Lessons learned from gene transfer approaches
© Current Science Ltd 1999
Received: 28 April 1999
Accepted: 1 June 1999
Published: 8 July 1999
Recent technological advances allow the transfer of genes to the synovial lining of joints. As well as opening novel opportunities for therapy, these techniques provide valuable new tools for the study of synovitis and other aspects of the biology of joints in health and disease. This article reviews briefly the results of experiments in which selected genes have been transferred to the knee joints of healthy rabbits and rabbits with antigen-induced arthritis.
Keywordsanimal model cartilage cytokine gene therapy growth factor rheumatoid arthritis synovium
One approach to the treatment of intra-articular pathologies involves the transfer of genes to synovium and their intrasynovial expression . The development of technologies with which to do this not only opens new therapeutic possibilities but also permits the study of selected gene products within the synovial microenvironment. Here we review briefly the results obtained from such investigations.
Most of these data have been obtained using the rabbit knee as a model. This joint has the advantage of being large enough for efficient intra-articular injection and serial lavage. Moreover, after euthanasia the various intra-articular tissues can be dissected and independently studied ex vivo.
We utilize gene delivery to the joint in two complimentary approaches to the study of joint diseases. In the first, genes whose products have been associated with arthritis are introduced into the synovium of a normal joint to determine whether their gene products can provoke synovitis or other joint disturbances. In the second, the genes are introduced into inflamed joints to determine whether their products have anti-inflammatory properties. Antigen-induced arthritis (aia), using ovalbumin as the inciting antigen, is often employed for these experiments.
Gene transfer to synovium
Ex vivo and in vivo gene transfer to the synovial lining of rabbits' knee joints*
Level of expression
Duration of expression
~ 2 weeks
Up to 6 weeks
Types A+B synoviocytes
Type B synoviocytes
Gene transfer to the normal synovium
Ex vivotransfer of the interleukin-1β gene
The first experiments of this kind were conducted by ex vivo transfer of a human interleukin (hIL)-1β complementary (c)DNA . Because the native molecule is not secreted efficiently, a leader sequence taken from the parathyroid hormone was fused to the N-terminus of the molecule. This molecule proved extraordinarily potent. Indeed in the first experiment where 107 autologous cells expressing IL-1β were returned to one knee joint, all the rabbits died or had to be euthanized.
In subsequent experiments, fewer cells were returned to the joint. Following intra-articular injection of 106 transduced cells, approximately 200pg hIL-1β could be lavaged from the joints for up to 2weeks, after which gene expression abruptly stopped. During this 2-week period, the rabbits developed fevers, lost weight, and their erythrocyte sedimentation rates rose dramatically. Knee joints receiving the hIL-1β construct were massively swollen and contained a voluminous, caseinous synovial fluid. Synovia were enormously hypertrophic and hyperplastic, not only invading the adjacent cartilage and bone but also penetrating the capsule into the adjacent muscle, areas of which became necrotic. It is also remarkable how all non-erosive components of these pathologies returned to normal once transgene expression was lost.
Later studies revealed that the transfer of as few as 105 IL-1β+ cells caused considerable intra-articular pathology despite the absence of detectable hIL-1β in the lavage fluid. This observation cautions against drawing strong conclusions concerning the pathophysiological roles of potential mediators based upon their synovial fluid concentrations.
Previous attempts to define intra-articular responses to IL-1β had relied upon intra-articular injection or pumps to deliver recombinant protein [7,8]. In neither case were the sequelae remotely as dramatic as those observed following transfer of the IL-1β gene. The greater potency of the gene transfer approach may not only result from its ability to deliver IL-1β in a sustained fashion, but also from the production of an authentically processed protein by synoviocytes within the synovial microenvironment.
In vivogene transfer
A variety of genes have been evaluated by in vivo adenoviral delivery (Table 2). Expression of a murine tumor necrosis factor (TNF) cDNA within the synovium of the rabbit knee produced moderate inflammatory infiltrates, which were particularly rich in polymorphonuclear leukocytes (our unpublished data, 1998). The release of glycosaminoglycans (GAG) from the articular cartilage was modestly elevated, while GAG synthesis was strongly depressed. When expressed in combination with an IL-1β transgene, TNF caused high levels of apoptosis in the synovium. This suggests that such a combination may limit synovitis by inducing cell death.
Growth factor genes are being evaluated for their ability to protect articular cartilage from degradation, as well as to enhance the repair of damaged cartilage. As discussed in , these genes may be delivered to synovium or to the cartilage itself. Transfer of a human transforming growth factor (TGF)-β1 gene to synovium provoked a massive hypertrophy and fibrosis of not only the injected joint, but also the soft tissues of the entire leg (our unpublished data, 1998). This proved fatal in several animals. When less virus was administered, pathology was restricted to the injected knee and included thickening and fibrosis of the synovium. This suggests that TGF-β may be a mediator of arthrofibrosis. Interestingly, the inflammatory infiltrate that often follows the intra-articular injection of adenoviral particles was virtually absent when the TGF-β1 gene was present.
Of particular interest was the formation of intrasynovial cartilage. Recent data suggest that cells within the synovium have the potential to differentiate into chondrocytes and to form cartilage. Our findings indicate that TGF-β can trigger this pathway and, in this way, lead to the human condition synovial osteochondromatosis .
Synovial delivery of genes encoding insulin-like growth factor (IGF)-1 and bone morphogenetic protein (BMP)-2, in contrast to the TGFβ gene, produced a modest elevation in cartilage GAG synthesis in the absence of major synovial changes (our unpublished data, 1998).
Nitric oxide (NO) is a radical with a physiological half-life of several seconds. Although there is literature to suggest an important role for this molecule in inflammation and joint disease , experimental studies have been hampered by various practical limitations. Among them is the near impossibility of administering NO to experimental animals in a pharmacologically useful manner. Transfer of genes encoding NO synthase solves this problem, and serves as a particularly good example of the value of the gene delivery approach to studying disease.
Intra-articular effects of various genes following direct delivery to the synovial lining of normal rabbits knees
TGF- β 1†
Gene transfer to the inflamed synovium
Ex vivotransfer of the interleukin-1 receptor antagonist(IL-1Ra)
Delivery of a cDNA encoding human IL-1Ra one day after induction of aia had no effect on synovitis, although it reduced the influx of leukocytes into the joint space and strongly protected the articular cartilage. For unknown reasons transgene expression was higher in joints with aia than in normal joints . Although synovitis was unaffected in this rabbit model, ex vivo transfer of human IL-1Ra genes did suppress synovitis in streptococcal cell wall arthritis in rats  and collagen-induced arthritis in mice . Ex vivo delivery of the IL-1Ra gene to joints is presently the subject of a human clinical trial .
In vivogene transfer
Adenoviral vectors have been used to transfer a number of additional potentially anti-inflammatory genes to the synovia of rabbits with aia (Table 3). A gene encoding bivalent IL-1 soluble type I receptor inhibited the influx of leukocytes into the joint space and protected the articular cartilage. Synovitis was moderately reduced .
Delivery of a gene encoding bivalent TNF soluble type I receptor had very little effect beyond a mild reduction in the inflammatory infiltrate into the joint space. Remarkably, a combination of both IL-1 and TNF soluble receptor genes not only protected cartilage and reduced the leukocytic influx into the joint space, but also inhibited joint swelling and strongly reduced the synovitis .
The viral (v)IL-10 gene was even more potent than the combination of IL-1 and TNF soluble receptors. Alone, it strongly reduced swelling, synovitis and leukocytic infiltration while protecting the articular cartilage . A similar effect has been noted in the paws of mice with collagen-induced arthritis .
In vivo delivery of human Fas ligand (FasL) was associated with large regions of cellular apoptosis within the synovium. It is not yet clear whether the level of cell killing observed under these circumstances is sufficient to produce a significant anti-arthritic effect in this model (our unpublished data, 1998). Adenoviral delivery of the FasL gene does, however, ameliorate disease in murine collagen-induced arthritis .
Intra-articular effects of various genes following direct delivery to the synovial lining of rabbit knees with antigen-induced arthritis
IL-1Ra + TNFRa
The contralateral effect
While performing these experiments, Ghivizzani et al  made an unexpected observation: injection of adenovirus carrying the soluble IL-1 receptor into one knee joint of animals with bilateral aia ameliorated disease not only in the injected knee but also in the contralateral knees which had received only saline or a marker gene. When a soluble TNF receptor gene was co-administered, this 'contralateral effect' was even stronger. The contralateral effect was particularly powerful when using the vIL-10 gene . A similar effect occurs in murine collagen-induced arthritis , and several other groups have reported similar findings suggesting that this is not some peculiarity of rabbit a.i.a .
The mechanism of the contralateral effect may reside with the ability of leukocytes to traffic between joints. Following injection of adenoviruses carrying marker genes into one knee joint of animals with bilateral disease, it is possible to recover cells expressing those marker genes from the contralateral knee joints and draining lymph nodes but not from other organs or the peripheral blood [16,17]. The identification of these cells and their possible role as mediators of the contralateral effect are under investigation.
The ability to transfer genes to the synovium and other tissues of interest offers unique opportunities for studying the biology of joints in health and disease. The data obtained by these means will aid the development of novel therapeutic approaches, irrespective of their direct application to gene therapy.
- Evans CH, Ghivizzani SC, Kang R, et al: Gene therapy for rheumatic disease. Arthritis Rheum. 1999, 42: 1-15. 10.1002/1529-0131(199901)42:1<1::AID-ANR1>3.0.CO;2-4.PubMedView ArticleGoogle Scholar
- Bandara G, Robbins PD, Georgescu HI, Mueller GM, Glorioso JC, Evans CH: Gene transfer to synoviocytes: prospects for gene treatment for arthritis. DNA Cell Biol. 1992, 11: 227-231.PubMedView ArticleGoogle Scholar
- Roessler BJ, Allen ED, Wilson JM, Hartman JW, Davidson BL: Adenoviral-mediated gene transfer to rabbit synovium in vivo. J Clin Invest. 1993, 92: 1085-1092.PubMedPubMed CentralView ArticleGoogle Scholar
- Nita I, Ghivizzani SC, Galea-Lauri J, et al: Direct gene delivery to synovium: an evaluation of potential vectors in vitro and in vivo. Arthritis Rheum. 1996, 39: 820-828.PubMedView ArticleGoogle Scholar
- Evans CH, Robbins PD, Ghivizzani SC, Herndon JH, Kang R: Clinical trial to assess the safety, feasibility and efficacy of transferring a potentially anti-arthritic cytokine gene to human joints with rheumatoid arthritis. Hum Gene Ther. 1996, 7: 1261-1280.PubMedView ArticleGoogle Scholar
- Ghivizzani SC, Kang R, Georgescu HI, et al: Constitutive intraarticular expression of human IL-1β following gene transfer to rabbit synovium produces all major pathologies of human rheumatoid arthritis. J Immunol. 1997, 159: 3604-3612.PubMedGoogle Scholar
- Pettipher ER, Higgs GA, Henderson B: Interleukin-1 induces leukocyte infiltration and cartilage proteoglycan degradation in the synovial fluid. Proc Natl Acad Sci U S A. 1986, 83: 8749-8753.PubMedPubMed CentralView ArticleGoogle Scholar
- Feige U, Karbowski C, Rordorf-Adam C, Pataki A: Arthritis induced by continuous infusion of hr-interleukin-1α into the rabbit knee joint. Int J Tissue React. 1989, 11: 225-238.PubMedGoogle Scholar
- Evans CH, Robbins PD: Potential treatment of osteoarthritis by gene therapy. In: Rheumatic Disease Clinics of North America. Edited by Brandt K. Philadelphia: WB Saunders Co,. 1999, 25: 333-344.View ArticleGoogle Scholar
- Milgram JW: Synovial osteochondromatosis: a histopathological study of thirty cases. J Bone Joint Surg. 1977, 59A: 792-801.Google Scholar
- Stefanovic-Racic M, Stadler J, Evans CH: Nitric oxide and arthritis. Arthritis Rheum. 1993, 36: 1036-1044.PubMedView ArticleGoogle Scholar
- Niu XF, Ibbotson G, Kubes P: A balance between nitric oxide and oxidants regulates mast cell-dependent neutrophil-endothelial cell interactions. Circ Res. 1996, 79: 992-999.PubMedView ArticleGoogle Scholar
- Otani K, Nita I, Macaulay W, Georgescu HI, Robbins PD, Evans CH: Suppression of antigen-induced arthritis by gene therapy. J Immunol. 1996, 156: 3558-3562.PubMedGoogle Scholar
- Makarov SS, lsen JC, Johnston WN, et al: Suppression of experimental arthritis by gene transfer of interleukin-1 receptor antagonist cDNA. Proc Natl Acad Sci U S A. 1996, 93: 402-406. 10.1073/pnas.93.1.402.PubMedPubMed CentralView ArticleGoogle Scholar
- Bakker AC, Joosten LAB, Arntz OJ, et al: Prevention of murine collagen-induced arthritis in the knee and ipsilateral paw by local expression of human interleukin-1 receptor antagonist protein in the knee. Arthritis Rheum. 1997, 40: 893-900.PubMedView ArticleGoogle Scholar
- Ghivizzani SC, Lechman ER, Kang R, et al: Direct adenoviral-mediated transfer of IL-1 and TNF-α soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects. Proc Natl Acad Sci U S A. 1998, 95: 4613-4618. 10.1073/pnas.95.8.4613.PubMedPubMed CentralView ArticleGoogle Scholar
- Lechman ER, Jaffurs D, Ghivizzani SC, et al: Direct adenoviral gene transfer of vIL-10 to rabbit knees with experimental arthritis ameliorates disease in both injected and contralateral knees. J Immunol. 1999Google Scholar
- Whalen JD, Lechman ER, Carlos CA, et al: Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws. J Immunol. 1999, 162: 3625-3632.PubMedGoogle Scholar
- Zhang H, Yang Y, Horton JL, et al: Amelioration of collagen-induced arthritis by CD95 (Apo-1/Fas)-ligand gene transfer. J Clin Invest. 1997, 100: 195-1957.Google Scholar
- Evans CH, Rediske JJ, Abramson SB, Robbins PD: Joint efforts: tackling arthritis using gene therapy. Mol Med Today. 1999, 5: 148-151. 10.1016/S1357-4310(99)01452-5.PubMedView ArticleGoogle Scholar