516. Analysis of AAV Serotype 8 Vector Integration in Normal and DNA-PKcs-Deficient Scid Mice by a Novel Strategy

Abstract

rAAV vectors integrate into host chromosomal DNA infrequently. However, we still may need to consider the possibility of adverse effects caused by rAAV vector integration because 1) rAAV2 vector integrations in animals accompany host chromosomal deletions or translocations, and occur preferentially in or near gene regulatory sequences; and 2) 3 to 4 % integrations occur in or near cancer-related genes. In order to further address this issue and understand the biology of rAAV vectors in vivo, we investigated rAAV8 vector integration in normal and DNA-PKcs-deficient scid mice. Scid mice were chosen because DNA-PKcs has been shown to be involved in rAAV vector genome processing. Here, we developed a novel strategy for isolation of a large number of rAAV proviruses directly from vector-transduced animal tissues with high efficiency and reliability, and without any selective pressure. The salient features of the strategy are: 1) the use of a shuttle rAAV8 vector carrying an ISceI site and AmpR/Ori (AO) cassette, 2) physical separation of integrated and extrachromosomal vector genomes following ISceI digestion of tissue DNA; 3) removal of unwanted genomic DNA by digestion with a DNA conformation-dependent nuclease; 4) mixing the tissue DNA with yeast genomic DNA at 1:1 ratio at the beginning of the procedure, with yeast DNA serving as a tag sequence to monitor for undesired intermolecular recombination that may arise during the procedure. We injected normal and scid mice with 7.2|[times]|10e12 vg of this rAAV8 shuttle vector via the tail vein, and made rAAV8 integration provirus libraries from transduced mouse liver DNA. Transformation of bacteria with |[sim]|0.3 |[mu]|g liver DNA generated over a thousand colonies in each library. Initial sequencing results of 121 and 69 clones from normal and scid mouse libraries demonstrated that 23 (19%) and 52 (75%) of the sequenced clones carried rAAV8 vector-cellular DNA junctions, respectively. Importantly, we have not seen any intermolecular recombination between rAAV8 vector and yeast genomes, establishing the high reliability of this strategy. A preliminary analysis suggested that integration sites in scid mice are more dispersed than in normal mice. In addition, we found that a majority of rAAV8 genomes in scid mouse liver form extrachromosomal double-stranded |[ldquo]|dog bone|[rdquo]| structures, i.e., with both terminal hairpin loops closed. Such dog bone forms are present at undetectable levels in rAAV2-injected mouse livers, but may represent intermediates toward various vector forms. Thus the isolation of a large number of rAAV proviral genomes from quiescent somatic cells in animals is feasible by our novel strategy, and high-throughput rAAV integration site analyses in normal and scid mouse tissues and detailed characterization of the dog bone forms will provide new insights into the mechanisms of rAAV vector transduction and integration in animal tissues.