Cas9-induced single cut enables highly efficient and template-free repair of a muscular dystrophy causing founder mutation.

With thousands of patients worldwide, CAPN3 c.550delA is the most frequent mutation causing severe, progressive, and untreatable limb girdle muscular dystrophy. We aimed to genetically correct this founder mutation in primary human muscle stem cells. We designed editing strategies providing CRISPR-Cas9 as plasmid and mRNA first in patient-derived induced pluripotent stem cells and applied this strategy then in primary human muscle stem cells from patients. Mutation-specific targeting yielded highly efficient and precise correction of CAPN3 c.550delA to wild type for both cell types. Most likely a single cut generated by SpCas9 resulted in a 5' staggered overhang of one base pair, which triggered an overhang-dependent base replication of an A:T at the mutation site. This recovered the open reading frame and the CAPN3 DNA sequence was repaired template-free to wild type, which led to CAPN3 mRNA and protein expression. Off-target analysis using amplicon sequencing of 43 in silico predicted sites demonstrates the safety of this approach. Our study extends previous usage of single cut DNA modification since our gene product has been repaired into the wild-type CAPN3 sequence with the perspective of a real cure.

Human primary muscle stem cells regenerate injured urethral sphincter in athymic rats.

BACKGROUND: The aim of the study was to demonstrate the efficacy of human muscle stem cells (MuSCs) isolated using innovative technology in restoring internal urinary sphincter function in a preclinical animal model. METHODS: Colonies of pure human MuSCs were obtained from muscle biopsy specimens. Athymic rats were subjected to internal urethral sphincter damage by electrocauterization. Five days after injury, 2 × 105 muscle stem cells or medium as control were injected into the area of sphincter damage (n = 5 in each group). Peak bladder pressure and rise in pressure were chosen as outcome measures. To repeatedly obtain the necessary pressure values, telemetry sensors had been implanted into the rat bladders 10 days prior to injury. RESULTS: There was a highly significant improvement in the ability to build up peak pressure as well as a pressure rise in animals that had received muscle stem cells as compared to control (p = 0.007) 3 weeks after the cells had been injected. Only minimal histologic evidence of scarring was observed in treated rats. CONCLUSION: Primary human muscle stem cells obtained using innovative technology functionally restore internal urethral sphincter function after injury. Translation into use in clinical settings is foreseeable.

Base editing repairs an SGCA mutation in human primary muscle stem cells.

Skeletal muscle can regenerate from muscle stem cells and their myogenic precursor cell progeny, myoblasts. However, precise gene editing in human muscle stem cells for autologous cell replacement therapies of untreatable genetic muscle diseases has not yet been reported. Loss-of-function mutations in SGCA, encoding α-sarcoglycan, cause limb-girdle muscular dystrophy 2D/R3, an early-onset, severe, and rapidly progressive form of muscular dystrophy affecting both male and female patients. Patients suffer from muscle degeneration and atrophy affecting the limbs, respiratory muscles, and heart. We isolated human muscle stem cells from 2 donors, with the common SGCA c.157G>A mutation affecting the last coding nucleotide of exon 2. We found that c.157G>A is an exonic splicing mutation that induces skipping of 2 coregulated exons. Using adenine base editing, we corrected the mutation in the cells from both donors with > 90% efficiency, thereby rescuing the splicing defect and α-sarcoglycan expression. Base-edited patient cells regenerated muscle and contributed to the Pax7+ satellite cell compartment in vivo in mouse xenografts. Here, we provide the first evidence to our knowledge that autologous gene-repaired human muscle stem cells can be harnessed for cell replacement therapies of muscular dystrophies.

Human muscle-derived CLEC14A-positive cells regenerate muscle independent of PAX7.

Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration.

In vitro characterization of hepatic toxicity of Alternaria toxins.

Alternaria mycotoxins are secondary fungal metabolites which can contaminate food and feed. They are produced by Alternaria species with alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and tentoxin (TEN) as the main representatives for Alternaria mycotoxins in food. Once passing the intestinal barrier, Alternaria toxins can reach the liver to exert yet uncharacterized molecular effects. Therefore, hepatic in vitro systems were used to examine selected Alternaria mycotoxins for their induction of metabolism-dependent cytotoxicity, phosphorylation of the histone H2AX as a surrogate marker for DNA double-strand breaks, and relevant marker genes for hepatotoxicity. Analysis of cell viability as well as the induction of H2AX phosphorylation in the hepatocarcinoma cell line HepG2 revealed a detoxification of 100 µmol/l AME and AOH by pre-treatment with S9 liver homogenate as shown by a decrease in cytotoxicity and H2AX histone phosphorylation to levels observed in control cells. Concentrations up to 100 µmol/l TeA and TEN did not induce H2AX phosphorylation whether metabolized or not. In the metabolically competent human hepatoma cell line HepaRG, no cytotoxicity of Alternaria toxins occurred even at high concentrations up to 100 µmol/l, which indicates a low cytotoxic potential. Induction of gene expression associated with liver toxicity was analyzed by quantitative real-time PCR using a specific hepatotoxicity PCR array in HepaRG cells: here, an evidence was found that 50 µmol/l of AOH, AME, TeA, and TEN might be associated with hepatotoxic effects, necrosis, and the development of diseases like cholestasis and phospholipidosis.