Transduction Efficiency Of AAV Vectors Research Articles

Site-Directed Mutagenesis of Surface-Exposed Lysine Residues Leads to Improved Transduction by AAV2, But Not AAV8, Vectors in Murine Hepatocytes In Vivo.

The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of recombinant adeno-associated virus 2 (AAV2) vectors, which negatively impacts the transduction efficiency of these vectors. Because ubiquitination occurs on lysine (K) residues, we performed site-directed mutagenesis where we replaced each of 10 surface-exposed K residues (K258, K490, K507, K527, K532, K544, K549, K556, K665, and K706) with glutamic acid (E) because of similarity of size and lack of recognition by modifying enzymes. The transduction efficiency of K490E, K544E, K549E, and K556E scAAV2 vectors increased in HeLa cells in vitro up to 5-fold compared with wild-type (WT) AAV2 vectors, with the K556E mutant being the most efficient. Intravenous delivery of WT and K-mutant ssAAV2 vectors further corroborated these results in murine hepatocytes in vivo. Because AAV8 vectors transduce murine hepatocytes exceedingly well, and because some of the surface-exposed K residues are conserved between these serotypes, we generated and tested two single mutants (K547E and K569E), and one double-mutant (K547 + 569E) AAV8 vector. However, no significant increase in the transduction efficiency of any of these mutant AAV8 vectors was observed in murine hepatocytes in vivo. These studies suggest that although targeting the surface-exposed K residues is yet another strategy to improve the transduction efficiency of AAV vectors, phenotypic outcome is serotype specific.

Effect of DNA topoisomerase I inhibitor on the transduction efficiency of an deno-associated virus vector in human airway epithelial cells

We tested the effects of a DNA topoisomerase inhibitor (camptothecin; CPT) on the transduction efficiency of AAV vectors in cultured human airway epithelial cells. The cells were treated with CPT for 24 hours, then exposed to AAV-CMV-LacZ for 1 hour at different multiplicities of infection (moi). Transduction efficiency of AAV vectors was assessed using X-gal staining as the percentage of LacZ-expressing cells. The transduction efficiency was approximately 1.5 to 10 fold increased by treatment with CPT prior to AAV vector exposure. However, treatment with CPT after AAV vector infection did not enhance the transduction efficiency of the vectors. These results suggest that pre-treatment with CPT increases the transduction efficiency of AAV vectors, probably by nodulating cellular function.

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

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.

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.

The use of adeno-associated virus type 2 (AAV) vectors has gained attention as a potentially useful alternative to the more commonly used retrovirus and adenovirus vectors for human gene therapy. However, 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 that binds the immunosuppressant drug FK506, termed the FK506-binding protein (FKBP52), interacts with the single-stranded D sequence within the AAV inverted terminal repeats, inhibits viral second-strand DNA synthesis, and consequently limits high-efficiency transgene expression (K. Qing, J. Hansen, K. A. Weigel-Kelley, M. Tan, S. Zhou, and A. Srivastava, J. Virol., 75: 8968-8976, 2001). 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 approximately 40% 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.

Adeno-associated virus type 2-mediated gene transfer: altered endocytic processing enhances transduction efficiency in murine fibroblasts.

Adeno-associated virus type 2 (AAV) is a single-stranded-DNA-containing, nonpathogenic human parvovirus that is currently in use as a vector for human gene therapy. However, the transduction efficiency of AAV vectors in different cell and tissue types varies widely. In addition to the lack of expression of the viral receptor and coreceptors and the rate-limiting viral second-strand DNA synthesis, which have been identified as obstacles to AAV-mediated transduction, we have recently demonstrated that impaired intracellular trafficking of AAV inhibits high-efficiency transduction of the murine fibroblast cell line, NIH 3T3 (J. Hansen, K. Qing, H. J. Kwon, C. Mah, and A. Srivastava, J. Virol. 74:992-996, 2000). In this report, we document that escape of AAV from the endocytic pathway in NIH 3T3 cells is not limited but processing within endosomes is impaired compared with that observed in the highly permissive human cell line 293. While virions were found in both early and late endosomes or lysosomes of infected 293 cells, they were localized predominantly to the early endosomes in NIH 3T3 cells. Moreover, treatment of cells with bafilomycin A1 (Baf), an inhibitor of the vacuolar H(+)-ATPase and therefore of endosomal-lysosomal acidification, decreased the transduction of 293 cells with a concomitant decrease in nuclear trafficking of AAV but had no effect on NIH 3T3 cells. However, after exposure of NIH 3T3 cells to hydroxyurea (HU), a compound known to increase AAV-mediated transduction in general, virions were detected in late endosomes and lysosomes, and these cells became sensitive to Baf-mediated inhibition of transduction. Thus, HU treatment overcomes defective endocytic processing of AAV in murine fibroblasts. These studies provide insights into the underlying mechanisms of intracellular trafficking of AAV in different cell types, which has implications in the optimal use of AAV as vectors in human gene therapy.

Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2.

Adeno-associated virus 2 (AAV)-based vectors have gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy. Although AAV uses the ubiquitously expressed cell surface heparan sulfate proteoglycan (HSPG) as a receptor, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We demonstrate here that cell surface expression of HSPG alone is insufficient for AAV infection, and that AAV also requires human fibroblast growth factor receptor 1 (FGFR1) as a co-receptor for successful viral entry into the host cell. We document that cells that do not express either HSPG or FGFR1 fail to bind AAV and, consequently, are resistant to infection by AAV. These non-permissive cells are successfully transduced by AAV vectors after stable transfections with cDNAs encoding the murine HSPG and the human FGFR1. Furthermore, AAV infection of permissive cells, known to express both FGFR1 and the epidermal growth factor receptor, is abrogated by treatment of cells with basic fibroblast growth factor, but not with epidermal growth factor. The identification of FGFR1 as a co-receptor for AAV should provide new insights not only into its role in the life cycle of AAV, but also in the optimal use of AAV vectors in human gene therapy.

Adeno-associated virus type 2-mediated gene transfer: role of epidermal growth factor receptor protein tyrosine kinase in transgene expression.

Adeno-associated virus type 2 (AAV), a single-stranded, DNA-containing, nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have recently documented that a cellular tyrosine phosphoprotein, designated the single-stranded D-sequence-binding protein (ssD-BP), plays an important role in AAV-mediated transgene expression (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879-10884, 1997) and that a strong correlation exists between the phosphorylation state of the ssD-BP and AAV transduction efficiency in vitro as well as in vivo (K. Y. Qing et al., J. Virol. 72:1593-1599, 1998). In this report, we document that treatment of cells with specific inhibitors of the epidermal growth factor receptor protein tyrosine kinase (EGF-R PTK) activity, such as tyrphostin, leads to significant augmentation of AAV transduction efficiency, and phosphorylation of the ssD-BP is mediated by the EGF-R PTK. Treatment of cells with EGF results in phosphorylation of the ssD-BP, whereas treatment with tyrphostin causes dephosphorylation of the ssD-BP and consequently leads to increased expression of the transgene. Furthermore, AAV transduction efficiency inversely correlates with expression of the EGF-R in different cell types, and stable transfection of the EGF-R cDNA causes phosphorylation of the ssD-BP, leading to significant inhibition in AAV-mediated transgene expression which can be overcome by the tyrphostin treatment. These data suggest that the PTK activity of the EGF-R is a crucial determinant in the life cycle of AAV and that further studies on the interaction between the EGF-R and the ssD-BP may yield new insights not only into its role in the host cell but also in the successful use of AAV vectors in human gene therapy.