Publicação
LAMA2-CMD: establishment of a new gene therapy strategy using an in vitro model
| Resumo: | In skeletal muscle tissue, the basement membrane lining the sarcolemma coats and protects myofibers from contraction-induced damage. A key component of this specialized structure is the Laminin-211 (LN211). Pathogenic mutations in the LAMA2 gene, which encodes the α2-chain of laminin-211, trigger a life-threatening and currently incurable disease, LAMA2-muscular dystrophy (LAMA2-MD). However, the classical gene therapy approaches are not feasible due to the size of the LAMA2 gene, which exceeds the packaging capacity of one of the most used viral vectors, the recombinant adeno-associated virus (rAAV). Instead of introducing the complete gene sequence into the cells, homology-directed repair (HDR) can theoretically correct the majority of pathogenic point mutations responsible for LAMA2-MD. Using the CRISPR/Cas9 system, we have established two C2C12 myoblast cell lines that carry point mutations in Lama2, which recapitulate the ones found in the dy2J/dy2J and dynmf417/dynmf417 mouse models for LAMA2-MD. Furthermore, the obtained single-cell clones were confirmed by nextgeneration sequencing (NGS) to be heterozygous for the intended mutations. The established cell lines were then characterized and had an impairment in myoblast differentiation and fusion into myotubes. The single-cell clone carrying the dynmf417 mutation was used to evaluate the reversion of the specific mutation with all-in-one rAAV vectors by HDR. The engineered vector encoded a smaller version of Cas9 from Neisseria meningitidis, Nme2Cas9, a single sgRNA and the HDR donor template. The rAAV production was also optimized and the AAV1 serotype was selected as the most suitable for the transduction of the C2C12 cells. The muscle-specific rAAV9:HDR system was not able to edit and revert the specific mutation in the Lama2 gene but gave critical insights into the best rAAV design strategies. This project will contribute to the development of a new therapeutic strategy targeting LAMA2-MD that will be preceded by the in vivo studies using the dy2J/dy2J mice. |
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| Autores principais: | Fernandes, Diogo Rodrigues |
| Assunto: | Distrofia muscular com deficiência em LAMA2 Terapia genética CRISPR Vetores adeno-associados recombinantes Edição de precisão do genoma Teses de mestrado - 2024 |
| Ano: | 2024 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | In skeletal muscle tissue, the basement membrane lining the sarcolemma coats and protects myofibers from contraction-induced damage. A key component of this specialized structure is the Laminin-211 (LN211). Pathogenic mutations in the LAMA2 gene, which encodes the α2-chain of laminin-211, trigger a life-threatening and currently incurable disease, LAMA2-muscular dystrophy (LAMA2-MD). However, the classical gene therapy approaches are not feasible due to the size of the LAMA2 gene, which exceeds the packaging capacity of one of the most used viral vectors, the recombinant adeno-associated virus (rAAV). Instead of introducing the complete gene sequence into the cells, homology-directed repair (HDR) can theoretically correct the majority of pathogenic point mutations responsible for LAMA2-MD. Using the CRISPR/Cas9 system, we have established two C2C12 myoblast cell lines that carry point mutations in Lama2, which recapitulate the ones found in the dy2J/dy2J and dynmf417/dynmf417 mouse models for LAMA2-MD. Furthermore, the obtained single-cell clones were confirmed by nextgeneration sequencing (NGS) to be heterozygous for the intended mutations. The established cell lines were then characterized and had an impairment in myoblast differentiation and fusion into myotubes. The single-cell clone carrying the dynmf417 mutation was used to evaluate the reversion of the specific mutation with all-in-one rAAV vectors by HDR. The engineered vector encoded a smaller version of Cas9 from Neisseria meningitidis, Nme2Cas9, a single sgRNA and the HDR donor template. The rAAV production was also optimized and the AAV1 serotype was selected as the most suitable for the transduction of the C2C12 cells. The muscle-specific rAAV9:HDR system was not able to edit and revert the specific mutation in the Lama2 gene but gave critical insights into the best rAAV design strategies. This project will contribute to the development of a new therapeutic strategy targeting LAMA2-MD that will be preceded by the in vivo studies using the dy2J/dy2J mice. |
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