Rescue of Dystrophin Protein Expression by Interfering of mRNA Processing

Majid  Mojarrad; Atieh  Eslahi; Hossein  Tanipur; Javad Zavar  Reza

doi:10.18502/ssu.v34i3.21721

Majid Mojarrad Department of Medical Genetics, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Atieh Eslahi Department of Medical Genetics, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Hossein Tanipur Department of Medical Genetics, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Javad Zavar Reza Department of clinical Biochemistry, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

DOI: https://doi.org/10.18502/ssu.v34i3.21721

Keywords: Dystrophin, gene skipping, mRNA processing.

Abstract

Introduction: Exon skipping has emerged as one of the most promising therapeutic strategies for Duchenne muscular dystrophy (DMD), because it restores the correct reading frame of dystrophin mRNA without directly modifying the genome. Delivery systems based on adeno-associated virus (AAV) enable long-term expression of exon-skipping components and improve therapeutic efficiency.

Methods: In this study, an AAV-based vector encoding a modified U7 snRNA designed to induce skipping of dystrophin exon 51 was constructed using a ligase-independent cloning (LIC) strategy. Recombinant AAV particles were produced and used to transduce immortalized human myoblasts. Exon 51 skipping efficiency was evaluated by RT-PCR, and dystrophin protein expression was assessed by western blotting.

Results: RT-PCR findings demonstrated effective exon 51 skipping in the treated cells, producing a shortened dystrophin mRNA transcript. Western blot analysis revealed increased expression of a truncated but functional dystrophin protein in AAV-treated myoblasts compared to controls.

Conclusion: These results indicate that the engineered AAV-U7 system effectively induces exon 51 skipping and restores dystrophin expression at the protein level. The LIC-based platform enables rapid generation of personalized exon-skipping vectors and represents a versatile and scalable approach for the development of RNA-based gene therapies for DMD.