1:Instituto de Salud Carlos III; 2:Fundación Andrés Marcio, niños contra la laminopatía

The LMNA c.745C>T p.R249W variant is the most frequent mutation found in LMNA-related congenital muscular dystrophy (L-CMD). L-CMD is an infantile, striated muscle, laminopathy characterized by prominent axial weakness, motor development loss, neck weakness and respiratory, cardiac and gastrointestinal alterations with fatal consequences. Since L-CMD is a rare genetic disease without cure, our main objective is to obtain effective therapies to fight against it. We have explored the therapeutic value of two different CRISPR-based strategies. One consists on the use of sgRNAs specific for the LMNA c.745C>T mutation, while the other depends on the homology independent targeted insertion (HITI) of a wild type, exon3-12 cassette upstream of the LMNA c.745C>T mutation. We have studied both approaches in human and mouse cells as well as in an Lmnaᴿ²⁴⁹ᵂ mouse model. In cultured cells, 745C>T-sgRNAs were highly efficient against the mutant allele and showed low activity for the wild type one. On the other hand, a low percentage of cells were edited by HITI. Importantly, for both approaches, AAV-mediated gene therapy studies showed significant survival improvement of the early lethality associated with the Lmnaᴿ²⁴⁹/ᴿ²⁴⁹ᵂ mice. In all the cases, mouse phenotype and cellular abnormalities induced by the LMNA-R249W mutation were reverted to a wild type status. These results confirm the therapeutic potential of these two CRISPR-based strategies for the treatment of L-CMD.