Developing gene-edited models to study mis-splicing in Hypertrophic Cardiomyopathy

• Autor(a) principal: Furtado, Marta Isabel Brandão
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/90817
• topic: cardiac differentiation; CRISPR/Cas9; human induced pluripotent stem cells; hyperthrophic cardiomyopathy; splicing; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Exonic variants that cause abnormal mRNA splicing have been reported in human disease. However, this phenomenon is relatively unstudied in Hypertrophic Cardiomyopathy, a complex genetic cardiac disease with more than 1500 pathogenic mutations identified in over 14 genes. The lack of suitable models to study the causal mechanism of HCM-associated variants poses a drawback to the understanding of the molecular basis underlying this disease. The recent emergence of CRISPR/Cas9 and hiPSCs technologies are an exciting approach to generate disease models that better recapitulate the characteristics of the human heart. Three gene-edited clones harbouring the MYBPC3 c.1090G>A variant were generated using the CRISPR/Cas9 system. This variant is predicted to disrupt the recognition of the donor splice-site and lead to the skipping of exon 12. However, throughout the gene-editing process and due to long time in culture, these cells lost expression of pluripotency markers and could not be differentiated into cardiomyocytes to perform further experiments. In parallel, a patient-derived HCM cell line (Xutl, p.I1250fs) and Gibco and TCLab control lines were differentiated into cardiomyocytes using a 2D protocol. The hiPSCs-CMs obtained expressed high levels of fetal sarcomeric-genes TNNT2 and TTN isoforms, when compared to the adult human heart. Morphologically, they also displayed characteristics that are compatible to those of embryonic cardiomyocytes. Therefore, these hiPSCs-CMs have an immature phenotype and could not fully recapitulate the characteristics of adult cardiomyocytes. For disease-modelling purposes, mature hiPSCs-CMs that are comparable to those found in the adult heart are required and 3D differentiation protocols are some of the approaches being implemented. Even though optimization of the gene-editing and cardiac differentiation protocols is essential for the success of subsequent experiments, the work performed in this thesis provided important advances in the development of improved cellular models to study complex genetic diseases, such as HCM.