Targeted integration of functional human ATM cDNA into genome mediated by HSV/AAV hybrid amplicon vector

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and sensitivity to ionizing radiation (IR). We have previously shown that a herpes simplex virus type 1 (HSV-1) amplicon vector carrying the human ataxia-telangiectasia mutated (ATM) complementary DNA (cDNA) is able to correct aspects of the cellular phenotype of human A-T cells in culture, and is also able to transfer the ATM cDNA to the Atm(-/-) mouse cerebellum. In order to achieve stable gene replacement, we have generated an HSV/adeno-associated virus (AAV) hybrid amplicon vector carrying the expression cassettes for the ATM cDNA [(9.2 kilobases (kb)] and enhanced green fluorescent protein (EGFP), flanked by AAV inverted terminal repeats (ITRs). This hybrid vector, in the presence of AAV Rep proteins, mediates site-specific integration into the AAVS1 site on chromosome 19 in human cells and in Atm(-/-) mice carrying that human locus. The functional activity of the vector-derived ATM was confirmed in vitro and in vivo by ATM autophosphorylation at Ser-1981 after IR. This proof-of-principle study establishes the ability of HSV/AAV hybrid amplicon vectors to mediate functional targeted integration of the ATM cDNA into A-T cells in culture and in Atm(-/-) mice in vivo, thus laying a foundation for possible gene therapy approaches in the treatment of A-T patients.