811. The Hotspots of Integration Targeted by the Site-Specific Integrase PhiC31 May Be Cell Line Dependent

Abstract

Recent 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 the phage integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudo attP sites in the host chromosome. However, a study which carefully analyzes the consequences of phage integrase mediated integration for each independent integration event has yet to be performed. In the present study we characterized the chromosomal integration sites after phiC31 mediated integration from extrachromosomal plasmids in the two liver derived cell lines Huh7 (human hepatoma cell line) and Hep1A (mouse hepatoma cell line) and the human embryonic kidney cell line 293. The number of cell clones selected in a colony forming assay from all three cell lines revealed a 6.0 fold, 17.2 fold and 4.5 fold increase in the number of colonies in Huh7 cells, Hep1A cells and 293 cells compared to the mutant integrase control group, respectively. Single cell clones from all three cell lines were amplified and the insertion sites were identified and characterized based on a plasmid rescue protocol. We found frequent smaller deletions of up to 26 bp in the chromosomal target sites (13 of 32 integrations) and deletions of up to 10 bp in the phage integrase attachment site attB (8 of 32 integrations) in both, Huh-7 and 293 cells. Surprisingly, we also detected insertions of up to 177 bp (7 of 32 integrants); most of the insertions were < 4 bp. The origin of these inserted DNA sequences remains to be determined. Out of the 32 independent integration events analyzed so far, three integrated into human chromosome 19 (19q13.31), a site not previously reported. Because these initial results do not yet have a statistically significant number of independent observations, further analyses of additional clones from all three cell lines are ongoing. In summary, these results suggest that hotspots in the host genome targeted by the site-specific integrase phiC31 may depend on the cell line and perhaps on the tissue from which the cell line is derived. These findings will have important implications on improving the safety of ex vivo and in vivo gene therapy approaches in which stable and long-term transgene expression levels are required.