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Chemically modified oligonucleotides for RNA editing

An antisense oligonucleotide, nucleotide technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of not showing the editing effect of target nucleotides

Pending Publication Date: 2021-12-03
プロキューアールセラピューティクスツーベスローテンフェンノートシャップ
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, specific editing effects on target nucleotides have not been shown without the use of recombinant ADAR enzymes with covalent bonds to AON

Method used

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  • Chemically modified oligonucleotides for RNA editing
  • Chemically modified oligonucleotides for RNA editing
  • Chemically modified oligonucleotides for RNA editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Antisense Oligonucleotides Comprising Methylphosphonate (MP) Linkage Modifications Using In Vitro Biochemical Disaggregation Assays (AON) is more stable than AON lacking such MP modifications

[0064]It is well known that the presence of a 2'-OMe modification of the nucleotide sugar moiety opposite to a target adenosine in a target RNA molecule reduces the inosine transfer of that particular target adenosine compared to an AON that does not carry such a 2'-OMe modification. of deamination. Unfortunately, the absence of such sugar modification at this particular position makes AON unstable. The inventors of the present invention questioned whether this problem could be solved by modifying the internucleotide linkage between two DNA nucleotides. To this end, two methylphosphonic acids were introduced between the two DNA nucleosides in the AON (one of which is opposite the target adenosine present in the target mouse IDUA RNA molecule) and their respective ...

Embodiment 2

[0067] Example 2: RNA Editing by AONs Carrying Stabilizing MP Bond Modifications

[0068] Next, the inventors questioned whether the MP modification, despite providing a more stable AON, would still prevent RNA editing, similar to the low RNA editing efficiency observed in AONs carrying PS modifications between DNA nucleotides of the AON (Data not shown). Therefore, it was investigated whether the MP modification - which is now known to increase the stability of AON - allows RNA editing, unlike the 2'-OMe modification (which, although providing stability), renders AON ineffective in RNA editing .

[0069] To this end, ADAR102-1 and ADAR102-13 were compared in RNA editing assays as follows. First, two AONs were annealed to the mouse IDUA target RNA. Annealing was performed at a 1:3 ratio of target RNA to AON in buffer (5 mM Tris-Cl pH 7.4, 0.5 mM EDTA, and 10 mM NaCl) (final concentrations in editing reactions were 6 nM AON and 2 nM target). The samples were heated at 95°...

Embodiment 3

[0080] Example 3: RNA Editing by AONs Carrying Stable MP Bond Modifications

[0081] Next, the inventors questioned where in the AON MP modifications could be made and still allow RNA editing. AONs with MP modifications at bond positions 0 to -6 relative to orphan nucleosides were synthesized and tested. Editing assays were performed as in Example 2, but with the following changes: final concentrations of 1 nM target RNA, 24 nM AON, and 3 nM ADAR2, and using 3 mM MgSO in editing reaction buffer 4 Instead of 3mM MgCl 2 . The reaction was carried out as described in Example 2, and by adding 95 μl of boiled 3 mM EDTA solution to 5 μl from Extract an aliquot from the reaction to stop the reaction.

[0082] A 6 μl aliquot of the stop reaction mixture was then used as template for cDNA synthesis using the Maxima Reverse Transcriptase Kit (Thermo Fisher) and a target RNA-specific primer (5'-GGAAACGTAGGTTGGGGTGTG-3'SEQ ID NO:8). RNA was initially denatured at 95 °C for 5 min in...

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Abstract

The invention relates to single-stranded RNA editing antisense oligonucleotides (AONs) for binding to a target RNA molecule for deaminating a target nucleotide, preferably an adenosine, present in the target RNA molecule and recruiting, in a cell, preferably a human cell, an enzyme with nucleotide deamination activity, preferably an ADAR enzyme, to deaminate the target nucleotide in the target RNA molecule. The AONs carry at least one methylphosphonate-modified internucleosidic linkage on a position that would render the AON more stable in comparison to an AON not carrying that methylphosphonate modification at that position.

Description

technical field [0001] The present invention relates to the field of medicine. Specifically, it relates to the field of RNA editing, in which RNA molecules in cells are targeted by antisense oligonucleotides (AONs) to specifically alter target nucleotides present in the target RNA molecule. The present invention aims to modify specific nucleotides in target RNA molecules, such as mutated nucleotides that may cause disease, by binding to an enzyme with deaminase activity. More specifically, the present invention relates to AONs that have been chemically modified at preferred locations to increase their in vivo and in vitro stability and thereby increase their RNA editing capacity. Background technique [0002] RNA editing is a natural process by which eukaryotic cells alter the sequence of their RNA molecules, often in a site-specific and precise manner, thereby increasing the genome-encoded RNA repertoire by orders of magnitude. RNA editing enzymes have been described in e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/11A61K31/7125
CPCC12N15/113C12N2310/11C12N2310/3125C12N2310/344C12N2310/315C12N2310/321C12N2310/322C12N2310/346
Inventor J·J·图鲁宁B·克莱恩L·范辛特菲特A·阿尔托C·P·克梅尔T·霍赫博姆L·A·范维森
Owner プロキューアールセラピューティクスツーベスローテンフェンノートシャップ
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