Advancing the Safety and Reach of RNAi Therapeutics

CONFIDENTIAL1 © 2020 Alnylam Pharmaceuticals, Inc.

Maja Janas De Angelis, PhD, DABT

Associate Director, Early Development

OTS 2020

Advancing the Safety and Reach

of RNAi Therapeutics

CONFIDENTIAL2

Thank You Alnylam Team, Past and Present

Medicinal Chemistry

RNA Synthesis and Annealing

RNAi Lead Discovery

RNAi Biology and Bioinformatics

In Vivo Sciences

Pathology

Bioanalytical Sciences and DMPK

Martin Maier Vasant Jadhav Scott Barros Natalie Keirstead Jing-Tao Wu Pete Smith Kevin Fitzgerald

Mark Schlegel Ivan Zlatev

Yongfeng Jiang Saket Agarwal Lei Johnson Onur Yilmaz Sam Chigas Annabelle SangreeShreya Jadhav

CONFIDENTIAL3

Agenda

Optimizing the safety of liver-targeted GalNAc-siRNA conjugates

Translating liver learnings to CNS-targeted siRNA conjugates

CONFIDENTIAL4

Safety Profile of GalNAc-siRNAs to DateORION-10+11 Pooled Data: Safety and Tolerability of InclisiranNo Evidence of Liver, Kidney, Muscle or Platelet Toxicity

1 Safety population includes all patients who received at least 1 dose of study medication and individual patients may be counted in more than one category2 No cases met Hy’s Law

CONFIDENTIAL5

Characteristics of the Human LFT Signal with Subset of GalNAc Conjugates:

Only Observed with Few Compounds Suggesting Sequence SpecificityALN-AAT01 Phase 1/2 Interim Results

Me

an

[+

/-S

EM

] A

AT

Re

lative

to

Ba

se

line

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

Days since dose

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

Placebo (N=5) 0.1 mg/kg (N=3) 0.3 mg/kg (N=3)

1.0 mg/kg (N=3) 3.0 mg/kg (N=3) 6.0 mg/kg (N=3)

PD: Serum AAT Protein Levels Placebo

1.0 mg/kg

3.0 mg/kg

6.0 mg/kg

Safety: Serum ALT Levels

-10 0 10 20 30 40 50 60 70

Days Since Dose

1

4

8

1

4

8

1

4

8

1

4

8

• Anecdotal evidence for RISC-mediated mechanism:

◦ Onset of LFT elevations coincides with onset of maximum RNAi activity

◦ High target knockdown appears necessary (but not sufficient) for LFT elevations

• Similar profile seen in few other programs with sporadic LFT elevations

CONFIDENTIAL6

Safety Considerations for RNAi Therapeutics

(1) Non-natural chemical modifications,

e.g. 2’-fluoro, phosphorothioate

(3) Hybridization-based off-target effects,

e.g. microRNA-like seed-based activity

(2) Dysregulation of natural RNAi pathways,

e.g. microRNAs

CONFIDENTIAL7

0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2

0

5

1 0

1 5

2 0

L iv e r - P u r in e s

T im e p o s t-d o s e (h rs )

Co

nc

en

tra

tio

n (

M)

2 '-F -A R e v u s ira n

2 '-F -A T T R S C 0 2

2 '-F -G R e v u s ira n

2 '-F -G T T R S C 0 2

2 '-F -I R e v u s ira n

2 '-F -I T T R S C 0 2

0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2

0

5

1 0

1 5

2 0

L iv e r - P y r im id in e s


Co

nc

en

tra

tio

n (

M)

2 '-F -U R e v u s ira n

2 '-F -U T T R S C 0 2

2 '-F -C R e v u s ira n

2 '-F -C T T R S C 0 2

0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2

0

1

2

3

4

P la s m a - P u r in e s


Co

nc

en

tra

tio

n (

M)

2 '-F -A R e v u s ira n

2 '-F -A T T R S C 0 2


2 '-F -G T T R S C 0 2


2 '-F -I T T R S C 0 2

0 2 4 4 8 7 2 9 6 1 2 0 1 4 4 1 6 8 1 9 2

0

1

2

3

4

P la s m a - P y r im id in e s


Co

nc

en

tra

tio

n (

M)


2 '-F -U T T R S C 0 2

2 '-F -C R e v u s ira n

2 '-F -C T T R S C 0 2

Monomer Generation Is Minimized with the ESC Design In Vivo

Rat, 30 mg/kg Single Subcutaneous Dose of Revusiran or ALN-TTRSC02

• 2'-F-monomer half-life of 1-2 days indicates

that no accumulation is expected with weekly

or less frequent dosing

• Only 2'-F-pyrimidines appear to re-distribute

systemically in plasma

CONFIDENTIAL8

2'-F-Monomers Are Not Detectable in Human Plasma or Urine at

Therapeutically-Relevant Doses of ESC-Conjugate

Revusiran: 7.5 mg/kg qd x 5 to healthy volunteers

ALN-TTRSC02: 50 mg (~0.83 mg/kg) single dose to healthy volunteers

• 2'-F-monomers reached steady state in plasma and

urine by Day 4, indicating a half-life of ~ 1 day and

therefore no accumulation with weekly or less frequent

dosing

0 4 8 1 2 1 6 2 0 2 4

0

2

4

6

P la s m a


Co

nc

en

tra

tio

n (

M)



2 '-F -U T T R S C 0 2

2 '-F -I T T R S C 0 2

0 -6 6 -1 2 1 2 -2 4

0

2

4

6

8

U r in e


mg




T T R S C 02

• Up to ~15 % of total monomer dose is excreted

in urine, and therefore even revusiran is likely

mainly excreted in oligomeric forms

0 4 8 1 2 1 6 2 0 2 4

0

2

4

6

P la s m a


Co

nc

en

tra

tio

n (

M)



2 '-F -U T T R S C 0 2

2 '-F -I T T R S C 0 2

0 -6 6 -1 2 1 2 -2 4

0

2

4

6

8

U r in e


mg




T T R S C 02

CONFIDENTIAL9 Janas MM, Zlatev, I et al. Nucleic Acids Res. 2019 Apr 23;47(7):3306-3320

2'-F-Monomer De-Risking

Monomer generation from STC siRNA in rat and human Low

Monomer generation from ESC siRNA in rat and human Mostly undetectable

Polymerase inhibition (Pol-γ, Pol-α, Pol-β, POLRMT) No

Polymerase substrate (Pol-γ, POLRMT) Poor

Obligate chain termination No

Non-obligate chain termination No

Cytotoxicity and mtDNA effects in vitroIn a subset of cell lines at concentrations > 16-fold higher

than generated in vivo from STC siRNA

2-year rat carcinogenicity study with STC siRNAsNo effects on survival or tumor incidence;

no apparent effects on mitochondrial function

CONFIDENTIAL10 Janas MM et al. Nucleic Acid Ther. 2017 Feb;27(1):11-22

Phosphorothioate Modification Is Well-Tolerated in

Double-Stranded Oligonucleotides In Vitro

DNA double stranded break assessment after transfection into HeLa cells:

Mock

CONFIDENTIAL11

*Nat Struct Mol Biol. 2013 Jul;20(7):789-95; Mol Biol Cell. 2010 May 1;21(9):1462-9; RNA. 2013 May;19(5):605-12

Potential Reasons:

• Relatively low % of cellular RISC pool occupied by siRNA

• Stabilization of additional RISC complexes in the presence of siRNA*

No Evidence of a Class Effect of siRNAs on microRNA Activity

- 5 10 15 30 45 60 90 120 - 5 10 15 30 45 60 90 120

100 nM PPIB 1 siRNA Transfection Mock Transfection

let-7 activity (HeLa cells)

Let-7 cleavage

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 5 10 15 30 45 60 90 120

Time (minutes)

% C

leavag

e

100 nM siRNA transfection

Mock Transfection

B A

D-6

49

58

G A

D-5

86

43

B A

D-5

77

27

B A

D-5

86

42

G A

D-6

81

11

B A

D-5

86

41

G A

D-7

23

92

G A

D-6

39

68

E+

AD

-72

78

8

G A

D-6

72

48

B A

D-6

42

72

G A

D-5

86

81

B A

D-6

68

08

G A

D-6

72

46

B A

D-6

54

21

G A

D-6

08

35

Ctr

l

G A

D-6

56

95

G A

D-6

68

10

G A

D-6

69

20

B A

D-6

68

16

G A

D-6

72

44

E+

AD

-75

99

5

G A

D-6

54

03

G A

D-6

87

26

B A

D-6

09

40

Ctr

l R

vr

G A

D-6

76

37

G A

D-6

73

27

B A

D-6

69

21

B A

D-6

66

78

B A

D-6

45

46

B A

D-6

69

22

G A

D-6

68

83

G A

D-6

14

44

Rv

r

G A

D-6

73

35

B A

D-6

66

86

B A

D-6

68

14

G A

D-6

40

08

G A

D-6

87

30

B A

D-6

45

43

B A

D-6

56

44

-8 0

-6 0

-4 0

-2 0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

1 6 0

1 8 0

2 0 0

2 2 0

m iR -2 2 -3 p - S e e d A c tiv ity

% t

arg

et

re

ma

inin

g

Increased

No impact

Decreased

Endogenous microRNA activity assessment after siRNA transfection:

miR-22 activity (COS cells)

siRNA ID

CONFIDENTIAL12 Janas MM, Schlegel, MK et al. Nat Commun. 2018 Feb 19;9(1):723

Seed-Based Off-Target Effects Are Important Drivers of

Rodent Hepatotoxicity for Subset of GalNAc-siRNAs

3’-UTR

miRNA-like seed mediated

binding to off-targets

Translation repression,

mRNA destabilization

Min/Marked hepatocellular degen, Mod

SCN, Min coagulative necrosis, Mod

↑mitoses, Min BDH, Mod vacuolation

Rat NOAEL: 10 mg/kgSeed enriched off-

target effects observed

CONFIDENTIAL13

ESC+ Seed Pairing Destabilization Strategy to Improve

Specificity and Therapeutic Index

Bramsen et. al. Nucleic Acids Res. 2010

Vaish et. al. Nucleic Acids Res. 2011

Lee et. al. Nat. Comm. 2015

Janas, Schlegel et al. Nat. Comm. 2018

Utilize seed-pairing

destabilization via novel

chemical modifications

to selectively destabilize

off-target binding

Off-target bindingPartial sequence match

Off-target mRNA 3’-UTR

Antisense loaded RISCpos. 2-8

Undesired off-

target activity Important Considerations

1. On-target potency must be maintained in vivo

2. Off-target activity should be minimized

(S)-GNA

CONFIDENTIAL14

ESC+ Demonstrates More Quiescent Off-Target Signature

Across Dose Levels in Rat Liver with Comparable On-Target KD

ES

CE

SC

+

3 mg/kg 10 mg/kg 30 mg/kg 60 mg/kg 120 mg/kg

DEGs (Differentially Expressed Genes), significant, 3’UTR match, significant, 3’UTR match, not significant

Dose: qw x 3

Necropsy: Day 16

Histopath

for 30 mg/kg dose

Parent ESC

AS7-GNA

= target mRNA

CONFIDENTIAL15

ALN- AAT01 ALN-AAT02

Structure*

Efficacy Up to 89% KD Up to 89% KD

Liver Safety

(ALT >3x ULN)1/15 (up to 6 mg/kg dose) 0/18 (up to 6 mg/kg dose)

Positive ESC+ Human POCALN-AAT02 Clinical Activity and Safety

* Images are representative

GalNAcGalNAc

GalNAc

GalNAcGalNAc

GalNAc

0 2 4 6 8 1 0 1 2

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

S t u d y W e e k

Se

ru

m A

AT

(g

/L)

0 . 3 m g / k g

1 . 0 m g / k g

3 . 0 m g / k g

P L A C E B O

L L O Q

S i n g l e D o s e A L N - A A T 0 2

0 2 4 6 8 1 0 1 2

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

S t u d y W e e k

Se

ru

m A

AT

(g

/L)

0 . 3 m g / k g

1 . 0 m g / k g

3 . 0 m g / k g

P L A C E B O

L L O Q


0 2 4 6 8 1 0 1 2

0 . 0

0 . 5

1 . 0

1 . 5

2 . 0

S t u d y W e e k

Se

ru

m A

AT

(g

/L)

0 . 3 m g / k g

1 . 0 m g / k g

3 . 0 m g / k g

L L O Q

P L A C E B O


CONFIDENTIAL16

GalNAc-siRNA Safety Summary

• Antisense strand-driven RNAi-mediated off-target effects are important drivers of hepatotoxicity

◦ No evidence for impact of chemical modifications on observed toxicity

◦ No evidence for microRNA competition

• ESC+ strategy mitigates seed-mediated off-target effects, improves specificity and further expands therapeutic window of siRNA conjugates

◦ Utilizes thermally destabilizing nucleotide, such as glycol nucleic acid (GNA), in seed region of antisense strand

◦ Multiple ESC+ conjugates have advanced into clinical development

◦ Positive human POC achieved

◦ Some sequences show an inherent ESC+ profile and may not require further chemical modification

Hybridization-based off-target effects, e.g.

microRNA-like seed-based activity

CONFIDENTIAL17

Agenda

Optimizing the safety of liver-targeted GalNAc-siRNA conjugates

Translating liver learnings to CNS-targeted siRNA conjugates

CONFIDENTIAL18

Knockdown Observed in Deep Brain Regions after a Single IT Injection

siRNA Conjugate Is Active Across CNS Regions

NHPTemporal

Cortex

Prefrontal

Cortex

Striatum

Lu

mb

ar

Sp

ine

Cerv

ical S

pin

e

Pre

fro

nta

l C

or t

ex

Tem

po

ral C

or t

ex

Str

iatu

m

0

2 0

4 0

6 0

8 0

1 0 0

T o o lk it A P P s iR N A

7 2 m g , D a y 2 9

AP

P m

RN

A R

em

ain

ing

(%

)

Lu

mb

ar

Sp

ine

Cerv

ical S

pin

e

Fro

nta

l C

or t

ex

Tem

po

ral C

or t

ex

Str

iatu

m

0

2 0

4 0

6 0

8 0

1 0 0

T o o lk it S O D 1 s iR N A

0 .9 m g , D a y 7 -8

SO

D1

mR

NA

Re

ma

inin

g (

%)

Rat

CONFIDENTIAL19

Lowest Activity in the Endothelium

siRNA Conjugate Is Active in Most CNS Cell Types in Rat

Neuron Astrocyte MicrogliaEndothelium

Green: MAP2

Purple: Duplex

Yellow: GFAP

Purple: Duplex

Yellow: IBA1

Purple: DuplexYellow: CD31

Purple: Duplex

OligodendrocyteYellow: MBP

Purple: Duplex

siR

NA

ac

cu

mu

lati

on

siR

NA

ac

tivit

y

GFAP siRNA IBA1 siRNAPECAM1 siRNA MBP siRNA

Lu

mb

ar

Sp

ine

Cerv

ical S

pin

e

Fro

nta

l C

or t

ex

Tem

po

ral C

or t

ex

Str

iatu

m

0

3 0

6 0

9 0

1 2 0

1 5 0

D a y 3 2

GF

AP

mR

NA

Re

ma

inin

g (

%)

Lu

mb

ar

Sp

ine

Th

ora

cic

Sp

ine

Cerv

ical S

pin

e

Cere

bellu

m

Rem

ain

ing

Hem

isp

here

0

3 0

6 0

9 0

1 2 0

1 5 0

D a y 1 4

PE

CA

M1

mR

NA

Re

ma

inin

g (

%)

Lu

mb

ar

Sp

ine

Cerv

ical S

pin

e

Hip

po

cam

pu

s

Tem

po

ral C

or t

ex

Bra

inste

m

0

3 0

6 0

9 0

1 2 0

1 5 0

D a y 3 2

IBA

1 m

RN

A R

em

ain

ing

(%

)

Lu

mb

ar

Sp

ine

Cerv

ical S

pin

e

Bra

inste

m

Cere

bellu

m

Rig

ht

Hem

isp

here

0

3 0

6 0

9 0

1 2 0

1 5 0

D a y 1 4

MB

P m

RN

A R

em

ain

ing

(%

)

MAP2 siRNA

Lu

m

ba

r S

pi n

e

Ce

rv

i ca

l S

pi n

e

Fro

nta

l C

orte

x

Te

m

po

ra

l C

orte

x

Stri a

tu

m

0

3 0

6 0

9 0

1 2 0

1 5 0

D a y 2 8

MA

P2

m

RN

A

Re

ma

in

in

g

(%

)

CONFIDENTIAL20

Single IT Injection, 60 mg

siRNA Conjugate Is Durable for > 6 Months in NHP

0

2 0

4 0

6 0

8 0

1 0 0

A D -4 5 4 8 4 4

T im e p o in t , (d a y s )

AP

P R

em

ain

ing

fro

m B

as

eli

ne

(%

)

1 0 0 1 -s A P P

1 0 0 2 -s A P P

1 0 0 1 -s A P P

1 0 0 2 -s A P P

1 7 13 29 55 93 120 153 184

Toolkit APP siRNA

CONFIDENTIAL21

• siRNA conjugates are active across CNS regions, including deep brain structures

• siRNA conjugates are active in most CNS cell types, including neurons, microglia, astrocytes, and

oligodendrocytes

• siRNA conjugates have long half-life in the CNS, resulting in durable activity (> 6 months in

monkey)

• No test article-related safety findings to date in rodent or monkey nonclinical studies

CNS Summary

CONFIDENTIAL22

Our Innovative Work Continues…with Physical Distancing

Documents

Advancing the Safety and Reach of RNAi Therapeutics