Dr. Alan J. Nixon
Joint diseases of many kinds, from simple swellings to advanced arthritic conditions, generally stem from cartilage developmental diseases or traumatic injury. They frequently follow an exacerbatory cycle to end in painful and dysfunctional joints. Prevention of arthritis is the major focus of research in cell based cartilage repair and drives the pharmaceutic industry to invest billions per year on R&D to abrogate pain in arthritic animals and man. Estimates of yearly cost to the horse industry reach as high as 100 million dollars which pales in comparison to the 2 billion spent annually on treatments for joint disease in man. Surgical methods to re-grow joint cartilage after injury aim to prevent arthritis and return horses to athletic potential. Research in the investigators lab has focused on cartilage and stem cell transplants bolstered by growth factor composites that are added to the transplant milieu. However, residence time for growth factor proteins is known to be limited to 1-to-2 weeks. To overcome this short-term depot, our research group has been exploring gene therapy methods used to introduce functional portions of the insulin-like growth factor-I (IGF-1) gene into joints damaged by OCD, acute injury, or the early stages of arthritis. Growth factors, particularly IGF-1, stimulate cartilage cell metabolism and thereby maintain healthy joints. After injury, the cartilage homeostatic balance is perturbed by a proliferation of degradatory enzymes and other bioactive peptides that insidiously damage the overall cartilage structure. The restoration of this balance normally depends on reduced exercise, surgical intervention, oral anti-inflammatory and pain relieving agents, along with extended periods of rest. The natural body check on rampant joint degradation comes primarily through antagonists to cytokine action, one of the most important of these being interleukin- I receptor antagonist AIL-l Rad. Untreated or severely damaged joints frequently develop osteoarthritis, which remains a leading cause of retirement of horses from active racing and often precludes even modest exercise programs. Enhanced levels of stimulatory growth factors, such as IGF-1, can be experimentally provided by articular injection or the use of slow-release polymers. However both result in only short periods of growth factor exposure, with little possibility of long-term impact on the joint. Methods to, permanently enhance growth factor concentrations in the joint are being explored in this grant and will utilize previous work on genetically engineered equine IGF-I constructs introduced to joints by viral vectors, resulting in the IGF-I gene being incorporated into the cell nuclei of joint lining and cartilage cells. A new generation of nonpathogenic viral vectors have been developed, and the effectiveness of their delivery of the IGF-I gene to articular structures is currently being tested. Recent untoward experiences in several human gene therapy trials using systemic adenovirus, gene therapy protocols have stimulated development of safer vectors for gene modulation of various genetic, developmental, and malignant diseases in man. This grant will continue to test the much-heralded adeno-associated virus (AAV) for its efficiency an long-term persistence for 1GF-I gene delivery. This focus on enhanced metabolism provides only one side of the homeostatic equation in joints. A reduced catabolic cascade would provide a complementary focus to reduce joint inflammation and cartilage degradation. Our collaborators at Colorado State University have developed an equine IL- I Ra and those at Harvard have refined construction of both 1GF-I and IL-1 Ra genes into new adeno-associated vectors. This grant tests these in lab conditions year 1) as well as in animal studies year 2).
Our previous Zweig-funded studies have allowed us to clone and sequence equine 16F-1, and provided proof of principle data showing that 1GF-I coding regions can be inserted into equine joint lining and cartilage repair cells using adenovirus, vectors. This genetic coding insert produced IGF-I protein for up to 4 weeks in equine tissue culture systems and, most importantly, in subsequent in vivo horse trials. Further, our evaluation of the native expression of this growth factor after cartilage injury showed two windows of time at 2 and 16 weeks when there are deficiencies in 1GF-I levels and supplemental endogenous IGF-I may be particularly useful in improving cartilage repair. IGF-I has minimal effects on cell division but significant impact on cartilage matrix proliferation. Application of composites of IGF-I and chondrocytes, to cartilage repair has resulted in significant joint regeneration and now represents common clinical practice for equine patients of the Cornell University Hospital for Animals. Initial research trials with IGF-I gene therapy indicated the relevant gene could be inserted into chondrocytes used for transplant procedures. These cells then carried the gene with them, which translocated to the cell nucleus and coded for IGF-I protein in effective quantities for up to 1 month. Further in vitro and in viva work at Colorado State University Orthopaedic Research Laboratories showed that synovial lining cells could be laden with the IL-1 Ra gene which provided clinical improvement in a knee chip model of arthritis. Despite the promising proof of principle results, adenovirus, vectors do not integrate their cargo gene with the host cell DNA material. Newer vectors, such as the adeno-associated virus, do integrate with the host cell genome and hold promise for months-to-years of gene expression. This, combined with a lack of any immune response to the vector, suggests a superior end result.
This will be the first experiment to use adeno-associated vectors to deliver growth factor and catabolic blacker genes to articular cells. Previous studies allowed us to develop a new AAV vector and streamline virus production methods, which will enhance the efficiency of integration and speed of onset of the viral vectors containing equine IGF-I and IL-IRA coding sequences in cartilage and joint lining cells. Previous work also confirms the equine IGF-I gene produces IGF-I protein when inserted into cells using the adeno-associated virus as vector. Further work is currently underway to optimize the virus penetration and gene insertion capability of AAV in equine chondrocytes and joint lining cells. Concurrent work is in progress at Colorado State to optimize the AAV.IL-IRa vector. The equine IL-lRa gene was sequenced and the protein used in animal studies which showed decreased lameness in horses with early arthritic disease. The current proposal combines the positive results of IL- I Ra to control joint degradation and IGF-I to stimulate joint repair. The first year of this 2-year grant will test the two AAV constructs in a novel laboratory culture model of a joint, complete with joint lining cells, joint fluid, and cartilage surface pieces. The second year of the project will then test whether IGF-I gene laden chondrocytes can produce a more durable cartilage surface than simple cells, and follow up with a study of additional benefit is derived from quelling the arthritic reaction in chondrocyte transplanted joints by direct injection of IL-1Ra. Preliminary data shows AAV vector can penetrate and deposit gene segments to equine cartilage cells and joint lining cells, so we expect the AAV.IGF-I and AAV.IL- I Ra to work similarly.
In summary, work in this grant cycle will involve a combination of in vitro and in vivo studies. Few labs have significant experience propagating an adeno-associated virus. Our long-term collaboration with Drs. Evans and Ghivizzani provides us unique access to adeno-associated lab techniques. Furthermore, additional volumes of our AAV.IGF-I construct have been ordered from a commercial lab at the University of North Carolina , Chapel Hill . Our hypothesis is that both IGF-I and IL- I Ra genes can be driven into the articular cells' permanent genome by adeno-associated viral vectors, without significant cell damage, and with a titer sufficiently high to establish local permanent production of IGF-I and IL-1Ra proteins and subsequent accelerated cartilage repair.