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Development of Monthly to Quarterly Subcutaneous Administration of RNAi Therapeutics Targeting the Metabolic Disease Genes
PCSK9, ApoC3 and ANGPTL3
Anna Borodovsky, William Querbes, Kristina Yucius, Abigail Liebow, Andrew Sprague, Stephanie Williams, Jessica Sutherland, Renta Hutabarat, Stuart Milstein, Satya Kuchimanchi, Rajeev Kallanthottathil, Klaus Charisse, Martin Maier, David Kallend,1 Jay Horton,2 Amy Simon, Kevin Fitzgerald
Alnylam Pharmaceuticals, Inc., Cambridge, MA, USA; 1The Medicines Company, Parsippany, NJ, USA; 2University of Texas South Western TX, USA
ALN-PCSsc Pre-Clinical Efficacy in NHP
Figure 8. Potent PCSK9 Knockdown and LDL-C Lowering: Single Dose Data
(0.150, 0.250, 0.400 mg/kg)
4
2
PCSK9
60
40
LDL-C
20
600
800
<0.5 below
(n=13)
1000 1200
within 0.5
(n=9)
1400
1600
0
1800
>0.5 above
(n=10)
Medium
<0.5 below
(n=7)
within 0.5
(n=3)
>0.5 above
(n=5)
Baseline PCSK9 levels
SD Relative to mean
Baseline PCSK9 (ng/mL)
SD Relative to mean
Fitzgerald, ATVB, April 2012
Figure 4. As published in LANCET, ALN-PCS02 was able to lower LDL-C to the same degree independent of PCSK9 baseline values
Figure 1. RNA Interference (RNAi)
6 mg/kg
-20
0
20
40
60
20
40
60
Natural
Process
of RNAi
Strand separation
RISC
mRNA
(A)n
Cleavage
mRNA
degradation
(A)n
Figure 1. RNA interference (RNAi) is a highly evolutionarily conserved mechanism of gene regulation. RNAi occurs at the post-transcriptional
level and is triggered by short double-stranded RNA (dsRNA), known as short interfering RNA (siRNA), which is endogenously processed from long
dsRNA by the RNase III enzyme Dicer or introduced into the cell exogenously as synthetic siRNAs. After being loaded into the RNA-inducing silencing
complex (RISC) in the cytoplasm, the siRNA causes sequence-specific degradation of its homologous mRNA sequences which in turn reduces
the protein encoded by the mRNA. By the introduction of synthetic therapeutic siRNA, this natural, endogenous mechanism may be utilized to
down-modulate any protein of interest.
PCSK9 role in both intracellular and
extracellular degradation of LDLR
protein
Recycling
ASGPR
Clathrin-coated
vesicle
RISC
Endosome
mRNA
Nucleus
Figure 5: An siRNA conjugate was developed to allow for targeted delivery to hepatocytes in vivo. The siRNA was chemically-modified to
enhance stability and conjugated at the 3’-end of the sense strand with a trivalent N-acetyl galactosamine (GalNAc) ligand to allow for targeting to the
asialoglycoprotein receptor (ASGPR) on hepatocytes. The GalNAc-siRNA is administered via subcutaneous injection.
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PCSK9
ALN-PCS
PCSK9 mRNA
A. Intracellular Pathway
B. Extracellular Pathway
Nucleus
0
20
40
60
80
Figure 2. ALN-PCSsc is a PCSK9 synthesis inhibitor that works by degrading PCSK9 mRNA inside the cell and preventing the production
of PCSK9 protein
100
ALN-TTRsc
dose groups
Placebo (n=3)
2.5 (n=3)
5.0 (n=3)
10.0 (n=3)
ALN-TTRsc (mg/kg), qd x5; qw x5
ALN-PCS Phase I Study Results
-20
PCSK9
0
20
40
60
ALN-PCS dose group
15
Day
20
60
80 100 120 140 160 180
5
10
15
0
5
10
15
Time (days)
D
LDL Cholesterol
0
20
40
HDL:TC
1.0
0.8
0.6
60
0.4
80
100
0
5
10
15
0
5
Time (days)
PBS
10
15
Time (days)
ALN-ANG, 0.3 mg/kg
ALN-ANG, 1 mg/kg
ALN-ANG, 3 mg/kg
ALN-ANG, 9 mg/kg
Figure 11. Effects of ANGPTL3 silencing on serum lipids in female ob/ob mice. Triglycerides (A), total cholesterol (B), and LDL cholesterol (C) levels
relative to PBS control group were determined for each time point and group averages ± SD are shown. N=4/group. (D) HDLc/TC ratio was determined
for each animal and group averages ± SD are shown. N=4/group.
ALN-AC3 Targeting ApoCIII
20
40
Biological Rationale in Hypertriglyceridemia
60
80
100
20
40
60
80
ANGPTL3
100
Day
120
140
160
180
200
ApoCIII is liver-expressed, genetically validated target
• 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
–– 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
Peterson et al., NEJM. 362:1082-1089 (2010)
Chang et al., Lipids in Health and Disease. 11:162 (2012)
Human
NHP
80
100
Days
0.150 mg/kg
0.250 mg/kg
0.400 mg/kg
25
-20
0
10
20
ALN-TTRsc
Single 10.0 mg/kg
Injection
30
Days
ALN-PCSsc
20
40
ALN-PCS dose group
0.015 mg/kg
0.045 mg/kg
0.090 mg/kg
60
siRNA
Dose
5
10
15
20
0.150 mg/kg
0.250 mg/kg
0.400 mg/kg
ALN-ANG
• Targets PCSK9
• Efficacy in multiple pre-clinical animal models
• CTA accepted; Phase 1 study to start this year
• Targets ANGPTL3, inhibitor of cellular lipases
• Efficacy in animal models of mixed hyperlipidemia
• Lead Candidates being optimized
25
Fitzgerald, ATVB, April 2012
Figure 3. 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
• Up to 94% KD of human ApoCIII achieved, with >60% KD sustained for at least 30 days following single 3 mg/kg SC dose
• Dosing once every 2 weeks results in 96% KD of human ApoCIII 35 days following first 3 mg/kg dose of AC3-GalNAc conjugate
• SAR for duration of action is underway
A
40
50
60
Duration of ApoCIII-GalNAc Activity Following Single Dose
in Mice Expressing Human ApoCIII
ApoCIII-GalNAc Activity With Q2W x3 Dosing
in Mice Expressing Human ApoCIII
B
Human ApoC3 Protein
Human ApoC3 Protein
PBS
-20
ANGPTL3 is liver-expressed, genetically validated target
• 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)
ALN-AC3, 3 mg/kg
0
20
40
60
80
100
0
10
20
30
Time (days)
40
ALN-AC3, 3 mg/kg
-40
-20
0
20
40
60
80
100
0
10
20
30
40
Time (days)
Figure 12. Duration of ApoCIII-GalNAc activity in mice transduced with 1 x1011 genome copies of AAV construct expressing human ApoCIII
gene. Serum hApoCIII levels relative to each animal’s pre-dose baseline were determined following (A) a single 3 mg/kg SC dose of ALN-AC3 or
(B) 3 mg/kg SC dose administered once every 2 weeks x 3. Group averages ± SD are shown. N=3/group.
ALN-ANG Targeting ANGPTL3
Figure 10. Potent Knockdown Achieved Following Single SC Dose in ob/ob Mice
Figure 7. Cardiovascular Metabolic Programs
0
Figure 12. Single and Multi-Dose Data in hAAV Model
Biological Rationale in Mixed Hyperlipidemia
60
*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
-20
10
100
0
40
LDL-C
*Data for n=6 from the 0.400 mg/kg group through Day 14 only
5
100
PCSK9
20
OTS 2014
Figure 6. 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
Figure 3. Pharmacodynamics and Clinical Efficacy
siRNA
Dose
80
% ApoC3 Knockdown
(Relative to Pre-dose)
PCSK9
synthesis
100
40
LDLc
• Single SC dose 0.3-9 mg/kg
• Up to 99% KD of serum ANGPTL3
• ED90 achieved with 1 mg/kg dose, ED70 ~0.3 mg/kg
A
Summary
B
GalNAc-siRNA ANGPTL3 Knockdown
(ob/ob female mice)
0
20
40
60
80
100
PBS
ALN-AC3
• Targets apoCIII, component of lipoprotein particles
• Efficacy in animal models of hypertriglyceridemia
• Lead Candidates being optimized
0.3
1
3
ANG-GalNAc Conjugate (mg/kg)
GalNAc-siRNA ANGPTL3 Knockdown
(ob/ob female mice)
ALN-ANG
Treatment Groups
% mANGPTL3 Knockdown
(Relative to Pre-dose)
A
0
% Mean TTR Knockdown
Relative to Baseline
% Mean TTR Knockdown
Relative to Baseline (± SEM)
LDLR
synthesis
0.015 mg/kg
0.045 mg/kg
0.090 mg/kg
20
-40
Drug Safety and Metabolism
• Non-GLP Studies in rat and NHP
–– NOAEL >300 mg/kg (QW*5)
• CTA-enabling, 4-Week Toxicology/TK Studies Completed
–– NOAEL >250 mg/kg (Q2W*3) in rats and NHP
–– NOEL >250 mg/kg for CV/Respiratory Safety Pharmacology in NHP
–– No Gene Tox. findings at ICH limit doses
-20
Lysosomal
degradation
80
0
Figure 9. Monthly dosing of ALN-PCSsc (6 mg/kg loading dose followed by Q1M 3 mg/kg) resulted in clamped nadir effects of up to a 92%
reduction in plasma PCSK9 protein and up to a 77% reduction in LDL-C. Moreover, the effect was maintained with very little fluctuation over
190 days of dosing.
Figure 6. Human POC for GalNAc-siRNA Conjugates
Inhibit only
extracellular
functions
-40
60
80
ALN-PCSsc achieves potent and stable PCSK9 knockdown and LDL-C lowering with SC dosing
Clathrin-coated pit
% mANGPTL3 Knockdown
(Relative to Pre-dose)
PCSK9
Blockers
Endosome
Inhibit PCSK9
synthesis and both
intracellular and
extracellular
functions
40
Summary of ALN-PCSsc Safety Studies
B
PCSK9
Synthesis
Inhibitors
60
20
Figure 9. Potent PCSK9 Knockdown and LDL-C Lowering: Multi-Dose Data
ASGPR
(pH>5)
ALN-TTRsc Phase 1 Study Results
LDL
40
0
-20
Day
0
Figure 2. PCSK9 Therapeutic Hypothesis
20
% ApoC3 Knockdown
(Relative to Pre-dose)
Targeted Gene
Silencing
Complementary pairing
0
-40
40
Figure 8. PCSK9 and LDL-C levels following SD treatment with ALN-PCSsc. Groups of 3 female cynos were treated with a single dose of ALN-PCSsc
at 0.1, 0.3, 1, 3, 6 and 10 mg/kg (data for pharmacologically active dose levels only shown). (A) PCSK9 protein levels in plasma, as determined by ELISA.
(B) Serum LDL-C group averages. Overall effect of ALN-PCSsc on PCSK9 and LDL-C level was highly statistically significant using an ANOVA model
P<0.001. Pairwise comparisons to the 0.1 mg/kg group were done via post hoc Tukey’s tests and were statistically significant for the 3, 6 and 10 mg/kg
groups with P<0.001.
GalNAc-siRNA
conjugate
Asialoglycoprotein Receptor (ASGPR)
• Highly expressed in hepatocytes
• High rate of uptake
• Recycling time ~15 minutes
• Conserved across species
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
C
20
80 100 120 140 160 180
% PCSK9 Knockdown and LDL-C Lowering
(Relative to Pre-bleed)
Cleavage
-20
Time (days)
Day
GalNAc3
-20
0
80
Figure 5. GalNAc-siRNA Conjugates for Systemic Subcutaneous RNAi
dsRNA
10 mg/kg
-40
0
80
0
dicer
6 mg/kg
Total Cholesterol
B
-40
-20
100
Synthetic siRNA
3 mg/kg
-40
10 mg/kg
-40
1 mg/kg
LDL-C
3 mg/kg
-60
High
B
1 mg/kg
PCSK9
Low
400
A
% PCSK9 Knockdown
(Relative to Pre-bleed)
6
0
ALN-PCSsc achieves highly durable PCSK9 knockdown and LDL-C reduction with single dose
• Single SC dose 1-10 mg/kg
–– 0.3 mg/kg no effect dose
• Up to 96% PCSK9 knockdown, up to 77% LDL-C lowering
• 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
80
Maximum Per-Subject
Percent Reduction
Number of Subjects
8
Triglycerides
A
% TG Knockdown
(Relative to PBS)
All Subjects
% LDL Lowering
(Relative to PBS)
Introduction: Cardiovascular disease remains the top cause of mortality in the United States. We have developed, and validated in human trials, a
platform for reducing the synthesis of genes expressed in the liver. The platform utilizes a GalNAc sugar ligand attached to the 3’-end of the sense strand
of an RNAi molecule to enable delivery specifically to the liver. Here we present data from this platform on multiple targets of interest in cardiovascular
disease, including PCSK9, ANGPTL3, and ApoC3.
Hypothesis: RNAi therapeutics targeting liver genes of interest (such as PCSK9, ApoC3, and ANGPTL3) will allow for control of lipid levels and reduce
the risk of cardiovascular disease.
Methods: Chemically modified siRNAs were designed using bioinformatic algorithms and were screened for potency in vitro. pM active siRNA molecules
were developed targeting PCSK9, ANGPTL3, and ApoC3.
Results: Monthly dosing of ALN-PCSsc (6 mg/kg loading dose followed by Q1M 3 mg/kg) resulted in clamped nadir effects of up to a 92% reduction in
plasma PCSK9 protein and up to a 77% reduction in LDL-C. Moreover, the effect was maintained with very little fluctuation over 190 days of dosing. Safety
studies in Rat and NHP at doses up to 300 mg/kg (multi-dose) showed that ALN-PCSsc was safe, demonstrating potential for a very wide therapeutic
index. ALN-PCSsc was selected as a development candidate and is being advanced towards an IND for the treatment of hypercholesterolemia.
ALN-ANG: Was tested in two models of hyperlipidemia, the ob/ob mouse and the hCETP-ApoB mouse. In the ob/ob model, treatment with ALN-ANG at
3 mg/kg resulted in a >99% lowering of ANGPTL3 protein, and a >80% lowering of LDL-C and triglycerides.
ALN-AC3: In a humanized ApoC3-AAV mouse model, treatment with 3 mg/kg ALN-AC3 resulted in a >90% lowering of ApoC3 protein after a single dose,
that was maintained at nadir with Q2W dosing.
Additional screening for development candidates for both programs is underway.
Conclusions: We have developed a modular, robust, and reliable platform for the delivery of RNAi therapeutics to the liver with a large therapeutic index.
This small volume, subcutaneous, platform has a remarkable duration of effect.
• Figure
A single
3 mg/kg dose of ALN-ANG results in ~80% reduction in serum LDL-C and TGs
11.
HDL: TC Ratio
PCSK9 Knockdown and LDL-C Reduction
by Baseline PCSK9 Levels
Baseline PCSK9 Distribution
Figure 11. TG and LDL-C Lowering With ANGPTL3 Silencing in ob/ob Mice
% TC Knockdown
(Relative to PBS)
Figure 4. Activity Independent of Baseline PCSK9 Levels
% LDL-C Lowering
(Relative to Pre-bleed)
Abstract
9
PBS
0.3 mg/kg
1 mg/kg
3 mg/kg
9 mg/kg
-50
0
GalNAc-siRNA conjugates provide platform for knockdown of genetically validated, liver-expressed target genes in cardio-metabolic disease
• Human translation validated in clinic with ALN-TTRsc
• New ESC chemistry improves potency and duration
• ALN-PCSsc CTA Accepted
–– 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
50
100
0
5
10
*
15
Time (days)
Figure 10. Dose-titration of ALN-ANG in female ob/ob mice. Mice received a single SC dose of PBS or ALN-ANG at 0.3, 1, 3, or 9 mg/kg on Day 0.
Serum ANGPTL3 levels relative to each animal’s pre-dose baseline were calculated and group averages ± SD are shown. N=4/group. (A) Day 13 serum
ANGPTL3 levels relative to pre-dose, (B) Time course of ANGPTL3 silencing in female ob/ob mice.
Disclosures
Kevin Fitzgerald, Ph.D.
A Subcutaneous, Potent and Durable RNAi Platform Targeting Metabolic Diseases, Genes PCSK9, ApoC3 and ANGPTL3
FINANCIAL DISCLOSURE
Employee of Alnylam Pharmaceuticals