348. Integration Site Analysis of a Helper-Dependent Adenovirus Vector Containing AAV ITRs and 22 kb of the Human Globin LCR

Abstract

We have previously shown that adenovirus (Ad) vectors containing the Ad serotype 35 fiber (Ad5/35) efficiently transduce human hematopoietic stem cells and that helper-dependent (HD) Ad5/35 vectors containing a 22kb -globin LCR as stuffer DNA and a GFP expression cassette flanked by AAV ITRs (HD-Ad.AAV-LCR) integrate into chromosomal DNA with high frequency. The high integration efficiency makes these vectors potential alternatives to rAAV or retrovirus vectors, which have been shown to preferentially integrate into genes. We performed a detailed integration site analysis of HD-Ad.AAV-LCR vectors in MO7e cells, a cell line that is used as a model for human hematopoietic cells. All integration sites contained the globin LCR and the GFP cassette, often as concatemers. We analyzed a total of 33 independent integration junctions between vector and chromosomal DNA. Ten junctions contained AAV ITR sequences and 23 junctions were formed by the Ad ITR and lacked varying numbers of terminal nucleotides. Based on these results we speculate that HD-Ad.AAV-LCR integration requires ss or ds DNA breaks in the vector genome and that the AAV and /or ITR are prone to such breaks. (Notably the termini of incoming Ad genomes are protected by the Terminal Protein, which hypothetically might prevent efficient Ad integration). Ten out of 33 integrations were within genes, all of them in introns. None of the integrations occured in oncogenes. For three integration sites we sequenced both the 5' and 3' junctions and did not find rearrangements in genomic sequences. A total of 11 out of 33 junctions were localized to chromosome 11 and 4 out of these 11 sites were within the globin LCR (a sequence that was also present in the HD-Ad.AAV-LCR vector). We speculate that tethering of integration to the globin locus/chromosome 11 involves homologous DNA sequences or proteins that are specific to DNA sequences or higher DNA structures. In an attempt to test this, we performed integration analyses after infection of MO7e cells with an HD-Ad5/35.AAV vector that contained short (shuffled) DNA sequences from human chromosome X as stuffer DNA (HD-Ad.AAV-X). None of the 12 analyzed integration sites of this vector were in chromosome 11 and only one was in chromosome X. Interestingly, 10 out 12 HD-Ad.AAV-X integration sites were in exons.