Mechanisms and optimization of in vivo delivery of lipophilic siRNAs

Cholesterol-conjugated siRNAs can silence gene expression in vivo. Here we synthesize a variety of lipophilic siRNAs and use them to elucidate the requirements for siRNA delivery in vivo. We show that conjugation to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo. Efficient and selective uptake of these siRNA conjugates depends on interactions with lipoprotein particles, lipoprotein receptors and transmembrane proteins. Highdensity lipoprotein (HDL) directs siRNA delivery into liver, gut, kidney and steroidogenic organs, whereas low-density lipoprotein (LDL) targets siRNA primarily to the liver. LDL-receptor expression is essential for siRNA delivery by LDL particles, and SR-BI receptor expression is required for uptake of HDL-bound siRNAs. Cellular uptake also requires the mammalian homolog of the Caenorhabditis elegans transmembrane protein Sid1. Our results demonstrate that conjugation to lipophilic molecules enables effective siRNA uptake through a common mechanism that can be exploited to optimize therapeutic siRNA delivery. RNA interference (RNAi) is an evolutionarily conserved biological process for specific silencing of gene expression. Synthetic smallinterfering (si)RNAs have emerged as an important tool for posttranscriptional gene silencing in mammalian cells and live animals owing to their unique properties, such as potency, specificity and lack of an interferon response 1 . Delivery remains the largest obstacle for in vivo applications of siRNAs, including their use as therapeutics following systemic administration. Delivery of siRNAs across plasma membranes in vivo has been achieved using vector-based delivery systems 2 , high-pressure intravenous injections of siRNA 3 and chemically modified siRNAs, including cholesterol-conjugated 4 , lipidencapsulated 5 and antibody-linked siRNAs 6 . Although these delivery approaches have been shown to be effective in mice, no functional studies elucidating the mechanism of siRNA uptake have yet been reported. Covalent conjugation of cholesterol to siRNAs facilitates cellular