探索靶向KRAS突变结直肠癌的多重基因编辑治疗及其对肿瘤侵润T细胞的影响

近50%结直肠癌患者携带KRAS突变，是预后不良独立危险因素。因KRAS/MEK/MAPK及PI3K/AKT通路存在交互作用且KRAS突变促进免疫抑制，目前尚缺乏有效分子靶向或免疫治疗手段，是结直肠癌治疗的难点。基于CRISPR/Cas9系统的多重基因编辑技术可对突变或有交互作用的多个基因同时高效敲除，为KRAS突变结直肠癌提供有效基因治疗策略。本项目拟构建靶向KRAS及其下游通路的多基因编辑载体，在KRAS突变结直肠癌细胞中筛选最佳抑瘤靶点组合；应用接头蛋白及抗病毒衣壳蛋白抗体技术改造腺病毒优化基因编辑递送系统；在KRAS突变PDX模型、肝转移模型及免疫系统人源化PDX模型中检测多基因编辑治疗结直肠癌的效果及安全性。并研究靶向KRAS的多基因编辑调节结直肠癌侵润T细胞的分子机制，探索基因编辑治疗与CAR-T细胞疗法联用的疗效，为KRAS突变结直肠癌的生物治疗提供新方法、思路和实验数据。

Nearly 50% cases of colorectal cancer (CRC) carry KRAS driver mutations, which is an independent risk factor for poor prognosis. The interaction between KRAS/MEK/MAPK and PI3K/AKT pathways and the immuno-suppression induced by KRAS mutation lead to the lack of effective targeted therapy or immunotherapy for KRAS mutated CRC. The third generation genome editing system, CRISPR/Cas9, could effectively deplete multiple mutated genes or interacted genes at the same time, therefore provide a potential therapeutic strategy for KRAS mutated CRC. In this project, we plan to construct and test multiplex gene-editing vectors targeting KRAS and several downstream genes in CRC cells, and obtain the best target combination whose depletion suppresses tumor growth most efficiently. The engineered bi-specific adaptor and anti-HEXON scFv will be applied to modify the adenovirus delivery system for multiplex gene editing. In addition, the therapeutic effect and safety of multiplex gene editing will be analyzed in KRAS mutant PDX model, liver metastasis model and humanized PDX model of CRC. In addition, the influence of multiplex gene therapy on tumor-infiltrating T cells, and the combining effects of multiplex gene therapy and CAR-T cell therapy will be both explored in KRAS mutated CRC. This project will be helpful to provide new technique, strategy and experimental data to the biotherapy of CRC carrying KRAS driver mutations.