Abstract TP368: Targeted Lipid Nanoparticles to Modulate Blood-Brain Barrier Transcytosis in Acute Ischemic Stroke

Ischemic stroke affects more than 600,000 Americans annually. Thrombectomy, where occluded arteries are cannulated for mechanical clot removal, can restore blood flow and improve survival, but still leaves 1/3 of patients functionally dependent, largely due to secondary ischemia-reperfusion injury. Brain edema in the acute phase of ischemia-reperfusion primarily associates with early-stage neurovascular damage and plays a major role in poor outcomes of stroke. Early on after ischemia/reperfusion, edema is mediated by increased transcytosis in the blood brain barrier (BBB) endothelium. Suppression of transcytosis has not yet been tested as a treatment for stroke. Major facilitator superfamily domain containing 2a (Mfsd2a) is a transporter protein that regulates caveolar transcytosis and is downregulated in ischemic stroke. We have previously demonstrated that 1) lipid nanoparticles loaded with mRNA (mRNA/LNP) enable rapid and robust protein expression; 2) vascular cell adhesion molecule (VCAM) targeting selectively delivers nanoparticles to stroke-affected BBB endothelium; 3) mRNA/LNP targeted to VCAM enabled protein expression in BBB endothelium. Therefore, we hypothesize that using LNPs containing Mfsd2a mRNA and targeted to vascular cell adhesion molecule (VCAM) can modulate Mfsd2a expression in inflamed BBB and improve outcome in ischemic stroke. In this work, we screened 10 LNP formulations and identified the optimized formulation on bEND.3 brain endothelial cells for Mfsd2a expression. The induction of Mfsd2a expression significantly enhanced Cav-1 expression and reduced the number of apical caveolae-formations by electron microscopy. Next, we used the acute ischemic stroke animal model (transient middle cerebral artery occlusion for 45 minutes) to assess the therapeutic outcome of VCAM-targeted LNP loaded with Mfsd2a mRNA. Right after reperfusion, we intravenously injected LNP and two additional dose every 24 hours (8ug RNA per dose). On day 3, we assessed brain edema by measuring the extravasation of radiolabeled albumin into the brain parenchyma. The percent of the extravasated albumin was significantly reduced after LNP treatment. In addition, using transmission electron microscopy, we observed less number of caveolae in the treated mice, compared to vehicle control. In conclusion, we have showed that VCAM targeted LNPs are able to deliver Mfsd2a mRNA to the inflamed brain vasculature and reduce brain edema by suppression of transcytosis.