JP2004344072A - Splicing control element for smn gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for controlling the expression of SMN gene. <P>SOLUTION: A nucleic acid for a splicing control factor or a variant thereof comprising a specific base sequence is provided. A nucleic acid-introducing carrier containing the nucleic acid is provided. The method for controlling the expression of SMN gene using the nucleic acid is provided. A pharmaceutical composition containing the nucleic acid or the carrier as active ingredient is provided. A method for retrieving a splicing-enhancing substance for SMN gene's exon 7 is also provided, comprising the following steps: in the presence/absence of a specimen, a cell is maintained which contains an exon trap vector construct operably bound with a nucleic acid construct comprising SMN2 gene's intron 6, SMN2 gene's exon 7 and SMN2 gene's intron 7 containing a nucleic acid for a splicing control element comprising a specific base sequence, and the amount of a transcription product corresponding to the SMN2 gene's exon 7 in the cell obtained in the presence of the specimen and the amount of a transcription product corresponding to the SMN2 gene's exon 7 in the cell obtained in the absence of the specimen are measured. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】図１は、ＳＭＮ１及びＳＭＮ２ エキソンのスプライシングパターンを示す。図中、各レーンの上側のバンドは、エキソン６からエキソン８の領域を含む全長転写産物を示し、下側のバンドは、エキソン７を欠損した転写産物を示す。また、図中、ＷＴ ＳＭＮ１（Ｃ）は、野生型ミニジーン、変異体ＳＭＮ１（Ｔ）は、エキソン７内部の６ヌクレオチドに位置するＣからＴへの転位を含むＳＭＮ１ミニジーン、ＷＴ ＳＭＮ２（Ｔ）は、野生型ＳＭＮ２ミニジーン、変異体ＳＭＮ２（Ｃ）は、エキソン７内の６ヌクレオチドに位置するＴからＣへの置換を含むＳＭＮ２ミニジーンを示す。
【図２】図２は、エキソントラップベクターの概略図を示す。図中、ＢＰは、分岐点、ＰＰＴは、ポリピリミジントラクトを示す。
【図３】図３は、インビトロスプライシングアッセイの結果を示す。上部パネルは、野生型ＳＭＮ１（Ｃ）から構築された欠失変異体の結果であり、下部パネルは、ＣからＴへの転位を含む変異体ＳＭＮ１から構築された欠失変異体の結果である。なお、下部パネルに示されるスコアは、全転写産物に対するエキソン７含有型の割合であり、値は、４回の解析の平均を示す。
【図４】図４は、イントロン７内におけるエレメント２の位置（上部パネル）及び構造（下部パネル）の概略図を示す。
【図５】図５は、ＣからＴへの転位を含む変異体ＳＭＮ１のエレメント２のステム−ループにおける変異による、エキソン７を含む転写産物の量に対する影響を調べた結果を示す。パネル（Ａ）のＡ〜Ｆは、Ｉ７ＤＭ１−Ｔミニジーンにおける種々の変異体の構造の概略図である。図中、○は、野生型エレメント２に対して、置換されたヌクレオチドを示す。パネル（Ｂ）は、イン・ビボスプライシング解析の結果を示す。図中、Ｉ７ＤＭ１−Ｔ、エキソントラップベクターにクローン化されたミニジーンＡからＦは、パネル（Ａ）に対応する。パネル（Ｃ）は、各構築物のスプライシングパターンの定量的解析の結果を示す。全転写産物に対するエキソン７含有型の割合は、４回の解析の平均±Ｓ．Ｄで表わされる。
【図６】図６は、エレメント２の挿入数による、エキソン７を含む転写産物の量に対する影響を調べた結果を示す。パネル（Ａ）は、ＳＭＮのイントロンにおけるエレメント２のタンデムリピートを有する構築物の概略図を示す。図中、Ｉ７ＤＭ１−Ｔは、１コピーのエレメント２を含むオリジナル構築物、エレメント ２−Ｔｘ３は、３コピーのエレメント２を含む構築物、エレメント ２−Ｔｘ４は、４コピーのエレメント２を含む構築物を示す。パネル（Ｂ）は、エレメント２の挿入数による、エキソン７を含む転写産物の量のＲＴ−ＰＣＲ解析（上部パネル）及び全転写産物に対するエキソン７ 含有型の割合（下部パネル）を示す。全転写産物に対するエキソン７ 含有型の割合は、４回の解析の平均±Ｓ．Ｄで表わされる。
【図７】図７は、エレメント２に結合する核タンパク質の同定のためのゲル移動度シフトアッセイの結果を示す図である。パネル（Ａ）は、ＳＫ−Ｎ−ＳＨ細胞核抽出物のゲル移動度シフトアッセイの結果を示す。パネル（Ｂ）は、野生型エレメント２又は変異体エレメント２の非標識ＲＮＡオリゴヌクレオチド（１、５、１４モル濃度超）を添加することにより競合アッセイを行なった結果を示す。
【図８】図８は、エレメント２に対するアンチセンスオリゴヌクレオチドによる、エキソン７を含む転写産物の発現への影響を調べた結果を示す。パネル（Ａ）は、用いた３つのアンチセンスオリゴヌクレオチド（Ａｓ−エレメント２、Ａｓ−ｃｏｎ、Ａｓ−ｐｙｒ）のＳＭＮ遺伝子上の位置を示す。パネル（Ｂ）は、Ａｓ−エレメント２について、エキソントラッピング系を用いたイン・ビボスプライシングのＲＴ−ＰＣＲ解析の結果を示す。エキソン７含有型の割合を、４回の実験の平均で示し、各レーンの下に示す。パネル（Ｃ）は、Ａｓ−ｃｏｎについて、エキソントラッピング系を用いたイン・ビボスプライシングのＲＴ−ＰＣＲ解析の結果を示す。エキソン７含有型の割合を、４回の実験の平均で示し、各レーンの下に示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a means for controlling the expression of SMN gene. More specifically, the present invention relates to a nucleic acid for a splicing regulatory element capable of regulating splicing of a SMN gene and a carrier for introducing the nucleic acid, a method for controlling the expression of the SMN gene, and a method for preventing or treating a disease caused by deletion or mutation of the SMN gene. And a method for screening a substance that enhances splicing of exon 7 of the SMN gene.
[0002]
[Prior art]
Spinal muscular atrophy is an autosomal recessive disorder, characterized by a loss of motor neurons in the spinal cord that results in muscle weakness in the proximal limbs and trunk, which ultimately results in death.
[0003]
As a gene considered to be a cause of the above-mentioned spinal muscular atrophy, a survivor motor neuron (SMN) gene located at the 5q13 locus of human chromosome 5 has been found (Non-Patent Documents 1 and 2).
[0004]
In humans, the SMN gene is known to generate two kinds of isoforms, ie, “SMNΔexon 7” containing no exon 7 and “SMN + exon 7” containing exon 7, by splicing. It is thought that the absence of “SMN + exon 7” including exon 7 causes the spinal muscular atrophy.
[0005]
For the development of a method for treating spinal muscular atrophy, for example, butyrate (sodium acetate, arginine butyrate, butyric acid, etc.), a histone deacetylase inhibitor such as trapoxin, trichostatin A, etc. Increasing exon 7 expression (see Patent Document 1), exposing a substance that controls exon 7 inclusion of the SMN2 gene to a mammalian cell transfected with exon 7-containing DNA of the SMN2 gene. Testing the substance that controls exon 7 inclusion in the SMN2 gene by examining the ratio of exon 7 inclusion and exon 7 skipping (see Patent Document 2), as a substance that controls exon 7 inclusion in the SMN2 gene The nucleic acid encoding the splicing regulator Htra2-beta1 protein is Transfected into object cells, thereby reducing the SMN2 transcripts that do not contain exon 7, thereby upregulate total length SMN2 expression (see Patent Document 2) have been attempted.
[0006]
However, when a histone deacetylase inhibitor is used, there is a drawback that cell division / proliferation may be impaired because deacetylation of histone is inhibited and the nucleosome formation of histone is affected. According to the method described in Patent Document 2, Htra2-beta1 is expressed in all cells of each organ / tissue and controls splicing of many genes. It has the disadvantage that Htra2-beta1 may affect the splicing of other genes that control splicing.
[0007]
[Patent Document 1]
JP-A-2002-338502, Example 4, etc.
[Patent Document 2]
European Patent Publication No. 1 133 993 (EP 1 133 993 A1)
[Non-patent document 1]
Lefebvre, S. et al., Cell, Vol. 80, pp. 155-165, published in 1995.
[Non-patent document 2]
Rochette, CT, et al., Hum. Genet. 108, pages 255-266, published in 2001.
[0008]
[Problems to be solved by the invention]
The present invention relates to expression of a full-length SMN gene, expression of an SMN2 gene-derived product that can supplement the function of the SMN1 gene, deletion of the SMN1 gene that causes spinal muscular atrophy, or mutation of the SMN1 gene. It is an object of the present invention to provide a nucleic acid having a splicing regulatory element of the SMN gene, which makes it possible to compensate for at least one of the functions of the SMN1 gene product caused by deficiency. Another object of the present invention is to provide a carrier for nucleic acid introduction that can efficiently introduce the nucleic acid having the splicing regulatory element into cells. Furthermore, an object of the present invention is to provide a method for controlling the expression of the SMN gene, which enables the expression of the full-length SMN gene, the expression of a product derived from the SMN2 gene that can supplement the function of the SMN1 gene, and the like. Further, the present invention provides a method for preventing or treating a disease caused by a deficiency or mutation of the SMN gene, in particular, a disease caused by a deficiency or mutation of the SMN gene, which can prevent or treat the development of spinal muscular atrophy. To provide a pharmaceutical composition for the same. Furthermore, the present invention provides a method for efficiently screening exon 7 of the SMN gene, which can efficiently screen for a substance capable of enhancing the expression of the full-length SMN gene, a substance capable of enhancing the expression of a product derived from the SMN2 gene that can supplement the function of the SMN1 gene, and the like. An object of the present invention is to provide a method for screening a splicing enhancer. It should be noted that other objects of the present invention are apparent from the description and all the teachings of this specification.
[0009]
[Means for Solving the Problems]
That is, the gist of the present invention is:
[1] a nucleic acid of a splicing regulatory element consisting of the nucleotide sequence of SEQ ID NO: 1 or 2,
[2] The nucleic acid of the nucleic acid variant according to [1], which comprises a nucleotide sequence different from the nucleotide sequence shown in SEQ ID NO: 1 or 2 through a nucleic acid polymorphism and can regulate splicing of the SMN gene. ,
[3] the nucleic acid of the above-mentioned [2], wherein the variant comprises a base sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25;
[4] a carrier for introducing a nucleic acid, comprising the nucleic acid according to any one of [1] to [3],
[5] a nucleic acid of the following a) or b):
a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 2, or
b) a nucleic acid of a variant of the nucleic acid according to a), comprising a nucleotide sequence different from the nucleotide sequence shown in SEQ ID NO: 2 through a nucleic acid polymorphism and capable of regulating splicing of the SMN gene
A method for controlling expression of the SMN gene, wherein at least one copy of
[6] Prevention of a disease caused by SMN gene deficiency or mutation, comprising the nucleic acid according to any one of [1] to [3] or the carrier for nucleic acid introduction according to [4] as an active ingredient. Or a pharmaceutical composition for treatment,
[7] (I) In the presence and absence of a test substance,
The following (1) to (3):
(1) With intron 6 of SMN2 gene
(2) With exon 7 of SMN2 gene
(3) SMN2 gene intron 7 containing the nucleic acid of the splicing regulatory element consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 2;
And maintaining a cell comprising an exon trap vector operably linked to the nucleic acid construct, wherein the nucleic acid constructs are arranged in the order of (1)-(2)-(3); and
(II) In step (I), the amount of a transcript corresponding to exon 7 of the SMN2 gene in the cell obtained in the presence of the test substance, and the amount of SMN2 in the cell obtained in the absence of the test substance Measuring the amount of a transcript corresponding to exon 7 of the gene,
A method for screening for a substance that enhances the splicing of exon 7 of the SMN gene, using as an index that the transcript corresponding to exon 7 of the SMN2 gene is increased by the presence of the test substance as compared to the absence thereof,
About.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
The nucleic acid of the splicing regulatory element of the present invention has one significant feature in that it comprises the nucleotide sequence (RNA) shown in SEQ ID NO: 1 or the nucleotide sequence (DNA) shown in SEQ ID NO: 2. The nucleic acid of the present invention is a sequence unit that regulates splicing to generate exon 7 in the sequence of the cis element (hereinafter, referred to as element 2) of the Survival motor neuron (SMN) gene, and is a nucleic acid that forms a stem-loop structure. It is.
[0011]
Therefore, according to the nucleic acid of the present invention, an excellent effect of generating a SMN transcript containing exon 7 is exhibited. Further, according to the nucleic acid of the present invention, even when the SMN1 gene is deficient or mutated, an excellent effect of generating a SMN transcript including exon 7 from SMN2 by splicing is exhibited.
[0012]
The survivor motor neuron (SMN) gene includes two types of SMN genes: an SMN1 gene [GenBank Accession Number: AH006635] and an SMN2 gene having a base sequence in which several nucleotides are different from the SMN1 gene. Is known to exist.
[0013]
In addition, the SMN1 gene usually generates transcripts of all exons from exon 1 to exon 8 by splicing, whereas the SMN2 gene has a property of generating a transcript not containing exon 7 by alternative splicing. Have. Furthermore, the translation product of the SMN2 gene has a property that it cannot complement the function of the translation product of the SMN1 gene. Therefore, according to the nucleic acid of the present invention, exon 7 is contained even when the original function of the SMN gene or the translation product of the SMN gene is deficient, or when the SMN1 gene is deficient or mutated. It produces an SMN transcript and exerts an excellent effect that it can supplement the original function of the SMN gene. Furthermore, according to the nucleic acid of the present invention, since a SMN transcript containing exon 7 can be generated, a disease caused by a deletion or mutation of the SMN gene, specifically, a disease such as spinal muscular atrophy, Can be used for treatment.
[0014]
In the natural world, even in the case of a naturally occurring variant of a natural gene caused by mutation, the product encoded by the variant may exert its original function. Therefore, the nucleic acid of the splicing regulatory element of the present invention may be any of the nucleic acids that regulate the splicing of the SMN gene, specifically, the splicing that causes exon 7, and specifically, the one that positively regulates SEQ ID NO: 1. Alternatively, the nucleotide sequence shown in 2 includes a nucleotide sequence different from the nucleotide sequence through a nucleic acid polymorphism and capable of regulating splicing of the SMN gene. The nucleic acid of the variant preferably has at least one base, ie, one or several bases, different from the base sequence shown in SEQ ID NO: 1 or 2 through a nucleic acid polymorphism, Desirably, the nucleic acid forms a stem-loop structure.
[0015]
The variants can also be artificially produced, for example, by a conventional site-specific mutagenesis method.
[0016]
The nucleotide sequence of a variant of the nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1 is one that regulates, specifically regulates, the splicing of SMN gene, specifically, the splicing that causes exon 7. If it is, specifically, for example,
A base sequence of a nucleic acid consisting of the base sequence shown in SEQ ID NO: 1 and a medium stringent, preferably a base sequence of an antisense strand of a nucleic acid which hybridizes under high stringent conditions;
The nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1 is optimally aligned by the BLAST algorithm [for example, in BLASTN, expected value 10, word size 3, gap cost (Existence: 11, Extension: 1)]. A base sequence having a calculated sequence identity of at least 70%, preferably 80% or more, more preferably 90% or more, and regulates splicing to generate exon 7, specifically, A base sequence of an element that positively regulates, preferably an element that forms a stem-loop,
A nucleotide sequence in which G at position 1 and U at position 2 in SEQ ID NO: 1 have been substituted with A and A, respectively.
A nucleotide sequence in which G at position 3 and G at position 20 in SEQ ID NO: 1 have been substituted with U and U, respectively;
A nucleotide sequence in which A at position 21 and A at position 22 of SEQ ID NO: 1 have been substituted with U and U, respectively.
And the like. The nucleotide sequence of the variant of the nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 2 regulates splicing of the SMN gene, specifically, splicing to generate exon 7, specifically, positively regulates splicing. Whatever is necessary, specifically, for example,
A base sequence of an antisense strand of a nucleic acid that hybridizes with a nucleic acid consisting of the base sequence shown in SEQ ID NO: 2 under moderately stringent conditions, preferably high stringency conditions;
The nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 2 is optimally aligned by the BLAST algorithm [for example, in BLASTN, expected value 10, word size 3, gap cost (Existence: 11, Extension: 1)]. A base sequence having a calculated sequence identity of at least 70%, preferably 80% or more, more preferably 90% or more, and regulates splicing to generate exon 7, specifically, A base sequence of an element that positively regulates, preferably an element that forms a stem-loop,
A nucleotide sequence in which G at position 1 and T at position 2 in SEQ ID NO: 2 have been substituted with A and A, respectively.
A nucleotide sequence in which G at position 3 and G at position 20 in SEQ ID NO: 2 have been substituted with T and T, respectively;
A nucleotide sequence in which A at position 21 and A at position 22 in SEQ ID NO: 2 have been substituted with T and T, respectively;
And the like.
[0017]
In the present specification, the above-mentioned “medium stringent conditions” refers to a composition: 6 × SSC (0.9 M NaCl, 0.09 M sodium citrate, pH 7.0) and 5 × Denhard (0.5% Ficoll)^TM, 0.5% polyvinylpyrrolidine, 0.5% bovine serum albumin) and 0.1% SDS at 37 ° C. overnight with a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 2. And low ionic strength, for example, 2 × SSC, more stringent for conditions such as 0.1 × SSC and / or higher temperature, 37 ° C. or higher, stringent for 50 ° C. or higher, for more stringent , 60 ° C. or higher, and even more stringent, can be achieved by washing under conditions such as 65 ° C. or higher.
[0018]
More specifically, the variant comprises, for example, a nucleic acid having the base sequence of SEQ ID NO: 21, a nucleic acid having the base sequence of SEQ ID NO: 22, and a base sequence of SEQ ID NO: 23. Examples include a nucleic acid, a nucleic acid having the base sequence shown in SEQ ID NO: 24, and a nucleic acid having the base sequence shown in SEQ ID NO: 25.
[0019]
The fact that the nucleic acid forms a stem-loop structure can be evaluated by analyzing the secondary structure using a conventional program such as the MFOLD program (manufactured by Michael Zucker Rensselaer Polytechnic Institute).
[0020]
Also, that the nucleic acid regulates, specifically, positively regulates splicing of the SMN gene, specifically, splicing that generates exon 7
(1) With intron 6 of SMN2 gene
(2) With exon 7 of SMN2 gene
(3) SMN2 gene intron 7 containing the nucleic acid of the splicing regulatory element consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 2;
And the nucleic acid constructs arranged in the order of (1)-(2)-(3) can be evaluated by using an exon trap vector operably linked thereto. Specifically, in the base sequence of intron 7 of (3) in the exon trap vector, the region of the base sequence shown in SEQ ID NO: 2 is replaced with a nucleic acid to be tested, and the resulting construct is The gene is introduced into a suitable host cell, for example, a COS-7 cell, SK-N-SH cell, or the like by a conventional gene transfer method, and the presence of the transcript corresponding to exon 7 in (2) above in the obtained cell is determined. It can be evaluated by detection (referred to as “splicing ability evaluation method”).
[0021]
Here, “positively regulates splicing that generates exon 7” means that splicing is performed so as to generate a transcript containing exon 7 (exon 7-containing type) or to increase the amount of the transcript. Means to control.
[0022]
Examples of the exon trap vector include a vector having an SV40 promoter and having a cloning site between two exons of the HIV-tat gene, and specifically, a pSPL3 vector (manufactured by Promega) and the like. .
[0023]
The intron 6 of (1), the exon 7 of (2), and the intron 7 of (3) can also be obtained based on the sequence of the Genebank Accession No .: AH006635.
[0024]
The nucleic acid construct is described, for example, in Molecular Cloning: A Laboratory Manual, 2nd edition [Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. , (1989)], etc.].
[0025]
In the present invention, in addition to the above-described exon trap vector construct, a plasmid containing a genome-derived sequence of the region from exon 6 to exon 8 of the SMN gene under the control of an appropriate promoter can also be used. Such an embodiment is also included in the present invention.
[0026]
Exon 7 was detected by analyzing the total RNA extracted from the cells by Northern blot analysis using a probe prepared based on the nucleotide sequence of exon 7, a primer pair specific to the vector used, and a specific pair of exon 7 It can be performed by subjecting to RT-PCR analysis or the like using a suitable primer pair. The probe or primer pair may be labeled with a labeling substance (fluorescent substance, radioisotope, etc.) that facilitates such detection.
[0027]
The chain length of the probe is not particularly limited, but is preferably at least 15 consecutive nucleotides, and more preferably at least 18 nucleotides, from the viewpoint of preventing nonspecific hybridization. Examples of the probe include a nucleic acid consisting of a base sequence of exon 7 or its antisense strand, and a nucleic acid consisting of a base sequence that does not exist in a known sequence in the database or an antisense strand thereof, of the base sequence of exon 7; And a nucleic acid having a continuous nucleotide sequence of 15 nucleotides or more in the above nucleotide sequence, or an antisense strand thereof.
[0028]
The chain length of the primer is not particularly limited, but is, for example, 15 to 40 nucleotides, and preferably 17 to 30 nucleotides.
[0029]
The sequence suitable for the probe or the primer can be obtained by, for example, conventional software that supports the design of the probe and / or the primer in consideration of the known sequence, formation of the secondary structure of the nucleic acid, and the like.
[0030]
Since the nucleic acid of the present invention has the above-described nucleotide sequence, it can express the full-length SMN gene, and can supplement the function of the SMN1 gene even when the SMN1 gene is defective or mutated to cause a functional defect. A product derived from the SMN2 gene can be expressed, and a defect in the SMN1 gene that causes spinal muscular atrophy or a defect in the function of the SMN1 gene product caused by mutation of the SMN1 gene can be compensated.
[0031]
Further, the nucleic acid of the present invention can be more efficiently introduced into cells or the like by using the carrier for nucleic acid introduction obtained by incorporating or retaining the nucleic acid into a carrier that can be introduced into cells. Therefore, such a nucleic acid introduction carrier is also included in the present invention.
[0032]
The nucleic acid introduction carrier of the present invention has one major feature in containing the nucleic acid of the present invention. Therefore, since the carrier for nucleic acid introduction of the present invention has the above-mentioned base sequence, it has an excellent effect that the full-length SMN gene can be expressed. Further, since the carrier for nucleic acid introduction of the present invention has the above-mentioned base sequence, even if the SMN1 gene is defective or mutated to cause a functional defect, a product derived from the SMN2 gene that can supplement the function of the SMN1 gene can be obtained. Can be expressed. Furthermore, since the carrier for nucleic acid introduction of the present invention has the above-mentioned nucleotide sequence, it compensates for the deficiency of the SMN1 gene that causes the onset of spinal muscular atrophy or the functional deficiency of the SMN1 gene product caused by mutation of the SMN1 gene. It has an excellent effect of being able to do so.
[0033]
Further, the carrier for nucleic acid introduction of the present invention has one major feature in that it contains the nucleic acid of the present invention, and that the nucleic acid is incorporated or held in a carrier that can be introduced into cells. Therefore, according to the carrier for introducing a nucleic acid of the present invention, the nucleic acid of the present invention can be more efficiently introduced into cells and the like, and more efficiently, a targeting site on a chromosome (for example, intron 7 of the SMN gene). Etc.).
[0034]
Examples of the “carrier that can be introduced into cells” include, for example, vectors, gold particles, liposomes, and the like suitable for cells to be introduced.
[0035]
Examples of the vector include mammalian cell vectors (retroviral vectors, semliki forest virus vectors, papilloma virus, etc.) including promoters that function in host cells (eg, cytomegalovirus (CMV) promoter, SV40 promoter, etc.). Vector, vaccinia virus vector, SV40-based vector, etc.).
[0036]
The carrier for introducing a nucleic acid of the present invention has an arrangement enabling homologous recombination with a targeting site on the chromosome (for example, intron 7 of the SMN gene on the chromosome), and a homologous set of the nucleic acid of the present invention. Alternatively, a nucleic acid construct in which a nucleic acid having a suitable sequence is arranged is also included. The nucleic acid construct may be further incorporated or held in the “carrier that can be introduced into cells”.
[0037]
Examples of the cells include human neuroblastoma SK-N-SH cells, SY-5Y cells, human primary cultured neurons, COS-7 cells, HeLa cells, and 293 cells.
[0038]
The “arrangement enabling homologous recombination” is not particularly limited, but is, in order, exon 7 of the SMN gene,
The nucleic acid of the following a) or b) on intron 7 of the SMN gene:
a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 2, or
b) a nucleic acid of a variant of the nucleic acid according to a), comprising a nucleotide sequence different from the nucleotide sequence shown in SEQ ID NO: 2 through a nucleic acid polymorphism and capable of regulating splicing of the SMN gene
A nucleic acid having at least one copy of
Exon 8 of SMN gene
And the like.
[0039]
According to the nucleic acid of the present invention and the carrier for introducing a nucleic acid of the present invention, splicing of the SMN gene can be regulated, and a method for controlling the expression of the SMN gene is provided.
[0040]
The SMN gene expression control method of the present invention is characterized in that the nucleic acid of the present invention is integrated into a chromosome. Therefore, according to the expression control method of the present invention, since the nucleic acid of the present invention is integrated into the targeting site on the chromosome (ie, intron 7 of the SMN gene), the full-length SMN gene can be expressed, and the SMN1 gene can be expressed. An excellent effect of being able to express a product derived from the SMN2 gene that can supplement the function is exhibited.
[0041]
The expression control method of the present invention includes the following nucleic acid a) or b):
a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 2, or
b) a nucleic acid of a variant of the nucleic acid according to a), comprising a nucleotide sequence different from the nucleotide sequence shown in SEQ ID NO: 2 through a nucleic acid polymorphism and capable of regulating splicing of the SMN gene
A method for controlling the expression of the SMN gene, wherein at least one copy of
(A) a step of introducing the nucleic acid or the carrier for introducing a nucleic acid of the present invention into cells, and
(B) maintaining the cells obtained in step (A) under conditions suitable for splicing;
And the like.
[0042]
The method for controlling the expression of the SMN gene of the present invention may be applied to a disease caused by a deficiency or mutation of the SMN gene, in particular, to an individual suffering from or possibly developing spinal muscular atrophy. Thus, prevention or treatment of the onset of the disease can be performed.
[0043]
In the step (A), from the viewpoint of allowing the nucleic acid of the present invention to efficiently integrate into a targeting site (ie, intron 7 of the SMN gene) by homologous recombination, preferably, a targeting site on a chromosome (for example, For the introduction of a nucleic acid, which is a nucleic acid construct in which the nucleic acid of the present invention and a nucleic acid having a sequence suitable for homologous recombination are arranged in a configuration that enables homologous recombination with the intron 7 of the SMN gene on the chromosome. It is preferable to use a carrier or a carrier for nucleic acid introduction obtained by incorporating or holding the nucleic acid construct in the “carrier capable of being introduced into cells”.
[0044]
Examples of the cells include human neuroblastoma SK-N-SH cells, SY-5Y cells, human primary cultured neurons, COS-7 cells, HeLa cells, and 293 cells. The cells may be cultured cells or cells of an individual (human, non-human mammal, etc.).
[0045]
The amount of the nucleic acid or the carrier for introducing a nucleic acid of the present invention is 100 ng or more, preferably 1 μg or more, more preferably 10 μg or more, and preferably 1000 μg or less, as the amount of nucleic acid. Is preferably 100 μg or less, more preferably 20 μg or less.
[0046]
Examples of the method of introducing the nucleic acid or the carrier for introducing a nucleic acid of the present invention into cultured cell cells include, for example, electroporation, transfection, a calcium phosphate method, a direct introduction method using a gene gun using gold particles, and a lipofection method. . Further, as a method for introducing the nucleic acid of the present invention or a carrier for introducing a nucleic acid into an individual, an in vivo method of directly introducing the individual into the body of the individual via a viral vector or a liposome, extracting certain cells from the individual, Examples of the method include the ex vivo method in which the nucleic acid or the carrier for introducing a nucleic acid of the present invention is introduced into a cell outside the body, and the obtained cell is returned to the body, as in the example of the method of introducing the cell into a cultured cell.
[0047]
Next, in the step (B), the cell into which the nucleic acid of the present invention or the carrier for introducing a nucleic acid of the present invention has been introduced is maintained so that splicing is performed in the cell.
[0048]
Here, when the cell is a cultured cell, splicing is performed by maintaining the cell under culture conditions according to the cell. When directly introduced into cells of an individual, splicing is performed by maintaining normal activity of the individual.
[0049]
In the expression control method of the present invention, mRNA is isolated from cells, and the presence of a transcript corresponding to exon 7 is detected by RT-PCR, Northern blot analysis, or the like, thereby controlling expression of the SMN gene, specifically Can evaluate the splicing of exon 7 of the SMN gene.
[0050]
In addition, according to the nucleic acid and the carrier for introducing a nucleic acid of the present invention, since the full-length SMN gene can be expressed as described above, a pharmaceutical composition for preventing or treating a disease caused by a deletion or mutation of the SMN gene. Things can be provided.
[0051]
The pharmaceutical composition of the present invention has one significant feature in that it contains the nucleic acid of the present invention and a carrier for introducing a nucleic acid as active ingredients. Therefore, the pharmaceutical composition of the present invention expresses a full-length SMN gene, expresses a product derived from the SMN2 gene that can supplement the function of the SMN1 gene, has a SMN1 gene deficiency that causes the onset of spinal muscular atrophy or Since it is possible to compensate for the functional deficiency of the SMN1 gene product caused by the mutation of the SMN1 gene, it is possible to prevent or treat the disease caused by the deficiency or mutation of the SMN gene, in particular, the development of spinal muscular atrophy. It has an excellent effect that it can be done.
[0052]
The content of the active ingredient in the pharmaceutical composition of the present invention can be appropriately set according to the disease to be treated and the degree of the disease state, the age of the patient, the weight, etc., for example, as the amount of the nucleic acid of the present invention, From the viewpoint of the dose at which a medicinal effect can be expected, 0.1 g or more, preferably 1.0 g or more, more preferably several g or more, and 100 g or less, preferably 30 g or less, more preferably 10 g or less. It is desirable.
[0053]
The pharmaceutical composition of the present invention may appropriately contain, in addition to the above-mentioned active ingredient, an auxiliary agent (for example, a buffer solution) capable of stably retaining a nucleic acid, a conventional auxiliary agent depending on the administration form, and the like.
[0054]
Administration of the pharmaceutical composition of the present invention can be performed, for example, in the same manner as in the method of introducing the nucleic acid of the present invention or the carrier for introducing a nucleic acid into an individual in the expression control method of the present invention. Specifically, for example, in the case of administration by the above-mentioned in vivo method, intravenous administration, arterial administration, subcutaneous administration, intradermal administration, muscle, etc., depending on the tissue, organ or the like to be administered via a viral vector, liposome or the like Internal administration, local administration and the like can be performed. When the ex vivo method is used, 1) cells are taken out from an individual, 2) the pharmaceutical composition of the present invention is introduced into the cells by the in vivo method, and 3) the cells are returned to the body of the individual again. It is done by doing.
[0055]
The pharmacological evaluation of the pharmaceutical composition of the present invention can be performed by performing the above-described method for evaluating splicing ability and confirming that exon 7 is included in the transcript of the SMN gene.
[0056]
Further, a splicing enhancer for exon 7 of the SMN gene can be screened using the kinetics and / or splicing ability of the nucleic acid of the present invention as an index. That is, the above-described method for evaluating splicing ability can be applied to screening for a splicing enhancer for exon 7 of the SMN gene. The present invention also includes a method for screening a substance that enhances splicing of exon 7 of the SMN gene.
[0057]
The screening method of the present invention comprises the following (1) to (3) in the presence and absence of (I) a test substance:
(1) With intron 6 of SMN2 gene
(2) With exon 7 of SMN2 gene
(3) SMN2 gene intron 7 containing the nucleic acid of the splicing regulatory element consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 2;
And maintaining a cell comprising an exon trap vector operably linked to the nucleic acid construct, wherein the nucleic acid constructs are arranged in the order of (1)-(2)-(3); and
(II) the amount of a transcript corresponding to exon 7 of the SMN2 gene in the cells obtained in the presence of the test substance in the step (I), and the amount of the transcript obtained in the absence of the test substance in the step (I). Measuring the amount of a transcript corresponding to exon 7 of the SMN2 gene in the cultured cells,
And using as an index the increase in the transcript corresponding to exon 7 of the SMN2 gene due to the presence of the test substance as compared to the absence thereof.
[0058]
The screening method of the present invention has one major feature in using the nucleic acid construct. Since the screening method of the present invention uses the nucleic acid construct, it has an excellent effect that a substance that acts on the splicing ability of the nucleic acid of the present invention can be efficiently screened. In addition, the screening method of the present invention uses the nucleic acid construct to evaluate the splicing ability of the nucleic acid of the present invention as an index. Therefore, a substance capable of enhancing the expression of the full-length SMN gene, SMN2 capable of complementing the function of the SMN1 gene, An excellent effect of efficiently screening a substance or the like capable of enhancing the expression of a gene-derived product is exhibited.
[0059]
The cells used in the screening method of the present invention are the same as those used in the method for evaluating splicing ability.
[0060]
The test substance includes, for example, low-molecular or high-molecular organic compounds, microbial metabolites, and the like.
[0061]
The step (I) can be performed by maintaining the cells in a medium containing a test substance or a medium containing no test substance. Here, examples of the medium include those suitable for cells, and examples include Dulbecco's Modified Eagle Medium (DMEM), α-Minimum Essential Medium (α-MEM), and RPMI1640.
[0062]
Next, the amount of a transcript corresponding to exon 7 of the SMN2 gene in the cells obtained in the presence of the test substance in the step (I) and the cells obtained in the absence of the test substance in the step (I) Then, the amount of a transcript corresponding to exon 7 of the SMN2 gene is measured [Step (II)].
[0063]
In the step (II), the transcript is obtained by a conventional method from the cells obtained in the step (I) to obtain total RNA, and the obtained total RNA is subjected to the same probe or primer pair as in the above-described method for evaluating splicing ability. Can be measured by Northern blot analysis and RT-PCR.
[0064]
In the screening method of the present invention, the presence of the test substance increases the transcript corresponding to exon 7 of the SMN2 gene as compared to the absence thereof, and the test substance is a substance that enhances the splicing of exon 7 of the SMN gene. It is an indicator that
[0065]
In addition, the substance that binds to the nucleic acid of the present invention is expected to regulate splicing of exon 7 of the SMN gene. Therefore, a substance that binds to the nucleic acid of the present invention, in particular, a substance that regulates splicing ability, for example, a binding protein, a mimetic thereof, a compound, and the like can also be screened using the nucleic acid of the present invention. In this case, 1) detecting the presence or absence of binding between the nucleic acid of the present invention and a test substance (protein-containing sample, cell extract, etc.)
2) obtaining a cell into which the test substance bound to the nucleic acid and the nucleic acid construct have been introduced;
3) By measuring the transcript corresponding to exon 7 of the SMN gene in the cell, it is possible to evaluate whether or not the test substance binds to the nucleic acid of the present invention and regulates splicing ability. As a control, cells into which the test substance has not been introduced but into which the nucleic acid construct has been introduced are used.
[0066]
The presence or absence of binding can be detected by a conventional method such as gel mobility shift assay, resonance plasmon interaction analysis and the like.
[0067]
Next, the bound substance is isolated from the test sample, and its structure (for example, amino acid sequence) is identified.
[0068]
Thereafter, cells into which the test substance bound to the nucleic acid and the nucleic acid construct have been introduced are obtained based on the obtained information on the structure.
[0069]
In the present invention, instead of using the above-described exon trap vector construct, a plasmid containing a genome-derived sequence of the region from exon 6 to exon 8 of the SMN gene under the control of an appropriate promoter can also be used. Such an embodiment is also included in the present invention.
[0070]
Furthermore, in the present invention, instead of using the above-described exon trap vector construct, a minigene having a genome-derived sequence of the region from exon 6 to exon 8 of the SMN gene is transcribed as pre-mRNA, and the obtained product is tested. By reacting with a nuclear extract or a transcription system in a tube and analyzing by electrophoresis or the like, a substance that enhances splicing of exon 7 of the SMN gene can also be screened. Such an embodiment is also included in the present invention.
[0071]
【Example】
Hereinafter, the present invention will be described specifically with reference to the following examples, but the present invention is not limited to these examples. In the following examples, unless otherwise specified, the composition of the culture medium is described in “Basic Experimental Methods for Molecular and Cellular Biology”, pages 50 to 51, published on September 1, 1994, published by Nankodo Co., Ltd., Molecular Cloning: A Laboratory Manual 2nd edition [Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. , (1989)].
[0072]
Example 1
For the in vivo splicing of the SMN gene, constructs of the SMN1 minigene containing the region from exon 6 to exon 8 in the pCI mammalian expression vector and the SMN2 minigene containing exon 6 to exon 8 in the pCI mammalian expression vector were examined. Constructs (gifted by Drs. Elliott Androphy (Tufts University) and Christian Lorson) were used.
[0073]
In addition, a minigene construct containing the SMN1 exon 6 to exon 8 region in the pCI vector and containing a C to T translocation in exon 7 was prepared by mutagenizing element 1 by a site-directed mutagenesis method. did.
[0074]
For transfection of the construct, COS-7 cells or SK-N-SH cells were used. The COS-7 cells were cultured in 10% fetal bovine serum (FBS) / Dulbecco's Modified Eagle Medium (DMEM) at 37 ° C. and 5% CO 2.₂SK-N-SH cells were cultured at 37 ° C., 5% CO 2 on α-Minimum Essential Medium (α-MEM) containing 10% FBS.₂Was performed under the following conditions. The cells were seeded on a 3.5 cm dish at a density of 60-80% confluency.
[0075]
Then, using the trade name: LipofectAMINE reagent or the trade name: LipofectAMINE ACE reagent (manufactured by Life Technologies) according to the manufacturer's protocol, the above-mentioned construct (1.0 μg equivalent) was added to COS-7 cells or SK-7 cells. -N-SH cells were transfected.
[0076]
The obtained transfected cells were dissolved in a buffer RLT (manufactured by Qiagen), and total RNA was obtained from the obtained lysate using a trade name: RNeasy Mini Kit (manufactured by Qiagen). Was isolated.
[0077]
The obtained total RNA, a random primer (manufactured by Takara Bio Inc.), and MMLV reverse transcriptase (manufactured by Life Technologies) were mixed with 20 μl of a reaction solution [composition: 2.7 μM random primer Hexa-deoxyribonucleotide] Mixture (manufactured by Takara Bio Inc.), 0.5 mM dNTP mixture, 12.5 mM DTT, 5 × first strand buffer, 200 UM-HLV reverse transcriptase) at 37 ° C. for 60 minutes to react the first strand cDNA. Was synthesized.
[0078]
Then, 1.2 μg of the first-strand cDNA generated from the construct, 0.4 μM of each pCI forward primer [5′-GCT AAC GCA GTC AGT GCT TC-3 ′ (SEQ ID NO: 3)] and pCI reverse primer A primer pair consisting of [5′-GTA TCT TAT CAT GTC TGC TCG-3 ′ (SEQ ID NO: 4)] and a trace amount of [α-³²P] PCR was carried out on a 50 μl reaction mixture containing 0.2 μM dNTP containing dNTP, 5 U rTaq DNA polymerase and 10 × PCR buffer (manufactured by Takara Bio Inc.), and splicing in vivo was examined. .
[0079]
The amplification conditions were as follows: a starting denaturation step by incubation at 94 ° C. for 2 minutes, followed by a 30-cycle reaction step consisting of 94 ° C. for 30 seconds and 56 ° C., 1.5 minutes and 72 ° C. for 1 minute. After that, conditions were set for performing a final extension step at 72 ° C. for 10 minutes.
[0080]
The obtained reaction products were separated by electrophoresis on a 5% polyacrylamide gel. The intensity of each band was quantified using a trade name: Densitography Program (manufactured by ATTO Corporation). The ratio of the exon 7-containing type was quantified and expressed as the ratio of the intensity of the exon 7-containing type to the intensity of all bands. The result is shown in FIG.
[0081]
As a result, as shown in FIG. 1, SMN1 mRNA expresses a full-length transcript, but SMN2 expresses a full-length transcript by transient transfection of each of the SMN1 minigene construct and the SMN2 minigene construct including the region from exon 6 to exon 8. It produced low levels of full-length transcripts and high levels of exon 7 deficient isoforms (SMNΔ7).
[0082]
Also, as shown in FIG. 1, the substitution of C to T present at 6 nucleotides in exon 7 of SMN1 results in the exclusion of exon 7, and the splicing pattern of exon 7 changes to exon 7 of wild-type SMN2. It turned out to be similar. In contrast, the mutant SMN2 (T to C substitution in SMN2) minigene was found to produce a full-length SMN transcript.
[0083]
Example 2
To determine cis-acting elements that can respond to SMN exon 7 skipping, a wild-type SMN1 minigene containing exon 7 and adjacent intron 6 and adjacent intron 7 and mutant SMN1 (C to T transposition in exon 7) Various deletion mutants, both minigenes, were constructed and these were cloned into the exon trap vector pSPL3. A schematic diagram of such a deletion mutant is shown in FIG.
[0084]
Various deletion mutants of SMN1 containing intron 6, exon 7 and intron 7 were identified using the following primer pairs:
For I7DM1, 600 Fwd primer [5'-AAG CTT GGC ATG AGC CAC TGC AAG AAA AC-3 '(SEQ ID NO: 5)] and OR primer [5'-GGA TCC GAG AAT TCT AGT AGG GAT GTAG G-3' ( A primer pair consisting of: SEQ ID NO: 6)];
For I7DM2, a primer pair consisting of the 600 Fwd primer and an OR-IR primer [5'-AAG CTT GTT TTA CAT TAA CCT TTC AAC T-3 '(SEQ ID NO: 7)];
For I7DM3, a primer pair consisting of the aforementioned 600Fwd primer and DR-11R primer [5′-GGA TCC GAA CTT TTT AAA TGT TCA AAA AC-3 ′ (SEQ ID NO: 8)];
I7DM4 was prepared by PCR using the 600 Fwd primer and IR primer [5'-GGA TCC CAA AAA CCA TAA AGT TTT AC-3 '(SEQ ID NO: 9)].
[0085]
In addition, deletion mutants constructed from wild-type SMN1 containing no translocation on exon 7 are referred to as I7DM1-C, I7DM1-C, I7DM1-C and I7DM1-C, respectively, Deletion mutants constructed from mutant SMN1 containing a transposition to T may be referred to as I7DM1-T, I7DM1-T, I7DM1-T, and I7DM1-T, respectively.
[0086]
Then, each of the obtained PCR products was digested with Not I and BamH I, and the obtained products were inserted into exon trap vector pSPL3 (manufactured by Life Technologies).
[0087]
The obtained PCR product was ligated to an exon trap vector pSPL3 (manufactured by Life Technologies) using a trade name: BKL kit (manufactured by Takara Bio Inc.). Before the experiment, all the obtained constructs were sequenced.
[0088]
COS-7 cells were transfected using the above-described construct, and total RNA was isolated from the transfected COS-7 cells 24 hours later using a trade name: RNeasy Mini Kit (manufactured by Qiagen). Released.
[0089]
Subsequently, the obtained total RNA (equivalent to 3.0 μg) was used as a primer for SA2 [5′-ATC TCA GTG GTA TTT GTG AGC-3 ′ (SEQ ID NO: 10)] and MMLV reverse transcriptase [Life Technology (Life Technology (Life Technology) Technologies)) to obtain a spliced product.
[0090]
The splicing product generated by the reverse transcription reaction is detected by PCR using the pSPL3 vector-specific primer pair, the SD6 primer [5′-TCT GAG TCA CCT GGA CAA CC-3 ′ (SEQ ID NO: 11)] and the SA2 primer. did. The PCR was performed using a reaction solution of a total volume of 40 μl [composition: 1.5 μl RT product, 1.0 μM SD6 primer, 1.0 mM SA2 primer, 0.2 mM dNTP mixture, 10 × PCR buffer, 5 U rTaq (manufactured by Takara Bio Inc.) )]. Amplification was performed by performing 30 cycles of a reaction consisting of 94 ° C. for 1 minute, 60 ° C. for 1 minute and 72 ° C. for 1 minute, followed by incubation at 72 ° C. for 5 minutes.
[0091]
The obtained PCR product was electrophoresed on a 5% polyacrylamide gel.
[0092]
As a result, as shown in the upper panel of FIG. 3, when various deletion mutants related to wild-type SMN1 (C) were transfected into COS-7 cells, all constructs MRNA (+ exon 7) was expressed, but no isoform lacking exon 7 was expressed.
[0093]
In contrast, as shown in the lower panel of FIG. 3, the longest construct of mutant SMN1 (I7DM1) containing the C to T transposition in SMN1 exon 7 and the flanking intron 6 of 235 bp and flanking intron 7 of 124 bp. , MRNA with both exon 7 containing and exclusion (51% containing).
[0094]
As shown in the lower panel of FIG. 3 (I7DM1), the percentage of exon-containing forms using the C to T transgene minigene containing exon 6 to exon 8 cloned into the exon trap vector was cloned into the pCI vector. The ratio was higher than that of the exon-containing form using the SMN minigene including the region of exon 6 to exon 8 which had been converted. Such differences indicate that there may be several cis-acting elements that regulate the splicing of SMN exon 7 in regions that differ from the sequence of the minigene cloned into the exon trap vector (about 400 bp). However, since these minigenes, including the C to T translocation in SMN1 exon 7 cloned into exon trap vector pSPL3, showed a marked increase in exon 7 exclusion, these constructs were associated with exon 7 in the 400 bp minigene. It appears to be useful in determining cis elements that can respond to elimination.
[0095]
For the deletion mutant in the flanking intron, as shown in FIG. 1, the deletion mutant I7DM4-T containing a 66 bp deletion of the 3 ′ flanking region of I7DM1-T increased the elimination of SMN exon 7 ( The splicing pattern of the deletion of mutant I7DM3-T, which contains a 52 bp deletion of I7DM1-T, was similar to that of I7DM1-T.
[0096]
Thus, the +59 to +72 14 bp region of flanking intron 7 is suggested to be a key element of the SMN exon-containing form involving the C to T transposition.
[0097]
FIG. 4 shows the position of the element 2 in the intron 7. In addition, as shown in FIG. 4, analysis of the higher-order structure by the MFOLD program GCG version revealed that element 2 (SEQ ID NO: 1) composed of 24 nucleotides had a unique stem-loop structure. . Furthermore, the sequence shows a perfect match between SMN1 and SMN2, suggesting that element 2 is important in regulating the splicing of SMN1 exon 7 and wild-type SMN2, including C to T transposition. Was.
[0098]
Example 3
To determine whether the stem-loop structure in element 2 is important in regulating the splicing of SMN exon 7, including the C to T transition, various mutations were made to the stem-loop structure in element 2 of I7DM1-T. Was introduced.
[0099]
Mutations in the stem-loop structure were performed using an I7DM1-T exon trap vector as a template, and nucleotides for introducing mutations [5'-CCC CTG AAC ATT TAA AAA GTT CAG ATG-3 '(SEQ ID NO: 12) and 5'-CAT TTG It was generated by PCR using TTT TCC ACA AAC CAT AAA GTT TTA-3 ′ (SEQ ID NO: 13)].
[0100]
COS-7 cells were transfected using the above-described construct, and total RNA was isolated from the transfected COS-7 cells 24 hours later using a trade name: RNeasy Mini Kit (manufactured by Qiagen). Released.
[0101]
Subsequently, the obtained total RNA (equivalent to 3.0 μg) was reverse-transcribed using the SA2 primer and MMLV reverse transcriptase (Life Technologies) to obtain a spliced product.
[0102]
The spliced product generated by the reverse transcription reaction was detected by PCR using the pSPL3 vector-specific primer pair, the SD6 and the SA2. The PCR was performed using a reaction solution of a total volume of 40 μl [composition: 1.5 μl RT product, 1.0 μM SD6 primer, 1.0 mM SA2 primer, 0.2 mM dNTP mixture, 10 × PCR buffer, 5 U rTaq (manufactured by Takara Bio Inc.) )]. Amplification was carried out by performing 30 cycles of a reaction at 94 ° C. for 1 minute, 60 ° C. for 1 minute and 72 ° C. for 1 minute, followed by incubation at 72 ° C. for 5 minutes.
[0103]
The obtained PCR product was electrophoresed on a 5% polyacrylamide gel.
[0104]
As a result, as shown in FIG. 5 panel (B), from the results of using construct A and construct B of FIG. 5 panel (A), the mutation or deletion of the loop region affected the splicing of exon 7. , Suggesting that the loop of element 2 is not important for splicing regulation.
[0105]
In contrast, the disruption of the stem structure, as shown in panel (B) of FIG. 5, resulted from the use of construct C and construct D of panel (A) of FIG. 5, as compared to the original structure of I7DM1-T. Cause a significant decrease in exon 7-containing form and the effect on exon 7-containing decrease was similar to that of deletion mutant I7DM4-T, indicating that the stem structure is important for the activity of element 2. all right.
[0106]
In addition, a G to C point mutation in the stem structure, as shown in panel (B) of FIG. 5, resulted in a dramatic increase in the exon 7 containing form as a result of using construct E of panel (A) of FIG. Due to the decrease, it was found that the CG base pair in the stem structure was also important for the splicing activity of element 2. On the other hand, as shown in panel (B) of FIG. 5, when construct F of panel (A) of FIG. 5 was used, when AU base pairs in the stem structure were replaced with GC base pairs, Exon 7 content was slightly reduced.
[0107]
Such findings suggest that the higher order stem structure of element 2 is important, but that the sequence of AU base pairs is not so important.
[0108]
Example 4
To test whether element 2 activates the splicing of SMN exon 7, including the C to T translocation, constructs containing the tandem repeats (3 or 4 repeats) of element 2 in the I7DM1-T vector [FIG. In panel (A) of No. 6, element 2x3 and element 2x4] were replaced with I7DM1-T vector and nucleotides for mutagenesis [5'-GTG GAA AAC AAA TGT TTT TGA ACA GTG GAA AAC AAA TGT TTT TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA T AAA TGT TTT TGA ACA TTT AAA AAG TTC-3 ′ (SEQ ID NO: 14) and 5′-AAA CCA TAA AGT TTT ACA AAA GTA AGA TTC AC-3 ′ (SEQ ID NO: 15) ] Was prepared by PCR.
[0109]
COS-7 cells were transfected using the above-described construct, and total RNA was isolated from the transfected COS-7 cells 24 hours later using a trade name: RNeasy Mini Kit (manufactured by Qiagen). Released.
[0110]
Subsequently, the obtained total RNA (equivalent to 3.0 μg) was reverse-transcribed using the SA2 primer and MMLV reverse transcriptase (Life Technologies) to obtain a spliced product.
[0111]
The spliced product generated by the reverse transcription reaction was detected by PCR using the pSPL3 vector-specific primer pair, the SD6 and the SA2. The PCR was performed using a reaction solution of a total volume of 40 μl [composition: 1.5 μl RT product, 1.0 μM SD6 primer, 1.0 mM SA2 primer, 0.2 mM dNTP mixture, 10 × PCR buffer, 5 U rTaq (manufactured by Takara Bio Inc.) )]. Amplification was performed by performing 30 cycles of a reaction consisting of 94 ° C. for 1 minute, 60 ° C. for 1 minute and 72 ° C. for 1 minute, followed by incubation at 72 ° C. for 5 minutes.
[0112]
The obtained PCR product was electrophoresed on a 5% polyacrylamide gel.
[0113]
As a result, as shown in FIG. 6 panel (B), it was found that the insertion of multicopy element 2 in intron 7 of SMN increased the content of SMN exon 7 depending on the number of insertions. Element 2 was shown to be an intron splicing enhancer of SMN exon 7 involving the C to T transposition.
[0114]
Example 5
Since disruption of element 2 shows a dramatic change in the splicing pattern of SMN exon 7, it was speculated that trans-acting factors would specifically bind to element 2 and regulate its splicing via direct binding. .
[0115]
Therefore,³²A gel shift assay was performed using the P-labeled oligo element 2 and a nuclear extract of neuroblastoma SK-N-SH cells.
[0116]
SK-N-SH cells (1.0 × 10⁷Cells) in 1 ml of 10 mM HEPES-NaOH (pH 7.9) containing 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 5 mM dithiothreitol (DTT) and 1 mM (p-amidinophenyl) methanesulfonyl fluoride (PMSF). Homogenized at 4 ° C. The buffer and other solutions used in this experiment were sterilized by filtration with Steritop (pore size: 220 nm, manufactured by Millipore Corporation) before use.
[0117]
After adding 10% Nonidet P-40 (trade name) to a final concentration of 0.6%, the homogenate was centrifuged at 15,000 rpm for 5 minutes. The pellet was resuspended in 10 volumes of 20 mM Tris-HCl (pH 7.5) containing 400 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 1 mM PMSF and centrifuged at 15,000 rpm for 5 minutes. The resulting pellet was stored at -80 ° C as a nuclear extract for a gel mobility shift assay.
[0118]
Then, a final volume of 20 μl (binding buffer: 20 mM HEPES, pH 7.9, 72 mM KCl, 1.5 mM MgCl₂, 0.78 mM magnesium acetate, 0.52 mM DTT, 3.8% glycerol, 0.75 mM ATP, 1 mM GTP) and various concentrations of nuclear extract mixed with RI-labeled RNA oligonucleotides (present as 6 nM final concentration) did. As the RI-labeled oligonucleotide, oligo-element 2 [5'-GUG GAA AAC AAA UGU UUU UGA ACA-3 '(SEQ ID NO: 16)] or oligo mutant element 2 [5'-GUG GAA AAC AAA UGC CCC] UGA ACA-3 '(SEQ ID NO: 17)] was used.
[0119]
The resulting mixture is then incubated at room temperature for 25 minutes, then loaded on a 4% native acrylamide gel in loading buffer (composition: 37.5 mM Tris-HCl, 315 mM glycine, 1.5 mM EDTA) and subjected to constant Electrophoresis was performed at 4 ° C. for 2 hours at a voltage of 100 V. The gel was then dried and exposed to an autoradiographic film.
[0120]
As a result, as shown in FIG. 7 panel (A), effective binding was observed, and the binding activity was increased corresponding to the amount of nuclear extract.
[0121]
Further, in order to confirm that the binding was specific, a competition experiment using unlabeled oligo element 2 was performed.
[0122]
As a result, as shown in FIG. 7 panel (B), pre-incubation of nuclear extracts with more than 25-fold molar concentrations of unlabeled oligo RNA resulted in almost complete removal of activity, but with TC Preincubation of the nuclear extract with an equal amount of unlabeled nucleotides for mutagenesis of element 2 (oligo mutant element 2) into which mutation was introduced by substitution did not inhibit the binding of RNA nucleoprotein. These data indicate that the trans-acting factor specifically binds to element 2 in intron 7 of the SMN pre-mRNA and that binding to cis-acting element 2 results in splicing of SMN exon 7, including C to T translocation. Indicates that it will enhance.
[0123]
Example 6
Element 2 of intron 7 of the SMN gene has been shown to play a key role in regulating exon 7 splicing and that trans-acting proteins can bind directly to this element. Thus, it was determined whether treatment of cells transfected with the I7DM1-T exon trap vector with an antisense oligonucleotide was sufficient to lead to a decrease in the SMN exon 7 containing type, including C to T translocation. .
[0124]
As the antisense oligonucleotide, an antisense oligonucleotide containing a 2'-O-methyl backbone modification and an antisense oligonucleotide containing a phosphorothioate backbone modification (synthesized by Japan Bio Service Inc) were used. The antisense oligonucleotide used was
Antisense oligonucleotide for element 2 [in FIG. 8, As-element 2; 5'-UGU UCA AAA ACA UUU GUU UUC-3 '(SEQ ID NO: 18)],
Antisense oligonucleotide to polypyrimidine tract of intron 6 of SMN1 [In FIG. 8, As-pyr; 5'-GAU AGC UAU AUA UAG AU-3 '(SEQ ID NO: 19)]
As a control, antisense oligonucleotides to other sequences of intron 6
[In FIG. 8, As-con; 5'-GAU AGC UAU AUA UAG AU-3 '(SEQ ID NO: 20)].
[0125]
These antisense oligonucleotides were co-transfected into COS-7 along with the exon trapping vector into which I7DM1-T was cloned. In addition, the concentration of the antisense oligonucleotide was adjusted to be in the range of 5 nM to 25 nM.
[0126]
Twenty-four hours after transfection, total RNA was extracted by a conventional method. RT-PCR using the primer pair [SD6 primer and SA2 primer] was performed to examine the effects of these antisense oligonucleotides on in vivo splicing of SMN exon 7.
[0127]
As a result, as shown in FIG. 8 panel (B), it was found that the As-element 2 reduced the SMN exon 7-containing type depending on the amount of the antisense oligonucleotide. As shown in FIG. 8 panel (C), the splicing of SMN exon 7 was not changed by As-con. In contrast, As-pyr slightly reduced the contained form.
[0128]
Since polypyrimidine tracts are essential for pre-mRNA splicing, it was expected that the As-pyr would effectively act as an antisense oligonucleotide blocking cis-acting elements.
[0129]
Similarly, As-element 2 caused an inhibition of SMN exon 7 splicing.
[0130]
These results indicate that element 2 is a key cis-acting element for SMN exon 7 splicing involving the C to T translocation.
[0131]
Sequence listing free text
SEQ ID NO: 1 is a sequence of the sequence unit of Element 2.
[0132]
SEQ ID NO: 2 is a sequence in a sequence unit of Element 2.
[0133]
SEQ ID NO: 3 is a sequence of a pCI forward primer.
[0134]
SEQ ID NO: 4 is a sequence of a pCI reverse primer.
[0135]
SEQ ID NO: 5 is a sequence of a 600 Fwd primer.
[0136]
SEQ ID NO: 6 is a sequence of an OR primer.
[0137]
SEQ ID NO: 7 is a sequence of an OR-IR primer.
[0138]
SEQ ID NO: 8 is a sequence of DR-11R primer.
[0139]
SEQ ID NO: 9 is a sequence of an IR primer.
[0140]
SEQ ID NO: 10 is a sequence of SA2 primer.
[0141]
SEQ ID NO: 11 is a sequence of SD6 primer.
[0142]
SEQ ID NO: 12 is a sequence of a mutation-introducing oligonucleotide.
[0143]
SEQ ID NO: 13 is a sequence of a mutation-introducing oligonucleotide.
[0144]
SEQ ID NO: 14 is a sequence of a mutation-introducing oligonucleotide.
[0145]
SEQ ID NO: 15 is a sequence of a mutation-introducing oligonucleotide.
[0146]
SEQ ID NO: 16 is the sequence of Oligo-Element 2.
[0147]
SEQ ID NO: 17 is the sequence of oligo-variant element 2.
[0148]
SEQ ID NO: 18 is the sequence of Antisense Oligonucleotide As-Element 2.
[0149]
SEQ ID NO: 19 is the sequence of antisense oligonucleotide As-pyr.
[0150]
SEQ ID NO: 20 is the sequence of antisense oligonucleotide As-con.
[0151]
SEQ ID NO: 21 is the sequence of a putative splicing regulatory element.
[0152]
SEQ ID NO: 22 is the sequence of the putative splicing regulatory element.
[0153]
SEQ ID NO: 23 is the sequence of a putative splicing regulatory element.
[0154]
SEQ ID NO: 24 is the sequence of a putative splicing regulatory element.
[0155]
SEQ ID NO: 25 is the sequence of a putative splicing regulatory element.
[0156]
SEQ ID NO: 26 is a sequence of Mutant A of Element 2
[0157]
SEQ ID NO: 27 is a sequence of mutant B of element 2.
[0158]
SEQ ID NO: 28 is a sequence of Mutant C of Element 2
[0159]
SEQ ID NO: 29 is a sequence of mutant D of element 2.
[0160]
SEQ ID NO: 30 is a sequence of mutant E of element 2.
[0161]
SEQ ID NO: 31 is a sequence of mutant F of element 2.
[0162]
【The invention's effect】
According to the nucleic acid of the present invention, a full-length SMN gene can be expressed, a product derived from the SMN2 gene capable of complementing the function of the SMN1 gene can be expressed, and the SMN1 gene causing the onset of spinal muscular atrophy can be expressed. An excellent effect of being able to compensate for the deficiency of the SMN1 gene product caused by the deficiency or mutation of the SMN1 gene. Further, the carrier for nucleic acid introduction of the present invention has an excellent effect that the nucleic acid of the present invention can be efficiently introduced into cells. Furthermore, according to the method for controlling the expression of the SMN gene of the present invention, the full-length SMN gene can be expressed, and a product derived from the SMN2 gene, which can complement the function of the SMN1 gene, can be expressed. The present invention has an excellent effect of compensating for the SMN1 gene deficiency that causes the above or the SMN1 gene product function deficiency caused by the mutation of the SMN1 gene. Further, the pharmaceutical composition of the present invention has an excellent effect of being able to prevent or treat a disease caused by a deletion or mutation of the SMN gene, in particular, the onset of spinal muscular atrophy. Further, according to the method for screening a substance for enhancing splicing of exon 7 of the SMN gene of the present invention, a substance capable of enhancing the expression of the full-length SMN gene and a substance capable of enhancing the expression of a SMN2 gene-derived product capable of complementing the function of the SMN1 gene Etc. can be efficiently screened.
[0163]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the splicing patterns of SMN1 and SMN2 exons. In the figure, the upper band of each lane indicates a full-length transcript containing the region from exon 6 to exon 8, and the lower band indicates a transcript lacking exon 7. In the figure, WT SMN1 (C) is a wild-type minigene, mutant SMN1 (T) is a SMN1 minigene containing a translocation of C to T located at 6 nucleotides inside exon 7, and WT SMN2 (T) is , Wild-type SMN2 minigene, mutant SMN2 (C) indicates a SMN2 minigene containing a T to C substitution located at 6 nucleotides in exon 7.
FIG. 2 shows a schematic diagram of an exon trap vector. In the figure, BP indicates a branch point, and PPT indicates a polypyrimidine tract.
FIG. 3 shows the results of an in vitro splicing assay. The upper panel is the result of a deletion mutant constructed from wild-type SMN1 (C) and the lower panel is the result of a deletion mutant constructed from a mutant SMN1 containing a C to T transposition. . The score shown in the lower panel is the ratio of the exon 7-containing type to the total transcripts, and the value indicates the average of four analyzes.
FIG. 4 shows a schematic diagram of the position (upper panel) and structure (lower panel) of the element 2 in the intron 7.
FIG. 5 shows the results of examining the effect of a mutation in the stem-loop of element 2 of mutant SMN1 containing a C to T transition on the amount of exon 7-containing transcripts. Panels (A) -A-F are schematic diagrams of the structures of various mutants in the I7DM1-T minigene. In the figure, ○ indicates nucleotides substituted for wild-type element 2. Panel (B) shows the results of in vivo boss pricing analysis. In the figure, minigenes A to F cloned into I7DM1-T, exon trap vector correspond to panel (A). Panel (C) shows the results of quantitative analysis of splicing patterns of each construct. The ratio of the exon 7-containing type to the total transcripts is the mean of four analyzes ± SEM. It is represented by D.
FIG. 6 shows the results of examining the effect of the number of inserted elements 2 on the amount of a transcript containing exon 7. Panel (A) shows a schematic of a construct with a tandem repeat of element 2 in the SMN intron. In the figure, I7DM1-T represents an original construct containing one copy of element 2, element 2-Tx3 represents a construct containing three copies of element 2, and element 2-Tx4 represents a construct containing four copies of element 2. Panel (B) shows RT-PCR analysis of the amount of transcripts containing exon 7 according to the number of inserted elements 2 (upper panel) and the ratio of exon 7-containing type to total transcripts (lower panel). The ratio of the exon 7-containing form to the total transcripts is the mean ± S.D. It is represented by D.
FIG. 7 is a diagram showing the results of a gel mobility shift assay for identifying a nucleoprotein that binds to element 2. Panel (A) shows the results of gel mobility shift assay of SK-N-SH cell nuclear extract. Panel (B) shows the results of a competition assay performed by adding unlabeled RNA oligonucleotides of wild-type element 2 or mutant element 2 (above 1, 5, and 14 molar concentrations).
FIG. 8 shows the results of examining the effect of an antisense oligonucleotide for element 2 on the expression of a transcript containing exon 7. Panel (A) shows the positions of the three antisense oligonucleotides used (As-element 2, As-con, As-pyr) on the SMN gene. Panel (B) shows the results of RT-PCR analysis of in-vivo splicing of As-Element 2 using an exon trapping system. The percentage of the exon 7 containing version is shown as an average of four experiments and is shown below each lane. Panel (C) shows the results of RT-PCR analysis of in-vivo splicing of As-con using an exon trapping system. The percentage of the exon 7 containing version is shown as an average of four experiments and is shown below each lane.

Claims (7)

A nucleic acid for a splicing regulatory element consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 2. 2. The nucleic acid variant nucleic acid according to claim 1, comprising a nucleotide sequence different from the nucleotide sequence shown in SEQ ID NO: 1 or 2 through a nucleic acid polymorphism and capable of regulating splicing of the SMN gene. The nucleic acid according to claim 2, wherein the variant comprises a base sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25. A carrier for introducing a nucleic acid, comprising the nucleic acid according to claim 1. A nucleic acid according to a) or b) below:
a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 2, or b) a nucleotide sequence different from the nucleotide sequence of SEQ ID NO: 2 through a nucleic acid polymorphism, and regulating the splicing of the SMN gene. A method for controlling the expression of the SMN gene, wherein at least one copy of the nucleic acid of the variant of the nucleic acid according to a) is integrated into a chromosome. A pharmaceutical composition for preventing or treating a disease caused by deletion or mutation of the SMN gene, comprising the nucleic acid according to any one of claims 1 to 3 or the carrier for introducing a nucleic acid according to claim 4 as an active ingredient. object. (I) In the presence and absence of a test substance,
The following (1) to (3):
(1) Intron 6 of the SMN2 gene and (2) exon 7 of the SMN2 gene and (3) intron 7 of the SMN2 gene containing the nucleic acid of the splicing regulatory element consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 2. And (1) maintaining a cell containing an exon trap vector construct operably linked to the nucleic acid construct arranged in the order of (2)-(3); and (II) in the step (I), The amount of the transcript corresponding to exon 7 of the SMN2 gene in the cells obtained in the presence of the test substance and the amount of the transcript corresponding to exon 7 of the SMN2 gene in the cells obtained in the absence of the test substance Measuring the amount and
A method for screening a substance that enhances the splicing of exon 7 of exon 7 of the SMN gene as an index, wherein the presence of the test substance increases the transcript corresponding to exon 7 of the SMN 2 gene as compared to the absence thereof.

Images