A Breath of Fresh Air: Optimizing Lungs for Transplantation Using siRNA-Based Therapies During Ex-Vivo Machine Perfusion

J. E.. Buchwald¹, H. H.. Fakih¹, C. E.. Tocheny², B. Tfayli³, H. Kharroubi³, S. Sanatkar⁴, S. De Taeye⁵, W. Michaud⁶, A. A.. Osho⁷, R. N.. Pierson III⁴, A. Khvorova¹, S. Rabi^8
1RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA, Worcester, Massachusetts ²Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA, Worcester, Massachusetts ³Division of Thoracic Surgery, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, Boston, Massachusetts ⁴Center for Transplantation Sciences, Division of Cardiac Surgery, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, Boston, Massachusetts ⁵Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, Boston, Massachusetts ⁶Division of Cardiac Surgery, Massachusetts General Hospital,, Boston, Massachusetts ⁷Division of Cardiac Surgery, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, Boston, Massachusetts ⁸Massachusetts General Hospital, Division of Cardiac Surgery, Boston, Massachusetts

Purpose: Ex vivo lung perfusion (EVLP) has emerged as a promising modality to recondition lungs using candidate therapeutics aimed at improving organ function and transplant suitability. Our group has previously identified that the lipophilic docosanoic acid (DCA) class of conjugated siRNA improves lung and heart delivery relative to other siRNA ligands.

Methods: Here, we evaluate the feasibility of EVLP as a rehabilitative platform for delivering candidate siRNA therapeutics to pre-transplant lungs ex vivo. Fully chemically modified JAK1 siRNAs targeting all mammalian species were conjugated to DCA and Cy3-tagged to visualize intracellular localization using fluorescence microscopy. Using a dual-lung perfusion system where each lung is ventilated and perfused independently, 1 uM of DCA-conjugated JAK1 siRNA was added to the experimental lung circuit while the control lung received no siRNA. Lungs were ventilated and perfused for 6-8 hours of normothermic (38C) perfusion with asanguinous perfusate. Lung biopsies and perfusate samples were collected every hour to assess siRNA biodistribution and silencing efficacy.

Results: Following 6 to 8 hours of perfusion, DCA-conjugated JAK1 siRNA was uniformly endocytosed by all porcine lung cell types as visualized by fluorescence microscopy, with uptake occurring in as early as 2 hours of perfusion and maximal signal detected at the conclusion of the perfusion. Quantification of JAK1 mRNA expression revealed trends towards suppressed JAK1 expression in siRNA-treated porcine lungs relative to lungs perfused without the siRNA. Studies are ongoing to measure the perfusate cytokine profiles and siRNA guide strand accumulation in treated and untreated lungs during EVLP and assess the durability of siRNA-mediated EVLP delivery and gene silencing in a porcine lung transplant model.

Conclusion: In this study, JAK1-siRNA is robustly and uniformly delivered to the porcine lung within 6 to 8 hours of EVLP. These studies represent a promising step towards our goal of targeting ischemia-reperfusion injury pathways using an siRNA cocktail to silence the family of pro-apoptotic and pro-inflammatory genes during EVLP, allowing for broader use of marginal lungs for human lung transplantation.

Identify the source of the funding for this research project: This project was supported by grants awarded to A.K. of the RNA Therapeutics Institute and the Perkins Fund awarded to S.A.R. of MGH.