Safety and Efficacy of Ultraviolet-A Light-Activated Gene Transduction for Gene Therapy of Articular Cartilage Defects

• Published in: Journal of bone and joint surgery. American volume 2006-04, Vol.88 (4), p.753-761
• Main Authors: Maloney, Michael D, Goater, J Jeffrey, Parsons, Richard, Ito, Hiromu, OʼKeefe, Regis J, Rubery, Paul T, Drissi, M Hicham, Schwarz, Edward M
• Format: Article
• Language: English
• ISSN: 0021-9355; 1535-1386
• DOI: 10.2106/JBJS.E.00400

BackgroundGene therapies for articular cartilage defects are limited by the absence of an in vivo delivery system that can mediate site-specific transduction restricted to within the margins of the defect during routine arthroscopy. We have proposed the use of ultraviolet light to stimulate gene expression following infection by recombinant adeno-associated virus (rAAV). However, research has demonstrated that short-wavelength ultraviolet light (ultraviolet C), while effective, is neither safe nor practical for this purpose. We evaluated the safety and efficacy of long-wavelength ultraviolet light (ultraviolet A) from a laser to induce light-activated gene transduction in articular chondrocytes in vitro and in vivo.MethodsThe effects of ultraviolet A from a 325-nm helium-cadmium laser, delivered through a fiberoptic cable, on cytotoxicity, mutagenesis, intracellular reactive oxygen species, and light-activated gene transduction of human articular chondrocytes were evaluated in dose-response experiments of primary cultures. Cytotoxicity was determined by trypan blue exclusion. The presence of pyrimidine dimers in purified genomic DNA was determined by enzyme-linked immunosorbent assays. Intracellular reactive oxygen species levels were determined by flow cytometry at one hour and twenty-four hours. In vitro light-activated gene transduction with rAAV vectors expressing the green fluorescent protein (eGFP) or β-galactosidase (LacZ) was determined by fluorescence microscopy and bioluminescence assays, respectively. In vivo light-activated gene transduction was quantified by stereotactic immunohistochemistry for β-galactosidase in rabbit articular cartilage defects in the patellar groove that had been irradiated with ±6000 J/m of ultraviolet A one week after direct injection of 10 transducing units of rAAV-eGFP.ResultsUltraviolet A failed to induce significant cytotoxicity at all fluencies below 6000 J/m. Dose-dependent cytotoxicity was observed at greater fluencies. In contrast to ultraviolet C, which induced significant (p < 0.05) pyrimidine dimer formation at all fluencies in a dose-dependent manner, ultraviolet A failed to induce DNA modifications. Conversely, ultraviolet C proved to be a poor inducer of intracellular reactive oxygen species, while ultraviolet A immediately induced high levels of intracellular reactive oxygen species, which were completely resolved twenty-four hours later. Ultraviolet A demonstrated significant light-activated gene transdu