title: Light-activated gene transduction of recombinant adeno-associated virus in human mesenchymal stem cells.
abstract: Deficiencies in skeletal tissue repair and regeneration lead to conditions like osteoarthritis, osteoporosis and degenerative disc disease. While no cure for these conditions is available, the use of human bone marrow derived-mesenchymal stem cells (HuMSCs) has been shown to have potential for cell-based therapy. Furthermore, recombinant adeno-associated viruses (rAAV) could be used together with HuMSCs for in vivo or ex vivo gene therapy. Unfortunately, the poor transduction efficiency of these cells remains a significant obstacle. Here, we describe the properties of ultraviolet (UV) light-activated gene transduction (LAGT) with rAAV in HuMSCs, an advance toward overcoming this limitation. Using direct fluorescent image analysis and real-time quantitative PCR to evaluate enhanced green fluorescent protein (eGFP) gene expression, we found that the optimal effects of LAGT with limited cytotoxicity occurred at a UV dose of 200 J/m(2). Furthermore, this UV irradiation had no effect on either the chondrogenic or osteogenic potential of HuMSCs. Significant effects of LAGT in HuMSCs could be detected as early as 12 h after exposure and persisted over 21 days, in a time and energy-dependent manner. This LAGT effect was maintained for more than 8 h after irradiation and required only a 10-min exposure to rAAV after UV irradiation. Finally, we show that the production of secreted TGFbeta1 protein from rAAV-TGFbeta1-IRES-eGFP infected to HuMSCs is highly inducible by UV irradiation. These results demonstrate that LAGT combined with rAAV is a promising procedure to facilitate gene induction in HuMSCs for human gene therapy.
authors.affiliation: The Center for Musculoskeletal Research, University of Rochester, Rochester, NY 14642, USA.
authors.forename: H
authors.initials: H
authors.lastname: Ito
authors.forename: J J
authors.initials: JJ
authors.lastname: Goater
authors.forename: P
authors.initials: P
authors.lastname: Tiyapatanaputi
authors.forename: P T
authors.initials: PT
authors.lastname: Rubery
authors.forename: R J
authors.initials: RJ
authors.lastname: O'Keefe
authors.forename: E M
authors.initials: EM
authors.lastname: Schwarz
mesh_terms: D002478:Cells, Cultured; D019902:Chondrocytes; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage*; D049452:Green Fluorescent Proteins; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D010011:Osteocytes; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells; D014161:Transduction, Genetic / Q000379:methods; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000235:genetics; D014466:Ultraviolet Rays
chemical_list: D008164:Luminescent Proteins; D016212:Transforming Growth Factor beta; D049452:Green Fluorescent Proteins