Journal of Medical Microbiology and Infectious Diseases

en Inhibition of HPV18 E6/E7 Protein-Expressing HeLa Cell Proliferation Using Optimized De Novo CRISPR/Cas9 Constructs Delivered by the LL-37 Peptide Host-pathogen interactions and susceptibility factors Host-pathogen interactions and susceptibility factors Original article Original article 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. Cervical cancer, HPV18 E6/E7 oncoproteins, CRISPR-Cas9 gene editing, Cell-penetrating peptide, LL-37 peptide, Tumor suppression 278 291 http://jommid.pasteur.ac.ir/browse.php?a_code=A-10-632-1&slc_lang=en&sid=1 Niloofar Khairkhah 0000-0003-0895-9913 No Cellular and Molecular Research Center, Institute for Prevention of Non-Communicable Diseases, Qazvin University of Medical Sciences, Qazvin, Iran Azam Bolhassani azam.bolhassani@yahoo.com 0000-0001-7363-7406 Yes Department of Hepatitis, AIDS and Blood-borne Diseases, Pasteur Institute of Iran, Tehran, Iran, Pasteur Institute of Iran, Tehran, Iran Reza Najafipour rnajafipour@gmail.com 0000-0001-6042-8314 No Cellular and Molecular Research Center, Institute for Prevention of Non-Communicable Diseases, Qazvin University of Medical Sciences, Qazvin, Iran; Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran Ali Namvar 0000-0002-2739-0518 No Blood Diseases Research Center (BDRC), Iranian Comprehensive Hemophilia Care Center, Iran University of Medical Sciences (IUMS), Tehran, Iran Alireza Milani a.milani@hotmail.com 0000-0003-4931-2753 No Department of Hepatitis, AIDS and Blood-borne Diseases, Pasteur Institute of Iran, Tehran, Iran, Pasteur Institute of Iran, Tehran, Iran Elnaz Agi 0000-0003-3587-2952 No Blood Diseases Research Center (BDRC), Iranian Comprehensive Hemophilia Care Center, Iran University of Medical Sciences (IUMS), Tehran, Iran Ali Anvar 0000-0003-4914-1659 No Blood Diseases Research Center (BDRC), Iranian Comprehensive Hemophilia Care Center, Iran University of Medical Sciences (IUMS), Tehran, Iran Mohammad Sadeqh Khosravy No WHO Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran, Tehran, Iran