siRNA Uptake During Ex Vivo Hypothermic and Normothermic Liver Machine Perfusion: A Promising New Therapy
Gillooly AR1, Moore C1, Bonaccorsi E2, Perry J1, Smith J1, Xue W1, Bruggenwirth I1, Thijssen M1, X. Wang1, X. E1, Mandrekar P1, Kowalik T1, Khvorova A1, Bozorgzadeh A1, Martins PN1
1University of Massachusetts Medical School, Worcester, MA; 2Université catholique de Louvain, Brussels, BE
Background: RNA interference therapy was recently approved for clinical use, with great potential for treatment of many diseases. The surface receptor FAS and tumor suppressor p53 are implicated in ischemic liver damage. Both may be silenced via RNAi with limited off-target effects. We determined the efficiency of FAS siRNA delivery with lipid nanoparticles during normothermic (37°C) and hypothermic (4°C) ex vivo machine perfusion. We show p53 silencing reduces inflammation/caspase expression during liver ischemia.
Methods: Rat portal veins were cannulated and perfused at 10mmHg on a closed-loop circuit regulated by circulating water bath. Controls were perfused with Williams E+10U insulin and nanoparticles. Normothermic livers were perfused with medium plus nanoparticle/FAS-siRNA complex with 3'-AlexaFluor-555 modification at 37°C. Hypothermic livers were perfused similarly at 4°C. Biopsies were collected at 4 hours, fixed, stained, then imaged with confocal microscopy. For p53 studies, siRNA was injected 24 hours before hilum clamping. Tissue/blood was collected 3 days later.
Results: Compared to empty vector controls, normothermic and hypothermic livers perfused with FAS siRNA demonstrate diffuse uptake in sinusoids and central veins. siRNA is observed in sinusoid membranes and in hepatocyte cytoplasm. Livers pretreated with p53 siRNA showed reduced cellular infiltration, vacuolization, and fewer caspase-3 positive cells.
Conclusions: By incorporating RNAi into preservation solutions or administering before procurement, we may reliably improve graft quality during the ischemic period. It is important to determine the ideal perfusion temperature. Further studies will quantify uptake with PNA hybridization at both perfusion temperatures and efficiency of FAS/p53 silencing in a transplant model.
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