Introduction

Rheumatoid arthritis (RA) is a debilitating, systemic autoimmune disease distinguished by chronic inflammation of the distal diarthriodial joints that affects almost 1% of the world's population. Affected joints exhibit inflammatory cell infiltration and synovial hyperplasia that contribute to the progressive degradation of cartilage and bone. Although there are many drugs on the market that alleviate some of the systems associated with arthritis, there are currently no effective pharmacological treatments for the disease. Recently, biological agents that modulate the proinflammatory activities of TNF- and IL-1 have shown efficacy as novel antiarthritic drugs.^1,2,3,4 However, arthritis therapies that employ biological agents are currently limited by possible systemic side effects such as the occurrence and re-emergence of viral and bacterial infections as well as their exorbitant expense. Moreover, immediately upon the termination of systemic therapy, disease pathology returns unabated. As a result of this clear need for alternative, novel therapies for treating arthritis, gene transfer has been evaluated as an approach to circumvent the inherent impediments associated with delivery of therapeutic proteins.

There are several different approaches that can be utilized for the treatment of arthritis.^5,6,7 Genes can be delivered locally at the site of disease pathology such as the joint by intra-articular injection. Alternatively, therapeutic genes can be delivered using specific circulating cell types such as T cells^8,9,10 or antigen-presenting cells (APCs) such as dendritic cells (DC).^11,12,13 Although these types of cells result in more systemic delivery of therapeutic proteins, the ability of certain immune regulatory cells to home to sites of inflammation can also allow for local treatments following systemic injection. It is also possible to increase the levels of circulating therapeutic proteins by delivery of the gene to tissues such as muscle or liver.^14,15

In regard to both systemic and local delivery, there are only two general approaches that can be employed, in vivo, direct and ex vivo, indirect gene delivery (Figure 1). The first approach involves the direct injection of the vector at the site where expression is needed, allowing for genetic modification of cells following injection. In contrast, the ex vivo approach involves the genetic manipulation of cells in culture followed by injection of the modified cells either locally such as by intra-articular injection or systemically such as by intravenous injection of genetically modified immune regulatory cells. Depending upon the gene to be delivered and the pathology to be treated, there are certain advantages and disadvantages to each approach.

Figure 1.

Approaches and vectors used for local gene transfer to joints.

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Vectors for intra-articular gene transfer

For ex vivo gene delivery, the vector used for gene transfer usually is one that can stably modify the target cells. For these purposes, retroviral-based vectors that integrate into the host DNA are usually employed. However, it is also possible to modify the target cell by one of the many different transfection methods of plasmid DNA. In contrast, for in vivo gene transfer such as to the synovium following intra-articular injection, a variety of vectors have been tested. For example, intra-articular injection of adenoviral vectors results in efficient, but transient expression of genes due in part to the immune response to the virally-infected cells.^16,17,18 In the absence of an immune response, adenoviral-mediated gene expression persists for months (C Evans, unpublished). Adeno-associated virus (AAV) vectors also are able to infect synovium, but the relative efficiency of AAV for infection of different synovial cells types and muscle as well as the duration of AAV gene expression is unclear.^19,20 However, the initial results with intra-articular gene transfer with AAV clearly warrant further investigation. Lentiviruses also are able to infect synovium efficiently, allowing for persistent gene expression.²¹ Finally, nonviral gene transfer methods using liposomes and DNA conjugates also allow for local delivery, but the efficiency is reduced compared to viral vectors and most of the formulations tested are themselves inflammatory.²² Thus, there is still no perfect vector for in vivo delivery, but there are at least several types of vectors that have the potential to confer high level, persistent gene delivery.

Antiarthritic effects of gene transfer to synovium

The pathophysiology of RA can be conveniently summarized in the manner shown in Figure 2. Inappropriate immune activation leads to the production of autoreactive immune cells that then stimulate the excessive synthesis of cytokines and other mediators that promote inflammation, the formation of pannus, and the destruction of articular tissues. Accordingly, it is possible to design therapeutic strategies that target immune function, cytokine activity, and pannus. A number of these have been the subject of experimental gene therapy approaches.

Figure 2.

Pathogenesis of arthritis. The steps involved in progression of rheumatoid arthritis are shown along with examples of possible biological agents that could be delivered by gene transfer to interfere with disease progression.

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Modulating immune responses

Immune responses are initiated by the presentation of antigen to T-lymphocytes. A productive immune reaction requires the interaction between costimulatory molecules of the B7 family on the APC, and CD28 on the surface of the T-lymphocyte, an interaction that can be blocked using soluble CTLA4. Similarly, an interaction between the TNF family member CD40L, expressed on CD4 T cells, and CD40, expressed on APC also is important for the initiation of antigen-specific immune stimulation. This CD40-CD40L interaction can be blocked with sCD40-Ig (PDR, unpublished). It is possible that interfering with costimulation in this manner not only inhibits the immediate immune response, but also induces anergy to the epitopes being presented in the absence of costimulation. To achieve antigen-specific anergy in this manner would have many obvious advantages for the treatment of autoimmune disorders, including RA.

The ability of CTLA4Ig to block the progression of arthritis was examined by injection of an Ad.CTLA4Ig, vector into the knee joints of mice with collagen-induced arthritis.²³ This treatment inhibited collagen-induced arthritis for at least 20 weeks. Oddly, this was accompanied by high levels of circulating CTLA4Ig even though intravenous, intramuscular, and subcutaneous injection of the vector failed to raise serum levels of the gene product to a significant degree. However, it appears that a state of anergy was not achieved. Moreover, it has been observed that local or systemic gene transfer of CTLA4Ig is effective, but not as effective as delivery of IL-1 and TNF inhibitors in reversing the onset and progression of disease (CHE and PDR, unpublished).

In general terms, the T-cell response to antigen presentation is either a T-helper-1 or -2 (Th-1 or Th-2) type. Th-1 and Th-2 cells can be distinguished on the basis of the cytokines they secrete. RA is considered to be a Th-1 driven disease and the intra-articular delivery of genes encoding type 2 cytokines, such as IL-4, -10, and -13 has antiarthritic activity in a variety of different animal models of RA.^{24,25,26,27,28,29,30} In particular, a powerful antiarthritic effect is achieved when the level of intraarticular IL-10 transgene expression is subject to endogenous regulation in a manner that follows disease activity.³¹ In addition to immune deviation, the antiarthritic activities of these gene products may benefit from their independent anti-inflammatory properties. IL-4 strongly protects bone and cartilage from destruction, although it does so at the expense of causing inflammation.²⁸

Considerable attention has been given to blocking the intra-articular activities of TNF and IL-1, the two most prominent downstream cytokines involved in the pathophysiology of arthritic joints. Such activities are encouraged by the success of the TNF antagonists Enbrel and Remicade as therapeutic agents for treating RA. Moreover, recombinant human IL-1Ra has just entered the market as the drug Kineret. The clinical efficacy of Kineret is constrained by its extremely short biological half-life,³² a problem that gene delivery is well positioned to overcome.

The intra-articular administration of cDNAs encoding type I and type II soluble TNF receptors, type I soluble IL-1 receptor and IL-1Ra, ameliorate disease in animal models of RA.^33,34,35,36 The comparative degree to which they do this reflects the animal model being used. Thus, the effects of genes encoding IL-1 antagonists are marked and comprehensive in collagen-induced arthritis, whereas in antigen-induced arthritis there is a strong antierosive effect, but only a limited anti-inflammatory effect. In the latter case, a comprehensive antiarthritic effect is achieved by the coadministration of genes encoding TNF and IL-1 antagonists. As described in a later section, the intra-articular delivery of a cDNA encoding human IL-1Ra has entered clinical trials. IL-1 may also be an important mediator of cartilage loss in OA. The IL-1Ra gene has shown chondroprotective activity in surgically induced models of OA in dogs, rabbits, and horses.^37,38,39

A cautionary note is introduced by the results of experiments in which a gene encoding TGF-, a potentially immunosuppressive, antiarthritic and chondroregenerative cytokine, has been evaluated intra-articularly. Contrary to expectations, this provoked extremely bad sequelae within the joint, including a massive fibrosis, osteophyte formation, loss of articular cartilage and, in some cases, death of the animal.⁴⁰ This contrasts with the antiarthritic consequences of the intramuscular and T-cell administration of plasmids encoding TGF-.^10,41

Blocking signal transduction

Makarov⁴² was the first to draw attention to the central role of NF-B in the expression of genes associated with inflammation and tissue destruction in arthritis. Gene-based approaches to blocking this transcription factor include using adenovirus to deliver 'super' IB (inhibitor of NF-B that is resistant to phosphorylation and thus proteosomal degradation),⁴³ or a dominant negative form of IKK- (IkB kinase-),⁴⁴ as well as transfection with decoy oligonucleotides.^45,43 All of these methods show efficacy in animal models of RA.

Another approach involving intracellular signaling utilizes an adenovirus encoding C-terminal Src kinase (Csk). This kinase is a negative regulator of Src family kinases, known to be important in osteoclast function. The intra-articular injection of adenovirus encoding Csk in rats with adjuvant arthritis not only prevents bone destruction but also reduces inflammation.⁴⁶ In addition, the JAK-STAT-3 pathway has been implicated in inflammatory responses. CIS3/SOCS3 inhibits JAK tyrosine kinase activity and negatively regulates STAT activity. When introduced by adenovirus into the joints of rats with antigen- or collagen-induced arthritis, CIS3 cDNA dramatically inhibits the severity of arthritis and joint swelling.⁴⁷

Targeting the pannus

In several important ways, the pannus of the rheumatoid joint resembles a tumor. Accordingly, one set of gene therapy strategies for treating RA borrow from the field of cancer gene therapy and attempt to limit the size of the pannus by killing synovial cells directly, inhibiting their division, or interfering with their blood supply.

The delivery of genes that induce the apoptosis of cells within the synovium produces a 'genetic synovectomy'. This is the latest evolution in a long clinical history of performing synovectomies in individual rheumatoid joints by surgical, chemical, and radiochemical means. Typically, these procedures relieve the symptoms of disease for as long as several years, after which time the synovium regenerates and the condition reappears.

Unlike most other gene therapy approaches to therapy, a genetic synovectomy does not require long-term gene expression and may thus be achievable using existing technology. Adopting a strategy from cancer gene therapy, investigators have used the herpes thymidine kinase gene in conjunction with gancilovir to kill synovial cells. The intra-articular injection of naked, plasmid DNA encoding herpes thymidine kinase is sufficient to generate a beneficial effect in the knee joints of rabbits with antigen-induced arthritis,⁴⁸ and this approach has been taken into a phase I clinical trial. Positive results were also obtained using a first-generation adenovirus to deliver the herpes thymidine kinase gene to the finger joints of monkeys with collagen-induced arthritis.⁴⁹

A variety of apoptotic genes that do not require a prodrug such as ganciclovir have also shown good results in animal models. These include FasL,^50,51 TRAIL,⁵² and p53.⁵³ The results obtained with p53 are interesting because, as well as producing the expected apoptosis of cells within synovium, this gene also had a dramatic anti-inflammatory effect in the knee joints of rabbits with antigen-induced arthritis.⁵⁴ The mechanism of the latter is unknown, but clearly of great potential value in the comprehensive treatment of inflammatory joint diseases. It should also be noted that delivery of the IB gene as a method of inhibiting the synthesis of inflammatory mediators provokes apoptosis in the inflamed synovium.⁴³

The transfer to synovium of genes that inhibit cell division can also inhibit the exuberant proliferation that produces pannus. In this regard, intra-articular delivery of the cyclin-dependent kinase inhibitors p21 and p16 ameliorate adjuvant arthritis in rats and mice.^54,55 Pannus formation also depends on neovascularization, and delivery of the angiogenesis inhibitors endostatin⁵⁶ or angiostatin⁵⁷ is effective in animal models of RA.

By and large, when a gene is transferred successfully to the synovial lining of a joint, it is expressed intra-articularly and its product behaves in the predicted manner. Several different classes of genes have been evaluated in the local therapy of animal models of arthritis and many show considerable promise as antiarthritic agents. Two genes, IL-1Ra and herpes thymidine kinase, have been taken forward into phase I clinical trials (see below). There is thus no shortage of candidate genes for use in the local gene therapy of arthritis. Proof of principle having been established, resources need to be concentrated on achieving long-term gene expression, the next hurdle to clinical application. For all applications, apart from a genetic synovectomy, it will probably be necessary to express transgenes for extended periods of time within the joint. Recent data strongly suggest that the prior inability to achieve this in animal models results from immune recognition of foreign transgene products synthesized within the joint.²¹ This may reflect a species mismatch between the therapeutic transgene and the recipient species, continued intra-articular synthesis of viral vector proteins, or both. Once these barriers are eliminated, it is possible to express transgenes intra-articularly for many months in experimental animals ²¹ (Gouze et al, unpublished).

Long-term transgene expression will set the stage for a concerted effort to regulate transgene expression in a clinically useful manner. Two general approaches are foreseen. One of them seeks to achieve endogenous regulation, such that the level of therapeutic transgene expression automatically reflects the level of disease activity. One such system, developed by Varley et al,⁵⁸ utilizes an acute-phase promoter for specificity coupled to a TAT-driven system for amplifying expression of the transgene. The therapeutic efficacy of this system intra-articularly has been shown using adenovirus engineered with a C3-Tat/HIV promoter expressing IL-1Ra⁵⁹ or IL-10.³¹ In these adenoviral vectors a feedback inhibition loop is activated when inflammation increases and vice versa. The observation that transgene expression following transduction with a lentivirus is amplified by cell division in response to an acute inflammatory insult may serve as the basis for an alternative approach to the endogenous regulation of transgene expression (Gouze et al, unpublished). The other approach to regulated expression makes use of promoters that respond to external stimuli, such as tetracycline, rapamycin, or RU486. None of these have yet been evaluated in the context of the intra-articular gene therapy of arthritis. However, Gould et al used i.m. expression of dTNF-R after muscle electroporation from a single plasmid vector Tet system⁶⁰ to inhibit CIA in DBA/1 mice.⁶¹

T cells and other approaches for systemic expression

Treating locally and selectively the inflamed arthritic joint has clear advantages. However, the possibility of treating each affected joint may prove difficult in a clinical setting because of the number of joints involved. Hence, alternative approaches have been developed to deliver therapeutic agents systemically.

T cells as carriers

Gene transfer experiments have been important to assess the role of mediators of pathogenesis and also in understanding the immunobiological mechanisms of action of potential therapeutic genes. The use of well-characterized animal models of arthritis, such as collagen-induced arthritis (CIA), has facilitated the development of engineered T cells that recognize joint autoantigens. Their use is based on the principle that autoantigen-specific T cells are capable of transferring disease in an antigen-specific manner. T cells are mobile and can cross endothelial barriers and most importantly, memory T cells are long lived and proliferate upon encounter with antigen hence amplifying the therapeutic biological effect if they have been genetically engineered to express a therapeutic gene. Nakajima et al⁸ using in vivo imaging of reporter gene-engineered T cells showed that as expected, collagen type II-specific T cells, derived from the collagen type II-induced arthritis (CIA) model, accumulate in the joints and draining lymph nodes of arthritic animals and remain there for some time.

Among the genes that have been introduced into joint-specific T cells using retroviruses are the soluble TNF receptor (sTNF-R),⁹ TGF-,¹⁰ p40 IL-12,⁸ soluble CD35 (CR1),⁶² and IL-10.⁶³ Interestingly, these studies have shown that T cells could both prevent arthritis transfer^64,65 as well as inhibit disease after onset in animal models.¹⁰ Affecting T-cell function also has an effect on the B-cell pool since the isotypes of anticollagen antibodies showed a shift from Th1-driven to Th2-driven isotypes using sTNF-R.⁶⁶ This effect of sTNF-R therapy requires cognate antigen recognition because when anti-TNF therapy is given systemically as protein therapy such an effect is not seen.⁶⁷ Importantly, by this delivery system the effect is antigen-specific and does not affect other immune reactions⁶³ that could be necessary such as in the case of an infectious pathogen. However, it is not understood how the T cells used, that are originally of a Th1 (proinflammatory) phenotype capable of transferring disease, are altered at the cellular and molecular level by the expression of a therapeutic gene.

Whereas in experimental animal models the antigen driving the arthritic reaction is an immunogen facilitating the derivation of arthritogenic T cells and the isolation of T-cell hybridomas, in the clinical setting such cell populations are not easy to obtain since the autoantigen may differ in different patients and it may change with time in the same patient because of epitope spreading. For these reasons, T cells have been engineered to recognize a joint antigen via a chimeric receptor comprised of an extracellular domain derived from an scFv of an anticollagen type II antibody fused to the signaling subunits of the chain of the FcR1⁶⁷ or the CD3 chain of the TCR.⁶⁸ These redirected T cells recognize unprocessed antigen in an MHC-independent fashion, secrete proinflammatory cytokines, and proliferate upon encounter with antigen. Upon transfer to naïve mice these cells can cause arthritis as do the Th1 arthritogenic T cells described above and can be recovered from lymph nodes up to three weeks post-transfer (YC, unpublished). Further engineering with therapeutic genes is necessary to prove the usefulness of this approach.

T cells and their function as targets

Pathogenic T cells and their functions in arthritis have also been targeted from syngeneic fibroblasts expressing galectin-1⁶⁹ to cause apoptosis, or inhibition of Th1 functions by secretion of Th2 cytokines,⁷⁰ or expressing soluble CD35.⁶² T cells have also been targeted by DNA immunization against TCR idiotypes,⁷¹ and blocking costimulatory molecules such as B7.1 and B7.2 using soluble CTLA-4-Ig⁷² from an adenovirus injected intravenously. IL-4 was expressed in APCs (ie B cells and macrophages) pulsed with collagen type II⁷³ and also in a nonantigen-specific manner in dendritic cells¹¹ both with therapeutic results.

Other cell types

Other systemic delivery of immunoregulatory molecules has been achieved using encapsulated xenogeneic cells expressing IL-10, IL-4 or IL-13,^74,75 syngeneic fibroblasts expressing IFN ,⁷⁶ or expressing a small molecular weight dimeric TNF receptor (dTNF-R).^77,78 The therapeutic effect of IFN is interesting because recently it has been shown that in mice the signaling pathways activated by IFN are different from that used in humans⁷⁹ because of the fact that the gene for STAT4 in mice is mutated.⁸⁰ It is possible that some of the IFN therapeutic effects shown are mediated via inhibition of the fos/AP-1 signaling pathway induced by osteoprotegerin ligand (OPL/RANK) gene expression.⁸¹

In addition, therapeutic effects have been reported using liposome-coated plasmid expressing IL-10 injected i.p.,⁸² intramuscular injection of TGF-,⁴¹ CR1-Ig,⁶² and soluble IL-1 receptor type II expressed from human keratinocytes.⁸³ To affect cytokine, adhesion molecules and MMP gene expression small dsDNA oligonucleotides containing the NF-B binding site were used to serve as decoys and compete for binding of NF-B at the transcriptional level.^84,43,45

Therapeutic interventions to ameliorate the formation of free radicals, which mediate part cartilage and bone destruction via oxidative stress, include expression of extracellular superoxide dismutase (EC-SOD) from syngeneic fibroblasts⁸⁵ injected s.c. or in combination with catalase from immortalized syngeneic synoviocytes injected i.a. or s.c.⁸⁶ To inhibit matrix metalloproteinase (MMP) activity, TIMP-1 was expressed from an adenovirus vector injected i.v.⁸⁷

Dendritic cells and the contralateral effect

It has been demonstrated that local intra-articular injection of adenoviral vectors expressing sTNF-alpha receptor, IL-1Ra, sIL-1 receptor Type I and Type II, IL-10, vIL-10, and IL-4 were able to confer a significant antiarthritis effect in murine, rat, or rabbit model of arthritis.^{11,12,13,17,26,29,30,88,89} Interestingly, local delivery of these agents to one joint or paw resulted in a therapeutic effect in the contralateral knee or in untreated paws. For example, intra-articular injection of an adenoviral vector expressing vIL-10, the Epstein-Barr virus encoded IL-10 gene, resulted in a reduction in disease pathology, reducing the white blood cell infiltrate and improving cartilage metabolism in the injected knee, but also in the contralateral control knee.⁹⁰ This effect was termed the contralateral effect because of the initial observation made in a rabbit knee model of arthritis. Although this contralateral effect was initially observed with intra-articular or periarticular injection of adenoviral vectors a similar effect has been observed with retroviral vectors, liposomes, and even with injection of genetically modified synovial fibroblasts into rabbit knees.⁸⁹ The possible mechanisms that could be involved in this effect include dissemination of virus following injection, systemic levels of protein, trafficking of genetically or functionally modified cells or possibly a neurologic component. However, recent analysis of the effect has suggested that APCs play a major role in conferring the antigen-specific effects to distal joints.⁸⁸ The role of DC in conferring a therapeutic effect has been further demonstrated by the observation that bone marrow-derived DC, genetically modified in culture, are effective agents to reverse established arthritis in murine models. In particular, it has been demonstrated that gene transfer of IL-4 or FasL to DC followed by injection into mice with established arthritis at day resulted in a significant regression of established diseased with more than half of the treated mice becoming disease free for at least 2 months post-treatment.^11,12,13

Clinical trials

Although patients with severe RA have a shortened life expectancy, it is not normally considered a life-threatening disease. This places severe constraints upon the types of clinical trials that can be developed.⁵⁹ In particular, risk: benefit ratios are skewed in a manner that requires particular attention to the potential risks of any clinical protocol. The two protocols that have so far entered the clinic deal with this reality in quite different ways. One utilizes ex vivo delivery to avoid introducing transducing agents directly into the individual and to permit safety testing of the transduced cells prior to injection. Moreover, the transduced cells are removed from the participant during joint replacement surgery 1 week after injection. Furthermore, the transgene encodes a protein that has been widely administered to humans, and shown to be safe. The other protocol minimizes risk by using nonviral gene delivery to transfer a gene that has been safely used in previous human gene therapy studies.

Since no system for the in vivo, intra-articular delivery of the IL-1Ra gene is yet suitable for human application, the first trials have utilized ex vivo gene delivery.^91,92 Based on preclinical evaluation in experimental models, a Moloney-based retroviral vector (MFG-IRAP), with expression of the human IL-1Ra cDNA under the transcriptional control of the retroviral LTR, was used for gene transfer. At the time, this was the first protocol to come before the RAC committee for the gene therapy of a nonlethal disease and safety was an over-riding consideration. To address this issue, the IL-1Ra gene was delivered to rheumatoid joints 1 week before they were removed during joint replacement surgery. This procedure also removed the transduced cells, because preclinical data suggest that few, if any, of them migrated from the joint after intra-articular injection. As an additional advantage, surgery also provided abundant tissue for subsequent analysis for the presence and expression of the transgene.

The final protocol approved by the RAC and FDA is summarized in Figure 3. Nine postmenopausal women with advanced RA were recruited to the study. The entry criteria required them to need surgical replacement of the 2nd–5th metacarpophalangeal (MCP) joints on one hand and at least one other joint surgery. The latter provided autologous synovial tissue from which were grown synovial fibroblasts. Cultures of these cells were divided into two. One-half of the cells were transduced with MFG-IRAP, while the other half remained as untransduced controls. After safety testing and confirming the production of sufficient IL-1Ra, the cells were injected intra-articularly into the MCP joints. In a double-blind, dose-escalation fashion, two MCP joints received control cells and two received transduced cells. After 1 week, the MCP joints were surgically replaced and the retrieved tissues examined for evidence of transgene expression.

Figure 3.

Schematic of clinical gene therapy trial for arthritis. Synovial cells were isolated following a regularly scheduled surgical procedure such as a thumb fusion as shown. The synovial fibroblasts were propagated in culture with half of the cells subjected to infection with the retroviral vector MFG-IL-1Ra, whereas the other half of the cells go untreated. Following extensive testing of the cells for the presence of adventitious agents, the cells were injected back into the 2nd–5th metacarpophalangeal (MCP) joints, 1 week prior to regularly scheduled joint replacement surgery. Two MCP joints received modified cells whereas the other two joints received unmodified synovial fibroblasts. At 1 week postinjection, the joints were surgically removed and the tissue analyzed for expression and pathophysiological effects of the IL-1Ra transgene. The completed trial was a dose-escalation study with the first three patients receiving 10⁶ cells per joint, the next three patients 5 10⁶ cells, and the last three patients receiving 10⁷ cells.

Full figure and legend (47K)

An analysis of synovial tissues retrieved from joint replacement surgery confirmed the expression of the transgene by those MCP joints receiving the transduced cells, but not by those receiving control cells. In situ hybridization and immunohistochemistry identified clusters of cells expressing high levels of IL-1Ra on the synovial surfaces of the relevant joints. No adverse events related to gene transfer were reported. This clinical trial is now closed.

A similar phase I study is underway in Dusseldorf, Germany (PI Peter Wehling), the major difference from the above protocol being a time of 1 month between gene transfer and surgery. So far three subjects have undergone the procedure, with results very similar to those described above. Another phase I protocol (PI Blake Roessler) involves the in vivo, intra-articular delivery of plasmid containing the thymidine kinase gene of herpes simplex virus. This transfects cells within the synovium, which renders them sensitive to the prodrug ganciclovir administered subsequently. As a result of a marked bystander effect, extensive death of cells within the synovium results. So far one individual has been treated in this manner.

Since RA is considered to be a nonlethal disease, considerable attention needs to be given to the design of clinical protocols. Nevertheless, one clinical study has been completed and two others are in progress. Based upon this limited experience of 13 subjects, it appears that genes can indeed be transferred safely to human rheumatoid joints. This encourages the pursuit of further clinical studies and one phase II protocol is undergoing regulatory review. Such studies require considerable resources and are expensive. If they are to be conducted outside of the pharmaceutical industry, suitable public funding mechanisms need to be established.

Current status and future directions

RA is a polygenic disease in which the MHC complex has an important contribution. The presence of a cytokine imbalance in the RA joint has been well described⁹³ and has served as the fundamental stepping stone for the development of novel therapeutic avenues that are independent of the MHC haplotype of the patient. Targeting the proinflammatory cytokine TNF has yielded impressive clinical results in particular in combination with methotrexate.⁹⁴ Yet, production of anti-TNF compounds is extremely expensive, disease relapses upon cessation of therapy, and 20% of patients do not respond. The mechanism involved in the lack of response to anti-TNF is still unknown. Delivery of therapeutic compounds by gene transfer has opened an important alternative avenue that can greatly improve on the limitations of protein therapy. The clinical studies pioneered by the group in Pittsburgh (CHE and PDR) using autologous synoviocytes expressing constitutively IL-1Ra from a retroviral vector demonstrated that such approach is safe^91,92 and feasible. Whether such ex vivo approach using these immobile cells will ultimately lead to a long-term benefit needs further investigation.

Animal models continue to provide different possible avenues for treatment and proof of principle. However, they have limitations⁹⁵ as many studies have used prophylactic interventions instead of treatment after disease onset, some models are short term (5–10 days) and do not reflect the long-term chronic nature of the human disease. Yet, some studies have been carried out for a few weeks post disease onset in rodents,^10,41 in xenotransplantation models of arthritis using human synoviocytes and cartilage,⁹⁶ in large animals such as horses,³⁹ and in a monkey CIA model.⁴³ It will be important to investigate the effects of gene therapy for long-term arthritis in chronic and spontaneous transgenic models of disease that can help elucidate and define new therapeutic targets, novel methods of delivery as well as possible systemic side effects.

RA has a complex pathology where chronic and acute inflammation may occur superimposed or as separate events. The continuous presence of TNF and IL-1 facilitate the chronic infiltration of inflammatory cells while complement-mediated effects increase the likelihood of rapid transvasation, free radical formation, and activation of MMPs. Free radicals may influence the formation of neoepitopes that could perpetuate the autoimmune response and cause mutations in DNA^97,98 that will affect both pathological processes and the ability of the cells to respond to therapy.⁹⁹ At present, it is unknown how these inflammation-acquired genetic changes affect the pathophysiology of the disease and whether they are irreversible or could be harnessed for therapeutic purposes.¹⁰⁰ More efforts need to be directed to elucidate these issues.

The effectiveness of gene therapy both by local in vivo or ex vivo applications or systemic delivery has proved beneficial in arthritis models. In many cases, short half-lived cytokines or cytokine inhibitors have been used and their plasma levels have been too low to be detected unless massive production was obtained such as during i.v. administration of adenovirus that infects primarily liver tissue. Despite the low plasma concentrations normally obtained, it is clear that low-level expression of a therapeutic gene for long periods can modify the pathological process. Whether this approach is sufficiently safe has not been thoroughly investigated and it is of concern that in cases of an infectious pathogen constitutive expression of a systemic nature could be detrimental. Advances in protein engineering, such as the development of novel TNF inhibitors,⁷⁷ latent cytokines that become activated at sites of disease,¹⁰¹ and the targeting of cytokines to specific sites via fusion to scFv moieties (immunocytokines),¹⁰² hold promise that systemic gene therapy can be made safer, targeted, and very effective.

The nature of therapeutic molecules is important. Molecules with a long half-life could accumulate in plasma and more readily cause side effects. Hence, secreted therapeutic molecules with short half-lives that are produced and consumed locally, such as cytokines having autocrine and paracrine effects, are more advantageous specially taken into account that in vivo delivery of vectors may not target all the cells involved in the pathogenic process.^103,104 However, some intracellular targets, such as suicide genes, that have bystander effects on nontransduced cells and the exploitation of the collateral effect may be further developed. Further investigation in new methods of delivery utilizing immobile cells versus mobile cells, or use of vectors that are nonimmunogenic should be carefully and quantitatively compared and assessed.

The main challenge for gene therapy in arthritis is to be able to provide a cost-effective long-term safe treatment that will improve dramatically the current therapeutic outcomes provided by protein therapy and provide for repair of joint tissue and function. Current therapeutic interventions have mainly dealt either with the autoimmune inflammation or with cartilage degradation. Little has been done to repair the damaged tissue and this in part may be because of the lack of appropriate arthritis animal models to test this.

Some studies have used combination therapy of anti-TNF and vIL-10,¹⁰⁵ anti-TNF and soluble IL-1 receptor,¹⁰⁶ TGF and anti-TNF,⁷⁸ EC-SOD and catalase.⁸⁶ These combination therapy studies indicated that targeting more than one pathway has synergistic if not at least additive clinical effects. New therapeutic targets are being discovered constantly including vasoactive intestinal peptide (VIP),¹⁰⁷ IL-17,¹⁰⁸ osteoprotegerin,²⁷ and IL-15¹⁰⁹ to name a few. Which combination therapy will be most effective needs further elucidation.

Stem cells have been proposed as potent therapeutic means for tissue repair in arthritic disease¹¹⁰ (see also Chapter by Jorgensen et al.) While mesenchymal stem cells capable of differentiating into cartilage and bone can be isolated from bone marrow and be genetically manipulated in vitro, it will be necessary to develop means to regulate their differentiation pathway in vivo as well as assuring their attachment to damaged areas for appropriate repair. These cells may also need to be engineered to stop the inflammatory reactions occurring in the diseased joint.

Amalgamating the new developments in transcriptionally regulated nonimmunogenic vectors with novel genetically engineered therapeutics as well as the new advances in stem cell research may provide improvements to current therapies. The hope is that gene therapy will bring long-term benefit to RA patients once the appropriate safety, ethical, and medical considerations are addressed.

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