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Double-stranded antisense nucleic acid with exon-skipping effect

A double-stranded nucleic acid and nucleotide technology, applied to medical preparations containing active ingredients, organic active ingredients, drug combinations, etc.

Inactive Publication Date: 2016-02-10
NAT UNIV CORP TOKYO MEDICAL & DENTAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Delivery of splice-switching oligonucleotides across the nuclear membrane remains a challenge (Non-Patent Document 6)

Method used

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  • Double-stranded antisense nucleic acid with exon-skipping effect
  • Double-stranded antisense nucleic acid with exon-skipping effect
  • Double-stranded antisense nucleic acid with exon-skipping effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0175] The accessibility of double-stranded antisense nucleic acid complexes to the nucleus of Huh-7 cells was tested and compared to the accessibility of single-stranded antisense oligonucleotides to the nucleus of Huh-7 cells. In experiments, to the extent that the antisense oligonucleotide (ASO) is able to reach the nucleus, the ASO should be able to inhibit the expression of the target gene ApoB, and thus, the amount of ApoB mRNA will show a corresponding decrease.

[0176] A single-stranded LNA / DNAgapmer antisense oligonucleotide (SEQ ID NO: 1 ) and a complementary RNA-based strand (SEQ ID NO: 2) were prepared with the following sequences and compositions:

[0177] 16merASO (targeting intron human apoB mRNA):

[0178] SEQ ID NO: 15'-C * T * C *c*c*a*c*c*a*c*a*t*a* G * C * A -3'

[0179] 16mercRNA (targeting intron human apoBmRNA):

[0180] SEQ ID NO: 25'-g*c*u*AUGUGGUGGG*a*u*g-3'

[0181] Lowercase italic letters represent DNA, underlined capital letter types repr...

Embodiment 2

[0188] The double-stranded antisense nucleic acid complex according to one embodiment of the present invention was tested for its ability to cause exon skipping during the processing of pre-mRNA of part of the dystrophin gene and compared it with a single-stranded antisense oligonucleotide This ability of glycosides was compared.

[0189] For this experiment, a stable expression plasmid for the human dystrophin gene fragment was constructed and a stable cell line containing the construct was established. The human dystrophin gene fragment has the full-length sequence of exon 57 to exon 59 except for intron 57, which is truncated for convenience due to its length.

[0190] Expression of dystrophin fragments in stable cell lines would generally be expected to produce mRNA comprising exons 57, 58 and 59. However, in the presence of a splice-switch oligonucleotide with the ability to cause exon 58 skipping during pre-mRNA processing, the expressed mRNA would be expected to contai...

Embodiment 3

[0240] Materials and methods

[0241] Synthesis of splicing-switching oligonucleotides (SSOs)

[0242]All SSOs used in this study are shown in Table 1. The sequence of the SSO was optimized by systematic screening as shown in the literature Shimo.T. et al. Two types of modifications, 2',4'-BNA and 2'-OMe, are incorporated into the SSO sequence, where the phosphodiester linkage is completely replaced by a phosphorothioate linkage. All SSOs were designed to have a sequence complementary to the human dystrophin gene, and were synthesized and purified by GeneDesign Inc. (Osaka, Japan).

[0243] Synthesis of modified complementary RNA

[0244] All modified complementary RNAs (modified cRNAs) used in this study are shown in Table 2. A 2'-OMe modification is incorporated into the modified cRNA sequence in which the phosphodiester linkage is partially replaced by a phosphorothioate linkage. Modified cRNA was synthesized and purified by GeneDesign Inc. (Osaka, Japan).

[0245] Pr...

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Abstract

Disclosed are double-stranded antisense nucleic acid complexes that can efficiently alter the processing of RNA in a cell via an antisense effect, and methods for using the same. One method comprises contacting with the cell a double-stranded nucleic acid complex comprising: a first nucleic acid strand annealed to a second nucleic acid strand, wherein: the first nucleic acid strand comprises (i) nucleotides independently selected from natural DNA nucleotides, modified DNA nucleotides, and nucleotide analogs, (ii) no regions that have 4 or more consecutive natural DNA nucleotides, (iii) the total number of natural DNA nucleotides, modified DNA nucleotides, and nucleotide analogs in the first nucleic acid strand is from 8 to 100, and (iv) the first nucleic acid strand is capable of hybridizing to RNA inside of the cell; and the second nucleic acid strand comprises nucleotides independently selected from natural RNA nucleotides, modified RNA nucleotides, and nucleotide analogs.

Description

technical field [0001] The present application relates to a double-stranded nucleic acid having the activity of altering the function of a coding RNA or a non-coding RNA, and more particularly, to the activity of the band by means of the antisense effect and by exon-skipping The effect of inhibiting the expression of the target gene, such as the effect of the coming effect. The double-stranded nucleic acid comprises one strand that acts as an antisense oligonucleotide that is complementary to RNA in the cell. Such RNA may be part of a coding or non-coding region, or may be part of an exon or intron. Background technique [0002] In recent years, oligonucleotides have been the subject of interest in the ongoing development of pharmaceutical products known as nucleic acid drugs, and in particular, the use of antisense The development of nucleic acid drugs of the method is actively underway. The antisense method is a method of selectively changing the mRNA (sense strand) enc...

Claims

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Application Information

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IPC IPC(8): A61K31/713A61P21/00
CPCA61K31/713C12N2310/11C12N2310/315C12N2310/3231C12N2310/341C12N2310/533C12N2320/33C12N15/113A61P21/00C12N2310/321C12N2310/3521C12N2310/351C12N2310/3513
Inventor 横田隆德仁科一隆吉冈耕太郎小比贺聪下刚典
Owner NAT UNIV CORP TOKYO MEDICAL & DENTAL UNIV
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