Schematic overview of the stepwise experimental approach to achieve siRNA-mediated allele-selective silencing of VWF; from in silico, to in vitro, and in vivo studies. (A) Using an alignment on the cDNA sequences of mouse inbred strains C57BL/6J (B6) and 129S1/SvImJ (129S), genetic differences between the strains’ Vwf gene were determined. Based on these genetic differences, an in-silico prediction analysis was performed to identify candidate strain-selective siRNAs. (B) Next, the candidate siRNAs were tested in vitro on activity and strain-selectivity. HEK293 cells were transiently co-transfected with a candidate siRNA and plasmids permitting expression of HA-tagged 129S-Vwf and MYC-tagged B6-Vwf using Lipofectamine 2000 as transfection reagent. VWF-MYC or VWF-HA protein was measured using an enzyme-linked immunosorbent assay (ELISA). siRNAs that were active and strain-selective in vitro were tested further in vivo on allele-selectivity. (C) For in vivo endothelial delivery, the lead candidate siRNAs were encapsulated in 7C1 LNPs. B6 or 129S mice were intravenously injected with nanoparticle-encapsulated siRNAs. Blood was drawn from the inferior vena cava, and plasma VWF protein levels were measured using ELISA. Lungs were harvested for measuring Vwf mRNA using quantitative polymerase chain reaction (qPCR) or VWF protein localization using immunofluorescent staining. This figure was created with Biorender.com.