A Subcutaneous, Potent and Durable RNAi Platform Targeting

ATVB 2014
A Subcutaneous, Potent and Durable
RNAi Platform Targeting Metabolic Diseases,
Genes PCSK9, ApoCIII and ANGPLT3
Kevin Fitzgerald
May 1, 2014
Presenter Disclosure Information
Kevin Fitzgerald, Ph.D.
A Subcutaneous, Potent and Durable RNAi Platform
Targeting Metabolic Diseases, Genes PCSK9, ApoCIII and
ANGPLT3
FINANCIAL DISCLOSURE
» Employee of Alnylam Pharmaceuticals
RNA Interference (RNAi)
Synthetic siRNA
dicer
dsRNA
Cleavage
Strand separation
RISC
Complementary pairing
Targeted Gene
Silencing
mRNA
(A)n
Cleavage
mRNA
degradation
(A)n
3
Natural
Process
of RNAi
PCSK9 Therapeutic Hypothesis
LDL
PCSK9 role in both intracellular and
extracellular degradation of LDLR
B
PCSK9
Blockers
Endosome
Inhibit only
extracellular
functions
Lysosomal
degradation
A
LDLR
synthesis
PCSK9
Synthesis
Inhibitors
Inhibit PCSK9
synthesis and both
intracellular and
extracellular
functions
PCSK9
synthesis
PCSK9
ALN-PCS
PCSK9 mRNA
Nucleus
A. Intracellular Pathway
B. Extracellular Pathway
4
ALN-PCS Phase I Study Results
Pharmacodynamics and Clinical Efficacy
PCSK9 knockdown and LDL-C reduction after single dose without statins
Randomized, placebo-controlled, single dose escalation study in healthy volunteers with elevated LDL (n=32)
Rapid, dose-dependent, and durable knockdown of PCSK9 of up to 84% with mean lowering of 68% at 0.4
mg/kg group (p<0.0001)
Major reductions in LDL-C of up to 50% with mean lowering of 41% at 0.4 mg/kg group (p<0.01)
No significant decreases in HDL-C
PCSK9
LDL-C
* Data for n=6 from the 0.400 mg/kg group through Day 14 only
-20
0
20
40
60
ALN-PCS dose group
80
0.015 mg/kg
0.045 mg/kg
0.090 mg/kg
100
siRNA
Dose
0.150 mg/kg
0.250 mg/kg
0.400 mg/kg
* Data for n=6 from the 0.400 mg/kg group through Day 14 only
% Knockdown LDL-C Serum Levels
Mean Relative to Baseline and Placebo
Mean Relative to Baseline and Placebo
% Knockdown PCSK9 Plasma Levels
-40
-20
0
20
40
ALN-PCS dose group
0.015 mg/kg
0.045 mg/kg
0.090 mg/kg
60
5
Fitzgerald, ATVB, April 2012
10
15
Day
20
25
siRNA
Dose
5
10
15
20
5
0.150 mg/kg
0.250 mg/kg
0.400 mg/kg
25
Activity Independent of Baseline PCSK9 Levels
PCSK9 Knockdown and LDL-C Reduction
by Baseline PCSK9 Levels
Baseline PCSK9 Distribution
All Subjects
(0.150, 0.250, 0.400 mg/kg)
80
Maximum Per-Subject
Percent Reduction
Number of Subjects
8
6
4
2
0
PCSK9
60
40
20
0
400
600
800
< 0.5 below
(n=13)
1000 1200
within 0.5
(n=9)
1400
1600
> 0.5 above
(n=10)
Baseline PCSK9 (ng/mL)
1800
LDL-C
Low
Medium
High
< 0.5 below
(n=7)
within 0.5
(n=3)
> 0.5 above
(n=5)
Baseline PCSK9 levels
SD Relative to mean
SD Relative to mean
Fitzgerald, ATVB, April 2012
6
GalNAc-siRNA Conjugates for Systemic
Subcutaneous RNAi
GalNAc3
Asialoglycoprotein Receptor (ASGPR)
GalNAc-siRNA
conjugate
Highly expressed in hepatocytes
High rate of uptake
Recycling time ~15 minutes
Conserved across species
ASGPR
(pH>5)
Clathrin-coated pit
protein
ALN-TTRsc ( ALN-PCSsc, ALN-ANG, ALN-AC3)
siRNA conjugated to N-acetylgalactosamine
(GalNAc) ligand
Efficient delivery to hepatocytes following
subcutaneous administration
“Enhanced stabilization chemistry” (ESC) used with
ALN-PCSsc, ALN-ANG, and ALN-AC3
» Significantly improved potency and durability compared
with ALN-TTRsc
Recycling
ASGPR
Clathrin-coated
vesicle
RISC
Endosome
mRNA
Nucleus
7
ALN-TTRsc Phase 1 Study Results
Human POC for GalNAc-siRNA Conjugates
Randomized, double-blind, placebo-controlled SAD and MAD study in healthy volunteers
Rapid, dose-dependent, consistent, and durable knockdown of serum TTR
» Significant knockdown of serum TTR (p<0.01) up to 94% TTR knockdown; Mean knockdown up to 92.4%
Generally well tolerated
» Only AEs associated with drug were generally mild ISRs, resolving within ~2 hours of onset
Duration of effect is longer in human vs. NHP
-20
0
20
40
60
ALN-TTRsc
dose groups
80
Placebo (n=3)
2.5 (n=3)
5.0 (n=3)
10.0 (n=3)
Relative to Baseline
% Mean TTR Knockdown
Relative to Baseline (± SEM)
% Mean TTR Knockdown
0
20
40
60
Human
NHP
80
100
100
0
ALN-TTRsc (mg/kg), qd x5; qw x5
Days
Zimmermann, Heart Failure Society of America, Sept. 2013
10
ALN-TTRsc
Single 10.0 mg/kg
Injection
20
30
Days
8
40
50
60
Cardiovascular Metabolic Programs
ALN-PCSsc
ALN-ANG
ALN-AC3
Targets PCSK9
Efficacy in multiple pre-clinical animal models
Development Candidate selected; IND late ’14 / early ’15
Targets ANGPTL3, inhibitor of cellular lipases
Efficacy in animal models of mixed hyperlipidemia
Lead Candidates being optimized
Targets apoCIII, component of lipoprotein particles
Efficacy in animal models of hypertriglyceridemia
Lead Candidates being optimized
9
ALN-PCSsc Pre-Clinical Efficacy in NHP
Potent PCSK9 Knockdown and LDL-C Lowering: Multi-Dose Data
ALN-PCSsc achieves potent PCSK9 knockdown and LDL-C lowering with SC dosing
% PCSK9 Knockdown or LDL-C Lowering
2 mg/kg dose, qdx5 load; qw maintenance
Up to 95% PCSK9 knockdown
Up to 67% LDL-C lowering in absence of statins
LDL-C
PCSK9
60
80
-20
0
20
40
60
80
100
0
20
40
ALN-PCSsc, 2 mg/kg
qd x5; qw x3
100
120
140
160
Day
10
ALN-PCSsc Pre-Clinical Efficacy in NHP
Potent PCSK9 Knockdown and LDL-C Lowering: Single Dose Data
ALN-PCSsc achieves highly durable PCSK9 knockdown and LDL-C reduction with single dose
Single SC dose 1-10 mg/kg
Up to 96% PCSK9 knockdown, up to 77% LDL-C lowering (in absence of statins)
Highly durable effects, supports once-monthly or possibly once-quarterly dosing
»
>50% LDL-C lowering maintained for over 3 months in 10 mg/kg group
PCSK9
3.0
6.0
10.0
1.0
-20
-20
0
0
% LDL-C Lowering
(relative to pre-bleed)
% PCSK9 Knockdown
(relative to pre-bleed)
1.0
LDL-C
20
40
60
80
3.0
6.0
10.0
20
40
60
80
100
100
0
ALN-PCSsc
(mg/kg)
20
40
Days
60
80
100
0
ALN-PCSsc
(mg/kg)
20
40
60
80
Days
11
100
120
ALN-PCSsc Pre-Clinical Safety Studies
Summary
Initial pre-clinical safety studies support wide therapeutic index
qWx5 dosing at 30, 100, and 300 mg/kg
3 Species: mouse, rat, and NHP
NOAEL >300mg/kg in all species
» No in-life findings
» No significant changes in serum chemistry, ALT/AST, cytokines etc.
– Significant reductions in LDL-C
» No adverse histopath findings
12
ANGPTL3
Biological Rationale in Mixed Hyperlipidemia
ANGPTL3 is liver-expressed, genetically validated
target
Phenotypic Effects of LOF
Angptl3 Mutations in Humans
Lipase inhibitor
Human loss-of-function Angptl3 mutations lead to
» Increased LPL activity, increased insulin sensitivity
» Decreased free fatty acids
Dallas Heart Study confirmed association
between polymorphisms near Angptl3 and lower
lipid traits
Mouse models match human genetics
» Mouse KO has lower circulating cholesterol and TGs
» Overexpression of ANGPTL3 in mice results in
markedly increased plasma lipid levels
» Obese, diabetic Angptl3 KO mouse is hypolipidemic
Musunuru et al., NEJM; 363:2220-2227 (2010)
Koishi, et al., Nat Gen; 30:151-157 (2002)
13
ALN-ANG Targeting ANGPTL3
GalNAc Conjugate Lead Optimization Ongoing
Demonstrated knockdown in ob/ob female mice
GalNAc-siRNA ANGPTL3 Knockdown
(ob/ob female mice)
1.4
1.4
1.2
Control
ALN-ANG
1.2
mANGPTL3 Protein
(Relative to Ave. Pre-dose)
mANGPTL3 Protein Relative to PBS=1
GalNAc-siRNA ANGPTL3 Knockdown
(ob/ob female mice)
1.0
0.8
0.6
0.4
0.2
0.0
PBS
10
3.0
1.0
ANG-GalNAc conjugate
0.3
1.0
0.8
0.6
0.4
0.2
0
0
2
qd x 5 (5 mg/kg)
Borodovsky, AHA., Nov. 2013
4
6
8
Days
14
10
ALN-ANG Pre-Clinical Efficacy
ANGPLT3 Knockdown; TG and LDL-C Lowering in ob/ob Mice
-40
-40
-40
Triglyceride
Protein
% Knockdown
% Knockdown
0
20
40
-20
-20
0
0
20
20
% Knockdown
-20
LDL-C
40
60
60
60
PBS
PBS
ANG-GalNAc
ANG-GalNAc
PBS
80
80
100
0
5
10
ALN-ANG, 3.0mg/kg
qd x5; BIW x3
15
20
25
Borodovsky, AHA., Nov. 2013
ANG-GalNAc
80
100
0
100
5
10
ALN-ANG, 3.0mg/kg
qd x5; BIW x3
Days
40
15
Days
20
25
0
5
10
ALN-ANG, 3.0mg/kg
qd x5; BIW x3
15
15
Days
20
25
ALN-AC3 Targeting ApoCIII
Biological Rationale in Hypertriglyceridemia
» Heterozygous individuals have lower TG
(-45%) and lower VLDL (-25%)
» Translates into less coronary artery
calcification and lower CAD risk
Mouse models match human genetics
1.0
Relative to PBS=1
mApoCIII/mGAPDH
1.2
0.8
0.6
0.4
0.2
0.0
PBS
2.5
1.25
1.0
0.8
0.6
0.4
0.2
1.25
2.5
5.0
0.0
0
Peterson et al., NEJM. 362:1082-1089 (2010)
Chang et al., Lipids in Health and Disease. 11:162 (2012)
5.0
ApoCIII--GalNAc, mg/kg
qd x5
% TG Lowering
Inhibits lipoprotein lipase and hepatic
lipase and hepatic uptake of TG-rich
particles, other mechanisms
Polymorphisms in ApoCIII have been
associated with hypertriglyceridemia
ApoCIII loss-of-function results in
greater TG hydrolysis into FFAs and
increased clearance
db/db mouse model
Relative to Individual Pre-dose =1
ApoCIII is liver-expressed, genetically
validated target
ApoCIII GalNAc3 Multi-Dose Dose Response
5
10
ApoCIII-GalNAc, mg/kg
qd x5
15
Days
16
20
25
Summary
GalNAc-siRNA conjugates provide platform for knockdown of
genetically validated, liver-expressed target genes in cardiometabolic disease
Human translation validated in clinic with ALN-TTRsc
New ESC chemistry improves potency and duration
ALN-PCSsc IND filing in late ’14/early ’15
» Robust lowering of plasma PCSK9 and serum LDL-C in NHPs
» Once-monthly or possibly once-quarterly SC dose regimen (LDL-C
and PCSK9 clamped at nadir)
» Wide therapeutic index with NOAEL ≥300 mg/kg in initial 5 dose
safety studies in mouse, rat and NHP
ALN-ANG and ALN-AC3 pre-clinical POC established
» Lead candidates undergoing optimization
17