Browse
Submit Data
Databases
API
Help

Dataset Information

6 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Restoration of the normal splicing pattern of the PLP1 gene by means of an antisense oligonucleotide directed against an exonic mutation.

ABSTRACT: An exonic missense mutation, c.436C>G, in the PLP1 gene of a patient affected by the hypomyelinating leukodystrophy, Pelizaeus-Merzbacher disease, has previously been found to be responsible for the alteration of the canonical alternative splicing profile of the PLP1 gene leading to the loss of the longer PLP isoform. Here we show that the presence of the c.436C>G mutation served to introduce regulatory motifs that appear to be responsible for the perturbed splicing pattern that led to loss of the major PLP transcript. With the aim of disrupting the interaction between the PLP1 splicing regulatory motifs and their cognate splicing factors, we designed an antisense oligonucleotide-based in vitro correction protocol that successfully restored PLP transcript production in oligodendrocyte precursor cells.

SUBMITTER: Regis S

PROVIDER: S-EPMC3760819 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Restoration of the normal splicing pattern of the PLP1 gene by means of an antisense oligonucleotide directed against an exonic mutation.

Regis Stefano S Corsolini Fabio F Grossi Serena S Tappino Barbara B Cooper David N DN Filocamo Mirella M

PloS one 20130903 9

An exonic missense mutation, c.436C>G, in the PLP1 gene of a patient affected by the hypomyelinating leukodystrophy, Pelizaeus-Merzbacher disease, has previously been found to be responsible for the alteration of the canonical alternative splicing profile of the PLP1 gene leading to the loss of the longer PLP isoform. Here we show that the presence of the c.436C>G mutation served to introduce regulatory motifs that appear to be responsible for the perturbed splicing pattern that led to loss of t ...[more]

PMID: 24019930

Similar Datasets

Antisense oligonucleotide-directed inhibition of nonsense-mediated mRNA decay.

Project description:Nonsense-mediated mRNA decay (NMD) is a cellular quality-control mechanism that is thought to exacerbate the phenotype of certain pathogenic nonsense mutations by preventing the expression of semi-functional proteins. NMD also limits the efficacy of read-through compound (RTC)-based therapies. Here, we report a gene-specific method of NMD inhibition using antisense oligonucleotides (ASOs) and combine this approach with an RTC to effectively restore the expression of full-length protein from a nonsense-mutant allele.

| S-EPMC4744113 | biostudies-literature

An exonic splicing enhancer within a bidirectional coding sequence regulates alternative splicing of an antisense mRNA.

Project description:The discovery of increasing numbers of genes with overlapping sequences highlights the problem of expression in the context of constraining regulatory elements from more than one gene. This study identifies regulatory sequences encompassed within two genes that overlap in an antisense orientation at their 3' ends. The genes encode the alpha-thyroid hormone receptor gene (TRalpha or NR1A1) and Rev-erbalpha (NR1D1). In mammals TRalpha pre-mRNAs are alternatively spliced to yield mRNAs encoding functionally antagonistic proteins: TRalpha1, an authentic thyroid hormone receptor; and TRalpha2, a non-hormone-binding variant that acts as a repressor. TRalpha2-specific splicing requires two regulatory elements that overlap with Rev-erbalpha sequences. Functional mapping of these elements reveals minimal splicing enhancer elements that have evolved within the constraints of the overlapping Rev-erbalpha sequence. These results provide insight into the evolution of regulatory elements within the context of bidirectional coding sequences. They also demonstrate the ability of the genetic code to accommodate multiple layers of information within a given sequence, an important property of the code recently suggested on theoretical grounds.

| S-EPMC2893273 | biostudies-literature

Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression.

Project description:While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases.

| S-EPMC7347940 | biostudies-literature

Effective exon skipping and dystrophin restoration by 2'-o-methoxyethyl antisense oligonucleotide in dystrophin-deficient mice.

Project description:Antisense oligonucleotide (AO)-mediated exon-skipping therapy is one of the most promising therapeutic strategies for Duchenne Muscular Dystrophy (DMD) and several AO chemistries have been rigorously investigated. In this report, we focused on the effect of 2'-O-methoxyethyl oligonucleotides (MOE) on exon skipping in cultured mdx myoblasts and mice. Efficient dose-dependent skipping of targeted exon 23 was achieved in myoblasts with MOE AOs of different lengths and backbone chemistries. Furthermore, we established that 25-mer MOE phosphorothioate (PS) AOs provided the greatest exon-skipping efficacy. When compared with 2'O methyl phosphorothioate (2'OmePS) AOs, 25-mer MOE (PS) AOs also showed higher exon-skipping activity in vitro and in mdx mice after intramuscular injections. Characterization of uptake in vitro corroborated with exon-skipping results, suggesting that increased uptake of 25-mer MOE PS AOs might partly contribute to the difference in exon-skipping activity observed in vitro and in mdx mice. Our findings demonstrate the substantial potential for MOE PS AOs as an alternative option for the treatment of DMD.

| S-EPMC3637291 | biostudies-literature

Oligonucleotide directed misfolding of RNA inhibits Candida albicans group I intron splicing.

Project description:RNA is becoming an important therapeutic target. Many potential RNA targets require secondary or tertiary structure for function. Examples include ribosomal RNAs, RNase P RNAs, mRNAs with untranslated regions that regulate translation, and group I and group II introns. Here, a method is described to inhibit RNA function by exploiting the propensity of RNA to adopt multiple folded states that are of similar free energy. This method, called oligonucleotide directed misfolding of RNA (ODMiR), uses short oligonucleotides to stabilize inactive structures. The ODMiR method is demonstrated with the group I intron from Candida albicans, a human pathogen. The oligonucleotides, (L)(TACCTTTC) and T(L)CT(L)AC(L)GA(L)CG(L)GC(L)C, with L denoting a locked nucleic acid residue, inhibit 50% of group I intron splicing in a transcription mixture at about 150 and 30 nM oligonucleotide concentration, respectively. Both oligonucleotides induce misfolds as determined by native gel electrophoresis and diethyl pyrocarbonate modification. The ODMiR approach provides a potential therapeutic strategy applicable to RNAs with secondary or tertiary structures required for function.

| S-EPMC123215 | biostudies-literature

Antisense oligonucleotide-induced alternative splicing of the APOB mRNA generates a novel isoform of APOB.

Project description:BackgroundApolipoprotein B (APOB) is an integral part of the LDL, VLDL, IDL, Lp(a) and chylomicron lipoprotein particles. The APOB pre-mRNA consists of 29 constitutively-spliced exons. APOB exists as two natural isoforms: the full-length APOB100 isoform, assembled into LDL, VLDL, IDL and Lp(a) and secreted by the liver in humans; and the C-terminally truncated APOB48, assembled into chylomicrons and secreted by the intestine in humans. Down-regulation of APOB100 is a potential therapy to lower circulating LDL and cholesterol levels.ResultsWe investigated the ability of 2'O-methyl RNA antisense oligonucleotides (ASOs) to induce the skipping of exon 27 in endogenous APOB mRNA in HepG2 cells. These ASOs are directed towards the 5' and 3' splice-sites of exon 27, the branch-point sequence (BPS) of intron 26-27 and several predicted exonic splicing enhancers within exon 27. ASOs targeting either the 5' or 3' splice-site, in combination with the BPS, are the most effective. The splicing of other alternatively spliced genes are not influenced by these ASOs, suggesting that the effects seen are not due to non-specific changes in alternative splicing. The skip 27 mRNA is translated into a truncated isoform, APOB87SKIP27.ConclusionThe induction of APOB87SKIP27 expression in vivo should lead to decreased LDL and cholesterol levels, by analogy to patients with hypobetalipoproteinemia. As intestinal APOB mRNA editing and APOB48 expression rely on sequences within exon 26, exon 27 skipping should not affect APOB48 expression unlike other methods of down-regulating APOB100 expression which also down-regulate APOB48.

| S-EPMC1784105 | biostudies-literature

RNA splicing process analysis for identifying antisense oligonucleotide inhibitors with padlock probe-based isothermal amplification.

Project description:RNA splicing, which mainly involves two transesterification steps, is a fundamental process of gene expression and its abnormal regulation contributes to serious genetic diseases. Antisense oligonucleotides (ASOs) are genetic control tools that can be used to specifically control genes through alteration of the RNA splicing pathway. Despite intensive research, how ASOs or various other factors influence the multiple processes of RNA splicing still remains obscure. This is largely due to an inability to analyze the splicing efficiency of each step in the RNA splicing process with high sensitivity. We addressed this limitation by introducing a padlock probe-based isothermal amplification assay to achieve quantification of the specific products in different splicing steps. With this amplified assay, the roles that ASOs play in RNA splicing inhibition in the first and second steps could be distinguished. We identified that 5'-ASO could block RNA splicing by inhibiting the first step, while 3'-ASO could block RNA splicing by inhibiting the second step. This method provides a versatile tool for assisting efficient ASO design and discovering new splicing modulators and therapeutic drugs.

| S-EPMC5621167 | biostudies-literature

Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4.

Project description:The discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.

| S-EPMC8124656 | biostudies-literature

Antisense Oligonucleotide- and CRISPR-Cas9-Mediated Rescue of mRNA Splicing for a Deep Intronic CLRN1 Mutation.

Project description:Mutations in CLRN1 cause Usher syndrome (USH) type III (USH3A), a disease characterized by progressive hearing impairment, retinitis pigmentosa, and vestibular dysfunction. Due to the lack of appropriate disease models, no efficient therapy for retinitis pigmentosa in USH patients exists so far. In addition, given the yet undefined functional role and expression of the different CLRN1 splice isoforms in the retina, non-causative therapies such as gene supplementation are unsuitable at this stage. In this study, we focused on the recently identified deep intronic c.254-649T>G CLRN1 splicing mutation and aimed to establish two causative treatment approaches: CRISPR-Cas9-mediated excision of the mutated intronic region and antisense oligonucleotide (AON)-mediated correction of mRNA splicing. The therapeutic potential of these approaches was validated in different cell types transiently or stably expressing CLRN1 minigenes. Both approaches led to substantial correction of the splice defect. Surprisingly, however, no synergistic effect was detected when combining both methods. Finally, the injection of naked AONs into mice expressing the mutant CLRN1 minigene in the retina also led to a significant splice rescue. We propose that both AONs and CRISPR-Cas9 are suitable strategies to initiate advanced preclinical studies for treatment of USH3A patients.

| S-EPMC7452116 | biostudies-literature

Altered PLP1 splicing causes hypomyelination of early myelinating structures.

Project description:ObjectiveThe objective of this study was to investigate the genetic etiology of the X-linked disorder "Hypomyelination of Early Myelinating Structures" (HEMS).MethodsWe included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients' fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs.ResultsAll patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio.InterpretationBrain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus-Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing.

| S-EPMC4479525 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data