Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 2.466
Filter
1.
Sci Rep ; 14(1): 684, 2024 01 06.
Article in English | MEDLINE | ID: mdl-38182646

ABSTRACT

Stargardt disease type 1 (STGD1), the most common form of hereditary macular dystrophy, can be caused by biallelic combinations of over 2200 variants in the ABCA4 gene. This leads to reduced or absent ABCA4 protein activity, resulting in toxic metabolite accumulation in the retina and damage of the retinal pigment epithelium and photoreceptors. Approximately 21% of all ABCA4 variants that contribute to disease influence ABCA4 pre-mRNA splicing. This emphasizes the need for therapies to restore disrupted ABCA4 splicing and halt STGD1 progression. Previously, QR-1011, an antisense oligonucleotide (AON), successfully corrected splicing abnormalities and restored normal ABCA4 protein translation in human retinal organoids carrying the prevalent disease-causing variant c.5461-10T>C in ABCA4. Here, we investigated whether QR-1011 could also correct splicing in four less common non-canonical splice site (NCSS) variants flanking ABCA4 exon 39: c.5461-8T>G, c.5461-6T>C, c.5584+5G>A and c.5584+6T>C. We administered QR-1011 and three other AONs to midigene-transfected cells and demonstrate that QR-1011 had the most pronounced effect on splicing compared to the others. Moreover, QR-1011 significantly increased full-length ABCA4 transcript levels for c.5461-8T>G and c.5584+6T>C. Splicing restoration could not be achieved in the other two variants, suggesting their more severe effect on splicing. Overall, QR-1011, initially developed for a single ABCA4 variant, exhibited potent splice correction capabilities for two additional severe NCSS variants nearby. This suggests the possibility of a broader therapeutic impact of QR-1011 extending beyond its original target and highlights the potential for treating a larger population of STGD1 patients affected by multiple severe ABCA4 variants with a single AON.


Subject(s)
ATP-Binding Cassette Transporters , Oligodeoxyribonucleotides, Antisense , Organoids , Stargardt Disease , Humans , ATP-Binding Cassette Transporters/genetics , Exons , Retina/cytology , RNA Splicing/drug effects , Stargardt Disease/drug therapy , Stargardt Disease/genetics , Oligodeoxyribonucleotides, Antisense/pharmacology , Organoids/drug effects
2.
Cancer Immunol Immunother ; 71(9): 2213-2226, 2022 Sep.
Article in English | MEDLINE | ID: mdl-35099588

ABSTRACT

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.


Subject(s)
Oligodeoxyribonucleotides, Antisense , Transforming Growth Factor beta2 , Triple Negative Breast Neoplasms , Animals , Disease Models, Animal , Humans , Leukocytes, Mononuclear/metabolism , Mice , Oligodeoxyribonucleotides, Antisense/pharmacology , T-Lymphocytes, Regulatory , Transforming Growth Factor beta2/antagonists & inhibitors , Triple Negative Breast Neoplasms/pathology
3.
Commun Biol ; 4(1): 1241, 2021 11 01.
Article in English | MEDLINE | ID: mdl-34725463

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/pharmacology , Extracellular Vesicles/physiology , Lysophospholipids/metabolism , Monoglycerides/metabolism , Oligodeoxyribonucleotides, Antisense/pharmacology , Cell Line, Tumor , Humans
4.
J Cardiovasc Pharmacol ; 78(1): e5-e11, 2021 07 01.
Article in English | MEDLINE | ID: mdl-34232223

ABSTRACT

ABSTRACT: Atherosclerotic cardiovascular disease (ASCVD) continues to be the leading cause of preventable death in the United States. Elevated low-density lipoprotein cholesterol (LDL-C) is well known to result in cardiovascular disease. Mainstay therapy for reducing LDL-C and ASCVD risk is statin therapy. Despite achieving desired LDL-C levels with lipid-lowering therapy, cardiovascular residual risk often persists. Elevated lipoprotein(a) [Lp(a)] levels have been highlighted as an inherent independent predictor of ASCVD, and decreasing Lp(a) levels may result in a significant reduction in the residual risk in high-risk patients. To date, there are no approved medications to lower Lp(a) levels. Nicotinic acid, proprotein convertase subtilisin/kexin 9 inhibitors, and antisense oligonucleotide have demonstrated modest to potent Lp(a) reduction. Spotlight has been placed on antisense oligonucleotides and their role in Lp(a) lowering. APO(a)LRx is in the frontline for selectively decreasing Lp(a) concentrations and ongoing research may prove that this medication may lower Lp(a)-mediated residual risk, translating into cardiovascular benefit.


Subject(s)
Atherosclerosis/drug therapy , Dyslipidemias/drug therapy , Hypolipidemic Agents/therapeutic use , Lipoprotein(a)/blood , Oligodeoxyribonucleotides, Antisense/therapeutic use , Oligonucleotides/therapeutic use , Atherosclerosis/blood , Atherosclerosis/genetics , Biomarkers/blood , Clinical Trials as Topic , Down-Regulation , Dyslipidemias/blood , Dyslipidemias/genetics , Evidence-Based Medicine , Humans , Hypolipidemic Agents/adverse effects , Oligodeoxyribonucleotides, Antisense/adverse effects , Oligonucleotides/adverse effects , Treatment Outcome
5.
ACS Synth Biol ; 10(8): 1847-1858, 2021 08 20.
Article in English | MEDLINE | ID: mdl-34283568

ABSTRACT

Regulatory RNA-based interactions are critical for coordinating gene expression and are increasingly being targeted in synthetic biology, antimicrobial, and therapeutic fields. Bacterial trans-encoded small RNAs (sRNAs) regulate the translation and/or stability of mRNA targets through base-pairing interactions. These interactions are often integral to complex gene circuits which coordinate critical bacterial processes. The ability to predictably modulate these gene circuits has potential for reprogramming gene expression for synthetic biology and antibacterial purposes. Here, we present a novel pipeline for targeting such RNA-based interactions with antisense oligonucleotides (ASOs) in order to reprogram gene expression. As proof-of-concept, we selected sRNA-mRNA interactions that are central to the Vibrio cholerae quorum sensing pathway, required for V. cholerae pathogenesis, as a regulatory RNA-based interaction input. We rationally designed anti-sRNA ASOs to target the sRNAs and synthesized them as peptide nucleic acids (PNAs). Next, we devised an RNA array-based interaction assay to allow screening of the anti-sRNA ASOs in vitro. Finally, an Escherichia coli-based gene expression reporter assay was developed and used to validate anti-sRNA ASO regulatory activity in a cellular environment. The output from the pipeline was an anti-sRNA ASO that targets sRNAs to inhibit sRNA-mRNA interactions and modulate gene expression. This anti-sRNA ASO has potential for reprogramming gene expression for synthetic biology and/or antibacterial purposes. We anticipate that this pipeline will find widespread use in fields targeting RNA-based interactions as modulators of gene expression.


Subject(s)
Gene Expression Regulation, Bacterial , Oligodeoxyribonucleotides, Antisense/chemistry , Peptide Nucleic Acids/chemistry , RNA, Bacterial/biosynthesis , Vibrio cholerae , RNA, Bacterial/genetics , Vibrio cholerae/genetics , Vibrio cholerae/metabolism
6.
Adv Drug Deliv Rev ; 175: 113809, 2021 08.
Article in English | MEDLINE | ID: mdl-34033819

ABSTRACT

Thanks to their abilities to modulate the expression of virtually any genes, RNA therapeutics have attracted considerable research efforts. Among the strategies focusing on nucleic acid gene inhibitors, antisense oligonucleotides and small interfering RNAs have reached advanced clinical trial phases with several of them having recently been marketed. These successes were obtained by overcoming stability and cellular delivery issues using either chemically modified nucleic acids or nanoparticles. As nucleic acid gene inhibitors are promising strategies to treat inflammatory diseases, this review focuses on the barriers, from manufacturing issues to cellular/subcellular delivery, that still need to be overcome to deliver the nucleic acids to sites of inflammation other than the liver. Furthermore, key examples of applications in rheumatoid arthritis, inflammatory bowel, and lung diseases are presented as case studies of systemic, oral, and lung nucleic acid delivery.


Subject(s)
Inflammation/drug therapy , Nanomedicine/methods , Nanoparticle Drug Delivery System , Nucleic Acids/administration & dosage , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/therapeutic use , Drug Delivery Systems/methods , Genes/drug effects , Humans , Inflammation/genetics , Nucleic Acids/therapeutic use , Oligodeoxyribonucleotides, Antisense/administration & dosage , Oligodeoxyribonucleotides, Antisense/therapeutic use , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/therapeutic use
7.
Eur J Pharm Biopharm ; 162: 92-98, 2021 May.
Article in English | MEDLINE | ID: mdl-33753212

ABSTRACT

This study describes the effective attack of oligonucleotides on the viral genome of highly pathogenic H5N1 influenza A virus (IAV) in vivo using for the first time the new delivery system consisting of biocompatible low-toxic titanium dioxide nanoparticles and immobilized polylysine-containing oligonucleotides with the native (ODN) and partially modified (ODNm) internucleotide bonds. Intraperitoneal injection of the TiO2•PL-ODN nanocomposite provided 65-70% survival of mice, while intraperitoneal or oral administration of TiO2•PL-ODNm was somewhat more efficient (~80% survival). The virus titer in the lung was reduced by two-three orders of magnitude. The nanocomposites are nontoxic to mice under the used conditions. TiO2 nanoparticles, unbound ODN, and the nanocomposite bearing the random oligonucleotide showed an insignificant protective effect, which indicates the ability of targeted oligonucleotides delivered in mice in the nanocomposites to site-specifically interact with complementary RNAs. The protection of oligonucleotides in nanocomposites by TiO2 nanoparticles and partial modification of the internucleotide bonds provides a continued presence of oligonucleotides in the body for the effective and specific action on the viral RNA. The proposed oligonucleotide delivery system can claim not only to effectively inhibit IAV genes but also to turn off other genes responsible for diseases caused by nucleic acids.


Subject(s)
Antiviral Agents/administration & dosage , Drug Carriers/chemistry , Influenza A Virus, H5N1 Subtype/drug effects , Influenza, Human/drug therapy , Oligodeoxyribonucleotides, Antisense/administration & dosage , Administration, Oral , Animals , Disease Models, Animal , Dogs , Female , Genome, Viral/drug effects , Humans , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/isolation & purification , Influenza, Human/virology , Injections, Intraperitoneal , Madin Darby Canine Kidney Cells , Male , Mice , Nanocomposites/chemistry , RNA, Viral/antagonists & inhibitors , Titanium/chemistry , Viral Load/drug effects
8.
ACS Appl Mater Interfaces ; 13(9): 10760-10767, 2021 Mar 10.
Article in English | MEDLINE | ID: mdl-33621039

ABSTRACT

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.


Subject(s)
Cell-Penetrating Peptides/chemistry , Drug Carriers/chemistry , Exosomes/chemistry , Oligodeoxyribonucleotides, Antisense/pharmacology , Thionucleotides/pharmacology , Cell-Penetrating Peptides/metabolism , Down-Regulation/drug effects , Drug Carriers/metabolism , Exosomes/metabolism , Gene Silencing/drug effects , Hep G2 Cells , Humans , Oligodeoxyribonucleotides, Antisense/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Thionucleotides/genetics
9.
Clin Cancer Res ; 27(7): 1912-1922, 2021 04 01.
Article in English | MEDLINE | ID: mdl-33500356

ABSTRACT

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).


Subject(s)
Brain Neoplasms/drug therapy , Glioblastoma/drug therapy , Oligodeoxyribonucleotides, Antisense/therapeutic use , Receptor, IGF Type 1/antagonists & inhibitors , Adult , Aged , Brain Neoplasms/immunology , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Female , Glioblastoma/immunology , Glioblastoma/mortality , Glioblastoma/pathology , Humans , Male , Middle Aged , Oligodeoxyribonucleotides, Antisense/adverse effects , Receptor, IGF Type 1/genetics
10.
Nucleic Acids Res ; 49(1): e5, 2021 01 11.
Article in English | MEDLINE | ID: mdl-33221877

ABSTRACT

RNA sequencing (RNA-seq) is extensively used to quantify gene expression transcriptome-wide. Although often paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA), many applications require alternate approaches to counteract the high proportion of ribosomal RNA (rRNA) in total RNA. Recently, digestion using RNaseH and antisense DNA oligomers tiling target rRNAs has emerged as an alternative to commercial rRNA depletion kits. Here, we present a streamlined, more economical RNaseH-mediated rRNA depletion with substantially lower up-front costs, using shorter antisense oligos only sparsely tiled along the target RNA in a 5-min digestion reaction. We introduce a novel Web tool, Oligo-ASST, that simplifies oligo design to target regions with optimal thermodynamic properties, and additionally can generate compact, common oligo pools that simultaneously target divergent RNAs, e.g. across different species. We demonstrate the efficacy of these strategies by generating rRNA-depletion oligos for Xenopus laevis and for zebrafish, which expresses two distinct versions of rRNAs during embryogenesis. The resulting RNA-seq libraries reduce rRNA to <5% of aligned reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion.


Subject(s)
Computational Biology/methods , Oligodeoxyribonucleotides, Antisense/genetics , RNA, Messenger/genetics , RNA, Ribosomal/genetics , RNA/genetics , Animals , Base Sequence , Female , Gene Expression Profiling/methods , Internet , Male , Oligodeoxyribonucleotides, Antisense/metabolism , Poly A/genetics , Poly A/metabolism , RNA/metabolism , RNA, Messenger/metabolism , RNA, Ribosomal/metabolism , Ribonuclease H/metabolism , Sequence Analysis, RNA/methods , Xenopus laevis/embryology , Xenopus laevis/genetics , Zebrafish/embryology , Zebrafish/genetics
11.
PLoS One ; 15(7): e0235433, 2020.
Article in English | MEDLINE | ID: mdl-32726316

ABSTRACT

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.


Subject(s)
Heart Ventricles/growth & development , Heart/growth & development , Muscle Development/genetics , N-Glycosyl Hydrolases/genetics , Animals , Animals, Genetically Modified/genetics , Animals, Genetically Modified/growth & development , Catalysis , Catalytic Domain/genetics , Heart/physiopathology , Heart Ventricles/pathology , Humans , Mice , Mice, Knockout , Morpholinos/genetics , Oligodeoxyribonucleotides, Antisense/genetics , Oligodeoxyribonucleotides, Antisense/pharmacology , Organogenesis/genetics , Xenopus laevis/genetics , Xenopus laevis/growth & development
12.
Nucleic Acids Res ; 48(13): 7454-7467, 2020 07 27.
Article in English | MEDLINE | ID: mdl-32520327

ABSTRACT

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.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/metabolism , Epithelial Cells/metabolism , Oligodeoxyribonucleotides, Antisense/pharmacology , RNA Splicing , Aminophenols/pharmacology , Bronchi/cytology , Cell Line, Tumor , Cells, Cultured , Chloride Channel Agonists/pharmacology , Chlorides/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Epithelial Cells/drug effects , Humans , Ion Transport/drug effects , Mutation , Quinolones/pharmacology
13.
Exp Neurol ; 332: 113389, 2020 10.
Article in English | MEDLINE | ID: mdl-32580014

ABSTRACT

Chronic cerebral hypoperfusion (CCH) promotes the development of Alzheimer's pathology. However, whether and how CCH impairs the synaptic vesicle trafficking is still unclear. In the present study, we found that the hippocampal glutamatergic vesicle trafficking was impaired as indicated by a significant shortened delayed response enhancement (DRE) phase in CA3-CA1 circuit and decreased synapsin I in CCH rats suffering from bilateral common carotid artery occlusion (2VO). Further study showed an upregulated miR-153 in the hippocampus of 2VO rats. In vitro, overexpression of miR-153 downregulated synapsin I by binding the 3'UTRs of SYN1 mRNAs, which was prevented by its antisense AMO-153 and miRNA-masking antisense oligodeoxynucleotides (SYN1-ODN). In vivo, the upregulation of miR-153 elicited similar reduced DRE phase and synapsin I deficiency as CCH. Furthermore, miR-153 knockdown rescued the downregulated synapsin I and shortened DRE phase in 2VO rats. Our results demonstrate that CCH impairs hippocampal glutamatergic vesicle trafficking by upregulating miR-153, which suppresses the expression of synapsin I at the post-transcriptional level. These results will provide important references for drug research and treatment of vascular dementia.


Subject(s)
Cerebrovascular Disorders/genetics , Cerebrovascular Disorders/physiopathology , Hippocampus/physiopathology , MicroRNAs/genetics , Synapsins/genetics , Synaptic Vesicles , 3' Untranslated Regions , Animals , Carotid Stenosis/physiopathology , Down-Regulation , Glutamates/metabolism , Male , Oligodeoxyribonucleotides, Antisense/pharmacology , Protein Processing, Post-Translational , Rats , Rats, Sprague-Dawley , Synapsins/biosynthesis
14.
Nucleic Acids Res ; 48(13): 7052-7065, 2020 07 27.
Article in English | MEDLINE | ID: mdl-32544249

ABSTRACT

Oligonucleotide-based therapeutics have become a reality, and are set to transform management of many diseases. Nevertheless, the modulatory activities of these molecules on immune responses remain incompletely defined. Here, we show that gene targeting 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) can have opposing activities on Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent suppression of TLR7 and activation of TLR8, in a sequence-dependent manner. Surprisingly, TLR8 potentiation by the gapmer ASOs was blunted by locked nucleic acid (LNA) and 2'-methoxyethyl (2'MOE) modifications. Through a screen of 192 2'OMe ASOs and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we identified core motifs preventing the immunosuppressive activities of 2'OMe ASOs on TLR7. Based on these observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve TLR7 function, and promote strong activation of phagocytes and immune cells. We also provide proof-of-principle data that gene-targeting ASOs can be selected to synergize with TLR8 agonists currently under investigation as immunotherapies, and show that rational ASO selection can be used to prevent unintended immune suppression of TLR7. Taken together, our work characterizes the immumodulatory effects of ASOs to advance their therapeutic development.


Subject(s)
Oligodeoxyribonucleotides, Antisense/pharmacology , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolism , Cells, Cultured , Humans , Imidazoles/metabolism , Leukocytes, Mononuclear , Oligonucleotides/metabolism , Toll-Like Receptor 7/agonists , Toll-Like Receptor 8/agonists
15.
J Immunol Res ; 2020: 6401978, 2020.
Article in English | MEDLINE | ID: mdl-32377535

ABSTRACT

Long noncoding RNAs (lncRNAs) have been increasingly recognized as key immune molecules that participate in the pathogenesis of autoimmune diseases. Previous studies have demonstrated that the lncRNA Ifng-AS1, a key scaffold that contributes to the transcription of IFN-γ, depends on T-bet for active transcription in Th1 cells. However, the effect of its human ortholog, IFNG-AS1, on the pathogenesis of rheumatoid arthritis (RA) remains unclear. In this study, we found that the transcript level of lncRNA IFNG-AS1 was increased in the peripheral blood of RA patients. IFNG, as a target gene of IFNG-AS1, was overexpressed and positively correlated with the transcript level of IFNG-AS1 in the RA patients. Our data also showed that the transcript level of T-bet was upregulated and positively correlated with IFNG-AS1 expression. T-bet regulated the transcription of IFNG-AS1 in human CD4+ T cells in vitro. Furthermore, strong positive correlations were observed between the increased transcript level of IFNG-AS1 and the serum level of rheumatoid factor, the erythrocyte sedimentation rate, and the C-reactive protein in RA patients, and patients positive for anticyclic citrullinated peptide antibodies had increased levels of IFNG-AS1. Finally, receiver operating characteristic (ROC) curve analysis suggested that IFNG-AS1 might be a potential biomarker of RA. Taken together, our findings indicated that IFNG-AS1, guided by T-bet, is augmented in the peripheral blood of RA patients and may play a critical role in the pathogenesis of RA by regulating the expression of IFNG.


Subject(s)
Arthritis, Rheumatoid/genetics , Interferon-gamma/genetics , Oligodeoxyribonucleotides, Antisense/genetics , RNA, Long Noncoding/genetics , Th1 Cells/immunology , Adult , Aged , Biomarkers/metabolism , C-Reactive Protein/metabolism , Female , Gene Expression Regulation , Humans , Male , Middle Aged , Rheumatoid Factor/blood , T-Box Domain Proteins/genetics , T-Box Domain Proteins/metabolism , Transcriptome , Up-Regulation
16.
Acta Microbiol Immunol Hung ; 67(1): 66-72, 2020 Feb 11.
Article in English | MEDLINE | ID: mdl-32043369

ABSTRACT

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.


Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Helicobacter Infections/drug therapy , Helicobacter pylori/drug effects , Oligodeoxyribonucleotides, Antisense/pharmacology , Peptide Nucleic Acids/pharmacology , Bacterial Load/drug effects , Cell Line, Tumor , HT29 Cells , Helicobacter pylori/genetics , Humans , Microbial Sensitivity Tests , Oligodeoxyribonucleotides, Antisense/genetics , Peptide Nucleic Acids/genetics , Real-Time Polymerase Chain Reaction
17.
Nucleic Acid Ther ; 30(2): 94-103, 2020 04.
Article in English | MEDLINE | ID: mdl-32043907

ABSTRACT

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.


Subject(s)
Amyloid Neuropathies, Familial/drug therapy , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides/administration & dosage , Thrombocytopenia/blood , Adult , Aged , Amyloid Neuropathies, Familial/genetics , Amyloid Neuropathies, Familial/immunology , Amyloid Neuropathies, Familial/pathology , Female , Genetic Predisposition to Disease , Humans , Immune System Diseases/chemically induced , Immune System Diseases/immunology , Immune System Diseases/pathology , Immunoglobulin G , Intracranial Hemorrhages/chemically induced , Intracranial Hemorrhages/immunology , Intracranial Hemorrhages/pathology , Male , Middle Aged , Oligodeoxyribonucleotides, Antisense/administration & dosage , Oligonucleotides/adverse effects , Oligonucleotides, Antisense/adverse effects , Quality of Life , Thrombocytopenia/chemically induced , Thrombocytopenia/immunology , Thrombocytopenia/pathology
18.
IUBMB Life ; 72(5): 855-871, 2020 05.
Article in English | MEDLINE | ID: mdl-31913572

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/therapeutic use , Drug Carriers , Drug Resistance, Neoplasm/drug effects , Molecular Targeted Therapy/methods , Nanotechnology/methods , Neoplasms/therapy , Animals , Antineoplastic Agents/metabolism , Cell Line, Tumor , Dendrimers/chemistry , Dendrimers/pharmacokinetics , Drug Compounding/methods , Humans , Liposomes/chemistry , Liposomes/pharmacokinetics , Mice , Micelles , Nanoparticles/chemistry , Nanoparticles/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Oligodeoxyribonucleotides, Antisense/administration & dosage , Oligodeoxyribonucleotides, Antisense/genetics , Oligodeoxyribonucleotides, Antisense/metabolism , Xenograft Model Antitumor Assays
19.
Mol Cell Biol ; 40(8)2020 03 30.
Article in English | MEDLINE | ID: mdl-31964754

ABSTRACT

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.


Subject(s)
Amyloidosis/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Amyloidosis/pathology , Animals , Apolipoproteins E/metabolism , Brain/metabolism , Cognition/physiology , Disease Models, Animal , Hippocampus/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/metabolism , Microglia/pathology , Oligodeoxyribonucleotides, Antisense/pharmacology , Receptors, LDL/metabolism
20.
J Neurol ; 267(4): 1070-1079, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31853709

ABSTRACT

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.


Subject(s)
Activities of Daily Living , Amyloid Neuropathies, Familial/drug therapy , Oligodeoxyribonucleotides, Antisense/pharmacology , Oligonucleotides/pharmacology , Outcome Assessment, Health Care , Polyneuropathies/drug therapy , Quality of Life , Severity of Illness Index , Adult , Aged , Aged, 80 and over , Amyloid Neuropathies, Familial/complications , Double-Blind Method , Drug Administration Schedule , Female , Humans , Male , Middle Aged , Mobility Limitation , Oligodeoxyribonucleotides, Antisense/administration & dosage , Oligonucleotides/administration & dosage , Polyneuropathies/etiology
SELECTION OF CITATIONS
SEARCH DETAIL
...