Division of Medical Biotechnology, Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Center for Cardiovascular Regeneration, Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, 77030, TX, USA.
Virol J. 2024 Sep 30;21(1):235. doi: 10.1186/s12985-024-02519-3.
Cell-penetrating peptides (CPPs) are effective for delivering therapeutic molecules with minimal toxicity. This study focuses on the use of penetratin, a well-characterized CPP, to deliver a DNA vector encoding short hairpin RNA (shRNA) targeting the respiratory syncytial virus (RSV) F gene into infected cells. RSV is known to cause severe lower respiratory infections in infants and poses significant risks to immunocompromised individuals and the elderly. We evaluated the antiviral efficacy of the penetratin-shRNA complex by comparing its ability to inhibit RSV replication and induce apoptosis with ribavirin treatment.
Penetratin-shRNA complexes were prepared at different ratios and analyzed using gel retardation assays, dynamic light scattering, and zeta potential measurements. The complexes were tested in HEp-2 and A549 cells for transfection efficiency, cytotoxicity, viral load, and apoptosis using plaque assays, real-time reverse transcription-polymerase chain reaction (RT-PCR), DNA fragmentation, propidium iodide staining, and caspase 3/7 activation assays.
The gel shift assay determined that a 20:1 CPP-to-shRNA ratio was optimal for effective complexation, resulting in particles with a size of 164 nm and a zeta potential of 8.7 mV. Transfection efficiency in HEp-2 cells was highest at this ratio, reaching up to 93%. The penetratin-shRNA complex effectively silenced the RSV F gene, reduced viral titers, and decreased DNA fragmentation and apoptosis in infected cells.
Penetratin effectively delivers shRNA targeting the RSV F gene, significantly reducing viral load and preventing apoptosis without toxicity. This approach surpasses Lipofectamine and shows potential for future therapeutic interventions, especially when combined with ribavirin, against RSV infection.
细胞穿透肽（CPPs）可有效递送治疗分子，而毒性最小。本研究专注于使用 penetratin，一种经过充分研究的 CPP，将携带针对呼吸道合胞病毒（RSV）F 基因的短发夹 RNA（shRNA）的 DNA 载体递送至感染细胞。RSV 已知会导致婴儿严重的下呼吸道感染，对免疫功能低下个体和老年人构成重大风险。我们通过比较 penetratin-shRNA 复合物抑制 RSV 复制和诱导凋亡的能力与利巴韦林治疗，评估了 penetratin-shRNA 复合物的抗病毒功效。
以不同比例制备 penetratin-shRNA 复合物，并通过凝胶阻滞分析、动态光散射和zeta 电位测量进行分析。使用噬斑分析、实时逆转录-聚合酶链反应（RT-PCR）、DNA 片段化、碘化丙啶染色和 caspase 3/7 激活测定，在 HEp-2 和 A549 细胞中测试复合物的转染效率、细胞毒性、病毒载量和凋亡。
凝胶迁移分析确定，20:1 的 CPP 与 shRNA 比例最有利于有效复合物形成，导致粒径为 164nm、zeta 电位为 8.7mV 的颗粒。在该比例下，HEp-2 细胞中的转染效率最高，可达 93%。penetratin-shRNA 复合物可有效沉默 RSV F 基因，降低病毒滴度，并减少感染细胞中的 DNA 片段化和凋亡。
penetratin 可有效递送至 RSV F 基因的 shRNA，显著降低病毒载量并预防凋亡，而无毒性。这种方法优于 Lipofectamine，并显示出针对 RSV 感染的未来治疗干预的潜力，特别是与利巴韦林联合使用时。
