动态超分子调控型CRISPR/Cas9基因编辑递送系统的构建及应用

小分子调控型CRISPR/Cas9基因编辑系统可以提高基因编辑的特异性和安全性，因而展现出极大的应用前景；但小分子和Cas9质粒分步递送方式复杂低效，且存在药物释放的不可控性，因而已成为制约其作为治疗工具发展的瓶颈问题。本项目构建具有肿瘤微环境响应性、可同步递送小分子和Cas9质粒的动态超分子聚合物载体，利用超分子与小分子的主客体识别作用及聚合物和Cas9质粒静电作用，实现两者的同步递送。同时，通过载体的化学结构调控，阐明其不同结构参数与转染效率和细胞毒性的关系，实现小分子和Cas9质粒的高效低毒递送；此外，利用肿瘤细胞里高表达的精胺/谷胱甘肽，实现小分子和Cas9质粒的响应性释放，完成小分子对Cas9活性的可控基因编辑。最后，通过对递送载体的功能化修饰，实现系统对肿瘤的精准基因治疗。此研究从给药系统角度为小分子调控型CRISPR/Cas9基因编辑系统实现体内精准治疗提供了新的思路。

Precision control of CRISPR/Cas9 using small molecules has shown great application potentials towards gene therapy due to it can improve the specificity of gene editing and the safety of gene therapy. However, the two-step delivery of small molecules and Cas9 plasmid is complicated and poor efficiency, which become a major bottleneck for the clinical treatment of CRISPR/Cas9 genome editing technologies. This project aims to develop dynamic supramolecular polymeric carrier that can have the co-delivery of small molecules and Cas9 plasmid and exhibit the stimuli-responsive in tumor microenvironment. In this delivery system, the co-delivery of small molecules and Cas9 plasmid have achieved by host-guest interaction with supramolecular and small molecules as well as electrostatic interaction with polymer and Cas9 plasmid. We propose to elucidate the relationship between the carrier chemical structure and genome editing efficiency as well as cytotoxicity in order to optimize the delivery system of CRISPR/Cas9 with high efficiency and low cytotoxicity. In cancer cells, overexperssion of spermine and GSH can trigger the stimuli-responsive release of small molecules and Cas9 plasmid , and achieve the precision control of CRISPR/Cas9 and provide the controllable gene editing. Finally, the functional modification have been introduced on carrier and improve the in vivo precision therapy of CRISPR/Cas9 on cancer therapy. Thus, the proposed project provides a new strategy for in vivo precision control of CRISPR/Cas9 using small molecules from the drug delivery point of view.