Investigating the use of translational readthrough inducing drugs in keratocytes from a mouse model of brittle cornea syndrome

Purpose: Brittle Cornea Syndrome (BCS) is a rare recessive condition characterized by extreme thinning of the cornea. The collagen‐rich corneal stroma accounts for 90% of corneal thickness in humans, and is a major determinant of visual acuity. BCS results from homozygous loss‐of‐function mutations in ZNF469 (BCS1) or PRDM5 (BCS2). Our mouse model of BCS1, caused by a premature termination codon (PTC) mutation in Zfp469, showed that the stroma is thinner due to of decreased type 1 collagen expression 1 . To elucidate disease mechanisms and test potential therapies, we established a primary keratocyte disease‐in‐a‐dish model of BCS1. Translational readthrough inducing drugs (TRIDs) can restore full‐length proteins truncated by PTC and are a potential treatment for 20% of BCS patients with PTC mutations. Primary keratocytes from our BCS1 mouse model were treated with TRIDs to assess their efficacy in improving the BCS phenotype in vitro. Methods: Zfp469 BCS1/BCS1 keratocytes were treated with TRIDs PTC124 (ataluren), amlexanox, gentamicin, geneticin or vehicle‐only, for 2 or 5 days. Following treatment, Zfp469 and Col1a1 mRNA levels were evaluated by RT‐qPCR. Changes to keratocyte cell‐derived matrices were assessed using immunostaining or the collagen dye CNA35. Results: Zfp469 BCS1/BCS1 keratocytes express 40% more Zfp469 mRNA than wildtype cells; this was unchanged by treatment with amlexanox, geneticin or gentamicin. PTC124 decreased expression of Zfp469 by 50%. Expression of Col1a1 , a key component of the stromal matrix, was significantly decreased by PTC124 treatment (<65% relative to vehicle‐only Zfp469 BCS1/BCS1 keratocytes). Collagen deposition, visualized using CNA35 dyes, was decreased by PTC124 treatment in BCS and wildtype keratocytes. Conclusions: BCS is a devastating condition. Extreme thinning of the corneal stroma, as a result of impaired collagen production by keratocytes, makes the cornea prone to rupture. This causes irreversible sight loss. There is currently no treatment for BCS. Our initial work suggests that TRIDs may not restore functional full‐length Zfp469 in primary keratocytes and are unlikely to be of therapeutic benefit in BCS. Reference 1. Stanton CM et al. A mouse model of brittle cornea syndrome caused by mutation in Zfp469. Dis Model Mech. 2021;14(9):dmm049175.