RESUMEN
Targeting oncogenes at the genomic DNA level can open new avenues for precision medicine. Significant efforts are ongoing to target oncogenes using RNA-targeted and protein-targeted platforms, but no progress has been made to target genomic DNA for cancer therapy. Here, we introduce a gamma peptide nucleic acid (γPNA)-based genomic DNA-targeted platform to silence oncogenes in vivo. γPNAs efficiently invade the mixed sequences of genomic DNA with high affinity and specificity. As a proof of concept, we establish that γPNA can inhibit c-Myc transcription in multiple cell lines. We evaluate the in vivo efficacy and safety of genomic DNA targeting in three pre-clinical models. We also establish that anti-transcription γPNA in combination with histone deacetylase inhibitors and chemotherapeutic drugs results in robust antitumor activity in cell-line- and patient-derived xenografts. Overall, this strategy offers a unique therapeutic platform to target genomic DNA to inhibit oncogenes for cancer therapy.
Asunto(s)
Neoplasias , Ácidos Nucleicos , Ácidos Nucleicos de Péptidos , Humanos , ADN/genética , Ácidos Nucleicos de Péptidos/farmacología , Ácidos Nucleicos de Péptidos/genética , ARN , Neoplasias/tratamiento farmacológico , Neoplasias/genéticaRESUMEN
RNA therapeutics have emerged as next-generation therapy for the treatment of many diseases. Unlike small molecules, RNA targeted drugs are not limited by the availability of binding pockets on the protein, but rather utilize Watson-Crick (WC) base-pairing rules to recognize the target RNA and modulate gene expression. Antisense oligonucleotides (ASOs) present a powerful therapeutic approach to treat disorders triggered by genetic alterations. ASOs recognize the cognate site on the target RNA to alter gene expression. Nine single-stranded ASOs have been approved for clinical use and several candidates are in late-stage clinical trials for both rare and common diseases. Several chemical modifications, including phosphorothioates, locked nucleic acid, phosphorodiamidate, morpholino, and peptide nucleic acids (PNAs), have been investigated for efficient RNA targeting. PNAs are synthetic DNA mimics where the deoxyribose phosphate backbone is replaced by N-(2-aminoethyl)-glycine units. The neutral pseudopeptide backbone of PNAs contributes to enhanced binding affinity and high biological stability. PNAs hybridize with the complementary site in the target RNA and act by a steric hindrance--based mechanism. In the last three decades, various PNA designs, chemical modifications, and delivery strategies have been explored to demonstrate their potential as an effective and safe RNA-targeting platform. This review covers the advances in PNA-mediated targeting of coding and noncoding RNAs for a myriad of therapeutic applications.
Asunto(s)
Ácidos Nucleicos de Péptidos , ARN , ARN/genética , ARN/uso terapéutico , ARN/química , Ácidos Nucleicos de Péptidos/farmacología , Ácidos Nucleicos de Péptidos/uso terapéutico , Ácidos Nucleicos de Péptidos/química , ADN/química , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/uso terapéutico , Emparejamiento BaseRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease. Recent findings have shown a marked metabolic reprogramming associated with changes in mitochondrial homeostasis and autophagy during pulmonary fibrosis. The microRNA-33 (miR-33) family of microRNAs (miRNAs) encoded within the introns of sterol regulatory element binding protein (SREBP) genes are master regulators of sterol and fatty acid (FA) metabolism. miR-33 controls macrophage immunometabolic response and enhances mitochondrial biogenesis, FA oxidation, and cholesterol efflux. Here, we show that miR-33 levels are increased in bronchoalveolar lavage (BAL) cells isolated from patients with IPF compared with healthy controls. We demonstrate that specific genetic ablation of miR-33 in macrophages protects against bleomycin-induced pulmonary fibrosis. The absence of miR-33 in macrophages improves mitochondrial homeostasis and increases autophagy while decreasing inflammatory response after bleomycin injury. Notably, pharmacological inhibition of miR-33 in macrophages via administration of anti-miR-33 peptide nucleic acids (PNA-33) attenuates fibrosis in different in vivo and ex vivo mice and human models of pulmonary fibrosis. These studies elucidate a major role of miR-33 in macrophages in the regulation of pulmonary fibrosis and uncover a potentially novel therapeutic approach to treat this disease.
Asunto(s)
Autofagia , Fibrosis Pulmonar Idiopática , Macrófagos , MicroARNs , Animales , Humanos , Ratones , Autofagia/genética , Bleomicina/efectos adversos , Homeostasis , Fibrosis Pulmonar Idiopática/metabolismo , Macrófagos/metabolismo , MicroARNs/genética , Mitocondrias/metabolismoRESUMEN
PURPOSE: MicroRNAs (miRNAs) are short (~ 22 nts) RNAs that regulate gene expression via binding to mRNA. MiRNAs promoting cancer are known as oncomiRs. Targeting oncomiRs is an emerging area of cancer therapy. OncomiR-21 and oncomiR-155 are highly upregulated in lymphoma cells, which are dependent on these oncomiRs for survival. Targeting specific miRNAs and determining their effect on cancer cell progression and metastasis have been the focus of various studies. Inhibiting a single miRNA can have a limited effect, as there may be other overexpressed miRNAs present that may promote tumor proliferation. Herein, we target miR-21 and miR-155 simultaneously using nanoparticles delivered two different classes of antimiRs: phosphorothioates (PS) and peptide nucleic acids (PNAs) and compared their efficacy in lymphoma cell lines. METHODS: Poly-Lactic-co-Glycolic acid (PLGA) nanoparticles (NPs) containing PS and PNA-based antimiR-21 and -155 were formulated, and comprehensive NP characterizations: morphology (scanning electron microscopy), size (differential light scattering), and surface charge (zeta potential) were performed. Cellular uptake analysis was performed using a confocal microscope and flow cytometry analysis. The oncomiR knockdown and the effect on downstream targets were confirmed by gene expression (real time-polymerase chain reaction) assay. RESULTS: We demonstrated that simultaneous targeting with NP delivered PS and PNA-based antimiRs resulted in significant knockdown of miR-21 and miR-155, as well as their downstream target genes followed by reduced cell viability ex vivo. CONCLUSIONS: This project demonstrated that targeting miRNA-155 and miR-21 simultaneously using nanotechnology and a diverse class of antisense oligomers can be used as an effective approach for lymphoma therapy.
Asunto(s)
Linfoma , MicroARNs , Ácidos Nucleicos de Péptidos , Humanos , Ácidos Nucleicos de Péptidos/farmacología , Antagomirs , MicroARNs/genética , Linfoma/tratamiento farmacológico , Linfoma/genética , Línea Celular , Línea Celular TumoralRESUMEN
BACKGROUND: Early and accurate diagnosis followed by timely treatment are the key prerequisites to fight tuberculosis (TB) and reduce its global burden. Despite scientific advances, the rapid and correct diagnosis of both pulmonary and extrapulmonary tuberculosis remains a challenge because of traditional reliance on detection of the elusive bacilli. Mycobacterium tuberculosis (Mtb)-specific host immune activation and cytokine production have shown significant promise as alternative means of detecting and distinguishing active disease from latent infection. We queried the diagnostic ability of phenotypic markers on Mtb-specific cytokine-producing immune cell subsets for identifying active TB. METHODS: Subjects belonging to the following groups were recruited: pulmonary and extrapulmonary TB, latent TB, cured TB, sick controls, and healthy controls. Polychromatic flow cytometry was used to identify host immune biomarkers in an exploratory cohort comprising 56 subjects using peripheral blood mononuclear cells. Clinical performance of the identified biomarker was evaluated using whole blood in a blinded validation cohort comprising 165 individuals. RESULTS: Cytokine secreting frequencies of Mtb-specific cluster of differentiation 4-positive (CD4+) T cells with CD38+CD27- phenotype clearly distinguished infected individuals with active tuberculosis from those without disease. Tumor necrosis factor-α (TNF-α) secretion from CD38+CD27-CD4+ T cells upon stimulation with ESAT6/CFP10 peptides had the best diagnostic accuracy at a cutoff of 9.91% (exploratory: 96.67% specificity, 88.46% sensitivity; validation: 96.15% specificity, 90.16% sensitivity). Additionally, this subset differentiated treatment-naive patients with TB from individuals cured of TB following completion of anti-TB therapy. CONCLUSIONS: Mtb-specific CD38+CD27-TNF-αâ+CD4+ T-cell subset is a robust biomarker both for diagnosing TB and assessing cure.
Asunto(s)
Tuberculosis Latente , Mycobacterium tuberculosis , Tuberculosis , Antígenos Bacterianos , Biomarcadores , Linfocitos T CD4-Positivos , Humanos , Tuberculosis Latente/diagnóstico , Leucocitos Mononucleares , Factor de Necrosis Tumoral alfaRESUMEN
Background: Early biomarkers of progression to severe dengue are urgently required to enable effective patient management and control treatment costs. Innate immune cells, which comprise the earliest responders to infection and along with the cytokines and chemokines they secrete, play a vital role in orchestrating the subsequent adaptive immune response and have been implicated in the enhancement of infection and "cytokine storm" associated with dengue severity. We investigated the early innate immune cytokine profile of dengue patients during acute phase of disease in a prospective blinded study that included subjects with acute dengue and febrile controls from four major hospitals in Bengaluru, India along with healthy controls. We used intracellular cytokine staining and flow cytometry to identify innate immune biomarkers that can predict progression to severe dengue. Results: Dengue infection resulted in enhanced secretion of multiple cytokines by all queried innate immune cell subsets, dominated by TNF-α from CD56+CD3+ NKT cells, monocyte subsets, and granulocytes along with IFN-γ from CD56+CD3+ NKT cells. Of note, significantly higher proportions of TNF-α secreting granulocytes and monocyte subsets at admission were associated with mild dengue and minimal symptoms. Dengue NS1 antigenemia used as a surrogate of viral load directly correlated with proportion of cytokine-secreting innate immune cells and was significantly higher in those who went on to recover with minimal symptoms. In patients with secondary dengue or those with bleeding or elevated liver enzymes who revealed predisposition to severe outcomes, early activation as well as efficient downregulation of innate responses were compromised. Conclusion: Our findings suggested that faulty/delayed kinetics of innate immune activation and downregulation was a driver of disease severity. We identified IFN-γ+CD56+CD3+ NKT cells and IL-6+ granulocytes at admission as novel early biomarkers that can predict the risk of progression to severity (composite AUC = 0.85-0.9). Strong correlations among multiple cytokine-secreting innate cell subsets revealed that coordinated early activation of the entire innate immune system in response to dengue virus infection contributed to resolution of infection and speedy recovery.