Characterization of a novel Duchenne muscular dystrophy conditional mouse model


Pre-Clinical Research

Poster Number: S39


Muthukumar Karuppasamy, PhD, University of Alabama at Birmingham, Katherine English, BS, Division of Neurology, Department of Pediatrics, University of Alabama at Birmingham, Jeffrey Widrick, PhD, Boston Children's Hospital, Louis Kunkel, PhD, Boston Children's Hospital, Matthew Alexander, PhD, University of Alabama at Birmingham

Background: Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disorder characterized by muscle weakness, cardiac arrhythmia, and respiratory weakness due to the mutations in the DMD gene resulting in a non-functional dystrophin protein. DMD predominantly affects 1:5000 males, although female carriers can show symptoms due to incomplete X-inactivation. Currently, many DMD animal models exist, but no effective conditional models to study the consequences of dystrophin loss in a tissue and temporal manner exist. Subsequently, we generated and analyzed a conditional Dystrophin fl52/Y; human alpha-skeletal actin muscle knockout (Dmd fl52/Y; HSA mKO) model by targeting exon 52 deletion in the Dystrophin gene to study the consequences of dystrophin loss in skeletal muscle lineages. The generated DMD mouse model ablates dystrophin protein expression after mating with a Cre recombinase transgenic mouse under the control of HSA promoter element that is restricted to the skeletal muscle.

Hypothesis: Deletion of dystrophin exon 52 in skeletal muscle is hypothesized to result in a severe phenotype recapitulating that observed in the mdx52 mouse model. The generated conditional DMD mKO mouse may have impaired locomotor activity, muscle architecture, pathophysiology, cardiac and respiratory systems similar to other mdx strains.

Methods: Using Cre/LoxP recombination system and CRISPR editing approach, we generated Dmd flox52/Y that was mated to an HSA-Cre+ mouse model. The resulting Dmd mKO mice have been assessed using histopathological, phenotypical, functional and biochemical assays based on TREAT-NMD standard operating protocols (SOPs) for mdx mouse models.

Results: Phenotypic analysis of these conditional Dmd mKO mice revealed a significant decline in locomotor activity and reduced muscle force, motor and muscular function. The histochemical analysis revealed an increase in centralized myonuclei and fibrotic area similar to mdx mice. Immunoassays including western blot and immunohistochemistry confirmed low expression levels of dystrophin in skeletal muscles of Dmd mKO mice.

Conclusions: This conditional Dmd mKO mouse model may yield invaluable insight into DMD disease pathology and possess similar phenotype found in DMD patients. The generated mouse model could be advantageous in preclinical, dystrophin gene therapy, and exon-skipping replacement studies.