Affiliation:
1. Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7352
Abstract
ABSTRACT
Marker rescue, the restoration of gene function by replacement of a defective gene with a normal one by recombination, has been utilized to produce novel adeno-associated virus (AAV) vectors. AAV serotype 2 (AAV2) clones containing wild-type terminal repeats, an intact
rep
gene, and a mutated
cap
gene, served as the template for marker rescue. When transfected alone in 293 cells, these AAV2 mutant plasmids produced noninfectious AAV virions that could not bind heparin sulfate after infection with adenovirus
dl
309 helper virus. However, the mutation in the
cap
gene was corrected after cotransfection with AAV serotype 3 (AAV3) capsid DNA fragments, resulting in the production of AAV2/AAV3 chimeric viruses. The
cap
genes from several independent marker rescue experiments were PCR amplified, cloned, and then sequenced. Sequencing results confirmed not only that homologous recombination occurred but, more importantly, that a mixed population of AAV chimeras carrying 16 to 2,200 bp throughout different regions of the type 3
cap
gene were generated in a single marker rescue experiment. A 100% correlation was observed between infectivity and the ability of the chimeric virus to bind heparin sulfate. In addition, many of the AAV2/AAV3 chimeras examined exhibited differences at both the nucleotide and amino acid levels, suggesting that these chimeras may also exhibit unique infectious properties. Furthermore, AAV helper plasmids containing these chimeric
cap
genes were able to function in the triple transfection method to generate recombinant AAV. Together, the results suggest that DNA from other AAV serotypes can rescue AAV capsid mutants and that marker rescue may be a powerful, yet simple, technique to map, as well as develop, chimeric AAV capsids that display different serotype-specific properties.
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology
Cited by
57 articles.
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