The surprising biology

of short RNAs and their relevance to

bioterrorism

RNAi Discovery

• C. elegans (Fire et al., 1998)• Drosophila (Carthew et al., 1998) • Planaria (Newmark et al., 1998)• Trypanosomes (Ullu et al., 1998) • Hydra (Lohmann et al., 1999)• Zebrafish (Wargelius et al., 1999)• Mice (Wianny & Zernicka-Goetz, 2000)• “cosuppression” in plants• “quelling” in Neurospora

5’ p

p3’

3’

5’

Short RNAs silence by mRNA

degradation

turnover

Drosophila and mammals worms and plants

Therapeutic gene silencing

DNA

Proteins

ReceptorsReceptorsEnzymesEnzymes

mRNA

Therapeutic gene silencing

Small Molecules

DNA

Proteins

ReceptorsReceptorsEnzymesEnzymes

Antibodies

mRNA

Protein Therapeutics

Therapeutic gene silencing

DNA

Proteins

ReceptorsReceptorsEnzymesEnzymes

Antibodies

mRNA

Protein Therapeutics

Small Molecules

Therapeutic gene silencing

DNA

mRNA

siRNA

Therapeutic gene silencingsiRNA

DNA

mRNA

siRNA silences mRNA

--blocks protein production

siRNA silences mRNA

--blocks protein production

Alnylam overview •Leader in RNAi therapeutics

Strong and experienced team• ~140 employees by year end; Cambridge, MA • Key founders: Sharp, Tuschl, Schimmel, Zamore, Bartel

Commitment to scientific excellence• Multiple papers in world’s top journals

– Nature, Nature Medicine, Cell, etc.

Strong product focus• ALN-RSV01

– Phase II Experimental Infection Study initiated June 2007• Rapidly developing pipeline

– 1 new IND by end ’07/early ’08– 1-2 addiitional INDs in ‘08– Multiple pre-clinical programs

Leading IP• Only company with access to all issued or granted fundamental IP

on siRNAs– Unambiguous FTO– Blocking position in entire field

• >150 issued chemistry/delivery patents (Isis, Tekmira, etc.)

Industry-leading collaborations and partnerships• 5 distinct partnerships with 4 major life science companies

– Medtronic, Novartis, Biogen Idec, and Roche• Significant federal funding• MicroRNA therapeutics with Regulus Therapeutics, LLC

Well-capitalized• 2004 IPO, NASDAQ: ALNY• Raised ~$750M to date

– ~$250M raised in private/public equity– ~$500M raised in realized partnership funding

• Q2 ’07 cash of ~$195M; Y-E ’07 cash >$435M• ~$1.25B market cap

Use of siRNA in therapy

1. Most diseases are the results of genes: virus, cancer (oncogenes and cell death), Parkinsons (alpha-synuclein).

2. Gene specific silencing but some off-target effects a) partially sequence-specific (positions 2-8 from 5’ end) b) 1.4 to 2 fold at mRNA level.

3. Delivery of siRNA to interior of cells in vivo is a challenge a) direct to organ (eye) b) systemic to organs.

Turning siRNAs into drugs

• Choose potent and specific sequence

• Stabilize against exonucleases

• Stabilize against endonucleases

• Add conjugate to improve biodistribution

Phosphorothioate 2’OMe, 2’F

Cholesterol

, others

» Phosphorothioate

» 2’OMe, 2’F

» Cholesterol, others

» Bioinformatics, in vitro assays

Incorporate minimal number of modifications required for appropriate pharmaceutical

properties

Incorporate minimal number of modifications required for appropriate pharmaceutical

properties

Respiratory Syncytial Virus (RSV) Program

•

•Harness RNAi to treat a major infectious disease– Significant unmet medical need

• >125,000 pediatric hospitalizations/yr in US• >170,000 adult hospitalizations/yr in US• Annual cost is >$750 million• Significant morbidity including asthma

– No effective therapies to treat RSV infection

• Synagis used for prevention

– ALN-RSV01 in Phase II clinical development• Target: RSV nucleocapsid “N” gene

– Highly conserved gene– Essential for viral replication

siRNA i.n. (25-100ug)

4 hrs Virus (RSV/A2)i.n. (106 pfu)

4 days Viral Titer in Lung

In Vivo anti-viral activity of siRNAsrodent prophylaxis model

P gene N gene L gene

0

1

2

3

4

5

6

P1 P2 P3 P4 P4-MM

Log

10

PFU

/g lu

ng

1 mg/kg2 mg/kg4 mg/kg

LLOD

ALN-RSV01

N2

L3L2L1

RSV Symposium, Aug. 2005

In Vivo anti-viral activity of siRNAsrodent treatment model

Virus (RSV/A2)i.n. (106 pfu)

Day 1 siRNASingle dose i.n. (2 mg/kg)

Day 5 Viral Titer in Lung

Day 2Day 3Day 4

0

1

2

3

4

5

6

Log

10 P

FU

/g lu

ng

Day 1

Day 2

Day 3

Day 4

PBS P1 ALN-RSV01 L3

LLOD

P4-MM

RSV Symposium, Aug. 2005

Alnylam biodefenseViral Hemorrhagic Fever Programs

• RNAi Therapeutic targeting the Ebola Virus– September, 2006– NIAID/NIH/DHHS– $23M– 39 month contract through IND filing

• Host-Directed RNAi Therapeutic for Ebola Virus and Other Hemorrhagic Fever Viruses– August, 2007 – DTRA/DoD– $38M– 33 month contract through Phase 1 studies

RNAi therapeutic targeting the Ebola virus

– Develop RNAi anti-viral targeting Ebola • Severe, often fatal infection• Collaboration with USAMRIID

(Drs. Bavari and Warfield)

– Target all pathogenic strains • Zaire and Sudan

– Non-biased approach to selecting viral gene targets

Structural:LVP30VP35NP

RNA

Membrane-associated:VP24VP40GP

Bray and Geisbert, Int. J. Biochem. Cell Biol. (2005) 37:1560-6.

● Macrophages and dendritic cells are primary cells involved in early infection

● Hepatocytes and other parenchymal cells involved in late infection

In Vivo delivery to appropriate cells

Protocol: siRNA was transfected into 293T cells and cells were infected next day. Viral titer was measured after 48h in the sup by plaque assay.

USAMRIID In vitro proof of concept Ebola siRNA reduces viral titer by over 90%

1 log reduction in viral titer with unmodified siRNA

EBOV-ZAIRE siRNA; 48h time point

1 000 10 000 100 000 1 000 000 10 000 000

L siRNA #2

L siRNA #1

NP siRNA #2

NP siRNA #1

VP35 siRNA #2

VP35 siRNA #1

VP24 siRNA #2

VP24 siRNA #1

Negative siRNA

No siRNA

Plaque Forming Units / ml

Data: Kelly Warfield and Sina Bavari, USAMRIID

Liposomal formulations for systemic RNAi

– Liposomes are organized lipid nanoparticles used in pharmaceutical drug delivery• Used in over 12 approved

drugs

– Enables delivery of siRNAs into cells• Highly efficient for liver

deliverysiRNA

● Guinea pig Zaire EBOV infection model

● Pooled Ebola or scrambled siRNA

● 1 mg/kg siRNA in SNALP2 liposomal formulation given i.p. 1 hr after EBOV infection and then daily on days 1-6.

● Sacrifice on day 7 for viremia (n=5 per group)

In vivo proof of concept Liposomally delivered Ebola siRNA protects from lethal infection

Reference: Geisbert et al., JID (2006) 193:1650-7.

•RNAi Therapeutic silencing of host genes involved in the pathogenesis and diathesis associated with viral hemorrhagic fever infections including hemorrhage and enhancement of viral assembly by host proteins

Coagulopathy

Host-directed viral assembly

and budding

Critical cell signaling

molecules

Broad spectrum RNAi therapeutic for Ebola virus targeting an endogenous host

factor

The discovery of 250 to 1000 new genes that encode microRNAs. These probably regulate 25-50% of all genes in vertebrates.

V. Ambros (Lee et al. 1993)L. Ruvkun (Wightman et al. 1993)T. Tuschl (Lagos-Quintana et al. 2001)D. Bartel (Lau et al. 2001)V. Ambros (Lee and Ambros, 2001)

MicroRNAs silence by translational inhibition and mRNA degradation

Proposed anatomy of miRNA/mRNA interactions

lin-14 mRNAlin-4 miRNA

8 nt “seed”

-5’3’-

V. Ambros (Lee et al. 1993)L. Ruvkun (Wightman et al. 1993)

The discovery of 250 to 1000 new genes that encode microRNAs. These probably regulate 25-50% of all genes in vertebrates.

V. Ambros (Lee et al. 1993)L. Ruvkun (Wightman et al. 1993)T. Tuschl (Lagos-Quintana et al. 2001)D. Bartel (Lau et al. 2001)V. Ambros (Lee and Ambros, 2001)

The expanding biology of miRNAs

• differentiation : miR-181, lsy-6, miR-1

• apoptosis : bantam, miR-14

• proliferation : miR-17-94 cluster, let-7

• developmental timing : lin-4, let-7

• morphogenesis : miR-430

“Control of stress response”

Literature on microRNA and stress Heart-specific miR-208 regulate stress-dependent cardiac growth

in mice (Rooij et al., Science 2007)

A model for the role of miR208 in cardiac gene regulation (Fig 6, Rooij et al., Science 2007)

Thank you for the opportunity to

present this lecture

Alnylam IP Leadership

Required for all siRNAtherapeutics

Fundamental IP

US 5,898,031: Crooke US 6,107,094: Crooke US 6,506,559: Fire and Mello EP 1144623: Kreutzer-Limmer EP 1214945: Kreutzer-Limmer EP 1230375: Glover EP 1407044: Tuschl II* US 7,056,704: Tuschl II US 7,078,196: Tuschl II Additional pending

Chemistry & Delivery IP

Required to introduce “drug-like” properties and

achieve delivery

>150 issued patents in-licensed from Isis, including

– US 7,138,517: Cook US 7,078,196: Tuschl II Key patents from Inex,

including– US 5,976,567: Wheeler– US 6,815,432: Wheeler– US 6,858,225: Semple

Many additional pending

Target IP

Relate to siRNAs for specific disease targets

US 7,056,704: Tuschl II– Covers any and all targets in

mammalian cells EP1352061: Kreutzer-Limmer II

– Covers >125 disease targetsVirusesCytokinesOncogenes/Cell cycleMany others

Many additional pending

Over 150 issued patents in major markets: US and EU

*Received Rule 51(4) EPC notification from EPO; equivalent to NOA from USPTO