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Investigational ALN-HBV Mediates Profound and

Durable HBsAg Silencing In Vitro and In Vivo

Laura Sepp-Lorenzino

November 15, 2015

The Liver Meeting 2015®

American Association for the Study of Liver Disease

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siRNA-GalNAc Conjugates SC-Administered Platform for Targeted Delivery to Hepatocytes

Chemically Modified siRNA High intrinsic potency

Metabolically stable

Long duration

Devoid of innate immune activation

Clinical safety

CMC

10

100

1000

10000

100000

1000000

0 50 100 150 200

Liv

er

Co

ncen

trati

on

(n

g/g

)

Time (h)

Hepatocyte Targeting N-acetyl galactosamine (GalNAc) ligand binds to

Asialoglycoprotein Receptor (ASGPR)

ASGPR is highly expressed in hepatocytes

High turnover (recycling time ~15 min)

Conserved across species

Enhanced Stabilization

Chemistry (ESC)

Standard Template

Chemistry (STC)

Liver siRNA exposure

5′-AS

5′-sense

3

Mean

(S

em

) %

AT

Kn

ockd

ow

n

40 100

80

60

40

20

0

-20

Time (Days)

0 10 20 30 50 60 70

15 mcg/kg (N=3)

45 mcg/kg (N=6)

75 mcg/kg (N=3)

Mean

(S

EM

) %

C5 K

no

ckd

ow

n

100

80

60

40

20

0

-20

0 10 20 30 40 50 60 70

50 mg

200 mg

400 mg

Placebo

Time (Days)

Mean

(S

EM

) P

CS

K9 K

no

ckd

ow

n

100

80

60

40

20

0

-20

-40

-60

Time (Months)

0 1 2 3 4 5 6

Placebo

25 mg

100 mg

300 mg

500 mg

800 mg

Potent and Durable Target Silencing in Humans

by ESC-GalNAc-siRNA Drug Candidates

ALN-AT3

ALN-CC5

ALN-PCSsc

Mean

(S

EM

) %

AL

A C

han

ge

-100

-75

-50

-25

0

25

50

0 5 10 15 20 25 30 35 40 45

Time (Days)

Placebo

0.035 mg/kg

0.1 mg/kg

0.35 mg/kg

1.0 mg/kg ALN-AS1

4

ASGPR Immunohistochemistry in Human Liver

Normal Liver

Hepatitis

Cirrhosis

MGH Collaboration

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Novel CHB Therapies Aim to Cure T

hera

peu

tic S

trate

gie

s

Goal: Enable Functional Cure

• Achieve state of immunological control of infection off all therapy

• Prevent progression of disease to cirrhosis, liver failure or HCC

• Clearance of circulating viral DNA, normalization of ALT and histology

Inhibit Viral Lifecycle = Direct Antiviral Agent (DAA)

• Reduce circulating virus and inhibit new/re-infection

• Entry, nucleocapsid assembly, cccDNA inhibitors

• Novel reverse transcriptase (NUCs) and RNaseH inhibitors

• Silencing of viral transcripts

Reduce Tolerogenic Proteins

• HBsAg secretion inhibitors

• Silencing of viral antigens

Break Immune Tolerance

• Boost immune response to aid in clearing virus

• TLR7 agonist

• Therapeutic vaccines

• Immune checkpoint inhibitors

• Silencing of PD-L1

RNAi

drugs

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HBV Targeting with RNAi Therapeutics

Compact genome

Overlapping transcripts

• 3.2 kb partially double stranded DNA

genome

• Replication occurs through RNA

intermediate

• 4 overlapping viral transcripts encoding 7

viral proteins translated across 3 reading

frames

Single siRNA can silence all viral

products

• pgRNA replication intermediate

• Polymerase, Core, HBsAg, HBeAg, X

protein

6 Kidd-Ljunggren K, J Gen Virol 83:1267–1280 (2002)

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RNAi Therapeutics for HBV

Dose-dependent antiviral response with intra-animal ascending doses • Mean 2.9 log10 decrease in viral DNA day 2-6 post 0.5 mg/kg dose ◦ >4 log10 reduction in circulating viral DNA achieved in highest titer animal

• Mean 2.0 log10 reduction in HBsAg at 0.5 mg/kg dose ◦ Up to 2.3 log10 reduction achieved

Meyers, TIDES, May 2014

Efficacy in HBV-Infected Chimpanzees (1/2)

Proof-of-concept

Single siRNA

Lipid Nanoparticle, IV

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ALN-HBV Targets a Highly Conserved

Sequence in HBV X Orf

3980 HBV complete genomes, A-H

pg RNA 3.5 kb PreS1 2.4 kb PreS2 2.1 kb

X 0.7 kb

ALN-HBV

Sequence homology (2-18) # of 3,980 Percentage

Perfect 3,868 97.2%

Allow 1 mismatch (mm) 3,963 (+95) 99.7%

No differential distribution across genotypes A-J

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X Orf Target Site is Downstream from Most

Prevalent Integration Hotspot

Linear double stranded HBV integration site map

from 81 HBV+ HCC patients

Wing-Kin Sung, et al., Nature Genetics 44:765 (2012)

ARWR 77: 1827-1845

ARWR 74: 1781-1799

Distribution of HBV integrated breakpoint

from 3 HBV+ HCC patients

Zhaoshi Jiang et al. Genome Res. 22:593-601 (2012)

ALN-HBV

ARWR 77: 1827-1845

ARWR 74: 1781-1799

ALN-HBV

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ALN-HBV Has pM Intrinsic Potency Against

Genotypes A-J in Cell Culture

ALN-HBV

# viral RNA silenced All (3.5, 2.4, 2.1, 0.7 kb)

HepG2.2.15 cells

Viral RNA Silencing

ED50

P ORF PCR

S ORF PCR

19 pM

12 pM

Genotype conservation A-J

5′-AS

5′-sense

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ALN-HBV Exhibits PK Consistent with ESC

Platform and Excellent Tolerability Profile

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Potent, Multi-Log HBsAg KD in Murine Model

• Mouse model with AAV-HBV vector, single SC dose of siRNA at 3 mg/kg

• ALN-HBV DC achieves potent knockdown of HBsAg

◦ Up to 3.6 log10 reduction; mean 1.6 log10 reduction 15 days after single dose

• Specificity confirmed with control siRNA

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Dose-Dependent and Durable HBsAg KD

in AAV-HBV Mouse Model

Pre-dose HBsAg titer range ~10-500 ng/mL

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Potent & Durable HBsAg KD after

Multiple Dosing

ALN-HBV

3 mg/kg

qwk x3, SC Pre-dose HBsAg titer range ~10-500 ng/mL

Control

ALN-HBV

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Phase 1 Phase 2

Clinical Development Plan

CTA Dec-2015

Robust development plan to maximize product opportunity

ALN-HBV for the Treatment of Chronic Hepatitis B

+ NUC SAD

HV

Key Objectives

• Safety

• PK

• HBsAg silencing: extent and duration

• Initial dose finding

SAD

CHB + NUC

MAD

CHB + NUC

+ NUC + Immune therapy

+ NUC + Other

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Summary

• Significant unmet need exists for novel HBV therapy resulting in “functional cure”

• Investigational RNAi therapeutic offers significant promise for treatment of HBV

◦ Novel mechanism for inhibiting all steps of HBV life cycle

◦ Decreases viral proteins including highly abundant non-infective HBsAg particles

• Proof of concept data in naturally HBV-infected chimps suggest robust

efficacy profile

• ASGPR-mediated delivery de-risked in Hepatitis

◦ Regulus RG-101 in HCV employing Alnylam’s GalNAc-conjugate platform

• ALN-HBV Development Candidate (DC) selected

◦ Potential with subcutaneous, qM profile and wide therapeutic index

◦ siRNA targets highly conserved site in genotypes A-J, and it is downstream from

integration hotspot

◦ Durable and dose-dependent HBsAg silencing observed in AAV-HBV mouse model

• Candidate on track for CTA in December 2015

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Thank You

www.alnylam.com

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Conflict of Interest Statement

All authors are employees or consultants* of

Alnylam Pharmaceuticals, Cambridge MA

Laura Sepp-Lorenzino, Andrew G. Sprague, Tara Mayo, Tuyen M. Nguyen, Svetlana

Shulga Morskaya, Huilei Xu, Stuart Milstein, Greg Hinkle, Pia Kasperkovitz, Richard G.

Duncan, Natalie Keirstead, Brenda Carito, Lauren Moran, Prasoon Chaturvedi, Krishna

C. Aluri, Husain Attarwala, Renta M. Hutabarat, Ju Liu, Chris Tran, Qianfan Wang,

Benjamin S. Brigham, Akin Akinc, Klaus B. Charisse, Vasant Jadhav, Satya Kuchimanchi,

Martin A. Maier, Muthiah Manoharan, Rachel Meyers, Tadeusz Wyrzykiewicz, Haroon

Hashmi, Julie Donovan, Tim Mooney, Daniel Freedman, Tanya P. Sengupta, Karin Galil*,

Eoin Coakley, Patrick Haslett