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The Journal of Bone and Joint Surgery 81:152-157 (1999)
© 1999 The Journal of Bone and Joint Surgery, Inc.

Arthritis: Is the Cure in Your Genes?*{dagger}

JAMES H. HERNDON, M.D.{ddagger}, BOSTON, MASSACHUSETTS, PAUL D. ROBBINS, PH.D.§ and CHRISTOPHER H. EVANS, PH.D., D.SC.§, PITTSBURGH, PENNSYLVANIA

*One or more of the authors has received or will receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article. In addition, benefits have been or will be directed to a research fund, foundation, educational institution, or other nonprofit organization with which one or more of the authors is associated. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were National Institutes of Health Grants PO1 DK44935 and RO1 AR43623.


   Introduction
Top
 Introduction
References
 
Rheumatoid arthritis is incurable and difficult to treat with traditional pharmacological approaches. Because conventional drug therapy has clearly failed to conquer this distressing condition, novel therapeutic strategies are required. We are not referring to the numerous dubious alternative methods touted by the tabloid press but to new possibilities arising from the results of serious laboratory investigation. Recent research into the biology of rheumatoid arthritis has identified a number of proteins with promising antiarthritic properties22, but their application to human disease is limited by difficulties in delivering them in a targeted, sustained fashion for extended periods of time.

Most investigators would agree, in general terms, that the natural history of rheumatoid arthritis is as follows. In genetically susceptible individuals, an unknown environmental agent triggers an autoimmune response, which leads to the production of mediators, particularly cytokines, that drive the pathophysiological processes leading to the clinical manifestations of rheumatoid arthritis. Therapeutic intervention in this chain of events is possible at a number of sites. However, most efforts have been concentrated on modulating either T-lymphocyte responses or the activities of various arthritogenic cytokines22.

Preclinical studies have confirmed that both of these approaches have merit. Agents that suppress the activities of lymphocyte costimulatory molecules have shown efficacy in animal models8,21. Antibodies directed against lymphocyte antigens, such as CD4, have also shown antiarthritic effects, and several are currently being used in clinical trials9. Most anticytokine strategies have centered on the suppression of interleukin-1 (IL-1) and tumor necrosis factor-{alpha} (TNF-{alpha})1. Promising agents that do this include neutralizing antibodies, soluble receptors, and the interleukin-1 receptor antagonist protein (IL-1Ra). All have shown encouraging activity in animal models, and several are currently being evaluated in clinical trials in humans. The short-term effects of administration of antibodies directed against tumor necrosis factor-{alpha}14 and soluble tumor necrosis factor-{alpha} receptors23 have been dramatic and have strongly justified the pursuit of novel therapies based on cytokine modulation.

Bringing these promising advances into common clinical use is restricted by one key fact: all of these novel antiarthritic agents are proteins and thus are almost impossible to deliver chronically as drugs. Proteins are digested by the gastrointestinal system and therefore cannot be delivered orally. Injection is possible, but most proteins are cleared from the serum rapidly. In the case of cytokines, the serum half-life is on the order of thirty minutes. Frequent subcutaneous self-administration is an inconvenient, unpleasant alternative, and complications have arisen, including local reactions at the site of the injection. These reactions have been severe enough to result in patients leaving clinical trials7. Intravenous administration is also possible but is more invasive and may require very high initial doses. We believe that gene therapy may obviate many of these problems and provide additional benefits, including the prospect of targeted, regulated delivery.

Our gene-therapy initiative was originally conceived as a response to the problem of delivering antiarthritic proteins in a clinically useful manner. The principle is to deliver a gene or genes that encode the antiarthritic proteins rather than to try to deliver the proteins themselves. Gene therapy is thus being developed as a biological drug-delivery system. There are several advantages to this approach. A stable, transferred gene under appropriate genetic control will permit sustained, and ultimately regulated, production of the therapeutic protein in situ. The tissues will then synthesize their own antiarthritic factors endogenously without the need for frequent, unpleasant, expensive, and inconvenient readministration. When the gene is delivered locally to diseased joints, the highest concentration of the gene product will occur intra-articularly with minimum exposure of nontargeted tissues and thus minimum side effects. For safety reasons, it is local delivery that we first tried in the clinical setting.

To use genes as a biological delivery system for the sustained expression of antiarthritic proteins, it is necessary to use gene-delivery vectors34. These are vehicles that facilitate the uptake and expression of genes by cells. Most vectors are based on viruses because the life cycles of viruses require them to transfer their genes to the cells that they infect and to express them intracellularly with high efficiency. For use in gene therapy, the viruses are modified to render them nonreplicating. In this way, the modified viruses will deliver their genetic payload to the cells with high efficiency, but because they cannot replicate they will not cause disease. Although a variety of viruses hold potential as future vectors for gene therapy, only a few have been sufficiently well developed for serious consideration30.

Retroviruses are by far the best developed of the viral vectors, and they are being used in the majority of human trials that are currently under way31. When engineered as vectors, they produce no viral proteins, and because they integrate into the host cell's genome they offer the best prospect for long-term expression. The main disadvantages of the retroviruses now being used in human trials are the requirement for the target cell to divide in order for the virus to insert itself into the host cell's DNA and the theoretical possibility of insertional mutagenesis.

Adenoviruses36 and herpes simplex virus18 hold considerable potential as alternative vectors because of their high infectivity toward nondividing cells and their high titer. Their use in humans has been severely limited by the inflammatory properties of adenoviral vectors and the cytotoxicity of vectors derived from herpes simplex virus. Adeno-associated virus may solve these problems, but difficulties in generating sufficient amounts of recombinant adeno-associated virus as vectors have limited their evaluation in this regard until recently35. Nonviral gene-delivery systems offer simpler, safer, less expensive alternatives to viral vectors, but they are currently much less efficient.

Vectors can be used to deliver genes to cells in either an ex vivo or an in vivo fashion. Ex vivo gene delivery requires the removal of cells from the body, their genetic alteration in vitro, and their subsequent reimplantation into the appropriate site. To accomplish in vivo gene delivery, the vector is injected directly into the site of interest.

In vivo gene delivery is less tedious and less expensive, but its use in humans is limited by safety concerns and the lack of suitable vectors. Although ex vivo delivery is laborious and complicated, it is much safer because no infectious agents are introduced directly into the body and because the genetically altered cells can be subjected to exhaustive safety testing before reimplantation.

Once the gene-delivery systems have been optimized, the next important questions with regard to use of gene therapy for arthritis are which genes to deliver and how to regulate their expression. At the present time, there is no way to predict which genes will have the best therapeutic properties, but the results of limited human trials with use of proteins indicate that interleukin-{alpha}1 receptor antagonist and tissue necrosis factor-{alpha} soluble receptor hold promise14,23,32. Studies of animal models of arthritis have suggested a number of additional cytokines, cytokine antagonists, and immunomodulatory proteins as potentially antiarthritic molecules.

Genes encoding these secreted proteins may be introduced either into extra-articular locations, where the gene products gain access to the systemic circulation (systemic delivery), or into individual affected joints (local delivery). There are advantages and disadvantages to both approaches, but the most progress has been made with local delivery10,12.

As initially postulated by Bandara et al. in 1992, antiarthritic genes can be transferred locally to joints by ex vivo or in vivo means4. Because of its large surface area and its intimate contact with the joint space, the synovial membrane was chosen as the target intra-articular tissue. Initial animal experiments were thus designed to evaluate available vectors for their ability to deliver the bacterial marker gene LacZ to synovial tissue4,26.

The results of these experiments led us to concentrate our subsequent efforts on use of an adenovirus-based vector for in vivo gene delivery to synovial tissue and on use of a retrovirus-based vector for ex vivo delivery. Both normal rabbit knees and the knees of rabbits with antigen-induced arthritis were used in a series of subsequent studies.

Antigen-induced arthritis was established by the intra-articular injection of ovalbumin into one or both knee joints of New Zealand White rabbits that had been previously sensitized to this antigen. Adenoviral vectors were used to transfer two different genes into the synovial tissue of the knee joints. One gene encoded a soluble form of the interleukin-1 type-I receptor. The other gene encoded a soluble form of the tumor necrosis factor-{alpha} type-I receptor. In both cases, the soluble receptors were dimerized by fusion to an immunoglobulin heavy chain17.

Direct introduction of the tumor necrosis factor-{alpha} type-I receptor gene had no dramatic effect on either the inflammatory or the chondrodestructive aspects of antigen-induced arthritis despite vigorous intra-articular gene expression. Direct introduction of the interleukin-1 type-I receptor gene, in contrast, had an inhibitory effect on the influx of leukocytes into the joint space and reduced the level of glycosaminoglycan release from the cartilage. Neither gene improved the histological appearance of the synovial tissue. When both tumor necrosis factor-{alpha} and interleukin-1 type-I receptors were expressed in the same knee joint, there was a strong anti-inflammatory and chondroprotective effect in the genetically altered knee accompanied by a marked normalization of the histological characteristics of the synovial tissue and an inhibition of swelling. Surprisingly, there was also a marked antiarthritic effect in the contralateral, control knee, which had not received the genes17.

These results are very important for two reasons. First, this combination of genes was shown to be powerfully antiarthritic. Second, it was discovered that local genetic treatment of one joint may be able to ameliorate the disease process in another joint. The mechanism underlying this observation is unknown. Nevertheless, this result suggests that local gene therapy for arthritis may be a more powerful and comprehensive approach than one would initially assume.

In preclinical evaluation of local, ex vivo gene therapy, we used a derivative of the Moloney murine leukemia virus, termed MFG, as the retroviral vector. The first potentially antiarthritic gene that was studied with this system was a cDNA encoding human interleukin-1Ra2. This gene was selected for a number of reasons. First, the protein has an outstanding safety profile. It is a naturally occurring inhibitor of the biological actions of interleukin-1 and has been administered at high doses, without any evidence of toxicity or other adverse responses, to healthy human volunteers and to patients with rheumatoid arthritis and infection. Moreover, this protein has shown antiarthritic activity in a number of animal models of rheumatoid arthritis and is being studied in human trials32.

A human interleukin-1Ra cDNA was cloned from a commercially available cDNA library derived from human monocytes and was inserted into the backbone of the MFG retrovirus. The resulting vector is called MFG-IRAP (interleukin-I receptor antagonist protein)5. To investigate further the potential of retrovirally mediated, ex vivo gene transfer to synovial tissue, we established a model using New Zealand White rabbits. This involved obtaining autologous synovial fibroblasts, propagating them in culture, and infecting them with MFG-IRAP. After confirming expression of the transgene (the gene that has been transferred to the cell), the transduced cells were returned to one or both knee joints of the donor rabbit by intra-articular injection. The cells continued to secrete human interleukin-1Ra intra-articularly at a declining rate for four to six weeks5. The reasons for this decline in expression are currently unknown, but some possibilities are that the genetically modified cells die after implantation, the gene is subject to promoter shutoff, or an immune response to human interleukin-1Ra is expressed in the rabbit knees. Loss of expression is clearly a barrier to the eventual widespread clinical use of this technology, and we are in the process of studying this problem.

Having confirmed the biological activity of human interleukin-1Ra after transfer of the gene to synoviocytes, we proceeded to evaluate the antiarthritic activity of the human interleukin-1Ra gene in antigen-induced arthritis in rabbits28. One day after injection, autologous synoviocytes that had been transduced with the human interleukin-1Ra gene were injected intra-articularly. An ELISA (enzyme-linked immunosorbent assay) of lavaged fluids led to the very unexpected but welcome observation that expression of the human interleukin-1Ra gene was increased in the arthritic joints.

Additional analysis of lavaged fluids showed that the production of rabbit interleukin-1ß and synovial fluid leukocytosis were reduced in the presence of the human interleukin-1Ra gene, although synovitis and joint-swelling were unaffected. Nevertheless, the human interleukin-1Ra gene had a dramatic chondroprotective effect on the cartilage of the joint, both inhibiting glycosaminoglycan release and maintaining glycosaminoglycan synthesis. It thus appears that the human interleukin-1Ra gene protects the articular cartilage in this model but has an incomplete anti-inflammatory effect. This implies that comprehensive gene therapy for arthritis will require the transfer of additional genes. Genes encoding tumor necrosis factor-{alpha} soluble receptors17 and interleukin-1033 show promise in this regard.

Having demonstrated the principle that human interleukin-1Ra could be transferred to the joint and bring about an antiarthritic effect in rabbits, we set about to construct a human trial based on this procedure. As a prelude, we conducted additional preclinical experiments to confirm that the procedure was safe and that it was likely to be effective in human tissues in addition to rabbit tissues.

In a collaborative study by two of us (P. D. R. and C. H. E.) and Dr. Steffan Gay of the University of Alabama in Birmingham, small pieces of normal human articular cartilage were implanted with human rheumatoid synovial fibroblasts under the kidney capsule of an SCID (severe combined immunodeficiency strain) mouse24. Because this type of mouse has no functional immune system, it cannot reject the human tissue xenograft. Instead, the human rheumatoid synovial cells form a pannus, which invades the cartilaginous fragments and secretes factors, which stimulate chondrocyte-mediated chondrolysis. This provides a convenient system with which to test the in vivo effects of gene transfer in human tissues without having to use human volunteers.

Human synovial fibroblasts were infected with MFG-IRAP, or, as a control, with MFG-LacZ, before coimplantation with the cartilage fragments in the SCID mice. Staining for MFG-LacZ demonstrated that approximately 70 percent of the human fibroblasts were expressing the transgene. After sixty days, in situ reverse transcriptase-polymerase chain reaction revealed intensive expression of the transgene mRNA. This is a very informative result because it indicates the potential for prolonged gene expression in immunosuppressed animals. This observation strongly suggests that the duration of gene expression in rabbit knees may be limited by an immune reaction to the heterologous human interleukin-1Ra produced by the transduced cells.

Chondrocyte-mediated chondrolysis was completely inhibited by the interleukin-1Ra gene, confirming that the data obtained with rabbit tissues also applied to human tissues. However, synovial invasion of cartilage was unaffected by interleukin-1Ra24. Nevertheless, additional experiments showed that the interleukin-10 gene strongly inhibited synovial invasion. Interleukin-10, however, had no effect on chondrocyte-mediated chondrolysis25. This confirms our earlier conclusion that comprehensive treatment of human rheumatoid arthritis will likely require the transfer of several genes, and interleukin-1Ra and interleukin-10 might be a useful combination to evaluate further.

As a final matter before initiating a human trial, it was necessary to undertake exhaustive testing in animals to ensure that the procedure would be safe. Systemic lifetime expression of high levels of human interleukin-1Ra through transduction of hematopoietic stem cells with MFG-IRAP had absolutely no adverse effects in mice6. Moreover, none of the rabbits used in the experiments ever showed overt signs of a pathological process resulting from the procedure, and no histopathological changes were seen in any of the extra-articular tissues11. Furthermore, when cells transduced with MFG-IRAP were used, no changes were observed in a battery of blood tests and no human interleukin-1Ra could be detected in the serum. Three rabbits have been given intravenous injections of MFG-IRAP-transduced autologous synoviocytes and are being retained for observation to determine whether later, long-term effects of the procedure occur. So far, we have maintained these rabbits for two years. They continue to have normal findings on blood tests (including sedimentation rate; complete blood-cell count; white-blood-cell differential; levels of cholesterol, electrolytes, glucose, blood urea nitrogen, creatine, amylase, alkaline phosphatase, and liver enzymes; platelet count; and levels of triglycerides, acid phosphatase, and messenger RNA from which the envelope of protein is made), and they remain healthy.

On the strength of the preceding data, we designed a human trial of gene therapy for rheumatoid arthritis. Because this had never been done before, it was essential to make the safety of the patients the overriding priority and to ensure that all patients gave detailed informed consent.

The safety margin of the procedure was maximized by the design of the human trial11. Specifically, cells are introduced into the joint shortly before the scheduled operation for insertion of the prosthetic joint is performed. This means that few, if any, transduced cells will remain in the patient to pose a possible long-term risk. Both the viral vector and the transduced cells are also subjected to exhaustive safety testing before use. Because of the desire to maximize safety, the goals of the trial are fairly modest. The specific goals are to determine: (1) whether ex vivo transfer of the human interleukin-1Ra gene to rheumatoid joints in humans can be successful with use of the techniques that we developed with animal models; (2) whether the transferred gene is expressed intra-articularly in human joints; (3) whether a local biological response to the transferred gene product occurs in human joints; and (4) whether the procedure is safe, feasible, and well tolerated by patients.

In order to be included in this trial, a patient: (1) must be a postmenopausal woman; (2) must have a diagnosis of rheumatoid arthritis according to the criteria of the American College of Rheumatology3, which include, for example, a positive rheumatoid factor, involvement of multiple joints, involvement of small joints for more than six months, and joint stiffness; (3) must be scheduled for replacement of the second through fifth metacarpophalangeal joints of at least one hand; and (4) must be scheduled for an operation on at least one other joint before the metacarpophalangeal joint replacement.

The last criterion for inclusion gives us the opportunity to obtain synovial tissue from the patient as a source of autologous synoviocytes. These cells are grown in vitro, and one-half are transduced with MFG-IRAP that has been prepared in a designated, specially constructed facility that meets the Food and Drug Administration guidelines for the preparation of vectors for human gene therapy. Once the patient's cells have been transduced, an enzyme-linked immunosorbent assay is performed to ensure that sufficient interleukin-1Ra is being produced. Most of the transduced and untransduced cells are then cryopreserved while the remainder are subjected to a battery of safety tests, which include assays for replication of competent retrovirus, endotoxin, and microbial agents such as mycoplasma, fungi, and bacteria. Once the cells have passed these tests, the stored cells are thawed, placed in culture, and, when they are confluent again, recovered.

The cells are suspended in saline solution and injected intra-articularly into the second through fifth metacarpophalangeal joints of one hand of the patient one week before the scheduled procedure. Two of the four joints receive untransduced, autologous control cells, and two receive cells expressing the interleukin-1Ra transgene. Both the surgeon and the patient are blinded as to which joints receive the transduced cells and which ones receive the untransduced cells. A week later, at the time of the replacement procedure, the synovial fluid, synovial tissue, and any residual articular cartilage are recovered and assayed for the presence and expression of the transgene and for evidence of a local biological response. Because this trial is limited to joints with end-stage disease that are scheduled for replacement and because the gene is present in the joint for only one week, clinical improvement is not an objective of this first trial.

As required by United States law, this trial was evaluated at several levels, including the local Institutional Review Board and the Institutional Biohazard Safety Committee, before it began. Next, the protocol was evaluated at the federal level by the Recombinant DNA Advisory Committee of the National Institutes of Health and the Food and Drug Administration, with which an Investigational New Drug Application was lodged. The National Institutes of Health also required us to establish an external monitoring board as a condition for providing financial support for this trial. This board comprises leaders in the fields of rheumatology, orthopaedic surgery, ethics, and gene therapy. Their additional review and approval were necessary before the trial could be commenced. In January 1996, we obtained approval to study nine patients.

The first patient was enrolled in the trial in March 1996. This patient was sixty-eight years old, had a twenty-year history of rheumatoid arthritis, and was scheduled to have an arthroplasty of the metacarpophalangeal joint of the right thumb as well as subsequent replacement of the second through fifth metacarpophalangeal joints. The first operation took place on April 4, 1996. Synovial tissue was retrieved at this time, and the cells were liberated by collagenase digestion. At the second passage, one-half of the fibroblastic cells that were infected with MFG-IRAP secreted high levels of interleukin-1Ra. The control cells that were not infected with the retrovirus secreted no detectable interleukin-1Ra.

The cells were then cryopreserved while aliquots were subjected to safety testing. The test results were favorable, and the cells were thawed, reseeded into culture, and, at the time of confluence, recovered. On July 17, 1996, 106 untransduced cells were injected into two of the metacarpophalangeal joints and 106 transduced cells suspended in saline solution were injected into the other two. A small volume of transduced and untransduced cell suspension was retained for postinjection testing for replication of competent retrovirus, endotoxin, mycoplasma, and bacteria.

One week later, the patient returned for joint lavage immediately before the replacement. All of the intra-articular tissues retrieved at the time of the operation were analyzed. We demonstrated, by in situ hybridization and reverse transcriptase-polymerase chain reaction, expression of the transgene in the joints that had been given an injection. The procedure was well tolerated by the patient, and no adverse short-term effects were noted. All patients have now been enrolled in the trial and have received treatment.

In an additional use of this technology, we are developing novel animal models of rheumatoid arthritis. The ability of genes to be expressed for long periods of time in selectively discrete tissues, such as the connective tissues of tendons, cartilage, bones, and ligaments, permits detailed study of the products in a manner that would be impossible with use of existing methods. One example of this, in which the interleukin-1ß gene was expressed in rabbit knees, was recently reported16. We believe that the new experimental information that can only be gained by gene-transfer methods justifies the further pursuit of these strategies irrespective of their direct application to the treatment of human disease.

It will take additional research to determine whether local or systemic delivery, or some combination of the two, is most appropriate for the treatment of rheumatoid arthritis in humans. Additional research is also needed to determine which gene or, more likely, which combinations of genes give the strongest antiarthritic effect. We have concentrated on using antagonists of interleukin-1 and tumor necrosis factor-{alpha} and have begun preliminary experiments using interleukin-10, CTLA4, and a variety of other candidate genes. Genes may also be used in conjunction with traditional drugs to provide superior combination therapies.

The concepts embodied in genetic therapy for rheumatoid arthritis can be applied to the treatment of osteoarthritis29 as well as to a variety of additional orthopaedic problems10, including the healing of bone13,27, cartilage20, meniscus19, and ligaments and tendons15.

The clinical trial in humans that we have initiated is important for a number of reasons. Not only is it the first gene-therapy trial for arthritis, it is also the first such trial for the treatment of any nonlethal disease. All previous gene-therapy protocols in humans were for conditions such as cancer, acquired immunodeficiency syndrome, and metabolic diseases with severe consequences. Our trial thus takes the field of gene therapy in an entirely new direction and has broken a very important psychological barrier in this regard. Because of its novelty, the overriding concern has been safety. Thus, the trial has been subjected to close scrutiny and modification by two local and two federal review bodies in addition to an independent monitoring panel. It is expected that this trial will make it easier to initiate future gene-therapy trials for arthritis in humans at medical centers around the world. In so doing, it will usher in an entirely new era of molecular orthopaedics.


   Footnotes
 
{dagger}Alfred Shands Lecture. Read at the Annual Meeting of the American Orthopaedic Association, Auckland, New Zealand, February 2, 1998.

{ddagger}Department of Orthopaedics, Massachusetts General Hospital, 55 Fruit Street, Gray 624, Boston, Massachusetts 02114-2617. E-mail address: jherndon@partners.org.

§Departments of Orthopaedic Surgery (C. H. E.) and Molecular Genetics and Biochemistry (P. D. R. and C. H. E.), Presbyterian-University Hospital, 200 Lothrop Street, Room C313, Pittsburgh, Pennsylvania 15213.


   References
Top
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