Development of a 3D in-vitro model of skeletal muscle involvement and proof-of-concept of a CRISPRa-based therapeutic approach in Late Onset Pompe disease

Humanised platforms are at the forefront of translation research as they potentially address the limitations of animal models, which often fail to fully replicate disease phenotypes. Previous research described the creation of an engineered muscle model for IOPD suitable for therapeutic testing, but thorough phenotypic and functional studies in LOPD models are still missing. The Tedesco group has pioneered the development of human iPSC-derived engineered muscles to model severe muscular dystrophies, including LMNA-related congenital muscular dystrophy [1, 3-6]. Building on this foundation, and harnessing the expertise gained during my internship in the Tedesco group, I generated and characterised an advanced patient-specific 3D muscle model for LOPD. This approach can provide a robust platform for disease modelling and screening of novel therapeutic strategies, aligned with the 3Rs animal welfare principles. Moreover, I developed and tested an innovative CRISPR-Cas9-based epigenome editing strategy, namely CRISPR-mediated transcriptional activation (CRISPRa), a powerful technique that uses a catalytically dead Cas9 fused to transcriptional activators, and sgRNAs, to induce gene expression from endogenous loci. This approach takes advantage of the residual GAA enzyme activity which is unique to LOPD patients and represents the first attempt to apply CRISPRa technology to PD treatment. Future extension of this study will see CRISPRa delivered via lipid nanoparticles (LNPs), a new delivery system that could help overcome the cargo limitations, pharmacokinetic challenges, toxicity and high cost of production posed by current gene delivery vectors. Overall, this project allowed the characterisation of LOPD biological and functional features of muscle, which is the most critically affected tissue, providing a valuable platform to test new therapeutic strategies and innovative delivery systems.