Chimeric single-stranded antisense polynucleotides and double-stranded antisense agent

a technology of antisense and polynucleotide, which is applied in the field of chimeric single-stranded polynucleotide and double-stranded antisense agents, can solve the problems of low potency, safety, and/or efficacy of antisense oligonucleotides using even these high-performance modified nucleic acids, and achieves high specificity, high efficiency, and high specificity.

Inactive Publication Date: 2016-04-21
NAT UNIV CORP TOKYO MEDICAL & DENTAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0032]According to certain embodiments, a chimeric antisense polynucleotide can be delivered to a target site with high specificity and high efficiency by associating a delivery moiety with the complex. According to cert

Problems solved by technology

Nonetheless, the designs of antisense oligonucleotides using even these high-performance modified nucleic acids still lack suitable efficiency, potency, and/or safety for use

Method used

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  • Chimeric single-stranded antisense polynucleotides and double-stranded antisense agent
  • Chimeric single-stranded antisense polynucleotides and double-stranded antisense agent
  • Chimeric single-stranded antisense polynucleotides and double-stranded antisense agent

Examples

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example 1

[0148]An experiment comparing the inhibition potency of a chimeric polynucleotide according to one embodiment of the invention with two conventional gapmer antisense oligonucleotide was conducted. The polynucleotide structures and the results of the experiment are shown in FIG. 7. The control gapmers are 13 bases (ApoB1 ASO 13mer; SEQ ID NO:6) and 20 bases (ApoB1 ASO 20mer-4; SEQ ID NO:2) in length. The 13mer control has a central region of 8 phosphorothioated DNA nucleotides, a first 5′-wing region of 2 LNA bases and a first 3′-wing region of 3 LNA bases, but does not have a second wing on either side. The 20mer control has the same structure as the 13mer, and adds 4 phosphorothioated 2′-O-Me RNA bases at the 5′ end and 3 phosphorothioated 2′-O-Me RNA bases at the 3′ end. According to the disclosure, these bases are not low protein-affinity nucleotides (because they are phosphorothioated) and thus they do not constitute a second 5′ or 3′-wing region. They would actually be classifi...

example 2

[0154]An experiment comparing the inhibition potency of a chimeric polynucleotide according to embodiments of the invention having either a second 5′-wing region, a second 3′-wing region, or both wing regions was conducted. The polynucleotide structures and the results of the experiment are shown in FIG. 8A. Two conventional gapmer controls were compared with the inhibition potency of 3 different chimeric polynucleotides.

[0155]The ASO's were tested in vivo using Hep 1-6 liver cells according to the procedure described in Example 1, and the results are shown in FIG. 8B. The ASOs were transfected at a concentration of 20 nM.

[0156]As shown by the results in FIG. 8B, all five ASOs show an antisense effect relative to the negative control (no ASO). However, comparing the inhibition potency of the 20mer chimeric polynucleotides with the 20mer control, the chimeric polynucleotide provides a greater degree of inhibition in all three cases. Significantly, the chimeric polynucleotide with a s...

example 3

[0157]Next, the chimeric polynucleotides were tested in vivo in mice. The sequences and design of the ASO probes and controls are shown in FIG. 9. The ASO were intravenously injected to a mouse in an amount of 3.0 mg / kg each through the tail vein. The mice were 4-week old female ICR mice with body weights of 20 to 25 g. The experiments using mice were all carried out with n=3. Also, as a negative control group, mice to which only PBS was injected instead of the single-stranded ASO or double-stranded nucleic acid complex were also prepared. Seventy-two hours after the injection, the mice were perfused with PBS, and then the mice were dissected to extract the liver. Subsequently, extraction of mRNA, synthesis of cDNA, and quantitative RT-PCR were carried out by the same methods as the methods described in Example 1, the amount of expression of mApoB / amount of expression of mGAPDH (internal standard gene) was calculated, and comparisons were made between the group administered with PBS...

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Abstract

Chimeric single-stranded polynucleotides and double-stranded antisense agents useful for modifying the expression of a target gene by means of an antisense effect are disclosed. The chimeric single-stranded antisense polynucleotide and double-stranded antisense agents comprise a central nucleotide region flanked by a first 5′-wing region and a first 3′-wing region of modified nucleotides, which are themselves flanked by a second 5′-wing region and/or a second 3′-wing region of nucleotides that have a low affinity for proteins and/or that have higher resistance to DNase or RNase than a natural DNA or RNA and are missing in a cell when the chimeric polynucleotide delivered. The double-stranded antisense agent further comprises a complementary strand annealed to the antisense strand. The polynucleotide can be used to modify RNA transcription levels, miRNA activity, or protein levels in cells.

Description

TECHNICAL FIELD[0001]The present application relates to a chimeric single-stranded polynucleotide and double-stranded antisense agent useful for modifying the expression of a target gene by means of an antisense effect. The chimeric single-stranded antisense polynucleotide comprises a central nucleotide region flanked by a first 5′-wing region and a first 3′-wing region of modified nucleotides, which are themselves flanked by a second 5′-wing region and / or a second 3′-wing region of nucleotides that have a low affinity for proteins and / or that have higher resistance to DNase or RNase than a natural DNA or RNA and are missing in a cell when the chimeric polynucleotide delivered. The double-stranded antisense agent comprises the chimeric single-stranded polynucleotide. The double-stranded antisense agent further comprises a complementary strand. The chimeric single-stranded polynucleotide and double-stranded antisense agent can be used to modify RNA transcription levels or protein lev...

Claims

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Application Information

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IPC IPC(8): C12N15/113
CPCC12N15/113C12N2310/3231C12N2310/11C12N2310/315A61K31/7088A61K31/712A61K31/7125A61K31/713C12N15/111C12N2310/14C12N2310/341C12N2310/53C12N2310/533A61P43/00
Inventor YOKOTA, TAKANORINISHINA, KAZUTAKAMIZUSAWA, HIDEHIROWADA, TAKESHI
Owner NAT UNIV CORP TOKYO MEDICAL & DENTAL UNIV
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