Inhibition of HPV۱۸ E۶/E۷ Protein-Expressing HeLa Cell Proliferation Using Optimized De Novo CRISPR/Cas۹ Constructs Delivered by the LL-۳۷ 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/Cas۹-mediated genome editing to disrupt the HPV۱۸ E۶ and E۷ oncogenes, which are critical drivers of tumorigenesis in HPV-associated cancers. Methods: Optimized single-guide RNA (sgRNA) sequences were designed to target the HPV۱۸ E۶ and E۷ oncogenes, along with the p۱۰۵ promoter region, for CRISPR/Cas۹-mediated genome editing. The sgRNA sequences were cloned into CRISPR/Cas۹ expression vectors. HPV۱۸-positive HeLa cells, were transfected in vitro with the recombinant vectors to assess gene editing efficiency. For the in vivo evaluation, C۵۷BL/۶ mice bearing HeLa-derived tumors received intravenous injections of LL-۳۷ peptide-complexed recombinant vectors. The therapeutic efficacy of this approach was quantitatively compared to cisplatin treatment. Results: The dual E۶/E۷-targeted group exhibited a statistically significant reduction in tumor volume compared to all other groups, including the single E۶-targeted group, the single E۷-targeted group, the cisplatin-treated group, and the untreated control group (P < ۰.۰۵). LL-۳۷ peptide demonstrated efficient delivery of CRISPR/Cas۹ vectors into HeLa tumor cells, with an optimal nitrogen-to-phosphate (N/P) ratio of ۵: ۱, achieving high transfection efficiency without systemic toxicity. Conclusion: These findings establish CRISPR/Cas۹-mediated gene editing as a potent therapeutic strategy for HPV-associated tumors and highlight LL-۳۷ as a promising non-viral delivery platform for CRISPR/Cas۹ constructs. This study is the first to demonstrate the in vivo efficacy of multiplexed sgRNA delivery targeting HPV۱۸ oncogenes in a preclinical model.

کلیدواژه ها:

Cervical cancer ، HPV۱۸ E۶/E۷ oncoproteins ، CRISPR-Cas۹ gene editing ، Cell-penetrating peptide ، LL-۳۷ peptide ، Tumor suppression