Abstract

Keratitis-ichthyosis-deafness (KID) syndrome is a severe, untreatable condition characterized by ocular, auditory, and cutaneous abnormalities, with major complications of infection and skin cancer. Most cases of KID syndrome (86%) are caused by a heterozygous missense mutation (c.148G>A, p.D50N) in the GJB2 gene, encoding gap junction protein Cx26, which alters gating properties of Cx26 channels in a dominant manner. We hypothesized that a mutant allele-specific small interfering RNA could rescue the cellular phenotype in patient keratinocytes (KCs). A KID syndrome cell line (KID-KC) was established from primary patient KCs with a heterozygous p.D50N mutation. This cell line displayed impaired gap junction communication and hyperactive hemichannels, confirmed by dye transfer, patch clamp, and neurobiotin uptake assays. A human-murine chimeric skin graft model constructed with KID-KCs mimicked patient skin in vivo, further confirming the validity of these cells as a model. In vitro treatment with allele-specific small interfering RNA led to robust inhibition of the mutant GJB2 allele without altering expression of the wild-type allele. This corrected both gap junction and hemichannel activity. Notably, allele-specific small interfering RNA treatment caused only low-level off-target effects in KID-KCs, as detected by genome-wide RNA sequencing. Our data provide an important proof-of-concept and model system for the potential use of allele-specific small interfering RNA in treating KID syndrome and other dominant genetic conditions.