Investigating RNA Interference-Mediated Inhibition of Liver Ischemia Reperfusion Injury During Liver Preservation

J. E. Buchwald¹, D. Echeverria², J. Sousa², N. McHugh², A. Khvorova², P. N. Martins³

¹RNA Therapeutics Institute, Department of Surgery, University of Massachusetts Chan Medical School, Worcester, MA, ²RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, ³Department of Surgery, University of Massachusetts Chan Medical School, Worcester, MA

Meeting: 2022 American Transplant Congress

Abstract number: 663

Keywords: Apoptosis, Gene expression, Ischemia, Liver transplantation

Topic: Basic Science » Basic Science » 14 - Ischemia Reperfusion

Session Information

Session Name: Ischemia Reperfusion

Session Type: Poster Abstract

Date: Saturday, June 4, 2022

Session Time: 5:30pm-7:00pm

Presentation Time: 5:30pm-7:00pm

Location: Hynes Halls C & D

*Purpose: Liver transplantation cures acute end-stage and chronic liver diseases, yet current strategies of organ preservation including static cold storage (SCS) and machine perfusion (MP) do not adequately protect procured organs from ischemia-reperfusion injury (IRI). The goal of this project is to use RNA interference (RNAi) technology in the form of small interfering RNAs (siRNAs) to improve the quality of suboptimal liver grafts by silencing liver IRI gene expression.

*Methods: Fully chemically-modified siRNAs targeting the apoptotic gene, Tp53, were synthesized and in vitro efficacy and potency were evaluated in both human (HepG2, ATCC) and rat (RH7777, ATCC) hepatocytes at 72 hr, with a top siRNA dose of 1.5 υM. Target mRNA expression was measured using the QuantiGene 2.0 Assay. In vivo silencing efficacy was validated by subcutaneously injecting 10 mg/kg siRNA into Lewis rats and measuring siRNA liver uptake, distribution, and silencing efficacy for up to 2 weeks. Ex vivo siRNA delivery was performed using chemically-modified, Cy3-labeled siRNA administered post-hepatectomy during SCS rat liver preservation over 24 hours, with liver uptake confirmed by fluorescence microscopy.

*Results: The initial in vitro screen identified 2 lead siRNA compounds targeting Tp53 mRNA with maximal silencing efficacy of >70% and potency in the nanomolar range. In vivo results demonstrate uniform siRNA delivery to the liver with silencing efficacy of >70%. Fluorescence microscopy confirmed siRNA rat liver uptake during ex vivo SCS preservation.

*Conclusions: Preliminary results identified chemically-modified siRNA compounds targeting Tp53 for RNAi-based modulation of IRI gene expression, and demonstrated in vivo silencing of target mRNA expression, and feasibility of ex vivo delivery during SCS. Future studies investigating the efficacy of lead siRNA compounds in a rat liver IRI model are ongoing. Once confirmed, ex vivo MP will be implemented to assess siRNA delivery and silencing efficacy.

To cite this abstract in AMA style:

Buchwald JE, Echeverria D, Sousa J, McHugh N, Khvorova A, Martins PN. Investigating RNA Interference-Mediated Inhibition of Liver Ischemia Reperfusion Injury During Liver Preservation [abstract]. Am J Transplant. 2022; 22 (suppl 3). https://atcmeetingabstracts.com/abstract/investigating-rna-interference-mediated-inhibition-of-liver-ischemia-reperfusion-injury-during-liver-preservation/. Accessed November 21, 2024.

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