Generation and characterization of IPSCS from a patient with muscular dystrophy with a missense mutation in FHL1

CASTAÑEDA, SHEILA; ZABALEGUI, FEDERICO; ABELLO POLO, VIRGINIA; BELLI, CAROLINA B; WAISMAN, ARIEL; LA GRECA, ALEJANDRO; SEVLEVER, GUSTAVO EMILIO; MIRIUKA, SANTIAGO GABRIEL; MORO, LUCIA

Mar del Plata

Congreso; Reunión Conjunta SAIC, SAI, FAIC, SAFIS; 2022

SAIC

The FHL1 gene locates in the Xq26 region, encodes four and a half LIM domain protein 1 and is expressed in skeletal and cardiac muscle. It has been related to cytoskeletal remodeling, myoblasts differentiation, sarcomere assembly and autophagy regulation. Mutations in FHL1 are related to muscular dystrophy (MD) with a limited life expectancy. The aim of this work was to generate and characterize an induced pluripotent stem cells (iPSCs) line derived from a patient with MD (MD-iPSC) that carries a pathogenic heterozygous missense mutation in FHL1 (c.377G>A, p.C126T) for in vitro disease modeling and personalized therapy development. In silico analysis revealed a misfolding of FHL1 p.C126T protein. To generate MD-iPSC, a blood sample was taken from the patient and erythroblasts were reprogrammed by transduction with STEMCCA lentiviral vector. After iPSCs clonal isolation and expansion, we confirmed the c.377G>A mutation, STEMCCA silencing and normal karyotype of MD-iPSCs. For pluripotency validation, alkaline phosphatase activity and pluripotency genes expression were assessed. Moreover, MD-iPSC was capable of differentiating into cells of the three germ layers by embryoid body formation. MD-iPSC derived cardiomyocytes were also obtained and characterized by observing high expression of cardiac markers by RT-qPCR, immunohistochemistry and western blot by day 23 of differentiation. As gene editing could be a possible in vivo therapy, we corrected the patient mutation in MD-iPSC by CRISPR-mediated homologous recombination. In summary, MD patient-derived iPSC line was efficiently generated and differentiated to muscle cells (cardiomyocytes), and the pathogenic mutation was corrected by CRISPR. With these results, we are able to study the patient's disease in vitro and develop a personalized therapy. In this sense, we are generating associated adenovirus (AAV) particles as a vector of the CRISPR system to transduce the patient cardiomyocytes simulating an in vivo therapy.