Development of allele specific gene silencing siRNAs for TGFBI Arg124Cys in Lattice Corneal Dystrophy Type I

Abstract

Purpose: This study aimed to investigate the potency and specificity of short-interfering RNA (siRNA) treatment for TGFBI-Arg124Cys lattice corneal dystrophy type I (LCDI) using both exogenous expression constructs in model systems and endogenous gene targeting in an ex vivo model using corneal epithelial cell cultures.

Methods: A panel of 19 TGFBI-Arg124Cys specific siRNAs were assessed by a dual-luciferase reporter assay. Further assessment using pyrosequencing and qRT-PCR was used to identify the lead siRNA by assessing both potency and specificity, while suppression of mutant TGFBIp was evaluated by western blot and Congo red aggregation assays. An ex vivo model of LCDI using corneal epithelial cell cultures was established using limbal biopsies from corneal dystrophy patients harbouring the Arg124Cys mutation. Treatment efficiency of the siRNA was assessed for the inhibition of the mutant allele in these corneal epithelial cells using pyrosequencing, qRT-PCR and a TGFBI ELISA.

Results: A lead siRNA was identified using exogenous constructs in a dual luciferase reporter assay, and by pyrosequencing and qRT-PCR. The potency and allele specificity of the siRNA was confirmed at the protein level in a western blot and a Congo red aggregation assay. This potency then translated to a 44% reduction of the endogenous Arg124Cys allele in the ex vivo model of LCDI, while still demonstrating allele specificity.

Conclusions: We have identified a lead siRNA specific to the TGFBI-Arg124Cys mutant allele associated with LCDI. Silencing of exogenous TGFBI was observed at both mRNA and protein levels and in an ex vivo model of LCDI with an efficient suppression of the endogenous mutant allele. This result indicates the potential of siRNA treatment as a personalized medicine approach for the management of heritable TGFBI-associated corneal dystrophies.

Keywords: 538 gene transfer/gene therapy • 482 cornea: epithelium • 519 extracellular matrix