DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Koshiro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Keisuke; Seth, Punit P.; Low, Audrey; Yoshida, Masayuki; Bennett, C. Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori

doi:10.1038/ncomms8969

Cited by 110 publications

(118 citation statements)

References 62 publications

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“…Wolfrum et al . (30) have demonstrated that a fraction of cholesterol modified siRNAs binds to different lipoproteins in blood and LDL/HDL complexes associate with cells and tissues, which has also been repeated with other hydrophobically modified oligonucleotides (31,32). It is unclear if the mechanism of cellular intrnalization in vivo and in vitro is similar since in vitro , serum and LDL/HDL strongly inhibit functional efficacy of cholesterol modified siRNAs, with the best efficacy observed in serum-free media (Supplementary Figure S1).…”

Section: Introductionmentioning

confidence: 89%

Visualization of self-delivering hydrophobically modified siRNA cellular internalization

et al. 2016

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siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs®) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention.

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Section: Introductionmentioning

confidence: 89%

Visualization of self-delivering hydrophobically modified siRNA cellular internalization

et al. 2016

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“…In the case of single-stranded ASO, the length of the linker in this study might be appropriate for effectively cleaving the phosphodiester bond between the ASO and these ligands. The efficacy of the Toc-heteroduplex oligonucleotide was recently found to be higher than the increased rate of the amount of ASO in the whole liver (Nishina et al, 2015a). Additional studies of structure-activity relationships are required to explore lipophilic ligand conjugations.…”

Section: Discussionmentioning

confidence: 99%

Comparative Characterization of Hepatic Distribution and mRNA Reduction of Antisense Oligonucleotides Conjugated with Triantennary N-Acetyl Galactosamine and Lipophilic Ligands Targeting Apolipoprotein B

Watanabe

Nakajima

Kasuya

et al. 2016

Journal of Pharmacology and Experimental Therapeutics

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Triantennary N-acetyl galactosamine (GalNAc, GN3) and lipophilic ligands such as cholesterol and a-tocopherol conjugations dramatically improve the distribution and efficacy of secondgeneration antisense oligonucleotides (ASOs) in the whole liver. To characterize ligands for delivery to liver cells based on pharmacokinetics and efficacy, we used a locked nucleic acid gapmer of ASO targeting apolipoprotein B as a model compound and evaluated the amount of ASO and apolipoprotein B mRNA in the whole liver, hepatocytes, and nonparenchymal (NP) cells as well as plasma total cholesterol after administration of ASO conjugated with these ligands to mice. Compared with unconjugated ASO, GN3 conjugation increased the amount (7-fold) and efficacy (more than 10-fold) of ASO in hepatocytes only and showed higher efficacy than the increased rate of the amount of ASO. On the other hand, lipophilic ligand conjugations led to increased delivery (3-to 5-fold) and efficacy (5-fold) of ASO to both hepatocytes and NP cells. GN3 and lipophilic ligand conjugations increased the area under the curve of ASOs and the pharmacodynamic duration but did not change the half-life in hepatocytes and NP cells compared with unconjugated ASO. In the liver, the phosphodiester bond between ASO and these ligands was promptly cleaved to liberate unconjugated ASO. These ligand conjugations reduced plasma total cholesterol compared with unconjugated ASO, although these ASOs were well tolerated with no elevation in plasma transaminases. These findings could facilitate ligand selection tailored to liver cells expressed in disease-related genes and could contribute to the discovery and development of RNA interference-based therapy.

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“…The next generation of ASO and siRNA agents is developed so as to improve both the biodistribution/efficacy and safety profiles of therapeutic ASOs or siRNAs for liver-directed applications [37]. It is based on ligand conjugation antisense technologies [28].…”

Section: Galnac Ligand Conjugation: the Next Generation Of Antisense mentioning

confidence: 99%

Gene-based therapies in lipidology

Gaudet

Brisson

2015

Current Opinion in Lipidology

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DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

Cited by 110 publications

References 62 publications

Visualization of self-delivering hydrophobically modified siRNA cellular internalization

Visualization of self-delivering hydrophobically modified siRNA cellular internalization

Comparative Characterization of Hepatic Distribution and mRNA Reduction of Antisense Oligonucleotides Conjugated with Triantennary N-Acetyl Galactosamine and Lipophilic Ligands Targeting Apolipoprotein B

Gene-based therapies in lipidology

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