The Inhibiting Effect And Mechanisms Of AAV-mediated EPO ShRNA In Choroidal Neovascularization

PURPOSE:To determine the effect of adeno-associated virus(AAV)-mediated gene therapy targeting Erythropoietin(EPO)gene in the progress of Neovascular Age-related Macular Degeneration(AMD)in murine laser-induced Choroidal Neovascularization(CNV)model.METHOD:Five Plasmids were designed and cloned in this study.Respectively,each of them is AAV based vector includes EPO shRNA or EPO miRNA for knocking down the expression of EPO,or EPOR76E which is a non-erythropoietic mutant of EPO,or Green Fluorescent Protein(GFP)as a control virus.Other than these,an EPO-T2A plasmid was designed for in vitro test.Self-complement AAV-1(ScAAV1)serotype was packaged and injected subretinally into C57/Bl6 mice to express EPO transgene specifically in RPE cells.Laser Photocoagulation(LP)was used to induce CNV lesions in wildtype C57/Bl6 mice 21 days post vector injection.Optical Coherence Tomography(OCT)and Funduscopy(Fundus)were then performed at different to qualify the transfection of virus,as well as to quantify the volume of CNV lesions.At the end,mice were euthanized and eyes were harvested for Choroidal Flatmount(Flatmount).Flatmount images were then captured under fluorescent microscope for quantifying the area of CNV lesions.RESULTS:At 7 days and 14 days after laser,in the EPO shRNA group,the average volume of CNV lesion had both statistically decreased in eyes injected with AAV1-Sc-smCBA-GFP-shRNA-EPO than eyes injected with AAV1-Sc-smCBA-GFP as control.In the EPOR76 E group,the average volume of CNV in eyes injected with AAV1-Sc-s CBA-EPOR76 E was significantly increased than in control eyes.Between 2 time points,the average volume of CNV lesions dropped with no significant difference.Similar results were also observed in the average area of CNV lesions in flatmount under fluorescent microscope.CONCLUSION:Sc-AAV1-mediated EPO knockdown could slow down the progress of CNV and might be a potential target for Neovascular AMD in the future.