Delivery Roundtable draft 7.ppt - Alnylam Pharmaceuticals · Jejunum 140 e nt group Liver Jejunum...

Delivery RoundtableDelivery RoundtableCurrent Status and Future Directions

J 25 2009June 25, 2009

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


• Direct RNAi

• Systemic RNAi


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


• Direct RNAi

• Systemic RNAi


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


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


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


41

Agendag


• Direct RNAi

• Systemic RNAi


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

PK/Biodistribution of LNPs Can Be Modified

JCI (2009) 119,661-673.

47

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

50

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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Importance of Mechanistic Insightsp g

RISC

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Multiple Ways into the CellCollaboration with Merino ZerialCollaboration with Merino Zerial


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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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visit our newly launchedvisit our newly launched

www.alnylam.com

Delivery Roundtable draft 7.ppt - Alnylam Pharmaceuticals · Jejunum 140 e nt group Liver Jejunum...

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Transcript of Delivery Roundtable draft 7.ppt - Alnylam Pharmaceuticals · Jejunum 140 e nt group Liver Jejunum...