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A Subcutaneous Platform for RNAi Therapeutics Targeting Metabolic Diseases: PCSK9 and ANGPTL3
Anna Borodovsky, William Querbes, Renta Hutabarat, Stuart Milstein, Rajeev Kallanthottathil, Satya Kuchimanchi, Klaus Charisse, Rachel Meyers, Muthiah Manoharan, Kristina Yucius, Abigail Liebow, Martin Maier,
Tracy Zimmermann, Verena Karsten, Jamie Harrop, Amy Chan, Rick Falzone, Jeffrey Cehelsky, Jessica Sutherland, Valerie Clausen, Julia Hettinger, Svetlana Shulga Morskaya, Jay Nair, David Kallend,1 Joseph Chiesa,2
Gary Peters,3 Saraswathy Nochur, Akshay Vaishnaw, Jared Gollob, Amy Simon, Kevin Fitzgerald
Alnylam Pharmaceuticals, Inc., Cambridge, MA, USA; 1The Medicines Company, Parsippany, NJ, USA; 2Covance Clinical Research Unit, Leeds, United Kingdom; 3Hammersmith Medicines Research, London, United Kingdom
Figure 3. ALN-TTRsc Phase I study results
A
Figure 6. ALN-ANG
A
Rapid, dose-dependent, consistent, and durable knockdown of serum TTR
• Excellent correlation of human to non-human primate TTR knockdown on mg/kg basis
• Up to 94% TTR knockdown; mean TTR knockdown of 87.5% and 92.4% at 5.0 and 10.0 mg/kg, respectively
– Confirmation of human translation of GalNAc-siRNA conjugate platform
ALN-TTRsc dose groups, mg/kg
1.4
Placebo MAD (n=4)
2.5 MAD (n=3)
100
5.0 MAD (n=3)
10.0 MAD (n=3)
5.0 mg/kg
10.0 mg/kg
Mean (SEM) % TTR KD in human
1.0
0.8
0.6
0.4
0.2
Synthetic siRNA
0.0
R2=0.83
p<0.001
25
0
5
10
15
20
25
30
35
40
45
50
55
60
B
Strand separation
0
Time (days)
25
50
75
B
Multiple Doses
1.25 mg/kg
(n=3)
2.5 mg/kg
(n=3)
5 mg/kg
(n=3)
10 mg/kg
(n=3)
2.5 mg/kg
(n=3)
5 mg/kg
(n=3)
10 mg/kg
(n=3)
Total
TTRsc
(n=21)
PBO
(n=7)
n (%)
n (%)
n (%)
n (%)
n (%)
n (%)
n (%)
n (%)
n (%)
0
0
0
0
1 (33%)
1 (33%)
3 (100%)
5 (24%)
1 (14%)
Headache
0
1 (33%)
1 (33%)
0
0
0
0
2 (10%)
1 (14%)
0
1 (33%)
0
0
0
0
0
1 (5%)
0
Local bruise
0
0
0
1 (33%)
0
0
0
1 (5%)
0
Local tenderness
0
0
0
1 (33%)
0
0
0
1 (5%)
0
Abdominal tenderness
0
0
0
0
0
0
1 (33%)
1 (5%)
0
Heart Failure Society of America, Sept. 2013
• All TEAEs mild or moderate in severity
mRNA
degradation
(A)n
• ISRs: clinically mild, consisting of transient erythema associated in some cases with edema and/or pain; self-limiting, resolved within ~2 hours of onset
• No study discontinuations, flu-like symptoms, or changes in cytokines, CRP, liver function tests, renal function and hematologic parameters
• No significant adverse events associated with drug at doses through 10.0 mg/kg
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.
0.0
1
0.8
0.6
0.4
0.2
PBS
10
100
Injection site reaction (ISRs)
Local erythema
(A)n
0.4
0.2
1.2
3
1
0
0.3
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Time (days)
5 mg/kg qd x5
Mean (SEM) % TTR KD in NHP
Single Dose
Reported AEs
mRNA
0.6
1.4
ANG-GalNAc conjugate
Safety and tolerability
Natural
Process
of RNAi
Complementary pairing
0.8
65
Heart Failure Society of America, Sept. 2013
Cleavage
Cleavage
50
ob/ob Ctrl
ob/ob ANG
1.6
1.0
0
ALN-TTRsc (mg/kg), qd x5; qw x5
Targeted Gene
Silencing
75
1.8
1.2
>95% Protein reduction in ANGPTL3-GalNAc3 treated (ob/ob) mice
1.4
>95% Triglyceride lowering in ANGPTL3-GalNAc3 treated (ob/ob) mice
C
1.4
PBS
PBS
GaINAc-ANG
GaINAc-ANG
1.2
1.2
1
1
TG, relative to pre-dose=1
Mean TTR relative to baseline (± SEM)
Figure 1. RNA interference (RNAi)
RISC
2.5 mg/kg
1.2
CONCLUSION: We have developed a robust and reliable platform for the delivery of RNAi therapeutics to the liver in vivo. This platform is administered as a small volume
subcutaneous dose and has been shown to be safe and effective in a recently reported Phase I trial in healthy human volunteers. We have extended this platform with
two targets of high interest in the cardiovascular field, PCSK9 and ANGPLT3. This platform is modular and enables targeting of any gene that is expressed in the liver
spanning cardiovascular and metabolic diseases such as diabetes, NASH and obesity.
dsRNA
ALN-TTRsc
GalNAc-siRNA targeting ANGPTL3
(ob/ob female mice)
1.4
mANGPTL3 protein,
relative to ave. pre-dose=1
• Consistent level of TTR knockdown with weekly dosing; durable effects lasting weeks after last dose
RESULTS: Highly potent and selective siRNAs were developed targeting both PCSK9 (ALN-PCSsc) and ANGPTL3 (ALN-ANGsc). In NHPs, ALN-PCSsc at a dose of
2mg/kg reduced circulating PCSK9 levels up to 94% with a subsequent lowering of LDL-C up to 67%. Moreover, the effect on both PCSK9 and LDL-C was durable, with
substantial lowering remaining 60 days post the last dose given. ALN-PCSsc was selected as a development candidate and will be advanced towards an IND in 2014.
ALN-ANGsc was tested in two models of hyperlipidemia: the ob/ob mouse and the hCETP-ApoB mouse. In both model systems, treatment with ALN-ANGsc resulted in a
significant lowering of total cholesterol, LDL-C and triglycerides. A dose response curve indicated that our current prototype molecule has a single dose ED50 of ~ 1mg/
kg. Additional SAR around this molecule continues towards a development candidate.
dicer
Demonstrated knockdown in ob/ob female mice
• Statistically significant knockdown of serum TTR at all doses evaluated (p<0.01)
METHODS: A set of chemically modified siRNAs were designed using bioinformatic algorithms and were synthesized and screened for potency by transfection in Hep3B
cells. In this manner, highly active siRNA molecules were developed targeting either PCSK9 or ANGPLT3. The siRNAs were tested in either rodent models or in nonhuman primates (NHPs) for activity,
mANGPTL3 protein relative to PBS=1
AIM: Despite treatment advances there remains a very high unmet medical need for therapies to treat cardiovascular and metabolic diseases. We have developed and
validated in human trials a platform for identifying potent and specific subcutaneously delivered RNA interference molecules targeting genes expressed in the liver. The
platform involves the utilization of a GalNAc sugar ligand attached to the 3’ end of the sense strand to enable ligand/receptor pair driven delivery of an siRNA molecule
specifically to the liver. Here we extend that platform to targets of interest in cardiovascular disease, mainly PCSK9 and ANGPLT3.
mANGPTL3 protein, relative to pre-dose=1
Abstract
0.8
0.6
0.8
0.6
0.4
0.4
0.2
0.2
Figure 4. Alnylam metabolic disease program
• Alnylam PCSK9 Phase I POC and TTRsc Phase I POC (with GalNAc-siRNA conjugate platform) opens significant opportunities in metabolic disease to address validated
but undruggable, liver-expressed targets
0
0
0
5
10
15
20
25
0
30
5
10
Time (days)
– Eg, ANGPTL3 for mixed hyperlipidemia, ApoC3 for hypertriglyceridemia, SCAP for Steatosis
15
20
25
Time (days)
ANG siRNA, 3 mg/kg qd x5 wk1; BIW wk 2-4
ANG siRNA, 3 mg/kg qd x5 wk1; BIW wk 2-4
Figure 2. GalNAc-siRNA conjugates for systemic RNAi
Alnylam
technology
ASGPR
(pH>5)
Clathrin-coated pit
D
protein
>85% LDL-C in ANGPTL3-GalNAc3 treated (ob/ob) mice
1.2
• Single and multiple genes (up to
10 at once)
• Develop lead conjugate
E
>60% Total cholesterol in ANGPTL3-GalNAc3 treated (ob/ob) mice
1.6
PBS
1.4
1
Human
genetics
1.2
0.8
PBS
GaINAc-ANG
GaINAc-ANG
• Silence all genes in candidate loci
• Validate directionality of genetics
GalNAc-siRNA
Human
genetics
• Specific gene silencing
1
Literature targets/
collaborators
GalNAc-siRNA
conjugate
e=1
GalNAc3
30
Tracy Zimmermann, Verena Karsten, Jamie Harrop, Amy Chan, Rick Falzone, Jeffrey Cehelsky, Jessica Suthe
Gary Peters,3 Saraswathy Nochur, Akshay Vaishnaw, Jared Gollob, Amy Simon, Kevin Fitzgerald
Alnylam Pharmaceuticals, Inc., Cambridge, MA, USA; 1The Medicines Company, Parsippany, NJ, USA; 2Covance Clinical Research U
Abstract
Figure 3. ALN-TTRsc Phase I
AIM: Despite treatment advances there remains a very high unmet medical need for therapies to treat cardiovascular and metabolic diseases. We have developed and
validated in human trials a platform for identifying potent and specific subcutaneously delivered RNA interference molecules targeting genes expressed in the liver. The
platform involves the utilization of a GalNAc sugar ligand attached to the 3’ end of the sense strand to enable ligand/receptor pair driven delivery of an siRNA molecule
specifically to the liver. Here we extend that platform to targets of interest in cardiovascular disease, mainly PCSK9 and ANGPLT3.
A
Rapid, dose-dependent, consi
• Statistically significant knockdo
METHODS: A set of chemically modified siRNAs were designed using bioinformatic algorithms and were synthesized and screened for potency by transfection in Hep3B
cells. In this manner, highly active siRNA molecules were developed targeting either PCSK9 or ANGPLT3. The siRNAs were tested in either rodent models or in nonhuman primates (NHPs) for activity,
• Consistent level of TTR knockd
RESULTS: Highly potent and selective siRNAs were developed targeting both PCSK9 (ALN-PCSsc) and ANGPTL3 (ALN-ANGsc). In NHPs, ALN-PCSsc at a dose of
2mg/kg reduced circulating PCSK9 levels up to 94% with a subsequent lowering of LDL-C up to 67%. Moreover, the effect on both PCSK9 and LDL-C was durable, with
substantial lowering remaining 60 days post the last dose given. ALN-PCSsc was selected as a development candidate and will be advanced towards an IND in 2014.
ALN-ANGsc was tested in two models of hyperlipidemia: the ob/ob mouse and the hCETP-ApoB mouse. In both model systems, treatment with ALN-ANGsc resulted in a
significant lowering of total cholesterol, LDL-C and triglycerides. A dose response curve indicated that our current prototype molecule has a single dose ED50 of ~ 1mg/
kg. Additional SAR around this molecule continues towards a development candidate.
• Excellent correlation of human
• Up to 94% TTR knockdown; m
– Confirmation of human trans
ALN-TTRsc dose groups, mg/k
1.4
5.0 MAD (
10.0 MAD
Placebo MAD (n=4)
2.5 MAD (n=3)
1.2
Mean TTR relative to baseline (± SEM)
CONCLUSION: We have developed a robust and reliable platform for the delivery of RNAi therapeutics to the liver in vivo. This platform is administered as a small volume
subcutaneous dose and has been shown to be safe and effective in a recently reported Phase I trial in healthy human volunteers. We have extended this platform with
two targets of high interest in the cardiovascular field, PCSK9 and ANGPLT3. This platform is modular and enables targeting of any gene that is expressed in the liver
spanning cardiovascular and metabolic diseases such as diabetes, NASH and obesity.
Figure 1. RNA interference (RNAi)
1.0
0.8
0.6
0.4
0.2
Synthetic siRNA
0.0
0
5
10
15
20
ALN-TTRsc (mg/kg), qd x5; qw x5
Heart Failure Society of America, Sept. 2013
dicer
dsRNA
Cleavage
B
Strand separation
Safety and tolerability
Natural
Process
of RNAi
1.25 m
(n=
Reported AEs
Targeted Gene
Silencing
RISC
Complementary pairing
mRNA
Cleavage
(A)n
n (%
Injection site reaction (ISRs)
0
Headache
0
Local erythema
0
Local bruise
0
Local tenderness
0
Abdominal tenderness
0
Heart Failure Society of America, Sept. 2013
• All TEAEs mild or moderate in severit
mRNA
degradation
(A)n
• ISRs: clinically mild, consisting of tran
• No study discontinuations, flu-like sym
• No significant adverse events associa
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.
Figure 4. Alnylam metabolic
• Alnylam PCSK9 Phase I POC and TTR
but undruggable, liver-expressed targ
– Eg, ANGPTL3 for mixed hyperlipid
Figure 2. GalNAc-siRNA conjugates for systemic RNAi
GalNAc3
Litera
col
GalNAc-siRNA
conjugate
ASGPR
(pH>5)
Clathrin-coated pit
GalNAc-siRNA
• siRNA conjugated to N-acetylgalactosamine (GalNAc) ligand targeting mRNA
• Efficient delivery to hepatocytes following subcutaneous administration
protein
Recycling
ASGPR
ASGPR
Clathrin-coated
vesicle
• Highly expressed in hepatocytes
– 0.5-1 million copies/cell
RISC
• Clears serum glycoproteins via clathrin-mediated endocytosis
• High rate of uptake
• Recycling time ~15 minutes
• Conserved across species
Endosome
Figure 5. ALN-PCSsc lowers
mRNA
140
Nucleus
ALN-TTRsc Phase 1 study
Study design
• Randomized, double-blinded, placebo-controlled SAD and MAD study in healthy volunteers
– 3:1 randomization (ALN-TTRsc:Placebo)
– 4 subjects/cohort
– Cohort 1-4: single doses of 1.25, 2.5, 5.0 and 10 mg/kg
– Cohort 5-7: multiple doses of 2.5, 5.0 and 10 mg/kg
▪ Multi-dose schedule: daily x 5, followed by weekly x 5
Primary objective
• Evaluate safety and tolerability of subcutaneously administered single and multiple doses of ALN-TTRsc
Secondary objectives
• Assess clinical activity
% of average pre-bleed
120
100
80
60
40
20
0
0
10
– Serum TTR, retinol binding protein (RBP), Vitamin A levels
Status
• Dosing completed; analysis ongoing
ALN-PCSsc, 2
qd x5; qw x3
ick Falzone, Jeffrey Cehelsky, Jessica Sutherland, Valerie Clausen, Julia Hettinger, Svetlana Shulga Morskaya, Jay Nair, David Kallend
b, Amy Simon, Kevin Fitzgerald
y, Parsippany, NJ, USA; 2Covance Clinical Research Unit, Leeds, United Kingdom; 3Hammersmith Medicines Research, London, United Kingdom
Figure 3. ALN-TTRsc Phase I study results
A
Figure 6. ALN-ANG
A
Rapid, dose-dependent, consistent, and durable knockdown of serum TTR
ed for potency by transfection in Hep3B
sted in either rodent models or in non-
• Consistent level of TTR knockdown with weekly dosing; durable effects lasting weeks after last dose
sc). In NHPs, ALN-PCSsc at a dose of
h PCSK9 and LDL-C was durable, with
be advanced towards an IND in 2014.
reatment with ALN-ANGsc resulted in a
cule has a single dose ED50 of ~ 1mg/
• Excellent correlation of human to non-human primate TTR knockdown on mg/kg basis
mANGPTL3 protein relative to PBS=1
• Up to 94% TTR knockdown; mean TTR knockdown of 87.5% and 92.4% at 5.0 and 10.0 mg/kg, respectively
– Confirmation of human translation of GalNAc-siRNA conjugate platform
ALN-TTRsc dose groups, mg/kg
1.4
ALN-TTRsc
100
5.0 MAD (n=3)
10.0 MAD (n=3)
Placebo MAD (n=4)
2.5 MAD (n=3)
2.5 mg/kg
5.0 mg/kg
1.2
10.0 mg/kg
Mean (SEM) % TTR KD in human
Mean TTR relative to baseline (± SEM)
tform is administered as a small volume
s. We have extended this platform with
any gene that is expressed in the liver
1.0
0.8
0.6
0.4
0.2
0.0
75
50
R2=0.83
p<0.001
25
0
5
10
15
20
25
ALN-TTRsc (mg/kg), qd x5; qw x5
B
GalNAc-siRN
(ob/o
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
PBS
10
0
30
35
40
45
50
55
60
ANG
65
0
Time (days)
25
50
75
100
Mean (SEM) % TTR KD in NHP
Heart Failure Society of America, Sept. 2013
Safety and tolerability
Natural
Process
of RNAi
Single Dose
B
Multiple Doses
1.25 mg/kg
(n=3)
2.5 mg/kg
(n=3)
5 mg/kg
(n=3)
10 mg/kg
(n=3)
2.5 mg/kg
(n=3)
5 mg/kg
(n=3)
10 mg/kg
(n=3)
Total
TTRsc
(n=21)
n (%)
n (%)
n (%)
n (%)
n (%)
n (%)
n (%)
n (%)
Reported AEs
PBO
(n=7)
n (%)
Injection site reaction (ISRs)
0
0
0
0
1 (33%)
1 (33%)
3 (100%)
5 (24%)
1 (14%)
Headache
0
1 (33%)
1 (33%)
0
0
0
0
2 (10%)
1 (14%)
Local erythema
0
0
0
0
0
1 (33%)
0
1 (5%)
0
Local bruise
0
0
0
1 (33%)
0
0
0
1 (5%)
0
Local tenderness
0
0
0
1 (33%)
0
0
0
1 (5%)
0
Abdominal tenderness
0
0
0
0
0
0
1 (33%)
1 (5%)
0
Heart Failure Society of America, Sept. 2013
• All TEAEs mild or moderate in severity
• ISRs: clinically mild, consisting of transient erythema associated in some cases with edema and/or pain; self-limiting, resolved within ~2 hours of onset
• No study discontinuations, flu-like symptoms, or changes in cytokines, CRP, liver function tests, renal function and hematologic parameters
• No significant adverse events associated with drug at doses through 10.0 mg/kg
post-transcriptional level and is triggered
dsRNA by the RNase III enzyme Dicer
C) in the cytoplasm, the siRNA causes
the introduction of synthetic therapeutic
Demonstrated knockdown in ob
• Statistically significant knockdown of serum TTR at all doses evaluated (p<0.01)
>95% Protein reduction in ANGP
1.4
1.2
mANGPTL3 protein, relative to pre-dose=1
bolic diseases. We have developed and
eting genes expressed in the liver. The
ir driven delivery of an siRNA molecule
3.
1
0.8
0.6
0.4
0.2
Figure 4. Alnylam metabolic disease program
• Alnylam PCSK9 Phase I POC and TTRsc Phase I POC (with GalNAc-siRNA conjugate platform) opens significant opportunities in metabolic disease to address validated
but undruggable, liver-expressed targets
0
0
5
10
Ti
– Eg, ANGPTL3 for mixed hyperlipidemia, ApoC3 for hypertriglyceridemia, SCAP for Steatosis
ANG siRNA, 3 mg/kg qd x5 wk1; BIW wk 2-4
Alnylam
technology
Literature targets/
collaborators
Human
genetics
D
1.2
• Specific gene silencing
• Single and multiple genes (up to
10 at once)
Clathrin-coated pit
1
• Silence all genes in candidate loci
• Develop lead conjugate
Human
genetics
Clathrin-coated
vesicle
RISC
Endosome
LDL-C, relative to pre-dose=1
• Validate directionality of genetics
Recycling
ASGPR
>85% LDL-C in ANGPTL3-GalNA
0.8
0.6
0.4
Figure 5. ALN-PCSsc lowers PCSK9 and LDL-C (in NHPs)
mRNA
140
Nucleus
0.2
ALN-PCSsc
LDL-C
% of average pre-bleed
120
0
PCSK9
0
5
10
Ti
100
Figure 5. ALN-PCSsc conjugate lowers PCSK9
and LDL-C in NHPs. ALN-PCSsc dosed at 2 mg/kg
demonstrated up to 95% lowering of plasma PCSK9
protein and up to 67% LDL-C lowering in cynomolgous
monkeys. Moreover, the effect lasted >50 days post
the last dose, indicating that a once monthly dosing
scheme is possible.
80
60
40
20
ANG siRNA, 3 mg/kg qd x5 wk1; BIW wk 2-4
Figure 6. ALN-ANG: An RNAi therape
responsive manner and is improved upon
(B) Multidose of 3 mg/kg eliminates circu
results in >95% reduction in TGs in the
the ob/ob mouse model of hyperlipidem
of hyperlipidemia.
Conclusions
• We have developed a robust and reliab
0
0
10
20
ALN-PCSsc, 2 mg/kg
qd x5; qw x3
30
40
Day
50
60
70
80
• This platform is administered as a sm
human volunteers.
• We have extended this platform to two
• This platform is modular and enables
NASH and obesity.
na Shulga Morskaya, Jay Nair, David Kallend,1 Joseph Chiesa,2
Research, London, United Kingdom
Figure 6. ALN-ANG
A
75
GalNAc-siRNA targeting ANGPTL3
(ob/ob female mice)
1.4
1.8
1.0
0.8
0.6
0.4
0.2
0.0
ob/ob Ctrl
ob/ob ANG
1.6
1.2
mANGPTL3 protein,
relative to ave. pre-dose=1
mANGPTL3 protein relative to PBS=1
R2=0.83
p<0.001
50
Demonstrated knockdown in ob/ob female mice
1.4
1.2
1
0.8
0.6
0.4
0.2
PBS
10
3
1
0
0.3
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
ANG-GalNAc conjugate
100
Time (days)
5 mg/kg qd x5
SEM) % TTR KD in NHP
B
n (%)
00%)
5 (24%)
1 (14%)
0
2 (10%)
1 (14%)
0
1 (5%)
0
0
1 (5%)
0
0
33%)
1 (5%)
0
1 (5%)
0
within ~2 hours of onset
parameters
1.4
-PCSsc conjugate lowers PCSK9
NHPs. ALN-PCSsc dosed at 2 mg/kg
p to 95% lowering of plasma PCSK9
o 67% LDL-C lowering in cynomolgous
over, the effect lasted >50 days post
ndicating that a once monthly dosing
ble.
1.4
PBS
PBS
GaINAc-ANG
metabolic disease to address validated
GaINAc-ANG
1.2
1.2
1
1
0.8
0.6
0.8
0.6
0.4
0.4
0.2
0.2
0
0
0
5
10
15
20
25
30
0
5
10
Time (days)
D
15
20
25
30
Time (days)
ANG siRNA, 3 mg/kg qd x5 wk1; BIW wk 2-4
ANG siRNA, 3 mg/kg qd x5 wk1; BIW wk 2-4
E
>85% LDL-C in ANGPTL3-GalNAc3 treated (ob/ob) mice
1.2
>60% Total cholesterol in ANGPTL3-GalNAc3 treated (ob/ob) mice
1.6
PBS
PBS
GaINAc-ANG
GaINAc-ANG
1.4
1
1.2
LDL-C, relative to pre-dose=1
an
cs
>95% Triglyceride lowering in ANGPTL3-GalNAc3 treated (ob/ob) mice
C
TG, relative to pre-dose=1
n (%)
>95% Protein reduction in ANGPTL3-GalNAc3 treated (ob/ob) mice
0.8
Tc, relative to pre-dose=1
PBO
(n=7)
(%)
mANGPTL3 protein, relative to pre-dose=1
mg/kg
=3)
Total
TTRsc
(n=21)
0.6
0.4
1
0.8
0.6
0.4
0.2
0.2
0
0
0
5
10
15
20
25
30
0
5
10
Time (days)
ANG siRNA, 3 mg/kg qd x5 wk1; BIW wk 2-4
15
20
25
30
Time (days)
ANG siRNA, 3 mg/kg qd x5 wk1; BIW wk 2-4
Figure 6. ALN-ANG: An RNAi therapeutic for the treatment of hyperlipidemia. (A) A novel GalNAc RNAi conjugate is able to reduce ANGPTL3 protein in dose
responsive manner and is improved upon multidose resulting in greater than 95% reduction of serum ANPTL3 protein in the ob/ob mouse model of mixed hyperlipidemia.
(B) Multidose of 3 mg/kg eliminates circulating serum ANGPLT3 protein as measured by ANGPTL3 specific ELISA assay. (C) Lowering of circulating serum ANGPTL3
results in >95% reduction in TGs in the ob/ob mouse model of hyperlipidemia. (D) Lowering of circulating serum ANGPTL3 results in >85% reduction in LDL-C in
the ob/ob mouse model of hyperlipidemia. (E) Lowering of circulating serum ANGPTL3 results in >60% reduction in total cholesterol in the ob/ob mouse model
of hyperlipidemia.
Conclusions
• We have developed a robust and reliable platform for the delivery of RNAi therapeutics to the liver in vivo.
• This platform is administered as a small volume subcutaneous dose and has been shown to be safe and effective in a recently reported Phase I trial in healthy
human volunteers.
• We have extended this platform to two targets of high interest in the cardiovascular field, PCSK9 and ANGPLT3.
• This platform is modular and enables targeting of any gene that is expressed in the liver spanning cardiovascular and metabolic diseases such as diabetes,
NASH and obesity.