1© 2018 Alnylam Pharmaceuticals, Inc.

March 29, 2019

Progress in Extrahepatic Silencing with siRNA

Conjugates

Stuart Milstein

2

2004

2014

2018

STC

ESC

Advanced

ESC

Potency/duration/

specificity

Stability, optimal

ligand orientation

Efficient delivery

to target cells

ESC+

siRNA

design/chemistryLinker Ligand

0.01

0.1

1

10

100

2006 2008 2010 2012 2014 2016 2018

ED

50

(m

g/k

g)

Year

Partially

modified

Evolution of conjugate potency

(mouse, SD ED50)

siRNA designs with enhanced potency and stability may extend to extrahepatic tissues

Transformative Advancements in Conjugate-Based Delivery

3

Extensive Durability and Safety Demonstrated in Liver Programs

STC

ESC

Advanced

ESC

Potency/duration/

specificity

Stability, optimal

ligand orientation

Efficient delivery

to target cells

ESC+

siRNA

design/chemistryLinker Ligand

0.01

0.1

1

10

100

2006 2008 2010 2012 2014 2016 2018

ED

50

(m

g/k

g)

Year

Partially

modified

Evolution of conjugate potency

(mouse, SD ED50)

4

Expanding Alnylam opportunities beyond liver

Investigational RNAi Therapeutics for CNS and Ocular Diseases

Devastating diseases with enormous burden and unmet need

• Alzheimer’s disease

• Amyotrophic lateral

sclerosis (ALS)

• Cerebral amyloid

angiopathy

• Frontotemporal

dementia

• Huntington’s disease

• Multi-system atrophy

• Parkinson’s disease

• Spinocerebellar ataxia

• AMD, dry

• AMD, wet

• Birdshot

chorioretinopathy

• Dominant retinitis

pigmentosa 4

• Fuch’s dystrophy

• hATTR amyloidosis

• Hereditary and

sporadic glaucoma

• Stargardt’s disease

Number of genetically validated targets known but few disease modifying therapies for these devastating or

life threatening disorders.

RNAi therapeutics directed to disease-causing, CNS- or ocular-expressed genes represent a potential

opportunity to address diseases with some of the greatest unmet need.

5

Single dose and dose response in rat

Intrathecal Delivery of CNS Optimized siRNA Conjugates

siRNAs targeting b-catenin or SOD1 in single dose or dose response

• Single siRNA conjugate doses of 0.9 mg, 0.3 mg, 0.07 mg

• Multidose arm- 0.3 mg monthly x 5

• Time points through 1 month for b-catenin and 6 months for SOD1

Day 0 427 28

Tissue

collection

16856 70 84 98 126

Tissues: Spinal cord: Lumbar, thoracic and cervical

Brain: prefrontal cortex, cerebellum and remaining brain

Fluids: CSF and plasma

Assays: mRNA, tissue siRNA levels, Histology

Strand (5'-3') Sequence

b-cateninS UACUGUUGGAUUGAUUCGAAA

AS TUUCGAAUCAAUCCAACAGUAGC

SOD1S CAUUUUAAUCCUCACUCUAAA

AS UUUAGAGUGAGGAUUAAAAUGAG

6

Silencing of b-catenin following a single IT dose

Robust and Durable Silencing Demonstrated Following a Single IT Dose

Lumbar

Thoracic

Cervical

Cerebellum

Frontal Cortex

Remaining Brain

Messag

e R

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ain

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7

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00

0.25

0.50

0.75

1.00

1.25

1.50

Time (Days)

SOD1 Parent - 0.9 mg

SOD1 VP - 0.3 mg Multi Dose

SOD1 VP - 0.9 mg

SOD1 VP - 0.3 mg

SOD1 VP - 0.075 mg

Lumbar Spinal Cord

Messag

e R

em

ain

ing

Control

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00

0.25

0.50

0.75

1.00

1.25

1.50

Time (Days)

SOD1 Parent - 0.9 mg

SOD1 VP - 0.9 mg

SOD1 VP - 0.3 mg

SOD1 VP - 0.075 mg

Control

Cervical Spinal Cord

Messag

e R

em

ain

ing

SOD1 VP - 0.3 mg Multi Dose

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00

0.25

0.50

0.75

1.00

1.25

1.50

Time (Days)

SOD1 Parent - 0.9 mg

SOD1 VP - 0.9 mg

SOD1 VP - 0.3 mg

SOD1 VP - 0.075 mg

Control

Cerebellum

Messag

e R

em

ain

ing

SOD1 VP - 0.3 mg Multi Dose

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00

0.25

0.50

0.75

1.00

1.25

1.50

Time (Days)

SOD1 Parent - 0.9 mg

SOD1 VP - 0.9 mg

SOD1 VP - 0.3 mg

SOD1 VP - 0.075 mg

Control

Frontal Cortex

Messag

e R

em

ain

ing

SOD1 VP - 0.3 mg Multi Dose

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00

0.25

0.50

0.75

1.00

1.25

1.50

Time (Days)

SOD1 Parent - 0.9 mg

SOD1 VP - 0.9 mg

SOD1 VP - 0.3 mg

SOD1 VP - 0.075 mg

Control

Thoracic Spinal Cord

Messag

e R

em

ain

ing

SOD1 VP - 0.3 mg Multi Dose

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 850.00

0.25

0.50

0.75

1.00

1.25

1.50

Time (Days)

SOD1 Parent - 0.9 mg

SOD1 VP - 0.9 mg

SOD1 VP - 0.3 mg

SOD1 VP - 0.075 mg

Control

Temporal Cortex

Messag

e R

em

ain

ing

SOD1 VP - 0.3 mg Multi Dose

Silencing of SOD1 following a single or multiple IT doses

Robust and Durable Silencing Demonstrated Following a Single IT Dose

Lumbar

Thoracic

Cervical

Cerebellum

Frontal Cortex

Temporal Cortex

Messag

e R

em

ain

ing

Messag

e R

em

ain

ing

Messag

e R

em

ain

ing

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em

ain

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ain

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ain

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8

Intrathecal delivery of siRNA provides durable knockdown throughout CNS

Robust Silencing Throughout the Brain

• Consistent lowering across animals in most regions of the brain

• PD comparison in liver across species together with extended duration seen in rodents expected to support

infrequent dosing in human

PD comparison in liver across species

9

Higher drug levels and robust silencing observed in brain with SOD1 siRNA conjugate

siRNA Conjugates Show Enhanced Uptake and Activity

• Rapid siRNA clearance from CSF

• Conjugate reveals superior uptake and

stability in brain over unconjugated

siRNA

• Increased uptake in brain results in

substantial improvement in mRNA

knockdown

Unconjugated siRNA

Conjugated siRNA

Unconjugated siRNA

Conjugated siRNA

Target silencing in cerebellum

Unconjugated siRNA

Conjugated siRNA

Control siRNA

10

CSF and Plasma PK in Rat Following IT dosing

0.9 mg SOD1, lumbar puncture

• Rapid disappearance from CSF

• Rapid appearance in plasma, tmax

5 min – 1 h

• Plasma concentrations parallel

CSF, but ~2 logs lower

0 6 1 2 1 8 2 4

0 . 0 0 0 1

0 . 0 0 1

0 . 0 1

0 . 1

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

S O D 1 - C 1 6 - V P , P l a s m a a n d C S F

T i m e ( h )

[s

iRN

A],

g/m

L

P l a s m a

C S F

U r in e

n=2

n=1n=2

n=2 n=3

n=3

Intrathecal (IT) dosing, 0.9 mg

Dose 42 8 24 h

5, 15, 30 min

Plasma

CSF

61

Sampling timeline

11

SOD1 IT Dose Response Reveals Minimal Silencing in the Liver and Kidney

Liver

Kidney

Though rapidly cleared from the CSF to the systemic circulation,

conjugated siRNAs do not show robust silencing in the liver or kidney

12

Rat IHC probed for b-catenin

b-cateninControl

Neocortex

Cerebellum

b-cateninControl

Basal forebrain

b-cateninControl

Striatum

b-cateninControl

Silencing Is Seen In All Regions of the Brain

13

Single dose NHP study design

Evaluating Translation of CNS siRNA Conjugate Delivery to NHP

Day 0 31

• siRNA conjugate dosed at 72 mg – IT bolus

• Evaluated a single target – b-catenin

Tissues: Spinal cord: Lumbar, thoracic and cervical

Brain: Prefrontal cortex, temporal cortex, cerebellum, brain stem

hippocampus

Fluids: CSF and plasmaHistology

Assays: mRNA, siRNA levels

Tissue

collection

Strand (5'-3') Sequence

b-cateninS UACUGUUGGAUUGAUUCGAAA

AS TUUCGAAUCAAUCCAACAGUAGC

14

siRNA uptake varies between CNS regions

siR

NA

ng

/gsiRNA levels

IT Dosed b-catenin siRNA is Detected Throughout the Spinal Cord and Brain

Regions of NHP

The day 31 time point shows the presence of significant siRNA

levels across all tissues tested

Lu

mb

ar

Th

ora

cic

Cerv

ical

Bra

in S

tem

Cere

bellu

m

Pre

fro

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or t

ex

Tem

po

ral C

or t

ex

Hip

po

cam

pu

s

Liv

er

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

5 0 0 0

6 0 0 0

7 0 0 0

8 0 0 0 s iR N A le v e ls - d a y 3 1

15

Robust Silencing Across CNS Demonstrating Successful Translation NHPb-catenin mRNA knockdown by tissue, Day 31

• The conjugate targeting b-catenin produces robust knockdown throughout the spinal cord

and brain at the 31 day time point.

b-catenin mRNA

Messag

e R

em

ain

ing

Lu

mb

ar

Sp

ine

Th

ora

cic

Sp

ine

Cerv

ical S

pin

e

Cere

bellu

m

Pre

fro

nta

l C

or t

ex

Tem

po

ral C

or t

ex

Hip

po

cam

pu

s

Bra

in S

tem

Liv

er

0 .0 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

P B S C o n tro l

C T N N B s iR N A

PBS

CTNNB

16

siRNAs Distribute Throughout the CNS in NHP Following IT Dosing

Lumbar cord Cervical cord Cerebellum

Neocortex Hippocampus Dentate Gyrus Basal Ganglia

17

Most neurons show siRNA uptake

siRNA Conjugates are Localized to Neurons Following IT Dosing

MAP2

siRNA

MAP2 is a neuronal marker

CTNNB siRNA probed with siRNA antibody

40X

100X

18

siRNA Conjugates are Localized to Microglia Following IT Dosing

Iba1 is a microglia marker

b-catenin siRNA probed with siRNA antibody

40X

100X

Iba1

siRNA40X

100X

19

siRNAs Conjugates are Localized to Astrocytes Following IT Dosing

GFAP is an Astrocyte marker

b-catenin siRNA probed with siRNA antibody

40X

100X

GFAP

siRNA40X

100X

40X

100X

20

siRNA vs ASO in hSOD1 (SOD1G93A) Rats

McCampbell et al, J Clin Invest. 2018;128(8):3558-3567

Study Purpose

• Head-to-head comparison of siRNA and ASO

– 3 siRNAs selected from mouse AAV-hsSOD1 screen

– ASO 1, based on McCampbell et al. (2018)◦ Demonstrated ~75% maximum silencing at 2 weeks in the same rat model

Study Design

• Dose: single IT injection of 0.9 mg in 30µl (Study Day 0)

– Same dose used for siRNA and ASO

• Dosed 13 week old hSOD1 rats (SOD1G93A)1

– Single Day 7 timepoint ◦ Single early time point was chosen to capture the highest expression of SOD1G93A before animals developed degenerative phenotype

Day 7 collection after single IT dose

Study Day

Animal Age (Weeks)13

Tissues collected

across spinal

cord and brain

0

14

7

IT injection

siRNA & ASO

SOD1G93A

Levels15

21

Improved hSOD1 mRNA Reduction with siRNA compared to ASO in CNS

siRNA vs ASO in hSOD1 (SOD1G93A) Rats

aC

SF

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

AS

O

siR

NA

0

20

40

60

80

100

120

% h

SO

D1 m

essage r

em

ain

ing

rela

tive to a

CS

F

Lumbar Thoracic CervicalBrain

StemCerebellum Hippo

Frontal

CortexTemporal

Cortex

An siRNA targeting hSOD1 showed superior silencing in all regions of the brain and spinal cord

22

Expanding Alnylam opportunities beyond liver

Investigational RNAi Therapeutics for CNS and Ocular Diseases

Devastating diseases with enormous burden and unmet need

• Alzheimer’s disease

• Amyotrophic lateral

sclerosis (ALS)

• Cerebral amyloid

angiopathy

• Frontotemporal

dementia

• Huntington’s disease

• Multi-system atrophy

• Parkinson’s disease

• Spinocerebellar ataxia

• AMD, dry

• AMD, wet

• Birdshot

chorioretinopathy

• Dominant retinitis

pigmentosa 4

• Fuch’s dystrophy

• hATTR amyloidosis

• Hereditary and

sporadic glaucoma

• Stargardt’s disease

Number of genetically validated targets known but few disease modifying therapies for these devastating or

life threatening disorders.

RNAi therapeutics directed to disease-causing, CNS- or ocular-expressed genes represent a potential

opportunity to address diseases with some of the greatest unmet need.

23

TTR is Produced in the Eye as Well as the Liver

Ocular manifestations of hATTR amyloidosis in patients

• Glaucoma (~12%-23%)

• Vitreous opacities (Wide range)

• Retinal abnormalities (~4% - 15%)

− retinal amyloid deposit

• Iris abnormalities (~14% - 38%)

− iris amyloid deposit

• Amyloid deposits on lens (~33%)

Pupillary deposits and abnormality predict onset of

glaucoma

• V30M and T114C study

− 62% eyes had high IOP

Liver transplant patients

• Study of Japanese liver transplant patients with V30M continue to

have ocular symptoms

24

Ocular TTR Silencing by Differentially Modified siRNA Conjugates in Rat

After Single Intravitreal InjectionOcular silencing of hTTR in transgenic mice

Specificity: No impact on expression of mTTR, Crx or Rhodopsin

PB

S

Par t

ially M

od

ifie

d

ES

C

EH

op

tim

ized

0 .2 5

0 .5

1

2

4

8

1 6

3 2

6 4

1 2 8

2 5 6

% m

es

sa

ge

re

ma

inin

g

Mouse TTR mRNA

Day 14, 50 g siRNA conjugate

25

Near complete reduction of TTR mRNA and protein 28 days following a single IVT dose

Robust Silencing of Ocular TTR by siRNA Conjugates in NHP

TTR protein analysis by IHC

TTR protein analysis by ELISA

TTR mRNA analysis by qPCR

Aqueous Humor

Vitreous Humor

0 0 .1 m g 0 .3 m g 1 .0 m g 3 .0 m g

0

5 0

1 0 0

1 5 0

D o s e

% R

em

ain

ing

TT

R P

ro

tein

0 0 .1 m g 0 .3 m g 1 .0 m g 3 .0 m g

0

5 0

1 0 0

1 5 0

D o s e

% R

em

ain

ing

TT

R P

ro

tein

Ciliary Body RPE

PB

S

0.1

mg

0.3

mg

1.0

mg

3.0

mg

PB

S

0.1

mg

0.3

mg

1.0

mg

3.0

mg

0

5 0

1 0 0

1 5 0

% T

TR

Me

ss

ag

e R

em

ain

ing

0.1

mg

/eye

PB

S

Dose

Dose

26

Expanding a pipeline of potentially transformative medicines

Alnylam CNS and Ocular Pipeline Strategy

Genetically validated

target gene

Biomarker for

POC in

Phase 1

Definable path

to approval and

patient access

27

• Durable silencing of target mRNA observed

across the CNS of rat and NHP following IT

administration

• Tissue uptake observed in all CNS tissues

examined with drug levels in the ng/g to g/g

range

• In both rat and NHP studies, intrathecal

administration of the novel siRNA conjugates

was found to be generally well tolerated

• Robust silencing throughout the brain observed

for an siRNA targeting SOD1

Successful delivery of siRNA conjugate to the CNS and eye

Consistent Silencing Observed Across Pre-clinical Species

• TTR silencing demonstrated in rodents and

NHP

• Target silencing seen in the CE and RPE

• Target silencing is specific

• Target knockdown demonstrated in NHP

following a single IVT dose of siRNA

• Equivalent silencing demonstrated for mRNA

and protein across the eye