Characterization of a Zebrafish Autosomal Recessive Limb-Girdle Muscular Dystrophy-1 (LGMDR1) Model for Drug Screening


Pre-Clinical Research

Poster Number: T359


Anna Cordeiro-Santanach, PhD, AGADA Biosciences Inc., Meagan McKenna, MSc, AGADA Biosciences, Pia Elustondo, PhD, AGADA Biosciences, Kanneboyina Nagaraju, PhD, Binghamton University, Eric Hoffman, PhD, Binghamton University, Binghamton, NY, USA

Autosomal recessive limb-girdle muscular dystrophy-1 (LGMDR1), previously symbolized LGMD2A, is a type of muscular dystrophy caused by mutations in the Calpain-3 (CAPN3) gene. These mutations cause progressive weakness in the muscles around the shoulders and the hips. It appears during childhood and early adulthood, resulting in loss of ambulation within 20 years of the diagnosis and it is currently untreatable. Animal models developed to date are not ideal to screen new drugs because of the late-onset and very mild disease features.
We have characterized three allelic zebrafish (Danio rerio) CAPN3 models generated using CRISPR/Cas9 genome editing technology. capn3b mRNA expression was characterized via whole mount in situ hybridization (WISH). All three models showed significantly reduced mRNA expression from the capn3b gene. Consistent null mRNA phenotypes were seen in the RNAless and mut1 lines. The third mut73 line displayed reduced but variable mRNA expression, with individual embryos showing mRNA expression ranging from undetectable to WT-like levels.
We characterized the phenotype of the calpain 3-deficient larvae using stressed swimming in 0.8% methylcellulose, a viscous compound that adds resistance to the water. The goal was to determine if specific endpoints, namely abnormalities in birefringence suggesting poor alignment of myofibers in myotome, and muscle membrane leakage in the null lines when the animals were injected with Evans Blue Dye, could be used for screening of potential therapeutic agents. We observed that mut1 (10.64%) and RNAless (3.90%) lines showed higher percentage of muscle phenotype under birefringence than the WT line (1.73%). Regarding muscular leakage, we also observed that mut1 (8.16%) and RNAless (6.06%) lines displayed an increased percentage of muscle leakage than the WT line (0%).
These capn3b-deficient lines set up a start for preclinical drug testing. Further behavioral testing to assess swimming and mobility abilities will provide additional information on the models.