利用CRISPR/Cas9靶向剪辑HPV16和PD1/PD-L1基因抑制宫颈癌的作用及机制研究

Current there still lack of effective treatment for cervical cancer. The breakthrough of genome editing technology and combination with immune regulation therapy in recent years brought the new light on cervical treatment. Our previous study demonstrated that the CRISPR/Cas9 specific to HPV16 oncogenes including targeting the Promoter-, E6-, E7-transcript could inhibit the cancer cell growth. Using a tumor transplant model, we demonstrated that the designed CRISPR/Cas9 could inhibit tumor growth. The application of CRISPR/Cas9 targeting HR-HPV key oncogenes in cervical cancer, as a new treatment strategy, has important scientific and clinical significance. In this study, we will screen to select the gRNAs could make mutations directly target to the oncogene, E6/E7 gene and the immune checkpoint regulator PD1/PD-L1 gene, respectively. Subsequently, the selected gRNA target different genes would be combined to knockdown the oncogene and immune checkpoint regulator gene simultaneously into the mice model of cervical cancer transplanted with TC-1 cell line. The efficacy would be evaluated to explore whether the combination can exert synergetic effect in cervical cancer. This study also could provide clues for exploring new strategy of other tumors.

目前对危害女性健康的中晚期浸润癌仍缺乏有效的治疗手段。高效的靶向基因剪接技术和肿瘤的联合免疫基因治疗已成为改善恶性肿瘤不良预后的候选手段。申请者前期利用CRISPR/Cas9技术成功修饰HPV16 E6、E7和启动子基因，引起HPV16+宫颈癌细胞体外的生长能力降低，裸鼠皮下移植瘤的生长受到显著抑制，这种方法能否成为宫颈癌的候选新方法值得进一步研究。本项目拟通过改进CRISPR/Cas9系统，分别修饰高危型HPV16 E6、E7和PD-1/PD-L1基因，观察单独修饰能否抑制宫颈癌的发生发展；采取联用修饰的高危型HPV16 E6、E7和PD1/PD-L1等多个分子的策略，探索联合免疫基因治疗在HPV16+宫颈癌治疗中的可行性和有效性。旨在为靶向高危型HPV16致癌基因和免疫调点基因的联合免疫基因治疗宫颈癌的策略提供实验数据，并为其它病毒源性肿瘤的治疗措施提供借鉴。

目前对危害女性健康的中晚期浸润癌仍缺乏有效的治疗手段。高效的靶向基因剪接技术和肿瘤的联合免疫基因治疗已成为改善恶性肿瘤不良预后的候选手段。我们通过改进CRISPR/Cas9系统，分别修饰高危型HPV16 E6/E7和PD1/PD-L1基因 ，后采取联用修饰高危型HPV16 HPV16 E6/E7和PD1/PD-L1多分子的策略，探索了联合免疫基因治疗在HPV16宫颈癌治疗中的可行性和有效性；同时探索CRISPR/Cas9-HPV和顺铂联合治疗达到更好治疗宫颈癌的效果；接下来，我们通过构建精确定位前列腺癌并靶向PLK1的适配子脂质体-CRISPR/Cas9复合物，可有效靶向前列腺癌细胞并有效抑制前列腺癌的发生及发展，为我们构建靶向剪接高危型HPV16的脂质体-.CRISRP/Cas9的制备提供了基础。最后，基于前期工作基础，构建靶向HPV16 E6/E7的CRISPR/Cas9系统，观察对HPV16阳性宫颈癌细胞生存的影响，利用脂质体剂型的生物相容性,稳定性，靶向性及大剂量药物装载率，继而制备靶向HPV16 E6/E7的阳离子脂质体，实现了CRISPR/Cas9-HPV基因药物高效体内靶向输送。为研制治疗HPV16阳性宫颈癌的基因药物奠定基础。