title: Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells.
abstract: 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 alpha2-3 sialic acid for transduction, as revealed by a competition experiment with an analog of alpha2-3 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.
authors.affiliation: Division of Genetic Therapeutics, Jichi Medical School, 3311-1 Yakushiji, Minami-kawachi, Tochigi 329-0498, Japan. murabe@jichi.ac.jp
authors.forename: Masashi
authors.initials: M
authors.lastname: Urabe
authors.forename: Takayo
authors.initials: T
authors.lastname: Nakakura
authors.forename: Ke-Qin
authors.initials: KQ
authors.lastname: Xin
authors.forename: Yoko
authors.initials: Y
authors.lastname: Obara
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mizukami
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kume
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kotin
authors.forename: Keiya
authors.initials: K
authors.lastname: Ozawa
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis / Q000235:genetics; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics / Q000254:growth & development*; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000502:physiology; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D018411:Spodoptera; D014161:Transduction, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology; D014776:Virus Cultivation
chemical_list: D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase; D019158:N-Acetylneuraminic Acid