CAPN3 Rescue and In vivo Regenerative Capacity of Gene Edited LGMD2A Patient-Specific iPSCs
Muscular dystrophies are inherited diseases characterized by progressive muscle weakness and vary in terms of severity, age of onset, tissues affected, and genetic basis. Currently, treatment is limited to palliative approaches targeting the secondary effects of inflammation and fibrosis. While these treatments aim to slow disease progression, they do not fix the root cause of the disease: gene mutations. Mutations in the dystrophin gene have been identified in the most common form of muscular dystrophy, Duchenne muscular dystrophy (DMD). Gene-based therapies to deliver functional dystrophin to diseased muscles have been evaluated or are in development to treat DMD. Previous studies also demonstrated the therapeutic potential of cell therapies to treat muscular dystrophy, including the generation of induced pluripotent stem cell (iPSC) derived myogenic progenitors. Limb girdle muscular dystrophies (LGMD) are a group of muscular dystrophies that consist of wide ranging genetic and clinical features. LGMD2A is caused by mutations in the calpain 3 gene (CAPN3) and is characterized by atrophy of proximal limb muscles. The Perlingeiro laboratory developed a strategy to produce a homozygous CAPN3 correction in LGMD2A iPSCs using CRISPR-cas9 based homology directed repair. The current study evaluated the in vitro rescue of the CAPN3 gene and CAPN3 protein expression as well as the in vivo capacity of iPSC derived myogenic progenitors, produced from CAPN3 gene edited homozygous clones, to engraft into mouse muscles. Homozygous CAPN3 gene correction in vitro was confirmed by RT-PCR of terminally differentiated myotubes and CAPN3 protein rescue was confirmed via western blot. Additionally, after transplantation of myogenic progenitors into the tibialis anterior muscles of mice, dystrophin positive, lamin a/c positive myofibers were seen by immunofluorescence analysis indicating the successful engraftment of the transplanted cells. These results suggest the potential of gene edited patient derived iPSCs as a treatment for muscular dystrophy.