Chen Zhu, Luo Bin, Cai Tian-Quan, Thankappan Anil, Xu Yiming, Wu Weizhen, DiMuzio Jillian, Lifsted Traci, DiPietro Marty, Disa Jyoti, Ng Bruce, Leander Karen, Clark Seth, Hoos Lizbeth, Zhou Yuchen, Jochnowitz Nina, Jachec Christine, Szczerba Peter, Gindy Marian E, Strapps Walter, Sepp-Lorenzino Laura, Seiffert Dietmar A, Lubbers Laura, Tadin-Strapps Marija
Thrombosis, Cardiometabolic Diseases, Merck Sharp & Dohme Corp, Kenilworth, New Jersey, USA.
Pharmacology (West Point), Merck Sharp & Dohme Corp, West Point, Pennsylvania, USA.
Mol Ther Nucleic Acids. 2015 Jan 27;4(1):e224. doi: 10.1038/mtna.2014.75.
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.
本研究旨在确定将小干扰RNA（siRNA）靶向大鼠和兔子凝血级联反应的可行性，这两种动物是研究血栓形成和止血常用的物种。通过体外筛选，鉴定出能使大鼠血浆前激肽释放酶和兔子凝血因子X（FX）的mRNA敲低超过90%的siRNA。一种用于体内递送siRNA的基于可电离氨基脂质的脂质纳米颗粒（LNP）制剂，在给药后第7天，在这两种动物中均表现出耐受性，且对凝血性无明显影响。前激肽释放酶siRNA-LNP和FX siRNA-LNP均导致肝脏中靶基因mRNA的剂量依赖性和选择性敲低，单次注射siRNA-LNP后第7天，最大敲低率超过90%。在大鼠动静脉分流血栓形成模型中，血浆前激肽释放酶的敲低与血栓重量适度减轻相关，且角质层出血时间未增加。兔子体内FX的敲低伴随着体外凝血时间延长。结果符合对两个靶点的预期，并首次证明了通过全身递送可电离LNP制剂包裹的siRNA在大鼠中靶向凝血因子的可行性，更广泛地说，在兔子中靶向感兴趣基因的可行性。
