αIIb启动子调控血小板靶向表达ADAMTS13治疗CRISPR/Cas9构建的TTP小鼠模型的实验研究

血栓性血小板减少性紫癜（TTP）是ADAMTS13基因突变或其自身抗体导致的血栓性微血管病。目前主要治疗方法是血浆置换与激素等，但相当部分患者仍无法治愈。我们课题组前期报道了αIIb启动子调控FVIII或FIX可使其靶向表达于血小板，并诱导对该凝血因子产生免疫耐受从而治疗血友病。我们研究还发现该启动子可调控血小板表达非凝血蛋白OVA并诱导对其产生免疫耐受。基于此，我们推测该启动子可调控血小板表达ADAMTS13蛋白，并诱导对该蛋白产生免疫耐受从而治疗TTP。为验证该假说，我们前期研究已发现αIIb启动子调控可使ADAMTS13蛋白储存于人巨核细胞Dami细胞内，并用CRISPR/Cas9构建出ADAMTS13基因敲除的杂合子小鼠。在此基础上，研究αIIb启动子调控血小板靶向表达ADAMTS13蛋白，并诱导对该蛋白产生免疫耐受，达到基因治疗TTP的目的，为TTP的治疗提供新的策略及理论依据。

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy characterized by loss of ADAMTS13 function which can be due to either genetic deficiency or inhibitory autoantibodies to ADAMTS13. Currently, the main treatment is plasma exchange and glucocorticoid and so on, but many patients still cannot be cured completely. Our research group has previously demonstrated that targeting FVIII or FIX expression to platelets under control of the αIIb promoter can not only result in coagulant protein storage in platelet, but also induce coagulant protein-specific immune tolerance for hemophilia treatment. We also found that targeting non-coagulant protein, ovalbumin (OVA), expression to platelets under control of the αIIb promoter can also result in OVA storage in platelet and induce OVA-specific immune tolerance. On the basis of these results, we hypothesize that targeting ADAMTS13 expression to platelets under control of the αIIb promoter can cure TTP by resulting in ADAMTS13 storage in platelet and inducing ADAMTS13-specific immune tolerance. To test our hypothesis, we have found that ADAMTS13 expression under control of the αIIb promoter can result in ADAMTS13 storage in human megakaryocytic cell line Dami in our previous study, and have constructed heterozygous ADAMTS13 knockout mice by using CRISPR/Cas9 technology. Based on the data and mouse model, we are going to study platelet-targeted gene transfer of ADAMTS13 under control of the αIIb promoter can result in targeting ADAMTS13 expression to platelets and induce ADAMTS13-specific immune tolerance for TTP gene therapy. This research will provide the novel strategy and theoretical basis for TTP treatment.