表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKIs）对EGFR基因突变型的非小细胞肺癌（NSCLC）治疗有效率较高，但几乎所有用TKIs治疗的病人最终都会由于基因突变而产生抗药性。因此针对基因突变这个根本问题开发针对EGFR突变型的基因编辑药物对于肺癌的个性化治疗具有重要意义。本项目根据递送重组Cas9蛋白进行基因编辑可以避免使用质粒带来严重的脱靶及伦理问题这一研究热点，结合实验室前期筛选获得的可高效递送蛋白的超电荷聚多肽，以递送Cas9蛋白特异性编辑NSCLC中的突变EGFR基因为目标，构建多种超电荷聚多肽与Cas9融合蛋白，并将分子内二硫键环肽融入其中，实现Cas9蛋白的高效递送、选择性释放及体内基因编辑，旨在获得安全、高效、稳定为一体的超电荷聚多肽和Cas9融合蛋白。同时首次针对NSCLC中突变的EGFR基因为靶点进行基因编辑，为肿瘤的个性化治疗提供新的技术基础和研究思路。

Although tyrosine kinase inhibitors (TKIs) have proven to have high efficacy in EGFR-mutant non-small-cell type lung cancer (NSCLC), nearly all patients unfortunately ultimately develop acquired resistance to the drugs. This acquired drug resistance often results from a secondary EGFR mutation. Rather than continuing to repeat a cycle of inducing new resistance mutations by additional targeted drug therapies, the development of entirely novel approaches seems appropriate. We propose a new approach-personalized molecular surgery-to correct or destroy the mutated EGFR using CRISPR/Cas9-mediated genome-editing technology. Genome editing through the delivery of Cas9 protein reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Here, we construct the supercharged polypeptides that can effectively deliver the protein into cells. This project focuses on the establishment of a highly efficient, low toxicity and steady supercharged polypeptide-Cas9 fusion proteins. We project to use this fusion protein to destroy the EGFR gene in EGFR-mutant NSCLC. We will construct various polypeptide-Cas9 fusion proteins. Meanwhile, we design an in vivo cleavable disulfide linker in recombinant fusion proteins. Our goal is to achieve highly efficient delivery and selective release of Cas9 protein. This project also first proposes the strategy for molecular surgical therapy to treat EGFR-mutant lung cancer with CRISPR/Cas9 technology. Altogether, this research will provide a new technology and strategy for the personalized treatment of tumor cells.