Role for highly regulated rep gene expression in adeno-associated virus vector production.
Recent success achieving long-term in vivo gene transfer without a significant immune response by using adeno-associated virus (AAV) vectors (X. Xiao, J. Li, and R. J. Samulski, J. Virol. 70:8098-8108, 1996) has encouraged further development of this vector for human gene therapy. Currently, studies focus on the generation of high-titer vectors by using the two-plasmid helper-vector system in adenovirus (Ad)-infected cells. To examine the effects of the AAV replication (rep) genes on recombinant AAV (rAAV) vector production, we have constructed a series of AAV helper plasmids that contain strong heterologous promoters in place of the endogenous p5 promoter. Although high-level rep gene expression was achieved, rAAV DNA failed to replicate in the absence of Ad infection. Moreover, unregulated overexpression of Rep78/68 led to substantially lower rAAV yields in the presence of Ad (10(4-5) versus 10(7-8)). In contrast, under similar conditions, reduced Rep78/68 expression resulted in much higher rAAV yields (10(9)). Molecular characterization showed that overexpression of the rep gene decreased rAAV DNA replication and severely inhibited capsid (cap) gene expression. Interestingly, a reduced rep level enhanced cap gene expression and supported normal rAAV DNA replication. These studies suggest a critical role for regulated rep gene expression in rAAV production and have facilitated the development of a new AAV helper plasmid that increases vector production eightfold over currently used constructs.
Li
J
J
Gene Therapy Center, University of North Carolina at Chapel Hill, 27599, USA.
Samulski
R J
RJ
Xiao
X
X
D000256:Adenoviridae; D002213:Capsid / Q000235:genetics; D002461:Cell Line, Transformed; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics* / Q000254:growth & development; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D006801:Humans; D011401:Promoter Regions, Genetic; D014764:Viral Proteins / Q000235:genetics*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2