ADENOASSOCIATED VIRUS TYPE 2-MEDIATED GENE-TRANSFER - ROLE OF EPIDERMAL GROWTH-FACTOR RECEPTOR PROTEIN-TYROSINE KINASE IN TRANSGENE EXPRESSION

Adeno-associated virus type 2 (AAV), a single-stranded, DNA-containing , nonpathogenic human parvovirus, has gained attention as a potentiall y useful vector for human gene therapy, However, the transduction effi ciency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have recently documented that a cellular tyrosi ne phosphoprotein, designated the single-stranded D-sequence-binding p rotein (ssD-BP), plays an important role in AAV-mediated transgene exp ression (K. Y. Qing et al,, Proc. Natl, Acad, Sci, USA 94:10879-10884, 1997) and that a strong correlation exists between the phosphorylatio n state of the ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing et at., J. Virol. 72:1593-1599, 1998). In this report, we document that treatment of cells with specific inhibitors of the epidermal growth factor receptor protein tyrosine kinase (EGF-R PTK) activity, such as tyrphostin, leads to significant augmentation of AAV transduction efficiency, and phosphorylation of the ssD-BP is m ediated by the EGF-R PTK. Treatment of cells with EGF results in phosp horylation of the ssD-BP, whereas treatment with tyrphostin causes dep hosphorylation of the ssD-BP and consequently leads to increased expre ssion of the transgene. Furthermore, AAV transduction efficiency inver sely correlates with expression of the EGF-R in different cell types, and stable transfection of the EGF-R cDNA causes phosphorylation of th e ssD-BP, leading to significant inhibition in AAV-mediated transgene expression which can be overcome by the tyrphostin treatment. These da ta suggest that the PTK activity of the EGF-R is a crucial determinant in the life cycle of AAV and that further studies on the interaction between the EGF-R and the ssD-BP may yield new insights not only into its role in the host cell but also in the successful use of AAV vector s in human gene therapy.