Wolfrum, C. et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nature Biotech. 25, 1149-1157

Institute of Molecular Systems Biology, Swiss Federal Institute of Technology, ETH Zürich, HPT E73.
Nature Biotechnology (Impact Factor: 41.51). 11/2007; 25(10):1149-57. DOI: 10.1038/nbt1339
Source: PubMed


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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    • "In the event that siRNAs are able to successfully penetrate the stratum corneum, siRNA-mediated gene regulation still requires keratinocyte uptake. " Self-delivery " siRNAs, like commercially available Accell™-siRNA, which carry chemical modifications that enhance cellular uptake, have been developed and effectively inhibit gene expression without the use of transfection reagents in vitro and in vivo [24] [25] [26] [27] [28]. "
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    ABSTRACT: Therapeutics based on short interfering RNA (siRNA), which act by inhibiting the expression of target transcripts, represent a novel class of potent and highly specific next-generation treatments for human skin diseases. Unfortunately, the intrinsic barrier properties of the skin combined with the large size and negative charge of siRNAs make epidermal delivery of these macromolecules quite challenging. To help evaluate the in vivo activity of these therapeutic and refine delivery strategies we generated an innovative reporter mouse model that predominantly expresses firefly luciferase (luc2p) in the paw epidermis - the region of murine epidermis that most closely models the tissue architecture of human skin. Combining this animal model with state-of-the-art live animal imaging techniques, we have developed a real-time in vivo analysis work-flow that has allowed us to compare and contrast the efficacies of a wide range nucleic acid-based gene silencing reagents in the skin of live animals. While inhibition was achieved with all of the reagents tested, only the commercially available “self-delivery” modified Accell-siRNAs (Dharmacon) produced potent and sustained in vivo gene silencing. Together, these findings highlight just how informative reliable reporter mouse models can be when assessing novel therapeutics in vivo. Using this work-flow, we developed a novel clinically-relevant topical formulation that facilitates non-invasive epidermal delivery of unmodified and “self-delivery” siRNAs. Remarkably, a sustained > 40% luc2p inhibition was observed after two 1-hour treatments with Accell-siRNAs in our topical formulation. Importantly, our ability to successfully deliver siRNA molecules topically brings these novel RNAi-based therapeutics one-step closer to clinical use.
    Journal of Controlled Release 10/2014; 196. DOI:10.1016/j.jconrel.2014.10.022 · 7.71 Impact Factor
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    • "Currently, the main treatment strategies are listed as follows: (1) necessary means should be taken to enhance expression of certain miRNA, which are known to be down-regulated during the disease state. miRNA simulative technology could be adopted, that is, the mimic double-stranded RNA can be synthesized based on the endogenous miRNA sequence, and then introduced into the body to maintain the miRNA level (Wolfrum et al. 2007); (2) knocking out or silencing the target miRNA which are known to be overexpressed in the pathological process . One way to do so is to use a target miRNA complementary antisense oligonucleotide chain, whose 3' end is coupled with a cholesterol molecule to improve its inhibition effect; alternatively , one could use the miRNA sponge technology to introduce many repeated copy and incomplete complementary oligonucleotide chain by the slow virus carrier into the body (van Rooij et al. 2008). "
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    ABSTRACT: Coronary artery disease (CAD) and pulmonary arterial hypertension (PAH) are two of the most dangerous vascular diseases. Their etiology and pathogenesis are not yet fully understood, thus it remains difficult to achive great advance in the diagnose, therapy and prognosis techniques. microRNAs (miRNAs), a class of highly conserved, small, noncoding RNAs, critically mediate the post-transcriptional gene modulation, which regulates an array of important physiopathological processes including those occurring in cardiac and pulmonary systems. Thereby manipulation of miRNA expression could potentially be applied therapeutically. In this review, we summarize the current knowledge on the roles of miRNAs in the development of vascular diseases, especially in CAD and PAH, providing a theoretical basis for potential uses of miRNA in diagnosis, prognosis, and therapy in these cardiovascular diseases.
    Biocontrol Science and Technology 09/2014; 69(9):643-7. DOI:10.1691/ph.2014.3982 · 0.94 Impact Factor
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    • "In cultured human hepatocytes, extracellularly applied cholesterol-conjugated lipoprotein-associated sRNA has been found to be able to induce RNAi [41]. This and other examples indicate that scavenger receptors are required for RNA uptake [34], [41], [42]. "
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    ABSTRACT: This review focuses on the mobility of small RNA (sRNA) molecules from the perspective of trans-kingdom gene silencing. Mobility of sRNA molecules within organisms is a well-known phenomenon, facilitating gene silencing between cells and tissues. sRNA signals are also transmitted between organisms of the same species and of different species. Remarkably, in recent years many examples of RNA-signal exchange have been described to occur between organisms of different kingdoms. These examples are predominantly found in interactions between hosts and their pathogens, parasites, and symbionts. However, they may only represent the tip of the iceberg, since the emerging picture suggests that organisms in biological niches commonly exchange RNA-silencing signals. In this case, we need to take this into account fully to understand how a given biological equilibrium is obtained. Despite many observations of trans-kingdom RNA signal transfer, several mechanistic aspects of these signals remain unknown. Such RNA signal transfer is already being exploited for practical purposes, though. Pathogen genes can be silenced by plant-produced sRNAs designed to affect these genes. This is also known as Host-Induced Genes Silencing (HIGS), and it has the potential to become an important disease-control method in the future.
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