RESUMEN
Mutations in the TECPR2 gene are the cause of an ultra-rare neurological disorder characterized by intellectual disability, impaired speech, motor delay, and hypotonia evolving to spasticity, central sleep apnea, and premature death (SPG49 or HSAN9; OMIM: 615031). Little is known about the biological function of TECPR2, and there are currently no available disease-modifying therapies for this disease. Here we describe implementation of an antisense oligonucleotide (ASO) exon-skipping strategy targeting TECPR2 c.1319delT (p.Leu440Argfs∗19), a pathogenic variant that results in a premature stop codon within TECPR2 exon 8. We used patient-derived fibroblasts and induced pluripotent stem cell (iPSC)-derived neurons homozygous for the p.Leu440Argfs∗19 mutation to model the disease in vitro. Both patient-derived fibroblasts and neurons showed lack of TECPR2 protein expression. We designed and screened ASOs targeting sequences across the TECPR2 exon 8 region to identify molecules that induce exon 8 skipping and thereby remove the premature stop signal. TECPR2 exon 8 skipping restored in-frame expression of a TECPR2 protein variant (TECPR2ΔEx8) containing 1,300 of 1,411 amino acids. Optimization of ASO sequences generated a lead candidate (ASO-005-02) with â¼27 nM potency in patient-derived fibroblasts. To examine potential functional rescue induced by ASO-005-02, we used iPSC-derived neurons to analyze the neuronal localization of TECPR2ΔEx8 and showed that this form of TECPR2 retains the distinct, punctate neuronal expression pattern of full-length TECPR2. Finally, ASO-005-02 had an acceptable tolerability profile in vivo following a single 20-mg intrathecal dose in cynomolgus monkeys, showing some transient non-adverse behavioral effects with no correlating histopathology. Broad distribution of ASO-005-02 and induction of TECPR2 exon 8 skipping was detected in multiple central nervous system (CNS) tissues, supporting the potential utility of this therapeutic strategy for a subset of patients suffering from this rare disease.
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This introduction charts the history of the development of the major chemical modifications that have influenced the development of nucleic acids therapeutics focusing in particular on antisense oligonucleotide analogues carrying modifications in the backbone and sugar. Brief mention is made of siRNA development and other applications that have by and large utilized the same modifications. We also point out the pitfalls of the use of nucleic acids as drugs, such as their unwanted interactions with pattern recognition receptors, which can be mitigated by chemical modification or used as immunotherapeutic agents.
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Ácidos Nucleicos , Ácidos Nucleicos/genética , Ácidos Nucleicos/uso terapéutico , Oligonucleótidos/genética , Oligonucleótidos/uso terapéutico , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéuticoRESUMEN
Mutations in KCNC3, the gene that encodes the Kv3.3 voltage dependent potassium channel, cause Spinocerebellar Ataxia type 13 (SCA13), a disease associated with disrupted motor behaviors, progressive cerebellar degeneration, and abnormal auditory processing. The Kv3.3 channel directly binds Hax-1, a cell survival protein. A disease-causing mutation, Kv3.3-G592R, causes overstimulation of Tank Binding Kinase 1 (Tbk1) in the cerebellum, resulting in the degradation of Hax-1 by promoting its trafficking into multivesicular bodies and then to lysosomes. We have now tested the effects of antisense oligonucleotides (ASOs) directed against the Kv3.3 channel on both wild type mice and those bearing the Kv3.3-G592R-encoding mutation. Intracerebroventricular infusion of the Kcnc3-specific ASO suppressed both mRNA and protein levels of the Kv3.3 channel. In wild-type animals, this produced no change in levels of activated Tbk1, Hax-1 or Cd63, a tetraspanin marker for late endosomes/multivesicular bodies. In contrast, in mice homozygous for the Kv3.3-G592R-encoding mutation, the same ASO reduced Tbk1 activation and levels of Cd63, while restoring the expression of Hax-1 in the cerebellum. The motor behavior of the mice was tested using a rotarod assay. Surprisingly, the active ASO had no effects on the motor behavior of wild type mice but restored the behavior of the mutant mice to those of age-matched wild type animals. Our findings indicate that, in mature intact animals, suppression of Kv3.3 expression can reverse the deleterious effects of a SCA13 mutation while having little effect on wild type animals. Thus, targeting Kv3.3 expression may prove a viable therapeutic approach for SCA13.
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Trastornos Motores/prevención & control , Mutación , Oligonucleótidos Antisentido/administración & dosificación , Proteínas Serina-Treonina Quinasas/metabolismo , Canales de Potasio Shaw/antagonistas & inhibidores , Ataxias Espinocerebelosas/complicaciones , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trastornos Motores/etiología , Trastornos Motores/metabolismo , Trastornos Motores/patología , Proteínas Serina-Treonina Quinasas/genética , Canales de Potasio Shaw/genética , Canales de Potasio Shaw/metabolismoRESUMEN
Over the last four decades, tremendous progress has been made in use of synthetic oligonucleotides as therapeutics. This has been possible largely by introducing chemical modifications to provide drug like properties to oligonucleotides. In this article I have summarized twists and turns on use of chemical modifications and their road to success and highlight areas of future directions.
RESUMEN
Many patients with advanced melanoma are resistant to immune checkpoint inhibition. In the ILLUMINATE-204 phase I/II trial, we assessed intratumoral tilsotolimod, an investigational Toll-like receptor 9 agonist, with systemic ipilimumab in patients with anti-PD-1- resistant advanced melanoma. In all patients, 48.4% experienced grade 3/4 treatment-emergent adverse events. The overall response rate at the recommended phase II dose of 8 mg was 22.4%, and an additional 49% of patients had stable disease. Responses in noninjected lesions and in patients expected to be resistant to ipilimumab monotherapy were observed. Rapid induction of a local IFNα gene signature, dendritic cell maturation and enhanced markers of antigen presentation, and T-cell clonal expansion correlated with clinical response. A phase III clinical trial with this combination (NCT03445533) is ongoing. SIGNIFICANCE: Despite recent developments in advanced melanoma therapies, most patients do not experience durable responses. Intratumoral tilsotolimod injection elicits a rapid, local type 1 IFN response and, in combination with ipilimumab, activates T cells to promote clinical activity, including in distant lesions and patients not expected to respond to ipilimumab alone.This article is highlighted in the In This Issue feature, p. 1861.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico , Ipilimumab , Melanoma , Neoplasias Cutáneas , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Protocolos de Quimioterapia Combinada Antineoplásica , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ipilimumab/administración & dosificación , Ipilimumab/uso terapéutico , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Resultado del Tratamiento , Estados UnidosRESUMEN
RNA therapeutics are finally taking their place as a main drug category alongside small molecules and proteins. Here, we follow the twists and turns on their road to success and highlight areas of ongoing research.
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Terapia Genética , ARN/genética , ARN/uso terapéutico , Desarrollo de Medicamentos , Terapia Genética/métodos , Humanos , MicroARNs , Oligonucleótidos , ARN sin Sentido , ARN Interferente PequeñoRESUMEN
Recently, it has become clear that the tumour microenvironment (TME) is important in cancer immunotherapy. While immune checkpoint inhibitors are effective for some patients, the heterogeneous nature and status of the TME ('cold' tumours) play a critical role in suppressing antitumour immunity in non-responding patients. Converting 'cold' to 'hot' tumours through modulation of the TME may enable expansion of the therapeutic efficacy of immunotherapy to a broader patient population. This paper describes advances in intratumoural immunotherapy, specifically activation of nucleic acid sensing pattern recognition receptors to modulate the TME.
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The objective of cancer immunotherapy is to prime the host's immune system to recognize and attack malignant tumor cells. IMO2125, a Tolllike receptor 9 (TLR9) agonist, exhibited potent antitumor effects in the murine syngeneic A20 lymphoma and the CT26 colon carcinoma models. IMO2125 exhibited superior A20 antitumor activity when injected intratumorally (i.t.) compared with equivalent subcutaneous doses. In mice bearing dual CT26 grafts, the i.t. injection of right flank tumors elicited infiltration of cluster of differentiation (CD)3+ T lymphocytes into tumors, resulting in the regression of injected and uninjected left flank tumors. Depletion of CD8+, but not CD4+, Tcells abrogated the IMO2125mediated antitumor response, suggesting that CD8+ lymphocytes are required for the antitumor activity. In mice harboring right flank CT26 and left flank ßgalactosidase (ßgal)expressing CT26.CL25 grafts, the i.t. administration of IMO2125 to the CT26 graft resulted in potent and dosedependent antitumor activity against the two grafts. Splenic Tcells isolated from these mice responded to AH1 antigen (present in the two tumors) and ßgal antigen (present only in CT26.CL25) in an interferon γ enzymelinked immunospot assay, suggesting the clonal expansion of Tcells directed against antigens from the two tumors. Mice with ablated CT26 tumors by previous IMO2125 treatment rejected reimplanted CT26 tumor cells, but not A20 tumor cells, demonstrating that the initial IMO2125 treatment created a longlived tumorspecific immune memory of CT26 antigens. A quantitative increase in CD3+ T lymphocytes in injected A20 tumors and an upregulation of selected checkpoint genes, including indoleamine 2,3dioxygenase (IDO)1, IDO2, programmed cell death protein-1 (PD-1); programmed cell death protein ligand 1 (PD-L1), carcinoembryonic antigenrelated cell adhesion molecule 1, tumor necrosis factor receptor superfamily member 4 (OX40), OX40 ligand, Tcell immunoglobulin and mucindomaincontaining 3 protein, lymphocyteactivation gene 3, cytotoxic Tlymphocyteassociated protein 4, were observed following IMO2125 treatment. IMO2125 also increased immune checkpoint gene expression in injected and uninjected contralateral CT26 tumors, suggesting that the coadministration of antiCTLA4, antiPD1 or antiPDL1 therapies with IMO2125 may provide additional therapeutic efficacy.
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Antineoplásicos Inmunológicos/farmacología , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Oligodesoxirribonucleótidos/farmacología , Oligonucleótidos Fosforotioatos/farmacología , Receptor Toll-Like 9/agonistas , Microambiente Tumoral/efectos de los fármacos , Animales , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Receptores Coestimuladores e Inhibidores de Linfocitos T/inmunología , Receptores Coestimuladores e Inhibidores de Linfocitos T/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Inyecciones Intralesiones , Ratones , Ratones Endogámicos BALB C , Neoplasias/inmunología , Neoplasias/patología , Oligodesoxirribonucleótidos/uso terapéutico , Oligonucleótidos Fosforotioatos/uso terapéutico , Células TH1/inmunología , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Resultado del Tratamiento , Microambiente Tumoral/inmunología , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Improving the efficacy of neovascularization is a promising strategy to restore perfusion of ischemic tissues in patients with peripheral arterial disease. The 14q32 microRNA cluster is highly involved in neovascularization. The Mef2a transcription factor has been shown to induce transcription of the microRNAs within this cluster. We inhibited expression of Mef2a using gene-silencing oligonucleotides (GSOs) in an in vivo hind limb ischemia model. Treatment with GSO-Mef2a clearly improved blood flow recovery within 3 days (44% recovery versus 25% recovery in control) and persisted until 14 days after ischemia induction (80% recovery versus 60% recovery in control). Animals treated with GSO-Mef2a showed increased arteriogenesis and angiogenesis in the relevant muscle tissues. Inhibition of Mef2a decreased expression of 14q32 microRNAs miR-329 (p = 0.026) and miR-494 (trend, p = 0.06), but not of other 14q32 microRNAs, nor of 14q32 microRNA precursors. Because Mef2a did not influence 14q32 microRNA transcription, we hypothesized it functions as an RNA-binding protein that influences processing of 14q32 microRNA miR-329 and miR-494. Mef2A immunoprecipitation followed by RNA isolation and rt/qPCR confirmed direct binding of MEF2A to pri-miR-494, supporting this hypothesis. Our study demonstrates a novel function for Mef2a in post-ischemic neovascularization via post-transcriptional regulation of 14q32 microRNAs miR-329 and miR-494.
RESUMEN
BACKGROUND AND AIMS: We aimed at investigating the role of 14q32 microRNAs in intimal hyperplasia and accelerated atherosclerosis; two major contributors to restenosis. Restenosis occurs regularly in patients treated for coronary artery disease and peripheral arterial disease. We have previously shown that inhibition of 14q32 microRNAs leads to increased post-ischemic neovascularization, and microRNA miR-494 also decreased atherosclerosis, while increasing plaque stability. We hypothesized that 14q32 microRNA inhibition has beneficial effects on intimal hyperplasia, as well as accelerated atherosclerosis. METHODS: Non-constrictive cuffs were placed around both femoral arteries of C57BL/6J mice to induce intimal hyperplasia. Accelerated atherosclerotic plaque formation was induced in hypercholesterolemic ApoE-/- mice by placing semi-constrictive collars around both carotid arteries. 14q32 microRNAs miR-329, miR-494 and miR-495 were inhibited in vivo using Gene Silencing Oligonucleotides (GSOs). RESULTS: GSO-495 administration led to a 32% reduction of intimal hyperplasia. Moreover, the number of macrophages in the arterial wall of mice treated with GSO-495 was reduced by 55%. Inhibition of miR-329 and miR-494 had less profound effects on intimal hyperplasia. GSO-495 administration also decreased atherosclerotic plaque formation by 52% and plaques of GSO-495 treated animals showed a more stable phenotype. Finally, cholesterol levels were also decreased in GSO-495 treated animals, via reduction of the VLDL-fraction. CONCLUSIONS: GSO-495 administration decreased our primary outcomes, namely intimal hyperplasia, and accelerated atherosclerosis. GSO-495 administration also favourably affected multiple secondary outcomes, including macrophage influx, plaque stability and total plasma cholesterol levels. We conclude that 14q32 microRNA miR-495 is a promising target for prevention of restenosis.
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Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/prevención & control , Colesterol/sangre , Arteria Femoral/patología , Silenciador del Gen , Hipercolesterolemia/prevención & control , MicroARNs/genética , Neointima , Enfermedad Arterial Periférica/prevención & control , Placa Aterosclerótica , Animales , Biomarcadores/sangre , Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/sangre , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/patología , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Arteria Femoral/metabolismo , Hipercolesterolemia/sangre , Hipercolesterolemia/genética , Hiperplasia , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , MicroARNs/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Enfermedad Arterial Periférica/sangre , Enfermedad Arterial Periférica/genética , Enfermedad Arterial Periférica/patología , Recurrencia , Remodelación VascularRESUMEN
Nucleic acid recognition is an important mechanism that enables the innate immune system to detect microbial infection and tissue damage. To minimize the recognition of self-derived nucleic acids, all nucleic acid-sensing signaling receptors are sequestered away from the cell surface and are activated in the cytoplasm or in endosomes. Nucleic acid sensing in endosomes relies on members of the TLR family. The receptor for advanced glycation end-products (RAGE) was recently shown to bind DNA at the cell surface, facilitating DNA internalization and subsequent recognition by TLR9. In this article, we show that RAGE binds RNA molecules in a sequence-independent manner and enhances cellular RNA uptake into endosomes. Gain- and loss-of-function studies demonstrate that RAGE increases the sensitivity of all ssRNA-sensing TLRs (TLR7, TLR8, TLR13), suggesting that RAGE is an integral part of the endosomal nucleic acid-sensing system.
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Endosomas/metabolismo , ARN/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Transducción de Señal , Receptores Toll-Like/inmunología , ADN/genética , ADN/metabolismo , Células HEK293 , Humanos , Inmunidad Innata , Reacción en Cadena de la Polimerasa , ARN/genética , Receptor para Productos Finales de Glicación Avanzada/genética , Receptor para Productos Finales de Glicación Avanzada/inmunología , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/inmunología , Receptor Toll-Like 8/metabolismoRESUMEN
Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 homolog B1 (UNC93B1) regulates the trafficking of nucleic acid-sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid-sensing TLRs in a receptor- and ligand-specific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)1- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1.
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Complejo 1 de Proteína Adaptadora/inmunología , Complejo 2 de Proteína Adaptadora/inmunología , Linfocitos B/inmunología , Proteínas de Transporte de Membrana/inmunología , Monocitos/inmunología , Receptores Toll-Like/inmunología , Complejo 1 de Proteína Adaptadora/genética , Complejo 2 de Proteína Adaptadora/genética , Secuencias de Aminoácidos , Linfocitos B/citología , Línea Celular Tumoral , Células HEK293 , Humanos , Proteínas de Transporte de Membrana/genética , Monocitos/citología , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Receptores Toll-Like/genéticaRESUMEN
Although the cause of Duchenne muscular dystrophy (DMD) is known, the specific factors that initiate and perpetuate disease progression are not well understood. We hypothesized that leaky dystrophin-deficient skeletal muscle releases endogenous danger signals (TLR ligands), which bind to Toll-like receptors (TLRs) on muscle and immune cells and activate downstream processes that facilitate degeneration and regeneration in dystrophic skeletal muscle. Here, we demonstrate that dystrophin-deficient mouse muscle cells show increased expression of several cell-surface and endosomal TLRs. In vitro screening identified ssRNA as a relevant endogenous TLR7 ligand. TLR7 activation led to myd88-dependent production of pro-inflammatory cytokines in dystrophin-deficient muscle cells, and cause significant degeneration/regeneration in vivo in mdx mouse muscle. Also, knockout of the central TLR adaptor protein, myd88 in mdx mice significantly improved skeletal and cardiac muscle function. Likewise, proof-of-concept experiments showed that treating young mdx mice with a TLR7/9 antagonist significantly reduced skeletal muscle inflammation and increased muscle force, suggesting that blocking this pathway may have therapeutic potential for DMD.
Asunto(s)
Glicoproteínas de Membrana/fisiología , Músculo Esquelético/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Miocardio/metabolismo , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 9/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Distrofina/deficiencia , Femenino , Humanos , Masculino , Glicoproteínas de Membrana/agonistas , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratones Noqueados , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Mioblastos Esqueléticos/inmunología , Mioblastos Esqueléticos/metabolismo , Miocardio/patología , Fenotipo , Receptor Toll-Like 7/agonistasRESUMEN
Systemic Lupus Erythematosus is an autoimmune disease characterized by production of autoantibodies against nucleic acid-associated antigens. Endogenous DNA and RNA associated with these antigens stimulate inflammatory responses through Toll-like receptors (TLRs) and exacerbate lupus disease pathology. We have evaluated an antagonist of TLR7, 8 and 9 as a therapeutic agent in lupus-prone NZBW/F1 mice. NZBW/F1 mice treated with the antagonist had lower serum levels of autoantibodies targeting DNA, RNP, Smith antigen, SSA and SSB than did untreated mice. Reduction in blood urea nitrogen and proteinuria and improvements in kidney histopathology were observed in antagonist-treated mice. The antagonist treatment also reduced serum IL-12 and IL-1ß and increased IL-10 levels. Levels of mRNA for IL-6, iNOS and IL-1ß were lower in the kidneys and spleen of antagonist-treated mice than in those of untreated mice. Levels of mRNA for IP-10, TNFRSF9 and FASL were lower and IL-4 mRNA were higher in spleens of antagonist-treated mice than in spleens of untreated mice. mRNA for the inflammasome component NLRP3 was lower and mRNA for the antioxidant enzymes, catalase and glutathione peroxidase 1 was higher in the kidneys of antagonist-treated mice than in those of untreated mice. These results show that the antagonist of TLR7, 8 and 9 effectively inhibits inflammatory pathways involved in the development of lupus in NZBW/F1 mice and constitutes a potential therapeutic approach for the treatment of lupus and other autoimmune diseases.
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Desoxirribonucleótidos/administración & dosificación , Desoxirribonucleótidos/antagonistas & inhibidores , Regulación hacia Abajo/inmunología , Lupus Eritematoso Sistémico/patología , Lupus Eritematoso Sistémico/prevención & control , Glicoproteínas de Membrana/antagonistas & inhibidores , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 8/antagonistas & inhibidores , Receptor Toll-Like 9/antagonistas & inhibidores , Animales , Autoanticuerpos/biosíntesis , Autoanticuerpos/sangre , Desoxirribonucleótidos/farmacología , Femenino , Mediadores de Inflamación/antagonistas & inhibidores , Riñón/efectos de los fármacos , Riñón/inmunología , Riñón/patología , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Endogámicos NZBRESUMEN
Psoriasis is a chronic inflammatory skin disease that involves the induction of T-helper 1 (Th1) and T-helper 17 (Th17) cell responses and the aberrant expression of proinflammatory cytokines, including IL-1ß. Copious evidence suggests that abnormal activation of Toll-like receptors (TLRs) contributes to the initiation and maintenance of psoriasis. We have evaluated an antagonist of TLR7, 8, and 9 as a therapeutic agent in an IL-23-induced psoriasis model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the ear or dorsum. IL-23-induced increase in ear thickness was inhibited in a dose-dependent manner by treatment with antagonist. Histological examination of ear and dorsal skin tissues demonstrated a reduction in epidermal hyperplasia in mice treated with the antagonist. Treatment with antagonist also reduced the induction of Th1 and Th17 cytokines in skin and/or serum, as well as dermal expression of inflammasome components, NLRP3 and AIM2, and antimicrobial peptides. These results indicate that targeting TLR7, 8, and 9 may provide a way to neutralize multiple inflammatory pathways that are involved in the development of psoriasis. The antagonist has the potential for the treatment of psoriasis and other autoimmune diseases.
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Inflamasomas/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , Oligonucleótidos/farmacología , Psoriasis/patología , Células TH1/patología , Células Th17/patología , Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 8/antagonistas & inhibidores , Receptor Toll-Like 9/antagonistas & inhibidores , Animales , Proteínas Portadoras/metabolismo , Citocinas/metabolismo , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Inyecciones Intradérmicas , Interleucina-1beta/metabolismo , Interleucina-23/administración & dosificación , Interleucina-23/efectos adversos , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas Nucleares/metabolismo , Oligonucleótidos/uso terapéutico , Psoriasis/inducido químicamente , Psoriasis/metabolismoRESUMEN
Oligonucleotides containing an immune-stimulatory motif and an immune-regulatory motif act as antagonists of Toll-like receptor (TLR)7 and TLR9. In the present study, we designed and synthesized oligonucleotide-based antagonists of TLR7, 8 and 9 containing a 7-deaza-dG or arabino-G modification in the immune-stimulatory motif and 2'-O-methylribonucleotides as the immune-regulatory motif. We evaluated the biological properties of these novel synthetic oligoribonucleotides as antagonists of TLRs 7, 8 and 9 in murine and human cell-based assays and in vivo in mice and non-human primates. In HEK293, mouse and human cell-based assays, the antagonist compounds inhibited signaling pathways and production of a broad range of cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-12, IL-6, interferon (IFN)-α, IL-1ß and interferon gamma-induced protein (IP)-10, mediated by TLR7, 8 and 9. In vivo in mice, the antagonist compounds inhibited TLR7- and TLR9-mediated cytokine induction in a dose- and time-dependent fashion. Peripheral blood mononuclear cells (PBMCs) obtained from antagonist compound-treated monkeys secreted lower levels of TLR7-, 8- and 9-mediated cytokines than did PBMCs taken before antagonist administration. The antagonist compounds described herein provide novel agents for the potential treatment of autoimmune and inflammatory diseases.
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Receptor Toll-Like 7/antagonistas & inhibidores , Receptor Toll-Like 8/antagonistas & inhibidores , Receptor Toll-Like 9/antagonistas & inhibidores , Animales , Células Cultivadas , Citocinas/biosíntesis , Femenino , Células HEK293 , Humanos , Lupus Eritematoso Sistémico/inmunología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Oligorribonucleótidos/química , Oligorribonucleótidos/farmacología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/agonistas , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Double-stranded RNA of viral origin and enzymatically synthesized poly I:C act as agonists of TLR3 and induce immune responses. We have designed and synthesized double-stranded synthetic oligoribonucleotides (dsORNs) which act as agonists of TLR3. Each strand of dsORN contains two distinct segments, namely an alignment segment composed of a heteronucleotide sequence and an oligo inosine (I) or an oligo cytidine (C) segment. We report here the results of studies of dsORNs containing varying lengths and compositions of alignment and oligo I/oligo C segments. dsORNs of 50-mer length with a 15-mer alignment segment and a 35-mer oligo I/oligo C segment form stable duplexes under physiological conditions and induce TLR3-mediated immune responses. dsORNs activated the IRF3 signaling pathway in J774 cells, induced production of cytokines, including IFN-ß, IFN-α, IP-10, IL-12 and IL-6, in murine and human cell-based assays and also induced multiple cytokines following systemic administration in mice and non-human primates.
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Diseño de Fármacos , Oligorribonucleótidos/síntesis química , Oligorribonucleótidos/farmacología , Receptor Toll-Like 3/agonistas , Animales , Secuencia de Bases , Western Blotting , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oligorribonucleótidos/química , Alineación de SecuenciaRESUMEN
Oligodeoxynucleotides (ODNs) containing a CpG or certain synthetic dinucleotides, referred to as immune-stimulatory dinucleotides, induce Toll-like receptor 9 (TLR9)-mediated immune responses. Chemical modifications such as 2'-O-methylribonucleotides incorporated adjacent to the immune-stimulatory dinucleotide on the 5'-side abrogate TLR9-mediated immune responses. In this study, we evaluated the effect of the location of immune-stimulatory dinucleotides in ODNs on TLR9-mediated immune responses. We designed and synthesized ODNs with two immune-stimulatory dinucleotides, one placed toward the 5'-end region and the other toward the 3'-end region, incorporated 2'-O-methylribonucleotides selectively preceding the 5'- or 3'-immune-stimulatory dinucleotide or both, and studied TLR9-mediated immune responses of these compounds in cell-based assays and in vivo in mice. These studies showed that an immune-stimulatory dinucleotide located closer to the 5'-end is critical for and dictates TLR9-mediated immune responses. These studies provide insights for the use of ODNs when employed as TLR9 agonists and antagonists or antisense agents.
RESUMEN
PURPOSE: Recent studies have shown that a new generation of synthetic agonist of Toll-like receptor (TLR) 9 consisting a 3'-3'-attached structure and a dCp7-deaza-dG dinucultodie shows more potent immunostimulatory effects in both mouse and human than conventional CpG oligonucleotides. Radiation therapy (RT) provides a source of tumor antigens that are released from dying, irradiated, tumor cells without causing systemic immunosuppression. We, therefore, examined effect of combining RT with a designer synthetic agonist of TLR9 on anti-tumoral immunity, primary tumor growth retardation and metastases in a murine model of lung cancer. METHODS: Grouped C57BL/6 and congenic B cell deficient mice (B(-/-)) bearing footpad 3LL tumors were treated with PBS, TLR9 agonist, control oligonucelotide, RT or the combination of RT and TLR9 agonist. Immune phenotype of splenocytes and serum IFN-γ and IL-10 levels were analyzed by FACS and ELISA, 24 h after treatment. Tumor growth, lung metastases and survival rate were monitored and tumor specific antibodies in serum and deposition in tumor tissue were measured by ELISA and immunofluorescence. RESULTS: TLR9 agonist expanded and activated B cells and plasmacytoid dendritic cells in wild-type mice and natural killer DCs (NKDCs) in B cell-deficient (B(-/-)) mice bearing ectopic Lewis lung adenocarcinoma (3LL). Combined RT with TLR9 agonist treatment inhibited 3LL tumor growth in both wild type and B(-/-) mice. A strong tumor-specific humoral immune response (titer: 1/3200) with deposition of mouse IgG auto-antibodies in tumor tissue were found in wildtype mice, whereas the number of tumor infiltrating NKDCs increased in B(-/-) mice following RT+ TLR9 agonist therapy. Furthermore, mice receiving combination therapy had fewer lung metastases and a higher survival than single treatment cohorts. CONCLUSIONS: Combination therapy with TLR9 agonist and RT induces systemic anti-tumoral humoral response, augments tumoral infiltration of NKDCs, reduces pulmonary metastases and improves survival in a murine model of 3LL cancer.