Chromosomal position effects on adeno -associated virus (AAV)-mediated gene targeting

Homologous recombination is essential for chromosome segregation and the preservation of genetic diversity, as well as repair of different types of DNA lesions. Gene targeting, in which a transgene recombines with a homologous chromosomal locus, presumably utilizes the same mechanisms as mitotic chromosomal recombination, but the effects of chromosomal position and neighboring genomic elements on gene targeting in human cells remain largely unexplored. To study these, we used a shuttle vector system in which murine leukemia virus (MLV)-based proviral targets present at different chromosomal locations and containing mutations in the neomycin phosphotransferase (neo) gene were corrected by adeno-associated virus (AAV)-mediated gene targeting. We performed a small-scale study in which sixteen identical target loci present in HT-1080 human fibrosarcoma cells were all successfully corrected by gene targeting. The gene targeting frequencies varied by as much as 10-fold, and there was a clear bias for correction of one of the targets in clones containing two target sites. Our results indicate that certain chromosomal positions are preferred sites for gene targeting in human cells. We next performed a large-scale study to determine which chromosomal elements promote gene targeting and recombination by creating a genome-wide AAV-mediated gene targeting map and comparing it to one of untargeted site locations. We found that a significantly greater percentage of targeted sites than control sites was present within RefSeq transcription units, and gene expression analysis of the parental HT-1080 cell line revealed that the median expression level of genes containing targeted sites was significantly higher than that of genes containing control integration events. Our results suggest that gene targeting occurs preferentially at sites that are present within transcriptionally active genes, possibly because the large-scale chromatin decondensation associated with target genes facilitates the accessibility of intragenic target sites to AAV and proteins necessary for homologous recombination. Importantly, we have shown that AAV-mediated gene targeting can occur throughout the genome and within many different types of chromosomal features, making the generation of cells with almost any desired mutation theoretically possible and holding promise for the therapeutic applications of AAV.