CRISPR脂样纳米片递送系统的构建及其在肝豆状核变性基因治疗中的研究

如何开发安全有效的递送载体以及提高基因编辑效率是CRISPR基因编辑技术应用于临床所面临的重大挑战。前期我们已经证明，对非病毒脂样纳米颗粒进行结构优化可显著提高其递送效率，实现核酸药物、CRISPR系统在体内的高效递送。另外，我们还发现工程化改造CRISPR-Cas12a mRNA和crRNA可最大化CRISPR-Cas12a系统的基因编辑效率。据此，本项目拟在前期基础上，制备既保留均苯脂样结构又引入有序片状结构的非病毒脂样纳米片；同时，将前期开发的工程化改造方案应用于可特异性靶向小鼠肝豆状核变性突变基因CRISPR-Cas12a系统；最后，构建小鼠肝豆状核变性模型，利用自组装脂样纳米片体内递送经工程化改造的CRISPR-Cas12a系统及同源修复模板，并评价该递送系统在基因治疗中的安全有效性。本项目有望开发新型的CRISPR脂样纳米片递送系统，为肝豆状核变性遗传性疾病的治疗提供新的策略。

How to develop safe and effective delivery systems and enhance the efficiency of genome editing remain huge challenges for CRISPR therapeutic applications in the clinic. Previously, we had demonstrated that optimization of lipid-like nanoparticles significantly increased their delivery efficiency, achieving robust nucleic acids and CRISPR delivery in vivo. Besides, we found that engineering CRISPR-Cas12a mRNA and crRNA was able to maximize genome editing efficiency. On the basis of our previous studies, the aim of this project is to construct non-viral lipid-like nanosheets containing the featured lipid-like structures and the introduced lamellar structures. Meanwhile, the engineering strategies that we previously established will be applied to the CRISPR-Cas12a system targeting the specific site of mouse Atp7b gene according to the PAM sequence adjacent to the hot spot mutation of the established Wilson Disease mouse model. Lastly, homology-directed repair templates will be co-delivered in the established Wilson Disease mouse model with the engineered CRISPR-Cas12a system by the self-assembled lipid-like nanosheets delivery system for Wilson Disease, and the therapeutic efficiency of such delivery system will be assessed in gene therapy. Overall, the novel lipid-like nanosheets merit further development for non-viral CRISPR delivery, and provide potential therapeutic strategies for Wilson Disease.