Dennis O. Pérez-López, Audrey A. Shively, Mohammed T. Abu-Salah, Zaid T. Abu-Salah, Jackson T. Veltrop, F. Javier Llorente Torres, Michael Garcia, W. David Arnold, Monique A. Lorson, Christian L. Lorson
Charcot-Marie-Tooth type 2 (CMT2) is a slow, progressive neuropathy characterized by axonal dysfunction, with clinical symptoms including distal muscle weakness and atrophy, sensory loss, foot deformities, and reduced nerve conduction velocity. CMT type 2E (CMT2E) is linked to mutations in the neurofilament light chain (NEFL) gene, which encodes NF-L, a critical component of the axonal cytoskeleton. To enhance our understanding of CMT2E pathogenesis and facilitate therapeutic development, we generated Nefl+/E397K and NeflE397K/E397K mouse models. Comprehensive phenotypic analyses, including motor function, balance, and strength assessments, revealed early axonal defects as early as postnatal day 21 (P21). These defects included reduced compound muscle action potential (CMAP), decreased negative area, and prolonged distal latency. Progressive disease features, such as worsening distal latency and functional impairments, were evident at 6 and 12 months. Muscle atrophy, denervation, and axonal morphology abnormalities were observed, with cross-sectional imaging of the sciatic nerve showing axonal defects starting at 3 weeks. Based upon these baseline studies, we investigated the efficacy of a “knock-down-and-replace” AAV9 vector in which the mutant Nefl gene was targeted with a shRNA and a shRNA-resistant “wildtype” Nefl cDNA was co-delivered within the same vector. While early timepoints did not demonstrate a significant rescue in the CMT phenotype, a significant improvement was observed in electrophysiological measures, skeletal muscle pathology, and axonal pathology.
These findings establish Nefl+/E397K and NeflE397K/E397K mice as robust models of CMT2E, exhibiting early and clinically relevant phenotypes. Furthermore, these were excellent contexts to demonstrate that an AAV-based therapeutic designed to effectively remove and replace the mutant nefl gene with a wildtype “resistant” copy.