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

Abstract

). FKBP52 can be phosphorylated at both tyrosine and serine/threonine residues, but only the phosphorylated forms of FKBP52 interact with the D sequence. Furthermore, the tyrosine-phosphorylated FKBP52 inhibits AAV second-strand DNA synthesis by greater than 90%, and the serine/threonine-phosphorylated FKBP52 causes ϳ40% inhibition, whereas the dephosphorylated FKBP52 has no effect on AAV second-strand DNA synthesis. In the present study, we have identified that the tyrosine-phosphorylated form of FKBP52 is a substrate for the cellular T-cell protein tyrosine phosphatase (TC-PTP). Deliberate overexpression of the murine wild-type (wt) TC-PTP gene, but not that of a cysteine-to-serine (C-S) mutant, caused tyrosine dephosphorylation of FKBP52, leading to efficient viral second-strand DNA synthesis and resulting in a significant increase in AAV-mediated transduction efficiency in HeLa cells in vitro. Both wt and C-S mutant TC-PTP expression cassettes were also used to generate transgenic mice. Primitive hematopoietic stem/ progenitor cells from wt TC-PTP-transgenic mice, but not from C-S mutant TC-PTP-transgenic mice, could be successfully transduced by recombinant AAV vectors. These studies corroborate the fact that tyrosine phosphorylation of the cellular FKBP52 protein strongly influences AAV transduction efficiency, which may have important implications in the optimal use of AAV vectors in human gene therapy.

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

“…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.…”

“…[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. administration of recombinant AAV vectors carrying the enhanced green fluorescent protein (AAV-EGFP) in FKBP52-knockout (FKBP52-KO) mice and in transgenic mice that express either the wild-type (wt) TC-PTP (wtTC-PTP-TG) or a mutant TC-PTP (mTC-PTP-TG) gene.…”

“…In mTC-PTP, the cysteine residue in the active site had been changed to a serine residue. 9,24 Approximately 1 Â 10 11 particles of recombinant AAV-EGFP vectors were injected i.v. via the tail-vein into groups of three mice each.…”

“…Transgenic mice overexpressing the murine wt or the C-S mutant TC-PTP gene were generated as described previously. 9 TC-PTP-TG mice were identified by PCR amplification of genomic DNA sample from tail snips using the murine TC-PTP cDNA-specific primerpair (5 0 -CGGTTAAATGTGCACAGTACTGGCC-3 0 and 5 0 -CTACAAC GAGAAGGTGCGAGAGC-3 0 ) followed by Southern blot hybridization using a 32 P-labeled TC-PTP DNA probe as described previously. 25,26 The generation and characterization of FKBP52-KO mice will be described elsewhere (Z Yang and W Shou, manuscript in preparation).…”