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Double-stranded lipid-modified RNA having high RNA interference effect

By connecting double-stranded lipids to the 5' end side of double-stranded RNA to construct double-stranded lipid-modified RNA, the problem of short-lived RNA interference effect of siRNA in cells is solved, and efficient nuclease resistance and intracellular introduction rate are achieved. , enhancing the stability of RNA interference effect and the safety of drug application.

Inactive Publication Date: 2011-02-23
NAT INST OF ADVANCED IND SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when double-stranded RNA, which is known to have RNA interference effects, is simply linked to lipids, the RNA interference effect is significantly weakened
The current status is that lipid-modified RNA that combines excellent RNA interference effects with useful effects based on lipids has not been constructed in the prior art

Method used

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  • Double-stranded lipid-modified RNA having high RNA interference effect
  • Double-stranded lipid-modified RNA having high RNA interference effect
  • Double-stranded lipid-modified RNA having high RNA interference effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 Inhibitory effect of 5' double-stranded lipid-modified RNA on luciferase gene expression

[0127] 1. Synthesis of double-stranded lipid-modified RNA targeting luciferase gene

[0128] 1-1. Sequences of sense strand RNA and antisense strand RNA

[0129] Design of double-stranded RNA containing sense strand RNA of 21 and 27 base lengths and antisense strand RNA of 21 and 27 base lengths with the same sequence, and can inhibit the gene expression of Renilla luciferase. When the antisense strand and the sense strand of 21 nucleotides are used for this double-stranded RNA, a 21-base double-stranded RNA having a 2-base overhang at the 3' end can be formed, and the double-stranded RNA uses 27 When the antisense strand and the sense strand are 2 bases in length, a double-stranded RNA of 27 bases in length with blunt ends can be formed. The sequences of the RNAs used are as follows.

[0130] 27nt dsRNA sense strand L27A: 5'-CUGGCCUUUCACUACUCCUACGAGCAC-3' (SE...

Embodiment 2

[0156] Example 2 Inhibitory effect of 5' double-stranded lipid-modified RNA on VEGF gene expression

[0157] 1. Synthesis of double-stranded lipid-modified RNA targeting VEGF gene

[0158] 1-1. Sequences of sense strand RNA and antisense strand RNA

[0159] Design double-stranded RNA containing sense strand RNA of 27 and 21 base lengths and antisense strand RNA of 27 and 21 base lengths. growth factor) and can inhibit the gene expression of VEGF. Using the above double-stranded RNA, the following experiments were performed. It should be noted that 21siRNA is a double-stranded RNA having a 2-base overhang at the 3' end of both the sense strand RNA and the antisense strand RNA, and the 27nt dsRNA is a complete double-stranded RNA having no overhang (single-stranded region). Strand RNA (a double-stranded RNA in which both the 5' end and the 3' end of the sense strand are blunt). The sequences of the 27nt dsRNA and 21si RNA used are as follows.

[0160] 27nt dsRNA sens...

Embodiment 3

[0193] Example 3 Synthesis of double-stranded lipid-modified RNA targeting WT1 gene

[0194] Using a method different from the above-mentioned Examples 1 and 2, a double-stranded lipid-modified sense compound capable of inhibiting WT1 gene expression, in which a double-stranded lipid is bonded to the 5' end of the sense strand RNA of the double-stranded RNA, was synthesized. stranded RNA.

[0195] First, sense strand RNA and antisense strand RNA were synthesized according to known methods. Next, use the 5'-amino-modifying agent C6 (Glen Research) to carry out aminoalkylation on the 5' end of the sense strand RNA according to the usual method (phosphoramidite synthesis method) to synthesize a 5'-terminal aminoalkyl-modified sense stranded RNA. Synthetic 5' terminal aminoalkyl modified sense strand RNA, the phosphate residue of the first nucleotide from the 5' terminal side is connected with -(CH 2 ) 6 -NH 2 Bond. The synthesized 5' terminal aminoalkyl-modified sense st...

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Abstract

Disclosed is novel double-stranded RNA which has high nuclease resistance, can be introduced into a cytoplasm at a high rate, and can exhibit an excellent RNA interference effect. Specifically disclosed is double-stranded lipid-modified RNA which is composed of sense-strand RNA comprising a nucleotide sequence complementary to a target sequence contained in a target gene and antisense-strand RNA comprising a nucleotide sequence complementary to the sense-strand RNA and which can inhibit the expression of the target gene, wherein a double-stranded lipid is bound to at least one nucleotide selected from the 1st to 6th nucleotides from the 5'-terminal of the sense-strand RNA directly or via a linker.

Description

technical field [0001] The invention relates to a double-stranded lipid-modified RNA capable of effectively inhibiting the expression of target genes. More specifically, the present invention relates to a double-stranded lipid-modified RNA that is resistant to nucleases, has a high rate of intracellular introduction, and can produce an excellent RNA interference effect. Furthermore, the present invention relates to a pharmaceutical composition using the double-stranded lipid-modified RNA and a method for inhibiting the expression of a target gene. Background technique [0002] Drug development for effective treatment of intractable diseases such as cancer and AIDS is a major topic in the field of life sciences. An effective method capable of overcoming the above-mentioned problems is the use of gene medicines that act only on specific genes. Among the above-mentioned gene medicines, in particular, the RNA interference (RNAi) method using a 21-base short double-stranded RNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7088C12N15/09A61K48/00
CPCC12N2320/32C12N2310/3515C12N15/111A61P31/18A61P35/00A61K31/7105A61K35/66A61K48/00C12N15/10C12N15/113C12N15/1135
Inventor 久保贵纪大庭英树丰福秀一林宏刚
Owner NAT INST OF ADVANCED IND SCI & TECH
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