Designing, testing, optimising and achieving delivery of gene silencing and editing therapies for genetic diseases

Abstract

Inherited corneal dystrophies such as Granular Corneal Dystrophy Type 2 (GCD2) and Meesmann Epithelial Corneal Dystrophy (MECD) are characterized by pathogenic mutations in the TGFBI and KRT12 genes, respectively. These autosomal dominant diseases can cause progressive visual impairment or ocular discomfort with limited treatment options currently available. This thesis aimed to develop allele-specific small interfering RNA (siRNA) therapies capable of silencing disease-causing mutations while preserving wild-type gene expression.

The research first optimized siRNA sequences targeting the R124H mutation in TGFBI, the primary cause of GCD2, and assessed knockdown efficiency in vitro. To overcome challenges in ocular delivery, various delivery systems—topical application, lipid nanoparticles, hydrogels, cell-penetrating peptides, and microneedles—were evaluated. MECD, caused by mutations such as L132P in KRT12, was chosen as a proof-of-concept disease model due to its rapid corneal epithelial turnover and available mouse models.

Topical delivery of siRNA to KRT12 in a transgenic mouse model demonstrated significant knockdown and phenotypic improvement, validating siRNA efficacy in vivo. Further, microneedle-mediated siRNA delivery showed promising results as a minimally invasive alternative that bypasses common ocular barriers. These findings support the feasibility of siRNA-based therapies for dominant ocular disorders and highlight MECD as an effective model to test siRNA constructs and delivery systems.

This thesis concludes that allele-specific RNA interference represents a viable therapeutic strategy for dominantly inherited corneal dystrophies. The successful use of siRNA in MECD models establishes a foundation for translation to GCD2 and potentially other dominant genetic diseases.

Thesis is embargoed until 30th September 2027.

Date of Award	Sept 2025
Original language	English
Sponsors	Avellino Labs Ltd
Supervisor	Tara Moore (Supervisor), Louise Robertson (Supervisor) & Andrew Nesbit (Supervisor)