Charcot-Marie-Tooth Disease type 4B3 (CMT4B3) is a recessive, clinically heterogenous and often severe form of hereditary peripheral neuropathy with onset during infancy or early childhood. Symptoms of CMT4B3 range from an isolated demyelinating sensorimotor polyneuropathy to a complex neurodevelopmental phenotype with axonal neuropathy, cranial nerve involvement, intellectual disability and facial dysmorphism. Mutations in the Sbf1 gene cause CMT4B3 via loss of the pseudo-phosphatase Myotubularin-Related Protein 5 (MTMR5). MTMR5 is an important yet poorly understood regulator of autophagy and endosomal sorting and is involved nervous system development. Gene replacement therapy would be appropriate for CMT4B3, however, the cDNA of Sbf1 is larger (5,679bp) than the size limit of adeno-associated viral vectors (~4,700 bp). 

To circumvent this challenge, we’ve devised several candidate minigenes based on comparative protein family studies, cross-species, and cross-domain investigations. Additionally, we have established a resource of four CMT4B3 patient iPSC lines and derived human motor neurons to both elucidate the mechanism of Sbf1 mutations on axonal degeneration and to validate the mini-gene replacement strategy. 

Functional studies in human motor neurons and iPSCs reveal that MTMR2 expression is decreased, while minimal MTMR5 expression remains, indicating that residual, truncated MTMR5 could produce toxic gain-of-function. Both an increase in supernatant NF-L, an axonal injury biomarker, and aberrances in autophagic flux were also identified in the patient motor neurons. We have identified one minigene that recapitulates proper subcellular distribution of MTMR5 in patient-derived fibroblasts, while both co-precipitate with the active binding partner of MTMR5, MTMR2.

Our studies provide evidence that CMT4B3 could occur via axonal mechanisms. Future directions include refinement of fluorescence-based autophagic trafficking assays, identification of novel MTMR5 binding partners and gene expression profiling. Insight from such studies will inform refinement of candidate minigenes and will be used to develop an image-based cellular phenotypic screening platform for CMT4B3.