Screening method for therapeutic agent for disease, fusion gene, and promoter

Abstract

To provide a screening method for prevention and / or treatment of aneurysm or rupture of aneurysm resulting from excessive release of calcium ions from the endoplasmic reticulum into the cytoplasm, as well as a fusion gene to be used therein.SOLUTION: A screening method introduces into cultured cells having an endogenous GON domain with a sequence different from that of an exogenous GON domain, a fusion gene comprising a KillerRed protein gene and a GON domain protein gene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for screening preventive and / or therapeutic agents for "diseases caused by elevated intracytoplasmic calcium levels," and to fusion genes, promoters, etc. suitable for use therein. [Background technology]

[0002] One of the causes of various diseases is the release of large amounts of calcium ions into the cytoplasm, but there are currently very few drugs that can effectively treat these diseases.

[0003] For example, drugs have been developed that directly inhibit the IP3 (inositol triphosphate) receptor, which is known as the calcium ion release pathway (Non-Patent Document 1). However, because a certain amount of calcium ions is necessary for life maintenance and the receptor also has other functions, there are concerns that inhibiting the pathway (receptor) itself may involve side effects and other risks.

[0004] Meanwhile, it has been discovered that a type of secretory metalloprotease called ADAMTS9 is involved in the release of calcium ions into the cytoplasm.

[0005] ADAMTS9 is a known protein belonging to the ADAMTS group that has the function of digesting extracellular matrix (ECM) proteins. However, it has unique functions due to a "characteristic sequence (GON domain)" at its C-terminus that is not found in most other ADAMTS. It is becoming clear that one of these unique functions is the inhibition of calcium ion release into the cytoplasm (Non-patent Document 2).

[0006] However, the current situation is that no method for searching for innovative drugs for the above diseases has yet been established. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Discovery of a cell membrane-permeable selective Ins(1,4,5)P3 antagonist, 2APB, and research into its specificity and usefulness (Takayuki Maruyama, https: / / repository.kulib.kyoto-.ac.jp / dspace / bitstream / 2433 / 149580 / 1 / yyakr00674.pdf) [Non-patent document 2] Identification of a novel ADAMTS9 / GON-1 function for protein transport from the ER to the Golgi,Molecular Biology of the Cell,Volume 23 May 1, 2012,P.1728-1741,Sawako Yoshina et al.) Summary of the Invention [Problem to be solved by the invention]

[0008] The inventors have succeeded in degrading the exogenous GON domain at any timing, independently of knockdown of ADAMTS9 expression in cultured cells, by deliberately introducing an exogenous GON domain (e.g., a GON domain derived from another species) having a GON domain sequence different from that of the GON domain gene endogenous to the cells, thereby arriving at the present invention.The objective of this invention is to establish a screening method for agents that prevent excessive calcium ion release using the GON domain itself as an indicator. [Means for solving the problem]

[0009] (First Invention) A method for screening for an agent that suppresses an increase in cytoplasmic calcium concentration or a preventive and / or therapeutic agent for a disease caused by such an increase, comprising the following steps (A) to (G):

[0010] (A) preparing cultured cells into which the fusion gene (I) below has been introduced and which have an endogenous GON domain with a sequence different from that of (I)-3; (B) A step of administering a candidate compound to the cultured cells of (A). (C) A step of introducing (II) into the cultured cells of (A). (D) A step of introducing (III) into the cultured cells of (A). (E) A step of irradiating the cultured cells of (A) with light having a wavelength capable of exerting the effect of (I)-2. (F) A step of measuring the amount of change in fluorescence before and after the irradiation of (E) using the detection function of (III). (G) A step of selecting a candidate compound having an effect of preventing excessive release of calcium ions into the cytoplasm by comparing the amount of change in fluorescence (F) between the group administered with the candidate compound and the group not administered with the candidate compound.

[0011] (I) A fusion gene comprising the following (I)-1 to (I)-3: (II) Substances that suppress ADAMTS9 gene expression (III) Substances with calcium ion detection function

[0012] (I)-1: Promoter for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein having photo-induced reactive oxygen species generation or diffusion activity, or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0013] (Second Invention) The screening method according to the first aspect of the present invention, wherein (I)-1 has the sequence described in the following (I)-1-1 or (I)-1-2:

[0014] (I)-1-1: Nucleotide sequence of SEQ ID NO: 1 (I)-1-2: A base sequence in which one or several bases are deleted, substituted, added, and / or inserted from the base sequence of SEQ ID NO: 1, and which has the same promoter activity as SEQ ID NO: 1.

[0015] (Third Invention) A screening kit for an agent for suppressing an increase in intracytoplasmic calcium concentration or an agent for preventing and / or treating a disease caused by said increase, comprising the following (I) to (IV):

[0016] (I) A fusion gene comprising the following (I)-1 to (I)-3: (II) Substances that suppress ADAMTS9 gene expression (III) Substances with calcium ion detection function (IV) A cultured cell for introducing (I), which has an endogenous GON domain with a sequence different from that of the GON domain of (I)-3 below.

[0017] (I)-1: Promoter for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein having photo-induced reactive oxygen species generation or diffusion activity, or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0018] (Fourth Invention) The kit according to the third aspect of the invention, wherein the fusion gene (I) further comprises the following (I)-5:

[0019] (I)-5: A sequence encoding a signal peptide that directs transport to the endoplasmic reticulum

[0020] (Fifth Invention) A promoter according to (I)-1 below, characterized in that it comprises the sequence according to (I)-1-1 or (I)-1-2 below.

[0021] (I)-1: Promoter for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein having photo-induced reactive oxygen species generation or diffusion activity, or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0022] (I)-1-1: Nucleotide sequence of SEQ ID NO: 1 (I)-1-2: A base sequence in which one or several bases are deleted, substituted, added, and / or inserted from the base sequence of SEQ ID NO: 1, and which has the same promoter activity as SEQ ID NO: 1.

[0023] (Sixth Invention) A fusion gene comprising the following sequences (I)-1 to (I)-3, wherein (I)-1 consists of the sequence described in the following sequence (I)-1-1 or (I)-1-2.

[0024] (I)-1: Promoter sequence for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein having photo-induced reactive oxygen species generation or diffusion activity, or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0025] (I)-1-1: Nucleotide sequence of SEQ ID NO: 1 (I)-1-2: A base sequence in which one or several bases are deleted, substituted, added, and / or inserted from the base sequence of SEQ ID NO: 1, and which has the same promoter activity as SEQ ID NO: 1.

[0026] (Seventh Invention) A fusion gene according to the sixth aspect of the invention, further comprising the following (I)-5:

[0027] (I)-5: A sequence encoding a signal peptide that directs transport to the endoplasmic reticulum

[0028] (Eighth Invention) The fusion gene according to the sixth aspect of the invention, wherein (I)-2 is a KillerRed protein gene or a mutant thereof.

[0029] (Ninth Invention) A fusion gene according to the sixth invention, characterized in that it has the sequence of SEQ ID NO:6.

[0030] (Tenth Invention) An expression cassette comprising the fusion gene according to the sixth aspect of the present invention.

[0031] (Eleventh Invention) A cultured cell of the following (IV) into which the fusion gene according to any one of the sixth to ninth inventions or the expression cassette according to the tenth invention has been introduced:

[0032] (IV) A cultured cell for introducing (I), which has an endogenous GON domain with a sequence different from that of the GON domain of (I)-3.

[0033] (Twelfth Invention) A cultured cell according to the eleventh aspect of the present invention, further comprising the following (II) and / or (III):

[0034] (II) Substances that suppress ADAMTS9 gene expression (III) Substances with calcium ion detection function

[0035] (Thirteenth Invention) A fusion protein produced by the fusion gene or expression cassette introduced into the cultured cell according to the eleventh invention.

[0036] (Fourteenth Invention) A method for measuring a biological sample, comprising the following steps (1) and (2):

[0037] (1) measuring the abundance of the following (X) protein or the expression level of the following (X) gene in a biological sample: (2) A step of determining that the biological sample is derived from a patient or potential patient group of (Y) below, when the abundance of the (X) protein or the expression level of the (X) gene measured in (1) is lower than the abundance of the (X) protein or the expression level of the (X) gene in a control sample derived from a healthy individual of the same species as the biological sample.

[0038] (X) GON domain protein or its gene (Y) Diseases caused by increased intracytoplasmic calcium concentration

[0039] (Fifteenth Invention) The method for measuring a biological sample according to the fourteenth invention, wherein the biological sample is a cell in which ADAMTS9 is expressed or a cultured cell thereof.

[0040] (16th invention) A method for measuring a biological sample according to the fifteenth invention, characterized in that the cells expressing ADAMTS9 are cells derived from vascular smooth muscle, and (Y) is a disease caused by apoptosis of vascular smooth muscle cells.

[0041] (Seventeenth Invention) The method for measuring a biological sample according to the sixteenth aspect of the present invention, wherein (Y) is an aneurysm or a disease caused by the rupture of an aneurysm.

[0042] (18th invention) The method for measuring a biological sample according to the seventeenth aspect of the present invention, wherein (Y) is cerebral hemorrhage or subarachnoid hemorrhage.

[0043] (19th invention) An inhibitor of an increase in cytoplasmic calcium concentration, characterized by comprising a gene comprising the sequence described in (I)-3 below, or a protein expressed from the gene.

[0044] (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0045] (20th Invention) The inhibitor according to the nineteenth aspect of the present invention, characterized in that the gene (I)-3 forms a fusion gene together with the promoter sequence according to the fifth aspect of the present invention.

[0046] (21st Invention) A preventive and / or therapeutic agent for a disease caused by an increase in intracytoplasmic calcium concentration, characterized by comprising a gene comprising the sequence described in (I)-3 below, or a protein expressed from the gene.

[0047] (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0048] (22nd Invention) A preventive and / or therapeutic agent according to the twenty-first aspect of the invention, characterized in that the gene (I)-3 forms a fusion gene together with the promoter sequence according to the fifth aspect of the invention. [Effects of the Invention]

[0049] The "screening method" and "screening kit" of the present invention make it possible to easily screen for "inhibitors of an increase in cytoplasmic calcium concentration" or "prophylactic and / or therapeutic agents for diseases caused by said increase" that have little risk of side effects such as those associated with IP3 receptor inhibitors. By using the "promoter" of the present invention, it is possible to properly express an exogenous GON domain protein without causing cell death in cultured cells, thereby further improving the screening accuracy of the "screening method" of the present invention. The "fusion gene," "expression cassette," "cultured cells" and "fusion protein" of the present invention can be used to screen for drugs for preventing and / or treating diseases caused by the release of calcium ions into the cytoplasm. The "method for measuring a biological sample" of the present invention makes it possible to detect diseases caused by an increase in intracytoplasmic calcium ion concentration at an early stage. The "inhibitor" of the present invention can inhibit an increase in intracytoplasmic calcium ion concentration. Furthermore, the "prophylactic and / or therapeutic agent" of the present invention can prevent and / or treat diseases caused by an increase in intracytoplasmic calcium ion concentration. [Brief explanation of the drawings]

[0050] [Figure 1] 1 is a diagram showing an example of the sequence of the "fusion gene" of the present invention (SEQ ID NO: 6). The sequences, listed for convenience, are actually connected. [Figure 2] Figure 2 shows an example of the amino acid sequence of the "fusion protein" of the present invention (SEQ ID NO: 7). This is the protein expressed from the fusion gene of Figure 1. The sequences, listed for convenience, are actually connected. [Figure 3] FIG. 3 shows an example of (II) a substance that suppresses the expression of the ADAMTS9 gene (siRNA against ADAMTS9 (a blend of four siRNAs)) used in the present invention. [Figure 4] Figure 4 shows the time course of the fluorescence intensity measured by the calcium sensor when the expression of the "ADAMTS9 gene having the GON domain" was inhibited (Example 6). The results indicate the process of calcium ion release into the cytoplasm. [Figure 5] Figure 5 shows the results of measurements similar to those in Figure 4, but after introducing an inhibitor of the "IP3 receptor," which is a calcium ion release pathway, into the culture medium before measurement. [Figure 6] FIG. 6 shows fluorescence photographs taken every 5 minutes when measuring the time-dependent changes in FIGS. [Figure 7] Figure 7 shows that cell viability, which is reduced by the addition of the inflammatory mediator "TXA2 analog (U-46619)," is further aggravated by the inhibition of ADAMTS9 (GON domain) expression (center), but is improved by the addition of the "IP3 receptor" inhibitor (2-APB) (right). [Figure 8] FIG. 8 shows a sequence (SEQ ID NO: 8) for producing guide RNA (gRNA) (a sequence complementary to gRNA) used when introducing the "expression cassette" of the present invention of Example 3 into cells using the CRISPR / Cas9 method in the production of the "cultured cells" of the present invention of Example 4. [Figure 9] FIG. 9 shows the sequences of the PCR primer set (SEQ ID NOs: 9 and 10) used in producing the "cultured cells" of the present invention in Example 4. [Figure 10]Fig. 10 shows the gene sequence of the region containing the "promoter sequence," "sequence encoding the C-terminus of IP3R (IP3 receptor)," and "sequence encoding the PA tag" in pCMV_IP3R-PA used in Test Example 4. The sequences, listed for convenience, are actually connected. [Figure 11] FIG. 11 shows that administration of ADAMTS9 siRNA (inhibition of GON domain expression) promotes IP3 receptor ubiquitination. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention will be described in detail below.

[0052] In the present invention, the "cytoplasmic calcium ion concentration" may also be simply referred to as the "cytoplasmic calcium concentration."

[0053] In addition, in the present invention, a "base sequence (or amino acid sequence) in which one or several bases (or amino acids) have been deleted, substituted, added, and / or inserted" from the original base sequence (or amino acid sequence) may be referred to as a "mutant."

[0054] Furthermore, in the present invention, unless otherwise specified, experimental techniques such as genetic manipulation methods are conventional methods in the relevant technical fields or methods based thereon.

[0055] [Screening method of the present invention] The "method for screening an agent for suppressing an increase in cytoplasmic calcium concentration or an agent for preventing and / or treating a disease caused by said increase" of the present invention is characterized by comprising the following steps (A) to (G):

[0056] (A) A step of preparing cultured cells into which the fusion gene (I) below has been introduced and which have an endogenous GON domain with a sequence different from that of (I)-3. (B) A step of administering a candidate compound to the cultured cells of (A). (C) A step of introducing (II) into the cultured cells of (A). (D) A step of introducing (III) into the cultured cells of (A). (E) A step of irradiating the cultured cells of (A) with light having a wavelength capable of exerting the effect of (I)-2. (F) A step of measuring the amount of change in fluorescence before and after the irradiation of (E) using the detection function of (III). (G) A step of selecting a candidate compound having an effect of preventing excessive release of calcium ions into the cytoplasm by comparing the amount of change in fluorescence (F) between the group administered with the candidate compound and the group not administered with the candidate compound.

[0057] (I) A fusion gene comprising the following (I)-1 to (I)-3: (II) Substances that suppress ADAMTS9 gene expression (III) Substances with calcium ion detection function

[0058] (I)-1: Promoter for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein having photo-induced reactive oxygen species generation or diffusion activity, or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0059] (Substances used in screening methods) Each constituent substance used in the "screening method" of the present invention will be explained below.

[0060] <<Fusion gene (I) including (I)-1 to (I)-3>>

[0061] <(I)-1: Promoter for Expression of (I)-2 and (I)-3> The promoters for expression of (I)-2 and (I)-3 preferably have the sequences described in (I)-1-1 or (I)-1-2 below, and details are described in the section on "Promoter" of the present invention below.

[0062] (I)-1-1: Nucleotide sequence of SEQ ID NO: 1 (I)-1-2: A base sequence in which one or several bases are deleted, substituted, added, and / or inserted from the base sequence of SEQ ID NO: 1, and which has the same promoter activity as SEQ ID NO: 1.

[0063] <(I)-2: A gene encoding a protein having the ability to generate or diffuse photoirradiation-dependent reactive oxygen species, or a mutant thereof> Genes encoding proteins that have the ability to generate or diffuse photoirradiation-dependent reactive oxygen species will be described in detail in the section on "fusion genes" of the present invention below, and examples thereof include those having the sequence of SEQ ID NO: 2.

[0064] <(I)-3: Gene encoding a GON domain protein or a mutant thereof> The GON domain gene or a mutant thereof will be described in detail in the section on "fusion gene" of the present invention below, and examples thereof include one having the sequence of SEQ ID NO:3.

[0065] <<(II) Substances that inhibit ADAMTS9 gene expression>> Substances that suppress the expression of the ADAMTS9 gene include, for example, siRNA genes and miRNA genes against ADAMTS9, but nucleic acids for RNAi such as siRNA are preferred because they are commercially available and can be easily used in experiments.

[0066] siRNA gene against endogenous ADAMTS9 gene: It is preferable to select an siRNA against the ADAMTS9 gene that is originally present in cultured cells (hereinafter sometimes referred to as "ADAMTS9 siRNA") that does not inhibit the expression of the "gene encoding the foreign GON domain protein of (I)-3" used in the form of a fusion gene in the "screening method" of the present invention.

[0067] ADAMTS9 siRNA can be appropriately designed based on the ADAMTS9 gene sequence and publicly known knowledge and produced by standard methods. Publicly known siRNAs are available commercially, and they can also be commissioned to be designed by companies such as Sigma.

[0068] Examples of commercially available siRNAs include "Dharmacon (registered trademark), Catalog ID: L-005779-00-0005" (a blend of four siRNAs: Figure 3), which can be purchased from the market. Products in the same series that differ only in volume include ID: L-005779-00-0010, ID: L-005779-00-0020, and ID: L-005779-00-0100, which are preferred in that they are guaranteed to knock down human ADAMTS9 with a high probability, but are not limited to these.

[0069] <<(III) Substances with calcium ion detection function>> There are no particular limitations on the substance (Ca sensor) that has the calcium ion detection function, as long as it has some kind of "detection function" (direct detection function) or "function to produce a substance with detection function" (indirect detection function).

[0070] An example of the "detection function" of calcium ions is the ability to emit fluorescence upon encountering calcium ions. Examples of substances with such a function include the "fluorescent protein Ca sensor (III)-1" and the "low-molecular-weight fluorescent Ca sensor (III)-2" described below.

[0071] An example of the "function to produce a substance with detection function" is the function to produce the "fluorescent protein Ca sensor (III)-1" described below, and an example of something that has such a function is a gene that encodes the "fluorescent protein Ca sensor (III)-1".

[0072] In addition, it is preferable to select a combination of (III) that does not overlap with the "protein having the photoirradiation-dependent reactive oxygen generating or diffusing activity" generated by the expression of the gene (I)-2 in terms of improving the accuracy of calcium ion detection. Another reason for selecting such a combination is that if the excitation wavelengths of (I)-2 and (III) are similar, when the excitation wavelength is irradiated onto (III) to measure the calcium ion concentration, (I)-2 is also excited at the same time, making it impossible to measure the calcium ion concentration before the excitation of (I)-2.

[0073] (III)-1: Fluorescent protein Ca sensor Examples of fluorescent protein Ca sensors include known GCaMP (a calcium sensor protein containing circularly permuted green fluorescent protein (cpGFP), calmodulin (CaM), and myosin light chain fragment (M13)).

[0074] There are multiple types of GCaMP, including, but not limited to, G-CaMP, G-CaMP1.6, GCaMP2, GCaMP3, G-CaMP4.1, GCaMP5, G-CaMP6, G-CaMP7, G-CaMP8, GCaMP6f, GCaMP6m, GCaMP6s, jGCaMP7f, jGCaMP7s, jGCaMP7b, jGCaMP7c, and GCaMP-X. Among the above, GCaMP6s is preferred because it has a proven track record of detecting calcium ions and is easy to use.

[0075] (III)-2: Small molecule fluorescent Ca sensor Examples of low-molecular-weight fluorescent Ca sensors include Fluo-3, Fluo-4, Fura2, Indo-1, Mag-Fula2, Rhod-2, and Rhod-590. All of these are commercially available, and it is sufficient to select one that is compatible with the fluorescence microscope set to be used. However, for the two reasons mentioned above, when KillerRed is used as (I)-2, it is preferable to use a sensor other than Rhod-2 or Rhod-590, which has an excitation wavelength close to that of KillerRed.

[0076] (III)-2 can be introduced by simply dissolving it in culture medium and adding it to cells, whereas (III)-1 is preferably introduced in the form of a gene encoding it inserted into a plasmid such as pCMV-GCaMP6s.

[0077] (Each step of the screening method) Hereinafter, each of the steps (A) to (G) used in the "screening method" of the present invention will be explained.

[0078] <<(A) A step of preparing cultured cells into which the fusion gene of (I) has been introduced and which have an endogenous GON domain with a sequence different from that of (I)-3>>

[0079] (A) "Preparation" includes not only creation but also methods such as acquiring something that has already been created by purchasing it.

[0080] The cultured cells used in (A) have an endogenous GON domain with a different sequence from the GON domain gene (I)-3 used in the "fusion gene" of the present invention because the exogenous GON domain protein can be destroyed by light irradiation at any time, independently of the siRNA-mediated suppression of the GON domain-containing gene (ADAMTS9 gene) endogenous to the introduced cells, and the effect of the GON gene can be measured more accurately.

[0081] The degree of sequence difference is not necessarily limited to a specific extent, as long as it does not suppress the expression of the GON domain in the fusion gene by suppressing ADAMTS9 (including the endogenous GON domain) in cultured cells (II). However, the important factor is the "position" of the different sequences, rather than the "number (proportion)" of the different sequences. For example, when (II) to be used is an siRNA for ADAMTS9, it is preferable that the sequence in the siRNA target region in the endogenous GON domain is as different as possible from the sequence in the corresponding region in the exogenous GON domain.

[0082] Cells that satisfy these conditions can be found relatively easily by selecting cells derived from a species other than that from which the foreign GON domain used in the fusion gene is derived. For example, when a GON domain derived from a nematode or a mutant thereof is used as (I)-3, it is preferable to select human cells as the cultured cells. The reason for this will be described in detail in the section <<(I)-3: Gene encoding a GON domain protein or a mutant thereof>> in the explanation of the "fusion gene" of the present invention.

[0083] The "fusion gene" of the present invention can be introduced, for example, by using the "expression cassette" of the present invention described below. In addition to direct introduction into the cultured genome, there are also methods of introducing the gene into the cytoplasm outside the genome using various vectors or plasmids, but direct introduction into the genome is preferred.

[0084] For more specific introduction methods and reasons why genomic introduction is preferred, The section (Use of the expression cassette) in the explanation of the "expression cassette" of the present invention and The section (method for producing cultured cells) in the explanation of the "cultured cells" of the present invention Further details are provided in the following.

[0085] <<(B) A step of administering a candidate compound to the cultured cells of (A)>>

[0086] The method of administering the candidate compound can be any common method for adding drugs to cells, and is not particularly limited to this. For example, the candidate compound may be directly sprinkled onto cultured cells (mixed into the culture medium) either alone or bound to another substance such as BSA (fetal bovine serum).

[0087] <<(C) A step of introducing (II) into the cultured cells of (A)>>

[0088] (II) can be introduced into cultured cells by a conventional method in the field of cell culture technology. For example, when (II) is siRNA, Lipofectamine TM This can be done using RNAiMAX Transfection Reagent (Thermo Fisher Scientific) or the like.

[0089] <<(D) A step of introducing (III) into the cultured cells of (A)>>

[0090] (III) can be introduced into cultured cells using conventional methods in the field of cell culture technology. For example, when (III) is pCMV-GCaMP6s, it can be introduced using Lipofectamine 2000 (Thermo Fisher Scientific) or the like.

[0091] <<(E) A step of irradiating the cultured cells of (A) with light having a wavelength capable of exerting the effect of (I)-2>>

[0092] After the gene (I)-2 is expressed, the expressed fluorescent protein is irradiated with light of a wavelength that can exert a photoirradiation-dependent generation or diffusion action of reactive oxygen.

[0093] <<(F) A step of measuring the amount of change in fluorescence before and after the irradiation of (E) using the detection function of (III)>>

[0094] Measurements may be taken twice, before and after irradiation, but it is preferable to measure periodically (e.g., every minute) for a certain period (e.g., about 20 minutes) before and after irradiation to observe changes in the amount of fluorescence as they occur. This is because it is possible to predict the timing at which a candidate compound will exhibit its effect (immediate effect, etc.).

[0095] <<(G) A step of selecting a candidate compound having an effect of preventing excessive release of calcium ions into the cytoplasm by comparing the amount of change in fluorescence (F) between the candidate compound-administered group and the unadministered group>>

[0096] "Excessive calcium ion release" refers to a state in which calcium ions are leaking out of the endoplasmic reticulum into the cytoplasm to such an extent that the calcium ion concentration in the cytoplasm exceeds the concentration in a normal (healthy) state.

[0097] The more the amount of change (increase) in fluorescence can be suppressed, the more effective it can be expected to be as an "inhibitor of an increase in intracytoplasmic calcium concentration" or an "agent for preventing and / or treating diseases caused by said increase."

[0098] As will be described later, the increase in cytoplasmic calcium ion concentration due to the suppression of GON domain expression is thought to be mainly due to "leakage" from the void created after the extraction (and degradation) of the "IP3 receptor," which is the calcium ion release pathway, from the endoplasmic reticulum membrane. Therefore, the "prevention of excessive release" in (G) is thought to be primarily the "prevention of leakage" caused by the ubiquitination and degradation of the IP3 receptor (see Test Example 4, Figure 11, etc.).

[0099] Therefore, by further adding the following step (H) to the above-mentioned "screening method" of the present invention, it can be confirmed that the "inhibitor" or "prophylactic and / or therapeutic agent" selected by the "screening method" of the present invention does not inhibit the IP3 receptor itself and has few side effects.

[0100] <<A step of determining whether the candidate compound inhibits the IP3 receptor itself or inhibits the ubiquitination of the IP3 receptor by comparing the change in the amount of ubiquitinated IP3 receptor between the candidate compound-administered group and the unadministered group before and after the irradiation of (H) and (E)>>

[0101] The amount of ubiquitinated IP3 receptor can be measured by common methods used for detecting proteins, such as Western blotting and ELISA.

[0102] Furthermore, by applying this step (H), it is also possible to screen for an agent that inhibits an increase in intracytoplasmic calcium concentration without carrying out steps (A) to (G). For example, by administering a "candidate compound" and "siRNA against ADAMTS9" (which triggers excessive release of calcium ions into the cytoplasm by suppressing the expression of the endogenous GON domain) to HEK293 cultured cells and comparing the amount of IP3 receptor ubiquitination "before and after administration of the candidate compound" or "between a group administered with the candidate compound and a group not administered with the candidate compound," it is thought that it would be possible to screen for "IP3 receptor ubiquitination inhibitors," i.e., a certain type of "inhibitor of the increase in cytoplasmic calcium concentration."

[0103] (Execution order of each process) (B), (C), and (D) do not have to be in this order, but they can be in any order. If it is desired to minimize the inhibitory effect of transfection reagents and the like on the cellular uptake of the candidate compound, it is preferable that at least (B) precedes (C) and (D), but this does not necessarily have to be the case.

[0104] In order to sufficiently suppress the influence of endogenous ADAMTS9, it is preferable to carry out (C), for example, four days before (D) and (E).

[0105] (D) is preferably carried out, for example, one day before (E) and (F).

[0106] Step (H) can be carried out before or after step (G), or in parallel with step (G).

[0107] In the "screening method" of the present invention, only the GON domain, rather than the full-length ADAMTS9, was used as the "foreign gene" (I)-3 to be introduced as a measurement index because ADAMTS9 has the "protease function" common to the ADAMTS group in addition to the "function of preventing calcium ion release (due to the GON domain)," making it difficult to determine whether the results obtained from the expression inhibition experiment were due to "inactivation of the GON domain function" or "loss of protease function."

[0108] In addition, we deliberately used the ``exogenous GON domain'' as an indicator rather than the ``endogenous GON domain (containing ADAMTS9)'' that is originally present in cells because if the ``endogenous GON domain'' is used as an indicator, it is not easy to confirm the accuracy of inhibition when inhibiting with siRNA. Therefore, it is necessary to control the expression of the "exogenous GON domain" by a means other than suppressing the expression of endogenous ADAMTS9.

[0109] Therefore, in the "screening method" of the present invention, a light irradiation-dependent suppression method using (I)-2 was also used.

[0110] Light irradiation-dependent suppression methods include the "CALI (Chromophore-assisted Light Inactivation)" method, which uses fluorescent molecules that generate reactive oxygen when irradiated with light of a specific wavelength, and can destroy molecules in a microscopic region near the fluorescent molecule at any time using the reactive oxygen.

[0111] The "screening method" of the present invention can be carried out using the "screening kit" of the present invention described below.

[0112] (Effect of screening method) The "screening method" of the present invention makes it possible to easily screen for "inhibitors of an increase in cytoplasmic calcium concentration" or "prophylactic and / or therapeutic agents for diseases caused by said increase" of the present invention, which have a low risk of side effects.

[0113] The "inhibitors of an increase in intracytoplasmic calcium concentration" screened by the "screening method" of the present invention include both those that suppress an increase in intracytoplasmic calcium ions in the short term and / or the long term. However, since it is possible to screen for those related to the suppression of GON domain expression, it is expected that in addition to symptomatic treatments, long-term inhibitors can also be screened.

[0114] "Suppressing an increase in cytoplasmic calcium concentration" primarily means preventing excessive release, but does not exclude the active elimination of calcium ions that have already been released into the cytoplasm. It also means that in the process of preventing excessive release, calcium ions that have already been released are utilized in the body, resulting in a reduction in calcium ion concentration.

[0115] The "increase inhibitors" to be specifically screened are not particularly limited, and may include any substances such as low molecular weight compounds, proteins, peptides, nucleic acids, and the like.

[0116] Specific examples of the disease include, but are not limited to, the diseases exemplified in the "Method for measuring a biological sample" of the present invention described below.

[0117] [Screening kit of the present invention] The "screening kit for an inhibitor of an increase in intracytoplasmic calcium concentration or a preventive and / or therapeutic agent for a disease caused by said increase" of the present invention is characterized by comprising the following (I) to (IV):

[0118] (Kit components) (I) A fusion gene comprising the following (I)-1 to (I)-3: (II) Substances that suppress ADAMTS9 gene expression (III) Substances with calcium ion detection function (IV) A cultured cell for introducing (I), which has an endogenous GON domain with a sequence different from that of the GON domain of (I)-3 below.

[0119] (I)-1: Promoter for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein having photo-induced reactive oxygen species generation or diffusion activity, or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0120] <About (I)> (I)-1 to (I)-3 will be described in detail in the section on "Fusion Genes" of the present invention below.

[0121] The fusion gene (I) preferably further contains the following (I)-5, and is also allowed to contain a sequence such as (I)-4.

[0122] (I)-4: Vector sequence (I)-5: A sequence encoding a signal peptide that directs transport to the endoplasmic reticulum

[0123] (I)-4 and / or (I)-5 will also be described in detail in the section on "Fused Genes" of the present invention below.

[0124] <Regarding (II) to (IV)> (II) and (III) are as described above in the section "Screening Method" of the present invention.

[0125] (IV) will be described in detail later in the section on "cultured cells" of the present invention.

[0126] <Other constituent materials> The "screening kit" of the present invention may contain various additives and other substances that are generally used in screening kits that use genetic material.

[0127] The fusion gene (I) as a kit material may be contained in a kit in a state where its constituent genes are separate. Furthermore, at least one of the kit materials (I), (II), and (III) may be in a form in which it has been previously introduced into (IV).

[0128] (Kit Use) The "screening kit" of the present invention can be used in the "screening method" of the present invention.

[0129] [Promoter of the present invention] The "promoter" of the present invention is the promoter of (I)-1 below, characterized in that it comprises the sequence of (I)-1-1 or (I)-1-2 below.

[0130] (I)-1: Promoter for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein having photo-induced reactive oxygen species generation or diffusion activity, or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0131] (Promoter sequence) (I)-1-1: Nucleotide sequence of SEQ ID NO: 1 (I)-1-2: A base sequence in which one or several bases are deleted, substituted, added, and / or inserted from the base sequence of SEQ ID NO: 1, and which has the same promoter activity as SEQ ID NO: 1.

[0132] SEQ ID NO:1: CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT

[0133] Furthermore, when the "fusion gene" of the present invention contains a "sequence encoding the signal peptide" of (I)-5, this "promoter for expression of (I)-2 and (I)-3" also functions as a promoter for expression of (I)-5.

[0134] (How to create a promoter) A promoter having the base sequence of SEQ ID NO: 1 in (1)-1-1 can be prepared by extracting the 3'-end region 305-508 (81 bp) of the promoter region of the known CMV (cytomegalovirus) promoter, but can also be artificially synthesized from scratch.

[0135] The CMV promoter used as a starting material is readily available on the market, but it can also be obtained commercially in the form of a vector, such as modified pEGFP-N3 manufactured by Addgene.

[0136] When a vector is used as a starting material, the "promoter" of the present invention can be prepared by amplifying by PCR only the above-mentioned portion necessary for the "promoter."

[0137] (Specific examples of mutants) (1)-1-2) The variant of the base sequence of SEQ ID NO: 1 may be any variant as long as it has a base sequence that has the same type of promoter activity as SEQ ID NO: 1. It can be prepared based on a gene having the base sequence of SEQ ID NO: 1 using conventional methods in the field of genetic engineering, but it can also be artificially synthesized from 1.

[0138] (Use of promoter) The "promoter" of the present invention can be used not only for the above-mentioned "screening method" of the present invention, but also for experiments on the expression of various foreign genes in cells and the expression of gene therapy drugs in vivo, and can be used, for example, as a constituent material of the "fusion gene," "expression cassette," "inhibitor," "preventive and / or therapeutic agent," etc. of the present invention described below.

[0139] (Promoter effect) The "promoter" of the present invention has the advantage of being easy to use, since it can appropriately regulate the expression level of the subsequent target gene in cells, compared to the known CMV promoter.

[0140] In foreign gene transfer technology, a promoter is used to express a protein encoded by a subsequent gene of interest, but there are problems if the expression level is too low or too high. If the expression level is too low, there is no practical benefit to gene transfer, but conversely, if the expression level is too high, it may have a detrimental effect on the transfected cells.

[0141] In particular, when the "fusion gene" of the present invention described below is used as the subsequent gene of interest, it has been found that the expression level is too high with the conventional CMV promoter, which can sometimes cause cell death.

[0142] The "promoter" of the present invention is a highly effective promoter that can exquisitely regulate the expression level of the fusion gene of the present invention in the cells into which it is introduced.

[0143] Therefore, among the "promoters" of the present invention, "promoter activity of the same type as SEQ ID NO: 1" in a mutant of SEQ ID NO: 1 means an activity that does not cause cell death in the introduced cells and can regulate the expression level of the target gene to be introduced, such as a "fusion gene," to a level sufficient for experiments, prevention, and / or treatment, etc.

[0144] [Fusion gene of the present invention] The "fusion gene" of the present invention comprises the following sequences (I)-1 to (I)-3, and (I)-1 is characterized by consisting of the sequence described in (I)-1-1 or (I)-1-2 below.

[0145] (Various sequences that make up the fusion gene) (I)-1: Promoter sequence for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein having photo-induced reactive oxygen species generation or diffusion activity, or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0146] (I)-1-1: Nucleotide sequence of SEQ ID NO: 1 (I)-1-2: A base sequence in which one or several bases are deleted, substituted, added, and / or inserted from the base sequence of SEQ ID NO: 1, and which has the same promoter activity as SEQ ID NO: 1.

[0147] <<(I)-1 promoter sequence>> The promoter sequence of (I)-1 is as described above in the section "Promoter" of the present invention.

[0148] <<(I)-2 Genes encoding proteins having the function of generating or diffusing reactive oxygen species or mutants thereof>> The "gene encoding a protein capable of generating or diffusing reactive oxygen species dependent on light irradiation" in (I)-2 refers to a gene encoding a protein that generates reactive oxygen species when irradiated with light of a specific wavelength, and also includes single nucleotide polymorphisms (SNPs) thereof.

[0149] Specific examples of proteins encoded by the gene (I)-2 include KillerRed and its mutants described below, among which KillerRed is preferred in that it has been confirmed that when used in the "screening method" of the present invention, there is almost no "adverse effect on cell survival due to the generated reactive oxygen species," which is generally assumed when KillerRed (or its mutants) is used.

[0150] <Examples of genes> Specific examples of the KillerRed gene (a base sequence encoding the KillerRed protein) include, but are not limited to, the sequence shown in SEQ ID NO: 2 below.

[0151] SEQ ID NO:2: ATGGGTTCAGAGGGCGGCCCCGCCCTGTTCCAGAGCGACATGACCTCAAAAATCTTCATCGACGGCGAGGTGAACGGCCAGAAGTTCACCATCGTGGCCGACGGCAGCAGCAAGTTCCCCCACGGCGACTTCAACGTGCACGCCGTGTGCGAGACCGGCAAGCTGCCCATGAGCTGGAA GCCCATCTGCCACCTGATCCAGTACGGCGAGCCCTTCTTCGCCCGCTACCCCGACGGCATCAGCCATTTCGCCCAGGAGTGCTTCCCCGAGGGCCTGAGCATCGACCGCACCGTGCGCTTCGAGAACGACGGCACCATGACCAGCCACcACACCTACGAGCTGGACGACACCTGCGTGG TGAGCCGCATCACCGTGAACTGCGACGGCTTCCAGCCCGACGGCCCCATCATGCGCGACCAGCTGGTGGACATCCTGCCCAACGAGACCCACATGTTCCCCCAGGCCCCAACGCCGTGCGCCAGCTGGCCTTCATCGGCTTCACCACCGCCGACGGCGGCCTGATGATGGGCCACTTC GACAGCAAGATGACCTTCAACGGCAGCCGCGCCATCGAGATCCCGGCCCACACTTCGTGACCATCATCACCAAGCAGATGAGGGACACCAGCGACAAGCGCGACCACGTGCCAGCGCGAGGTGGCCTACGCCCACAGCGTGCCCCGCATCACCAGCGCCATCGGTAGCGACGAGGat

[0152] <Mutant> Examples of mutants of the gene (I)-2 include base sequences that encode "amino acid sequences in which one or more amino acids are deleted, substituted, added, and / or inserted from the protein sequence described in (I)-2, and which have the same protein inactivation function as the protein of (I)-2."

[0153] <Protein inactivation function similar to that of the protein (I)-2> For example, the KillerRed protein has the function of emitting red fluorescence and generating reactive oxygen species when irradiated with green light of approximately 530-550 nm.

[0154] In other words, the "protein inactivation function similar to that of the protein of (I)-2" in the description of the mutant of (I)-2 means, like the KillerRed protein, "the function of generating reactive oxygen species when irradiated with light of a specific wavelength, and using that reactive oxygen species to inactivate (destroy) the protein fused to KillerRed."

[0155] In the description of the present invention, "KillerRed protein (or its gene)" may be used as a "representative example of (I)-2," but this does not necessarily mean that "(II)" is limited to "KillerRed."

[0156] <<(I)-3: Genes encoding GON domain proteins or mutants thereof>> The "gene encoding a GON domain protein" in (I)-3 refers to a gene encoding a known protein corresponding to the domain present at the C-terminus of ADAMTS9, and also includes single nucleotide polymorphisms (SNPs) thereof.

[0157] Furthermore, the positions of the C-terminus and N-terminus of GON domain proteins in the genome do not necessarily match due to "differences in biological species" or "differences in interpretation" based on the analytical software used, but the "GON domain (protein)" referred to in this invention includes all of these.

[0158] <Examples of genes> A specific example of a GON domain gene (a base sequence encoding a GON domain protein) is the nematode-derived GON domain "CeGON" shown in sequence number 3 below, but is not limited to this.

[0159] SEQ ID NO:3: ACAAAGAAACCACGTCGAACTCAATATTGTTTTGAAAGAAATTGCCTTCCGTCAACTTGTCAGGAGCTTAAATCTCAGAATGTTAAGGCTAAAGATGGAAATTACACTATTCTTCTTGACGGATTCACTATTGAAATTTATTGTCATCGAATGAATTCAACCATTCCTAAAGCTTATTTGAACGTTAATCCAAGAACCAATTTTGCAGAGGTTTATGGAAAAAAATTAATATACCCTCATACTTGCCCATTTAATGGTGATCGTAATGATTCATGCCATTGTTCAGAAGACGGCGATGCAAGTGCTGGATTGACGAGATTCAATAAAGTTCGAATAGATTTGTTGAATAGAAAGTTCCATCTGGCGGATTATACATTTGCAAAACGAGAATATGGTGTTCATGTGCCATATGGTACTGCCGGTGATTGCTACAGTATGAAAGATTGTCCACAGGGAATATTCTCAATTGATTTAAAATCTGCTGGTCTGAAATTAGTTGACGATCTGAATTGGGAGGATCAAGGTCATCGAACATCCTCTCGAATCGATCGTTTTTATAACAATGCAAAAGTTATTGGTCACTGTGGTGGTTTTTGTGGAAAATGCTCTCCTGAGCGGTACAAAGGACTAATCTTTGAAGTTAATACAAAATTATTAAATCATGTGAAAAATGGTGGACACATTGATGATGAATTGGATGATGATGGTTTCTCTGGTGACATGGATtaa

[0160] <Variant> Examples of the variant of the GON domain gene of (I)-3 include "a base sequence encoding an amino acid sequence in which one or several amino acids in the GON domain protein are deleted, substituted, added, and / or inserted and having an IP3 receptor degradation inhibitory function of the GON domain", etc.

[0161] <IP3 receptor degradation inhibitory function of the GON domain> In Test Example 2 described below, the release of calcium ions into the cytoplasm was prevented by administering an IP3 receptor inhibitor (2-APB) in addition to disruption of the GON domain, confirming that IP3 receptors are involved in the release of calcium ions into the cytoplasm due to disruption of the GON domain.

[0162] On the other hand, it is known that when calcium ions pass through the IP3 receptor (the receptor functions as a calcium ion channel), it is pulled out of the endoplasmic reticulum membrane and degraded via polyubiquitination. Test Example 4, which uses ADAMTS9 siRNA (described below), confirmed that this phenomenon occurs to an extreme extent when the GON domain is destroyed (Figure 11).

[0163] These results suggest that the GON domain mediates the signal transduction pathway through the IP3 receptor, either directly or indirectly. Degradation of IP3 receptor via polyubiquitination, and resulting from it Excessive release of calcium ions ("leakage" from the cavity after the IP3 receptors are pulled out) This suggests that the

[0164] When the GON domain functions normally, the IP3 receptor naturally releases calcium ions (Ca 2+ ) channel performs its regulatory function normally, and excessive release (leakage) of calcium ions into the cytoplasm is suppressed.

[0165] In other words, the "function of the GON domain to inhibit IP3 receptor degradation" in the description of the mutant (I)-3 means the "function of the GON domain to generate a signal to inhibit IP3 receptor degradation."

[0166] From the above, it is considered that the "excessive release-preventing effect" in step (G) of the "screening method" of the present invention, which selects candidate compounds having the "excessive release-preventing effect" of calcium ions, is also mainly a "leakage-preventing effect" resulting from ubiquitination and degradation of IP3 receptors.

[0167] Furthermore, when the "fusion gene" of the present invention or an "expression cassette" containing the same is used in the above-mentioned "screening method" of the present invention, the gene encoding the GON domain protein of (I)-3 must have a gene sequence different from the GON domain sequence endogenous to the cells into which it is introduced.

[0168] In order to carry out the "screening method" of the present invention using the activity of an exogenous GON domain protein as an indicator, it is preferable to suppress the expression of the GON domain in ADAMTS9 that is originally present in the cell. However, when using siRNA technology, for example, if the same gene sequence is used, the expression of the exogenous GON domain gene will also be suppressed by siRNA against endogenous ADAMTS9.

[0169] In other words, in order to prevent the expression of the exogenous GON domain gene of (I)-3 from being suppressed by siRNA of endogenous ADAMTS9, the gene sequence must be different from that of the GON domain gene in the ADAMTS9 endogenous to the transfected cells.

[0170] The degree of difference in the sequence may be such that the expression of the GON domain in the fusion gene is not suppressed by suppressing ADAMTS9 (including the endogenous GON domain) in cultured cells (II).

[0171] Such an exogenous GON domain that is not suppressed by ADAMTS9 siRNA can be created by modifying the sequence of (I)-3, focusing on the position corresponding to the "sequence targeting the endogenous GON domain" in the siRNA of (II).

[0172] On the other hand, a foreign GON domain that meets these conditions can be produced relatively easily by using a GON domain from a different species than the cell into which it is to be introduced, by artificially synthesizing it based on that GON domain, or by designing a mutant. If a GON domain currently exists in another biological species, it is not only easy to find one that can function as a GON domain in cultured cells, but also because it has a different amino acid sequence from the endogenous GON domain, or even if it has the same amino acid sequence, it has a different gene (codon) that encodes it, and therefore there is a high possibility that its expression will not be suppressed even by siRNA of endogenous ADAMTS9.

[0173] However, as long as such a difference in sequence is ultimately present, the gene sequence may be modified based on a GON domain derived from the same species as the cells to be introduced, and used as the foreign GON domain gene of (I)-3.

[0174] Specifically, when introducing the "fusion gene" of the present invention or an "expression cassette" containing the same into human-derived cells, it is preferable to use, as (I)-3, for example, one having the sequence of a nematode-derived GON domain gene (or a mutant thereof).

[0175] This is because the GON domains of nematodes and humans differ in some of their amino acid sequences, and also in some of the codons for the same amino acids.

[0176] Furthermore, the "fusion gene" of the present invention preferably further comprises the following (I)-5, and is also allowed to further comprise a sequence such as (I)-4.

[0177] (I)-4: Vector sequence (I)-5: A sequence encoding a signal peptide that directs transport to the endoplasmic reticulum

[0178] <<(I)-4: Vector sequence>> The "vector sequence" in (I)-4 refers to a sequence derived from a vector used as a material when creating the "fusion gene" or "expression cassette" of the present invention, and refers to a residual sequence or the like that is generated due to the position of a restriction enzyme. However, there are no particular restrictions on the sequence as long as it does not contain a start codon, and this vector sequence does not need to be included in the fusion gene. A specific example of (I)-4 is the sequence of SEQ ID NO: 4, which is located at the position shown in the fusion gene in FIG.

[0179] SEQ ID NO:4: GGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTCGAATTC

[0180] <<(I)-5: Sequence encoding a signal peptide that directs transport to the endoplasmic reticulum>> A sequence encoding a signal peptide is a sequence encoding a protein that guides a co-expressed polypeptide or protein to a desired location, and (I)-5 of the present invention is a sequence (hereinafter sometimes simply referred to as a "signal sequence") that can transport a GON domain protein expressed in an introduced cell to the endoplasmic reticulum.

[0181] Specific examples of (I)-5 include the sequence of SEQ ID NO: 5 derived from a nematode, which is located at the position shown in the fusion gene in Figure 1, but are not limited to this. As long as it functions as a signal sequence, it may be a sequence in which a part of it has been mutated by a standard method in the field of gene-related technology.

[0182] SEQ ID NO:5 ATGCGCTCCATCGGCGGCTCATTCCATCTGCTGCAGCCCGTCGTCGCCGCTCTCATACTCCTCGTCGTCTGCCTCGTTTATGCG

[0183] <<Other Arrays>> In addition to the above sequences, the "fusion gene" of the present invention may include other known sequences used in fusion genes, as well as other sequences used in the field of gene transfer technology, such as antibiotic genes that facilitate the selection of cells into which the fusion gene has been introduced.

[0184] (Method for creating fusion genes) The fusion gene of the present invention can be prepared using a conventional method in the field of gene-related technology, specifically, for example, by the following procedures i) to vi).

[0185] i) Preparation of promoter sequence of (I)-1 The promoter sequence of (I)-1 can be prepared in the same manner as the "promoter" of the present invention described above.

[0186] ii) Creation of the KillerRed gene (the base sequence encoding the KillerRed protein) of (I)-2 The KillerRed gene, an example of (I)-2, can be artificially synthesized based on the sequence information of a gene encoding a known KillerRed protein, but it is also possible to obtain it commercially in the form of a vector, such as FP961 manufactured by Evrogen, through distributors such as Wako.

[0187] The gene (I)-2 obtained in the form of a vector can be excised from the vector and used, or alternatively, other sequences constituting the fusion gene can be introduced into the vector without excision to form the "expression cassette" of the present invention.

[0188] iii) Creation of the GON gene (a base sequence encoding a GON domain protein) of (I)-3 The base sequence encoding the GON domain protein of (I)-3 can be prepared, for example, as follows. (cDNA creation) It can be produced as cDNA using the "GON gene (mRNA)" purified from total RNA collected from an organism (preferably a nematode, etc.) and "reverse transcriptase," or it can be artificially synthesized based on the sequence information of a gene encoding a known GON domain protein.

[0189] (cDNA amplification) Next, the GON gene obtained above, such as cDNA, is amplified separately from the KillerRed gene by PCR or other methods using it as a template.

[0190] iv) Construction of vector sequence (I)-4 As mentioned above, a "vector sequence" is a residual strand or the like when a vector is used as a material for each sequence constituting the "fusion gene" of the present invention, and therefore there is no need to deliberately create or insert it. However, in cases where the sequences that may be included in the "fusion gene" are known in advance, it is also possible to add an artificially synthesized sequence based on such sequence information to the constituent sequences of the fusion gene.

[0191] v) Construction of the signal sequence of (I)-5 A known signal peptide portion that directs transport to the endoplasmic reticulum can be produced by reverse transcribing it from a genomic gene (RNA) using cDNA, or it can be artificially synthesized based on the sequence information of such a known signal peptide gene.

[0192] vi) Creation of fusion genes The fusion gene can be produced by amplifying each of the sequences that make up the fusion gene, fusing the sequences using PCR or the like, and then inserting the resulting sequence into the expression cassette of the present invention, which will be described later. Alternatively, they can be designed and constructed so that the three are connected when inserted into an expression cassette.

[0193] Alternatively, the fusion gene can be prepared by purchasing a commercially available plasmid into which some of the sequences constituting the fusion gene have already been incorporated, and then incorporating the remaining sequences. For example, it can be produced by purchasing a commercially available plasmid that already contains the "KillerRed sequence" (a type of (I)-2) and a "promoter sequence" capable of expressing it, and then inserting the remaining sequences, such as the GON domain gene (I)-3) and the signal sequence (I)-5), into it.

[0194] The order of each sequence is as shown in FIG. 1 (SEQ ID NO: 6).

[0195] The inventors have confirmed that the expressed GON domain protein does not function even if the sequences are connected in the order of "(1)-5: signal sequence," "(1)-3: GON domain sequence," and "(1)-2: KillerRed sequence" from the 5' end, meaning that the GON domain protein must be located at the C-terminus of the expressed "fusion protein."

[0196] (Specific examples of fusion genes) Specific examples of the "fusion gene" of the present invention include, but are not limited to, those set forth in SEQ ID NO: 6 below.

[0197] Note that the sequences listed below for convenience are actually connected.

[0198] SEQ ID NO: 6: (Figure 1) CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT SEQ ID NO: 1 (promoter) GGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTCGAATTC SEQ ID NO: 4 (vector sequence) ATGCGCTCCATCGGCGGCTCATTCCATCTGCTGCAGCCCGTCGTCGCCGCTCTCATACTCCTCGTCGTCTGCCTCGTTTATGCG SEQ ID NO: 5 (Signal Peptide) ATGGGTTCAGAGGGCGGCCCCGCCCTGTTCCAGAGCGACATGACCTTCAAAATCTTCATCGACGGCGAGGTGAACGGCCAGAAGTTCACCATCGTGGCCGACGGCAGCAGCAAGTTCCCCCACGGCGACTTCAACGTGCACGCCGTGTGCGAGACCGGCAAGCTGCCCATGAGCTGGAAGCCCATCTGCCACCTGATCCAGTACGGCGAGCCCTTCTTCGCCCGCTACCCCGACGGCATCAGCCATTTCGCCCAGGAGTGCTTCCCCGAGGGCCTGAGCATCGACCGCACCGTGCGCTTCGAGAACGACGGCACCATGACCAGCCACcACACCTACGAGCTGGACGACACCTGCGTGGTGAGCCGCATCACCGTGAACTGCGACGGCTTCCAGCCCGACGGCCCCATCATGCGCGACCAGCTGGTGGACATCCTGCCCAACGAGACCCACATGTTCCCCCACGGCCCCAACGCCGTGCGCCAGCTGGCCTTCATCGGCTTCACCACCGCCGACGGCGGCCTGATGATGGGCCACTTCGACAGCAAGATGACCTTCAACGGCAGCCGCGCCATCGAGATCCCCGGCCCACACTTCGTGACCATCATCACCAAGCAGATGAGGGACACCAGCGACAAGCGCGACCACGTGTGCCAGCGCGAGGTGGCCTACGCCCACAGCGTGCCCCGCATCACCAGCGCCATCGGTAGCGACGAGGat SEQ ID NO: 2 (KillerRed) ACAAAGAAACCACGTCGAACTCAATATTGTTTTGAAAGAAATTGCCTTCCGTCAACTTGTCAGGAGCTTAAATCTCAGAATGTTAAGGCTAAAGATGGAAATTACACTATTCTTCTTGACGGATTCACTATTGAAATTTATTGTCATCGAATGAATTCAACCATTCCTAAAGCTTATTTGAA CGTTAATCCAAGAACCAATTTTGCAGAGGTTTATGGAAAAAAATTAATATACCCTCATACTTGCCCATTTAATGGTGATCGTAATGATTCATGCCATTGTTCAGAAGACGGCGATGCAAGTGCTGGATTGACGAGATTCAATAAAGTTCGAATAGATTTGTTGAATAGAAAGTTCCATCTGG CGGATTATACATTTGCAAAACGAGAATATGGTGTTCATGTGCCATATGGTACTGCCGGTGATTGCTACAGTATGAAAGATTGTCCACAGGGAATATTCTCAATTGATTTAAAATCTGCTGGTCTGAAATTAGTTGACGATCTGAATTGGGAGGATCAAGGTCATCGAACATCCTCTCGAATC GATCGTTTTTATAACAATGCAAAAGTTATTGGTCACTGTGGTGGTTTTTGTGGAAAATGCTCTCCTGAGCGGTACAAAGGACTAATCTTTGAAGTTAATACAAAATTATTAAATCATGTGAAAAATGGTGGACACATTGATGATGAATTGGATGATGATGGTTTCTCTGGTGACATGGATtaa Sequence number 3 (CeGON)

[0199] (Mutant (gene)) In addition to the above-mentioned SEQ ID NOs: 2 and 3, the genes used in the "fusion gene" of the present invention may also be genes capable of producing the same proteins as the proteins they produce, i.e., genes containing "different codons capable of producing the same amino acid sequence." Furthermore, it may be a "gene encoding a mutant (protein)" of the protein produced by SEQ ID NO: 2 or 3.

[0200] The mutant (gene) can be created based on the base sequence of SEQ ID NO: 2 or 3 using a standard method in the field of genetic engineering, but it can also be artificially synthesized from scratch.

[0201] Specific examples of "mutants (proteins)" will be described in detail in the section (Types of Mutants) in the explanation of the "fusion protein" of the present invention below.

[0202] (Use of fusion gene) By using it as a constituent material of the "expression cassette" of the present invention described below and introducing it into cultured cells, etc., the "cultured cells" of the present invention can be produced.

[0203] (Effect of fusion gene) The "fusion gene" of the present invention can express "a protein having the photoirradiation-dependent reactive oxygen generating or diffusing activity (I)-2" such as "KillerRed protein" together with "a GON domain protein."

[0204] Therefore, due to the effect of KillerRed protein, etc., it is possible to freely inactivate (destroy) nearby expressed GON domain proteins at any timing when green light is irradiated.

[0205] In other words, it can be used as an "experimental reagent" to investigate the role of GON domain proteins in introduced cells, and has the extremely advantageous effect of being able to be used in the above-mentioned "screening method of the present invention" to confirm the calcium ion release prevention effect of drug candidate compounds.

[0206] Furthermore, by using a "protein that has the ability to generate or diffuse photoinduced reactive oxygen species" such as KillerRed protein to inactivate the GON domain, it becomes possible to use this method in combination with gene inactivation methods using RNAi techniques such as siRNA.

[0207] Furthermore, as described above, the GON gene used in the fusion gene of the present invention has a sequence different from the GON domain in the ADAMTS9 present in the cells to be transfected. Therefore, the exogenous GON domain protein can be destroyed by light irradiation at any timing, independently of the siRNA-mediated suppression of the endogenous ADAMTS9 gene in the transfected cells, allowing the effect of the GON gene to be measured more accurately.

[0208] In other words, by using the "fusion gene" of the present invention, it becomes easier to find an "inhibitor" or "prophylactic and / or therapeutic agent" of the present invention with fewer side effects in the "screening method" of the present invention.

[0209] [Expression cassette of the present invention] The "expression cassette" of the present invention is characterized by using the above-mentioned "fusion gene" of the present invention.

[0210] The term "expression cassette" refers to a gene fragment for introducing a foreign gene, which is composed of, for example, a promoter sequence, an open reading frame (translatable region), and a terminator sequence. In the present invention, the "expression cassette" itself can be directly introduced into the genome, or it can be in the form of a viral vector, a phage vector, a plasmid, or the like.

[0211] Examples of viral vectors include adeno-associated viruses (AAV) and lentiviruses (LV).

[0212] A candidate for a plasmid would be, for example, a plasmid called pEGFP-N3 from which the EGFP sequence has been removed.

[0213] (Construction of expression cassette)

[0214] Furthermore, the "expression cassette" of the present invention can include, in addition to the "fusion gene" of the present invention, a general sequence used to function as an expression cassette.

[0215] (Method for creating an expression cassette) The "expression cassette" of the present invention can be prepared by joining the above-mentioned constituent sequences together using a conventional method in the field of gene-related technology.

[0216] (Use of expression cassette) As described above, the "expression cassette" of the present invention can be introduced into cultured cells, etc. in the form of a viral vector, phage vector, plasmid, etc., but for stable expression, it is preferable to introduce it into the genome of the cultured cells themselves.

[0217] Methods for making them into the form of a virus vector, phage vector, plasmid, etc., and methods for introducing them into the genome of the target cell can be carried out by conventional methods in the field of gene transfer technology.

[0218] Introduction into the genome can be carried out using genome editing techniques, which have been widely used in recent years. Specifically, for example, the "CRISPR / Cas9 plasmid" and the "expression cassette" of the present invention can be simultaneously transfected into target cells, and the genome can be cleaved by the expressed Cas9 and gRNA. Then, the expression cassette can be inserted into the genome by a method that utilizes "homologous recombination" that occurs between the "expression cassette" and the same sequence in the genome.

[0219] (Effect of expression cassette) The "expression cassette" of the present invention can be used in the above-mentioned "screening method" of the present invention.

[0220] [Cultured cells of the present invention] The "cultured cells" of the present invention are characterized in that the "fusion gene" or "expression cassette" of the present invention has been introduced into the cultured cells described below in (IV).

[0221] (IV) A cultured cell for introducing (I), which has an endogenous GON domain with a sequence different from that of the GON domain of (I)-3.

[0222] (cultured cells) In order to investigate the influence of the GON domain as a cause of diseases caused by an increase in calcium ion concentration in the cytoplasm, it is preferable that the cells used as the source of the "cultured cells" of the present invention are those in which the GON domain is expressed under normal conditions. However, in screening experiments, it is more convenient to suppress the expression of GON domain genes that are originally endogenous to the cells, so the fewer the types of "endogenous GON domain-containing genes" that are expressed under normal conditions, the better.

[0223] On the other hand, when the "cultured cells" of the present invention are used in the "method for screening inhibitors of an increase in cytoplasmic calcium concentration and preventive and / or therapeutic agents for diseases caused by said increase" of the present invention, as described above, it is preferable to select cells derived from a biological species different from the biological species from which the foreign GON domain gene is derived. For example, when the GON domain gene is derived from a nematode, human-derived cells are preferred.

[0224] For the above reasons, the "cultured cells" of the present invention are preferably human cultured cells, particularly "HEK293 cells," which express ADAMTS9 but not ADAMTS20, among the ADAMTS genes having the GON domain.

[0225] HEK293 cells have the advantages of high transfection efficiency in all of the following areas, ease of handling, and the ability to be cultured in monolayers as adherent cells.

[0226] ADAMTS9 siRNA transfection Transfection of calcium sensors such as GCaMP6s Transfection of the fusion gene of the present invention into the genome

[0227] The "cultured cells" of the present invention may further contain the following (II) and / or (III): However, when cultured cells are used in the "screening method" of the present invention, it is preferable that (II) and / or (III) be introduced into the cells after administration of the candidate compound for the "prophylactic and / or therapeutic agent" so as not to inhibit the uptake of the candidate compound into the cells.

[0228] (II) Substances that suppress ADAMTS9 gene expression (III) Substances with calcium ion detection function

[0229] The "substance that suppresses the expression of the ADAMTS9 gene" in (II) and the "substance that has calcium ion detection function" in (III) are as described in the "screening method" of the present invention above.

[0230] (Method for producing cultured cells) The "cultured cells" of the present invention can be prepared by introducing the "fusion gene" of the present invention or the "expression cassette" of the present invention into the cultured cells using conventional methods in the field of gene-related technology, as described above, but it is preferable to introduce them into the genome of the target cells themselves.

[0231] This is because introduction into the genome does not result in transient transfection, as compared with plasmids, and the fusion gene is stably replicated in other proliferated cells, and because data can be measured stably if the expression cassette is reliably introduced into all cells.

[0232] Introduction into the genome The method for direct introduction into the genome can be any conventional method in the field of gene transfer technology, such as genome editing technology or recombination technology, and is not particularly limited.

[0233] However, from the viewpoint of precise location and improved introduction efficiency, it is preferable to use genome editing technology. For example, CRISPR / Cas9, the prime editing method using an evolved reverse transcriptase, or improved versions of these methods are possible candidates.

[0234] (Uses of cultured cells) The "cultured cells" of the present invention can be further used in the above-mentioned "screening method" of the present invention by introducing (II) a "substance that suppresses the expression of ADAMTS9" or (III) a "substance having calcium ion detection function." The procedures for introducing these are as described in the explanation of the "screening method" of the present invention.

[0235] (Effect of cultured cells) The "cultured cells" of the present invention have different sequences for the endogenous GON domain gene and the exogenous GON domain gene, and therefore the expression of the endogenous GON domain and the exogenous GON domain can be controlled separately and independently. In addition, because they have the "promoter" of the present invention, the expression level of the exogenous GON domain gene can be exquisitely maintained, making them extremely suitable as cultured cells to be used in the above-mentioned "screening method" of the present invention.

[0236] [Fusion protein of the present invention] The "fusion protein" of the present invention is characterized by being a protein produced by the "fusion gene" or "expression cassette" of the present invention that has been introduced into the "cultured cell" of the present invention.

[0237] Furthermore, the "fusion protein" of the present invention more preferably further contains a "signal peptide" that directs translocation to the endoplasmic reticulum.

[0238] This fusion protein preferably contains a "KillerRed protein" and a "Nematode GON domain (CeGON) protein."

[0239] Specific examples of the "fusion protein" of the present invention include the sequence shown in SEQ ID NO: 7 below (see FIG. 2), which is expressed from the fusion gene of SEQ ID NO: 6.

[0240] Note that the sequences listed below for convenience are actually connected.

[0241] SEQ ID NO:7: MRSIGGSFHLLQPVVAALILLVVCLVYA: signal peptide MGSEGGPALFQSDMTFKIFIDGEVNGQKFTIVADGSSKFPHGDFNVHAVCETGKLPMSWKPICHLIQYGEPFFARYPDGISHFAQECFPEGLSIDRTVRFENDGTMTSHHTYELDDTCVVSRITVN CDGFQPDGPIMRDQLVDILPNETHMFPHGPNAVRQLAFIGFTTADGGLMMGHFDSKMTFNGSRAIEIPGPHFVTIITKQMRDTSDKRDHVCQREVAYAHSVPRITSAIGSDED:KillerRed protein TKKPRRTQYCFERNCLPSTCQELKSQNVKAKDGNYTILLDGFTIEIYCHRMNSTIPKAYLNVNPRTNFAEVYGKKLIYPHTCPFNGDRNDSCHCSEDGDASAGLTRFNKVRIDLLNRKFHLADYTFAKREYGVHVPYGTAGDCYSMKDCPQGIFSIDLKSAGLKLVDDLNWEDQGHRTSSRIDRFYNNAKVIGHCGGFCGKCSPERYKGLIFEVNTKLLNHVKNGGHIDDELDDDGFSGDMD (the end is a stop codon): CeGON protein

[0242] (Type of mutant) Among the "fusion proteins" of the present invention, those that correspond to "mutants" of the base protein (those having a known KillerRed or GON domain protein) include, as described in the explanation of the "fusion gene" of the present invention, mutant proteins in which one or several amino acids have been deleted, substituted, added, and / or inserted in the amino acid sequence of the "KillerRed protein" and / or the "GON domain protein," and which do not lose at least the functions of the original proteins (such as the protein inactivation function of KillerRed or the IP3 receptor degradation inhibitory function of the GON domain).

[0243] The type and position of the amino acid mutation are not particularly limited, but preferred examples include conservative substitutions that are similar to the original amino acid in properties such as charge, side chain, polarity, and aromaticity, and that have little effect on the function of the protein (see, for example, Molecular Biology of the Cell, Garland Science; 6th edition).

[0244] Specifically, amino acids can be classified as follows, for example, depending on the type of side chain. Conservative substitutions include, but are not limited to, substitutions within these groups and subgroups.

[0245] Acidic amino acid group: aspartic acid, glutamic acid

[0246] Basic amino acid group: lysine, arginine, histidine

[0247] Neutral amino acid group: (Below, subgroups are classified according to structural characteristics)

[0248] Amino acids with hydrocarbon chains: glycine, alanine, valine, leucine, isoleucine, proline

[0249] Amino acids containing hydroxy groups: serine and threonine

[0250] Sulfur-containing amino acids: cysteine, methionine

[0251] Amino acids containing amide groups: asparagine, glutamine

[0252] Amino acid containing an imino group: Proline

[0253] Amino acids containing aromatic groups: phenylalanine, tyrosine, tryptophan

[0254] Uncharged polar amino acids with low polarity side chains: glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine

[0255] Branched-chain amino acids: leucine, valine, isoleucine

[0256] Neutral amino acids with hydrophilic side chains: asparagine, glutamine, threonine, serine, tyrosine, cysteine

[0257] The following mutant of KillerRed (Supernova-Red) can also be used as a mutant in the present invention.

[0258] That is, in the amino acid sequence of KillerRed, the 5th glycine (G) is replaced with valine (V), the 147th asparagine (N) is replaced with serine (S), the 162nd leucine (L) is replaced with threonine (T), the 164th phenylalanine (F) is replaced with threonine (T), the 174th leucine (L) is replaced with lysine (K), and the 206th methionine (M) is replaced with threonine (T).

[0259] Other examples of KillerRed mutants include HyperNova.

[0260] (Method of creating mutants) Among the "fusion proteins" of the present invention, those corresponding to mutants can also be synthesized by directly linking amino acids, but when used in the "screening method" of the present invention, it is preferable to express those mutated at the gene stage, as this allows for easy introduction into cells.

[0261] Gene mutants can be created by mutating a gene encoding a basic protein using standard methods in the field of genetic engineering, or by artificially synthesizing from scratch a sequence corresponding to the mutated gene sequence.

[0262] Specifically, mutants can be created by substituting bases using commercially available kits (e.g., PrimeSTAR (registered trademark) (TaKaRa), PrimeSTART™ (CosmoBio)) using known methods or methods similar thereto.

[0263] (Effect of fusion protein) The "fusion protein" of the present invention can play a very important role in the "screening method" of the present invention by being expressed in the "cultured cells" of the present invention.

[0264] [Method for measuring (diagnosing) biological samples of the present invention] The "method for measuring a biological sample" of the present invention is characterized by comprising the following steps (1) and (2):

[0265] (1) measuring the abundance of the following (X) protein or the expression level of the following (X) gene in a biological sample: (2) A step of determining that the biological sample is derived from a patient or potential patient group of (Y) below, when the abundance of the (X) protein or the expression level of the (X) gene measured in (1) is lower than the abundance of the (X) protein or the expression level of the (X) gene in a control sample derived from a healthy individual of the same species as the biological sample.

[0266] (X) GON domain protein or its gene (Y) Diseases caused by increased intracytoplasmic calcium concentration

[0267] <Biosamples> Specific biological samples include parts of organs or tissues extracted accidentally or artificially from a living body, cells derived from a living body, or cultured cells thereof. However, as for cells, cells immediately after collection are preferable to cultured cells because more accurate information can be obtained from them.

[0268] The above-mentioned "biological sample" is primarily a sample derived from an individual organism suspected of having disease (Y) or diagnosed with said disease, but is not necessarily limited to such a condition. It may also be a sample derived from an individual organism that has not been tested for said disease (e.g., a sample collected during a health checkup), and cells derived from organs, tissues, etc. of such individuals may be used.

[0269] Specifically, it is preferable that the cultured cells express ADAMTS9 and are suitable for investigating the disease (Y).

[0270] Specifically, for example, when (Y) is a disease caused by apoptosis of vascular smooth muscle cells, the biological sample is preferably vascular smooth muscle-derived cells or cultured cells thereof.

[0271] Specific examples of (Y) "diseases caused by increased intracytoplasmic calcium concentration" include, but are not limited to, diseases caused by aneurysms or aneurysm rupture, particularly cerebral hemorrhage and subarachnoid hemorrhage.

[0272] <Control sample> The "control sample derived from a healthy individual of the same species as the biological sample" used for the determination is a sample derived from an individual (healthy individual) who has been confirmed to be healthy at least with respect to (Y), and "the same species" means that the biological sample of the subject is derived from the same "biological species," "organ," "tissue," "biologically derived cells," or "cultured cells," etc.

[0273] Data such as "abundance of (X) protein or expression level of (X) gene" obtained from a sample from a healthy individual does not need to be obtained for each measurement of a (patient) biological sample; previously obtained data may be used.

[0274] [Inhibitor of the present invention] The "inhibitor of an increase in cytoplasmic calcium concentration" of the present invention is characterized by containing a gene comprising the sequence described in (I)-3 below, or a protein expressed from the gene.

[0275] (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0276] The mutants are as described above.

[0277] (Inhibitor composition)

[0278] Protein or gene: The GON domain protein or the gene encoding it can be derived from either a human or non-human organism, but it is preferable to use a GON domain protein or its gene from the target organism in which the calcium ion release inhibition experiment using the inhibitor will be conducted, as this allows for more accurate reproduction of the inhibitory function within the organism and confirmation of the inhibitory effect.

[0279] Other ingredients: When the active ingredient of the inhibitor of the present invention is the above-mentioned "gene," it is preferable that it further contains sequences that are useful for expressing the above-mentioned "gene" within cells, such as a promoter sequence and / or a signal sequence, and these sequences are also preferably derived from the target organism in which an experiment to inhibit calcium ion release using the inhibitor is to be conducted.

[0280] Promoter sequence: Although the known CMV promoter and the like can be used as the promoter sequence, the "promoter" of the present invention, which is an improved CMV promoter, is preferred.

[0281] When the "promoter" of the present invention is introduced into cells as an experimental or research reagent, it is useful for more stable maintenance of the introduced cells and their cultured cell lines.

[0282] Signal sequence: The signal sequence is preferably a gene sequence encoding a signal peptide that directs translocation to the endoplasmic reticulum, such as that shown in SEQ ID NO: 5 above, but is not limited to SEQ ID NO: 5.

[0283] In addition to the above components, the experimental and research reagents may contain solvents and various other additives that are generally used in experimental and research reagents containing genetic materials.

[0284] (Inhibitor manufacturing method) The "inhibitor" of the present invention can be produced by a conventional method.

[0285] (Effect of inhibitors) Research by the present inventors has revealed that the GON domain is involved in the release of calcium ions into cells. Therefore, the "inhibitor" of the present invention inhibits the release of calcium ions (Ca 2+ It is a highly useful experimental and research reagent that inhibits the increase in the release concentration of calcium ions (cytoplasmic calcium ion concentration). In particular, it has been found that the GON domain is involved in "leakage from the void" caused by the removal of IP3 receptors from the endoplasmic reticulum membrane, which is separate from the "normal calcium ion release route" via the IP3 receptor. Therefore, it is also very useful as an experimental and research reagent for this leakage. The inhibitor of the present invention can also be used as an active ingredient of the "agent for preventing and / or treating diseases caused by an increase in intracytoplasmic calcium concentration" of the present invention, which will be described later.

[0286] [Preventive and / or therapeutic agent of the present invention] The "prophylactic and / or therapeutic agent for diseases caused by an increase in intracytoplasmic calcium concentration" of the present invention is characterized by containing a gene comprising the sequence described in (I)-3 below, or a protein expressed from the gene.

[0287] (I)-3: A gene encoding a GON domain protein or a mutant thereof

[0288] The mutants are as described above.

[0289] (Constitution of preventive and / or therapeutic agent) The "proteins," "genes," "promoter sequences," "signal sequences," etc. used in the "prophylactic and / or therapeutic agent" of the present invention are as explained above in the "inhibitor" of the present invention.

[0290] In particular, it is preferable that the active ingredient of the "prophylactic and / or therapeutic agent" of the present invention is derived from the organism that is the target of prevention and / or treatment, since there is less risk of side effects and the like.

[0291] Other ingredients: In addition to the above-mentioned components, the "prophylactic and / or therapeutic agent" may contain, in addition to the above-mentioned components, solvents such as sterile water and physiological saline, excipients, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, etc., which are generally used in prophylactic and / or therapeutic agents.

[0292] (Dosage form) The dosage form of the "prophylactic and / or therapeutic agent" is not particularly limited, and may be solid, liquid, granular, powder, etc., and examples of dosage forms by administration include injections, transdermal agents, inhalants, nasal drops, and oral agents.

[0293] (Method for producing preventive and / or therapeutic agent) The "prophylactic and / or therapeutic agent" of the present invention can be formulated into each of the above dosage forms by a conventional method.

[0294] (Dosage of prophylactic and / or therapeutic agent) The dosage of the "prophylactic and / or therapeutic agent" of the present invention is not particularly limited and may be appropriately selected depending on the age, weight, health condition, etc. of the subject to be administered.

[0295] When the active ingredient is a GON domain protein, the amount may be that of a typical protein preparation, and the amount may be set, for example, as the amount per kg per day for an adult, and may be administered in a single dose or in multiple doses (e.g., 2, 3, or 4 times).

[0296] When the active ingredient is a gene encoding a GON domain protein, the amount may be any amount commonly used for gene therapy drugs, and this amount may be set, for example, as the amount per kg per day for an adult, and may be administered in a single dose or in multiple doses (e.g., 2, 3, or 4 times).

[0297] The frequency of administration may be a single dose, daily administration, or administration every few days (e.g., 2, 3, or 4 days), once a week, once a few weeks (e.g., 2, 3, or 4 weeks), once a month, or once a few months (e.g., 2, 3, or 4 months).

[0298] (Administration route of prophylactic and / or therapeutic agents) In the case of proteins, they may be administered systemically or locally, for example, intravenously, intramuscularly, subcutaneously, transdermally, intranasally, via the lungs, or orally.

[0299] (Effects of preventive and / or therapeutic agents) The "prophylactic and / or therapeutic agent" of the present invention can prevent and / or treat diseases caused by an increase in intracytoplasmic calcium ion concentration. Such diseases include, but are not limited to, the diseases exemplified in the "method for measuring a biological sample" of the present invention described above. [Example]

[0300] Example 1: "Promoter" of the present invention The promoter of the known cytomegalovirus CMV was purchased, and the C-terminal portion (approximately 81 bases) was extracted to create the "promoter" of the present invention (pCMV(-81)) represented by SEQ ID NO: 1.

[0301] [Example 2: "Fusion gene" of the present invention] Using the sequences of "SEQ ID NO: 1," "SEQ ID NO: 2," "SEQ ID NO: 3," and "SEQ ID NO: 5" as constituent materials and using standard methods in gene-related technology, the "fusion gene" of the present invention (pCMV(-81)_sig-KillerRed-CeGON) shown in SEQ ID NO: 6 (Figure 1) was created.

[0302] In FIG. 1, the sequences of SEQ ID NOs: 1 to 5 are listed in itemized form for convenience, but they are all connected.

[0303] The above "pCMV" refers to the promoter sequence of "SEQ ID NO: 1" in Figure 1 (improved CMV promoter). The above "sig" refers to the sequence encoding the signal peptide of "SEQ ID NO: 5" in FIG. The above "KillerRed" refers to the sequence encoding the KillerRed protein of "SEQ ID NO: 2" in FIG. The above-mentioned "CeGON" refers to the gene encoding the GON domain protein of C. elegans, "SEQ ID NO: 3" in FIG.

[0304] In addition, the fusion gene of Example 2 above actually also contains "SEQ ID NO: 4: vector sequence" at the position shown in Figure 1, which corresponds to the residual strand derived from the vector.

[0305] Example 3: "Expression cassette" of the present invention A plasmid was prepared for inserting the "fusion gene" of Example 2 into the genome of a target cell by homologous recombination.

[0306] Specifically, in Example 4 described below, at the position on the genome where the fusion gene is to be introduced (human RPL34P18 gene), approximately 600 bp of a "guide RNA (gRNA) recognition sequence" as shown in SEQ ID NO: 8 below was added before and after the "fusion gene," thereby creating an "expression cassette" (plasmid for homologous recombination) of the present invention.

[0307] gRNA recognition sequence: gtacccaggccgacttcgag: SEQ ID NO: 8 (Figure 8)

[0308] A gRNA is an RNA having a sequence complementary to SEQ ID NO: 8, which is used to guide Cas9 to the fusion gene introduction site (gRNA recognition sequence) in the genome in the CRISPR / Cas9 method described below. It is generated by transcription from the DNA sequence in the CRISPR / Cas9 plasmid described below.

[0309] [Example 4: "Cultured Cells" of the Present Invention (Cultured Cells Introduced with an Expression Cassette)] Creation of CRISPR / Cas9 plasmids A plasmid for introducing (transfecting) the "expression cassette" of the present invention was prepared according to the following procedure.

[0310] 1) Cas9 SmartNuclease TM A DNA for constructing gRNA with the same sequence as SEQ ID NO: 8 was inserted into an All-in-one Vector (SBI System Biosciences, California, USA), which acts as a guide for the pseudogene "human RPL34P18" that does not affect cell growth. This DNA for constructing gRNA is transcribed into RNA within the cell, generating a gRNA complementary to SEQ ID NO: 8, which acts as a guide.

[0311] 2) Expression cassette The expression cassette of Example 3 was used.

[0312] 3) Transfection One day before transfection, HEK293 cells were seeded onto a 35 mm dish. Cells were transfected with 1) a CRISPR / Cas9 plasmid containing a gRNA sequence targeting the RPL34P18 gene and 2) an expression cassette (plasmid for homologous recombination) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0313] One week later, a portion of the cells was collected and PCR was used to confirm that some cells had pCMV(-81)_sig-KillerRed-CeGON inserted into their genome.

[0314] The following primer set was used for PCR (FIG. 9).

[0315] SEQ ID NO: 9: CACTTTGGGAGGGAGTTCAA

[0316] SEQ ID NO: 10: ACACGGCGTGCACGTTGAAGTC

[0317] A group containing cells in which it was confirmed that the "fusion gene" had been introduced into the genome was extracted, and the cells were further diluted and cultured, and subjected to PCR again to extract cells in which the "fusion gene" had been introduced into the genome. This procedure was repeated 10 times to establish a cell line in which the "fusion gene" of the present invention had been introduced into the genome, and this was used as the "cultured cell" of the present invention.

[0318] Example 5: "Cultured Cells" of the Present Invention (Cultured Cells Transfected with Expression Cassette and ADAMTS9 siRNA Gene) siRNA against human ADAMTS9 was administered to cultured cells (HEK293 cells) into whose genome the "fusion gene" of Example 4 had been introduced.

[0319] The ADAMTS9 siRNA used was "Dharmacon (registered trademark), Catalog ID: L-005779-00-0005" (Figure 3).

[0320] (II)-1 to (II)-4 are all sequences that are not found in the nematode-derived GON domain gene, nor in the fusion genes of the present invention such as sequence number 6, and are therefore preferred in that the inventors have confirmed that when the above siRNAs are used, the fusion genes of the present invention having the nematode-derived GON domain are not knocked down.

[0321] siRNA administration was performed using Lipofectamine TM RNAiMAX Transfection Reagent (Thermo Fisher Scientific) was used and performed according to the manufacturer's instructions.

[0322] [Example 6: "Cultured Cells" of the Present Invention (Expression Cassette, ADAMTS9 siRNA Gene, Calcium Sensor-Introduced Cultured Cells)] By further introducing a calcium sensor (GCaMP6s) into the cultured cells of Example 5, the "cultured cells" of the present invention capable of measuring the release of calcium ions into the cytoplasm were prepared.

[0323] Specifically, three days after the administration of siRNA in Example 5, pCMV-GCaMP6s was transfected.

[0324] Transfection was performed using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0325] [Comparative Example 1: Cultured cells] Cultured cells of Comparative Example 1 were prepared in the same manner as in Example 6, except that a non-target control siRNA (negative control) was used instead of "ADAMTS9 siRNA."

[0326] The above siRNA was also purchased from Dharmacon as the following product. Product name: ON-TARGETplus Non-targeting siRNAs Number:D-001810-0X

[0327] [Example 7: "Fusion Protein" of the Present Invention] The protein expressed from the fusion gene of Example 2 in the cultured cells of Example 6 is shown in FIG. 2 (SEQ ID NO: 7).

[0328] In Figure 2, the proteins listed for convenience are actually all connected.

[0329] [Example 8: Establishment of a screening system] The following procedure for the "screening method" of the present invention was established.

[0330] 1) A candidate compound is administered to the cultured cells (HEK293 cells) into which the expression cassette of Example 4 has been introduced. 2) ADAMTS9 siRNA is administered to the cells of 1). 3) Three days after ADAMTS9 siRNA administration, cells were transfected with pCMV-GCaMP6s. 4) The calcium ion concentration is measured the day after 3).

[0331] 4) is carried out as follows:

[0332] For example, 5 minutes after the start of calcium ion measurement (0 minutes), green light (excitation wavelength 530-550 nm) is irradiated for approximately 3 minutes, inducing the expression of reactive oxygen species from the KillerRed protein in the fusion protein and thereby inactivating the GON domain expressed nearby.

[0333] The calcium ion concentration in the cytoplasm is measured at 1-minute intervals for 20 minutes before and after the start of the measurement, including the irradiation time.

[0334] As a control, the calcium ion concentration in the cytoplasm in the absence of the candidate compound is also measured.

[0335] The calcium ion concentrations before and after irradiation, or compared with the above-mentioned control, are compared, and those that are able to suppress calcium ion release into the cytoplasm are selected as preventive and / or therapeutic agents or candidate lead compounds for diseases (such as cerebral infarction) caused by excessive release of calcium ions from the endoplasmic reticulum into the cytoplasm.

[0336] [Example 9: "Inhibitor of increase in intracytoplasmic calcium concentration" of the present invention]

[0337] A "fusion gene" containing each of the following sequences is defined as an "inhibitor of an increase in intracytoplasmic calcium concentration" of the present invention.

[0338] The promoter sequence of the present invention according to SEQ ID NO: 1 Sequence encoding the signal peptide of SEQ ID NO:5 Sequence encoding the CeGON domain of SEQ ID NO: 3

[0339] The "inhibitor of an increase in intracytoplasmic calcium concentration" is used in the form of an expression cassette introduced into a vector such as the one described below.

[0340] A vector in which the EGFP sequence has been removed from the pEGFP-N3 plasmid

[0341] [Example 10: "Disease preventive and / or therapeutic agent" of the present invention] The "inhibitor of an increase in cytoplasmic calcium concentration" of Example 9 described above is used as the "prophylactic and / or therapeutic agent for diseases caused by an increase in cytoplasmic calcium concentration" of the present invention.

[0342] [Test Example 1: Test to confirm calcium ion release by disruption of foreign GON domain] The "cultured cells" of Example 6 or Comparative Example 1 were transfected with pCMV-GCaMP6s and then cultured for 12 days. 2+ Measurements were carried out.

[0343] (preparation) Before the measurement, the culture medium was replaced with the following:

[0344] D-MEM (High Glucose) with L-Glutamine and HEPES (phenol red-free, Wako)

[0345] This is to prevent the fluorescent background from becoming stronger due to phenol red, which weakens the contrast with the fluorescence from the target calcium ions.

[0346] (Ca 2+ (measurement of Fluorescence was measured using a 40x lens.

[0347] The fluorescence of GCaMP6s was photographed using a GFP filter at 1-minute intervals for 20 minutes. From 5 minutes to 8 minutes after the start of imaging, green light passed through the RFP filter of the microscope was irradiated, and the exogenous CeGON domain was destroyed by the generation or diffusion of reactive oxygen species dependent on the light irradiation of KillerRed.

[0348] (result) The results are shown as "Changes in fluorescence intensity over time (fluorescence change) over 20 minutes" (Figure 4: Example 6, Comparative Example 1) and "Fluorescence photographs taken after 0, 5, 10, 15, and 20 minutes" (second row of Figure 6: Example 6, first row of Figure 6: Comparative Example 1).

[0349] As can be seen from Figures 4 and 6, the destruction of the exogenous GON-1 domain protein by the action of KillerRed resulted in an increase in fluorescence intensity over time and the release of large amounts of calcium ions into the cytoplasm.

[0350] On the other hand, as can be seen from Figures 4 and 6, when Control siRNA was used as the siRNA, the exogenous GON-1 domain protein was destroyed by KillerRed, but the expression of endogenous ADMTS9 was not suppressed, and therefore the expression of the endogenous GON domain in ADAMTS9 prevented the release of calcium ions.

[0351] (Consideration)

[0352] In other words, the GON domain is directly involved in the release of calcium ions into the cytoplasm, and the "screening method" of the present invention, which utilizes such an exogenous GON domain gene, has been proven to be highly effective in selecting "inhibitors of increases in cytoplasmic calcium concentration" and "preventive and / or therapeutic agents for diseases caused by such increases."

[0353] Furthermore, in this test example, the amount of fluorescence did not increase in the "cultured cells" of Example 6 to which ADAMTS9 siRNA was administered until green light was irradiated (0 to 5 minutes), which also proves that the foreign GON domain gene (or protein) in the fusion gene was functioning as an "inhibitor of an increase in cytoplasmic calcium concentration" or a "prophylactic and / or therapeutic agent for diseases caused by said increase" of the present invention. In other words, this also strongly suggests the effectiveness of the "inhibitor" of the present invention, which contains only a foreign GON domain as an active ingredient, as in Example 9.

[0354] [Test Example 2: Rescue test by inhibiting calcium ion release pathway] When conducting a test similar to that in Test Example 1, a known inhibitor (2-APB) capable of inhibiting the action of the calcium ion release pathway (IP3 receptor) into the cytoplasm was added in advance, and it was confirmed that the IP3 receptor is involved in the release of calcium ions due to disruption of the GON domain.

[0355] (Administration of 2-APB) In the preparation step of Test Example 1, the following was used in place of the culture medium used for replacement.

[0356] Culture medium: D-MEM (High Glucose) with L-Glutamine and HEPES (phenol red-free, Wako) supplemented with 2-APB to a final concentration of 100 μM

[0357] (Ca 2+ (measurement of After the replacement, the mixture was left to stand in the dark at 37°C for 5 minutes, and then the fluorescence of GCaMP6s was photographed in the same manner as in Test Example 1.

[0358] (result) The results are shown as "Change in fluorescence intensity over time over 20 minutes (fluorescence change)" (FIG. 5: Example 6, Comparative Example 1) and "Fluorescence photographs after 0, 5, 10, 15, and 20 minutes" (fourth row of FIG. 6: Example 6, third row of FIG. 6: Comparative Example 1).

[0359] As can be seen from Figures 5 and 6, the amount of fluorescence, which should have increased over time due to the destruction of the exogenous GON-1 domain protein, was reduced by the addition of the IP3 receptor inhibitor (2-APB), indicating that the release of calcium ions into the cytoplasm was prevented.

[0360] (Consideration)

[0361] This indicates that the IP3 receptor is involved in the release of calcium ions due to disruption of the GON domain, and also supports (proves) that "2-APB, a known IP3 receptor inhibitor," could be selected by the "screening method" of the present invention, i.e., supports (proves) that the screening method of the present invention can be used as a screening method for "inhibitors of increases in cytoplasmic calcium concentration."

[0362] [Test Example 3: Test to confirm the effect of ADAMTS9 siRNA on the viability of vascular smooth muscle cells] We investigated the effect of GON domain disruption on aneurysms or aneurysm rupture, which are important diseases caused by the release of calcium ions into the cytoplasm. Specifically, the effect of ADAMTS9 siRNA on cell viability when vascular smooth muscle was administered with an inflammatory mediator (TXA2) that induces inflammation was confirmed using an MTS assay.

[0363] (siRNA administration) Vascular smooth muscle cells (P53LMACO1) were administered siRNA against ADAMTS9 (mouse) or non-target control siRNA (negative control).

[0364] The control siRNA used was the same as that used in Comparative Example 1 and was purchased from Dharmacon. siRNA against mouse ADAMTS9 was purchased from Dharmacon under Catalog ID: L-167415-00-0005 and used.

[0365] siRNA was administered using Lipofectamine TM RNAiMAX Transfection Reagent (Thermo Fisher Scientific) was used and performed according to the manufacturer's instructions.

[0366] (Addition of IP3 receptor inhibitor [2-APB]) Two days after siRNA administration, the IP3 receptor inhibitor 2-APB (5 μM) was added and the cells were cultured at 37°C for 1 hour (Figure 7: right). As a control, cells were also cultured without the addition of 2-APB (FIG. 7: left, center).

[0367] (Addition of inflammatory mediator [TXA2]) Then, 10 μM of U-46619, an analog of TXA2 (thromboxane A2), a type of inflammatory mediator, was added (Figure 7: center, right).

[0368] Inflammatory mediators are substances that cause inflammatory reactions in the body and maintain the inflammatory state.

[0369] (Measurement of cell viability) One hour after the addition of TXA2, an MTS assay was performed.

[0370] The MTS assay is a method for measuring cell viability in vitro. TM The assay was performed using the AQueous Nonradioactive Cell Proliferation Assay Kit according to the manufacturer's instructions.

[0371] (result) The results are shown in Figure 7.

[0372] As can be seen from Figure 7, administration of ADAMTS9 siRNA further reduced the cell viability that had been reduced by TXA2 (Figure 7: center), but administration of 2-APB restored the cell viability (Figure 7: right side).

[0373] (Consideration)

[0374] This suggests that suppression of GON domain protein expression (located at the C-terminus of ADAMTS9), together with inflammatory mediators, is involved in vascular smooth muscle cell death via the release of calcium ions into the cytoplasm, leading to vascular fragility. This supports the previously known finding that when vascular smooth muscle is damaged by inflammation within the blood vessels, the strength of the blood vessel wall decreases, causing aneurysms to expand. The above results also indicate that GON domain proteins or genes encoding them may be useful as preventive and / or therapeutic agents for diseases caused by cerebral aneurysms and their rupture.

[0375] [Test Example 4: Confirmation test of promotion of IP3 receptor ubiquitination by GON domain knockdown] (siRNA administration) ADAMTS9 siRNA or control siRNA was administered to HEK293 cells.

[0376] Each siRNA was the same as that used in Example 5 and Comparative Example 1 above, and was purchased from Dharmacon.

[0377] (Creation of tagged IP3 receptors and tagged ubiquitin proteins) The cDNA sequence of the IP3 receptor or ubiquitin and the respective peptide tag genes were introduced into cells using CMV promoters. Specifically, two days after administration of each siRNA, the siRNA-administered cells were simultaneously transfected with the following two types of plasmids a) and b) using Lipofectamine 3000.

[0378] a) pCMV_IP3R-PA: A plasmid for expressing a PA-tagged portion (C-terminal portion) of IP3R (IP3 receptor). b) pCMV_Ub-HA: a plasmid for expressing HA-tagged ubiquitin

[0379] PA tag: a peptide tag derived from the human podoplanin PLAG sequence HA tag: a peptide tag derived from human influenza virus (hemagglutinin) Ub: Ubiquitin protein

[0380] The gene sequence of the region in a) pCMV_IP3R-PA containing the "CMV promoter sequence," "sequence encoding the C-terminus of IP3R," "sequence encoding the PA tag," etc. is shown in SEQ ID NO: 11 and FIG. 10.

[0381] Note that the sequences listed below for convenience are actually connected.

[0382] SEQ ID NO:11: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG CMV enhancer array GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT CMV promoter sequence GGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCGCCACC Vector-derived sequence Atg start codon GGATCC ATCGCCACC vector-derived sequence (underlined: BamHI recognition site) GGCGTTGCCATGCCAGGTGCCGAAGATGATGTGGTGTAA PA tag gene + stop codon

[0383] (Inhibition of degradation of ubiquitinated IP3 receptors) The day after transfection in a) and b), lactacystin (a proteasome inhibitor) was added and the cells were cultured for 1 hour to prevent the ubiquitinated IP3 receptor from being degraded by the proteasome.

[0384] After 1 hour, the cells were harvested and immunoprecipitated with anti-PA antibody. IP3 receptor and ubiquitized IP3 receptor were detected by Western blotting using the following two antibodies.

[0385] Anti-PA antibody (anti-PA) Anti-HA antibody (anti-HA)

[0386] (result) The detection results are shown in FIG.

[0387] As can be seen from Figure 11, although the amount of IP3 receptor detected by anti-PA was equivalent in control siRNA- and ADAMTS9 siRNA-administered cells, the amount of ubiquitinated IP3 receptor detected by anti-HA was greater in HEK293 cells administered with ADAMTS9 siRNA, confirming that suppression of ADAMTS9 (GON domain) expression increases IP3 receptor ubiquitination.

[0388] (Consideration) This suggests that suppression of the GON domain expression results in excessive release and ubiquitination of IP3 receptors from the endoplasmic reticulum membrane. In other words, the increase in cytoplasmic calcium ion concentration due to inhibition of GON domain expression is thought to be largely due to excessive release (leakage) of calcium ions from the void created by the withdrawal of IP3 receptors from the endoplasmic reticulum membrane. [Industrial Applicability]

[0389] The "screening method" and "screening kit" of the present invention make it possible to easily screen for "inhibitors of an increase in cytoplasmic calcium concentration" or "prophylactic and / or therapeutic agents for diseases caused by said increase" that have little risk of side effects such as those associated with IP3 receptor inhibitors. By using the "promoter" of the present invention, it is possible to properly express an exogenous GON domain protein without causing cell death in cultured cells, thereby further improving the screening accuracy of the "screening method" of the present invention. The "fusion gene," "expression cassette," "cultured cells" and "fusion protein" of the present invention can be used to screen for drugs for preventing and / or treating diseases caused by the release of calcium ions into the cytoplasm. The "method for measuring a biological sample" of the present invention makes it possible to detect diseases caused by an increase in intracytoplasmic calcium ion concentration at an early stage. The "inhibitor" of the present invention can inhibit an increase in intracytoplasmic calcium ion concentration. Furthermore, the "prophylactic and / or therapeutic agent" of the present invention can prevent and / or treat diseases caused by an increase in intracytoplasmic calcium ion concentration.

Claims

1. A method for screening for an agent that inhibits an increase in cytoplasmic calcium concentration or a preventive and / or therapeutic agent for a disease caused by said increase, comprising the following steps (A) to (G): (A) preparing cultured cells into which the fusion gene (I) below has been introduced and which have an endogenous GON domain with a sequence different from that of (I)-3: (B) A step of administering a candidate compound to the cultured cells of (A). (C) introducing (II) into the cultured cells of (A); (D) introducing (III) into the cultured cells of (A); (E) A step of irradiating the cultured cells of (A) with light having a wavelength capable of exerting the effect of (I)-2. (F) A step of measuring the amount of change in fluorescence before and after the irradiation of (E) by utilizing the detection function of (III). (G) A step of selecting a candidate compound having an effect of preventing excessive release of calcium ions into the cytoplasm by comparing the amount of change in fluorescence (F) between the candidate compound administration group and the non-administration group. (I) A fusion gene comprising the following (I)-1 to (I)-3: (II) Substances that suppress ADAMTS9 gene expression (III) Substances with calcium ion detection function (I)-1: Promoter for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein that generates or diffuses photoinduced reactive oxygen species or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof

2. The screening method according to claim 1, wherein (I)-1 has the sequence shown in (I)-1-1 or (I)-1-2 below. (I)-1-1: Base sequence of SEQ ID NO: 1 (I)-1-2: A base sequence in which one or several bases are deleted, substituted, added, and / or inserted from the base sequence of SEQ ID NO: 1, and which has the same promoter activity as SEQ ID NO:

1.

3. A screening kit for an agent for suppressing an increase in intracytoplasmic calcium concentration or a preventive and / or therapeutic agent for a disease caused by said increase, comprising the following (I) to (IV): (I) A fusion gene comprising the following (I)-1 to (I)-3: (II) Substances that suppress ADAMTS9 gene expression (III) Substances with calcium ion detection function (IV) A cultured cell for introducing (I), which has an endogenous GON domain with a sequence different from that of the GON domain described in (I)-3 below. (I)-1: Promoter for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein that generates or diffuses photoinduced reactive oxygen species or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof

4. The kit according to claim 3, wherein the fusion gene (I) further comprises the following (I)-5: (I)-5: A sequence encoding a signal peptide that directs transport to the endoplasmic reticulum

5. A promoter according to (I)-1 below, characterized in that it comprises the sequence described in (I)-1-1 or (I)-1-2 below. (I)-1: Promoter for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein that generates or diffuses photoinduced reactive oxygen species or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof (I)-1-1: Base sequence of SEQ ID NO: 1 (I)-1-2: A base sequence in which one or several bases are deleted, substituted, added, and / or inserted from the base sequence of SEQ ID NO: 1, and which has the same promoter activity as SEQ ID NO:

1.

6. A fusion gene comprising the following (I)-1 to (I)-3, wherein (I)-1 consists of the sequence described in the following (I)-1-1 or (I)-1-2. (I)-1: Promoter sequence for expression of (I)-2 and (I)-3 (I)-2: A gene encoding a protein that generates or diffuses photoinduced reactive oxygen species or a mutant thereof (I)-3: A gene encoding a GON domain protein or a mutant thereof (I)-1-1: Base sequence of SEQ ID NO: 1 (I)-1-2: A base sequence in which one or several bases are deleted, substituted, added, and / or inserted from the base sequence of SEQ ID NO: 1, and which has the same promoter activity as SEQ ID NO:

1.

7. The fusion gene according to claim 6, further comprising the following (I)-5: (I)-5: A sequence encoding a signal peptide that directs transport to the endoplasmic reticulum

8. The fusion gene according to claim 6, wherein (I)-2 is a KillerRed protein gene or a mutant thereof.

9. The fusion gene according to claim 6, characterized in that it has the sequence of SEQ ID NO:

6.

10. An expression cassette comprising the fusion gene according to claim 6.

11. A cultured cell according to (IV) below, into which the fusion gene according to any one of claims 6 to 9 or the expression cassette according to claim 10 has been introduced. (IV) A cultured cell for introducing (I), which has an endogenous GON domain with a sequence different from that of the GON domain of (I)-3.

12. The cultured cell according to claim 11, further comprising the following (II) and / or (III): (II) Substances that suppress ADAMTS9 gene expression (III) Substances with calcium ion detection function

13. A fusion protein produced by the fusion gene or expression cassette introduced into the cultured cell according to claim 11.

14. A method for measuring a biological sample, comprising the following steps (1) and (2): (1) measuring the amount of the following (X) protein present or the expression level of the following (X) gene in a biological sample: (2) A step of determining that the biological sample is derived from a patient or potential patient group of (Y) below, when the abundance of the (X) protein or the expression level of the (X) gene measured in (1) is lower than the abundance of the (X) protein or the expression level of the (X) gene in a control sample derived from a healthy individual of the same species as the biological sample. (X) GON domain protein or its gene (Y) Diseases caused by increased intracytoplasmic calcium concentration

15. The method for measuring a biological sample according to claim 14, wherein the biological sample is a cell in which ADAMTS9 is expressed or a cultured cell thereof.

16. The method for measuring a biological sample according to claim 15, wherein the cells expressing ADAMTS9 are cells derived from vascular smooth muscle, and (Y) is a disease caused by apoptosis of vascular smooth muscle cells.

17. 17. The method for measuring a biological sample according to claim 16, wherein (Y) is an aneurysm or a disease caused by the rupture of an aneurysm.

18. 18. The method for measuring a biological sample according to claim 17, wherein (Y) is cerebral hemorrhage or subarachnoid hemorrhage.

19. An inhibitor of an increase in cytoplasmic calcium concentration, characterized by comprising a gene comprising the sequence described in (I)-3 below, or a protein expressed from the gene. (I)-3: A gene encoding a GON domain protein or a mutant thereof

20. The inhibitor according to claim 19, wherein the gene (I)-3 forms a fusion gene together with the promoter sequence according to claim 5.

21. A preventive and / or therapeutic agent for a disease caused by an increase in intracytoplasmic calcium concentration, characterized by comprising a gene comprising the sequence described in (I)-3 below, or a protein expressed from the gene. (I)-3: A gene encoding a GON domain protein or a mutant thereof

22. 22. The preventive and / or therapeutic agent according to claim 21, wherein the gene (I)-3 forms a fusion gene together with the promoter sequence according to claim 5.

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