739. rAAV Delivered MicroRNA Therapeutics Towards Efficacious Treatment of Corneal Neovascularization
Authors: Yi Lu, Jianzhong Ai, Phillip Tai, Dominic J. Gessler, Qin Su, Qiang Zheng, Phillip D. Zamore, Xun Xu, Guangping Gao
Journal: Molecular Therapy (2016)

Corneal diseases are the second major cause of blindness worldwide. Corneal neovascularization (NV) is one of the most common pathologies in corneal diseases, leading to vision loss or even blindness. However, even though different treatments for corneal NV are available, 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. We hypothesize that modulating expression of angiogenesis related miRNAs might be an effective approach for treating corneal NV. To this end, we first set out to identify target miRNAs that play roles in corneal NV using Nanostring technologies and the classic alkali-burn induced corneal NV mouse model. Among 618 mouse miRNAs profiled, we found 35 up-regulated and 3 down-regulated miRNAs in the neovascularized corneas, which were confirmed by qRT-PCR. We selected miR-184 and miR-204, two miRNAs that were down-regulated > 10-folds in neovascularized corneas, as our candidate targets to test the concept of in vivo gene delivery for therapeutic miRNA to treat corneal NV. As recombinant adeno-associated virus (rAAV) holds promise for highly efficient and safe in vivo gene delivery to different target tissues in a serotype dependent manner, we opted to evaluate 14 serotypes of rAAV.EGFP for gene transfer efficiency in mouse corneas by eye drops (ED), intra-stromal (IS) and subconjunctival (SC) injections to search for the suitable vector(s) for delivering miRNA therapeutics to the corneas. Among the 4 leading serotype vectors (i.e. rAAVrh.8, rh.10, rh.39 and rh.43) identified, we chose rAAVrh.10 for overexpressing pri miR-184 and pri miR-204 in the corneas with alkali-burn induced corneal NV. rAAVrh.10-mediated overexpression of miR-184 or miR-204 by IS and SC injections but not ED efficiently inhibited angiogenesis and was efficacious and safe for either prevention (IS) or treatment (SC) of corneal NV. We further invested potential target genes and pathways to elucidate the anti-angiogenic mechanism of these two miRNA therapeutics. We revealed that the anti-angiogenic effect of rAAV.pri-miR-184 was achieved by targeting Fzd4 gene, thus suppressing the canonical Wnt signaling, a well-known pathway playing vital roles in angiogenesis; while rAAV.pri-miR-204 exerted its anti-angiogenic effect by targeting angiopoietin-1 gene (Angpt1), a well-acknowledged pro-angiogenic factor. Meanwhile, the transcriptome analysis is under the way to search for novel gene targets of the miRNA therapeutics. Our study has great clinical relevance and demonstrated, for the first time, that 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 efficacious and safe clinical option for treating corneal NV and other angiogenesis-related diseases including age-related macular edema (AMD) and cancer.