Dr. Alan J. Nixon
A more effective means to induce cartilage regrowth after injury aims to prevent arthritis and holds promise to return horses to athletic potential. Research to date in this field has focused on cell transplants bolstered by growth factor composites that are added to the transplant milieu. However, residence time for growth factor proteins is known to have a 1-to-2 week limit. To overcome this short-term flux, this research program will explore gene therapy procedures used to introduce functional portions of the insulin-like growth factor-I (IGF-I) gene into joints damaged by OCD, acute injury, or the early stages of arthritis. Growth factors, particularly IGF-I, predominantly stimulate cartilage cell metabolism to maintain healthy cartilage. 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 antiinflammatory and pain relieving agents, along with extended periods of rest. Untreated or severely damaged joints frequently develop osteoarthritis, which remains a leading cause of horses' retirement 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 in only 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 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 will be 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 cycle will test the heralded adeno-associated virus for its efficiency and long-term persistence for IGF-I gene delivery.
Our previous Zweig-funded studies have cloned and sequenced equine IGF-I, and provided proof of principle data showing that IGF-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 2 months 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 shows two windows of time at 2 and 16 weeks when there are deficiencies in IGF-I levels and supplemental endogenous IGF-I may be particularly useful in improving cartilage repair. Our studies suggest TGF-P 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 significant impact on 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 the IGF-I gene indicated chondrocytes used for transplant procedures could carry the gene with them, and the gene translocated to the cell nucleus where it became functional and produced IGF-I protein in effective quantities for up to 2 months. Further in vitro and in vivo work showed that synovial lining cells could be laden with the same IGF-I gene and used to provide a "second-wave" of IGF-I synthesis when local IGF-I formation from transplanted chondrocytes was declining. This new bi-modal gene approach in gene therapy trials avoids an immune reaction to the initial adenovirus and gene insert in chondrocytes by sequestering the vector inside the cell nucleus before the cells are transplanted to the body. 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 genes to chondrocytes. The initial year of the grant will focus on maximizing virus production and enhancing efficiency of the viral vector containing equine IGF-I coding sequence in integrating into chondrocytes and synovial cells. Previous work confirms the equine IGF-I gene produces ample IGF-I protein from transformed chondrocyte and synovial cells. Further, this protein drives chondrocyte matrix forming functions, and the current research (Zweig 2001 grant) will test whether the IGF-I gene laden transplanted chondrocytes can produce a more durable cartilage surface than previously evaluated cells implanted with a depot of IGF-I protein. The proposed work will verify how long the IGF-I gene persists in chondrocytes and synovial cells and evaluate the effects of this gene expression on cell function. Importantly, the possible complications of unabated IGF-I gene expression on cell function, including depletion of other molecules vital for cartilage synthesis or significant diminution of intrinsic production of IGF-I, need to be examined.
The initial year will involve only in vitro studies. Few labs have significant experience propagating an adeno-associated virus. Our long-term collaboration with Drs. Evans and Robbins provides us unique access to adeno-associated lab techniques, and the equine IGF-I gene has been inserted into adeno-associated vectors in Dr Robbins' lab. Preliminary data suggests the gene is functional when inserted into cartilage cells, and major work is proposed for 2002 to further optimize gene delivery doses and examine longevity of gene persistence in the cell. Our hypothesis is that IGF-I genes can be driven into the 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 proteins.