OC 23.3 - Naked miRNA mimics and siRNAs selectively transfect megakaryocytes and platelets in vivo and durably suppress target gene expression to modulate megakaryocyte and platelet function in mice.

Background: We showed previously that washed platelets internalize and utilize naked double-stranded miRNA mimics at high efficiency upon direct exposure ex vivo.

Aims: Develop efficient, tissue-specific, tunable, non-integrative gene therapy in megakaryocytes and platelets, via selective in vivo transfection with naked mi/siRNAs.

Methods: We tested transfection and functional effects of naked modified-base mi/siRNAs (“siLNAs”) ex vivo, and in vivo in mice.

Results: Murine bone marrow and splenic megakaryocytes and blood platelets showed rapid, high efficiency transfection with naked siLNAs both ex vivo, and in vivo following either intravenous or subdermal administration. Surprisingly, no other blood, marrow or splenic cells or endothelium showed naked siLNA transfection. Cultured MEG-01 cells only became transfectable upon megakaryocytic differentiation, coincident with CD41 onset, further confirming megakaryocyte/platelet-specific transfection. Intravenously administered naked RhoA siLNAs suppressed RhoA selectively in megakaryocytes, and modulated ploidy and platelet production reflecting established RhoA functions, demonstrating siLNA regulation of megakaryocyte function in vivo. Naked siLNA targeting P2y12 ADP receptor dose-dependently suppressed P2y12 in vivo selectively in blood platelets within 24 hours, reduced ADP-stimulated reactivity compared to controls, and yielded protection from FeCl3-induced carotid artery thrombosis. Therapeutic window for P2y12 siLNA in mice, assessed by thrombosis (FeCl3 injury) and bleeding (tail clip) across a dose range, was strikingly wide: >3-fold that observed for acute intravenous ticagrelor (off-label/Selleckchem) in this model. Platelet P2y12 and reactivity remained suppressed over multiple days using siLNA-loaded subdermal pumps, demonstrating both efficient subdermal siLNA delivery to platelets, and durable target gene suppression. Mi/siLNAs were non-immunogenic.

Conclusion(s): Naked siLNAs support facile and rapid gene functional analysis in megakaryocytes and platelets in vivo and ex vivo, for accelerated, multiplex-able testing in animal models without necessity and caveats of traditional reverse genetics. This platform may also provide opportunities for non-integrative, tissue-targeted, precision medicine-based gene therapeutics to modulate megakaryocyte and platelet functions across a broad range of clinical scenarios.