Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks.
The use of adeno-associated virus (AAV) to package gene-targeting vectors as single-stranded linear molecules has led to significant improvements in mammalian gene-targeting frequencies. However, the molecular basis for the high targeting frequencies obtained is poorly understood, and there could be important mechanistic differences between AAV-mediated gene targeting and conventional gene targeting with transfected double-stranded DNA constructs. Conventional gene targeting is thought to occur by the double-strand break (DSB) model of homologous recombination, as this can explain the higher targeting frequencies observed when DSBs are present in the targeting construct or target locus. Here we compare AAV-mediated gene-targeting frequencies in the presence and absence of induced target site DSBs. Retroviral vectors were used to introduce a mutant lacZ gene containing an I-SceI cleavage site and to efficiently deliver the I-SceI endonuclease, allowing us to carry out these studies with normal and transformed human cells. Creation of DSBs by I-SceI increased AAV-mediated gene-targeting frequencies 60- to 100-fold and resulted in a precise correction of the mutant lacZ reporter gene. These experiments demonstrate that AAV-mediated gene targeting can result in repair of a DNA DSB and that this form of gene targeting exhibits fundamental similarities to conventional gene targeting. In addition, our findings suggest that the selective creation of DSBs by using viral delivery systems can increase gene-targeting frequencies in scientific and therapeutic applications.
Miller
Daniel G
DG
Department of Medicine, Division of Hematology, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195, USA.
Petek
Lisa M
LM
Russell
David W
DW
D015139:Blotting, Southern; D002460:Cell Line; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D000229:Dependovirus / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
D004247:DNA