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UV-aided gene therapy promises improved repair of damaged knees

Jul 2006
UVA wavelength helps trigger DNA repair mechanism without harming cartilage cells

Kevin Robinson

The human knee is a very sophisticated joint; it also is one of the most vulnerable to injury. Each year, some 3 million Americans twist their knees badly enough to need surgery. However, even with surgery, some patients never recover total knee function. Many will go on to develop severe osteoarthritis because of damaged cartilage that never fully heals.

Unlike skin and other tissue, cartilage does not receive blood and so does not have the materials to regenerate on its own. Now researchers at the University of Rochester Medical Center in New York have announced that they have taken a significant step toward developing gene therapy that will enable knee cartilage to regenerate. They hope to use long-wavelength ultraviolet (UVA) radiation to switch on genes to make new cartilage instead of scar tissue. They report that UVA is effective at stimulating cartilage cells to incorporate and express a reporter gene delivered by a recombinant virus. Eventually, they hope to deliver a therapeutic gene the same way.

According to Edward M. Schwarz of the university’s Center for Musculoskeletal Research, many animals, such as newts or salamanders, can regenerate entire lost body parts. When their tail is lopped off, for example, genes in the cells at the injury site become active and begin to grow a new appendage. Mammals, on the other hand, are not so lucky. We can regenerate some types of tissue, but we cannot express the genes needed to regrow body parts or cartilage.

For several years, researchers have looked for a gene therapy that could stimulate cartilage growth. The idea is to reprogram the genetic instructions in a cell to switch on the right genes to grow tissue. Research has shown that recombinant adeno-associated viruses work well to get the genes into the cell. Researchers engineer the viruses to contain the genes that cartilage cells need to divide and create more cells.

Unlike other viruses that go wild with self-replication, thus triggering the immune system to respond, the adeno-associated viruses have only a single strand of DNA instead of the usual two strands. This improves the safety because, to use the DNA, the cell must first synthesize a second strand. Unless their DNA is damaged, cartilage cells do not readily make new DNA.

To stimulate the production of DNA using the genes added by the viruses, the researchers use UV irradiation to damage the cells’ DNA and cause them to set about repairing it. In the process, the viral DNA is incorporated into the mix and begins to produce cartilage.

“In our approach,” Schwarz said, “the genes are delivered by a single-stranded DNA virus, which is not active until the cell it infects has been stimulated with UV light. This stimulation turns on the DNA polymerases that are required to convert the single strand of DNA into double-stranded DNA.”

Most research has examined the use of short-wavelength (254 nm) UVC radiation to activate the polymerases. However, according to Schwarz, this wavelength is not good at the job because it has a poor absorption profile and causes too much cell damage at the fluences needed to activate the therapy. He and his group decided to try UVA at 325 nm.

In a paper published in the April issue of the Journal of Bone and Joint Surgery, Schwarz asserted that UVA has several benefits over UVC: It does not actually damage the DNA, it can be transmitted through a fiber optic cable that is compatible with standard surgical instruments, and it can be produced by a laser, enabling short exposure times.

He and his colleagues tested the method in vitro on living human cartilage cells removed during surgery. They also conducted tests in vivo on rabbits. For the in vitro model, they irradiated the cartilage cells with either UVA or UVC light. For UVA light, they used a helium-cadmium laser from Omnichrome Corp. (acquired by Melles Griot Inc.) operating at 325 nm and coupled to a multimode optical fiber. The operating output at the fiber’s end was approximately 12 W. For UVC light, they used a UV cross-linker made by Spectronics Corp. of Westbury, N.Y., operating at 254 nm and approximately 15 W.

To examine the effectiveness of the light at stimulating cartilage cells to incorporate the viral DNA, the scientists exposed the irradiated cells to the virus vector carrying either enhanced GFP or the bacterial ∋-galactosidase reporter gene LacZ. They also looked at the levels of reactive oxygen species, which are believed to play a key role in stimulating the gene therapy response in UVA-treated cells. They measured UV cytotoxic effects at 24 hours after irradiating cells with doses of UVA light escalating from 300 to 15,000 J/m2. Lastly, to measure the ability of the UV radiation to damage the cellular DNA, they measured the levels of pyrimidine dimer formation.

They found that the UVA radiation did not cause cell death at fluences below 6000 J/m2. UVC light, by contrast, kills cells at fluences as low as 100 J/m2. They also found that UVA was effective at stimulating cells to express the adeno-associated viral DNA. The effects are dose-dependent starting at 600 J/m2.

In measuring the pyrimidine dimer formation, the group discovered that UVA does not damage DNA. Even at the highest fluences used for treatment, the light did not generate any pyrimidine dimers beyond a background level. However, it is much more effective than UVC at generating the reactive oxygen species needed for the therapy.

In the rabbit model, the scientists created a knee cartilage injury that they then treated with light-activated gene therapy employing the UVA radiation at various fluences and using viruses laden with enhanced GFP. After a week, the rabbits were sacrificed, and reviewers evaluated the expression of the GFP gene in the cartilage cells.

To test light-activated gene therapy, the researchers irradiated damaged rabbit cartilage with 0 or 6000 J/m2 of UVA light and delivered an adeno-associated virus, tagged with enhanced GFP, to the damaged tissue. Photographs of the defective tissue (A, B, F, G) were taken at 4x magnification, of the edges of the tissue (C, D, H, I) were taken at 10x, and of a region 1.3 mm away from the edge of the damaged tissue (E, J) were taken at 40x magnification.

They found that exposure to UVA radiation increased the expression of adeno-associated viral DNA in the rabbit’s cartilage cells by tenfold. In addition, they observed no UV-light-induced problems with wound healing. Because the virus did not contain a gene designed to repair the damage, they saw no improvement in healing.

Schwarz said that the researchers are experimenting with various therapeutic genes in rabbits and that they hope to move to human clinical trials in four or five years.

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