CRISPR/Cas9 Genome Editing in LGMD2A/R1 Patient-Derived Induced Pluripotent Stem and Skeletal Muscle Progenitor Cells

Area of Science:

• Biochemistry
• Genetics
• Regenerative Medicine

Background:

• Calpain 3 (CAPN3) mutations cause limb-girdle muscular dystrophy (LGMD2A/LGMDR1), a recessive disorder characterized by proximal limb muscle atrophy.
• Patient-derived induced pluripotent stem cells (iPSCs) offer a valuable model for studying LGMD2A.

Purpose of the Study:

• To develop strategies for correcting CAPN3 mutations in patient-derived iPSCs and myogenic progenitor cells.
• To generate isogenic LGMD2A-corrected iPSCs for disease modeling.
• To explore potential translational approaches for LGMD2A therapy.

Main Methods:

• Generated patient-specific iPSCs with CAPN3 mutations (W130C, 550delA).
• Employed CRISPR/Cas9 genome editing combined with FACS and Tet transactivator-based selection for biallelic correction in iPSCs.
• Utilized CRISPR/Cas9 for targeted mutation rescue in iPSC-derived CD82+/Pax7+ myogenic progenitor cells.

Main Results:

• Successfully generated a functional chimeric exon 3-4 in iPSCs, correcting both CAPN3 mutations.
• Demonstrated CRISPR/Cas9-mediated rescue of the common exon 4 CAPN3 mutation in myogenic progenitor cells.
• Established isogenic LGMD2A-corrected iPSCs suitable for disease modeling.

Conclusions:

• Developed two distinct CRISPR/Cas9-based strategies for CAPN3 mutation correction in LGMD2A models.
• The generated isogenic iPSCs provide a valuable resource for studying LGMD2A pathogenesis.
• The rescue strategy in myogenic progenitor cells holds promise for future therapeutic applications.