基于CRISPR/Cas9技术的苯丙酮尿症患胎来源的羊水iPSCs基因突变修复的研究

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism caused by a deficiency in the hepatic enzyme phenylalanine hydroxylase (PAH). Dietary restriction of phenylalanine remains to be the mainstay treatment for PKU but it remains difficult due to progressive decrease in adherence to diet and the presence of neurocognitive defects despite therapy. Gene therapy seems to be an experimental, yet promising approach for PKU treatment in the near future. Human amniotic fluid-derived stem cells-induced pluripotent stem cells (hAF-iPSCs), a novel source of stem cells, could be used for genomic engineering and cellular gene therapy. CRISPR/Cas9, has been recently applied into genomic editing in various species and has been proved to be an effective tool for genomic engineering. The mutation p.Arg243Gln of PAH gene is the most prevalent mutation in Chinese PKU patients. In the present study, we established hAF-iPSCs from PKU patients, and precisely correct the p.Arg243Gln homozygous mutant cells to normal cells via the use of CRISPR/Cas9 system, and further differentiate them into hepatocytes both in vitro and in vivo experiments. Accomplishment of this project would construct the techniques and methods for constructing hAF-iPSCs from PKU patients. Most importantly, it may provide a theoretical and technological basis for the clinical gene therapy of PKU as well as enlightenments for other genetic disorders’ gene therapy.

苯丙氨酸羟化酶（PAH）基因突变所致的苯丙酮尿症（PKU）是新生儿期最常见的氨基酸代谢病，低苯丙氨酸饮食是目前主要的治疗方法，但部分患儿治疗后仍有智力发育障碍。编辑修复突变的PAH基因是PKU治疗的方向。患者来源的羊水诱导多能干细胞（hAF-iPSCs）提供理想的疾病细胞模型，CRISPR/Cas9技术可以精准高效地编辑修复特定基因，其有效性已在多种单基因遗传病研究中被证实。本研究拟建立PKU患胎来源的hAF-iPSCs，利用CRISPR/Cas9系统对PAH基因p.Arg243Gln纯合突变的hAF-iPSCs进行基因组编辑修复，进一步诱导分化其为肝脏细胞，检测PAH活性，同时观察基因修复后的hAF-iPSCs用于肝脏人源化小鼠模型的效果。本研究期望建立PKU患胎来源的hAF-iPSCs，基因编辑修复后诱导iPSCs获得治疗细胞，为下一步开展PKU临床基因治疗提供理论和技术支持。

课题以苯丙酮尿症（PKU）患胎（携带PAH基因p.Arg241Cys和p.Ala434Asp变异）来源的羊水为研究对象，首先建立PKU患胎来源的诱导多能干细胞（PKU-hAF-iPSCs），构建体外PKU疾病细胞模型，为后续实验奠定基础。采用基于同源修复的CRISPR/Cas9技术对PKU-hAF-iPSCs的p.Arg241Cys变异进行编辑修复，进一步诱导其分化为肝脏细胞。实验结果：（1）本课题建立了一套成熟和稳定的PKU患胎羊水细胞来源iPSCs诱导体系。采用小分子联合miRNA法通过电转转染诱导羊水细胞产生iPSCs，经染色体核型分析、STR连锁分析和Sanger测序证实hAF-iPSCs与患胎羊水细胞遗传背景一致。PKU-hAF-iPSCs克隆碱性磷酸酶染色呈阳性，免疫荧光染色表达干细胞多能性标志物Oct4,SSEA4,TRA-1-60及TRA-1-81，体内分化实验结果显示PKU-hAF-iPSCs在体内具有向三个胚层的细胞多向分化潜能，证实所构建的PKU-hAF-iPSCs可作为PKU基因治疗研究的疾病细胞模型。（2）采用CRISPR/Cas9系统成功修复hAF-iPSCs细胞中p.Arg241Cys变异。Sanger 测序方法证实p.Arg241Cys 位点成功修复。目标序列捕获高通量测序技术检测易感脱靶位点及其附近区域基因序列均无脱靶发生。（3）基因修复后的hAF-iPSCs体外成功诱导分化为肝细胞。诱导后的肝细胞表达AFP、ALB和HNF4A，在人诱导多能干细胞定向肝细胞的诱导过程中，干细胞特异的多潜能基因OCT3/4和NANOG 逐渐丧失，而肝细胞特异性基因ASGPR1、CYP1A1和CYP7A1表达水平明显升高，F7、G6PC、TAT、ALB、FOXA2和HNF4A也在不同程度上表达。基因编辑后hAF-iPSCs用于肝脏人源化小鼠模型效果的工作正在进行。综合上述结果，本研究为下一步开展PKU临床基因治疗提供理论和技术支持。项目负责人及参与人在研究期间发表SCI论文4篇，中文核心论文7篇，获得省级及厅级科技成果2项。培养硕士研究生4名。项目投入经费20万元，支出12.0964万元。各项支出与预算基本相符。剩余经费万元，剩余经费计划用于本项目研究后续支出。

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