Artificial mirtron-mediated gene knockdown: Functional DMPK silencing in mammalian cells

Seow, Yiqi; Sibley, Christopher R.; Wood, Matthew

doi:10.1261/rna.030601.111

Cited by 13 publications

(27 citation statements)

References 49 publications

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“…Whether ageing is intrinsic to the cells or is a reflection of the cellular response to the ageing environment is under debate. (Seow et al, 2012;reviewed in Overby et al, 2018); Mexiletine (Nguyen and Campbell, 2016); adding muscleblind-like protein 1 (reviewed in Konieczny et al, 2017…”

Section: Satellite Cell Defects In Muscular Dystrophiesmentioning

confidence: 99%

Skeletal muscle in health and disease

Morgan

Partridge

2020

Disease Models &Amp; Mechanisms

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Skeletal muscle fibres are multinucleated cells that contain postmitotic nuclei (i.e. they are no longer able to divide) and perform muscle contraction. They are formed by fusion of muscle precursor cells, and grow into elongating myofibres by the addition of further precursor cells, called satellite cells, which are also responsible for regeneration following injury. Skeletal muscle regeneration occurs in most muscular dystrophies in response to necrosis of muscle fibres. However, the complex environment within dystrophic skeletal muscle, which includes inflammatory cells, fibroblasts and fibro-adipogenic cells, together with the genetic background of the in vivo model and the muscle being studied, complicates the interpretation of laboratory studies on muscular dystrophies. Many genes are expressed in satellite cells and in other tissues, which makes it difficult to determine the molecular cause of various types of muscular dystrophies. Here, and in the accompanying poster, we discuss our current knowledge of the cellular mechanisms that govern the growth and regeneration of skeletal muscle, and highlight the defects in satellite cell function that give rise to muscular dystrophies.

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Section: Satellite Cell Defects In Muscular Dystrophiesmentioning

confidence: 99%

Skeletal muscle in health and disease

Morgan

Partridge

2020

Disease Models &Amp; Mechanisms

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“…The first class, mirtrons [30, 31] and mirtron-based shRNAs [32, 33] are precisely located within introns, where the splicing machinery generates pre-miRNA, eluding the need for Drosha processing. In the second class, Ago-shRNAs, which are based on miR-451 [34–36], Dicer cleavage is substituted by Ago2 endonucleolytic activity for maturation (Fig.…”

Section: Promoter Selectionmentioning

confidence: 99%

Guidelines for the optimal design of miRNA-based shRNAs

Ros

2016

Methods

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RNA interference (RNAi) is an extremely useful tool for inhibiting gene expression. It can be triggered by transfected synthetic small interfering RNA (siRNA) or by expressed small hairpin RNA (shRNA). The cellular machinery processes the latter into siRNA in vivo. shRNA is preferred or required in genetic screens and specific RNAi approaches in gene therapy settings. Despite its many successes, the field of shRNAs faces many challenges. Insufficient knockdowns and off-target effects become obstacles for shRNA usage in many applications. Numerous failures are triggered by pitfalls in shRNA design that is often associated with impoverished biogenesis. Here, based on current understanding of the miRNA maturation pathway, we discuss the principles of different shRNA design (pre-miRNA-like, pri-miRNA-like and Ago-shRNA) with an emphasis on the RNA structure. We also provide detailed instructions for an optimal design of pre-miRNA-like shRNA.

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“…An algorithm ( 43 ) selected putative mirtron target sites within human ataxin 7 cDNA ( 48 ) such that antisense molecules may fulfil splicing requirements when incorporated as either the 5΄ strand or 3΄ strand of a mirtron hairpin ( 49 ). A Basic Local Alignment Search Tool (BLAST) was used to ascertain target sequences’ specificity among human transcripts ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ).…”

Section: Methodsmentioning

confidence: 99%

“…Natural intronic miRNAs and mirtrons can be adapted to successfully silence a gene of interest ( 41 – 45 ). A potential benefit of mirtrons in gene therapy is the strict definition of pre-miRNA through the splicing reaction, perhaps minimizing off-target effects by producing a well-defined seed region, compared to microprocessed miRNA ( 43 ). Further, it has been revealed that in some nucleotide expansion disorders, both Drosha and its cofactor DGCR8 can be sequestered in nuclear RNA aggregates in patient brain cells leading to potentially harmful decreases in processing of endogenous miRNAs and showing that this system is indeed saturable ( 46 , 47 ).…”

Section: Introductionmentioning

confidence: 99%

Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7

Curtis

Seow

Wood

et al. 2017

Nucleic Acids Research

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We evaluate a knockdown-replacement strategy mediated by mirtrons as an alternative to allele-specific silencing using spinocerebellar ataxia 7 (SCA7) as a model. Mirtrons are introns that form pre-microRNA hairpins after splicing, producing RNAi effectors not processed by Drosha. Mirtron mimics may therefore avoid saturation of the canonical processing pathway. This method combines gene silencing mediated by an artificial mirtron with delivery of a functional copy of the gene such that both elements of the therapy are always expressed concurrently, minimizing the potential for undesirable effects and preserving wild-type function. This mutation- and single nucleotide polymorphism-independent method could be crucial in dominant diseases that feature both gain- and loss-of-function pathologies or have a heterogeneous genetic background. Here we develop mirtrons against ataxin 7 with silencing efficacy comparable to shRNAs, and introduce silent mutations into an ataxin 7 transgene such that it is resistant to their effect. We successfully express the transgene and one mirtron together from a single construct. Hence, we show that this method can be used to silence the endogenous allele of ataxin 7 and replace it with an exogenous copy of the gene, highlighting the efficacy and transferability across patient genotypes of this approach.

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Artificial mirtron-mediated gene knockdown: Functional DMPK silencing in mammalian cells

Cited by 13 publications

References 49 publications

Skeletal muscle in health and disease

Skeletal muscle in health and disease

Guidelines for the optimal design of miRNA-based shRNAs

Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7

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