Targeting Factor 12 (F12) with a novel RNAi delivery platform as a prophylactic treatment for Hereditary Angioedema (HAE)
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Stacey Melquist1, Darren Wakefield1, Holly Hamilton1, Qili Chu1, Aaron Almeida1, Lauren Almeida1, Megan Walters1, Jessica Montez1, Julia Hegge1, Jason Klein1, Christine Hazlett1,
Stephanie Bertin1, Tracie Milarch1, Edie Doss1, Rachael Schmidt1, Linda Goth1, Sheryl Ferger1, David Rozema1, James Hamilton2, David Lewis1 and Steven Kanner1
RESEARCH POSTER PRESENTATION DESIGN © 2015
www.PosterPresentations.com
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36
Day
• Dose-dependent F12 knockdown
• 4 mpk dose showed >1 log10 knockdown at nadir (Day 15), with
80% knockdown for 1 month
• ARC-F12 = AD01520 + ARC-EX1 dosed at 1:1 ratio
•
•
•
Transverse cut of tail vein, monitor time to clotting
Factor 7 (F7) is a key clotting factor in extrinsic pathway, and a F7-targeted RNAi
trigger is used as a positive control in this study
8 mpk ARC-F12 or 8 mpk F7 trigger + 8 mpk ARC-EX1, single IV dose seven
days prior to challenge
10
-1
10
0
10
1
[RNAi trigger], nM
ACKNOWLEDGEMENTS
The authors would like to thank Aaron Andersen, Crystal Klas, Molly Zeller, Leah Staley, Felicia Beauprey and
Christina Furseth for technical assistance.
This publication was made possible in part by Grant Number P51 RR000167 from the National Center for
Research Resources (NCRR), a component of the National Institutes of Health (NIH), to the Wisconsin National
Primate Research Center, University of Wisconsin-Madison. This research was conducted in part at a facility
constructed with support from Research Facilities Improvement Program grant numbers RR15459-01 and
RR02141-01. This publication’s contents are solely the responsibility of the authors and do not necessarily
represent the official views of NCRR or NIH.
F12 knockdown
10
Inhibition of thrombus formation
1
0.1
0.01
40
30
0.1
0.01
250
80
200
Seconds
1
100
60
40
150
100
20
50
0
0
20
10
0
0.001
F7 Activity
Induced by exposure of carotid artery to ferric chloride
Measure time to blood flow occlusion, thrombi
Clinically relevant indicator of physiological response to F12 knockdown
Treatment with 4 mpk ARC-F12 seven days prior to challenge
Bleeding Time
F7 Levels
10
-F
12
10
-2
FeCl3 thromboembolism mouse model
C
10
-3
F12 Levels
-F
12
22
C
Day
15
A
R
8
A
R
10
-4
• Dramatic increase in occlusion times observed in mice receiving
ARC-F12 is similar to results obtained in FXII-/- mice5
90
10
0
11
0
12
0
13
0
80
70
60
50
40
30
20
aPTT
1.2
1.0
60
0.8
0.6
40
0.4
80
20
Days
F7
4
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36
-F
12
29
C
22
Sa
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0.0
10 -5
0
10
Bleeding risk assessment in mouse model
A
R
15
F12
80
90
10
0
11
0
12
0
13
0
0.4
F7
8
1.4
0.0
0.6
0.0
4
4 mpk ARC-F12, 5xq4w dosing, IV injection
0.2
F12 (normalized)
0.5
Hour
70
0.8
0.2
0.0
•
•
•
•
Pharmacodynamic effect of F12 knockdown in NHP
6
60
1.0
-F
12
1.0
18.6
69.5
81.5
133.4
145.0
261.0
4
50
0.4
1.2
C
1.5
EC50 (pM)
2
Saline
1 mpk
2 mpk
4 mpk
A
R
Relative Rluc-FXII
Expression
2.0
RNAi
Trigger ID
AD00466
AD00469
AD00468
AD00467
AD00470
AD00474
0
•
Summary of EC50 values (in vitro screening)
AD00469
0.0
40
0.6
Controls
• Huh7 cells transfected with RNAi triggers at 1 nM or 0.1 nM
• Data expressed as Renilla/firefly luciferase ratio
0.001
F7
F12 RNAi trigger ID, AD#
0.01
0.5
• ~90% F12 knockdown achieved after first dose, even greater
knockdown following subsequent doses
• >80% inhibition of F12 levels maintained between dosing
• No changes in toxicity markers (clin chem, CBC) after dosing
30
0.8
0.4
0
10
F12 (normalized)
F12 (normalized)
Saline
2 mpk AD00900
2 mpk AD01520
0.2
Top candidate
RNAi triggers
0.1
1.0
1.4
1.0
0.6
20
AD01520
1.2
0.8
Days
Normalized F12
F12 RNAi trigger dosed 1:1 with ARC-EX1, single dose, IV injection
1.4
1
Saline
ARC-F12
• ARC-F12 treatment significantly reduced edema from carrageenan
challenge (p<0.001)
• Reduction in swelling in ARC-F12 treated rats is similar to those
seen in rats treated with kallikrein-targeted antibody7
0.1nM
•
 baseline volume (mL)
0.2
Iteration of RNAi trigger modification
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.2
1.5
10
• Single high dose of chol-RNAi triggers showed significant and
sustained knockdown of F12 levels
• AD00900 (AD00469 family) exhibited >1 log10 knockdown of F12
for >1 month
Two-point in vitro screen of F12 RNAi triggers
2 mpk
4 mpk
1.2
0.0
Day
RESULTS
1.4
Change in Paw Volume
-F
12
Normalized F12
0.4
Time to Occlusion (min)
A. et al. (2004) J. Thromb. Thrombolysis 17:139–143
Koster A. et al. (1994) Br. J. Haematol. 87:422–424
3 Zeerleder S. et al. (1999) Thromb. Haemost. 82:1240–1246
4 Pauer, H. U. et al. (2004) Thromb. Haemost. 92:503
5 Renne, T. et al. (2005) J. Exp. Med. 202:271
6 Figure modified from Albert-Weissenberger, C. et al. (2014) Front. Cell Neurosci. 8:345
7 Kenniston, J.A. et al. (2014) J. Biol. Chem. 289:23596–23608
0.6
F12 knockdown
2 or 4 mpk ARC-F12, 5xq4w dosing, IV injection
aPTT (seconds)
REFERENCES
0.8
Lead candidate
“ARC-F12”
DPC™
RNAi trigger
Peptide
• Amphipathic
• Canonical siRNA
backbone
peptide for
or other format
endosomal escape
• Liver-tropic
• Peptide amines
targeting ligand
“masked” with pH- NAG
(eg. cholesterol)
(N-acetyl
labile moiety,
galactoseunmasked in
amine)
endosome
• Targeted to liver
with NAG
CDM Bond
• Co-injected IV with (masking
Cholesterol
RNAi trigger
chemistry)
• For ARC-F12,
DPC™ and RNAi trigger do NOT form a
DPC™ is ARC-EX1
complex, they are separately targeted to
the liver
1.0
14 8
kininogen
angioedema
1.2
0.0
1nM
Dynamic PolyConjugate (DPC™) for liver delivery
2
Non-GLP
toxicology
Sa
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contact
(intrinsic)
coagulation
cascade
FXIIa
bradykinin
BK-B2
receptor
1 Girolami
Efficacy testing in diseaserelevant animal models
FXII6
prekallikrein
kallikrein
In vivo screening
in NHP
F12 (normalized)
ARC-F12
SAR on lead candidate chol-RNAi triggers: synthesis of modified cholRNAi triggers and in vivo screening
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468
474
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AAT
Aha1
--
–
F12-deficient mice:
•
viable and fertile4
•
do not show bleeding defects4,5
•
protected from thromboembolic disease (stroke, pulmonary
embolism)5
F12 deficiency in humans is not associated with either bleeding or
thrombotic disorders1,2,3
Relative Renilla
Luciferase Expression
–
•
•
Inflammation caused by subcutaneous injection of l carrageenan
Induced by multiple mediators of inflammation/edema including pro-inflammatory
cytokines, histamine, prostaglandins, and others in addition to bradykinin
Treatment with 8 mpk ARC-F12 seven days prior to carrageenan challenge
Saline
AD00897
AD00898
AD00899
AD00900
AD00901
AD00902
1.4
F12 inhibition is genetically validated
•
•
C
Synthesis of chol-RNAi triggers and in vivo screening in WT mice
Evaluating ARC-F12 in multi-dose NHP studies
Relative F12 ELISA
Serum F12 levels measured by ELISA, values were normalized to pre-treatment
and saline control.
Mice were dosed at 8 mg/kg (mpk) F12 RNAi trigger/ARC-EX1, single dose, IV
injection
A
R
•
RESULTS
Carrageenan-induced paw edema in rats
F12 (normalized)
RNAi trigger synthesis and in vitro screening
•
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Key component of contact activation pathway (thrombosis) and kininkallekrein system (angioedema)
•
Cleavage of FXII by kallikrein generates FXIIa: FXIIa generates
FXIa (coagulation) and kallikrein (angioedema)
•
Predominantly expressed in the liver; circulates in plasma
RESULTS
Initial F12 chol-RNAi trigger in vivo screen
-F
12
–
Bioinformatic selection of RNAi trigger sequences specific for F12 – filter
to identify rodent/human/non human primate (NHP) cross-reactive triggers
C
Factor XII (F12)
RESULTS
ARC-F12 screening funnel
A
R
INTRODUCTION
Research Corporation, Madison, WI 53711 2Arrowhead Research Corporation, Pasadena, CA 91101
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• Bleeding times do not differ between saline and ARC-F12 treated
mice
• Lack of bleeding observed in ARC-F12 treated mice is similar to
results obtained in FXII-/- mice5
• Increased activated partial thromboplastin time (aPTT) in ARC-F12treated Cynomolgus monkeys is observed in parallel with
decreased serum F12 levels
• No changes in Prothrombin Time (PT)
• aPTT is elongated in individuals with F12 deficiency
CONCLUSIONS
• Screening of in vitro-active F12 RNAi triggers in wild type mice
identified those triggers that exhibited significant and sustained
knockdown of serum F12 levels
• SAR studies allowed identification of a lead RNAi trigger that
demonstrated >97% maximum knockdown after a single 2 mg/kg
dose
• Studies in mice showed reduced FeCl3-induced thromboembolism
consistent with the expected physiological effects of F12
knockdown
• High dose ARC-F12 treatment showed no evidence of increased
bleeding in mice
• Carrageenan-induced paw edema is reduced in ARC-F12 treated
rats
• A multi-dose study in NHPs showed >90% sustained knockdown of
serum F12 levels without toxicity for monthly doses up to 4 mg/kg
ARC-EX1
• NHPs in these studies showed changes in aPTT, a physiological
response consistent with F12 deficiency