Phosphorylation of surface-exposed tyrosine residues negatively impacts the transduction efficiency of recombinant AAV2 vectors. Pre-treatment of cells with specific cellular serine/threonine kinase inhibitors also significantly increased the transduction efficiency of AAV2 vectors. We reasoned that site-directed mutagenesis of surface-exposed serine residues might allow the vectors to evade phosphorylation and thus lead to higher transduction efficiency. Each of the 15 surface-exposed serine (S) residues was substituted with valine (V) residues, and the transduction efficiency of three of these mutants, S458V, S492V and S662V, was increased by up to ∼20-fold in different cell types. The S662V mutant was efficient in transducing human monocyte-derived dendritic cells (moDCs), a cell type not readily amenable to transduction by the conventional AAV vectors, and did not induce any phenotypic changes in these cells. Recombinant S662V-AAV2 vectors encoding a truncated human telomerase (hTERT) gene were generated and used to stimulate cytotoxic T cells (CTLs) against target cells. S662V-AAV2-hTERT vector-transduced DCs resulted in rapid, specific T-cell clone proliferation and generation of robust CTLs, which led to specific cell lysis of K562 cells. These studies suggest that high-efficiency transduction of moDCs by serine-modified AAV2 vectors is feasible, which supports the potential utility of these vectors for future human DCs vaccine studies.
Bibliographical noteFunding Information:
We thank Drs. Kenneth I. Berns and Nicholas Muzyczka for a critical reading of this manuscript. We thank Robert Ng for assistance in the preparation of Fig. 3 . 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 P01 DK-058327 – Project 1 (to AS), R01 HL-097088 (to AS and SZ), and R01 GM-082946 (to SZ and MA-M) from the National Institutes of Health. GRJ was supported in part by an ‘Overseas Associate Fellowship-2006’ from the Department of Biotechnology, Government of India.
- Adeno-associated virus vectors
- Capsid proteins
- Dendritic cells
- Gene expression
- Serine/threonine kinase