Dr. Alan J. Nixon
The continuing focus of this research is the investigation of methods to enhance cartilage repair by the introduction of functional portions of the insulin-like growth factor- I (IGF-1) gene into joints damaged by 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, the cartilage homeostatic balance is disturbed 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 badly 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, without the 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 endogamous 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 6 months of this project, IGF-adenoviral constructs were developed and preliminary testing showed enhanced local IGF-I production, which subsequently resulted in stimulation of cartilage cell synthetic activity. 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. Two different types of viral-IGF-I construct will be developed and tested, however, the work to date has concentrated on a combination of adenovirus and the equine IGF-I gene that was cloned and sequenced in our previous studies. 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 viral composites have been started and the adenoviral construct tested in vitro to determine its infectivity of joint cartilage cells. Successful transfer was verified by the detection of elevated IGF-I messenger RNA and by the subsequent stimulation of the cartilage cells to synthesize new cartilage matrix products.
These trials suggest the adenovirus achieves high incorporation rates- certainly higher than our previous studies with another viral vector (retrovirus), where preliminary studies of genetically tagged viral invasion of equine chondrocytes indicated a retrovirus infection rate of up to only 24%. While our initial adenovirus infection rates are much higher than this, 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 was apparently quite significant, it would be more rapidly attenuated, and experiments to test this are planned for 2000. 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 project, and the remainder of 1998 will allow further testing of the longevity of this response in cartilage cell and synovial cell cultures. The current renewal will examine retroviral constructs to insert equine IGF-I genomic material into cartilage cells before transplantation, and subsequent grant cycles will test the ability of both viruses to infect synovial and cartilage cells in living animals, and the potential impact of gene therapy on cartilage lesions as well as 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. However, a serious detriment to the use of retroviral constructs has been the relative inability to "infect" enough cells in the joint or other tissue to generate a significant pool of synthetically active cells. We propose this can be overcome by selecting the retrovirus-IGF-I labeled cells through insertion of a drug resistance gene along with IGF-I DNA. Cartilage cell cultures infected with this retroviral-IGF-I composite will then be exposed to the drug in the growth media, which is toxic only to cartilage cells not containing the retroviral-IGF vector. The result should be elimination of unlabeled cells, with a subsequently high "infection" rate, allowing these selected cells to be further propagated prior to transplant to articular defects. Our work on the adenoviral-IGF-I vector provides a quick "hit" to joints that is easy to administer by injection, while the retroviral construct will be used to label cartilage cells destined for grafting procedures, where a long-term impact is desired. The differing problems in equine joint disease require this dual approach, and the current grant adds the second phase to the structured development of equine gene therapy approaches to joint disease. Additionally, these experiments form a natural progression of our previous work cloning equine IGF-1. Previous studies also define the time periods when supplemental IGF-I is required, and both of these research results are critical preliminary data for this gene therapy initiative. 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.