Gene transfer as therapy for rheumatoid arthritis: why, what and how?

Y. Chernajovsky

Kennedy Institute of Rheumatology, 1 Aspenlea Road, Hammersmith, London W6 8LH, UK

Historically, chemically derived drugs have been pursued because of the relative ease of mass production and simple delivery. Chemical modification of drugs in order to increase their effectiveness and lower the side-effects has provided the majority of the drugs in use today. However, synthetic drugs target cellular reactions which are also part of homeostatic/physiological functions, and so they may have non-specific secondary effects in healthy tissues and organs which could be detrimental to patients.

Over the last decade, biomedical research has increased our knowledge regarding the pathological processes and soluble factors involved in the autoimmune and inflammation-mediated destruction of cartilage and bone. The identification of pathogenic factors such as cytokines, matrix metalloproteases, chemokines and angiogenic factors has led to the search for chemical, biological and genetically engineered antagonists. Also, the cells which produce these soluble factors have been targeted in order to inhibit their pathogenic function or induce their death by apoptosis. Therapy using biological agents is thought to be one rational answer to achieving specificity as it targets specific molecules whose biological functions are understood at least in vitro. These molecules are overexpressed in disease and `natural' compounds are used to antagonize or modulate their activity. Chronic diseases such as rheumatoid arthritis require long-term therapy and due to the high costs in production of biological agents, as well as the potential secondary effects due to long-term exposure, their use may be restricted.

Treatment by gene transfer could overcome the limitations of protein therapy with biologicals. Certain biological compounds, such as cytokines, have a very short half-life in vivo and in order to achieve effective therapeutic response locally they may need to be delivered systemically at toxic concentrations. Antibody-based molecules have been designed to increase the half-life of biologicals in blood, but lamentably these molecules have increased immunogenicity which decreases their potency over time. By transfer of therapeutic genes locally, we can surmount these limitations. Therapeutic genes can be delivered locally to the diseased tissue. The soluble mediators are then expressed and consumed locally, having very limited effects systemically. In addition, by choosing either constitutively active or transcriptionally regulated promoters, therapeutic genes could be expressed long term or induced only as required.

Current work in animal models of arthritis and in vitro studies with synovial explants from patients with rheumatoid arthritis indicate that inhibition of pro-inflammatory cytokines, matrix metalloproteases together with trophic factors for cartilage and bone are effective therapeutic agents. Molecules which target the pannocyte, or macrophage, T- and B-cell function can also protect and ameliorate disease. Combination therapy with peptides with anti-inflammatory properties [soluble tumour necrosis factor (TNF) receptors, anti-inflammatory cytokines such as interferon (IFN) beta, interleukin (IL)-10, IL-4], chondroprotective IL-1 receptor antagonist (IL-1ra), tissue inhibitor of metalloprotease (TIMPs), cartilage anabolic factors (transforming growth factor ß1) and antibodies, is feasible and relatively easy using current genetic engineering methods. These molecules have been tested successfully in animal models of arthritis.

One advantage of using animal models is that the natural history of the disease can be followed from its induction. In these models, therapeutic modalities can be evaluated. However, most models studied to date are acute self-limiting models which do not resemble the human chronic disease. Accordingly, the biological basis for chronicity has to be understood and recent developments using transgenic mice which develop a chronic arthritis may be instrumental in this regard [1]. The challenge of effective gene therapy in chronic models of arthritis may require different delivery systems than those employed to date.

It could be argued that rheumatoid arthritis represents a spectrum of different diseases whose end outcome is cartilage and bone erosion, with extra-articular involvement in the more aggressive cases. Furthermore, it is quite possible that the pathogenic mechanisms leading to similar clinical findings are different for each type of disease. Thus, each type may require a different gene for successful cure. Some patients develop a self-limiting disease, some a chronic relapsing type and others a progressive type that leads to joint replacement within 3–5 yr of onset. An indication for the diversity in rheumatoid arthritis, and that could be subdivided as having evolved by different pathological mechanisms, is suggested by the lack of therapeutic response in patients treated with disease-modifying anti-rheumatic synthetic drugs and also after biological therapy such as anti-TNF antibodies [2]. Not all patients with rheumatoid arthritis are rheumatoid factor positive and 20% of patients have lymphoid follicles in the pannus, suggesting several pathobiological mechanisms.

Thus, it is imperative that more basic research is undertaken to understand the molecular basis for the differences amongst these diseases, taking into account whether pathogenic factors such as viruses exist and if possible additional hereditary factors located outside the MHC locus are also involved. The classification of rheumatoid arthritis in molecular terms, such as cytokine profiles, complement factors, proteolytic enzymes, enzymes involved in free radical metabolism, levels of their natural inhibitors/regulators must be determined, so that an efficient patient-tailored therapy can be devised. Developmental cell and molecular biology of endothelial, B and T cells, antigen-presenting cells, chondrocytes, mesenchymal stem cells, synovial and bone cells will increase our understanding of the pathophysiology and help identify new therapeutic targets.

The myriad of developments in the area of gene therapy vectors encompass chemical advances to improve liposome technology and molecular genetics to improve virus properties.

Currently, local delivery of therapeutic genes has been pursued using in vivo intra-articular injection of viruses expressing therapeutic genes such as IL-1ra, TNF antagonists and anti-inflammatory cytokines or apoptosis-inducing agents such as the herpes simplex thymidine kinase and FAS ligand genes. The direct in vivo use of viruses has limitations, such as immunogenicity, which leads to short-term expression using adenoviruses, or lack of effective infection by retroviruses which only infect dividing cells. Some of these problems can be solved using other viral vectors such as adeno-associated virus (AAV) and lentiviral vectors which can infect non-dividing cells and also incorporate into the genome of the cell.

A simple method of i.m. injection of naked DNA or delivery of cationic liposome/plasmid DNA complexes has also been used in animal models. The limitation of the plasmid DNA approach in vivo is again the inefficiency of the method and transient expression obtained because the DNA is not incorporated into the genome of the cell (for recent reviews, see [3, 4]).

Ex vivo therapy has been pursued by genetically engineering cells to produce therapeutic genes. Non-mobile cells such as synoviocytes or fibroblasts, which after infection in vitro are transplanted locally into joints or used as `secreting factories' i.p., have been used successfully [5]. A clinical trial using retrovirally engineered autologous synoviocytes to secrete IL-1ra implanted in joints is under way [6]. This clinical study represents an important breakthrough both ethically and scientifically, as it was the first trial approved for a non-hereditary, non-fatal disease. However, whether the injection of all affected joints in a patient will be feasible by this method remains to be established since the number of cells to be expanded for such therapy may prove prohibitive.

Another ex vivo method used in animal models of arthritis is the use of arthritogenic T cells. We have engineered arthritogenic T cells to express inhibitory cytokines and cytokine antagonists with retroviral vectors, and have shown that we can inhibit collagen-induced arthritis transfer from arthritic DBA/1 to SCID mice, as well as ameliorate established disease [7, 8]. The advantage of this approach is the use of the unique biological properties of these cells, which include: specific antigen recognition and proliferation, migration through endothelial barriers and in some cases long half-life. T cells traffic through the body, enter inflammatory sites and, if their specific antigen is present, proliferate, thus amplifying locally the biological therapeutic effect. This method has a great advantage when compared with the non-mobile cell carrier approach, since using lymphocytes will facilitate the treatment of multiple affected joints. The therapeutic effects in collagen-induced arthritis, using arthritogenic T cells expressing TNF antagonists, are impressive since very few cells are infected (0.2%) yet they are capable of downregulating inflammation, inhibiting cell infiltration to joints and also shown to decrease B-cell function by inhibiting antibody production. This latter biological effect has not been observed using protein therapy with anti-TNF antibodies or TNF receptor–Ig fusion proteins. Importantly, the T lymphocytes used are arthritogenic and capable of transferring the disease to naive animals. How the biology of the T cell is altered by the expression of the therapeutic gene is unknown and is the focus of intensive investigation. Tissue-specific pathogenic T cells have also been genetically engineered to treat animal models of diabetes and multiple sclerosis.

In a clinical setting, the antigen(s) driving the autoimmune response in the joints at a particular time point is unknown. This important difference with our experimental work in arthritis models will limit our capability to expand arthritogenic human cells for ex vivo genetic engineering. We have therefore decided to retarget peripheral blood T cells to cartilage antigens and use them as therapeutic gene carriers. This was achieved by cloning and genetically engineering a chimeric receptor comprised of an extracellular domain from an anti-collagen type II monoclonal antibody scFv and transmembrane and intracellular domains of T-cell receptor signalling subunits [9]. Importantly, these engineered cells recognize antigen via the antibody moiety and not the T-cell receptor/MHC/peptide complex; the chimeric receptor is `universal' and could be used to retarget T cells irrespective of the patient's MHC genotype. T cells engineered to express this chimeric receptor with a retroviral vector recognize native (unprocessed) collagen type II and respond in vitro by secreting cytokines and proliferating. We envisage a scenario in which T cells can be isolated from a patient and, after ex vivo engineering with a retrovirus expressing as transgenes a chimeric receptor and therapeutic gene(s), they can be reinfused for therapeutic purposes. This approach is also being pursued by other investigators for cancer treatment by retargetting T cells to tumour antigens.

In summary, the advantage of biological therapy over chemical therapy appears obvious. However, the targets and methods of gene delivery, which will be simpler, economic and available to all patients according to their specific needs, will require further efforts and discoveries. The molecular methodologies are in place for this to happen and we look forward, working with enthusiasm, to the finding of the appropriate cures.

Acknowledgments

The critical review and comments of D. Isenberg, R. A. Mageed, M. Londei, A. Cope, O. L. Podhajcer and G. Adams are greatly appreciated.

Notes

Present address: Bone and Joint Research Unit, St Bartholomew's and Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, Charterhouse Square, London EC1M 6BQ, UK.

References

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