目的:已经开发了蜂毒素及其衍生物来支持有效的基因传递系统。它们促进内体释放的能力增强了基于纳米颗粒的基因治疗的递送。然而,其在病毒载体方面的潜在应用尚未受到太多关注。因此,我们希望通过蜂毒素类似物优化rAAV载体,以提高rAAV在肝癌细胞中的转导效率,并探讨蜂毒素类似物对rAAV的作用机制。
方法:将多种源自蜂毒素的肽插入到重组腺相关病毒载体中的衣壳蛋白的第VIII环中。将这些携带gfp或fluc基因的载体在人胚肾293(HEK293T)细胞中进行定量聚合酶链反应测定和转导测定,以研究载体生产和基因递送的效率。此外,通过不同培养细胞的体外转导和对C57BL/6小鼠的体内尾静脉给药,检查了特定p5RHH-rAAV载体递送基因的能力。最后,通过使用rAAV2细胞内生命周期每个阶段的药理学抑制剂,探索了载体介导的转导机制的复杂细节.
结果:从现有文献中鉴定出76种蜂毒素相关肽。其中,发现CMA-3、p5RHH和aAR3显著抑制rAAV2载体粗裂解物的转导。p5RHH-rAAV2载体不仅有效转导rAAV潜能细胞系,而且有效转导先前认为对rAAV具有抗性的细胞系。机械上,巴弗洛霉素A1,一种液泡内体酸化抑制剂,完全抑制p5RHH-rAAV2载体介导的转基因表达。最重要的是,p5RHH-rAAV8载体也增加了C57BL/6小鼠体内的肝转导。
结论:将蜂毒素类似物掺入rAAV衣壳导致rAAV介导的转基因表达的显著改善。虽然进一步的修改仍然是一个感兴趣的领域,我们的研究大大拓宽了蜂毒素在病毒载体介导的基因传递中的药理学前景.
OBJECTIVE: Melittin and its derivative have been developed to support effective gene delivery systems. Their ability to facilitate endosomal release enhances the delivery of nanoparticle-based gene therapy. Nevertheless, its potential application in the context of viral vectors has not received much attention. Therefore, we would like to optimize the rAAV vector by Melittin analog to improve the transduction efficiency of rAAV in liver cancer cells and explore the mechanism of Melittin analog on rAAV.
METHODS: Various melittin-derived peptides were inserted into loop VIII of the capsid protein in recombinant adeno-associated virus vectors. These vectors carrying either gfp or fluc genes were subjected to quantitative polymerase chain reaction assays and transduction assays in human embryonic kidney 293 (HEK293T) cells to investigate the efficiency of vector production and gene delivery. In addition, the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice. Finally, the intricate details of the vector-mediated transduction mechanisms were explored by using pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.
RESULTS: A total of 76 melittin-related peptides were identified from existing literature. Among them, CMA-3, p5RHH and aAR3 were found to significantly inhibit transduction of rAAV2 vector crude lysate. The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV. Mechanistically, bafilomycin A1, a vacuolar endosome acidification inhibitor, completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors. Most importantly, p5RHH-rAAV8 vectors also increased hepatic transduction in vivo in C57BL/6 mice.
CONCLUSIONS: The incorporation of melittin analogs into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression. While further modifications remain an area of interest, our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery. Please cite this article as: Meng J, He Y, Yang H, Zhou L, Wang S, Feng X, Al-shargi OY, Yu X, Zhu L, Ling, C. Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency. J Integr Med. 2024; 22(1): 72-82.