Reyhane Najafi, Azam Bolhassani, Maryam Montazeri, Elnaz Agi,
Volume 11, Issue 3 (9-2023)
Abstract
Introduction: Developing potent therapeutic vaccines against human papillomaviruses (HPVs) is crucial for the effective management of various HPV-associated cancers. DNA-based vaccines are attractive due to their safety, stability, and capacity to elicit a targeted immune response against specific antigens. Heat shock proteins (HSPs) can enhance the efficacy of DNA vaccines when used as adjuvants. In this study, we created a recombinant DNA molecule by fusing the HPV16 e7 gene with either the hspB1 or hsp27 gene and assessed its expression in a eukaryotic cell line. Methods: Initially, we constructed a recombinant eukaryotic expression vector by inserting the hsp27-e7 fusion gene into the pcDNA3.1 (-) vector. The concentration and purity of the sample were evaluated using NanoDrop spectrophotometry. We cultured human embryonic kidney 293T (HEK-293T) cells in RPMI 1640 medium and transfected them with the pcDNA3.1-hsp27-e7 construct using Lipofectamine 2000 transfection reagent. After 48 hours, we assessed the expression of the Hsp27-E7 fusion protein by western blotting using an anti-E7 monoclonal antibody. Results: We successfully subcloned the hsp27-e7 fusion gene into the pcDNA3.1 (-) vector, and enzymatic digestion confirmed a distinct ~975 bp band on an agarose gel. The concentration and purity of the recombinant DNA vector in a 10 mL culture were measured to be 210 ng/µL and 1.86, respectively. Furthermore, the expression of the Hsp27-E7 fusion protein in HEK-293T cells was confirmed by Western blot analysis, which detected a distinct band of approximately 38 kDa. Conclusion: Our in vitro findings demonstrate successful expression of the DNA construct encoding the hsp27-e7 gene, which can be utilized as a DNA vaccine for future in vivo investigations.
Niloofar Khairkhah, Azam Bolhassani, Reza Najafipour, Ali Namvar, Alireza Milani, Elnaz Agi, Ali Anvar, Mohammad Sadeqh Khosravy,
Volume 12, Issue 4 (12-2024)
Abstract
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.
