The Harry M. Zweig Memorial Fund for Equine Research

Joint Repair Through Targeted RNA Interference and
Anabolic Gene Induction

Dr. Alan J. Nixon

Damage to cartilage in high-motion joints can result from either acute injury during racing and training or from developmental orthopedic diseases such as osteochondritis dissecans (OCD). Both generally follow a cycle of acute cartilage deficit, followed by insidious arthritis characterized by increasing pain and dysfunction. Estimates of the annual cost of joint injury and arthritis to the equine industry approach $100 million. Prevention of arthritis is the major focus of our research in growth factor and cell-based cartilage repair to drive new cartilage deposition in injured joints. Surgical methods to resurface cartilage after injury can to some extent prevent arthritis and return horses to athletic potential. Research in the investigator's laboratory has focused on both cartilage and adult stem cell transplants, bolstered by growth factor composites added at surgery. However, these systems add cells and growth factors to stimulate cartilage cell function, without regard for the many degradation pathways involved in joint surface erosion. Over the past year, and in the new work proposed for 2007-08, we seek ways to control the degradation systems that flourish in joint disease, and in so doing push existing methods for joint repair to the next level. Insulin-like growth factor-1 (IGF-1) is a valuable adjunct to cell-based therapies in joint repair. Application of mixtures of cartilage cells and IGF-1 has been used in over 120 equine cases suffering from stifle, shoulder, fetlock, and knee injuries and developmental syndromes such as OCD. However, little attention has been directed at combining IGF-1 therapy with agents to improve the local host environment through knockdown of inducers of arthritis, predominantly interleukin-1 (IL-1)

Osteoarthritis is a complex disease with numerous precipitating causes. However, it follows a common pathway after perturbation of the cartilage metabolic balance, with proliferation of degradatory enzymes and other inflammatory proteins that erode the joint. The body's natural check on joint degradation comes primarily through antagonists to the erosion-inducing protein, interleukin-1 (IL-1). Control of cartilage destruction may result from blocking IL-1 before it is synthesized. This can be done by new technology that employs small specific fragments of RNA that destroy coding RNA before it forms protein in the cell. RNA interference relies on a newly discovered natural mechanism that evolved in plants and non-mammalian species to allow cells to reduce the load of pathologic organisms such as invading viruses. Recent investigations suggest it can be used in mammals to control diseases as varied as cancer, neurodegenerative conditions, and AIDS. We have shown in the past year that it can be useful in treating musculoskeletal syndromes. The key discovery was that short segments of RNA target full length messenger RNAs for removal within the cytoplasm. This process provides a balance to messenger RNA production.

Our research using Zweig funding for the past 18 months has established the potent benefit of local silencing of genes coding for degradatory enzymes before they can be synthesized. By harnessing gene synthesis methods we have shown that cartilage and joint lining cells can continually dampen production of the enzymes that erode cartilage. Current studies suggest that despite being able to quell destructive enzymes, concurrently suppressed cartilage matrix formation does not bounce back as completely as we had expected. The current proposal combines genes for silencing destructive enzymes (using RNA interference) with a growth factor gene that will build up cartilage integrity.

We hypothesize that damaged and arthritic cartilage can be repaired and stabilized by the use of RNA interference techniques to reduce enzyme-mediated cartilage softening combined with IGF-I gene insertion to provide concurrent repair action to restore cartilage density.

By utilizing predictive computer programs, we have designed and tested 8 interfering RNAs that reduce the level of the RNA coding for the highly destructive protein IL-1. One of these 8 was extraordinarily effective, bordering on 100% efficient in quelling this destructive gene. We now want to help re-build cartilage by using our previously developed IGF-I gene insertion techniques in combination with IL-1 knockdown. This 2-year proposal will allow further testing of IL-1 knockdown constructs in joint lining cells and the potential synergism with stimulatory IGF-I gene function.

The experiments will use laboratory culture techniques to examine the efficiency of the RNA interference products designed by the investigators. The initial year will continue on the past 18 months work, using simple cell culture systems to better determine knock-down success of IL-1 b in joint lining cells. We have screened numerous interfering RNA constructs for their knockdown effects in cartilage cells. The complete joint has considerable cross-talk between joint lining and cartilage, and examination of damaging enzyme levels at the joint lining level, and then by combined culture of joint lining and cartilage (co-culture) will be vital in establishing the value of RNA interference methods. Arthritic cartilage and synovial lining cell co-cultures will duplicate action in the live animal. The final phase will develop vectors that permanently seed target cells with coding sequence to produce these short interfering RNAs as well as carry in sequence used previously to induce IGF-I synthesis to assist in joint recovery. Both coding regions will be assembled onto the one DNA backbone, and eventually tested in animals with cartilage injury and finally in horses with arthritic joints. Ultimately, we hope to return horses with career-ending joint disease back to competitive athletes, and at the very least, improve their quality of life in retirement or less athletic pursuits.