Myoblots: dystrophin quantification by in‐cell western assay for a streamlined development of Duchenne muscular dystrophy (DMD) treatments

Albiasu-Arteta

Anton-Martinez

et al. 2021

Sci Rep

Self Cite

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient’s immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and β-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

Section: Discussionmentioning

confidence: 99%

“…Myoblots were performed as described before 52 . In short, clones were seeded in 96-well plates and incubated for 24 h in SMMC, after which they were treated with MyoD virus and incubated in differentiation media for 7 days.…”

Section: Methodsmentioning

confidence: 99%

Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening

Albiasu-Arteta

Anton-Martinez

et al. 2021

Sci Rep

Self Cite

“…Our choice to target this particular UTR region, increases basal overexpression in DMD cultures, and the amount of overexpression varies significantly when evaluated by western blot (more than 2.5 times) or our preferred method, myoblots (close to 1.5 times). We like to consider that myoblot evaluation reflects more closely the actual protein expression, as it is not subjected to many of the inherent problems of western blotting when evaluating very large proteins (Ruiz-Del-Yerro et al, 2018). This is why we cannot comment yet on the differences in expression between our study and other published studies that also aimed to overexpress utrophin by gene edition, but which 1) targeted different promoters regions ( UTRN A or UTRN B) of utrophin and 2) evaluated their results by western blot analysis (Wojtal et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

“…Immortalized myoblasts derived from muscle biopsies from healthy controls and DMD patients were provided by the CNMD Biobank, London, UK and the Institut de Myologie Paris, France, and cultured using skeletal muscle medium (SMM) (Promocell, Germany) and differentiation media as previously described (Ruiz-Del-Yerro et al, 2018).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Duchenne Muscular Dystrophy Cell Culture Models Created By CRISPR/Cas 9 Gene Editing And Their Application To Drug Screening

Albiasu-Arteta

Anton-Martinez

et al. 2020

Preprint

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CRISPR/Cas9-mediated gene editing may allow treating and studying rare genetic disorders by respectively, correcting disease mutations in patients, or introducing them in cell cultures. Both applications are highly dependent on Cas9 and sgRNA delivery efficiency. While gene editing methods are usually efficiently applied to cell lines such as HEK293 or hiPSCs, CRISPR/Cas9 editing in vivo or in cultured myoblasts prove to be much less efficient, limiting its use. After a careful optimisation of different steps of the editing protocol, we established a consistent approach to generate human immortalised myoblasts disease models through CRISPR/Cas9 editing. Using this protocol we successfully created a coding deletion of exon 52 of the DYSTROPHIN (DMD) gene in wild type immortalised myoblasts modelling Duchenne muscular dystrophy (DMD), and a microRNA binding sites deletion in the regulatory region of the UTROPHIN (UTRN) gene leading to utrophin upregulation in in Duchenne muscular dystrophy patient immortalised cultures. Sanger sequencing confirmed the presence of the corresponding genomic alterations and protein expression was characterised using myoblots. To show the utility of these cultures as platforms for assessing the efficiency of DMD treatments, we used them to evaluate the impact of exon skipping therapy and ezutromid treatment. Our editing protocol may be useful to others interested in genetically manipulating myoblasts and the resulting edited cultures for studying DMD disease mechanisms and assessing therapeutic approaches.SummaryWe report two novel immortalised myoblast culture models for studying Duchenne muscular dystrophy (DMD), generated through CRISPR/Cas9 gene editing: one recapitulates a common DYSTROPHIN (DMD) deletion and the other a regulatory mutation leading to UTROPHIN (UTRN) ectopic upregulation.

Evaluation of Exon Skipping and Dystrophin Restoration in In Vitro Models of Duchenne Muscular Dystrophy

López-Martínez

Methods in Molecular Biology

Arechavala‐Gomeza

2022

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Several exon skipping antisense oligonucleotides (eteplirsen, golodirsen, viltolarsen, and casimersen) have been approved for the treatment of Duchenne muscular dystrophy, but many more are in development targeting an array of different DMD exons. Preclinical screening of the new oligonucleotide sequences is routinely performed using patient-derived cell cultures, and evaluation of their efficacy may be performed at RNA and/or protein level. While several methods to assess exon skipping and dystrophin expression in cell culture have been developed, the choice of methodology often depends on the availability of specific research equipment.In this chapter, we describe and indicate the relevant bibliography of all the methods that may be used in this evaluation and describe in detail the protocols routinely followed at our institution, one to evaluate the efficacy of skipping at RNA level (nested PCR) and the other the restoration of protein expression (myoblot), which provide good results using equipment largely available to most research laboratories.