Development of Allele-specific Therapeutic siRNA In Meesmann Epithelial Corneal Dystrophy

Abstract

Purpose: : To investigate the cause of severe Meesmann epithelial corneal dystrophy (MECD) and develop siRNA therapy for dominant corneal disorders.

Methods: : An extended family with MECD where the clinical presentation was consistently severe was studied. The genes encoding keratins K3 and K12 were analyzed and the causative mutation identified. The relative ability of various mutant keratins to form normal filaments versus pathogenic aggregates was measured. The ability of mutation-specific siRNA species to block mutant K12 expression without affecting wild-type K12 expression was investigated using a K12-luciferase reporter gene assay. In addition, the ability of lead siRNAs to inhibit mutant K12 at a protein level and reverse cytoskeletal aggregation phenotype in a cell-based model system were assessed.

Results: : Affected members of the severe MECD family were shown to carry heterozygous missense mutation c.419T>C in exon 1 of the KRT12 gene. This mutation predicts the amino acid change leucine to proline at codon 132 (designated L132P for brevity; full designation p.Leu132Pro) in the K12 gene. Using a cell-culture assay of keratin filament formation, L132P was shown to be significantly more disruptive than R135T, which is associated with typical, mild MECD (p<0.05). A siRNA sequence walk identified a number of potent inhibitors for the mutant allele, which had no appreciable effect on wild-type K12 at concentrations of 0.01 to 6.25nM siRNA. The most specific and potent inhibitors were shown to completely block mutant K12 protein expression, even at the lowest siRNA concentration of 0.01 nM, with negligible effect on wild-type K12. The lead inhibitor was able to significantly reverse the filament aggregation phenotype in cultured cells (p<0.001).

Conclusions: : Unusually severe MECD can be caused by particular mutations that are highly disruptive to keratin filament assembly. It is possible to design highly potent siRNA inhibitors against mutant alleles for future treatment of MECD and other corneal dystrophies caused by dominant-negative mutations.

Keywords: cornea: epithelium • degenerations/dystrophies • gene transfer/gene therapy