An ultraviolet-A (UVA) laser beam transmitted down a fiber-optic cable might permit safe, accurate, site-specific gene therapy for damaged joint cartilage, University of Rochester researchers report in the April issue of the Journal of Bone & Joint Surgery.¹ As with all gene therapy, attempts to fix damaged knee cartilage by gene engineering requires the ability to get the repair gene to the right place, keep it there, and turn it on. According to the investigators, "This is the first demonstration of in vivo site-directed gene delivery to articular defects [using] a method that is highly compatible with standard arthroscopy."

The treatment is designed to mimic the embryonic gene expression that results in chondrogenesis during fetal development. This is done by removing articular chondrocytes, infecting them with recombinant adeno-associated virus (rAAV), then inducing gene expression with ultraviolet light.

Prior attempts to achieve this using short-wavelength UV-C light successfully induced DNA polymerases and induced tissue regrowth, but also destroyed DNA in nearby healthy tissues. The researchers then turned to UV-A as a possibly safer alternative, senior author Edward M. Schwarz, PhD, told CIAOMed. UV-C activates genes by creating DNA damage and triggering cellular repair mechanisms. UV-A apparently creates free radicals, which then induce gene expression without damaging DNA. UV-A has the additional advantage of being transmissible via fiber-optic cable, making it potentially applicable as an arthroscopic procedure.

The new study provides proof of concept that this approach is feasible in vivo. The researchers first demonstrated that UV-A light can be used to activate genes injected into cartilage defects, using cultured chondrocytes transfected with rAAV carrying a reporter gene that makes a visible product when expressed. The procedure is extremely safe and causes no cytotoxicity.

The in vivo work was done using a rabbit model in which a standardized articular defect was made on the patellar trochlea. Following anesthesia and creation of the defect, the damaged area was exposed to 0–6000 J/m2 of UV-A; 10⁷ transducing units of rAAV carrying the marker-enhanced green fluorescent protein (eGFP) were then injected into the injured area.

One week later both the experimental knee and the uninvolved contralateral control knee were sectioned and examined in 3-mm slices. A score was derived for each animal based on the total number of articular chondrocytes, the number of eGFP-positive chondrocytes, and the number of empty lacunae in the fields immediately adjacent to the defects in 40X photomicrographs.

While similar studies using UV-C produced significant cell death 24 hours after treatment, the UV-A induction produced no significant cytotoxicity, leading researchers to conclude that human articular chondrocytes are able to tolerate much higher doses of UV-A compared with UV-C. Chondrocytes expressing the eGFP marker gene derived from the injection were only present in the areas immediately surrounding the cartilage defect.

According to Dr. Schwarz, the safety results had been expected, but the researchers were surprised by a dramatic improvement in the efficiency of gene transfer to chondrocytes in the area around the injury, which was 10-fold higher in the knees exposed to UV-A. Future experiments will determine whether UV-A-activated genes can actually increase the production of new chondrocytes and the formation of new cartilage in this animal model. "We have yet to use a 'therapeutic' gene," he said.

Reference

1. Maloney MD, Goater JJ, Parsons R, et al. Safety and efficacy of ultraviolet-A light-activated gene transduction for gene therapy of articular cartilage defects. J Bone Joint Surg. 2006;88:753-761.