Late Breaking: LAMA2-CMD: establishment of a new gene therapy strategy using an in vitro model


Pre-Clinical Research

Poster Number: Virtual


Diogo Fernandes, BSc, Faculty of Sciences of University of Lisbon, Sólveig Thorsteinsdóttir, PhD, Faculty of Science of University of Lisbon, Jose Jose Escandell, PhD, Instituto de Biologia Experimental e Tecnológica, Ana Rita Carlos, PhD, Faculty of Science of University of Lisbon

Pathogenic mutations in LAMA2, which encodes the α2-chain of laminins 211 and 221, trigger a life-threatening and currently incurable disease, the LAMA2-congenital muscular dystrophy (LAMA2-CMD). Several studies have tried to design possible strategies for LAMA2-CMD therapeutics. However, classical gene therapy approaches are not feasible due to the size of the LAMA2 gene, which exceeds the packaging capacity of the most viral vector used in gene therapy. According to the Leiden Open Variation Database (LOVD), over 70% of the LAMA2 mutations in patients correspond to nucleotide substitutions. This opens the possibly to establish new therapeutic strategies that are based on efficient genome editing of base substitutions. Instead of introducing the complete gene sequence into cells, homology-directed repair (HDR) putatively allows to correct the majority of pathogenic point mutations. Using the CRISPR-Cas9 system, we have established two C2C12 myoblast cell lines that carry point mutations in Lama2, which recapitulate the ones found on the dy2J/dy2J and dynmf417/dynmf417 mouse models for LAMA2-CMD. To generate these cell lines, the transfection protocol of the CRISPR-Cas9 system delivery either as plasmid DNA or ribonucleoprotein complexes was optimized using cationic lipids. Furthermore, the obtained single cell clones were confirmed by DNA sequencing and allele-specific PCR to be heterozygous for the intended mutations. After being phenotypically characterized, these cell lines will be used to test the reversion of the specific mutations in the Lama2 gene with all-in-one recombinant adeno-associated virus (rAAV). By in silico optimization, this engineered vector will we able to encode a smaller version of Cas9 from Neisseria meningitidis, Nme2Cas9 , a single sgRNA and the HDR donor template. This project will be a proof-of-concept that precedes the in vivo studies using dy2J/dy2J mice and will contribute to establish a new possible therapeutic strategy targeting LAMA2-CMD.