Effect of modular conjugation strategy for<i>N</i>-acetylgalactosamine-targeted antisense oligonucleotides

Yamamoto, Tsuyoshi; Sawamura, Motoki; Terada, Chisato; Kashiwada, Koki; Wada, Fumito; Yamayoshi, Asako; Obika, Satoshi; Harada‐Shiba, Mariko

doi:10.1080/15257770.2019.1677911

Cited by 6 publications

(8 citation statements)

References 34 publications

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“…This result was consistent with a few previous observations of GalNAc-conjugated RNase H-mediated ASOs [18,33]. According to our research reported elsewhere [21], we speculate that this difference in activity between the 5 -and 3 -congeners originated from their differences in sensitivity (tolerability) towards nucleases. The predominant nuclease activity in the serum is known to be 3 -exonuclease, while the predominant activity in cells is endonuclease [29,34].…”

Section: Configuration-activity Study Of Galnac-conjugated Tiny Lnasupporting

confidence: 94%

“…tL-5G3 is an miR122-targeting tiny LNA carrying three GalNAc hp units on its 5 terminus. The GalNAc hp units were loaded onto the tiny LNA through a conventional phosphoramidite solid-phase DNA synthesis method, in which a phosphodiester bond was adopted (rather than a phosphorothioate bond) as the linker chemistry of an inter-GalNAc hp and ASO-GalNAc hp based on our previous notion of gapmer-type ASOs [18,21]. Mice were subcutaneously injected with CtrASO at a single dose of 17.5 nmol/kg and with tinyLNA or tL-5G3 at a single dose of 5 mg/kg (1.84 and 1.20 µmol/kg, respectively) and sacrificed 7 days after injection.…”

Section: Effect Of Galnac Conjugation On In Vivo Activity Of Tiny Lnamentioning

confidence: 99%

“…Moreover, it is quite intriguing to see that, using a biolabile phosphodiester bond on the tiny LNA is more advantageous than a metabolically stable phosphorothioate linkage (e.g., tL-5G3 vs. tL-5G3-PS). This implies the involvement of endogenous endonucleases to digest the PO linkage of the conjugate [21]. If this is the case, even a tiny LNA, a very small ASO fully armed with nuclease resistant 2 ,4 -BNA/LNA, is meant to be a substrate of the responsible endonuclease (s).…”

Section: Configuration-activity Study Of Galnac-conjugated Tiny Lnamentioning

confidence: 99%

“…Among the congeners of conjugatable GalNAc ligands reported so far, the monovalent GalNAc phosphoramidite unit, including ours, has some advantages such as a shorter synthetic scheme, solid-phase synthesis applicability, and increased freedom in configuration design. In previous studies, we demonstrated the usefulness and flexibility of this type of GalNAc unit on gapmer-type ASOs and optimized the structure of the building block to maximize the effect of the ligand [18,21,22]. Specifically, we have realized that a biolabile phosphodiester linkage is more favorable than a biostable phosphorothioate bond as a ligand-connecting linker, indicating that after being taken up by the hepatocytes, the conjugate should be digested quickly to liberate its parent ASO, although some endonucleases are thought to be responsible for this cleavage.…”

Section: Introductionmentioning

confidence: 99%

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Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides

et al. 2021

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The development of clinically relevant anti-microRNA antisense oligonucleotides (anti-miRNA ASOs) remains a major challenge. One promising configuration of anti-miRNA ASOs called “tiny LNA (tiny Locked Nucleic Acid)” is an unusually small (~8-mer), highly chemically modified anti-miRNA ASO with high activity and specificity. Within this platform, we achieved a great enhancement of the in vivo activity of miRNA-122-targeting tiny LNA by developing a series of N-acetylgalactosamine (GalNAc)-conjugated tiny LNAs. Specifically, the median effective dose (ED50) of the most potent construct, tL-5G3, was estimated to be ~12 nmol/kg, which is ~300–500 times more potent than the original unconjugated tiny LNA. Through in vivo/ex vivo imaging studies, we have confirmed that the major advantage of GalNAc over tiny LNAs can be ascribed to the improvement of their originally poor pharmacokinetics. We also showed that the GalNAc ligand should be introduced into its 5′ terminus rather than its 3′ end via a biolabile phosphodiester bond. This result suggests that tiny LNA can unexpectedly be recognized by endogenous nucleases and is required to be digested to liberate the parent tiny LNA at an appropriate time in the body. We believe that our strategy will pave the way for the clinical application of miRNA-targeting small ASO therapy.

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Section: Configuration-activity Study Of Galnac-conjugated Tiny Lnasupporting

confidence: 94%

Section: Effect Of Galnac Conjugation On In Vivo Activity Of Tiny Lnamentioning

confidence: 99%

Section: Configuration-activity Study Of Galnac-conjugated Tiny Lnamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides

et al. 2021

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“…One reliable approach to avoiding nephrotoxicity is to minimize exposure of the drug to the kidney, given that short oligonucleotides such as high-affinity gapmer ASOs have been shown to preferentially accumulate in the kidney, especially in the proximal tubules. 3 , 4 , 5 We and other groups previously have reported strategies for efficient hepatic delivery of ASOs; 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 specifically, the conjugation of a N -acetylgalactosamine (GalNAc) ligand has proven to remarkably improve the targeting of ASOs to the liver by reinforcing cellular uptake via receptor-mediated endocytosis while reducing renal accumulation of ASOs. The trimeric form of GalNAc-conjugated ligands is known to serve as an excellent ligand for the asialoglycoprotein receptor that is expressed selectively on hepatocytes.…”

Section: Introductionmentioning

confidence: 99%

Drug discovery and development scheme for liver-targeting bridged nucleic acid antisense oligonucleotides

Wada

Yamamoto

Kobayashi

et al. 2021

Molecular Therapy - Nucleic Acids

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Antisense oligonucleotides (ASOs) containing bridged nucleic acids (BNAs) have been proven to be very powerful. However, ensuring a reliable discovery and translational development scheme for this class of ASOs with wider therapeutic windows remains a fundamental challenge. We here demonstrate the robustness of our scheme in the context of the selection of ASOs having two different BNA chemistries (2, 0 4 0 -BNA/ locked nucleic acid [LNA] and amido-bridged nucleic acid [AmNA]) targeting human proprotein convertase subtilisin/ kexin type 9 (PCSK9). The scheme features a two-step process, including (1) a unique and sensitive in vitro screening approach, called Ca 2+ enrichment of medium (CEM) transfection, and (2) a ligand-targeted drug delivery approach to better reach target tissues, averting unintended accumulation of ASOs. Using CEM screening, we identified a candidate ASO that shows >70% cholesterol-lowering action in monkeys. An N-acetylgalactosamine (GalNAc) ligand then was appended to the candidate ASO to further broaden the therapeutic margin by altering the molecule's pharmacokinetics. The Gal-NAc conjugate, HsPCSK9-1811-LNA, was found to be at least ten times more potent in non-human primates (compared with the unconjugated counterpart), with reduced nephrotoxicity in rats. Overall, we successfully showed that our drug development scheme is better suited for selecting clinically relevant BNA-based ASOs, especially for the treatment of liver-associated diseases.

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Nano‐LYTACs for Degradation of Membrane Proteins and Inhibition of CD24/Siglec‐10 Signaling Pathway

Wang

Liu

et al. 2023

Advanced Science

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Lysosome‐targeting chimeras (LYTACs) are an emerging therapeutic modality that effectively degrade cancer cell membranes and extracellular target proteins. In this study, a nanosphere‐based LYTAC degradation system is developed. The amphiphilic peptide‐modified N‐acetylgalactosamine (GalNAc) can self‐assemble into nanospheres with a strong affinity for asialoglycoprotein receptor targets. They can degrade different membranes and extracellular proteins by linking with the relevant antibodies. CD24, a heavily glycosylated glycosylphosphatidylinositol‐anchored surface protein, interacts with Siglec‐10 to modulate the tumor immune response. The novel Nanosphere‐AntiCD24, synthesized by linking nanospheres with CD24 antibody, accurately regulates the degradation of CD24 protein and partially restores the phagocytic function of macrophages toward tumor cells by blocking the CD24/Siglec‐10 signaling pathway. When Nanosphere‐AntiCD24 is combined with glucose oxidase, an enzyme promoting the oxidative decomposition of glucose, the combination not only effectively restores the function of macrophages in vitro but also suppresses tumor growth in xenograft mouse models without detectable toxicity to normal tissues. The results indicate that GalNAc‐modified nanospheres, as a part of LYTACs, can be successfully internalized and are an effective drug‐loading platform and a modular degradation strategy for the lysosomal degradation of cell membrane and extracellular proteins, which can be broadly applied in the fields of biochemistry and tumor therapeutics.

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Effect of modular conjugation strategy forN-acetylgalactosamine-targeted antisense oligonucleotides

Cited by 6 publications

References 34 publications

Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides

Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides

Drug discovery and development scheme for liver-targeting bridged nucleic acid antisense oligonucleotides

Nano‐LYTACs for Degradation of Membrane Proteins and Inhibition of CD24/Siglec‐10 Signaling Pathway

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