A.C. Bakker

Intra-articular IL-10 gene transfer regulates the expression of collagen-induced arthritis (CIA) in the knee and ipsilateral paw

We studied the effects of local IL‐10 application, introduced by a recombinant human type 5 adenovirus vector, in the mouse knee joint during the early phase of CIA. One intra‐articular injection with the IL‐10‐expressing virus (Ad5E1mIL‐10) caused substantial over‐expression of IL‐10 in the mouse knee joint, using virus dosages which did not induce distracting inflammation. High expression of IL‐10 was noted for a few days, being maximal at day 1. One intra‐articular injection of Ad5E1mIL‐10 in the knee joints of collagen type II (CII)‐immunized mice, before onset of CIA was noted, reduced the incidence of collagen arthritis in that knee. Of high interest, the protective effect of local IL‐10 expression by Ad5E1mIL‐10 was not restricted to the knee joint alone. The arthritis incidence in the ipsilateral paw was highly suppressed. In contrast, local IL‐10 over‐expression was not effective when treatment was started after onset of CIA. Further analysis in the acute streptococcal cell wall‐induced arthritis model revealed that local IL‐10 over‐expression markedly suppressed the production of tumour necrosis factor‐alpha (TNF‐α) and IL‐1α, but had no significant effect on IL‐1β and IL‐12 production in the inflamed synovium. These data indicate that local over‐expression of IL‐10 in the knee joint of mice regulates the expression of collagen arthritis, probably through down‐regulation of TNF‐α.

An inflammation-inducible adenoviral expression system for local treatment of the arthritic joint.

To achieve a disease-regulated transgene expression for physiologically responsive gene therapy of arthritis, a hybrid promoter was constructed. The human IL-1β enhancer region (−3690 to −2720) upstream of the human IL-6 promoter region (−163 to +12) was essential in mounting a robust response in HIG-82 synovial fibroblasts and in RAW 264,7 macrophages. A replication-deficient adenovirus was engineered with luciferase (Luc) controlled by the IL-1/IL-6 promoter (Ad5.IL-1/IL-6-Luc). LPS caused a 23- and 4.6-fold induction of Luc. activity in RAW cells infected with Ad5.IL-1/IL-6-Luc or the conventional Ad5.CMV-Luc construct, respectively. Next, adenoviruses (106 ffu) were injected into the knees of C57Bl/6 mice. An intra-articular injection of zymosan, 3 days after Ad5.IL-1/IL-6-Luc, increased Luc. activity by 39-fold but had no effect in the Ad5.CMV-Luc joints. The constitutive CMV promoter was rapidly silenced and could not be reactivated in vivo. In contrast, the IL-1/IL-6 promoter could be reactivated by Streptococcal cell wall (SCW)-induced arthritis up to 21 days after infection. Next the IL-1/IL-6 promoter was compared to the C3-Tat/HIV-LTR two-component system in wild-type, IL-6−/− and IL-1−/− gene knockout mice. Both systems responded well to LPS-, zymosan- and SCW-induced arthritis. However, the basal activity of the IL-1/IL-6 promoter was lower and IL-6 independent. This study showed that the IL-1/IL-6 promoter is feasible to achieve disease-regulated transgene expression for treatment of arthritis.

A tropism-modified adenoviral vector increased the effectiveness of gene therapy for arthritis

Adenoviral vectors (AdV) are used for anti-inflammatory cytokine therapy in experimental arthritis. Cell entry of AdV is dependent on the initial recognition of the coxsackie-adenovirus receptor (CAR) on cells. Recently, an Arg-Gly-Asp (RGD) motif was introduced in the HI loop of the fiber knob, this enables the adenovirus to bypass CAR and mediate cell entry via RGD binding integrins. In this study, we explored the transduction efficiency of the RGD-modified adenovirus in synovium and compared the RGD-modified with the conventional adenoviral vector for their effectiveness to modulate the murine collagen-induced arthritis (CIA) model when used to overexpress mIL-1Ra in the knee joint. Twenty-four hours after intra-articular injection of 107 fluorescent forming units (ffu) virus, luciferase (luc) activity in Ad5LucRGD-injected joints was up to 38 times higher than in AdCMVLuc-injected joints, and in arthritic joints the transduction efficiency was up to 69 times higher for the Ad5LucRGD viruses. Transduction of the synovial lining by the RGD-modified adenovirus containing the mIL-1Ra transgene, markedly improved the inhibition of CIA compared with the conventional virus in both a prophylactic and therapeutic treatment protocol. These results show that targeting integrins with the RGD-modified AdV improved the outcome of gene therapy for arthritis.

inflammation-inducible intra-articular production of human IL-1 receptor antagonist results in a more efficient inhibition of collagen-induced arthritis than does constitutive expression of the same transgene

Background Achieving biologically effective yet safe levels of recombinant anti-inflammatory proteins by gene therapy may require three regulatory features: 1) a basal level that is sufficiently low to avoid or minimise chronic immunosuppression 2) transcriptional regulation over a wide dynamic range, and 3) induced expression levels that are sufficiently high to achieve the desired biological effects. The C3-Tat/HIV promoter construct seems to have these properties.1 In this two-component expression system the complement factor 3 (C3) promoter regulates production of the HIV transactivator of transcription, and the Tat protein then stimulates expression of the desired transgene, which is regulated by the HIV-LTR promoter. Both C3 and HIV LTR promoters are inducible by proinflammatory cytokines. Objectives To achieve disease-inducible expression of recombinant anti-inflammatory proteins in order to allow autoregulation of drug dose by natural homeostatic mechanisms. Methods We compared a disease-inducible, two component expression system (C3-Tat/HIV) with the constitutive immediate early cytomegalovirus (ieCMV) promoter in the polyarticular Collagen-Induced Arthritis (CIA) model in mice. DBA/I mice were immunised with bovine type II collagen and boostered on day 22. On day 22, mice without any clinical signs of arthritis were selected and adenoviral vectors (Ad. CMV-Luc, Ad. CMV-IL-1Ra, or Ad. C3-Tat/HIV-IL-1Ra) that contained luciferase or the human IL-1Ra gene under control of one of the two promoters were used to transfect the synovial lining of both knees. The injected knee joints and ipsilateral paws were then scored for signs of arthritis and at the end histology was taken. Results Inducible promoter-driven IL-1Ra expression resulted in significantly improved inhibition of CIA than did CMV-driven IL-1Ra production. Moreover, overexpression of IL-1Ra in the knee joints also prevented CIA in the ipsilateral paws. Conclusion Our data (1) demonstrate the feasibility of an inducible expression system for producing a recombinant transgene for treatment of arthritis and (2) show that this system is more effective than strong, constitutive transgene expression for preventing collagen-induced arthritis in mice. Reference Varley AW, Geiszler SM, Gaynor RB, Munford RS. A two-component expression system that responds to inflammatory stimuli in vivoin vivo. Nat Biotechnol. 1997;15(10):1002–6

Dualistic role of TGFβ in osteoarthritis cartilage destruction and osteophyte formation

Osteoarthritis is a condition of focal cartilage lesions, combined with alterations in the subchondral bone and new hone formation (osteophytes) at the joint margins.