Delivery and expression of mRNA therapeutics during ex vivo kidney perfusion: A feasibility study in porcine and human donor kidneys
Achieving efficient mRNA delivery and expression in intact solid organs beyond the liver remains a major challenge for advancing mRNA therapeutics. Normothermic machine perfusion (NMP) provides a clinically relevant ex vivo platform for targeted intervention during donor organ preservation, minimizing systemic exposure and enabling organ-confined mRNA delivery. Here, we demonstrate that intra-arterial infusion of lipid nanoparticles (LNPs) encapsulating reporter mRNAs during kidney NMP enables robust, organ-wide protein expression in human-sized porcine donor kidneys. Engineered LNP formulations were first evaluated for stability and efficacy in kidney cell lines, where LNPs pegylated with TPGS (D--tocopherol polyethylene glycol 1000 succinate) achieved the highest reporter protein expression levels. Next, the LNP-encapsulated mRNAs were introduced into porcine kidneys via a 5-minute infusion during NMP. mCherry and human erythropoietin (hEPO) mRNAs were delivered via the renal artery, serving as intracellular and secreted reporters, respectively. The perfusion was continued for 6-12h after LNP-mRNA infusion with the erythrocyte-based perfusate at 37 {degrees}C. LNP-mRNA administration resulted in widespread parenchymal mRNA uptake and rapid, robust reporter protein expression that continued to increase over the perfusion period. hEPO protein was detected in perfusate and urine, while mCherry expression localized to endothelial and tubular cells within the renal parenchyma. Consistent with the in vitro data, TPGS-LNPs produced the highest expression during NMP. Importantly, LNP-mediated mRNA delivery during NMP did not affect perfusion parameters or histological integrity. These findings demonstrate successful mRNA-driven protein expression in intact donor kidneys, establishing NMP as a clinically relevant route for organ-specific RNA delivery and protein expression modulation. One sentence summaryUsing a clinically compatible normothermic machine perfusion model, we demonstrate that lipid nanoparticle-mediated mRNA delivery enables rapid, organ-wide protein expression in human-sized porcine donor kidneys.