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Rna interference mediated inhibition of btb and cnc homology 1, basic leucine zipper transcription factor 1 (bach 1) gene expression using short interfering nucleic acid (sina) sequence listing

A technology for interfering nucleic acids, nucleotides, applied in the field of RNA interference-mediated inhibition of BTB and CNC homolog 1, basic leucine zipper transcription factor 1 (Bach1) gene expression using short interfering nucleic acid (siNA) , able to address issues such as Nrf2-dependent antioxidant loss

Inactive Publication Date: 2012-05-02
SCHERING AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Taken together, the human lung tissue expression data support the hypothesis that regulators of Nrf2 activity in the COPD lung include an altered balance between positive (DJ-1) and negative (Keap1 and Bach1) factors, leading to Nrf2-dependent antioxidant lost

Method used

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  • Rna interference mediated inhibition of btb and cnc homology 1, basic leucine zipper transcription factor 1 (bach 1) gene expression using short interfering nucleic acid (sina) sequence listing
  • Rna interference mediated inhibition of btb and cnc homology 1, basic leucine zipper transcription factor 1 (bach 1) gene expression using short interfering nucleic acid (sina) sequence listing
  • Rna interference mediated inhibition of btb and cnc homology 1, basic leucine zipper transcription factor 1 (bach 1) gene expression using short interfering nucleic acid (sina) sequence listing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0577] Example 1: Design, synthesis and identification of siNAs active against Bach1

[0578] Bach1 siNA synthesis

[0579] A series of 42 siNA molecules were designed, synthesized and evaluated for efficacy against Bach1. The main criteria for the design of Bach1 for human siNAs were (i) homology between the 2 species (human and mouse), and (ii) high efficacy as determined by a proprietary algorithm Score. Mouse sequences were also investigated for use in animal models. The effect of siNA on Bach1 RNA levels and its effect on heme oxygenase-1 (HMOX-1) mRNA levels were also examined. The siNA sequences designed, synthesized and evaluated for efficacy against Bach1 are described in Table 1a (target sequences) and Table 1b (modified sequences).

[0580] Table 1a: Bach1 Target Sequences, Target Sites Indicated. The Homology column indicates the complete homology of the siNA to the human transcript (h), the mouse transcript only (m), and the human and mouse transcript (hm),...

Embodiment 2

[0651] Example 2: In vitro evaluation of siNA in human bronchial epithelial cells

[0652] Duplexes corresponding to 29961-DC, 29964-DC, 29984-DC, 29988-DC, 29957-DC, 29947-DC and 29956-DC were tested for maximal Bach1 mRNA knockdown and potency in human bronchial epithelial cells as follows siNA by ID number.

[0653] Cell culture preparation:

[0654] Human bronchial epithelial cells (NHBE cells) from Lonza (Cat. No. CC-2540) were grown at 37 °C in the presence of 5% CO2 and grown in BEBM minimal medium (Lonza, Cat. No. CC-3171) on Biocoat collagen 1 coated culture flask (Becton Dickinson).

[0655] Transfection:

[0656] NHBE cells were plated in collagen 1-coated plates and cultured in appropriate media. Cells were incubated at 37° C. for 24 hours in the presence of 5% CO 2 after plating. siNA was diluted to 1uM in OptiMEM 1 and transfection reagent was diluted to 25ug / ml. For siNA formulations, equal volumes of diluted siNA and delivery lipids were combined and ...

Embodiment 3

[0673] Example 3: Testing of siNAs for TLR3, TLR7 and TLR8-mediated immune stimulation

[0674] NHBE cells were processed as described above in Example 2 and used to measure endosomal TLR3-mediated immune stimulation, including poly I:C as a positive control for OAS1 mRNA upregulation. For the measurement of membrane-bound TLR3-mediated immune stimulation, NHBE cells were cultured at 1200 cells / 96 wells and treated with (100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM, 0.03nM, 0.01nM) siRNA administered in PBS.

[0675] Cell surface TLR3 mediated immunostimulatory responses were measured by noting the % increase in OAS1 mRNA levels when NHBE cells were treated with siNA in the absence of delivery vehicle. (% increase in OAS1 mRNA levels relative to PBS diluent).

[0676] To determine the % increase in OAS1 (immunostimulatory biomarker) mRNA levels mediated by immunostimulatory TLR3, seven siNAs 29961-DC, 29964-DC, 29947-DC, 29988-DC, 29984-DC, 29956-DC and 29957-DC were use...

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PUM

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Abstract

The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of Bach1 gene expression and / or activity, and / or modulate a Bach1 gene expression pathway. Specifically, the invention relates to double-stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules that are capable of mediating or that mediate RNA interference (RNAi) against Bach1 gene expression.

Description

[0001] This application claims the benefit of US Provisional Application No. 61 / 161,699, filed March 19, 2009. The applications listed above, including the drawings, are hereby incorporated by reference in their entirety. [0002] sequence listing [0003] Pursuant to 37 CFR §1.52(e)(5), the sequence listing submitted via EFS is incorporated herein by reference. Sequence Listing text files submitted via EFS include file "SequenceListing75WPCT" created on February 25, 2010, which is 109,874 bytes in size. Background of the invention [0004] Bach1 is a transcriptional repressor of heme oxygenase-1 (HO-1) and other Nrf2-dependent phase II genes. The transcription factor Bach1 belongs to the cap'n'collar (CNC) and basic region leucine zipper superfamily of transcriptional regulators (termed CNC-bZip). Furthermore, Bach1 represents in its N-terminal region the so-called broad complex, tram-track, bric-a-brac (BTB) domain (also known as poxvirus and zinc finger (POZ) domain). ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113
CPCC12N2310/317C12N15/113C12N2310/14C12N2310/321C12N2310/322A61P11/00A61P11/02A61P11/06A61P11/08A61P11/14A61P17/00A61P43/00C12N2310/3521C12N2310/353C07H21/00A61K31/7088A61K48/00
Inventor W.斯特拉普斯V.皮克林J.沙
Owner SCHERING AG
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