Viral delivery approaches for CRISPR/Cas9-based knockdown of the Myocilin gene in Trabecular Meshwork

Abstract

Purpose : Elevated intraocular pressure (IOP) is a major risk factor for the progression of primary open angle glaucoma (POAG). Damage to the trabecular meshwork (TM) is responsible for increased aqueous humor outflow resistance that raises IOP. Of the factors that are involved in TM dysfunction, mutation of the myocilin gene (MYOC) was the first identified. MYOC mutations contribute to ~4% of POAG cases. Although, the normal role of myocilin is unknown, mutations in MYOC cause a deleterious gain-of-function leading to lack of extracellular secretion and endoplasmic reticulum (ER) stress within TM cells. Our goal is to develop genome editing methods to modify genes associated with TM pathophysiology, which are not addressed by current IOP lowering therapies. We previously demonstrated the feasibility of targeting the mutant (mut)-MYOC gene using CRISPR/Cas9 genome editing in mice and human donor eyes using the adenovirus (Ad)-5 gene transfer system. However, Ad5 is not a suitable vector for clinical use. Therefore, we aim to study the efficacy of adeno-associated virus (AAV) and lenti virus (LV) vectors expressing CRISPR/Cas9 to target the TM and reverse ocular hypertension (OHT) in mut-MYOC associated POAG.

Methods : We compared the TM specific tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes with the LV vector that express eGFP driven by the CMV promoter. These vectors were injected via intravitreal (IVT) and intracameral (IC) routes to determine expression in ocular tissues. Efficient CRISPR-Cas9 constructs that selectively knockdown MYOC (cr-MYOC) were designed to evaluate the effect on ER stress markers in mut-MYOC expressing transformed TM cells and the effect on IOP in the transgenic (Tg)-MYOC^Y437H mouse model of OHT.

Results : Of the AAV serotype tested, Trp-mutant scAAV2 had prominent expression of eGFP in the TM via slow-IC route. However, expression was also observed in other retinal and optic nerve head tissues. In contrast, LV_eGFP expression was more specific to the TM injected via the IVT route. Cr-MYOC transduced cells were shown to have reduced expression of MYOC and ER stress markers GRP78, ATF4 and CHOP. LV_cr-MYOC injected mice show significantly reduced IOP in Tg-MYOC^Y437H mice.

Conclusions : LV was found to be more specific and efficient in mediating target gene expression in the TM and facilitate MYOC gene knockdown.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.