Unlocking Therapeutic Potential: Enhanced shRNA delivery with Tat peptide in the Human Respiratory Syncytial Virus Treatment

Purpose: This research investigated the development of short hairpin RNA (shRNA) molecules designed to target specific regions of the HRSV M and F genes. The study aimed to assess the therapeutic potential of these shRNAs and evaluate the effectiveness of Tat peptide-mediated delivery in enhancing their functionality. Methods: To investigate the therapeutic potential of short hairpin RNA against HRSV, we acquired isolates from pediatric patients experiencing respiratory illness. These isolates were then cultured in human epidermoid carcinoma cells (HEp-2). To target the M and F genes of HRSV, we constructed plasmids expressing shRNAs. We further investigated the Tat peptide as a facilitator for shRNA plasmid delivery. The cytotoxicity of ribavirin, shRNA constructs, and control agents was assessed using the MTT assay. Next, we compared the transfection efficiency of Tat peptide-mediated shRNA delivery with that of lipofectamine 3000™. Finally, real-time PCR was employed to quantify HRSV replication in the treated cells. Results: Our study successfully demonstrated that Tat peptide-mediated delivery of shRNA plasmids significantly suppressed the expression of the M and F genes of HRSV compared to lipofectamine 3000™. This suppression was evident in both short-term experiments and scenarios involving stable shRNA expression. Furthermore, the combination of ribavirin with shRNA treatment resulted in a substantial reduction in viral load. Notably, the most pronounced antiviral effect was observed when both shRNAs were employed simultaneously. Conclusion: Our findings suggest that Tat peptide-mediated delivery of shRNA plasmids holds significant potential for achieving stable suppression of HRSV genes. This approach warrants further investigation as a potential gene therapy strategy for HRSV. By demonstrating promising results in vitro, this study highlights the need for future in vivo studies to comprehensively evaluate the therapeutic potential of this approach in a clinical setting.