Abstract
Purpose :
Primary open angle glaucoma (POAG) is common and is characterized by poor drainage of Aqueous humor (AH) through the conventional outflow pathway. Previous studies have shown overexpression of fibrous proteins in glaucomatous trabecular meshwork (TM) is related to increased stiffness of TM. Here, we present an engineered mechanosensitive channel (EMC) that directly senses tension in the membrane lipid bilayer of TM cells and in response, transiently opens its large non-specific pore to release cytoplasmic fluid. Since more than hundred genes have been identified to be associated with glaucoma, and no common convergent pathway(s) have been identified at this point, the goal is to develop a gene-agnostic gene therapy for enhancing aqueous humor outflow facility to lower the intraocular pressure (IOP).
Methods :
Reporter (mCherry) immunofluorescence imaging was conducted to determine EMC localization on membrane of transfected cells. Several EMC variants were screened in-vitro via pressure clamp electrophysiology to identify the one that is activated at physiologically relevant pressures. Dexamethasone (DEX) treated adult C57BL/6J mice were used as a glaucoma model. Longitudinal measurement of IOP was measured by tonometer. AAV-carried EMC was delivered via intracameral injection into the IOP elevated eyes. Confocal fluorescence microscopy was carried out to image expression of EMC in TM. Image analysis was performed using ImageJ software and was compared to control.
Results :
The heterologously expressed EMC was found to act as a tension activated pressure release valve in trabeculocytes, as measured by pressure clamp. Via intracameral delivery of AAV2/8, EMC was efficiently transduced in vivo in TM cells, as quantified by reporter expression. EMC expression in TM led to enhanced aqueous humor outflow facility as compared to control. Longitudinal measurements showed lowering of IOP after AAV-EMC injection in the DEX-mouse model of ocular hypertension. Quantification of retinal ganglion cell counts showed higher survival in EMC-treated DEX-group, which was attributed to lowering of IOP by EMC.
Conclusions :
The use of heterologously expressed engineered mechanosensitive channel, acting as macromolecular pressure sensor and outflow actuator in TM cells, led to regulation of IOP. The development of a safe and effective long-lasting treatment for glaucoma would improve the lives of many individuals.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.