UV-aided gene therapy promises improved repair of damaged knees
UVA wavelength helps trigger DNA repair mechanism without harming cartilage cells
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
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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