Correction of the Exon 2 Duplication in DMD Myoblasts by a Single CRISPR/Cas9 System

Lattanzi, Annalisa; Duguez, Stéphanie; Moiani, Arianna; Izmiryan, Araksya; Barbon, Elena; Martin, Samia; Mamchaoui, Kamel; Mouly, Vincent; Bernardi, Francesco; Mavilio, Fulvio; Bovolenta, Matteo

doi:10.1016/j.omtn.2017.02.004

Cited by 43 publications

(31 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…CRISPR/Cas9 as a possible therapeutic approach for DMD has been explored in the past in in vitro models of the disease. In most of these cases, a “permanent” exon skipping approach was selected where a shorter protein would be produced (Ousterout et al, 2015; Young et al, 2016), while in some others full length dystrophin was the result of the edition (Lattanzi et al, 2017; Li et al, 2014; Wojtal et al, 2015). After several studies showed efficacy also in mice models (Long et al, 2016; Nelson et al, 2016; Tabebordbar et al, 2016), a recent study in dogs is currently the most advanced example of its application to DMD (Amoasii et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

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

Soblechero-Martín

Albiasu-Arteta

Anton-Martinez

et al. 2020

Preprint

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

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

Soblechero-Martín

Albiasu-Arteta

Anton-Martinez

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Other permanent gene replacement therapies using CRISPR‐based gene editing are currently being developed (Long et al, ). The latter, which is a mutation‐specific approach, has been used to correct exon deletions, and duplications as well as sequence mutations (Lattanzi et al, ; Li et al, ; Young et al, ).…”

Section: Discussionmentioning

confidence: 99%

Mutational spectrum of dystrophinopathies in Singapore: Insights for genetic diagnosis and precision therapy

Tomar

Moorthy

Sethi

et al. 2019

American J of Med Genetics Pt C

View full text Add to dashboard Cite

Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive disorders caused by mutations in the DMD gene. Emerging therapies targeting patients with specific mutations are now becoming a reality for many of these patients. Precise molecular diagnosis is essential to facilitate the identification of possible new treatments for patients in the local context. In this study, we screened 145 dystrophinopathic patients in Singapore and assessed their molecular status for eligibility to current emerging genetic therapies. Overall, 140 (96.5%) of all patients harbored pathogenic DMD mutations comprising 95 exonic deletions (65.5%), 14 exonic duplications (9.7%), and 31 pathogenic small mutations (21.4%). Nonsense and frameshift mutations constitute 83.9% of all the small mutations. We found 71% (103/145) of all Singaporean dystrophinopathy patients to be theoretically amenable for exon skipping, either through skipping of single (53.1%) or multiple exons (17.9%).This approach is applicable to 81.1% (77/95) of patients carrying deletions and 83.9% (26/31) of those with small mutations. Eteplirsen induced skipping of exon 51 is applicable to 12.4% of local patients. Nonsense read-through therapy was found to be applicable in another 12.4% of all patients. Mutation screening is crucial for providing insights into the underlying genetic signature of the disease in the local population and contributes toward existing information on DMD mutations in Asia and globally. This will guide future targeted drug development and clinical trial planning for this disease. K E Y W O R D SBecker muscular dystrophy, Duchenne muscular dystrophy, dystrophinopathy, genetic diagnosis, mutation spectrum

show abstract

“…In fact, exons 2-7, which encode part of the ABD-1, are the most frequently mutated portion of the 5′-proximal hot spot (8). Two reports have described genomic editing of a DMD mutation comprising a duplication of exon 2 (30,31), and other reports showed editing of an exon 7 splice site mutation in the golden retriever dog model of DMD (32,33). In-frame deletions and missense mutations of the 5′ region of the DMD gene that affect the ABD-1 structure have been associated with a decrease in dystrophin protein stability, reduced actin binding affinity, and protein mis-folding and degradation (21,27,(34)(35)(36).…”

Section: Introductionmentioning

confidence: 99%

Functional correction of dystrophin actin binding domain mutations by genome editing

Kyrychenko¹,

Kyrychenko²,

Tiburcy³

et al. 2017

JCI Insight

View full text Add to dashboard Cite

Dystrophin maintains the integrity of striated muscles by linking the actin cytoskeleton with the cell membrane. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD) that result in progressive, debilitating muscle weakness, cardiomyopathy, and a shortened lifespan. Mutations of dystrophin that disrupt the amino-terminal actin-binding domain 1 (ABD-1), encoded by exons 2-8, represent the second-most common cause of DMD. In the present study, we compared three different strategies for CRISPR/Cas9 genome editing to correct mutations in the ABD-1 region of the DMD gene by deleting exons 3-9, 6-9, or 7-11 in human induced pluripotent stem cells (iPSCs) and by assessing the function of iPSC-derived cardiomyocytes. All three exon deletion strategies enabled the expression of truncated dystrophin protein and restoration of cardiomyocyte contractility and calcium transients to varying degrees. We show that deletion of exons 3-9 by genomic editing provides an especially effective means of correcting disease-causing ABD-1 mutations. These findings represent an important step toward eventual correction of common DMD mutations and provide a means of rapidly assessing the expression and function of internally truncated forms of dystrophin-lacking portions of ABD-1.

show abstract

Correction of the Exon 2 Duplication in DMD Myoblasts by a Single CRISPR/Cas9 System

Cited by 43 publications

References 50 publications

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

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

Mutational spectrum of dystrophinopathies in Singapore: Insights for genetic diagnosis and precision therapy

Functional correction of dystrophin actin binding domain mutations by genome editing

Contact Info

Product

Resources

About