Targeting Antithrombin to
Treat Hemophilia
siRNA-AT3
Margaret Ragni, MD, MPH
University of Pittsburgh
THSNA, Chicago IL
April 14, 2016
Disclosures
• Consultancy:Baxalta;Biogen,Biomarin; ShireDevelopmentLL
• ResearchFunding:Alnylam; Baxalta;Baxter; Biogen;CSLBehring;
Dimensions;GenentechRoche;Novartis;SPARK;VascularMedicine
Institute
• Honoraria:Alnylam,Baxalta,Biomarin,Medscape/WebMD/Biogen
• Advisorycommittees: Baxalta; Biogen;FoundationforWomenand
GirlswithBloodDisorders;NationalHemophiliaFoundation,Tacere
Benitec
Hemophilia
Background:
Hemophilia is an X-linked bleeding disorder
§ Caused by defective, deficient FVIII, FIX, leading to the
inability to generate thrombin
§ Results in spontaneous, traumatic bleeds - joints, muscles
§ Prophylaxis 2-3/week to prevent bleeds
- Effective, but invasive, expensive, and 50% abandon as adults
- Inhibitor formation in 25% - neutralizes FVIII, morbidity, mortality
Goal: simpler, less invasive, less costly treatment
Targeting Antithrombin
Background:
§ Exciting time in the treatment of hemophilia
§ Novel therapeutics in development
§ Decrease clot inhibition – AT3
§ Moderate the clinical severity of hemophilia
§ Provide simpler, effective therapy for hemophilia
Targeting Antithrombin
Why target AT?
§ AT deficiency moderates clinical severity of hemophilia
§ Concept: reduce AT levels to prevent bleeds
§ Hemophilia bleeding phenotype is less severe
Preclinical Studies: HA, HB mouse and NHP models
Clinical Studies: HA, HB with and without inhibitors
Three Rivers Coagulation Cascade
Allegheny River
Monongahela River
THE POINT (PITTSBURGH)
Ohio River
Three Rivers Coagulation Cascade
Allegheny River
Monongahela River
X
THE POINT (PITTSBURGH)
Ohio River
Three Rivers Coagulation Cascade
XI
VII
Allegheny River
Monongahela River
X
THE POINT (PITTSBURGH)
Ohio River
II
Three Rivers Coagulation Cascade
XI
VII
Allegheny River
Monongahela River
X
THE POINT (PITTSBURGH)
Ohio River
II
Inhibiting AT3
XI
VII
Allegheny River
Monongahela River
X
THE POINT (PITTSBURGH)
Ohio River
II
AT3
Inhibiting AT3
XI
VII
Allegheny River
Monongahela River
X
THE POINT (PITTSBURGH)
Ohio River
II
AT3
Inhibiting AT3
XI
VII
Allegheny River
Monongahela River
X
THE POINT (PITTSBURGH)
Ohio River
II
AT3
siRNA-AT3
What is siRNA?
§ In 1990’s:
Plant biologists inserted two copies of gene
encoding purple color of petunias - flowers
turned white or patchy purple-white
§ Concept:
Color change attributed to silencing of purple genes by
messenger mRNA, shut down by silencing siRNAs.
§ RNAi:
RNA interference (RNAi) is a natural mechanism to
regulate gene expression. It is mediated by “small
interfering RNAs” or “siRNAs” that stabilize genome
by silencing repetitive genes, foreign viruses.
§ siRNAs:
Complementary to cellular mRNA sequence, disrupt
synthesis of target mRNA, which is degraded, silenced.
Napoli C et al, Plant Cell , 1990; Novina CD, Sharp PA, Nature 2004
siRNA-AT3
How does siRNA work?
• Harnesses natural synthetic pathway
siRNA-AT3
GalNAc
◦ Interferes with translation of mRNA
ASGPR
◦ Mediated by small interfering RNAs, siRNA
• Silences a therapeutic gene
◦ Can target any gene in genome
◦ Targets AT3 mRNA in liver
◦ Interferes with translation of mRNA- AT3
◦ Binds to mRNA -AT3
◦ Degrades mRNA-AT3
◦ Silences gene expression
◦ Prevents AT3 synthesis
AT3
Recycling
ASGPR
Clathrin-coated
Vesicle
RISC
Endosome
AT3 mRNA
Nucleus
Hepatocyte
17
RISC = RNA silencing complex
siRNA-AT3
siRNA: Targets antithrombin (AT3)
siRNA-AT3
Weekly Dosing in Mice Suppresses AT3
1.2
ASGPR
AT3
Recycling
ASGPR
Clathrin-coated
Vesicle
RISC
Endosome
AT3 mRNA
Nucleus
Hepatocyte
Relative Antithrombin Level
(Normalized to pre-dose and PBS)
GalNAc
3 mg/kg
1.5 mg/kg
0.75 mg/kg
1.0
0.8
0.6
0.4
0.2
0.0
0
4
qw x5
8
12
16
20
24
Day
Circulation
GalNAc = N-acetyl galactosamine; ASGPR = asialoglycoprotein receptor
28
32
Repeat-Dose PK in NHP
1.0
(Pre-dose = 1)
Relative Plasma AT Activity
Preclinical Studies: siRNA-AT3 is titratable, reversible.
0.8
0.6
0.4
0.2
0.0
-20
0
20
40
0.5 mg/kg qw x 8
1 mg/kg q2w x 4
1.5 mg/kg qw x 5
Seghal A et al, Nat Med 2015
60
80
Day
100
120
Recovery
0.125 mg/kg qw x 12
Recovery
0.25 mg/kg qw x 12
Recovery
NHP = non-human primates
140
160
180
Thrombin Generation: Hemophilia Severity
Thrombin Generation: Clot formation in hemophilia plasma
§ Sensitive to coagulation cascade
§ Correlates with hemophilia severity
Tissue factor = 1 pM
Phospholipids = 4 µM
Control
Relative Thrombin Generation
450400-
Thrombin (nM)
Peak Thrombin
1.2
500-
350300250-
FVIII = 10%
200-
FVIII = 2.7%
150100-
FVIII < 1%
50-
ETP
1.0
0.8
0.6
0.4
0.2
0-
0
10
20
30
40
50
0
N=40
Time (min)
Normal
Dargaud Y et al, T&H 2005; Akinc A et al, ISTH 2013
Severe
N=23
Moderate Mild
N=10
N=13
_______Hemophilia_______
Thrombin Generation: AT3-Depleted Plasmas
Thrombin generation in AT3-depleted hemophilia A and B plasmas.
Control with VIII (IX) and AT3 added back.
HA Donor Plasma
HB Donor Plasma
AT3 depletion increases peak thrombin and delays thrombin inhibition.
Seghal A et al, Nat Med 2015
Titration: AT3 and Factor Replacement
HA Donor Plasma
HB Donor Plasma
With increasing AT3 reduction:
§ There is increasing thrombin generation response to factor replacement.
§ Peak thrombin after factor dosing to 20-60% levels for bleeds does not exceed that of
heterozygous deficiency, dotted line, (and VTE risk), even when AT3 is as low as 20%.
Seghal A et al, Nat Med 2015
siRNA-AT3: Hemostasis in Laser Injury Model
Preclinical Proof of Concept
Untreated HB Mice
•
•
•
•
•
siRNA-AT3 Treated HB Mice
In a cremaster muscle microvessel laser injury model: real-time clot formation
Hemophilia B mice treated with single-dose 30 mg/kg siRNA-AT3
Laser injury induced 8 days post siRNA-AT3 treatment
2 Hemophilia B mice, 3 injury sites each (6 total injuries)
At 6/6 injury sites: there is platelet (red) and fibrin deposition (green)
Ivanciu L ,Camire R Akinc A, ISTH 2013
siRNA-AT3: Laser Injury Model (HA Mice)
Deposition of Platelets
WT
HA + PBS
400
WT
HA + PBS
300
HA + 1 mg/kg
HA + 30 mg/kg
HA + 100U/kg rFVIII
Fibrin Area (microns2)
Platelets Area (microns2)
500
Deposition of Fibrin
300
200
HA + 1 mg/kg
HA + 30 mg/kg
HA + 100U/kg rFVIII
200
100
100
0
0
0
30
60
90
120
Time (sec)
Group
WT
HA + PBS
HA + 1 mg/kg siRNA-AT3
HA + 30 mg/kg siRNA-AT3
160
Animals (N)
5
5
5
6
180
Injuries (N)
25
25
25
30
0
30
60
Stable Thrombus (N)
25
0
25
30
90
120
Time (sec)
160
Percent AT mRNA in Liver
100%
100%
50%
5%
Results: As siRNA-AT3 increases, AT falls, platelet and fibrinogen deposition increases.
Sehgal A et al., Nat Med 2015
180
siRNA-AT3: Saphenous Vein Model (HA Mice)
Saphenous vein bleeding model
Study results
• Treated animals: single SC dose siRNA-AT3
to yield ~70% AT knockdown at time of
model initiation
• Control animals: single IV dose of rFVIII
25 IU/kg 15 min prior to model initiation
20
Number of Hemostatic Events
• Saphenous vein exposed and transected to
initiate bleeding
• 30 seconds after cessation of blood loss,
clot dislodged to re-initiate bleeding
• Outcome: Number of hemostatic events in
30 min observation period recorded
N=8
N=6
N = 15
N=9
p < 0.0001
15
10
~70%
AT
KD
5
0
Conclusion
--
WT
Saline
siRNA-AT3
rFVIII
25 IU/kg
HA
Single-dose sc siRNA-AT3 results in 70% AT3 knockdown, with more hemostatic events than
saline or control, comparable to rFVIII dosing.
Buyue Y et al, Blood 2008; Sehgal A et al, Nat Med 2015
siRNA-AT3: Thrombin Generation in Inhibitor NHP
Inhibitor Model: In NHP HA inhibitor model, induced by sheep VIII Ab, there is a dosedependent increase in thrombin generation, even in presence of neutralizing FVIII inhibitor.
Induction of Hemophilia A
Pre Ab
140
4 hr Post Ab
1.2
120
1.0
100
Peak Thrombin (nM)
Relative FVIII Levels
1.4
0.8
0.6
0.4
80%
AT reduction
** (p<0.01)
80
60%
AT reduction
60
40
20
0.2
0.0
Normalization of Thrombin Generation
<0.01
Saline
<0.01
0.25
<0.01
0.50
0
Pre-dose
0.25
0.50
siRNA-AT3 (mg/kg) qw
siRNA-AT3 (mg/kg) qw
Normal
Seghal A et al, Nat Med 2015
Saline
Induced HA
siRNA-AT3: Survival Benefit in HA Mice
26-Week Chronic Toxicity Study
• Mice:
HA (B6;129S4-F8tm1Kaz)
• Dosing: siRNA-AT3 10 or 30 mg/kg
sc; 30 mg/kg weekly dosing
• Control: Saline
• Groups: N=70; 35 per sex) powered
for spontaneous loss due to
HA mouse strain sensitivity
Results
•
•
•
•
•
No adverse clinical signs
No changes in body weight
No changes in hematology
No changes in clinical chemistry
Survival benefit in treated animals
(p <0.0001, Log-rank, Mantel-Cox)
Survival of HA Mice
100
80
Percent Survival
Groups
60
40
0 mg/kg
10 mg/kg
30 mg/kg
20
0
0
50
100
150
200
Days
Conclusion
There was a significant survival benefit, p<0.0001, in animals receiving siRNA-AT3, consistent
with hypothesis that a reduction in AT3 levels leads to improved hemostasis.
Sehgal A et al, Nat Med 2015
siRNA-AT3: Phase I Clinical Trial
Aim: siRNA-AT3 Dose Escalation
1° Objective: Safety, tolerability
2° Objective: AT knockdown, thrombin generation
Dose Group
SC Dose (mcg/kg)
Subjects
No./Part
Single-dose
30 mcg/kg
Volunteers
N=4 (Part A)
Weekly x 3
15, 45, 75 mcg/kg
Hem A, B
N=12 (Part B)
Monthly x 3
225, 450, 900, 1800 mcg/kg
Hem A, B
N=12 (Part C)
Age:
Type:
Severity:
Weight:
Mean 38 years (27-46)
Hemophilia A, B (20,4)
Severe, moderate (22, 2)
Mean 78 kg (71-85)
28
Phase 1 Single-Dose Study in Volunteers
Single-dose 30 mcg/kg sc siRNA-AT3:
Part A
· AT knockdown: Max 19%±4.4% vs. placebo, p<0.01
Durable for over 60 days
· Well tolerated, no SAE, injection site findings, thrombosis
-20
Treatment Group
siRNA-AT3
30 mcg/kg
Placebo
Mean % AT Knockdown
-10
0
10
20
30
40
0
10
20
30
40
Days
50
60
70
80
29
Phase 1 Weekly Dosing in Hemophilia A, B
Weekly subcutaneous siRNA-AT3:
Part B
Cohort
% Mean (+/- SEM) AT3 Activity
Relative to Baseline
125
15 mcg/kg
100
AT Lowering
53%
(N=3)
45 mcg/kg
75
86%
(N=6)
50
75 mcg/kg
Dose Group
25
(N=3)
15 mcg/kg (N=3)
45 mcg/kg (N=6)
75 mcg/kg (N=3)
0
0
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
Day
30
74%
Phase 1 Monthly Dosing in Hemophilia A, B
Monthly
subcutaneous siRNA-AT3:
A
% Mean (+/- SEM) AT3 Activity
Relative to Baseline
125
Part C
Cohort
AT3 Lowering
Dose Group
225 mcg/kg (N=3)
450 mcg/kg (N=3)
100
225 mcg/kg
900 mcg/kg (N=3)
1800 mcg/kg (N=3)
80%
(N=3)
450 mcg/kg
85%
(N=3)
75
900 mcg/kg
50
88%
(N=3)
0.4
0.3
25
1800 mcg/kg
(N=3)
0
0
10
20
30
40
50
60
70
80
Day
90
100 110 120 130 140 150
84%
Phase 1 siRNA-AT3: Safety
Safety, Tolerability
§ Generally well tolerated
§ No discontinuation
§ AEs: 3 drug-related - all mild
Injection site reactions, resolved at 24 hours
One patient (45 mcg/kg) –mild transient pain
One patient (1800 mcg/kg) – mild transient erythema, pain
One patient – headache, transient
§
§
§
§
§
No SAEs related to drug
Bleeds successfully treated with standard factor replacement
No thromboembolic events or significant D-dimer increases
No anti-drug antibody (ADA) formation
Normal LFTs, CBC, platelets, fibrinogen, EKG
32
Phase I siRNA-AT3: Thrombin Generation
Thrombin Generation by % AT Lowering
250
Peak Thrombin Generation (nM)
% AT
Lowering
Peak Thrombin
nM
200
150
100
Control
110
<25%
18
25-50%
26
50-75%
47
>75%
62*
*p<0.001, compared with <25%
50
0
N=4
Healthy
Volunteers
Pasi KJ et al, ASH 2015
AT Lowering
< 25%
N=24
AT Lowering
25-50%
N=21
AT Lowering
50-75%
N=18
Patients with Hemophilia
AT Lowering
>75%
N=9
Results: With AT lowering,
thrombin generation improves
in hemophilia patients.
Phase I siRNA-AT3: Thrombin Generation
Hem A, B Inhibitor Plasma:
In children:
Increase in mean peak thrombin
generation, 22nM to 80nM
Comparable responses in
children (≤12 years) as in adults
Conclusion: With AT lowering, peak thrombin improves in inhibitor patients and children.
Kenet G et al, ASH 2015
Phase I siRNA-AT3: Bleed Events
Bleed Events by % AT Lowering
ABR Estimate, Mean (SEM)
(Bleeds Per Year)
40
30
20
10
0
Patients
ABR, Mean (SEM)
ABR, Median
AT Lowering
<25%
AT Lowering
25-50%
AT Lowering
50-75%
AT Lowering
>75%
N=24
N=21
N=18
N=9
34 ± 10
20 ± 7
14 ± 4
6±3
13
11
10
0
p<0.05
Conclusions: With AT lowering by quartile, <25% to >75%, there is reduction in ABR.
Pasi KJ, et al. Blood 2015
ABR = Annualized bleed rate
Phase I siRNA-AT3 : Bleed Events
Bleed Events: Onset and Observation Periods
Conclusion: As AT3 is lowered, bleeds decrease, from d. 0-28 (onset) to d. 29-112 (observation).
(900 mcg/kg qM)
120
70
AT
Peak Thrombin
Bleed Event
60
Factor Administration
50
80
40
60
30
40
20
20
10
0
0
0
28
Onset
Pasi KJ et al, Blood 2015
Day
56
Observation
84
Peak Thrombin (nM)
% Relative AT Activity
100
Phase 1 siRNA-AT3: Bleed Events
Bleed Events: AT3 lowering in Cohorts 1, 2, 3
Summary of Median ABR
ABR Estimate (Bleeds Per Year)
30
25
20
-85%
15
-92%
Cohorts
1-3
2&3
% AT
Lowering
85%
92%
Median
ABR
4.3
2.2
10
5
0
Cohort
1-3
Historical
On-Demand
Cohort
1-3
Onset
Cohort
1-3
Cohort
2-3
Observation
Conclusion: With progressive AT3 lowering, there is reduction in ABR.
Pasi KJ et al, Blood 2015
Phase 1 siRNA-AT3: Bleed Events
Bleed-Free Period Correlates with AT3 Lowering
-20
350
AT % Knockdown
Peak Thrombin
300
Bleed Event
Factor Administration
250
20
200
40
150
60
100
80
50
100
0
0
14
28
42
56
70
84
98
126
154
182
D-dimer
(mcg/mL)
Time (Days)
0.4
0.2
0
0
14
28
42
56
70
84
98 112 126 140 154 168 182
Time (Days)
210
Peak Thrombin (nM)
% AT Knockdown
0
Comparison: ABR in Selected Prospective Studies
50
On Demand
ABR - Median
Prophylaxis
40
30
20
10
0
These are not head-to-head studies, thus direct comparisons cannot be made.
Haemophilia 2013, N Engl J Med 2013; Blood 2014; J Thromb Haemost 2012; Haemophilia 2014; Blood 2015; ASH 2015
siRNA-AT3: Summary
Conclusions:
§ Potential new therapeutic for hemophilia, rare bleeding disorders
§ Monthly, subcutaneous administration
§ Preclinical and clinical studies support safety, preliminary efficacy
§ With AT3 lowering, thrombin generation (peak, ETP) increases
§ With AT3 lowering, bleeds (ABR) decrease
§ Open questions: how to manage trauma, surgery
§ Future studies: inhibitor patients, children, other rare bleeding disorders
40
Preclinical Studies
Clinical Studies
Alnylam, Cambridge:
A Seghal, S Barros, J Qin, T Racie,
J Hettinger, M Carioto, Y Jiang,
J Brodsky, H Prabhala, X Zhang,
H Attarwala, R Hutabarat, D Foster,
S Milstein, K Charisse, S Kuuchimanchi,
M Maier, L Nechev, P Kandasamy,
A Kel’in, J Nair, K Rajeev, M Manoharan, R
Meyers, B Sorensen, A Simon, A Akinc
London, UK:
J Pasi, S Rangarajan, D Bevan
S Austin, PChowdary
Philadelphia PA
L Invanciu, R Camire
Chapel Hill, NC
B Cooley
Lyon, France
Y Dargaud, C Negrier
Manchester UK:
Charles Hay
Southhampton UK:
Rashid Kazmi
Truro, UK:
Michael Creagh
Plovdiv, Bulgaria:
Pencho Georgiev
Sofia, Bulgaria:
Toshko Lissitchkov
Zurich, Switzerland:
Brigit Brand
Quintile, London:
Tim Mant
Alnylam,Cambridge:
A Akinc, J Chong, B Sorensen
41