RESUMO
Transforming growth factor-beta (TGF-ß) pathway mediates suppression of antitumor immunity and is associated with poor prognosis in triple-negative breast cancer (TNBC). In this study, we generated a humanized animal model by transplanting human peripheral blood mononuclear cells into immunodeficient mice followed by inoculation of MDA-MB-231 cells and subsequently analyzed the role of TGF-ß2 in the interaction between human T cells and human tumor cells. Following reconstitution of the human immune system, inhibition of TGF-ß signaling by TGF-ß2 antisense oligodeoxynucleotide (TASO) resulted in accelerated tumor growth inhibition. TGF-ß2 inhibition also resulted in downregulation of peripheral Foxp3 + regulatory T cells (Treg), whereas no effect was seen in the expression of CD8 + cytotoxic T cells. Analysis of the TASO-treated mice serum revealed elevated levels of human IFN-γ and reduced levels of human IL-10 and TGF-ß2. Moreover, TGF-ß2 inhibition resulted in increased CD8 + T cell infiltration, whereas the reduced infiltration of Tregs into the tumor partly resulted from decreased expression of CCL22. Decreased intratumoral Tregs facilitated the activation of cytotoxic T cells, associated with increased granzyme B expression. These results indicate that TASO potentiated T cell-mediated antitumor immunity, and it is proposed that TGF-ß2 may be a promising target in the immunotherapeutic strategy of TNBC.
Assuntos
Oligodesoxirribonucleotídeos Antissenso , Fator de Crescimento Transformador beta2 , Neoplasias de Mama Triplo Negativas , Animais , Modelos Animais de Doenças , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Linfócitos T Reguladores , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Next generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application. Here we compare intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in tumour cell lines PC9 and LK2, with good and poor productive uptake, respectively. We find that the majority of AZD4785 is rapidly delivered to CD63+late endosomes (LE) in both cell lines. Importantly, lysobisphosphatidic acid (LBPA) that triggers ASO LE escape is presented in CD63+LE in PC9 but not in LK2 cells. Moreover, both cell lines recycle AZD4785 in extracellular vesicles (EVs); however, AZD4785 quantification by advanced mass spectrometry and proteomic analysis reveals that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells thus offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs.
Assuntos
Antineoplásicos/farmacologia , Vesículas Extracelulares/fisiologia , Lisofosfolipídeos/metabolismo , Monoglicerídeos/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Linhagem Celular Tumoral , HumanosRESUMO
Exosomes are natural delivery vehicles because of their original feature such as low immunogenicity, excellent biocompatibility, and migration capability. Engineering exosomes with appropriate ligands are effective approaches to improve the low cellular uptake efficiency of exosomes. However, current strategies face considerable challenges due to the tedious and labor-intensive operational process. Here, we designed a novel peptides-equipped exosomes platform which can be assembled under convenient and mild reaction condition. Cell-penetrating peptides (CPPs) was conjugated on HepG2 cells-derived exosomes surface which can not only enhance the penetrating capacity of exosomes but also assist exosomes in loading antisense oligonucleotides (ASOs). The cellular uptake mechanism was investigated and we compared the difference between natural exosomes and modified exosomes. The resulting nanosystem demonstrated a preferential tropism for cells that are parented to their source tumor cells and could remarkably increase the cellular delivery of G3139 with efficient downregulation of antiapoptotic Bcl-2. This work developed a rapid strategy for intracellular delivery of nucleic acids, thus providing more possibilities toward personalized cancer medicine.
Assuntos
Peptídeos Penetradores de Células/química , Portadores de Fármacos/química , Exossomos/química , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Tionucleotídeos/farmacologia , Peptídeos Penetradores de Células/metabolismo , Regulação para Baixo/efeitos dos fármacos , Portadores de Fármacos/metabolismo , Exossomos/metabolismo , Inativação Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Oligodesoxirribonucleotídeos Antissenso/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tionucleotídeos/genéticaRESUMO
PURPOSE: Despite standard of care (SOC) established by Stupp, glioblastoma remains a uniformly poor prognosis. We evaluated IGV-001, which combines autologous glioblastoma tumor cells and an antisense oligonucleotide against IGF type 1 receptor (IMV-001), in newly diagnosed glioblastoma. PATIENTS AND METHODS: This open-label protocol was approved by the Institutional Review Board at Thomas Jefferson University. Tumor cells collected during resection were treated ex vivo with IMV-001, encapsulated in biodiffusion chambers with additional IMV-001, irradiated, then implanted in abdominal acceptor sites. Patients were randomized to four exposure levels, and SOC was initiated 4-6 weeks later. On the basis of clinical improvements, randomization was halted after patient 23, and subsequent patients received only the highest exposure. Safety and tumor progression were primary and secondary objectives, respectively. Time-to-event outcomes were compared with the SOC arms of published studies. RESULTS: Thirty-three patients were enrolled, and median follow-up was 3.1 years. Six patients had adverse events (grade ≤3) possibly related to IGV-001. Median progression-free survival (PFS) was 9.8 months in the intent-to-treat population (vs. SOC, 6.5 months; P = 0.0003). In IGV-001-treated patients who met Stupp-eligible criteria, PFS was 11.6 months overall (n = 22; P = 0.001) and 17.1 months at the highest exposure (n = 10; P = 0.0025). The greatest overall survival was observed in Stupp-eligible patients receiving the highest exposure (median, 38.2 months; P = 0.044). Stupp-eligible patients with methylated O6-methylguanine-DNA methyltransferase promoter (n = 10) demonstrated median PFS of 38.4 months (P = 0.0008). Evidence of immune activation was noted. CONCLUSIONS: IGV-001 was well tolerated, PFS compared favorably with SOC, and evidence suggested an immune-mediated mechanism (ClinicalTrials.gov: NCT02507583).
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Receptor IGF Tipo 1/antagonistas & inibidores , Adulto , Idoso , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Feminino , Glioblastoma/imunologia , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos Antissenso/efeitos adversos , Receptor IGF Tipo 1/genéticaRESUMO
ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibian Xenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of the adprhl1 gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lacking Adprhl1 exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.
Assuntos
Ventrículos do Coração/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , N-Glicosil Hidrolases/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Catálise , Domínio Catalítico/genética , Coração/fisiopatologia , Ventrículos do Coração/patologia , Humanos , Camundongos , Camundongos Knockout , Morfolinos/genética , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Organogênese/genética , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimentoRESUMO
Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5' splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Splicing de RNA , Aminofenóis/farmacologia , Brônquios/citologia , Linhagem Celular Tumoral , Células Cultivadas , Agonistas dos Canais de Cloreto/farmacologia , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Transporte de Íons/efeitos dos fármacos , Mutação , Quinolonas/farmacologiaRESUMO
Helicobacter pylori is the most common cause of chronic infection in human and is associated with gastritis, peptic ulcer disease, and adenocarcinoma of mucosa-associated lymphoid tissue cells. Peptide nucleic acid (PNA) is a synthetic compound, which can inhibit the production of a particular gene. This study aimed to investigate the effect of PNA on inhibiting the expression of cagA. After confirmation of the desired gene by polymerase chain reaction (PCR), the antisense sequence was designed against cagA gene. The minimum inhibitory concentrations of conjugated PNA against H. pylori was determined. The effect of the compound on the expression level of the cagA was investigated in HT29 cell culture using real-time PCR. The results showed 2 and 3 log reduction in bacterial count after 8- and 24-h treatment with 4 and 8 µM of the compound, respectively. The lowest expression level of the cagA gene was observed at a concentration of 8 µM after 6 h. The results of this study showed that cell-penetrating peptide antisense can be employed as effective tools for inhibiting the target gene mRNA for various purposes. Moreover, further research is necessary to assess the potency, safety, and pharmacokinetics of CPP-PNAs for clinical prevention and treatment of infections due to H. pylori.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Ácidos Nucleicos Peptídicos/farmacologia , Carga Bacteriana/efeitos dos fármacos , Linhagem Celular Tumoral , Células HT29 , Helicobacter pylori/genética , Humanos , Testes de Sensibilidade Microbiana , Oligodesoxirribonucleotídeos Antissenso/genética , Ácidos Nucleicos Peptídicos/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Inotersen, a 2'-O-methoxyethyl (2'-MOE) phosphorothioate antisense oligonucleotide, reduced disease progression and improved quality of life in patients with hereditary transthyretin amyloidosis with polyneuropathy (hATTR-PN) in the NEURO-TTR and NEURO-TTR open-label extension (OLE) trials. However, 300 mg/week inotersen treatment was associated with platelet count reductions in several patients. Mean platelet counts in patients in the NEURO-TTR-inotersen group remained ≥140 × 109/L in 50% and ≥100 × 109/L in 80% of the subjects. However, grade 4 thrombocytopenia (<25 × 109/L) occurred in three subjects in NEURO-TTR trial, and one of these suffered a fatal intracranial hemorrhage. The two others were treated successfully with corticosteroids and discontinuation of inotersen. Investigations in a subset of subjects in NEURO-TTR (n = 17 placebo; n = 31 inotersen) and OLE (n = 33) trials ruled out direct myelotoxicity, consumptive coagulopathy, and heparin-induced thrombocytopenia. Antiplatelet immunoglobulin G (IgG) antibodies were detected at baseline in 5 of 31 (16%) inotersen-treated subjects in NEURO-TTR, 4 of whom eventually developed grade 1 or 2 thrombocytopenia while on the drug. In addition, 24 subjects in the same group developed treatment-emergent antiplatelet IgG antibodies, of which 2 developed grade 2, and 3 developed grade 4 thrombocytopenia. Antiplatelet IgG antibodies in two of the three grade 4 thrombocytopenia subjects targeted GPIIb/IIIa. Plasma cytokines previously implicated in immune dysregulation, such as interleukin (IL)-23 and a proliferation-inducing ligand (APRIL) were often above the normal range at baseline. Collectively, these findings suggest an underlying immunologic dysregulation predisposing some individuals to immune-mediated thrombocytopenia during inotersen treatment.
Assuntos
Neuropatias Amiloides Familiares/tratamento farmacológico , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/administração & dosagem , Trombocitopenia/sangue , Adulto , Idoso , Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/imunologia , Neuropatias Amiloides Familiares/patologia , Feminino , Predisposição Genética para Doença , Humanos , Doenças do Sistema Imunitário/induzido quimicamente , Doenças do Sistema Imunitário/imunologia , Doenças do Sistema Imunitário/patologia , Imunoglobulina G , Hemorragias Intracranianas/induzido quimicamente , Hemorragias Intracranianas/imunologia , Hemorragias Intracranianas/patologia , Masculino , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/efeitos adversos , Oligonucleotídeos Antissenso/efeitos adversos , Qualidade de Vida , Trombocitopenia/induzido quimicamente , Trombocitopenia/imunologia , Trombocitopenia/patologiaRESUMO
Brain lipoprotein receptors have been shown to regulate the metabolism of ApoE and ß-amyloid (Aß) and are potential therapeutic targets for Alzheimer's disease (AD). Previously, we identified E3 ubiquitin ligase IDOL as a negative regulator of brain lipoprotein receptors. Genetic ablation of Idol increases low-density lipoprotein receptor protein levels, which facilitates Aß uptake and clearance by microglia. In this study, we utilized an antisense oligonucleotide (ASO) to reduce IDOL expression therapeutically in the brains of APP/PS1 male mice. ASO treatment led to decreased Aß pathology and improved spatial learning and memory. Single-cell transcriptomic analysis of hippocampus revealed that IDOL inhibition upregulated lysosomal/phagocytic genes in microglia. Furthermore, clustering of microglia revealed that IDOL-ASO treatment shifted the composition of the microglia population by increasing the prevalence of disease-associated microglia. Our results suggest that reducing IDOL expression in the adult brain promotes the phagocytic clearance of Aß and ameliorates Aß-dependent pathology. Pharmacological inhibition of IDOL activity in the brain may represent a therapeutic strategy for the treatment of AD.
Assuntos
Amiloidose/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidose/patologia , Animais , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Cognição/fisiologia , Modelos Animais de Doenças , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Receptores de LDL/metabolismoRESUMO
Multidrug resistance (MDR), defined as the ability of cancer cells to gain resistance to both conventional and novel chemotherapy agents, is an important barrier in treating malignancies. Initially, it was discovered that cellular pumps dependent on ATP were the cause of resistance to chemotherapy, and further studies have found that other mechanisms such as increased metabolism of drugs, decreased drug entry, and defective apoptotic pathways are involved in this process. MDR has been the focus of numerous initiatives and countless studies have been undertaken to better understand MDR and formulate strategies to overcome its effects. The current review highlights various nano-drug delivery systems including polymeric/solid lipid/mesoporous silica/metal nanoparticles, dendrimers, liposomes, micelles, and nanostructured lipid carriers to overcome the mechanism of MDR. Nanoparticles are novel gateways to enhance the therapeutic efficacy of anticancer agents at the target site of action due to their tumor-targeting abilities, which can limit the unwanted systemic effects of chemotherapy agents and also reduce drug resistance. Additionally, other innovative strategies including RNA interference as a biological process used to inhibit or silence specific gene expression, natural products as MDR modulators with little systemic toxic effects, which interfere with the functions of proteins involved in drug efflux, and physical approaches such as combination of conventional drug administration with thermal/ultrasound/photodynamic strategies are also highlighted.
Assuntos
Antineoplásicos/uso terapêutico , Portadores de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , Nanotecnologia/métodos , Neoplasias/terapia , Animais , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Dendrímeros/química , Dendrímeros/farmacocinética , Composição de Medicamentos/métodos , Humanos , Lipossomos/química , Lipossomos/farmacocinética , Camundongos , Micelas , Nanopartículas/química , Nanopartículas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To examine the impact on quality of life (QOL) of patients with hATTR amyloidosis with polyneuropathy treated with inotersen (Tegsedi™) versus placebo. METHODS: Data were from the NEURO-TTR trial (ClinicalTrials.gov Identifier: NCT01737398), a phase 3, multinational, randomized, double-blind, placebo-controlled study of inotersen in patients with hATTR amyloidosis with polyneuropathy. At baseline and week 66, QOL measures-the Norfolk-QOL-Diabetic Neuropathy (DN) questionnaire and SF-36v2® Health Survey (SF-36v2)-were assessed. Treatment differences in mean changes in QOL from baseline to week 66 were tested using mixed-effect models with repeated measures. Responder analyses compared the percentages of patients whose QOL meaningfully improved or worsened from baseline to week 66 in inotersen and placebo arms. Descriptive analysis of item responses examined treatment differences in specific activities and functions at week 66. RESULTS: Statistically significant mean differences between treatment arms were observed for three of five Norfolk-QOL-DN domains and five of eight SF-36v2 domains, with better outcomes for inotersen than placebo in physical functioning, activities of daily living, neuropathic symptoms, pain, role limitations due to health problems, and social functioning. A larger percentage of patients in the inotersen arm than the placebo arm showed preservation or improvement in Norfolk-QOL-DN and SF-36v2 scores from baseline to week 66. Responses at week 66 showed more substantial problems with daily activities and functioning for patients in the placebo arm than in the inotersen arm. CONCLUSION: Patients with hATTR amyloidosis with polyneuropathy treated with inotersen showed preserved or improved QOL at 66 weeks compared to those who received placebo.
Assuntos
Atividades Cotidianas , Neuropatias Amiloides Familiares/tratamento farmacológico , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Avaliação de Resultados em Cuidados de Saúde , Polineuropatias/tratamento farmacológico , Qualidade de Vida , Índice de Gravidade de Doença , Adulto , Idoso , Idoso de 80 Anos ou mais , Neuropatias Amiloides Familiares/complicações , Método Duplo-Cego , Esquema de Medicação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Limitação da Mobilidade , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/administração & dosagem , Polineuropatias/etiologiaRESUMO
Amyloidosis is a group of diseases characterized by extracellular deposition of amyloid fibril complexes. Fibril deposition results in organ dysfunction and possible failure. Amyloidosis is regarded as a rare disease, but in general is underdiagnosed. The two main types of systemic amyloidosis are immunoglobulin light chain and transthyretin amyloidosis. The increased availability of noninvasive cardiac imaging, genetic testing and improved laboratory assays and protein identification methods have led to increased diagnosis. However, in many cases, the diagnosis is not made until the patient develops organ impairment. Earlier diagnosis is required to prevent irreversible organ failure. Novel treatments for immunoglobulin light chain and transthyretin amyloidosis that halt disease progression, prolong and increase quality of life have recently become available.
Assuntos
Amiloidose/diagnóstico , Amiloidose/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Erros de Diagnóstico/prevenção & controle , Amiloidose/genética , Bortezomib/uso terapêutico , Erros de Diagnóstico/tendências , Diagnóstico Precoce , Testes Genéticos/métodos , Testes Genéticos/tendências , Humanos , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos/uso terapêuticoRESUMO
We evaluated the effect of microRNA (miR)-9 inhibition on fracture healing in a rat model of femoral fracture. The rats were divided into sham, negative control and miR-9 inhibitor groups. The miR-9 inhibitor group received 30 pmol/mL inhibitor intrathecally for 8 consecutive weeks following surgery-induced femoral fracture. The effect of miR-9 inhibition on fracture healing was estimated by determining the bone mineral density (BMD) and by performing X-ray analysis of the fractured bone. The serum levels of markers of bone formation were estimated by enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction, and western blotting and immunohistochemical analysis were performed to assess the effect of miR-9 inhibition on fracture healing. The BMD at the fracture site was significantly higher in the miR-9 inhibitor group than in the negative control group. Inhibition of miR-9 blocked the fracture gap and resulted in new callus formation at the fracture site. The serum levels of osteocalcin and bone GLA protein were increased and that of alkaline phosphatase was decreased by inhibition of miR-9 compared to levels in the negative control. However, inhibition of miR-9 significantly increased the mRNA levels of runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 7 (BMP-7) in the bone tissue at the fracture site compared to the negative control group; this result was confirmed by western blotting. In conclusion, -miR-9 inhibition enhanced fracture healing by modulating the BMP-7/Runx2 signalling pathway in a rat model of femoral fracture.
Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Consolidação da Fratura/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Animais , Proteína Morfogenética Óssea 7/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fêmur/efeitos dos fármacos , Fêmur/metabolismo , Fêmur/patologia , Masculino , Osteogênese/efeitos dos fármacos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: Transthyretin-mediated hereditary amyloidosis (hATTR amyloidosis) is a multisystemic disease with heterogeneous clinical presentation. Hallmarks of the disease are sensory-motor and autonomic neuropathy and cardiomyopathy. Two disease-modifying drugs, inotersen (an antisense oligonucleotide) and patisiran (a small interfering RNA agent), were recently approved for the treatment of hATTR polyneuropathy. We here review the results of the RNA-targeted therapy clinical trials with special emphasis on the endpoints measuring autonomic symptoms and function. METHODS: Literature review. We used the terms "autonomic neuropathy", "dysautonomia", "autonomic symptoms", "oligonucleotides", "inotersen" and "patisiran" in patients with hATTR amyloidosis. RESULTS: In the NEURO-TTR (inotersen) clinical trial, the modified NIS+7 score (mNIS+7) remained stable in 36% of the patients in the inotersen arm (defined as a change of less than 2 points), and 50% of patients had improved quality of life (Norfolk-QOL-DN score) after 15 months. In the APOLLO patisiran trial, 74% of the patients showed stabilization of the neuropathy, defined as a < 10 points increase on mNIS+7, and 51% of patients showed an improvement of quality of life (Norfolk QOL-DN), favoring patisiran at 18 months. Patients on patisiran had a reduced burden of autonomic dysfunction as measured by the COMPASS-31, and a stabilization of nutritional status, suggesting an effect on gastrointestinal autonomic function. CONCLUSIONS: Clinical trials of inotersen and patisiran showed that these agents were able to halt the progression of the disease, with some patients even reducing the burden of polyneuropathy, and improving qualify of life. The information on their impact on autonomic parameters is limited, warranting further dedicated studies.
Assuntos
Neuropatias Amiloides Familiares/tratamento farmacológico , Doenças do Sistema Nervoso Autônomo/tratamento farmacológico , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos/uso terapêutico , RNA Interferente Pequeno/uso terapêutico , Neuropatias Amiloides Familiares/complicações , Sistema Nervoso Autônomo/efeitos dos fármacos , Doenças do Sistema Nervoso Autônomo/etiologia , Ensaios Clínicos como Assunto , Humanos , Polineuropatias/tratamento farmacológico , Polineuropatias/etiologiaRESUMO
Nonviral delivery of nucleic acids to the cell nucleus typically requires chemical methods that do not guarantee specific delivery (e.g., transfection agent) or physical methods that may require extensive fabrication (e.g., microfluidics) or an elevated pressure (e.g., 105 Pa for microneedles). We report a method of delivering oligonucleotides to the nucleus with high specificity (relative to the cytosol) by synergistically combining chemical and physical approaches. Particularly, we demonstrate that DNA oligonucleotides appended with a polythymidine [poly(T)] segment (chemical) profusely accumulate inside the nucleus when the cells are under gentle compression imposed by the weight of a single glass coverslip (physical; â¼2.2 Pa). Our "compression-cum-poly(T)" delivery method is simple, can be generalizable to three "hard-to-transfect" cell types, and does not induce significant levels of cytotoxicity or long-term oxidative stress to the treated cells when provided the use of suitable compression times and oligonucleotide concentrations. In bEnd.3 endothelial cells, compression-aided intranuclear delivery of poly(T) is primarily mediated by importin ß and nucleoporin 62. Our method significantly enhances the intranuclear delivery of antisense oligonucleotides to bEnd.3 endothelioma cells and the inhibition of two target genes, including a reporter gene encoding the enhanced green fluorescent protein and an intranuclear lncRNA oncogene (metastasis-associated lung adenocarcinoma transcript 1), when compared with delivery without gentle compression or poly(T) attachment. Our data underscore the critical roles of pressure and nucleotide sequence on the intranuclear delivery of nucleic acids.
Assuntos
Núcleo Celular/metabolismo , Sistemas de Liberação de Medicamentos , Oligodesoxirribonucleotídeos Antissenso , Poli T , Animais , Camundongos , Oligodesoxirribonucleotídeos Antissenso/química , Oligodesoxirribonucleotídeos Antissenso/farmacocinética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Poli T/química , Poli T/farmacocinética , Poli T/farmacologia , Células RAW 264.7RESUMO
Introduction: Hereditary transthyretin-mediated amyloidosis (ATTRv; v for variant) is an underdiagnosed, progressive, and fatal multisystemic disease with a heterogenous clinical phenotype that is caused by TTR gene mutations that destabilize the TTR protein, resulting in its misfolding, aggregation, and deposition in tissues throughout the body. Areas covered: Inotersen, an antisense oligonucleotide inhibitor, was recently approved in the United States and Europe for the treatment of the polyneuropathy of ATTRv based on the positive results obtained in the pivotal phase 3 trial, NEURO-TTR. This review will discuss the mechanism of action of inotersen and its pharmacology, clinical efficacy, and safety and tolerability. A PubMed search using the terms 'inotersen,' 'AG10,' 'antisense oligonucleotide,' 'hereditary transthyretin amyloidosis,' 'familial amyloid polyneuropathy,' and 'familial amyloid cardiomyopathy' was performed, and the results were screened for the most relevant English language publications. The bibliographies of all retrieved articles were manually searched to identify additional studies of relevance. Expert opinion: Inotersen targets the disease-forming protein, TTR, and has been shown to improve quality of life and neuropathy progression in patients with stage 1 or 2 ATTRv with polyneuropathy. Inotersen is well tolerated, with a manageable safety profile through regular monitoring for the development of glomerulonephritis or thrombocytopenia.
Assuntos
Neuropatias Amiloides Familiares/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Polineuropatias/tratamento farmacológico , Adulto , Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/fisiopatologia , Animais , Humanos , Mutação , Oligodesoxirribonucleotídeos Antissenso/efeitos adversos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos/efeitos adversos , Oligonucleotídeos/farmacologia , Polineuropatias/etiologia , Pré-Albumina/genética , Qualidade de VidaRESUMO
Specific gene knockdown mediated by the antisense oligodeoxynucleotides (AODNs) strategy emerged as a rapid and effective tool for probing gene role in plant cells, particularly tip-growing pollen tubes. Here, we describe the protocol for the successful employment of AODN technique in growing tobacco pollen tubes, covering AODN design, application, and analysis of the results. We also discuss the advantages and drawbacks of this method.
Assuntos
Técnicas de Silenciamento de Genes/métodos , Nicotiana/genética , Oligodesoxirribonucleotídeos Antissenso/genética , Tubo Polínico/genética , Regulação da Expressão Gênica de PlantasRESUMO
Hereditary transthyretin amyloidosis is a fatal autosomal dominant disorder characterized by deposition of transthyretin amyloid into the peripheral nervous system, heart, kidney, and gastrointestinal tract. Previous treatments using liver transplantation and small molecule stabilizers were not effective in stopping disease progression. Inotersen, a 2'-O-methyoxyethyl-modified antisense oligonucleotide, which acts by reducing the production of transthyretin, was recently demonstrated to improve disease course and quality of life in early hereditary transthyretin amyloidosis polyneuropathy in a 15-month Phase III study.
Assuntos
Neuropatias Amiloides Familiares/tratamento farmacológico , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos/uso terapêutico , Animais , HumanosRESUMO
Multidrug-resistant (MDR) Acinetobacter baumannii is a difficult pathogen due to its propensity to develop resistance to antibiotics. Antisense nucleic acid analogs have been proposed as a potential alternative anti-infective approach. We developed a peptide nucleic acid (PNA) compound that targets the essential Acinetobacter gene carA. The PNA oligomer was conjugated to the cell-penetrating peptide (RXR)4XB. In vitro testing of the PNA conjugate against four clinical strains of MDR-A. baumannii in minimal medium demonstrated that all four strains were inhibited at a concentration of 1.25 µM. In vivo testing of the PNA conjugate was done using a Galleria mellonella model of sepsis caused by one of the clinical strains. Preliminary testing of a variety of inocula demonstrated that an inoculum of 1 × 106 cfu was lethal to the majority of caterpillars by day 3, but not within 24 hours. The PNA compound was administered 30 minutes after an inoculum of 1 × 106 cfu at doses estimated to produce concentrations of â¼5 and 20 µM. The PNA compound had no effect at the lower dose. However, the higher dose reduced mortality from 5/28 (18%) to 0/28 (0%) at day 1 (p = 0.051) and from 19/28 (68%) to 9/28 (32%) at day 6 (p = 0.015). Antisense therapy is a novel approach to dealing with difficult MDR pathogens that could circumvent the problem of progressive resistance to available antibiotics. Further studies need to be done with additional strains and more complex in vivo model systems.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Ácidos Nucleicos Peptídicos/farmacologia , Peptídeos/farmacologia , Antibacterianos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Testes de Sensibilidade Microbiana/métodosRESUMO
Osteosarcoma is the most common malignant tumor of bone with a high potential for metastasis and poor prognosis. This study intends to explore the effect of tankyrase1 (TANK1) in the development of osteosarcoma cells and the underlying mechanism. The osteosarcoma cell line MG-63 cells were cultured and transfected with tankyrase1 antisense oligodeoxynucleotides (TANK1-ASODN). Cell proliferation was detected with CCK-8 and immunofluorescence. Cell migration and invasion were examined by wound healing assay and Transwell assay, respectively. Reverse transcription-quantitative polymerase chain reaction was performed to detect the mRNA level of TANK1 and western blot was conducted to detect relative protein expression during the research. As a result, we demonstrated that TANK1 was upregulated in osteosarcoma. The TANK1-ASODN inhibited MG-63 cell proliferation, migration and invasion. The progress of epithelial-mesenchymal transition (EMT) was also suppressed in TANK1-ASODN transfected MG-63 cells compared to control group. Besides, the TANK1-ASODN activated and modulated the Hippo/YAP signaling which might be the pathway that TANK1 depended on. Overall, our finding supported that TANK1-ASODN slowed down the progress of osteosarcoma by suppressing cell proliferation, migration, invasion and EMT through Hippo/YAP pathway.