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Journal of Virology, April 2000, p. 3555-3565, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Kinetics of Recombinant Adeno-Associated Virus-Mediated Gene Transfer

Ajay K. Malik,1 Paul E. Monahan,2 David L. Allen,1 Bing-Guan Chen,1 R. Jude Samulski,3 and Kotoku Kurachi1,*

Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618,1 and Departments of Pediatrics2 and Pharmacology,3 University of North Carolina, Chapel Hill, North Carolina 27599

Received 1 September 1999/Accepted 18 January 2000

Recombinant adeno-associated virus (rAAV) vectors have been shown to be useful for efficient gene delivery to a variety of dividing and nondividing cells. Mechanisms responsible for the long-term, persistent expression of the rAAV transgene are not well understood. In this study we investigated the kinetics of rAAV-mediated human factor IX (hFIX) gene transfer into human primary myoblasts and myotubes. Transduction of both myoblasts and myotubes occured with a similar and high efficiency. After 3 to 4 weeks of transduction, rAAV with a cytomegalovirus (CMV) promoter showed 10- to 15-fold higher expression than that with a muscle-specific creatine kinase enhancer linked to beta -actin promoter. Factor IX expression from transduced myoblasts as well as myotubes reached levels as high as approximately 2 µg of hFIX/106 cells/day. Southern blot analyses of high-molecular-weight (HMW) cellular genomic and Hirt DNAs isolated from rAAV/CMVhFIXm1-transduced cells showed that the conversion of single-stranded vector genomes to double-stranded DNA forms, but not the level of the integrated forms in HMW DNA, correlated with increasing expression of the transgene. Together, these results indicate that rAAV can transduce both proliferating and terminally differentiated muscle cells at about the same efficiency, that expression of transgenes increases linearly over their lifetime with no initial lag phase, and that increasing expression correlates with the appearance of double-stranded episomal rAAV genomes. Evidence showing that the rAAV virions can copackage hFIX, presumably nonspecifically, was also obtained.

* Corresponding author. Mailing address: Department of Human Genetics, C570, MSRB II, Box 0618, University of Michigan Medical School, Ann Arbor, MI 48109-0618. Phone: (734) 647-3153. Fax: (734) 647-3158. E-mail: kkurachi{at}umich.edu.

Journal of Virology, April 2000, p. 3555-3565, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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