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EMBO Molecular Medicine Nov 2023Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality. The advent of approved treatments for this devastating condition has significantly changed...
Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality. The advent of approved treatments for this devastating condition has significantly changed SMA patients' life expectancy and quality of life. Nevertheless, these are not without limitations, and research efforts are underway to develop new approaches for improved and long-lasting benefits for patients. Protein arginine methyltransferases (PRMTs) are emerging as druggable epigenetic targets, with several small-molecule PRMT inhibitors already in clinical trials. From a screen of epigenetic molecules, we have identified MS023, a potent and selective type I PRMT inhibitor able to promote SMN2 exon 7 inclusion in preclinical SMA models. Treatment of SMA mice with MS023 results in amelioration of the disease phenotype, with strong synergistic amplification of the positive effect when delivered in combination with the antisense oligonucleotide nusinersen. Moreover, transcriptomic analysis revealed that MS023 treatment has minimal off-target effects, and the added benefit is mainly due to targeting neuroinflammation. Our study warrants further clinical investigation of PRMT inhibition both as a stand-alone and add-on therapy for SMA.
Topics: Animals; Humans; Infant; Mice; Exons; Muscular Atrophy, Spinal; Oligonucleotides; Quality of Life; Survival of Motor Neuron 2 Protein
PubMed: 37724723
DOI: 10.15252/emmm.202317683 -
The Journal of Biological Chemistry Aug 2023Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation, and tissue remodeling. Aberrant proteolytic activity of HNE contributes to organ...
Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation, and tissue remodeling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used the systematic evolution of ligands by exponential enrichment to develop ssDNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models.
Topics: Humans; Cystic Fibrosis; Emphysema; Leukocyte Elastase; Neutrophils; Serine Proteinase Inhibitors; Aptamers, Nucleotide; Sensitivity and Specificity; Enzyme Activation; Proteolysis; Cells, Cultured
PubMed: 37286041
DOI: 10.1016/j.jbc.2023.104889 -
Nature Communications Feb 2024Drugs that target pre-mRNA splicing hold great therapeutic potential, but the quantitative understanding of how these drugs work is limited. Here we introduce...
Drugs that target pre-mRNA splicing hold great therapeutic potential, but the quantitative understanding of how these drugs work is limited. Here we introduce mechanistically interpretable quantitative models for the sequence-specific and concentration-dependent behavior of splice-modifying drugs. Using massively parallel splicing assays, RNA-seq experiments, and precision dose-response curves, we obtain quantitative models for two small-molecule drugs, risdiplam and branaplam, developed for treating spinal muscular atrophy. The results quantitatively characterize the specificities of risdiplam and branaplam for 5' splice site sequences, suggest that branaplam recognizes 5' splice sites via two distinct interaction modes, and contradict the prevailing two-site hypothesis for risdiplam activity at SMN2 exon 7. The results also show that anomalous single-drug cooperativity, as well as multi-drug synergy, are widespread among small-molecule drugs and antisense-oligonucleotide drugs that promote exon inclusion. Our quantitative models thus clarify the mechanisms of existing treatments and provide a basis for the rational development of new therapies.
Topics: Humans; RNA Splicing; Azo Compounds; Oligonucleotides; Oligonucleotides, Antisense; RNA Splice Sites; Muscular Atrophy, Spinal; Pyrimidines
PubMed: 38424098
DOI: 10.1038/s41467-024-46090-5 -
Biomolecules Jul 2023Poly(ADP-ribose) (PAR) Polymerase 1 (PARP-1), also known as ADP-ribosyl transferase with diphtheria toxin homology 1 (ARTD-1), is a critical player in DNA damage repair,... (Review)
Review
Poly(ADP-ribose) (PAR) Polymerase 1 (PARP-1), also known as ADP-ribosyl transferase with diphtheria toxin homology 1 (ARTD-1), is a critical player in DNA damage repair, during which it catalyzes the ADP ribosylation of self and target enzymes. While the nuclear localization of PARP-1 has been well established, recent studies also suggest its mitochondrial localization. In this review, we summarize the differences between mitochondrial and nuclear Base Excision Repair (BER) pathways, the involvement of PARP-1 in mitochondrial and nuclear BER, and its functional interplay with other BER enzymes.
Topics: Poly(ADP-ribose) Polymerase Inhibitors; DNA Repair; Mitochondria; Poly Adenosine Diphosphate Ribose
PubMed: 37627260
DOI: 10.3390/biom13081195 -
Nature Communications Aug 2023DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases....
DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.
Topics: Polymerase Chain Reaction; DNA; DNA Methylation; Temperature; Oligonucleotides; CpG Islands
PubMed: 37620381
DOI: 10.1038/s41467-023-40873-y -
Nucleic Acids Research Sep 2023Programmable site-specific nucleases promise to unlock myriad applications in basic biology research, biotechnology and gene therapy. Gene-editing systems have...
Programmable site-specific nucleases promise to unlock myriad applications in basic biology research, biotechnology and gene therapy. Gene-editing systems have revolutionized our ability to engineer genomes across diverse eukaryotic species. However, key challenges, including delivery, specificity and targeting organellar genomes, pose barriers to translational applications. Here, we use peptide nucleic acids (PNAs) to facilitate precise DNA strand invasion and unwinding, enabling prokaryotic Argonaute (pAgo) proteins to specifically bind displaced single-stranded DNA and introduce site-specific double-strand breaks (DSBs) independent of the target sequence. We named this technology PNA-assisted pAgo editing (PNP editing) and determined key parameters for designing PNP editors to efficiently generate programable site-specific DSBs. Our design allows the simultaneous use of multiple PNP editors to generate multiple site-specific DSBs, thereby informing design considerations for potential in vitro and in vivo applications, including genome editing.
Topics: CRISPR-Cas Systems; DNA; DNA Breaks, Double-Stranded; Gene Editing; Genome; Peptide Nucleic Acids; Argonaute Proteins
PubMed: 37560931
DOI: 10.1093/nar/gkad655 -
Cells Sep 2023The potential of oligonucleotide therapeutics is undeniable as more than 15 drugs have been approved to treat various diseases in the liver, central nervous system... (Review)
Review
The potential of oligonucleotide therapeutics is undeniable as more than 15 drugs have been approved to treat various diseases in the liver, central nervous system (CNS), and muscles. However, achieving effective delivery of oligonucleotide therapeutics to specific tissues still remains a major challenge, limiting their widespread use. Chemical modifications play a crucial role to overcome biological barriers to enable efficient oligonucleotide delivery to the tissues/cells of interest. They provide oligonucleotide metabolic stability and confer favourable pharmacokinetic/pharmacodynamic properties. This review focuses on the various chemical approaches implicated in mitigating the delivery problem of oligonucleotides and their limitations. It highlights the importance of linkers in designing oligonucleotide conjugates and discusses their potential role in escaping the endosomal barrier, a bottleneck in the development of oligonucleotide therapeutics.
Topics: Endosomes; Central Nervous System; Liver; Muscles; Oligonucleotides
PubMed: 37759475
DOI: 10.3390/cells12182253 -
Nucleic Acids Research Aug 2023Nucleic acids not only form the basis of heredity, but are increasingly a source of novel nano-structures, -devices and drugs. This has spurred the development of...
Nucleic acids not only form the basis of heredity, but are increasingly a source of novel nano-structures, -devices and drugs. This has spurred the development of chemically modified alternatives (xeno nucleic acids (XNAs)) comprising chemical configurations not found in nature to extend their chemical and functional scope. XNAs can be evolved into ligands (XNA aptamers) that bind their targets with high affinity and specificity. However, detailed investigations into structural and functional aspects of XNA aptamers have been limited. Here we describe a detailed structure-function analysis of LYS-S8-19, a 1',5'-anhydrohexitol nucleic acid (HNA) aptamer to hen egg-white lysozyme (HEL). Mapping of the aptamer interaction interface with its cognate HEL target antigen revealed interaction epitopes, affinities, kinetics and hot-spots of binding energy similar to protein ligands such as anti-HEL-nanobodies. Truncation analysis and molecular dynamics (MD) simulations suggest that the HNA aptamer core motif folds into a novel and not previously observed HNA tertiary structure, comprising non-canonical hT-hA-hT/hT-hT-hT triplet and hG4-quadruplex structures, consistent with its recognition by two different G4-specific antibodies.
Topics: Ligands; Aptamers, Nucleotide; G-Quadruplexes; Nucleic Acids; Molecular Dynamics Simulation; SELEX Aptamer Technique
PubMed: 37439359
DOI: 10.1093/nar/gkad592 -
The Journal of Biological Chemistry Jul 2023Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25 nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature...
Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25 nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature cleavage and polyadenylation of thousands of genes, a phenomenon known as U1 snRNP telescripting; however, the underlying mechanism remains elusive. In this study, we demonstrated that U1 AMO could disrupt U1 snRNP structure both in vitro and in vivo, thereby affecting the U1 snRNP-RNAP polymerase II interaction. By performing chromatin immunoprecipitation sequencing for phosphorylation of Ser2 and Ser5 of the C-terminal domain of RPB1, the largest subunit of RNAP polymerase II, we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high phosphorylation of Ser2 signal at intronic cryptic polyadenylation sites (PASs). In addition, we showed that core 3'processing factors CPSF/CstF are involved in the processing of intronic cryptic PAS. Their recruitment accumulated toward cryptic PASs upon U1 AMO treatment, as indicated by chromatin immunoprecipitation sequencing and individual-nucleotide resolution CrossLinking and ImmunoPrecipitation sequencing analysis. Conclusively, our data suggest that disruption of U1 snRNP structure mediated by U1 AMO provides a key for understanding the U1 telescripting mechanism.
Topics: Morpholinos; Oligonucleotides, Antisense; Polyadenylation; Ribonucleoprotein, U1 Small Nuclear; RNA Polymerase II; RNA Precursors; Humans; HeLa Cells; Gene Knockdown Techniques; Cleavage And Polyadenylation Specificity Factor; Cleavage Stimulation Factor; Transcription, Genetic
PubMed: 37224962
DOI: 10.1016/j.jbc.2023.104854 -
Proceedings of the National Academy of... Jun 2023We previously demonstrated that the polycomb repressive complex 2 chromatin-modifying enzyme can directly transfer between RNA and DNA without a free-enzyme intermediate...
We previously demonstrated that the polycomb repressive complex 2 chromatin-modifying enzyme can directly transfer between RNA and DNA without a free-enzyme intermediate state. Simulations suggested that such a direct transfer mechanism may be generally necessary for RNA to recruit proteins to chromatin, but the prevalence of direct transfer capability is unknown. Herein, we used fluorescence polarization assays and observed direct transfer for several well-characterized nucleic acid-binding proteins: three-prime repair exonuclease 1, heterogeneous nuclear ribonucleoprotein U, Fem-3-binding factor 2, and MS2 bacteriophage coat protein. For TREX1, the direct transfer mechanism was additionally observed in single-molecule assays, and the data suggest that direct transfer occurs through an unstable ternary intermediate with partially associated polynucleotides. Generally, direct transfer could allow many DNA- and RNA-binding proteins to conduct a one-dimensional search for their target sites. Furthermore, proteins that bind both RNA and DNA might be capable of readily translocating between those ligands.
Topics: DNA-Binding Proteins; Polynucleotides; RNA; RNA-Binding Proteins; DNA; Chromatin
PubMed: 37339225
DOI: 10.1073/pnas.2220537120