Gene Correction of Limb Girdle Muscular Dystrophy Type 2A Patient-Specific Induced Pluripotent Stem Cells

University of Minnesota Ph.D. dissertation. September 2019. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Rita Perlingeiro. 1 computer file (PDF); x, 138 pages.Targeted differentiation of pluripotent stem (PS) cells into myotubes enables in vitro disease modeling of skeletal muscle diseases. Although various protocols achieve myogenic differentiation in vitro, resulting myotubes invariably display an embryonic identity. This is a major hurdle for accurately recapitulating disease phenotypes in vitro, as disease typically does not manifest in the embryonic muscle, but at more mature stages. To address this problem, we identified four factors from a small molecule screen whose combinatorial treatment resulted in myotubes with enhanced maturation, as shown by increased expression of fetal, neonatal and adult myosin heavy-chain isoforms. These molecular changes were confirmed by global chromatin accessibility and transcriptome studies. Importantly, we also observed this maturation in three-dimensional muscle bundles, which displayed improved in vitro contractile force generation in response to electrical stimulus. Thus, we established a model for in vitro muscle maturation from PS cells. We applied this maturation model for in vitro validation of Calpain 3 (CAPN3) protein expression. CAPN3 mutations are associated with Limb Girdle Muscular Dystrophy type 2A (LGMD2A), which is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. Using a gene knock-in approach, here we applied CRISPR-Cas9 mediated genome editing to induced pluripotent stem (iPS) cells from three LGMD2A patients carrying three different CAPN3 mutations, to enable correction of mutations in the CAPN3 gene. CAPN3 protein rescue upon gene correction was validated in myotube-derivatives in vitro following the small molecule treatment. Transplantation of gene corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof concept for the integration of genome editing and iPS cell technologies to develop a novel autologous cell therapy for LGMD2A