WO2005078093A1

WO2005078093A1 - siRNA MOLECULE INHIBITING THE EXPRESSION OF PRION GENE

Info

Publication number: WO2005078093A1
Application number: PCT/JP2005/002572
Authority: WO
Inventors: Hirohiko Hohjoh
Original assignee: Japan Health Sciences Foundation
Priority date: 2004-02-18
Filing date: 2005-02-18
Publication date: 2005-08-25
Also published as: JPWO2005078093A1

Abstract

An siRNA molecule for inhibiting the expression of a prion gene. This siRNA molecule is a double-stranded RNA molecule capable of inhibiting the expression of the prion gene in a cell by RNAi. In this double-stranded RNA molecule, an antisense strand constituting the same contains a sequence specifically hybridizable with a target sequence consisting of the 66- to 85-th residues or the 513- to 532-residues in the mouse prion gene mRNA sequence represented by SEQ ID NO:1 or a target sequence corresponding thereto in another organism.

Description

Specification

SiRNA molecule that suppresses prion gene expression

Reference to related application

[0001] This patent application is accompanied by a claim of priority based on Japanese Patent Application No. 2004-41157 (filed on Feb. 18, 2004), which was a previously filed patent application in Japan. The entire disclosure content of this subsequent patent application is incorporated herein by reference. Background of the Invention

[0002] Field of the Invention

The present invention relates to gene silencing by the RNAi phenomenon, and more particularly, to an siRNA molecule for suppressing prion gene expression and a method for suppressing prion gene expression using the same.

[0003] Background art

RNAi (RNA interference) is a sequence-specific post-transcriptional repression mechanism induced by double-stranded RNA (dsRNA). This phenomenon has been observed in various species such as flies, insects, protozoa, vertebrates, and higher plants. Regarding the molecular mechanism of action of RNAi, many studies in Drosophila and Caenorhabditis elegans have shown that a 21-25 nucleotide long RNA fragment called siRNA (short interfering RNA) has been (Hammond, SM et al., Nature 404, 293-296, 2000; Parrish, S. et al, Mol. Cell. 6, 1077-1087, 2000; Zamore, PD et al., Cell 101, 25-33, 2000), and the fact that this siRNA is produced from long double-stranded RNA by an RNase III-like nuclease called Dicer (Brenstein, E. et al., Nature 409, 363-366, 2001; Elbashir, SM et al "Genes Dev. 15, 188-200, 2001).

[0004] In mammals, RNAi was initially considered to be a phenomenon found only in oocytes and preimplantation embryos. In general, mammalian cells undergo rapid and non-specific RNA degradation pathways by RNase L, a sequence non-specific RNase, and rapid translation by double-stranded RNA-dependent protein kinase (PKR) induced by interferon. It has an inhibitory pathway, both of which are activated by double-stranded RNA of 30 nucleotides or more. Therefore, in mammalian cells other than undivided cells and those lacking PKR, sequence-specific RNAi activity is masked by the above-described response to double-stranded RNA of 30 nucleotides or more. There is. It has recently been reported that a synthesized 21-nucleotide siRNA duplex can specifically inhibit expression of endogenous genes in mammalian cell culture (Elbashir, SM et al., Nature 411). , 494-498, 2001).

[0005] The principle of gene expression suppression by RNAi is considered as follows! First, when the siRNA is introduced into cells, it binds to the multiprotein complex, forming RISC (RNA-induced silencing complex). This RISC then binds to the mRNA of the target gene and cleaves the portion of the mRNA to which the siRNA has bound by its nuclease activity. Thereby, the expression of the protein by the target gene is inhibited.

[0006] On the other hand, prion protein is known as a causative substance causing various neurodegenerative diseases. Aberrant prion proteins are small, infectious proteinaceous particles having non-nucleic acid components that are resistant to nucleases and proteases. Known human diseases caused by the prion protein include Kuru- spongiform encephalopathy, Creutzfeldt-Jakob disease, Gerstmann-Stroisler syndrome, and fatal familial insomnia.

[0007] It has been reported that when a prion gene (PrP gene) knockout mouse is inoculated with a prion pathogen, prion disease does not develop (Nature 356, 577-582, 1992). This is thought to be because the normal prion protein required for the amplification of the abnormal prion protein is not expressed, so that the amount of the abnormal prion protein required for the onset of prion disease does not accumulate in cells. I have. It is known that the knockout mouse is almost morphologically the same as a normal mouse. Therefore, it is considered that prion disease can be treated or prevented by knocking out or knocking down the prion gene.

Summary of the Invention

[0008] The present inventors have remarkably suppressed the expression of the prion gene in cells by using an siRNA designed with a specific region in the mRNA of the prion gene as a target sequence. I found out that The present invention is based on this finding.

Therefore, an object of the present invention is to provide an siRNA molecule for suppressing the expression of a prion gene.

[0010] The siRNA molecule according to the present invention is a double-stranded RNA molecule capable of suppressing the expression of a prion gene in a cell by RNAi, and has an antisense chain strength of not more than the antisense chain force constituting the double-stranded RNA molecule. Target sequence of: (a) a target sequence having residues 66-85 in the mouse prion gene mRNA sequence represented by SEQ ID NO: 1, and (b) a prion gene mRNA sequence derived from an organism other than a mouse. (A) a target sequence having a sequence strength corresponding to the target sequence of (a); (c) a target sequence of a 513-532 residue in the mouse prion gene mRNA sequence represented by SEQ ID NO: 1; or (d) ) A prion gene mRNA sequence derived from an organism other than a mouse, wherein the sequence comprises a sequence that specifically hybridizes to a target sequence consisting of a sequence corresponding to the target sequence of (c). It is a strand RNA molecule.

According to the present invention, it is possible to significantly suppress the expression of a prion gene by utilizing the RNAi phenomenon.

Brief Description of Drawings

[FIG. 1] FIG. 1 is a diagram showing the positions of target sequences 114 in the mRNA sequence of mouse PrP gene (SEQ ID NO: 1) and the mRNA sequence of human PrP gene (SEQ ID NO: 2).

FIG. 2 is a graph showing the effect of various siRNAs on the expression of endogenous PrP gene in N2a cells.

Detailed description of the invention

[0013] As used herein, the term "double-stranded RNA" refers to an RNA molecule obtained by hybridizing all or part of two single-stranded RNA molecules. The number of nucleotides constituting each single-stranded RNA may be different from each other. One or both nucleotide chains constituting the double-stranded RNA may include a single-stranded portion (overhang).

[0014] As used herein, the term "double-stranded portion" refers to a portion of a double-stranded RNA in which nucleotides of both strands are paired, that is, a single-stranded portion in the double-stranded RNA. It means the part excluding the chain part. [0015] As used herein, the "sense strand" refers to a nucleotide chain having a sequence homologous to the gene coding strand, and the "antisense strand" refers to a sequence complementary to the gene coding strand. It means having a nucleotide chain.

[0016] As used herein, the term "complementary" means that two nucleotides can be paired under hybridization conditions. For example, adenine (A) and thymine ( T) or peracyl (U), and the relationship between cytosine (C) and guanine (G).

[0017] The double-stranded RNA molecule according to the present invention is a double-stranded RNA molecule capable of suppressing the expression of a prion gene in a cell by RNAi, and the double-stranded RNA molecule is contained in the mRNA sequence of the prion gene. It is designed to target a specific sequence. The target sequence used in the present invention is a sequence having 66-85 residues and a sequence having 513-532 residues in the mouse prion gene mRNA sequence represented by SEQ ID NO: 1. Furthermore, the target sequence used in the present invention is not limited to that derived from a mouse, and the prion gene mRNA sequence derived from another organism is not limited to that derived from a mouse. Things. The target sequence in an organism other than the mouse is determined by comparing the mouse prion gene mRNA sequence with the prion gene mRNA sequence derived from the target organism, and identifying a region homologous to the target sequence in the mouse. Is determined. Identification of a homologous region by comparing a plurality of sequences can be performed by standard methods well known in the art. For example, FIG. 1 shows an alignment of the prion gene mRNA sequences derived from mouse and human. According to this, the target sequence in human is expressed in the human prion gene mRNA sequence represented by SEQ ID NO: 2. It is clear that the sequence is also a sequence with residues 66-85 and a sequence with residues 516-535.

[0018] The antisense strand constituting the double-stranded RNA molecule according to the present invention comprises a sequence that specifically hybridizes to the above-described target sequence. Here, "specifically hybridize" means that the antisense strand does not hybridize to a nucleic acid molecule other than the prion gene and its transcript in a cell that suppresses the expression of the prion gene. Means that. The antisense strand is intended to include a sequence corresponding to the target sequence, but does not need to have a sequence completely complementary to the target sequence. As long as it can specifically hybridize to the target sequence, it may contain nucleotides that are not complementary. However, the antisense strand preferably comprises a sequence complementary to the target sequence.

[0019] The sense strand constituting the double-stranded RNA molecule according to the present invention includes a sequence capable of forming a double strand with the antisense strand in a cell in which prion gene expression is to be suppressed. Just fine. Those skilled in the art can easily determine whether or not two ribonucleotide chains form a double strand in a cell. For example, the hybridization conditions in a target cell can be reproduced in vitro, and two ribonucleotide chains can be added thereto to determine whether or not these hybridize.

[0020] According to a preferred embodiment of the present invention, the sense strand corresponds to nucleotide residues located at positions 117 and 918 from the 5 'end to the 3' side of the target sequence. It is assumed that each of the positions further comprises a nucleotide residue complementary to the antisense strand. Therefore, according to this embodiment, the sense strand is located at positions corresponding to the nucleotide residues located at positions 8 and 19 to 20 from the 5 ′ end to the 3 ′ side of the target sequence. And may contain nucleotide residues that are not complementary to the antisense strand.

According to another preferred embodiment of the present invention, the sense strand is located at a position corresponding to a nucleotide residue located at position 118 from the 5 ′ end to the 3 ′ side of the target sequence. And a nucleotide residue complementary to the antisense strand. Therefore, according to this embodiment, the sense strand is linked to the antisense strand at a position corresponding to the nucleotide residue located at position 191-220 from the 5 ′ end to the 3 ′ side of the target sequence. It may contain nucleotide residues that are not complementary.

According to another preferred embodiment of the present invention, the sense strand comprises a nucleotide residue complementary to the antisense strand at a position corresponding to the target sequence. You.

The antisense strand constituting the double-stranded RNA molecule according to the present invention may have a protruding portion (overhang) on the 3′-terminal side of the double-stranded portion paired with the nucleotide of the sense strand. Good. The nucleotide sequence of this overhang may be the sequence or The length of any of the non-contiguous sequences is not particularly limited, but is preferably a two-nucleotide long sequence, and more preferably two peracil residues (UU). The sense strand may also have a similar overhang at the 3 ′ end of the double-stranded portion paired with the nucleotide of the antisense strand. However, according to a preferred embodiment of the present invention, the sense strand is free of overhangs at its 3 'end.

[0024] The double-stranded portion of the double-stranded RNA molecule according to the present invention may be any as long as it comprises a sequence corresponding to the above-mentioned target sequence. Therefore, this double-stranded portion can further contain another sequence, for example, a nucleotide residue corresponding to a sequence located around the target sequence in the prion gene mRNA. However, according to a preferred embodiment of the present invention, the double-stranded portion is composed of a sequence corresponding to the target sequence, that is, includes only a sequence corresponding to the target sequence. You.

When a long double-stranded RNA molecule is introduced into mammalian cells, double-stranded RNA-dependent protein kinase ゃ 2,15,1-oligoadenylate synthase is induced. It is known to lead to death. Therefore, the double-stranded RNA molecule according to the present invention does not induce double-stranded RNA-dependent protein kinase or 2'-5,1-oligoadenylate synthetase in mammalian cells. I prefer that. The chain length of the double-stranded RNA molecule satisfying this condition is easily understood by those skilled in the art. It is generally known that the above cell death is caused by a double-stranded RNA molecule of 30 nucleotides or more, and therefore, the double-stranded RNA molecule according to the present invention is preferably 29 nucleotides or less, more preferably It has a chain length of 25 nucleotides or less. As used herein, the term “chain length” refers to the nucleotide length of a double-stranded RNA molecule including a single-stranded portion instead of only a double-stranded portion.

[0026] The double-stranded RNA molecule according to the present invention can suppress prion gene expression in cells. Therefore, according to the present invention, there is provided a method for suppressing the expression of a prion gene in a cell, comprising the step of introducing the double-stranded RNA molecule of the present invention into the cell.

[0027] The cell may be any cell as long as it can cause an RNAi phenomenon by siRNA and expresses a prion gene, and is preferably a mammalian cell. It is. Examples of mammalian cells include those derived from mouse, rat, rat, muster, egret, dog, monkey, human and the like. Suitable cultured cells include, for example, He La cells, NIHZ3T3 cells, COS-7 cells, and 293 cells.

[0028] The method for introducing a double-stranded RNA molecule into a cell according to the present invention is not particularly limited as long as it can introduce a double-stranded nucleic acid molecule into a cell. In the method according to the present invention, a particularly suitable method for introducing a double-stranded RNA molecule into cells is a transfection method using a liposome, preferably a cationic ribosome, which is a Lipofectamine 2000 transfection reagent. (Invitrogen), Oligofectamin transfection reagent (Invitrogen), jetSI transfection reagent (Polyplus-transfection), TransMessenger (Qiagen) and other commercially available transfection reagents. Can be.

Alternatively, prion gene expression suppression using the double-stranded RNA molecule of the present invention is performed by introducing a vector that expresses the double-stranded RNA molecule of the present invention in a cell into a target cell. You can also. In this method, two types of vectors expressing each of the sense strand and the antisense strand of the double-stranded RNA molecule of the present invention may be used, or the sense strand of the double-stranded RNA molecule of the present invention may be used. A vector comprising both DNA encoding DNA and DNA encoding the antisense strand may be used. Therefore, according to another aspect of the present invention, there is provided a vector comprising DNA encoding the sense strand of the double-stranded RNA molecule according to the present invention, and a DNA encoding the antisense strand of the double-stranded RNA molecule. Or a vector comprising both a DNA encoding the sense strand and a DNA encoding the antisense strand of the double-stranded RNA molecule according to the present invention, into a cell. A method for suppressing the expression of a prion gene in a cell is provided.

[0030] The above-described vector may be any vector as long as it can express one or both chains of the double-stranded RNA molecule according to the present invention in a cell. Thus, the above vectors comprise DNA encoding the strand of the double-stranded RNA molecule in a form that allows its transcription in the cell. Such vectors can be easily constructed by those skilled in the art.For example, if necessary, elements such as a promoter and a terminator can be operably linked. it can. Any promoter can be used as long as it can function in a target cell, and for example, any of a constitutive promoter and an inducible promoter may be used. Also, a prion gene promoter can be used, whereby a transcript of the prion gene can be generated, and at the same time, a double-stranded RNA molecule according to the present invention for degrading the transcript can be generated. Introduction of the constructed vector into a target cell can be appropriately performed by those skilled in the art by a method known in the art.

[0031] In particular, it is preferable to use a single vector capable of expressing both strands of the double-stranded RNA molecule according to the present invention. Such vectors, even those that independently express the two strands of a double-stranded RNA molecule, are expressed as a hairpin-type double-stranded RNA in which the two strands are linked by a linker sequence. It may be something ヽ. Those skilled in the art can appropriately construct a vector that expresses the double-stranded RNA molecule of the present invention as a hairpin double-stranded RNA, for example, as described in the literature (Bass, BL, Cell 101, 235). -238, 2000; Tavernarakis, N. et al, Nat.Genet. 24, 180-183, 2000; Malagon, F. et al, Mol.Gen. Genet. 259, 639-644, 1998; Parrish, S. et. al., Mol. Cell 6, 1077-1087, 2000).

The double-stranded RNA molecule and the method for suppressing the expression of a prion gene using the same according to the present invention can be used only for analyzing the function of a prion gene and a prion protein. It can also be used to suppress the expression of prion genes in E. coli, thereby treating or preventing prion diseases such as Kourou spongiform encephalopathy, Creutzfeldt-Jakob disease, Gerstmann-Stroisler syndrome, and fatal familial insomnia. It is also possible to do.

Example

Hereinafter, the present invention will be described more specifically with reference to Examples, but these Examples do not limit the present invention.

Example 1: Inhibition of mouse PrP gene expression in N2a cells by each bran siRNA

In N2a cells derived from mouse neuroblastoma, various siRNAs targeting four regions (target sequences 114) in the endogenous mouse PrP gene were designed, and these siRNAs were used. The effect of suppressing the expression of the gene was examined.

[0035] The nucleotide sequences of the designed various siRNAs are shown in Table 1 below. In Table 1, the sense strands of each siRNA are aligned on the upper side and the antisense strand on the lower side. Nucleotides complementary to each other are marked with a colon ":" between both strands. In addition, in the name of each siRNA, the number added after “PrP” means a region in the targeted PrP gene, and the position of each region in the mRNA sequence of the gene is as shown in FIG. is there.

[0036] [Table 1] Table 1: Various siRNAs designed for mouse PrP gene Name Sequence SEQ ID NO:

5 '-GAUGUCGGCCUCUGCAAAAAUU-3' 3

PrPl

3 '-UUCUACAGCCGGAGACGUUUUU-5' 4

5 '-GAUGUCGUCCUCUGCAAAUU-3' 5

F. PrPl

3 '-UUCUACAGCCGGAGACGUUUUU-5' 4

5 '-GAGGGGGUACCCAUAAUCAUU-3' 6

PrP2

3 '-UUCUCCCCCAUGGGUAUUAGU- 5, 7

5, -CGCUACUACCGUGAAAACAUU-3 '8

PrP3

3,-UUGCGAUGAUGGCACUUUUGU- 5, 9

5 '-CAGUACAGCAACCAGAACAAUU- 3' 1 0

PrP4

3 '-UUGUCAUGUCGUUGGUCUUGUU-5' 1 1

5 '-C AGUAC AUCAACCAGAACUU-3' 1 2

F. PrP4

3 '-UUGUCAUGUCGUUGGUCUUGUU-5' 1 1

5 '-CAGUACAGCAACCAGAACUU-3' 1 3

F. PrP4-2

3 '-UUGUCAUGUCGUUGGUCUUGUU-5' 1 1

5 '-CAGUACAGCAACCAGAACAA-3, 1 4

PrP4B

3 '-UUGUCAUGUCGUUGGUCUUGUU-5' 1 1 Each oligoribonucleotide pair described above was synthesized, and 20 μl of each was added to an annealing buffer (30 mM HEPES pH 7.0, 100 mM potassium acetate, and 10 mM magnesium acetate), and the mixture was added at 90 ° C. After heat denaturation for 3 minutes, a ring was performed all day and night at 37 ° C. From this, each siRNA was obtained.

[0038] N2a cells were obtained from 10% fetal calf serum (Life Technologies), lOOUZml penicillin (Life Technologies) and 100 μl

5% CO atmosphere in Dulbecco's Modified Eagle Medium (Sigma) supplemented with (Life Technologies)

Grow at 37 ° C under 2 atmospheres.

[0039] Transfection of N2a cells with each siRNA (IOOnM) was performed using jetSI transfection reagent (Polyplus-transfection).

[0040] 48 hours after the transfection, total RNA was isolated, and cDNA synthesis was performed using reverse transcriptase. The expression level of the PrP gene was measured by real-time PCR using this cDNA as type II. These series of expression level measurements were performed using a SYBR green PCR kit (Molecular Probe).

FIG. 2 shows the effect of various siRNAs on the expression of endogenous PrP gene in N2a cells. FIG. 2 shows the ratio of the expression level of the PrP gene to the expression level of the control gene (G3PDH gene) when each siRNA was used! / ヽ. This expression level ratio is standardized as 1.0, which is obtained for a control sample using double-stranded RNA (siCont) (manufactured by Qiagen) which does not suppress gene expression. Each data is the average from at least four independent experiments, and the error bars assigned to each data represent the standard deviation.

[0042] According to FIG. 2, the expression of the mouse PrP gene was suppressed by various siRNAs, and in particular, the expression of the gene was significantly suppressed by the siRNAs designed for target sequences 1 and 4. Puru.

[0043] Furthermore, cell lysates were prepared 48 hours after transflector Ekushi Yong, examined the expression of p _r p protein by Western blotting using anti-PrP antibodies. As a result, m

As well as the suppression of RNA expression, it was evident that the expression level of the protein was also suppressed.

Claims

The scope of the claims

[1] A double-stranded RNA molecule capable of suppressing the expression of a prion gene in a cell by RNAi, wherein the antisense strand constituting the double-stranded RNA molecule has the following target sequence:

(a) a target sequence consisting of residues 66 to 85 in the mouse prion gene mRNA sequence represented by SEQ ID NO: 1,

(b) a prion gene mRNA sequence derived from an organism other than a mouse, the target sequence having a sequence strength corresponding to the target sequence of (a);

(c) a target sequence consisting of residues 513-532 in the mouse prion gene mRNA sequence represented by SEQ ID NO: 1, or

(d) In the mRNA sequence of a prion gene derived from an organism other than a mouse, a target sequence having a sequence strength corresponding to the target sequence of (c)

A double-stranded RNA molecule comprising a sequence that specifically hybridizes to

[2] The double-stranded RNA molecule according to claim 1, wherein the double-stranded RNA molecule comprises a sequence complementary to the target sequence.

[3] The position where the sense strand constituting the double-stranded RNA molecule corresponds to the nucleotide residues located at positions 17 and 911 from the 5 ′ end to the 3 ′ side of the target sequence The double-stranded RNA molecule according to claim 1, wherein the double-stranded RNA molecule comprises a nucleotide residue complementary to the antisense strand.

[4] The sense strand constituting the double-stranded RNA molecule is complementary to the antisense strand at a position corresponding to the nucleotide residue at position 118 from the 5 ′ end to the 3 ′ side of the target sequence. The double-stranded R according to claim 1, which comprises a basic nucleotide residue.

NA molecule.

[5] The sense strand constituting the double-stranded RNA molecule is located at a position corresponding to the target sequence.

2. The double-stranded RNA molecule according to claim 1, wherein the double-stranded RNA molecule comprises a nucleotide residue complementary to the antisense strand.

[6] The double-stranded RNA molecule according to claim 1, wherein the sense strand constituting the double-stranded RNA molecule does not include a protruding portion at its 3 ′ end.

[7] In mammalian cells, double-stranded RNA-dependent protein kinase or 2'-5 2. The double-stranded RNA molecule according to claim 1, which does not induce one oligoadenylate synthase.

[8] The double-stranded RNA molecule according to claim 7, which has a chain length of 29 nucleotides or less.

[9] A method for suppressing the expression of a prion gene in a cell, comprising a step of introducing the double-stranded RNA molecule according to any one of claims 18 to the cell.

[10] The method according to claim 9, wherein the cell is a mammalian cell.

[11] A vector comprising both a DNA encoding a sense strand and a DNA encoding an antisense strand of the double-stranded RNA molecule according to any one of claims 18 to 18.

[12] A vector comprising a DNA encoding the sense strand of the double-stranded RNA molecule according to any one of claims 18 to 18, and a DN encoding the antisense strand of the double-stranded RNA molecule. A method for suppressing expression of a prion gene in a cell, comprising a step of introducing the vector according to claim 11 into a cell, or a step of introducing the vector according to claim 11 into the cell.

[13] The method according to claim 12, wherein the cell is a mammalian cell.