Cartilage injury, which often can lead to osteoarthritis, is a major cause of debility among young athletic horses. Osteoarthritis frequently limits a horse's use, causes chronic disability, and sometimes even necessitates euthanasia. To help heal damaged joints and minimize or reverse arthritic changes in traumatized joints, Alan Nixon BVSc, MS, Dipl ADVS, an equine orthopedist and director of the Comparative Orthopedics Laboratory, is continuing to improve gene therapy for repairing equine cartilage damage.
In his previous work, largely supported by grants from the Harry M. Zweig Memorial Fund, Nixon has successfully cloned a substance called the insulin-like growth factor (IGF-I), which is important for cartilage maintenance in healthy joints. He did so in collaboration with Christopher Evans, a professor of orthopedic surgery at the Center for Molecular Orthopaedics at Harvard University School of Medicine who is an expert inusing gene therapy for treating arthritis in humans. Together they cloned IGF-I because it stimulates chrondrocyte cells to help build new cartilage inside a deteriorating joint. Nixon found IGF-I effective for promoting cartilage growth and the matrix synthesis of the cartilage surface in horses.
In 1998 and 1999, Nixon's work showed that by modifying adenoviruses IGF-I could be inserted into chondrocytes; the researchers found they were able to incorporate the genetically engineered IGF-I gene inside the adenovirus vector backbone. The modified adenoviruses are capable of penetrating living cells without causing any harmand deliver IGF-I DNA into the host-cell genome. Nixon calls the process transfection to distinguish it from infection, which implies having made a disease. Nixon found that this process could result in the production of active IGF-I protein for as long as 30 days.
In fact, largely because of this work, composites of IGF-I and chondro-cytes are now used at the equine hospital at the Cornell University Hospital for Animals to repair cartilage and help improve joint regeneration. But the impact of IGF-I on the activity of transplanted chondrocytes is only short term.
Nixon's work in 2000 confirmed that the equine IGF-I gene could be injected into the horse's fetlock and seed the joint's synovial lining. Evidence of the IGF-I gene expression was still found in joint fluid 90 days later.
Recent work by Nixon also has shown that after cartilage injury, the joint shows an early deficiency of IGF-I, which then peaks at eight weeks, only to decline again beginning at 16 weeks and thereafter.
"This means there's an early and a late window of opportunity when administering supplemental IGF-I may be particularly useful for improving cartilage repair," says Nixon.
With a renewed grant from the Zweig Fund, Nixon and Evans are examining the effects of inserting the gene for IGF-I into chondrocytes just as they're being transplanted into the joint. They are optimistic that this will extend the impact of IGF-I beyond the four weeks previously reported. By inserting the gene for IGF-I, which stimulates chondrocyte metabolism, it is hoped that the healing substances will continue to be produced month after month, much like a manufacturing pump inside the joint, to promote cartilage repair and minimize or reverse arthritic changes in traumatized joints.
The 2001 grant funds an experiment with eight horses. The researchers will compare how cartilage defects in the horses' stifles compare when they are either filled with chondrocytes that have been enhanced with the IGF-I gene or with chondrocytes exposed to a null gene (the control). The first stage of the experiment was to produce more adenoviral IGF-I compounds.
The second stage, which the researchers are completing this summer, involves implanting the adenovira l IGF-I transfected chondrocytes into the horses. In the final stage, they will compare the healing process with arthroscopic examinations and biopsies at one and two months after implants and then will conduct a tissue analysis eight months after the transplants. They will determine how persistent the IGF-I gene is at each point and whether direct injections of IGF-I gene vectors to the joint fluid would be necessary in clinical cases.
"This dual approach builds on our ability to treat not only generalized joint disease by stimulating the production of new cartilage over long periods of time using direct gene therapy approaches, but also specific cartilage injuries with gene-enhanced chondrocyte implantations," explains Nixon. "Both approaches diminish the likelihood of arthritis and may possibly reverse the early stages of arthritis in horses and other animals."
