Keller, A. K. | Search bioRxiv

bioRxiv2025

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.

bioengineering↗

bioRxiv2024

End of preservation normothermic machine perfusion of porcine kidneys after ischaemic injury reprograms metabolism and induces fibrosis after transplant despite unchanged function: insights from the renal proteome.

Normothermic machine perfusion (NMP) after initial hypothermic preservation of donor kidneys prior to transplantation is becoming a clinical reality, but the precise molecular mechanisms through which the graft is impacted remain only partially characterised. Using an unbiased proteomic methodology, we found that auto transplantation of is chaemically injured porcine kidneys resulted in an activation of the stress response 14 days after transplantation, as well as in selective changes in the proteins responsible for the metabolism of organic acids. The addition of 4 hours of NMP at the end of organ preservation (endNMP) resulted in coordinated changes to the renal proteome at 14 days when compared with the effect of transplant after preservation by hypothermic machine perfusion alone: most notably increased fibrosis and widespread additional reprogramming of metabolism. These findings were supported by intersection with single cell transcriptomics data which suggested an enrichment of proteins predominantly expressed in fibroblasts in kidneys with end of preservation NMP 14 days post-transplant compared to healthy kidneys. Our data showed that the addition of endNMP to existing preservation strategies resulted in a different molecular phenotype after transplantation, despite unchanged filtration function. In addition to potentially conferring benefits, NMP may also result in potentially detrimental molecular changes and thus protocols should be carefully evaluated to derive optimal clinical outcomes.

physiology↗