A knock down strategy for rapid, generic, and versatile modelling of muscular dystrophies in 3D-tissue-engineered-skeletal muscle

Stijn L.M. in ‘t Groen, Marnix Franken, Theresa Bock, Marcus Krüger, Jessica C. de Greef, W. W.M.Pim Pijnappel

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Human iPSC-derived 3D-tissue-engineered-skeletal muscles (3D-TESMs) offer advanced technology for disease modelling. However, due to the inherent genetic heterogeneity among human individuals, it is often difficult to distinguish disease-related readouts from random variability. The generation of genetically matched isogenic controls using gene editing can reduce variability, but the generation of isogenic hiPSC-derived 3D-TESMs can take up to 6 months, thereby reducing throughput. Methods: Here, by combining 3D-TESM and shRNA technologies, we developed a disease modelling strategy to induce distinct genetic deficiencies in a single hiPSC-derived myogenic progenitor cell line within 1 week. Results: As proof of principle, we recapitulated disease-associated pathology of Duchenne muscular dystrophy and limb-girdle muscular dystrophy type 2A caused by loss of function of DMD and CAPN3, respectively. shRNA-mediated knock down of DMD or CAPN3 induced a loss of contractile function, disruption of tissue architecture, and disease-specific proteomes. Pathology in DMD-deficient 3D-TESMs was partially rescued by a candidate gene therapy treatment using micro-dystrophin, with similar efficacy compared to animal models. Conclusions: These results show that isogenic shRNA-based humanized 3D-TESM models provide a fast, cheap, and efficient tool to model muscular dystrophies and are useful for the preclinical evaluation of novel therapies.

Original languageEnglish
Article number3
JournalSkeletal Muscle
Volume14
Issue number1
DOIs
StatePublished - Dec 2024
Externally publishedYes

Keywords

  • 3D-organ-on-a chip
  • Calpain-3
  • Disease modelling
  • Duchenne muscular dystrophy
  • Dystrophin
  • Gene knockdown
  • LGMD2A
  • Skeletal muscle-on-a-chip
  • Tissue engineering

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