Normothermic machine perfusion (NMP) is a novel clinical approach to overcome the limitations of traditional hypothermic organ preservation. NMP can be used to assess and recondition organs prior to transplant and is the subject of clinical trials in solid organ transplantation. In addition, NMP provides an opportunity to deliver therapeutic agents directly to the organ, thus avoiding many limitations associated with systemic treatment of the recipient. We report the delivery of oligonucleotide‐based therapy to human kidneys during NMP, in this case to target microRNA function (antagomir). An antagomir targeting mir‐24‐3p localized to the endothelium and proximal tubular epithelium. Endosomal uptake during NMP conditions facilitated antagomir co‐localization with proteins involved in the RNA‐induced silencing complex (RISC) and demonstrated engagement of the miRNA target. This pattern of uptake was not seen during cold perfusion. Targeting mir‐24‐3p action increased expression of genes controlled by this microRNA, including heme oxygenase‐1 and sphingosine‐1‐phosphate receptor 1. The expression of genes not under the control of mir‐24‐3p was unchanged, indicating specificity of the antagomir effect. In summary, this is the first report of ex vivo gymnotic delivery of oligonucleotide to the human kidney and demonstrates that NMP provides the platform to bind and block detrimental microRNAs in donor kidneys prior to transplantation.

Background: Although liver normothermic machine perfusion is increasingly used clinically, there are few reports of complications or adverse events. Many centers perform liver NMP to viability test suboptimal grafts, often for prolonged periods. In addition, several researchers are investigating NMP as a drug delivery platform, which usually necessitates prolonged perfusion of otherwise non-viable liver grafts. We describe two instances of methaemoglobinaemia during NMP of suboptimal livers.Methods: The NMP of eight human livers rejected for transplantation is described. Methaemoglobinaeima developed in two; one perfused using generic Medtronic™ perfusion equipment and one using the OrganOx Metra®.Results: The first liver (53 years DBD) developed methaemoglobinaemia (metHb = 2.4%) after 13 h of NMP, increasing to metHb = 19% at 16 h. Another liver (45 years DBD) developed methaemoglobinaemia at 25 h (metHb = 2.8%), which increased to metHb = 28.2% at 38 h. Development of methaemoglobinaemia was associated with large reductions in oxygen delivery and oxygen extraction. Both livers were steatotic and showed several suboptimal features on viability testing. Delivery of methylene blue failed to reverse the methaemoglobinaemia. Compared to a matched cohort of steatotic organs, livers which developed methaemoglobinaemia showed significantly higher levels of hemolysis at 12 h (prior to development of methaemoglobinaemia).Conclusions: Methaemglobinaemia is a complication of NMP of suboptimal liver grafts, not limited to a single machine or perfusion protocol. It can occur within 13 h (a timepoint frequently surpassed when NMP is used clinically) and renders further perfusion futile. Therefore, metHb should be monitored during NMP visually and using blood gas analysis.

Introduction Normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for delivery of novel therapeutics to optimize organ quality. This includes RNA interference (RNAi) therapeutics e.g. antisense oligonucleotides (ASO) that block detrimental microRNAs. The intracellular kinetics of RNAi therapeutics are crucial for their pharmacological effect, however, it remains poorly understood. NMP provides an ideal platform to investigate this further. Method During NMP, human kidneys (n = 12) were treated for 6 hours with a fluorescently-labelled ASO designed to block microRNA-24-3p activity. Biopsies were taken at 0, 2, 4, and 6 hours. Kidney sections were stained with antibodies against early endosomes (Rab5), late endosomes (Rab7), RNA-induced silencing complexes (GW182) and lysosomes (LAMP2). Confocal microscopy images were obtained and co-localisation quantified using Hugyens™ software following batch deconvolution. The global transcriptomic impact of ASO therapy was also assessed using RNA sequencing. Result Following 2 hours of NMP, ASO was primarily found in tubular epithelial cells. Co-localisation studies revealed ASO uptake via endocytosis and endosomal sorting occurring during NMP. This was followed by cytoplasmic escape and co-localisation of ASO with GW182 proteins. This pattern of co-localisation was not seen in scrambled sequence or cold perfusion controls. RNAseq analysis revealed a decrease in inflammatory pathways and upregulation of microRNA-24-3p targets. Discussion This is the first study to demonstrate NMP facilitates gymnotic ASO delivery directly into the RISC, whereby, it blocks microRNA-mediated mRNA silencing and increases bioavailability of protective targets. This study highlights the capacity of NMP to re-programme gene expression in donor kidneys using RNAi therapeutics. Take-home Message Ex vivo normothermic machine perfusion of donor kidneys provides a unique window of opportunity prior to transplantation when we can deliver therapies to improve the quality of the organ. Novel genetic therapies designed to protect kidneys against ischemia reperfusion injury could potentially increase organ utilisation and improve post-transplant outcomes for the many patients on the kidney transplant waiting list.

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