Quantitation of physiological and biochemical barriers to siRNA liver delivery via lipid nanoparticle platform.

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.

Biodistribution of small interfering RNA at the organ and cellular levels after lipid nanoparticle-mediated delivery.

Chemically stabilized small interfering RNA (siRNA) can be delivered systemically by intravenous injection of lipid nanoparticles (LNPs) in rodents and primates. The biodistribution and kinetics of LNP-siRNA delivery in mice at organ and cellular resolution have been studied using immunofluorescence (IF) staining and quantitative polymerase chain reaction (qPCR). At 0.5 and 2 hr post tail vein injection of Cy5-labeled siRNA encapsulated in LNP, the organ rank-order of siRNA levels is liver > spleen > kidney, with only negligible accumulation in duodenum, lung, heart, and brain. Similar conclusions were drawn by using qPCR to measure tissue siRNA levels as a secondary end point. siRNA levels in these tissues decreased by more than 10-fold after 24 hr. Within the liver, LNPs delivered siRNA to hepatocytes, Kupffer cells, and sinusoids in a time-dependent manner, as revealed by IF staining and signal quantitation methods established using OPERA/Columbus software. siRNA first accumulated in liver sinusoids and trafficked to hepatocytes by 2 hr post dose, corresponding to the onset of target mRNA silencing. Fluorescence in situ hybridization methods were used to detect both strands of siRNA in fixed tissues. Collectively, the authors have implemented a platform to evaluate biodistribution of siRNA across cell types and across tissues in vivo, with the objective of elucidating the pharmacokinetic and pharmacodynamic relationship to guide optimization of delivery vehicles.