Xie Yi-Lin, Wang Ji-Yao, He Yun, Yu Xiao-Min, Zheng Qing-Yun, Ling Chen, Feng Xi-Lin, Zhu Li-Qing
State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China.
State Key Laboratory of Genetic Engineering and Engineering Research Center of Gene Technology (Ministry of Education), School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai 200438, China; Department of Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China; Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, Wenzhou 325000, Zhejiang Province, China.
J Integr Med. 2023 Jan;21(1):106-115. doi: 10.1016/j.joim.2022.10.003. Epub 2022 Oct 10.
Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy.
The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.
rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.
Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.
蜂毒肽是一种细胞穿透肽，可提高许多非病毒基因递送载体的效率，但其在病毒载体中的应用尚未得到充分研究。非致病性重组腺相关病毒（rAAV）载体是一种理想的体内基因递送载体。然而，只有在提高其转导效率后才能充分发挥其潜力。为了提高rAAV2载体的转导效率，我们尝试开发一种基于蜂毒肽的rAAV2载体递送策略。
将蜂毒肽插入所有病毒蛋白（VP）的环VIII或VP2的N端的rAAV2衣壳中。对各种rAAV2-gfp或-fluc载体进行定量实时聚合酶链反应和蛋白质印迹分析，分别测定其滴度和衣壳蛋白的完整性。或者，将基于野生型衣壳的载体与蜂毒肽预孵育，然后转导培养细胞或将混合物经尾静脉注射给C57BL/6和BALB/c裸鼠。进行体内生物发光成像以评估转基因表达。
将蜂毒肽插入VP环VIII的rAAV2载体转导效率较低，可能是由于与靶细胞结合的能力显著降低。在VP2的N端融合蜂毒肽产生的载体没有VP2亚基。有趣的是，在常用的rAAV载体中，rAAV2和rAAV6载体与蜂毒肽预孵育显著提高了它们在体外HEK293和Huh7细胞中的转导效率。蜂毒肽还能够在体内增加小鼠肝脏中rAAV2介导的转基因表达。从机制上讲，蜂毒肽没有改变载体-受体相互作用。此外，培养细胞的细胞计数试剂盒-8分析和血清转氨酶水平表明蜂毒肽几乎没有细胞毒性。
与将蜂毒肽插入rAAV2衣壳不同，与蜂毒肽预孵育显著提高了rAAV2介导的转基因表达。尽管需要进一步的体内评估，但本研究不仅扩展了蜂毒肽的药理学潜力，还提供了一种改善rAAV载体介导的基因治疗的新策略。
