Another more drastic approach to silencing the inflammatory and destructive process is by killing resident cells in the arthritic joint. This approach is called genetic synovectomy, and it is the latest development in the tradition of synovectomy, as performed surgically, chemically or by radiation therapy. In RA the fibroblast-like type B synoviocyte is a transformed cell that exhibits unrestrained proliferation, lacking contact inhibition in vitro, and elevated expression of the pro-oncogene c-myc.
A gene therapeutic approach is to induce apoptosis of RA transformed synoviocytes using TRAIL (Apo2L) or FasL (CD95L). TRAIL and FasL are type II membrane proteins and activate death receptors for transduction of apoptotic signals. Activated lymphocytes and synoviocytes from RA patients express TRAIL receptors, and adenoviral TRAIL gene transfer induced significant apoptosis in RA synoviocytes [71
]. High Fas expression was identified in inflamed synovium, and in infiltrating leucocytes in RA and animal models of arthritis. Okamoto and coworkers [72
] showed that FasL is capable of Fas-mediated apoptosis of human RA synoviocytes. Adenoviral gene transfer of TRAIL into rabbit knee joints 4 days after transplantation of IL-1β-expressing synoviocyte allografts resulted in pronounced apoptosis of synoviocytes, and inhibited local IL-1 effects [71
]. Also, adenoviral FasL gene transfer induced apoptosis of the synoviocytes and markedly ameliorated CIA in mice [73
A more selective approach is to target the tumor suppressor molecule p53. The p53 gene is upregulated in the RA joint, and evidence is accumulating that this gene is mutated and acts as a dominant negative inhibitor of wild-type p53 in the RA synovium [74
]. It is postulated that the same oxidative stress that induces DNA damage and apoptosis in cells can also cause p53 mutations that transform cells resulting in aberrant proliferation [76
]. Mutated p53 is considered responsible for the transformation of synovial fibroblasts into invasive phenotypes that are destructive to cartilage and bone [77
]. The p53 protein is a key regulator of inflammation, because induction of CIA in p53 gene knockout DBA1/J resulted in the development of more severe arthritis [78
]. Adenoviral transfer of the p53 gene into rabbit knee joints with IL-1β-expressing synoviocyte allografts resulted in pronounced apoptosis of synoviocytes, and marked reduction in inflammatory exudates [79
]. The p53 protein is strictly maintained in its inactive form under normal conditions, and the tight post-translational control also implies for exogenously added p53 [80
]. This could suggest that local p53 gene delivery to arthritic joints is a safe strategy.
An alternative approach to induction of p53-mediated cell death is to use conditionally replicative adenoviruses (CRAds) [81
]. CRAds can only propagate in the absence of a functional p53 gene, and thus they can selectively kill transformed synovial fibroblasts in a self-limiting process. Furthermore, CRAds can be used for intra-articular viral propagation, and so extend the range of transgene activity and link the duration of expression to the presence of transformed synoviocytes. The somatic p53 mutation has not been described in experimental models of arthritis, and therefore this treatment with CRAds might not be effective in animals. However, inducing cell toxicity by introducing the HSV thymidine kinase (TK) gene is effective in animal models. HSV-TK can phosphorylate the nontoxic prodrug ganciclovir into ganciclovir triphosphate, which causes chain termination and single-strand breaks upon incorporation into DNA [82
]. As a consequence, cells go into apoptosis by a process that is largely unknown. An advantage is the so-called 'bystander effect', by which HSV-TK can affect even cells in which the gene is not introduced [83
This suicide gene system was evaluated in experimental arthritis by intra-articular injection of HSV-TK expression plasmids and adenoviruses in AIA in rabbits and in CIA in rhesus monkeys, respectively [84
]. Subsequent treatment with intravenous ganciclovir increased apoptotic cell death in the synovium and cytolysis of the synovial lining layer in both arthritis models. An alternative approach for suicide gene therapy is to inhibit cell proliferation. The cell cycle is controlled by the kinase activity of cyclin/cyclin-dependent kinases and their inhibitors p16 (INK4a) and p21 (Cip1). Forced overexpression of either p16 or p21 by adenoviral vector transduction of the synovium inhibited in vitro
growth of RA synovial fibroblasts and inhibited pathology in rat adjuvant arthritis [86
] and murine CIA [88
]. These cyclin-dependent kinase inhibitors not only prevented synovial growth (pannus formation) but also suppressed the expression of the proinflammatory cytokines IL-1β, IL-6 and TNF-α [88
] and of degrading proteinases [89
]. At least for p21, it has been shown that it also can inactivate NF-κB and activator protein-1, showing that these cyclin-dependent kinase inhibitors may have a much broader therapeutic spectrum.
The therapeutic efficacy of genetic synovectomy can be improved by cell-specific targeting of transformed synoviocytes. Fibre knob modification by introducing a RGD motif changes the tropism of the adenovirus and transduced the synovial fibroblast more efficiently than did the conventional adenovirus in vitro
(Table ) and in rat ankle joint [90
]. We previously showed that IL-1Ra gene therapy was more efficacious in the treatment of murine CIA when using RGD modified adenoviral vectors [35
]. RGD modification may direct the adenovirus to the transformed synovial fibroblast, and studies are in progress to combine cell targeting with genetic synovectomy in experimental arthritis.
Increased transduction of synovial fibroblasts with recombinant adenoviruses with RGF-modified fibre knobs