This meta-analysis of case–control studies investigating the association between SLE and serological markers of EBV infection shows a significant association between the disease and anti-VCA IgG (OR = 2.08, 95% CI 1.15 to 3.76) but not between SLE and anti-EBNA1 IgG (1.45, 95% CI 0.70 to 2.96). The immune complexes from an antibody response can also be deposited at sites of viral infection or in the synovium. Serial titers measured included antibodies to Epstein-Barr virus (EBV) antigens, group B coxsackieviruses, rubella virus, cytomegalovirus, and herpes simplex virus. Of all tested viruses, cytomegalovirus and parvovirus B19 were positive (each in 10 patients, two double-positives), whereas herpes simplex virus was positive in two patients. These viral arthritis syndromes can be diagnosed by recognition of well defined clinical signs and detection of viral antibodies and nucleic acids. The joint samples were negative for viral DNA from adenovirus and varicella-zoster virus. In some cases, antiviral treatment is available for the underlying systemic disease.
The cause of this relatively common syndrome is not known, but it is presumed to be a viral or postinfectious arthritis. The Epidemiology Unit at the German Rheumatism Research Center was charged with maintaining the register, and an advisory board was established by the German Society of Rheumatology. This required a 3‐day hospital admission and treatment with intravenous followed by high‐dose oral (60 mg) and topical steroids, which resulted in gradual improvement. This study confirmed that genes could be safely transferred to human rheumatoid joints and expressed within them, at least for 1 week . Other herpes viruses, such as cytomegalovirus (CMV), herpes simplex virus (HSV) and varicella zoster virus (VZV) are unusual causes of arthritis. Viruses are one type of pathogen that can cause an infection in the body. 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 .
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 , Roessler and colleagues treated one subject with plasmid DNA encoding herpes simplex virusthymidine 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. The basic concept – gene transfer to the synovium. In animal models of osteoarthritis (OA), treatment has tended to focus on cDNAs encoding anti-inflammatory proteins and cartilage growth factors. A further 294,000 individuals have the juvenile form of the disease.
Forty-eight hours following admission, the patient became haemodynamically unstable again (temperature 38.7°C, BP 109/90mmHg, HR 125 beats per minute, RR 16 breaths per minute, SAO2 96% on air) with a CRP level of 292mg/l and a WCC of 11.8 × 109/l. The trials have been discussed in detail . In the first phase I study, 14 subjects with RA and 1 with ankylosing spondylitis were administered vector . Acetaminophen are found in many nonprescription medications such as aspirin-free Anacin*, Excedrin caplets, Panadol, Tylenol, and Tylenol Arthritis. 141. 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 .
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. In summary, these data indicate that intramuscular electroporation with the gene encoding POMC can mediate potent antinociceptive effects. A dose-dependent increase in neutralizing antibody to the AAV2 capsid was noted. Research is focusing on the choice of serotype and the host immune response to the vectors. Cross-linking of immunoglobulins on latently infected memory B cells has been shown to precipitate the lytic cycle of viral replication , and this is seen in other disease states such as malaria .
According to Apparailly and colleagues , 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 . Another study indicates the following order of preference: AAV2 > 1 > 5 > 8 . 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  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. In adulthood, HBV is transmitted through sexual activity or needle exposures. AAV has been used to express soluble TNF receptors , beta interferon (IFN-β) , angiostatin , dominant negative Iκκβ (inhibitor of kappa B kinase β) , and IL-1Ra  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 second-strand synthesis . 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 . 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 (104 to 105 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 . In agreement with this, mutations to the AAV coat protein that prevent ubiquitination also increase transduction efficiency .
According to Traister and colleagues , 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 . For intra-articular gene therapy to be a clinical success, there is a need for extended periods of transgene expression. The whole field of gene therapy was therefore shocked when a subject with RA died shortly after the injection of recombinant AAV into her right knee joint . The safety bar for gene therapeutics seems to be set at a far higher level than for other therapeutics, despite the ability of non-genetic medicines to do just as much harm. Barriers to delivery are overcome, however, by the genetic transduction of cells within the joint, whereby the transgene product is synthesized and secreted locally for an extended period, accumulating in the synovial fluid and articular tissues to provide sustained, therapeutic concentrations lacking the peaks and troughs of intermittent application. Of interest, long-term 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 ). Figure 3 Fibroblasts resident in fibrous articular tissues support stable expression of exogenous transgenes. Periodic urine and blood tests are needed to check for side effects. (2000) CEA-Regulated Replication-Selective Adenovirus for Treatment of CEA-Positive Tumors Third Annual Am.Soc. 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 . 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 . Some investigators have recommended giving corticosteroids at low to moderate doses to control symptoms and viremia. 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 .
So far, there have been no adverse events and some evidence of a clinical response in certain subjects (Tetsuya Tomita, personal communication). 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 ). 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 [55–61]. Vector genomes were detected in the peripheral blood cells of certain subjects, especially at the highest dose, suggesting leakage of vector from the joint. See the article online for full details of the relationships. This provided tissue for subsequent analysis of gene expression and other functions (). 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 , or mediators of arthritis, such as TNF , are protective.
It is also possible to induce tolerance by DNA immunization in the absence of adjuvant . Though effective in animal models, such strategies may be risky in humans. 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-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 ). Figure 4 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 . Studies using the murine delayed-type hypersensitivity reaction as a model  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 .
The anti-arthritic 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-dioxy-genase, 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. Clinical Context: Hydroxychloroquine inhibits chemotaxis of eosinophils and locomotion of neutrophils and impairs complement-dependent antigen-antibody reactions. 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 . 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 . 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  engineered a T-cell receptor whose extracellular domain contains a type II collagen-binding motif. There was evidence of herpes simplex virus in certain tissues, but not adenovirus or cytomegalovirus. Anti-sense RNA was shown to traffic to the synovium of diseased joints, suggesting facilitated local delivery. 49,50 However, only Gouze et al 26 have specifically identified the requirements of a successful vector for achieving long-term expression in joints and demonstrated how AAV uniquely satisfies these requirements. 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  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  have delivered short interfering RNA molecules targeted to IL-1, IL-6, and IL-18 in murine collagen-induced arthritis . 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.