Ex vivo machine perfusion enables robust and widespread lipophilic-conjugated siRNA delivery to the porcine lung.

BACKGROUND

Normothermic ex vivo lung perfusion (EVLP) provides a controlled opportunity for direct molecular intervention in donor lungs before transplantation. Small interfering RNAs (siRNAs) enable sequence-specific gene silencing and, when chemically stabilized and conjugated, can be engineered to optimize tissue delivery and intracellular activity following transient exposure. We evaluated whether fully chemically modified docosanoic acid (DCA)-conjugated siRNA could be delivered to donor lungs during EVLP.

METHODS

Porcine paired lungs underwent normothermic EVLP for 6-to-8-hours with administration of fully chemically modified, DCA-conjugated siRNA targeting Janus kinase 1 (JAK1). Pulmonary siRNA distribution, tissue accumulation, and cellular uptake were assessed using molecular and imaging approaches. Complementary in vivo mouse experiments using the same JAK1-targeting siRNA provided a benchmark for pulmonary accumulation following systemic administration.

RESULTS

EVLP resulted in widespread pulmonary distribution of DCA-conjugated siRNA, with detectable uptake within 2 hours and sustained tissue accumulation throughout perfusion. Cellular uptake was demonstrated in multiple pulmonary cell populations, with prominent localization to alveolar epithelial cells and macrophages. Tissue accumulation levels following EVLP was comparable to that observed following systemic administration in mice. Despite efficient tissue accumulation and cellular uptake, functional gene silencing was not detected during the 6-to-8-hour EVLP period.

CONCLUSION

Normothermic EVLP enables widespread delivery and cellular uptake of fully chemically modified, DCA-conjugated siRNA in donor lungs during clinically relevant perfusion times. These findings establish proof-of-concept for EVLP as a controlled platform for ex vivo delivery of programmable oligonucleotide therapeutics. Further studies are needed to determine whether EVLP-delivered siRNA produces sustained post-perfusion gene silencing and improves graft injury and post-transplant outcomes.