Adeno-Associated Virus Type 2-Mediated Gene Transfer: Role of Cellular T-Cell Protein Tyrosine Phosphatase in Transgene Expression in Established Cell Lines In Vitro and Transgenic Mice In Vivo

Adeno-associated virus type 2 (AAV) is a nonpathogenic human parvovirus that contains a single-stranded DNA as its genome and requires coinfection with a helper virus, usually adenovirus, for its optimal replication (2, 28). In the absence of coinfection with the helper virus, the wild-type (wt) AAV establishes a latent infection and the viral genome integrates into human chromosomal DNA in a site-specific manner (19, 20, 38). The nonpathogenicity of AAV and the remarkable site specificity of its integration have led to the development of recombinant AAV vectors for gene transfer and gene therapy. Although recombinant AAV genomes appear not to integrate site specifically, AAV vectors have been successfully used to deliver genes to a wide variety of cells and tissues in vitro and in vivo (3, 4, 10, 11, 14-18, 26, 29-33, 39-41, 44-46, 48). AAV vectors have also been used in phase I clinical trials for gene therapy of cystic fibrosis and hemophilia B (10, 16). However, the transduction efficiency of AAV vectors has been reported to vary widely in different cell types. Two independent laboratories have reported that the rate-limiting step in transduction by AAV vectors is viral second-strand DNA synthesis (8, 9). We have previously documented the existence of a host cell protein that we designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence within the inverted terminal repeat of the AAV genome, is phosphorylated at tyrosine residues by the cellular epidermal growth factor receptor protein tyrosine kinase EGFR-PTK, and inhibits viral second-strand DNA synthesis leading to inefficient transgene expression (22, 23, 34, 36, 37). We subsequently identified the ssD-BP to be FKBP52, a cellular chaperone protein (36). In this report, we present evidence to document that the cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein (FKBP52), is dephosphorylated at tyrosine residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP) (21, 47). Stable transfection of a murine TC-PTP expression plasmid catalyzes tyrosine dephosphorylation of FKBP52, leads to efficient viral second-strand DNA synthesis, and results in a significant increase in AAV-mediated transduction efficiency in established human cell lines as well as in primary cells from TC-PTP-transgenic mice. These studies have important implications in the optimal use of AAV vectors in human gene therapy.