AAV-mediated genome editing to normalize PMP22 gene duplication responsible for Charcot-Marie-Tooth disease type 1A

Charcot-Marie-Tooth disease type 1A (CMT1A) is one of the most common hereditary peripheral neuropathies, for which there exists no radical therapy. In the present study, we developed a method to correct duplication of PMP22 gene, which causes CMT1A, based on genome editing with gRNA that targets a unique sequence present at two sites that sandwich only a single copy of duplicated PMP22 genes. Quantitative PCR and Southern blot analyses revealed that infection of iPS cells (iPSCs) or iPSC-derived Schwann cells with an AAV2 vector expressing both hSaCas9 and gRNA targeting the tandem repeat sequence decreased PMP22 transcript levels by 20 - 40%. CMT1A-iPSC-derived Schwann cell precursors and Schwann cells exhibited decreased proliferation and increased apoptosis. These characteristics extended to impaired myelination in co-culture with normal iPSC-derived neurons. Increased apoptosis and impaired myelination ability of human CMT1A-iPSC-derived Schwann cell precursors were normalized by infection with the newly-developed AAV2 genome-editing vector. In vivo transfer of AAV2 vectors to peripheral nerves is efficient, so this potential therapeutic modality for CMT1A is of significant clinical interest.