This objective of this project is to optimize the nanoparticle delivery system for effective ocular delivery of siRNA in animal models in order to validate the therapeutic targets developed at USAMRICD. In collaboration with the Target Discovery Core and the In Vivo Target Validation Core led by Dr. Albert Ruff at USAMRICD, we have developed new method to control the size and shape of siRNA nanoparticles. More importantly, this method can compact nanoparticles to smaller size with higher stability in physiological media, optimized a protocol to surface-coat nucleic acid nanoparticles with hyaluronic acid and retained the stability of the nanoparticles, and identified the conditions using reversible crosslinking density to stabilize siRNA nanoparticles. In addition, we have demonstrated gene knockdown in several cell lines, and confirmed that disulfide reversible crosslinks are essential to successful transfection and gene knockdown. In a pilot study with Dr. Ruff, we have identified feasible injection parameters and analysis protocols, although initial testing did not show positive gene knockdown with PEGylated spherical nanoparticles following sub-conjunctival injection, we plan to continue test the newer generation of rod and worm-like nanoparticles, which have shown promising results in other animal models.