Familial Support Group Meeting Overview of ALN-TTR, a

Familial Support Group Meeting

Overview of ALN-TTR, a Novel RNAi

Therapeutic for TTR Amyloidosis October 29, 2011

Jared Gollob, M.D.

Agenda

Background on RNAi and TTR Amyloidosis

ALN-TTR Program: Preclinical Data

Natural History Study of Serum TTR Levels

ALN-TTR01 Phase 1 Study Design and Status

Summary and Next Steps

2

RNAi Cellular Mechanism

mRNA

mRNA degradation

dsRNA dicer

Cleavage

Strand separation

Complementary pairing

Cleavage

(A)n

(A)n

Natural Process of RNAi

Synthetic siRNA

Targeted Gene

Silencing

RISC

3

Lipid Nanoparticles (LNPs) for Systemic RNAi

Multi-component lipid formulation

» Amino lipid

» Structural lipid

» PEG lipid

» Cholesterol

Highly efficient for liver delivery

» Hepatocyte-specific gene

silencing achieved

• Low surface charge

• Small uniform size particle <100 nm

DLin-KC2-DMA

Cholesterol mPEG2000-C14 glyceride

O

ON

DSPC

4

TTR Biology

TTR synthesized predominantly in liver, circulates as ~55 KDa tetramer

Binds and transports serum retinol binding protein (RBP)/vitamin A and

minor fraction of serum thyroxine (T4)

Knock-out mice have mild peripheral phenotype » Reduced levels of serum retinol, RBP, thyroid hormone without significant

physiological effects – Normal vitamin A metabolism, total liver retinol unchanged (Wei et al., 1995)

– Thyroid function normal (Palha et al., 1994)

» Modest sensorimotor changes may represent developmental effect (Fleming et al.,

2007)

Native TTR

tetramer

Rearranged

tetramer

Monomer Oligomeric, non-fibrillar

and protofibrillar species

Amyloid fibril

n

Adapted from Hou X. et al., 2007

5

Transthyretin (TTR) Amyloidosis

Autosomal dominant disease

Deposition of mutant and wild-type TTR in tissues outside of liver » Nerves and/or heart are main target organs depending on mutation

Familial amyloidotic polyneuropathy (FAP) » Prevalence of ~10,000 cases worldwide

» V30M most common mutation

Familial amyloidotic cardiomyopathy (FAC) » Prevalence of ~40,000 cases worldwide

» V122I most common mutation

Liver transplant current standard of care for early stage V30M FAP

Drugs in development » TTR tetramer stabilizers (tafamidis, diflunisal)

» Agents targeting hepatic TTR synthesis (siRNA, ASO)

6

Agenda






7

ALN-TTR Summary

8

Molecule Anti-TTR siRNA, modified duplex

Formulation and Dosing

Formulated in lipid nanoparticle

Molecular Hypothesis

An RNAi therapeutic targeting TTR gene will inhibit mutant and wild type TTR production in the liver

Therapeutic Hypothesis

Decreasing hepatic production of mutant and wild type TTR will decrease amyloid deposition in tissues and facilitate amyloid clearance from tissues, thereby halting progression or improving end-organ dysfunction

Potential Indications

Familial amyloidotic polyneuropathy (FAP) Familial cardiac amyloidosis (FAC)

Stage Status ALN-TTR01 Phase I trial initiated in July 2010; Advancing ALN-TTR02 with 2nd generation LNP

ALN-TTR siRNA Selection

>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

Val122Ile Val122 (-) Cys10Arg

Ser23Asn Phe44Ser

Ser52Pro

Ala91Ser Thr118Met

Val30Ala

Val30Gly

Val30Leu

Val30 Met

5’ UTR

3’ UTR

9

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

TT

R/G

AP

DH

mR

NA

(

rela

tive t

o c

on

tro

l)

80% cut-off

ALN-TTR siRNA Lead Selection In Vitro Screen

~140 siRNAs screened to identify lead candidate

Assays performed in HepG2 cells

TTR mRNA quantified by RT-PCR 24hr post-transfection

AALSD: Liver Meeting, Nov 2009

IC50 = 3 pM

1x10 -5

0.001 0.1 10 0

0.2

0.4

0.6

0.8

1

1.2

1.4

siRNA (nM)

TT

R/G

AP

DH

mR

NA

(re

lati

ve

to

co

ntr

ol)

10

ALN-TTR Silences Mutant Human TTR V30M TTR Transgenic Mouse Model

ALN-TTR silences human V30M TTR mRNA and suppresses mutant protein levels Single i.v. dose of ALN-TTR or control siRNA Liver mRNA and serum TTR levels measured 48hr post-dose

Collaboration with M. Saraiva

ED50 ~ 0.15 mg/kg RNAi, MicroRNAs, 2009

Liver mRNA

TT

R/G

AP

DH

mR

NA

(re

lati

ve

to

co

ntr

ol s

iRN

A)

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 le

ve

ls

(re

lati

ve

to

co

ntr

ol s

iRN

A)

ALN-TTR

(mg/kg)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0.03

***

0.3

***

3 3

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)

11

ALN-TTR Silencing is Durable V30M TTR Transgenic Mouse Model

ALN-TTR efficacy is both rapid and durable Single i.v. dose 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

TT

R/G

AP

DH

mR

NA

(rel

ativ

e to

Co

ntr

ol s

iRN

A)

Control

siRNA

Day 3

TT

R S

eru

m P

rote

in

(rel

ativ

e to

co

ntr

ol s

iRN

A)

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

1.6 Serum 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

AALSD: Liver Meeting, Nov 2009 Collaboration with M. Saraiva

12

ALN-TTR Prophylactic Efficacy V30M TTR Transgenic Mouse Model

ALN-TTR blocks pathogenic accumulation of mutant human TTR in peripheral tissues >95% Reduction of V30M hTTR deposition Multi-dose i.v. injections of ALN-TTR or control siRNA, 3 mg/kg (days 0, 14, 28) Quantitation of V30M hTTR deposition by immunohistochemistry (day 56)

0

5

10

15

20

25

30

Re

lati

ve

TT

R T

iss

ue

Le

ve

ls

Esophagus Colon Sciatic

nerve

Dorsal root

ganglion Stomach

Control siRNA ALN-TTR

Keystone: RNA Silencing, Jan. 2010

8 weeks

ALN-TTR or control siRNA

16 weeks ALN-TTR

Inte

sti

ne

Sto

mach

S

cia

tic n

erv

e

DR

G

control

siRNA


100% 100%

92%

95%

100%


13

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-TTR or control siRNA, 3 mg/kg (d0, 14, 28, 42, 56, and 70)

Quantitation of TTR deposition by immunohistochemistry (day 77)

21 months

ALN-TTR or control siRNA

24 months

ALN-TTR

Control siRNA

0

10

20

30

40

50

60

70

Sciatic

nerve

Dorsal root

ganglion

Esophagus Colon Stomach

Re

lati

ve

TT

R T

iss

ue

Le

ve

ls

Control siRNA ALN-TTR

Dorsal Root Ganglion

100%

99%

99%

97%

99%

XII International Symposium on Amyloidosis, Apr. 2010 Collaboration with M. Saraiva

14

ALN-TTR Reduces TTR mRNA Non-Human Primates

ALN-TTR shows dose dependent silencing of TTR mRNA Single i.v. infusion of ALN-TTR or control siRNA

Liver mRNA levels measured 48hr post-dose

Keystone RNAi, Feb 2009

ALN-TTR

(mg/kg)

1.0 Control siRNA

(mg/kg)

3 0. 3 3.0

*** ***

***

47% 62%

82% TT

R/G

AP

DH

mR

NA

(Rela

tive t

o C

ontr

ol)

0.0

0.2

0.4

0.6

0.8

1.0 ED50 ~ 0.3 mg/kg

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

15

2nd Generation ALN-TTR Program ALN-TTR02

ALN-TTR02 shows >10-fold improved in vivo efficacy Single i.v. infusion

Liver mRNA levels measured 48 hr post-dose

Potent, dose-dependent TTR silencing

ED50 ~ 0.03 mg/kg

43%

85%

94%

0.0

0.2

0.4

0.6

0.8

1.0

ALN-TTR02

(mg/kg)

0.1

Control

0. 03 0.3 ALN-TTR01

(mg/kg)

1.0 Control

0. 3 3.0

* *

*

47% 62%

82%

TT

R m

RN

A L

eve

ls

(Re

lative

to

Co

ntr

ol)

0.0

0.2

0.4

0.6

0.8

1.0 * p< 0.05

*

* *

* p< 0.05

ED50 ~ 0.3 mg/kg

1st Generation 2nd Generation

BRI Translational Genetics Series, Feb. 2011

16

0

0.2

0.4

0.6

0.8

1.0

1.2

0 4 8 12 16 20 24 28

Suppression of TTR Protein by ALN-TTR02 is Durable Non-Human Primates

TT

R S

eru

m P

rote

in

(re

lati

ve

to

pre

ble

ed

)

Days post-treatment

ALN-TTR02 ED50 ~ 0.03 mg/kg, ED80 ~ 0.2 mg/kg

Maximal TTR protein suppression at ~ 7-14 days

ALN-TTR02 demonstrates durable, dose-dependent suppression of TTR serum protein

levels greater than 28 days in NHP Single 15 min IV infusion (0.03, 0.1, 0.3 mg/kg)

Serum TTR protein levels measured by ELISA on days 0, 1, 2, 4, 7, 14, 21, and 28

BRI Translational Genetics Series, Feb. 2011

17

0.03 mg/kg 0.1 mg/kg 0.3 mg/kg

Agenda






18

ALN-TTR-NT Study Design / Demographics / Mutations

Study Design / Objective » Non-intervention / natural history

study

» Assess variability of serial serum

TTR levels in patients and carriers

using:

– ELISA-based assay

– Isoelectric focusing gel (IEF)

– LCMS/MS

» Weekly blood draws

– 4 consecutive weeks at clinic or at

subject’s home

» PI: Dr. John Berk (Boston University)

Status/Demographics

» 27 subjects enrolled

» 26 completers

Carrier (11)

Patient (15)

Disease State

M (13) F (13)

Gender

Pro 49 (4)

Met 30 (10) Ala 60 (5)

Ile 122 (4)

Met 107 (1)

Ser 97 (1) GLY 42/ASN90

(1)

ALN-TTR-NT-001 Mutations

19

Coefficient of Variation for Serum TTR All versus V30M Only

Group

included in

analysis

Visits

included in

analysis

Number of

Subjects

Number of

Observations Mean

Variance

Component

Estimate of

Within

Subject SD

Variance

Component

Estimate of

Between

Subject SD

Carrier (All) All visits 11 44 194.9 33.2 42.6

Patient (All) All visits 15 57 207.9 47.8 52.3

Overall (All) All visits 26 101 202.2 42.0 47.7

Carrier

(V30M) All visits 4 16 230.7 26.5 55.1

Patient

(V30M) All visits 6 24 230.2 19.0 65.5

Overall

(V30M) All visits 10 40 230.4 22.3 58.2

17%

23%

21%

11%

8%

10%

20

Summary

Serum TTR shows relatively modest intrapatient variability (~10-20% fluctuation from week to week)

LCMS/MS analysis on V30M and V122I samples in progress

Additional key factors to be analyzed in assessment of response to ALN-TTR include: » Serum RBP

» Vitamin A levels

» Kinetics of TTR/RBP/Vitamin A suppression

21

Agenda






22

ALN-TTR01 Phase I Study

Study Design Randomized, placebo-controlled, single-blind, single-

dose escalation study » 3:1 randomization » 4 patients/cohort

Up to 36 patients with ATTR » Conducted in Portugal (T. Coelho), Sweden (O. Suhr),

France (D. Adams) and UK (P. Hawkins)

Primary objective » Safety and tolerability

Secondary objectives » Characterize plasma pharmacokinetics » Assess preliminary pharmacodynamic activity

– Serum TTR, RBP, Vitamin A levels » TTR measured by ELISA and LCMS/MS

23

ALN-TTR01 Phase I Study (Cont’d)

Study Design Treatment regimen

» Single 15-minute ALN-TTR01 i.v. infusion

» Premedication with corticosteroids, H1/H2 blockers,

acetaminophen

» Dose levels: 0.01-1.0 mg/kg

Status » Initiated Q3 ’10; actively enrolling

» To present safety and pharmacodynamic data at VIIIth International

Symposium on Familial Amyloidotic Polyneuropathy in Kumamoto,

Japan on November 21st

24

Agenda






25

TTR Program Summary and Next Steps

ALN-TTR01 In Phase I clinical trial, actively enrolling

» Targets wild-type TTR and all mutant forms of TTR

» LNP formulation

» Potent reduction of TTR in vitro

» Potent and durable silencing of TTR in vivo – Wild-type TTR in rodents and non-human primates

– Mutant human V30M TTR in transgenic mouse

» Therapeutic efficacy measured by reduced TTR pathogenic tissue deposition in V30M transgenic mouse

ALN-TTR02 Drug candidate using same siRNA as ALN-TTR01

formulated in second-generation LNP with enhanced potency, in development; IND/CTA filing in 2011

26

Acknowledgments TTR Amyloidosis Program

Scientific Collaborators

Maria Saraiva, Institute of Cellular and Molecular Biology, Porto,

Portugal

Yukio Ando and Hiro Jono, Kumamoto University, Japan

ALN-TTR01 Clinical Investigators

Teresa Coelho

» Hospital Geral de Santo Antonio, Porto, Portugal

Ole Suhr

» Umea University Hospital, Umea, Sweden

David Adams

» CHU Hospital Bicetre, Le Kremlin-Bicetre, France

Tim Mant/Philip Hawkins

» Quintiles Drug Research Unit at Guy’s Hospital, London

27

Documents

Familial Support Group Meeting Overview of ALN-TTR, a