Allele-Specific Small Interfering RNA Corrects Aberrant Cellular Phenotype in Keratitis-Ichthyosis-Deafness Syndrome Keratinocytes

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.

Figure 1.. Genotype, morphology, GJB2 expression, and subcellular localization in keratinocytes.

(a) cDNA sequences of GJB2 from the patient with KID syndrome and the healthy donor. (b) Morphology of keratinocytes from healthy donor and patient with KID syndrome at early passages (P1 or P5) and late passages (P35 or P45). Bar = 100 μm. (c) mRNA expression of total GJB2, the WTallele, and the mutant allele in keratinocytes from healthy donor and patient with KID syndrome determined by qRT-PCR. (d) Total C×26 protein (asterisk, at26 kDa) expression was examined by immunoprecipitation and immunoblotting, which shows decreased C×26 expression in patient cells. (e) Expression of C×26 detected by immunofluorescence staining, where gap junction plaques can be found at cell-cell junctions in normal keratinocytes (arrows), whereas C×26 in keratinocytes from the patient with KID syndrome was localized discretely in the cytoplasm (arrowheads). Bar = 40 μm. (f) E-Cad was stained in green color. C×26 expression in the skin is shown, where punctate staining of C×26 was observed (arrows). The dotted lines show dermal-epidermal junction. Bar = 40 μm. ***P < 0.001. Cont, keratinocytes derived from the healthy donor; E-Cad, E-cadherin; KID, keratitis-ichthyosis-deafness; N.S., not significant; qRT-PCR, quantitative reverse transcriptase-PCR; WT, wild-type.

Figure 2.. Abnormal gap junction and hemichannel behavior in KID-KCs.

The gap junction intercellular communication in KID-KCs (KID) or control-KCs (Cont) was examined by the SLDT assay, and the hemichannel activity was examined by whole-cell patch clamp and NB uptake. (a) Representative images of SLDT in the keratinocytes using NB tracer (red). (b) Data analysis shows that KID-KCs had impaired ability to transfer NB to adjacent cells. (c) Representative patch clamp records from single keratinocytes in response to the voltage step protocol from −110 mV to +110 mV in 20 mV increments. (d) The plot of current density against membrane voltage reveals aberrantly enhanced hemichannel activity in KID-KCs. (e) Representative images of NB uptake (red), with the nuclei stained with DAPI (blue). (f) Data analysis shows increased uptake of NB in KID-KCs. All data are presented as the mean ± SEM. Bar = 200 μm. *P < 0.05; ***P < 0.001. Control-KC, control keratinocyte; KID-KC, keratitis-ichthyosis-deafness syndrome-derived keratinocyte; NB, neurobiotin; SEM, standard error of the mean; SLDT, scrape-loading dye transfer.

Figure 3.. Epidermal morphology of grafted skin in human-murine chimeric skin graft model.

Primary fibroblasts and immortalized keratinocytes derived from the patient with KID syndrome harboring a heterozygous GJB2 c.148G>A mutation or a healthy donor were used to generate bioengineered skin sheets, which were grafted onto NOD-severe combined immunodeficiency mice (NSG mice). Eight weeks after grafting, regenerated skin grafts were harvested. (a, d) Macroscopic examination showed fine, dry scales in the graft generated from (d) patient cells compared with that generated from (a) control cells. (c, f) Histological examination showed hyperkeratosis and spongiosis in (f) the patient skin graft, resembling that seen in (c) the patient skin. (b, e) The skin architecture of (e) the control skin graft was also similar to (b) healthy donor skin. Bar = 100 μm. KID, keratitis-ichthyosis-deafness.

Figure 4.. Allele-specific GJB2 knockdown by S7.

(a) The mRNA expression of total GJB2, WT, and MUT GJB2 alleles in control-KCs (Control) and KID-KCs (KID) treated with AS-siRNA S7 was examined by qRT-PCR and compared with UT cells. (b) Total C×26 protein expression in S7-treated cells from three independent IP/IB experiments. (c) The expression was quantified using densitometry. β-actin was used as a loading control and HeLa cells were used as a negative control. A reduction in total C×26 expression was detected in KID-KCs after S7 treatment, but such a change was not detected in control-KCs. **P < 0.01. AS-siRNA, allele-specific small interfering RNA; control-KC, control keratinocyte; IB, immunoblotting; IP, immunoprecipitation; KID-KC, keratitis-ichthyosis-deafness syndrome-derived keratinocyte; MUT, mutant; N.S., not significant; UT, untreated; WT, wild-type.

Figure 5.. S7 treatment corrected abnormal GJ and hemichannel functions in KID-KCs.

(a, b) SLDT was performed in siRNA-treated KID-KCs (KID) and control-KCs (Cont) to assess GJ activity, (c, d) Analysis of NB transfer (red). Each dot in panels (c) and (d) represents the average NB transfer from a single image. Three independent experiments were carried out and at least 10 images were analyzed from each experiment. Restoration of GJ activity was detected in KID-KCs following S7 treatment. Whole-cell patch clamp and NB uptake were carried out to examine hemichannel activity. (e–g) Records of currents from single cells under the voltage step protocol. (h) The plot of current density against membrane voltage shows correction of hyperactive hemichannels in KID-KCs after S7 treatment. (i) Representative NB (red) uptake images. The nuclei were stained with DAPI (blue). (j) Data analysis shows reversal of aberrantly enhanced NB uptake in KID-KCs. Data are presented as the mean ± SEM. Bar = 200 μm. *P < 0.05; **P < 0.01; ***P < 0.001. Control-KC, control keratinocyte; KID-KC, keratitis-ichthyosis-deafness syndrome-derived keratinocyte; NB, neurobiotin; N.S., not significant; SEM, standard error of the mean; si-cont, control small interfering RNA; SLDT, scrape-loading dye transfer.