The surprising
biology
of short RNAs and
their relevance to
bioterrorism
RNAi Discovery
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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
3’
5’ p
3’
p 5’
Short RNAs silence
by mRNA degradation
turnover
Drosophila and mammals
worms and plants
Therapeutic gene silencing
DNA
mRNA
Proteins
Receptors
Enzymes
Therapeutic gene silencing
Protein
Therapeutics
DNA
mRNA
Proteins
Receptors
Antibodies
Enzymes
Small
Molecules
Therapeutic gene silencing
Protein
Therapeutics
DNA
mRNA
Proteins
Receptors
Antibodies
Enzymes
Small
Molecules
Therapeutic gene silencing
siRNA
DNA
mRNA
siRNA
Therapeutic gene silencing
DNA
mRNA
siRNA silences mRNA
--blocks protein
production
Alnylam overview
•Leader in RNAi therapeutics
Strong and experienced team
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~140 employees by year end; Cambridge, MA
Key founders: Sharp, Tuschl, Schimmel, Zamore, Bartel
Commitment to scientific excellence
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Multiple papers in world’s top journals
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Nature, Nature Medicine, Cell, etc.
Strong product focus
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ALN-RSV01
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Phase II Experimental Infection Study initiated June 2007
Rapidly developing pipeline
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1 new IND by end ’07/early ’08
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1-2 addiitional INDs in ‘08
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Multiple pre-clinical programs
Leading IP
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Only company with access to all issued or granted fundamental IP on
siRNAs
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Unambiguous FTO
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Blocking position in entire field
>150 issued chemistry/delivery patents (Isis, Tekmira, etc.)
Industry-leading collaborations and partnerships
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5 distinct partnerships with 4 major life science companies
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Medtronic, Novartis, Biogen Idec, and Roche
Significant federal funding
MicroRNA therapeutics with Regulus Therapeutics, LLC
Well-capitalized
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2004 IPO, NASDAQ: ALNY
Raised ~$750M to date
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~$250M raised in private/public equity
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~$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
Cholesterol,
others
» Bioinformatics, in vitro assays
• Stabilize against
exonucleases
» Phosphorothioate
• Stabilize against
endonucleases
Phosphorothioate
2’OMe, 2’F
Incorporate minimal number of
modifications required for
appropriate pharmaceutical
properties
» 2’OMe, 2’F
• Add conjugate
to improve
biodistribution
» Cholesterol, others
Respiratory Syncytial Virus (RSV) Program
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•Harness RNAi to treat a major
infectious disease
– Significant unmet medical need
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>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
In Vivo anti-viral activity of siRNAs
rodent prophylaxis model
Log10 PFU/g lung
6
1 mg/kg
2 mg/kg
4 mg/kg
5
4
3
2
1
0
LLOD
P1
P2
P3
P gene
siRNA
i.n. (25-100ug)
RSV Symposium, Aug. 2005
ALNRSV01
N2
N gene
4 hrs
L1
L2
L3
P4
P4-MM
L gene
Virus (RSV/A2)
i.n. (106 pfu)
4 days
Viral Titer in Lung
In Vivo anti-viral activity of siRNAs
rodent treatment model
Log10 PFU/g lung
6
Day 1
Day 2
Day 3
Day 4
5
4
3
2
1
LLOD
0
PBS
P1
Virus (RSV/A2)
i.n.
(106
pfu)
RSV Symposium, Aug. 2005
ALN-RSV01
Day 1
Day 2
Day 3
Day 4
siRNA
Single dose
i.n. (2 mg/kg)
L3
Day 5
P4-MM
Viral Titer in Lung
Alnylam biodefense
Viral Hemorrhagic Fever Programs
• RNAi Therapeutic targeting the Ebola Virus
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September, 2006
NIAID/NIH/DHHS
$23M
39 month contract through IND filing
• Host-Directed RNAi Therapeutic for Ebola Virus and
Other Hemorrhagic Fever Viruses
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August, 2007
DTRA/DoD
$38M
33 month contract through Phase 1 studies
RNAi therapeutic targeting the Ebola virus
– Develop RNAi anti-viral targeting
Ebola
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Severe, often fatal infection
Collaboration with USAMRIID
(Drs. Bavari and Warfield)
– Target all pathogenic strains
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Zaire and Sudan
– Non-biased approach
to selecting viral gene
targets
RNA
Structural:
L
VP30
VP35
NP
Membrane
-associated:
VP24
VP40
GP
In Vivo delivery to appropriate cells
● Macrophages and
dendritic cells are
primary cells
involved in early
infection
● Hepatocytes and
other parenchymal
cells involved in
late infection
Bray and Geisbert, Int. J. Biochem. Cell Biol. (2005) 37:1560-6.
USAMRIID In vitro proof of concept
Ebola siRNA reduces viral titer by over 90%
EBOV-ZAIRE siRNA; 48h time point
No siRNA
Negative siRNA
VP24 siRNA #1
1 log reduction in
viral titer with
unmodified siRNA
VP24 siRNA #2
VP35 siRNA #1
VP35 siRNA #2
NP siRNA #1
NP siRNA #2
L siRNA #1
L siRNA #2
1 000
10 000
100 000
1 000 000
10 000 000
Plaque Forming Units / ml
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.
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
siRNA
• Highly efficient for liver
delivery
In vivo proof of concept
Liposomally delivered Ebola siRNA protects from lethal infection
● 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)
Reference: Geisbert et al., JID (2006) 193:1650-7.
Broad spectrum RNAi therapeutic for
Ebola virus targeting an endogenous host
factor
Coagulopathy
Host-directed viral
assembly
and budding
Critical cell
signaling
molecules
•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
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
8 nt “seed”
3’-
-5’
lin-14 mRNA
lin-4 miRNA
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
Over 150 issued patents in major markets: US and EU
Fundamental IP
Required for all siRNA
therapeutics
 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
*Received Rule 51(4) EPC notification from EPO; equivalent to NOA from USPTO
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 targets
 Viruses
 Cytokines
 Oncogenes/Cell cycle
 Many others
 Many additional pending