Enhanced gene transfer to arthritic joints using adeno-associated virus type 5: implications for intra-articular gene therapy.

BACKGROUND: Gene therapy of the joint has great potential as a new therapeutic approach for the treatment of rheumatoid arthritis (RA). The vector chosen is of crucial importance for clinical success. OBJECTIVE: To investigate the tropism and transduction efficiency in arthritic joints in vivo, and in synovial cells in vitro, using five different serotypes of recombinant adeno-associated virus (rAAV) encoding beta-galactosidase or green fluorescent protein genes. METHODS: rAAV was injected into the ankle joints of rats with adjuvant arthritis after the onset of disease. Synovial tissue was examined at different time points for beta-galactosidase protein and gene expression by in situ staining and polymerase chain reaction (PCR) analysis, respectively. In addition, the ability of rAAV to transduce primary human fibroblast-like synoviocytes from patients with RA was investigated in vitro. RESULTS: Intra-articular injection of the rAAV5 serotype resulted in the highest synovial transduction, followed by much lower expression using rAAV2. Expression of the transgene was already detectable 7 days after injection and lasted for at least 4 weeks. Only background staining was seen for serotypes 1, 3, and 4. Importantly, there was a minimal humoral immune response to rAAV5 compared with rAAV2. Additionally, it was found that both rAAV2 and rAAV5 can efficiently transduce human fibroblast-like synoviocytes obtained from patients with RA. CONCLUSION: Intra-articular rAAV mediated gene therapy in RA might be improved by using rAAV5 rather than other serotypes.

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-1 beta 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 (10(6) 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.

C3-Tat/HIV-regulated intraarticular human interleukin-1 receptor antagonist gene therapy results in efficient inhibition of collagen-induced arthritis superior to cytomegalovirus-regulated expression of the same transgene.

OBJECTIVE: To achieve disease-inducible expression of recombinant antiinflammatory proteins in order to allow autoregulation of drug dose by natural homeostatic mechanisms. METHODS: We compared the inducible 2-component expression system (C3-human immunodeficiency virus/transactivator of transcription [C3-Tat/HIV]) with the constitutive cytomegalovirus (CMV) promoter in the polyarticular collagen-induced arthritis (CIA) model in mice. DBA/1 mice were immunized with bovine type II collagen and were given boosters on day 21. On day 22, mice were injected intraarticularly with the adenoviral vectors AdCMVLuc, AdCMVhIL-1Ra, AdC3-Tat/HIV-Luc, or AdC3-Tat/HIV-hIL-1Ra. The injected knee joints and hind paws were then scored for signs of arthritis, and knee joint histology was compared. RESULTS: The CMV-driven interleukin-1 receptor antagonist (IL-1Ra) expression resulted in a high constitutive expression and amelioration of CIA. C3-Tat/HIV-driven IL-1Ra expression could be detected only on days 24, 29, and 35. Fourteen days after injection of the vectors, CIA was significantly better inhibited by the C3-Tat/HIV-driven IL-1Ra expression compared with the CMV-driven IL-1Ra expression. Moreover, prevention of CIA in the knee joints also prevented CIA in the untreated hind paws. CONCLUSION: Our data demonstrate for the first time the feasibility of an inducible expression system for local production of IL-1Ra for treatment of arthritis in the CIA model.

Overexpression of active TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation.

OBJECTIVE: To investigate the impact of a prolonged and constant active TGF-beta expression by the synovial lining cells on cartilage and ligamentous joint structures in vivo. DESIGN: An adenoviral vector (AdTGF-beta1(223,225)) was used for the overexpression of active TGF-beta1 in knee joints of C57Bl/6 mice. RESULTS: It was found that physiological relevant levels of active TGF-beta1 produced by the synovial lining layer resulted in histopathological changes: hyperplasia of synovium and chondro-osteophyte formation at the so-called chondro-synovial junctions. No histological changes were seen after intra-articular injection of an empty control vector (AdDL70-3) or by overexpression of latent TGF-beta1 (AdTGF-beta1). The predominant site of TGF-beta production in osteoarthritis (OA) and rheumatoid arthritis (RA) is the synovial lining layer. To address the question whether the TGF-beta-induced changes were related to the expression site in the synovial lining, the synovial lining layer was depleted by local treatment with liposomes encapsulating clodronate. Depletion of the lining resulted in a dramatic change of TGF-beta1-induced pathology: markedly reduced chondro-osteophyte formation and increased accumulation of extracellular matrix in the synovium. CONCLUSION: This study shows that overexpression of active TGF-beta1 in the knee joint results in OA-like changes and suggests the synovial lining cells contribute to the chondro-osteophyte formation.

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 10(7) 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.

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-alpha) and IL-1alpha, but had no significant effect on IL-1beta 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-alpha.

Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction.

Rheumatoid arthritis is a chronic inflammatory joint disease, leading to cartilage and bone destruction. In this study, we investigated the effects of local IL-4 application, introduced by a recombinant human type 5 adenovirus vector, in the knee joint of mice with collagen-induced arthritis. One intraarticular injection with an IL-4-expressing virus caused overexpression of IL-4 in the mouse knee joint. Enhanced onset and aggravation of the synovial inflammation were found in the IL-4 group. However, despite ongoing inflammation, histologic analysis showed impressive prevention of chondrocyte death and cartilage erosion. In line with this, chondrocyte proteoglycan synthesis was enhanced in the articular cartilage. This was quantified with ex vivo 35S-sulfate incorporation in patellar cartilage and confirmed by autoradiography on whole knee joint sections. Reduction of cartilage erosion was further substantiated by lack of expression of the stromelysin-dependent cartilage proteoglycan breakdown neoepitope VDIPEN in the Ad5E1 mIL-4-treated knee joint. Reduced metalloproteinase activity was also supported by markedly diminished mRNA expression of stromelysin-3 in the synovial tissue. Histologic analysis revealed marked reduction of polymorphonuclear cells in the synovial joint space in the IL-4-treated joints. This was confirmed by immunolocalization studies on knee joint sections using NIMP-R14 staining and diminished mRNA expression of macrophage-inflammatory protein-2 in the synovium tissue. mRNA levels of TNF-alpha and IL-1beta were suppressed as well, and IL-1beta and nitric oxide production by arthritic synovial tissue were strongly reduced. Our data show an impressive cartilage-protective effect of local IL-4 and underline the feasibility of local gene therapy with this cytokine in arthritis.

Homotypic fusion between aggregated lysosomes triggered by elevated [Ca2+]i in fibroblasts.

Previous studies demonstrated that microinjection of antibodies to the cytoplasmic domain of the lysosomal glycoprotein lgp120 induces aggregation of lysosomes in NRK cells. Here we show that the antibody-clustered vesicles do not co-localize with MPR and ss-COP-containing organelles, confirming their lysosomal nature. Observations by transmission and high voltage electron microscopy indicated that, although tightly apposed to each other, aggregated lysosomes remained as separate vesicles, with an average diameter of 0.3-0.4 micron. However, when cells microinjected with antibody were exposed to the Ca2+ ionophore ionomycin, large vesicles were formed within the lysosome clusters, suggesting the occurrence of lysosome-lysosome fusion. Stereological measurements of lysosome diameters on confocal and transmission electron microscopy indicated that the large lgp120-positive vesicles could have originated from the fusion of 3 up to 15 individual lysosomes. To verify if agents that mobilize Ca2+ from intracellular stores had the same effect, anti-lgp120-microinjected cells were treated with thapsigargin, and with the receptor-mediated agonists bombesin and thrombin. Thapsigargin also induced the formation of large lgp120-containing vesicles, detected by both confocal and transmission electron microscopy. Analysis of antibody-clustered lysosomes in streptolysin O-permeabilized cells indicated that an intracellular free Ca2+ concentration of 1 microM was sufficient to trigger formation of large lysosomes.

Prevention of murine collagen-induced arthritis in the knee and ipsilateral paw by local expression of human interleukin-1 receptor antagonist protein in the knee.

OBJECTIVE: To determine the efficacy of local human interleukin-1 receptor antagonist (HuIL-1Ra) gene therapy in murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were immunized against bovine type II collagen. Before the onset of arthritis, NIH/3T3 fibroblasts transfected with pMFG-IRAP were transplanted into the knee cavity. Normal NIH/3T3 cells served as controls. Paws were evaluated macroscopically for redness, swelling, and deformities during the course of arthritis. Swelling of the knee joints was measured by external gamma counting of 99mtechnetium accumulation in the joint. Paws and knee joints were dissected and processed for histologic studies to evaluate inflammation and cartilage destruction. RESULTS: The NIH/3T3 fibroblasts survived in the joint cavity of DBA mice for at least 7 days. The transduced cells expressed immunoreactive and bioactive HuIL-1Ra in the knee joint, and produced sufficient amounts to block the effect of 1 ng of recombinant murine IL-1alpha on chondrocyte proteoglycan synthesis. The onset of CIA was almost completely prevented in knee joints containing HuIL-1Ra-producing cells, whereas joints containing normal cells showed severe inflammation and destruction of cartilage. Moreover, onset of CIA in the draining joints (ipsilateral paws) of the HuIL-1Ra gene-bearing knees was also prevented. CONCLUSION: Local production of HuIL-1Ra in the knee was able to ameliorate the effects of IL-1 on cartilage and could prevent the onset of CIA not only in that knee, but also in the "draining" paw. This indicates the feasibility of gene transfer as a therapeutic approach to modulating arthritis.

Role of interleukin 1 in antigen-induced exacerbations of murine arthritis.

The mechanism underlying the chronic and intermittent course of rheumatoid arthritis is not elucidated. In the present study, the role of interleukin 1 (IL-1) was investigated in exacerbations of antigen-induced arthritis in mice. A flare-up of smoldering inflammation (weeks 3 to 4 of antigen-induced arthritis) was inducible by injection of a small amount of methylated bovine serum albumin into the hypersensitive knee joint. Immunohistochemistry showed IL-1 expression in the synovial lining layer and in focal areas of the inflamed synovium during the flare-up. IL-1 was also measured in 1-hour culture supernatant of synovial tissue taken during the flare-up by a bioassay. The expression of both immunoreactive and bioactive IL-1 in the hypersensitive joint peaked around 6 hours after antigen (2 micrograms of methylated bovine serum albumin) injection and declined thereafter. Antigen rechallenge induced an acute joint swelling of the arthritic joint but not in the naive joint of the sensitized mouse, yet synovia of both joints produced IL-1 after antigen injection. Remarkably, a single intravenous injection of rabbit anti-IL-1 alpha and -beta antibodies 1 hour before antigen rechallenge neutralized IL-1 in the joint. Anti-IL-1 treatment significantly reduced the antigen-induced joint swelling (30 to 40%) but did not affect the profound influx of polymorphonuclear cells in the onset of the exacerbation. However, a profound relief of the inflammation (synovitis) was obtained by IL-1 blockade on day 4 of the exacerbation. Chondrocyte proteoglycan synthesis was markedly suppressed in the antigen-challenged naive knee joints suggesting that this was a direct IL-1 effect as the inflammation was insignificant. Anti-IL-1 treatment was able to maintain chondrocyte proteoglycan synthesis in the antigen-rechallenged joint, which was highly suppressed in the control group. Furthermore, the enhanced proteoglycan breakdown in the antigen-rechallenged joints was significantly decreased in the anti-IL-1 group. We concluded that IL-1 is an important mediator in exacerbations of murine arthritis, and amelioration of cartilage pathology was obtained with anti-IL-1 antibody treatment.

Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.