RESUMO
Immune checkpoint blockade (ICB) therapy is an important treatment option for individuals with cancer, but it has certain limitations. Identifying a better target that can overcome tumor immune escape and stimulate T cell activity is critical. This research aimed to delve into the molecular mechanism underlying the immunoregulatory function of metadherin (MTDH), which is a novel and potential therapeutic target in hepatocellular cancer (HCC). A small interfering RNA library was screened using the luciferase reporter assay and PD-L1 promoter. The Cancer Genome Atlas database and HCC tissues were used to investigate the relationship between MTDH and PD-L1. The association between MTDH and ß-catenin/lymphoid enhancer binding factor (LEF-1) was discovered by co-immunoprecipitation. The chromatin immunoprecipitation assay was used to investigate the interaction of MTDH with the PD-L1 promoter when LEF-1 expression was silenced. Locked nucleic acid antisense oligonucleotides (ASOs) were used to inhibit MTDH. We utilized in vitro co-cultures and in vivo syngeneic tumor development experiments to confirm the effectiveness of MTDH ASO combined with PD-1 monoclonal antibody (mAb). MTDH was demonstrated to be a PD-L1 modulator. MTDH increased PD-L1 expression and upregulated PD-L1 transcriptional activity through ß-catenin/LEF-1 signaling. More importantly, MTDH ASO improved the anti-PD-1 response and increased cytotoxic T-cell infiltration in PD-1 mAb-treated malignancies. MTDH effectively predicts the therapeutic efficacy of ICB therapy. Our results imply that combining MTDH ASO with PD-1 mAb could be a promising therapeutic strategy for HCC. In addition, MTDH is a potential novel biomarker for predicting the effectiveness of immune checkpoint inhibitor treatment.
Assuntos
Anticorpos Monoclonais , Antígeno B7-H1 , Carcinoma Hepatocelular , Inibidores de Checkpoint Imunológico , Neoplasias Hepáticas , Proteínas de Membrana , Oligonucleotídeos Antissenso , Proteínas de Ligação a RNA , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Linhagem Celular Tumoral , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Oligonucleotídeos Antissenso/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Microambiente Tumoral , beta Catenina/genética , beta Catenina/imunologiaRESUMO
Spinal muscular atrophy (SMA) is an inherited lower motor neuron disease. SMA occurs secondary to alterations in the survival motor neuron 1 gene (SMN1), which is the main driver of SMN protein production. The severity of the disease is determined by the number of copies of the SMN2 gene, which is a homolog to SMN1 but not as efficient in protein production. Three medications have recently been approved for the treatment of SMA. Nusinersen is an intrathecal antisense oligonucleotide that alters SMN2 pre-mRNA, onasemnogene abeparvovec-xioi is an intravenous SMN1 gene replacement therapy, and risdiplam is an oral small molecule splicing modifier of SMN2. No head-to-head studies have been conducted comparing these medications, so selection of one of these medications for an individual with SMA can be challenging. In this article we outline the efficacy, safety, and other pertinent factors to consider when selecting a therapy for an individual with SMA. The age of the individual and comorbidities, such as liver or kidney disease, help guide treatment choices. All three of these medications are efficacious, and early initiation is critical for obtaining the best outcomes.
Assuntos
Atrofia Muscular Espinal/tratamento farmacológico , Fármacos Neuromusculares/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/administração & dosagem , Animais , Compostos Azo/administração & dosagem , Compostos Azo/imunologia , Produtos Biológicos/administração & dosagem , Produtos Biológicos/imunologia , Humanos , Atrofia Muscular Espinal/epidemiologia , Atrofia Muscular Espinal/imunologia , Fármacos Neuromusculares/imunologia , Oligonucleotídeos/imunologia , Oligonucleotídeos Antissenso/imunologia , Pirimidinas/administração & dosagem , Pirimidinas/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Resultado do TratamentoRESUMO
Modification of specificity of T cells for the use in adoptive transfer (CAR- or TCR-redirected T cells) has revolutionized the therapy of liquid tumors and some infectious diseases. However, several obstacles are still hampering the efficacy of such potent therapy, hence concurrent modification of the function is also required to obtain successful results. Here we show the use of splice-switching antisense oligonucleotides (SSOs) as a tool to transiently modify T cell function. We demonstrate the possibility to transfect SSOs and an exogenous TCR into primary human T cells in the same electroporation reaction, without affecting viability and function of the transfected T lymphocytes. Moreover, we show that SSOs targeting T cell-specific mRNAs induce the skipping of the targeted exons, and the reduction of the protein and consequent modification of T cell function. This technical work paves the way to the use of SSOs in immune cells, not only for the knockdown of the functional isoform of the targeted proteins, but also for the protein manipulation by elimination of specific domains encoded by targeted exons.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Linfócitos T/imunologia , Sobrevivência Celular/imunologia , Éxons/efeitos dos fármacos , Éxons/genética , Humanos , Mutação/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/imunologia , Splicing de RNA/genética , Splicing de RNA/imunologia , RNA Mensageiro/genética , Linfócitos T/efeitos dos fármacosRESUMO
: Antisense oligonucleotides (ASOs) are synthetically prepared short single-stranded deoxynucleotide sequences that have been validated as therapeutic agents and as a valuable tool in molecular driving biology. ASOs can block the expression of specific target genes via complementary hybridization to mRNA. Due to their high specificity and well-known mechanism of action, there has been a growing interest in using them for improving vaccine efficacy. Several studies have shown that ASOs can improve the efficacy of vaccines either by inducing antigen modification such as enhanced expression of immunogenic molecules or by targeting certain components of the host immune system to achieve the desired immune response. However, despite their extended use, some problems such as insufficient stability and low cellular delivery have not been sufficiently resolved to achieve effective and safe ASO-based vaccines. In this review, we analyze the molecular bases and the research that has been conducted to demonstrate the potential use of ASOs in vaccines.
Assuntos
Adjuvantes Imunológicos/farmacologia , Imunidade/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Adjuvantes Imunológicos/farmacocinética , Animais , Humanos , Oligonucleotídeos Antissenso/imunologia , Oligonucleotídeos Antissenso/farmacocinética , Vacinação , Vacinas/imunologia , Vacinas/farmacocinética , Vacinas/farmacologiaAssuntos
Oligonucleotídeos Antissenso/efeitos adversos , Oligonucleotídeos Antissenso/imunologia , Segurança , Vias de Administração de Medicamentos , Humanos , Funções Verossimilhança , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/uso terapêutico , Medição de RiscoRESUMO
Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types.
Assuntos
Anticorpos/farmacologia , Imunoconjugados/química , Oligonucleotídeos Antissenso/imunologia , RNA Interferente Pequeno/imunologia , Anticorpos/química , Anticorpos/imunologia , Azidas/química , Linhagem da Célula/efeitos dos fármacos , Reação de Cicloadição , Ciclo-Octanos/química , Sistemas de Liberação de Medicamentos , Endossomos/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Humanos , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Oligonucleotídeos Antissenso/antagonistas & inibidores , Oligonucleotídeos Antissenso/química , Peptídeos/química , Peptídeos/farmacologia , RNA Interferente Pequeno/antagonistas & inibidores , RNA Interferente Pequeno/química , Transglutaminases/química , Transglutaminases/imunologia , Transglutaminases/farmacologiaRESUMO
PURPOSE: Prostate cancers show remarkable resistance to emerging immunotherapies, partly due to tolerogenic STAT3 signaling in tumor-associated myeloid cells. Here, we describe a novel strategy combining STAT3 inhibition with Toll-like Receptor 9 (TLR9) stimulation to unleash immune response against prostate cancers regardless of the genetic background. EXPERIMENTAL DESIGN: We developed and validated a conjugate of the STAT3 antisense oligonucleotide (ASO) tethered to immunostimulatory TLR9 agonist (CpG oligonucleotide) to improve targeting of human and mouse prostate cancer and myeloid immune cells, such as myeloid-derived suppressor cells (MDSC). RESULTS: CpG-STAT3ASO conjugates showed improved biodistribution and potency of STAT3 knockdown in target cells in vitro and in vivo. Systemic administration of CpG-STAT3ASO (5 mg/kg) eradicated bone-localized, Ras/Myc-driven, and Ptenpc -/- Smad4pc -/- Trp53c -/- prostate tumors in the majority of treated mice. These antitumor effects were primarily immune-mediated and correlated with an increased ratio of CD8+ to regulatory T cells and reduced pSTAT3+/PD-L1+ MDSCs. Both innate and adaptive immunity contributed to systemic antitumor responses as verified by the depletion of Gr1+ myeloid cells and CD8+ and CD4+ T cells, respectively. Importantly, only the bifunctional CpG-STAT3ASO, but not control CpG oligonucleotides, STAT3ASO alone, or the coinjection of both oligonucleotides, succeeded in recruiting neutrophils and CD8+ T cells into tumors. Thus, the concurrence of TLR9 activation with STAT3 inhibition in the same cellular compartment is indispensable for overcoming tumor immune tolerance and effective antitumor immunity against prostate cancer. CONCLUSIONS: The bifunctional, immunostimulatory, and tolerance-breaking design of CpG-STAT3ASO offers a blueprint for the development of effective and safer oligonucleotide strategies for treatment of immunologically "cold" human cancers.
Assuntos
Heterogeneidade Genética , Imunomodulação/efeitos dos fármacos , Oligodesoxirribonucleotídeos/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , Neoplasias de Próstata Resistentes à Castração/etiologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Fator de Transcrição STAT3/genética , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Tolerância Imunológica , Imunofenotipagem , Masculino , Camundongos , Camundongos Transgênicos , Oligodesoxirribonucleotídeos/imunologia , Oligonucleotídeos Antissenso/imunologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Fator de Transcrição STAT3/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptor Toll-Like 9/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antisense oligonucleotides (ODNs) are therapeutic molecules that hybridize to complementary target mRNA sequences. To further overcome the poor cellular uptake of ODNs, we proposed a novel strategy to deliver ODNs by conjugating the anti-influenza A virus (IAV) ODN with a peptide showing high affinity to the hemagglutinin (HA) on the surface of IAV particles or the IAV-infected host cells. The HA-specific binding peptides were selected by phage display, and the individual binding clones are characterized by DNA sequencing, and the selected phage was further assayed by enzyme-linked immunosorbent assay. The final selected HA-binding peptide, SHGRITFAYFAN, was conjugated to an anti-IAV ODN. The delivery efficiency and the anti-IAV effects of the conjugated molecule were evaluated in a cell-culture and a mouse-infection model. The conjugated molecule was successfully delivered into IAV-infected host cells more efficiently than the anti-IAV ODN in vitro and in vivo. Furthermore, the conjugated molecule protected 80% of the mice from lethal challenge and inhibited the plaque count by 75% compared to the unconjugated molecule (60% and 40%). These findings demonstrate that the delivery of antisense oligodeoxynucleotides to infected tissues by a virus-binding peptide-mediated system is a potential therapeutic strategy against IAV.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Vírus da Influenza A Subtipo H1N1/imunologia , Oligonucleotídeos Antissenso/uso terapêutico , Animais , Hemaglutininas/metabolismo , Humanos , Imunoconjugados , Vírus da Influenza A Subtipo H1N1/química , Alphainfluenzavirus/química , Camundongos , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/imunologia , Biblioteca de Peptídeos , Peptídeos/metabolismoRESUMO
Activated and regulatory T cells express the negative co-stimulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds B7 on antigen-presenting cells to mediate cellular responses. Single nucleotide polymorphisms in the CTLA-4 gene have been found to affect alternative splicing and are linked to autoimmune disease susceptibility or resistance. Increased expression of a soluble splice form (sCTLA-4), lacking the transmembrane domain encoded by exon 3, has been shown to accelerate autoimmune pathology. In contrast, an exon 2-deficient form lacking the B7 ligand binding domain (liCTLA-4), expressed by diabetes resistant mouse strains has been shown to be protective when expressed as a transgene in diabetes susceptible non-obese diabetic (NOD) mice. We sought to employ an antisense-targeted splice-switching approach to independently produce these CTLA-4 splice forms in NOD mouse T cells and observe their relative impact on spontaneous autoimmune diabetes susceptibility. In vitro antisense targeting of the splice acceptor site for exon 2 produced liCTLA-4 while targeting exon 3 produced the sCTLA-4 form in NOD T cells. The liCTLA-4 expressing T cells exhibited reduced activation, proliferation and increased adhesion to intercellular adhesion molecule-1 (ICAM-1) similar to treatment with agonist α-CTLA-4. Mice treated to produce liCTLA-4 at the time of elevated blood glucose levels exhibited a significant reduction in the incidence of insulitis and diabetes, whereas a marked increase in the incidence of both was observed in animals treated to produce sCTLA-4. These findings provide further support that alternative splice forms of CTLA-4 affects diabetes susceptibility in NOD mice and demonstrates the therapeutic utility of antisense mediated splice-switching for modulating immune responses.
Assuntos
Autoimunidade/genética , Antígeno CTLA-4/genética , Diabetes Mellitus Tipo 1/genética , Suscetibilidade a Doenças/imunologia , Oligonucleotídeos Antissenso/genética , Abatacepte , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Adesão Celular , Proliferação de Células , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Éxons , Regulação da Expressão Gênica , Imunoconjugados/farmacologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/imunologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Índice de Gravidade de Doença , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
Antisense oligonucleotides (ASO) containing 2'-O-methoxyethyl ribose (2'-MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. 2'-MOE ASOs are generally well tolerated, displaying minimal to mild proinflammatory effect at doses far exceeding therapeutic doses. Although the vast majority of 2'-MOE ASOs are safe and well tolerated, a small subset of ASOs inducing acute inflammation in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. One of those ASOs, ISIS 147420, causes a severe inflammatory response atypical of this class of oligonucleotides characterized by induction in interferon-ß (IFN-ß) at 48 h followed by acute transaminitis and extensive hepatocyte apoptosis and necrosis at 72 h. A large number of interferon-stimulated genes were significantly up-regulated in liver as early as 24 h. We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA, thus triggering an acute innate immune response. ISIS 147420 toxicity was independent of Toll-like receptors, because there was no decrease in IFN-ß in Toll/interleukin-1 receptor-domain-containing adapter-inducing IFN-ß or Myd88-deficient mice. The involvement of cytosolic retinoic acid-inducible gene (RIG)-I-like pattern recognition receptors was also investigated. Pretreatment of mice with melanoma differentiation-associated gene 5 (MDA5) and IFN-ß promoter stimulator-1 ASOs, but not RIG-I or laboratory of genetics and physiology 2 (LGP2) ASOs, prevented the increase in IFN-ß and alanine aminotransferase induced by ISIS 147420. These results revealed a novel mechanism of oligonucleotide-mediated toxicity requiring both MDA5 and IPS-1 and resulting in the activation of the innate immune response.
Assuntos
RNA Helicases DEAD-box/imunologia , DNA/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Oligonucleotídeos Antissenso/imunologia , Ribose/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Alanina Transaminase/genética , Alanina Transaminase/imunologia , Alanina Transaminase/metabolismo , Animais , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA/genética , DNA/metabolismo , Hepatócitos/imunologia , Hepatócitos/metabolismo , Imunidade Inata/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon , Interferon beta/genética , Interferon beta/imunologia , Interferon beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Oligonucleotídeos Antissenso/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Receptores de Interleucina-1/metabolismo , Ribose/genética , Ribose/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismoRESUMO
CONTEXT: This article reviews the use of albumin microcapsules 3-4 µm in size containing cytokine inhibiting drugs which include neutralizing antibodies to TNF and IL1, CNI-1493, antisense oligonucleotides to TNF and NF-kappaB, and the antioxidant catalase. OBJECTIVE: Describe the effects, cellular uptake and distribution of microencapsulated drugs and the effect in both a peritonitis model of infection and a model of adjuvant-induced arthritis. METHODS: The studies performed by our group are reviewed, the only such studies available. RESULTS: Microencapsulation of these compounds produced high intracellular drug concentrations due to rapid uptake by phagocytic cells, including endothelial cells, without toxicity. All compounds produced excellent inhibition of TNF and IL1 resulting in improved animal survival in a peritonitis model of septic shock and inflammation in an arthritis model. CONCLUSION: Albumin microencapsulated pro-inflammatory cytokine inhibiting compounds are superior to equivalent concentration of these compounds administered in solution form.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Cápsulas/análise , Citocinas/antagonistas & inibidores , Imunossupressores/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , Albuminas/química , Animais , Anticorpos Neutralizantes/imunologia , Antioxidantes/metabolismo , Catalase/antagonistas & inibidores , Catalase/genética , Citocinas/imunologia , Composição de Medicamentos/métodos , Glucocorticoides/administração & dosagem , Glucocorticoides/imunologia , Humanos , Hidrazonas/administração & dosagem , Hidrazonas/imunologia , Imunossupressores/imunologia , Oligonucleotídeos Antissenso/imunologiaRESUMO
The effect of IL-10 antisense oligodeoxynucleotides (ODN) on the susceptibility of burned mice to intradermal (i.d.) infection of methicillin-resistant Staphylococcus aureus (MRSA) was studied. Abscesses formed and sepsis did not develop in normal mice infected i.d. with 10(8)CFU/mouse of MRSA. Similarly, sepsis caused by MRSA i.d. infection did not develop and abscesses formed in burned mice treated with IL-10 antisense ODN. However, all of the burned mice treated with scrambled ODN (control group) died by infectious complications stemming from MRSA i.d. infection, and an MRSA-abscess did not form in these mice. Macrophages (MÏ) isolated from the infection site tissue of burned mice that were treated with IL-10 antisense ODN were identified as M1MÏ, while MÏ isolated from burned mice that were treated with scrambled ODN were shown to be M2MÏ. MRSA-abscesses formed in burned mice inoculated with M1MÏ, and these mice resisted a lethal dose of MRSA i.d. infection. However, an abscess did not form, and sepsis caused by MRSA i.d. infection developed in burned mice that were inoculated with M2MÏ. These results indicate that severely burned mice treated with IL-10 antisense ODN are resistant against i.d. infection with MRSA. M1MÏ appeared in the infection site tissues of severely burned mice that were treated with IL-10 antisense ODN may play a role on the abscess formation and inhibiting sepsis caused by MRSA i.d. infection.
Assuntos
Abscesso/tratamento farmacológico , Antibacterianos/administração & dosagem , Queimaduras/tratamento farmacológico , Macrófagos/imunologia , Oligonucleotídeos Antissenso/administração & dosagem , Sepse/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológico , Abscesso/complicações , Abscesso/imunologia , Animais , Antibacterianos/imunologia , Antibacterianos/uso terapêutico , Queimaduras/complicações , Queimaduras/imunologia , Injeções Intradérmicas , Interleucina-10/antagonistas & inibidores , Interleucina-10/imunologia , Macrófagos/efeitos dos fármacos , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Oligonucleotídeos Antissenso/imunologia , Oligonucleotídeos Antissenso/uso terapêutico , Sepse/complicações , Sepse/imunologia , Índice de Gravidade de Doença , Infecções Estafilocócicas/complicações , Infecções Estafilocócicas/imunologiaRESUMO
Originally conceived as a method to silence transcription/translation of nascent RNA, nucleic acids aimed at downregulating gene expression have been shown to act at multiple levels. Some of the intriguing features of these gene-silencing nucleic acids include activation of molecular signals in immune cells which confer tolerogenic properties. We have discovered a method to induce stable tolerogenic ability to dendritic cells ex vivo using a mixture of phosphorothioate-modified antisense DNA targeting the primary transcripts of CD40, CD80 and CD86. Autologous human dendritic cells generated in the presence of these oligonucleotides prevent and reverse type 1 diabetes (T1D) in the non-obese diabetic (NOD) strain mouse model of the human disease, and have been shown to be safe in established diabetic human patients. Even though this ex vivo approach is clinically feasible, we have gone beyond a cell therapy approach to develop a "population-targeting" microsphere formulation of the three antisense oligonucleotides. Effectively, such a product could constitute an "off-the-shelf" vaccine. In this paper, we describe the progress made in developing this approach, as well as providing some insight into potential molecular mechanisms of action.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , Vacinas de DNA/uso terapêutico , Animais , Química Farmacêutica , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Humanos , Tolerância Imunológica , Camundongos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
In recent years, a new view of dendritic cells (DCs) as a main regulator of immunity to induce and maintain tolerance has been established. In vitro manipulation of their development and maturation is a topic of DC therapeutic application, which utilizes their inherent tolerogenicity. In this field, the therapeutic potential of antisense, siRNA, and blocking antibody are an interesting goal. In the present study, the efficiency of these three methods--siRNA, antisense, and blocking antibody--against CD40 molecule and its function in DCs and BCL1 cell line are compared. DCs were separated from mouse spleen and then cultured in vitro using Lipofectamine 2000 to deliver both silencers; the efficacy of transfection was estimated by flow cytometry. mRNA expression and protein synthesis were assessed by real time-PCR and flow cytometry, respectively. By Annexin V and propidium iodine staining, we could evaluate the viability of transfected cells. Knocking down the CD40 gene into separate groups of DCs by siRNA, antisense, and blocking antibody treated DCs can cause an increase in IL-4, decrease in IL-12, IFN-γ production, and allostimulation activity. Our results indicated that, in comparison to antisense and blocking antibody, siRNAs appear to be quantitatively more efficient in CD40 downregulation and their differences are significant.
Assuntos
Anticorpos Bloqueadores/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Tolerância Imunológica/imunologia , Oligonucleotídeos Antissenso/imunologia , RNA Interferente Pequeno/imunologia , Animais , Anexina A5 , Anticorpos Bloqueadores/farmacologia , Antígenos CD40/genética , Antígenos CD40/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Técnicas In Vitro , Lipídeos , Camundongos , Oligonucleotídeos Antissenso/farmacologia , Reação em Cadeia da Polimerase , Propídio , RNA Interferente Pequeno/farmacologiaAssuntos
Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Indústria Farmacêutica/tendências , Neoplasias/tratamento farmacológico , Fator de Crescimento Transformador beta/efeitos adversos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Proliferação de Células/efeitos dos fármacos , Ensaios Clínicos como Assunto/tendências , Terapia Combinada , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Transgênicos , Neoplasias/metabolismo , Neoplasias/patologia , Oligonucleotídeos Antissenso/imunologia , Medicina de Precisão , Inibidores de Proteínas Quinases/farmacologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
The prevalence of allergies and asthma among the world's population has been steadily increasing due to environmental factors. It has been described that exposure to ozone, diesel exhaust particles, or tobacco smoke exacerbates allergic inflammation in the lungs. These environmental oxidants increase the levels of cellular reactive oxygen species (ROS) and induce mitochondrial dysfunction in the airway epithelium. In this study, we investigated the involvement of preexisting mitochondrial dysfunction in the exacerbation of allergic airway inflammation. After cellular oxidative insult induced by ragweed pollen extract (RWE) exposure, we have identified nine oxidatively damaged mitochondrial respiratory chain-complex and associated proteins. Out of these, the ubiquinol-cytochrome c reductase core II protein (UQCRC2) was found to be implicated in mitochondrial ROS generation from respiratory complex III. Mitochondrial dysfunction induced by deficiency of UQCRC2 in airway epithelium of sensitized BALB/c mice prior the RWE challenge increased the Ag-induced accumulation of eosinophils, mucin levels in the airways, and bronchial hyperresponsiveness. Deficiency of UQCRC1, another oxidative damage-sensitive complex III protein, did not significantly alter cellular ROS levels or the intensity of RWE-induced airway inflammation. These observations suggest that preexisting mitochondrial dysfunction induced by oxidant environmental pollutants is responsible for the severe symptoms in allergic airway inflammation. These data also imply that mitochondrial defects could be risk factors and may be responsible for severe allergic disorders in atopic individuals.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Inflamação/imunologia , Mitocôndrias/imunologia , Pólen/imunologia , Espécies Reativas de Oxigênio/metabolismo , Hipersensibilidade Respiratória/imunologia , Alérgenos/imunologia , Ambrosia/imunologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Complexo III da Cadeia de Transporte de Elétrons/imunologia , Humanos , Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Oligonucleotídeos Antissenso/imunologia , Oligonucleotídeos Antissenso/metabolismo , Extratos Vegetais/imunologia , Espécies Reativas de Oxigênio/imunologia , Explosão Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismoRESUMO
High-risk Neuroblastoma (NB) has still a poor prognosis. Liposomes targeted to NB cells and encapsulating antisense CpG-containing oligonucleotides (TL-asCpG) had increased anti-tumour efficacy in NB xenografts compared to free asCpG. Interleukin 10 (IL-10) suppresses antigen presenting cell activation contributing to tumour-mediated immune suppression. In principle, combination of TL-asCpG and antibodies against IL-10 receptor (aIL-10R) could prolong immune system activation, leading to better therapeutic results. Mice treated with TL-asCpG 4 h after human NB cell inoculation survived significantly longer than controls. An increased life span was achieved also in mice receiving TL-asCpG 24 and 72 h after NB cell challenge. The addition of aIL-10R to TL-asCpG in the 4-h protocol significantly increased the percentage of long term survivors compared to TL-asCpG only. Surviving mice treated with the combined strategy were completely cured. In contrast, long term surviving mice treated only with TL-asCpG presented lymph node infiltration with NB cells. TL-asCpG plus aIL-10R treatment was significantly superior to TL-asCpG alone also for the 24-h protocol. Ex vivo experiments demonstrated that the combined therapy evoked a stronger and more prolonged immune system activation compared to monotherapy. These results support the feasibility of a clinical trial with TL-asCpG and aIL-10R in advanced NB patients.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Interleucina-10/antagonistas & inibidores , Neuroblastoma/prevenção & controle , Oligonucleotídeos Antissenso/uso terapêutico , Receptores de Interleucina-10/antagonistas & inibidores , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ilhas de CpG/imunologia , Feminino , Gangliosídeos/imunologia , Humanos , Interleucina-10/imunologia , Lipossomos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neuroblastoma/imunologia , Neuroblastoma/patologia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/imunologia , Proteínas Proto-Oncogênicas c-myb/imunologia , Receptores de Interleucina-10/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The recent discovery of IL-17-producing CD4(+) Th subset significantly revised the Th1/Th2 dichotomy model proposed by Mosmann and Coffman almost two decades ago. Th17 cells are involved in the pathogenesis of many human autoimmune diseases. Th17 cells, their developmental pathways and their effector functions, therefore, provide novel therapeutic targets.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Citocinas/imunologia , Interleucina-17/imunologia , Neoplasias/tratamento farmacológico , Linfócitos T Auxiliares-Indutores/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Doenças Autoimunes/imunologia , Ensaios Clínicos como Assunto , Citocinas/antagonistas & inibidores , Humanos , Interleucina-17/antagonistas & inibidores , Interleucina-17/genética , Interleucina-17/metabolismo , Neoplasias/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Oligonucleotídeos Antissenso/imunologia , Oligonucleotídeos Antissenso/uso terapêutico , Receptores de Hidrocarboneto Arílico/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores do Ácido Retinoico/imunologia , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/imunologia , Receptores dos Hormônios Tireóideos/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
TLRs represent the first line of defense against invading pathogens in the innate immune system. Certain cytokines are important mediators and essentially necessary to assure an appropriately regulated immune response. Recent data gave initial evidence that IL-1beta is one of the most relevant members of these regulating cytokines. We investigated the induction of IL-1beta production in monocytes and pDCs stimulated with ligands for TLR7 and TLR8 and with antiphospholipid antibodies (aPL). Using human monocytes and pDCs for stimulation with specific TLR7 and TLR8 ligands such as resiquimod (R848) and single stranded RNA (RNA42) as well as with a human monoclonal aPL HL5B resulted in a specific upregulation of IL-1beta mRNA and protein in these cells. Determination of expression-levels using real-time RT-PCR showed significantly augmented TLR-dependent IL-1beta and caspase-1 expression. This increase could be substantially enhanced by adding the monoclonal aPL HL5B. To demonstrate the direct dependency between TLR stimulation and IL-1beta production, specific TLR inhibitors were applied and the IL-1beta and caspase-1 secretion could be explicitly decreased. The respective protein levels were determined using Western Blot, FACS analysis or ELISA assays. In conclusion we demonstrated that the downstream signaling pathway of TLR7 and TLR8 in monocytes and pDCs after stimulation with specific ligands included not only the secretion of cytokines such as TNFalpha and IL-1beta but as well the activation of necessary regulating proteins like caspase-1. APL seem to enforce this process hinting that endogenous stimulation of TLRs in the Antiphospholipid Syndrome (APS) patients resulted in enhanced secretion of proinflammatory cytokines.
Assuntos
Anticorpos Antifosfolipídeos/imunologia , Caspase 1/metabolismo , Células Dendríticas/metabolismo , Interleucina-1beta/metabolismo , Monócitos/metabolismo , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Anticorpos Antifosfolipídeos/metabolismo , Síndrome Antifosfolipídica/imunologia , Caspase 1/genética , Caspase 1/imunologia , Separação Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/patologia , Indução Enzimática/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Imidazóis/farmacologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Ligantes , Masculino , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/patologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/imunologia , RNA/imunologia , Sequências Reguladoras de Ácido Nucleico/genética , Sequências Reguladoras de Ácido Nucleico/imunologia , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/genética , Receptor 8 Toll-Like/agonistas , Receptor 8 Toll-Like/genéticaRESUMO
Alternative splicing of pre-mRNA leads to variation in the exons that form the mRNA, and provides eukaryotic organisms with an additional qualitative control of gene expression. Disruptions in the regulation of pre-mRNA splicing caused by heritable genomic mutations or quantitative shifts in the regulation of exon inclusion can lead to disease. Alternative exon inclusion (pre-mRNA splicing) produces different proteins with alternative activities, derived from the same pre-mRNA, and is utilized by the immune system to expand gene function. Recent advances in the delivery of splice switching oligomers to lymphoid cells, combined with the ability to manipulate mRNA splicing to either correct mis-splicing or to alter the balance of different splice forms, holds great promise for the development of new therapeutic strategies for the treatment of immune-related disease. Antisense-based targeted manipulation of various immune modulating molecules as therapeutic approaches are discussed in this review.