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Proof-of-concept Studies for siRNA-mediated Gene Silencing for Coagulation Factors in Rat and Rabbit
Zhu Chen1, Bin Luo2, Tian-Quan Cai3
1Thrombosis, Cardiometabolic Diseases, Merck Sharp & Dohme Corp, Kenilworth, New Jersey, USA.
Abstract:
The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target the coagulation cascade in rat and rabbit, two commonly used species for studying thrombosis and hemostasis. siRNAs that produced over 90% mRNA knockdown of rat plasma prekallikrein and rabbit Factor X (FX) were identified from in vitro screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day 7 postdosing in both species. Both prekallikrein siRNA-LNP and FX siRNA-LNP resulted in dose-dependent and selective knockdown of target gene mRNA in the liver with maximum reduction of over 90% on day 7 following a single dose of siRNA-LNP. Knockdown of plasma prekallikrein was associated with modest clot weight reduction in the rat arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. Knockdown of FX in the rabbit was accompanied with prolongation in ex vivo clotting times. Results fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat, and, more broadly, targeting a gene of interest in rabbit, via systemic delivery of ionizable LNP formulated siRNA.
Insights
This study shows that lipid nanoparticle (LNP) delivered short interfering RNA (siRNA) can effectively reduce target genes in rats and rabbits. This demonstrates a feasible method for gene targeting in thrombosis and hemostasis research.
Area of Science:
- Biochemistry
- Pharmacology
- Molecular Biology
Background:
- The coagulation cascade is crucial for hemostasis and thrombosis.
- Targeting specific coagulation factors offers potential therapeutic strategies.
- Developing effective delivery systems for gene silencing agents is essential.
Purpose of the Study:
- To establish the feasibility of delivering short interfering RNA (siRNA) targeting the coagulation cascade in rats and rabbits.
- To evaluate the efficacy and safety of an ionizable amino lipid-based lipid nanoparticle (LNP) formulation for in vivo siRNA delivery.
- To demonstrate the potential for targeting coagulation factors via systemic delivery of LNP-formulated siRNA.
Main Methods:
- In vitro screening to identify siRNAs targeting rat plasma prekallikrein and rabbit Factor X (FX) with >90% mRNA knockdown.
- Characterization of an ionizable amino lipid-based LNP formulation for siRNA delivery in rats and rabbits.
- In vivo administration of prekallikrein siRNA-LNP and FX siRNA-LNP to assess dose-dependent gene knockdown in the liver.
- Evaluation of the impact of siRNA-LNP on coagulation parameters using the rat arteriovenous shunt thrombosis model and ex vivo clotting time assays in rabbits.
Main Results:
- Identified siRNAs achieving >90% mRNA knockdown for rat plasma prekallikrein and rabbit Factor X in vitro.
- The LNP formulation was well-tolerated and did not significantly affect coagulability at day 7 postdosing in either species.
- Systemic administration of siRNA-LNPs resulted in dose-dependent and selective target gene mRNA knockdown in the liver (>90% reduction).
- Prekallikrein knockdown in rats led to modest clot weight reduction without increasing bleeding time; FX knockdown in rabbits prolonged ex vivo clotting times.
Conclusions:
- Ionizable LNP-formulated siRNA enables effective and selective gene knockdown of coagulation factors in rats and rabbits.
- This study demonstrates the feasibility of targeting coagulation factors in rats and genes of interest in rabbits through systemic siRNA delivery.
- The findings support the potential of LNP-siRNA technology for developing novel therapeutics in thrombosis and hemostasis.
