Moore, Daniel; (2024) Editing the Nuclear Envelope: Using Human Induced Pluripotent Stem Cells and CRISPR-Cas Technology to Develop Genetic Therapies for Skeletal Muscle Laminopathies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Laminopathies are a clinically heterogenous group of diseases that are caused by mutations in nuclear lamin protein genes, particularly LMNA. Nuclear lamins are type V intermediate filament proteins that assemble to form the nuclear lamina, a meshwork-like structure that protects the nucleus from mechanical damage, coordinates chromatin organisation and regulates gene expression. LMNA encodes LAMIN A/C, while LMNB1 and LMNB2 encode LAMIN B1 and LAMIN B2 respectively. The most severe laminopathies include LMNA-related congenital muscular dystrophy, a severe, paediatric disorder for which there is currently no cure. Efforts to research both disease mechanisms and therapeutic strategies are hindered by a lack of clear genotype-phenotype correlation, limited access to patient samples and a lack of effective humanised disease models. Previous work in our laboratory demonstrated the potential of patient-derived induced pluripotent stem cells (iPSCs) to model hallmark laminopathy-associated phenotypes, including abnormal nuclear shape and mislocalisation of nuclear envelope proteins, in myogenic cells using a transgene-based protocol for differentiation. In this thesis, three patient-derived LMNA-mutant lines are used to validate this disease modelling platform, using a transgene-free protocol for myogenic differentiation, which more accurately mimics in vivo development. Utilising this approach, this work investigates the impact of LMNA mutations on early myogenic differentiation, nuclear shape, nuclear envelope protein localisation, and transcriptomics in skeletal muscle cells. Moreover, these models are used to develop CRISPR-based specific correction and permanent exon removal strategies for therapeutic targeting of mutations residing in exon 5. Targeting of an amenable LMNA-mutant with these approaches ameliorated disease-associated phenotypes. Overall, the work presented in this thesis demonstrates the potential of this platform for the development and assessment of patient-specific therapeutic strategies for skeletal muscle laminopathies.

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