Mechanisms and optimization of in vivo delivery of lipophilic siRNAs

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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. High-density 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.

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Figure 1: Lipophilic siRNA conjugates have different in vivo activities.
Figure 2: Lipid-conjugated siRNAs (siRNA-apoM) associate with lipoproteins and albumin in blood.
Figure 3: Cholesterol-siRNAs associated with lipoproteins are taken up by the liver more efficiently than in free or albumin-bound cholesterol-siRNAs.
Figure 4: Cholesterol-siRNA is taken up by different tissues depending on its association with specific lipoproteins.
Figure 5: Cholesterol-siRNA associated with lipoproteins is taken up independently of lipoprotein particle endocytosis.
Figure 6: LDL receptor (Ldlr) mediates LDL-associated cholesterol-siRNA delivery in vivo.
Figure 7: Sid1 is required for uptake of lipid-conjugated siRNAs in vitro.


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We would like to thank J. Maraganore, H.-P. Vornlocher, M.A. Maier and P. Sharp for helpful discussion and suggestions. These studies were supported by National Institutes of Health grant 1 P01 GM073047-01 (M.S.).

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Correspondence to Markus Stoffel.

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M.S. is a member of the scientific advisory board of Alnylam Pharmaceuticals, Inc.

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