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Modulation of toll-like receptor 4 expression by antisense oligonucleotides

An antisense oligonucleotide and oligonucleotide technology, applied in the field of Toll-like receptor 4, can solve the problems of non-specificity, inactivity, toxicity, etc.

Inactive Publication Date: 2011-12-07
IDERA PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Those skilled in the art will also recognize that antisense oligonucleotides may have off-target effects if the correct sequence, correct length, and proper nucleic acid and oligonucleotide chemistry are not used, And may lead to molecular instability, inactivity, non-specificity and toxicity
Due to this unpredictability of antisense oligonucleotides, to date only one antisense oligonucleotide has been licensed for use in humans, and no antisense oligonucleotides have been marketed for human use

Method used

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  • Modulation of toll-like receptor 4 expression by antisense oligonucleotides
  • Modulation of toll-like receptor 4 expression by antisense oligonucleotides
  • Modulation of toll-like receptor 4 expression by antisense oligonucleotides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Preparation of TLR4-specific antisense oligonucleotides

[0120] Using an automated DNA synthesizer (OligoPilot II, AKTA, (Amersham) and / or Expedite 8909 (Applied Biosystem)) followed figure 1 Chemical entities according to the invention were synthesized on a 1 μmol to 0.1 mM scale using the linear synthetic protocol outlined in .

[0121] 5'-DMT dA, dG, dC and T phosphoramidites were purchased from Proligo (Boulder, CO). 5'-DMT 7-deaza-dG and araG phosphoramidites were obtained from Chemgenes (Wilmington, MA). DiDMT-glycerol linker solid phase support was obtained from Chemgenes. 1-(2'-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine amidite was obtained from Glen Research (Sterling, VA), 2 '-O-methyl ribonucleoside imide was obtained from Promega (Obispo, CA). All compounds according to the invention are phosphorothioate backbone modified.

[0122] pass 31 P and 1 H NMR spectroscopy to characterize all nucleoside phosphoramidites. Modified nucleo...

Embodiment 2

[0124] Cell Culture Conditions and Reagents

[0125] HEK293 Cell Culture Assay for TLR4 Antisense Activity

[0126] at 5% CO 2 HEK293 cells stably expressing human TLR4 / CD14 / MD-2 (Invivogen, San Diego, CA) were dispensed in 48-well plates at 250 μL / well in DMEM supplemented with 10% heat-inactivated FBS in an incubator. At 80% confluency, cells were treated with 400 ng / mL secreted human embryonic alkaline phosphatase (SEAP) reporter plasmid (pNifty2-Seap) (Invivogen) in the presence of 4 μL / mL Lipofectamine (Invitrogen, Carlsbad, CA) in culture medium. Transiently transfect cultures. The SEAP reporter plasmid can be induced by NF-κB. Plasmid DNA and Lipofectamine were diluted separately in serum-free medium and incubated at room temperature for 5 minutes. After incubation, the diluted DNA and Lipofectamine were mixed, and the mixture was incubated for an additional 20 minutes at room temperature. Aliquots of 25 µL of the DNA / Lipofectamine mixture containing 100 ng of pl...

Embodiment 3

[0129] In Vivo Activity of TLR4 Antisense Oligonucleotides

[0130] Subcutaneously inject 5 mg / kg of mouse TLR4 antisense oligonucleotide or PBS according to an example of the present invention to 5-6 week-old female C57BL / 6 mice (N=3 / group), once a day for 3 days . Following administration of TLR4 antisense oligonucleotides, mice were injected subcutaneously with 0.25 mg / kg TLR4 agonist. Two hours after TLR agonist administration, blood was collected and TLR-mRNA, TLR4 protein and IL-12 concentrations were determined by ELISA.

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Abstract

Antisense oligonucleotide compounds, compositions and methods for downregulating TLR4 expression are provided. The composition comprises an antisense oligonucleotide targeted to a nucleic acid encoding TLR4. The composition may also comprise an antisense oligonucleotide targeted to a TLR4-encoding nucleic acid in combination with other therapeutic and / or prophylactic compounds and / or compositions. Methods of using these compounds and compositions to downregulate TLR4 expression and prevent or treat diseases that would benefit from modulating TLR4 expression are provided.

Description

[0001] This application claims priority to US Provisional Patent Application Serial No. 61 / 111,148, filed November 4, 2008, the disclosure of which is expressly incorporated herein by reference. Background of the invention [0002] field of invention [0003] The present invention relates to Toll-like receptor 4 (TLR4). In particular, the present invention relates to antisense oligonucleotides that specifically hybridize to nucleic acids encoding TLR4, thereby modulating TLR4 expression and activity, and their use in the treatment or prevention of diseases associated with TLR4 or that may benefit from modulation of TLR4 expression. use. [0004] Overview of related technologies [0005] Toll-like receptors (TLRs) are present on many cells of the immune system and have been shown to be involved in the innate immune response (Hornung, V. et al. (2002) J. Immunol. 168:4531-4537). TLR is a key means for mammals to recognize foreign molecules and launch an immune response to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/70C12N5/00
CPCC12N15/1138C07H21/00C12N2310/11A61P1/04A61P1/14A61P1/16A61P11/00A61P11/06A61P11/08A61P13/10A61P13/12A61P15/00A61P15/02A61P17/00A61P17/02A61P17/06A61P17/14A61P19/02A61P21/00A61P21/04A61P25/00A61P25/18A61P27/02A61P27/14A61P29/00A61P3/00A61P31/00A61P31/04A61P33/06A61P35/00A61P37/00A61P37/06A61P37/08A61P43/00A61P5/00A61P5/14A61P7/04A61P7/06A61P9/00A61P9/10A61P9/14A61P3/10C12N15/113A61K31/70
Inventor 马里卡朱纳.普塔埃坎巴.坎迪马拉拉克什米.巴加特王大庆郁东萨德西尔.阿格拉沃尔
Owner IDERA PHARMA INC
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