Pérez-Ramos,S1; Badosa, C1; Elias-Tersa, A1; Trifunov, S1; Balcells, S2; Grinberg, D2; Nascimiento, A1; Palau, F3; Domínguez, L3; Jimenez-Mallebrera, C1.
1 Laboratory of Translational Research in Neuromuscular Diseases. Neuromuscular Unit. Institute of Pediatric Research Sant Joan de Deu/ Sant Joan de Deu Hospital/ University of Barcelona, Barcelona, Spain.
2 Department of Genetics, Microbiology and Statistics. IBUB; CIBERER; IRSJD/Faculty of Biology/ University of Barcelona, Barcelona, Spain
3 Neurogenetics and Molecular Medicine Group. Institute of Pediatric Research Sant Joan de Deu/ Sant Joan de Deu Hospital/ University of Barcelona, Barcelona, Spain.
Congenital muscular dystrophies (CMD) are a wide group of rare, often fatal and untreatable diseases. CMDs are regularly caused by dominant mutations affecting genes that code for extracellular matrix proteins, such as collagen-VI. Collagen-VI related myopathies are the most common subgroup of ultra-rare early-onset CMD, consisting of a heterogeneous spectrum of phenotypes ranging from the mild Bethlem form to the severe Ullrich presentation. Mutations are mainly dominant negative (50-70%), located in any of the three genes encoding the collagen-VI alpha chains (COL6A1, COL6A2, COL6A3). The changes are usually point mutations affecting glycine residues that may cause supramolecular alterations, as well as exert a dominant negative effect on the wild type collagen VI molecules and their organization in the extracellular matrix. Thus, any gene editing approach to silence the mutated allele or to correct the pathogenic alteration would be very helpful. The recently discovered CRISPR-Cas9 system is a potent technology for site-specific genome engineering. In this study, we want to test CRISPR-Cas9 efficiency to target the COL6A1 c.877 G>A dominant mutation in fibroblasts of heterozygous patients. The aim is to demonstrate as a proof of concept the viability of this gene-editing tool within the area of rare CMDs. For the delivery of Cas9 and sgRNA into the cells, both plasmids and ribonucleoprotein complexes will be tested. The editing effectiveness will be assessed both by genetic analysis and functional studies of the treated fibroblasts to assess the potential restoration of collagen VI expression and function and its consequences within the edited cells