8. Adeno-Associated Virus 2-Mediated Gene Transfer: A Complex Interaction between Epidermal Growth Factor Receptor Protein Tyrosine Kinase Signaling and Ubiquitin/Proteasome Pathway in Transgene Expression

Abstract

The transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular protein, FKBP52, inhibits the viral second-strand DNA synthesis, and consequently, transgene expression (J. Virol., 75: 9818, 2001). FKBP52 is phosphorylated at tyrosine residues by epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK), which inhibits AAV second-strand DNA synthesis by greater than 90% (J. Virol. 72: 9835, 1998; J. Virol., 75: 9818, 2001). Tyrosine-phosphorylated FKBP52 is dephosphorylated by T-cell protein tyrosine phosphatase (TC-PTP), which negatively regulates EGFR-PTK signaling, and leads to a significant increase in AAV transduction (J. Virol. 77: 2741, 2003). We have documented high-efficiency transduction of hepatocytes in TC-PTP-transgenic mice (Gene Ther., 11: 1165, 2004). We have also documented that intracellular trafficking of AAV from cytosol to nucleus is improved in hematopoietic cells from TC-PTP transgenic mice (Hum. Gene Ther., 15: 1207, 2004). In the present studies, we studied the role of EGFR-PTK signaling in AAV-mediated transgene expression. Consistent with our previous studies, less than 5% of HeLa cells were transduced by single-stranded (ss) AAV-EGFP vectors, and pretreatment of cells with Tyrphostin 23 (Tyr23), a specific inhibitor of EGFR-PTK, or stable expression of TC-PTP resulted in respectively |[sim]|12-fold and |[sim]|15-fold increase in AAV transduction. Although transduction by self-complementary (sc) AAV2-EGFP vectors was |[sim]|8-fold higher compared with their ss counterparts, pretreatment with Tyr23 or stable expression of TC-PTP resulted in a further |[sim]|10-fold increase in transduction. These data suggested that Tyr23 and TC-PTP affected other aspects of EGFR-PTK signaling involved in regulating vector transduction. We next examined whether Tyr23 or TC-PTP modulated the ubiquitin/proteasome pathway, both because proteasomes have been implicated in the regulation of EGFR endocytosis, and proteasome inhibitors have been shown to augment AAV transduction. Treatment of HeLa cells with MG132, a specific inhibitor of proteasome, resulted in respectively |[sim]|6-fold and |[sim]|8-fold increase in transduction efficiency of ss and scAAV vectors, However, neither a synergistic nor an additive effect was observed following pretreatment with MG132+Tyr23 or MG132+TC-PTP with either ss or scAAV vectors in HeLa cells. Similar results were obtained with murine fibroblast cell line NIH3T3, adult mouse hepatocyte cell line H2.35, and fetal mouse hepatocyte cell line FL83B. Taken together, these data indicate that complex interactions between EGFR-PTK signaling and ubiquitin/ proteasome pathway affect various aspects of intracellular trafficking and second-strand DNA synthesis of AAV vectors. A better understanding of these interactions is likely to be important in yielding new insights in the optimal use of recombinant AAV vectors in human gene therapy.