Adeno-Associated Virus Type 2-Mediated Gene Transfer: Role of Epidermal Growth Factor Receptor Protein Tyrosine Kinase in Transgene Expression

Abstract

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.

Transduction of ovarian cancer cells: a recombinant adeno-associated viral vector compared to an adenoviral vector

“…The transduction efficiency on these cells could however be improved in various ways: to a minor extent with continuous exposure of the cells to rAAV-GFP or by repeated administration as studied on NIH-OVCAR3 cell line. With the help from adenovirus (wild-type or mutant) a larger improvement in transduction efficiencies was observed, as reported by others (Ferrarri et al, 1996;Fisher et al, 1996; Qing et al, 1997Qing et al, , 1998Mah et al, 1998). On NIH-OVCAR3 cells this increase also proved to be independent of the rAAV-GFP concentration.…”

“…Several cellular factors play a role in this conversion. Recent publications could demonstrate a correlation between the transduction efficiency by rAAV vectors and the phosphorylation status of a cellular tyrosine phospho-protein (ssD-BP) (Ferrari et al, 1996;Qing et al, 1997Qing et al, , 1998Mah et al, 1998). This protein binds to the 3′ end of the AAV genome.…”

High-efficiency Transduction and Correction of Murine Hemophilia B Using AAV2 Vectors Devoid of Multiple Surface-exposed Tyrosines

“…[33][34][35] In the past years, we have shown that specific inhibition of epidermal growth factor receptor protein tyrosine kinase dramatically increases transduction efficiency with both ssAAV and scAAV vectors. 18,[36][37][38][39] In a recent series of studies, we documented that during the process of navigation through the late endosome, epidermal growth factor receptor protein tyrosine kinase-mediated tyrosine phosphorylation of AAV2 capsid proteins promotes ubiquitination and degradation of AAV2, thus leading to impairment of viral nuclear transport and decrease in transduction efficiency in intact cells. 20,22 Based on these studies, 20 we mutated each of the seven surface-exposed tyrosine residues on viral capsids, which yield AAV2 vectors with significantly increased transduction efficiency in vitro as well as in vivo, compared with their WT counterpart.…”

A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5′ End or within the Genome of Feline Parvovirus and Can Modify Virus Replication

“…The protein may bind the viral genome and be involved in regulation of transcription or replication. The effect on replication or fill-in of the viral DNA may be similar to that seen for a cellular protein which binds the AAV genome just inside the 3Ј hairpin and blocks viral DNA fill-in during infection (22,23,41), where binding is regulated by tyrosine phosphorylation of the protein (34,39). Some hnRNP proteins are also phosphorylated on tyrosine by tyrosine kinases, or on serine or threonine by protein kinase C or casein kinase, and those modifications can regulate the binding or FIG.…”