20 Human Human vs. NHP 0 20 40 60 80 15 mcg/kg (N=3) 45 mcg/kg (N=6) 75 mcg/kg (N=3) 100 0 10 20 30 40 50 60 70 Time (Days) 25 *Data as of 2 June 2015

Mean (SEM) % PCSK9 Knockdown % PCSK9 Knockdown (relative to pre-bleed) ALN-PCSsc Phase 1 Study Results (SAD) Highly durable PCSK9 knockdown in both human and NHP Potent, dose-dependent, and durable PCSK9 knockdown observed upon SC single dose Mean maximum knockdown of 82 ± 2% (p<0.05); durable, lasting ~4 months (120 days) ALN-PCSsc generally well tolerated Enhanced potency observed in man compared to NHP; rapid onset of effect and longer duration -40-20 0 Treatment Placebo 25 mg 100 mg Human SAD 300 mg 500 mg 800 mg -20 0 20 NHP SAD 1.0 3.0 6.0 10.0 20 40 40 60 60 80 26 80 100 0 1 2 3 4 5 Months Day/Treatment combinations where N=1 not displayed Data in database as of 04 August 2015 100 0 20 40 60 80 100 ALN-PCSsc Days (mg/kg)

Summary sirna-galnac molecules can successfully be delivered predominantly to the liver at high exposure levels by virtue of GalNAc binding to ASGPR. Pharmacokinetic properties of sirna-galnac molecules developed so far, appear similar across programs including Short plasma half-life (plasma Tmax ~1 hour upon SC dosing), but long liver half-life, high liver partitioning and exposure corresponding to durable PD effects. At toxicological doses, exposure in liver and kidney become disproportional, potentially due to reduced efficiency of receptor mediated uptake. Maximum PD effects are achieved at doses < 5mg/kg. Several sirna-galnac conjugates administered to humans ( e.g. ALN-TTRsc, ALN- AT3, ALN-CC5, ALN-PCS) have been shown to achieve a rapid, dose-dependent and durable knockdown of target protein. Data achieved so far confirm human translation of the sirna-galnac conjugate platform and demonstrates PoC for Alnylam s approach of developing sirna-galnac as RNAi therapeutics. - Enhanced potency observed in the clinic for second generation molecules 27

Acknowledgements Drug Safety & Metabolism Michael Placke Renta Hutabarat Yuanxin Xu Prasoon Chaturvedi Natalie Keirstead Luke Utley Mustafa Varoglu Husain Attarwala Ju Liu Minggeng Gao Valerie Clausen Qianfan Wang Xuemei Zhang Krishna Aluri Sean Dennin Program Advisors Akshay Vaishnaw Rachel Meyers Muthiah Manoharan Sara Nochur Program Teams Tracy Zimmermann Alfica Sehgal Kevin Fitzgerald Akin Akinc Benny Sorensen Vasant Jadhav Chemistry Klaus Charisse Martin Maier Rubina Parmar 28

29 Thank You!