Promoter-Targeted CRISPR Correction of PMP22 Gene Dosage Restores Schwann Cell Homeostasis in Charcot-Marie-Tooth Disease Type 1A.

BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A), the most common inherited peripheral neuropathy, arises from a 1.4 Mb duplication of the Peripheral Myelin Protein 22 (PMP22) gene, causing gene-dosage-dependent toxic overexpression that drives Schwann cell dysfunction and demyelination. We developed a gene-dosage-targeted therapeutic strategy using an sgRNA-mediated bidirectional promoter microdeletion (sgRNA-BPM) approach to selectively suppress pathogenic PMP22 overexpression while preserving physiological expression levels. METHODS: Clinical evaluation of an index patient (pedigree analysis, muscle MRI, electrophysiological assessment) confirmed an inherited demyelinating neuropathy with distal muscle atrophy. Patient-derived pluripotent stem cells (iPSCs) were used to establish a single-cell-derived clone in which all three PMP22 P1 promoter TATA boxes were disrupted (- 2, - 2, and - 17 bp deletions). This promoter-targeted editing attenuated PMP22 expression during directed differentiation into Schwann cell lineage. RESULTS: Edited cells exhibited partial restoration of Schwann cell maturation-associated, promyelinating-like transcriptional features, accompanied by upregulation of SOX9 and AP2α, attenuation of aberrant PMP22 overexpression, and improvement of Schwann cell morphology. Quantitative imaging revealed ~ 50% reduction (p < 0.01) in PMP22/S100β co-expression index, indicating substantial molecular and structural correction. Despite P1 promoter disruption, basal PMP22 expression was preserved, consistent with transcription driven by the downstream TATA-less P2 promoter. This promoter-level repression of PMP22 was reproducible in mature human primary Schwann cells, where quantitative PCR confirmed reduced PMP22 expression (p < 0.05), supporting translational feasibility of this gene-dosage modulation strategy beyond iPSC-derived systems. CONCLUSIONS: Collectively, patient-anchored, promoter-targeted genome editing can potentially correct pathogenic PMP22 dosage while preserving basal expression, establishing a translational iPSC-based framework for treating dosage-sensitive demyelinating neuropathies such as CMT1A.