The Harry M. Zweig Memorial Fund for Equine Research


Pro-Inflammatory Cytokine Targets in Joint
Disease and Check-Points for Gene Inhibition

Dr. Alan J. Nixon

NixonAthletic horses often start to develop insidious concussive joint erosions as the intensity of training and competition increases. The early warning signs in 2-year-old racehorses include joint fluid build-up and pain on manipulation of the affected joints. Alert trainers treat these signs by changes in exercise patterns and intensity, cold therapy, oral anti-inflammatory agents, and occasionally systemic joint medication. Rarely, flaking and chip fragmentation of the joint surface requiring surgery develops. As 3- to 4-year-olds, actual cartilage loss limits the response to most therapies. Steroids are introduced to more potently quell the symptoms, but often accelerate structural damage to the joint and can lead to insidious arthritic degradation with increasing pain and dysfunction. Estimates of the annual cost of joint injury and arthritis to the equine industry approach $100 million. It has become increasingly clear that control of arthritis will not progress until we understand the key degradatory cytokines in equine joint disease. Prevention of arthritis is the major focus of our research in cell-based cartilage repair using growth factor gene or protein methods to drive healing of traumatized joints. Research in the investigator’s laboratory has focused on both cartilage and adult stem cell transplants, bolstered by growth factor composites added at surgery.  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, these systems add cells and growth factors to stimulate cartilage cell function, but do not quell the enzymes and degradation pathways that continue to erode the joint surface.

Osteoarthritis has a complex of precipitating traumatic causes.  However, it follows a common pathway after perturbation of cartilage homeostasis, with proliferation of degradatory enzymes and other inflammatory proteins that erode the joint.

acute stifle injury

 

 

Figure 1. Acute stifle injury often leads to chronic degeneration and arthritis, with meniscal tearing and joint surface erosions (left).  The number and type of degradatory enzymes and other destructive agents in equine joint disease that weaken and fibrillate the cartilage (right), are largely unknown. This grant profiles these agents, develops mechanisms to control their flux, and tests them separately and in combination.

 

 

Our research program aims to better understand the fundamental interaction of these inflammatory mediators in acute joint injury, chronic surface erosion and chip fracture, and subsequent arthritis.  Targeted treatment with cytokine gene silencing may halt the progression from joint surface injury and fragmentation to arthritis. Two cytokines, interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), are thought to be the principal messengers of destruction in arthritis. Our previous work suggests that controlling IL-1 through over-expression of its own natural inhibitor or gene silencing through IL-1 RNA interference restores cartilage function for several weeks. Despite the benefit of local knockdown of the IL-1 gene product (before it can be synthesized into the active cytokine), our studies indicate continued expression of other degradatory players such as TNF-α may partly account for the poor recovery of suppressed cartilage structural components. The current proposal will examine the fundamental interactions of cytokines in natural joint disease, develop a tier of inflammatory targets for treatment, examine the repercussions of single gene knockdown on other cytokine profiles, and finally develop multimodal gene silencing constructs that may provide a comprehensive therapeutic product.

We hypothesize that numerous inflammatory cytokines and enzymes are elevated in arthritis and that IL­1 and TNF are the master regulators, both of which need to be silenced before cartilage structural genes can function to rebuild cartilage matrix.

Our previous studies show catabolic IL-1 gene fluxes can be controlled in articular tissues by gene knockdown. Despite reduced IL-1 formation, our experiments show a recalcitrant suppression of cartilage component formation, essentially failing to rebuild the damage to the joint surface. This grant will examine the fundamental interactions of degradatory cytokines in natural joint disease, develop a tier of inflammatory targets, examine the repercussions of single gene knockdown on other cytokine profiles, and finally develop multicomponent gene silencing constructs that may provide a therapeutic modality.  Previous Zweig Funded grants during 2005-2006 screened IL-1 silencing motifs and in 2007 allowed development of stable IL-1 gene induced silencing suitable for clinical application. This proposal uses the Cornell equine genome project gene chip, developed in our lab, to deepen our understanding of the inflammatory agent profile in equine traumatic joint disease, and assesses the interplay of inflammatory cytokines/enzymes and growth factors. Until we understand these molecular events, we can not hope to appropriately target the most destructive cytokines, nor which cytokine group more profoundly suppresses active cartilage reformation.

This 2-year proposal aims to:
1) Use gene chip and other molecular expression assays to profile damaging gene expression in equine joint disease, specifically horses with knee chips.
2) Test IL-1 and TNF gene silencing motifs alone and in combination to determine how completely they can control joint reaction.
3) Evaluate expression of other cytokines and pro-inflammatory mediators suppressing cartilage rebound after silencing IL-1 or TNF.

Previous work has identified one highly efficient IL-1 gene knockdown agent from dozens tested in cartilage and joint lining cells. This coding segment has been assembled into a DNA fragment capable of self-sustained production of new silencing agents from the individual target cells. This “silencing microfactory” is unique in medicine and has cross-over to arthritis in man. Moreover, we have established a collaboration with Dr John Rossi in California, a leader in the field of gene silencing to control HIV infection, who has guided our work in developing multiple coding regions in the one construct.  Additionally, through another collaboration with Dr Perry Hackett of the University of Minnesota, we have further formed this coding region into a gene package (coined Sleeping Beauty™) capable of permanent integration into target cells so the gene is not lost during cell division. Both plasmids effectively knockdown IL-1, but do little to help rebuild cartilage. We have developed two strategies to overcome this. 1) Supplement stimulatory growth factors through gene therapy which has been a focus of our lab for 6 years. (now the major part of a pending NIH grant). 2) Verify the target hierarchy in naturally occurring joint disease and widen the search by assessing expression of all major degradatory agents.

The experiments in this two-year grant will use gene expression arrays and histologic techniques to profile pro-inflammatory cytokine expression in the joint lining from a range of Thoroughbred and Standardbred knee chip and arthritis cases, employ laboratory culture techniques to examine the efficiency of IL-1 and TNF cytokine RNA interference products, and use gene and protein assays to measure cross-talk between these cytokines and other agents in the joint lining and adjacent cartilage. The project long-term goal is to assemble multiple coding regions targeting the key elements in joint destruction onto the one DNA backbone, and eventually to test this gene based anti-inflammatory motif in animals with cartilage injury and finally in horses with arthritic joints. Ultimately, we hope to return horses with possible career-ending joint injury back to being competitive athletes.