Intra-articular adenoviral-mediated gene transfer of trail induces apoptosis of arthritic rabbit synovium.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that primarily affects joints. In rheumatoid joints there is extensive synovial proliferation with diseased synovium becoming highly aggressive, attaching to the articular cartilage and bone to form what is termed a pannus. The formation of active pannus is central to erosive disease and resulting joint destruction. In this study, we examined the ability to eliminate the hyperplastic synovium by adenoviral-mediated gene transfer of human TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family that is able to induce apoptosis through interaction with receptors containing death domains, DR4 and DR5. Infection of synovial cells derived from RA patients with Ad.TRAIL resulted in significant apoptosis in three out of five lines. Moreover, primary rabbit synovial fibroblasts were also sensitive to Ad.TRAIL-mediated gene transfer. In a rabbit model of arthritis, intra-articular gene transfer of TRAIL induced apoptosis in cells within the synovial lining, reduced leukocytic infiltration and stimulated new matrix synthesis by cartilage. These results demonstrate that TRAIL can affect the viability of the cells populating the activated synovium in arthritic joints and suggest that the delivery of TRAIL to arthritic joints may represent a non-invasive mechanism for inducing pannus regression.

Gene transfer of p53 to arthritic joints stimulates synovial apoptosis and inhibits inflammation.

Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects joints. During the pathogenesis of rheumatoid arthritis, the synovial lining becomes dramatically thickened and hyperplastic. This highly aggressive tissue invades and destroys articular cartilage and bone. Several lines of evidence suggest that the proliferation of the synovial tissue may be due to disruption in the control of the cell cycle or apoptotic pathways. In particular, mutations in the tumor suppressor protein p53 have been found in synovial tissue from RA joints. We have examined the effects of overexpression of p53 by adenoviral infection in synovial cells in culture and in synovial tissue in vivo in a rabbit model of arthritis. Here we demonstrate that p53 overexpression resulted in significant apoptosis in human and rabbit synovial cells in culture. Furthermore, intraarticular injection of Ad-p53 resulted in extensive and rapid induction of synovial apoptosis in the rabbit knee without affecting cartilage metabolism. Interestingly, a significant reduction in the leukocytic infiltrate was observed within 24 h postinfection of Ad.p53. These results suggest that intraarticular gene transfer of p53 is able to induce synovial apoptosis as well as reduce inflammation and thus may be useful clinically for the treatment of RA.

Direct gene delivery strategies for the treatment of rheumatoid arthritis.

Gene therapy offers a novel and innovative approach to the delivery of therapeutic proteins to the joints of patients with arthritis. Several viral vectors, including adenovirus, adeno-associated virus, retrovirus and herpes simplex virus, are capable of delivering exogenous cDNAs to the synovial lining, enabling effective levels of intra-articular transgene expression following direct injection to the joint. The expression of certain gene products has proven to be sufficient to inhibit the progression of disease in animals with experimental arthritis. Non-viral methods of gene transfer, however, are less satisfactory, and are limited by toxicity and transience of expression. Although the principle of direct gene delivery to the joint has been demonstrated, maintaining persistent intra-articular transgene expression remains a challenge.

Gene therapy for autoimmune disorders.

Although many autoimmune disorders do not have a strong genetic basis, their treatment may nevertheless be improved by gene therapies. Most strategies seek to transfer genes encoding immunomodulatory products that will alter host immune responses in a beneficial manner. Used in this fashion, genes serve as biological delivery vehicles for the products they encode. By this means gene therapy overcomes obstacles to the targeted delivery of proteins and RNA, and improves their efficacy while providing a longer duration of effect, and, potentially, greater safety. Additional genetic strategies include DNA vaccination and the ablation of selected tissues and cell populations. There is considerable evidence from animal studies that gene therapies work: examples include the treatment of experimental models of rheumatoid arthritis, multiple sclerosis, diabetes, and lupus. Pre-clinical success in treating animal models of rheumatoid arthritis has led to the first clinical trial of gene therapy for an autoimmune disease. In this Phase I study, a cDNA encoding the interleukin-1 receptor antagonist was transferred to the knuckle joints of patients with advanced rheumatoid arthritis. Two additional clinical trials are in progress. It is likely that gene therapy will provide effective new treatments for a wide range of autoimmune disorders.

Vector systems for gene transfer to joints.

The prospects for the development of gene therapy treatments for certain orthopaedic diseases have been fueled by advances in the understanding of the molecular components of these disorders. These studies have identified molecules that could have therapeutic or reparative effects in certain settings. The ability to transfer and appropriately express the genes encoding these molecules is dependent on the availability of effective gene transfer vectors. Numerous vector systems have been used to transfer and express genes in joints with varied levels of success. The current review is designed to briefly outline the basics of the different gene transfer vector systems available for use by researchers in the orthopaedic fields.

Gene therapy approaches for treating rheumatoid arthritis.

Current gene therapy approaches for treating rheumatoid arthritis have made use of gene transfer technology as an improved delivery system for emerging proteins and other biologicals whose activities may have therapeutic value. Preclinical research has focused on two primary directions, evaluation of methods of gene delivery and identification of gene products with antiarthritic potential. Although there are reports involving systemic gene delivery, the bulk of effort has focused on local, intraarticular administration using ex vivo and in vivo methods. Viral-based vectors, including adenovirus, adeno-associated virus and herpes simplex virus have the greatest efficiency of gene delivery after intraarticular injection and are capable of generating relevant levels of gene products in several animal models of disease. However, there are limitations to existing generations of these systems that currently preclude their clinical application. Those gene products found to be efficacious in animal models of rheumatoid arthritis include proteins that specifically block the activity of the primary inflammatory cytokines, and include interleukin-1 receptor antagonist and soluble receptors for tumor necrosis factor and interleukin-1. Delivery and expression of genes encoding certain cytokines such as interleukins -4, -10, and -13 and viral interleukin-10, that block synthesis of inflammatory mediators and downregulate aspects of cellular and humoral immune pathways have been found beneficial. Although significant progress has been made, leading to Phase I clinical trials, there remain several hurdles to the routine practice of gene therapy for treatment of rheumatoid arthritis.

Adenoviral mediated delivery of FAS ligand to arthritic joints causes extensive apoptosis in the synovial lining.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease where the synovial lining layer of the joint becomes thickened, hypercellular, and highly aggressive. Invading synovial tissue erodes cartilage and subchondral bone and leads to loss of joint function. FasL, a cell-surface molecule on activated T-cells interacts with its receptor, Fas, to induce apoptosis in target cells. We addressed the feasibility of using adenoviral gene transfer of FasL therapeutically to mediate apoptosis in arthritic joints similar in size to the small joints of the hands and feet that are the primary sites of RA in humans. METHODS: Adenoviral vectors were used to transfer FasL and LacZ cDNAs into human RA and rabbit synovial fibroblasts in culture where apoptosis was evaluated using MTT and TUNEL analyses. The ability of Ad.FasL to mediate synovial apoptosis in vivo was then addressed in an IL-1-induced arthritis model in the rabbit knee. RESULTS: In culture, delivery of FasL was found to efficiently induce apoptosis in both human RA and rabbit synovial fibroblasts. The ability of Ad.FasL to induce synovial apoptosis was then evaluated in rabbit knee joints. 24 h after intra-articular injection of 10(11) Ad.FasL particles, large regions of synovial tissue were observed histologically consisting primarily of fibrous matrix and cellular debris. TUNEL staining of corresponding sections was highly positive for fragmented DNA. Glycosaminoglycan (GAG) synthesis from cartilage shavings from treated joints suggests that Ad.FasL does not induce significant apoptosis in resident articular chondrocytes. CONCLUSIONS: Infection of human and rabbit synovial fibroblasts with Ad.FasL results in significant apoptotic cell death in vitro. Direct intra-articular injection of Ad.FasL in the arthritic rabbit knee results in extensive apoptosis in the synovium without affecting chondrocyte viability.

Cartilage injury and repair.

Articular cartilage is a complex structure that, once damaged, has little capacity for permanent repair. The problem lies in the inability of the body to regenerate tissue with the appropriate macromolecular constituents and architecture of normal hyaline cartilage. Although full-thickness defects are capable of stimulating a repair response, the resulting fibrocartilage is inferior and cannot withstand long-term, repetitive use. Numerous surgical approaches that involve penetration of subchondral bone offer short-term to moderate-term relief of symptoms, whereas other approaches have seen significant improvement through transplantation of osteochondral and periosteal tissue and implantation of autologous chondrocytes. Despite these procedures, articular cartilage damage continues to be an unmet clinical problem. Improvements in biochemical and molecular biologic techniques may allow advances in the understanding of chondrocyte and cartilage biology and may provide innovative and novel approaches to stimulating the repair of articular cartilage through biologic means.

Development of herpes simplex virus replication-defective multigene vectors for combination gene therapy applications.

Some gene therapy applications will require simultaneous expression of multiple gene products to achieve a therapeutic effect. In this study we describe the generation and characterization of replication incompetent herpes simplex virus type 1 (HSV-1) vectors (HX86Z or HX86G) carrying distinct and independently regulated expression cassettes for five transgenes (hIL-2, hGM-CSF, hB7.1, HSV-tk and lacZ or hIFN gamma). The transgenes, representing 12 kb of DNA sequence, were recombined into separate loci of a single mutant virus vector deleted for 11.6 kb of vector sequences representing portions of nine viral genes, ICP4, ICP22, ICP27, ICP47, UL24, UL41, UL44, US10 and US11. Deletion of the immediate--early genes ICP4, ICP22 and ICP27 substantially reduced vector cytotoxicity, prevented early and late viral gene expression and left intact MHC class I antigen expression. Simultaneous expression of multiple transgenes was obtained for up to 7 days in primary human melanoma cells with peak expression at 2-3 days after infection. The transgenes were chosen for their potential to function synergistically in tumor destruction and vaccine gene therapy applications, but the method and vector employed could be applied to other multigene therapy strategies. This study demonstrates the potential for engineering large transgene capacity DNA viruses such as HSV-1 for expression of multiple transgenes.