title: High-efficiency transduction of human monocyte-derived dendritic cells by capsid-modified recombinant AAV2 vectors.
abstract: 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.
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, College of Medicine, Gainesville, FL, USA. aslanidi@peds.ufl.edu
authors.forename: George V
authors.initials: GV
authors.lastname: Aslanidi
authors.forename: Angela E
authors.initials: AE
authors.lastname: Rivers
authors.forename: Luis
authors.initials: L
authors.lastname: Ortiz
authors.forename: Lakshmanan
authors.initials: L
authors.lastname: Govindasamy
authors.forename: Chen
authors.initials: C
authors.lastname: Ling
authors.forename: Giridhara R
authors.initials: GR
authors.lastname: Jayandharan
authors.forename: Sergei
authors.initials: S
authors.lastname: Zolotukhin
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
authors.forename: Arun
authors.initials: A
authors.lastname: Srivastava
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics*; D019943:Amino Acid Substitution; D036022:Capsid Proteins / Q000235:genetics*; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000821:virology*; D005822:Genetic Vectors; D006801:Humans; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics; D012694:Serine / Q000235:genetics; D014161:Transduction, Genetic
chemical_list: D036022:Capsid Proteins; D050505:Mutant Proteins; D012694:Serine