Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors.

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by approximately 25-fold in WT MEFs, but only by approximately 4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency approximately 23-fold in WT MEFs, but only approximately 4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, approximately 59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only approximately 28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

SU5416 is a potent inhibitor of hepatocyte growth factor receptor (c-Met) and blocks HGF-induced invasiveness of human HepG2 hepatoma cells.

BACKGROUND/AIMS: SU5416 is a potent inhibitor of receptor tyrosine kinases, including those of the vascular endothelial growth factor receptor, stem cell factor receptor, and platelet-derived growth factor receptor. Because of the overwhelming evidence favoring the role of aberrant hepatocyte growth factor (HGF)/Met signaling in the pathogenesis of various human cancers, various inhibitor strategies have been employed to therapeutically target this receptor. METHODS: Cell proliferation was determined by incorporation of [(3)H] thymidine. Invasiveness was assayed in Boyden Chambers with 8 microm Matrigel coated filters. Phosphorylation of ERK1/2, Akt by HGF stimulation was detected by Western blotting. RESULTS: We found that SU5416 inhibited motility scattering and the invasive activity of a hepatocellular carcinoma cell line HepG2 in vitro and growth in primary cultured hepatocytes induced by HGF. Consequently, tyrosine autophosphorylation of the c-met induced by HGF was inhibited in these cells by SU5416 in a dose-dependent manner. Furthermore, ERK1/2 and Akt phosphorylation, the signaling events down-stream of c-met activation were reduced. Moreover, SU5416 caused reversion in NIH3T3 fibroblasts transformed by the oncogenic form of the receptor, Tpr-Met. CONCLUSIONS: Inhibition of various solid tumors growth and metastasis by SU5416 may be partially attributed to blocking activation of the hepatocyte growth factor receptor.

Impaired nuclear transport and uncoating limit recombinant adeno-associated virus 2 vector-mediated transduction of primary murine hematopoietic cells.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.