We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.

Special (lipid) delivery: The role of the ionizable lipid pKa in the in vivo delivery of siRNA by lipid nanoparticles has been studied with a large number of head group modifications to the lipids. A tight correlation between the lipid pKa value and silencing of the mouse FVII gene (FVII ED50) was found, with an optimal pKa range of 6.2–6.5 (see graph). The most potent cationic lipid from this study has ED50 levels around 0.005 mg kg−1 in mice and less than 0.03 mg kg−1 in non‐human primates.

Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

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Der Einfluss des pKS‐Werts von ionisierbaren Lipiden auf den siRNA‐Transport durch Lipidnanopartikel in vivo wurde anhand zahlreicher Modifikationen der Lipid‐Kopfgruppen studiert. Dabei wurde ein Zusammenhang zwischen pKS‐Wert und Stummschaltung des Maus‐FVII‐Gens (FVII ED50) gefunden: pKS‐Werte von 6.2–6.5 erwiesen sich als optimal (siehe Diagramm). Für das wirksamste kationische Lipid betrug ED50 etwa 0.005 mg kg−1 in Mäusen und <0.03 mg kg−1 in Primaten (außer Menschen).