P12

Modelling therapeutic strategies in Duchenne muscular dystrophy by CRISPR/Cas-based approaches using iPSC-derived cardiomyocytes

J Dulak(1)

1: Department of Medical Biotechnology; Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University; Gronostajowa 7, Kraków, Poland

Duchenne muscular dystrophy is X-linked genetic disease affecting about 1:5,000 boys. The   mutations in DMD gene are usually out of frame and lead to absence of dystrophin, the structural protein in striated muscles. The patients loose ambulation in their teens, while heart failure is the major cause of death usually in the 3rd decade of life.

Human iPSC have been generated by reprogramming of blood leukocytes of DMD boys and healthy subjects. DMD mutations were corrected by CRISPR/Cas9 editing and HDR repair while relevant mutations have been introduced by DMD gene editing in healthy cells  (J Mol Cell Cardiol, 2021;  doi: 10.1016/j.yjmcc.2021.07.007). The studies revealed disturbance of structure and function of iPSC-derived DMD cardiomyocytes, Specifically, RNAseq and proteomic analysis showed disturbed gene expression of iron handling pathways. CISD1, encoding mitoNEET, the outer mitochondrial membrane exporting iron was identified as potential modulatory target. mitoNEET is decreased in DMD cardiomyocytes and CRISPR/Cas9 correction of DMD mutation restored mitoNEET level and corrected iron handling and ROS generation (Cardiovasc Res 2024;  doi: 10.1093/cvr/cvad182).

In another study deactivated Cas9 (SadCas9) coupled with VP64 transcriptional activator delivered with AAV6 in a single vector harbouring also sgRNAs, upregulated utrophin expression in DMD cardiomyocytes, improving calcium handling and electrophysiological properties  (Mol Ther Nucleic Acids, doi: 10.1016/j.omtn.2024.102247).

In sum, CRISPR/Cas-edited iPSC-derived cardiac cells allow to investigate DMD disease mechanisms revealing novel pathways for potential therapeutic exploitation.

Supported by grants from the National Science Center: MAESTRO 2018/30/A/NZ3/00412; and DAINA #2024/52/L/NZ3/00142.