Adeno-associated 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

Abstract

Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, 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 synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the beta-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. 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; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1+ lin- primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, 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 cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.

FIG. 1

Comparative analyses of transduction efficiencies of vCMVp-lacZ in established human cell lines. Approximately equivalent numbers of HeLa (A), KB (B), and 293 (C) cells were infected with vCMVp-lacZ at an MOI of 20 under identical conditions. Forty-eight hours p.i., cells were fixed and stained with X-Gal, and blue cells were enumerated as described in the text. Magnification, ×100.

FIG. 2

Analysis of binding of AAV to HeLa, KB, and 293 cells. Equivalent numbers of cells from each cell line, along with a negative (M07e) control, were analyzed in virus-binding assays with ³⁵S-radiolabeled AAV as described in the text.

FIG. 3

EMSA with WCE prepared from human HeLa, KB, and 293 cells. Equivalent amounts of WCE prepared from each cell type were used in EMSA with the D(−) probe as described in the text. The phosphorylated and dephosphorylated forms of the ssD-BP are indicated by the arrow and the arrowhead, respectively.

FIG. 4

EMSA with the D(−) and D(±) probes and effect of treatment with NaOV and genistein on the ssD-BP and the dsD-BP. (A) WCE prepared from the indicated cell types were used in EMSA with the D(−) probe and the D(±) probe as described in the text. (B) HeLa cells were treated either with 1 mM NaOV or with 150 μM genistein, and equivalent amounts of WCE prepared from these cells were used in EMSA with the D(−) probe and the D(±) probe as described in the text.

FIG. 5

Transduction efficiency of recombinant vTc-Neo in HeLa cells following treatment with HU, genistein, or NaOV. Equivalent numbers of cells were infected with the recombinant vector at an MOI of 20 following either no treatment or treatment with the indicated compounds. Forty-eight hours p.i., G418 was added, and G418-resistant colonies were enumerated 14 days p.i. following staining with methylene blue as described in the text.

FIG. 6

EMSA with WCE prepared from HeLa cells expressing the Ad E4orf6 protein. Equivalent amounts of WCE prepared from mock-transfected cells or cells transfected with plasmid pAdE4orf6 or plasmid pKY-4 were used in EMSA with the D(−) probe as described in the text. The phosphorylated and dephosphorylated forms of the ssD-BP are indicated by the arrow and the arrowhead, respectively.