Understanding pathological mechanisms and developing novel therapeutic approaches for CMT1B disease

About This Grant

Project Summary/Abstract Mutations in the myelin protein zero (MPZ) gene cause one of the most common forms of hereditary neuropathy known as Charcot-Marie-Tooth type 1B (CMT1B) disease with phenotypes ranging from severe early-onset demyelinating to late-onset axonal neuropathy. CMT1B is a debilitating disorder with patients showing progressive distal weakness and atrophy, sensory loss, classical steppage gait, and painful and disfiguring lower limb contractures. MPZ is expressed mainly in myelinating Schwann cells and functions to compact myelin sheath in peripheral nerves. Here we propose two aims to elucidate the cellular and molecular disease mechanism and develop a novel gene therapy approach. In Aim 1, we will determine molecular and cellular mechanisms by which MPZ variants lead to a spectrum of phenotype from demyelination to axonal form using patient-derived peripheral nerve organoids (PNOs). Our team has the largest known cohort of patient Fibroblasts with various MPZ mutations where we are developing induced pluripotent stem cell (iPSC) lines. We demonstrate that CMT1B PNOs display reduced myelination, elevated ER stress and UPR, and Schwann cell survival, emphasizing the potential of PNOs to mimic CMT1B patient pathology. We also found that PNOs replicate disease severity, as R98C mutant PNOs show greater demyelination, axonal loss, and lower MPZ expression than S63del, mirroring patient phenotypes. We will use various cutting-edge technologies including single-cell RNAseq, MEA array, high-resolution imaging, and CRISPR gene correction to investigate disease mechanisms in patient PNOs. In Aim 2, we will develop a novel gene therapy approach for CMT1B and evaluate its efficacy in rescuing disease phenotypes in MPZR98C mice and our newly established patient-derived PNOs toward establishing essential preclinical data for translation. Considering that MPZ is an essential protein, and many MPZ mutations act through dominant negative, toxic-gain-of-function mechanisms, we hypothesize that a successful therapeutic strategy will eliminate the mutant MPZ while replacing it with wild-type form to maintain healthy myelination. Therefore, here we propose to develop a universal knockdown-and-replace gene therapy strategy that can be applied to virtually all patients with a dominant MPZ-associated neuropathy, regardless of the causative mutation. To do this, we will generate bi-functional AAV vectors designed to: (1)_knock down both wild-type and mutant MPZ using designed artificial miRNAs, and (2) deliver a miRNA-resistant wild-type MPZ replacement gene within the same vector. Our goal is to restore normal MPZ to Schwann cells, improving myelination and Schwann-axon interaction. This multidisciplinary research will uncover the pathomechanisms of MPZ-related neuropathy by pioneering the patient-specific CMT1B PNO model and generating preclinical data to support the translation of a novel AAV-based knockdown-and-replace gene therapy with strong clinical potential. The methods and findings from this study may also broaden the application of this technology to other inherited diseases.