Gene Therapy For Arthritis Research Articles

Computational Design and Application of Endogenous Promoters for Transcriptionally Targeted Gene Therapy for Rheumatoid Arthritis

The promoter regions of genes that are differentially regulated in the synovial membrane during the course of rheumatoid arthritis (RA) represent attractive candidates for application in transcriptionally targeted gene therapy. In this study, we applied an unbiased computational approach to define proximal-promoters from a gene expression profiling study of murine experimental arthritis. Synovium expression profiles from progressing stages of collagen-induced arthritis (CIA) were classified into six distinct groups using k-means clustering. Using an algorithm based on local over-representation and comparative genomics, we identified putatively functional transcription factor-binding sites (TFBS) in TATA-dependent proximal-promoters. Applying a filter based on spacing between TATA box and transcription start site (TSS) combined with the presence of over-represented nuclear factor kappaB (NFkappaB), AP-1, or CCAAT/enhancer-binding protein beta (C/EBPbeta) sites, 382 candidate murine and human promoters were reduced to 66, corresponding to 45 genes. In vitro, 9 out of 10 computationally defined promoter regions conferred cytokine-inducible expression in murine cells and human synovial fibroblasts. Under these conditions, the serum amyloid A3 (Saa3) promoter showed the strongest transcriptional induction and strength. We applied this promoter for driving therapeutically efficacious levels of the interleukin-1 receptor antagonist (Il1rn) in a disease-regulated fashion. These results demonstrate the value of bioinformatics for guiding the selection of endogenous promoters for transcriptionally targeted gene therapy.

Progress and Prospects: genetic treatments for disorders of bones and joints

Gene therapies directed toward the treatment of arthritis and tissue repair continue to be the most active areas of research for bone and joint diseases. In the past 2 years, two trials in rheumatoid arthritis have been completed. a Phase I study reporting safety and a Phase I/II study that has yet to be published. An additional, small study has reported the first evidence of clinical efficacy. Two Phase I trials of gene therapy for osteoarthritis have also been initiated. There is much preclinical activity in developing AAV vectors for future trials in the gene therapy of arthritis. Research into tissue repair and regeneration remains at the preclinical stage, but a considerable volume of research attests to the promise of gene transfer in this arena, especially in the context of bone healing. For tissue repair, the major research questions are still which genes to use and how best to deliver them.

RNA interference-based gene therapy for successful treatment of rheumatoid arthritis

Background: RNA interference (RNAi) is a powerful endogenous process initiated by short double-stranded RNAs, which results in sequence-specific posttranscriptional gene silencing. The possibility of blocking the expression of any protein carries huge expectations for potential therapeutic applications in a wide range of diseases. For clinical development, however, the use of RNAi-based therapeutics has to overcome major obstacles, mainly targeted delivery and safety issues. Objective/methods: In this short review, we provide an overview of specifications for RNAi-based gene therapy in rheumatoid arthritis (RA) and discuss recent progresses in the development of efficient silencing, focusing on expression of short hairpin RNAs. Results/conclusions: Combining advances in RNAi methodology with gene therapy technology opens avenues for rapid applications to RA.

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.

Clinical Responses to Gene Therapy in Joints of Two Subjects with Rheumatoid Arthritis

This paper provides the first evidence of a clinical response to gene therapy in human arthritis. Two subjects with rheumatoid arthritis received ex vivo, intraarticular delivery of human interleukin-1 receptor antagonist (IL-1Ra) cDNA. To achieve this, autologous synovial fibroblasts were transduced with a retrovirus, MFG-IRAP, carrying IL-1Ra as the transgene, or remained as untransduced controls. Symptomatic metacarpophalangeal (MCP) joints were injected with control or transduced cells. Joints were clinically evaluated on the basis of pain; the circumference of MCP joint 1 was also measured. After 4 weeks, joints underwent surgical synovectomy. There were no adverse events in either subject. The first subject responded dramatically to gene transfer, with a marked and rapid reduction in pain and swelling that lasted for the entire 4 weeks of the study. Remarkably, joints receiving IL-1Ra cDNA were protected from flares that occurred during the study period. Analysis of RNA recovered after synovectomy revealed enhanced expression of IL-1Ra and reduced expression of matrix metalloproteinase-3 and IL-1beta. The second subject also responded with reduced pain and swelling. Thus, gene transfer to human, rheumatoid joints can be accomplished safely to produce clinical benefit, at least in the short term. Using this ex vivo procedure, the transgene persisted within the joint for at least 1 month. Further clinical studies are warranted.

Current status of gene therapy for rheumatoid arthritis

Gene therapy offers great possibilities for treating rheumatoid arthritis (RA). Traditional surgical and pharmaceutical methods of treating RA have met with limited therapeutic success and have failed to produce a cure, but the past several years have seen extensive progress toward development of a gene therapy for arthritis. Numerous vectors and therapeutic genes have been investigated in animal models of arthritis, and the potential of gene therapy to treat or manage RA has been demonstrated in several clinical studies. Gene therapy offers the possibility of overcoming many of the limitations of current biologic therapies by providing long-term, high-level localized expression of therapeutic genes, potentially in as little as a single dose. In this review, we explore the advances in gene therapy for RA and summarize the recent preclinical and clinical data. In addition, we provide an overview of vectors and targets for RA gene therapy.

Gene therapy of arthritis and orthopaedic disorders: current experimental approaches in China and in Canada

Background: Gene therapy offers new possibilities for the clinical management of orthopaedic conditions that are difficult to treat by traditional surgical or medical means. Objective: This review discusses the different gene therapy strategies available today, the safety issues with which they may be associated, and the potential for cooperation between China and Canada. Results/conclusion: In the last decade, extensive improvements have been made to optimize gene therapy and have been tested on several orthopaedic conditions. China is a pioneer in some fields of gene therapy; the first anticancer gene therapy drug has been licensed in China. Canada has a very active research establishment and a populace that embraces new scientific developments, especially in medical sciences.

Advances in local targeted gene therapy for arthritis: towards clinical reality

Margriet J Vervoordeldonk, Caroline J Aalbers & Paul P Tak† †Author for correspondence Academic Medical Center/University of Amsterdam, Division of Clinical Immunology and Rheumatology, F4–218, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands Tel.: +31 205 662 171; Fax: +31 206 919 658; p.p.tak@amc.uva.nl and, Arthrogen BV, Amsterdam, The Netherlands ‘[There is] a strong rationale for IFN-β gene therapy as a novel therapeutic approach for arthritis.’

Cartilage proteoglycan-specific T cells as vectors of immunomodulatory biologicals in chronic proteoglycan-induced arthritis

Systemic administration of agents that neutralize or antagonize T h1-mediated pro-inflammatory responses has been demonstrated to ameliorate inflammation in chronic autoimmune disease. However, systemic administration of such immunosuppressive biologicals causes serious side effects and has only limited success. To minimize these side effects, autoantigen-specific lymphocytes have been proposed as a carrier to deliver immunosuppressive agents to sites of inflammation. Here we studied the effects of primary cartilage proteoglycan-specific CD4 + T cells that were transduced using an efficient method of viral transduction with active genes encoding IL-1β receptor antagonist, soluble TNF-α receptor-Ig, IL-4 or IL-10 in chronic proteoglycan-induced arthritis in mice. This is the first study describing such gene therapy using primary CD4 + T cells in a chronic arthritis. Moreover, the impact of proteoglycan-specific T h1, T h2 or naïve T cells was studied. Although proteoglycan-TCR transgenic CD4 + T cells can transfer arthritis to lymphopenic recipients, none of the proteoglycan-TCR transgenic T cell phenotypes that were tested induced worsening of arthritis in wild type hosts. Proteoglycan-specific T cells ameliorated arthritis when expressing the transduced IL-10 gene, and not when expressing the other transgenes/phenotypes. Although all of the tested biologicals can suppress in a wide range of different inflammatory disorders, especially IL-10 would therefore serve as a promising candidate to be used in cellular gene therapy for chronic arthritis.

Arthritis gene therapy at an inflection point

Gene therapy is not for the faint of heart. Since its launch in 1989 [1], human gene therapy has been variously cast as visionary, feckless, a panacea and a dangerous folly. One of its pioneers is in jail, and another was sued successfully in civil court following the death of a clinical trial subject. Skeptics like to point out that it has killed approximately as many people as it has cured and, apart from two cancer gene therapies approved recently in China, there is little to show for nearly two decades of promises and expensive research. Against these headwinds, arthritis gene therapy has made laudable progress. Proof of principle is well established in animal models, and a small number of clinical trials have been implemented (Table 1). However, we have reached the stage of diminishing returns with preclinical research. Pivotal clinical trials are needed to maintain momentum and confirm promise in human disease [2]. These will require a considerable injection of funding, without which research will stagnate and become largely condemned to re-inventing the wheel in animal models. Arthritis gene therapy emerged in the early 1990s (Figure 1) and made quite rapid progress, leading, within a few years, to proof of concept in animal models of rheumatoid arthritis and osteoarthritis, and a Phase I clinical trial – remarkable achievements for a new area of research. As reflected in the publication history shown in Figure 1, the number of investigators remains limited, with approximately 30 papers appearing in the refereed literature each year at a remarkably constant rate since 1999. The fact that almost half of these publications are review articles is telling. Arthritis was the subject of the first human gene-therapy protocol for a nonlethal disease [3], and consequently attracted much scrutiny from the regulatory agencies. The data from this trial confirmed that genes could be safely transferred to human joints and expressed within them [4]. Moreover, in a similar small, German study, each of two subjects treated with this gene therapy appeared to mount a clinical response, one of them dramatically so [5] [Wehling et al., Unpublished Data]. Nevertheless, the ex vivo, retrovirus-based approach used in these two studies is unlikely to find wide clinical application; ex vivo gene transfer using autologous cell culture is too cumbersome and costly, and concerns regarding insertional mutagenesis with retrovirus vectors have resurfaced. Subsequent clinical trials (Table 1) based on this approach have used allogeneic cells lines to reduce costs and, in one case, irradiation of cells to prevent cell division and thus tumorigenicity. In the search for an efficient vector that can be introduced safely into joints by intra-articular injection, most investigators have converged on adeno-associated virus (AAV). Recombinant AAV has risen in popularity as a gene therapy vector because it is perceived to be safe and new technologies permit easier production of clinical-grade material [6]. Wild-type AAV causes no known disease and recombinant AAV vectors have been used safely in gene therapy trials of a number of single-gene disorders, as well as Parkinson’s disease, Alzheimer’s disease and cancer. Two large Phase III trials for prostate cancer using AAV are underway, and orphan drug status has been granted by the EU for AAV-mediated gene therapy for familial lipoprotein lipase deficiency.

CLONING OF HUMAN INTERLEUKIN-10 GENE AND TRANSGENE EXPRESSION IN RABBIT SYNOVIAL CELLSIN VITRO

In this work, the full-length open reading frame of the human interleukin-10 (hIL-10) gene was amplified through reverse transcription–polymerase chain reaction (RT-PCR), and then PCR products were inserted into pcDNA4/HisMax to construct an eukaryotic expression vector. After optimization by green fluorescent protein (GFP), recombinant hIL-10 genes were transfected and expressed in rabbit synovial cells compounded with liposome in vitro. In cell culture supernatant, rhIL-10 was detected using enzyme-linked immunosorbent assay (ELISA) at time intervals of 12 hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days. After 12 hours of transfection, ELISA showed that transgene expression of hIL-10 in rabbit synovial cells was elevated; at 72 hours, hIL-10 expression reached its peak value; and then it declined gradually until 7 days, compared with the control. After 14 days, transgene expression ceased. Gene cloning of hIL-10 and its transgene expression in synovial cells therefore gives a basis for the gene therapy of rheumatoid arthritis.

Gene therapy for arthritis

Arthritis is among the leading causes of disability in the developed world. There remains no cure for this disease and the current treatments are only modestly effective at slowing the disease's progression and providing symptomatic relief. The clinical effectiveness of current treatment regimens has been limited by short half-lives of the drugs and the requirement for repeated systemic administration. Utilizing gene transfer approaches for the treatment of arthritis may overcome some of the obstacles associated with current treatment strategies. The present review examines recent developments in gene therapy for arthritis. Delivery strategies, gene transfer vectors, candidate genes, and safety are also discussed.

Arthritis gene therapy's first death

In July 2007 a subject died while enrolled in an arthritis gene therapy trial. The study was placed on clinical hold while the circumstances surrounding this tragedy were investigated. Early in December 2007 the Food and Drug Administration removed the clinical hold, allowing the study to resume with minor changes to the protocol. In the present article we collate the information we were able to obtain about this clinical trial and discuss it in the wider context of arthritis gene therapy.

Application of a disease-regulated promoter is a safer mode of local IL-4 gene therapy for arthritis

The application of disease-regulated promoters in local gene therapy for rheumatoid arthritis potentiates the development of a sophisticated treatment that relies on a restricted and fine-tuned supply of biologicals. Although several studies have investigated regulated promoters for achieving effective transgene expression during arthritis, none have explored their potential for minimizing deleterious effects arising from constitutive overexpression of transgenes under naive conditions. Using naive and collagen-induced arthritic mice, we examined the applicability of a hybrid interleukin-1 enhancer/interleukin-6 proximal promoter for achieving efficacious murine interleukin-4 gene therapy under arthritic conditions, while minimizing interleukin-4-induced inflammation under naive conditions. We found strong upregulation of transgene expression in virally transduced knee joints under arthritic conditions compared to levels in naive animals. Besides its responsiveness, the promoter strength proved sufficient for generating therapeutically efficacious levels interleukin-4, as demonstrated by the successful protection against cartilage erosion in collagen-induced arthritis. Most importantly, promoter-mediated restriction of the potent chemotactic interleukin-4 in naive animals strongly reduced the amounts of inflammatory cell influx. This study suggests the suitability of the interleukin-1 enhancer/interleukin-6 proximal promoter for the development of a local gene therapy strategy for rheumatoid arthritis that requires fine-tuned and restricted expression of transgenes with a pleiotrophic nature.

Death Prompts a Review of Gene Therapy Vector

CLINICAL RESEARCHThe death last week of a patient receiving experimental gene therapy for arthritis has triggered a federal review of all trials using the same vector.

Transgene Persistence and Cell Turnover in the Diarthrodial Joint: Implications for Gene Therapy of Chronic Joint Diseases

Local gene therapy for chronic joint diseases requires prolonged transgenic expression, but this has not been reliably achieved in animal models. Using normal and immunocompromised animals, we examined the capacity of various cell types in joint tissues to maintain and express exogenous transgenes after direct intra-articular gene delivery. We found that transgenic expression could persist for the lifetime of the animal but required precise immunological compatibility between the vector, transgene product, and host. It was not dependent on vector integration or promoter origin. We identified two phenotypically distinct sub-populations of genetically modified cells within the joint: (i) transient cells, with a half-life of a few weeks, and (ii) stable cells that reside in the joint tissues indefinitely. Contrary to the prevailing assumption, the transient sub-population was composed almost exclusively of synovial fibroblasts, indicating that the synovium is not an appropriate tissue upon which to base a long-term therapy. Instead, fibroblasts in the ligaments, tendons, and capsule emerged as the primary cell types capable of sustained therapeutic transgene expression. This study sheds new light on the cellular dynamics of articular tissues and suggests that cell turnover and immune reactivity are the key determinants in achieving sustained transgenic expression intra-articularly.

Gene Therapy of Arthritis with TCR Isolated from the Inflamed Paw

In recent years, the treatment of autoimmune diseases has been significantly advanced by the use of biological agents. However, some biologics are accompanied with severe side effects, including tuberculosis and other types of infection. There is thus a critical need for nonsystemic and lesion-specific methods of delivering these therapeutic agents. We attempted to treat a mouse model of arthritis by using T cells that expressed a regulatory molecule and were specifically directed to the inflamed paw. To this end, we first identified the TCR alphabeta genes accumulating in the inflamed paw of mice with collagen-induced arthritis (CIA) by a combination of single-strand chain polymorphism analysis of TCR and single-cell sorting. We identified an expanded clone B47 which is autoreactive but is not specific to type II collagen. In vivo, TCR genes from B47-transduced T cells accumulated in the inflamed paw. Injection of cells cotransduced with the B47 and soluble TNFRIg genes resulted in a significant suppression of CIA. The suppression was correlated with the amount of TNFRIg transcripts in the hind paw, not with the serum concentrations of TNFRIg. Moreover, T cells cotransduced with the B47 and intracellular Foxp3 genes significantly suppressed CIA with reductions in TNF-alpha, IL-17A, and IL-1beta expression and bone destruction. T cells cotransduced with B47 and Foxp3 genes also suppressed the progression of established CIA. Therefore, immunosuppressive therapy with autoreactive TCR is a promising therapeutic strategy for arthritis whether the TCRs are used to deliver either soluble or intracellular suppressive molecules.

Gentherapie in der Orthopädie

Gene therapy in orthopaedic surgery is being intensively studied in the context of different genetic and nonmendelian diseases. Experimental progress in this area is characterized by the complexity of gene vector selection and production, gene transfer technology, application routes, choice of animal models, and evaluation of its efficacy using structural and functional parameters. The first clinical studies for gene therapy of rheumatoid arthritis already demonstrated their practical feasibility. Current data indicate that gene-based therapies are effective in promoting the repair of articular cartilage, and bone defects. Such strategies may lead to the development of novel molecular therapies treatments in orthopaedic surgery.

Intraarticular Interferon-βGene Therapy Ameliorates Adjuvant Arthritis in Rats

Interferon (IFN)-beta has significant immunomodulatory properties and has received much interest as a potentially therapeutic agent for rheumatoid arthritis (RA). Systemic IFN-beta treatment of patients with RA was not effective, probably because of pharmacokinetic issues. Therefore, we studied the effect of local IFN-beta production by adenovirus-mediated gene transfer to the ankle joints of arthritic rats. Adjuvant arthritis (AA) in rats was used as a model to study intraarticular gene therapy with an adenoviral vector encoding the rat IFN-beta gene (Ad.IFN-beta). The effect on paw swelling was measured by water displacement plethysmometry. Synovial tissue of the hind paws was examined by immunohistochemistry. Bone destruction was analyzed on the basis of radiographs. In addition, quantitative real-time polymerase chain reaction was used to assess IFN-beta expression. Levels of IFN-beta mRNA and protein peaked 2 days after intraarticular injection and declined thereafter. Local delivery of Ad.IFN-beta after the onset of disease reduced paw swelling significantly. This was accompanied by a reduction in synovial inflammation. The clinical effects in rat AA lasted up to 9 days. Strikingly, Ad.IFN-beta treatment protected bone from erosion, reduced levels of c-Cbl and Cbl-b (both signaling molecules essential for osteoclast activity), and reduced the matrix metalloproteinase-3:tissue inhibitor of metalloproteinase-1 ratio in the joint. Immunohistochemical analysis of the synovial tissue revealed a clear shift toward a more antiinflammatory cytokine profile. Local overexpression of IFN-beta inhibits arthritis progression and protects against bone destruction in rat AA. These findings validate IFN-beta as a therapeutic molecule for intraarticular gene therapy of arthritis.

Gene therapy works in animal models of rheumatoid arthritis so what!

Rheumatoid arthritis (RA) is a systemic disease with polyarticular manifestation of chronic inflammation in the knees and small joints of hand and feet. The current systemic anti-tumor necrosis factor (TNF)-alpha therapies with biologics ameliorate disease in 60% to 70% of RA patients. However, biologics must be given systemically in relatively high dosages to achieve constant therapeutic levels in the joints, and side effects have been reported. To this end, local gene delivery can provide an alternative approach to achieve high, long-term expression of biologics, optimizing the therapeutic efficacy and minimizing systemic exposure. Evidence from animal models convincingly supports the application of local gene therapy in rheumatoid arthritis, but preclinical studies remain necessary to evaluate the merge of cell-specific targeting, viral vector development, and disease-regulated transgene expression to optimize efficacy and safety.