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

Abstract

T+49 (0)30 450540501 (Translational design, muscle stem cells). One sentence summary: Patient primary muscle stems cells gene repaired with >90% efficiency by base editing maintain their regenerative properties for autologous cell replacement therapies of muscular dystrophy.

P2X7 Receptor Antagonist Reduces Fibrosis and Inflammation in a Mouse Model of Alpha-Sarcoglycan Muscular Dystrophy

“…Immunomodulatory regimens become even more relevant as new gene therapies or gene editing approaches are being developed for alpha-sarcoglycanopathy [67][68][69], with gene therapy being in Phase I/II clinical trials for LGMD R3 patients (NCT01976091; NCT00494195). Since the success of gene therapy in the muscle tissue has to challenge the pre-existing status of chronic tissue inflammation typically identified in this diseases [67][68][69][70][71], our data suggest that P2X7 antagonism might represent a good strategy to dampen chronic inflammation, possibly leading to a better delivery of gene therapy. To date P2X7 selective antagonists have already been tested in Phase I/II clinical trials for the treatment of Crohn's disease, rheumatoid arthritis, basal cell carcinoma with an overall good tolerability and variable efficacy [72] and a new trial is currently ongoing assessing the effects of JNJ-54175446, a potent, brain-penetrant, selective P2X7 antagonist [73] in patients with major depressive disorder (ClinicalTrials.gov Identifier: NCT04116606).…”

mRNA-mediated delivery of gene editing tools to human primary muscle stem cells

“…The MD-causing SGCA c.157G>A mutation can be repaired highly efficiently in human MuSCs by plasmid-based delivery of ABE7.10 and a suitable sgRNA, following the enrichment of transfected cells using a fluorescence reporter. 20 We asked whether a comparable repair efficacy could be achieved through the mRNA-mediated delivery of ABE7.10 in the absence of any selection marker. We transfected human MuSCs carrying a heterozygous SGCA c.157G>A mutation with ABE7.10 mRNA and the corresponding sgRNA ( Figure 5 A).…”

“…Indeed, despite their limited availability and local migratory potential, it is becoming increasingly clear that well-defined and highly myogenic MuSCs could be a feasible, safe, and efficacious cell source for autologous cell replacement therapies to treat muscle-wasting disorders. 13 , 20 , 34 , 35 , 36 , 37 Obtaining large numbers of highly regenerative ex vivo expanded primary MuSCs for autologous therapies is still a challenge, but significant progress has been made in recent years. 13 , 34 , 38 , 39 The number of cells needed for the functional reconstitution of human muscles has yet to be determined and may depend on many factors related to both the fitness of the transplanted cells and the environment of the recipient muscle.…”

“…Codon-optimized ABE7.10 mRNA based on the amino acid sequence of ABE7.10_4.1 20 was purchased from AmpTec (Hamburg, Germany). SpCas9 and GFP mRNA were purchased from Aldevron (North Dakota, USA).…”

“…The corrected cells exhibited their full myogenic and regenerative potential in xenografts. 20 However, plasmid-based delivery carries a risk of transgene integration and leads to a relatively long exposure to gene-modifying enzymes, which in turn increases the probability of off-target mutagenesis. mRNA-mediated transgene delivery could provide transient expression while eliminating the risk of transgene integration.…”