Maroun Khoury

Efficient suppression of murine arthritis by combined anticytokine small interfering RNA lipoplexes.

OBJECTIVEnBlocking tumor necrosis factor (TNF) effectively inhibits inflammation and joint damage in rheumatoid arthritis (RA), but 40% of RA patients respond only transiently or not at all to the current anti-TNF biotherapies. The purpose of this study was to develop an alternative targeted therapy for this subgroup of RA patients. As proof of concept, we tested the efficiency of an RNA interference (RNAi)-based intervention that targets proinflammatory cytokines in suppressing murine collagen-induced arthritis (CIA).nnnMETHODSnTwo synthetic short interfering RNA (siRNA) sequences were designed for each of the proinflammatory cytokines interleukin-1 (IL-1), IL-6, and IL-18. Their silencing specificity was assessed according to lipopolysaccharide-induced messenger RNA expression in J774.1 mouse macrophages as compared with control siRNA. For in vivo administration, siRNA were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA and were injected intravenously (0.5 mg/kg) into DBA/1 mice with CIA.nnnRESULTSnWeekly injections of anti-IL-1, anti-IL-6, or anti-IL-18 siRNA-based lipoplexes significantly reduced the incidence and severity of arthritis, abrogating joint swelling and destruction of cartilage and bone, both in the preventative and the curative settings. The most striking therapeutic effect was observed when the 3 siRNA were delivered in combination. The siRNA lipoplex cocktail reduced all pathologic features of RA, including inflammation, joint destruction, and the Th1 response, and overall parameters of RA were improved as compared with anti-TNF siRNA lipoplex-based treatment.nnnCONCLUSIONnOur results present a novel option for in vivo RNAi-based antiinflammatory immunotherapy. Our findings indicate that intravenous administration of a lipoplex cocktail containing several anticytokine siRNA is a promising novel antiinflammatory therapy for RA, as well as a useful and simple tool for understanding the pathophysiology of RA and for evaluating new therapeutic candidates.

Inflammation‐inducible anti‐TNF gene expression mediated by intra‐articular injection of serotype 5 adeno‐associated virus reduces arthritis

The tumor necrosis factor (TNF)‐α plays a central role in rheumatoid arthritis (RA) and current biotherapies targeting TNF‐α have a major impact on RA treatment. The long‐term safety concerns associated with the repetitive TNF blockade prompt optimization of therapeutic anti‐TNF approaches. Since we recently demonstrated that intra‐articular gene transfer using a recombinant adeno‐associated virus serotype 5 (rAAV5) efficiently transduces arthritic joints, we evaluate its effect on collagen‐induced arthritis (CIA) when encoding TNF antagonists.

Reduction of arthritis following intra-articular administration of an adeno-associated virus serotype 5 expressing a disease-inducible TNF-blocking agent

Background: In the context of preclinical development, we studied the potential of intra-articular gene delivery using a recombinant adeno-associated virus 5 (rAAV5) encoding a chimeric human tumour necrosis factorα (TNFα) soluble receptor I linked to a mouse immunoglobulin heavy chain Fc portion (TNF receptor I; TNFRI-Ig). Methods: Expression was under control of a nuclear factor kappa B (NFκB)-responsive promoter and compared with a cytomegalovirus (CMV) promoter (rAAV5.NFκB-TNFRI-Ig and rAAV5.CMV-TNFRI-Ig, respectively). Results: Fibroblast-like synoviocytes transduced in vitro with rAAV5.NFκB-TNFRI-Ig were able to produce TNFRI-Ig protein in response to several stimuli, and this was inhibited upon treatment with a specific NFκB blocking agent. A bioassay revealed that the synthesised TNFRI-Ig was bioactive, showing a higher affinity for human than for rat TNFα. Transcription of the transgene and protein production were detectable in joints injected with both constructs. No dissemination of the vector was observed outside the joints. A significant reduction in paw swelling was seen in rats treated with rAAV5.NFκB-TNFRI-Ig. This clinical effect was accompanied by a decrease in pro-inflammatory cytokine levels and an increase in IL10 expression in the synovium. Conclusion: These results provide evidence that intra-articular gene therapy using rAAV5 encoding TNFRI-Ig may be a safe and feasible approach for the treatment of rheumatoid arthritis. The higher affinity for human TNFα suggests that in patients with rheumatoid arthritis the therapeutic effect might be even more pronounced than in rat adjuvant arthritis.

A comparative study on intra-articular versus systemic gene electrotransfer in experimental arthritis

Electric pulse mediated gene transfer has been applied successfully in vivo for increasing naked DNA administration in various tissues. To achieve non‐viral gene transfer into arthritic joint tissue, we investigated the use of electrotransfer (ET). Because anti‐inflammatory cytokine strategies have proven efficient in experimental models of arthritis, we compared the therapeutic efficiency of local versus systemic delivery of the interleukin‐10 (IL‐10) using in vivo ET.

Adeno-Associated Virus Type 5-Mediated Intraarticular Administration of Tumor Necrosis Factor Small Interfering RNA Improves Collagen-Induced Arthritis

OBJECTIVEnRNA interference (RNAi) is a powerful tool for sequence-specific gene silencing, and interest in its application in human diseases is growing. Given the success of recent strategies for administering gene therapy in rheumatoid arthritis using recombinant vectors such as adeno-associated virus type 5 (rAAV5) for optimized intraarticular gene transfer, we undertook the present study to determine the feasibility of using rAAV5-mediated RNAi-based therapy in arthritis.nnnMETHODSnWe developed rAAV5 vectors expressing short hairpin small interfering RNA (shRNA) against tumor necrosis factor alpha (TNFalpha) under H1 promoter, and carrying the enhanced green fluorescent protein (eGFP) reporter gene under cytomegalovirus promoter (rAAV5-shTNF). TNFalpha gene silencing was validated in vitro with mouse macrophages. Mice with collagen-induced arthritis were injected in the ankle and knee joints, at disease onset, with either rAAV5-shTNF or control rAAV5-eGFP vectors (5 x 10(9) particles). Arthritis severity was assessed clinically and histologically, and immunologic response was examined. Local and systemic transgene expression was monitored using quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical analysis, and enzyme-linked immunosorbent assay.nnnRESULTSnAfter a single injection of rAAV5-shTNF into inflamed joints, local TNFalpha gene silencing provided rapid and long-term suppression of arthritis progression and reduced joint damage compared with that observed in control groups. Treatment with rAAV5-shTNF was associated with decreased proliferation and interferon-gamma production by antigen-stimulated T cells from draining lymph nodes, and the potency of this treatment was similar to that observed with other treatment strategies targeting TNFalpha at the protein level, either locally or systemically.nnnCONCLUSIONnOur data present the first proof-of-concept for the application of rAAV5-mediated RNAi-based gene therapy for local blockade of inflammation in experimental arthritis.

Mesenchymal stem cells in osteoarticular pediatric diseases: an update

Cellular therapy has gained an increasing popularity in recent years. Mesenchymal stem cells (MSCs) have the potential to differentiate into bone, cartilage, or fat tissue. In recent studies, these cells have also shown healing capability by improving angiogenesis and preventing fibrosis, which could have a role in tissue repair and tissue regeneration. Preclinical and clinical orthopedic studies conducted in the adult population support the use of MSCs for bone-healing problems, early stages of osteonecrosis, and local bone defects. Only a few published studies support the use of MSCs in pediatric osteoarticular disorders, probably due to the unknown long-term results of cellular therapy. The purpose of this review is to explain the mechanism by which MSCs could exhibit a therapeutic role in pediatric osteoarticular disorders.

MicroRNA Profiling of B Cell Subsets from Systemic Lupus Erythematosus Patients Reveals Promising Novel Biomarkers

MicroRNAs control the differentiation and function of B cells, which are considered key elements in the pathogenesis of systemic lupus erythematosus (SLE). However, a common micro(mi)RNA signature has not emerged since published data includes patients of variable ethnic background, type of disease, and organ involvement, as well as heterogeneous cell populations. Here, we aimed at identifying a miRNA signature of purified B cells from renal and non-renal severe SLE patients of Latin American background, a population known to express severe disease. Genome-wide miRNA expression analyses were performed on naive and memory B cells and revealed two categories of miRNA signatures. The first signature represents B cell subset-specific miRNAs deregulated in SLE: 11 and six miRNAs discriminating naive and memory B cells of SLE patients from healthy controls (HC), respectively. Whether the miRNA was up or down-regulated in memory B cells as compared with naive B cells in HC, this difference was abolished in SLE patients, and vice versa. The second signature identifies six miRNAs associated with specific pathologic features affecting renal outcome, providing a further understanding for SLE pathogenesis. Overall, the present work provided promising biomarkers in molecular diagnostics for disease severity as well as potential new targets for therapeutic intervention in SLE.

Additional file 3: Figure S3. of Combination therapy of menstrual derived mesenchymal stem cells and antibiotics ameliorates survival in sepsis

Effect of MenSCs treatment in mice with polymicrobial sepsis. Serum was isolated 24xa0hours after sepsis induction and administration of different treatments with AB or MenSCs or both (sham, nu2009=u20093; saline, nu2009=u20095; AB, nu2009=u20092–4; MenSCs, nu2009=u20094; MenSCsu2009+u2009AB, nu2009=u20094) to determine the concentrations of alkaline phosphatase (ALP) (left panel) and albumin (right panel). Dot plots represent individual values, horizontal bars represent mean values, and vertical bars represent standard error values. **Pu2009≤u20090.01. AB antibiotics, MenSCs menstrual derived mesenchymal stem cells, ns not significant. (PDF 180 kb)

A7.12 Micro-RNA signature in systemic lupus erythematosus

Background and objectives Several microRNAs are known to control the differentiation and function of B cells, that are considered key elements in the pathogenesis of systemic lupus erythematosus (SLE). However, a common miRNA signature has not emerged in SLE, since published data includes patients of variable ethnic background, type of disease and organ involvement. Moreover, published gene expression studies are often limited since they target only unfractionated, heterogeneous cell populations. Here, we aimed at identifying a miRNA-signature of purified B cells from renal and non renal severe (Bilag A) SLE patients of hispanic ethnic background, a population know to express severe disease. Materials and methods Blood samples were obtained from healthy sex, age and ethnically matched controls (HC) with no history of autoimmune diseases and from SLE patients of chilean hispanic descent fulfilling SLICC and ACR criteria, with or without severe lupus nephritis (LN) (mean Bilag count respectively 17.8 and 16.8). Naive and memory B cells were sorted using CD27 surface marker. Total RNAs extraction, reverse transcription, and amplification steps were performed to fulfil quality and integrity criteria of the MIQE guidelines. The genome-wide miRNA expression study was perfomed using the TaqMan® Human MicroRNA Array Cards v3.0 (Applied Biosystems). Data were normalised using ExpressionSuite software (Life technologies) and analysed using RT2 profiler PCR array data analysis version 3.5 (Qiagen). Results TLDA analyses of naive and memory B cells from HC and 2 subsets of SLE patients revealed two categories of miRNA-based signatures. The first signature represents miRNAs with potential as diagnostic biomarkers : 11 miRNAs discriminating all SLE patients from HC, and 8 miARNs discriminating patient subsets (SLE versus SLE-LN). The second signature identifies 13 miRNAs with therapeutic potential in lupus. Clustering analyses of TLDA datasets evidenced that the main differences in miRNA expression profilings between SLE patients were between naive and memory B cells, independently of disease severity. In all cases, whether the miRNA was up or down-regulated in memory B cells as compared with naive B cells in HC, this difference was abolished in SLE patients. Among these, we found miR-223 that was previously reported as deregulated in SLE, as well as in other autoimmune disorders and B cell leukaemia. Array data were further validated on individual samples (n = 6). Conclusions Overall, the present work identified two types of miRNA-based signatures in circulating B cells isolated from Chilean SLE patients, providing promising biomarkers in molecular diagnostics for disease severity as well as potential new targets for therapeutic intervention in SLE. References Wu HJ, Ivanov II, Darce J, et al. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity 2010; 32(6):815–27. doi: 10.1016/j.immuni.2010.06.001. Lécuyer E, Rakotobe S, Lengliné-Garnier H, et al. Segmented filamentous bacterium uses secondary and tertiary lymphoid tissues to induce gut IgA and specific T helper 17 cell responses. Immunity 2014; 40(4):608–20. doi: 10.1016/j.immuni.2014.03.009. Detert J, Bastian H, Listing J, et al. Induction therapy with adalimumab plus methotrexate for 24 weeks followed by methotrexate monotherapy up to week 48 versus methotrexate therapy alone for DMARD-naive patients with early rheumatoid arthritis: HIT HARD, an investigator-initiated study. Ann Rheum Dis 2013; 72(6):844–50. doi: 10.1136/annrheumdis-2012-201612. Epub 2012 Jun 27.