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.