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Journal of Virology, February 2006, p. 1874-1885, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1874-1885.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Scalable Generation of High-Titer Recombinant Adeno-Associated Virus Type 5 in Insect Cells

Masashi Urabe,1* Takayo Nakakura,1 Ke-Qin Xin,2 Yoko Obara,1 Hiroaki Mizukami,1 Akihiro Kume,1 Robert M. Kotin,3 and Keiya Ozawa1

Division of Genetic Therapeutics, Jichi Medical School, Tochigi 329-0498, Japan,1 Department of Molecular Biodefense Research, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan,2 Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland3

Received 14 June 2005/ Accepted 27 November 2005

We established a method for production of recombinant adeno-associated virus type 5 (rAAV5) in insect cells by use of baculovirus expression vectors. One baculovirus harbors a transgene between the inverted terminal repeat sequences of type 5, and the second expresses Rep78 and Rep52. Interestingly, the replacement of type 5 Rep52 with type 1 Rep52 generated four times more rAAV5 particles. We replaced the N-terminal portion of type 5 VP1 with the equivalent portion of type 2 to generate infectious AAV5 particles. The rAAV5 with the modified VP1 required {alpha}2-3 sialic acid for transduction, as revealed by a competition experiment with an analog of {alpha}2-3 sialic acid. rAAV5-GFP/Neo with a 4.4-kb vector genome produced in HEK293 cells or Sf9 cells transduced COS cells with similar efficiencies. Surprisingly, Sf9-produced humanized Renilla green fluorescent protein (hGFP) vector with a 2.4-kb vector genome induced stronger GFP expression than the 293-produced one. Transduction of murine skeletal muscles with Sf9-generated rAAV5 with a 3.4-kb vector genome carrying a human secreted alkaline phosphatase (SEAP) expression cassette induced levels of SEAP more than 30 times higher than those for 293-produced vector 1 week after injection. Analysis of virion DNA revealed that in addition to a 2.4- or 3.4-kb single-stranded vector genome, Sf9-rAAV5 had more-abundant forms of approximately 4.7 kb, which appeared to correspond to the monomer duplex form of hGFP vector or truncated monomer duplex SEAP vector DNA. These results indicated that rAAV5 can be generated in insect cells, although the difference in incorporated virion DNA may induce different expression patterns of the transgene.

* Corresponding author. Mailing address: Division of Genetic Therapeutics, Jichi Medical School, 3311-1 Yakushiji, Minami-kawachi, Tochigi 329-0498, Japan. Phone: 81-285-58-7402. Fax: 81-285-44-8675. E-mail: murabe{at}jichi.ac.jp.

Journal of Virology, February 2006, p. 1874-1885, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1874-1885.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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