Replacing the genes that lead to a cloudy cornea

Knockout-replacement gene therapy for dominantly inherited corneal dystrophy using snoRNA-mediated gene silencing.

The corneal dystrophies (CDs) are a heterogeneous group of inherited eye diseases. The snoMEN system is a new form of highly potent gene silencing vector, based on small nucleolar RNA. Expressed from small introns, the snoMEN molecules are ideally suited to a knockout-replacement method of gene therapy in dominantly inherited disorders, where both alleles of the endogenous target gene are silenced and replaced by modified version of the gene that is resistant to silencing.

Here, snoMEN vectors are being optimized to silence the genes encoding keratins K3, K12, and the BIGH3 gene, all of which are involved in dominant-negative forms of CD. The 3’UTR sequences of these genes are being targeted. Replacement genes are using a foreign 3’UTR and are therefore immune to silencing. A luciferase reporter gene system is being used to assay test various snoMEN constructs to identify those that are most efficacious and specific. Knockout-replacement constructs are being made where the most efficacious snoMEN is expressed from an intron. A minimal version of the human K12 promoter is being defined by deletion mapping and used to direct tissue-specific expression in corneal epithelial cells. The effect of using more than one snoMEN vector on efficacy of gene silencing is being explored. Delivery is initially by plasmid transfection and subsequently, lentiviral vector versions are being be made.

The outcome of this project is intended to be proof-of-concept of this novel system, which will enable the team to attract major grant funding to make GMP-compliant versions of the lentiviral vectors and move towards clinical trials.

The aim of this project was to develop a gene therapy for Meesmann epithelial corneal dystrophy that might also be extended later for other inherited causes of sight loss.

One approach is to switch off the faulty gene in the patient’s cornea that makes an unhealthy protein, and then replace it with a new gene that has been engineered to make healthy protein. To do so, you would have to find a way to switch off the gene, and then develop a replacement gene that is not affected by the on-off switch.

At the start of the project the team investigate the snoMEN system. Unfortunately it turned out not to be good enough at switching off the K12 gene. But the team went on to investigate another system to do the same task. And by the end of the project they had successfully developed all the elements and were ready to test the system in animals in a future project.

The research team also took a second approach to treating corneal dystrophy. They developed small ‘silencer’ molecules called siRNA that can get inside cells and switch off the faulty copy of the gene and were also ready to test this approach in animals.

Related research

• Liao H, Irvine AD, Macewen CJ, Weed KH, Porter L, Corden LD, et al. Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy. PLoS ONE. 2011;6(12):e28582.
• Allen EHA, Atkinson SD, Liao H, Moore JE, Leslie Pedrioli DM, Smith FJD, et al. Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy. Invest Ophthalmol Vis Sci. 2013 Jan;54(1):494–502.
• Courtney DG, Atkinson SD, Moore JE, Maurizi E, Serafini C, Pellegrini G, et al. Development of allele-specific gene-silencing siRNAs for TGFBI Arg124Cys in lattice corneal dystrophy type I. Invest Ophthalmol Vis Sci. 2014 Feb;55(2):977–85.