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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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