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Journal of Integrative Medicine ›› 2023, Vol. 21 ›› Issue (1): 106-115.doi: 10.1016/j.joim.2022.10.003

• Original Experimental Research • Previous Articles    

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo

Yi-lin Xiea*, Ji-yao Wanga,b,c,*, Yun Hea, Xiao-min Yub,c, Qing-yun Zhenga, Chen Linga,b,c, Xi-lin Fenga,d, Li-qing Zhub,c   

  1. a. 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
    b. Department of Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China
    c. Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, Wenzhou 325000, Zhejiang Province, China
    d. Yantai Fuheng Biological Technology Co., Ltd., Yantai 264006, Shandong Province, China
  • Received:2022-04-09 Accepted:2022-06-26 Online:2023-01-19 Published:2022-10-14
  • Contact: Li-qung Zhu

Objective: 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.
Methods: 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.
Results: 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.
Conclusion: 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.

Key words: rAAV, Melittin, Capsid engineering, Co-administration, Transduction efficiency

[1] Chen Zhang, Bai Li, Shu-qin Lu, Yong Li, Yong-hua Su, Chang-quan Ling. Effects of melittin on expressions of mitochondria membrane protein 7A6, cell apoptosis-related gene products Fas and Fas Ligand in hepatocarcinoma cells. Journal of Chinese Integrative Medicine, 2007, 5(5): 559-563.
[2] Yong-qiang Chen, Zhen-an Zhu, Yong-qiang Hao, Ke-rong Dai, Chen Zhang. Effect of melittin on apoptsis and necrosis of U2OS cells. Journal of Chinese Integrative Medicine, 2004, 2(3): 208-209.
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