PROJECT SUMMARY Nanoscale drug carriers (i.e. nanocarriers) have attracted much attention for their ability to transport diverse therapeutic and diagnostic agents and to selectively target specific cells and tissues. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Schlemm's canal (SC) endothelial cells hold promise as a cellular target for glaucoma therapy, as their mechanical stiffness is associated with modulation of intraocular pressure (IOP). Rho kinase inhibitors and actin- depolymerizing agents reduce endothelial cell stiffness and significantly lower IOP in animals and humans with several now approved for clinical use. However, these agents are associated with significant side effects, including conjunctival hyperemia and corneal verticillata. Studies show that >50% of patients treated with these therapeutics exhibit adverse side effects. Targeted nanocarrier delivery systems may address these issues but are not currently capable of passing through the corneal epithelium and must therefore be administered via intraocular injection. As frequent eye injections would not be well tolerated by patients, sustained intraocular delivery systems are needed to minimize the frequency of drug administration. Gene therapy targets for treatment of ocular hypotension have emerged, holding promise for a future glaucoma cure following a single intraocular injection, but a targeted gene delivery system is needed to enhance selective transfection of SC cells. A significant need therefore exists for both sustained nanocarrier delivery systems and gene delivery systems for intraocular strategies targeting the SC. With these needs in mind, the objective of this proposal is to engineer a scalable, customizable, synthetic nanocarrier platform that can be adapted to transport diverse therapeutic agents to outflow pathway cells with controllable release rates. Successful completion of this work will result in the first delivery system for sustained intraocular release of nanocarriers, a novel nonviral gene delivery platform for selective transfection of SC cells, and completion of nonhuman primate studies to justify clinical trials of these delivery systems in humans. The following Specific Aims will be completed: Aim 1: Optimize the duration of therapeutic effect for nanocarriers targeting Schlemm’s canal cells while avoiding side effects and toxicity within the cornea and vascular tissues in mouse eyes. Aim 2: Demonstrate nonviral transfection of Schlemm's canal cells in vivo using targeted nanocarriers without affecting nearby ocular tissues in mice Aim 3: Demonstrate that targeted nanocarriers containing latrunculin-A significantly increase conventional outflow facility and lower IOP in nonhuman primates without adverse effects.