GENE EDITING AND MOLECULAR THERAPY: P.285Effective gene editing of the human DYSF founder mutation c.1624delG in patient-derived cells and generation of analogous "humanized" mouse models for in vivo treatment

Abstract

Dysferlinopathy, also known as limb-girdle muscular dystrophy type 2B (LGMD2B), is caused by mutations in DYSF. It is one of the most frequent of > 40 untreatable muscular dystrophies leading to progressive muscle degeneration and wheel-chair-dependency. We generated hiPSC from two patients carrying a founder frameshift mutation (c.1624delG) in DYSF exon 44 that results in a complete absence of dysferlin protein and developed various gene editing approaches to repair this mutation using CRISPR/Cas9. First, using mutation-specific sgRNAs in combination with the enhanced specificity Cas9 variant eSpCas9(1.1), we were able to restore the mutant sequence to its wild-type form via homology directed repair without introducing any silent mutations in the repair template. Second, we used Cas9-induced NHEJ, in particular the indel signature bias of specific sgRNAs, to restore the DYSF reading frame in patient-derived cells with a high efficiency. We could readily demonstrate rescue of the re-framed dysferlin protein by western blot after editing. In parallel, we generated two related transgenic mouse lines, one with the murine Dysf exon 44 exchanged for the human wild-type DYSF exon 44 and the other containing the human c.1624delG founder mutation within the human DYSF exon 44. The latter lacks dysferlin protein and recapitulates the progressive muscular dystrophy phenotype seen in LGMD2B patients. Using these models, we are assessing ways to re-create in vivo our DYSF editing approaches directly on the human sequence, utilizing genetic tools that can be applied to patient cells.