Systemic delivery of specific and efficient CRISPR/Cas9 system targeting HPV16 oncogenes using LL-37 antimicrobial peptide in C57BL/6 mice

Human papillomavirus (HPV) type 16 is the most common sexually transmitted virus related to cervical cancer. Among different types of advanced novel therapies, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-mediated gene editing holds great promise for cancer treatment. In this research, optimal gRNA sequences targeting HPV16 E5, E6, E7, and p97 promoter for CRISPR/Cas9-mediated genome editing were designed by in silico prediction. After cloning, delivery of the recombinant vectors into C3, TC1 and HeLa tumor cells was evaluated by Lipofectamine 2000, and LL-37 antimicrobial peptide. Then, the levels of cell cycle proteins (p21, p53, and Rb) were investigated after treatment by western blot analysis. Finally, C57BL/6 mice were inoculated with C3 tumor cells, and treated with recombinant vectors and cisplatin. Based on the tumor size reduction and IHC results, the E6 + E7-treated group with a high percentage of cleaved caspase-3 positive cells (45.75%) and low mitotic index of 2-3 was determined as the best treatment among other groups. Moreover, the potential of LL-37 peptide to overcome the CRISPR/Cas9 delivery challenge was shown for the first time. Overall, our study suggests that the CRISPR/Cas9-mediated gene editing of pre-existing tumors is effective, specific and nontoxic, and the outlook for precise gene therapy in cancer patients is very bright.

Automated summary

Human papillomavirus (HPV) type 16 is the most common sexually transmitted virus associated with cervical cancer. In this study, optimal gRNA sequences targeting HPV16 E5, E6, E7, and p97 promoter for CRISPR/Cas9-mediated genome editing were designed. The E6 + E7-treated group showed the best treatment efficacy, with a high percentage of cleaved caspase-3 positive cells (45.75%) and a low mitotic index of 2-3. Additionally, the potential of LL-37 peptide to overcome the CRISPR/Cas9 delivery challenge was demonstrated for the first time. Overall, the study suggests that CRISPR/Cas9-mediated gene editing is an effective, specific, and nontoxic treatment for pre-existing tumors, and the future of precise gene therapy in cancer patients is very promising.