利用CRISPR/Cas9系统同源性修复蛋白C基因突变治疗血栓性疾病

Thrombosis and its complications is a common disease with highest morbidity and mortality worldwide. As a result, it has a strong impact on social development and economic construction. There is an ever-changing and complex balance between procoagulant system and anticoagulant system. Inadequate antithrombotic therapy will give rise to recurrent thrombosis, but the excessive treatment will cause hemorrhage. Therefore, the safe and effective treatment of the thrombosis diseases is a matter of urgent concern. Protein C is synthesized in the hepatocytes and circulated in the blood as a natural anticoagulant factor. Hereditary protein C deficiency caused by mutations in protein C gene (PROC) is one of the most common thrombotic diseases. Through the previous research, we discovered that PROC mutations (PROC c.565C>T和PROC c.574_576del) are the most common thrombosis-causing mutations among Chinese population. Recent years, the CRISPR/Cas9 system has been demonstrated to have the function of correct mutations based on efficient, locus-specific homologous recombination, and has been used as genetic therapy in some diseases caused by monogenic mutations. In this study, we intended to use CRISPR/Cas9 system to correct the PROC mutations in human cells, and then monitored the transcription, synthesis and secretion, as well as the anticoagulation function of Protein C. This strategy may one day allow correction of disease-causing mutations in thrombosis patients.

血栓形成及其并发症已成为世界范围内致残率和致死率最高的疾病之一，严重影响社会发展和经济建设。人体促凝系统与抗凝系统的动态平衡复杂多变，抗栓治疗措施不足时血栓易复发，抗栓药物使用过量时易出血，相关疾病的安全、有效治疗成为亟需解决的医学难题。蛋白C是体内重要的天然抗凝因子，蛋白C基因突变导致的蛋白C缺乏症是遗传性易栓症最常见的病因之一。本课题组通过前期研究发现，蛋白C基因的2种突变（c.565C>T和c.574_576del）是中国人群血栓形成的常见危险因素。CRISPR/Cas9基因编辑系统通过sgRNA与DNA的特异性结合将Cas9蛋白靶向至特定基因区域实现对基因的编辑，已成功应用于单基因突变性遗传病的基因治疗研究。本项目拟利用sgRNA/Cas基因编辑技术（gRNA/Cas9）在活体细胞同源性修复蛋白C基因突变，从根源上移除致病突变，实现血栓性疾病的防治。

血栓形成及其并发症已成为世界范围内致残率和致死率最高的疾病之一，严重影响社会发展和经济建设。人体促凝系统与抗凝系统的动态平衡复杂多变，抗栓治疗措施不足时血栓易复发，抗栓药物使用过量时易出血，相关疾病的安全、有效治疗成为亟需解决的医学难题。蛋白C是体内重要的天然抗凝因子，蛋白C基因突变导致的蛋白C缺乏症是遗传性易栓症最常见的病因之一。本课题组通过前期研究发现，蛋白C基因的2种突变（c.565C>T和c.574_576del）是中国人群血栓形成的常见危险因素。CRISPR/Cas9基因编辑系统通过sgRNA与DNA的特异性结合将Cas9蛋白靶向至特定基因区域实现对基因的编辑，已成功应用于单基因突变性遗传病的基因治疗研究。本项目成功构建sgRNA/Cas基因编辑体系在模型细胞体系中实行同源性修复蛋白C基因突变，体外实验证实我们构建的sgRNA/Cas基因编辑体系对PROC c.565C>T及PROC c.574_576del的稳转蛋白C表达体系HDR修复效率分别为15%和9.9%。对细胞培养上清及细胞裂解液行WB及Elisa试验证实，PC:Ag含量较突变型明显增加，对细胞培养上清浓缩液行蛋白C活性检测及TGT试验证实蛋白C抗凝活性修复。有望通过此项技术，从根源上移除致病突变，实现血栓性疾病的防治。