Targeting TGFBeta-mediated IOP dysregulation in a large animal model of glaucoma

Summary: Current therapies for glaucoma are often limited in their efficacy by poor patient compliance with frequent medications. There is a critical need for new glaucoma therapies that are less arduous for patients, particularly for children and very elderly people with glaucoma. By providing lasting effects from a single treatment, and directly addressing underlying pathophysiology of glaucoma, gene therapy represents an attractive strategy for the management of this chronic and blinding disease. High concentrations of TGFβ2 in the aqueous humor contribute to fibrogenic extracellular matrix and stiffening of the trabecular meshwork (TM) in glaucoma, which in turn reduces aqueous humor outflow and increases intraocular pressure (IOP). Our goal is to provide initial proof of concept for a novel therapeutic approach to glaucoma, that will target progressive TGFβ2-associated aqueous outflow pathway pathology in a feline model of early onset glaucoma due to LTBP2 mutation. This project builds on a previously reported strategy of CRISPR interference to suppress the pathologic TGFβ signaling that is a common feature in glaucomatous eyes. In a recently published study, lentiviral vector delivery of KRAB-dCAS9 and sgRNAs targeting the promoter region of TGFβ2 led to effective CRISPR interference of TGFβ2 expression in human TM cells cultured in vitro, and the same strategy showed IOP-lowering efficacy in a TGFβ2 over-expressing mouse model of ocular hypertension. In this proposal, in Aim 1, we will optimize viral vector mediated transduction of feline TM cells in vitro and in feline glaucomatous eyes in vivo. We will identify the lentivirus (Feline Immunodeficiency Virus, FIV) vector system and dose with optimal transduction, ocular tissue specificity for feline TM, and safety profile in vivo. In Aim 2, We will design and optimize a panel of sgRNAs targeting the promoter regions of TGFβ2 and TGFβ receptor 1 (TGFβR1) for KRAB-dCAS9 CRISPR interference in feline TM cells in vitro, to advance to evaluate safety and efficacy in pre-clinical pilot studies in vivo. Unlike conventional Cas9 which is used for genome editing, dCAS9 lacks endonuclease activity and instead serves as a “blocker” or repressor in the promoter region of the targeted gene. KRAB further enhances this inhibition by its histone deacetylase activity. We will use the top candidate sgRNAs to conduct a small scale proof of concept study for CRISPR interference of TGFβ2 and TGFβR1 in vivo to provide a foundation for future, larger scale pre-clinical studies. Outcomes will include quantitation of TGFβ2 and TGFβR1 by qPCR and immunoblotting and/or ELISA as appropriate, clinical evaluation of IOP and ocular toxicity, and screening for off- target effects. Accomplishing these aims will provide the validated and optimized tools necessary for future in vivo testing of this promising, mutation-agnostic gene therapy approach in our highly translationally relevant spontaneous large animal model of early onset glaucoma due to LTBP2 mutation.