Abstract
Reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that integrase øC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cellsin vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudoattP sites in the host chromosomes. In the present study we investigated whether cryptic øC31 recognition sites in the host genome may result in chromosomal rearrangements. Of 69 independent integration events analyzed in human cells, 6 (8.7%) integrated into human chromosome 19 (19q13.31) and 10 (14.49%) integrated into human chromosome 12 (12q22). Most importantly, of all integration sites analyzed, 15% were found to contain an integrated transgene that was flanked by DNA sequences originating from two different chromosomes. To confirm chromosomal translocations we performed a polymerase chain reaction analysis of chromosomal DNA flanking the transgene and also performed limited studies to determine the genotype of single-cell clones. Although the mechanism responsible for chromosomal translocations needs to be further characterized, we speculate that cryptic øC31 attachment sites flanking the transgene and cryptic øC31 attachment sites in the host genome recombine with each other.
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