It is estimated that 1 in 100,000 people will experience a central retinal artery occlusion (CRAO) accompanied by acute monocular vision loss. Even when resolved by reperfusion, 80% of patients have a visual acuity of 20/400 or worse. The body?s natural response to ocular trauma is the induction of heat shock protein 72, Hsp72, to combat the acute effects of protein denaturation and aggregation and the chronic effects of apoptosis and secondary necrosis. Unfortunately, Hsp72 induction can require several valuable hours while a patient is in jeopardy of permanent vision loss. A cell-penetrating antibody, mAb 3E10, is proposed as an intracellular transporter to deliver Hsp72 into cells. Previous work with this 3E10-Hsp72 fusion (?Fv-Hsp70?) has shown cytoprotection against apoptotic cell death in vitro and in vivo. Our long-term goal is to determine the clinical effectiveness of this targeted Hsp72 in countering cell death in the retina following a CRAO. The objective here is to show our Fv-Hsp70, administered intravitreally, blocks cell death using a cerebral artery occlusion method to generate the CRAO in mice. The central hypothesis for the proposed research is that 3E10 can deliver Hsp72 directly into energy-deficient cells to minimize protein denaturation and aggregation, as well as inhibit both ATP-dependent and independent apoptosis, thus preventing significant retinal damage, particularly to the sensitive retinal ganglion cells. The rationale for the proposed research is that the cell penetrating antibody, 3E10, binds extracellular DNA and nucleosides, targets that are quite accessible where there are damaged cells, and it penetrates still viable cells through an equilibrative nucleoside salvage pathway. 3E10 is unique in that it penetrates cells without apparent harm and has been administered to humans without evidence of toxicity. Fv-Hsp70 has already been created and shown to be an effective cytoprotectant in vivo, minimizing by 68% the infarct volume in brain tissue when administered to rats after a stroke. It has also resulted in 45% less cardiomyocyte death at the heart apex of post-reperfusion treated rabbits versus 3 different sham controls. We are requesting funding to achieve the following specific aims: 1) Characterize the Fv-Hsp70?s pharmacokinetics and pharmacodynamics delivered to the vitreous humor. Fv- Hsp70 will be formulated in suitable ophthalmological solutions. 2) Evaluate Fv-Hsp70 efficacy at the maximum tolerated dose (MTD) by intravitreal delivery to mice in a CRAO model. 3) Evaluate the Fv-Hsp70 efficacy at the MTD at several time points after reperfusion to determine a maximal window of administration that measurably improves retinal cell survival and can be implemented in a clinical setting such as the ER. The proposed research is innovative because it utilizes a unique antibody-mediated, energy-independent intracellular delivery system for protein therapeutics. Inducing heat shock proteins in vivo takes time, whereas the impact of our product is the rapid delivery of Hsp72 into damaged cells to prevent cell death.
?Relevance of the proposed project to public health The research proposal is relevant to the NEI because the development of ocular cytoprotectants to help prevent loss of vision from a central retinal artery occlusion is an unmet medical need. Thus, the proposed research is relevant to the mission of NEI to reducing the incidence of vision loss, even blindness, by developing a novel, intravitreally administered cytoprotectant that prevents significant retinal cell death following a central retinal artery occlusion. Ultimately, the treatment proposed by Rubicon Biotechnology will be the first of its kind to target vulnerable retinal cells and help them survive.
