基于CRISPR/Cas9基因编辑技术治疗肝豆状核变性的实验研究

Inherited rare diseases are a group of serious life-threatening diseases that caused by genetic lesions in the genome, most of which do not have effective treatment. Gene therapy is a promising strategy to cure these diseases. Recent rapid advances in engineered nuclease-mediated genome editing, especially CRISPR/Cas9, provide efficient and convenient tools for gene therapy, which may make it possible to correct disease-related gene mutations or put a wild type gene into the genome. Gene-editing based therapy therefore hopefully can overcome short-comings of traditional gene therapy in that this strategy leads to permanent expression of therapeutic genes. However, as to Wilson’s disease, whose pathogenic gene, ATP7B, is larger than 4 kb, current gene-editing strategy is not able to fit the needs of complicated mutation spectrum. To overcome this challenge, we previously developed a micro-homologue mediated end joining technology base on CRISPR/Cas9 system, which we demonstrated to be able to induce integration of large DNA fragment into safe harbor of genome. In this proposal, we plan to treat Wilson’s disease with two different strategies, with fully considering most patients’ mutation spectrum, current editing strategy and our own novel technology. In the first strategy, we plan to use our own micro-homologue mediated end joining technology to integrate wildtype ATP7B expression cassette into genome safe harbor. The other strategy we use long arm homologue directed recombination to paste artificial fusion exon 8-21 of ATP7B downstream of the endogenous intron 7 of ATP7B locus. Meanwhile, we will test these two strategies both in mouse in vivo and in cells derived from human patient. Hopefully, these strategies will maintain ATP7B activity to such a level that requires no additional medicine to keep ideal concentration of copper ions. We hope our study will generate a promising therapeutic strategy against Wilson’s disease with our own intellectual property rights.

遗传性罕见病是当前严重危害人民健康的重要疾病之一;基因治疗有望根治此类疾病。近年来以CRISPR/Cas9为代表的基因编辑技术发展迅速，可在染色体水平纠正或替代致病基因，克服传统基因治疗不能长期表达的弊端,为基因治疗开辟新的领域。然而对于肝豆状核变性，由于致病基因ATP7B太大、突变位点复杂，目前的基因编辑策略难以形成普适性治疗方案。前期研究中,为解决这一问题，我们发展了基于CRISPR/Cas9的微同源末端连接技术，可以介导大片段DNA定点插入基因组安全位点。本申请根据临床ATP7B突变特征、现有编辑技术和项目组前期基础，设计两种针对性基因编辑方案：基于CRISPR/Cas9的微同源末端连接介导的全基因插入和长片段同源重组介导的外显子替代。同时在肝豆状核变性小鼠和病人IPS细胞中测试这两种方案，希望能够恢复ATP7B活性至理想水平，最终形成一个有应用前景、具有自主知识产权的治疗方案。

本项目针对一种肝脏相关遗传病，肝豆状核变性，利用小鼠疾病模型进行基于基因编辑技术的基因治疗研究。肝豆状核变性(Wilson’s disease)是一种由铜代谢异常引起的常染色体隐性遗传性疾病，铜排泄减少导致铜在肝、中枢神经系统（CNS）、角膜等脏器沉着，损害器官功能从而引发疾病。目前对肝豆状核变性的治疗方案，主要是通过锌制剂、螯合剂等药物治疗，旨在减少铜离子的吸收以及促进铜离子的排泄；药物治疗方案常伴随有严重的副作用，部分病人依从性差，且需要终身服药等缺点；亟需开发新的治疗方案。近年来，以CRISPR/Cas9为代表的基因编辑技术发展迅速，可在染色体水平纠正或替代致病基因，克服传统基因治疗不能长期表达的弊端,为基因治疗开辟新的领域。在本项目中，我们针对中国人群肝豆状核变性致病基因ATP7b的突变特征，设计一系列基于基因编辑技术的治疗方案，结合AAV病毒、非病毒导入体系，我们在体内验证了部分方案的有效性。首先，在体外筛选、优化了基于Cas9介导的同源重组编辑方案；并在小鼠体内验证了该方案的有效性。同时，我们也结合基因编辑领域的最新研究进展，针对小鼠模型ATP7b突变特点，开发了一系列新型的碱基编辑工具，包括在国际上首次提出了基于Cas9内部插入脱氨酶的碱基编辑器构建方案，基于sgRNA骨架结构优化改造的碱基编辑器，通用型的AAV-碱基编辑器导入方案，以及活性可接受安全药物控制的可诱导碱基编辑器等等。在此基础上，我们还验证了AAV兼容的编辑工具导入体系，并最终通过体内实验证明了基因编辑工具在遗传性肝豆状核变性基因治疗中的效果；最后，我们还开发了一系列具有肝脏靶向的非病毒导入系统，其中部分已经在动物模型中观察到治疗效果。以上研究成果为进一步开发基于基因编辑技术的肝脏相关遗传性疾病的基因治疗方案研究奠定了坚实的基础。

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