Proof-of-concept Studies for siRNA-mediated Gene Silencing for Coagulation Factors in Rat and Rabbit

Chen, Zhu; Luo, Bin; Cai, Tian-Quan; Thankappan, Anil; Xu, Yiming; Wu, Wei; DiMuzio, Jillian; Lifsted, Traci; DiPietro, Marty; Disa, Jyoti; Ng, Bruce; Leander, Karen; Clark, Seth; Hoos, Lizbeth; Zhou, Yuchen; Jochnowitz, Nina; Jachec, C.; Szczerba, Peter; Gindy, Marian E.; Strapps, Walter; Sepp‐Lorenzino, Laura; Seiffert, Dietmar; Lubbers, Laura S.; Tadin‐Strapps, Marija

doi:10.1038/mtna.2014.75

Cited by 22 publications

(25 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…siRNA design, synthesis, and formulation, and dosing Methods of siRNA oligo design, synthesis, in-vitro screening, formulation, and dosing were as reported before [23,24]. Briefly, 84 siRNAs were designed against rat FXII mRNA (NM_001014006) and synthesized at Merck.…”

Section: Fxii Knockout Ratsmentioning

confidence: 99%

“…The core target sequences of the lead FXII siRNA and the nontargeting control siRNA (nt control) used in this report are 5 0 -ATACTGCTTTCTAAGTAAC-3 0 and 5 0 -GTCGCCTTATATCGGTCGA-3 0 , respectively. For in-vivo studies, the FXII siRNA and the nt control siRNA were formulated into cationic lipid nanoparticles (LNPs), dosed in the buffer of 10 mmol/l TRIS, 70 mmol/l NaCl, 5 wt% Sucrose, pH 7.5, and administered to male SpragueDawley (SD) rats (Charles River Laboratories, weighted approximately 300 g) via a bolus tail vein injection (2 ml/ kg) as described before [23,24].…”

Section: Fxii Knockout Ratsmentioning

confidence: 99%

“…Analysis of mRNA knockdown from liver samples Methods for RNA extraction from liver and RT-PCR analysis were as described previously [23,24]. All Taqman probes and primers were purchased from Applied Biosystems (Foster City, California, USA) as prevalidated sets for rat (sequences not shown but available upon request.…”

Section: Fxii Knockout Ratsmentioning

confidence: 99%

See 2 more Smart Citations

Factor XII full and partial null in rat confers robust antithrombotic efficacy with no bleeding

Cai¹,

Wu²,

Shin

et al. 2015

Blood Coagulation &Amp; Fibrinolysis

Self Cite

View full text Add to dashboard Cite

This report aims at exploring quantitatively the relationship between FXII inhibition and thromboprotection. FXII full and partial null in rats were established via zinc finger nuclease-mediated knockout and siRNA-mediated knockdown, respectively. The rats were subsequently characterized in thrombosis and hemostasis models. Knockout rats exhibited complete thromboprotection in both the arteriovenous shunt model (∼100% clot weight reduction) and the FeCl3-induced arterial thrombosis model (no reduction in blood flow), without any increase in cuticle bleeding time compared with wild-type control rats. Ex-vivo aPTT and the ellagic acid-triggered thrombin generation assay (TGA) exhibited anticoagulant changes. In contrast, ex-vivo PT or high tissue factor-triggered TGA was indistinguishable from control. Rats receiving single doses (0, 0.01, 0.03, 0.1, 0.3, 1 mg/kg) of FXII siRNA exhibited dose-dependent knockdown in liver FXII mRNA and plasma FXII protein (95 and 99%, respectively, at 1 mg/kg) at day 7 post dosing. FXII knockdown was associated with dose-dependent thromboprotection (maximal efficacy achieved with 1 mg/kg in both models) and negligible change in cuticle bleeding times. Ex-vivo TGA triggered with low-level (0.5 μmol/l) ellagic acid tracked best with the knockdown levels and efficacy. Our findings confirm and extend literature reports of an attractive benefit-to-risk profile of targeting FXII for antithrombotic therapies. Titrating of FXII is instructive for its pharmacological inhibition. The knockout rat is valuable for evaluating both mechanism-based safety concerns and off-target effects of FXII(a) inhibitors. Detailed TGA analyses will inform on optimal trigger conditions in studying pharmacodynamic effects of FXII(a) inhibition.

show abstract

Section: Fxii Knockout Ratsmentioning

confidence: 99%

Section: Fxii Knockout Ratsmentioning

confidence: 99%

See 1 more Smart Citation

Factor XII full and partial null in rat confers robust antithrombotic efficacy with no bleeding

Cai¹,

Wu²,

Shin

et al. 2015

Blood Coagulation &Amp; Fibrinolysis

Self Cite

View full text Add to dashboard Cite

show abstract

“…2015). In vivo siRNA qualification was done in 8 weeks old male Sprague Dawley rats weighing approximately 144–170 g purchased from Charles River Laboratories.…”

Section: Methodsmentioning

confidence: 99%

Preclinical and translational evaluation of coagulation factor IXa as a novel therapeutic target

Ankrom

Wood

et al. 2016

Pharmacology Res & Perspec

View full text Add to dashboard Cite

The benefits of novel oral anticoagulants are hampered by bleeding. Since coagulation factor IX (fIX) lies upstream of fX in the coagulation cascade, and intermediate levels have been associated with reduced incidence of thrombotic events, we evaluated the viability of fIXa as an antithrombotic target. We applied translational pharmacokinetics/pharmacodynamics (PK/PD) principles to predict the therapeutic window (TW) associated with a selective small molecule inhibitor (SMi) of fIXa, compound 1 (CPD1, rat fIXa inhibition constant (Ki, 21 nmol/L) relative to clinically relevant exposures of apixaban (rat fXa Ki 4.3 nmol/L). Concentrations encompassing the minimal clinical plasma concentration (C min) of the 5 mg twice daily (BID) dose of apixaban were tested in rat arteriovenous shunt (AVS/thrombosis) and cuticle bleeding time (CBT) models. An I max and a linear model were used to fit clot weight (CW) and CBT. The following differences in biology were observed: (1) antithrombotic activity and bleeding increased in parallel for apixaban, but to a lesser extent for CPD1 and (2) antithrombotic activity occurred at high (>99%) enzyme occupancy (EO) for fXa or moderate (>65% EO) for fIXa. translational PK/PD analysis indicated that noninferiority was observed for concentrations of CPD1 that provided between 86% and 96% EO and that superior TW existed between 86% and 90% EO. These findings were confirmed in a study comparing short interfering (si)RNA‐mediated knockdown (KD) modulation of fIX and fX mRNA. In summary, using principles of translational biology to relate preclinical markers of efficacy and safety to clinical doses of apixaban, we found that modulation of fIXa can be superior to apixaban.

show abstract

“…Finally, although plasma kallikrein inhibition or deficiency only confers modest thromboprotection (Bird et al, 2012;Chen et al, 2015), one could not exclude the possibility that some off-target activity on plasma kallikrein from compound 1 may have contributed to efficacy (based on ∼6-fold selectivity of FXIa over plasma kallikrein; Table 1). Thus, we evaluated the ex vivo plasma kallikrein activity for our dose-dependent study on MES (Supplemental Fig.…”

Section: Fxia Inhibitor Reduces Cerebral Microembolic Signalsmentioning

confidence: 99%

Inhibition of Factor XIa Reduces the Frequency of Cerebral Microembolic Signals Derived from Carotid Arterial Thrombosis in Rabbits

Wang¹,

Kurowski²,

Wu³

et al. 2016

J Pharmacol Exp Ther

Self Cite

View full text Add to dashboard Cite

Factor XI (FXI) is an integral component of the intrinsic pathway of the coagulation cascade and plays a critical role in thrombus formation. Because its role in the pathogenesis of cerebral microembolic signals (MES) is unclear, this study used a potent and selective small molecule inhibitor of FXIa, compound 1, to assess the effect of FXI blockade in our recently established preclinical model of cerebral MES induced by FeCl 3 injury of the carotid artery in male New Zealand White rabbits. Ascending doses of compound 1 were evaluated simultaneously for both carotid arterial thrombosis by a Doppler flowmeter and MES in the middle cerebral artery by a transcranial Doppler. Plasma drug exposure and pharmacodynamic responses to compound 1 treatment were also assessed. The effective dose for 50% inhibition (ED 50 ) of thrombus formation was 0.003 mg/kg/h compound 1, i.v. for the integrated blood flow, 0.004 mg/kg/h for reduction in thrombus weight, and 0.106 mg/kg/h for prevention of MES. The highest dose, 3 mg/kg/h compound 1, achieved complete inhibition in both thrombus formation and MES. In addition, we assessed the potential bleeding liability of compound 1 (5 mg/kg/h, i.v., .1250-fold ED 50 levels in arterial thrombosis) in rabbits using a cuticle bleeding model, and observed about 2-fold (not statistically significant) prolongation in bleeding time. Our study demonstrates that compound 1 produced a robust and dose-dependent inhibition of both arterial thrombosis and MES, suggesting that FXIa blockade may represent a novel therapeutic strategy for the reduction in MES in patients at risk for ischemic stroke.

show abstract

Proof-of-concept Studies for siRNA-mediated Gene Silencing for Coagulation Factors in Rat and Rabbit

Cited by 22 publications

References 38 publications

Factor XII full and partial null in rat confers robust antithrombotic efficacy with no bleeding

Factor XII full and partial null in rat confers robust antithrombotic efficacy with no bleeding

Preclinical and translational evaluation of coagulation factor IXa as a novel therapeutic target

Inhibition of Factor XIa Reduces the Frequency of Cerebral Microembolic Signals Derived from Carotid Arterial Thrombosis in Rabbits

Contact Info

Product

Resources

About