CMT2 presents as a slow, progressive disorder associated with axonal dysfunction. CMT type 2E (CMT2E) is associated with mutations in the gene neurofilament light chain (NEFL). The protein, NF-L, is one of five subunits that comprise neurofilaments and contribute to the axonal cytoskeleton. To advance the development of therapeutics for CMT2E and to better understand the underlying biology of disease progression, we generated and characterized Nefl+/E397K and NeflE397K/E397K mice. These models display early quantifiable functional deficits, including reduced compound muscle action potential (CMAP) area, negative area, and increased distal latency. Consistent with this, neuronal and skeletal muscle pathology is also observed. We have now used these experimental contexts for the development of a gene therapy vector for CMT2E that utilizes the “knockdown-and-replace” strategy, in which a single vector delivers: 1) a shRNA to reduce the expression of the disease allele; and 2) a shRNA-resistant cDNA encoding wild type NF-L. identified rescue in electrophysiology, nerve morphology, muscle pathology, and motor function deficits.
We screened a series of shRNAs in culture and identified several candidates that reduce mouse Nefl and human NEFL efficiently. Additionally, we generated a NEFL cDNA sequence that is modified (resistant to the shRNA) at the nucleotide level without altering the amino acid sequence. Importantly, since this approach is not specific for any particular NEFL mutation, it can be broadly applied to the patient population. AAV9 was used to deliver the dual-cargo vector via an intracerebroventricular injection of 1 x1012 vector genomes on postnatal day (P) 0 and 1. Vector expression was confirmed in disease-relevant tissue. We now demonstrate that a significant correction of electrophysiology parameters, axon and muscle pathology, and motor function assessments in vector-treated Nefl-E397K mice but not untreated Nefl-E397K mice. Additionally, individual components (shRNA- or cDNA-alone) did not significantly improve the phenotype, demonstrating that delivery and expression of both components was important for the efficacious activity of the knockdown-and-replace vector. Collectively, these results provide important pre-clinical evidence of an effective gene therapy for CMT2E that could be used in a mutation-agnostic manner to address the breadth of disease-causing mutations.