Light-activated gene transduction enhances adeno-associated virus vector-mediated gene expression in human articular chondrocytes.
OBJECTIVE
To evaluate the effects of ultraviolet (UV) light as an adjuvant for recombinant adeno-associated virus (rAAV) transduction in human articular chondrocytes.


METHODS
Primary articular chondrocytes and immortalized chondrocytes (tsT/AC62) were exposed to various doses of UV light (0-1,000 J/m(2)) and infected at various multiplicities of infection (MOIs) with rAAV containing the enhanced green fluorescent protein (EGFP) gene. Cells were analyzed for viability and EGFP expression by fluorescence-activated cell sorting on days 2, 4, and 8 following infection. To evaluate the transduction efficiency in intact articular cartilage, full-thickness explants were exposed to UV light (0-200 J/m(2)), infected with rAAV-eGFP, and analyzed for transduction via immunohistochemistry.


RESULTS
Toxicity from UV exposure was observed at doses > or =500 J/m(2) and > or =200 J/m(2) in primary and immortalized chondrocyte cultures, respectively. Transduction efficiency was dependent on the UV dose, MOI, and time. In the cell line, the adjuvant effect of UV on the percentage of cells transduced was modest, but 100 J/m(2) increased the mean fluorescence intensity (MFI) of the transduced cells 4-fold. In contrast, UV treatment had a profound effect on the transduction efficiency of primary chondrocytes, which reached approximately 100% after exposure to 100 J/m(2) of UV light and 10(3) MOIs for 8 days. Under the same conditions, 200 J/m(2) of UV light enhanced the MFI 7-fold. In cartilage explants, there was no difference in the number of transduced chondrocytes at the edge of the explants in the superficial, intermediate, or basal zones; however, 200 J/m(2) of UV light increased the transduction efficiency 2-fold at a low MOI. In the center of the explants, the superficial chondrocytes were efficiently transduced; those in the intermediate and basal zones could not be efficiently transduced under any condition. In the superficial chondrocytes, a low MOI and 200 J/m(2) of UV light increased the transduction efficiency 3-fold (to 100%).


CONCLUSIONS
UV light at doses of up to 200 J/m(2) (which do not significantly affect cell viability) significantly enhances the transduction efficiency and expression of the transduced gene in cultures of rAAV-infected primary chondrocytes and in chondrocytes in the superficial zone of intact articular cartilage. These findings support the concept that UV-activated gene transduction could be used as an adjuvant for in vivo rAAV articular cartilage gene therapy with low viral titers to prevent and/or treat arthritis.
Ulrich-Vinther
Michael
M
The Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York 14642, USA. Edward_Schwarz@URMC.Rochester.edu
Maloney
Michael D
MD
Goater
J Jeffrey
JJ
Søballe
Kjeld
K
Goldring
Mary B
MB
O'Keefe
Regis J
RJ
Schwarz
Edward M
EM
D000293:Adolescent; D000328:Adult; D002358:Cartilage, Articular / Q000378:metabolism / Q000528:radiation effects* / Q000821:virology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism / Q000528:radiation effects* / Q000821:virology; D000229:Dependovirus / Q000235:genetics*; D004307:Dose-Response Relationship, Radiation; D005260:Female; D015870:Gene Expression / Q000528:radiation effects*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D014161:Transduction, Genetic; D019076:Transgenes; D014466:Ultraviolet Rays
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins