586. Development of Anti-Angiogenic miRNA Therapeutics for Corneal Neovascularization

Abstract

GG and XX are co-corresponding authorsCorneal diseases are the second major cause of blindness worldwide. Corneal neovascularization (CNV) is one of the most common pathological processes in corneal diseases, leading to vision loss or even blindness in patients. However, even though different treatments for CNV are available in clinic, a safe and effective therapy remains to be an unmet medical challenge. MicroRNAs (miRNAs) play roles in regulating more than half of all protein-coding genes in mammals including those involved with angiogenesis. Therefore, modulating expression of angiogenesis-related miRNAs might be an effective approach for treating this blindness-causing corneal lesion. To this end, we first tested four angiogenesis-related miRNAs (i. e. miR-126, miR-10, miR-17a and miR-92) for their role in angiogenesis in vitro by assessing cell proliferation and tube formation of VEGF-stimulated human umbilical vein endothelial cells (HUVECs) transfected with the plasmid constructs overexpressing the pri-miRNAs or Tough Decoy (Tud) RNAs inhibitory to the corresponding target miRNAs. We found that TuD-miR-10b, pri-miR-17a and pri-miR-92 were significantly inhibitory for the cell proliferation and tube formation of HUVECs, while TuD-miR-126-5p could impede HUVEC tube formation but not proliferation. Our data reveal that miR-126 and miR-10 are pro-angiogenic regulators, whereas miR-17a and miR-92 are negative regulators of angiogenesis, suggesting the feasibility of using miRNA as therapeutic targets to modulate angiogenesis in CNV. We next set out to identify more target miRNAs that play roles in CNV. To date, there has been no report on miRNA expression profile of CNV. Using Nanostring technologies and the classic alkali-burn induced CNV mouse model, we profiled small RNAs prepared from the corneal tissues harvested from the study mice before and days 5, 10 and 15 after alkali injury for expression levels of 618 mouse miRNAs. We found 35 up-regulated and 3 down-regulated miRNAs in the mouse neovascularized corneas. Currently, we are in the process of confirming expression profiles of those candidate target miRNAs by qRT-PCR. For proof-of-concept, we proposed to use rAAV vector for efficient delivery of those pri-miRNAs or their corresponding Tud RNAs to the corneas of alkali-burn induced CNV mice and evaluate their potency in preventing or treating CNV. To this end, we first assessed several serotypes of rAAV EGFP for gene transfer efficiency in mouse corneas after delivery by ectopic eye drops, or intrastromal or subconjunctival injections to identify the most efficient AAV serotype and method of delivery for miRNA therapeutics. The results of those experiments will be presented. In sum, our study has great clinical relevance; miRNA-targeted novel therapeutics that can be delivered as either rAAV or synthetic nucleic acid drugs (i. e. miRNA mimics and antagomir) might offer an additional clinical option for treating CNV.