Zweig support is helping Nixon determine the most appropriate delivery system for a genetically engineered growth factor that stimulates cartilage-producing cells.
Alan Nixon's Gene Therapy Techniques Repair Equine Cartilage Damage
Zweig support is helping Nixon determine the most appropriate delivery system for a genetically engineered growth factor that stimulates cartilage-producing cells.
Rather than treating the pain of an arthritic joint with anti-inflammatory agents, Alan J. Nixon, an equine orthopedist and director of the Comparative Orthopedics Laboratory in the College of Veterinary Medicine, is developing gene therapy methods to induce a virtual "drug-manufacturing pump" within the joint. Such a mechanism could produce healing substances month after month to repair cartilage and minimize or reverse arthritic changes in traumatized joints.
"Instead of reducing the inflammatory response in a horse's knee with the injection of steroids or other joint stabilizing agents, we're taking the opposite tack," explains Nixon, an associate professor of surgery. "We want to prevent the joint from further deterioration by stimulating the production of new cartilage over long periods of time."
"We want to prevent the joint from further deterioration by stimulating the production of new cartilage over long periods of time."
Toward this end, Nixon's first task was to build the manufacturing plant by cloning IGF-I, the insulin-like growth factor that stimulates chrondrocyte (cartilage-producing) cells to help build a new cartilage surface inside a deteriorating joint. IGF-I is a strong growth-promoting and matrix-synthesizing stimulant and is the major factor involved in cartilage maintenance in healthy joints.
In the course of isolating and cloning the growth-factor gene for use in the equine gene therapy program, Nixon joined forces with Christopher Evans, the Henry J. Mankin Professor of Orthopedic Surgery at the University of Pittsburgh School of Medicine. Evans has a thriving gene therapy program for treating arthritis in humans.
"There are many small promoter areas that make the machinery of the IGF-I DNA more effective in instructing the cells to produce the proteins that build cartilage," Nixon points out. "Evans has developed several DNA enhancers that can boost this production by 10 to 20 times what is normally seen."
Now in the early phases of an award from the Harry M. Zweig Memorial Fund for Equine Research, Nixon is looking for the most appropriate delivery system-a vector capable of incorporating the genetically engineered IGF-I gene inside the cell nuclei. The two top candidates are modified viruses: a retrovirus and an adenovirus. In addition to evaluating their penetrating abilities, Nixon wants to be sure that these viruses, though no longer virulent, have no negative side effects inside the delicate joint area.
Using simple gene splicing techniques, Nixon will create combinations of the viruses and the IGF-I gene to yield a virion (a virus particle isolated from host cells consisting of nucleic acid within a protein coat) capable of penetrating living cells to deliver viral DNA and IGF-I DNA into the host-cell genome. He chose viruses because they remain the best vectors for getting a large number of virions through the cell membrane without causing it any harm. He calls this process, transfection, to distinguish it from infection, which implies having made a disease.
"If we took a syringe of this material and injected it into the joint of a horse, we'd confidently expect most cells to be transfected," he says.
There is one discrete downside to the use of an adenovirus, however. While the adenovirus penetrates the cell wall to insert the IGF-I gene, it never becomes fully incorporated into the cell's own DNA. Hence, when the cells divide, the growth-promoting factor isn't carried over to the daughter cells. This results in strong initial therapeutic effects that are unfortunately of short duration, perhaps only a few months.
Retroviruses, on the other hand, become locked inside a cell's DNA, thus fixing the growth-promoting factor in place, potentially for years. Preliminary studies in horse cartilage cells show retrovirus transfection rates of up to 24 percent.
In subsequent phases of his research, Nixon plans to use retrovirus vectors to transfect chondrocyte cells he grows for transplants being done in the college's Equine Hospital. These grafted cells would thereby have an enhanced capability of synthesizing a new cartilage surface once inside the joint.
The final research phase will test the ability of both virus vectors to transfect synovial and cartilage cells in living animals and the impact transfection has on cartilage lesions as well as on several models of early osteoarthritis in horses.
This is a very exciting time in gene therapy, and Nixon is the first to use cartilage enhancing agents in treating equine arthritis through gene therapy approaches.
"We now know the little segments of DNA that are essential for initiating cartilage cell function and can make them by simply programming a machine in the Biotechnology Building on campus," he says. "By joining them with the right vector, we hope not only to improve cartilage healing in acute injury, but also for the first time, anticipate some reversal of the early stages of arthritis in horses and other animals."
