The Harry M. Zweig Memorial Fund for Equine Research

Enhanced Joint Function Through Targeted RNA Interference

Dr. Alan J. Nixon

Damage to cartilage in high-motion joints can result from either acute injury during athletic events or from developmental orthopedic diseases such as osteochondritis dissecans (OCD). Both generally follow a cycle of persisting cartilage deficit, insidious arthritic degradation, and increasing pain and dysfunction. Prevention of arthritis is the major focus of our research in cell-based cartilage repair and the use of protein-directed methods to diminish degradatory enzyme levels. Estimates of the annual cost of joint injury and arthritis to the equine industry approach $100 million, which pales in comparison to the $2 billion directed at treating osteoarthritis in man. Surgical methods to resurface joint 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 other enzymes and degradation pathways involved in joint surface erosion. In this grant 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 100 equine cases suffering from stifle, shoulder, fetlock, and knee injuries and developmental syndromes such as OCD. However, little attention has been directed toward the IGF binding proteins, which can diminish IGF-I interaction with receptors. Extension of the impact of IGF-1 may be derived by diminishing the levels of these natural binding proteins that sequester IGF-1. The most important of these binding proteins is IGF binding protein-3 (IGFBP-3). Down-regulation of this binding protein will unshackle IGF-1 and increase ligand levels for receptor activation and positive signaling.

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 rapid joint degradation comes primarily through antagonists to the erosion-inducing protein, interleukin-1 (IL-1). Permanent abrogation 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.

Our previous Zweig-funded studies allowed us to clone and sequence equine IGF binding proteins. Similarly, animal studies have verified the impact of IGF-1 on cartilage cell function and the resulting enhanced cartilage repair. IGF binding protein sequence data will allow us to design and synthesize a specific nucleic acid interference fragment. 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 expect it will be extraordinarily useful in treating musculoskeletal syndromes. The key discovery was that short segments of RNA target full length messenger RNAs for destruction within the cytoplasm. This process provides a balance to messenger RNA production. By utilizing predictive computer programs, we have designed several interfering RNAs that should reduce the level of the IGF binding protein-3. In so doing, the action and longevity of natural IGF-1 synthesized by chondrocytes will be enhanced. This represents an indirect way of bolstering IGF-1 actions within the joint. The second and complementary phase of RNA interference will come from a reduction in the primary agent of cartilage loss, interleukin 1 (IL-1). The gene sequence for equine IL-1 is known, and this has allowed us to design interfering RNAs that will diminish RNA coding for the highly destructive protein IL-1 b . These two interfering RNAs target totally different aspects of cartilage homeostasis; one the inflammatory cascade and the other the control of stimulatory molecules. They are expected to have complementary actions in restoring joint function.

The experiments in this two-year grant will use laboratory culture techniques to examine the efficiency of the RNA interference products designed by the investigators. The initial year will use simple cell culture systems to individually determine the knock-down success of IGFBP-3 and then later IL-1 b . These systems of RNA interference have not been examined previously in joint disease, therefore screening techniques will be used in culture systems to identify the interfering sequences that perform best for both target RNA molecules. The second year of the study will extend the information on the interference of IGFBP-3 and IL-1 b to an arthritic joint model, using arthritic cartilage and synovial lining cell co-cultures to 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. Ultimately the investigators' long-term goal is to examine RNA interference in models of natural joint disease in the living animal.