Gene therapy of the rheumatic diseases: 1998 to 2008

During the decade since the launch of Arthritis Research, the application of gene therapy to the rheumatic diseases has experienced the same vicissitudes as the field of gene therapy as a whole. There have been conceptual and technological advances and an increase in the number of clinical trials. However, funding has been unreliable and a small number of high-profile deaths in human trials, including one in an arthritis gene therapy trial, have provided ammunition to skeptics. Nevertheless, steady progress has been made in a number of applications, including rheumatoid arthritis and osteoarthritis, Sjögren syndrome, and lupus. Clinical trials in rheumatoid arthritis have progressed to phase II and have provided the first glimpses of possible efficacy. Two phase I protocols for osteoarthritis are under way. Proof of principle has been demonstrated in animal models of Sjögren syndrome and lupus. For certain indications, the major technological barriers to the development of genetic therapies seem to have been largely overcome. The translational research necessary to turn these advances into effective genetic medicines requires sustained funding and continuity of effort.


Introduction
When Arthritis Research was launched, the field of gene therapy was going from strength to strength. The preceding decade had seen the number of human gene therapy trials grow, since the first properly authorized gene transfer to a human in 1989, to a total of 368 by 1998. Despite the worst predictions of the skeptics, there had been no serious adverse events and the field looked forward, like the economy that was fuelling much speculation in the area, to continued rapid growth. Optimists predicted that the first genetic medicines would be on the market within a few years. Rheumatoid arthritis (RA) had become an early target for gene therapy (Figure 1), capturing the optimism of the early 1990s and beginning clinical trials in 1996. The first Inter-national Meeting on the Gene Therapy of Arthritis and Related Disorders (GTARD) was held at the National Institutes of Health (NIH) (Bethesda, MD, USA) in 1998 [1] and attracted over 200 participants.
Matters then changed abruptly. The 1999 death of Jesse Gelsinger [2] reopened safety concerns. This, in turn, made it more difficult to obtain funding from traditional sources, such as the NIH, as well as the biotechnology industry, which was also dealing with a rapidly slowing economy. Many rheumatic diseases, though serious, are not considered to be lifethreatening, a factor that further reduced enthusiasm for gene therapy research in this area under these circumstances.
Although the first flush of enthusiasm is over, the past decade has seen steady progress in developing genetic therapies for several conditions, and the number of clinical trials worldwide is approaching 1,500. The first commercial gene therapeutic, Gendicin for cancer of the head and neck, has been launched in China [3], and gene therapy for familial lipoprotein lipase deficiency is available as an orphan drug in Europe and the US. Cures have been reported for X-linked severe combined immunodeficiency disease (SCID) [4], adenosine deaminase-SCID [5], and X-linked chronic granulomatous disease [6]. Striking success in treating Leber's congenital amaurosis has recently been reported by two independent groups [7,8].
recently held in Seattle. As discussed below, there have been a number of clinical trials in the area of arthritis gene therapy, one of which has entered phase II, and some other areas are in an advanced preclinical stage of development.

Advances in technology
Central to any successful gene therapy is the ability to transfer genes efficiently and safely to the target cells. The same basic viral and nonviral vectors available now were available 10 years ago, but there have been developments in their engineering and application.

Viral vectors
Although oncoretroviruses, such as the Moloney murine leukemia virus, were the first to be used in clinical trials and dominated applications in human gene therapy for some years, they are less popular now. Pseudotyping the retroviral coat has overcome, to some degree, the problem of modest titers, but the inconvenience and expense of ex vivo gene transfer remain. Furthermore, the occurrence of insertional mutagenesis during human gene therapy trials [11] has generated a huge barrier to the use of oncoretroviruses in nonlethal nonmendelian diseases. The US Food and Drug Administration (FDA), for example, requires a 15-year followup on all clinical trials using integrating vectors.
Because lentivirus vectors are also integrating retroviruses, they are covered by the same restrictions. This is unfortunate because vesicular stomatitis virus-pseudotyped lentiviruses are extremely efficient and do not require host cell division; they transduce synovium very effectively after intra-articular injection [12,13]. Although lentiviruses are being engineered to remain episomal, their human use in rheumatology seems unlikely within any reasonable time frame.
Adenovirus vectors have overtaken retroviruses as the most commonly used in human clinical trials. Indeed, the first commercially available gene therapeutic, Gendicine [3], which is available in China for cancer, uses adenovirus. Engineering adenovirus vectors has taken the direction of deleting larger and larger segments of the viral genome, leading to high-capacity 'gutted' vectors that lack all viral coding sequences [14]. This reduces the immunogenicity of transduced cells, but not that of the virions themselves. Although gutted adenovirus vectors have several advantages, including a theoretical carrying capacity of over 30 kb, they are difficult to produce and purify. Other modifications to adenovirus include mutating coat proteins to enhance transduction efficiency or to alter tropism. The inclusion of an arginine-glycine-aspartate sequence, for instance, greatly enhances the transduction of synovium [15].
Adeno-associated virus (AAV) has seen the greatest recent development. AAV was previously hampered by difficulties in manufacturing large amounts of clinical-grade vector and modest levels of transgene expression in many cell types. The latter reflects, in part, the single-stranded DNA genome of AAV, which requires the host cell to synthesize a complementary second strand; this process is inefficient in many cells.
The production problems have been eased by new technologies to facilitate the generation of recombinant AAV [16]. Most significantly, transgene expression has been made much higher, quicker, and more reliable by the development of self-complementary (sc) vectors containing positive-and negative-strand viral genomes linked at one terminal repeat [17]. The drawback is that the packaging capacity is reduced by half to about 2 kb. Nevertheless, many of the cytokines and other modulatory molecules of interest in rheumatic diseases have cDNAs that are small enough to fit into this space. Recent data confirm the superiority of scAAV as a means of transferring genes to joints and expressing them intra-articularly [18].
There has been a rapid increase in the number of different recombinant AAV serotypes [19]. Some of these offer altered tropisms and enhanced transduction efficiencies. AAV1, for instance, has a much greater ability to transduce skeletal muscle than the prototypical AAV2 serotype [20]. It is unclear which, if any, of these new serotypes will find applications in rheumatology, although this is an active area of research (see next section). English language publications on arthritis gene therapy in the refereed literature. The data are based on a PubMed search using 'arthritis gene therapy' as the search term. The first paper on arthritis gene therapy was published in 1992 [27]. The first efficacy data for animal models of rheumatoid arthritis (RA) appeared in 1996 [103,104], and the first efficacy data for animal models of osteoarthritis (OA) followed a year later [79]. The first human trial for RA began in 1996 [29]. Seven clinical trials for RA and OA have been initiated, one of them reaching phase II ( Table 1). The first evidence of possible clinical responses to gene transfer was published this year [31]. Reprinted with permission [105].
Until recently, little attention was paid to the immune reaction to AAV given its perceived low immunogenicity. This rapidly changed when data from a clinical trial using AAV to treat hemophilia noted a neutralizing immune reaction [21] involving the generation of cytotoxic T lymphocytes [22]. This led to transient transaminitis and curtailed transgene expression. In light of these sorts of findings, the immune response to AAV is undergoing reevaluation.
The perceived safety of AAV vectors has also contributed to their increased popularity. As noted, vector-related deaths have occurred in trials using recombinant retrovirus and adenovirus. Although a fatality occurred last year in an arthritis trial using recombinant AAV [23], the FDA determined that the vector was not to blame and allowed the trial to continue. The number of human trials using AAV has risen to over 50, most of these being approved in the last few years. Two large phase III trials for prostate cancer using AAV are under way. As noted earlier, orphan drug status has been granted for AAV-mediated gene therapy for familial lipoprotein lipase deficiency, and eyesight has been restored to patients with Leber's congentital amaurosis using AAV vectors [7,8].
Although a number of other viral vectors have been used in clinical trials, they are less relevant to rheumatic diseases. Herpes simplex virus, for instance, is still troubled by cytotoxicity and its use is increasingly restricted to the nervous system, where it has a natural latency. Viral vectors are reviewed in reference [24].

Nonviral vectors
Nonviral vectors continue to be of interest because they are simpler, safer, and less expensive than viruses and offer very large carrying capacities. The simplest vectors are plasmids. Transfection efficiency can be increased by associating the DNA with a carrier, such as a liposome or a polymer, or through the use of a physical stimulus, such as an electric pulse (electroporation). Although a very large number of formulations exist, nonviral gene delivery (transfection) remains much less efficient than viral gene delivery (transduction) and this remains a barrier to its wider use. Despite this, nonviral vectors remain of interest because of persistent reports in the refereed literature of success when using them to treat animal models of rheumatic disease (discussed in the next sections). Nonviral vectors are reviewed in reference [25].
Available online http://arthritis-research.com/content/11/1/209 All of these target rheumatoid arthritis, except for the TissueGene Inc. and Kolon Life Science trials that target osteoarthritis. The Targeted Genetics Corporation trial can also recruit subjects with psoriatic arthritis and ankylosing spondylitis. AAV, adeno-associated virus; FDA, US Food and Drug Administration; HSV-tk, herpes simplex virus thymidine kinase; IL-1Ra, interleukin-1 receptor antagonist; N/A, not applicable; OBA, Office of Biotechnology Activities; PI, principal investigator; TGF-β 1 , transforming growth factor-beta-1; TNFR, tumor necrosis factor receptor. Reprinted with permission [23].

Rheumatoid arthritis Local therapy
Interest in applying gene therapy to the treatment of rheumatic diseases began in the early 1990s with attempts to deliver cDNAs to the synovial linings of joints [26,27]. The basic premise is quite simple (Figure 2). The sustained intraarticular expression of a cDNA encoding a secreted antiarthritic product will treat the joint locally without the need for readministration and avoid the peaks and troughs of traditional routes of drug delivery. No competing technology is able to do this. If gene transfer is sufficiently efficient, cDNAs encoding nonsecreted products are also possible. Treating individual diseased joints rather than the entire patient reduces costs and lowers opportunities for adverse systemic side effects. A number of different types of transgene have been suggested for this purpose, including those encoding cytokine antagonists, immunomodulators, antiangiogenic factors, apoptotic agents, antioxidants, inhibitors of mitosis, as well as molecules that modulate cell signalling and the activities of transcription factors (reviewed in [28]).
By the time the first issue of Arthritis Research appeared, a phase I clinical trial was under way ( Figure 1 and Table 1). This used a retrovirus (MFG-IRAP) to deliver the human interleukin-1 receptor antagonist (IL-1Ra) cDNA by an ex vivo protocol to the metacarpophalangeal (MCP) joints of patients with advanced RA [29]. Among the strict safety requirements of this study was the need to recruit subjects who needed MCP joint replacement surgery, so that the genetically modified cells could be surgically removed 1 week after injection.
This study confirmed that genes could be safely transferred to human rheumatoid joints and expressed within them, at least for 1 week [30]. Although several subjects reported symptomatic improvement, the study was not designed to measure efficacy. A small similar German study, involving just two subjects, is thus of interest because it included preliminary outcome measures based on pain and swelling, using a joint that did not receive the IL-1Ra cDNA as an intrapatient control. Both subjects responded to gene transfer, one of them dramatically so, and the clinical improvement lasted for the entire 4 weeks of the study, despite one subject experiencing flares in nontreated joints [31].
The occurrence of leukemia in humans as a result of insertional mutagenesis using retrovirus vectors, coupled to the high cost of ex vivo gene therapy using passaged autologous cells, has curtailed future trials of this kind. Instead, investigators are concentrating on in vivo gene delivery to joints. Based upon promising preclinical data in rabbits [32], Roessler and colleagues treated one subject with plasmid DNA encoding herpes simplex virus-thymidine kinase and followed this with administration of ganciclovir to effect a genetic synovectomy. Although there were no adverse events associated with this procedure, the trial overlapped with the death of Jesse Gelsinger in 1999, which hindered recruitment, and the study was terminated. Since then, the emphasis for in vivo delivery to joints has shifted to AAV for the reasons described in the previous section.
There have been two clinical trials using AAV, both sponsored by Targeted Genetics Corporation (Seattle, WA, USA). The vector (tgAAC94) comprises AAV2 with a singlestranded DNA genome encoding etanercept. Expression is driven by a human cytomegalovirus immediate early promoter. The trials have been discussed in detail [23]. In the first phase I study, 14 subjects with RA and 1 with ankylosing spondylitis were administered vector [33]. Fourteen knee joints and one ankle were injected with 10 10 or 10 11 virus particles per milliliter; knee joints received 5 mL and the ankle received 2 mL. A subsequent phase I/II study enrolled 127 subjects with a dose escalation of 10 11 , 10 12 , or 10 13 virions per milliliter to be injected into symptomatic knee, ankle, wrist, MCP, or elbow joints. The protocol allowed subjects to receive a second injection of tgAAC94.
The phase I/II trial attracted considerable attention last year when a subject died soon after receiving a second injection of vector into her knee joint [23]. The case aroused controversy because, in addition to receiving cDNA encoding etanercept, the subject was on adalimumab, having previously taken etanercept until this was discontinued because of a flare. The subject died from histoplasmosis, a known risk factor with anti-tumor necrosis factors (anti-TNFs), in conjunction with a massive retroperitoneal hematoma. After a lengthy investigation by the FDA and the Recombinant DNA Advisory Committee of the NIH, the trial was allowed to proceed in a slightly modified fashion. Preliminary efficacy data suggest that some subjects had symptomatic improvement in response to the gene treatment [34].
A number of groups are now interested in using AAV to deliver genes to joints. Research is focusing on the choice of serotype and the host immune response to the vectors. Serotypes 1, 2, 5, and 8 have attracted the most scrutiny. According to Apparailly and colleagues [35], AAV5 is superior to AAV1 or 2 in the knee joints of mice. This was confirmed in rats, and AAV2 and 5 were shown to have equal efficiency in transducing cultures of human synovial fibroblasts [36]. Another study indicates the following order of preference: AAV2 > 1 > 5 > 8 [37]. However, when human synovial fluids were screened for pre-existing immunity to AAV, neutralizing antibodies to serotypes 1 and 2 were more common than antibodies to serotype 5, suggesting to Boissier and colleagues [37] that AAV5 may be more useful in humans, despite lower transduction efficiency. Humoral reactions to AAV2 were noted in the trial of tgAAC94, mentioned above, but possible cell-mediated immunity was not measured. Studies of AAV2-mediated gene delivery to the knee joints of rabbits confirm a neutralizing immune reaction that prevents redosing [18]. AAV has been used to express soluble TNF receptors [38], beta interferon (IFN-β) [39], angiostatin [40], dominant negative Iκκβ (inhibitor of kappa B kinase β) [41], and IL-1Ra [18] in the joints of experimental animals, with an associated antiarthritic effect.
In most species, conventional AAV vectors containing a single-stranded DNA genome have only a modest ability to transduce articular tissues. Transduction efficiency can be enhanced by irradiation, a process that provokes secondstrand synthesis [42]. The need for the latter can be obviated with the use of scAAV, and recent findings confirm the superiority of these vectors in the rabbit knee joint [18]. According to data from the same study, only 10% to 20% of AAV genomes that enter synovial fibroblasts appear in the nucleus. This identifies a second constraint to transduction efficiency that helps to account for the relatively high number of AAV virions (10 4 to 10 5 particles per cell) needed for useful levels of transgene expression. Proteosome inhibitors improve the nuclear uptake of AAV genomes in human synovial cells, leading to greatly enhanced transgene expression [43]. In agreement with this, mutations to the AAV coat protein that prevent ubiquitination also increase transduction efficiency [44]. According to Traister and colleagues [45], transgene expression from AAV vectors is increased in human synovial fibroblasts in the presence of inflammatory cytokines. A similar effect was reported some years ago by Pan and colleagues [46,47] in rat knee joints but this has been difficult to reproduce [18].
For intra-articular gene therapy to be a clinical success, there is a need for extended periods of transgene expression. This has proved difficult in animal models. Recent work by Gouze and colleagues [48] identified immune reactions to nonhomologous proteins as the major barrier to prolonged transgene expression. Using the rat knee joint as a model system, they showed that cDNA encoding a rat protein delivered by an immunologically silent vector can be expressed in a stable prolonged fashion. Of interest, longterm transgene expression does not require an integrating vector and is independent of the promoter. Instead, it relies on the presence of long-lived nonmitotic cells within certain dense collagenous tissues in and around the joint (Figure 3).
An ability to achieve long-term transgene expression opens the way for regulated expression. Two approaches have been investigated. One makes use of endogenous cues to ensure that the level of expression tracks disease activity within the joint. These strategies use inducible promoters based upon upstream regulatory sequences that control the expression of acute-phase proteins and inflammatory cytokines, such as IL-1 and IL-6 [38,49,50]. A related method uses a sequence containing multiple nuclear factor-kappa-B (NF-κB)-binding sites [38]. A second approach to regulated transgene expression uses exogenous molecules, such as doxycyline, to manipulate the level of production [51][52][53]. The latter approach provides greater insurance against inappropriate transgene expression as might occur during an infection. Though not strictly gene therapy, a related clinical trial injects decoy oligonucleotides that inhibit the activity of the transcription factor NF-κB into rheumatoid joints [54]. So far, there have been no adverse events and some evidence of a clinical response in certain subjects (Tetsuya Tomita, personal communication).

Systemic therapy
In a polyarticular condition such as RA, an intra-articular gene therapy might require the injection of large numbers of joints. Moreover, a local gene therapy might not address systemic extra-articular manifestations of the disease. Thus, there is interest in a more general approach to therapy in which a transgene is introduced into a site where a secreted gene product will have access to the systemic circulation ( Figure 2). Proof of principle has been established using intramuscular, intravenous, intraperitoneal, and subcutaneous routes of delivery by in vivo and ex vivo methods (reviewed in [28]). Although this approach has obvious attractions, it provides only an incremental advance over what is already achieved by traditional methods of protein delivery and is accompanied by increased risk of adverse events. For these reasons, it has not achieved widespread popularity. One interesting possible exception, however, is the parenteral administration of naked DNA.
There are several reports in the refereed literature ascribing potent antiarthritic properties to plasmid DNA when delivered by intramuscular, intraperitoneal, intravenous, and intranasal routes [55][56][57][58][59][60][61]. Because levels of transgene expression are low when DNA is administered in these ways, an alternative explanation for their efficacy in animal models of RA is needed. One possibility is the uptake of DNA by antigenpresenting cells (APCs) which then travel to sites of antigen presentation where sufficient transgene is expressed to modulate immune reactivity locally. This is an example of facilitated local therapy, described in the next section.
DNA can also be used to vaccinate. There are several examples using animal models of RA in which DNA vaccines that express arthritogenic antigens, such as heat-shock proteins [62], or mediators of arthritis, such as TNF [63], are protective. It is also possible to induce tolerance by DNA immunization in the absence of adjuvant [64]. Though effective in animal models, such strategies may be risky in humans.

Facilitated local therapy
The ability to target multiple diseased joints selectively by a single parenteral injection is known as facilitated local therapy ( Figure 2). This was first noted as a contralateral therapeutic effect in the knee joints of rabbits with bilateral antigen- Fibroblasts resident in fibrous articular tissues support stable expression of exogenous transgenes. Following intra-articular injection of lentivirus-GFP or Ad.GFP into the knees of nude rats, groups of animals were sacrificed at days 5 and 168. The knee joints and surrounding tissues were harvested intact, decalcified, and processed for histology. For each joint, the approximate positions of fluorescent cells identified in serial, sagittal whole-knee sections were tabulated in green on knee joint diagrams similar to those shown on the left. The diagrams shown are representative of the results observed with both viruses at the respective times. On the right, images characteristic of the appearance of the GFP + cells in tissue sections at the different times are shown (×20 magnification). Lines indicate the approximate regions represented by the tissue sections. The numbers of GFP + cells in the synovium and subsynovium were reduced dramatically at day 168. The density and distribution of GFP + cells in the tendon, ligament, and fibrous synovium were largely unchanged over the duration of the experiment. No fluorescent cells were seen in the articular cartilage with either virus at any time point. B, bone; GFP, green fluorescent protein; M, muscle; P, patella. Reprinted with permission [48].
induced arthritis [65]. It occurs with both in vivo and ex vivo [66] gene delivery and is thought to reflect immune modulation via APCs that are exposed to appropriate transgene products as they present arthritogenic antigens to T lymphocytes ( Figure 4). Studies using the murine delayed-type hypersensitivity reaction as a model [67] showed that genetically modified dendritic cells and macrophages could migrate to sites of inflammation and inhibit immune-driven pathology in an antigen-specific manner. In subsequent studies, dendritic cells expressing IL-4 were shown to migrate to the paws of MHC-matched mice with collagen-induced arthritis and quell disease activity, even in established disease [68]. The antiarthritic effect was stronger than when the same adenovirus vector was used to deliver IL-4 systemically. A variety of additional transgenes, including IL-10, indoleamine 2,3-dioxygenase, and IκB (inhibitor of kappa-B), are effective in this manner.
A related strategy produces selective ablation of autoreactive T lymphocytes by modifying APCs to express inducers of apoptosis on their cell surfaces. When the APC expresses an arthritogenic antigen to reactive T lymphocytes, the latter undergo apoptosis. Although this has been shown in murine models using Fas ligand [69,70] as the transgene, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a better candidate because its receptor has more limited distribution, thus reducing opportunities for unwanted side effects. Impressive proof of principle has been demonstrated in murine collagen-induced arthritis using dendritic cells that express TRAIL [71]. The therapeutic effect was improved by pulsing the dendritic cells with type II collagen before injection. The equivalent manipulation in RA will be complicated because the inciting antigens are not known, although a regulatory bystander effect could be achieved. The response to TRAIL gene transfer is enhanced when RNA interference is used to knock down expression of its decoy receptor, DcR2 [72].
T lymphocytes also home to sites of inflammation and immune reactivity. Like APCs, they may be genetically modified and used to target multiple sites of disease by parenteral administration, although lymphocytes are more difficult to transduce than APCs. However, proof of principle has been shown in several animal experiments using IL-4, IL-10, IL-12 p40, and anti-TNF single-chain antibody as transgenes [73,74]. In most animal models, the arthritogenic antigen is known and T lymphocytes with the appropriate T-cell receptor can be used to maximize the effect. In RA, however, the inciting antigen is not known and enrichment is difficult. As one response to this limitation, Annenkov and Chernajovsky [75] engineered a T-cell receptor whose extracellular domain contains a type II collagen-binding motif.  A model based upon trafficking of antigen-presenting cells (APCs) to explain the contralateral effect. Introduction of a suitable vector, in this example one encoding viral interleukin-10 (vIL-10), into an inflamed joint transduces synovium and APCs. Lymphocytes are very difficult to transduce, as reflected in the figure. Intra-articular antigen presentation thus occurs in the presence of a high local concentration of vIL-10 produced by the synovium, the APC, or both. Under these conditions, the immune response deviates toward a therapeutic Th2 response. Lymphocytes and APCs then traffic to other joints via the blood stream or lymphatics, where they suppress disease. Reprinted with permission [28].
Isis Pharmaceuticals (Carlsbad, CA, USA) sponsored phase I, IIa, and II clinical trials in which anti-sense RNA directed against TNF was injected intravenously and subcutaneously into subjects with RA. Anti-sense RNA was shown to traffic to the synovium of diseased joints, suggesting facilitated local delivery. The phase II study involved 157 subjects with RA who received 200 mg of anti-sense RNA twice a week, once a week, or once a fortnight. Subjects in the two highest dosing groups showed improvement in ACR20 (American College of Rheumatology 20% improvement criteria) scores. These studies were reported on the company's website [76] but were never published in the peer-reviewed literature. The company is no longer pursuing this project.
The recent emergence of RNA interference provides the ability to knock down cytokine synthesis in a highly specific fashion. Khoury and colleagues [77] have delivered short interfering RNA molecules targeted to IL-1, IL-6, and IL-18 in murine collagen-induced arthritis [77]. Knockdown of each cytokine was effective in reducing the incidence and severity of disease, but a dramatic therapeutic effect was observed when all three were inhibited together.

Osteoarthritis
Osteoarthritis (OA) is highly prevalent, incurable, and difficult to treat, imposing an enormous socioeconomic burden. Because it affects a limited number of joints and has no known systemic components, OA is well suited to local gene therapy [78]. Several preclinical studies confirm the efficacy of local gene delivery in the treatment of experimental models of OA [79][80][81][82]. Nearly all of these have used IL-1Ra as the transgene product, reflecting the importance of IL-1 as a mediator in the osteoarthritic joint. The equine study of Frisbie and colleagues [83] is of interest because, in addition to using the conventional histological outcome measures, they noted a reduction in lameness in response to gene therapy. This is an encouraging result for a disease in which pain is the overriding presenting clinical symptom. OA is common in horses, dogs, and other companion animals, suggesting a role for gene therapy in veterinary medicine.
Because destruction of the articular cartilage is the most obvious pathological lesion in the affected joints of individuals with OA, studies on the treatment of OA overlap with those on cartilage regeneration. Discussion of gene transfer approaches to cartilage regeneration lies beyond the scope of this article, but reference [84] provides a good recent review. Collectively, cDNAs encoding insulin-like growth factor-1, fibroblast growth factor-2, bone morphogenetic protein (BMP)-2, BMP-4, BMP-7, TGF-β, and sonic hedgehog have shown promise in cartilage repair. Clinical trials are under way in Korea and the US using a retrovirally transduced, human chondrocyte cell line as a vehicle for the ex vivo delivery of TGF-β 1 to joints with OA (Table 1). This protocol is based upon preclinical data showing a surprising restorative effect in animal models of cartilage damage when TGF-β 1 is delivered in an ex vivo fashion using allograft or even xenograft cells [85]. In the human trial, the cells are irradiated prior to injection to prevent cell division and thus eliminate the risk of cancer from these aneuploid retrovirally transduced cells. So far, 16 subjects have been treated in this fashion without incident. Elevation of TGF-β 1 levels has not been observed in serum, but two subjects have presented with synovial effusion. Eight patients have demonstrated symptomatic improvement, and evaluation via magnetic resonance imaging has found evidence of cartilage regeneration.

Gout
When urate crystals are injected into subcutaneous air pouches on mice, they induce inflammatory responses of the type seen in human gout. Ex vivo delivery of prostaglandin D synthetase has a strong anti-inflammatory effect, suggesting that this could serve as the basis of a gene treatment for crystal induced arthropathy [86]. A small clinical study indicates that recombinant IL-1Ra (Kineret ® ) has a beneficial effect in human gout [87]. This suggests an additional clinical target for the genetic therapies, discussed above, that presently use IL-1Ra cDNA to treat RA and OA.

Other rheumatic diseases
Sjögren syndrome Like diarthrodial joints, salivary glands are discrete isolated structures that lend themselves to local gene transfer. Vectors can be introduced through a cannulated duct and reach the luminal surfaces of the epithelial cells. Because the salivary gland is well encapsulated, there is little risk of vector escaping to nontarget organs. Many of the principles described above in the context of RA also apply to Sjögren syndrome [88].
Although adenovirus vectors transfer genes to the salivary glands very efficiently, AAV is proving to be the vector of choice because it is safe and noninflammatory. Serotypes 2 and 5 show promise, and efficacy has been demonstrated in animal models using IL-10 [89] and vasoactive intestinal peptide [90] as the transgene products. Because the salivary gland has an exocrine function, it can also be used as a site of gene transfer for systemic delivery purposes [91]. Gene therapy for Sjögren syndrome is reviewed in references [88].

Lupus
Several of the strategies we have already discussed in the context of RA have also been applied to lupus. Unlike RA therapy, lupus therapy has not benefitted in a dramatic fashion from the introduction of biologics. Moreover, because lupus is accompanied by a large increase in mortality, the risk-to-benefit ratio is more favorable toward gene therapy. Lupus is thought to involve excess production of type 2 cytokines, so a number of investigators have introduced type 1 cytokines, such as IL-2 and IL-12. Encouraging results followed the intramuscular injection of plasmids encoding these cytokines in murine models [92,93]. Injection of plasmids encoding an IFN-γ receptor: Fc construct has also shown promise [94]. In some experiments, the efficiency of transfection has been increased by electroporation, leading to efficacy with a cDNA encoding a dominant negative mutation of monocyte chemoattractant protein-1 [95]. The DNA vaccination approach has also worked using a cDNA encoding a consensus peptide from anti-DNA immunoglobulins.
Other investigators have used adenovirus to deliver the immunoinhibitory receptor PD-L1, TACI (transmembrane activator and CAML [calcium modulator and cyclophilin ligand] interactor, an inhibitor of B-lymphocyte stimulator [BLyS]), CTLA4, and a soluble form of the TGF receptor II [96][97][98][99]. CTLA4 and CD40Ig have also been delivered in animal models using AAV8 [100,101]. Data from animal models thus provide numerous examples of successful gene therapy in murine models of lupus. The challenge is to translate these into clinically useful human protocols. Gene therapy for lupus is reviewed in reference [101].
Antiphospholipid syndrome DNA vaccination has been used to generate antibodies to TNF with associated improvement in an animal model of antiphospholipid syndrome [102].

Summary and future directions
During the decade under review, the application of gene therapy in rheumatic diseases has undergone several mood swings. Nevertheless, a small group of investigators in this area has maintained a remarkably steady output of research papers (Figure 1), leading to several phase I clinical trials and one phase II trial in RA. There is evidence of a clinical response in certain subjects, suggesting that additional trials to establish efficacy are merited. Their implementation is not aided by the high cost of clinical trials. Moreover, there are widespread concerns about safety, and many question the use of gene therapy to treat nongenetic nonlethal diseases. These concerns are amplified by the clinical and commercial success of protein-based therapies for RA. Nevertheless, conventional biologics are very expensive, and an effective intra-articular gene therapy administered only rarely is likely to be far less costly.
OA, in contrast, responds poorly to conventional treatments and is a leading and growing cause of morbidity. The pressing need for better ways to control this common, debilitating, and expensive condition could be met by responsible gene therapy protocols using safe vectors. Two clinical trials exploring the use of gene therapy in OA are under way and another is in the pipeline. If successful, they could lead to wide human and veterinary application and pave the way for additional protocols in other arthritides.
Proof of principle has been established in animal models of Sjögren syndrome and lupus, pointing to the need for trans-lational research to develop clinical trials. Because these diseases, unlike RA, do not respond well to present biologics, alternative approaches, such as gene therapy, seem worthwhile. Their success could encourage further investigations in serious, intractable, rheumatic diseases, such as scleroderma. The technology of gene transfer has developed to the point where it is no longer the rate-limiting step for many purposes. Instead, there is a need for considerable funding, persistence, and continuity of effort to bring gene therapy into rheumatologic clinical practice.

Competing interests
CHE and PDR are on the scientific advisory board of TissueGene Inc. (Rockville, MD, USA), for which they receive an honorarium but no stock. TissueGene Inc. is developing gene therapies for osteoarthritis. CHE is on the supervisory board of Orthogen AG (Düsseldorf, Germany), and PDR is on the scientific advisory board. Neither individual receives an honorarium, but CHE owns stock in the company. Orthogen AG is not developing gene therapies for arthritis. PDR and SCG are cofounders of Molecular Orthopaedics Incorporated (Chapel Hill, NC, USA), which is developing gene therapies for osteoarthritis. The authors are developing a clinical protocol using AAV to treat osteoarthritis by gene therapy.