title: High-efficiency transduction of fibroblasts and mesenchymal stem cells by tyrosine-mutant AAV2 vectors for their potential use in cellular therapy.
abstract: Adeno-associated virus 2 (AAV2) vectors transduce fibroblasts and mesenchymal stem cells (MSCs) inefficiently, which limits their potential widespread applicability in combinatorial gene and cell therapy. We have reported that AAV2 vectors fail to traffic efficiently to the nucleus in murine fibroblasts. We have also reported that site-directed mutagenesis of surface-exposed tyrosine residues on viral capsids leads to improved intracellular trafficking of the mutant vectors, and the transduction efficiency of the single tyrosine-mutant vectors is ∼10-fold higher in human cells. In the current studies, we evaluated the transduction efficiency of single as well as multiple tyrosine-mutant AAV2 vectors in murine fibroblasts. Our results indicate that the Y444F mutant vectors transduce these cells most efficiently among the seven single-mutant vectors, with >30-fold increase in transgene expression compared with the wild-type vectors. When the Y444F mutation is combined with additional mutations (Y500F and Y730F), the transduction efficiency of the triple-mutant vectors is increased by ∼130-fold and the viral intracellular trafficking is also significant improved. Similarly, the triple-mutant vectors are capable of transducing up to 80-90% of bone marrow-derived primary murine as well as human MSCs. Thus, high-efficiency transduction of fibroblasts with reprogramming genes to generate induced pluripotent stem cells, and the MSCs for delivering therapeutic genes, should now be feasible with the tyrosine-mutant AAV vectors.
authors.affiliation: Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Mengxin
authors.initials: M
authors.lastname: Li
authors.forename: Giridhara R
authors.initials: GR
authors.lastname: Jayandharan
authors.forename: Baozheng
authors.initials: B
authors.lastname: Li
authors.forename: Chen
authors.initials: C
authors.lastname: Ling
authors.forename: Wenqin
authors.initials: W
authors.lastname: Ma
authors.forename: Arun
authors.initials: A
authors.lastname: Srivastava
authors.forename: Li
authors.initials: L
authors.lastname: Zhong
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D022021:Tacrolimus Binding Proteins / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes; D014443:Tyrosine / Q000235:genetics*
chemical_list: D014443:Tyrosine; D022021:Tacrolimus Binding Proteins; C403485:tacrolimus binding protein 4