ADENOASSOCIATED VIRUS TYPE 2-MEDIATED GENE-TRANSFER - CORRELATION OF TYROSINE PHOSPHORYLATION OF THE CELLULAR SINGLE-STRANDED D-SEQUENCE-BINDING PROTEIN WITH TRANSGENE EXPRESSION IN HUMAN-CELLS IN-VITRO AND MURINE TISSUES IN-VIVO

Although the adeno-associated virus type 2 (AAV)-based vector system h as gained attention as a potentially useful alternative to the more co mmonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the r ate-limiting second-strand viral DNA synthesis, significantly affect i ts transduction efficiency. We have identified a cellular tyrosine pho sphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3' end of the AAV genome and may prevent viral second-strand DNA synthesi s in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:1087 9-10884, 1997). In the present studies, we examined whether the phosph orylation state of the ssD-BP correlates with the ability of AAV to tr ansduce various established and primary cells in vitro and murine tiss ues in vivo. The efficiencies of transduction of established human cel ls by a recombinant AAV vector containing the beta-galactosidase repor ter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP w hich interacted with the minus-strand D probe were also as follows: 29 3 > KB > KeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line , and in CD34(+) primary human hematopoietic progenitor cells; consequ ently, the efficiencies of AAV-mediated transgene expression were sign ificantly lower in these cells. Murine Sca-1(+) lin-primary hematopoie tic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced b y AAV vectors. Dephosphorylation of the ssD-BP also correlated with ex pression of the adenovirus E4orf6 protein, known to induce AAV gene ex pression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from m ouse brain, heart, liver, lung, and skeletal-muscle tissues, all of wh ich are known to be highly permissive for AAV-mediated transgene expre ssion, contained predominantly the dephosphorylated form of the ssD-BP . Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cel lular gene that encodes the ssD-BP may promote the successful use of A AV vectors in human gene therapy.