JP4242128B2 - Screening method for cerebral amyloidosis preventive and therapeutic drug - Google Patents

Description

【図面の簡単な説明】
【図１】コントロール及びAD脳におけるHMG1の発現レベル及び分布を示す図及び写真である。ａ及びｂ：側頭皮質の細胞質（ａ）及び粒子（ｂ）画分のイムノブロット写真（上図）及び２９ｋＤａのバンド濃度を示すグラフ（下図；縦軸はコントロールに対する％割合；各数値は６検体の平均±標準誤差；個々の数値は丸印で示す）。ｃ−ｈ：コントロール及びAD脳切片のHMG1（ｃ−ｅ，ｈ）、HLA-DR（ｆ，ｈ）又はAβ（ｇ）に対する抗体を用いた免疫染色写真。切片（ｅ−ｈ）は同一個体由来の一連のものであり、＊（ｅ−ｇ）は同一の脈管を示す。矢印はインセットに高倍率で示されたと同じ老人斑を示す。ｈはHMG1及びHLA-DRに対する抗体を用いた老人斑の二重染色。スケールバー：２５０μｍ（ｅ−ｇ）及び５０μｍ（ｈ及びｅ−ｇのインセット）。
【図２】トランスジェニックマウス脳におけるＨＭＧ１の局在性を示す写真である。12月齢のPS1（ａ，ｃ，ｅ，ｇ）及びPS/APPマウス（ｂ，ｄ，ｆ，ｈ）の前頭脳切片のCD11b（MAC1，ａ−ｄ）、Aβ（ｅ−ｈ）又はHMG1（ｇ，ｈ）に対する抗体を用いた免疫染色。ボックスの高倍率像をインセットで示す（ｃ−ｈ）。スケールバー：１ｍｍ（ａ，ｂ）、２００μｍ（ｃ−ｈ）及び５０μｍ（ｃ−ｈ中のインセット）。
【図３】 KAを注射したラット脳におけるHMG1の局在性を示す写真である。海馬切片のMAP2（ａ，ｂ）、HMG1（ｃ−ｅ，ｇ，ｉ−ｌ）、Aβ（ｆ，ｈ）、CD11b（OX42，ｊ，ｌ）又はGFAP（ｉ，ｋ）に対する抗体を用いた免疫染色。脳室内KA注射と対側（ａ，ｃ，ｅ，ｆ，ｉ，ｊ）及び同側（ｂ，ｄ，ｇ，ｈ，ｋ，ｌ）。海馬CA3中のボックスの高倍率像（ｅ−ｌ）。ｉ−ｌ：HMG1及びCD11b（ｊ，ｌ）又はGFAP（ｉ，ｋ）に対する抗体を用いた二重染色。スケールバー：５００μｍ（ａ−ｄ）及び５０μｍ（ｅ−ｌ）。
【図４】 Aβ42を注射したラット海馬におけるHMG1の局在性を示す写真である。海馬切片のAβ（ａ，ｅ，ｇ）HMG1（ｃ，ｅ，ｆ）、MAP2（ｂ）又はCD11b（OX42，ｄ，ｆ，ｇ）に対する抗体を用いた免疫染色。ｅ，ｆ：HMG1及びAβ（ｅ）又はCD11b(ｆ)に対する抗体を用いた二重染色。免疫染色された各切片（ａ−ｆ）中のボックスの高倍率像をインセットで示す。矢印は抗HMG1抗体による瀰漫性染色を示す。ｇ：Aβ及びCD11bに対する抗体を用いた海馬中のAβ沈着周辺の二重染色の共焦点顕微鏡像。スケールバー：５００μｍ（ａ−ｆ）及び５０μｍ（ａ−ｆ中のインセット，ｇ）。
【図５】ミクログリアによるAβ42食作用に対するHMG1の阻害作用を示す写真及び図である。ａ及びｂ：ビヒクル（ａ中のレーンＣ及びｂ中のControl）又は種々の濃度のHMG1の存在下でAβ42とインキュベートしたラットミクログリアライセートのイムノブロット写真（ａ）及び総Aβ42量を示すグラフ（縦軸はコントロールに対する％割合；各数値は３つの実験の平均±標準誤差；**、***はコントロールの数値に対してそれぞれp<0.01及びp<0.001を示す）。ｃ及びｄ：ビヒクル（ｃ中のレーンTGF-β1(-)及びｄ中のVehicle）又はTGF-β1（ｃ中のレーンTGF-β1(+)及びｄ中のTGF-β1）の存在下で、Aβ42（ｃ中のHMG1(-)及びｄ中のControl）又はAβ42及びHMG1（ｃ中のHMG1(+)及びｄ中のHMG1）とインキュベートしたラットミクログリアライセートのイムノブロット写真（ａ）及び総Aβ42量を示すグラフ（縦軸はコントロールに対する％割合；各数値は３つの実験の平均±標準誤差；***、†††はそれぞれビヒクル及びTGF-β1単独の数値に対してp<0.001を示す）。
【図６】ミクログリアにおけるAβ40の取込み及びAβ40による活性化に及ぼすHMG1の影響を示す写真及び図である。ａ及びｂ：ビヒクル、TGF-β1又はHMG1存在下でAβ40とインキュベートしたラットミクログリアのライセートのイムノブロット写真（ａ）及び総Aβ40量を示すグラフ（縦軸はビヒクルに対する％割合；各数値は３つの実験の平均±標準誤差を示す）。ｃ−ｅ：Aβ40存在下又は非存在下で、ビヒクル（HMG1(-)）又はHMG1（HMG1(+)）とインキュベートしたラットミクログリアのライセートの抗iNOS抗体に対するイムノブロット写真（ｃ上図）、並びに培養上清中のNO₂ ^-量（ｃ下図）TNF-α量（ｄ）及びIL-6量（ｅ）を示すグラフ（各数値は３つの実験の平均±標準誤差；***、†††は、それぞれビヒクル及びAβ40処理の数値に対してp<0.001であることを示す）。
【図７】インビトロでのHMG1とAβペプチドとの結合及びAβペプチドの凝集を示す写真である。ａ及びｂ：Aβ40又はAβ42の存在下又は非存在下でHMG1をインキュベートしたサンプルについてのHMG1（ａ）又はAβに対する抗体を用いたイムノブロット（矢印はHMG1とAβ40又はAβ42との複合体を示す）。ｃ：Aβ40又はAβ42とHMG1とをインキュベートした後、反応液のHMG1（上図）又はAβ（下図）に対する抗体によって免疫沈降させ、HMG1又はAβに対する抗体を用いてイムノブロットしたもの。
【図８】老人斑形成過程における細胞外HMG1の関与を示す模式図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a screening means for a substance having a senile plaque formation inhibitory activity, characterized by using High Mobility Group Protein-1 (hereinafter abbreviated as HMG1). The present invention also relates to the pharmaceutical use of the substance obtained by the screening, particularly the prevention / treatment of brain amyloidosis such as Alzheimer's disease and Down's syndrome.
[0002]
[Prior art]
Senile plaques are one of the major neuropathological changes in Alzheimer's disease and are detected from the earliest stages of the onset process. The main component constituting senile plaques is amyloid β (hereinafter abbreviated as Aβ) peptide, which is produced by cleaving amyloid precursor protein (APP) with two types of (β- and γ-) secretases. . As its main molecular species, there are Aβ40 consisting of 40 amino acid residues and Aβ42 having 2 residues longer on the C-terminal side.
[0003]
Recent studies suggest that Aβ peptide deposition is controlled by complex interactions between regulators of the acellular and cellular (glia) excision pathways. Non-cellular pathways include 1) secretory peptidases that degrade extracellular Aβ peptides, such as neprilysin and insulin-degrading enzyme (IDE) (for example, Non-Patent Document 1), and 2) from the brain parenchyma to the circulatory system. There is an outflow route of Aβ by transportation and discharge (for example, Non-Patent Document 2). In Alzheimer's disease, extracellular deposits of Aβ peptide accumulate significantly and are associated with microglia. The involvement of such microglia suggests that they perform Aβ phagocytic function (for example, Non-Patent Document 3) and / or are involved in inflammatory activation (for example, Non-Patent Document 4). . Based on the discovery that anti-Aβ antibodies produced by Aβ vaccine therapy induce microglial phagocytosis through Fc receptors (eg, Non-Patent Document 5) and / or remove plasma Aβ peptides (eg, Non-Patent Document 6). The importance of Aβ clearance has received much attention. In addition, cytokines such as transforming growth factor β1 (TGF-β1) promote Aβ clearance by microglia (for example, Non-Patent Document 7). Recently, the inventors have shown that extracellular stress proteins such as Hsp90, Hsp70, Grp78 promote Aβ clearance by activating phagocytosis by microglia and Aβ degradation by NFκB activation and / or by chaperone activity. (For example, nonpatent literature 8).
[0004]
Many of the proteins associated with amyloidosis are known to share similar structures and sequences and are believed to contribute to amyloid fibril formation. Such amyloidogenic consensus sequences are also found in proteins that are not known to be associated with amyloidosis. For example, Kallijarvi et al., HMG1, a non-histone chromosomal protein, has a sequence motif that is homologous to an Aβ peptide, and an HMG1 fragment containing the sequence motif forms amyloid-like fibrils in vitro and polymerizes Aβ40. It is reported to promote (Non-patent Document 9).
[0005]
On the other hand, Toyama et al. Detected an abnormal presenilin 2 protein (known as one of the causative genes of familial Alzheimer's disease (FAD)) in the brain of sporadic Alzheimer's disease (SAD). It is intended to interfere with normal splicing by binding to the transcript, and that when a decoy substance that binds to HMG1 is allowed to act on a cell model of Alzheimer's disease, normal presenilin 2 mRNA is synthesized and no abnormal presenilin 2 protein is produced. Announced (eg, Non-Patent Document 10) is expected to lead to the development of second-generation Alzheimer's disease therapeutics that exceed the current symptomatic therapeutics.
[0006]
[Non-Patent Document 1]
Journal of Biological Chemistry (J. Biol. Chem.), 273, p32730-32738 (1998)
[Non-Patent Document 2]
Proc. Of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), Vol. 96, p14088-14093 (1999)
[Non-Patent Document 3]
American Journal of Pathology, Am. J. Pathol., 148, 399-403 (1996)
[Non-Patent Document 4]
Neurobiological Aging, Vol. 21, p383-421 (2000)
[Non-Patent Document 5]
Nature, vol.400, p173-177 (1999)
[Non-Patent Document 6]
Proc. Of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), Vol. 98, p. 8850-8855 (2001)
[Non-Patent Document 7]
Nature. Med., Volume 7, p612-618 (2001)
[Non-Patent Document 8]
FASEB Journal (FASEB J.), Volume 16, p601-603 (2002)
[Non-patent document 9]
Biochemistry, Vol. 40, p10032-10037 (2001)
[Non-Patent Document 10]
Tokyo Yomiuri Shimbun, January 9, 2002 evening issue
[0007]
[Problems to be solved by the invention]
However, in brain amyloidosis such as Alzheimer's disease, a useful substance that inhibits aggregation / deposition of already produced Aβ peptide and promotes its degradation and removal has not yet been found, and brain amyloidosis from such a viewpoint has not been found yet. There is almost no research and development of therapeutic drugs.
Accordingly, the object of the present invention is to (1) promote the aggregation of Aβ and (2) inhibit the Aβ phagocytosis by microglia to inhibit the mechanism of Aβ removal, thereby promoting the formation of senile plaques and the brain To identify a factor causing amyloidosis and to provide a screening method for a substance having a preventive / therapeutic effect on cerebral amyloidosis, a kit for the same, etc. by promoting Aβ clearance by inhibiting the action or expression of the factor .
[0008]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have found that HMG1 levels are increased in the brain of Alzheimer's disease patients, and that extracellular HMG1 coexists with Aβ42 in senile plaques. It has been found that HMG1 inhibits phagocytosis of Aβ42 by microglia in an in vitro microglia culture system, and that HMG1 forms a complex with Aβ42 and promotes the formation of Aβ42 aggregates in vitro. As a result of further studies based on these findings, the present inventors have completed the present invention.
[0009]
That is, the present invention
[1] A vaccine for promoting amyloid β42 aggregation inhibitory action and / or amyloid β42 phagocytosis by microglia, comprising HMG1 or a partial peptide thereof or a salt thereof,
[2] A diagnostic agent for amyloid β42 aggregation and / or amyloid β42 phagocytosis caused by microglia, comprising a nucleotide containing a base sequence encoding HMG1 or a partial peptide thereof,
[3] An inhibitor of amyloid β42 aggregation and / or amyloid β42 phagocytosis by microglia, comprising an antibody against HMG1 or a partial peptide thereof or a salt thereof,
[4] A diagnostic agent for amyloid β42 aggregation and / or amyloid β42 phagocytosis caused by microglia, comprising an antibody against HMG1 or a partial peptide thereof or a salt thereof,
[5] An inhibitor of amyloid β42 aggregation and / or amyloid β42 phagocytosis by microglia, comprising a nucleotide sequence that is complementary to a base sequence encoding HMG1 or a partial peptide thereof or a part thereof.
[6] A screening method for a substance that inhibits HMG1 and inhibits amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia, comprising using HMG1 or a partial peptide thereof or a salt thereof,
[7] A screening method for a substance that inhibits HMG1 and exhibits amyloid β42 aggregation inhibitory activity, comprising contacting amyloid β42 and a test substance with HMG1 or a partial peptide thereof or a salt thereof, and assaying aggregation of amyloid β42 ,
[8] The method according to [7] above, wherein comparison is made with aggregation of amyloid β42 when amyloid β42 is contacted with HMG1 or a partial peptide thereof or a salt thereof.
[9] Inhibiting HMG1 and promoting amyloid β42 phagocytosis by microglia, comprising contacting HMG1 or a partial peptide thereof or a salt thereof, amyloid β42 and a test substance with microglia, and assaying amyloid β42 phagocytosis by microglia A screening method for substances having an effect,
[10] The method according to [9] above, wherein comparison is made with amyloid β42 phagocytosis by microglia when HMG1 or a partial peptide thereof or a salt thereof and amyloid β42 are contacted with microglia.
[11] A kit for screening a substance comprising HMG1 or a partial peptide thereof or a salt thereof, which inhibits HMG1 and suppresses amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia,
[12] The kit according to the above [11], further comprising amyloid β42,
[13] A substance that inhibits amyloid β42 aggregation and / or exhibits amyloid β42 phagocytosis promoting action by microglia, obtained by using the method according to [6] or the kit according to [11],
[14] A substance that inhibits HMG1 and suppresses amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia,
[15] A medicament comprising the substance described in [13] or [14] above,
[16] The medicament according to [15] above, which is a prophylactic / therapeutic agent for Alzheimer's disease, Down's syndrome or amyloid angiopathy,
[17] A substance that suppresses the expression of HMG1 and suppresses amyloid β42 aggregation and / or promotes phagocytosis of amyloid β42 by microglia, characterized by using a nucleotide containing a base sequence encoding HMG1 or a partial peptide thereof Screening method,
[18] A test substance is added to a system capable of producing HMG1 or a partial peptide thereof from a nucleotide containing a base sequence encoding HMG1 or a partial peptide thereof, and the expression of HMG1 or the partial peptide is assayed The method according to [17] above,
[19] A test substance and amyloid β42 are added to a system capable of generating HMG1 or a partial peptide thereof from a nucleotide containing a base sequence encoding HMG1 or a partial peptide thereof, and expression of HMG1 or a partial peptide thereof and aggregation of amyloid β42 A method for screening a substance that suppresses the expression of HMG1 and suppresses amyloid β42 aggregation, characterized in that
[20] A test substance, amyloid β42 and microglia are added to a system capable of producing HMG1 or a partial peptide thereof from a nucleotide containing a base sequence encoding HMG1 or a partial peptide thereof, and expression of HMG1 or a partial peptide thereof and microglia are caused. A screening method for a substance that suppresses the expression of HMG1 and exhibits amyloid β42 phagocytosis promoting action by microglia, characterized by assaying amyloid β42 phagocytosis,
[21] For screening for a substance comprising a nucleotide containing a base sequence encoding HMG1 or a partial peptide thereof and suppressing amyloid β42 aggregation by suppressing expression of HMG1 and / or amyloid β42 phagocytosis promoting action by microglia kit,
[22] The kit according to the above [21], further comprising amyloid β42,
[23] A substance that suppresses the expression of HMG1 and suppresses amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia, obtained by using the method according to [17] or the kit according to [21],
[24] A substance that suppresses the expression of HMG1 and inhibits amyloid β42 aggregation and / or promotes amyloid β42 phagocytosis by microglia,
[25] A medicament comprising the substance described in [23] or [24] above,
[26] A medicament or the like according to the above [25], which is a prophylactic / therapeutic agent for Alzheimer's disease, Down's syndrome or amyloid angiopathy.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
HMG1 used in the present invention is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. HMG1 is, for example, any cell (eg, spleen cell, etc.) of a human or other mammal (eg, cow, monkey, pig, horse, sheep, goat, dog, cat, guinea pig, rat, mouse, rabbit, hamster, etc.). Nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells) , B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells Hepatocytes or stromal cells, or precursor cells of these cells, stem cells or cancer cells, etc.) or blood cells, or any tissue in which these cells are present, eg, brain, brain Site (eg, olfactory bulb, cephalic nucleus, basal ganglia, hippocampus, thalamus, hypothalamus, hypothalamic nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain , Substantia nigra), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, It may be a protein derived from the spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testis, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc., chemical synthesis or cell-free translation system It may be a protein synthesized in Alternatively, it may be a recombinant protein produced from a transformant introduced with a polynucleotide encoding HMG1.
[0011]
The “amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2” is, for example, about 85% or more, preferably 90% or more, more preferably, the amino acid sequence represented by SEQ ID NO: 2. Examples include amino acid sequences having about 95% or more, most preferably about 98% or more identity.
Alternatively, the “amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2” includes: a) one or more of the amino acid sequences represented by SEQ ID NO: 2 (preferably 1 to 30) Amino acid sequence from which about 1 amino acid, more preferably about 1 to 10 amino acids, more preferably several (1 to 5) amino acids are deleted, b) 1 or 2 in the amino acid sequence represented by SEQ ID NO: 2 An amino acid sequence to which the above (preferably about 1 to 30, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids are added, c) represented by SEQ ID NO: 2. One or more amino acids in the amino acid sequence (preferably about 1 to 30, more preferably about 1 to 10, more preferably several (1 to 5)) are substituted with other amino acids. Amino acid sequence, or d) Such as proteins comprising the amino acid sequence which is a combination of these is also used.
The “protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2” of the present invention means “an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2”. And a protein having substantially the same activity as the protein having the amino acid sequence represented by SEQ ID NO: 2.
Examples of “substantially the same quality of activity” include Aβ42 aggregation promoting activity and Aβ42 phagocytosis inhibiting activity by microglia. “Substantially homogeneous” indicates that their activities are homogeneous in nature. Therefore, it is preferable that the above activities are equivalent, but the degree of these activities (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times) And quantitative factors such as the molecular weight of the protein may be different.
The aggregation promoting activity of Aβ42 and the activity of inhibiting Aβ42 phagocytosis by microglia can be measured according to a method known per se. For example, it can be measured according to the method described in the screening method of the present invention described later.
[0012]
In the present specification, HMG1 is described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation. HMG1 including human HMG1 containing the amino acid sequence represented by SEQ ID NO: 2 has a carboxyl group (—COOH), carboxylate (—COO) at the C-terminus.⁻), Amide (-CONH₂) Or ester (—COOR).
Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl or n-butyl._1-6Alkyl groups such as C, such as cyclopentyl, cyclohexyl, etc._3-8A cycloalkyl group such as phenyl, α-naphthyl and the like_6-12Aryl groups such as phenyl-C such as benzyl, phenethyl and the like_1-2Alkyl groups or α-naphthyl-C such as α-naphthylmethyl_1-2C such as alkyl group_7-14In addition to the aralkyl group, a pivaloyloxymethyl group that is widely used as an oral ester is used.
When HMG1 has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in HMG1. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
Further, in HMG1, in the above-described protein, the amino group of the N-terminal methionine residue is a protecting group (for example, C-form such as formyl group, acetyl, etc.)._2-6C such as alkanoyl group_1-6An acyl group and the like, a glutamyl group produced by cleavage of the N-terminal side in vivo, pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (for example, —OH, —SH, An amino group, an imidazole group, an indole group, a guanidino group, etc.) are suitable protecting groups (for example, C such as formyl group and acetyl)._2-6C such as alkanoyl group_1-6Examples thereof include those protected with an acyl group or the like, or complex proteins such as so-called glycoproteins to which sugar chains are bound.
As a specific example of HMG1, for example, human-derived HMG1 containing the amino acid sequence represented by SEQ ID NO: 2 is used. Human HMG1 is described in Nucleic Acids Res., Vol. 17, No. 3, p1197-1214 (1989), and GenBank has an accession number CAA31110.1 (cDNA is X12597). It is a known protein that has been registered and published.
[0013]
As a partial peptide of HMG1 (hereinafter sometimes simply referred to as “partial peptide of the present invention”), it is a peptide having the above-mentioned partial amino acid sequence of HMG1 and has substantially the same activity as HMG1. Any of the HMG1 protein molecules having a partial amino acid sequence containing a sequence motif homologous to the amyloidogenic consensus sequence of the Aβ peptide described in Non-Patent Document 9 above may be used. Used.
The number of amino acids in the partial peptide of the present invention is preferably a peptide having an amino acid sequence of at least 20 or more, preferably 50 or more, more preferably 100 or more of the amino acid sequence of HMG1.
Here, “substantially the same quality of activity” has the same meaning as described above. The measurement of “substantially the same quality of activity” can be performed in the same manner as described above.
[0014]
In the partial peptide of the present invention, the C-terminus is carboxyl group (—COOH), carboxylate (—COO).⁻), Amide (-CONH₂) Or ester (—COOR). When the partial peptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
Further, in the partial peptide of the present invention, as in the case of HMG1, the amino group of the N-terminal methionine residue is protected with a protecting group, and Gln produced by cleaving the N-terminal side in vivo is pyroglutamic acid. In which a substituent on the side chain of an amino acid in the molecule is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound.
Examples of the salt of HMG1 or a partial peptide thereof include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
[0015]
HMG1 or a salt thereof can be produced from the above-mentioned human or other mammalian cells or tissues by a known protein purification method, or a transformant containing DNA encoding HMG1 described later is cultured. Can also be manufactured. Moreover, it can also manufacture according to the protein synthesis method described later or this.
When producing from human or mammalian tissue or cells, the human or mammalian tissue or cells are homogenized and then extracted with acid, etc., and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.
[0016]
For the synthesis of HMG1 or a partial peptide thereof, a salt thereof or an amide thereof, a commercially available resin for protein synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein is cut out from the resin, and at the same time, various protecting groups are removed, and further, intramolecular disulfide bond forming reaction is carried out in a highly diluted solution to obtain the target protein or its amide.
For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. Examples of carbodiimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide, and the like. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (for example, HOBt, HOBt), or the protected amino acid is activated in advance as a symmetric acid anhydride, HOBt ester or HOBt ester. Can then be added to the resin.
[0017]
The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range known to be usable for protein bond forming reaction, and is usually selected appropriately from a range of about −20 ° C. to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.
[0018]
The protection of the functional group that should not be involved in the reaction of the raw material, the protection group, the removal of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
Examples of the protective group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include groups derived from carbonic acid such as lower alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like are used.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.
[0019]
Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.
Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 Addition of a cation scavenger such as -butanedithiol, 1,2-ethanedithiol, etc. is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.
[0020]
As another method for obtaining an amide form of a protein, for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide (protein) chain is extended to the desired chain length on the amino group side. A protein from which only the N-terminal α-amino protecting group of the peptide chain is removed and a protein from which only the C-terminal carboxyl protecting group has been removed are prepared, and both proteins are mixed in a mixed solvent as described above. To condense. The details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-described method to obtain the desired crude protein. This crude protein can be purified using various known purification means, and the amide form of the desired protein can be obtained by lyophilizing the main fraction.
In order to obtain a protein ester, for example, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired protein ester is obtained in the same manner as the protein amide. be able to.
[0021]
A partial peptide of HMG1 or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving HMG1 with an appropriate peptidase. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the peptide of interest can be produced by condensing the partial peptide or amino acid capable of constituting HMG1 and the remaining part, and removing the protective group when the product has a protective group. Examples of known condensation methods and protecting group elimination include the methods described in the following a) to e).
a) M. Bodanszky and M.A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
b) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
c) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
d) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
e) Supervised by Yajima Haruaki, Development of follow-up medicines Volume 14 Peptide synthesis Hirokawa Shoten
Further, after the reaction, the partial peptide of the present invention can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted into a free form by a known method. Can do.
[0022]
Any nucleotide containing a base sequence encoding the above-mentioned HMG1 protein may be used as the nucleotide containing the base sequence encoding HMG1 (hereinafter sometimes abbreviated as “the nucleotide of the present invention”). It may be DNA, RNA, or a DNA / RNA chimera, preferably DNA. The nucleotide may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
[0023]
Examples of DNA containing a base sequence encoding HMG1 (hereinafter sometimes abbreviated as “DNA of the present invention”) include genomic DNA, genomic DNA library, human or other mammals (for example, bovine, monkey, Any cell (eg, spleen cell, nerve cell, glial cell, pancreatic β cell, bone marrow cell, mesangial cell, Langerhans cell) of horse, pig, sheep, goat, dog, cat, guinea pig, rat, mouse, rabbit, hamster, etc. , Epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils , Eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, or Progenitor cells of these cells, stem cells or cancer cells, etc., blood cells, or any tissue in which these cells exist, for example, the brain, each part of the brain (eg, olfactory bulb, buccal nucleus, basal sphere, hippocampus) , Thalamus, hypothalamus, hypothalamic nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra), spinal cord, pituitary gland, stomach, pancreas, kidney , Liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cell, prostate, testicle Any of cDNA derived from the testis, ovary, placenta, uterus, bone, joint, skeletal muscle (particularly each part of the brain and brain), cDNA library derived from the cells / tissues described above, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid, retrovirus vector, adenovirus vector, adeno-associated virus vector, and the like. Moreover, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells / tissues described above.
[0024]
Specifically, examples of DNA encoding HMG1 include DNA containing the base sequence represented by SEQ ID NO: 1, or hybridizing with the base sequence represented by SEQ ID NO: 1 under highly stringent conditions. Any DNA may be used as long as it encodes a protein having a base sequence for soybean and having substantially the same quality as HMG1 (eg, Aβ42 aggregation promoting activity, Aβ42 phagocytosis inhibiting activity by microglia). The DNA capable of hybridizing with the base sequence represented by SEQ ID NO: 1 is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, with the base sequence represented by SEQ ID NO: 1. Most preferably, DNA containing a base sequence having a homology of about 95% or more is used.
[0025]
Hybridization should be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning Second Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can do. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual. More preferably, it can be carried out according to highly stringent conditions.
The highly stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
[0026]
More specifically, as the DNA encoding human-derived HMG1 containing the amino acid sequence represented by SEQ ID NO: 2, DNA containing the base sequence represented by SEQ ID NO: 1 is used.
[0027]
A nucleotide containing a base sequence complementary to the target region of the target nucleic acid, that is, a nucleotide that can hybridize with the target nucleic acid can be said to be “antisense” to the target nucleic acid. On the other hand, a nucleotide containing a base sequence having homology with the target region of the target nucleic acid (that is, when the target nucleic acid encodes a protein, a nucleotide encoding a partial peptide of the protein) is “sense” to the target nucleic acid. It can be said that. As used herein, the term “corresponding” means homologous to or complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. Here, “having homology” or “complementary” means about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more between base sequences. Having homology or complementarity. Also, “corresponding” between a nucleotide, base sequence or nucleic acid and peptide (protein) means that the peptide (protein) has an amino acid sequence translated from the nucleotide (nucleic acid) or its complement sequence. Usually pointing.
[0028]
A nucleotide containing all or part of a base sequence complementary to the nucleotide of the present invention (hereinafter also referred to as “antisense nucleotide of the present invention”) is a cloned or determined nucleotide of the present invention. It can be designed and synthesized based on the base sequence information. Such nucleotides can inhibit the replication or expression of a gene containing the nucleotide sequence of the nucleotide of the present invention. That is, the antisense nucleotide of the present invention can hybridize with RNA transcribed from the HMG1 gene, can inhibit mRNA synthesis (processing) or function (translation into protein), or can be associated with HMG1. The expression of the HMG1 gene can be regulated and controlled through interaction with RNA. Nucleotides complementary to selected sequences of HMG1-related RNA and nucleotides that can specifically hybridize with HMG1-related RNA are useful for regulating and controlling the expression of HMG1 gene in vivo and in vitro. It is also useful for treating or diagnosing diseases.
[0029]
The target region of the antisense nucleotide of the present invention is not particularly limited as long as the antisense nucleotide is hybridized, and as a result, the translation of the HMG1 protein is inhibited. The length of the target region is the entire sequence of HMG1 mRNA. The short sequence may be about 15 bases, and the long sequence may be the entire mRNA or initial transcription product sequence. In view of ease of synthesis and antigenicity, an oligonucleotide consisting of about 15 to about 30 bases is preferable, but not limited thereto. Specifically, for example, 5 ′ end hairpin loop of HMG1 mRNA, 5 ′ end 6-base pair repeat, 5 ′ end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon, 3 ′ An end untranslated region, 3 ′ end palindromic region, and 3 ′ end hairpin loop can be selected as target regions, but any region within the HMG1 gene can be selected as a target. For example, it is also preferable to use the intron portion of the gene as a target region.
Furthermore, the antisense nucleotide of the present invention not only hybridizes with HMG1 mRNA or an initial transcription product to inhibit translation into protein, but also binds to the HMG1 gene, which is a double-stranded DNA, to form a triplex. May be formed so that RNA transcription can be inhibited.
[0030]
Antisense nucleotides include deoxynucleotides containing 2-deoxy-D-ribose, deoxynucleotides containing D-ribose, other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or non-nucleotides Other polymers with backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a pair of bases as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a ring or a base). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and unmodified polynucleotides (or unmodified oligonucleotides), and more publicly known Modified, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotides Modified, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged or sulfur-containing bonds (eg, phosphorothioates, phosphorodithioates, etc.) ), For example, proteins (nucleases, nuclease inhibitors, and toxins) , Antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelates Compounds containing compounds (for example, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) It may be. Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.
[0031]
Antisense nucleotides are RNA, DNA, or modified nucleic acids (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, nucleic acid sulfur derivatives and thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to increase the affinity for the target sense strand, and to be antitoxic if toxic Make sense nucleic acids less toxic. Many such modifications are known in the art, such as J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. Ed ., Antisense Research and Applications, CRC Press, 1993, etc.
[0032]
Antisense nucleotides may be altered or contain modified sugars, bases, linkages, donated in specialized forms such as liposomes, microspheres, applied by gene therapy, or added Could be given in form. In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( For example, phospholipid, cholesterol, etc.) may be used. Preferred lipids to be added include cholesterol and derivatives thereof (for example, cholesteryl chloroformate, cholic acid, etc.). Such can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of the nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
[0033]
Ribozymes capable of specifically cleaving HMG1 mRNA or the initial transcript of the HMG1 gene within the coding region (including the intron portion in the case of the initial transcript) can also be included in the antisense nucleotide of the present invention. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligo DNA having the base sequence of the enzyme active site also has a nucleic acid cleavage activity. As long as it has sequence-specific nucleic acid cleavage activity, it is used as a concept including DNA. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and a few bases at both ends adjacent to the part having the hammerhead structure (about 10 bases in total) are made into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme has the additional advantage of not attacking genomic DNA since it only uses RNA as a substrate. When the mRNA corresponding to the nucleotide of the present invention has a double-stranded structure by itself, a target sequence can be identified by using a hybrid ribozyme linked with an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. It can be made into a strand [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked with a tRNA-modified sequence. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
[0034]
A double-stranded oligo RNA complementary to a partial sequence within the coding region of HMG1 mRNA or the initial transcript of the HMG1 gene (including the intron portion in the case of the initial transcript) can also be included in the antisense nucleotide of the present invention. . When a short double-stranded RNA is introduced into a cell, a phenomenon called RNA interference (RNAi), in which mRNA complementary to the RNA is degraded, has been known in nematodes, insects, plants, etc. Recently, it has been confirmed that this phenomenon also occurs in mammalian cells [Nature, 411 (6836): 494-498 (2001)], and has attracted attention as an alternative to ribozyme.
The antisense oligonucleotide and ribozyme of the present invention determine the target region of mRNA or initial transcript based on HMG1 cDNA sequence or genomic DNA sequence information, and are commercially available DNA / RNA automatic synthesizers (Applied Biosystems, Beckman et al.) Can be prepared by synthesizing a complementary sequence thereto. Double-stranded oligo RNA having RNAi activity is prepared by synthesizing a sense strand and an antisense strand with a DNA / RNA automatic synthesizer, and in an appropriate annealing buffer, for example, at about 90 to about 95 ° C. for about 1 minute. After modification, it can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. In addition, longer double-stranded polynucleotides can be prepared by synthesizing complementary oligonucleotide strands so as to alternately overlap, annealing them, and then ligating with ligase.
[0035]
The HMG1 gene expression inhibitory activity of the antisense nucleotide of the present invention is determined using a transformant containing the nucleotide of the present invention, an in vivo or in vitro HMG1 gene expression system, or an in vivo or in vitro translation system for HMG1 protein. You can investigate.
[0036]
The DNA encoding the partial peptide of the present invention may be any DNA as long as it contains the base sequence encoding the partial peptide of the present invention described above. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by RT-PCR using mRNA fractions prepared from the cells / tissues described above.
Specifically, examples of the DNA encoding the partial peptide of the present invention include (1) DNA having a partial base sequence of DNA having the base sequence represented by SEQ ID NO: 1, or (2) SEQ ID NO: 1 having a base sequence that hybridizes with the base sequence represented by 1 under high stringency conditions, and substantially the same activity as HMG1 (eg, aggregation promoting activity of Aβ42, Aβ42 phagocytosis inhibiting activity by microglia, etc.) For example, DNA having a partial base sequence of DNA encoding a protein having
Examples of DNA that can hybridize to the base sequence represented by SEQ ID NO: 1 include, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, with the base sequence represented by SEQ ID NO: 1. Most preferably, DNA containing a base sequence having a homology of about 95% or more is used.
[0037]
As a means for cloning DNA that completely encodes HMG1 or a partial peptide thereof (hereinafter sometimes abbreviated as “HMG1” in general), a synthetic DNA primer having a partial base sequence of HMG1 is used for PCR. Amplification or DNA incorporated into an appropriate vector can be selected by hybridization with a DNA fragment encoding part or all of HMG1 or labeled with synthetic DNA. The method of hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.
[0038]
DNA base sequence conversion can be performed by PCR or a known kit such as Mutan.^TM-Super Express Km (Takara Shuzo Co., Ltd.), Mutan^TM-K (Takara Shuzo Co., Ltd.) can be used according to a method known per se such as the ODA-LA PCR method, the Gapped duplex method, the Kunkel method, or the like.
The cloned DNA encoding HMG1 can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 'end side, and may have TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
An expression vector for HMG1 can be produced, for example, by (a) cutting out a target DNA fragment from DNA encoding HMG1, and (b) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. it can.
[0039]
Examples of vectors include E. coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used.
The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when animal cells are used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
Of these, the CMV promoter, SRα promoter and the like are preferably used. When the host is Escherichia, trp promoter, lac promoter, recA promoter, λP_LWhen the host is Bacillus, such as a promoter, lpp promoter, etc., SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable.
[0040]
In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like is used as desired. it can. Examples of selection markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter Amp).^rA neomycin resistance gene (hereinafter Neo)^rG418 resistance) and the like. In particular, CHO (dhfr⁻) When using the dhfr gene as a selection marker using cells, the target gene can also be selected using a medium not containing thymidine.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention. When the host is Escherichia, PhoA signal sequence, OmpA, signal sequence, etc., and when the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc. are used in yeast. In some cases, MFα • signal sequence, SUC2 • signal sequence, etc. When the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used.
A transformant can be produced using a vector containing DNA encoding HMG1 constructed in this manner.
[0041]
As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
As a specific example of the genus Escherichia, Escherichia coli K12 / DH1 [Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology]. 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], etc. are used. .
Examples of the genus Bacillus include Bacillus subtilis MI114 [Gen, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984). )] Etc. are used.
Examples of yeast include Saccharomyces cerevisiae AH22, AH22R.⁻NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris, and the like.
[0042]
As insect cells, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five derived from eggs of Trichoplusia ni^TMCells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, sputum-derived cell lines (Bombyx mori N; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, J.L. et al., In Vivo, 13, 213-217, (1977)).
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter referred to as CHO (dhfr).⁻Abbreviated cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, human HEK293 cells and the like.
[0043]
For transforming Escherichia, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (Proc. Natl. Acad. Sci. USA) 1972), Gene, Vol. 17, 107 (1982), and the like.
Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, 168, 111 (1979).
To transform yeast, for example, Methods in Enzymology, 194, 182-187 (1991), Proceedings of the National Academy of Sciences of -The method can be carried out according to the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
In order to transform animal cells, for example, cell engineering separate volume 8 new cell engineering experiment protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973).
In this way, a transformant transformed with an expression vector containing DNA encoding HMG1 is obtained.
When cultivating a transformant whose host is an Escherichia bacterium or Bacillus genus, a liquid medium is suitable as a medium used for the culture, including a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, and potato extract. Examples of inorganic or organic substances and inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.
[0044]
As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics] 431-433, Cold Spring Harbor Laboratory, New York 1972]. In order to make the promoter work efficiently as necessary, a drug such as 3β-indolylacrylic acid can be added.
When the host is an Escherichia bacterium, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation can be added.
When the host is Bacillus, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration or agitation can be added.
When cultivating a transformant whose host is yeast, examples of the medium include a Burkholder minimal medium [Bostian, KL et al., Proceedings of the National Academy of Sciences of Science. The USA (Proc. Natl. Acad. Sci. USA), vol. 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Proceedings of the National -Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary.
[0045]
When cultivating a transformant whose host is an insect cell or insect, 10% bovine serum that has been immobilized on Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)), etc. Those appropriately added with these additives may be used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.
When cultivating a transformant whose host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], DMEM medium. [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)] is used. The pH is preferably about 6-8. The culture is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary.
As described above, HMG1 can be generated inside the cell, the cell membrane, or extracellularly of the transformant.
[0046]
Separation and purification of HMG1 from the culture can be performed, for example, by the following method.
When extracting HMG1 from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and cultured by ultrasound, lysozyme and / or freeze-thawing. Or after destroying a cell, the method of obtaining the crude extract of HMG1 by centrifugation or filtration etc. are used suitably. Protein denaturants such as urea and guanidine hydrochloride in the buffer, Triton X-100^TMA surfactant such as may be contained. When HMG1 is secreted into the culture solution, the cells or cells are separated from the supernatant by a method known per se after completion of the culture, and the supernatant is collected.
Purification of HMG1 contained in the thus obtained culture supernatant or extract can be performed by appropriately combining known separation / purification methods. These known separation and purification methods include mainly molecular weights such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method using difference in charge, method using difference in charge such as ion exchange chromatography, method utilizing specific newness such as affinity chromatography, and difference in hydrophobicity such as reversed phase high performance liquid chromatography And a method using a difference in isoelectric point, such as isoelectric focusing method.
[0047]
When the obtained HMG1 is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when obtained as a salt, a method known per se or a method analogous thereto Can be converted to the free form or other salts.
In addition, HMG1 produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by allowing an appropriate protein modifying enzyme to act before or after purification. Examples of protein modifying enzymes include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase, and the like.
The activity of HMG1 thus produced can be measured by an enzyme immunoassay using a specific antibody.
[0048]
The antibody against HMG1 may be either a polyclonal antibody or a monoclonal antibody as long as it can recognize HMG1.
An antibody against HMG1 can be produced according to a method for producing an antibody or antiserum known per se using HMG1 as an antigen.
[0049]
[Production of monoclonal antibodies]
(A) Preparation of monoclonal antibody-producing cells
HMG1 is administered to a mammal at a site where antibody production is possible by administration, together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2 to 6 weeks, 2 to 10 times in total. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats, and mice and rats are preferably used. When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with an antigen, for example, an individual with a confirmed antibody titer from a mouse, collect spleen or lymph nodes 2 to 5 days after the final immunization, A monoclonal antibody-producing hybridoma can be prepared by fusing the antibody-producing cells contained with myeloma cells. The antibody titer in the antiserum can be measured, for example, by reacting a labeled HMG1 described later with an antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used.
Examples of myeloma cells include NS-1, P3U1, SP2 / 0, etc., and P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to be used and the number of myeloma cells is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. The cell fusion can be efficiently carried out by incubating at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes.
[0050]
Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which HMG1 antigen is adsorbed directly or with a carrier, and then radioactive substances or An anti-immunoglobulin antibody labeled with an enzyme or the like (when the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A, and a method for detecting a monoclonal antibody bound to a solid phase; Examples include a method in which a hybridoma culture supernatant is added to a solid phase on which a globulin antibody or protein A is adsorbed, HMG1 labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected.
Selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto, but can usually be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium may be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1-10% fetal calf serum, or serum-free for hybridoma culture A culture medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) etc. can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.
[0051]
(B) Purification of monoclonal antibody
Separation and purification of monoclonal antibodies can be carried out in the same way as in the separation and purification of ordinary polyclonal antibodies (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchanger (eg, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method for obtaining an antibody by dissociating the binding using an active adsorbent such as protein A or protein G) Can be done according to
[0052]
[Preparation of polyclonal antibody]
The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (HMG1 antigen) and a carrier protein is prepared, a mammal is immunized in the same manner as the above monoclonal antibody production method, and an antibody-containing substance against HMG1 is collected from the immunized animal, It can manufacture by performing isolation | separation purification.
Regarding the complex of immune antigen and carrier protein used to immunize mammals, the type of carrier protein and the mixing ratio of carrier and hapten should be such that antibodies can be efficiently produced against haptens that are immunized by cross-linking to carriers. Any ratio may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin and the like are about 0.1 to 20 in weight ratio with respect to hapten 1. Preferably, a method of coupling at a ratio of about 1 to 5 is used.
Various coupling agents can be used for coupling of the hapten and the carrier, but active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and dithiobilidyl group are used.
The condensation product is administered to warm-blooded animals at the site where antibody production is possible, or with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration can usually be carried out about once every 2 to 6 weeks, about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, etc. of mammals immunized by the above method, preferably from blood.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.
[0053]
HMG1 is upregulated in both the particle fraction of Alzheimer's disease brain (including nuclei, membranes and insoluble proteins) and the cytoplasmic fraction (including cytosolic and extracellular soluble proteins), and extracellular HMG1 is aged Coexists with spots. Furthermore, HMG1 inhibits the phagocytosis of Aβ42 by microglia and also binds to Aβ42 to promote the formation of Aβ42 aggregates. Accordingly, HMG1 or a partial peptide thereof or a salt thereof (hereinafter also referred to as “HMG1 etc.”), DNA encoding HMG1 etc., an antisense nucleotide of the present invention, an antibody against HMG1 etc. (hereinafter abbreviated as “antibody of the present invention”). Have the following uses:
[0054]
[1] Use of HMG1 or a partial peptide thereof or a salt thereof
(A) A vaccine for promoting Aβ42 aggregation inhibitory action and / or Aβ42 phagocytosis by microglia
When HMG1 or a partial peptide thereof or a salt thereof is administered as a vaccine, an anti-HMG1 antibody can be produced in the body. Since the anti-HMG1 antibody can neutralize the Aβ42 aggregation promoting action of HMG1 and the Aβ42 phagocytosis inhibiting action by microglia, the HMG1 vaccine has an Aβ42 aggregation inhibiting action and / or an effect of promoting Aβ42 phagocytosis by microglia. It is useful as a prophylactic / therapeutic agent for diseases associated with senile plaque formation by Aβ deposition using Aβ42 as a seed, specifically, brain amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.) .
When HMG1 or the like is used for the prevention or treatment of the above diseases, it can be vaccinated according to conventional means.
For example, a) HMG1 or the like is sterilized orally as a tablet, capsule, elixir, microcapsule or the like with sugar coating as necessary, or with water or other pharmaceutically acceptable liquid It can be used parenterally in the form of injections such as solutions or suspensions. For example, b) HMG1 etc. are mixed in a unit dosage form generally required for the practice of the formulation, together with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. Can be manufactured. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.
[0055]
Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavorings such as peppermint, red oil and cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80)^TM, HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
[0056]
In addition, the vaccine includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin, Polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection solution is usually filled in a suitable ampoule.
Since the preparation thus obtained is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can do.
The dose of HMG1 and the like varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, in an Alzheimer's disease patient (as 60 kg), about 0.01 to 1000 mg at a time, Preferably it is about 0.1-100 mg, More preferably, it is about 1-50 mg. When administered parenterally, it varies depending on the target organ, symptoms, administration method, etc. For example, in the form of an injection, for example, about 0.01 to 1000 mg at a time in Alzheimer's disease patients (as 60 kg) It is convenient to administer about 0.1 to 100 mg, more preferably about 1 to 50 mg. In the case of other animals, an amount converted per 60 kg can be administered. Administration can be performed 1 to 10 times at intervals of 1 to 21 days.
[0057]
[2] Use of nucleotide encoding HMG1 or a partial peptide thereof
(A) Gene diagnostic agent
The nucleotides and antisense nucleotides of the present invention can be used as probes to detect HMG1 or a partial peptide thereof in humans or mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Since it is possible to detect abnormalities (gene abnormalities) in the encoded DNA or mRNA, for example, as a genetic diagnostic agent such as damage, mutation or decreased expression of the DNA or mRNA, increase or excessive expression of the DNA or mRNA, etc. Useful.
The above gene diagnosis using the nucleotide or antisense nucleotide of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proc. Of the National Academy of Sciences of USA, Vol. 86, pp. 2766-2770 (1989)) be able to.
If excessive expression of HMG1 is detected by Northern hybridization, for example, overexpression of HMG1 may cause aggregation of Aβ42 and / or abnormal Aβ42 phagocytosis due to microglia, and hence formation and accumulation of senile plaques. Therefore, it can be diagnosed that there is a high possibility of suffering from a disease involving senile plaques, such as brain amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
[0058]
[3] Use of antibody against HMG1 or a partial peptide thereof or a salt thereof
(A) Quantification of HMG1 and diagnosis of abnormal Aβ42 phagocytosis by Aβ42 aggregation and / or microglia
Since the antibody of the present invention can specifically recognize HMG1, it can be used for diagnosis of Aβ42 aggregation and / or diagnosis of Aβ42 phagocytosis by microglia by detecting extracellular HMG1.
That is, the present invention
(I) In a test solution characterized by competitively reacting the antibody of the present invention with a test solution and labeled HMG1, and measuring the ratio of labeled HMG1 bound to the antibody A method for quantifying the receptor of
(Ii) The test solution and the antibody of the present invention insolubilized on the carrier and the labeled another antibody of the present invention are reacted simultaneously or successively, and then the activity of the labeling agent on the insolubilized carrier is measured. A method for quantifying HMG1 in a test solution is provided.
[0059]
In the quantification method of (ii) above, when one antibody is an antibody that recognizes the N-terminal part of HMG1, the other antibody is preferably an antibody that recognizes another part of HMG1, for example, the C-terminal part. .
In addition to quantification of the protein using a monoclonal antibody against HMG1, detection by tissue staining or the like can also be performed. For these purposes, the antibody molecule itself may be used, or the F (ab ′) of the antibody molecule.₂, Fab ′, or Fab fractions may be used.
[0060]
The method for quantifying HMG1 using the antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (eg, amount of HMG1) in the solution to be measured is chemically determined. Any measurement method may be used as long as it is a measurement method that is detected by a standard or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but the sandwich method described later is particularly preferable in view of sensitivity and specificity.
[0061]
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. Examples of radioisotopes include [¹²⁵I], [¹³¹I], [³H], [¹⁴C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.
For insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize and immobilize proteins or the like may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further labeled with another monoclonal antibody of the present invention (secondary reaction), and then on the insolubilized carrier. By measuring the activity of the labeling agent, the amount of HMG1 in the test solution can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times. The labeling agent and the insolubilization method can be the same as those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
[0062]
In the method for measuring HMG1 by the sandwich method of the present invention, antibodies having different HMG1 binding sites are preferably used as the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of HMG1, the antibody used in the primary reaction is preferably the C-terminal. For example, an antibody that recognizes the N-terminal is used.
The monoclonal antibody of the present invention can be used in measurement systems other than the sandwich method, for example, competitive method, immunometric method, nephrometry, and the like.
In the competition method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid-phased antibody is used as the first antibody, or As the first antibody, a soluble method is used, and a solid phase method using a solid phase antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test solution is separated. Are reacted with an excess amount of labeled antibody, and then a solid phased antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the test solution.
In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained.
In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of HMG1 by adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Sequel Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (3rd edition) (Medical Shoin, Showa) 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Id. Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)), Id. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) )) (End of academic pre Company issued) and the like can be referred to.
As described above, HMG1 can be quantified with high sensitivity by using the antibody of the present invention.
[0063]
In the above quantification method using the antibody of the present invention, the concentration of the protein is determined by quantifying the concentration of extracellular HMG1 in the brain parenchyma or cerebral vascular wall using the extracellular fluid of the brain parenchyma or cerebral vascular wall as a test solution Is detected, for example, HMG1 overproduction and / or leakage is likely to cause aggregation of Aβ42 and / or abnormal Aβ42 phagocytosis due to microglia, and hence formation and accumulation of senile plaques. It can be diagnosed that there is a high possibility of suffering from a disease involving senile plaques, for example, brain amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
[0064]
(B) Aβ42 phagocytosis promoter by inhibiting aggregation of Aβ42 and / or microglia
Since the antibody of the present invention can neutralize the Aβ42 aggregation promoting action of HMG1 and the Aβ42 phagocytosis inhibiting action by microglia, it has the effect of suppressing Aβ42 aggregation and / or promoting Aβ42 phagocytosis by microglia. It is useful as a prophylactic / therapeutic agent for diseases associated with senile plaque formation by Aβ deposition as a seed, specifically, brain amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
When the antibody of the present invention is used for the prevention / treatment of the above-mentioned diseases, it can be formulated according to conventional means.
For example, a) the antibody of the present invention can be administered orally as a tablet, capsule, elixir, microcapsule, etc., sugar-coated as necessary, or with water or other pharmaceutically acceptable liquid It can be used parenterally in the form of sterile solutions or injections such as suspensions. For example, b) a unit dosage form required to practice a generally accepted formulation with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. Can be produced by mixing with. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.
[0065]
Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavorings such as peppermint, red oil and cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80)^TM, HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
[0066]
The preventive / therapeutic agent includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.
Since the preparation thus obtained is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can do.
The dose of the antibody of the present invention varies depending on the administration subject, target organ, symptom, administration method, and the like, but in the case of oral administration, for example, in Alzheimer's disease patients (60 kg), about 0.1 to about 0.1 per day. 100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in Alzheimer's disease patients (60 kg) It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.
[0067]
Furthermore, since the antibody of the present invention specifically recognizes HMG1, it can also be used as a targeting drug for delivering these drugs to HMG1 in combination with an HMG1 inhibitor described below.
[0068]
[4] Use of the antisense nucleotide of the present invention
(A) Aβ42 phagocytosis promoter by Aβ42 aggregation inhibition and / or microglia
Since the antisense nucleotide of the present invention inhibits the synthesis of HMG1 mRNA and translation into HMG1 protein, it has the effect of suppressing Aβ42 aggregation and / or promoting Aβ42 phagocytosis by microglia, and uses Aβ42 as a seed. It is useful as a prophylactic / therapeutic agent for diseases involving senile plaque formation due to Aβ deposition, specifically brain amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
When the antisense nucleotide of the present invention is used as the above-mentioned prophylactic / therapeutic agent, the antisense nucleotide is inserted alone or into an appropriate vector such as a retroviral vector, adenoviral vector, adenoviral associated viral vector, etc. It can be formulated. The antisense nucleotide can be formulated as it is or with a physiologically recognized carrier such as an adjuvant for promoting intake, and can be administered by a gene gun or a catheter such as a hydrogel catheter.
[0069]
For example, the nucleotide may be used as a tablet, capsule, elixir, microcapsule or the like, sugar-coated as necessary, or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of an injection such as a suspension. For example, the nucleotides are admixed in a unit dosage form generally required for the practice of the approved formulation with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. Can be manufactured by. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.
[0070]
Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavorings such as peppermint, red oil and cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80)^TM, HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
[0071]
The preventive / therapeutic agent includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.
Since the preparation thus obtained is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can do.
[0072]
The dosage of the antisense nucleotide varies depending on the administration subject, target organ, symptom, administration method, and the like. However, in the case of oral administration, for example, in Alzheimer's disease patients (as 60 kg), about The amount is 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in Alzheimer's disease patients (as 60 kg) It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.
[0073]
[5] HMG1 inhibitor cleaning method and screening kit
A compound or a salt thereof that inhibits the activity of HMG1 or a partial peptide thereof or a salt thereof (eg, Aβ42 aggregation promoting activity, Aβ42 phagocytosis inhibitory activity by microglia) is an effect of suppressing Aβ42 aggregation and / or promoting Aβ42 phagocytosis by microglia. It is useful as a prophylactic / therapeutic agent for diseases associated with senile plaque formation due to Aβ deposition, specifically Aβ42 seeds, such as brain amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.) It is.
Therefore, HMG1 and the like are useful as a reagent for screening a compound or a salt thereof that inhibits the activity of HMG1 or the like (eg, Aβ42 aggregation promoting activity, Aβ42 phagocytosis inhibitory activity by microglia).
[0074]
That is, the present invention
(1) Screening for a substance that promotes Aβ42 aggregation-promoting activity of HMG1 and / or Aβ42 phagocytosis-inhibiting activity by microglia (hereinafter, sometimes simply abbreviated as “HMG1 inhibitor”), characterized by using HMG1 or the like Provide a method, more specifically, for example,
(2a) Screening for an HMG1 inhibitor, characterized by comparing the aggregation of Aβ42 when (i) Aβ42 is brought into contact with HMG1 or the like and (ii) when Aβ42 and a test substance are brought into contact with HMG1 or the like Method, and
(2b) It is characterized by comparing Aβ42 phagocytosis by microglia when (i) HMG1 etc. and Aβ42 are brought into contact with microglia and (ii) when HMG1 etc., Aβ42 and a test substance are brought into contact with microglia. , A method for screening an HMG1 inhibitor is provided.
[0075]
A specific description of the screening method (2a) of the present invention will be given below.
HMG1, its partial peptide or a salt thereof may be any one as described above. Aβ42 is isolated or purified by a known method from the brain parenchyma or cerebral vascular wall of human or other mammal, chemically synthesized based on the known Aβ42 amino acid sequence, and encodes Aβ42 Any of those produced using a recombinant cell or a cell-free protein synthesis system may be used.
Examples of test substances include peptides, proteins, nucleic acids, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like, which are novel compounds. It may be a known compound.
In order to carry out the above screening method, a preparation such as HMG1 is prepared by suspending HMG1 or the like in a buffer suitable for screening. The buffer may be a buffer that does not inhibit the binding between HMG1 and the like and Aβ42 and / or aggregation of Aβ42, such as a phosphate buffer having a pH of about 4 to 10 (preferably about pH 6 to 8) and a Tris-HCl buffer. Any may be used.
Aβ42 aggregation-promoting activity such as HMG1 can be achieved by, for example, incubating HMG1 and Aβ42 (and test substance) at room temperature to about 40 ° C. for about 1 to about 48 hours, and then subjecting the reaction mixture to immunoblotting with an anti-Aβ antibody. Can be measured.
For example, in the case of (ii) above, a substance that inhibits Aβ42 aggregation promoting activity such as HMG1 as a test substance that inhibits Aβ42 aggregation promoting activity by about 20% or more, preferably 50% or more, compared to the case of (i) above Can be selected.
The screening kit of the present invention contains HMG1 or a partial peptide thereof or a salt thereof. Examples of the screening kit of the present invention include the following.
[Reagent for screening]
(1) Measurement buffer
pH 7.0 phosphate buffered saline (PBS)
(2) Protein preparation
Purified HMG1 (eg from calf thymus)
(3) Aβ42
10 μM PBS solution
(4) Anti-Aβ polyclonal antibody (derived from rabbit)
[0076]
The specific description of the screening method (2b) of the present invention is as follows.
As for HMG1 and the like, Aβ42 and the test substance, the same method as in the above (2a) is used. For example, microglia is prepared by suspending mixed glial cells (mixture of astrocytes and microglia) prepared from brain hemispheres excised from mammals such as rats in a medium such as DMEM supplemented with FBS, and plating them on a culture dish or the like. For example, 5% CO₂It is obtained by culturing at about 30 to about 40 ° C. in / 95% air and recovering the floating microglia from the mixed glia culture.
In order to carry out the above screening method, a preparation such as HMG1 is prepared by suspending HMG1 or the like in a buffer or medium suitable for screening. As the buffer, a phosphate buffer, Tris-hydrochloric acid buffer or the like having a pH of about 4 to 10 (preferably, a pH of about 6 to 8) is preferably used, and a microglia culture medium such as MEM or DMEM is preferably used as the medium.
Aβ42 phagocytosis inhibitory activity by microglia such as HMG1 is obtained by adding, for example, HMG1 and Aβ42 (and test substance) to a microglia suspension culture, for example, 5% CO 2.₂After incubation for about 1 to about 48 hours in / 95% air at about 30 to about 40 ° C., microglia can be collected and dissolved, and the lysate can be measured by immunoblotting with anti-Aβ antibody.
For example, in the case of (ii) above, the test substance that inhibits Aβ42 phagocytosis inhibition activity by microglia by about 20% or more, preferably 50% or more, compared to the case of (i) above, is Aβ42 phagocytosis by microglia such as HMG1. It can be selected as a substance that inhibits the inhibitory activity.
The screening kit of the present invention contains HMG1 or a partial peptide thereof or a salt thereof. Examples of the screening kit of the present invention include the following.
[Reagent for screening]
(1) Measurement buffer
pH 7.0 phosphate buffered saline (PBS)
(2) Protein preparation
Purified HMG1 (eg from calf thymus)
(3) Aβ42
10 μM PBS solution
(4) Microglia culture medium
DMEM with 10% FBS
(5) Anti-Aβ polyclonal antibody (derived from rabbit)
[0077]
The substance obtained using the screening method or screening kit of the present invention is a compound selected from the above-mentioned test substances or a salt thereof, and has Aβ42 aggregation promoting activity such as HMG1 and / or Aβ42 phagocytosis inhibiting activity by microglia. It is a compound that inhibits. A representative example of such a compound is HMG1-binding nucleic acid. HMG1 is a non-histone chromosomal protein and has a DNA binding domain in its amino acid sequence. Therefore, a nucleic acid having a base sequence that can bind to HMG1 inhibits AMG42 aggregation-promoting activity of HMG1 and / or Aβ42 phagocytosis inhibition activity by microglia by capturing HMG1.
As a salt of a compound that is an HMG1 inhibitor, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). , Tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
[0078]
[6] Use of HMG1 inhibitor
A compound that inhibits Aβ42 aggregation promoting activity and / or Aβ42 phagocytosis inhibiting activity by microglia, such as HMG1, has an effect of suppressing Aβ42 aggregation and / or Aβ42 phagocytosis by microglia, and uses Aβ42 as a seed. It is useful as a prophylactic / therapeutic agent for diseases involving senile plaque formation due to Aβ deposition, specifically brain amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
[0079]
When the HMG1 inhibitor obtained using the screening method or screening kit of the present invention is used as the prophylactic / therapeutic agent, it can be carried out according to conventional means. For example, the compound may be used as a tablet, capsule, elixir, microcapsule or the like, sugar-coated as needed, or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of an injection such as a suspension. For example, the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., in a unit dosage form as required for generally accepted formulation practice. Can be manufactured by. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.
[0080]
Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavorings such as peppermint, red oil and cherry. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80)^TM, HCO-50) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
[0081]
The preventive / therapeutic agent includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.
Since the preparation thus obtained is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can do.
[0082]
The dose of the HMG1 inhibitor varies depending on the administration subject, target organ, symptom, administration method, and the like. However, in the case of oral administration, for example, an HMG1 inhibitor is generally administered to an Alzheimer's disease patient (as 60 kg). About 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of injection, for example, for Alzheimer's disease patients (as 60 kg) Thus, it is convenient to administer the HMG1 inhibitor in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.
[0083]
[7] Screening method and screening kit for expression inhibitor such as HMG1
Furthermore, the present invention provides a screening method for a substance that suppresses the expression of HMG1 and the like and suppresses Aβ42 aggregation and / or promotes Aβ42 phagocytosis by microglia (hereinafter also referred to as “HMG1 expression inhibitor”) and a kit therefor provide. In this method, a change in the amount of mRNA such as HMG1 is measured using a nucleotide containing the base sequence encoding HMG1 or the like as a probe or primer. Preferably, a change in Aβ42 aggregation promoting activity by microglia or Aβ42 phagocytosis inhibitory activity by HMG1 or the like is further measured by the same method described above for the screening method for HMG1 inhibitor.
[0084]
Specifically, the amount of mRNA such as HMG1 is measured as follows.
(I) Administration of a test substance to a normal or disease model non-human mammal (for example, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc., more specifically, demented rat, etc.) After a certain period of time (for example, 30 minutes to 3 days later, preferably 1 hour to 2 days later, more preferably 1 hour to 24 hours later), specific tissues such as brain parenchyma and cerebral blood vessels are excised. The mRNA such as HMG1 contained in the tissue can be quantified by, for example, extracting mRNA from cells by a conventional method and using a technique such as TaqMan PCR. It is also possible to analyze by performing.
(Ii) After culturing a transformant expressing HMG1 or the like prepared according to the above method in the presence of a test substance and after a certain period of time (for example, 1 day to 7 days later, preferably 1 day to 3 days later, more preferably 1 day to 3 days later) mRNA such as HMG1 contained in the transformant can be quantified and analyzed in the same manner.
(Iii) A nucleotide containing a base sequence encoding HMG1 or the like prepared according to the above method is added to a known in vitro transcription system in the presence of a test substance and incubated for a certain time (for example, after 30 minutes to 3 The mRNA such as HMG1 contained in the reaction solution can be quantified and analyzed in the same manner after 1 day, preferably after 1 hour to 2 days, and more preferably after 1 hour to 24 hours.
[0085]
When Aβ42 aggregation promoting activity such as HMG1 is further measured, Aβ42 is administered (added) in addition to the test substance, and a part of the cell extract or reaction solution is subjected to immunoblotting with an anti-Aβ antibody. When the Aβ42 phagocytosis inhibitory activity by microglia is further measured, (i) Aβ42 is administered in addition to the test substance, and after a certain period of time, microglia is separated and dissolved from tissues such as brain parenchyma, and the lysate is anti-Aβ (Ii) (iii) In addition to the test substance, which is subjected to immunoblotting with an antibody, or a brain slice is prepared and the presence of Aβ42 in microglia is detected by immunohistochemical staining (for example, see Examples below) Aβ42 and microglia are added, and after a certain period of time, microglia is recovered and dissolved, and the lysate may be subjected to immunoblotting with an anti-Aβ antibody.
[0086]
The screening kit used in the screening method comprises a nucleotide containing a base sequence encoding HMG1 or a partial peptide thereof. Examples of the screening kit of the present invention include the following.
[Reagent for screening]
(1) Nucleotide preparation
Isolated HMG1 cDNA (eg, from calf thymus mRNA)
(2) Aβ42
10 μM PBS solution
(3) Microglia culture medium
DMEM with 10% FBS
(4) Anti-Aβ polyclonal antibody (derived from rabbit)
[0087]
The compound obtained by using this screening method / screening kit or a salt thereof is a compound that suppresses the expression of HMG1 or the like and thereby suppresses Aβ42 aggregation and / or promotes Aβ42 phagocytosis by microglia.
As a salt of a compound that is an HMG1 expression inhibitor, a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). , Tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
[0088]
[8] Use of HMG1 expression inhibitor
A compound that suppresses the expression of HMG1 or the like has an effect of suppressing Aβ42 aggregation and / or promoting Aβ42 phagocytosis by microglia, and a disease involving senile plaque formation by Aβ deposition using Aβ42 as a seed, specifically Is useful as a prophylactic / therapeutic agent for brain amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
[0089]
When the HMG1 expression inhibitor is used as the prophylactic / therapeutic agent, it can be carried out according to conventional means. For example, it can be made into a tablet, capsule, elixir, microcapsule, sterile solution, suspension and the like in the same manner as the preparation containing the above-mentioned HMG1 inhibitor.
Since the preparation thus obtained is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can do.
[0090]
Although the dose of the HMG1 expression inhibitor varies depending on the administration subject, target organ, symptom, administration method, etc., in the case of oral administration, generally, for example, suppression of HMG1 expression for Alzheimer's disease patients (60 kg) The agent is about 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of injection, for example, for Alzheimer's disease patients (as 60 kg) Thus, it is convenient to administer the HMG1 expression inhibitor at about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day. In the case of other animals, an amount converted per 60 kg can be administered.
[0091]
[9] Method for screening a compound that decreases the promoter activity of the HMG1 promoter
Furthermore, the present invention relates to a reporter gene assay method using the HMG1 promoter, wherein the promoter activity is measured when the test substance is administered and when the test substance is not administered. Provide screening methods.
Reporter gene assays can be performed by methods known per se, such as The Journal of Biological Chemistry (JBC), 272, 22800-22808, 1997, Development, 124, 793-804, 1997. For example).
Examples of test substances include peptides, proteins, biologically derived non-peptide compounds (such as carbohydrates and lipids), synthetic compounds, microbial cultures, cell extracts, plant extracts, animal tissue extracts, and the like. The compound may be a novel compound or a known compound.
The promoter activity can be measured by examining the expression level of the reporter gene. For example, the expression level of the reporter gene can be measured according to a method such as Northern blotting, reverse transcription-polymerase chain reaction (RT-PCR), TaqMan polymerase chain reaction, or the like.
In this method, a compound that reduces the expression level of a reporter gene when a test substance is administered by about 20%, preferably 30% or more, more preferably about 50% or more, compared to the case where the test substance is not administered. , And can be selected as a compound that decreases the promoter activity of the HMG1 promoter.
[0092]
A compound or a salt thereof that decreases the promoter activity of the HMG1 promoter has an effect of suppressing Aβ42 aggregation and / or promoting Aβ42 phagocytosis by microglia, and senile plaque formation by Aβ deposition using Aβ42 as a seed is involved. It is useful as a preventive / therapeutic agent for diseases, specifically, brain amyloidosis (eg, Alzheimer's disease, Down's syndrome, amyloid angiopathy, etc.).
[0093]
When a compound or a salt thereof that decreases the promoter activity of the HMG1 promoter is used as the prophylactic / therapeutic agent, it can be carried out according to conventional means. For example, it can be made into a tablet, capsule, elixir, microcapsule, sterile solution, suspension and the like in the same manner as the preparation containing the above-mentioned HMG1 inhibitor.
Since the preparation thus obtained is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can do.
[0094]
The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like. However, in the case of oral administration, generally, for example, HMG1 promoter for Alzheimer's disease patients (as 60 kg) The amount of the compound that decreases the promoter activity is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of injection, for example, for Alzheimer's disease patients (as 60 kg) It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound that decreases the promoter activity of the HMG1 promoter per day. . In the case of other animals, an amount converted per 60 kg can be administered.
[0095]
[10] Production of DNA-introduced animal of the present invention
The present invention relates to a non-human mammal having an exogenous DNA of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the exogenous mutant DNA of the present invention). provide.
That is, the present invention
[1] A non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
[2] The animal according to [1], wherein the non-human mammal is a rodent
[3] The animal according to [2], wherein the rodent is a mouse or a rat, and
[4] An object of the present invention is to provide a recombinant vector that contains the exogenous DNA of the present invention or a mutant DNA thereof and can be expressed in mammals.
A non-human mammal having an exogenous DNA of the present invention or a mutant DNA thereof (hereinafter abbreviated as a DNA-transferred animal of the present invention) can be used for embryos including unfertilized eggs, fertilized eggs, spermatozoa and primordial cells thereof. Preferably, at the stage of embryonic development in non-human mammal development (more preferably at the single cell or fertilized egg cell stage and generally prior to the 8-cell stage), the calcium phosphate method, electric pulse method, lipofection method, aggregation method, It can be produced by transferring the target DNA by the microinjection method, particle gun method, DEAE-dextran method or the like. Further, by the DNA transfer method, the target exogenous DNA of the present invention can be transferred to somatic cells, living organs, tissue cells, etc., and can be used for cell culture, tissue culture, etc. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned embryo cells with a cell fusion method known per se.
Examples of non-human mammals include cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like. Among them, rodents, especially mice (for example, C57BL / 6 strain, DBA2 strain, etc. as pure strains) are relatively short in terms of ontogeny and biological cycle from the viewpoint of creating a disease animal model system, and are easy to reproduce. As a system, B6C3F₁System, BDF₁System, B6D2F₁Strain, BALB / c strain, ICR strain, etc.) or rat (for example, Wistar, SD, etc.) is preferred.
Examples of the “mammal” in the recombinant vector that can be expressed in the mammal include humans and the like in addition to the above non-human mammals.
[0096]
The exogenous DNA of the present invention is not the DNA of the present invention inherently possessed by a non-human mammal but the DNA of the present invention once isolated and extracted from the mammal.
Examples of the mutant DNA of the present invention include those in which a mutation (for example, mutation or the like) has occurred in the base sequence of the original DNA of the present invention, specifically, addition of a base, deletion, substitution to another base, etc. The resulting DNA is used, and abnormal DNA is also included.
The abnormal DNA means DNA that expresses abnormal HMG1, and for example, DNA that expresses a protein that suppresses the function of normal HMG1 is used.
The exogenous DNA of the present invention may be derived from mammals of the same or different species as the target animal. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct bound downstream of a promoter that can be expressed in animal cells. For example, when transferring the human DNA of the present invention, the expression of DNA derived from various mammals (for example, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology with this. The DNA of the present invention is highly expressed by microinjecting a DNA construct (eg, a vector) conjugated with the human DNA of the present invention downstream of various promoters into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA transfer mammals can be created.
[0097]
As an expression vector for HMG1, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus or baculovirus, etc. are used. It is done. Of these, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis or plasmids derived from yeast are preferably used.
Examples of promoters that regulate the above DNA expression include: (1) promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.); ▼ Promoters from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), such as albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidity Protein, glutathione S-transferase, platelet-derived growth factor β, keratin K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dystrophin, Tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (commonly abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, Metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF-1α), β actin, α And β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, Prep Enkephalin A, such as a promoter, such as vasopressin is used. Among these, a cytomegalovirus promoter capable of being highly expressed throughout the body, a human peptide chain elongation factor 1α (EF-1α) promoter, human and chicken β-actin promoters, and the like are preferable.
The vector preferably has a sequence (generally called a terminator) that terminates transcription of the target messenger RNA in a DNA-transferred mammal. For example, the sequence of each DNA derived from viruses and various mammals The SV40 terminator of simian virus or the like is preferably used.
[0098]
In addition, the splicing signal of each DNA, enhancer region, part of intron of eukaryotic DNA, etc. 5 ′ upstream of the promoter region, between the promoter region and the translation region, or the translation region for the purpose of further expressing the target foreign DNA It is also possible to connect it 3 ′ downstream of the target.
Further, by adding a sequence encoding a secretion signal 5 ′ upstream of the translation region, the translation product of the foreign DNA can be secreted out of the cell (removing the nuclear translocation signal portion of the DNA encoding HMG1). Preferably). A transgenic animal that secretes exogenous HMG1 extracellularly will be extremely useful in that it can be a model of Alzheimer's disease that better reflects pathological changes in Alzheimer's disease than conventionally known PS1 mice and PS / APP mice.
[0099]
Normal HMG1 translation regions include liver, kidney, thyroid cell, fibroblast-derived DNA and various commercially available genomes derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). A complementary DNA prepared by a known method can be obtained as a raw material from the DNA library as all or part of the genomic DNA or from a liver, kidney, thyroid cell, or fibroblast-derived RNA. In addition, exogenous abnormal DNA can produce a translation region obtained by mutating a normal HMG1 translation region obtained from the above cells or tissues by a point mutagenesis method.
The translation region can be prepared as a DNA construct that can be expressed in a metastasized animal by an ordinary DNA engineering technique in which it is linked downstream of the promoter and optionally upstream of the transcription termination site.
The transfer of the foreign DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the subject mammal. The presence of the foreign DNA of the present invention in the embryo cells of the produced animal after DNA transfer means that all the progeny of the produced animal retain the foreign DNA of the present invention in all the germ cells and somatic cells. means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has the foreign DNA of the present invention in all of its germ cells and somatic cells.
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as a DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. .
The transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be excessively present in all germ cells and somatic cells of the target mammal. Excessive exogenous DNA of the present invention is present in the embryo cells of the produced animal after the DNA transfer. This means that all the offspring of the produced animal have the exogenous DNA of the present invention in all the germ cells and somatic cells. Means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has an excess of the foreign DNA of the present invention in all germ cells and somatic cells.
By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred and passaged so as to have the DNA in excess.
[0100]
The non-human mammal having the normal DNA of the present invention has a high expression of the normal DNA of the present invention, and eventually develops hyperfunction of HMG1 by promoting the function of the endogenous normal DNA. And can be used as a model animal for the disease state. For example, by using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathologic mechanism of HMG1 hyperfunction and HMG1-related diseases and to examine methods for treating these diseases.
Further, since the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of free HMG1, it can be used in screening tests for therapeutic agents for diseases related to HMG1.
On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as the DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. Furthermore, the target foreign DNA can be incorporated into the aforementioned plasmid and used as a raw material. A DNA construct with a promoter can be prepared by a normal DNA engineering technique. The abnormal DNA transfer of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the embryo cell of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all the germ cells and somatic cells. The offspring of this type of animal that has inherited the foreign DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred and passaged so as to have the DNA.
[0101]
The non-human mammal having the abnormal DNA of the present invention has a high expression of the abnormal DNA of the present invention, and finally inhibits the function of the endogenous normal DNA to cause a functional inactive refractory of HMG1. Can be used as a model animal for the disease state. For example, by using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of HMG1 functional inactive refractory and to examine a method for treating this disease.
Further, as a specific applicability, the abnormal DNA highly expressing animal of the present invention is a model for elucidating the function inhibition (dominant negative action) of normal HMG1 by the abnormal HMG1 of the present invention in HMG1 functional inactive refractory disease. It becomes.
In addition, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of free HMG1, it can be used in a therapeutic drug screening test for HMG1 functional inactive refractory disease.
Further, as other applicability of the above-mentioned two kinds of DNA transfer animals of the present invention,
(1) Use as a cell source for tissue culture,
(2) Between HMG1 and a protein specifically expressed or activated by HMG1 by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or analyzing HMG1 expressed by DNA Relevance analysis,
(3) Cell of tissue having DNA is cultured by a standard tissue culture technique, and using these, research on the function of cells from tissue generally difficult to cultivate,
(4) Screening for drugs that enhance cell function by using the cells described in (3) above, and
(5) Isolation and purification of the mutant HMG1 of the present invention and production of antibodies thereof can be considered.
Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of diseases related to HMG1, including functional inactive refractories of HMG1, etc., and in each organ of a disease model related to HMG1 More detailed pathological findings can be obtained, which can contribute to the development of new treatment methods and to the study and treatment of secondary diseases caused by the diseases.
Further, each organ can be taken out from the DNA-transferred animal of the present invention, and after minced, free DNA-transferred cells can be obtained, cultured, or the cultured cells can be organized using a protease such as trypsin. . Furthermore, it is possible to investigate the relationship between the specification of HMG1-producing cells, apoptosis, differentiation or proliferation, or the signal transduction mechanism in them, and to investigate their abnormalities. It becomes.
Furthermore, in order to develop a therapeutic agent for a disease related to HMG1, including a functionally inactive refractory of HMG1, using the DNA-transferred animal of the present invention, using the test method and the quantitative method described above, It is possible to provide an effective and rapid screening method for a therapeutic drug for the disease. Moreover, it is possible to examine and develop a DNA therapy for diseases associated with HMG1 using the DNA-transferred animal of the present invention or the exogenous DNA expression vector of the present invention.
[0102]
[11] Knockout animal
The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and the DNA expression-deficient non-human mammal of the present invention.
That is, the present invention
[1] A non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated,
[2] The embryonic stem cell according to item [1], wherein the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli),
[3] The embryonic stem cell of [1], which is neomycin resistant,
[4] The embryonic stem cell of [1], wherein the non-human mammal is a rodent
[5] The embryonic stem cell according to item [4], wherein the rodent is a mouse,
[6] The DNA expression-deficient non-human mammal in which the DNA of the present invention is inactivated,
[7] The DNA can be inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli), and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention [6] The non-human mammal according to Item,
[8] The non-human mammal according to item [6], wherein the non-human mammal is a rodent.
[9] The non-human mammal according to item [8], wherein the rodent is a mouse, and
[10] A screening method for a compound or a salt thereof that promotes or inhibits promoter activity against the DNA of the present invention, which comprises administering a test compound to the animal of [7] and detecting the expression of a reporter gene I will provide a.
The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated refers to suppressing the DNA expression ability by artificially adding mutation to the DNA of the present invention possessed by the non-human mammal, or By substantially losing the activity of HMG1 encoded by the DNA, the DNA has substantially no ability to express HMG1 (hereinafter sometimes referred to as the knockout DNA of the present invention). An embryonic stem cell (hereinafter abbreviated as ES cell).
As the non-human mammal, the same ones as described above are used.
As a method for artificially adding a mutation to the DNA of the present invention, for example, a part or all of the DNA sequence can be deleted or another DNA can be inserted or replaced by a genetic engineering technique. With these mutations, for example, the knockout DNA of the present invention may be prepared by shifting the reading frame of the codon or destroying the function of the promoter or exon.
[0103]
Specific examples of non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated (hereinafter abbreviated as the DNA inactivated ES cell of the present invention or the knockout ES cell of the present invention) A DNA of the present invention possessed by a non-human mammal is isolated and a neomycin resistance gene, a drug resistance gene typified by a hygromycin resistance gene, or lacZ (β-galactosidase gene), cat (chloramphenicol acetyl) Inserting a reporter gene or the like typified by a transferase gene) to disrupt the function of the exon, or inserting a DNA sequence (for example, a polyA addition signal) that terminates the transcription of the gene into an intron between the exons, By preventing the synthesis of complete messenger RNA As a result, a DNA strand having a DNA sequence constructed so as to disrupt the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cell Southern hybridization analysis using the DNA sequence of the present invention or the vicinity thereof as a probe, or PCR method using the DNA sequence of the targeting vector and the DNA sequence of the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as primers And can be obtained by selecting the knockout ES cell of the present invention.
In addition, as the original ES cell for inactivating the DNA of the present invention by homologous recombination method, for example, those already established as described above may be used, or according to the known Evans and Kaufma method. It may be newly established. For example, in the case of mouse ES cells, currently 129 ES cells are generally used, but since the immunological background is unclear, an alternative purely immunologically genetic background For example, for the purpose of obtaining ES cells with clear clarification, for example, B57 improved by reducing the number of eggs collected in C57BL / 6 mice and C57BL / 6 by crossing with DBA / 2₁Mouse (F of C57BL / 6 and DBA / 2₁) Can be used well. BDF₁In addition to the advantage that the number of eggs collected is high and the eggs are strong, the mouse has C57BL / 6 mice as a background. Therefore, when ES cells obtained using these mice are used to produce pathological model mice, C57BL / 6 mice can be advantageously used in that the genetic background can be changed to C57BL / 6 mice by backcrossing.
When establishing ES cells, blastocysts on the third day after fertilization are generally used, but in addition to this, many blastocysts can be efficiently obtained by collecting eggs and culturing them to blastocysts. Early embryos can be obtained.
Although both male and female ES cells may be used, male ES cells are usually more convenient for producing germline chimeras. In addition, it is desirable to discriminate between males and females as soon as possible in order to reduce troublesome culture.
[0104]
One example of a method for determining the sex of an ES cell is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. If this method is used, it is conventionally about 10 times for karyotype analysis.⁶In contrast to the number of cells required, the number of ES cells of about 1 colony (about 50) is sufficient, so primary selection of ES cells in the early stage of culture can be performed by male / female discrimination. Yes, the early labor of the culture can be greatly reduced by enabling the selection of male cells at an early stage.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes of the obtained ES cell is preferably 100% of the normal number. However, if it is difficult due to physical operations during establishment, the gene of the ES cell is knocked out, and then normal cells (for example, chromosomes in mice) It is desirable to clone again into cells where the number is 2n = 40.
The embryonic stem cell line obtained in this way is usually very proliferative, but it tends to lose its ontogenic ability, so it needs to be subcultured carefully. For example, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5%) in the presence of LIF (1-10000 U / ml) on suitable feeder cells such as STO fibroblasts. Culturing is carried out by a method such as culturing at about 37 ° C. with carbon dioxide gas, 90% air, and at the time of passage, for example, a trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, Preferably, the cells are converted into single cells by treatment with about 0.1% trypsin / 1 mM EDTA and seeded on newly prepared feeder cells. Such passage is usually carried out every 1-3 days. At this time, it is desirable to observe the cells, and when morphologically abnormal cells are found, the cultured cells should be discarded.
ES cells can be differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by monolayer culture to high density or suspension culture until a cell conglomerate is formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embrology and Experimental Morphology, 87, 27, 1985], ES cells of the present invention. The DNA expression-deficient cells of the present invention obtained by differentiation are useful for in vitro cell biology of HMG1 or HMG1.
[0105]
The DNA expression deficient non-human mammal of the present invention can be distinguished from normal animals by measuring the mRNA level of the animal using a known method and comparing the expression level indirectly.
As the non-human mammal, those similar to the above can be used.
The DNA expression-deficient non-human mammal of the present invention is a DNA in which, for example, a targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector of the present invention is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination.
A cell in which the DNA of the present invention is knocked out is obtained by analyzing the DNA sequence of the Southern hybridization analysis or targeting vector using the DNA sequence of or near the DNA of the present invention as a probe and the mouse used for the targeting vector of the present invention. It can be determined by analysis by a PCR method using a DNA sequence in a neighboring region other than DNA as a primer. When a non-human mammalian embryonic stem cell is used, a cell line in which the DNA of the present invention is inactivated by gene homologous recombination is cloned, and the cell is placed at a suitable time, for example, an 8-cell non-human mammal. It is injected into animal embryos or blastocysts, and the produced chimeric embryos are transplanted into the uterus of the non-human mammal that is pseudopregnant. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When some of the germ cells of the chimeric animal have the mutated DNA locus of the present invention, all tissues are artificially mutated from the population obtained by mating such a chimeric individual with a normal individual. It can be obtained by selecting an individual composed of the added cells having the DNA locus of the present invention, for example, by determining the coat color. The individual thus obtained is usually an HMG1 hetero-expression deficient individual, and HMG1 hetero-expression deficient individuals can be mated to obtain HMG1 homo-expression deficient individuals from their offspring.
[0106]
When using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into the chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by a microinjection method. Compared to mammals, it can be obtained by selecting those having a mutation at the DNA locus of the present invention by gene homologous recombination.
Thus, an individual in which the DNA of the present invention is knocked out can be reared in a normal breeding environment after confirming that the individual animal obtained by mating has also been knocked out.
In addition, germline acquisition and retention may be followed in accordance with conventional methods. That is, a homozygous animal having the inactivated DNA in both homologous chromosomes can be obtained by mating male and female animals possessed by the inactivated DNA. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in a state where there are 1 normal individual and multiple homozygous animals. By breeding male and female heterozygotes, homozygotes and heterozygotes having the inactivated DNA are bred and passaged.
The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated is very useful for producing the non-human mammal deficient in DNA expression of the present invention.
Further, since the non-human mammal deficient in DNA expression of the present invention lacks various biological activities that can be induced by HMG1, it can be a model for diseases caused by inactivation of the biological activity of HMG1. It is useful for investigating the cause of diseases and examining treatment methods.
[0107]
(11a) Screening method for compounds having therapeutic / preventive effects on diseases caused by deficiency or damage of the DNA of the present invention
The DNA expression-deficient non-human mammal of the present invention can be used for screening a compound having a therapeutic / preventive effect on a disease caused by the deficiency or damage of the DNA of the present invention.
That is, the present invention is caused by the deficiency or damage of the DNA of the present invention, which comprises administering a test compound to the non-human mammal deficient in DNA expression of the present invention and observing and measuring changes in the animal. Provided is a screening method for a compound having a therapeutic / preventive effect on a disease or a salt thereof.
Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include those described above.
Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. These compounds are novel compounds. It may be a known compound.
Specifically, the non-human mammal deficient in DNA expression of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptom, etc. of the animal as an index. The therapeutic / prophylactic effect of the compound can be tested.
As a method for treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dosage of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
[0108]
In the screening method, when a test compound is administered to a test animal, when the index value of the test animal changes by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound is It can be selected as a compound having a therapeutic / preventive effect against other diseases.
The compound obtained by using the screening method is a compound selected from the above-mentioned test compounds, and is used as a medicament for a safe and low-toxic therapeutic / preventive agent for a disease caused by HMG1 deficiency or damage. Can do. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). And the like, and physiologically acceptable acid addition salts are particularly preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
A medicament containing the compound obtained by the screening method or a salt thereof can be produced in the same manner as the aforementioned medicament containing an HMG1 inhibitor.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered.
[0109]
(11b) Screening method for compounds that reduce the activity of the promoter for the DNA of the present invention
According to the present invention, there is provided a compound or a salt thereof for reducing the activity of a promoter for the DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in DNA expression of the present invention and detecting expression of a reporter gene. A screening method is provided.
In the screening method described above, the DNA expression-deficient non-human mammal of the present invention is inactivated by introducing a reporter gene among the DNA expression-deficient non-human mammals of the present invention described above, A gene capable of expressing the reporter gene under the control of a promoter for the DNA of the present invention is used.
Examples of the test compound are the same as described above.
As the reporter gene, the same ones as described above are used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene, or the like is preferable.
In the non-human mammal of the present invention in which the DNA of the present invention is replaced with a reporter gene, the expression of the substance encoded by the reporter gene is traced because the reporter gene exists under the control of the promoter for the DNA of the present invention. By doing so, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding HMG1 is replaced with the β-galactosidase gene (lacZ) derived from E. coli, β-galactosidase is expressed instead of HMG1 in the tissue where HMG1 is originally expressed. Therefore, for example, by staining with a reagent that is a substrate of β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal), HMG1 The expression state in the animal body can be observed. Specifically, HMG1-deficient mice or tissue sections thereof were fixed with glutaraldehyde, washed with phosphate buffered saline (PBS), and then stained with X-gal at about room temperature or around 37 ° C. After reacting for 30 minutes to 1 hour, the tissue specimen is washed with a 1 mM EDTA / PBS solution to stop the β-galactosidase reaction and observe the coloration. Moreover, you may detect mRNA which codes lacZ according to a conventional method.
The compound obtained by using the screening method or a salt thereof is a compound selected from the test compounds described above, and is a compound that decreases the promoter activity for the DNA of the present invention.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). In particular, physiologically acceptable acid addition salts are preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
[0110]
Thus, the DNA expression deficient non-human mammal of the present invention is extremely useful for screening a compound or a salt thereof that reduces the activity of the promoter for the DNA of the present invention, and the excessive expression of the DNA of the present invention is involved. It can greatly contribute to the investigation of the causes of various diseases and the development of preventive and therapeutic drugs.
[0111]
In the present specification and drawings, bases, amino acids, and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.
DNA: Deoxyribonucleic acid
cDNA: complementary deoxyribonucleic acid
A: Adenine
T: Thymine
G: Guanine
C: cytosine
RNA: Ribonucleic acid
mRNA: Messenger ribonucleic acid
dATP: Deoxyadenosine triphosphate
dTTP: Deoxythymidine triphosphate
dGTP: Deoxyguanosine triphosphate
dCTP: Deoxycytidine triphosphate
ATP: Adenosine triphosphate
EDTA: Ethylenediaminetetraacetic acid
SDS: sodium dodecyl sulfate
[0112]
Gly: Glycine
Ala: Alanine
Val: Valine
Leu: Leucine
Ile: Isoleucine
Ser: Serine
Thr: Threonine
Cys: Cysteine
Met: Methionine
Glu: Glutamic acid
Asp: Aspartic acid
Lys: Lysine
Arg: Arginine
His: Histidine
Phe: Phenylalanine
Tyr: Tyrosine
Trp: Tryptophan
Pro: Proline
Asn: Asparagine
Gln: Glutamine
pGlu: pyroglutamic acid
*: Corresponds to the stop codon
Me: methyl group
Et: ethyl group
Bu: butyl group
Ph: phenyl group
TC: thiazolidine-4 (R) -carboxamide group
[0113]
In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
Tos: p-toluenesulfonyl
CHO: Formyl
Bzl: benzyl
Cl₂Bzl: 2,6-dichlorobenzyl
Bom: benzyloxymethyl
Z: benzyloxycarbonyl
Cl-Z: 2-chlorobenzyloxycarbonyl
Br-Z: 2-bromobenzyloxycarbonyl
Boc: t-butoxycarbonyl
DNP: dinitrophenol
Trt: Trityl
Bum: t-butoxymethyl
Fmoc: N-9-fluorenylmethoxycarbonyl
HOBt: 1-hydroxybenztriazole
HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide
DCC: N, N'-dicyclohexylcarbodiimide
[0114]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
SEQ ID NO: 1
The base sequence of cDNA encoding human-derived HMG1 is shown.
SEQ ID NO: 2
The amino acid sequence of human-derived HMG1 is shown.
[0115]
【Example】
EXAMPLES The present invention will be described in more detail with reference to the following examples, but these do not limit the scope of the present invention. In addition, the result regarding the densitometric analysis of an immunoblot is shown as an average +/- standard error. Statistical significance of differences was determined by analysis of variance (ANOVA). Further statistical analysis for post hoc comparisons was performed using the Bonferroni / Dunn test (StatView, Abacus Concepts, Berkley, CA). Moreover, the confocal microscope observation was performed as follows. AD brain sections were incubated overnight at room temperature with antibodies against HMG1 and HLA-DR, and hippocampal sections from rats injected with Aβ42 were incubated with antibodies against Aβ and CD11b. The primary antibody was detected with rhodamine or FITC-labeled anti-rabbit IgG antibody and anti-mouse IgG antibody, and the fluorescence was observed using a laser scanning confocal microscope LSM410 (Carl Zeiss, Germany).
[0116]
Example 1 Up-regulation of HMG1 in the brain of Alzheimer's disease (AD)
6 patients diagnosed clinically and histopathologically as AD (age 81.2 ± 3.5 years; autopsy delay 3.8 ± 0.7 hours) and 6 controls without clinical and morphological evidence of brain lesions ( Brain tissue was obtained by autopsy from age 71.0 ± 4.2 years; autopsy delay 8.0 ± 2.9 hours). Age and delay in necropsy were not significantly different between AD and control groups. The temporal cortex was used for the experiment. Neuropathological evaluation of AD was performed according to the criteria of the Consortium to Establish a Registry for Alzheimer ’s Disease (CERAD). Cytoplasmic fraction (including cytosol and extracellular soluble protein) and particle fraction (including nucleus, membrane and insoluble protein) were prepared according to the method described in Brain Res. 780: 260-269 (1998). . Both fractions (10 μg protein) were subjected to SDS-PAGE. After immunoblotting with an antibody against HMG1 or HMG2 (BD Pharmingan, San Diego, Calif.), Protein bands were detected using a chemiluminescence assay (ECL kit, Amersham Pharmacia Biotech, Buckinghamshire, U.K.). For semi-quantitative analysis of protein bands, X-ray film was scanned with a CCD color scanner (DuoScan, AGFA) and the density was measured.
Immunohistochemical analysis was also performed essentially according to the method described in Brain Res. 780: 260-269 (1998). Specifically, brain slices were incubated in 0.3% hydrogen peroxide-containing phosphate buffered saline (PBS) for 30 minutes, washed with 0.3% Triton X-100-containing PBS (PBS-T), and then an antibody against HMG1, Aβ ( Chemicon, Temecula, CA) and PBS-T containing HLA-DR (Dako, Glostrup, Denmark) were incubated overnight at room temperature. After washing with PBS-T and incubation with Vectastatin ABC elite kit (Vector Laboratories, Burlingame, Calif.), The brain slices were visualized using 3,3′-diaminobenzidine (DAB) with nickel enhancement and hydrogen peroxide .
As a result, 29 kDa HMG1 was detected in both the cytoplasmic and particle fractions of the control and AD brain temporal cortex (FIG. 1). It was present in the cytoplasm fraction more than the particle fraction. In AD brain, HMG1 expression level in both cytoplasmic and particle fractions was significantly higher than in control brain (Figs. 1a and b, P <0.05 and P <0.01, respectively), and HMG1 expression was up-regulated in AD brain. It shows that it is rate. 28 kDa HMG2 was also detected in both the cytoplasmic and particle fractions, but its expression (lower than HMG1) was not significantly different in AD brain (data not shown).
In the control brain, HMG1 immunoreactivity was faintly detectable in the cytoplasm and nucleus of neurons and glial cells (FIG. 1c). In AD brain, HMG1 immunoreactivity was increased at these locations (FIG. 1d). Furthermore, HMG1 immunoreactivity was observed in a plaque-like manner and was strongly detected in the region of Aβ immunoreactivity (compare FIG. 1e with FIG. 1g). Human leukocyte antigen (HLA) -DR, a marker of activated microglia, was also observed (FIG. 1f). Double immunostaining revealed that HMG1 is located both inside and around the microglial cells expressing HLA-DR (FIG. 1h). These immunocytochemical results indicate that extracellular HMG1 accumulates around the area where microglia accumulate in senile plaques.
[0117]
Reference Example 1 Distribution of HMG1 in transgenic mice
Recently, several transgenic mice that overproduce Aβ peptide have been created. Tg2576 mice and mutant presenilin-1 (PS1) mice express K670N / M671L (Swedish) mutant human amyloid precursor protein (APP) and M146L mutant human PS1, respectively. Furthermore, a double transgenic PS / APP system has been created by crossing Tg2576 mice with PS1 mice. In PS1 mice, mouse Aβ42 production is increased, but no Aβ deposits are formed, whereas in PS / APP mice, significant human and mouse Aβ40 and Aβ42 production and accelerated Aβ deposit formation occurs. However, extensive neuronal loss is not detected in either PS1 or PS / APP mice. Thus, we examined the immunoreactivity of HMG1 and Aβ in transgenic mice as a model showing a massive production of Aβ42 without neuronal loss.
12 months old PS1 and PS / APP mice were used for the experiment. Frontal brain sections from these mice were obtained from Am. J. Pathol., 158: 1345-1354 (2001), and then immunostained with antibodies against HMG1, Aβ and CD11b (MAC1, Caltag Laboratories, San Francisco, Calif.). For double-labeled immunohistochemical staining, brain sections were incubated for 4 days at 4 ° C. with rabbit anti-HMG1 antibody with mouse monoclonal Aβ antibody in PBS-T. After washing, sections were incubated with anti-rabbit IgG antibody and HMG1 immunoreactivity was detected using DAB and nickel ammonium. After completion of HMG1 immunostaining, sections were treated with PBS-T containing 0.5% hydrogen peroxide for 30 minutes to destroy residual peroxidase from the first cycle. Thereafter, a second immunohistochemical cycle of sections and anti-mouse IgG antibody was performed. The DAB reaction was performed without nickel enhancement.
As a result, in PS1 mice, branched microglia that were immunostained with rat anti-CD11b antibody (MAC1) were observed, but Aβ deposition and reactive microglia were not detected even at 12 months of age (FIGS. 2a and c). And e). In 12-month-old PS / APP mice, excessive Aβ deposition associated with bush and amoeba microglia was detected in a pattern similar to the immunoreactivity of Aβ and HLA-DR in AD brain (FIGS. 2b, d and f). Compared to FIGS. 1f and g). However, HMG1 immunoreactivity in these transgenic mice was slightly different from that in AD brain. That is, HMG1 immunoreactivity was prominently detected in neurons and glial nuclei in both PS1 and PS / APP mice (FIGS. 2g and h). Although observed in the vicinity of Aβ deposition in PS / APP mice, this immunoreactivity was probably found in glial micronuclei rather than extracellularly (FIG. 2h). That is, PS / APP mice showed Aβ deposition, but no diffuse HMG1 immunoreactivity was detected. These results suggest that the formation of Aβ deposits in PS / APP mice can be caused primarily by overproduction of Aβ peptides not involving extracellular HMG1.
[0118]
Reference Example 2 Distribution of HMG1 in a rat model
To elucidate the association between extracellular HMG1 and Aβ in the brain, we further developed two rat models: 1) Hippocampal CA3 without Aβ deposition as a result of intraventricular (icv) injection of kainic acid (KA) 2) HMG1 immunoreactivity was examined in rats that caused massive neuronal loss and 2) rats that produced large Aβ deposits with moderate neuronal loss as a result of intra-hippocampal injection of Aβ42. Microtubule-associated protein-2 (MAP2) was used as an indicator of neuronal survival.
Male Wistar rats weighing approximately 280 g were fasted overnight with free water supply. Rats were anesthetized for microinjection of stereotaxic brain fixation (50 mg / kg of pentobarbital sodium was injected intraperitoneally) and fixed in a Kopf stereotaxic brain fixation frame. Thereafter, 1 μg / 2 μl KA was injected into the right ventricle (ie, 3.8 mm tail side, 1.2 mm right side, 3.8 mm bottom surface from Bregma). Alternatively, the right hippocampus (ie, 0.2 mm tail side, 2.0 mm right side, 4.0 mm bottom side) of the rat was injected with 4.5 μg / 2 μl of Aβ42 (AnaSpec, San Jose, Calif.) Via a motor-driven 10 μl Hamilton syringe. . Three days later, treated rats are cold-fixed with 150 ml of 10 mM phosphate buffered saline (PBS) through the artery followed by 4% paraformaldehyde, 0.35% glutaraldehyde and 0.2% picric acid (in 100 mM phosphate buffer). 300 ml of the solution was perfused under deep anesthesia with pentobarbital (100 mg / kg ip). After perfusion, the brain was quickly removed and fixed with paraformaldehyde in 100 mM PB for 2 days, and then transferred to 15% sucrose solution (in 100 mM PB containing 0.1% sodium azide) at 4 ° C. Brain slices were cut into 20 μm sections in a cryostat and collected in PBS-T. Thereafter, hippocampal sections were immunostained with antibodies against MAP2, HMG1, Aβ, CD11b (OX42, Harlan Sera-Lab, Loughbrough, U.K.) and GFAP (Chemicon International, Temecula, Calif.).
As a result, i.c.v. injection of KA caused neuronal loss in the ipsilateral hippocampal CA3 after 3 days (Fig. 3b) but not the contralateral (Fig. 3a). No Aβ deposition was observed (FIGS. 3f and h), but activated microglia and astrocytes (which are immunostained with an antibody against CD11b (OX42) and an antibody against glial fibrillary acidic protein (GFAP), respectively) Accumulated in CA3 (Figure 3i-l). HMG1 immunoreactivity was prominently detected in neurons and glial nuclei (FIGS. 3e, g and i-l). HMG1 immunoreactivity was also detected in glial processes of neurodegenerative CA3 (FIGS. 3g, k and l), but no diffuse staining was detected.
In rats injected microscopically with Aβ42 into the hippocampus, amoeba-like microglia (Figs. 4d, f and g) immunostained with mouse anti-CD11b antibody are observed around large Aβ deposits (Figs. 4a, e and g) in the hippocampus It was done. In addition, a moderate loss of MAP2 immunoreactivity was detected in the hippocampal dentate gyrus (DG) around Aβ deposits (Figure 4b). HMG1 immunoreactivity was observed in neurons and glial nuclei, and glial processes (FIGS. 4c, e and f). Furthermore, HMG1 immunoreactivity was detected as extracellular diffuse staining around Aβ deposits (FIGS. 4c, e and f). From these observations, extracellular HMG1 leaks primarily from dead neurons, and diffuse HMG1 deposition may be caused by both massive neuronal death and Aβ accumulation.
[0119]
Example 2 Inhibition of Aβ42 phagocytosis by microglia induced by HMG1
In order to investigate the mechanistic significance of the presence of HMG1, the effect of HMG1 on the function of microglia was examined using a pure culture of rat microglia. In the experiment, Aβ40 and Aβ42 were not trifluoroacetic acid (TFA) salts, but easily aggregated hydrochlorides. The present inventors recently found that Aβ40 induces production of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) at 10 μM (FASEB J. 16: 601-603 ( 2002)). In contrast, extracellular Aβ42 significantly caused phagocytosis by microglia at lower concentrations (0.03-0.3 μM) 6-24 hours after Aβ42 exposure. Insulin or pepstatin A suppressed the decrease in Aβ42 levels in microglia after 3 days, but did not suppress phosphoramidon (neprilysin inhibitor), and phagocytosed Aβ42 was stimulated by IDE-like peptidase and / or cathepsin D-like aspartic proteinase in the microglia. It suggests that it will be decomposed. Although relatively low concentrations (less than 1 μM) of Aβ40 did not cause microglia uptake, at higher concentrations (3-10 μM) microglia began to phagocytose Aβ40. Thus, rat microglia show different responses to Aβ40 and Aβ42.
Mixed glial cells (a mixture of astrocytes and microglia) were prepared from brain hemispheres carefully removed from the meninges of newborn Wistar rats (SLC Inc., Shizuoka, Japan). The tissue suspension was filtered through a 50 μm diameter nylon mesh (cell strainers, Falcon) into a 50 ml tube and centrifuged at 200 × g for 10 minutes to collect the cells. Cells are resuspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, plated on 100 mm dishes and 5% CO₂Incubated at 37 ° C in / 95% air. With great care to avoid endotoxin contamination, suspended microglia were collected from the mixed glia culture and plated onto new culture dishes.
The resulting rat microglia (purity greater than 97%) is present in the presence or absence of 0.015-1.5 μM purified calf HMG1 and / or 0.4 nM recombinant human TGF-β1 (Gibco BRL, Life Technologies, Tokyo, Japan). Were incubated with 0.3 μM Aβ42 or 3 μM Aβ40. After 24 hours, microglia were collected and lysed, and then samples were subjected to immunoblotting with anti-Aβ antibody (Chemicon, Temecula, CA). Monomers and aggregated Aβ were detected using a chemiluminescence assay, and the total amount of Aβ was measured with a densitometer.
When HMG1 purified from calf thymus was added to rat microglia cultures containing low concentrations (0.3 μM) of Aβ42, HMG1 inhibited Aβ42 phagocytosis in a concentration-dependent manner to over 60% (FIGS. 5a and b). Since it has been reported that astrocyte-derived TGF-β1 is important for Aβ clearance, the effect of HMG1 on Aβ clearance induced by TGF-β1 was further investigated. 0.4nM recombinant human TGF-β1 significantly increased Aβ42 phagocytosis, whereas HMG1 markedly displayed TGF-β1-induced Aβ phagocytosis up to the level produced by HMG1 treatment in the absence of TGF-β1 (FIGS. 5c and d). In contrast, Aβ40 phagocytosis was not altered by TGF-β1 and HMG1 at high concentrations (3 μM) (FIGS. 6a and b). These results suggest that if extracellular HMG1 accumulates in senile plaques, Aβ42 clearance by microglia activators such as TGF-β1 will be inhibited, but Aβ40 clearance will not be inhibited. .
[0120]
Reference Example 3 Effect of HMG1 on microglial activation induced by Aβ40
Microglia was treated with 10 μM Aβ40 (AnaSpec) in the presence or absence of 1.5 μM HMG1 (Wako Pure Chemical Industries) purified from calf thymus. Medium was removed from each well 24 hours after treatment. NO₂ ^-The amount was measured spectrophotometrically (DU-640 spectrophotometer, Beckman) using Griess reagent. The amount of TNF-α and IL-6 released from the cells was measured using an ELISA kit for rat TNF-α and IL-6 (BioSource, Camarillo, CA) according to the manufacturer's instructions.
As a result, 10 μM Aβ40 was found to be nitrite (NO₂ ^-) Was induced to induce inducible nitric oxide synthase (iNOS) expression, and TNF-α and IL-6 production was induced (FIGS. 6c, d and e). However, HMG1 did not affect the production of these compounds in the presence and absence of Aβ40 (FIGS. 6c, d and e). Thus, HMG1 does not affect the production of NO, TNF-α and IL-6 by microglia in the presence and absence of Aβ40.
[0121]
Example 3 Binding of HMG1 to Aβ peptides in vitro and their aggregation
Purified calf HMG1 (1.5 μg) was incubated at 37 ° C. with 3 μg Aβ40 or Aβ42 (in PBS (final volume 20 μl)). Samples were subjected to immunoblotting with antibodies to HMG1 or Aβ after 0, 6 and 24 hours. Further, 1.5 μg of HMG1 was incubated with 3 μg of Aβ40 or Aβ42 at 37 ° C. for 24 hours, then an antibody against HMG1 or Aβ (10 μg) was added to the mixture, and further incubated at 4 ° C. for 2 hours. Protein A-Sepharose (50 μl of 50% slurry) was then added and incubated overnight at 4 ° C. After centrifugation, the immunoprecipitates were washed 3 times in 1 ml of cold buffer, resuspended in Laemmli sample buffer and subjected to immunoblotting with antibodies against SDS-PAGE and HMG1 or Aβ.
When HMG1 was incubated with Aβ42, complexes of these proteins (approximately 33 kDa) were detected after 6 hours (FIGS. 7a and b). Incubation of Aβ42 with HMG1 did not change the oligomer formation after 24 hours and significantly increased the formation of higher molecular aggregates (FIG. 7b). In contrast, incubation with Aβ42 alone decreased its oligomer level and increased aggregates after 6 hours. On the contrary, after 24 hours, no oligomers and aggregates of Aβ40 were detected, but a complex of HMG1 and Aβ40 was detected (FIGS. 7a and b).
After incubating HMG1 with Aβ40 or Aβ42 in vitro for 24 hours, the mixture was immunoprecipitated with antibodies against HMG1 or antibodies against Aβ. These antibodies precipitated both HMG1 and Aβ peptide (FIG. 7c). In addition, complexes of HMG1 with either Aβ40 or Aβ42 were also immunoprecipitated. Therefore, HMG1 bound to Aβ peptide and promoted the formation of Aβ42 aggregates, but did not promote the formation of Aβ40 aggregates.
[0122]
In conclusion, HMG1 protein levels are upregulated in both cytoplasmic and particulate fractions of AD brain, and HMG1 coexists with senile plaques. Diffuse HMG1 immunoreactivity was observed in the hippocampus of rats injected with Aβ42, but not in the hippocampus and PS / APP mice of rats injected with KA, and extracellular HMG1 and Aβ42 interact synergistically. This suggests the formation of senile plaques. Extracellular HMG1 inhibited phagocytosis of Aβ42 by microglia in the presence and absence of TGF-β1. Furthermore, HMG1 bound to Aβ peptide and promoted the formation of Aβ42 aggregates (FIG. 8). These results suggest that up-regulation of HMG1 inhibits Aβ42 clearance by microglia, so that Aβ deposition is maintained. Thus, it has been shown that extracellular HMG1 may play a pathogenic role in AD through the inhibition of Aβ clearance and promotion of Aβ aggregation by microglia.
[0123]
【The invention's effect】
HMG1 or a partial peptide thereof or a salt thereof is used as a vaccine for the prevention / treatment of brain amyloidosis such as Alzheimer's disease, and nucleotides complementary to nucleotides encoding HMG1 etc. and antibodies against HMG1 etc. are used for the prevention / treatment of brain amyloidosis. Useful as an agent.
Nucleotides encoding HMG1 and the like and antibodies against HMG1 and the like are useful as diagnostic agents for brain amyloidosis.
A screening method for an HMG1 inhibitor using HMG1 and the like and a screening method for an HMG1 expression inhibitor using a nucleotide encoding HMG1 and the like are useful as a search tool for a compound having prophylactic / therapeutic activity for brain amyloidosis.
[0124]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram and a photograph showing the expression level and distribution of HMG1 in control and AD brains. a and b: immunoblot photographs of the cytoplasm (a) and particle (b) fractions in the temporal cortex (upper figure) and a graph showing the band concentration of 29 kDa (lower figure; vertical axis is% ratio relative to control; each numerical value is 6 Specimen mean ± standard error; individual numbers are circled). hh: Immunostaining photographs using antibodies against HMG1 (ce, h), HLA-DR (f, h) or Aβ (g) of control and AD brain sections. Sections (e-h) are from the same individual, and * (eg) indicates the same vessel. The arrows show the same senile plaques as shown at high magnification on the inset. h is double staining of senile plaques using antibodies against HMG1 and HLA-DR. Scale bar: 250 μm (eg) and 50 μm (inset of h and eg).
FIG. 2 is a photograph showing the localization of HMG1 in the transgenic mouse brain. CD11b (MAC1, ad), Aβ (e-h) or HMG1 (12 month old PS1 (a, c, e, g) and frontal brain sections of PS / APP mice (b, d, f, h) ( Immunostaining with antibodies against g, h). A high magnification image of the box is shown inset (ch). Scale bars: 1 mm (a, b), 200 μm (c-h) and 50 μm (inset in c-h).
FIG. 3 is a photograph showing the localization of HMG1 in the rat brain injected with KA. Antibodies against hippocampal slices MAP2 (a, b), HMG1 (ce, g, il), Aβ (f, h), CD11b (OX42, j, l) or GFAP (i, k) were used. Immunostaining. Intraventricular KA injection and contralateral (a, c, e, f, i, j) and ipsilateral (b, d, g, h, k, l). High magnification image (e-l) of the box in hippocampus CA3. il: Double staining using antibodies against HMG1 and CD11b (j, l) or GFAP (i, k). Scale bars: 500 μm (ad) and 50 μm (e-1).
FIG. 4 is a photograph showing the localization of HMG1 in the rat hippocampus injected with Aβ42. Immunostaining using antibodies against Aβ (a, e, g) HMG1 (c, e, f), MAP2 (b) or CD11b (OX42, d, f, g) of hippocampal slices. e, f: Double staining using antibodies against HMG1 and Aβ (e) or CD11b (f). A high magnification image of the box in each immunostained section (af) is shown inset. Arrows indicate diffuse staining with anti-HMG1 antibody. g: Confocal microscopic image of double staining around Aβ deposits in the hippocampus using antibodies against Aβ and CD11b. Scale bars: 500 μm (af) and 50 μm (inset in af, g).
FIG. 5 is a photograph and drawing showing the inhibitory action of HMG1 on Aβ42 phagocytosis by microglia. a and b: immunoblot photographs (a) of rat microglia lysate incubated with Aβ42 in the presence of vehicle (Control in lanes C and b in a) or HMG1 at various concentrations (longitudinal) (longitudinal) Axes are percentages relative to the control; each value is the mean ± standard error of three experiments; ** and *** indicate p <0.01 and p <0.001, respectively, for the control values). c and d: in the presence of vehicle (lane TGF-β1 (−) in c and vehicle in d) or TGF-β1 (lane TGF-β1 (+) in c and TGF-β1 in d), Immunoblot photograph (a) and total Aβ42 amount of rat microglia lysate incubated with Aβ42 (HMG1 (−) in c and Control in d) or Aβ42 and HMG1 (HMG1 (+) in c and HMG1 in d) (The vertical axis is the percentage relative to the control; each value is the mean ± standard error of three experiments; *** and ††† indicate p <0.001 for the vehicle and TGF-β1 alone values, respectively) .
FIG. 6 is a photograph and drawing showing the effect of HMG1 on Aβ40 uptake and activation by Aβ40 in microglia. a and b: immunoblot photograph of rat microglia lysate incubated with Aβ40 in the presence of vehicle, TGF-β1 or HMG1 (a) and a graph showing the total amount of Aβ40 (the vertical axis is the percentage relative to the vehicle; Mean of experiment ± standard error is shown). ce: immunoblot photographs of anti-iNOS antibody of rat microglia lysate incubated with vehicle (HMG1 (-)) or HMG1 (HMG1 (+)) in the presence or absence of Aβ40, and (c top panel) NO in the culture supernatant₂ ^-Amount (c lower figure) Graph showing TNF-α amount (d) and IL-6 amount (e) (each value is the mean ± standard error of three experiments; *** and ††† are vehicle and Aβ40 treatment, respectively) P <0.001 for the numerical value of).
FIG. 7 is a photograph showing binding of HMG1 and Aβ peptide and aggregation of Aβ peptide in vitro. a and b: immunoblot using antibodies against HMG1 (a) or Aβ for samples incubated with HMG1 in the presence or absence of Aβ40 or Aβ42 (arrows indicate the complex of HMG1 and Aβ40 or Aβ42) . c: Incubated Aβ40 or Aβ42 and HMG1, immunoprecipitated with an antibody against HMG1 (upper figure) or Aβ (lower figure) of the reaction solution, and immunoblotted with an antibody against HMG1 or Aβ.
FIG. 8 is a schematic diagram showing the involvement of extracellular HMG1 in the senile plaque formation process.

Claims (15)

A diagnostic agent for amyloid β42 aggregation and / or amyloid β42 phagocytosis caused by microglia, comprising a nucleotide containing a base sequence encoding HMG 1 . HMG 1 or the antibodies comprises an antibody to salts thereof, amyloid β42 aggregation and / or diagnostic agents of amyloid β42 meals acting abnormally by microglia. HMG 1 or is characterized by using a salt thereof, a screening method for a substance showing inhibition to amyloid β42 aggregation inhibiting and / or amyloid β42 meals action promoting action by microglia HMGl. HMG 1 or is brought into contact with amyloid beta 42 and the test substance in a salt thereof, characterized by assaying the aggregation of amyloid beta 42, a screening method of a substance showing inhibition to amyloid beta 42 aggregation inhibiting action of HMGl. HMG 1 or is characterized by performing the comparison with the aggregation of amyloid β42 when contacting the amyloid β42 salt thereof, The method of claim 4. HMG 1 or is contacted salt thereof, amyloid β42 and test substance microglia, and wherein the assaying amyloid β42 meals action by microglia, the substance having an amyloid β42 meals action promoting action by microglia by inhibiting HMG1 Screening method. HMG 1 or is characterized by performing the comparison between amyloid β42 meals effect of microglia in the case where a salt thereof and amyloid β42 contacting the microglial The method of claim 6 wherein. HMG 1 or is comprising a salt thereof, kit for screening substances that show inhibition to amyloid β42 aggregation inhibiting and / or amyloid β42 meals action promoting action by microglia HMGl. The kit according to claim 8, further comprising amyloid β42. Which comprises using a nucleotide containing a base sequence encoding the HMG 1, the screening method for a substance showing an amyloid β42 meals action promoting action by amyloid β42 aggregation inhibition and / or microglia to suppress the expression of HMGl. The test substance is added from nucleotides containing the nucleotide sequence encoding the HMG 1 in a system capable of producing HMG 1, characterized in that testing the expression of HMG 1, method of claim 10. It was added a test substance and amyloid beta 42 nucleotides to a system capable of producing HMG 1 containing the base sequence encoding the HMG 1, characterized by assaying the aggregation of the expression of HMG 1 and amyloid beta 42, expression of HMG1 Screening method for substances that suppress amyloid β42 aggregation. Test substance from nucleotides to a system capable of producing HMG 1 containing the base sequence encoding the HMG 1, was added amyloid β42 and microglia, and wherein the assaying amyloid β42 meals action by expression and microglia HMG 1 The screening method of the substance which suppresses the expression of HMG1 and shows the amyloid (beta) 42 phagocytosis promoting effect by microglia. A kit for screening a substance comprising a nucleotide containing a base sequence encoding HMG 1 and suppressing amyloid β42 aggregation suppression and / or amyloid β42 phagocytosis promoting action by microglia. The kit according to claim 14, further comprising amyloid β42.

Images