PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints

BACKGROUND: Gene transfer to synovium in joints has been shown to be an effective approach for treating pathologies associated with rheumatoid arthritis (RA) and related joint disorders. However, the efficiency and duration of gene delivery has been limiting for successful gene therapy for arthritis. The transient gene expression that often accompanies non-viral gene delivery can be prolonged by integration of vector DNA into the host genome. We report a novel approach for non-viral gene therapy to joints that utilizes phage phiC31 integrase to bring about unidirectional genomic integration. METHODS: Rabbit and human synovial cells were co-transfected with a plasmid expressing phiC31 integrase and a plasmid containing the transgene and an attB site. Cells were cultured with or without G418 selection and the number of neo-resistant colonies or eGFP cells determined, respectively. Plasmid rescue, PCR query, and DNA sequence analysis were performed to reveal integration sites in the rabbit and human genomes. For in vivo studies, attB-reporter gene plasmids and a plasmid expressing phiC31 integrase were intra-articularly injected into rabbit knees. Joint sections were used for histological analysis of beta-gal expression, and synovial cells were isolated to measure luciferase expression. RESULTS: We demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the transgene and attB increased the frequency of transgene expression in rabbit synovial fibroblasts and primary human RA synoviocytes. Plasmid rescue and DNA sequence analysis of plasmid-chromosome junctions revealed integration at endogenous pseudo attP sequences in the rabbit genome, and PCR query detected integration at previously characterized integration sites in the human genome. Significantly higher levels of transgene expression were detected in vivo in rabbit knees after intra-articular injection of attB-reporter gene plasmids and a plasmid expressing phiC31 integrase. CONCLUSION: The ability of phiC31 integrase to facilitate genomic integration in synovial cells and increase transgene expression in the rabbit synovium suggests that, in combination with more efficient DNA delivery methods, this integrase system could be beneficial for treatment of rheumatoid arthritis and other joint disorders.     

Plasmid DNA electrotransfer for intracellular and secreted proteins expression: new methodological developments and applications

In vivo electrotransfer is a physical method of gene delivery in various tissues and organs, relying on the injection of a plasmid DNA followed by electric pulse delivery. The importance of the association between cell permeabilization and DNA electrophoresis for electrotransfer efficiency has been highlighted. In vivo electrotransfer is of special interest since it is the most efficient non-viral strategy of gene delivery and also because of its low cost, easiness of realization and safety. The potentiality of this technique can be further improved by optimizing plasmid biodistribution in the targeted organ, plasmid structure, and the design of the encoded protein. In particular, we found that plasmids of smaller size were electrotransferred more efficiently than large plasmids. It is also of importance to study and understand kinetic expression of the transgene, which can be very variable, depending on many factors including cellular localization of the protein, physiological activity and regulation. The most widely targeted tissue is skeletal muscle, because this strategy is not only promising for the treatment of muscle disorders, but also for the systemic secretion of therapeutic proteins. Vaccination and oncology gene therapy are also major fields of application of electrotransfer, whereas application to other organs such as liver, brain and cornea are expanding. Many published studies have shown that plasmid electrotransfer can lead to long-lasting therapeutic effects in various pathologies such as cancer, blood disorders, rheumatoid arthritis or muscle ischemia. DNA electrotransfer is also a powerful laboratory tool to study gene function in a given tissue.     

Review. The status of plasmapheresis and apheresis

The paper surveys the technical and clinical development of plasmaexchange therapy. Until today there is no agreement on a unique treatment protocol, the variation ranges from 3-4 treatments in multiple sclerosis to biweekly chronic treatment in hypercholesterolaemia. An exchange volume of 1-1.5 of the plasma volume is recommended. Despite occasional complications, mostly due to replacement fluid, the method is regarded as safe. The first clinical application dates back to 1952, when Adams reported on the treatment of hyperviscosity syndrome. With rheumatoid arthritis plasmaexchange therapy of immunological mediated diseases started in 1963. In the following years a lot of enthusiastic case reports caused a general therapeutic optimism which was to be corrected by controlled trials. There is no proven benefit of plasmapheresis in rheumatoid arthritis and rapid glomerulonephritis for instance. For lymphoplasmapheresis in rheumatoid arthritis and for plasmapheresis in multiple sclerosis and renal graft rejection the results are contradictive. The paper discusses the different indications for apheresis therapy including hematologic disorders to be treated with plasmapheresis or cytapheresis.     

The metabolism of synthetic leukotriene B4 in synovial fluid and whole human blood

The metabolism in vitro of synthetic leukotriene B4 (LTB4) in synovial fluid from rheumatoid arthritis and osteoarthritis patients and in whole blood from these same patient groups and from normal volunteers has been studied. A linear relationship existed between a plot of the time of incubation of samples with LTB4 and the percentage of the initial concentration of LTB4 at each time point. The slope of this line, the rate constant for metabolism, has been used to compare different samples. LTB4 was metabolised more rapidly in the synovial fluid of rheumatoid arthritis patients than osteoarthritis patients. Furthermore, LTB4 was metabolised more rapidly in the blood of rheumatoid arthritis patients than either osteoarthritis patients or normal volunteers. These differences in metabolism correlate with the polymorphonuclear leukocyte (PMN) and albumin content of samples. It is suggested that binding of LTB4 to albumin in vivo will in part determine the available concentration of LTB4 in inflammatory lesions.     

Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws

Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery to diseased joints offers the prospect of achieving high, local concentrations of a therapeutic gene product in a sustained manner, while minimizing exposure of nontarget organs. We report that a single administration of a modified adenovirus encoding the Epstein-Barr-derived homologue of IL-10 can suppress the development of disease for extended periods of time when injected locally within the periarticular tissue surrounding the ankle joints of mice with collagen type II-induced arthritis. Furthermore, we show that injection of an adenoviral vector carrying the IL-10 gene into a single paw can suppress development of arthritis in other, noninjected paws of the same individual. The systemic protection resulting from local gene therapy occurred in the absence of detectable levels of viral IL-10 in the serum. Circulating Ab levels to heterologous collagen were unaffected; however, treatment with viral IL-10 significantly suppressed the development of Abs to autologous mouse type II collagen. Thus, the treatment of a single joint by local delivery of the vIL-10 gene may protect multiple joints of the same individual while avoiding deleterious side effects often associated with systemic therapy.     

Long non‐coding RNAs in rheumatoid arthritis

Rheumatoid arthritis, a disabling autoimmune disease, is associated with altered gene expression in circulating immune cells and synovial tissues. Accumulating evidence has suggested that long non‐coding RNAs (lncRNAs), which modulate gene expression through multiple mechanisms, are important molecules involved in immune and inflammatory pathways. Importantly, many studies have reported that lncRNAs can be utilized as biomarkers for disease diagnosis and prognostication. Recently, dysregulation of lncRNAs in rheumatoid arthritis and other autoimmune diseases has been revealed. Experimental studies also confirmed their crosstalk with matrix metalloproteinases, nuclear factor‐κB signalling and T‐cell response pertinent to autoimmunity and inflammation. Circulating lncRNAs, such as HOTAIR, differentiated patients with rheumatoid arthritis from healthy subjects. Taken together, lncRNAs are good candidates as biomarkers and therapeutic targets in rheumatoid arthritis. Further investigation on in vivo delivery of these regulatory molecules and large‐cohort validation of their clinical applicability may be useful.     

Gene therapy of arthritis

Gene therapy can offer a new approach to arthritis treatment which acts at an inflammation site. Numerous studies show high efficacy of gene therapy in different models of arthritis in humans. Even a single injection of a recombinant vector results in a stable prolonged expression of a therapeutic gene and a longterm therapeutic effect. In contrast to biologic therapy involving numerous systemic injections of recombinant anti-inflammatory proteins, gene therapy does not produce systemic side effects. Vectors based on retroviruses, adenoviruses, adeno-associated viruses, and recombinant plasmids could provide delivery of target genes. Of significant importance is the development of noninvasive methods of gene therapy: intranasal and peroral. The current state of research in arthritis gene therapy is discussed in this review.     

A novel approach for gene therapy: engraftment of fibroblasts containing the artificial chromosome expression system at the site of inflammation

BACKGROUND: Rheumatoid arthritis is characterized by inflammation of the synovial tissue. High systemic doses are necessary to achieve therapeutic levels of anti-rheumatic drugs in the joints. Gene transfer might provide a more efficient delivery system for genes encoding therapeutic proteins. METHODS: The artificial chromosome expression system (ACE System) is a new non-integrating, non-viral gene expression system which functions like a natural chromosome. This technology offers advantages over current expression systems because it allows stable and predictable expression of proteins encoded by single or multiple genes over long periods of time. We are developing ex vivo gene therapy using murine artificial chromosomes containing a reporter gene (LacZ and red fluorescent protein (RFP)) for local delivery of genes in rats with adjuvant arthritis (AA). RESULTS: The delivery of the intact ACE System into rat fibroblast-like synoviocytes (FLS) and rat skin fibroblasts (RSF) was detected within 24 to 48 h post-transfection. After growing cells under selection, clones expressing LacZ and RFP were identified. Furthermore, we investigated the feasibility of local delivery of a reporter gene to the joints of rats with AA by ex vivo gene therapy. This resulted in engraftment of the injected cells in the synovial tissue microarchitecture and expression of the reporter gene. CONCLUSIONS: This work demonstrates the potential feasibility of treating arthritis and other inflammatory diseases using fibroblasts containing the ACE System as a non-viral vector for gene therapy.     

NONSPECIFIC ANTI-INFLAMMATORY AGENTS—Some Notes on Their Practical Application,Especially in Rheumatic Disorders

A number of acute and chronic inflammatory disorders are amenable to varying degrees of therapeutic control with the administration of nonspecific anti-inflammatory drugs. An evaluation of these suppressive agents in the field of rheumatic diseases and practical suggestions regarding their administration are presented.Eight synthetically modified corticosteroid compounds are available commercially. Each of them exhibits qualitative differences in one or several physiologic actions, each has certain advantages and disadvantages in therapy, and each shares the major deterrent features of corticosteroids. Prednisone, prednisolone, methylprednisolone, fluprednisolone and paramethasone have similar therapeutic indices, and there is little choice between them for the usual rheumatoid patient requiring steroid therapy. Conversely, the therapeutic indices of dexamethasone, betamethasone and triamcinolone are lower than that of prednisolone; they are less desirable for routine use and should be reserved for specially selected cases.Salicylates are preferred to adrenocortical steroids in the treatment of the ordinary patient with acute rheumatic fever. Steroid therapy should be reserved for resistant cases and for those with significant carditis. Salicylates are mainstays for pain relief in rheumatoid arthritis, but with the analgesic doses usually employed their anti-inflammatory action is slight.Phenylbutazone is a highly useful anti-inflammatory agent, especially in management of acute gouty arthritis and ankylosing (rheumatoid) spondylitis; its metabolite, oxyphenylbutazone, does not exhibit clear-cut advantages.Colchicine specifically suppresses acute gouty arthritis. Its analogues, desacetylcolchicine and desacetylthiocolchicine, produce fewer unpleasant gastrointestinal symptoms, but may promote agranulocytosis and alopecia.A number of indole preparations with anti-inflammatory activity have been tested clinically. One of them, indomethacin, has received extensive therapeutic trial; with dosages that can be tolerated the drug is fairly effective in the symptomatic control of ankylosing (rheumatoid) spondylitis but it is of questionable value in peripheral rheumatoid arthritis.     

Chemokine blockade: a new era in the treatment of rheumatoid arthritis?

Blockade of chemokines or chemokine receptors is emerging as a new potential treatment for various immune-mediated conditions. This review focuses on the therapeutic potential in rheumatoid arthritis, based on studies in animal models and patients. Several knockout models as well as in vivo use of chemokine antagonists are discussed. Review of these data suggests that this approach might lead to novel therapeutic strategies in rheumatoid arthritis and other chronic inflammatory disorders.     

Cutaneous gene transfer and therapy: the present and the future

The easy accessibility of the skin as a therapeutic target provides an exciting potential for this organ for the development of gene therapy protocols for cutaneous diseases and a variety of metabolic disorders. Thus far, full phenotypic reversion of a diseased phenotype has been achieved in vivo for junctional epidermolysis bullosa and X-linked or lamellar ichthyosis and in vitro for xeroderma pigmentosum. These recessive skin diseases are characterized by skin blistering, abnormalities in epidermal differentiation and increased development of skin cancers, respectively. Corrective gene delivery at both molecular and functional levels was achieved by transduction of cultured skin cells using retroviral vectors carrying the specific curative cDNA. These positive results should prompt clinical trials based on transplantation of artificial epithelia reconstructed ex vivo using genetically modified keratinocytes. Promising results have also been obtained in phenotypic reversion of cells isolated from patients suffering from a number of metabolic diseases such as gyrate atrophy, familial hypercholesterolemia or phenylketonuria. In these diseases transplantation of autologous artificial epithelia expressing the transgenes of interest or direct transfer of the DNA to the skin represents a potential therapeutic approach for the systemic delivery of active molecules. Successful cutaneous gene therapy trials, however, require development of protocols for efficient gene transfer to epidermal stem cells, and information about the host immune response to the recombinant polypeptides produced by the implanted keratinocytes. The availability of spontaneous animal models for genodermatoses will validate the gene therapy approach in preclinical trials.     

Induction of the p16INK4a senescence gene as a new therapeutic strategy for the treatment of rheumatoid arthritis.

Synovial tissue affected by rheumatoid arthritis is characterized by proliferation, which leads to irreversible cartilage and bone destruction. Current and experimental treatments have been aimed mainly at correcting the underlying immune abnormalities, but these treatments often prove ineffective in preventing the invasive destruction. We studied the expression of cyclin-dependent kinase inhibitors in rheumatoid synovial cells as a means of suppressing synovial cell proliferation. Synovial cells derived from hypertrophic synovial tissue readily expressed p16INK4a when they were growth-inhibited. This was not seen in other fibroblasts, including those derived from normal and osteoarthritis-affected synovial tissues. In vivo adenoviral gene therapy with the p16INK4a gene efficiently inhibited the pathology in an animal model of rheumatoid arthritis. Thus, the induction of p16INK4a may provide a new approach to the effective treatment of rheumatoid arthritis.     

Rationale of using different biological therapies in rheumatoid arthritis

Due to ongoing developments of novel agents in the field of biological pharmacotherapy, there are now more arrows available in clinicians' quivers for the treatment of rheumatic conditions. As a consequence, however, clear treatment strategies have to be defined in order to guarantee a qualitatively high and individually stage-adapted, state-of-the-art regimen for affected patients. This review summarizes recent evidence regarding the rationale of using different biological therapies to treat rheumatoid arthritis, the most common inflammatory joint disorder after activated osteoarthritis, and draws an actual picture of a possible standardized therapeutic algorithm without claiming exclusive appropriateness.     

MicroRNA——关节炎治疗的新靶点

关节炎是一种较为常见的累及运动系统的慢性疾病,可分为骨性关节炎和风湿性关节炎等．我国北方地区冬季寒冷,该病发病率较高．小干扰RNA（small interfering RNA, siRNA）是一类长度约为21～23对核苷酸的双链小分子RNA,参与基因沉默,已经应用于关节炎治疗的研究中. MicroRNA(miRNA）是一类内源性小分子RNA．其常规作用方式是通过与靶基因的3′UTR结合抑制靶基因的表达．目前,已有研究表明miRNA与关节炎发病联系密切．通过局部注射等多种途径,向体内递送各种经过修饰的miRNA能够发挥改善关节炎症状等作用．这些研究提示miRNA将成为关节炎治疗的新靶点．     

High-efficiency gene transfer into nontransformed cells: utility for studying gene regulation and analysis of potential therapeutic targets

The elucidation of the signalling pathways involved in inflammatory diseases, such as rheumatoid arthritis, could provide long sought after targets for therapeutic intervention. Gene regulation is complex and varies depending on the cell type, as well as the signal eliciting gene activation. However, cells from certain lineages, such as macrophages, are specialised to degrade exogenous material and consequently do not easily transfect. Methods for high-efficiency gene transfer into primary cells of various lineages and disease states are desirable, as they remove the uncertainties associated with using transformed cell lines. Significant research has been undertaken into the development of nonviral and viral vectors for basic research, and as vehicles for gene therapy. We briefly review the current methods of gene delivery and the difficulties associated with each system. Adenoviruses have been used extensively to examine the role of various cytokines and signal transduction molecules in the pathogenesis of rheumatoid arthritis. This review will focus on the involvement of different signalling molecules in the production of tumour necrosis factor alpha by macrophages and in rheumatoid synovium. While the NF-kappaB pathway has proven to be a major mediator of tumour necrosis factor alpha production, it is not exclusive and work evaluating the involvement of other pathways is ongoing.     

Parvovirus vectors: use and optimisation in cancer gene therapy

With the increasing incidence and mortality of cancer worldwide, there is an urgent need for new therapeutic approaches. Gene therapy is one such approach and preliminary data are promising. Viral and nonviral vector systems for gene delivery are available, but most of the current systems suffer from disadvantages such as low transfection efficiencies, in vivo instability, targeting problems, mutagenic potential and immunogenicity. Viruses of the Parvoviridae family, which are characterised by their oncotropism, oncosuppression, long-term gene expression and human apathogenicity, potentially offer advantages as viral vectors. This article evaluates their usefulness in gene therapy strategies for cancer.     

Adenoviral vectors for gene transfer and therapy

Due to the very efficient nuclear entry mechanism of adenovirus and its low pathogenicity for humans, adenovirus-based vectors have become gene delivery vehicles that are widely used for transduction of different cell types, especially for quiescent, differentiated cells, in basic research, in gene therapy applications, and in vaccine development. As an important basis for their use as gene medicine, adenoviral vectors can be produced in high titers, they can transduce cells in vivo with transgenes of more than 30 kb, and they do not integrate into the host cell genome. Recent advances in the development of adenoviral vectors have brought considerable progress on issues like target cell specificity and tropism modification, long-term expression of the transgene, as well as immunogenicity and toxicity in vivo, and have suggested that the different generations of non-replicative and replicative vectors available today will each suit best for certain applications.     

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.     

Delivery of electric pulses for DNA electrotransfer to mouse muscle does not induce the expression of stress related genes

In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic delivery of therapeutic proteins. Electrotransfer is a powerful method for DNA transfer into skeletal muscle. In view of the broad potential gene therapy clinical application of electrotransfer offers, it is important to perform toxicology studies on electrotransfered muscle tissue. We have investigated if the delivery of square wave electric pulses of low field strength and long duration to mouse tibial cranial muscle induced the expression of stress related genes. We have profiled gene expression patterns in muscles at different times after delivery of electric pulses using Stress/Toxicology microarrays. No significant variation in the expression of stress related-genes was detected between treated and non-treated muscles. This suggests that application of adequate, fine-tuned, electric pulses to the skeletal muscle is a non-toxic technique for gene therapy.     

Paradoxical effects of adenovirus-mediated blockade of TNF activity in murine collagen-induced arthritis.

Collagen-induced arthritis (CIA) is an experimental model of arthritis widely used to dissect the pathogenesis of human rheumatoid arthritis and to identify potential therapeutic targets. Among these, TNF-alpha has been recognized to play an important role. Here we investigate the feasibility and therapeutic efficacy of prolonged blockade of TNF-alpha activity through the adenovirus-mediated gene delivery of a dimeric chimeric human p55 TNFR-IgG fusion protein and compare it to protein therapy in established CIA. A single i.v. administration of the replication-deficient adenovirus yielded microgram serum levels of the chimeric fusion protein and ameliorated CIA for 10 days. Subsequently, benefit was lost and a rebound to greater inflammatory activity was observed despite the continual presence of bioactive TNFR fusion protein. A similar trend was also observed in mice injected directly with comparable amounts of a human TNFR-IgG fusion protein, whereas the administration of a control adenovirus-encoding beta-galactosidase or of a control human IgG1 protein did not significantly affect the disease course. The mechanisms of the rebound of CIA were investigated, and augmented Ab response to collagen type II and TNFR were identified as potential causes. Our results confirm the feasibility of adenovirus-mediated gene delivery of cytokine inhibitors in animal models of autoimmune diseases for investigational purposes and highlight the importance of prolonged studies. Further investigations are needed to optimize ways of exploiting the potential of adenoviral gene therapy in RA.