Initial report of siRNA uptake during normothermic and hypothermic liver machine perfusion: A promising new therapy
(2019) 25th Annual Congress of the International Liver Transplantation, ILTS 2019 — Location: Toronto, ON, Canada (15.May.2019)
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- Background: RNA interference (RNAi) is a natural gene modulation process that was recently approved for clinical use and has great potential for treatment of many pathological conditions. 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 also show silencing p53 via siRNA reduces inflammation and caspase expression during liver ischemia. Methods: Rat portal veins were cannulated (Fig. 1A) and perfused at 10mmHg on a closed-loop pump circuit regulated by circulating water bath (Fig. 1B). Controls were perfused with Williams E+10U insulin and lipid nanoparticles (Fig. 1C). 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, formalin fixed, stained, then imaged with confocal microscopy. For p53 studies, siRNA was delivered 24 hours before hilum clamping. Tissue/blood was collected 3 days after siRNA injection. Results: Compared to empty vector controls (Fig. 2A-D), normothermic (Fig. 2E-H) and hypothermic livers (Fig. 2I-L) perfused with FAS siRNA demonstrate diffuse uptake in sinusoids and surrounding central veins. SiRNA is observed together 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 delivering before procurement, we may reliably improve the quality of livers during the ischemic period. It will be important to determine the ideal perfusion temperature. Further studies will quantify uptake with peptide-nucleic acid hybridization at both perfusion temperatures and the degree of FAS or p53 silencing in a transplant model.
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Gillooly, A., Bonaccorsi Riani, E., Moore, C., Perry, J., Bruggenwirth, I., Thijssen, M., Wang, X., Mandrekar, P., Kowalik, T., Porte, R., Leuvenink, H., & Martins, P. N. (2019). Initial report of siRNA uptake during normothermic and hypothermic liver machine perfusion: A promising new therapy. Transplantation, 103(8 Supplement 1), 232-233. https://hdl.handle.net/2078.5/218590 (Original work published 2019)