11. An Additional Role of Cellular FKBP52 in Recombinant Adeno-Associated Virus 2 Vector-Mediated Gene Transfer and Transgene Expression

Abstract

We have reported that transduction efficiency of recombinant AAV vectors is impaired in a murine fibroblast cell line as well as in primary murine hematopoietic stem/progenitor cells (J. Virol., 74: 992, 2000; Hum. Gene Ther., 15: 1207, 2004) because AAV fails to traffic efficiently into the nucleus. We have also reported that a cellular protein, FKBP52, phosphorylated at tyrosine or serine/threonine residues, inhibits viral second-strand DNA synthesis and limits transgene expression (J. Virol., 75: 8968, 2001), and that in hematopoietic stem/progenitor cells from transgenic mice overexpressing TC-PTP, a cellular protein tyrosine phosphatase, AAV transduction efficiency is significantly augmented (J. Virol., 77: 2741, 2003). A significant augmentation is also achieved following treatment of these cells with hydroxyurea (HU) (J. Virol., 75: 4080, 2001; Hum. Gene Ther., 15: 1207, 2004), because HU facilitates nuclear transport of AAV in these cells. In FKBP52-knockout (FKBP52-KO) mice, however, following HU-treatment, transduction efficiency in hematopoietic stem/progenitor cells was significantly less pronounced than that from TC-PTP-TG mice (Hum. Gene Ther., 15: 1207-1218, 2004). To further explore whether FKBP52 plays an additional role(s) in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) from wild-type (WT), FKBP52-heterozygous (HE), and FKBP52-KO (KO) mice. As expected, AAV-mediated transgene expression was low in WT MEFs, and was not significantly increased in HE or KO MEFs. The low transduction efficiency was not due to lack of expression of heparan sulfate proteoglycan and fibroblast growth factor receptor 1, the cellular receptor and co-receptor, respectively, for AAV. Southern blot analyses further confirmed that AAV could efficiently enter MEFs from each of three genotypes. However, whereas HU-treatment increased AAV transduction efficiency in WT MEFs |[sim]|25-fold, the transduction efficiency in KO MEFs was increased only |[sim]|4 fold. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU-treatment increased transduction efficiency |[sim]|23 fold in WT MEFs, but only |[sim]|4 fold in KO MEFs. Thus, the differential transduction efficiency following HU- treatment was independent of viral second-strand DNA synthesis. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU-treatment significantly increased transduction efficiency of scAAV vectors compared with mock-transfected KO cells. Taken together, these studies suggest that in addition to inhibiting viral second-strand DNA synthesis, FKBP52, being a cellular chaperone protein, may facilitate intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.