ALN-TTR, an RNAi Therapeutic for the Treatment of Transthyretin-Mediated Amyloidosis

Rene Alvarez, Ph.D.

TTR-Mediated Amyloidosis (ATTR) ProgramALN-TTR

RNAi to treat significant orphan disease

Transthyretin (TTR)-mediated amyloidosis (ATTR)» Caused by mutation in TTR gene

» Amyloid deposits in nerves and heart

– Familial Amyloid Polyneuropathy (FAP)

– Familial Amyloid Cardiomyopathy (FAC)

» ~50,000 patients with significant morbidity and mortality

Clinical pathology » Typical onset ~40 to >60 yr

» Fatal within 5-15 years

» Loss of autonomic function, painful neuropathy

» Congestive heart failure

Liver transplant current standard-of-care for

subset of FAP patients

Initiate Phase I trial H1, 2010

22

RNA Interference

mRNA

Target-specific

mRNA degradation

dsRNAdicer

Cleavage

Strand separation

Complementary pairing

Cleavage

(A)n

(A)n

Natural Process of RNAi

Synthetic siRNA

Reduced protein

expression

RNA-induced

silencing complex

3

Systemic RNAi DeliveryLipid Nanoparticles

Unformulated siRNAs are rapidly cleared from circulation Lipid nanoparticle formulations (SNALP) of siRNAs prolong half-life and enable hepatic deliverySystemic administration of SNALP-formulated siRNAs results in» Dose-dependent mRNA and protein

suppression of hepatocyte-expressed disease targets

» Suppression maintained for 2 to 4 weeks» Has been demonstrated in multiple species

(rodents, non-human primate) for multiple targets (apoB, TTR, Factor VII, PCSK9)*

4

*Zimmerman et al., Nature, 2006; Akinc et al., Nature Biotech, 2008; Frank-Kamenetsky et al., PNAS, 2008

ATTR Program Rationale

Effective delivery of siRNA to hepatocytes with

current LNP platform

» Chemically modified TTR siRNA

» Formulated in SNALP for systemic delivery

Hepatocytes primary site of TTR expression

» Mutant and wild-type TTR proteins pathogenic

– Liver transplant can stabilize or improve V30M FAP patients

– However, cardiac disease accelerates in other ATTR

patients due to increased production of wild-type TTR

» Production of both wild-type and mutant TTR

ideally targeted

Target well validated with human genetics

» ~90% FAP caused by V30M mutation

» FAC caused primarily by V122I mutation

5

Transthyretin crystal structure

ALN-TTR siRNA Selection

6

>100 Mutations identified in TTR gene

ALN-TTR targets region of TTR mRNA common to wild-type and all

known mutant forms of TTR

Asp18Glu

Asp18Gly

Ala25Ser

Ala25Thr

Arg34Thr

Ala36Pro

Trp41Leu

Gly47Ala

Gly47Arg

Gly47Glu

Gly47Val

Ser50Arg

Glu54Gln

Glu54Gly

Leu58Arg

Leu58His

Thr60Ala

Phe64Leu

Phe64Ser

Tyr69His

Tyr69Ile

Val71Ala

Asp74His

Ile84Asn

Ile84Ser

His90Asn

Gln92Lys

Gly101Ser

Arg104Cys

Arg104His

Ala109Thr

Ala109Val

Ala109Ser

Ser112Ile

Tyr116Ser

Thr119Met

Gly6Ser

Leu12Pro

Val20Ile

Pro24Ser

Val28Met

Phe33Cys

Phe33Ile

Phe33Leu

Phe33Val

Lys35Asn

Asp38Ala

Glu42Asp

Glu42Gly

Ala45Asp

Ala45Ser

Ala45Thr

Thr49Ala

Thr49Pro

Glu51Gly

Gly53Glu

Leu55Arg

Leu55Pro

Leu55Gln

Thr59Lys

Glu61Lys

Ile68Leu

Lys70Asn

Ile73Val

Ser77Phe

Ser77Thy

Glu89Gln

Glu89Lys

Ala97Gly

Ala97Ser

Pro102Arg

Ile107Val

Leu111Met

Tyr114Cys

Tyr114His

Ala120Ser

Pro125Ser

Val122Ala

Val122IleVal122 (-)Cys10Arg

Ser23Asn Phe44Ser

Ser52Pro

Ala91Ser Thr118Met

Val30Ala

Val30Gly

Val30Leu

Val30 Met

5’ UTR

3’ UTR

Durable Suppression of TTR In Vivo

Durable reduction of TTR mRNA with rodent TTR siRNA analog Single i.v. infusion of rodent TTR analog or control siRNA; 1 mg/kg dose

Liver mRNA levels measured on Days 3, 8, 11, 15, 19, 22, 25 and 29 post-dose

7

** p < 0.01, *** p < 0.001(one-way ANOVA, Bonferroni post- hoc test)

** *

***

** **

0 1 3 8 11 15 19 22 25 290.0

0.2

0.4

0.6

0.8

1.0

1.2

Days after administration

Rela

tive T

TR

mR

NA

Level

(no

rmalized

to

day 0

)

Control siRNA

Rodent TTR analog

ALN-TTR Reduces TTR mRNA Non-Human Primates

ALN-TTR shows dose dependent silencing of TTR mRNASingle i.v. infusion of ALN-TTR or control siRNALiver mRNA levels measured 48 hr post-dose

8

ALN-TTR

(mg/kg)

1.0Control siRNA

(mg/kg)

3 0. 3 3.0

*** ***

***

47% 62%

82%TT

R/G

AP

DH

mR

NA

(Rela

tive

to

Con

tro

l)

0.0

0.2

0.4

0.6

0.8

1.0ED50 ~ 0.3 mg/kg

*** p< 0.001 (one-way ANOVA, Dunn’s post-hoc test)

ALN-TTR

0.3 mg/kg

1 mg/kg

3 mg/kg

Day 0 1 2 3 4 5 7 10 14

Durability of Reduction of Wild-Type TTR In Vivoin NHP

9

Control siRNA 3 mg/kg

ALN-TTR Silences Mutant Human TTRV30M TTR Transgenic Mouse Model

ALN-TTR silences human V30M TTR mRNA and suppresses mutant protein levelsSingle i.v. dose of ALN-TTR or control siRNA

Liver mRNA and serum TTR levels measured 48 hr post-dose

10

ED50 ~ 0.15 mg/kg

Liver mRNA

TT

R/G

AP

DH

mR

NA

(rela

tive t

o c

on

tro

l siR

NA

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

3

Control

siRNA

(mg/kg)

0.03

***

0.3

ALN-TTR

(mg/kg)

***

3

Serum Protein

TT

R s

eru

m l

evels

(rela

tive t

o c

on

tro

l siR

NA

)

ALN-TTR

(mg/kg)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0.03

***

0.3

***

33

Control

siRNA

(mg/kg)

*** p< 0.001 (one-way ANOVA,

Dunn’s post-hoc test)

*** p< 0.001 (one-way ANOVA,

Dunn’s post-hoc test)

ALN-TTR Silencing is DurableV30M TTR Transgenic Mouse Model

ALN-TTR efficacy is both rapid and durable

Single i.v. bolus of ALN-TTR or control siRNA; 1 mg/kg

Liver mRNA and serum protein levels measured on Days 3, 8, 15 and 22 post-dose

11

TT

R/G

AP

DH

mR

NA

(rel

ativ

e to

Co

ntr

ol

siR

NA

)

Control

siRNA

Day 3

TT

R S

eru

m P

rote

in

(rel

ativ

e to

co

ntr

ol

siR

NA

)

ALN-TTR ALN-TTR

Day 8 Day 15 Day 22

Liver mRNA

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6Serum Protein

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Control

siRNA

Day 3 Day 8 Day 15 Day 22

Therapeutic Efficacy for ALN-TTR

V30M TTR Transgenic Mouse Model

ALN-TTR blocks pathogenic accumulation of mutant human TTR in peripheral tissuesMulti-dose i.v. bolus of ALN-TTR or control siRNA, 3 mg/kg (d0, 14, 28)Quantitation of TTR deposition by immunohistochemistry on day 56

12

0

5

10

15

20

25

30

Mea

n T

TR

IHC

In

ten

sity

Esophagus Colon Sciatic

nerve

Dorsal root

ganglionStomach

Control siRNAALN-TTR

Collaboration with M. Saraiva

8 weeks

ALN-TTR

or control siRNA

16 weeks

Therapeutic Efficacy for ALN-TTR

V30M TTR Transgenic Mouse Model

13

Marked reduction of mutant TTR in tissues associated with human diseaseMutant V30M TTR quantified with immunohistochemistry

ALN-TTR

IntestineStomachSciatic NerveDorsal Root Ganglion

control

siRNA

ALN-TTR Therapeutic Efficacy V30M TTR Transgenic Mouse Model

ALN-TTR promotes regression of pathogenic mutant human TTR deposits in peripheral tissues>90% Regression of existing V30M hTTR tissue deposits

Multi-dose IV bolus of ALN-TTR01 or control siRNA, 3 mg/kg (week 0, 2, 4, 6, 8, 10)

Quantitation of TTR deposition by immunohistochemistry (week 11)

14

21 months

ALN-TTR

or control siRNA

24 months

ALN-TTR01

Control siRNA

0

10

20

30

40

50

60

70

Sciatic

nerve

Dorsal root

ganglion

Esophagus Colon Stomach

Rela

tive T

TR

Tis

su

e L

evels

Control siRNAALN-TTR

Dorsal Root Ganglion

100%

98.8%

98.5%

97%

98.8%

TTR-Mediated Amyloidosis (ATTR) Program Summary

We have identified a lead siRNA targeting wild-type and all mutant

forms of TTR with an IC50 of 3 pM in hepatocyte cell lines

ALN-TTR reduced TTR mRNA levels (ED50 ~ 0.3 mg/kg) in non-

human primate, with significant reduction of TTR serum protein levels

and durability of suppression >14 days post-single administration

ALN-TTR reduced mutant TTR mRNA and plasma protein levels >

90% in the hTTR V30M transgenic mouse model, with suppression

lasting > 21 days post-single administration

ALN-TTR, when administered to hTTR V30M transgenic mice,

prevents TTR deposition (young mice) and leads to regression (old

mice) of pre-existing TTR deposits in key target tissues, including:

dorsal root ganglia, sciatic nerve, stomach, and intestines

These findings demonstrate the potential therapeutic benefit of an

RNAi therapeutic targeting TTR for the treatment of TTR-mediated

amyloidosis (ATTR)

15

TTR TeamProgram Advisors and Extended Team

Victor KotelianskiDave KonysStu PollardStu MilsteinGreg HinkleRichard DuncanTuyen NguyenGang WangAkin AkincMartin MaierMara BrobergWill CantleyKlaus Charisse Jason CostiganPei GeMartin Maier Rachel MeyersKathleen Mills Jonathan O'SheaChang PengBill QuerbesJeffrey Rollins Sarfarz ShaikhChristopher SherrillMarjie SolomonSergey Shulga-MorskoyMark TracyLigang ZhangAbby CapobiancoFrances WongMark TracyTony RossomandoChunhua WangJeff KopaczCaroline Stark-BeerRebecca Howard

16

Core TeamRene Alvarez (Program Leader)

Dinah Sah (Program Leader)

Rena Denoncourt (Program Manager)

Christina Gamba-Vitalo

Jeff Cehelsky

Renta Hutabarat

Jared Gollob

Klaus Charisse

Lubo Nechev

Matthias Kretschmer

Mano Manoharan

Sara Nochur

Lauri Binne

Akshay Vaishnaw

Rick Falzone

David Watkins

Research TeamTodd Borland

Qingmin Chen

Saraiva Lab, Tekmira