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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

doi: 10.1097/TP.0000000000002515
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1Division of Organ Transplantation, Department of Surgery, University of Massachusetts, Worcester, MA.

Received 4 September 2018. Revision received 18 October 2018.

Accepted 28 October 2018.

A.R.G. participated in the performance of the research, writing of the article, and data analysis. J.P. participated in the performance of the research. P.N.M. participated in the research design, writing of the article, and data analysis.

The authors declare no conflicts of interest.

American Society of the Studies of Liver Diseases (Career Development Award in Liver Transplantation), UMass Faculty Development Grant.

Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com).

Correspondence: Paulo N. Martins, MD, PhD, Transplant Division, Department of Surgery, University of Massachusetts Memorial Medical Center, 55 Lake Ave North, Worcester, MA 01605. (paulo.martins@umassmemorial.org).

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.

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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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