Mutation-targeted siRNA therapy for connexin 26-associated keratitis-ichthyosis-deafness syndrome

Abstract

Background & Aim Keratitis-ichthyosis-deafness (KID) syndrome is a severe, currently untreatable condition characterized by ocular, auditory and cutaneous abnormalities, with major complications from infection to skin cancer. 86% of cases are caused by a heterozygous missense mutation (c.148G>A, p.D50N) in gene GJB2, encoding gap junction protein connexin 26 (Cx26), which alters gating properties of Cx26 channels and gap junction intercellular communication in a dominant manner. There is a pressing need to develop new treatments for the condition. We hypothesized that design and optimization of a mutant-allele-specific siRNA (AS-siRNA) should rescue the cellular phenotype in patient epidermal keratinocytes, and as such could form the basis of a future gene therapy. Methods, Results & Conclusion To that end, a KID syndrome cell line (KID-KC) was established from primary keratinocytes of a KID syndrome patient with heterozygous p.D50N mutation, which displayed impaired gap junction intercellular communication and hyperactive hemichannels, confirmed by dye transfer, patch clamp and neurobiotin uptake assays. In KID-KCs, treatment with AS-siRNA led to robust inhibition of the mutant GJB2 allele without altering expression of the wildtype allele. This resulted in correction of both gap junction intercellular communication and hemichannel activity. Furthermore, AS-siRNA 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 for the potential use of AS-siRNA in treating KID syndrome, and in other genetic conditions due to dominant mutations.