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First Report of siRNA Uptake (for RNA Interference) During Ex Vivo Hypothermic and Normothermic Liver Machine Perfusion

Gillooly, Andrew R. BS1; Perry, Jessica BS1; Martins, Paulo N. MD, PhD1

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doi: 10.1097/TP.0000000000002515

RNA interference is a naturally occurring specific method to silence genes with wide potential for treating human disease. Engineered RNA oligonucleotides targeting proprotein convertase subtilisin-kexin type 9 were recently shown to reliably lower LDL cholesterol, with a single injectable dose lasting 6 to 12 months.1 Applying short interfering RNA (siRNA) therapy to livers via ex vivo machine perfusion before transplantation may open the door to using organs from extended criteria donors that would otherwise be discarded. Treating isolated livers would also reduce costs compared with systemic therapy. We show for the first time that siRNA against the Fas receptor added directly to perfusion solution is uptaken into rat livers during hypothermic (4°C) and normothermic (37°C) perfusion. The Fas receptor expressed in liver signals hepatocytes to apoptose after binding its respective ligand. In mice, reduced FAS expression via siRNA confers protection against chemically induced acute liver failure.2 We aim to silence FAS during the ischemic period before transplantation and thus reduce or even reverse graft damage. Transfection into hepatocytes is achieved by coating siRNA with lipid nanoparticles, which facilitate endocytosis across cell membranes and release siRNA into the cytoplasm.3 SiRNA-lipid complexes were delivered in perfusion solution via portal vein cannulation, and distribution was observed with fluorescent confocal microscopy (Figure 1). Full methods are described in Supplemental Materials and Methods (SDC,http://links.lww.com/TP/B651). Further studies will quantify FAS knockdown and the effect of FAS in a rat transplant model. SiRNA therapy during organ machine perfusion is an exciting frontier with transformational potential to improve clinical transplant outcomes.

FIGURE 1
FIGURE 1:
FAS short interfering RNA (siRNA) uptake in rat hepatocytes during hypothermic (4°C) and normothermic (37°C) ex vivo machine perfusion. Scale represents 20 μm. Adult male rat livers were harvested and the portal vein cannulated (arrow) with a modified angiocatheter (A). Livers were perfused via the cannula on a closed-loop circuit with a roller pump, warmed or cooled by a circulating water bath. Portal vein pressure was maintained at 10 mm Hg. B, Empty lipid nanoparticles were imaged with electron microscopy; scale represents 200 nm (C). Perfusion solution was composed of Williams medium E supplemented with 10 U of insulin. Control livers were perfused with medium + Invivofectamine (ThermoFisher) transfection lipid nanoparticles (D-G) for 4 hours. Livers perfused with medium + Invivofectamine complexed with 50 nM FAS siRNA (Qiagen) at 37°C for 4 hours demonstrate diffuse uptake in sinusoids and surrounding central veins (H-K) compared with controls. This effect is even more pronounced in livers perfused with siRNA at 4°C for 4 hours (L-O). Nuclei are visualized in cyan with DAPI, FAS siRNA in yellow with an AlexaFluor-555 3´ modification, and cell membranes in red with wheat germ agglutinin conjugated to AlexaFluor-488 (ThermoFisher).

REFERENCES

1. Fitzgerald K, White S, Borodovsky A, et al. A highly durable RNAi therapeutic inhibitor of PCSK9. N Engl J Med. 2017;376:41–51.
2. Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med. 2003;9:347–351.
3. Jayaraman M, Ansell S, Mui B, et al. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew Chem Int Ed Engl. 2012;51:8529–8533.

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