Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy, affecting ~1 in 2,500 individuals worldwide. Patients with CMT disease display motor and sensory loss in the distal limbs, which can lead to ambulatory loss, limb amputation, and morbidity, incurring significant public health costs. The CMT2 subtype is difficult to understand at the mechanism level. Not only does it manifest impaired neuronal axon function, it is also often associated with mutations in ubiquitously expressed aminoacyl-tRNA synthetases (ARSs), the enzymes that charge tRNAs with an amino acid to generate aminoacyl-tRNAs for protein synthesis. While CMT2 pathogenic mutations are known, whether these mutations drive the disease progression by a loss- or gain-of-function effect is inconclusive. Without this knowledge, our efforts to combat CMT2 are limited. Most studies of CMT2 use vertebrate models, which while useful, differ considerably from human patients in genotypes. We hypothesize that the best approach to elucidate the pathogenic mechanism of CMT2 is to use induced pluripotent stem cells (iPSC)-derived motor neurons as a cell model. With the ability to differentiate into peripheral motor neurons, iPSC models represent a unique opportunity to study CMT2 in a platform of the human disease. We will focus on the R329H mutation in AARS (alanyl aminoacyl-tRNA synthetase), which reduces tRNA charging and is expected to cause defective protein synthesis. While we already have an iPSC model for the mutation generated from the human embryonic H9 line, we propose here to generate a patient- derived model for comparison, which will provide critical insight into whether the disease mechanism is impacted by other factors associated with the genetic background of the patient.
In Aim 1, we will generate a control line based on the fibroblasts of the patient to restore the wild-type (wt) sequence at the AARS-R329H locus. We will use CRISPR/Cas9 for this restoration and will validate it by assays for the AARS enzyme. We expect that the restored wt sequence will confer full enzyme activity.
In Aim 2, we will establish pluripotent iPSC lines from the control and patient fibroblasts and validate each by assays for AARS. We will then differentiate each line to motor neurons and identify phenotypes specific to the mutation. Combined, this work will produce an isogenic pair of wt and AARS-R329H iPSC lines that maintain the genetic background of the patient. The phenotype of this patient-derived pair, relative to the H9-derived pair, will provide new insight into the disease within the genetic context of the patient. Both pairs will be shared with the research community of CMT2 and related peripheral neuropathies.
While studies of CMT2 on vertebrate models have advanced our understanding of the disease mechanism, the translation of such studies to clinical therapies has not been successful. We propose to generate an iPSC model from the fibroblasts of a patient with the CMT2 mutation AARS-R329H as a platform that most closely reproduces the human disease. We expect that this patient-derived model, together with our already-made H9- derived model, will provide new clues to the disease mechanism and guide the development of new treatments.
