Delayed Expression of Adeno-Associated Virus Vector DNA

• Published in: Intervirology Vol. 42; no. 4; pp. 213 - 220
• Main Authors: Afione, Sandra A., Wang, Jianming, Walsh, Scott, Guggino, William B., Flotte, Terence R.
• Format: Journal Article
• Language: English
• Published: Basel, Switzerland S. Karger AG 1999
• Subjects: Adeno-associated virus; Adenoviridae - physiology; Adenovirus; Cell Line, Transformed; Dependovirus - genetics; Dependovirus - physiology; DNA, Recombinant - metabolism; Genetic Vectors - genetics; Genetic Vectors - physiology; green fluorescent protein; HeLa Cells; Humans; Original Paper; Time Factors; Transfection; Transformation, Genetic; Tumor Cells, Cultured; Virus Integration; Virus Latency; Gene therapy; Adenovirus; Adeno-associated virus
• ISSN: 0300-5526; 1423-0100
• DOI: 10.1159/000024980

Two previous reports indicated that recombinant adeno-associated virus (rAAV) vectors were dependent on helper adenovirus (Ad) for efficient conversion of single-stranded (ss) rAAV DNA to the double-stranded (ds) form. This finding is somewhat paradoxical, however, since during a latent infection wild-type (wt)-AAV is rapidly converted to a ds form in the absence of Ad. Our hypothesis was that the effect observed in the previous studies was due to kinetic factors, i.e. to a relative delay in conversion to ds-DNA rather than to an absolute requirement for Ad. To test this, Hela cells were infected with a rAAV-CMV-green fluorescent protein (GFP) vector either in the presence or absence of Ad. Within the first 2 days, Ad infection resulted in a 4-fold increase in AAV vector expression and an augmentation of conversion to a ds-AAV DNA. By 6 days, however, the total number of GFP-expressing cells in the Ad-free culture had exceeded the original number in the Ad co-infected cells, and the conversion to ds-DNA episomes was substantial and ongoing.