Development of lipid nanoparticle formulations of siRNA for hepatocyte gene silencing following subcutaneous administration

Recently developed lipid nanoparticle (LNP) formulations of siRNA have proven to be effective agents for hepatocyte gene silencing following intravenous administration with at least three LNP–siRNA formulations in clinical trials. The aim of this work was to develop LNP–siRNA systems for hepatocyte gene silencing that can be administered subcutaneously (s.c.). Three parameters were investigated, namely LNP size, residence time of the polyethylene glycol (PEG)-lipid coating and the influence of hepatocyte-specific targeting ligands. LNP sizes were varied over the range of 30 to 115nm in diameter and PEG-lipid that dissociates rapidly (PEG-DMG) and slowly (PEG-DSG) were employed. In mice, results show that large (~80nm) LNP exhibited limited accumulation in the liver and poor Factor VII (FVII) gene silencing at 1mg siRNA/kg body weight. Conversely, small (~30nm) LNP systems showed maximal liver accumulation yet still had minimal activity. Interestingly, intermediate size (~45nm) LNP containing PEG-DSG exhibited nearly equivalent liver accumulation as the smaller systems following s.c. administration but reduced FVII levels by 80% at 1mg siRNA/kg body weight. Smaller systems (~35nm diameter) containing either PEG-DMG or PEG-DSG were less active; however addition of 0.5mol.% of a GalNAc-PEG lipid to these smaller systems improved activity to levels similar to that observed for the ~45nm diameter systems. In summary, this work shows that appropriately designed LNP–siRNA systems can result in effective hepatocyte gene silencing following s.c administration. [Display omitted]