Diabetic retinopathy (DR) is one of the leading causes of visual impairment and blindness worldwide. Current therapies for DR primarily focus on inhibiting vascular endothelial growth factor A (VEGFA); however, their efficacy remains limited due to drug resistance and the requirement for repeated intravitreal injections. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing technology enables specific targeting and knockout of the VEGFA gene, offering a novel therapeutic approach for DR. In this study, we synthesized a novel ionizable lipid, M3, and assembled the optimal-performing M3-F4 into lipid nanoparticles (M3-F4 LNP) for codelivery of VEGFA-targeting Cas9 mRNA (mCas9) and single guide RNA (sgRNA). The optimized formulation, composed of M3:cholesterol:DSPC:DMG-PEG at a molar ratio of 45:42.5:10:2.5, exhibited a particle size below 100 nm, a PDI below 0.2, and an encapsulation efficiency above 80%. Sanger sequencing-based indel analysis confirmed VEGFA editing in HRMECs, with sgRNA1 achieving an indel frequency of approximately 28.7%. In high glucose-induced human retinal microvascular endothelial cells (HRMECs), the mCas9/sgVEGFA@M3-F4 LNP reduced cell proliferation, migration, invasion, and tube formation, while restoring endothelial barrier integrity and exerting anti-inflammatory effects. A single intravitreal injection of mCas9/sgVEGFA@M3-F4 LNP effectively inhibited pathological neovascularization and retinal leakage in both oxygen-induced retinopathy mice and streptozotocin-induced diabetic mice. Furthermore, it markedly attenuated VEGFA-induced inflammation while maintaining excellent biocompatibility. This study demonstrates M3-F4 LNP as a promising method for efficient CRISPR/Cas9 delivery and provides robust support for gene therapy strategies in DR treatment. in vivo
