Scn9a antisense pain killer

一种药学、药用盐的技术，应用在非中枢性止痛剂、重组DNA技术、DNA / RNA片段等方向

Active Publication Date: 2019-12-03
OLIPASS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0043] Antisense Inhibition of SCN9A Pre-mRNA Splicing: So far, there are no reported cases of SCN9A ASO-induced alternative splicing of SCN9A pre-mRNA

Method used

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  • Scn9a antisense pain killer
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  • Scn9a antisense pain killer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0343] Example 1. Exon skipping induced by "ASO 7" in PC3 cells (A)

[0344] "ASO 7" designated in Table 1 is a 14-mer ASO that binds complementary to a 14-mer sequence at the 3' splice site spanning the junction of intron 3 and exon 4 within the human SCN9A pre-mRNA . 14-mer target sequence within the 3' splice site at [(5' → 3') uugu guuuag ┃ GUACACUU UU] are marked as "bold" and "underlined". "ASO 7" has a 6-mer complementary overlap with intron 3 and an 8-mer complementary overlap with exon 4.

[0345] Given that PC3 cells (Cat. No. CRL1435, ATCC) are known to express abundant human SCN9A mRNA [ Br. J. Pharmacol vol 156, 420-431 (2009)], "ASO 7" was evaluated for its ability to induce exon 4 skipping in PC3 cells as follows.

[0346] [Cell culture and ASO treatment] PC3 cells grown in a 60 mm dish containing 5 mL of F-12K medium were treated with "ASO 7" at 0 (negative control), 1, 10 or 100 zM.

[0347] [RNA extraction and cDNA synthesis by one-step PCR] After ...

Embodiment 2

[0351] Example 2. Exon skipping induced by "ASO 7" in PC3 cells (B)

[0352] The ability of "ASO 7" to induce "exon 4" skipping in PC3 cells was evaluated as in "Example 1", unless otherwise indicated. In this experiment, PC3 cells were treated with "ASO 7" at 0 (negative control), 1, 10, 100 and 1,000 aM for 24 hours. Nested PCR reactions were performed against a set of primers [exon 3 / 6_forward: (5' → 3') GGACCAAAAATGTCGAGCCT; and exon 9_reverse: (5' → 3') GCTAAGAAGGCCCAGCTGAA], the The primers described above were designed to amplify products with the sequence at the junction of exon 3 and exon 6.

[0353] The primer sequence for "exon 3 / 6_forward" more selectively recognizes the junction of "exon 3 and exon 6" than the "exon 3 and exon 6" found in the full-length SCN9A mRNA The junction of child 4". Primer sequences were designed to detect SCN9A splice variants missing exons 4–5 more sensitively than full-length SCN9A mRNA. Such exon-skipping primers will help detect m...

Embodiment 3

[0355] Example 3. qPCR of SCN9A mRNA in PC3 cells treated with "ASO 7" (A)

[0356] "ASO 7" was evaluated by SCN9A nested qPCR for its ability to induce changes in human SCN9A mRNA levels in PC3 cells as described below.

[0357] [ASO Treatment] PC3 cells were treated with "ASO 7" at 0 (negative control), 0.1, 1 or 10 aM (2 culture dishes for each ASO concentration).

[0358] [RNA extraction and cDNA synthesis by one-step PCR] After incubation with "ASO 7" for 24 hours, total RNA was extracted and subjected to one-step PCR amplification as described in "Example 1".

[0359] [Nested qPCR amplification] Dilute the cDNA solution 100-fold, and make 1 μL of each diluted PCR products were subjected to a set of exon-specific primers for [exon 3_forward: (5'→3') TGACCATGAATAACCCAC; and exon 4_reverse: (5'→3')GCAAGGATTTTTACAAGT] 20 μL real-time PCR reaction. qPCR reactions were performed with a TaqMan probe of [(5' → 3') 5,6-FAM-GGACCAAAA-Zen-ATGTCGAGTACAC-3IABkFQ], which targets ex...

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PUM

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Abstract

The current invention provides peptide nucleic acid derivatives targeting the 3' splice site of exon 4 in the human SCN9A pre-mRNA. The peptide nucleic acid derivatives potently induce SCN9A mRNA splice variant(s) lacking the SCN9A exon 4 in cells, and are useful to safely treat pains or conditions involving Nav1.7 activity.

Description

[0001] The present invention relates to peptide nucleic acid derivatives that complementarily target human SCN9A pre-mRNA for the treatment of v 1.7 Subtype-Mediated Pain and Disorders, and claims benefit of priority to US Provisional Application No. 62 / 449,738, filed January 24, 2017, which is hereby incorporated by reference in its entirety. [0002] Background of the invention [0003] Voltage-gated sodium channels (VGSCs) are transmembrane proteins composed of α and β subunits. VGSC functions as a channel for sodium ions across the cell membrane. Sodium channel activity is produced by the α-subunit. VGSC subtypes are defined according to subtypes of the α-subunit. To date, at least 10 subtypes of VGSC, the Na v 1.1, Na v 1.2,...,Na v 1.9 and Na x . [0004] Each VGSC subtype has a distinct α subunit and is destined to display characteristic physiological roles depending on the tissue in which it is expressed. For example, Na v The 1.2 isoform is expressed in centra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/113
CPCC07K14/00C07K14/003C12N15/1138C12N2310/11C12N2310/3181C12N2310/333C12N2310/334C12N2310/335C12N2310/336C12N2320/33A61P25/00A61P29/00A61P43/00A61K38/00C12N15/113C12N2310/3513
Inventor 郑信郑多览曹峰准张降愿田玹珠裵真英裵泰渊Y.全李俊演朴善花D.B.安
Owner OLIPASS CORP
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