Dr. Alan J. Nixon
The continuing focus of this research is the use of gene therapy procedures to introduce functional portions of the insulin-like growth factor-I (IGF-1) gene into joints damaged by OCD, acute injury, or those in the early stages of arthritis. Growth factors, particularly IGF-1, 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 antiinflammatory 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.
Our previous Zweig funded studies have cloned and sequenced both equine IGF-I and transforming growth factor-beta (TGF-b). These gene products produce IGF-I and TGF-b 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-b may be particularly useful in improving cartilage repair. Our studies suggest TGF-b 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-b may have an earlier application in deep cartilage injuries when numbers of differentiated cartilage cells are inadequate.
This experiment continues a series of trials evaluating biologic delivery mechanisms to transport the active portion of equine growth factor genes to joint structures. During the first 12 months of this project, human IGF-adenoviral constructs were developed and evaluated. Summary data showed enhanced but moderately low levels of local IGF-I production. Studies performed this year used an equine-specific IGF-adenoviral transgene, which has improved the production of IGF-I to beyond the physiological maximum working in cartilage systems during growth and development. Additionally, work has started on the development of a new equine IGF-I retrovirus construct, which will extend the impact of the IGF-I gene for months to years rather than the weeks to months evident in current adenovirus-IGF trials. These gene transfer experiments use a viral piggyback system, where the gene coding IGF-I "infects" cells in the articular cartilage as well as those 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. The adenoviral construct penetrated joint cartilage cells quite successfully in trials done earlier this year. Successful transfer was verified by the detection of elevated IGF-I messenger RNA and protein, and by the subsequent stimulation of the cartilage cells to synthesize new cartilage matrix products.
These trials suggest that the adenovirus achieves high incorporation rates, and provides a solid foundation for 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. This combination of benefits has made adenoviral constructs a suitable starting point for this in vivo project, and the remainder of 1999 will allow further in vitro testing of the longevity of this response in cartilage cell and synovial cell cultures. The remainder of this year will also allow culture evaluation of retroviral constructs to insert equine IGF-I genomic material into cartilage cells before transplantation, and subsequent grant cycles (Year 2001+) will test the ability of retroviruses to infect cartilage cells in living animals, and to evaluate the potential impact of gene therapy on several models of early osteoarthritis in horses. The use of retroviral vectors provides the longevity of response that is lacking in adenoviral vectors used previously. While our initial adenovirus infection rates are high, the viral DNA and accompanying IGF-I DNA are not incorporated into the host cell genome and therefore are not passed on to daughter cells in the process of normal cell turnover. Therefore, despite the fact that the initial impact of the introduced gene is quite significant, it is more rapidly attenuated, and in vitro experiments so far show the peak effects at 4 and 8 days, before a decline at 14 days and only moderate persisting levels by 28 days. Our in vitro work on the adenoviral-IGF-I vector shows it should provide a quick "hit" to joints that is simple to administer by injection. The proposed study for 2000 tests this hypothesis by adeno-IGF-I injections to joints, followed by serial harvesting of synovial fluid for IGF-I assay, and harvesting of cartilage and synovial biopsies for assessment of transfected cell numbers. This will then be followed by a second study in 2000 using biopsied synovial lining cells to reinsert IGF-I gene fragments back into joints. Comparison of these two approaches will determine the appropriate clinical routine that will be necessary for practice application of these materials. While the current proposal focuses on in vivo testing of adenoviral transgenes, the retroviral construct started earlier this year will be evaluated by years end for labeling cartilage cells destined for grafting procedures. The differing problems in equine joint diseases require this dual approach, and the current grant adds critical in vivo information to the structured development of equine genetherapy approaches to joint disease. The implementation of both vector programs will allow us to not only improve cartilage healing in acute injury, but also to possibly reverse the early stages of arthritis in horses and other animals.