Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE134926 | GEO | 2019/07/27
REPOSITORIES: GEO
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Project description:Myotonic dystrophy type 1 (DM1) is an RNA dominant disease in which mutant transcripts containing an expanded CUG repeat (CUGexp) cause muscle dysfunction by interfering with biogenesis of other mRNAs. The toxic effects of mutant RNA are mediated partly through sequestration of splicing regulator Muscleblind-like 1 (Mbnl1), a protein that binds to CUGexp RNA. A gene that is prominently affected encodes chloride channel 1 (Clcn1), resulting in hyperexcitability of muscle (myotonia). To identify DM1-affected genes and study mechanisms for dysregulation, we performed global mRNA profiling in transgenic mice that express CUGexp RNA, as compared to Mbnl1 knockout and Clcn1 null mice. We found that the majority of changes induced by CUGexp RNA in skeletal muscle can be explained by reduced activity of Mbnl1, including many changes that are secondary to myotonia. The pathway most affected comprises genes involved in calcium signaling and homeostasis. Some effects of CUGexp RNA on gene expression are caused by abnormal alternative splicing or downregulation of Mbnl1-interacting mRNAs. However, several of the most highly dysregulated genes showed altered transcription, as indicated by parallel changes of the corresponding premRNAs. These results support the idea that trans-dominant effects of CUGexp RNA on gene expression in this transgenic model may occur at the level of transcription, RNA processing, and mRNA decay, and are mediated mainly but not entirely through sequestration of Mbnl1. Experiment Overall Design: All experiments involved generating expression profiles of quadriceps muscles taken from mice. Experiments 1 and 2: samples were hybridized to Moe430A and Moe430B arrays. Experiment 3: samples were hybridized to Mouse Genome 430 2.0 array. Experiment 1 compared expression profiles of wild-type mice (FVB strain) with two lines, designated 20b and 41, of CUGexp transgenic mice with FVB background. Experiment 2 compared expression profiles of Clcn1-null (myotonic) mice with the wild-type background strain (BALB). Experiment 3 compared expression profiles of Mbnl1-null mice with the wild-type background strain (FVB).
2009-02-13 | E-GEOD-14691 | biostudies-arrayexpress
Project description:Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes bear great potential in the fight against antimicrobial resistance and for precision microbiome editing. The development of such antisense antibiotics has primarily focused on using phosphoramidite morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based therapy in general. Here, we compare seven chemically distinct 10mer-ASOs, all designed to target the same essential acpP mRNA upon delivery with a KFF-peptide carrier into Salmonella, comparing PNA, PMO, phosphorothioate modified-DNA (PTO), 2’-methoxylated RNA (RNA-OMe), 2’-methoxyethylated RNA (RNA-MOE), 2’-fluorinated RNA (RNA-F) and 2’-locked RNA (LNA). Our systematic analysis using a variety of in vitro and in vivo methods to evaluate ASO uptake, target pairing and bacterial killing suggests that only PNA and PMO make it into Salmonella to cause bacterial growth inhibition. However, given their high target binding and translational repression activity in vitro, ASOs based on LNA and RNA-MOE appear to be promising candidates for antisense antibiotics that will require the identification of an efficient carrier.
2023-07-11 | GSE232819 | GEO
Project description:Myotonic dystrophy type 1 (DM1) is an RNA dominant disease in which mutant transcripts containing an expanded CUG repeat (CUGexp) cause muscle dysfunction by interfering with biogenesis of other mRNAs. The toxic effects of mutant RNA are mediated partly through sequestration of splicing regulator Muscleblind-like 1 (Mbnl1), a protein that binds to CUGexp RNA. A gene that is prominently affected encodes chloride channel 1 (Clcn1), resulting in hyperexcitability of muscle (myotonia). To identify DM1-affected genes and study mechanisms for dysregulation, we performed global mRNA profiling in transgenic mice that express CUGexp RNA, as compared to Mbnl1 knockout and Clcn1 null mice. We found that the majority of changes induced by CUGexp RNA in skeletal muscle can be explained by reduced activity of Mbnl1, including many changes that are secondary to myotonia. The pathway most affected comprises genes involved in calcium signaling and homeostasis. Some effects of CUGexp RNA on gene expression are caused by abnormal alternative splicing or downregulation of Mbnl1-interacting mRNAs. However, several of the most highly dysregulated genes showed altered transcription, as indicated by parallel changes of the corresponding premRNAs. These results support the idea that trans-dominant effects of CUGexp RNA on gene expression in this transgenic model may occur at the level of transcription, RNA processing, and mRNA decay, and are mediated mainly but not entirely through sequestration of Mbnl1. Keywords: comparison of normal and transgenic mice
2009-02-05 | GSE14691 | GEO
Project description:Distinct RNA-mediated impacts on alternative splicing and extracellular matrix gene expression in a mouse model of myotonic dystrophy. Myotonic dystrophy (DM1) is associated with expression of expanded CTG DNA repeats as RNA (CUGexp RNA). To test whether CUGexp RNA creates a global splicing defect, we compared skeletal muscle of two mouse DM1 models, one expressing a CTGexp transgene, and another homozygous for a defective Mbnl1 gene. Strong correlation in splicing changes for ~100 new Mbnl1-regulated exons indicates loss of Mbnl1 explains >80% of the splicing pathology due to CUGexp RNA. In contrast, only about half of mRNA level changes can be attributed to loss of Mbnl1, indicating CUGexp RNA has Mbnl1-independent effects, particularly on mRNAs for extracellular matrix (ECM) proteins. We propose that CUGexp RNA causes two separate effects: loss of Mbnl1 function, disrupting splicing, and loss of another function that disrupts ECM mRNA regulation, possibly mediated by MBNL2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies. MBNL1 knockout and HSALR mice on FVB background. To test whether CUGexp RNA creates a global splicing defect, we compared skeletal muscle of two mouse DM1 models, one expressing a CTGexp transgene, and another homozygous for a defective Mbnl1 gene. These samples were compared to the skeletal muscle of a wildtype mouse.
2010-01-23 | E-GEOD-17986 | biostudies-arrayexpress
Project description:Myotonic dystrophy type 1 (DM1) is a dominantly inherited disorder due to a toxic gain of function of RNA transcripts containing expanded CUG repeats (CUGexp). Patients with DM1 present with multisystemic symptoms, such as muscle wasting, cognitive impairment, cataract, frontal baldness, and endocrine defects, which resemble accelerated aging. Although the involvement of cellular senescence, a critical component of aging, was suggested in studies of DM1 patient-derived cells, the detailed mechanism of cellular senescence caused by CUGexp RNA remains unelucidated. Here, we developed a DM1 cell model that conditionally expressed CUGexp RNA in human primary cells so that we could perform a detailed assessment that eliminated the variability in primary cells from different origins. To identify the pathway that induced cellular senescence, we conducted comprehensive gene expression analysis using RNA sequencing (RNA-Seq) in CUGexp+ and CUGexp− cells.
2022-04-19 | GSE196265 | GEO
Project description:Myotonic dystrophy type 1 (DM1) is a dominantly inherited disease that affects multiple organ systems. Cardiac dysfunction is the second leading cause of death in DM1. We quantified gene expression in heart tissue from a heart-specific DM1 mouse model (EpA960/MCM) which inducibly expresses human DMPK exon 15 containing 960 CUG expanded repeats and that reproduced Celf1 up regulation. To assess if, in addition to splicing and miRNA defects, CUGexp RNA also perturbed the steady state mRNA levels of genes, we carried out a microarray study on wildtype E14, adult, MCM controls and DM1 mouse hearts. As anticipated we noted a large number of genes to be developmentally regulated in wildtype hearts, however, within 72h of induction of CUGexp RNA there appeared to be a coordinate adult-to-embryonic shift in steady state levels of many genes. We identified transcripts over-expressed or under-expressed in hearts of wildtype adult mice, wildtype embryonic day 14 (E14), and DM1 mice induced to express CUGexp RNA for 72h and 1wk, when compared to MCM controls. Multiple group comparison.
2014-01-24 | E-GEOD-48991 | biostudies-arrayexpress
Project description:DM1 (OMIM #160900) is a chronic, slowly progressing multisystemic disease, with symptoms that include loss of muscle strength, myotonia and excessive fatigue. DM1 is caused by a dynamic expansion of CTG repeats, ranging from 50 to several thousands, in the 3’ untranslated region of the DMPK gene. Characteristic molecular features of the disease have been associated with a toxic RNA gain of function of the CUG expansions. Expanded CUG repeats have been demonstrated to be toxic per se in several cell types and animal models disrupting gene transcription and pre-mRNA alternative splicing. Deep sequencing is here used to characterize the deregulation of the RNAs associated to the RISC complex in the skeletal muscle of DM1 patients.
2018-07-15 | GSE108590 | GEO
Project description:DM1 (OMIM #160900) is a chronic, slowly progressing multisystemic disease, with symptoms that include loss of muscle strength, myotonia and excessive fatigue. DM1 is caused by a dynamic expansion of CTG repeats, ranging from 50 to several thousands, in the 3’ untranslated region of the DMPK gene. Characteristic molecular features of the disease have been associated with a toxic RNA gain of function of the CUG expansions. Expanded CUG repeats have been demonstrated to be toxic per se in several cell types and animal models disrupting gene transcription and pre-mRNA alternative splicing. Deep sequencing is here used to characterize the deregulation of the RNAs associated to the RISC complex in the skeletal muscle of DM1 patients.
2018-07-15 | GSE108589 | GEO
Project description:DM1 (OMIM #160900) is a chronic, slowly progressing multisystemic disease, with symptoms that include loss of muscle strength, myotonia and excessive fatigue. DM1 is caused by a dynamic expansion of CTG repeats, ranging from 50 to several thousands, in the 3’ untranslated region of the DMPK gene. Characteristic molecular features of the disease have been associated with a toxic RNA gain of function of the CUG expansions. Expanded CUG repeats have been demonstrated to be toxic per se in several cell types and animal models disrupting gene transcription and pre-mRNA alternative splicing. Deep sequencing is here used to characterize the deregulation of the RNAs associated to the RISC complex in the skeletal muscle of DM1 patients.
2018-07-15 | GSE108591 | GEO
Project description:Antisense oligonucleotides (ASOs) are being actively investigated as potential therapeutics for a broad range of neurodegenerative diseases. While these small oligonucleotides have been effective in the clinic, many basic questions regarding ASO internalization, trafficking, and modes of enhancing delivery remain. To address these questions, we investigated how lipid nanoparticle (LNP) delivery affects ASO uptake and distribution in the brain. We show that ASOs are internalized and active in central nervous system (CNS) cell types both in vivo and in vitro. While differential cellular activity and polarization states do not affect ASO potency, encapsulating ASOs in LNPs increases ASO activity up to 100-fold in cultured CNS cells. This dramatic increase in efficacy is facilitated by the intracellular trafficking of ASOs away from lysosomes or enhanced ASO uptake, which is cell-type-specific. We assessed the translatability of these results by screening ASO-LNPs in vitro and intracerebroventricularly injecting top performing formulations in mice. ASO-LNP delivery induced a strong ASO-dependent microgliosis response in the brain revealing that LNP encapsulation cannot mask ASO-mediated toxicity. However, LNP-delivered ASOs did not downregulate mRNA levels in bulk tissue due to changes in ASO distribution. While ASOs distribute widely across the brain after bulk injection, ASO-LNPs are preferentially internalized by cells lining the blood vessels and ventricles. Consistent with our findings in cells, ASOs localize to the endolysosomal system following both ASO and ASO-LNP delivery in the brain as determined using immunoelectron microscopy. These data provide valuable insights into how LNPs regulate ASO uptake and distribution in the brain, and support further development of ASO-LNPs for treating CNS disorders.
2023-05-15 | GSE220777 | GEO