252. Targeted Transgene Integration of the Human ATM cDNA Mediated by HSV/AAV Hybrid Amplicon Vector
Authors: Jocelyn D. Sanford, Angelika Oehmig, Katherine F. Perry, Xandra O. Breakefield, Maria L. Cortes
Journal: Molecular Therapy (2006)

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and radiation sensitivity. The large size of the cDNA (9.2 kb) required to correct the hereditary deficiency in A-T has limited use of many vector types for genetic treatment. HSV-1 amplicon vectors provide a means to deliver large genes to cells efficiently and without toxicity. We have previously reported the construction of an HSV-1 amplicon vector carrying the full-length human ATM cDNA, and shown the ability of this vector to correct aspects of the cellular phenotype of human A-T cells in culture. In order to restore stable gene function in A-T cells, it would be optimal to employ vectors that have the capacity for efficient genomic integration in a site-specific manner. We have generated an HSV/ AAV hybrid amplicon vector carrying the ATM cDNA flanked with AAV ITR sequences, which should be able, in the presence of AAV Rep proteins, to mediate site-specific recombination into the AAVS1 site on chromosome 19 in human cells. The hybrid vector, designated as pHyRIGN-ATM, contains the ATM cDNA under the CBA promoter, the GFP reporter gene under the CMV promoter, and a neomycin resistance cassette, all between the ITRs. The integrity of the amplicon construct has been verified by restriction analysis and sequencing. To characterize integration sites in transduced clones we have used human 293 control cells, and SV40-transformed human fibroblast lines from a healthy A-T heterozygote and a patient with A-T. Following infection with the pHyRIGN-ATM hybrid amplicon vector, or a hybrid control vector, stable clones expressing EGFP have been selected with G418. Positive clones have been expanded and evaluated for FLAG- ATM expression. Using immunoprecipitation with FLAG M2 antibody and western blot analysis with ATM antibody, we have been able to detect FLAG-tagged ATM protein in the A-T clones transduced with the pHyRIGN-ATM hybrid amplicon vector. In addition, we determined the functional activity of the vector- derived FLAG-ATM by autophosphorylation of ATM on Ser-1981 after ionizing radiation. These experiments show that clones obtained after infection with the HSV/AAV vector express functional ATM. Clones are currently being analyzed for the frequency and site of genomic integration by PCR and Southern blot analysis using different primers and probes to distinguish random versus AAVS1 integrations, and for the integration of ITR-flanked and/or amplicon backbone sequences. Transgenic mice bearing the human AAVS1 site, which has been shown to function as a target for this integration event, have been crossed to Atm +/- mice to create Atm -/- bearing the AAVS1 site. Current efforts are focused on the delivery of this hybrid vector to different tissues of Atm -/- AAVS1 +/+ mice. These mice will allow us to evaluate whether we can achieve targeted integration of the ATM cDNA into the Atm -/- genome for gene replacement in cells in vivo, thereby demonstrating the feasibility of HSV/AAV amplicon vectors as a gene therapy vector for A-T. This work was supported by A-T Children's Project and NINDS R21.