title: Quantitation of physiological and biochemical barriers to siRNA liver delivery via lipid nanoparticle platform.
abstract: Effective delivery of small interfering RNA (siRNA) requires efficient cellular uptake and release into cytosol where it forms an active complex with RNAi induced silencing complex (RISC). Despite rapid developments in RNAi therapeutics, improvements in delivery efficiency of siRNA are needed to realize the full potential of this modality in broad therapeutic applications. We evaluated potential physiological and biochemical barrier(s) to the effective liver delivery of siRNA formulated in lipid nanoparticle (LNP) delivery vehicles. The comparative siRNA delivery performance of three LNPs was investigated in rats. They were assembled with either C14- or C18-anchored PEG-lipid(s), cationic lipid(s), and various helper lipid(s) and contained the same siRNA duplex. These LNPs demonstrated differentiated potency with ED50's ranging from 0.02 to 0.25 mg/kg. The two C14-PEG-LNPs had comparable siRNA exposure in plasma and liver, while the C18-PEG-LNP demonstrated a higher plasma siRNA exposure and a slower but sustained liver uptake. RISC bound siRNA within the liver, a more proximal measure of the pharmacologically active siRNA species, displayed loading kinetics that paralleled the target mRNA knockdown profile, with greater RISC loading associated with more potent LNPs. Liver perfusion and hepatocyte isolation experiments were performed following treatment of rats with LNPs containing VivoTag-fluorescently labeled siRNA. One hour after dosing a majority of the siRNA within the liver was associated with hepatocytes and was internalized (within small subcellular vesicles) with no significant cell surface association, indicating good liver tissue penetration, hepatocellular distribution, and internalization. Comparison of siRNA amounts in hepatocytes and subcellular fractions of the three LNPs suggests that endosomal escape is a significant barrier to siRNA delivery where cationic lipid seems to have a great impact. Quantitation of Ago-2 associated siRNA revealed that after endosomal escape further loss of siRNA occurs prior to RISC loading. This quantitative assessment of LNP-mediated siRNA delivery has highlighted potential barriers with respect to endosomal escape and incomplete RISC loading for delivery optimization efforts.
authors.affiliation: Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, ‡RNA Discovery Biology, ∥Laboratory Animal Resources, and #Pharmaceutical Sciences, Merck Research Laboratories, Merck & Co., Inc. , 770 Sumneytown Pike, West Point, Pennsylvania 19446, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.forename: Mei
authors.initials: M
authors.lastname: Ou
authors.forename: Ed
authors.initials: E
authors.lastname: Keough
authors.forename: Jeff
authors.initials: J
authors.lastname: Roberts
authors.forename: Ken
authors.initials: K
authors.lastname: Koeplinger
authors.forename: Mike
authors.initials: M
authors.lastname: Lyman
authors.forename: Scott
authors.initials: S
authors.lastname: Fauty
authors.forename: Ed
authors.initials: E
authors.lastname: Carlini
authors.forename: Melissa
authors.initials: M
authors.lastname: Stern
authors.forename: Rena
authors.initials: R
authors.lastname: Zhang
authors.forename: Suzie
authors.initials: S
authors.lastname: Yeh
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Mahan
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Don
authors.initials: D
authors.lastname: Slaughter
authors.forename: Marian
authors.initials: M
authors.lastname: Gindy
authors.forename: Conrad
authors.initials: C
authors.lastname: Raab
authors.forename: Charles
authors.initials: C
authors.lastname: Thompson
authors.forename: Jerome
authors.initials: J
authors.lastname: Hochman
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005260:Female; D022781:Hepatocytes / Q000378:metabolism; D008055:Lipids / Q000737:chemistry*; D008099:Liver / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D053758:Nanoparticles / Q000008:administration & dosage* / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D008055:Lipids; D034741:RNA, Small Interfering