1

Preclinical Development of ALN-AS1, an

Investigational RNAi Therapeutic for the

Treatment of the Hepatic Porphyrias

Amy Chan, Abigail Liebow, Makiko Yasuda, Scott Barros, Tim Racie, Martin Maier, Satya Kuchimanchi, Don Foster, Stuart Milstein, Klaus Charisse, Muthiah Manoharan, Robert Desnick, Rachel Meyers, Kevin Fitzgerald, Amy Simon, and William Querbes

International Conference of Poprhyrins and Porphyrias

15 September 2015

2

Therapeutic Approach to Harnessing RNAi

dsRNA dicer

Cleavage

Natural Process of RNAi

Synthetic siRNA

mRNA

mRNA degradation

Strand separation

Complementary pairing

Cleavage

(A)n

(A)n

Targeted Gene

Silencing

RISC

3

Receptor Targeted siRNA Conjugates for Delivery to Hepatocytes

Lead Selection & Optimization Subcutaneous Delivery

siRNA sequence » Specificity » Crossreactivity » Potency

Introduce chemical modifications for

“drug-like” properties

Small Scale Gene walks In vitro assays

Chemistry, Manufacturing and Controls

Medium Scale In vivo biology

Large Scale GMP Production Clinical trials

Selection

Optimization

GalNac-siRNA

Conjugates

siRNA chemistry » Stability » Immuno-silence » Potency

ASGPR Receptor » Highly expressed

on hepatocytes » Rapidly recycles » Conserved across

species

4

Clinical Experience with GalNac-siRNA Conjugates

GalNAc Conjugate Platform

• Currently utilized in 6 clinical stage ALNY

programs

• Over 250 patients and >1500 doses

administered

• Generally well tolerated

Human Proof of Concept

Ex: TTR Lowering in TTR Amyloidosis Program

Human Clinical Efficacy

Ex: Reduced Bleed Rate in Hemophilia Program

Gilmore, ACC, March 2015, Sorenson, ISTH, June 2015

Alnylam Development Pipeline

5

ALN-AS1 Therapeutic Hypothesis

Brennan et al. Nature 1979, Hermes-Lima et al. Biochem Biophys Acta 1991, Dar et al., Hepatobiliary Pancreat Dis

Int.; 9:93-6 (2010), Dowman et al., Ann Intern Med; 154:571-2 (2011)

Targeting Liver ALAS1 to Lower Heme Intermediate Production

ALN-AS1

Role of Liver in Disease

• Liver and domino transplant

experience

◦ ALA/PBG and symptoms

improve or come on rapidly

after transplant

• ALN-AS1 potential to treat

all acute hepatic porphyrias

including AIP, VP and HCP

6

ALN-AS1 Preclinical Development Overview

Lead ID Disease

Model POC NHP

Evaluation

Drug Safety and

Metabolism

Phase 1 Testing

• Identify ALAS1 target sequence and optimized chemistry

• Liver ALAS1 knockdown in rodents

• Impact of ALN-AS1 on ALA/PBG

• Define ALAS1 and ALA/PBG relationship

• Assay Development

• ALAS1 lowering kinetics

• Dose and dose regimen exploration

• Tolerability of ALN-AS1 in different species

• PK/PD relationships

• Evaluate any potential on target risks

Key

Data

7

ALN-AS1 Multidose Prophylaxis in Rat AIP Model

ALAS-GalNAc 3, 1, 0.3mg/kg (QWx4)

Phenobarb

D18 PBGD

LNP

D0 D7 D14 D21 D25 (sac)

ALAS1 mRNA

0.0

1.0

2.0

3.0

4.0

4.5

PBS ALN-AS1

3mg/kg 1mg/kg 0.3mg/kg

- + + + +

+ + + + +

rAL

AS

1/r

GA

PD

H,

Re

lative

to

pb

s=

1

PBGD LNP

siRNA

PB

ALA/PBG

0

2

4

6

8

10

PBS 3 mg/kg 1 mg/kg 0.3 mg/kg

- + + + +

+ + + + +

Urinary

ALA

or

PB

G

(m

mol/m

ol c

reatinin

ine)

PB

S=

1

-

PBGD LNP

ALN AS1

PB

PBG ALA

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ALN-AS1 Tissue Distribution in Rats

Exposure is the highest in the liver, followed by kidney; remaining tissues have

<2% of delivered dose deposited

Single Dose 10mg/kg 4 hours

0

1

10

100

1000

AL

N-A

S1 (

µg

/g)

Less than 2% of total exposure combined

~12X lower tissue concentration kidney vs. liver

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Circulating Extracellular RNA Detection (cERD)

Monitoring RNAi Activity in Liver

• mRNA or 5’RACE product in tissue

• Circulating secreted protein

Detection of Circulating ALAS1 mRNA

• Exosomes are shed into bodily fluids from many

different cell types and contain mRNA and miRNA

derived from tissue of origin

• Exosomes can be used to monitor ALAS1 mRNA

levels after ALN-AS1 dosing in serum/urine without

need for biopsy

Method for Circulating ALAS1 mRNA Detection

0

20

40

60

80

100

120

PBS 1.25 2.5 5.0

ALAS-GalNAc

(mg/kg) N

orm

ali

ze

d A

LA

S-1

by cERD

by liver biopsy

AS1 Transcript

QDx5, EOD, d15 (not DC)

Sehgal et al. RNA 2012

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Use of cERD Assay to Monitor ALN-AS1

Pharmacology in NHPs

QW-weekly

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Liver ALN-AS1 Concentrations and ALAS1

mRNA Knockdown in NHP

Single Dose 10mg/kg Multiple Dose 2.5mg/kg

• ALN-AS1 liver drugs levels correlate with extent and duration

of ALAS1 mRNA reduction

Weekly x8

12

0

100

200

300

400

500

600

700

NH

V 1

NH

V 2

NH

V 3

NH

V 4

11304

11313

11305

11309

11311

11319

11337

11513

11320

12049

Re

lati

ve

AL

AS

1 m

RN

A L

eve

ls (

% N

HV

)

Serum Urine

cERD Assay Demonstrates ALAS1 Upregulation

in Porphyria Patients vs. Healthy Volunteers

• ~3-fold upregulation of ALAS1 mRNA in AIP patients (n=10) vs. NHV (n=4)

• Good correlation of ALAS1 mRNA in urine and serum

• Chart review suggests trend of ALAS1 upregulation correlating with worse disease

severity

AIP Patients Normal Healthy

Volunteers

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Day to Day Variability in ALAS1 Levels with

cERD Assay in AIP Patient Samples

Human cERD Proof of Concept

• Minimal day to day variability

• Potential clinical utility

◦ ALAS1 changes during attacks

◦ Compare patient populations

◦ Monitoring PD with ALN-AS1 treatment

• Exploratory use in our EXPLORE natural

history study and in Phase 1

0

50

100

150

200

250

300

350

113

04-

Colle

ction 1

113

04-

Colle

ction 2

113

13-

Colle

ction 1

113

13-

Co

llectio

n 2

Rela

tive

AS

1 m

RN

A L

eve

ls (

% N

HV

)

Donor ID

Serum Urine

AIP Patient 1 AIP Patient 2

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ALN-AS1 Nonclinical Safety and Metabolism

Safety margins of >300x (rat) and >1500x (NHP), based on respective

no adverse effect levels (NOAELs) and clinical starting doses (mg/kg)

CTA-Enabling Studies for Start of Phase I Clinical Trial

Exploratory Studies Cytokine screening in vitro (hWBA) and in vivo (mouse) No acute pro-inflammatory

effects

Dose range-finding toxicity studies in mouse, rat, NHP

(repeat dose, 3-5 weeks in duration)

Establish dose levels for GLP

studies

GLP-Compliant

Safety Studies

Safety pharmacology (cardiovascular and respiratory) in

telemetered NHPs

NOAEL ≥150 mg/kg

Genetic toxicology in vitro (Ames, chromosomal

aberrations in hPBL) and in vivo (rat BM micronucleus)

All negative up to limit doses

General Toxicology studies in rat and NHP

• 13 week studies + 13 week recovery

• Toxicokinetics and CNS evaluation (NHP)

Rat NOAEL ≥ 30 mg/kg

NHP NOAEL ≥ 150 mg/kg

Absorption,

Distribution,

Metabolism,

Excretion (ADME)

Studies

PK/PD in rat, NHP

Toxicokinetics included in all Tox studies (plasma; liver and kidney in rodents)

Metabolic profiling in rat, NHP (plasma, liver)

Plasma protein binding (mouse, rat, NHP, human)

In vitro, ex vivo CYP studies

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ALN-AS1 Impact on P450s No Signs of Heme Deficiency in Preclinical Studies

Study/Species Doses CYPs Results

In vitro direct CYP

inhibition, human liver

microsomes

0.01-

10,000

ug/mL

1A2, 2B6,

2C8, 2C9,

2C19, 2D6,

3A4/5

No inhibition of

any isoform

In vitro CYP induction,

primary human

hepatocytes

0.1, 1, 10,

100 ug/mL

1A2, 2B6,

3A4

No changes in

CYP levels or

enzyme activity

Ex vivo CYP Activity

after ALN-AS1 dosing,

rat liver microsomes

30, 100,

300 mg/kg,

weekly x5

1A, 2B, 2D,

2E, 3A

No biologically

significant

changes

Ex vivo CYP Activity

after ALN-AS1 dosing,

NHP liver microsomes

30, 100,

300 mg/kg,

weekly x5

1A, 2B, 3A,

2D, 2E

No biologically

significant

changes

In vivo DDI with CYP

substrate probes

following ALN-AS1

dosing, NHP

5 mg/kg,

daily x5

1A, 2D, 2E,

3A

No reduction in

metabolism of

any CYP

substrate probes

No significant reduction

in P450 function or signs

of heme deficiency

detected with ALAS1

lowering

3A4,5 29%

2C8,9,18

18% 1A2 13%

2E1 7%

2D6 2%

2B6 1%

2A6 4%

Other 26%

~75% of total human liver CYPs

evaluated in these studies

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Conclusions and Next Steps for ALN-AS1

Conclusions

• ALN-AS1 is a subcutaneous administered RNAi therapeutic targeting ALAS1 in development for

the treatment of the hepatic porphyrias

◦ Potent silencing of liver ALAS1 mRNA and reduction in ALA/PBG in preclinical models

◦ Robust safety profile supporting the initiation of Phase 1

• The cERD assay is a non invasive method for monitoring circulating ALAS1 mRNA

◦ ALAS1 mRNA upregulated in serum/urine from AIP patients consistent with role in disease

pathophysiology

◦ Potential utility of the assay to understand ALAS1 levels during attacks, in different patient

populations, and following ALN-AS1 dosing in clinical trials

ALN-AS1 Clinical Development Plan

• EXPLORE natural history study (ongoing)

◦ Non interventional study in AIP patients with recurrent attacks

• Phase 1 Study (ongoing)

◦ Part A/B- Single ascending dose (SAD) and multiple ascending dose (MAD) study in AIP

asymptomatic high excreter patients (ASHE)

◦ Part C- Multiple dose study in AIP patients with multiple recurrent attacks

• Pivotal study in patients with recurrent attacks

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Acknowledgements

ALN-AS1 Core Team

• Bill Querbes (PL)

• Tanya Sengupta (PM)

• Amy Simon

• Craig Penz

• Amy Chan

• Scott Barros

• Lauri Binne

• Jason Costigan

• Satya Kuchimanchi

• Anshul Gupta

• Rena Denoncourt

• Nate Taneja

• Jon O’Shea

• Sarfraz Shaikh

• Mano Manoharan

• Rajeev Kallanthottathil

• Jeff Rollins

• Lubo Nechev

• John Frenz

• Jolly Bhatia

• Patrick Igwenagu

• Husain Attarwala

• Renta Hutabarat

• Ju Liu

• Krishna Aluri

• Chris Tran

• Yongli Gu

• Minggeng Gao

• Qianfan Wang

• Xuemei Zhang

• Mary Carioto

• Julia Hettinger

• Garvin Warner

• Mike Placke

• Matt Algarin

• Dave Mosher

• Svetlana Shulga Morskaya

• Meghan Collins

• Tim Mooney

EXPLORE Investigators

• Karl Anderson

• Herb Bonkovsky

• Montgomery Bissel

• John Phillips

• Charles Parker

• Manisha Balwani

• Joseph Bloomer

• Pauline Harper

• Eliane Sardh

• David Rees

• Mike Badminton

• Penelope Stein

• Raili Kauppinen

• Ulrich Stölzel

• Jorge Frank

• Elisabeth Minder

• Jean Charles Deybach

• Laurent Gouya

• Pavel Martesek

• Janne Langendonk

• Sverre Sandberg

• Delia D’Avola

• Maria Ivanova

• Paulo Ventura

• Maria Capellini

• Jerzy Wingyga

• Peter Meissner

• Joanne Marsden

ALN-AS1 Advisors and Extended Team

• Abigail Liebow

• Tim Racie

• Brian Bettencourt

• Kirsten McCarthy

• Siddharth Jain

• Don Foster

• Stu Milstein

• Rick Duncan

• Jared Gollob

• Martin Maier

• Klaus Charisse

• Matthias Kretschmer

• Lauren Melton

• Akshay Vaishnaw

• Jeff Cehelsky

• Kevin Fitzgerald

• Rachel Meyers

• Dave Watkins

• Oved Amitay

• Sara Nochur

• Carol Pitcher Towner

• Donna Mackey

• Ligang Zhang

• Lubomir Tchangov

• Karen Dobson

• Jeff Kopacz

• Jennifer Pena

• Kelley Hanson

Preclinical

Collaborators

• Robert Desnick

• Makiko Yasuda

• John Phillips

APF

• Desiree Lyon

• Jessica Hungate

• Natalia Sturza

APC Coordinator

• Hetanshi Naik

Phase 1 Investigators

• Eliane Sardh

• Nabil AL-Tawil

• Pauline Harper

• David Rees

• Penelope Stein

• Manisha Balwani

• Karl Anderson

• Monty Bissell

• Joseph Bloomer