Remodeling the Neuroimmune Microenvironment in Retinal Ischemia‐Reperfusion Injury via Combined CRISPR/Cas9 Targeting of ACSL4 and Isorhamnetin

Abstract Retinal ischemia‐reperfusion (RIR) injury induces oxidative stress, excitotoxicity, inflammation, and ferroptosis, which interact through complex crosstalk, forming a retinal pathological microenvironment (RPMe) that drives retinal ganglion cell (RGC) death. Central to these processes is the dysregulation of the neuroimmune microenvironment (NiMe), characterized by aberrant microglial activation around RGCs and immune signaling imbalances. Here, through analysis of single‐cell RNA sequencing, it identifies significant activation of the sphingolipid signaling pathway in RIR‐injured retinal microglia, which crosstalks with immune signaling pathways. This crosstalk disrupts NiMe homeostasis. To address this, a biomimetic nanoparticle system coated with retinal precursor cell membranes is developed. This system co‐delivers a CRISPR/Cas9‐based Acyl‐CoA synthetase long‐chain family member 4 (ACSL4) inhibitor to suppress ferroptosis and sphingolipid signaling and isorhamnetin, a natural molecule is identified to directly bind protein kinase A to inhibit the glutamatergic synapse signaling pathway involved in oxidative stress. By targeting sphingolipid signaling and its crosstalk with PI3K/AKT and ASK1/JNK/NF‐κB pathways in microglia, as well as glutamatergic synapse signaling and ferroptosis in RGCs, this system restores NiMe balance. The retinal precursor cell membrane‐coated nanoparticle offers a novel, synergistic, and targeted therapeutic strategy for RIR‐related retinal diseases by integrating CRISPR/Cas9 technology with natural product molecule therapy.