Supplementary MaterialsSupplementary Video 1. reagents is certainly accompanying this publication. Abstract Frameshift mutations in the gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the reading frame, resulting in expression of a shortened, but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of (gene encoding dystrophin, which are mainly exon deletions3,4. The X-chromosomal location of transcripts6 has already been translated into clinical studies7,8. However, AONs – though in the beginning efficient in a dose-dependent manner6 – offer only temporary and limited efficacy of expression9. Endonuclease-based gene editing strategies provide a more efficient and permanent genomic correction, as exhibited in mouse models10C14. Recently, intravenous (i.v.) application of AAV9 delivering CRISPR/Cas9 components in a beagle model of DMD (exon 50 deficiency) proved successful in restoring expression of a shortened dystrophin in various muscles, including the heart15. However, functional data have not been reported as of yet. We have generated a DMD pig model lacking exon 521, resulting in a complete loss of dystrophin expression (Fig. 1a, Methods). First, we assessed whether local application of Cas9 and selected gRNAs targeting exon 51 (Extended Data Fig. 1a-c, Source Data Extended Data Fig. 1c) induces expression of a shortened, but stable dystrophin (Fig. 1a). Ten to fourteen-day-old piglets were subjected to unilateral fore- and hindlimb intramuscular (i.m.) injection of a pair of intein-split (Sp)-Cas92 and gRNA-encoding computer virus particles (AAV9-Cas9-gE51, 2×1013 vp/kg each) (Extended Data Fig. 1d-f, Source Data Extended Data Fig. 1f). After six weeks, histological analysis revealed restitution of membrane-localized dystrophin in the treated areas, and – due to leakage of the vector – in low levels at the contralateral limb. Successful removal of exon 51 and expression of DMD51-52 was confirmed at genomic, transcript, and protein levels (Fig. 1b-d and Extended Data Itgam Fig. 2a, Source Data Extended Data Fig. 2a), though full Cefepime Dihydrochloride Monohydrate congruence could not be reached due to sample variability. Mass spectrometry analysis (Supplementary Fig. 1a-c) of treated muscle tissue indicated partial normalization of proteins dysregulated in DMD (Fig.1e), with several fibrosis-related proteins significantly reduced (Supplementary Fig. 2a). Principal component analysis of the proteome confirmed that Cefepime Dihydrochloride Monohydrate this global protein profile of AAV9-Cas9-gE51-injected muscle tissue resided closer to healthy than DMD animals (Fig. 1f). Open in a separate window Physique 1 Genome editing of (E51-52) in WT limb muscle mass or in the indicated muscle tissue of improves survival and reduces cardiac arrhythmogenic vulnerabilitya, Kaplan-Meier curve of the survival time of untreated intracellular Ca2+ analysis of single cardiomyocytes within 300 m-thick heart slices managed in biomimetic chambers (Methods, Extended Data Fig. Cefepime Dihydrochloride Monohydrate 5d)26. Compared to wildtype heart samples, cells from untreated exon 52 (hrescues disease phenotypes of skeletal and cardiac muscle mass cells from patient-specific iPSCsa, Schematic indicating strategy to rescue defective skeletal myotube development in myoblasts differentiated from hDMD52 hiPSCs by transduction with two AAV6 vectors formulated with an intein-split Cas9 and gRNAs made to induce exon 51 excision. b, RT-qPCR evaluation of skeletal myotube markers 7-14 times after myotube induction in charge (n=8 indie differentiations), hDMD52 (n=7), hDMD52+AAV (n=6) and hDMD51-52 (n=4) myoblasts Cefepime Dihydrochloride Monohydrate (cf. Supply Data Fig. 4), indicated as mean fold changeSEM with p beliefs from a one-way ANOVA from the logarithmized beliefs with Bonferronis multiple evaluation check (F=26.21, df=3; F=14.32, df=21; F=10.84, df= 21). c, Immunofluorescence evaluation of myosin large chain (MyHC-), -actinin and dystrophin 2 weeks after myotube induction in myoblasts of Cefepime Dihydrochloride Monohydrate most mixed groupings, representative of >30 pictures gathered in 3 indie differentiations except hDMD51-52 n=2. Range pubs, 100 m. Insets present multinucleation (best) and sarcomeric striations (bottom level). Scale pubs, 25 m. d, Percentage of MyHC-+ cells 7-14 times after myotube induction of myoblasts of every from the indicated groupings (cf. Supply Data Fig. 4), symbolized as mean fold changeSEM with p beliefs from a.