Lee, MY;
Wang, H-Z;
White, TW;
Brooks, T;
Pittman, A;
Halai, H;
Petrova, A;
... Di, W-L; + view all
(2020)
Allele-Specific Small Interfering RNA Corrects Aberrant Cellular Phenotype in Keratitis-Ichthyosis-Deafness Syndrome Keratinocytes.
Journal of Investigative Dermatology
, 140
(5)
1035-1044.e7.
10.1016/j.jid.2019.09.022.
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Accepted version-siRNA and KIDJID-2019-0132.R2_Proof_hi.pdf - Accepted Version Download (3MB) | Preview |
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. 86% of cases are caused by a heterozygous missense mutation (c.148G>A, p.D50N) in the GJB2 gene, encoding gap junction protein connexin 26 (Cx26), which alters gating properties of Cx26 channels in a dominant manner. We hypothesized that a mutant-allele-specific siRNA (AS-siRNA) could rescue the cellular phenotype in patient keratinocytes. A KID syndrome cell line (KID-KC) was established from primary patient keratinocytes with a heterozygous p.D50N mutation. This 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-KC mimicked patient skin in vivo, further confirming the validity of these cells as a model. In vitro treatment with AS-siRNA led to robust inhibition of the mutant GJB2 allele without altering expression of the wildtype. This corrected both gap junction and hemichannel activity. Notably, AS-siRNA treatment caused only low-level off-target effects in KID-KC, as detected by genome-wide RNA sequencing. Our data provide an important proof-of-concept and model system for the potential use of AS-siRNA in treating KID syndrome, and other dominant genetic conditions.
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