A novel minimally humanized mouse model of aniridia for preclinical evaluation of CRISPR gene editing strategies

Introduction Aniridia is a rare congenital vision-loss disorder primarily caused by variants in the paired box 6 ( PAX6 ) gene. Current treatments do not maintain vision long term, nor address the underlying disease mechanism. CRISPR-mediated genome editing holds great promise for treating genetic diseases but requires demonstration of efficacy on human DNA to support translational relevance. The aim of this work was to generate and test a minimally humanized mouse model of aniridia, carrying a FLAG-tagged Pax6 and the most common recurring pathogenic aniridia-patient variant, introduced for the first time in mice. Methods Ocular assessments (gross examination, slit lamp imaging, and fluorescent and confocal microscopy) were performed in mice homozygous for humanization and heterozygous for the variant, paralleling patients. Mice were examined immediately post-wean and at adulthood, with the former chosen as a likely time for treatment initiation. As an exploratory proof-of-principle, a lipid nanoparticle-delivered adenine base editor (ABE8e) was tested to demonstrate the utility of the model. Results Ocular assessments revealed the expected phenotypes for an aniridia mouse model, supporting successful generation and phenotypic validation of the humanized mouse. The ABE8e treatment resulted in a modest correction (4.8% ± 0.4% SEM) of the pathogenic variant in clinically relevant ex vivo cortical neurons. Discussion Although designed for the development of genome editing therapies, this humanized mouse is also applicable to other therapies requiring interaction with human DNA or RNA. It also enables evaluation of nonsense-suppression drugs since the readthrough proteins retain the FLAG tag. More broadly, this methodology is applicable to other pathogenic variants for aniridia, CRISPRa approaches, and other genetic diseases for which CRISPR-based therapy holds promise.