Gene therapy for the ocular surface

Abstract

Meesmann’s epithelial corneal dystrophy (MECD), which clinically presents with microcysts that can cause irritation, blurred vision or photophobia, is a genetic disorder caused by dominant-negative mutations in the KRT3 and KRT12 genes. Eradicating the mutant protein or tipping the balance strongly in favour of the wildtype allele are viable options for therapeutic intervention. Here we studied two therapeutic approaches for suppression of the mutant KRT12 allele and have developed, characterised and initiated in vivo testing using two novel KRT12 mouse models.

For a transient therapeutic approach, short interfering RNAs (siRNAs) were designed and proved capable of mutation-specific inhibition of the alleles responsible for two MECD causative mutations (p.Leu132Pro and p.Arg135Thr; 70-90%) in vitro. No off-target issues were observed and suppression of endogenously expressed p.Leu132Pro was also shown in an ex vivo model. For a more generic, yet potentially permanent therapeutic approach, total KRT12 was suppressed (~50%) with an siRNA expressed from a short hairpin by targeting a region homologus to both the WT and mutant mRNAs. KRT12 was replaced with a co-expressed recoded allele made resistant to the siRNA.

To further develop these potential therapeutics, two novel mouse models were generated allowing evaluation of gene modulation in vivo. (1) A humanised dominant negative mutant model that expresses K12 p.Leu132Pro revealed major changes to corneal phenotype in homozygous animals. Microcysts were observed and keratin expression patterns disrupted. Additionally, RNAseq analysis highlighted over 1600 dysregulated genes, which could feature other potential therapeutic targets for the treatment of symptomatic MECD. Heterozygous mice presented with a subtler phenotype. (2) A Krt12 luciferase reporter mouse model was optimised and will facilitate live animal corneal imaging, thus aiding the development of topical siRNA delivery formulations. These mouse models in conjunction with our gene silencing development programme pave the way for in vivo assessment of RNAi-based therapeutics for the cornea.

Date of Award	Jun 2014
Original language	English
Sponsors	Medical Research Council, Wellcome Trust & University of Dundee
Supervisor	Tara Moore (Supervisor), Tony Bjourson (Supervisor), Sarah Atkinson (Supervisor), Jonathan Moore (Supervisor) & W.H. Irwin McLean (Supervisor)