Dr. Alan J. Nixon
The objective of this program is the continued exploration of gene therapy procedures to introduce functional portions of the insulin-like growth factor-I (IGF-I) gene into joints damaged by OCD, acute injury, or those in the early stages of arthritis. Growth factors, particularly IGF-I, are predominantly involved in the maintenance of healthy cartilage by stimulating cartilage cell metabolism. After injury, this cartilage homeostatic balance is perturbed by a proliferation of degradatory enzymes and other bioactive peptides which insidiously damage the cartilage structure. The restoration of this balance normally depends on reduced exercise, surgical intervention, oral anti-inflammatory and pain relieving agents, and extended periods of rest. 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-I can be experimentally provided by articular injection or the use of slow-release polymers. However, both result only in short periods of growth factor exposure, with little possibility of long-term impact on the joint. Methods to permanently enhance growth factor articular concentrations are being explored in this grant and utilize previous work on genetically engineered equine IGF-I constructs which will be introduced to joints by viral vectors, resulting in incorporation of the IGF-I gene into the cell nuclei of joint lining and cartilage cells. Despite recent untoward experiences in several human systemic gene therapy trials, the use of local gene therapy protocols, such as local joint delivery, remains a safe and effective mechanism to improve joint health in the long-term.
Our previous Zweig funded studies have cloned and sequenced both equine IGF-I and transforming growth factor-beta (TGF-beta). These gene products produce IGF-I and TGF-beta proteins that have been extensively evaluated in equine tissue culture systems. Further, our evaluation of the expression of these growth factors after cartilage injury shows that an early deficiency is followed by a transitory peak at 8 weeks, only to decline again at 16 weeks and beyond. This information indicates an early and a late window of opportunity when supplemental endogenous IGF-I or TGF-beta may be particularly useful in improving cartilage repair. Our studies suggest TGF-beta enhances cellular division among chondrocytes and stem cells, but has a limited potential to drive up cartilage matrix synthesis. Fortunately, IGF-I has largely complementary activity, with minimal effects on cell division, but a significant impact on matrix proliferation. As a result, selection of IGF-I may be useful when chondrocytes are already present in adequate numbers, while TGF-beta may have an earlier application in deep cartilage injuries when numbers of differentiated cartilage cells are inadequate. Application of composites of IGF-I and chondrocytes to cartilage repair has resulted in significant joint regeneration, and now represents common clinical practice in the equine hospital of the Cornell University Hospital for Animals. However, longevity of the IGF-I impact on transplant chondrocyte activity is limited by protein residence. This grant seeks to examine the effect of inserting the gene for IGF-I into chondrocytes at the time of implantation, which is proposed to extend the impact of IGF-I on the anabolic activity of the new chondrocytes beyond the 2 weeks previously reported in trials of IGF-I protein laden chondrocyte transfers.
This experiment continues a series of trials evaluating biologic delivery mechanisms to transport the active portion of equine growth factor genes to joint structures. Studies in 1999 showed that the IGF-I gene can be inserted into chondrocytes and synovial cells forming the lining of joints, and that once inside cells the gene produces active IGF-I protein for as long as 30 days. In vivo studies performed in 2000 confirmed that the equine IGF-I gene could be directly injected to the fetlock and take up residence by seeding the joint synovial lining. Expression of IGF-I was found in joint fluid over 90 days, which would provide significant prolonged effects on healing cartilage. These gene transfer experiments use a viral piggyback system, where the gene coding IGF-I "infects" cells forming the interior lining of the knee joint. The combination of the equine IGF-I gene and a modified adenovirus used simple gene splicing techniques to yield a virion particle capable of penetrating living cells and delivering IGF-I DNA to the host cell genome.
These trials suggested that the adenovirus achieved high incorporation rates, and provided a solid foundation for additional in vivo testing of adeno-IGF-I in equine joints. Clinically, the adenoviral construct does have a major practical advantage in that it can be administered to a joint by injection, thereby providing a relatively non-invasive method for growth factor gene delivery. Gene enhanced chondrocytes for use in transplant systems is the logical next step, since this would allow local autostimulation of cartilage matrix synthesis in newly grafted cartilage defects. Subsequent followup joint injections of further vector would then bolster chondrocyte function via synovial seeding and IGF-I production which would enter the joint fluid and influence cartilage healing by diffusion. Our year 2000 in vivo studies show the adenoviral-IGF-I vector provides a quick "hit" to joints that is simple to administer by injection. The proposed study for 2001 tests the hypothesis that adeno-IGF-I transfer to chondrocytes at the time of implantation will enhance chondrocyte anabolic functions sufficiently to drive significant new cartilage repair, Such a system is a natural progression for the current chondrocyte transplant program, and actually simplifies that protocol by eliminating the need for IGF-I addition to the milieu at implant. Rather, the cells will make their own IGF-I; a simple, safe and perhaps more efficacious means to bolster cartilage healing.
To test this hypothesis, the present proposal plans a study in the horse stifle, where cartilage defects are either filled with IGF-I gene enhanced chondrocytes or chondrocytes exposed to a null gene in the same vector. The healing response will be evaluated by arthroscopic examination and biopsy at 1 and 2 months after implant, and final tissue analysis 8 months after repair. Persistence of the IGF-I gene at each time point will be determined by sensitive PCR techniques, which will determine whether additional direct injection of IGF-I gene vectors to the joint fluid will be required in clinical cases. Such a dual approach combines previous year 2000 study results with current proposals in a practical way, and builds on our ability to treat not only generalized joint disease by direct gene therapy approaches, but also focal cartilage injury by gene-enhanced chondrocyte implantation. Both approaches diminish the likelihood of arthritis, and may possibly reverse the early stages of arthritis in horses and other animals.