May 14, 2014

Muthiah Manoharan

TIDES 2014

GalNAc-siRNA

with Enhanced Stabilization Chemistry:

ESC-GalNAc-siRNA

Agenda

siRNA-GalNAc Conjugates: Background

Standard Template Chemistry (STC)

» Human POC of ALN-TTRsc

Enhanced Stabilization Chemistry (ESC)

» mTTRsc

» ALN-AT3

» ALN-PCSsc

Summary

2

GalNAc-siRNA Conjugates for Systemic RNAi Receptor and the Ligand

ASGPR

(pH>5)

GalNAc-

siRNA

conjugate

Clathrin-coated pit

Clathrin-

coated vesicle

Endosome

Recycling

ASGPR

mRNA

Nucleus

protein

RISC

Asialoglycoprotein Receptor (ASGPR) Highly expressed in hepatocytes » 0.5-1 million copies/cell

Clears serum glycoproteins via

clathrin-mediated endocytosis

High rate of uptake

Recycling time ~15 minutes

Conserved across species

Valency Gal GalNAc

Mono- ~4 mM ~50 μM

Bi- ~3 μM ~24 nM

Tri- ~100 nM ~3 nM

Valency and Affinity

3

Making Drugs Out of siRNA Conjugates Role of Chemical Modifications

4

Ligand

GalNAc mediates binding and internalization of conjugate by hepatocytes

siRNA Chemistry

Stabilize conjugate in subcutaneous space, lymphatics, plasma, and

hepatocytes

Increase specificity

Reduce immune stimulation

Enhance cellular delivery

Improve RISC activity

siRNA

= 2′OH = 2′-O-methyl = PS

Ligand

5′-sense

= 2′-F (GalNAc)3

Schröder et. al., The Proteome of the Lysosome, Proteomics.10:4053-4076 (2010)

(adapted from Mark Cancilla, Merck)

siRNA Conjugates Encounter Various Metabolic

Enzymes En Route to Site of Action

5

SC

Injection site Capillary Uptake

Lymphatics

Central

Compartment

(systemic

circulation)

D/M

Kidney

D/M

Liver

Effect Site

Biliary excretion

Endosome

RISC

Lysosome1

Hepatocyte

Nucleases (5′ exo, 3′ exo, endo) Peptidases

Excretion

Phosphatases

Nucleases

Peptidases

N-acetylgalactosidase

many others

P-bodies and associated

exonucleases

Kinases - Clp1

Phosphatases

Cytosol

GalNAc-siRNA

conjugate

GalNAc-siRNA Conjugates for Systemic RNAi Molecular Targets Discussed Today

ALN-TTRsc, ALN-AT3, and ALN-PCSsc

6

Potent, rapid, dose-dependent, and durable target

knockdown with SC administration

Hepatocyte target genes

» ALN-TTRsc targeting TTR for treatment of transthyretin-

mediated amyloidosis

» ALN-AT3 targeting AT to restore thrombin generation in

hemophilia and rare bleeding disorders

» ALN-PCSsc targeting PCSK9 to lower cholesterol for

treatment of hypercholesterolemia

ED80 levels for target knockdown from 0.5 – 5 mg/kg

» Generally achieved with qw dosing regimen

More potent conjugates reflect advances in Enhanced

Stabilization Chemistry (ESC)

Used with ALN-AT3 and ALN-PCSsc

Significantly improved potency and durability

Maintains wide therapeutic index and safety

Agenda

siRNA-GalNAc Conjugates: Background

Standard Template Chemistry (STC)

» Human POC of ALN-TTRsc

Enhanced Stabilization Chemistry (ESC)

» mTTRsc

» ALN-AT3

» ALN-PCSsc

Summary

7

ALN-TTRsc Phase 1 Study Results Human POC for GalNAc-siRNA Conjugates

Randomized, double-blind, placebo-controlled SAD and MAD study in healthy volunteers Rapid, dose-dependent, consistent, and durable knockdown of serum TTR

» Significant knockdown of serum TTR (p<0.01) up to 94% TTR knockdown; mean knockdown up to 92.4%

Generally well tolerated » Only AEs associated with drug were generally mild ISRs resolved within ~2 hours of onset

Duration of effect longer in human vs. NHP

Zimmermann, Heart Failure Society of America, Sept. 2013

100

80

60

40

20

0

-20

Days ALN-TTRsc (mg/kg), qd x5; qw x5

% M

ean

TT

R K

no

ckd

ow

n

Rela

tive t

o B

aselin

e (

± S

EM

)

2.5 (n=3)

5.0 (n=3)

10.0 (n=3)

Placebo (n=3)

ALN-TTRsc dose groups

% M

ean

TT

R K

no

ckd

ow

n

Rela

tive t

o B

aselin

e

100

80

60

40

20

0

0 10 20 30 40 50 60

Days

Human

NHP

ALN-TTRsc Single 10.0 mg/kg

Injection

8

Conjugates More stable in Human than NHP In vitro Metabolic Profile at 24 hr

NHP – 24 hour (5µM)

Peak Intensity

Human – 24 hour (5µM)

Peak intensity

9

Metabolite -1

Metabolite -2

Metabolite -3

Metabolite -4

Metabolite -5

Metabolite -6

Metabolite -7

Metabolite -8

Metabolite -9

Metabolite -10

Metabolite -11

Metabolite -12

Metabolite -1

Metabolite -2

Metabolite -3

Metabolite -4

Metabolite -5

Metabolite -6

Metabolite -7

Metabolite -8

Metabolite -9

Metabolite -10

Metabolite -11

Metabolite -12

Full Length AS

Full Length Sense

Human vs. NHP metabolic profile explains extended

duration of action in human for TTRsc

5′-sense

= 2′-F = 2′-O-methyl (GalNAc)3

Standard Template Chemistry (ALN-TTRsc)

Metabolic Profile

Sense Strand (5′-3′)

Antisense Strand (5′-3′)

Agenda

siRNA-GalNAc Conjugates: Background

Standard Template Chemistry (STC)

» Human POC of ALN-TTRsc

Enhanced Stabilization Chemistry (ESC)

» mTTRsc

» ALN-AT3

» ALN-PCSsc

Summary

10

Conjugate Platform Transitioning Standard Template Chemistry (STC) to Enhanced Stabilization Chemistry (ESC)

5′-sense

5′-antisense

= 2′-F = 2′-O-methyl (GalNAc)3

Standard Template Chemistry, STC

(ALN-TTRsc)

5′-sense

5′-antisense

= 2′-F = 2′-O-methyl (GalNAc)3

Enhanced Stabilization Chemistry, ESC

(mTTR, ALN-AT3, ALN-PCS)

Lead

Optimization

by

Further

Stabilization

Chemistries

11

ESC Improves Efficacy of mTTRsc

12

ESC design improves in vivo efficacy by 5-fold over STC design in mTTRsc

STC ESC

0

20

40

60

80

100

120

PBS 25 mg/kg 5 mg/kg 1 mg/kg 5 mg/kg 1 mg/kg 0.2 mg/kg

TT

R m

RN

A l

eve

ls D

ep

icte

d

as

% o

f P

BS

Co

ntr

ol

(mT

TR

/mG

apd

h)

Subcutaneous Single Dose

GalNAc Conjugates Are a Repeat-Dose Platform Chronic Dosing in Mice with ESC-mTTRsc

Steady knockdown maintained with chronic dosing Sustained knockdown at both ED50 (1 mg/kg) and ED80 (2.5 mg/kg) dose levels

Absence of tachyphylaxis or sensitization

No changes in serum TTR levels in PBS control group

GalNAc Conjugates are a multi-dose platform, showing dose additive effect

13

% K

no

ck

do

wn

Se

rum

TT

R

(Fra

ction

P

re-d

ose

)

0 30 60 90 120 150 180 210 240 270

Long-Term Dosing: QW Dosing with ESC-mTTRsc

100

80

60

40

20

0

-20

PBS 1.0 mg/kg 2.5mg/kg

Day

siRNA-GalNAc Lead Optimization Tuning STC-siRNA-GalNAc to ESC-siRNA-GalNAc

siRNA

synthesis

In vitro

screening

in vivo exposure/

PD in rodent or hTG

Design chemically

modified siRNAs

Xn

ESC-siRNA-GalNAc

STC-siRNA-GalNAc

14

Select 3-6 conjugates

for high dose

rat toxicity screen

NHP PK/PD

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

= 2′-F = 2′-O-methyl

(GalNAc)3

S 5′

= Enzymatic cleavage site (thickness reflects frequency

of corresponding cleavage products observed)

S 5′

Standard Template Chemistry (STC)

Enhanced Stabilization Chemistry (ESC)

AS 5′

AS 5′

Liver Exposure

Target Compound Tmax

(h)

Cmax

(µg/g)

AUC0-t

(h·µg/g)

AUC0-48

(h·µg/g)

AT3 STC 2 59.5 735 735

AT3 ESC 8 285 21546 9697

10

100

1000

10000

100000

1000000

0 50 100 150 200Liv

er

Co

ncen

trati

on

(n

g/g

)

Time (h)

SC

ESC

Liver Exposure

STC

15

ESC Significantly Enhances Efficacy and Duration Reduction of AT Protein After Single SC Dose in NHP

16

Potent and durable silencing achieved after single SC dose >10-fold improvement in efficacy over standard template chemistry

Substantially extended duration of effect

% K

no

ck

do

wn

Se

rum

AT

(R

ela

tive

to

Pre

-do

se

)

Day

100

80

60

40

20

1.0

1.2

-10 0 10 20 30 40

STC-AT3 (10 mg/kg)

ESC-AT3 (10 mg/kg)

ESC-AT3 (1 mg/kg)

Weekly SC administration of ALN-AT3 in WT mice results in potent and consistent AT suppression

Repeat dose ED50 for AT knockdown <0.75 mg/kg

Nadir reached at ~day 14

Maintenance of AT Knockdown with Repeat Dosing ALN-AT3 Pre-Clinical Studies

Akinc, WFH, July 2012 17

100

80

60

40

20

0

-20

4 8 12 16 20 24 28 32

Day

% A

nti

thro

mb

in K

no

ck

do

wn

Norm

aliz

ed

to

Pre

-Dose

an

d P

BS

0

3.00

1.50

0.75

ALN-AT3

(mg/kg)

ALN-AT3, qw x5

Drug Levels in Tissue During Chronic Dosing (ALN-AT3) No Evidence for Accumulation in Tissue (Mouse Liver)

ALN-AT3, dosing

(qw x 14)

Post-dose

(4 h)

Tissue

collection Pre- and post-

dose (4 h)

Pre- and post-

dose (4 h, 3 days)

Pre- and post-

dose (4 h)

Pre- and post-

dose (4 h)

18

10

100

1000

10000

0 10 20 30 40 50 60 70 80 90

Liv

er

siR

NA

(n

g/g

)

Day

Actual Data with Interpolated Data Added Actual Data

1.0 mg/kg qw

0.5 mg/kg qw

0.2 mg/kg qw

Drug Levels in Tissue During Chronic Dosing (ALN-AT3) No Evidence for Accumulation in Tissue (Mouse Liver)

Additional peak and trough concentration data added by interpolation; additional 3-day

post-dose data added by assuming fixed ratio relative to peak concentration

Liver levels are approximately dose proportional

Mean steady state liver drug load of ~3 g/g per mg/kg cumulative weekly dosing

ALN-AT3, dosing

(qw x 14)

Post-dose

(4 h)

Tissue

collection Pre- and post-

dose (4 h)

Pre- and post-

dose (4 h, 3 days)

Pre- and post-

dose (4 h)

Pre- and post-

dose (4 h)

19

10

100

1000

10000

0 10 20 30 40 50 60 70 80 90

Liv

er

siR

NA

(n

g/g

)

Day

1100 ng/g

400 ng/g

3400 ng/g

Actual Data with Interpolated Data Added Actual Data

1.0 mg/kg qw

0.5 mg/kg qw

0.2 mg/kg qw

ALN-AT3 Pre-Clinical Efficacy Potent AT Knockdown and Normalization of Thrombin Generation

20

ALN-AT3 achieves potent AT knockdown and fully corrects thrombin generation in non-human primates (NHP)

Weekly SC doses for >5 months result in potent, dose-dependent, and durable AT knockdown

In NHP hemophilia “inhibitor” model, ALN-AT3 fully restores thrombin generation to normal levels

Akinc, ISTH, July 2013

Recovery

Recovery 0.25 mg/kg qw x 12

Recovery 1.5 mg/kg qw x 5

100

80

60

40

20

0

-20 0 20 40 60 80 100 120 140 160

% M

ea

n A

T K

no

ck

do

wn

(P

re-d

ose

= 1

)

Day

1 mg/kg q2w x 4

0.125 mg/kg qw x 12 0.5 mg/kg qw x 8

Normal Hemophilia A (Induced)

Pre-dose

ALN-AT3 (mg/kg) qw

Rela

tive

Th

rom

bin

Ge

nera

tio

n

(Pe

ak T

hro

mb

in)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Saline

1.6

0.25

~60%

AT

KD

0.5

p<0.01

~80%

AT

KD

What About Species-Related Differences? ALN-AT3 Single Dose Efficacy and Recovery

21

Single-dose efficacy: mouse ≈ rat > NHP

Time to nadir and recovery kinetics: NHP > mouse > rat

Day

100

80

60

40

20

0

-20

-40

-10 0 10 20 30 40 50 60

Re

lati

ve

AT

Le

ve

l

Mouse 2.5 mg/kg

Rat 3.0 mg/kg

NHP 3.0 mg/kg

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Fra

cti

on

PC

S

messag

e r

em

ain

ing

siRNA rank order +/- SD

0.1 nM, average activity across 3 human cell lines

Screen of ca. 200 PCSK9 siRNAs identified initial lead

Tested Lead as STC and ESC versions in vivo SD

ALN-PCSsc Lead Selection Use What You Know, Then Iterate

PCSsc

Lead

22

STC-PCS ESC-PCS 0.0

0.2

0.4

0.6

0.8

1.0

1.2

PBS

hP

CS

K9 p

rote

in

(Rela

tive t

o p

re-d

ose)

STC-PCS vs. ESC-PCS hPCSK9 tg, mice 72h, 3 mg/kg

ED90 of ESC-PCS ~1 mg/kg BIW (12 doses)

Reduction in PCSK9 results in >60% lowering of LDL-C (data not shown)

Duration of nadir PCSK9 post-last dose (BIW) is ~20 days

The compound potency was sufficient but stability/dose frequency not sufficient

to meet TPP Goal of once-monthly dosing

Another round of ESC lead optimization was used!

Multi-Dose ESC-PCS Results in >90% Lowering of PCSK9 But Duration can be Improved (NHP)

23

Day

0 20 40 60 80 100 120

100

80

0

-20

q2w

q1w

60

40

20

-40

-60

% K

no

ckd

ow

n P

CS

K9

(r

ela

tive t

o p

re-b

leed)

ESC-PCS

1 mg/kg

Dosing

Schedule BIW

siRNA-GalNAc Lead Optimization Tuning ESC-siRNA-GalNAc to Development Candidate for PCSK9

siRNA

synthesis

In vitro

screening

in vivo exposure/

PD in rodent or hTG

Design chemically

modified siRNAs

X2

Select 3-6 conjugates

for high dose

rat toxicity screen

ALN-PCSsc Development Candidate

NHP PK/PD

ESC-siRNA-GalNAc

24

ALN-PCSsc Achieves Potent, Highly Durable PD Pre-Clinical Efficacy in NHP with Single Dose

LDL-C PCSK9

Days

0 20 40 60 80 100

1.0 3.0 6.0 10.0

% P

CS

K9

Kn

oc

kd

ow

n

(re

lative

to

pre

-ble

ed

)

Days

0 20 40 60 80 100 120

% L

DL

-C L

ow

eri

ng

(re

lative

to

pre

-ble

ed

)

100

80

60

40

20

0

-20

80

60

40

20

0

-20

100

ALN-PCSsc (mg/kg)

ALN-PCSsc (mg/kg)

1.0 3.0 6.0 10.0

ALN-PCSsc achieves highly durable PCSK9 knockdown and LDL-C reduction with single dose Single SC dose 1-10 mg/kg

Up to 96% PCSK9 knockdown

Up to 77% LDL-C lowering in absence of statins

Highly durable effects, supports once-monthly and possibly once-quarterly dosing » >50% LDL-C lowering maintained for over 3 months in 10 mg/kg group

25 Fitzgerald, ATVB, May 2014

Comparing STC- to ESC-GalNAc-siRNA Conjugates Key Data in NHP

26

STC

(ALN-TTRsc)

ESC

(ALN-PCSsc)

Potency

ED50

SD 10.0 mg/kg 1.0 mg/kg

MD (qw) ca. 2.0 mg/kg 0.2 mg/kg

Stability

t1/2

Liver ~24 h ~120 h

Duration ~40 days ~120 days

Safety

Cytokine/Complement

Activation None None

Adverse ISRs None None

NOAEL (MD) >300 mg/kg >300 mg/kg

Agenda

siRNA-GalNAc Conjugates: Background

Standard Template Chemistry (STC)

» Human POC of ALN-TTRsc

Enhanced Stabilization Chemistry (ESC)

» mTTRsc

» ALN-AT3

» ALN-PCSsc

Summary

27

Summary

28

Alnylam has developed an effective, well-tolerated, SC-administered

GalNAc-conjugate platform with proven human clinical translation

Enhanced Stabilization Chemistry (ESC) of siRNA-GalNAc conjugates

has led to increased metabolic stability resulting in:

» Increased liver exposure

» Improved potency: Up to 10-fold SD potency improvement and less

injection volume

» Increased duration of effect, which further increases from rodents to

NHP

» No compromise in safety

ESC-conjugate design translates well across multiple siRNAs and

targets

Expect favorable translation of potency and duration in humans for

ESC-GalNAc-conjugates