title: Selective Rep-Cap gene amplification as a mechanism for high-titer recombinant AAV production from stable cell lines.
abstract: Gene transfer vectors based on adeno-associated virus mediate high-level, stable gene expression in a variety of postmitotic tissues; thus, there is interest in developing improved production systems. We previously described the generation of rAAV producer cell lines that, upon infection with adenovirus, yielded biologically active rAAV particles. In these studies we show that the adenovirus multiplicity of infection (m.o.i.) is a critical variable for efficient production of cell line-derived rAAV and can affect yields by over 200-fold. Moreover, a threshold level of adenovirus was found necessary for high-titer vector production. To define the possible factors responsible for adenovirus m.o.i. -dependent rAAV yields, we analyzed rep and cap expression as a function of adenovirus m.o.i. High-level AAV capsid protein synthesis was observed in rAAV producer cells at adenovirus m.o.i. > or =10. This prompted us to analyze the rep-cap copy number following adenovirus infection. We documented robust episomal DNA amplification (100-fold) of integrated rep-cap sequences. Interestingly, no amplification of rep-cap sequences was observed when the sequences (in plasmid form) were transfected into adenovirus-infected HeLa cells. These data suggest that adenovirus-dependent rep-cap gene amplification is a critical process responsible for efficient rAAV synthesis in stable cell lines.
authors.affiliation: Children's Research Institute, Children's Hospital Inc., Ohio State University, Columbus, Ohio 43205, USA.
authors.forename: X
authors.initials: X
authors.lastname: Liu
authors.forename: F
authors.initials: F
authors.lastname: Voulgaropoulou
authors.forename: R
authors.initials: R
authors.lastname: Chen
authors.forename: P R
authors.initials: PR
authors.lastname: Johnson
authors.forename: K R
authors.initials: KR
authors.lastname: Clark
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured / Q000166:cytology / Q000821:virology; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005784:Gene Amplification; D018014:Gene Transfer Techniques; D005814:Genes, Viral / Q000235:genetics*; D005822:Genetic Vectors / Q000008:administration & dosage; D006367:HeLa Cells / Q000166:cytology / Q000821:virology; D006378:Helper Viruses / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014162:Transfection; D014779:Virus Replication; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D017931:DNA Primers; D004274:DNA, Recombinant; D004279:DNA, Viral; D001616:beta-Galactosidase