Lee, Ming Yang; (2018) Allele-specific siRNA Therapy for Keratitis-Ichthyosis-Deafness Syndrome. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Dominant mutations in the gene GJB2 cause keratitis-ichthyosis-deafness (KID) syndrome, a severe condition affecting the skin, cornea and inner ear. GJB2 encodes the protein connexin-26 (Cx26) which forms hemichannels or gap junction channels allowing the passage of signalling molecules. Approximately 80% of KID syndrome patients carry a c.148G>A (p.D50N) mutation in GJB2, which results in aberrant channel function. We hypothesised that silencing of the mutant allele in patient keratinocytes using allele-specific siRNA could correct the channel function. First, to confirm whether patient keratinocytes with only one wildtype GJB2 allele formed functional channels following allele-specific siRNA treatment, GJB2+/- keratinocytes were generated using CRISPR/Cas9. The scrape-loading dye transfer (SLDT) assay showed no distinguishable difference in gap junction intercellular communication (GJIC) between GJB2+/- and GJB2+/+ cells, suggesting normal GJIC in GJB2+/- keratinocytes. Nineteen siRNAs were designed and tested in HeLa cells expressing wildtype or mutant GJB2-GFP transgene. A lead siRNA, was discovered, which potently inhibited the mutant mRNA and protein without affecting wildtype GJB2 expression. The efficacy of the lead siRNA was assessed using keratinocytes derived from a KID syndrome patient (KID-KC) harbouring heterozygous c.148G>A mutation. These cells displayed pathological features of KID syndrome, with reduced gap junction plaque formation, impaired GJIC and hyperactive hemichannels confirmed by immunostaining, SLDT, patch clamp and neurobiotin uptake assays. Following treatment with the siRNA, selective silencing of mutant GJB2 allele in KID-KCs was confirmed at mRNA and protein levels. Significant improvement of GJIC and reversal of hemichannel activity were detected, with the latter corrected to a level comparable to that recorded in normal keratinocytes. Furthermore, RNA-Seq analysis showed that only six genes in the KID-KC transcriptome were significantly altered by the siRNA treatment, suggesting low-level off-target effects. In conclusion, allele-specific siRNA silencing of pathogenic dominant GJB2 mutation could be a potential therapeutic intervention for KID syndrome.

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