CRISPR-Cas9 editing of a TNPO3 mutation in a muscle cell model of limb-girdle muscular dystrophy type D2

Poyatos-García, Javier; Blázquez-Bernal, Águeda; Selva-Giménez, Marta; Bargiela, Ariadna; Espinosa-Espinosa, Jorge; Vázquez‐Manrique, Rafael P.; Bigot, Anne; Artero, Rubén; Vílchez, Juan J.

doi:10.1016/j.omtn.2023.01.004

Cited by 1 publication

(2 citation statements)

References 80 publications

(112 reference statements)

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“…Alternatively, other strategies such as ribonucleoprotein (RNPs) complexes have demonstrated greater transfection and cleavage efficiencies as well as reduced off-target effects than plasmids (linked to the reduced time of active RNP complexes compared to plasmids) (Kim et al, 2014; Zhang et al, 2021). Indeed, our experience with RNPs in immortalized myoblast (but different loci) revealed cleavage efficiencies above 87% (Poyatos-García et al, 2023).…”

Section: Discussionmentioning

confidence: 99%

“…A total of 5000 primary and immortalized myoblasts (derived from the patient 45 – 55) per well were seeded in 96-well plates and were incubated at 37 C in a humidified chamber with 5% CO 2 for 24, 48, 72, and 96 h in SMC medium. Cell proliferation was measured using the CellTiter 96 aqueous non-radioactive cell proliferation assay (MTS) (Promega) as previously described (Poyatos-García et al, 2023).…”

Section: Methodsmentioning

confidence: 99%

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Deletion of exons 45 to 55 in theDMDgene: from the therapeutic perspective to thein vitromodel

Poyatos-García,

Soblechero-Martín,

Liquori

et al. 2023

Preprint

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Current gene therapies for DMD aim to match benign spontaneous deletions for correcting out-of-frame DMD mutations. Deletion of 45-55 DMD exons (del45-55) is one of the candidates, as it has been described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering the mechanisms that transform a benign mutation into a severe one is crucial for understanding the pathophysiological mechanisms of dystrophinopathies and may condition the strategies of emerging therapies. Cellular models are essential for this purpose, but its availability is compromised by the need of a muscle biopsy. Here, we have introduced into a DMD cell line a del45-55 mimicking the intronic breakpoints of a subgroup of patients, through the CRISPR-Cas9 system. We observed the edited myoblasts recovered the dystrophin expression and ameliorated the myogenic defects. Besides, we confirmed the potential of CRISPR-Cas9 to create tailored mutations, being an useful approach to generate in vitro models. Also, we generated an immortalized myoblast line derived from a patient with the specific del45-55. Thus, we provide helpful resources to deep into unknown factors responsible of DMD severity.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%