Fatemeh Namazi, Azam Bolhassani, Seyed Mehdi Sadat, Shiva Irani,
Volume 7, Issue 4 (10-2019)
Abstract
Introduction: The Nef accessory protein is an attractive antigenic candidate in the development of HIV-1 DNA- or protein-based vaccines. The most crucial disadvantage of DNA and protein-based vaccines is their low immunogenicity, which can be improved by cell-penetrating peptides (CPPs) as effective carrier molecules. Methods: In this study, the HIV-1 Nef protein was generated in the Escherichia coli expression system for in vitro delivery using a novel CPP, Latarcin 1 peptide, in a non-covalent manner. Also, the Histidine-rich nona-arginine peptide was utilized to transfer the HIV-1 Nef gene. The size, morphology, and zeta potential of the complexes were evaluated by scanning electron microscopy (SEM) and Zetasizer. The efficiency of cell transfection was studied using a fluorescence microscopy and flow cytometry for the DNA/CPP complexes and western blot analysis for the protein/CPP complexes. Results: The Nef protein generated in the BL21 strain migrated as a dominant band of ~30 kDa in SDS-PAGE. The SEM data confirmed the formation of stable complexes with a size below 200 nm. MTT assay demonstrated that the complexes did not represent any considerable cytotoxic effect compared to untreated HEK-293T cells. The results of fluorescence microscopy, flow cytometry, and western blotting revealed that the Nef DNA and protein constructs could be significantly transfected into HEK-293T cell line using these CPPs. Conclusion: These data suggest that the Histidine-rich nona-arginine peptide and Latarcin 1 peptide as CPPs can be considered as a promising approach in the development of the HIV-1 vaccine for gene or protein delivery.
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.
