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Chronic pain involves sensitization of nociceptors and synaptic transmission of painful signals in nociceptive circuits in the dorsal horn of the spinal cord. We investigated the contribution of clathrin-dependent endocytosis to sensitization of nociceptors by G protein-coupled receptors (GPCRs) and to synaptic transmission in spinal nociceptive circuits. We determined whether therapeutic targeting of endocytosis could ameliorate pain. mRNA encoding dynamin (Dnm) 1-3 and adaptor-associated protein kinase 1 (AAK1), which mediate clathrin-dependent endocytosis, were localized to primary sensory neurons of dorsal root ganglia of mouse and human and to spinal neurons in the dorsal horn of the mouse spinal cord by RNAScope®. When injected intrathecally to mice, Dnm and AAK1 siRNA or shRNA knocked-down Dnm and AAK1 mRNA in dorsal root ganglia neurons, reversed mechanical and thermal allodynia and hyperalgesia, and normalized non-evoked behavior in preclinical models of inflammatory and neuropathic pain. Intrathecally administered inhibiters of clathrin, Dnm and AAK1 also reversed allodynia and hyperalgesia. Disruption of clathrin, Dnm and AAK1 did not affect normal motor functions of behaviors. Patch clamp recordings of dorsal horn neurons revealed that Dnm1 and AAK1 disruption inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing release of synaptic vesicles from presynaptic primary afferent neurons. Patch clamp recordings from dorsal root ganglion nociceptors indicated that Dnm siRNA prevented sustained GPCR-mediated sensitization of nociceptors. By disrupting synaptic transmission in the spinal cord and blunting sensitization of nociceptors, endocytosis inhibitors offer a therapeutic approach for pain treatment.

International audience; Vectorized small interfering RNAs (siRNAs) are widely used to induce gene silencing. Among the delivery systems used, lipid-based particles are the most effective. Our objective was the development of novel lipid-polymer hybrid nanoparticles, from lipoplexes (complexes of cationic lipid and siRNAs), and poly (lactic-co-glycolic acid) (PLGA), using a simple modified nanoprecipitation method. Due to their morphology, we called these hybrid nanoparticles Spheroplexes. We elucidated their structure using several physico-chemical techniques and showed that they are composed of a hydrophobic PLGA matrix, surrounded by a lipid envelope adopting a lamellar structure, in which the siRNA is complexed, and they retain surface characteristics identical to the starting nanoparticles, i.e. lipoplexes siRNA. We analyzed the composition of the particle population and determined the final percentage of spheroplexes within this population, 80 to 85% depending on the preparation conditions, using fluorescent markers and the ability of flow cytometry to detect nanometric particles (approximately 200 nm). Finally, we showed that spheroplexes are very stable particles and more efficient than siRNA lipoplexes for the delivery of siRNA to cultured cells. We administered spheroplexes contain siRNAs targeting TNF-α to mice with ulcerative colitis induced by dextran sulfate and our results indicate a disease regression effect with a response probably mediated by their uptake by macrophages / monocytes at the level of lamina propria of the colon. The efficacy of decreased level of TNF-α in vivo seemed to be an association of spheroplexes polymer-lipid composition and the specific siRNA. These results demonstrate that spheroplexes are a promising hybrid nanoparticle for the oral delivery of siRNA to the colon.

Hepatic fibrosis is a major consequence of chronic liver disease such as non-alcoholic steatohepatitis which is undergoing a dramatic evolution given the obesity progression worldwide, and has no treatment to date. Hepatic stellate cells (HSCs) play a key role in the fibrosis process, because in chronic liver damage, they transdifferentiate from a “quiescent” to an “activated” phenotype responsible for most the collagen deposition in liver tissue. Here, using a diet-induced liver fibrosis murine model (choline-deficient amino acid-defined, high fat diet), we characterized a specific population of HSCs organized as clusters presenting simultaneously hypertrophy of retinoid droplets, quiescent and activated HSC markers. We showed that hypertrophied HSCs co-localized with fibrosis areas in space and time. Importantly, we reported the existence of this phenotype and its association with collagen deposition in three other mouse fibrosis models, including CCl4-induced fibrosis model. Moreover, we have also shown its relevance in human liver fibrosis associated with different etiologies (obesity, non-alcoholic steatohepatitis, viral hepatitis C and alcoholism). In particular, we have demonstrated a significant positive correlation between the stage of liver fibrosis and HSC hypertrophy in a cohort of obese patients with hepatic fibrosis. These results lead us to conclude that hypertrophied HSCs are closely associated with hepatic fibrosis in a metabolic disease context and may represent a new marker of metabolic liver disease progression.

The receptor for advanced glycation end products (RAGE) plays a central role in the chronic inflammatory process associated with atherosclerosis development. We aimed to develop lipoplexes carrying RAGE-short hairpin (sh) RNA, targeted to the adhesion molecule P-selectin, selectively expressed on the surface of activated endothelium (Psel-lipo/shRAGE) to down-regulate RAGE expression as a therapeutic strategy for atherosclerosis. In vitro, Psel-lipo/shRAGE lipoplexes were efficiently taken up by activated endothelial cells (EC), decreased the expression of RAGE protein, and proved to be functional by reducing the monocyte adhesion to activated EC. In ApoE-deficient mice, the targeted lipoplexes accumulated specifically and efficiently transfected the aorta. The repeated administration of Psel-lipo/shRAGE lipoplexes, twice per week for one month: i) reduced the expression of RAGE protein in the aorta by decreasing the expression of NF-kB and TNF-α; ii) diminished the plasma levels of TNF-α, IL6, IL-1β, and MCP-1; iii) inhibited the atherosclerotic plaque development and iv) had no significant adverse effects. In conclusion, the newly developed Psel-lipo/shRAGE lipoplexes reduce the inflammatory processes associated with RAGE signaling and the progression of atherosclerosis in ApoE-deficient mice. Downregulation of RAGE employing these lipoplexes may represent a promising new targeted therapy to block atherosclerosis progression.

International audience; Vectorized small interfering RNAs (siRNAs) are widely used to induce specific mRNA degradation in the intracellular compartment of eukaryotic cells. Recently, we developed efficient cationic lipid-based siRNA vectors (siRNA lipoplexes or siLex) containing sodium alginate (Nalg-siLex) with superior efficiency and stability properties than siLex. In this study, we assessed the physicochemical and some biological properties of Nalg-siLex compared to siLex. While no significant differences in size, ζ potential and siRNA compaction were detected, the addition of sodium alginate modified the particle morphology, producing smoother and heterogeneous particles characterized by transmission electron microscopy. We also noted that Nalg-siLex have surface differences observed by X-ray photoelectron spectroscopy. These differences could arise from an internal reorganization of components induced by the addition of sodium alginate, that is indicated by Small-Angle X-ray Scattering results. Moreover, Nalg-siLex did not trigger significant hepatotoxicity nor inflammatory cytokine secretion compared to siLex. Taken together these results suggest that sodium alginate played a key role by structuring and reinforcing siRNA lipoplexes, leading to more stable and efficient delivery vector.

Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biologic functions, the contribution of endothelial TXNIP has not been well defined. To investigate the endothelial function of TXNIP, we generated a TXNIP knockout mouse on the Cdh5-cre background (TXNIPfl/fl cdh5cre). Control (TXNIPfl/fl) and TXNIPfl/fl cdh5cre mice were fed a high protein-low carbohydrate (HP-LC) diet for 3 mo to induce metabolic stress. We found that TXNIPfl/fl and TXNIPfl/fl cdh5cre mice on an HP-LC diet displayed impaired glucose tolerance and dyslipidemia concretizing the metabolic stress induced. We evaluated the impact of this metabolic stress on mice with reduced endothelial TXNIP expression with regard to arterial structure and function. TXNIPfl/fl cdh5cre mice on an HP-LC diet exhibited less endothelial dysfunction than littermate mice on an HP-LC diet. These mice were protected from decreased aortic medial cell content, impaired aortic distensibility, and increased plasminogen activator inhibitor 1 secretion. This protective effect came with lower oxidative stress and lower inflammation, with a reduced NLRP3 inflammasome expression, leading to a decrease in cleaved IL-1β. We also show the major role of TXNIP in inflammation with a knockdown model, using a TXNIP-specific, small interfering RNA included in a lipoplex. These findings demonstrate a key role for endothelial TXNIP in arterial impairments induced by metabolic stress, making endothelial TXNIP a potential therapeutic target.-Bedarida, T., Domingues, A., Baron, S., Ferreira, C., Vibert, F., Cottart, C.-H., Paul, J.-L., Escriou, V., Bigey, P., Gaussem, P., Leguillier, T., Nivet-Antoine, V. Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

International audience; INTRODUCTION:Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biological functions, the contribution of endothelial TXNIP has not been well-defined in regards to endothelial and vascular function or in post-ischemic revascularisation. We postulated that inhibition of endothelial TXNIP with siRNA or in a Cre-LoxP system could be involved in protection from high fat, high protein, low carbohydrate (HFHPLC) diet-induced oxidative stress and endothelial dysfunction, leading to vascular damage and impaired revascularisation in vivo.METHODS AND RESULTS:To investigate the role of endothelial TXNIP, the TXNIP gene was deleted in endothelial cells using anti-TXNIP siRNA treatment or the Cre-LoxP system. Murine models were fed a HFHPLC diet, known to induce metabolic disorders. Endothelial TXNIP targeting resulted in protection against metabolic disorder-related endothelial oxidative stress and endothelial dysfunction. This protective effect mitigates media cell loss induced by metabolic disorders and hampered metabolic disorder-related vascular dysfunction assessed by aortic reactivity and distensibility. In aortic ring cultures, metabolic disorders impaired vessel sprouting and this alteration was alleviated by deletion of endothelial TXNIP. When subjected to ischemia, mice fed a HFHPLC diet exhibited defective post-ischemic angiogenesis and impaired blood flow recovery in hind limb ischemia. However, reducing endothelial TXNIP rescued metabolic disorder-related impairment of ischemia-induced revascularisation.CONCLUSION:Collectively, these results show that targeting endothelial TXNIP in metabolic disorders is essential to maintaining endothelial function, vascular function and improving ischemia-induced revascularisation, making TXNIP a potential therapeutic target for therapy of vascular complications related to metabolic disorders.

International audience; Currently, most nonviral nucleic acid vectors are in the form of colloidal suspensions administered primarily parenterally. This type of formulation and the mode of administration impose strong constraints such as the size of the administered vectors or the production of sterile preparations. The tablet form provides access to easy oral administration, well accepted by patients; As regards nucleic acid vectors, a dry form represents an advance in terms of stability. Using an optimized lipid-based small interfering RNA-delivery system, we studied the tabletability of a liquid suspension of these vectors. We optimized the conditions of freeze-drying by choosing excipients and process, allowing for the conservation of both the gene-silencing efficacy of the formulated siRNAs and the supramolecular structure of the lipid particulate system. Gene-silencing efficacy was assayed on luciferase-expressing cells and the structure of the siRNA vector in freeze-dried and tablet forms was examined using small-angle X-ray scattering (SAXS) synchrotron radiation. The freeze-dried powders were then mixed with excipients necessary for the good progress of the compression by allowing for a regular supply of the matrix and the reduction of friction. The compression was carried out using a rotary press simulator that allows for complete monitoring of the compression conditions. After compression, formulated siRNAs retained more than 60% of their gene-silencing efficacy. Within the tablets, a specific SAXS signal was detectable and the lamellar and cubic phases of the initial liquid suspension were restored after resuspension of siRNA vectors by disintegration of the tablets. These results show that the bilayer lipid structures of the particles were preserved despite the mechanical constraints imposed by the compression. If such a result could be expected after the freeze-drying step, it was never shown, to our knowledge, that siRNA-delivery systems could retain their efficacy and structure after mechanical stress such as compression. This opens promising perspectives to oral administration of siRNA as an alternative to parenteral administration.

International audience; Classical osteogenesis imperfecta (OI) is an inherited rare brittle bone disease caused by dominant mutations in the COL1A1 or COL1A2 genes, encoding for the alpha chains of collagen type I. The definitive cure for the disease will require a gene therapy approach, aimed to correct or suppress the mutant allele. Interestingly, individuals lacking alpha2(I) chain and synthetizing collagen alpha1(I)3 homotrimers do not show bone phenotype, making appealing a bone specific COL1A2 silencing approach for OI therapy. To this aim, three different Col1a2-silencing RNAs (siRNAs), -3554, -3825 and -4125, selected at the 3'-end of the murine Col1a2 transcript were tested in vitro and in vivo. In murine embryonic fibroblasts Col1a2-siRNA-3554 was able to efficiently and specifically target the Col1a2 mRNA and to strongly reduce alpha2(I) chain expression. Its efficiency and specificity were also demonstrated in primary murine osteoblasts, whose mineralization was preserved. The efficiency of Col1a2-siRNA-3554 was proved also in vivo. Biphasic calcium phosphate implants loaded with murine mesenchymal stem cells were intramuscularly transplanted in nude mice and injected with Col1a2-siRNA-3554 three times a week for three weeks. Collagen alpha2 silencing was demonstrated both at mRNA and protein level and Masson's Trichrome staining confirmed the presence of newly formed collagen matrix. Our data pave the way for further investigation of Col1a2 silencing and siRNA delivery to the bone tissue as a possible strategy for OI therapy.

Classical osteogenesis imperfecta (OI) is an inherited rare brittle bone disease caused by dominant mutations in the COL1A1 or COL1A2 genes, encoding for the α chains of collagen type I. The definitive cure for the disease will require a gene therapy approach, aimed to correct or suppress the mutant allele. Interestingly, individuals lacking α2(I) chain and synthetizing collagen α1(I)3 homotrimers do not show bone phenotype, making appealing a bone specific COL1A2 silencing approach for OI therapy. To this aim, three different Col1a2-silencing RNAs (siRNAs), −3554, −3825 and −4125, selected at the 3′-end of the murine Col1a2 transcript were tested in vitro and in vivo. In murine embryonic fibroblasts Col1a2-siRNA-3554 was able to efficiently and specifically target the Col1a2 mRNA and to strongly reduce α2(I) chain expression. Its efficiency and specificity were also demonstrated in primary murine osteoblasts, whose mineralization was preserved. The efficiency of Col1a2-siRNA-3554 was proved also in vivo. Biphasic calcium phosphate implants loaded with murine mesenchymal stem cells were intramuscularly transplanted in nude mice and injected with Col1a2-siRNA-3554 three times a week for three weeks. Collagen α2 silencing was demonstrated both at mRNA and protein level and Masson's Trichrome staining confirmed the presence of newly formed collagen matrix. Our data pave the way for further investigation of Col1a2 silencing and siRNA delivery to the bone tissue as a possible strategy for OI therapy., Highlights • Identification of a specific and efficient Col1a2 siRNA • Silencing of Col1a2 allows osteoblasts mineralization. • Col1a2 silencing is not impairing matrix deposition in vivo.

International audience; The progress in small-interfering RNA (siRNA) therapeutics depends on the development of suitable nanocarriers to perform specific and effective delivery to dysfunctional cells. In this paper, we questioned whether P-selectin, a cell adhesion molecule specifically expressed on the surface of activated endothelial cells (EC) could be employed as a target for nanotherapeutic intervention. To this purpose, we developed and characterized P-selectin targeted PEGylated cationic liposomes able to efficiently pack siRNA and to function as efficient vectors for siRNA delivery to tumour necrosis factor-α (TNF-α) activated EC. Targeted cationic liposomes were obtained by coupling a peptide with high affinity for P-selectin to a functionalized PEGylated phospholipid inserted in the liposomes' bilayer (Psel-lipo). As control, scrambled peptide coupled cationic liposomes (Scr-lipo) were used. The lipoplexes obtained by complexation of Psel-lipo with siRNA (Psel-lipo/siRNA) were taken up specifically and at a higher extent by TNF-α activated b.End3 endothelial cells as compared to non-targeted Scr-lipo/siRNA. The Psel-lipo/siRNA delivered with high efficiency siRNA into the cells. The lipoplexes were functional as demonstrated by the down-regulation of the selected gene (GAPDH). The results demonstrate an effective targeted delivery of siRNA into cultured activated endothelial cells using P-selectin directed PEGylated cationic liposomes, which subsequently knock-down the desired gene.

Effective vaccine formulations consist of several components: an antigen carrier, the antigen, a stimulator of cellular immunity such as a Toll-like Receptors (TLRs) ligand, and a stimulator of humoral response such as an inflammasome activator. Here, we investigated the immunostimulatory and adjuvant properties of lipopolyamines, cationic lipids used as gene carriers. We identified new lipopolyamines able to activate both TLR2 and TLR4 and showed that lipopolyamines interact with TLRs via a mechanism different from the one used by bacterial ligands, activating a strong type-I IFN response, pro-inflammatory cytokines and IL-1β secretion. The TLR and inflammasome stimulations, together with the antigen carrier properties of lipopolyamines, resulted in both humoral and cellular immunity in mice vaccinated against OVA and make lipopolyamines promising one-component vaccine adjuvants., SCOPUS: ar.j, info:eu-repo/semantics/published

Rationale: Monocytes play critical roles in the pathogenesis of arthritis by contributing to the inflammatory response and bone erosion. Among genes involved in regulating monocyte functions, miR-146a negatively regulates the inflammatory response and osteoclast differentiation of monocytes. It is also the only miRNA reported to differentially regulate the cytokine response of the two classical Ly6Chigh and non-classical Ly6Clow monocyte subsets upon bacterial challenge. Although miR-146a is overexpressed in many tissues of arthritic patients, its specific role in monocyte subsets under arthritic conditions remains to be explored. Methods: We analyzed the monocyte subsets during collagen-induced arthritis (CIA) development by flow cytometry. We quantified the expression of miR-146a in classical and non-classical monocytes sorted from healthy and CIA mice, as well as patients with rheumatoid arthritis (RA). We monitored arthritis features in miR-146a-/- mice and assessed in vivo the therapeutic potential of miR-146a mimics delivery to Ly6Chigh monocytes. We performed transcriptomic and pathway enrichment analyses on both monocyte subsets sorted from wild type and miR-146a-/- mice. Results: We showed that the expression of miR-146a is reduced in the Ly6Chigh subset of CIA mice and in the analogous monocyte subset (CD14+CD16-) in humans with RA as compared with healthy controls. The ablation of miR-146a in mice worsened arthritis severity, increased osteoclast differentiation in vitro and bone erosion in vivo. In vivo delivery of miR-146a to Ly6Chigh monocytes, and not to Ly6Clow monocytes, rescues bone erosion in miR-146a-/- arthritic mice and reduces osteoclast differentiation and pathogenic bone erosion in CIA joints of miR-146a+/+ mice, with no effect on inflammation. Silencing of the non-canonical NF-κB family member RelB in miR-146a-/- Ly6Chigh monocytes uncovers a role for miR-146a as a key regulator of the differentiation of Ly6Chigh, and not Ly6Clow, monocytes into osteoclasts under arthritic conditions. Conclusion: Our results show that classical monocytes play a critical role in arthritis bone erosion. They demonstrate the theranostics potential of manipulating miR-146a expression in Ly6Chigh monocytes to prevent joint destruction while sparing inflammation in arthritis.

OBJECTIVE: The nuclear protein heterogeneous nuclear RNP A2/B1 (hnRNP A2/B1) is involved in posttranscriptional regulation of gene expression. It is constitutively expressed in lymphoid organs and highly up-regulated in the synovial tissue of patients with rheumatoid arthritis (RA), who may also generate autoantibodies to this protein. This study was undertaken to investigate the potential involvement of hnRNP A2/B1 in the pathogenesis of autoimmune arthritis, by silencing hnRNP A2/B1 expression in 2 animal models of RA. METHODS: Collagen-induced arthritis (CIA) and the K/BxN serum-transfer model were used as animal models of RA. Efficient silencing of hnRNP A2/B1 was achieved using a liposome-based carrier system for delivery of small interfering RNAs. Expression of hnRNP A2/B1 was analyzed by flow cytometry, reverse transcription-quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. The number of osteoclasts was determined by tartrate-resistant acid phosphatase staining. Cytokine levels and anticollagen antibody levels were measured by enzyme-linked immunosorbent assay. RESULTS: Efficient silencing of hnRNP A2/B1 was achieved in all lymphoid organs. In both experimental models, the incidence and severity of arthritis were largely reduced and bone erosion was not detectable as compared to the control groups. Down-modulation of hnRNP A2/B1 significantly interfered with the production of proinflammatory cytokines from monocyte/macrophages, but not from T cells. Consistent with these findings, production of T cell cytokines was not impaired when cells were restimulated in vitro with type II collagen. Furthermore, levels of anticollagen antibodies were not affected by hnRNP A2/B1 silencing. CONCLUSION: Our findings suggest that hnRNP A2/B1 has an important role in regulation of the innate immune system, especially at the level of monocyte/macrophage activation. Therefore, down-modulation of hnRNP A2/B1 seems to affect primarily the effector phase of autoimmune arthritis.

In higher vertebrates, the primordium of the nervous system, the neural tube, is shaped along the rostrocaudal axis through two consecutive, radically different processes referred to as primary and secondary neurulation. Failures in neurulation lead to severe anomalies of the nervous system, called neural tube defects (NTDs), which are among the most common congenital malformations in humans. Mechanisms causing NTDs in humans remain ill-defined. Of particular interest, the thoracolumbar region, which encompasses many NTD cases in the spine, corresponds to the junction between primary and secondary neurulations. Elucidating which developmental processes operate during neurulation in this region is therefore pivotal to unraveling the etiology of NTDs. Here, using the chick embryo as a model, we show that, at the junction, the neural tube is elaborated by a unique developmental program involving concerted movements of elevation and folding combined with local cell ingression and accretion. This process ensures the topological continuity between the primary and secondary neural tubes while supplying all neural progenitors of both the junctional and secondary neural tubes. Because it is distinct from the other neurulation events, we term this phenomenon junctional neurulation. Moreover, the planar-cell-polarity member,Prickle-1, is recruited specifically during junctional neurulation and its misexpression within a limited time period suffices to cause anomalies that phenocopy lower spine NTDs in human. Our study thus provides a molecular and cellular basis for understanding the causality of NTD prevalence in humans and ascribes toPrickle-1a critical role in lower spinal cord formation.

International audience; We report here the proof of concept of the use of new assembled arrays of ultramicroelectrodes (UMEs) for the simultaneous and dynamic sensing of reactive nitrogen species such as nitric oxide (NO) and peroxynitrite (ONOO-, so-called PUN) released by cultured cells. These devices fill the need to study the key roles of these two reactive nitrogen species where a chemical imbalance is implicated in several pathologies and physiological disorders. Arrays containing over 2400 gold UMES were designed in such a way to increase sensitivity and allow detection of low concentrations of the target analytes in vivo. These disposable platforms are then integrated within cell culture wells and plates, resulting in an easy-to-handle tool for cellular biology. They were successfully used to detect in real time PUN formed in situ upon the reaction of NO released from a NO donor molecule ((Z)-1-[N-(2-aminoepropyl)-N-(2-ammoniopropyl)amino]-diazen-1-ium-1,2-diolate) and superoxide (O-2(-)) released by PMA (phorbol-12-myristate-13-acetate)-induced HL60 cells. (C) 2014 Elsevier Ltd. All rights reserved.

Raising high titer antibodies in animals is usually performed by protein immunization, which requires the long and sometimes difficult step of production of the recombinant protein. DNA immunization is an alternative to recombinant proteins, only requiring the building of an eukaryotic expression plasmid. Thanks to efficient DNA delivery techniques such as in vivo electroporation, DNA vaccination has proven useful the last few years. In this work, we have shown that it is possible to raise very high antibody titers in rabbit by DNA electroporation of an antigen encoding plasmid in the skeletal muscle with the right set of electrodes and rabbit strain. In a model of botulinum toxins types A and E, the neutralizing titers obtained after three treatments were high enough to fit the European Pharmacopeia, while it did not for type B toxin. Furthermore, the raised antibodies have high avidity and are suitable for in vitro and in vivo immunodetection of proteins.

Inhibition of vascular endothelial growth factor (VEGF) has become the standard of care for patients presenting with wet age-related macular degeneration. However, monthly intravitreal injections are required for optimal efficacy. We have previously shown that electroporation enabled ciliary muscle gene transfer results in sustained protein secretion into the vitreous for up to 9 months. Here, we evaluated the long-term efficacy of ciliary muscle gene transfer of three soluble VEGF receptor-1 (sFlt-1) variants in a rat model of laser-induced choroidal neovascularization (CNV). All three sFlt-1 variants significantly diminished vascular leakage and neovascularization as measured by fluorescein angiography (FA) and flatmount choroid at 3 weeks. FA and infracyanine angiography demonstrated that inhibition of CNV was maintained for up to 6 months after gene transfer of the two shortest sFlt-1 variants. Throughout, clinical efficacy was correlated with sustained VEGF neutralization in the ocular media. Interestingly, treatment with sFlt-1 induced a 50% downregulation of VEGF messenger RNA levels in the retinal pigment epithelium and the choroid. We demonstrate for the first time that non-viral gene transfer can achieve a long-term reduction of VEGF levels and efficacy in the treatment of CNV.

Objective Glucocorticoid-induced leucine zipper (GILZ) has effects on inflammatory pathways that suggest it to be a key inhibitory regulator of the immune system, and its expression is exquisitely sensitive to induction by glucocorticoids. We undertook this study to test our hypothesis that GILZ deficiency would exacerbate experimental immune-mediated inflammation and impair the effects of glucocorticoids on inflammation and, correspondingly, that exogenous GILZ would inhibit these events. Methods GILZ−/− mice were generated using the Cre/loxP system, and responses were studied in delayed-type hypersensitivity (DTH), antigen-induced arthritis (AIA), K/BxN serum–transfer arthritis, and lipopolysaccharide (LPS)–induced cytokinemia. Therapeutic expression of GILZ via administration of recombinant adeno-associated virus expressing the GILZ gene (GILZ-rAAV) was compared to the effects of glucocorticoid in collagen-induced arthritis (CIA). Results Increased T cell proliferation and DTH were observed in GILZ−/− mice, but neither AIA nor K/BxN serum–transfer arthritis was affected, and GILZ deficiency did not affect LPS-induced cytokinemia. Deletion of GILZ did not impair the effects of exogenous glucocorticoids on CIA or cytokinemia. In contrast, overexpression of GILZ in joints significantly inhibited CIA, with an effect similar to that of dexamethasone. Conclusion Despite effects on T cell activation, GILZ deficiency had no effect on effector pathways of arthritis and was unexpectedly redundant with effects of glucocorticoids. These findings do not support the hypothesis that GILZ is central to the actions of glucocorticoids, but the efficacy of exogenous GILZ in CIA suggests that further evaluation of GILZ in inflammatory disease is required.

Since the discovery of RNA interference by Fire et al. (1998) (Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C., 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811), small-interfering RNA, or siRNA, has become an indisputable tool for specifically silencing a target gene. The main obstacle for the development of siRNA as an efficient drug is to deliver it in whole organism to target tissues and across the cell membrane to the cytoplasm, where the RNAi machinery is localized, to trigger the sequence-specific mRNA degradation. In the absence of transfection reagents or physical treatment that may damage the cellular membrane, most cells do not spontaneously take up siRNAs. Several delivery systems capable of increasing the stability of siRNA in biological medium and cellular uptake have been reported so far. In this review, we will introduce the biological barriers to siRNA delivery in vivo and discuss recent advances in material sciences, nanotechnology, and nucleic acid chemistry that have yielded promising nonviral delivery systems, some of which are currently undergoing testing in clinical trials. The diversity of these systems highlights the recent progress of siRNA-based therapy using nonviral approaches.

International audience; Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK1R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK1R in endosomes with membrane-anchored drug conjugates providesmore effective and sustained pain relief than conventional plasmamembrane-targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and b-arrestin blocked SP-induced NK1R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK1R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK1R antagonists. These results reveal a critical role for endosomal signaling of the NK1R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.

The promise of using nucleic acid as a drug has opened new therapeutics strategy for a large variety of diseases. Several types of therapeutic nucleic acid exist, ranging from short synthetic RNA- or DNA-based oligonucleotides to plasmid-based DNA-expressing constructs. According to their functional activity, they can either induce the expression of a protein (gene therapy strategy) or trigger the silencing of a gene (RNA interference or antisense strategy). Current nucleic acid–based treatment strategies are largely injection-based, but effective oral delivery platforms for nucleic acids may result in improved patient comfort and compliance. In this chapter, we propose to discuss the barriers to oral delivery of nucleic acids as well as review the various technologies already tested in preclinical study and the experimental models available to assay oral delivery systems.

International audience; In the RNA interference process, the catalytic degradation of an endogenous mRNA results from the Watson-Crick complementary recognition by either a small silencing synthetic double-stranded ribonucleotide (siRNA) or by a small hairpin RNA (shRNA) produced in the cell by transcription from a DNA template. This interference process ideally results in an exquisitely specific mRNA suppression. The present review is dedicated to siRNAs. It describes the mechanism of RNA silencing and the main siRNA delivery techniques, with a focus on siRNA self-complexing to cationic lipids to form nanoparticles, which are called lipoplexes. The addition to lipoplexes of an anionic polymer leads to the ternary formulation APIRL (Anionic-Polymer-Interfering-RNA-Lipoplexes) with increased in vivo stability and biological efficacy. In terms of clinical development, the review focuses on therapeutic applications by intravenous delivery to the liver and inflammatory joints, and to localized siRNA delivery to the ocular sphere.

Lipopolyamines (LPAs) are cationic lipids; they interact spontaneously with nucleic acids to form lipoplexes used for gene delivery. The main hurdle to using lipoplexes in gene therapy lies in their immunostimulatory properties, so far attributed to the nucleic acid cargo, while cationic lipids were considered as inert to the immune system. Here we demonstrate for the first time that di-C18 LPAs trigger pro-inflammatory responses through Toll-like receptor 2 (TLR2) activation, and this whether they are bound to nucleic acids or not. Molecular docking experiments suggest potential TLR2 binding modes reminiscent of bacterial lipopeptide sensing. The di-C18 LPAs share the ability of burying their lipid chains in the hydrophobic cavity of TLR2 and, in some cases, TLR1, at the vicinity of the dimerization interface; the cationic headgroups form multiple hydrogen bonds, thus crosslinking TLRs into functional complexes. Unravelling the molecular basis of TLR1 and TLR6-driven heterodimerization upon LPA binding underlines the highly collaborative and promiscuous ligand binding mechanism. The prevalence of non-specific main chain-mediated interactions demonstrates that potentially any saturated LPA currently used or proposed as transfection agent is likely to activate TLR2 during transfection. Hence our study emphasizes the urgent need to test the inflammatory properties of transfection agents and proposes the use of docking analysis as a preliminary screening tool for the synthesis of new non-immunostimulatory nanocarriers., Graphical abstract Image 2

RNA interference is an invaluable tool in biology to specifically silence a given gene. Synthetic duplexes of RNA oligonucleotides are widely used to induce mRNA degradation in cultured cells or in whole organisms. They have to be vectorized to reach their target site. Here, we describe the preparation of highly efficient siRNA vectors based on cationic liposomes and polyanionic polymers and their application in cultured cells to silence reporter and/or endogenous genes.

The development of osteosarcoma, the most common malignant primary bone tumor is characterized by a vicious cycle established between tumor proliferation and paratumor osteolysis. This osteolysis is mainly regulated by the receptor activator of nuclear factor κB ligand (RANKL). Preclinical studies have demonstrated that Rankl blockade by soluble receptors is an effective strategy to prevent osteolytic lesions leading to osteosarcoma inhibition. A new therapeutic option could be to directly inhibit Rankl expression by small interfering RNAs (Rkl-siRNAs) and combine these molecules with chemotherapy to counteract the osteosarcoma development more efficiently. An efficient siRNA sequence directed against both mouse and rat mRNAs coding Rankl was first validated in vitro and tested in two models of osteosarcoma: a syngenic osteolytic POS-1 model induced in immunocompetent mice and a xenograft osteocondensant model of rat OSRGA in athymic mice. Intratumor injections of Rankl-directed siRNAs in combination with the cationic liposome RPR209120/DOPE reduced the local and systemic Rankl production and protected bone from paratumor osteolysis. Although Rkl-siRNAs alone had no effect on tumor development in both osteosarcoma models, it significantly blocked tumor progression when combined with ifosfamide compared with chemotherapy alone. Our results indicate that siRNAs could be delivered using cationic liposomes and thereby could inhibit Rankl production in a specific manner in osteosarcoma models. Moreover, the Rankl inhibition mediated by RNA interference strategy improves the therapeutic response of primary osteosarcoma to chemotherapy.

Energetic failure which occurs in both ischemia/reperfusion and acute drug-induced hepatotoxicity is frequently associated with oxidative stress. This study displays the setting of a new cell culture model for hepatic energetic failure, i.e., HepG2 models modified by etomoxir [ETO] addition [0.1 mM to 1 mM] and compares the cell impact versus tert-butylhydroperoxide [TBOOH; 0.2 mM], an oxidative stress inducer. As it was observed with Minimum Essential Medium (MEM) without any interfering agent, decreasing temperature drastically lowered adenosine triphosphate (ATP) levels, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) viability test, and protein content, compared to 37 °C (p=0.02, p

Item does not contain fulltext OBJECTIVE: Several lines of evidence implicate cytosolic phospholipase A(2)alpha (cPLA(2)alpha) as a critical enzyme in inflammatory disorders, including rheumatoid arthritis. Since cells from the myeloid compartment regulate local and systemic disease pathogenesis, the present study was undertaken to examine the effect of cPLA(2)alpha inhibition in experimental arthritis, using a delivery system tailored to target monocyte functions by RNA interference (RNAi). METHODS: Mice with collagen-induced arthritis (CIA) were injected intravenously with an anti-cPLA(2)alpha small interfering RNA (siRNA) sequence (siPLA2) formulated as lipoplexes with the RPR209120/DOPE cationic liposome and a carrier DNA. The clinical course of joint inflammation was assessed, and the immunologic balance was analyzed by measuring T helper cell frequencies and cytokine expression. Biodistribution studies of siRNA were also performed. RESULTS: Weekly systemic injection of siPLA2 lipoplexes significantly reduced the incidence and severity of CIA, in both preventive and curative settings, as compared with findings in control animals. Histologic scores for inflammation and cartilage damage were reduced. The clinical effect was associated with local inhibition of tumor necrosis factor alpha secretion and lower cPLA(2)alpha expression and activity. The siPLA2 lipoplexes enabled triggering of in vivo RNAi-mediated gene silencing of cPLA(2)alpha in CD11b+ cells recovered from the spleen. While the treatment had no effect on anti-type II collagen (anti-CII) antibodies, CII-specific T helper cells producing interferon-gamma, but not interleukin-17, in draining lymph node cells were decreased. CONCLUSION: Our findings indicate that systemic RNAi-mediated cPLA(2)alpha gene silencing in CD11b+ cells is effective in the treatment of CIA, and Th1 suppression is one of the potential underlying mechanisms, whereas Th17 suppression is not.

Cells from the mononuclear phagocyte system (MPS) act as systemic and local amplifiers that contribute to the progression of chronic inflammatory disorders. Transforming growth factor-β–activated kinase 1 (TAK1) is a pivotal upstream mitogen-activated protein kinase-kinase-kinase acting as a mediator of cytokine expression. It remains critical to determine in vivo the implication of TAK1 in controlling the innate immune system. Here, we describe a vehicle tailored to selectively deliver siRNAs into MPS cells after intravenous administration, and validate in vivo the potential of the RNAi-mediated TAK1 knock down for immunomodulation. In a mouse model of immune-mediated inflammatory disorder, we show that anti-TAK1 siRNA lipoplexes efficiently alleviate inflammation, severely impair the downstream c-Jun N-terminal kinase and nuclear factor-κB signaling pathways, and decrease the expression of proinflammatory mediators. Importantly, the systemic TAK1 gene silencing decreases the frequency of Th1 and Th17 cells, both mediating autoimmunity in experimental arthritis, demonstrating the immunomodulatory potential of TAK1. Finally, in vitro inhibition of TAK1 in myeloid cells decreases interferon-γ–producing T cells, suggesting that a delivery sys-tem able to target MPS cells and to silence TAK1 impacts on pathogenic T effector cells in autoimmunity.

Objective Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid-induced protein, the reported molecular interactions of which suggest that it functions to inhibit inflammation. However, the role of endogenous GILZ in the regulation of inflammation in vivo has not been established. This study was undertaken to examine the expression and function of GILZ in vivo in collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis (RA), and in RA synoviocytes. Methods GILZ expression was detected in mouse and human synovium by immunohistochemistry and in cultured cells by real-time polymerase chain reaction and permeabilization flow cytometry. GILZ function was assessed in vivo by small interfering RNA (siRNA) silencing using cationic liposome–encapsulated GILZ or control nontargeting siRNA and was assessed in vitro using transient overexpression. Results GILZ was readily detectable in the synovium of mice with CIA and was up-regulated by therapeutic doses of glucocorticoids. Depleting GILZ expression in vivo increased the clinical and histologic severity of CIA and increased synovial expression of tumor necrosis factor and interleukin-1 (IL-1), without affecting the levels of circulating cytokines or anticollagen antibodies. GILZ was highly expressed in the synovium of patients with active RA and in cultured RA synovial fibroblasts, and GILZ overexpression in synovial fibroblasts inhibited IL-6 and IL-8 release. Conclusion Our findings indicate that GILZ functions as an endogenous inhibitor of chronic inflammation via effects on cytokine expression and suggest that local modulation of GILZ expression could be a beneficial therapeutic strategy.

An original ligand (Lac-10-Chol) designed to interact with asialoglycoprotein receptors to potentially target hepatocyte was synthesised by grafting a lactose head to a cholesteryl structure, which was then included in liposomes. Preliminary formulation tests led to the selection of conventional formulations based on soybean phosphatidylcholine/cholesterol/DOTAP (+/- DOPE) (+/- Lac-10-Chol) that present reproducible absolute entrapment value (1.45 +/- 0.10%), with a size of 109 +/- 7 nm and a slight positive charge (3.77 +/- 1.59 mV). Cell viability (via the MTT test), expressed as the percentage of nontreated cells in HepG2 cells, was very close to the control. Internalization tests evidenced an intracellular penetration of fluorescent liposomes, but no specific ligand effect was demonstrated (P0.05). Nevertheless, regarding the adenosine triphosphate (ATP) assay, a slight increase was obtained with liposome loaded with ATP incorporating Lac-10-chol after 24 hours (P0.05).

This study aimed at restricting expression of DNA vaccine to specific cell types by using skin-specific promoters (i) to contribute to the understanding of the mechanism of intradermal DNA vaccines and (ii) to address safety concerns associated with DNA vaccines. The expression and immune response after delivery of plasmids encoding luciferase by intradermal DNA electroporation were assessed. Two ubiquitous promoters, CMV and CAG, and three tissue-specific promoters were studied. Keratin 14 promoter restricts gene expression to keratinocytes of the epidermal basal layer, CD11c promoter to dendritic cells and fascin promoter only to mature dendritic cells. The use of plasmids with tissue-specific promoter resulted in significant, but very low protein expression, as compared to that obtained with ubiquitous promoter plasmids. Immunisation with both ubiquitous promoter plasmids elicited humoral and cellular anti-luciferase immune response. No immune response was observed after delivery of CD11c plasmid while fascin and keratin 14 plasmids induced IFN-gamma response suggesting that the targeting of skin-specific cells could be a suitable approach but only for the treatment of pathologies or pathogens requiring mainly cellular and not humoral immune response.

Background We recently reported an efficient formulation of siRNA targeting TNF-α, that was able to restore immunological balance in a mouse arthritis model following intravenous injection. Method Since this efficient formulation included the pre association of siRNA with a DNA cargo, we decided to extensively characterise siRNA lipoplexes with or without DNA cargo, in order to better understand the DNA cargo enhancing effect. Results We showed that addition of DNA cargo to siRNA lipoplexes led to specific gene extinction in vitro , using reduced siRNA concentration. This procedure is also applicable to other lipid vectors, like Lipofectamine or DMRIE-C. No structural modification could be observed in siRNA lipoplexes upon addition of DNA cargo using dynamic light scattering or transmission electronic microscopy. Nevertheless, we observed some slight differences, in the amount of lipid required to obtain neutrality of the complex and in stability of the complex towards incubation with heparan sulfate. Conclusions These results suggest that the addition of DNA cargo to siRNA complexes is an easy procedure that leads to more efficient complexes to transfer siRNA at low concentration and in the presence of serum.

RNA interference is an invaluable tool in biology to specifically silence a given gene. Synthetic duplexes of RNA oligonucleotides are widely used to induce mRNA degradation in cultured cells or in whole organisms. They have to be vectorized to reach their target site. Here, we describe the preparation of highly efficient siRNA vectors based on cationic liposomes and polyanionic polymers and their application in cultured cells to silence reporter and/or endogenous genes.

International audience; Even though RNA interference is a highly specific technique to selectively silence the expression of any gene, the delivery of RNAi molecules remains a challenge. Recently, we developed a clinically acceptable efficient formulation of siRNA. This delivery system consisted of the cationic lipid 2-{3-[Bis-(3-amino-propyl)-amino]-propylamino}-N-ditetradecyl carbamoyl methyl-acetamide, or DMAPAP, (ii) the neutral lipid 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine or DOPE and (iii) an anionic polymer that enhances lipoplexes efficiency. We show here that this enhancement of efficiency is due to higher stability of polymer containing-siRNA lipoplexes, leading to longer retention of siRNA integrity. We assayed siRNA integrity upon incubation of siRNA lipoplexes in various biological media using electrophoresis and Fluorescence Correlation Spectroscopy. We show that the addition of anionic polymer provided enhanced stability of incorporated siRNA mainly in saline buffer at room temperature and in human serum at 37 degrees C and did not interfere with the release of siRNA from lipoplexes in modeled cellular media. (C) 2015 Elsevier B.V. All rights reserved.

International audience; Chronic infection with hepatitis B virus (HBV) occurs commonly and complications that arise from persistence of the virus are associated with high mortality. Available licensed drugs have modest curative efficacy and advancing new therapeutic strategies to eliminate the virus is therefore a priority. HBV is susceptible to inactivation by exogenous gene silencers that harness RNA interference (RNAi) and the approach has therapeutic potential. To advance RNAi-based treatment for HBV infection, use in vivo of hepatotropic lipoplexes containing siRNAs with guanidinopropyl (GP) modifications is reported here. Lipoplexes contained polyglutamate, which has previously been shown to facilitate formulation and improve efficiency of the non-viral vectors. GP moieties were included in a previously described anti-HBV siRNA that effectively targeted the conserved viral X sequence. Particles had physical properties that were suitable for use in vivo: average diameter was approximately 50-200 nm and surface charge (zeta potential) was + 65 mV. Efficient hepatotropic delivery of labeled siRNA was observed following systemic intravenous injection of the particles into HBV transgenic mice. Good inhibition of markers of viral replication was observed without evidence of toxicity. Efficacy of the GP-modified siRNAs was significantly more durable and formulations made up with chemically modified siRNAs were less immunostimulatory. An RNAi-mediated mechanism was confirmed by demonstrating that HBV mRNA cleavage occurred in vivo at the intended target site. Collectively these data indicate that GP-modified siRNAs formulated in anionic polymer-containing lipoplexes are effective silencers of HBV replication in vivo and have therapeutic potential. (C) 2015 Elsevier B.V. All rights reserved.

We report here a method that allows fast, efficient, and low-cost screening for gene function in the vascular system of the vertebrate embryo. Through intracardiac delivery of nucleic acids optimally compacted by a specific cationic lipid, we are able to induce in vivo endothelial cell-specific gain-of-function during development of the vascular network in the chick embryo. When the nucleic acids are delivered during the period of intraembryonic hematopoiesis, aortic hemangioblasts, the forerunners of the hematopoietic stem cells known to derive from the aortic endothelium, are also labeled. Similarly, we show that siRNA could be used to induce loss-of-function in vascular endothelial cells. This gene transfer technique was also applied to the mouse embryo with a high efficiency. The present method allows large-scale analysis and may represent a new and versatile tool for functional genomics.

(-)-Quinic acid was used as a starting material for the preparation of sialyl Lewis(x) mimetics in order to target E-selectin. Spatial orientation of the hydroxyl groups of quinic acid could mimic the l-fucose ones. Introduction of a side chain ending with a carboxylic acid was effected to replace the sialic acid interaction at the carbohydrate recognition domain. A first series of derivatives, incorporating amino acids linked to quinic acid, were tested for their affinity and found to interact with E-selectin with IC(50) within the millimolar range.