Delivery Roundtable

Delivery Roundtable
Current Status and Future Directions
J
June
25,
25 2009
Agenda
g
• RNAi and the Role for Delivery
• Direct RNAi
• Systemic RNAi
• Future Strategies
5
RNA Interference
Synthetic siRNA
dicer
dsRNA
Cleavage
Strand separation
Targeted Gene
Silencing
RISC
Complementary pairing
mRNA (A)n
Cleavage
mRNA
degradation
6
Natural
Process
of RNAi
(A)n
Making Drugs Out of siRNAs
The Challenge
Characteristics
•
•
•
•
•
•
•
Structure adapted from
Klosterman,P. S.; Shah, S. A.;
Steitz, T. A Biochemistry (1999),
38, 14784-14792.
M.W 12,000-14,000
Size: 2 turns of helix
40 negative charges
Hydrophilic
Hydrated heavily
ca. 5.5 nm X 2 nm
Biostability
y
Sense strand
2 bp (double) 3’ overhangs
21-23 base pairs long
dsRNA
Seed
sequence
(residues 2-8)
Anti-sense or
‘Guide’ strand
7
Achievingg RNAi as Therapy
py
• Introducing “drug-like” properties into siRNAs
» Potency
» Selectivity
» Stability
• Achieving delivery to target tissues/cells
» PK/PD/Biodistribution
» Cellular uptake
8
Delivery of RNAi Therapeutics
Key driver of success
• Delivery
» PK/PD/Biodistribution
» Cellular uptake
M j progress achieved
Major
hi d
• Robust in vivo efficacy
Delivery Approaches
• Conjugates
• Liposomal NPs
• Peptides
p
• Antibodies
» >25
25 Targets
– Includes many “un-druggable”
» >5 Organs
– Includes lung, liver, and CNS
» 6 Species
– Includes humans
9
RNAi Delivery Strategies
Direct RNAi
Respiratory
» RSV/Influenza
» Cystic fibrosis
» Asthma/COPD
Ocular
» AMD/Retinopathy
» Glaucoma
CNS
»
»
»
»
Neuropathic pain
Spinal cord injury
Huntington’s
Huntington
s disease
Parkinson’s disease
Topical/Mucosal
» Infectious disease
10
Systemic RNAi
Multiple major diseases
»
»
»
»
»
Metabolic
Viral disease
Cancer
Inflammation
Cardiovascular
RNAi Therapeutics Opportunity
Large N
Number
mber of Undr
Undruggable
ggable Targets
New Drug
g Opportunities
pp
World of targets
Accessible targets for
small molecules/antibodies
RNAi accessible targets
Oral drug space
Mab/Protein
Drug space
New targets and disease
11
Non-oral
Non
oral SM
»
»
»
»
»
»
»
Undruggable
U
d
bl ttargets
t
Potent, selective lead
Rapid; 3-6 month to lead
Cross species active
M lti t
Multi-targeting
ti
Allelic specificity
Mab-like PD
Alnylam Development Pipeline
Key Programs
Discovery
Development
Phase I
Phase II
Phase III
RSV Infection
Liver Cancers
PCSK9/Hypercholesterolemia
TTR Amyloidosis
IND 2009 candidate
Huntington’s Disease
Alnylam Proprietary Programs
Co-development Programs
3 programs in clinical trials in 2009
12
Agenda
g
• RNAi and the Role for Delivery
• Direct RNAi
• Systemic RNAi
• Future Strategies
13
Direct Delivery in CNS
E ample C
Example:
Cy3-Htt
3 Htt siRNA Distrib
Distribution
tion After Intrastriatal Inf
Infusion
sion
HDSA (2008)
14
Cortex
Striatum
Thalamus
Substantia Nigra
Direct Delivery in CNS
Example:
p An Oligodendrocyte
g
y Gene
Silencing of Rat CNPase
Norm
malized CNP/MBP ((% PBS)
100
Piece 0
Piece 1
• Infusion Duration: ~3 d
• Infusion rate: 10 µl/hr
80
*** p< 0.001
** p<0.01 vs PBS
**
Two way ANOVA
60
siRNA cleavage site
***
40
RNAi-Mediated Cleavage
of CNPase in Rats
Step I.
Ligation of adaptor
1118/1119
RTGSP
RNA adaptor
3’
20
~299bp
p Predicted product
p
PBS siCNP
0
PBS
7.5
3.8
1.9
Step II. cDNA Synthesis
GSP
GR5’
Step III. PCR
PBS siCNP
0.9
AD-12436 (mg/mL)
Silencing
g of Primate CNPase
Relative CNPase/MB
R
BP mRNA (%)
100
Injection Site
300
299 nt
Adjacent Site
80
60
y Infusion Duration: 7 days
y Infusion Rate: 20 µl/hr
40
Regular PCR
20
0
TD PCR
Verified by cloning and sequencing of amplified products (18/20 clones)
Control
Animal #1
Querbes et al., Oligonucleotides (2008)
15
500
Animal #2
Direct Delivery in CNS
Therapeutic
p
Silencingg of Huntingtin
g
Strong pre-clinical data support advancement toward clinic
• RNAi therapeutic targeting huntingtin corrects disease in animal model
» Silencing of huntingtin gene
» Decreased disease pathology and behavioral changes
Behavior Improvement in Disease Model
7
6
Normal mice
Control siRNA
HTT siRNA
* p ≤ 0.01
5
*
4
3
2
1
Luc
HTT siRNA
7
6
5
4
3
*
*
2
1
0
0
Normal
7 Days
Pre-Disease
Induction
Post-Disease
Induction
PNAS (2007) 104, 17204-209
16
Improved Histopathology
8
M
Mean
number neuropil
aggregates/25
500 μm2
Mean # of ffoot slips
8
9
Group 1
Group 2
FISH Staining in Lung
4 Hours after Single 10 mg/kg Dry Powder or Liquid siRNA Installation
Luc siRNA DP, 4 Hrs, x10
Neg Ctrl siRNA DP,
DP 4 Hrs,
Hrs x10
17
No Treatment, x10
Luc siRNA D5W
D5W, 4 Hrs
Hrs, x10
Direct Delivery in Lung
Example: Anti
Anti-viral
viral Activity of RSV siRNAs
6
Log10 PFU/g lung
5
1 mg/kg
2 mg/kg
4 mg/kg
4
3
2
1
LLOD
0
P1
P2
P3
P gene
siRNA
i.n. (25-100ug)
RSV Symposium, Aug. 2005
18
4 hrs
ALN-RSV01
N2
N gene
Virus (RSV/A2)
i.n. (106 pfu)
L1
L2
L3
P4
P4-MM
L gene
4 days
Viral Titer in Lung
ALN-RSV01 Phase II GEMINI Study
Human POC for RNAi Therapeutics
ALN-RSV01 shows statistically significant reduction in RSV infection
•
•
•
Randomized, double-blind, placebo-controlled study (n=88)
~40% Relative reduction in infection rate (P<0
(P<0.01)
01)
~95% Increase in number of uninfected subjects (P<0.01)
Plaque Assay
80
Placebo
P<0.01
70
~95%
Increase
20
60
~40%
Reduction
No. of Unin
nfected
% Infec
cted
Plaque Assay
25
ALN-RSV01
50
40
30
20
15
10
5
P=0.0069
10
0
0
1
2
3
4
5
6
7
Study Day
8
Int’l Symp Res Vir Infect, Feb 2008
19
9
10
11
Placebo
ALN-RSV01
siRNA Conjugates Enhance Delivery
5´
O
3´
O
O
P
O
O
OH
NH
O
N
O
RNAi Activity in vitro without transfection reagent
140
Unmodified
Cholesterol conjugated
120
% Gen
ne expression
E.g., Cholesterol
• Promotes cellular uptake
» Direct cell permeation
100
80
60
40
20
0
500
1000
siRNA dose (nM)
Nature (2004) 432, 173-178
20
1500
2000
Direct Delivery to Mucosal Tissue
Intravaginal Application of Chol-siRNA
Chol siRNA against Nectin 1
Cell Host Microbe (2009) 5,84-94
21
Agenda
g
• RNAi and the Role for Delivery
• Direct RNAi
• Systemic RNAi
• Future Strategies
22
Systemic RNAi
• Parenteral administration increases
access to multiple major diseases
»
»
»
»
»
Metabolic
Viral disease
Cancer
Inflammation
Cardiovascular
• Validated Systemic
y
RNAi approaches
pp
» Conjugates
» Liposomal nanoparticles
• Multiple
p approaches
pp
»
»
»
»
23
Polymers
Small molecules
Peptides
Antibodies
% ApoB
B mRNA conte
ent relative to Saline treatme
ent group
Chol-siRNAs Silence apoB in Liver and
Jejunum
140
Liver
120
100
80
*
60
*
40
*
*
20
0
saline
Chol-luc-
Chol-MM
unconjugated Chol-apoB-1
apoB-1
* P<0.0001 compared to saline control animals
Nature (2004) 432, 173-178
24
Jejunum
j
Chol-apoB-2
Lipophilic Conjugates
SAR for Gene Silencing In Vivo
OH
HO
HO
O
P O
3'
O
N
OH Y
Y = O or S O
5'
H
N
O
O
L
O
Cholesterol (C27)
140
* P<0.05
** P<0.005
Rel. apoB m
mRNA (%)
120
O
O
N
H
Lithocholic-oleyl (C43)
O
100
O
*
80
Stearoyl (C18)
*
Docosanoyl (C22)
60
O
**
**
Lauroyl (C12)
O
40
Myristoyl (C14)
20
O
Palmitoyl (C16)
0
PBS
C27
MM
C27
C18
C22
C43
C12
C14
C16
O
Linoleoyl (C18, two C=C bond)
Wolfrum et al., Nat. Biotech. Sep 2007
25
Biodistribution of Chol-siRNAs is Lipoprotein-Mediated
Chol-siRNAs bind to circulating lipoproteins and
traffic to tissue in a receptor-mediated
receptor mediated fashion
Nature Biotech (2007). 25:1149-1157
26
Lipoprotein-mediated uptake of chol-siRNAs
into tissues of Ldlr–/–
Ldlr / and wildtype mice
Stoffel, Keystone March 2008
27
Liposomal Nanoparticles for Systemic RNAi
• Multi-component lipid formulation
»
»
»
»
Cationic lipid
Fusogenic lipid
PEG lipid
Cholesterol
• Highly efficient for liver delivery
» Hepatocyte-specific gene
silencing achieved
• Low
Lo surface
s rface charge
• Small uniform size particle < 100 nm
28
Liposomal siRNA Delivery to Liver
Mouse
Detection of Cy3-siRNA in Liver
Clearance from Plasma
% Injected Dose
100
10
1
0.1
0
4
8
12
Time
16
20
24
30 mins
Detection in Tissues
100
10
1
0.1
29
Fat
Muscle
Small
intestine
Thymus
Femur
Heart
Kidney
Control
Co
to
Lungs
0 01
0.01
Liver
% Injecte
ed Dose
Composite
10
0 mg/kg
g/ g
3 mg/kg
g/ g
1 mg/kg
g/ g
Dose-Dependent Silencing of Factor VII
1.4
Rat
1.4
Plasma Factor VII Protein
1.2
Relative FV
VII Protein
Relative Live
er FVII mRNA
Liver Factor VII mRNA
1.2
1.0
08
0.8
0.6
1.0
08
0.8
0.6
0.4
0.4
0.2
0.2
0.0
0.0
PBS
siCont
10mg/kg
siFVII
10mg/kg
siFVII
5mg/kg
siFVII
siFVII
2.5mg/kg 1.25mg/kg
40
PBS
siCont
10mg/kg
siFVII
10mg/kg
siFVII
5mg/kg
siFVII
siFVII
2.5mg/kg 1.25mg/kg
1.6
1.4
30
1.2
25
20
15
1.0
0.8
0.6
10
0.4
5
0.2
0
PBS
siCont
10mg/kg
0
siFVII
10mg/kg
siFVII
5mg/kg
siFVII
2.5mg/kg
Nature Biotechnology (2008) 26,561-569.
30
Durability
y
35
Relative FVII protein
Prothro
ombin Time (s)
Prothrombin Time
siFVII
1.25mg/kg
***
***
**
***
5
***
10
*
siCont
15
Time (d)
20
siFVII
25
30
Repeat Silencing of Factor VII
Rat
Repeat dosing over 3 months highly effective
• Comparable potency and durability of silencing with repeat dosing
oe
evidence
de ce for
o tac
tachyphylaxis
yp y a s o
or immunogenicity
u oge c ty
• No
1.6
siCont
siFVII
Relative FVII protein
1.4
12
1.2
1.0
0.8
0.6
0.4
0.2
0.0
20
D
Dose
1
Molecular Therapy (2009) 17,872-879.
31
40
Time (days)
D
Dose
2
60
80
D
Dose
3
100
Silencing apoB
Non-Human
Non
Human Primate
Efficacy in monkeys with Systemic RNAi after single IV injection
• Effects are rapid, potent, dose-dependent and durable
• RNAi effects are specific
p
and lead to measurable therapeutic
p
benefit
• RNAi mechanism proven in vivo
100
* P <.05 ** P < .005
mRNA
Protein
80
**
%C
Control
% Co
ontrol
60
60.5
**
**
80
40
*
23.2
**
1mg/kg
2.5 mg/kg
2 day
Nature (2006) 441, 111-114
32
8.9
1 mg/kg
11 day
20
**
14.2
11.7
2.5 mg/kg
>85%
Inhibition
*
34.1
9.2
0
>65%
Inhibition
60
31.7
20
109.8
100
79.0
72.4
40
* P <.05 ** P < .005
Cholesterol
LDL
HDL
Day 11 Post-Dose
Post Dose (2.5
(2 5 mg/kg)
Alnylam Systemic Programs
ALN PCS ALN
ALN-PCS,
ALN-TTR,
TTR ALN-VSP
ALN VSP
ALN-PCS to treat hypercholesterolemia
• Targets
T
t PCSK9
• IND candidate for 2009
PCS
ALN-TTR
ALN
TTR to treat TTR amyloidosis
• Targets liver expressed TTR
• IND candidate for 2009
ALN VSP to treat liver
ALN-VSP
li er cancer
• Target two key genes
» KSP – critical for cell division
» VEGF – critical for angiogenesis
• Phase I
33
TTR
KSP/
VEGF
Liver Cancer Program
ALN VSP
ALN-VSP
RNAi to treat liver cancers
• Prevalent solid tumor and common
site of metastatic disease
» ~700,000/yr Incidence of HCC worldwide
» ~500,000/yr
500,000/yr Patients with liver
metastasis
• ALN-VSP is dual-target product
» Targeting 2 pathways increases potential
therapeutic impact
–
–
Proliferation: Kinesin Spindle Protein (KSP)
Angiogenesis: VEGF
» Liposomal nanoparticle formulation
–
With Tekmira Pharmaceuticals
• ALN-VSP in clinical development
» Phase I liver cancer patient study
34
RNAi-Mediated Cell Cycle Arrest
Murine Liver Cancer Model
Orthotopic tumor model with intrahepatic Hep3B seeding in SCID mice
• Single IV bolus injection of ALN-VSP or control siRNA
• Mitotic arrest (monoasters) clearly detected in VSP-treated animals
• KSP and VEGF target mRNAs cleaved in tumors confirming RNAi mechanism
Control siRNA
hVEGF mRNA
hKSP mRNA
RNA
adapter
hKSP mRNA 3’ cleavage product
RNA
adapter
hKSP mRNA 3’ cleavage product
hKSP mRNA 3
cleavage product
ALN-VSP
hVEGF mRNA 3’ cleavage product
GR5N
GR5N
hVEGF mRNA 3’ cleavage product
hVEGF mRNA 3
cleavage product
Rev3 Rev2 cDNA
Rev4 Rev3 cDNA
PCR product 380 nt
PCR product 210 nt
VSP 0 2
VSP 0 2
4
35
1
4
mg/kg
400
300
400
300
200
200
100
100
Keystone: RNAi, Feb 2009
2
2
1
mg/kg
ALN-VSP Anti-Tumor Activity
Murine Liver Cancer Model
Orthotopic tumor model with intrahepatic Hep3B seeding in SCID mice
• ALN-VSP demonstrates clear anti-tumor activity compared with controls
Control siRNA, n=6
Keystone: RNAi, Feb 2009
36
ALN-VSP, n=7
ALN-VSP Anti-Tumor Activity
Comparative
p
Efficacyy vs. Sorafenib
Orthotopic tumor model with intrahepatic Hep3B seeding in SCID mice
• Significant survival benefit of ALN-VSP treatment vs. controls
• Superior survival advantage of ALN
ALN-VSP
VSP vs
vs. sorafenib treatment
100
Control
Sorafenib
Soraf/Control
S
f/C t l
Soraf+ALN-VSP
ALN-VSP
Survival (%)
80
60
40
log-rank Control
Soraf
NS
20
Soraf
Soraf/Cntrl
NS
NS
Soraf/Cntrl
VSP p=0.002 p=0.012 p=0.020 VSP
Soraf/VSP p=0.0006 p=0.003 p=0.025 NS
0
12
Keystone: RNAi, Feb 2009
37
21
30
39
48
57
66
PCSK9/Hypercholesterolemia Program
ALN PCS
ALN-PCS
RNAi to treat hypercholesterolemia
yp
• Significant unmet medical need
» >500,000 Patients with severe
hypercholesterolemia
–
I d
Inadequately
t l managed
d by
b statins
t ti and
d other
th drugs
d
• PCSK9 is regulator of LDL metabolism
» Controls expression of LDL receptor
» Validated in human genetics
• Attractive opportunity for Systemic RNAi
» PCSK9 expressed in liver
p
» Earlyy clinical markers of activityy possible
• Collaboration with UT Southwestern
» Horton, Hobbs, Brown, and Goldstein
38
RNAi Silencing of PCSK9 Protein and LDLc
Non Human Primates
Non-Human
Efficacy of PCSK9 silencing in non-human primates
• PCSK9 plasma levels reduced by up to 70% of pre-dose levels
• Rapid
R id reductions
d ti
iin LDL cholesterol
h l t l llevels
l b
by 40
40-60%
60%
• Durable effects after single injection
PCSK9 protein
LDL Cholesterol
2.0
1.4
Day 4
1.8
Day 7
Day 14
Da 4
Day
Day 21
* p ≤ 0.05
Da 7
Day
Da 14
Day
* p ≤ 0.05
1.2
relative to Pre-d
dose
rellative to pre-d
dose
1.6
1.4
1.2
1.0
0.8
06
0.6
*
0.4
0.0
PBS
Control
siRNA
PNAS (2008) 105,11915-920
39
1.0
0.8
*
0.6
*
*
04
0.4
0.2
0.2
PCSK9
siRNA
0.0
Da 21
Day
PBS
Control
siRNA
PCSK9
siRNA
TTR Amyloidosis Program
ALN TTR
ALN-TTR
RNAi to treat significant orphan disease
• Transthyretin (TTR) Amyloidosis
» Caused by mutation in TTR gene
» Amyloid deposits in nerves and heart
–
–
Familial Amyloid Polyneuropathy (FAP)
Familial Amyloid Cardiomyopathy (FAC)
» ~10,000
10 000 patients WW with FAP
• Clinical pathology
» Typical onset ~30-50 yr
» Fatal within 5-15 years
» Severe
S
pain/loss
i /l
off autonomic
t
i nervous function
f
ti
• TTR is well-validated target
» Human and mouse genetics
–
No pathology in knock-out mouse
» Produced
P d
d almost
l
t exclusively
l i l iin liliver (95%)
• Liver transplant current standard of care
40
RNAi Silencing of Mutant and Wild-Type TTR
Mo se and Non
Mouse
Non-Human
H man Primate
Efficacy in transgenic mouse model and non-human primates
• Reduced
educed mutant
uta t V30M-TTR
30
plasma
p
as a levels
e esa
and
d liver
e mRNA >90%
90% in transgenic
t a sge c mice
ce
• Reduced liver TTR mRNA levels ~80% in non-human primates
Non-human primate**
hTTR transgenic mouse*
1.4
1 mg/kg
3 mg/kg
1.6
6 mg/kg
TTR/GAPH
H
TTR/GAPD
DH
3 mg/kg
g g
1.2
1.0
0.8
0.6
04
0.4
10
1.0
0.8
0.6
0.4
0.2
0.2
PBS
Control
C
t l
siRNA
Keystone: RNAi, Feb 2009
41
1 mg/kg
g g
1.4
1.2
0.0
0.3 mg/kg
g g
TTR
siRNA
0.0
Control
C
t l
siRNA
TTR
siRNA
*Lipidoid formulation, w/ MIT; **SNALP formulation, Tekmira
Agenda
g
• RNAi and the Role for Delivery
• Direct RNAi
• Systemic RNAi
• Future Strategies
42
Future Strategies
g
• Optimizing LNPs
» New formulations of lipids
» New lipids
» Tailoring PK/PD and biodistribution
• Targeting ligands
» New conjugates
» Targeted liposomes
• ssRNAi
» New physical properties
43
Liposome Formulation Development Progress
Efficacy Improvement Over Time
120
% R
Residual Factor VII
100
LNP01
LNP01 80
LNP A
60
ED50
40
20
LNP B
LNP D
LNP C
0
0.1
1
FVII siRNA Dose (mg/kg)
Int’l Symp Athero, June 2009
44
10
“Lipidoid” Library for Novel Nanoparticles
Aln lam MIT Collaboration
Alnylam-MIT
Library Components
1
O
O
O10
6
O
O
O12
O13
10
11
O15
O
O
13
O18
O
O
15
N
H
N
H
N11
N
H
N12
22
O
NH2
60
O
O
O
NH2
NH2
O
31 HO
N
H
N
H
91
N
H
H
N
75
94
95
NH2
OH
77
NH2
O
NH2
NH
NH2
NH2
N
76
NH2
80
O
O
N
H2N
NH2
N
HN
112
114
NH2
N
H
NH2
H2N
N
H
N
109 H2N
7
10
11
13
21
22
N
H
24
NH2
NH2
25
26
28
31
116
H
N
NH2
H2N
NH2
NH2
32
33
34
36
NH2
117
H
N
N
H
6
20
115
NH2
HO
1
17
NH2
NH2
103
NH2
N
NO TEST
0-20
20-40
40-60
60-80
80-100
15
NH2
N
N
H
99
NH2
N
H
N
H
H2N
H2N
NH2
N
H
H
N
113
NH2
N
H2N
100
NH2
N
111
N
H
NH2
N
H2N
N
NH2
N
98
N
NH2
79
110
O10 O12 O13 O14 O15 O18 N12 N14 N15 N16 N18 Q10 Q12 Q13 Q14 Q18
H2N
NH2
N
N
H
N
N
HO
HO
93
H
N
N
H
HO
In Vitro Screen
NH2
NH2
H2N
H
N
70 HN
NH2
28 HO
NH2
H
N
NH2
N
HO
96
NH2
26 HO
87
H
N
N
H
64
OH
O
86
NH2
HO
62
63
NH2
NH2
HO
82
NH2
HO
61
HO
25
NH2
HO
90
NH2
24 HO
N
H
N18
38
OH
O
N16
O
21
O
N
H
N
H
NH2
O
OH
O
N15
O
HO
HO
36
20 HO
N
H
N14
34
NH2
O
O
N10
33
17
O
N9
NH2
O
O
O
O14
NH2
O
O
O
7
O
O
O
OH
38
NH2
60
61
62
63
OH
64
70
32 HO
NH2
NH2
N
81
75
76
77
Synthetic Scheme
79
80
O
O
+
H2N R2
R1 O
ΔT
81
O
R1 O
82
86
R1 O
MeI
N R2
R1 O
Me
N
R2
R1 O
87
90
91
93
94
O
O
95
96
O
O
O
R1 N
H
+
H2N
NH2
ΔT
R1 N
H
H
N
Nature Biotechnology (2008) 26, 561-569.
45
H
N R1
O
O
99
100
103
N
N
R1
98
N R1
H
109
Factors That Impact
p LNP Pharmacology
gy
Stability of PEGylation
Molecular Therapy (2009) 17, 872-879.
46
Particle Size
PK/Biodistribution of LNPs Can Be Modified
JCI (2009) 119,661-673.
47
Initial Dosing and Lower Maintenance Dose
Multiple Injections
relative to PBS=1
1.2
PCSK9/GAPDH
Cholesterol
1.0
0.8
1.2
0.6
0.4
1.0
0.0
0.1
1
10
LNP formulated PCS‐A2
(mg/kg)
To
otal Cholesterol
Re
elative to PBS=1
1 0.2
0.8
0.6
0.4
PBS
3mg/kg bolus+ PBS once a week
0.2
3mg/kg bolus+ 0. 3 mg/kg per week
0
5
.
Initial
3mg/kg
3
/k b
bolus
l
Int’l Sym of Athero, June 2009
48
10
15
20
25
30
35
40
days post initial bolus
Maintenance: 1 x wk
Rats were bled one day prior to repeated dosing
45
50
55
60
Targeted
g
Deliveryy
• The use of targeting ligands may substantially
develop delivery solutions to increase
» Efficiency
» Access to other tissues
• Applicable to both conjugate and LNP strategies
• Targeting
T
ti approaches
h
» Small molecules
» Peptides
» Monoclonal antibodies
49
Alnylam
y
Deliveryy Strategy
gy
• Two key systemic delivery strategies at Alnylam
» Lipid nanoparticles
– Including targeting with ligands
» Conjugates
C j
t
– Including targeting with ligands
• A third delivery strategy
» Single stranded siRNAs
– An early an alternate approach to delivery of siRNAs
– Agreement
A
t with
ith IIsis
i
50
Single Stranded and Duplex si-RNAs
Physiochemical Properties
Single-stranded
g
siRNA
•
•
•
•
MW ~ 7000
19 formal negative charges
Flexible with ~ 1 nm width
Hydrophobic surfaces accessible
for protein interactions
» Aromatic bases (green) are
exposed
Double-stranded siRNA
•
•
•
•
•
MW ~13,000
Two molecules (one strand pink)
40 formal negative charges
Rigid with ~ 2 nm diameter
Very little exposed hydrophobic
surface
» Aromatic bases (green) are paired
and buried in duplex
These different physical/chemical properties will likely
result in marked differences in pharmacokinetic profiles
51
Importance
p
of Mechanistic Insights
g
RISC
52
Multiple Ways into the Cell
Collaboration with Merino Zerial
Keystone: RNAi, Feb 2009
53
RNAi Deliveryy
Alnylam has broad and long-term commitment to improving
and expanding
p
g deliveryy technology
gy
• Continue development of direct and systemic approaches
» Many technologies under evaluation
– Direct RNAi
– Systemic RNAi
» New Liposomes and lipidoids
» New conjugates including peptides and antibodies
» Single
g strand siRNA
• Basic research
» Marino Zerial
• Alnylam
y
approached
pp
pro-activelyy byy academics and
p
companies
» >25 evaluations ongoing with external groups at any one time
54
visit our newly launched
www.alnylam.com