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Updated: Apr 2, 2026
CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors
Published on: September 14, 2019
Stem Cell-Mediated Exon Skipping of the Dystrophin Gene by the Bystander Effect
Mirella Meregalli, Andrea Farini, Clementina Sitzia
1Laboratorio Cellule Staminali- Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti - Universita degli Studi di Milano Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122 Milan, Italy. yvan.torrente@unimi.it.
Abstract:
Duchenne muscular dystrophy (DMD) is characterized by the loss of a functional dystrophin protein; the muscles of DMD patients progressively degenerate as a result of mechanical stress during contractions, and the condition eventually leads to premature death. By means antisense oligonucleotides (AONs), it is possible to modulate pre-mRNA splicing eliminating mutated exons and restoring dystrophin open reading frame. To overcome the hurdles in using AONs for therapeutic interventions, we exerted engineered human DMD stem cells with a lentivirus, which permanently and efficiently delivered the cloned AONs. Here we describe for the first time the exosome-mediated release of AONs from engineered human DMD CD133+ stem cells allowing the rescue of murine dystrophin expression. Finally, upon release, AONs could be internalized by host cells suggesting a potential role of exosomes acting as vesicular carriers for DMD gene therapy.
Insights
Antisense oligonucleotides delivered via engineered stem cells and exosomes show promise for Duchenne muscular dystrophy (DMD) gene therapy by restoring dystrophin expression and offering a potential new treatment avenue.
Area of Science:
- Biotechnology
- Gene Therapy
- Stem Cell Biology
Background:
- Duchenne muscular dystrophy (DMD) results from a lack of functional dystrophin protein, leading to progressive muscle degeneration and premature death.
- Antisense oligonucleotides (AONs) offer a therapeutic strategy by modulating pre-mRNA splicing to restore the dystrophin reading frame.
Purpose of the Study:
- To develop a novel delivery system for AONs to overcome therapeutic challenges in DMD.
- To investigate the exosome-mediated release of AONs from engineered human stem cells for potential DMD gene therapy.
Main Methods:
- Engineering human DMD CD133+ stem cells with a lentivirus to permanently deliver cloned AONs.
- Characterizing the exosome-mediated release of AONs from these engineered stem cells.
- Assessing the internalization of released AONs by host cells and the rescue of dystrophin expression in a murine model.
Main Results:
- Successful engineering of human DMD stem cells for sustained AON delivery.
- Demonstration of exosome-mediated release of AONs from engineered stem cells.
- Evidence of AON internalization by host cells and restoration of murine dystrophin expression.
Conclusions:
- Exosomes can act as effective vesicular carriers for delivering AONs in the context of DMD gene therapy.
- This exosome-mediated delivery system represents a promising advancement for treating Duchenne muscular dystrophy.
- Further research into this approach could lead to novel therapeutic strategies for DMD patients.
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