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LAMA2-MD: establishment of a new gene therapy strategy using patient- derived cell lines
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Resumo(s)
LAMA2-related muscular dystrophy (LAMA2-MD) is a neuromuscular disease caused by mutations in the LAMA2 gene, leading to the complete absence or expression of a mutated laminin-α2 chain. This chain is part of a laminin isoform (laminin-211, LN211) abundantly found in the basement membrane of skeletal muscle, having a critical role in its’ structure and function. Currently, there is no cure available for LAMA2- MD, with existing therapies focusing on managing symptoms rather than addressing the underlying structural defects. Recent advances in gene therapy offer a promising approach to treat this disease, with one of the most promising delivery systems being the recombinant adeno-associated virus (rAAV) due to its lack of apparent pathogenicity, high cellular tropism, and persistence of expression. This delivery vector combined with the CRISPR-Cas9 gene editing technology offers a potential cure for this disorder. With this project we aimed to generate an all-in-one rAAV-CRISPR-Cas9 vector targeting LAMA2 mutations found in LAMA2-MD patients and test its ability to correct them using 2D and 3D in vitro cellular models. For that, we characterized the LAMA2-deficient patient-derived cells identifying phenotypic markers of the disease in this model such as impaired myotube fusion and DNA damage. For the viral vector two different serotypes were tested, AAV9 and the rMyoAAV, and the last one was chosen due to its higher transduction efficiency. The all-in-one vector was designed encoding a sgRNA, a smaller version of Cas9 from Neisseria meningitidis, iNme2Cas9, and the HDR donor template, with the selected rAAV capsid serotype. While the vector led to a small percentage of HDR genome editing we observed the reversion of one phenotypic marker: the impaired myotube fusion. Furthermore, we were able to generate and characterize 3D myospheroids using patient-derived cells. Overall, this project shall contribute to the development of safer and more effective therapeutic strategies for LAMA2-MD, and potentially other genetic disorders.
Descrição
Tese de mestrado, Biologia Molecular e Genética, 2025, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
LAMA2-related muscular dystrophy all-in-one rAAV vector Gene therapy CRISPRCas9 Myospheroids