Rational Design of Cationic Lipids for siRNA Delivery.

S. C. Semple et. al, Nat Biotechnol. 2010 Feb;28(2):172-6. Epub 2010 Jan 17.


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.


The study describes the discovery of a novel lipid, known as “KC2,” based on a medicinal chemistry effort to explore the structure-activity relationships in the lipid “DLinDMA,” which is used in certain first generation LNPs such as Tekmira’s stable nucleic acid-lipid particles (SNALP) formulations. A large number of novel lipids were synthesized to probe the relationship of lipid structure, such as the alkyl chain, linker, and head group moieties, with function as determined by screening for in vivo gene silencing activity. Additional measurements were performed to characterize the ability of the novel lipids to mediate certain physicochemical changes in lipid bilayers consistent with needed disruption of endosomal membranes. In order to explore its suitability for systemic delivery, the novel KC2 lipid was formulated with siRNA in an LNP formulation. Specifically, the in vivo data showed that: Gene silencing in rodents was achieved following a single injection at doses as low as 0.01mg/kg; potent and selective silencing of the clinically relevant gene transthyretin (TTR) was achieved at doses as low as 0.1 mg/kg in non-human primates; and, the formulation was found to be well tolerated in both rodents and non-human primates.