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Journal of Virology, November 1999, p. 9468-9477, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Concatamerization of Adeno-Associated Virus Circular Genomes Occurs through Intermolecular Recombination

Jusan Yang,1 Weihong Zhou,1 Yulong Zhang,1 Terese Zidon,1 Terry Ritchie,1 and John F. Engelhardt1,2,*

Department of Anatomy and Cell Biology1 and Department of Internal Medicine, Center for Gene Therapy,2 School of Medicine, University of Iowa, Iowa City, Iowa

Received 15 April 1999/Accepted 26 July 1999

Long-term recombinant AAV (rAAV) transgene expression in muscle has been associated with the molecular conversion of single-stranded rAAV genomes to high-molecular-weight head-to-tail circular concatamers. However, the mechanisms by which these large multimeric concatamers form remain to be defined. To this end, we tested whether concatamerization of rAAV circular intermediates occurs through intra- or intermolecular mechanisms of amplification. Coinfection of the tibialis muscle of mice with rAAV alkaline phosphatase (Alkphos)- and green fluorescent protein (GFP)-encoding vectors was used to evaluate the frequency of circular concatamer formation by intermolecular recombination of independent viral genomes. The GFP shuttle vector also encoded ampicillin resistance and contained a bacterial origin of replication to allow for bacterial rescue of circular intermediates from Hirt DNA of infected muscle samples. The results demonstrated a time-dependent increase in the abundance of rescued plasmids encoding both GFP and Alkphos, which reached 33% of the total circular intermediates by 120 days postinfection. Furthermore, these large circular concatamers were capable of expressing both GFP- and Alkphos-encoding transgenes following transient transfection in cell lines. These findings demonstrate that concatamerization of AAV genomes in vivo occurs through intermolecular recombination of independent monomer circular viral genomes and suggest new viable strategies for delivering multiple DNA segments at a single locus. Such developments will expand the utility of rAAV for splicing large gene inserts or large promoter-gene combinations carried by two or more independent rAAV vectors.

* Corresponding author. Mailing address: Department of Anatomy and Cell Biology, University of Iowa, School of Medicine, 51 Newton Rd., Room 1-101 BSB, Iowa City, IA 52242. Phone: (319) 335-7753. Fax: (319) 335-7198. E-mail: john-engelhardt{at}uiowa.edu.

Journal of Virology, November 1999, p. 9468-9477, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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