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Tlr inhibitory oligonucleotides and their use

A technology of oligonucleotides and nucleotides, applied in the field of TLR inhibitory oligonucleotides and their applications, can solve problems such as inability to perform nucleotide sequences

Pending Publication Date: 2018-08-21
SBI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with regard to the oligonucleotides described in the present invention as CxTy(CCT)nCm, the above strategy for altering the nucleotide sequence cannot be carried out since the potency of the oligonucleotide is entirely dependent on the unique nucleotide sequence

Method used

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  • Tlr inhibitory oligonucleotides and their use
  • Tlr inhibitory oligonucleotides and their use
  • Tlr inhibitory oligonucleotides and their use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0212]Stability of partially phosphorothioated oligonucleotides compared to oligonucleotides with normal phosphodiester bonds or phosphorothioated oligonucleotides

[0213]

[0214] Oligonucleotides (10 ng / uL) were incubated with 20% (v / v) human serum (Cosmo Bio, Cat. 12181201) in distilled water (DW) at 37°C. The oligonucleotide-containing solution was collected after 1 day incubation and checked for the concentration of remaining undegraded oligonucleotide. The concentration of oligonucleotides in solution was determined by hybridization assays using complementary oligonucleotides of (CCT)12 or (TCC)12 as probes that detect only Oligonucleotides for full-length oligonucleotides.

[0215]

[0216] figure 1 The proportion of intact oligonucleotides remaining after 1 day incubation in serum-containing medium is shown; this proportion is expressed in % compared to the concentration of oligonucleotides at the beginning of the study. Such as figure 1 As shown in a, CCT12PO...

Embodiment 2

[0218] In Vivo Toxicity Examination of Oligonucleotides

[0219]

[0220] Oligonucleotides were dissolved in saline at 2 mg / ml and injected subcutaneously into mice (BALB / c) at 100 uL / 10 g body weight once daily for 7 consecutive days to give 20 mg / kg (mpk). Check the body weight of the mice to see if the oligonucleotide shows any effect on body weight. exist Figures 2a to 2c In the graph of , the X-axis represents the number of days from the first dose (ie, day 0 is the first day of dosing; day 6 is the last day of dosing). The mean body weight (3 animals per group) is shown on the Y-axis with the scale set to 100% from the beginning of the study.

[0221] The day after the last dose (day 7), blood collected from the mice was biochemically analyzed for effects on markers indicative of liver damage such as alanine aminotransferase (ALT). Figure 2d with 2e The blood ALT concentration is shown on the Y axis. ALT is often measured clinically as part of the diagnostic eva...

Embodiment 3

[0229] Inhibits NF-kB transcriptional activity

[0230]

[0231] The CAL-1 / NF-kB-GFP cell line was established for monitoring the activity of the NF-kB transcription factor in a cell-based assay (WO2014 / 082254). The vector encoding the GFP reporter gene driven by the NF-kB consensus transcriptional response element was transfected into the human plasmacytoid DC cell line by electroporation; CAL-1 (human pDC cell line, Maeda et al., Int J Hematol., 2005 , 81, 148-54, JP5011520). Transfected cells were further selected with antibiotics. Then, CAL-1 / NF-kB-GFP cells (1×10 5 / well) were seeded in 96-well flat-bottomed plates (Costar) and cultured with TLR agonists with or without oligonucleotides of the present invention. Cells at 37 °C in 5% CO 2 Incubate for 4 hours (TLR7 / 8) or 6 hours (TLR9) in a humidified incubator. The ratio of GFP-positive cells detected by flow cytometry (FACSCalibur, BD Bioscience Co., Ltd) shows NF-kB as data from cells without inhibitors (Medium) ...

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Abstract

The inhibitory oligonucleotides with partial phosphorothioation with reduced toxicity strongly block NF-kB activation induced by TLR9 agonists and TLR7 / 8 agonists. The production of proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFa), is inhibited by the inhibitory-oligonucleotides. Interferon (IFN) production from human PBMC induced by TLR9 agonist is prevented by the inhibitory-oligonucleotides. These oligonucleotides can be used as a remedy for the treatment of immune-mediated disorders such as rheumatoid arthritis, systemic lupus erythematosus (SLE), sepsis, multiple organ dysfunction syndromes and inflammatory cytokine-mediated inflammatory disease.

Description

technical field [0001] The present invention relates to oligonucleotides and remedies for the treatment of immune-mediated disorders using oligonucleotides. Immune-mediated disorders include autoimmune diseases, graft rejection, hypersensitivity, diseases associated with self-antigens, microbial overstimulation of the host immune system, Toll-like receptor (TLR)-mediated diseases, NF-kB-mediated diseases, interferon-mediated diseases and inflammatory cytokine-mediated inflammatory diseases. Background technique [0002] The immune system protects the body from bacterial, parasite, fungal, viral infections and from growing tumor cells. Immunity can be divided into innate immunity and adaptive immunity. The innate immune response typically occurs immediately after infection, providing an early barrier against infectious disease, while the adaptive immune response subsequently generates antigen-specific long-term protective immunity. [0003] However, unwanted immune respons...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/117A61K31/7088A61P37/02
CPCC12N15/117A61K45/06C12N2310/17C12N2310/315C12N2320/51C12N2320/53A61K31/7125A61P3/04A61K2300/00
Inventor 江指永二石田晃司细泽拓美奥山惠小泷步
Owner SBI BIOTECH CO LTD
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