Mutation-targeted gene therapy for Keratitis-Ichthyosis-Deafness syndrome - proof of conceptfor topical siRNA delivery

Supervisors: Dr Wei-Li Di, Dr Veronica Kinsler and Professor Stephen Hart

This inter-disciplinary PhD project is a collaboration between the Skin Biology Centre, the Molecular Genetics Unit, and the Gene therapy Unit. It contains all the elements of an excellent PhD, training the student in a range of laboratory techniques including molecular biology, cell biology and nanoparticle technology, whilst demonstrating clear clinical applicability and the potential for broader application in other genetic skin diseases.

1. Allele specific siRNA can knock-down mutant gene GJB2
2. A designed nanoparticle-siRNA can effectively deliver siRNA into skin

Aims and methods:
Keratitis-ichthyosis-deafness syndrome (KID) (OMIM 148210) is a currently untreatable genetic disease characterized by congenital erythrokeratoderma, sensorineural deafness and ocular abnormalities(1). Patients with this rare condition have a poor quality of life due to thickened inflamed skin, deafness and poor vision. Furthermore, they have an increased risk of cancer: 20% of patients between the ages of 15 and 40 develop carcinoma of the skin and/or tongue(2). In this context, there is a pressing need to develop a specific treatment for this disease to improve the quality of life and, most importantly, potentially to decrease the risk of squamous cell carcinoma.

The disease-associated gene is GJB2, which encodes protein connexin 26. Missense mutations in GJB2 can result in amino acid replacement in connexin 26, consequently changing the gating properties of connexin 26 and affecting intercellular communication. 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)(3).

We propose to develop a topical gene therapy for KID syndrome, centred on knock-down of the mutant allele of gene GJB2 using small interfering RNA (siRNA) technology, and nanoparticle delivery into the skin. This research is driven by the absence of treatment for this debilitating and often fatal condition, and if successful will provide proof of concept for many genetic skin disorders.

In this project, the student will be expected to test a tiled set of functionally asymmetric siRNAs in cells expressing wild-type or mutant GJB2/GFP, in order to select the most potent siRNA for knocking-down the mutant allele. The functional discrimination of GJB2 followed by treatment of siRNA will be performed. Furthermore, the potent siRNA will be conjugated with the a customised nanoparticle which the student will develop and optimise, and the delivery efficiency of the nanoparticle-siRNA complex into keratinocytes and the epidermis of the skin will be evaluated in in vitro 3D culture model and in vivo skin graft model.

All the techniques required in the project are well established in our department, and a critical core group of post-doctoral scientists, research assistants and students work within the research group. The student will have regular meetings with supervisors and will receive training in wide range of molecular and cellular techniques. The experience gained will be applicable to a wide range of disorders that are amenable to ex-vivo stem cell gene modification.

1) Gilliam,A., Williams,M.L. (2002) Fatal septicemia in an infant with keratitis, ichthyosis, and deafness (KID) syndrome. Pediatr. Dermatol., 19, 232-236.
2) Natsuga,K., Akiyama,M., Shimizu,H. (2011) Malignant skin tumours in patients with inherited ichthyosis. Br. J. Dermatol., 165, 263-268.
3) Mazereeuw-Hautier,J., Bitoun,E., Chevrant-Breton,J., Man,S.Y., Bodemer,C., Prins,C., Antille,C., Saurat,J.H., Atherton,D., Harper,J.I., et al. (2007) Keratitis-ichthyosis-deafness syndrome: disease expression and spectrum of connexin 26 (GJB2) mutations in 14 patients. Br. J. Dermatol., 156, 1015-1019.