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Alnylam RNAi Roundtable
March 4, 2010
Agenda & Introductions
Welcome
• Cynthia Clayton
» Senior Director, Investor Relations and Corporate
Communications
Recent Progress with Systemic Delivery of RNAi
Therapeutics
• Stuart Pollard, Ph.D.
» Vice President, Scientific and Business Strategy
• Akin Akinc, Ph.D.
» Associate Director, Research
2
RNAi Roundtable
• In-depth discussion around key topics relating to
Alnylam and the development of RNAi
therapeutics
• Interactive environment
• Recurring event
• Virtual and on-site RNAi Roundtables
3
Reminders
• Event will run until ~1:00 PM
• Q&A session will be held at end of presentation
» Submit questions at bottom of webcast screen
» Questions may be submitted at any time
• Replay, slides and audio available at
www.alnylam.com
4
Alnylam Forward Looking Statements
This presentation contains forward-looking statements.
There are a number of important factors that could cause
actual results to differ materially from the results anticipated
by these forward-looking statements. These important
factors include those that we discuss in our most recent
quarterly report on Form 10-K under the caption “Risk
Factors.” If one or more of these factors materialize, or if
any underlying assumptions prove incorrect, our actual
results, performance or achievements may vary materially
from any future results, performance or achievements
expressed or implied by these forward-looking statements.
5
Alnylam RNAi Roundtable
March 4, 2010
Recent Progress with Systemic
Delivery of RNAi Therapeutics
Agenda
• Introduction
• Next Generation LNP Delivery
• Future Strategies
• Summary
7
Achieving Delivery
Modification and Formulation
Naked siRNA
Key challenge
Cholesterol Conjugated siRNA
Antibody-Protamine
Complex siRNA
• PK/PD/Biodistribution
• Cellular uptake
• Key to broad application of RNAi
Broad and significant effort at Alnylam
• Internal and external collaborations
Major progress achieved
• Direct and Systemic delivery
• Robust in vivo efficacy
(Alnylam experience)
LNP siRNA
» >25 Targets
–
Includes many “un-druggable”
» >5 Organs
–
» 6 Species
siRNA Dynamic PolyConjugates
Aptamer Conjugated siRNA
8
Includes lung, liver, gut, and CNS
–
Includes humans
RNAi Delivery Strategies
Direct RNAi
Respiratory
» RSV/Influenza
» Cystic fibrosis
» Asthma/COPD
Ocular
» AMD/Retinopathy
» Glaucoma
CNS
»
»
»
»
Neuropathic pain
Spinal cord injury
Huntington’s disease
Parkinson’s disease
Topical/Mucosal
» Infectious disease
9
Systemic RNAi
Multiple major diseases
»
»
»
»
»
Metabolic
Viral disease
Cancer
Inflammation
Cardiovascular
Lipid Nanoparticles (LNPs) for Systemic RNAi
• Multi-component lipid formulation
»
»
»
»
Cationic lipid
Fusogenic lipid
PEG lipid
Cholesterol
• Highly efficient for liver delivery
O
N
O
» Hepatocyte-specific gene
silencing achieved
DLinDMA
mPEG2000-C14 glyceride
• Low surface charge
• Small uniform size particle <100 nm
10
Cholesterol
Rapid Advancements in Delivery
Publications and Presentations
Presentations
• 2009 Alnylam R&D Day (Nov 2009)
• “Advances in Biopharmaceuticals”
Keystone Symposia (Jan 2010)
Publications
• Lipid-like materials for low-dose in vivo
gene silencing
» Love et al., Proc. Natl. Acad. Sci. USA,
107:1864-9 (Jan 2010)
• Rational design of cationic lipids for
siRNA delivery
» Semple et al., Nature Biotechnology,
28: 172-178 (Jan 2010)
11
Agenda
• Introduction
• Next Generation LNP Delivery
• Future Strategies
• Summary
12
1st Generation LNP Systemic Delivery
In Vivo Silencing of apoB in Primates
Efficacy in primates with Systemic RNAi after single IV injection
• Rapid, potent, dose-dependent and durable effects
• RNAi specific and leads to measurable therapeutic benefit
• RNAi mechanism proven in vivo
* P <.05 ** P < .005
100
* P <.05 ** P < .005
mRNA
Protein
80
% Control
**
60
40
>65%
Inhibition
>85%
Inhibition
60
40
0
**
**
*
**
1mg/kg
2.5 mg/kg
2 day
Nature, 441, 111-114, Mar 2006
13
80
*
**
20
20
0
% Control
100
1 mg/kg
Cholesterol
LDL
HDL
Day 11 Post-Dose (2.5 mg/kg)
Saline
siRNA-Treated
Cyno-1 Cyno-2
1000
750
500
2.5 mg/kg
11 day
250
225 nt
Next Generation LNPs
Remarkable Potency Improvements with Novel Lipids
Novel LNPs set new benchmark for systemic RNAi with ~100 fold improved potency
• Efficacy in rodents following single IV injection
• Each LNP comprised of distinct cationic lipid component
• Improvement in potency has resulted in ED50<0.03 mg/kg
% Residual Factor VII
120
100
C12-200
80
DLinDMA
DLinDAP
60
DLin-KC2-DMA-a
ED50
40
DLin-KC2-DMA-b
98N12-5(I)
DLin-K-DMA
20
0
0.01
Keystone: Advance in Biopharm., Jan 2010
14
0.1
1
FVII siRNA Dose (mg/kg)
10
Next Generation LNPs
In Vivo Silencing of Factor VII in Rodent
•
•
Effects are rapid, potent, dose-dependent and durable
ED50 ~ 0.01 mg/kg
1.4
mRNA Silencing
1.2
1.0
0.8
0.6
0.4
0.2
0.0
PBS
0.3
0.3
0.1
siCont
(mg/kg)
0.03
0.01
0.003
Relative FVII Protein Level
FVII/GAPDH relative mRNA Level
Efficacy in mouse with Systemic RNAi after single IV injection
1.0
0.8
0.6
0.4
0.2
PBS
siFVII
(mg/kg)
0.3
0.3
0.1
0.03
siCont
(mg/kg)
0.01
0.003
0.001
siFVII
(mg/kg)
1.4
20
15
10
5
0
PBS
0.3
siCont
(mg/kg)
0.3
0.1
0.03
0.01
siFVII
(mg/kg)
Keystone: Advance in Biopharm., Jan 2010
0.003
0.001
Relative FVII Protein Level
Pharmacological Effect
Prothrombin Time (s)
Protein Suppression
1.2
0.0
0.001
25
15
1.4
Durability
1.2
1.0
0.8
0.6
0.4
PBS
siFVII 1.0 mg/kg
siFVII 0.1mg/kg
0.2
0.0
0
5
10
15
20
25
Day
30
35
40
45
Next Generation LNPs
Demonstrate Potent and Durable Silencing in Primates
Novel LNPs demonstrate enhanced potency in primates
• Single IV injection in primates
• Potent, rapid, and durable silencing of target genes (e.g., transthyretin, or TTR)
• Provides back-up LNPs for current development programs
Serum TTR Protein
1.2
1.0
0.8
0.6
*ED50 <0.03 mg/kg
0.4
0.2
0.0
PBS
0.03
0.1
0.3
DLin-KC2-DMA-b
(mg/kg)
Keystone: Advance in Biopharm., Jan 2010
17
0.03
0.1
0.3
* C12-200
(mg/kg)
Serum TTR Protein, %Pre-Dose
Relative TTR mRNA Levels
0.03 mg/kg
0.1 mg/kg
0.3 mg/kg
1.00
0.75
0.50
0.25
0.00
0
Single
siRNA dose
5
10
15
Day
20
25
PCSK9 Gene Silencing with 2nd Generation LNPs
Primate Model
ALN-PCS demonstrates potent efficacy in primates
90
800
80
700
70
600
60
0.03 mpk
500
400
0.10 mpk
0.30 mpk
1.00 mpk
50
40
300
30
200
20
100
10
0
-3
0
-1
1
3
5
7
Day
siRNA
Dosing
Keystone: Advance in Biopharm., Jan 2010
18
LNP-control
(1.00 mpk)
9
11
13
-3
-1
1
siRNA
Dosing
3
5
7
Day
9
11
13
LNP-siPCS
900
LDLc, mg/dl
PCSK9 protein, ng/ml
• Employs 2nd generation LNPs
• Rapid and durable dose-dependent reduction in PCSK9 protein
• PCSK9 silencing results in >50% reductions in LDLc
Combinatorial Chemistry Approach for Novel Materials
Alnylam-MIT Collaboration
Library Components
Synthetic Scheme
Nature Biotechnology (2008) 26:561-569.
19
In Vitro Screen
Headgroup
Linker
Hydrocarbon
Chains
Bilayer
Hexagonal HII
DLinDMA
20
Evolution and Mechanistic Insights into LNP Delivery
Two classes of formulations
Ionizable LNP (iLNP)
Cationic LNP (cLNP)
(Tekmira, AlCana, UBC)
EC50
(MIT)
In vivo Hepatocyte Silencing
DLinDMA
TETA-5-LAP
C12-200
DLin-KC2-DMA
•
•
•
•
21
Neutral surface charge at physiological pH
Becomes positively charged at low endosomal pH
Enters via clathrin-mediated endocytosis
ApoE-dependent
• Slightly positively charged
• Enters via macropinocytosis
• ApoE-independent
Mechanism of Liver Silencing with LNPs
ApoE Dependence in Cultured Primary Hepatocytes
Cellular uptake of siRNA requires ApoE for iLNPs but not cationic LNPs
• Primary mouse hepatocytes exposed to 20 nM cLNP- or iLNP- AF647-siRNA
• Cultures fixed and stained with DAPI after 4 hours
Control
+ ApoE
cLNP
(e.g. TETA-5LAP, C12-200)
iLNP
(e.g. DLinDMA,
Dlin-KC2-DMA)
40X
Keystone: Advance in Biopharm., Jan 2010
22
Mechanism of Silencing in Liver with iLNPs
ApoE Dependence In Vivo
iLNP silencing of FVII is absent in ApoE KO mice but restored by premixing with ApoE
• Single IV bolus of iLNP-siFVII at 0.2 mg/kg, with or without rhApoE at 0.0003 - 0.1 mg/kg
• FVII liver mRNA and serum protein levels measured 48 hours post-dose
ApoE KO
WildType
1.2
1.2
1.0
1.0
0.8
0.6
0.4
0.2
0.8
0.6
0.4
0.2
0.0
0.0
PBS
-
ApoE
0.1 mg/kg
LNP-siFVII (0.2 mg/kg)
Keystone: Advance in Biopharm., Jan 2010
23
rhApoE
FVII Serum Protein
Relative to control
Relative to control
FVII Liver mRNA
-
0.1
0.03
0.01
0.003
ApoE (mg/kg)
LNP-siFVII (0.2 mg/kg)
0.001
0.0003
Silencing in Liver with iLNPs
Mouse Primary Hepatocytes
WT
iLNP-siRNA
iLNP-AF647siRNA
LDLR KO
iLNP-siRNA
24
Internalization of iLNPs is impaired in
LDLR KO primary hepatocytes
• WT and LDLR KO mouse
primary hepatocytes
incubated with iLNPs for 4h
• Imaging by automated
confocal microscopy
In Vivo Silencing Activity of Ionizable LNPs
Diminished in LDLR KO Mice
iLNP activity is LDLR dependent in vivo
• Factor VII gene silencing activity in WT and LDLR KO mice
• Serum Factor VII levels determined in animals 48h post dose
1.4
Relative Serum FVII Protein Level
WT
LDLR KO
1.2
1.0
0.8
0.6
0.4
0.2
0.0
PBS
CHI: RNAi, Jan 2010
25
0.10
0.03
iLNP (mg/kg)
0.01
Asialoglycoprotein Receptor (ASGPR) Targeting
• Tissue specific
» High receptor expression on liver
hepatocytes
• Readily endocytosed
(high turnover)
• Well-studied design
requirements for high affinity
ligands
• Well conserved across species
» Mouse and human are 89% and
80% identical to rat (HL-1)
• Proven delivery of cargo to liver
» E.g., recombinant proteins with
incomplete sialylation
29
Asialoglycoprotein receptor (ASGPR)
Impact on rate of clearance
ASGPR Targeting by GalNAc LNPs
iLNP silencing of FVII in ApoE KO mice can be rescued by incorporation of GalNAc3
• Single IV bolus of iLNP-siFVII at 0.2 mg/kg, with or without 0.005 – 0.5% GalNAc3
incorporated
• FVII serum protein levels measured 48 hours post dose
1.2
GalNAc3-PEG2000-DSG
Relative Serum FVII Level
1.0
0.8
0.6
0.4
0.2
0.0
PBS
iLNP
FVII
siRNA
Keystone: Advance in Biopharm., Jan 2010
30
0.005%
0.015%
0.05%
iLNP FVII-siRNA
+ %GalNAc
0.15%
0.5%
GalNAc Targeted iLNPs
WT and LDLR KO Mice
GalNAc-targeted iLNPs not LDLR dependent
• Factor VII gene silencing activity 0.5% GalNAc-iLNP in WT and LDLR KO mice
• Serum Factor VII levels determined in animals 48h post dose
WT
Relative Serum FVII Protein Level
1.2
LDLR -/-
1.0
0.8
0.6
0.4
0.2
0.0
PBS
CHI: RNAi, Jan 2010
31
0.1
0.03
0.5% GalNAc-iLNP
(mg/kg)
0.01
Activity of GalNAc-Targeted LNPs is Abolished
in ASGPR H2 KO Mice
GalNAc-targeted iLNPs demonstrate GalNAc-specific gene silencing activity in vivo
•
Relative FVII Serum Protein Level
•
Wildtype or ASGR2 KO mice were administered PBS, non-targeted shielded-iLNPs, or
0.5% GalNAc-shielded iLNPs
Serum Factor VII levels were determined in animals 48h post dose
1.2
1.0
0.8
0.6
0.4
0.2
0.0
PBS
3
iLNP
CHI: RNAi, Jan 2010
32
3
1
0.5% GalNAc-iLNP
WT mice
0.3
PBS
3
3
iLNP
0.5% GalNAciLNP
ASGPR H2 KO
Translating Delivery Beyond the Liver
• Main focus of LNP research has been on
hepatic applications
• Initial extension of technology was to tumors
residing in the liver
• Currently, efforts are underway to further extend
technology to extrahepatic tumor and immune
cell applications
33
ALN-VSP Anti-Tumor Activity
RNAi-Mediated Cell Cycle Arrest
• Single IV injection of ALN-VSP or
control siRNA
• Mitotic arrest (monoasters) in
VSP-treated animals
• KSP and VEGF mRNAs cleaved,
confirming RNAi mechanism
Control siRNA, n=6
Orthotopic Tumor Model
Control siRNA
ALN-VSP
ALN-VSP, n=7
Potent Anti-Tumor Efficacy
• ALN-VSP demonstrates
anti-tumor activity
compared with controls
• Anti-tumor activity
associated with
improved survival
Keystone: RNAi, Feb 2009
34
Tumor Delivery Extended to Extra-Hepatic Tumors
Mitotic arrest (monoasters) in metastatic colorectal tumors arising from intrahepatic
HCT116 seeding in mice
• Multi-dose IV bolus of ALN-VSP or control siRNA (SNALP-siCont), 4 mg/kg
(2x/week for 3 weeks)
• H&E histology of tumor bearing lymph nodes 48 hours post-last dose
mitotic figures per FOV
25
20
15
10
5
0
SNALP-siCont
AACR-NCI-EORTC, Nov. 2009
35
ALN-VSP
monoasters
simple mitotic figures
SNALP-siCont
ALN-VSP
Lymph node
Gene Silencing Extended to Immune Cells
Novel KC2-based LNPs achieves
silencing in immune cells
CD11b+
16000
14000
Relative CD45 Levels
• CD45 and GFP protein suppression
observed in peritoneal leukocytes
24 hrs after IV administration of
LNP-KC2-siCD45 at 3 mg/kg
• Some silencing seen in dendritic
cells in spleen and bone marrow
CD45 KD in Peritoneal Cavity
Control siRNA
CD45 siRNA
12000
10000
8000
6000
4000
siCtrl
siCD45
2000
0
CD45
Keystone: Advance in Biopharm., Jan 2010
36
Macrophages
CD11b+
Dendritic Cells
CD11c+
Delivery of RNAi with LNPs
Summary of Research Progress
• Systemic delivery to liver has advanced
» Improved potency with LNPs by ~100-fold over prototype
» Mechanistic understanding of LNPs beginning to be elucidated
– PK/PD relationship defined for LNPs
– ApoE mechanism demonstrated
– Proof of concept for ASGPR-targeted LNPs demonstrated with
GalNAc
• Mechanistic understanding of systemic delivery with
LNPs provides basis for design of next generation
formulations
• LNP delivery extended from liver and liver tumor to
extrahepatic tumors and immune cells
37
Agenda
• Introduction
• Next Generation LNP Delivery
• Future Strategies
• Summary
38
Future Strategies
Optimizing LNPs
• New formulations of lipids
• New lipids
• Tailoring PK/PD and biodistribution
Targeting ligands
• New conjugates
• Targeted liposomes
Single-stranded RNAi (ssRNAi)
• New physical properties
39
Agenda
• Achieving Systemic Delivery and Role of LNPs
• Next Generation LNP Delivery
• Future Strategies
• Summary
40
Systemic Delivery of RNAi Therapeutics
Summary
Alnylam has a broad and long-term commitment
to improving and expanding delivery technology
• Continued optimization of LNP platform for
Systemic delivery, as well as Direct delivery
• Alnylam approached proactively by academics
and companies
» >25 Evaluations ongoing with external groups at any
one time
• These developments enable more programs
and broaden the potential impact of RNAi
therapeutics
41
Alnylam Development Pipeline
Key Programs
Discovery
Development
Phase I
Phase II
Phase III
ALN-RSV01 (Adult)
RSV Infection
ALN-RSV02 (Pediatric)
ALN-VSP
Liver Cancers
ALN-TTR01
TTR-mediated Amyloidosis
PCSK9/Hypercholesterolemia
Huntington’s Disease
Phase I start 1H’10
ALN-TTR02
ALN-PCS
ALN-HTT
Alnylam Proprietary Programs
42
Co-development Programs
Alnylam RNAi Roundtable
Q&A
43
www.alnylam.com
44
Confidential