rAAV-mediated multi-miRNA delivery targeting MAPK pathway suppresses laser-induced choroidal neovascularization in mice

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss, with neovascular AMD (nAMD) characterized by choroidal neovascularization (CNV) and subretinal fibrosis. Although anti-VEGF therapy is the current standard, frequent injections and limited efficacy necessitate alternative treatments. The mitogen-activated protein kinase (MAPK) pathway contributes to AMD by promoting oxidative stress-induced apoptosis, inflammation, and angiogenesis. We evaluated the therapeutic potential of MAPK-targeting microRNAs (miRNAs) delivered via recombinant adeno-associated virus (rAAV). Two rAAV2.7m8-based strategies were developed: a MAPK-miR mixture (three individually packaged miRNAs: hsa-miR-122-5p, let-7a-5p, and miR-26a-5p) and an MAPK-miR tandem (all three miRNAs in one vector under separate U6 promoters). Both formulations effectively inhibited MAPK activation in oxidative stress-induced RPE cells and laser-induced CNV mouse models. Treatment reduced CNV leakage and angiogenesis, as confirmed by fluorescein angiography and histology. The MAPK-miR mixture showed enhanced efficacy in suppressing epithelial-mesenchymal transition (EMT) and subretinal fibrosis. This study demonstrates the therapeutic benefit of rAAV-mediated multi-miRNA delivery targeting MAPK signaling in AMD. Sustained expression from rAAV vectors may overcome the limitations of anti-VEGF therapy, offering a novel gene therapy approach for nAMD. Further optimization and safety evaluation are warranted for clinical translation.