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 cellbased 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 TGFb1 protein from rAAV-TGFb1-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.

Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

“…14 In comparison to lentiviral vectors, retrovirus-based systems tend to lose transgene expression over time 15 and AAV-based systems are less efficiently transduced into MSCs. 16 Alternatively, when MSCs are used to treat noninherited diseases and are only required to express the therapeutic gene for a short period of time, nonintegrating vectors including adenoviruses or herpes saimirii viruses (HSV) are preferred. Although the first generation of adenovirus vectors was successfully modified to enhance MSC transduction efficiency, 17 the development of a strong immune response directed against adenoviral proteins still limits their overall efficacy.…”

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases

“…To overcome these shortcomings, Ito et al [33] used an ultraviolet (UV) lightactivated transduction system to improve the delivery of AAV vectors into human MSCs. This procedure involving UV irradiation had no effect on either the chondrogenic or osteogenic potential of MSCs.…”

Biological activation of bone‐related biomaterials by recombinant adeno‐associated virus vector

“…[34][35][36] Gene therapy for bone regeneration has not been studied sufficiently with rAAV, probably because of the weak expression from these vectors. However, with several modalities available to enhance gene expression from rAAV, 21,25 further research is warranted.…”

“…24 Moreover, rAAV has powerful effects on many cell types, including mesenchymal stem cells and chondrocytes. 18,25,26 We previously investigated the use of lyophilized rAAV on allografts with promising results in bone allograft healing. 18,27 We also tested the use of the lyophilization method on bone-related biomaterials as a means of expanding their use.…”