UCL Great Ormond Street Institute of Child Health


Great Ormond Street Institute of Child Health


Mutation-targeted small interfering RNA gene therapy for Keratitis-Ichthyosis-Deafness syndrome

Supervisors: Professor Wei-Li Di, Professor Veronica Kinsler

Keratitis-Ichthyosis-Deafness syndrome (KID) is a currently untreatable genetic disease with an incidence of 1 in 100,000 new births and characterized by congenital erythrokeratoderma, sensorineural deafness and ocular abnormalities. Patients with this rare condition have a poor quality of life due to thickened inflamed skin, deafness and poor vision. KID patients also have an increased risk of cancer: 20% of patients between the ages of 15-40 develop carcinoma of the skin and/or tongue (1). In this context, there is a pressing need to develop a specific treatment for this disease to improve symptoms, quality of life and, most importantly, potentially to decrease the risk of squamous cell carcinoma by normalizing skin phenotype.

KID is caused by heterozygous missense mutations in the gene GJB2 (2). Although several missense mutated alleles in GJB2 have been reported, around 87% of KID syndrome patients have a heterozygous c.148G>A mutation, causing the substitution of an aspartic acid residue to an asparagine at position 50 (p. D50N) (2). GJB2 encodes connexin 26 (cx26), a junctional protein able to assemble to form hemi-channels (or connexons) in cell membrane. Two hemichannels pair to form intercellular channels (the gap junction) between adjacent cells, permitting the direct exchange of electric impulses, metabolites and second messenger molecules (<1200Da) between cells (3). Studies have shown that mutant cx26 was incapable of inducing intercellular coupling in vitro, resulting in impairment of intercellular communication.

We have been developing a therapeutic strategy for this debilitating KID syndrome, in which the mutant copy is silenced by mutation-targeted small interfering RNA (siRNA), allowing the wild-type copy to play a dominant role (4). A functional correction on gap junction channel activity following treatment of the siRNA was showed the hemi-channel activity of the connexin was corrected by 35% in KID keratinocytes, compared to either untreated KID cells or KID cells treated with a non-specific siRNA. The reversal of aberrant hemi-channel activity suggests that the mutation-targeted siRNA is a potential therapeutic intervention for KID syndrome. Based on these encouraging results, we are now well placed to translational develop an effective siRNA treatment for this condition.

We propose to develop an in vivo disease model to test the siRNA therapy strategy with the ultimate aim to translational bring this therapy to the patients.

Study plan:
There will be three major studies:
i) The uptake of siRNA into patient’s skin cells (keratinocytes) will be investigated to achieve a high siRNA delivery efficiency with low cytotoxicity. We used research-grade transfectant reagents previously to facilitate uptake of siRNA into cells. For translational development, we will adapt an existing receptor-targeted liposome-peptide nanocomplex developed by Prof Hart to improve the stability and enhance the uptake of siRNA into keratinocytes. Cell transfection conditions will be optimised, and efficiency of siRNA uptake will be evaluated by mRNA level of GJB2 and cx26 protein expression, and gap junction channel activity in KID keratinocytes by dye coupling.
ii) Characterisation of in vivo humanised KID disease model. We have commercially been developing a humanised KID mouse model, and we will evaluate the therapy strategy of mutation-targeted siRNA in this in vivo disease model. As this is a newly developed mouse model, the student is expected to characterise the disease model by conducting studies including geno- pheno-type checking and skin barrier function assessment (5).
iii) Evaluate the efficacy of mutation-targeted siRNA therapy for KID syndrome in vivo. We plan to topically delivery of siRNA into the skin. The mutation targeted siRNA will be carried into the skin by various formulations/approaches such as ointments, cell-penetrating peptide, spherical nucleic acid nanoparticle conjugates or microneedle arrays. The efficacy of topical siRNA treatment for KID syndrome will be assessed in the in vivo disease model. The parameters including application concentration of siRNA, stability of siRNA and time course of application will be tested by evaluating the level of mutant GJB2 mRNA knocking-down. Functional assessment for gap junction channel activity will be performed in cells isolated from the disease model.

We are expecting that the student will carry out the first part of study during first 8 to 10 months and the second and third studies during rest of time (18 to 20 months).

1. Mazereeuw-Hautier, J. et al. Keratitis-ichthyosis-deafness syndrome: disease expression and spectrum of connexin 26 (GJB2) mutations in 14 patients. Br.J.Dermatol. 156, 1015-1019 (2007)
2. Richard, G. et al. Missense mutations in GJB2 encoding connexin-26 cause the ectodermal dysplasia keratitis-ichthyosis-deafness syndrome. Am.J.Hum.Genet. 70,1341-1348 (2002).
3. Laird, D. W. Life cycle of connexins in health and disease. Biochem.J. 394, 527-543 (2006).
4. Lee, M. Y. et al. Allele-Specific Small Interfering RNA Corrects Aberrant Cellular Phenotype in Keratitis-Ichthyosis-Deafness Syndrome Keratinocytes. The Journal of investigative dermatology 140, 1035-1044.e1037, doi:10.1016/j.jid.2019.09.022 (2020).
5. Press, E. et al. Disease-linked connexin26 S17F promotes volar skin abnormalities and mild wound healing defects in mice. Cell Death Dis 8, e2845, doi:10.1038/cddis.2017.234 (2017).