Mechanism of LNP-mediated Alas1 silencing in hepatocytes. The LNPs encapsulating the double-stranded Alas1-siRNA consist of cationic, ionizable lipids that are positively charged at acidic pH but neutral at physiologic pH. While in circulation, the LNPs acquire apolipoprotein E on their surface, which is bound primarily by low-density lipoprotein receptors that are highly expressed on the surface of hepatocytes. This induces clathrin-mediated endocytosis and the formation of early endosomes. As the endosomes mature and the pH becomes highly acidic, the ionizable lipids turn positive and bind to the negative lipids that are exposed on the endosomal membrane, leading to membrane fusion and disruption of the LNP. This allows escape of the Alas1-siRNA into the hepatocyte cytoplasm, where it is loaded onto the RISC. The passenger (sense) strand is degraded, leaving only the guide (antisense) strand on the RISC, which binds endogenous Alas1 mRNA in a complementary manner and degrades it. Professional illustration by Patrick Lane, ScEYEnce Studios.