基于腺嘌呤单碱基编辑系统的肝豆状核变性ATP7B基因点突变修复研究

肝豆状核变性（HLD），又称Wilson病，是一种以铜代谢障碍为表现的常染色体隐性遗传病，其发病主要由于致病基因ATP7B序列突变造成其编码的铜转运蛋白功能减弱或丧失。目前主要通过间断性的驱铜治疗维持体内的负铜平衡，但该方式需终身治疗且所使用药物存在一定的不良反应，因此开发新的Wilson病治疗手段显得尤为必要。基于CRISPR的单碱基编辑系统可实现基因组特定碱基类型的转换，可被应用于对单核苷酸致病突变的逆转。申请人前期研究筛选发现国内人群高频突变位点ATP7B-p.P992L突变可通过腺嘌呤单碱基编辑器(ABE)靶向逆转。本项目拟通过RNA-seq、WGS等技术评估ABE逆转突变的RNA和DNA脱靶，并在序列人源化小鼠模型及人体遗弃胚胎中评估ABE逆转突变对于Wilson病治疗效果。本研究将明确基于单碱基编辑器逆转致病突变位点的可行性和安全性，为建立Wilson病治疗新策略提供理论依据。

Hepatolenticular degeneration (HLD) , also called as Wilson disease, is an autosomal recessive genetic disease characterized by copper metabolism disorder. Its pathogenesis is predominantly due to the weakening or loss of the function of copper transporter encoded by ATP7B mutation. At present, the negative copper balance in the body is maintained mainly through intermittent copper-repellent therapy, but this method requires life-long treatment and the drugs used to have certain adverse reactions, so it is particularly necessary to develop new Wilson disease treatment methods. CRISPR-based base editing systems can implement the transformation of specific base types in the genome and can be applied to the reversal of single nucleotide pathogenic mutations. The applicant's previous study found that ATP7B-p.P992L mutation which is high frequency mutation sites in Chinese population could be targeted for reversal through adenine base editor (ABE). In this project, we will evaluate the RNA and DNA off-target by RNA-seq and WGS, and evaluate the therapeutic effect of ABE reversal mutation on Wilson disease in sequence humanized mouse models and human abandoned embryos. This study will clarify the feasibility and safety of reversing pathogenic mutation sites based on ABE base editor and provide a theoretical basis for the establishment of new therapeutic strategies for Wilson disease.