Our recent studies have revealed that among the 10 different commonly used adeno-associated virus (AAV) serotypes, AAV3 vectors transduce human liver cancer cells extremely efficiently because these cells express high levels of human hepatocyte growth factor receptor (hHGFR), and AAV3 utilizes hHGFR as a cellular co-receptor for viral entry. In this report, we provide further evidence that both extracellular as well as intracellular kinase domains of hHGFR are involved in AAV3 vector entry and AAV3-mediated transgene expression. We also document that AAV3 vectors are targeted for degradation by the host cell proteasome machinery, and that site-directed mutagenesis of surface-exposed tyrosine (Y) to phenylalanine (F) residues on AAV3 capsids significantly improves the transduction efficiency of Y701F, Y705F and Y731F mutant AAV3 vectors. The transduction efficiency of the Y705+731F double-mutant vector is significantly higher than each of the single mutants in liver cancer cells in vitro. In immunodeficient mouse xenograft models, direct intratumoral injection of AAV3 vectors also led to high-efficiency transduction of human liver tumor cells in vivo. We also document here that the optimized tyrosine-mutant AAV3 vectors lead to increased transduction efficiency following both intratumoral and tail-vein injections in vivo. The optimized tyrosine-mutant AAV3 serotype vectors containing proapoptotic genes should prove useful for the potential gene therapy of human liver cancers.
Bibliographical noteFunding Information:
We thank Drs R Jude Samulski and Xiao Xiao for their kind gifts of recombinant AAV3 and AAV2–CBAp–EGFP plasmids, respectively, and Dr Gail E Tomlinson for generously providing the Hep293TT cell line. We also thank Drs Regino Gonzalez-Peralta and Satish K Walia for a critical review of the manuscript as well as for their general counsel. The expert technical assistance of Mr Baozheng Li and Ms Wenqin Ma is gratefully acknowledged. This research was supported, in part, by a Junior Investigator Award from the University of Florida Shands Cancer Center, American Cancer Society Chris DiMarco Institutional Research Grant (to GVA), and Public Health Service Grants R01 HL-076901, R01 HL-097088 and P01 DK-058327 (Project 1) from the National Institutes of Health (to AS). BC was supported by a state-sponsored program for Graduate Students from China Scholarship Council, Government of China.
- AAV vectors
- human hepatocyte growth factor receptor
- human liver cancer
- tyrosine mutants