Organ transplantation is the only cure for end-stage disease, yet many patients die while waiting for a transplant due to a perpetual shortage of donor organs. Insufficient donor supply is further diminished by ischemia-reperfusion injury (IRI) during procurement and preservation of organs, driving primary graft dysfunction and resulting in significant patient morbidity and mortality. Mitigating IRI is critically needed to increase the number of viable donor organs and improve patient survival. Temporarily reducing, not eradicating, organ expression levels of IRI mediators during the transplant period may provide an opportunity for therapeutic modulation of pathogenic gene expression and subsequently, promote organ rehabilitation to enable suitability for transplant.

Small interfering RNAs (siRNAs) are a revolutionary new class of medicine that enable potent, temporary, yet durable modulation of gene expression by leveraging the endogenous RNA interference (RNAi) pathway to degrade mRNA, rendering downstream protein translation ineffective. The use of chemically-stabilized siRNAs in organ transplantation is ideal due to their robust duration of effect, where a single systemic injection supports 6-12 months of efficacy, overlapping with the critical period of primary graft dysfunction and acute organ rejection. Here, we leverage siRNA-based technologies to prophylactically intervene during the transplantation process to rehabilitate and optimize organs for transplantation.

First, we establish normothermic ex vivo machine perfusion as a feasible and robust platform for functional delivery of lipophilic docosanoic acid conjugated (DCA)-siRNA targeting the master inflammatory regulator, JAK1, in rat, pig, and discarded human heart models whereby we efficiently transduce all major cardiac cell types. We translated these results to a porcine orthotopic heart transplant model, demonstrating successful transplantation of the siRNA-treated organs during ex vivo machine perfusion and confirm lack of recipient secondary organ exposure. Next, we validated the platform of ex vivo lung perfusion for robust and widespread delivery of DCA-conjugated siRNA in a porcine model. Finally, we investigated the impact of silencing known IRI mediators pre-transplant in a rat donor pre-treatment model using hepatocyte-specific GalNAc-conjugated siRNAs. We identified lead siRNAs targeting mediators of IRI cell death and inflammation, Fas and Hmgb1, and employed lead candidates in a model of rat liver IRI, validating that therapeutic silencing of these targets modulates liver expression post-ischemia and drives improvement in liver function post-injury.

These studies support the application of targeted and programmable therapies using siRNA technologies in solid organ transplantation, enabling organ-specific treatment for rehabilitation and recovery, and establishes an RNAi platform for translational and clinical transplant applications.