Development of a new gene therapy strategy for LAMA2-MD Development and Evolutionary Morphogenesis

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A key component of the basement membrane of skeletal muscle tissue 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 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 C2C12 myoblast cell lines that carry point mutations in Lama2, which recapitulate the ones found in two different mouse models for LAMA2-MD. The established cell lines were characterized to have impairment in myoblast differentiation and fusion into myotubes. The single-cell clones were then used to evaluate the mutation reversion 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 a HDR donor template. 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.