Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Allele-selective inhibition of huntingtin expression by switching to an miRNA-like RNAi mechanism.

ABSTRACT: Inhibiting expression of huntingtin (HTT) protein is a promising strategy for treating Huntington's disease (HD), but indiscriminant inhibition of both wild-type and mutant alleles may lead to toxicity. An ideal silencing agent would block expression of mutant HTT while leaving expression of wild-type HTT intact. We observe that fully complementary duplex RNAs targeting the expanded CAG repeat within HTT mRNA block expression of both alleles. Switching the RNAi mechanism toward that used by miRNAs by introducing one or more mismatched bases into these duplex RNAs leads to potent (<10 nM) and highly selective (>30-fold relative to wild-type HTT) inhibition of mutant HTT expression in patient-derived cells. Potent, allele selective inhibition of HTT by mismatched RNAs provides a new option for developing HD therapeutics.

SUBMITTER: Hu J

PROVIDER: S-EPMC3021381 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Allele-selective inhibition of huntingtin expression by switching to an miRNA-like RNAi mechanism.

Hu Jiaxin J Liu Jing J Corey David R DR

Chemistry & biology 20101101 11

Inhibiting expression of huntingtin (HTT) protein is a promising strategy for treating Huntington's disease (HD), but indiscriminant inhibition of both wild-type and mutant alleles may lead to toxicity. An ideal silencing agent would block expression of mutant HTT while leaving expression of wild-type HTT intact. We observe that fully complementary duplex RNAs targeting the expanded CAG repeat within HTT mRNA block expression of both alleles. Switching the RNAi mechanism toward that used by miRN ...[more]

PMID: 21095568

Similar Datasets

Mechanism of allele-selective inhibition of huntingtin expression by duplex RNAs that target CAG repeats: function through the RNAi pathway.

Project description:Huntington's disease is an incurable neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat within one allele of the huntingtin (HTT) gene. Agents that block expression of mutant HTT and preserve expression of wild-type HTT target the cause of the disease and are an alternative for therapy. We have previously demonstrated that mismatch-containing duplex RNAs complementary to the expanded trinucleotide repeat are potent and allele-selective inhibitors of mutant HTT expression, but the mechanism of allele selectivity was not explored. We now report that anti-CAG duplex RNA preferentially recruits argonaute 2 (AGO2) to mutant rather than wild-type HTT mRNA. Efficient inhibition of mutant HTT protein expression requires less AGO2 than needed for inhibiting wild-type expression. In contrast, inhibiting the expression of mutant HTT protein is highly sensitive to reduced expression of GW182 (TNRC6A) and its two paralogs, a protein family associated with miRNA action. Allele-selective inhibition may involve cooperative binding of multiple protein-RNA complexes to the expanded repeat. These data suggest that allele-selective inhibition proceeds through an RNA interference pathway similar to that used by miRNAs and that discrimination between mutant and wild-type alleles of HTT mRNA is highly sensitive to the pool of AGO2 and GW182 family proteins inside cells.

| S-EPMC3526262 | biostudies-literature

Allele-selective inhibition of mutant huntingtin expression with antisense oligonucleotides targeting the expanded CAG repeat.

Project description:Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD.

| S-EPMC2991413 | biostudies-literature

Single-stranded RNAs use RNAi to potently and allele-selectively inhibit mutant huntingtin expression.

Project description:Mutant huntingtin (HTT) protein causes Huntington disease (HD), an incurable neurological disorder. Silencing mutant HTT using nucleic acids would eliminate the root cause of HD. Developing nucleic acid drugs is challenging, and an ideal clinical approach to gene silencing would combine the simplicity of single-stranded antisense oligonucleotides with the efficiency of RNAi. Here, we describe RNAi by single-stranded siRNAs (ss-siRNAs). ss-siRNAs are potent (>100-fold more than unmodified RNA) and allele-selective (>30-fold) inhibitors of mutant HTT expression in cells derived from HD patients. Strategic placement of mismatched bases mimics micro-RNA recognition and optimizes discrimination between mutant and wild-type alleles. ss-siRNAs require Argonaute protein and function through the RNAi pathway. Intraventricular infusion of ss-siRNA produced selective silencing of the mutant HTT allele throughout the brain in a mouse HD model. These data demonstrate that chemically modified ss-siRNAs function through the RNAi pathway and provide allele-selective compounds for clinical development.

| S-EPMC3444165 | biostudies-literature

Allele-selective inhibition of expression of huntingtin and ataxin-3 by RNA duplexes containing unlocked nucleic acid substitutions.

Project description:Unlocked nucleic acid (UNA) is an acyclic analogue of RNA that can be introduced into RNA or DNA oligonucleotides. The increased flexibility conferred by the acyclic structure fundamentally affects the strength of base pairing, creating opportunities for improved applications and new insights into molecular recognition. Here we test how UNA substitutions affect allele-selective inhibition of expression of trinucleotide repeat genes Huntingtin (HTT) and Ataxin-3 (ATX-3). We find that the either the combination of mismatched bases and UNA substitutions or UNA substitutions alone can improve potency and selectivity. Inhibition is potent, and selectivities of >40-fold for inhibiting mutant versus wild-type expression can be achieved. Surprisingly, even though UNA preserves the potential for complete base pairing, the introduction of UNA substitutions at central positions within fully complementary duplexes leads to >19-fold selectivity. Like mismatched bases, the introduction of central UNA bases disrupts the potential for cleavage of substrate by argonaute 2 (AGO2) during gene silencing. UNA-substituted duplexes are as effective as other strategies for allele-selective silencing of trinucleotide repeat disease genes. Modulation of AGO2 activity by the introduction of UNA substitutions demonstrates that backbone flexibility is as important as base pairing for catalysis of fully complementary duplex substrates. UNA can be used to tailor RNA silencing for optimal properties and allele-selective action.

| S-EPMC3893079 | biostudies-literature

Exploring the effect of sequence length and composition on allele-selective inhibition of human huntingtin expression by single-stranded silencing RNAs.

Project description:Mutant huntingtin (HTT) protein is the cause of Huntington's disease (HD), an incurable neurological disorder. Almost all patients are heterozygous for mutant HTT and approaches that reduce levels of mutant HTT while leaving expression of wild-type HTT intact might be ideal options for therapeutic development. We have developed several allele-selective strategies for silencing HTT, including single-stranded silencing RNAs (ss-siRNAs). ss-siRNAs are oligonucleotides containing chemical modifications that permit action through the RNA interference (RNAi) pathway. Modified ss-siRNAs chosen to test the effects of varying oligomer length, lipid modification, the introduction of mismatched bases, and variation of chemical modification. We find that several modified ss-siRNA are potent and allele-selective inhibitors of HTT expression. An ss-siRNA with three mismatched bases relative to the CAG repeat was an allele-selective inhibitor of HTT expression in the HdhQ175 mouse model. Multiple allele-selective ss-siRNAs provide a wide platform of modifications to draw on for further optimization and therapeutic development. Our data provide insights into how ss-siRNAs can be modified to improve their properties and facilitate the discovery of the lead compounds necessary for further development.

| S-EPMC4026301 | biostudies-literature

Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells.

Project description:Post-transcriptional gene silencing is a promising therapy for the monogenic, autosomal dominant, Huntington's disease (HD). However, wild-type huntingtin (HTT) has important cellular functions, so the ideal strategy would selectively lower mutant HTT while sparing wild-type. HD patients were genotyped for heterozygosity at three SNP sites, before phasing each SNP allele to wild-type or mutant HTT. Primary ex vivo myeloid cells were isolated from heterozygous patients and transfected with SNP-targeted siRNA, using glucan particles taken up by phagocytosis. Highly selective mRNA knockdown was achieved when targeting each allele of rs362331 in exon 50 of the HTT transcript; this selectivity was also present on protein studies. However, similar selectivity was not observed when targeting rs362273 or rs362307. Furthermore, HD myeloid cells are hyper-reactive compared to control. Allele-selective suppression of either wild-type or mutant HTT produced a significant, equivalent reduction in the cytokine response of HD myeloid cells to LPS, suggesting that wild-type HTT has a novel immune function. We demonstrate a sequential therapeutic process comprising genotyping and mutant HTT-linkage of SNPs, followed by personalised allele-selective suppression in a small patient cohort. We further show that allele-selectivity in ex vivo patient cells is highly SNP-dependent, with implications for clinical trial target selection.

| S-EPMC5402279 | biostudies-literature

Allele-selective inhibition of mutant huntingtin by peptide nucleic acid-peptide conjugates, locked nucleic acid, and small interfering RNA.

Project description:The ability to inhibit expression of a mutant allele while retaining expression of a wild-type protein might provide a useful approach to treating Huntington's Disease (HD) and other inherited pathologies. The mutant form of huntingtin (HTT), the protein responsible for HD, is encoded by an mRNA containing an expanded CAG repeat. We demonstrate that peptide nucleic acid conjugates and locked nucleic acids complementary to the CAG repeat selectively block expression of mutant HTT. The selectivity of inhibition is at least as good as that shown by a small interfering RNA targeted to a deletion polymorphism. Our data suggest that antisense oligomers are promising subjects for further development as an anti-HD therapeutic strategy.

| S-EPMC3908448 | biostudies-literature

RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression.

Project description:Abasic substitutions within DNA or RNA are tools for evaluating the impact of absent nucleobases. Because of the importance of abasic sites in genetic damage, most research has involved DNA. Little information is available on the impact of abasic substitutions within RNA or on RNA interference (RNAi). Here, we examine the effect of abasic substitutions on RNAi and allele-selective gene silencing. Huntington's disease (HD) and Machado Joseph Disease (MJD) are severe neurological disorders that currently have no cure. HD and MJD are caused by an expansion of CAG repeats within one mRNA allele encoding huntingtin (HTT) and ataxin-3 (ATX-3) proteins. Agents that silence mutant HTT or ATX-3 expression would remove the cause of HD or MJD and provide an option for therapeutic development. We describe flexible syntheses for abasic substitutions and show that abasic RNA duplexes allele-selectively inhibit both mutant HTT and mutant ATX-3. Inhibition involves the RNAi protein argonaute 2, even though the abasic substitution disrupts the catalytic cleavage of RNA target by argonaute 2. Several different abasic duplexes achieve potent and selective inhibition, providing a broad platform for subsequent development. These findings introduce abasic substitutions as a tool for tailoring RNA duplexes for gene silencing.

| S-EPMC3794577 | biostudies-literature

Universal RNAi Triggers for the Specific Inhibition of Mutant Huntingtin, Atrophin-1, Ataxin-3, and Ataxin-7 Expression.

Project description:The expansion of CAG repeats within the coding region of associated genes is responsible for nine inherited neurodegenerative disorders including Huntington's disease (HD), spinocerebellar ataxias (SCAs), and dentatorubral-pallidoluysian atrophy (DRPLA). Despite years of research aimed at developing an effective method of treatment, these diseases remain incurable and only their symptoms are controlled. The purpose of this study was to develop effective and allele-selective genetic tools for silencing the expression of mutated genes containing expanded CAG repeats. Here we show that repeat-targeting short hairpin RNAs preferentially reduce the levels of mutant huntingtin, atrophin-1, ataxin-3, and ataxin-7 proteins in patient-derived fibroblasts and may serve as universal allele-selective reagents for polyglutamine (polyQ) diseases.

| S-EPMC6957814 | biostudies-literature

Allele-selective inhibition of mutant atrophin-1 expression by duplex and single-stranded RNAs.

Project description:Dentatorubral-pallidoluysian atrophy (DRPLA) is a progressive neurodegenerative disorder that currently has no curative treatments. DRPLA is caused by an expansion of a CAG trinucleotide repeat region within the protein-encoding sequence of the atrophin-1 (ATN-1) gene. Inhibition of mutant ATN-1 protein expression is one strategy for treating DRPLA, and allele-selective gene silencing agents that block mutant expression over wild-type expression would be lead compounds for therapeutic development. Here we develop an assay for distinguishing mutant from wild-type ATN-1 protein by gel electrophoresis. We use this assay to evaluate duplex RNAs and single-stranded silencing RNAs (ss-siRNAs) for allele-selective inhibition of ATN-1 protein expression. We observed potent and allele-selective inhibition by RNA duplexes that contain mismatched bases relative to the CAG target and have the potential to form miRNA-like complexes. ss-siRNAs that contained mismatches were as selective as mismatch-containing duplexes. We also report allele-selective inhibition by duplex RNAs containing unlocked nucleic acids or abasic substitutions, although selectivities are not as high. Five compounds that showed >8-fold allele selectivity for mutant ATN-1 were also selective for inhibiting the expression of two other trinucleotide repeat disease genes, ataxin-3 (ATXN-3) and huntingtin (HTT). These data demonstrate that the expanded trinucleotide repeat within ATN-1 mRNA is a potential target for compounds designed to achieve allele-selective inhibition of ATN-1 protein, and one agent may allow the targeting of multiple disease genes.

| S-EPMC4108179 | 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