Selective Rep-Cap Gene Amplification as a Mechanism for High-Titer Recombinant AAV Production from Stable Cell Lines
| Autor: | Ruju Chen, K. Reed Clark, Philip R. Johnson, Xingluo Liu, Frosso Voulgaropoulou |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
DNA Replication
Genes Viral viruses Blotting Western Genetic Vectors DNA Recombinant Fluorescent Antibody Technique Biology Transfection Virus Replication Polymerase Chain Reaction Adenoviridae law.invention Multiplicity of infection Plasmid law Drug Discovery medicine Genetics Humans Adenovirus infection Molecular Biology Cells Cultured In Situ Hybridization DNA Primers Pharmacology Gene Amplification Gene Transfer Techniques Dependovirus beta-Galactosidase medicine.disease Virology Molecular biology Viral replication Capsid Cell culture DNA Viral Recombinant DNA Molecular Medicine Helper Viruses HeLa Cells Plasmids |
| Zdroj: | Molecular Therapy. 2(4):394-403 |
| ISSN: | 1525-0016 |
| DOI: | 10.1006/mthe.2000.0132 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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