Adeno-Associated Virus Type 2-Mediated Gene Transfer: Role of Cellular FKBP52 Protein in Transgene Expression

Abstract

Here, we report that a partial amino acid sequence of ssD-BP purified from HeLa cells is identical to a portion of a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein 52 (FKBP52). FKBP52 was purified by using a prokaryotic expression plasmid containing the human cDNA. The purified protein could be phosphorylated at both tyrosine and serine or threonine residues, and only the phosphorylated forms of FKBP52 were shown to interact with the AAV single-stranded D-sequence probe. Furthermore, in in vitro DNA replication assays, tyrosine-phosphorylated FKBP52 inhibited AAV second-strand DNA synthesis by greater than 90%. Serine-or threoninephosphorylated FKBP52 caused Ϸ40% inhibition, whereas dephosphorylated FKBP52 had no effect on AAV second-strand DNA synthesis. Deliberate overexpression of FKBP52 effectively reduced the extent of tyrosine phosphorylation of the protein, resulting in a significant increase in AAV-mediated transgene expression in human and murine cell lines. These studies corroborate the idea that the phosphorylation status of the cellular FKBP52 protein correlates strongly with AAV transduction efficiency, which may have important implications for the optimal use of AAV vectors in human gene therapy.Adeno-associated virus type 2 (AAV) is a small, nonpathogenic, single-stranded DNA-containing virus which requires coinfection with a helper virus, usually adenovirus, for its optimal replication (1, 31). In the absence of coinfection with the helper virus, the wild-type AAV establishes a latent infection in which the viral genome integrates into human chromosomal DNA in a site-specific manner (22,23,45). The nonpathogenic nature of AAV coupled with the remarkable site specificity of integration prompted the development of recombinant AAV vectors for gene transfer and gene therapy. Although recombinant AAV genomes do not appear to integrate site specifically, AAV vectors have been successfully used for gene delivery to a wide variety of cells and tissues in vitro and in vivo (2, 3, 11, 12, 16-19, 21, 32-36, 47, 48, 51, 55, 57), as well as in phase I clinical trials for gene therapy of cystic fibrosis and hemophilia B (11, 18). However, the transduction efficiencies of AAV vectors vary greatly in different cell types. Studies from two independent laboratories have suggested that following infection, the viral second-strand DNA synthesis is a ratelimiting step in efficient transduction by AAV vectors (8,9). We have documented that a host cell protein, designated the single-stranded D-sequence binding protein (ssD-BP), interacts specifically and preferentially with the D sequence within the inverted terminal repeat (ITR) at the 3Ј end of the AAV genome and, in its tyrosine-phosphorylated form, prevents viral second-strand DNA synthesis, resulting in inhibition of AAV-mediated transgene expression. ssD-BP is phosphorylated at tyrosine residues by the epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK), and the phosphorylation state of ssD-BP corre...

Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo

“…Regardless of the mechanism involved, the extent of viral second-strand DNA synthesis was consistent with the observed transcriptional activity of the vector genomes. [4][5][6][7][8][9][10][11] These data strongly argue that rather than strand-annealing, TC-PTP-mediated tyrosine dephosphorylation of FKBP52, or the absence of FKBP52, leads to efficient AAV second-strand DNA synthesis resulting in an increase in AAV-mediated transgene expression in hepatocytes in vivo. Although our data might appear at odds with the model proposed by Nakai et al, 2 which favors DNA strand-annealing as the mechanism of hepatocyte transduction, the simplest explanation is that viral secondstrand DNA synthesis is the rate-limiting step in B95% of hepatocytes, whereas DNA strand-annealing might be operative in B5% of hepatocytes.…”

“…Phosphorylated FKBP52 inhibits the viral second-strand DNA synthesis leading to inefficient transgene expression. [6][7][8][9][10][11] We have also documented that FKBP52 is dephosphorylated at tyrosine residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP), 22,23 which leads to efficient viral …”

“…2,3 Others and we, on the other hand, have suggested that viral second-strand DNA synthesis is the rate-limiting step in AAV-mediated transgene expression. [4][5][6][7][8][9][10][11] We previously observed that following intravenous (i.v.) administration into the tail-vein, AAV vectors selectively target the liver in mice, but less than 5% of hepatocytes express the transgene.…”

“…13,[17][18][19] In our previous studies, we have identified that a 52 kDa cellular protein, FKBP52, which binds the immunosuppressant drug FK506, 20,21 interacts specifically with the D-sequence within the inverted terminal repeat (ITR) of the AAV genome. 8 FKBP52 is phosphorylated at tyrosine residues by the cellular epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK). Phosphorylated FKBP52 inhibits the viral second-strand DNA synthesis leading to inefficient transgene expression.…”

“…Phosphorylated FKBP52 inhibits the viral second-strand DNA synthesis leading to inefficient transgene expression. [6][7][8][9][10][11] We have also documented that FKBP52 is dephosphorylated at tyrosine residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP), 22,23 which leads to efficient viral second-strand DNA synthesis and a significant increase in AAV-mediated transduction efficiency in established human cell lines as well as in primary hematopoietic stem cells from TC-PTP-transgenic mice. 9 In our current studies, we have examined the efficiency of AAV-mediated transgene expression in murine hepatocytes following i.v.…”