Inhibition of HPV18 E6/E7 Protein-Expressing HeLa Cell Proliferation Using Optimized De Novo CRISPR/Cas9 Constructs Delivered by the LL-37 Peptide

Introduction: CRISPR/Cas-mediated gene editing has emerged as a transformative therapeutic modality for targeting oncogenic pathways in cancer. This technology enables precise disruption of oncogenic processes, such as tumor cell migration and invasion, and facilitates targeted tumor eradication. This study employed CRISPR/Cas9-mediated genome editing to disrupt the HPV18 E6 and E7 oncogenes, which are critical drivers of tumorigenesis in HPV-associated cancers. Methods: Optimized single-guide RNA (sgRNA) sequences were designed to target the HPV18 E6 and E7 oncogenes, along with the p105 promoter region, for CRISPR/Cas9-mediated genome editing. The sgRNA sequences were cloned into CRISPR/Cas9 expression vectors. HPV18-positive HeLa cells, were transfected in vitro with the recombinant vectors to assess gene editing efficiency. For the in vivo evaluation, C57BL/6 mice bearing HeLa-derived tumors received intravenous injections of LL-37 peptide-complexed recombinant vectors. The therapeutic efficacy of this approach was quantitatively compared to cisplatin treatment. Results: The dual E6/E7-targeted group exhibited a statistically significant reduction in tumor volume compared to all other groups, including the single E6-targeted group, the single E7-targeted group, the cisplatin-treated group, and the untreated control group (P < 0.05). LL-37 peptide demonstrated efficient delivery of CRISPR/Cas9 vectors into HeLa tumor cells, with an optimal nitrogen-to-phosphate (N/P) ratio of 5: 1, achieving high transfection efficiency without systemic toxicity. Conclusion: These findings establish CRISPR/Cas9-mediated gene editing as a potent therapeutic strategy for HPV-associated tumors and highlight LL-37 as a promising non-viral delivery platform for CRISPR/Cas9 constructs. This study is the first to demonstrate the in vivo efficacy of multiplexed sgRNA delivery targeting HPV18 oncogenes in a preclinical model.