Oligonucleotides for modulating scn9a expression

A technology of oligonucleotides and nucleotides, used in recombinant DNA technology, DNA/RNA fragments, medical preparations with non-active ingredients, etc.

Pending Publication Date: 2021-03-16
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These findings have stimulated the recognition of many Na v 1.7 Small molecule drug discovery projects for modu

Method used

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  • Oligonucleotides for modulating scn9a expression
  • Oligonucleotides for modulating scn9a expression
  • Oligonucleotides for modulating scn9a expression

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example

[0423] Oligonucleotide Synthesis

[0424] The compounds are listed in the compound table, which also shows the nucleobase sequence, complementary target sequence regions (start and end) on SEQ ID NO: 1, gap polymer design, Tm (dG) and after receiving the compound treatment The level of remaining mRNA in the cells (see Example 1 below).

[0425] Oligonucleotide synthesis is well known in the art. Below are scenarios that may apply. The oligonucleotides of the present invention can be produced by slightly different methods in terms of equipment, vectors and concentrations used.

[0426] Oligonucleotides were synthesized on an Oligomaker 48 using the phosphoramidite method on a uridine universal carrier at a 1 μmol scale. At the end of the synthesis, the oligonucleotides were cleaved from the solid support using ammonia at 60°C for 5-16 hours. Oligonucleotides were purified by reverse phase HPLC (RP-HPLC) or by solid phase extraction and characterized by UPLC and further conf...

example 1

[0443] Example 1: In vitro reduction of Nav1.7 in the SK-N-AS human cell line using oligonucleotides.

[0444] LNA-modified oligonucleotides targeting human Nav1.7 were tested for their ability to reduce Nav1.7 mRNA expression in human SK-N-AS neuroblastoma cells purchased from ATCC (CRL-2137). SK-N-AS cells were cultured in Dulbecco's modified Eagle's medium (ECACC-94092302) supplemented with 0.1 mM non-essential amino acids (NEAA) and a final concentration of 10% fetal bovine serum. Cells were incubated in an active evaporation incubator (Thermo C10) at 37°C, 5% CO 2 and 95% humidity for cultivation. Cells were inoculated into 95 μL of SK-N-AS cell culture medium at a density of 9300 cells / well (96-well plate), and left in an incubator for 24 hours. Oligonucleotides diluted in PBS (5.0 μL) to a final concentration of 5 μM were then added to the cell cultures from precast 96-well dilution plates. The cell culture plates were incubated in the incubator for 96 hours.

[04...

example 2

[0452] Example 2: Reduction of Nav1.8 alone or in combination with reduction of Nav1.7 in a modified SK-N-AS human cell line (CRISPR activated Nav1.8) using oligonucleotides in vitro.

[0453] Activation of Nav1.8 expression in SK-N-AS cells:

[0454] SK-N-AS cells were transduced with hCMV-Blast-dCas9-VPR encoded lentiviral particles (#VCAS11918, Dharmacon) at 0.5 MOI and selected with 2 μg / mL elastin for 10 days. SK-N-AS stably expressing dCas9-VPR protein was subsequently engineered to express Nav1.8-specific sgRNA. Briefly, the expression cassette driven by the U6 promoter was synthesized as a gBlock and then subcloned in the PiggyBac vector (#PB511B-1, System Biosciences). The identity of the vector was verified by Sanger sequencing. Stable integration of the U6-driven cassette expressing the Nav1.8-specific sgRNA was obtained using a transposase expression plasmid (#PB210PA-1, System Biosciences) following the manufacturer's instructions. Cells stably expressing the s...

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Abstract

The present invention relates to oligonucleotides (oligomers) that are complementary to voltage-gated sodium ion channel encoding nucleic acids, such as SCN9A, which encodes the voltage-gated sodium channel Nav1.7. The oligonucleotides of the invention are capable of inhibiting the expression of voltage-gated sodium ion channels, such as Nav1.7, and are useful in the prevention or the treatment ofpain.

Description

technical field [0001] The present invention relates to oligonucleotides (oligomers) that are complementary to a voltage-gated sodium ion channel encoding a nucleic acid, such as SCN9A, which encodes a voltage-gated sodium channel Na v 1.7. Oligonucleotides of the present invention are capable of inhibiting voltage-gated sodium ion channels such as Na v 1.7, and for the prevention or treatment of pain. Background technique [0002] Voltage-gated sodium channels (Na v s) Plays a crucial role in excitable tissues, its activation and switching on leading to the initial phase of the action potential. Na v The cycling of s in open, closed, and inactive states and their intimate relationship to the activity of other ion channels lead to precise control of intracellular ion concentrations. [0003] Na v 1.7 is a related voltage-activated ion channel expressed almost exclusively in parenchymal peripheral sensory nerves. Mice with conditional knockout of Nav1.7 in sensory neur...

Claims

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Application Information

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IPC IPC(8): C12N15/113
CPCC12N15/1138C12N2310/321C12N2310/322C12N2310/341C12N2310/14C12N2310/11C12N2320/31A61K31/7088A61K48/00A61P25/04A61K47/549C12N2310/315C12N2310/3231C12N2310/351
Inventor 吕克·彼泽森索伦·拉斯穆森詹路易吉·利辛奇克里斯托弗·桑德加
Owner F HOFFMANN LA ROCHE & CO AG
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