背景:凝血因子V(FV)缺乏症是一种罕见的出血性疾病,通常使用新鲜冰冻的血浆进行治疗。无义突变的患者可能会对通读剂的治疗产生反应。
目的:为了研究F5p.Arg1161Ter突变,导致几个患者严重的FV缺乏,可以接受通读疗法。
方法:在F5p.Arg1161Ter纯合患者中评估F5mRNA和蛋白表达。五种具有不同作用机制的通读剂,即G418、ELX-02、PTC-124、2,6-二氨基嘌呤(2,6-DAP)和Amlexanox(AMX),在突变的体外和离体模型中进行了测试。
结果:F5p.Arg1161Ter纯合子患者显示残留的F5mRNA和功能性血小板FV,指示可检测的自然读数水平。与野生型相比,用FV-Arg1161TercDNA转染的COS-1细胞表达0.7%FV活性。用0-500μMG418,ELX-02和2,6-DAP剂量依赖性地增加FV活性高达7.0倍,3.1倍和10.8倍,分别,而PTC-124和AMX(单独或联合使用)无效。这些发现通过用处理细胞的条件培养基重构的FV耗尽的血浆中的凝血酶生成测定得到证实。除ELX-02外的所有化合物均显示一定程度的细胞毒性。F5p.Arg1161Ter纯合患者的离体分化巨核细胞,FV免疫染色为阴性,用所有五种连读剂治疗后转为阳性。值得注意的是,他们还能够内化G418或2,6-DAP拯救的突变体FV,这将需要在体内维持关键的血小板FV池。
结论:这些发现为F5p.Arg1161Ter突变的复读介导的解救提供了体外和离体的原理证明。
BACKGROUND: Coagulation factor V (FV) deficiency is a rare bleeding disorder that is usually managed with fresh-frozen plasma. Patients with nonsense mutations may respond to treatment with readthrough agents.
OBJECTIVE: To investigate whether the F5 p.Arg1161Ter mutation, causing severe FV deficiency in several patients, would be amenable to readthrough therapy.
METHODS: F5 mRNA and protein expression were evaluated in a F5 p.Arg1161Ter-homozygous patient. Five readthrough agents with different mechanisms of action, i.e. G418, ELX-02, PTC-124, 2,6-diaminopurine (2,6-DAP), and Amlexanox, were tested in in vitro and ex vivo models of the mutation.
RESULTS: The F5 p.Arg1161Ter-homozygous patient showed residual F5 mRNA and functional platelet FV, indicating detectable levels of natural readthrough. COS-1 cells transfected with the FV-Arg1161Ter cDNA expressed 0.7% FV activity compared to wild-type. Treatment with 0-500 μM G418, ELX-02, and 2,6-DAP dose-dependently increased FV activity up to 7.0-fold, 3.1-fold, and 10.8-fold, respectively, whereas PTC-124 and Amlexanox (alone or in combination) were ineffective. These findings were confirmed by thrombin generation assays in FV-depleted plasma reconstituted with conditioned media of treated cells. All compounds except ELX-02 showed some degree of cytotoxicity. Ex vivo differentiated megakaryocytes of the F5 p.Arg1161Ter-homozygous patient, which were negative at FV immunostaining, turned positive after treatment with all 5 readthrough agents. Notably, they were also able to internalize mutant FV rescued with G418 or 2,6-DAP, which would be required to maintain the crucial platelet FV pool in vivo.
CONCLUSIONS: These findings provide in vitro and ex vivo proof-of-principle for readthrough-mediated rescue of the F5 p.Arg1161Ter mutation.