Deletion of exons 45 to 55 in the DMD gene: from the therapeutic perspective to the in vitro model

Autores de IIS La Fe

• Javier Poyatos García

  Autor

• Alessandro Liquori

  Autor

• Pilar Maestre Fernandez-Flores

  Autor

• Elisa González Romero

  Autor

• Rafael Vázquez Manrique

  Autor

• Nuria Muelas Gómez

  Autor

• Gema García García

  Autor

• Juan Jesús Vílchez Padilla

  Autor

Participantes ajenos a IIS La Fe

• Soblechero-Martín, P
• López-Martínez, A
• Ohana, J
• Arechavala-Gomeza, V

Grupos

• 

  Biomedicina Molecular, Celular y Genómica

• Hematología y Hemoterapia

• Patología Neuromuscular y Ataxias

Abstract

Background Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. Methods We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status. Results Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. Conclusions Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.

Keywords

• Duchenne muscular dystrophy; Becker muscular dystrophy; CRISPR-Cas9; Gene therapy; Cell model; Dystrophin