Engineered virus-like particle-assembled VEGF-targeting Cas9 ribonucleoprotein treatment alleviates neovascularization in wet age-related macular degeneration

Background Age-related macular degeneration (AMD), particularly the wet form, is a leading cause of vision loss, characterized by abnormal blood vessel growth in the retina. Targeting vascular endothelial growth factor (VEGF) has proven effective in mitigating the disease. While knock-out VEGF expression using genome editing is a proven therapeutic strategy, conventional delivery systems such as adeno-associated virus (AAV) and lipid nanoparticles (LNPs) face limitations, including prolonged expression, potential genome integration, and reduced efficiency for macromolecule delivery. Engineered virus-like particles (eVLPs) offer an alternative, combining the efficiency of viral systems with the transient nature of non-viral platforms. Here, we investigated the therapeutic efficacy of eVLPs for the transient delivery of VEGF-targeting Cas9 ribonucleoprotein (RNP) in a laser-induced choroidal neovascularization (LI-CNV) mouse model of wet AMD. Methods EVLPs were produced by co-transfecting HEK293T cells with plasmids encoding VSV-G, MMLVgag–pro–pol, MMLVgag–3xNES–Cas9, and gN19-Vegfa sgRNA. The delivery efficiency and VEGF disruption were initially assessed in NIH/3T3 cells through immunocytochemistry, enzyme-linked immunosorbent assay, and deep sequencing. In vivo , Cas9-eVLPs were administered to the LI-CNV mouse model to evaluate the delivery efficiency and therapeutic efficacy. Insertion and deletion (INDEL) frequency, VEGF expression change, therapeutic efficacy, and safety issus were evaluated using deep sequencing, ELISA, histological analyses, and ocular functional tests. Results Cas9-eVLPs demonstrated efficient intracellular delivery in vitro , achieving up to 99% indel frequency at the VEGF target locus and significant VEGF downregulation without cytotoxicity. Single subretinal injection of Cas9-eVLPs into the retinal pigment epithelium (RPE) effectively disrupted VEGF, with expression nearly abolished by day seven. In LI-CNV mouse model, this intervention reduced CNV area and volume by 32.9% and 26.5%, respectively, while significantly decreasing VEGF protein levels in the RPE. Furthermore, Cas9-eVLPs delivery did not cause any retinal anatomical or functional toxicity. Conclusion This study highlights the potential of eVLPs as a safe and efficient delivery platform for Cas9 RNPs, achieving precise VEGF knockout and significant reductions in neovascularization in a mouse model of wet AMD. With transient Cas9 expression, high editing efficiency, and minimal risk of genomic integration, eVLPs present a promising alternative to conventional delivery systems for advancing genome editing therapies.