The adeno-associated virus 2 (AAV) vectors have gained attention as an alternative to the more commonly used retrovirus- and adenovirus-based vectors. Recombinant AAV vectors have been shown to target the liver efficiently, but the transgene expression is restricted to approximately 5% of the hepatocytes. Because the viral genome is a single-stranded DNA, and single strands of both polarities are encapsidated with equal frequency, it has been suggested that failure to undergo DNA strand-annealing accounts for the lack of efficient transgene expression. Others and we, on the other hand, have proposed that failure to undergo viral second-strand DNA synthesis contributes to the observed low efficiency of transgene expression. We have previously documented that a cellular protein, designated FKBP52, inhibits AAV second-strand DNA synthesis, when present in phosphorylated forms, and consequently, limits transgene expression in non-hepatic cells, whereas unphosphorylated forms of FKBP52 have no effect. To further evaluate whether phosphorylated FKBP52 is also involved in regulating AAV-mediated transgene expression in murine hepatocytes, we generated transgenic mice over-expressing the cellular T cell protein tyrosine phosphatase (TC-PTP) protein, known to catalyze dephosphorylation of FKBP52, as well as mice deficient in FKBP52. We have demonstrated that dephosphorylation of FKBP52 in TC-PTP transgenic (TC-PTP-TG) mice, and removal of FKBP52 in FKBP52-knockout (FKBP52-KO) mice, results in efficient transduction of murine hepatocytes following tail-vein injection of recombinant AAV vectors. We have also documented efficient viral second-strand DNA synthesis in hepatocytes from both TC-PTP-TG and FKBP52-KO mice. Thus, our data strongly support the contention that the viral second-strand DNA synthesis, rather than DNA strand-annealing, is the rate-limiting step in the efficient transduction of hepatocytes. This proposal will test the following hypotheses: 1. FKBP52 is phosphorylated at serine/threonine residues by DNA-PKcs, and dephosphorylated by PP5; 2. Optimal transduction of primary hepatocytes is limited by serine/threonine phosphorylated forms of FKBP52, but can be overcome by deliberate overexpression of PP5; 3. DNA-PKcs-deficient mice overexpressing TC-PTP allow efficient transduction of murine hepatocytes; and 4. Efficient integration of the AAV proviral genome occurs in primary hepatocytes in DNA-PKcs-deficient/ TC-PTP-transgenic mice in vivo. The knowledge gained from these studies will not only shed light on the AAV-hepatocyte interactions, but will also be applicable in further improvements in recombinant AAV vectors for their potential use in gene therapy of human liver diseases in general, and al-antitrypsin deficiency (AATD) and glycogen storage disease (GSD) in particular, the main focus of this PPG application.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Program Projects (P01)
Project #
5P01DK058327-07
Application #
7311629
Study Section
Special Emphasis Panel (ZDK1)
Project Start
Project End
Budget Start
2006-08-01
Budget End
2007-07-31
Support Year
7
Fiscal Year
2006
Total Cost
$217,146
Indirect Cost
Name
University of Florida
Department
Type
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
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