Generation of mutant human SOD1 knock-in mouse lines at the Rosa26 locus as a platform for developing genome-editing...

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the degeneration of upper and lower motor neurons, resulting in progressive paralysis and death within a few years of symptom onset. Although current treatments modestly slow the disease progression, effective disease-modifying and curative therapies remain an urgent unmet need. SOD1 mutations are one of the major genetic causes of familial ALS. The p.Leu127Ser (L126S) and p.Gly94Ser (G93S) variants are clinically relevant pathogenic variants for which appropriate animal models are needed for preclinical evaluation of gene-editing therapies. However, most existing SOD1 models rely on high copy overexpression of mutant SOD1. Therefore, animal models carrying a single copy mutant human SOD1 allele are required for evaluating the in vivo efficacy of genome editing therapies. Here, we used CRISPR/Cas9-mediated homology-directed repair to generate a knock-in mouse line at the Gt(ROSA)26Sor (Rosa26) locus carrying a single-copy, 11-kb human SOD1 genomic fragment, including all exons and introns, with the L126S mutation. The Rosa26-hSOD1^L126S mice did not develop ALS-like phenotypes during the limited observation period. However, they faithfully retained a single-copy mutant human SOD1 genomic allele, providing a valuable preclinical platform for evaluating genome-editing therapies. We also generated Rosa26-hSOD1^G93S mice carrying the SOD1 G93S mutation with comparable efficiency. Together, these mutant human SOD1 knock-in mouse lines provide a versatile and clinically relevant platform for the preclinical evaluation of genome-editing therapies targeting heterozygous SOD1 mutations.