LNP-mediated EglN siRNA delivery is liver specific and down-regulates EglN via an RNAi mechanism. (A) Bioluminescent images of HIF1α-Luc mice 72 hours after a single intravenous dose of LNPs targeting all 3 EglN family members or, as a negative control, green fluorescent protein (GFP). Total dose was 1 mg/kg (0.33 mg/kg per family member). (B-C) Quantification of EglN mRNA levels in livers (B) and kidneys (C) of mice 1 week after treatment with LNPs targeting all 3 EglN family members or firefly luciferase. mRNA levels were normalized to GAPDH mRNA and then to corresponding values in mice treated with luciferase siRNA. (D) Quantification of Epo mRNA levels in liver and kidney in mice treated as in panels B and C. (E) Overview of 5′RACE assay to monitor the cleavage site of target mRNA. (F) 5′RACE of mouse liver total RNA isolates from mice treated with the indicated siRNAs. Specific cleavage sites were confirmed by sequencing of excised bands from gel. For panels B through D, n = 5; for panel F, n = 3. Error bars represent 1 SD. **P < .01.