Cre-mRNA-mediated induction of mutant myocilin in trabecular meshwork leads to ocular hypertension in an inducible transgenic mouse model of myocilin glaucoma

Abstract

Purpose : Myocilin (MYOC) mutations are the leading genetic cause of Primary Open Angle Glaucoma (POAG). Recently, we have developed a Cre-inducible transgenic mouse that expresses a DsRed-tagged Y437H mutant of human myocilin (Tg.Cre-MYOC^Y437H). Here, we explored whether intraocular injections of mRNA expressing Cre can induce mutant MYOC selectively in the trabecular meshwork and further characterized the glaucoma phenotypes of Tg.Cre-MYOC^Y437H mice

Methods : In this model, we utilized TARGATT^TM site-specific knock-in strategy to generate a Cre recombinase inducible mutant MYOC transgenic mouse tagged with DsRed fluorescent protein. An intravitreal (IVT) or intracameral (IC) injection of mRNA expressing Cre recombinase was performed in adult Tg.Cre-MYOC^Y437H mice to induce the expression of DsRed tagged mutant human myocilin. Myocilin expression in various ocular tissues was analyzed using fluorescence microscopy, western blotting, and immunostaining. Glaucoma phenotypes including IOP, RNFL thickness, functional (PERG and VEP) and structural (RBPMS staining) loss of retinal ganglion cells (RGCs) and optic nerve degeneration (PPD staining) was performed at 10 and 15 weeks of injections.

Results : A single IC injection of mRNA expressing Cre recombinase selectively induced human mutant myocilin protein in TARGATT^TM mouse TM tissue resulted in a gradual and sustained increase in IOP that lasts for 15 weeks post-injection (37% increase in IOP; n=8 eyes; p-value < 0.0005). Pattern ERG and VEP demonstrated significant RGC functional loss and damage to the brain’s visual centers respectively (n=6 eyes p-value < 0.005) at 10 and 15 weeks post injections. Interestingly, intravitreal injections (IVT) of Cre-mRNA also induced mutant MYOC in TM and ciliary muscle inducing IOP elevation. However, IVT injections led to MYOC expression in other ocular muscles, resulting in lens collapse on the retina and degeneration of ocular tissues.

Conclusions : Our findings suggest that IC injection of Cre-mRNA selectively induced mutant myocilin in TM, resulting in glaucoma phenotypes similar to POAG patients with Y437H mutation. This model may be useful in studying pathological mechanisms of glaucomatous TM damage.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.