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Transcript of Delivery Roundtable draft 7.ppt - Alnylam Pharmaceuticals · AD-12436 (mg/mL) PBS 7.5 3.8 1.9 0.9...
Agendag
• RNAi and the Role for Delivery• RNAi and the Role for Delivery
• Direct RNAi
• Systemic RNAi
• Future StrategiesFuture Strategies
5
RNA InterferenceSynthetic siRNA
dsRNA dicer
Cleavage
NaturalStrand separation
Natural Process of RNAi
mRNA
Complementary pairing
(A)
Targeted GeneSilencing
RISC
mRNA
mRNAdegradation
Cleavage
(A)n
(A)degradation (A)n
6
Making Drugs Out of siRNAsThe ChallengeThe Challenge
Characteristics• M.W 12,000-14,000• Size: 2 turns of helix• 40 negative charges• Hydrophilic• Hydrated heavily• ca. 5.5 nm X 2 nm• Biostabilityy
Structure adapted from Klosterman,P. S.; Shah, S. A.;
Steitz, T. A Biochemistry (1999), 38, 14784-14792.
21-23 base pairs long2 bp (double) 3’ overhangsSense strand
dsRNA
Seed sequence
(residues 2-8)
Anti-sense or‘Guide’ strand
7
Achieving RNAi as Therapyg 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• Delivery
» PK/PD/Biodistribution» Cellular uptake
M j hi dMajor progress achieved• Robust in vivo efficacy
» >25 TargetsDelivery Approaches » 25 Targets – Includes many “un-druggable”
» >5 Organs– Includes lung, liver, and CNS
Delivery Approaches• Conjugates• Liposomal NPs• Peptides Includes lung, liver, and CNS
» 6 Species– Includes humans
p• Antibodies
9
RNAi Delivery Strategies
Direct RNAi Systemic RNAi
Multiple major diseases» Metabolic» Viral disease
Respiratory» RSV/Influenza» Cystic fibrosis» Asthma/COPD Viral disease
» Cancer» Inflammation» Cardiovascular
» Asthma/COPDOcular» AMD/Retinopathy» Glaucoma
CNS» Neuropathic pain» Spinal cord injury» Huntington’s disease» Huntington s disease» Parkinson’s disease
Topical/Mucosal» Infectious disease
10
RNAi Therapeutics OpportunityLarge N mber of Undr ggable TargetsLarge Number of Undruggable Targets
New Drug Opportunities
Accessible targets for
g pp
World of targets
small molecules/antibodies
RNAi accessible targetsU d bl t t
Mab/ProteinDrug space
» Undruggable targets» Potent, selective lead» Rapid; 3-6 month to lead» Cross species active
M lti t tiNon-oral SM
Oral drug space
Drug space » Multi-targeting» Allelic specificity» Mab-like PDNew targets and disease
Non oral SM
11
Alnylam Development PipelineKey Programs
Discovery Development Phase I Phase II Phase III
Key Programs
RSV Infection
Liver Cancers
PCSK9/Hypercholesterolemia
TTR Amyloidosis IND 2009 candidateTTR Amyloidosis
Huntington’s Disease
IND 2009 candidate
3 programs in clinical trials in 2009
Alnylam Proprietary Programs Co-development Programs
3 programs in clinical trials in 2009
12
Agendag
• RNAi and the Role for Delivery• RNAi and the Role for Delivery
• Direct RNAi
• Systemic RNAi
• Future StrategiesFuture Strategies
13
Direct Delivery in CNSE ample C 3 Htt siRNA Distrib tion After Intrastriatal Inf sionExample: Cy3-Htt siRNA Distribution After Intrastriatal Infusion
Cortex Striatum
Thalamus Substantia NigraThalamus Substantia Nigra
HDSA (2008)
14
Direct Delivery in CNSExample: An Oligodendrocyte Gene p g y
*** p< 0.001** p<0.01 vs PBS**
80
100
(% P
BS)
Piece 0Piece 1• Infusion Duration: ~3 d
• Infusion rate: 10 µl/hr
Silencing of Rat CNPase
RNAi-Mediated Cleavage
GR5’
RNA adaptor
siRNA cleavage site
3’
Step I. Ligation of adaptor
Step II. cDNA Synthesis
1118/1119
GSP
RTGSP
Step III. PCR~299bp Predicted product
Two way ANOVA
***
20
40
60
mal
ized
CN
P/M
BP
(
of CNPase in Rats
PBS siCNP PBS siCNP
500
p p
0
Nor
m
AD-12436 (mg/mL)PBS 7.5 3.8 1.9 0.9
Silencing of Primate CNPase 500300 299 nt
60
80
100
BP
mR
NA
(%) Injection Site
Adjacent Site
g
Regular PCR TD PCRVerified by cloning and sequencing of amplified products (18/20 clones)
Infusion Duration: 7 daysInfusion Rate: 20 µl/hr
0
20
40
Rel
ativ
e C
NPa
se/M
B
0 Control Animal #1 Animal #2R
Querbes et al., Oligonucleotides (2008)
1515
Direct Delivery in CNS Therapeutic Silencing of Huntingtinp g g
Strong pre-clinical data support advancement toward clinic • RNAi therapeutic targeting huntingtin corrects disease in animal model
» Silencing of huntingtin gene» Silencing of huntingtin gene» Decreased disease pathology and behavioral changes
Behavior Improvement in Disease Model8 Normal mice
Improved Histopathology9
Luc
5
6
7
8
Control siRNAHTT siRNA
foot
slip
s
*
* p ≤ 0.01
Normal mice
neur
opil
500 μm
2
5
6
78
LucHTT siRNA
1
2
3
4
Mea
n #
of f *
Mea
n nu
mbe
r ag
greg
ates
/25
**
1
2
34
5
1
0Normal
Pre-DiseaseInduction
7 Days Post-Disease
Induction
Group 1 Group 2
M 1
0
PNAS (2007) 104, 17204-209
16
FISH Staining in Lung 4 Hours after Single 10 mg/kg Dry Powder or Liquid siRNA Installation4 Hours after Single 10 mg/kg Dry Powder or Liquid siRNA Installation
Luc siRNA DP, 4 Hrs, x10 No Treatment, x10
Neg Ctrl siRNA DP 4 Hrs x10 Luc siRNA D5W 4 Hrs x10Neg Ctrl siRNA DP, 4 Hrs, x10 Luc siRNA D5W, 4 Hrs, x10
17
Direct Delivery in LungExample: Anti-viral Activity of RSV siRNAsExample: Anti viral Activity of RSV siRNAs
61 mg/kg
4
5
U/g
lung
2 mg/kg4 mg/kg
1
2
3
Log1
0 PF
P gene N gene L gene
0
1
P1 P2 P3 P4 P4-MM
LLOD
ALN-RSV01 N2 L3L2L1
siRNA i.n. (25-100ug)
4 hrs Virus (RSV/A2)i.n. (106 pfu)
4 days Viral Titer in Lung
P gene N gene L gene
RSV Symposium, Aug. 2005
18
ALN-RSV01 Phase II GEMINI StudyHuman POC for RNAi TherapeuticsHuman 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 01)• ~40% Relative reduction in infection rate (P<0.01)• ~95% Increase in number of uninfected subjects (P<0.01)
Plaque Assay25
Plaque Assay
P<0.0180
ALN-RSV01Placebo
15
20
nfec
ted
~95%Increase
50
60
70
cted
~40%Reduction
10
No.
of U
nin
20
30
40
% In
fec
0
5
Placebo ALN-RSV010
10
20
1 2 3 4 5 6 7 8 9 10 11
P=0.0069
Int’l Symp Res Vir Infect, Feb 2008
Study Day
19
siRNA Conjugates Enhance DeliveryP
O
O
OO
N
O HN H
O
O
5´3´
N
O
O
E.g., Cholesterol• Promotes cellular uptake
» Direct cell permeation
RNAi Activity in vitro without transfection reagent
120
140Unmodified Cholesterol conjugated
60
80
100ne
exp
ress
ion
0
20
40
% G
en
500 1000 1500 2000siRNA dose (nM)
Nature (2004) 432, 173-178
20
Direct Delivery to Mucosal TissueIntravaginal Application of Chol siRNA against Nectin 1Intravaginal Application of Chol-siRNA against Nectin 1
Cell Host Microbe (2009) 5,84-94
21
Agendag
• RNAi and the Role for Delivery• RNAi and the Role for Delivery
• Direct RNAi
• Systemic RNAi
• Future StrategiesFuture Strategies
22
Systemic RNAi• Parenteral administration increases
access to multiple major diseases» Metabolic» Viral disease» Cancer» Inflammation» Cardiovascular
• Validated Systemic RNAi approachesy pp» Conjugates» Liposomal nanoparticles
• Multiple approachesp pp» Polymers» Small molecules» Peptides» Antibodies
23
Chol-siRNAs Silence apoB in Liver and JejunumJejunum
140
ent g
roup
Liver Jejunum
100
120
Salin
e tr
eatm
e j
60
80
ent r
elat
ive
to
** *
20
40
B m
RN
A co
nte *
0saline Chol-luc- Chol-MM unconjugated
apoB-1Chol-apoB-1 Chol-apoB-2%
Apo
B
* P<0.0001 compared to saline control animals
Nature (2004) 432, 173-178
24
Lipophilic Conjugates SAR for Gene Silencing In VivoSAR for Gene Silencing In Vivo
HOHO OH
OP
O
Y NO
OH
H5' 3'
O OHO OH Y N HN
OLY = O or S
O
O
O
O
Cholesterol (C27)
140
*** P<0.05
NH
O
O
O
Lithocholic-oleyl (C43)
Stearoyl (C18)
mR
NA
(%)
80
100
120
* *
** P<0.005
O
O Lauroyl (C12)
Docosanoyl (C22)
Rel
. apo
B m
40
60 ** **
O
O
Palmitoyl (C16)
Myristoyl (C14)
Linoleoyl (C two C=C bond)
PBS C27MM
C27 C18 C22 C43 C12 C14 C160
20
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 fashiontraffic to tissue in a receptor mediated fashion
Nature Biotech (2007). 25:1149-1157
26
Lipoprotein-mediated uptake of chol-siRNAsinto tissues of Ldlr / and wildtype miceinto tissues of Ldlr–/– and wildtype mice
Stoffel, Keystone March 2008
27
Liposomal Nanoparticles for Systemic RNAi
• Multi-component lipid formulation» Cationic lipid» Cationic lipid» Fusogenic lipid» PEG lipid» Cholesterol» Cholesterol
• Highly efficient for liver delivery» Hepatocyte-specific gene
silencing achieved
Lo s rface charge• Low surface charge• Small uniform size particle < 100 nm
28
Liposomal siRNA Delivery to LiverMouse
10
100
ose
Clearance from Plasma Detection of Cy3-siRNA in Liver
1
10
% In
ject
ed D
100
0.1%
0 4 8 12 16 20 24Time
30 minsDetection in Tissues
1
10
100
ed D
ose
Composite
0 01
1
% In
ject
e
0.1
Control 10 mg/kg 3 mg/kg 1 mg/kg0.01
Live
r
Lung
s
Kid
ney
Hea
rt
Fem
ur
Smal
lin
test
ine
Mus
cle
Thym
us Fat
Co t o 0 g/ g 3 g/ g g/ g
29
Dose-Dependent Silencing of Factor VII RatRat
0 8
1.0
1.2
1.4
er F
VII m
RN
A
Liver Factor VII mRNA
0 8
1.0
1.2
1.4
VII P
rote
in
Plasma Factor VII Protein
0.2
0.4
0.6
0.8
Rel
ativ
e Li
ve
0.2
0.4
0.6
0.8
Rel
ativ
e FV
1.6Durability
0.0PBS siCont
10mg/kgsiFVII
10mg/kgsiFVII
5mg/kgsiFVII
2.5mg/kgsiFVII
1.25mg/kg
0.0PBS siCont
10mg/kgsiFVII
10mg/kgsiFVII
5mg/kgsiFVII
2.5mg/kgsiFVII
1.25mg/kg
40Prothrombin Time
0.8
1.0
1.2
1.4
FVI
I pro
tein
y
20
25
30
35
ombi
n Ti
me
(s)
*
***** ***
***
***
0
0.2
0.4
0.6
Rel
ativ
e
siCont siFVII5
10
15
Prot
hro
05 10 15 20 25 30
Time (d)
0PBS siCont
10mg/kgsiFVII
10mg/kgsiFVII
5mg/kgsiFVII
2.5mg/kgsiFVII
1.25mg/kg
Nature Biotechnology (2008) 26,561-569.
30
Repeat Silencing of Factor VIIRatRat
Repeat dosing over 3 months highly effective • Comparable potency and durability of silencing with repeat dosing• No evidence for tachyphylaxis or immunogenicityo e de ce o tac yp y a s o u oge c ty
1 2
1.4
1.6
n
siCont siFVII
0.8
1.0
1.2
FVII
prot
ei
0.2
0.4
0.6
Rel
ativ
e
0.0
20 40 60 80 100
Time (days)D 1 D 2 D 3Dose 1 Dose 2 Dose 3
Molecular Therapy (2009) 17,872-879.
31
Silencing apoBNon-Human PrimateNon Human Primate
Efficacy in monkeys with Systemic RNAi after single IV injection • Effects are rapid, potent, dose-dependent and durable• RNAi effects are specific and lead to measurable therapeutic benefit
100
p p• RNAi mechanism proven in vivo
ProteinmRNA** P < .005* P <.05
100109.8
** P < .005* P <.05
60
80
ontro
l
60.5
**72.4
Protein
79.0
Con
trol
80
100
>65%Inhibition >85%
Inhibition
40% C
o
**31.7
** % C
40
60
34.1*
**
0
20
1mg/kg 2.5 mg/kg 1 mg/kg 2.5 mg/kg
8.9 11.7
*9.2**
23.2
Day 11 Post Dose (2 5 mg/kg)Cholesterol LDL HDL
2014.2**
Nature (2006) 441, 111-114
2 day 11 dayDay 11 Post-Dose (2.5 mg/kg)
32
Alnylam Systemic ProgramsALN PCS ALN TTR ALN VSPALN-PCS, ALN-TTR, ALN-VSP
ALN-PCS to treat hypercholesterolemiaT t PCSK9• Targets PCSK9
• IND candidate for 2009ALN-TTR to treat TTR amyloidosis
PCS
ALN TTR to treat TTR amyloidosis• Targets liver expressed TTR• IND candidate for 2009
ALN VSP to treat li er cancerALN-VSP to treat liver cancer• Target two key genes
» KSP – critical for cell division
TTR
» VEGF – critical for angiogenesis• Phase I
KSP/VEGF
33
Liver Cancer ProgramALN VSPALN-VSP
RNAi to treat liver cancers• Prevalent solid tumor and common• Prevalent solid tumor and common
site of metastatic disease» ~700,000/yr Incidence of HCC worldwide» ~500,000/yr Patients with liver» 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» 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 ModelMurine 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
hKSP mRNA
hKSP mRNA 3’ cleavage productRNA adapter
hKSP mRNA 3’ cleavage product
GR5N
hVEGF mRNA
hVEGF mRNA 3’ cleavage product
hVEGF mRNA 3’ cleavage product
GR5N
RNA adapter
ALN-VSPhKSP mRNA 3 cleavage product
cDNARev2Rev3
hVEGF mRNA 3 cleavage product
cDNARev3Rev4
4 2 1 mg/kgVSP 02
4 2 1 mg/kgVSP 02
PCR product 380 nt PCR product 210 nt
400300200100
400300200
Keystone: RNAi, Feb 2009
100100
35
ALN-VSP Anti-Tumor Activity Murine Liver Cancer ModelMurine 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 ALN-VSP, n=7
Keystone: RNAi, Feb 2009
36
ALN-VSP Anti-Tumor ActivityComparative Efficacy vs. Sorafenibp y
Orthotopic tumor model with intrahepatic Hep3B seeding in SCID mice• Significant survival benefit of ALN-VSP treatment vs. controls
Superior survival advantage of ALN VSP vs sorafenib treatment
100 ControlSorafenibS f/C t l
• Superior survival advantage of ALN-VSP vs. sorafenib treatment
60
80
ALN-VSP
Soraf/ControlSoraf+ALN-VSP
(%)
40Survival
log-rank Control
0
20log-rank Control
SorafSoraf/Cntrl
VSP
Soraf NSSoraf/Cntrl NS NS
VSP p=0.002 p=0.012 p=0.020Soraf/VSP p=0.0006 p=0.003 p=0.025 NS
12 21 30 39 48 57 66
Keystone: RNAi, Feb 2009
37
PCSK9/Hypercholesterolemia ProgramALN PCSALN-PCS
RNAi to treat hypercholesterolemiayp• Significant unmet medical need
» >500,000 Patients with severe hypercholesterolemia
I d t l d b t ti d th d– Inadequately managed by statins and other drugs
• PCSK9 is regulator of LDL metabolism» Controls expression of LDL receptor» Validated in human genetics» Validated in human genetics
• Attractive opportunity for Systemic RNAi» PCSK9 expressed in liver» Early clinical markers of activity possibley y p
• Collaboration with UT Southwestern» Horton, Hobbs, Brown, and Goldstein
38
RNAi Silencing of PCSK9 Protein and LDLcNon Human PrimatesNon-Human Primates
Efficacy of PCSK9 silencing in non-human primates • PCSK9 plasma levels reduced by up to 70% of pre-dose levels
R id d ti i LDL h l t l l l b 40 60%• Rapid reductions in LDL cholesterol levels by 40-60%• Durable effects after single injection
2.0PCSK9 protein
1.4LDL Cholesterol
Da 4 Da 7 Da 14 Da 21
dose 1.4
1.6
1.8Day 4 Day 7 Day 14 Day 21
dose 1.0
1.2 * p ≤ 0.05Day 4 Day 7 Day 14 Day 21
* p ≤ 0.05
* **
lativ
e to
pre
-d
0 6
0.8
1.0
1.2
ativ
e to
Pre
-d0 4
0.6
0.8
PBS Control PCSK9 PBS Control PCSK9
rel
0.0
0.2
0.40.6
rel
0.0
0.2
0.4*
siRNA siRNAsiRNA siRNA
PNAS (2008) 105,11915-920
39
TTR Amyloidosis ProgramALN TTRALN-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 patients WW with FAP» ~10,000 patients WW with FAP• Clinical pathology
» Typical onset ~30-50 yr» Fatal within 5-15 years
S i /l f t i f ti» Severe pain/loss of autonomic nervous function• TTR is well-validated target
» Human and mouse genetics– No pathology in knock-out mouseP d d l t l i l i li (95%)» Produced almost exclusively in liver (95%)
• Liver transplant current standard of care
40
RNAi Silencing of Mutant and Wild-Type TTRMo se and Non H man Primate Mouse and Non-Human Primate
Efficacy in transgenic mouse model and non-human primates• Reduced mutant V30M-TTR plasma levels and liver mRNA >90% in transgenic mice educed uta t 30 p as a e e s a d e 90% t a sge c ce• Reduced liver TTR mRNA levels ~80% in non-human primates
1.41 mg/kg 3 mg/kg 6 mg/kg
hTTR transgenic mouse* Non-human primate**1.6 0.3 mg/kg 1 mg/kg 3 mg/kg
1.0
1.2
DH
1 mg/kg 3 mg/kg 6 mg/kg
1 0
1.2
1.4
H
g g g g g g
0 4
0.6
0.8
TTR
/GA
PD
0.6
0.8
1.0
TTR
/GA
PH
0.0
0.2
0.4
PBS C t l TTR0.0
0.2
0.4
C t l TTR
*Lipidoid formulation, w/ MIT; **SNALP formulation, Tekmira
PBS ControlsiRNA
TTRsiRNA
ControlsiRNA
TTRsiRNA
Keystone: RNAi, Feb 2009
41
Agendag
• RNAi and the Role for Delivery• RNAi and the Role for Delivery
• Direct RNAi
• Systemic RNAi
• Future StrategiesFuture Strategies
42
Future Strategiesg
• Optimizing LNPsOptimizing LNPs» New formulations of lipids» New lipids» Tailoring PK/PD and biodistribution
• Targeting ligands» New conjugates» New conjugates» Targeted liposomes
• ssRNAissRNAi» New physical properties
43
Liposome Formulation Development ProgressEfficacy Improvement Over TimeEfficacy Improvement Over Time
120
80
100
120r V
II
LNP01
ED50
40
60
80
Residu
al Facto
LNP01 LNP A
0
20
40
% R LNP B
LNP CLNP D
FVII siRNA Dose (mg/kg)
0.1 1 100
Int’l Symp Athero, June 2009
44
“Lipidoid” Library for Novel NanoparticlesAln lam MIT CollaborationAlnylam-MIT Collaboration
OO
OO
O12
O13
O NH21
O
O NH2
O
O
NH2
O NH2
HO NH2
HO
HO NH2
HO NH2
NNH2
NHO
HO
NH2
NHONH2
6
7
10
82
86
87
33
34
36
N
NH2
H2N
NH2
H NH2
110O
OO10 O10 O12 O13 O14 O15 O18 N12 N14 N15 N16 N18 Q10 Q12 Q13 Q14 Q18
NO TEST 10-20 620-40 740-60 10
Library ComponentsIn Vitro Screen
OO
OO
NH
O
OO
O15
O NH2
OO
NH2
O NH2
O
ONH2
HO NH2
OH
HO NH2
NH
HN
NH
HN
NH
HN
HN
HN
NH
NH
HO
O N NH2
O N NH2
N
N
HNN
NH2
NH2
NH
NH2
15
17
20
21
11
1390
91
93
94
95
60
61
62
63
64
38 NH
N NH
H2N NH2
NH
NH2
N NH2H2N
NH
NH2H2N
N
NH2
H2N
111
112
113
114
115
NH
O
O14
O18
N9
N10
60-80 1180-100 13
151720212224252628NH2
NH2OH
HO NH2
OH
HO
HONH2
HO OHNH2
HO NH2
HO O NH
H H
HN NH
N NH2
N NH2
NNH2
NNH2
N NH2
H
NH
NH2
NH
HN NH2
H2N
H2N NH2
H2N NH2
NH
HN OHHO
H2NHN OH
NH
O
NH
O
NH
O
O
NH
O
21
22
77
79
80
96
98
99
100
103
109
70
75
76
24
25
26
28
31
NH2
N
NH2
NH2NH2
116
117
H2NNH
O
N15
N12
N14
N16
N11
283132333436386061626364HO O NH2
HO NH2
N NH2
N NH2
2
NH
O 80
81
31
32N18
647075767779808182868790
OH N R
R1 O
O
R1 O
O
MeI MeΔT
Synthetic Scheme
9091939495969899100103109
R1 O+ H2N R2
R1 O
O
N R2
R1 O
O
NR2
Me
R1 N
O+
R1 NH
O
NH2NNH2
R1NH
O
HNΔT
R1 NH
R1HN
O
R1HN
O
Nature Biotechnology (2008) 26, 561-569.
45
Factors That Impact LNP Pharmacologyp gy
Stability of PEGylation Particle Size
Molecular Therapy (2009) 17, 872-879.
46
Initial Dosing and Lower Maintenance DoseMultiple Injections
0.8
1.0
1.2
PBS=1
PCSK9/GAPDHCholesterol
Multiple Injections
0.2
0.4
0.6
relative to
1.0
1.2
1 0.0
0.1 1 10
LNP formulated PCS‐A2(mg/kg) 0.6
0.8otal Cho
lesterol
elative to PBS
=1
0.2
0.4To Re PBS
3mg/kg bolus+ PBS once a week
3mg/kg bolus+ 0. 3 mg/kg per week
Maintenance: 1 x wkInitial 3 /k b l
0 5 10 15 20 25 30 35 40 45 50 55 60days post initial bolus.
Rats were bled one day prior to repeated dosing3mg/kg bolus
Int’l Sym of Athero, June 2009
48
Targeted Deliveryg y
• The use of targeting ligands may substantiallyThe use of targeting ligands may substantially develop delivery solutions to increase» Efficiency» Access to other tissues
• Applicable to both conjugate and LNP strategiesT ti h• Targeting approaches» Small molecules» Peptides» Peptides» Monoclonal antibodies
49
Alnylam Delivery Strategyy y gy
• Two key systemic delivery strategies at AlnylamTwo key systemic delivery strategies at Alnylam» Lipid nanoparticles
– Including targeting with ligandsC j t» Conjugates– Including targeting with ligands
• A third delivery strategyA third delivery strategy» Single stranded siRNAs
– An early an alternate approach to delivery of siRNAsA t ith I i– Agreement with Isis
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Single Stranded and Duplex si-RNAsPhysiochemical Properties Physiochemical Properties
Single-stranded siRNA Double-stranded siRNAg• MW ~ 7000• 19 formal negative charges• Flexible with ~ 1 nm width
Hydrophobic surfaces accessible
• MW ~13,000 • Two molecules (one strand pink)• 40 formal negative charges
Rigid with 2 nm diameter• Hydrophobic surfaces accessible for protein interactions » Aromatic bases (green) are
exposed
• Rigid with ~ 2 nm diameter• Very little exposed hydrophobic
surface» Aromatic bases (green) are paired
These different physical/chemical properties will likely result in marked differences in pharmacokinetic profiles
and buried in duplex
result in marked differences in pharmacokinetic profiles
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Multiple Ways into the CellCollaboration with Merino ZerialCollaboration with Merino Zerial
Keystone: RNAi, Feb 2009
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RNAi DeliveryyAlnylam has broad and long-term commitment to improving and expanding delivery technology p g y 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 strand siRNAg
• Basic research» Marino Zerial
• Alnylam approached pro-actively by academics and y pp p y ycompanies» >25 evaluations ongoing with external groups at any one time
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