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BioRxiv : the Preprint Server For... Apr 2024Antisense oligonucleotides (ASOs) are widely used as therapeutics for neurodegenerative diseases, cancers, and virus infections. One class of ASOs functions to enhance...
Antisense oligonucleotides (ASOs) are widely used as therapeutics for neurodegenerative diseases, cancers, and virus infections. One class of ASOs functions to enhance protein expression by sequestering the mature microRNA (miRNA) in a double-stranded structure within the RNA-induced silencing complex (RISC). An alternative approach for the targeted control of gene expression is to use ASOs that bind to the pre-elements of miRNAs (pre-miRNAs) and modulate their enzymatic processing. Here, we demonstrate that ASOs can be used to disrupt a specific structural feature, "junction," within pre-miR-31 that is important in directing efficient processing by the Dicer/TRBP complex. Furthermore, we extend and validate this strategy to pre-miR-144, which has a similar junction-dependent structure-function relationship. We found that a significant number of human pre-miRNAs are predicted to contain junctions, and validated our ASO approach on several members of this group. Importantly, we also verified the application of junction-targeting ASOs for the specific inhibition of pre-miRNA processing . Our study reemphasizes the important roles of RNA structure in regulating Dicer/TRBP processing of pre-miRNAs and provides the framework to develop structure-informed ASOs that serve to inhibit miRNA production.
PubMed: 38645194
DOI: 10.1101/2024.04.08.588531 -
Biochimica Et Biophysica Acta.... Jun 2024TGF-β is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-β co-receptor endoglin (ENG) tends to be...
TGF-β is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-β co-receptor endoglin (ENG) tends to be upregulated in kidney fibrosis. ENG has two membrane bound isoforms generated via alternative splicing. Long-ENG was shown to enhance the extent of renal fibrosis in an unilateral ureteral obstruction mouse model, while short-ENG inhibited renal fibrosis. Here we aimed to achieve terminal intron retention of endoglin using antisense-oligo nucleotides (ASOs), thereby shifting the ratio towards short-ENG to inhibit the TGF-β1-mediated pro-fibrotic response. We isolated mRNA from kidney biopsies of patients with chronic allograft disease (CAD) (n = 12) and measured total ENG and short-ENG mRNA levels. ENG mRNA was upregulated 2.3 fold (p < 0.05) in kidneys of CAD patients compared to controls, while the percentage short-ENG of the total ENG mRNA was significantly lower (1.8 fold; p < 0.05). Transfection of ASOs that target splicing regulatory sites of ENG into TK173 fibroblasts led to higher levels of short-ENG (2 fold; p < 0.05). In addition, we stimulated these cells with TGF-β1 and measured a decrease in upregulation of ACTA2, COL1A1 and FN1 mRNA levels, and protein expression of αSMA, collagen type I, and fibronectin. These results show a potential for ENG ASOs as a therapy to reduce interstitial fibrosis in CKD.
Topics: Humans; Fibrosis; Endoglin; Oligonucleotides, Antisense; Introns; Transforming Growth Factor beta1; Kidney; Male; Fibronectins; Female; Actins; Middle Aged; Animals; Collagen Type I; Alternative Splicing; Fibroblasts; RNA, Messenger; Mice; Cell Line
PubMed: 38642778
DOI: 10.1016/j.bbadis.2024.167186 -
Advanced Drug Delivery Reviews Jun 2024Fibrotic diseases are characterised by myofibroblast differentiation, uncontrolled pathological extracellular matrix accumulation, tissue contraction, scar formation... (Review)
Review
Fibrotic diseases are characterised by myofibroblast differentiation, uncontrolled pathological extracellular matrix accumulation, tissue contraction, scar formation and, ultimately tissue / organ dysfunction. The cornea, the transparent tissue located on the anterior chamber of the eye, is extremely susceptible to fibrotic diseases, which cause loss of corneal transparency and are often associated with blindness. Although topical corticosteroids and antimetabolites are extensively used in the management of corneal fibrosis, they are associated with glaucoma, cataract formation, corneoscleral melting and infection, imposing the need of far more effective therapies. Herein, we summarise and discuss shortfalls and recent advances in in vitro models (e.g. transforming growth factor-β (TGF-β) / ascorbic acid / interleukin (IL) induced) and drug (e.g. TGF-β inhibitors, epigenetic modulators) and gene (e.g. gene editing, gene silencing) therapeutic strategies in the corneal fibrosis context. Emerging therapeutical agents (e.g. neutralising antibodies, ligand traps, receptor kinase inhibitors, antisense oligonucleotides) that have shown promise in clinical setting but have not yet assessed in corneal fibrosis context are also discussed.
Topics: Humans; Fibrosis; Corneal Diseases; Animals; Genetic Therapy; Cornea; Transforming Growth Factor beta
PubMed: 38642593
DOI: 10.1016/j.addr.2024.115317 -
ELife Apr 2024We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail...
We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism. SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.
Topics: Animals; Cell Movement; Fibroblast Growth Factors; High-Temperature Requirement A Serine Peptidase 1; Neural Crest; Serpin E2; Signal Transduction; Xenopus laevis; Xenopus Proteins
PubMed: 38634469
DOI: 10.7554/eLife.91864 -
MedRxiv : the Preprint Server For... Apr 2024Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by the selective and progressive death of motor neurons (MNs)....
Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by the selective and progressive death of motor neurons (MNs). Understanding the genetic and molecular factors influencing ALS survival is crucial for disease management and therapeutics. In this study, we introduce a deep learning-powered genetic analysis framework to link rare noncoding genetic variants to ALS survival. Using data from human induced pluripotent stem cell (iPSC)-derived MNs, this method prioritizes functional noncoding variants using deep learning, links cis-regulatory elements (CREs) to target genes using epigenomics data, and integrates these data through gene-level burden tests to identify survival-modifying variants, CREs, and genes. We apply this approach to analyze 6,715 ALS genomes, and pinpoint four novel rare noncoding variants associated with survival, including chr7:76,009,472:C>T linked to . CRISPR-Cas9 editing of this variant increases expression in iPSC-derived MNs and exacerbates ALS-specific phenotypes, including TDP-43 mislocalization. Suppressing with an antisense oligonucleotide (ASO), showing no toxicity, completely rescues ALS-associated survival defects in neurons derived from sporadic ALS patients and from carriers of the ALS-associated G4C2-repeat expansion within ASO targeting of may be a broadly effective therapeutic approach for ALS. Our framework provides a generic and powerful approach for studying noncoding genetics of complex human diseases.
PubMed: 38633814
DOI: 10.1101/2024.03.30.24305115 -
The Journal of Clinical Investigation Apr 2024Renal interstitial fibrosis is an important mechanism in the progression of chronic kidney disease (CKD) to end-stage kidney disease. However, we lack specific...
Renal interstitial fibrosis is an important mechanism in the progression of chronic kidney disease (CKD) to end-stage kidney disease. However, we lack specific treatments to slow or halt renal fibrosis. Ribosome profiling identified upregulation of a secreted micropeptide, C4orf48 (Cf48), in mouse diabetic nephropathy. Cf48 RNA and protein levels were upregulated in tubular epithelial cells in human and experimental CKD. Serum Cf48 levels were increased in human CKD and correlated with loss of kidney function, increasing CKD stage, and the degree of active interstitial fibrosis. Cf48 overexpression in mice accelerated renal fibrosis, while Cf48 gene deletion or knockdown by antisense oligonucleotides significantly reduced renal fibrosis in CKD models. In vitro, recombinant Cf48 (rCf48) enhanced TGF-β1-induced fibrotic responses in renal fibroblasts and epithelial cells independently of Smad3 phosphorylation. Cellular uptake of Cf48 and its profibrotic response in fibroblasts operated via the transferrin receptor. RNA immunoprecipitation-sequencing identified Cf48 binding to mRNA of genes involved in the fibrotic response, including Serpine1, Acta2, Ccn2, and Col4a1. rCf48 binds to the 3'UTR of Serpine1 and increases mRNA half-life. We identify the secreted Cf48 micropeptide as a potential enhancer of renal fibrosis that operates as an RNA-binding peptide to promote the production of extracellular matrix.
Topics: Animals; Humans; Male; Mice; 3' Untranslated Regions; Diabetic Nephropathies; Fibrosis; Kidney; Mice, Knockout; Renal Insufficiency, Chronic; RNA-Binding Proteins; Smad3 Protein; Transforming Growth Factor beta1; Nerve Tissue Proteins
PubMed: 38625739
DOI: 10.1172/JCI178392 -
Nucleic Acids Research May 2024Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular...
Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO.
Topics: Humans; Oligonucleotides, Antisense; TRPC Cation Channels; Calcium; A549 Cells; Animals; Mice; Imidazoles; TRPC6 Cation Channel; Egtazic Acid; Endosomes; Cell Line, Tumor
PubMed: 38621757
DOI: 10.1093/nar/gkae245 -
Proceedings of the National Academy of... Apr 2024Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different...
Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.
Topics: Humans; C9orf72 Protein; Alleles; Amyotrophic Lateral Sclerosis; Frontotemporal Dementia; Motor Neurons; Mutation; DNA Repeat Expansion; Dipeptides
PubMed: 38621131
DOI: 10.1073/pnas.2307814121 -
Biological & Pharmaceutical Bulletin 2024In this study, we prepared antisense oligonucleotide (ASO)-encapsulated nanoparticles (NPs) with a suitable profile for oral administration for the treatment of...
In this study, we prepared antisense oligonucleotide (ASO)-encapsulated nanoparticles (NPs) with a suitable profile for oral administration for the treatment of inflammatory bowel disease (IBD). We chose a water-in-oil-in-water (w/o/w) method to prepare the NPs using poly(lactide-co-glycolide) as a matrix and Pluronic as a stabilizer. The obtained NPs had a suitable diameter (158 nm) for the penetration of the mucus layer, endocytic uptake by enterocytes, and accumulation in inflammatory lesions in the intestine. The amount of ASOs in the NPs was relatively large (6.41% (w/w)). When the NPs were stably dispersed in solutions that mimicked gastrointestinal (GI) juice, minimal leakage of ASOs was demonstrated over the required period. The NPs were administered orally to mice with colitis induced by dextran sodium sulfate, which reduced target gene expression in the colons and rectums of the mice, whereas naked ASO administration caused no reduction in gene expression. Thus, the NPs have the potential of promising oral carriers of ASOs for the treatment of IBD that specifically target inflammatory lesions in the GI tract, thereby reducing the non-specific toxic effects of ASOs.
Topics: Animals; Mice; Oligonucleotides, Antisense; Inflammatory Bowel Diseases; Administration, Oral; Nanoparticles; Water
PubMed: 38616115
DOI: 10.1248/bpb.b23-00769 -
Nucleic Acids Research May 2024Glioblastoma multiforme is a universally lethal brain tumor that largely resists current surgical and drug interventions. Despite important advancements in understanding...
Glioblastoma multiforme is a universally lethal brain tumor that largely resists current surgical and drug interventions. Despite important advancements in understanding GBM biology, the invasiveness and heterogeneity of these tumors has made it challenging to develop effective therapies. Therapeutic oligonucleotides-antisense oligonucleotides and small-interfering RNAs-are chemically modified nucleic acids that can silence gene expression in the brain. However, activity of these oligonucleotides in brain tumors remains inadequately characterized. In this study, we developed a quantitative method to differentiate oligonucleotide-induced gene silencing in orthotopic GBM xenografts from gene silencing in normal brain tissue, and used this method to test the differential silencing activity of a chemically diverse panel of oligonucleotides. We show that oligonucleotides chemically optimized for pharmacological activity in normal brain tissue do not show consistent activity in GBM xenografts. We then survey multiple advanced oligonucleotide chemistries for their activity in GBM xenografts. Attaching lipid conjugates to oligonucleotides improves silencing in GBM cells across several different lipid classes. Highly hydrophobic lipid conjugates cholesterol and docosanoic acid enhance silencing but at the cost of higher neurotoxicity. Moderately hydrophobic, unsaturated fatty acid and amphiphilic lipid conjugates still improve activity without compromising safety. These oligonucleotide conjugates show promise for treating glioblastoma.
Topics: Glioblastoma; Animals; RNA, Small Interfering; Humans; Mice; Cell Line, Tumor; Xenograft Model Antitumor Assays; Brain Neoplasms; Oligonucleotides, Antisense; Gene Silencing; Mice, Nude
PubMed: 38613388
DOI: 10.1093/nar/gkae260