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Journal of Integrative Medicine ›› 2024, Vol. 22 ›› Issue (1): 72-82.doi: 10.1016/j.joim.2023.10.030

• Original Experimental Research • Previous Articles     Next Articles

Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency

Jing-shun Menga,b,c, Yun Hed, Heng-bin Yangd, Li-ping Zhoud, Si-yuan Wangd, Xi-lin Fengd,e, Omar Yahya Al-shargid, Xiao-min Yuf, Li-qing Zhuf, Chang-quan Linga,b,c   

  1. a. Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
    b. Oncology Department of Traditional Chinese Medicine, Changhai Hospital, Naval Medical University, Shanghai 200433, China
    c. Faculty of Traditional Chinese Medicine, Naval Medical University, Shanghai 200043, China
    d. 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
    e. Yantai Fuheng Biological Technology Co., Ltd., Yantai, Shandong Province 264006, China.
    f. Department of Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China
  • Received:2023-02-20 Accepted:2023-05-22 Online:2024-01-31 Published:2023-11-12
  • Contact: Chang-quan Ling


Melittin and its derivatives have been characterized to establish effective gene delivery systems. Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery. Nevertheless, little investigation has been conducted to explore its potential application in the context of viral vectors.


Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors. These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of 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 revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.


A total of 76 melittin-related peptides were compiled from existing literature. Among them, cMA2, Melt13, p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors. 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 demonstrated increased hepatic transduction in vivo in C57BL/6 mice.


The incorporation of melittin analogues 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.

Key words: Melittin, Recombinant adeno-associated virus, Capsid engineering, Transduction efficiency

[1] Yi-lin Xie, Ji-yao Wang, Yun He, Xiao-min Yu, Qing-yun Zheng, Chen Ling, Xi-lin Feng, Li-qing Zhu. The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo. Journal of Integrative Medicine, 2023, 21(1): 106-116.
[2] Gai Ran, Xi-lin Feng, Yi-lin Xie, Qing-yun Zheng, Peng-peng Guo, Ming Yang, Ying-lu Feng, Chen Ling, Li-qing Zhu, Chen Zhong. The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy. Journal of Integrative Medicine, 2021, 19(6): 515-525.
[3] Chen Ling, Yuan Wang, Ying-lu Feng, Ya-ni Zhang, Jun Li, Xin-rui Hu, Li-na Wang, Mao-feng Zhong, Xiao-feng Zhai, Irene Zolotukhin, Arun SrivastavaChang-quan Ling. Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions. Journal of Integrative Medicine, 2015, 13(5): 341-346.
[4] Yuan-hui Zhang, Yuan Wang, Yusufali Ali Hussein, Frederick Ashby, Daniel Zhang, Zi-fei Yin, George V. Aslanidi, Arun Srivastava, Chang-quan Ling, Chen Ling. Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo. Journal of Integrative Medicine, 2014, 12(6): 483-494.
[5] 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.
[6] 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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