Development of a corneal bioluminescence mouse for real-time in vivo evaluation of gene therapies

Abstract

Purpose: The purpose of this study was to develop and characterize a novel biolumi-nescence transgenic mouse model that facilitates rapid evaluation of genetic medicine delivery methods for inherited and acquired corneal diseases. Methods: Corneal expression of the firefly luciferase transgene (luc2) was achieved via insertion into the Krt12 locus, a type I intermediate filament keratin that is exclusively expressed in the cornea, to generate the Krt12luc2 mouse. The transgene includes a multiple target cassette with human pathogenic mutations in K3 and K12. Results: The Krt12luc2 mouse exclusively expresses luc2 in the corneal epithelium under control of the keratin K12 promoter. The luc2 protein is enzymatically active, can be readily visualized, and exhibits a symmetrically consistent readout. Moreover, structural integrity of the corneal epithelium is preserved in mice that are heterozygous for the luc2 transgene (Krt12+/luc2 ). Conclusions: This novel Krt12luc2 mouse model represents a potentially ideal in vivo system for evaluating the efficacies of cornea-targeting gene therapies and for estab-lishing and/or validating new delivery modalities. Importantly, the multiple targeting cassette that is included in the Luc2 transgene will greatly reduce mouse numbers required for in vivo therapy evaluation.