345. Sensitive Molecular Detection of Various Stable Episomal Concatemeric and Integrated Forms of rAAV2 after Long-Term Expression In Vivo
Authors: Karim Zaoui, Manfred Schmidt, Denise E. Sabatino, Fayaz R. Khazi, Hanno Glimm, Junwei Sun, Katherine A. High, Christof von Kalle
Journal: Molecular Therapy (2004)

rAAV vectors can lead to the long term expression of their double stranded DNA transgenes and are considered a promising vector for gene therapy. The recombinant adeno-associated virus serotype 2 (rAAV2) has been demonstrated to confer long-term gene expression as well as a lack of pathogenicity and toxicity. Successful gene transfer and expression of human coagulation factor IX were shown in hemophilia B patients after muscle-directed gene transfer of an AAV2 vector. Trials are ongoing for muscular dystrophy and cystic fibrosis. AAV has been known for a long time to persist in the form of episomal concatemers (circular and linear). The possibility of in vivo concatemerization of rAAV genomes may have interesting consequences and applications. Therapeutic vector designs with split expression cassettes across two separate vectors have been described, which expressed the combined gene product after concatemerization in the cell nucleus (Nakai H et al. Nat. Biotechnol. 2000, Duan D et al. Nat. Med. 2000, Sun L et al. Nat. Med. 2000). The specific relations and interactions leading to concatimerization are not completely understood. The existence of episomal concatemer forms has so far also hampered precise AAV- integration analysis. To detect existing concatemeric and integrated forms, we have used a combination of a Multiplex- PCR and LAM-PCR for the analysis of rAAV2 transduced long-term cell lines and long-term in vivo murine tissue samples. Interestingly, in an assay set up to detect all possible permutations of head-to-head, head-to-tail and tail-to-tail concatemeric sequences in a cell line, we found a defined number and configuration of partial deletions of 5'-, 3'- ITRs, complete deletions of both ITRs and additional deletions of the ori- region of the transgene, presumably in episomal forms of the vector. We detected more than 20 additional variant amplicons of rAAV2 up to 15 month after systemic in vivo delivery into liver and muscle tissue of mice. No complete HT-, TT-, or HT form could be observed at any time point after injection. In the presence of this defined abundance of concatemeric forms, a high resolution integration site analysis by LAM- PCR of AAV2 transduced murine liver and muscle tissue could both confirm the presence of more than 20 similar concatemeric forms as detected by Multiplex- PCR, and furthermore identify 9 genomically integrated forms with partial vector ITR deletions and exact mapping to the murine genome. We are currently analyzing how completely all present sequences were represented by this analysis. We conclude that highly sensitive LAM PCR for the detection of AAV integration can be performed successfully on in vitro and in vivo samples to detect concatemeric and genomic rAAV fusion sequences. The combination of Multiplex- and LAM- PCR can provide essential precise information on the molecular status of AAV vectors, their insertions and deletions in target cells of gene therapy.