Nucleic acid construct capable of measuring homologous recombination activity and use thereof

Abstract

To provide a means for simply and rapidly detecting the presence or absence and the degree of homologous recombination activity in an individual, and to provide a means useful for the detection and treatment of a homologous recombination-recovering cancer having no therapeutic means at present.SOLUTION: The nucleic acid construct of the present invention includes (1) a promoter region, (2) a mutant gene sequence having a cleavage site inside a gene sequence encoding a protein, and (3) a nucleotide sequence composed of a first homologous region and a second homologous region, which are partial regions in the mutant gene sequence of (2), and in which the partial region including the cleavage site can be substituted by homologous recombination. The nucleic acid construct of the present invention can be used as a reagent or kit for measuring homologous recombination activity, a diagnostic agent for homologous recombination deficient cancer, a therapeutic agent for homologous recombination recovery cancer, and the like.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid construct that enables measurement of homologous recombination activity, and the use of the nucleic acid construct as a reagent or kit for measuring homologous recombination activity, an agent for detecting homologous recombination-deficient cancer, etc. [Background technology]

[0002] Double-strand breaks in genomic DNA are the most mutagenic and dangerous form of DNA damage. Therefore, homologous recombination (HR), which can accurately repair double-strand breaks, is essential for maintaining genome stability (Non-Patent Document 1). HR occurs only during the S to G2 phases, when sister chromatids are present as templates. However, non-homologous end joining (NHEJ), another double-strand break repair mechanism, functions regardless of the cell cycle (Non-Patent Document 2). Other minor repair pathways, such as alternative end-joining (AEOJ) and single-strand annealing (SEA), involving PolQ, exist, but both methods inevitably induce mutations after repair (Non-Patent Document 3).

[0003] HR is a highly complex reaction involving many proteins, with Rad51, which mediates strand exchange, playing a central role (Non-Patent Document 4). Factors that support the function of Rad51, such as BRCA1 and BRCA2, are also essential for HR, and human cells lacking BRCA1 or BRCA2 exhibit defects in the repair of double-strand breaks by HR (Non-Patent Document 5).

[0004] HR deficiency causes various cancers. Hereditary breast and ovarian cancer syndrome (HBOC) is the most common hereditary tumor, and its main cause is genetic abnormalities in BRCA1 or BRCA2 (Non-patent Documents 6, 7). Mutations in HR-related genes have also been reported in various types of sporadic (non-hereditary) cancers (Non-patent Documents 8, 9).

[0005] Diagnosis of HR-deficient cancers is performed by genetic testing of BRCA1 and BRCA2 (Non-Patent Document 10). However, given that several dozen proteins are involved in the HR reaction, current genetic testing is not sufficient. It is considered preferable to detect HR activity itself, and several methods have been proposed for this purpose (Non-Patent Documents 11, 12). However, all of these involve a relative assessment of HR activity. While it would be most effective and reliable to assess the HR activity of individual cells as an absolute value rather than a relative assessment, no such method has yet been developed.

[0006] Platinum-based drugs or poly(ADP-ribose) polymerase (PARP) inhibitors are used to treat HR-deficient cancers (Non-Patent Documents 13, 14). The latter, PARP inhibitors, have attracted particular attention due to their "synthetic lethal" relationship with HR. However, the frequent occurrence of resistant cancers in PARP inhibitor therapy is a major problem, and no treatments for these resistant cancers currently exist (Non-Patent Documents 15, 16). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Moynahan ME and Jasin M. Nat Rev Mol Cell Biol. 2010 Mar;11(3):196-207. [Non-patent document 2] Chang HHY et al. Nat Rev Mol Cell Biol. 2017 Aug;18(8):495-506. [Non-patent document 3] Saito S et al. Nat Commun. 2017 Jul 11;8:16112 [Non-patent document 4] Morrical SW. Cold Spring Harb Perspect Biol. 2015 Feb 2;7(2):a016444. [Non-Patent Document 5] Prakash R et al. Cold Spring Harb Perspect Biol. 2015 Apr 1;7(4):a016600.

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[0008] If it were possible to easily and quickly detect the presence or absence and level of HR activity in cells, tissues, and individuals, it would be an extremely useful method for the diagnosis and treatment of HR-deficient cancers, including HBOC, regardless of the type of cancer. Furthermore, in the case of BRCA1-deficient cancers, BRCA1 restoration (reversion mutation) is one of the factors that contribute to the acquisition of PARP inhibitor resistance (Non-Patent Documents 17, 18). Therefore, if cancer cells with restored HR activity could be detected and killed, this would contribute to the treatment of resistant cancers. However, there are currently no known methods for easily and quickly detecting the presence or level of HR activity in individuals, or for selectively detecting and killing cancer cells with restored HR activity.

[0009] An object of the present invention is to provide a means for easily and quickly detecting the presence or absence and level of HR activity in an individual. Another object of the present invention is to provide a means useful for detecting and treating homologous recombination-mediated cancers, for which no treatment is currently available. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors have developed a nucleic acid construct with a structure designed so that in cells that have homologous recombination activity, a gene sequence encoding a protein is reproduced and the protein is expressed, but in cells that have lost homologous recombination activity, the gene sequence is not reproduced and the protein is not expressed.They have also found that, with this nucleic acid construct, the presence or absence of homologous recombination activity can be detected in a very short time using a transient expression system, and that homologous recombination activity can be evaluated and measured in absolute values ​​rather than relative evaluations, and that if the nucleic acid construct of the present invention is prepared using a gene sequence that encodes a protein that has the effect of reducing cell viability, such as a suicide gene, a therapeutic effect can be obtained by killing cancer cells that have recovered from homologous recombination, and have completed the present invention, which includes the following aspects.

[0011] [1] A nucleic acid construct comprising the following (1) to (3): (1) Promoter region. (2) A mutant gene sequence that has a cleavage site within the gene sequence that codes for a protein. (3) A partial region in the mutant gene sequence of (2), which is a base sequence consisting of a first homologous region and a second homologous region that can replace the partial region containing the cleavage site by homologous recombination, and is any of the following base sequences (i) to (iii): (i) A continuous subsequence in the gene sequence encoding the protein, which subsequence includes the upstream and downstream regions adjacent to the cleavage site in the mutant gene sequence of (2). (ii) A nucleotide sequence that is homologous to the partial sequence of (i) and encodes the same amino acid sequence as the partial sequence. (iii) A contiguous partial sequence in a gene sequence encoding a protein having 80% or more sequence identity with the protein and the same activity as the protein, said partial sequence having homology with the partial sequence of (i). [2] The nucleic acid construct of claim 1, wherein (2) comprises a stop codon upstream of the cleavage site. [3] The nucleic acid construct according to [1], wherein the sequence (3) is a continuous partial sequence in the gene sequence encoding the protein, and is a partial sequence including the upstream and downstream regions adjacent to the cleavage site in the mutant gene sequence of (2). [4] The nucleic acid construct according to [2], wherein the sequence (3) is a continuous partial sequence in the gene sequence encoding the protein, and is a partial sequence including the upstream and downstream regions adjacent to the stop codon and cleavage site in the mutant gene sequence of (2). [5] The nucleic acid construct according to any one of [1] to [4], wherein the sequences (ii) and (iii) have 90% or more homology with the partial sequence of (i). [6] The nucleic acid construct according to any one of [1] to [5], wherein in the sequence (3), the first homologous region and the second homologous region each have a chain length of at least 20 bases. [7] The nucleic acid construct according to any one of [1] to [6], which comprises a poly(A) addition signal between the mutant gene sequence of (2) and the sequence of (3). [8] The nucleic acid construct according to any one of [1] to [7], wherein the cleavage site is a restriction enzyme recognition site. [9] The nucleic acid construct according to any one of [1] to [8], wherein the nucleic acid construct is a circular nucleic acid construct comprising (2) and (3) in this order downstream of (1), or a linear nucleic acid construct obtained by cleaving the circular nucleic acid construct at the cleavage site, or a linear nucleic acid construct comprising (2) and (3) in this order downstream of (1).

[10] The nucleic acid construct according to any one of [1] to [8], wherein the nucleic acid construct is a circular nucleic acid construct comprising (2) downstream of (1) and (3) upstream of (1), or a linear nucleic acid construct obtained by cleaving the circular nucleic acid construct at the cleavage site, or a linear nucleic acid construct comprising (2) downstream of (1) and (3) upstream of (1).

[11] The nucleic acid construct according to any one of [1] to

[10] , wherein the gene sequence is a gene sequence encoding a protein that has the effect of reducing cell viability or a protein whose expression in cells can be detected.

[12] The nucleic acid construct according to

[11] , wherein the gene sequence is the sequence of a suicide gene, a DNA damage-inducing gene, a DNA repair inhibitor gene, a luciferase gene, a fluorescent protein gene, a cell surface antigen gene, a secreted protein gene, or a membrane protein gene.

[13] A linear nucleic acid construct comprising the following (a) to (d) in the order (c), (d), (a), and (b) from upstream to downstream: (a) Promoter region. (b) The upstream region of a protein-coding gene sequence. (c) a downstream region of said gene sequence; (d) A base sequence consisting of a first homologous region and a second homologous region that can replace the upstream region and the downstream region by homologous recombination, which is any of the base sequences (d-1) to (d-3) below: (d-1) A continuous partial sequence in a gene sequence encoding the protein, the partial sequence including a region spanning the upstream region and the downstream region. (d-2) A base sequence that is homologous to a partial sequence of (d-2) and encodes the same amino acid sequence as the partial sequence. (d-3) A contiguous partial sequence in a gene sequence that encodes a protein having 80% or more sequence identity with the protein and the same activity as the protein, said partial sequence having homology with the partial sequence of (d-1).

[14] A reagent for measuring homologous recombination activity, comprising the nucleic acid construct according to any one of [1] to

[13] , wherein the gene sequence is a gene sequence encoding a protein whose expression in a cell can be detected.

[15] A kit for measuring homologous recombination activity, comprising the measurement reagent described in

[14] .

[16] A screening system for drugs that affect homologous recombination activity, comprising the nucleic acid construct according to any one of [1] to

[13] .

[17] A diagnostic agent for homologous recombination-deficient cancer, comprising the nucleic acid construct according to any one of [1] to

[13] .

[18] A detection agent for detecting homologous recombination restored cancer cells, comprising the nucleic acid construct described in any one of [1] to

[13] , wherein the gene sequence is a gene sequence encoding a protein whose expression in the cell can be detected.

[19] The detection agent described in

[18] , wherein the homologous recombination restored cancer cells are PARP inhibitor-resistant cancer cells.

[20] A companion diagnostic agent for predicting the effect of an anticancer drug on a homologous recombination-deficient cancer, comprising the nucleic acid construct according to any one of [1] to

[13] .

[21] The companion diagnostic described in

[20] , wherein the anticancer agent is a DNA-damaging anticancer agent.

[22] The companion diagnostic described in

[21] , wherein the DNA-damaging anticancer agent is a PARP inhibitor.

[23] A therapeutic agent for homologous recombination-recovered cancer, comprising the nucleic acid construct according to any one of [1] to

[13] , wherein the gene sequence is a gene sequence encoding a protein that has the effect of reducing cell viability.

[24] The therapeutic agent described in

[23] , wherein the homologous recombination-restoring cancer is a PARP inhibitor-resistant cancer.

[25] Introducing the nucleic acid construct according to any one of [1] to

[13] , wherein the gene sequence is a gene sequence encoding a protein whose expression in cells can be detected, into test cells in which homologous recombination activity is to be measured and HR-non-deficient control cells having normal homologous recombination activity; measuring the expression level of the protein in the test cells and in non-HR deficient control cells; and Comparing the expression levels in the test cells with those in HR-non-deficient control cells 10. A method for measuring homologous recombination activity in a test cell, comprising:

[26] Introducing the nucleic acid construct according to any one of [1] to

[13] into a cell having normal homologous recombination activity, and then treating the cell with an individual compound from the group of compounds, or treating a cell having normal homologous recombination activity with an individual compound from the group of compounds, and then introducing the nucleic acid construct according to any one of [1] to

[13] into the cell; and measuring the expression of said protein A method for identifying candidate drugs that affect homologous recombination activity, comprising:

[27] The method described in

[26] , wherein the protein is a protein whose expression in cells can be detected as a signal, and when a lower protein signal is detected in cells treated with the compound than in cells not treated with the compound, the method comprises selecting the compound as a candidate inhibitor that inhibits homologous recombination activity, or when a faster rise in the protein signal or a higher signal is detected in cells treated with the compound than in cells not treated with the compound, the method comprises selecting the compound as a candidate promoter that promotes homologous recombination activity.

[28] The method described in

[26] , wherein the protein is a protein having the effect of reducing cell viability, and when the decrease in cell viability is suppressed in cells treated with the compound compared to cells not treated with the compound, the method comprises selecting the compound as a candidate inhibitor that inhibits homologous recombination activity, or when the decrease in viability of cells treated with the compound occurs more quickly than the decrease in viability of cells not treated with the compound, the method comprises selecting the compound as a candidate promoter that promotes homologous recombination activity.

[29] Introducing the nucleic acid construct according to any one of [1] to

[13] into cancer cells of a cancer patient; and measuring the expression of said protein A method for diagnosing homologous recombination deficient cancer, comprising:

[30] The method described in

[29] , wherein the cancer cells are cells isolated from the cancer patient, and the introduction of the nucleic acid construct into the cancer cells is carried out ex vivo.

[31] The method described in

[29] , wherein the protein is a protein whose expression within cells can be detected as a signal, the introduction of the nucleic acid construct into the cancer cells is carried out by administering the nucleic acid construct to the cancer patient, and whether a signal of the protein can be detected from the cancer lesion is examined.

[32] The method described in

[29] , wherein the protein is a secreted protein whose expression in cells can be detected, the introduction of the nucleic acid construct into the cancer cells is carried out by administering the nucleic acid construct to the cancer patient, and the activity of the protein in blood isolated from the patient after administration of the nucleic acid construct is measured.

[33] Introducing the nucleic acid construct according to any one of [1] to

[13] into cancer cells of a cancer patient who is currently undergoing PARP inhibitor treatment or who has previously been diagnosed with homologous recombination deficient cancer; and measuring the expression of said protein A method for detecting homologous recombination-recovered cancer cells, comprising:

[34] The method described in

[33] , wherein the cancer cells are cells isolated from the patient, and the introduction of the nucleic acid construct into the cancer cells is carried out ex vivo.

[35] The method described in

[33] , wherein the protein is a protein whose expression within cells can be detected as a signal, the introduction of the nucleic acid construct into the cancer cells is carried out by administering the nucleic acid construct to the patient, and whether a signal from the protein can be detected from the cancer lesion is examined.

[36] Introducing the nucleic acid construct according to any one of [1] to

[13] into cancer cells of a cancer patient; and measuring the expression of said protein A method for predicting the efficacy of an anticancer drug against a homologous recombination-deficient cancer, comprising:

[37] The method described in

[36] , wherein the anticancer drug is a DNA-damaging anticancer drug.

[38] The method described in

[37] , wherein the DNA damaging anticancer drug is a PARP inhibitor.

[39] The method according to any one of

[36] to

[38] , wherein the cancer cells are cells isolated from the patient, and the introduction of the nucleic acid construct into the cancer cells is carried out ex vivo.

[40] A method according to any one of

[36] to

[38] , wherein the protein is a protein whose expression within a cell can be detected as a signal, the introduction of a nucleic acid construct into the cancer cells is carried out by administering the nucleic acid construct to the patient, and whether a signal of the protein is detected from the cancer lesion is examined.

[41] The method according to any one of

[36] to

[38] , wherein the protein is a secreted protein whose expression in cells can be detected, the introduction of the nucleic acid construct into the cancer cells is carried out by administering the nucleic acid construct to the cancer patient, and the activity of the protein in blood isolated from the patient after administration of the nucleic acid construct is measured.

[42] A method for treating homologous recombination-restoring cancer, comprising administering to a patient having homologous recombination-restoring cancer a nucleic acid construct according to any one of [1] to

[13] , wherein the gene sequence is a gene sequence encoding a protein that has the effect of reducing cell viability.

[43] The method described in

[42] , wherein the nucleic acid construct is administered locally to the patient's tumor or in the vicinity of the tumor.

[44] The method described in

[42] or

[43] , wherein the homologous recombination restored cancer is a PARP inhibitor resistant cancer.

[45] A method for predicting whether a genetic mutation identified in a cancer patient is a pathogenic mutation that impairs homologous recombination activity, comprising: constructing an expression vector that expresses a mutant gene having the same mutation as the mutation; preparing an expression vector that expresses a wild-type gene that does not have the mutation; introducing a mutant gene expression vector and a wild-type gene expression vector into the gene-deficient cell, and also introducing the nucleic acid construct according to any one of claims 1 to 11; measuring the expression of the protein in cells into which any of the expression vectors and nucleic acid constructs has been introduced; wherein, when the expression level of the protein in a cell into which a mutant gene expression vector has been introduced is lower than the expression level of the protein in a cell into which a wild-type gene expression vector has been introduced, it is indicated that the mutation is a pathogenic mutation that impairs homologous recombination activity. [Effects of the Invention]

[0012] The present invention provides a novel method for evaluating the homologous recombination activity of cells in absolute terms, rather than in relative terms. The nucleic acid construct of the present invention allows the presence or absence of homologous recombination activity to be detected in a very short time using a transient expression system. The nucleic acid construct of the present invention is useful as a reagent or kit for measuring homologous recombination activity, as well as a screening system for drugs that affect homologous recombination activity, a diagnostic agent for homologous recombination-deficient cancers, a detector for homologous recombination-recovered cancer cells, and a companion diagnostic agent for predicting the effectiveness of anticancer drugs against homologous recombination-deficient cancers. Furthermore, by using a gene sequence encoding a protein that reduces cell viability, such as a suicide gene, a nucleic acid construct that selectively exerts a cytocidal effect on cells with homologous recombination activity can be obtained. Anticancer drug-resistant cancers with restored homologous recombination activity, which are a major problem in PARP inhibitor therapy, currently have no treatment available. However, the nucleic acid construct of the present invention makes it possible to treat such resistant cancers. Furthermore, the construct of the present invention can be used to determine or predict whether a mutation identified in a cancer patient is a pathogenic mutation. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram illustrating the nucleic acid construct (version 1) of the present invention prepared in the Examples. [Figure 2] FIG. 1 is a structural diagram of pIRES used in the preparation of a plasmid vector-type nucleic acid construct in the Examples (the structural diagram of Clontech's pIRES Vector Information has been partially modified). [Figure 3] This shows an example of the results of a luciferase assay using a nucleic acid construct (DR-Nluc construct, version 1) prepared using a luciferase gene. [Figure 4] This is an example of the results of examining the cell-killing effect of a nucleic acid construct (DR-DTA construct, version 1) prepared using the suicide gene DT-A, using a colony formation assay. [Figure 5] This is an example of the results of examining the cell-killing effect of the version 1 DR-DTA construct by a growth inhibition test. [Figure 6] This shows an example of the results of luciferase assays in which the version 1 DR-Nluc construct was introduced into HT1080 cells, MDA-MB-436 cells, and HCC1937 cells. [Figure 7] FIG. 1 illustrates the differences between versions 1 and 2 of the DR-Nluc construct. [Figure 8] This is an example of the results of a luciferase assay using the DR-Nluc-v2 construct. [Figure 9] This shows an example of the results of examining the cell-killing effect of the DR-DTA-v2 construct by colony formation assay. [Figure 10] FIG. 1 is a diagram illustrating the structure of the DR-TK construct prepared using the suicide gene HSV-TK. [Figure 11] This shows an example of the results of examining the cell-killing effect of a DR-TK construct by a growth inhibition test. [Figure 12] This shows an example of the results of a luciferase assay using the DR-SecNluc construct. DETAILED DESCRIPTION OF THE INVENTION

[0014] A first aspect of the nucleic acid construct of the present invention includes the following (1) to (3). (1) Promoter region. (2) A mutant gene sequence that has a cleavage site within the gene sequence that codes for a protein. (3) A partial region in the mutant gene sequence of (2), which is a base sequence consisting of a first homologous region and a second homologous region that can replace the partial region containing the cleavage site by homologous recombination, and which is any of the base sequences (i) to (iii) below. (i) A continuous subsequence in the gene sequence encoding the protein, which subsequence includes the upstream and downstream regions adjacent to the cleavage site in the mutant gene sequence of (2). (ii) A sequence that is homologous to the partial sequence of (i) and encodes the same amino acid sequence as the partial sequence. (iii) A contiguous subsequence in a gene sequence encoding a protein having 80% or more sequence identity with the protein and the same activity as the protein, said subsequence being homologous to the subsequence of (i).

[0015] The promoter (1) is not particularly limited and may be any promoter that can exert promoter activity in cells (typically mammalian cells such as human cells). Generally, promoters that constitutively exert strong promoter activity in cells into which the nucleic acid construct of the present invention is introduced are preferably used, but inducible promoters that exert promoter activity under certain conditions may also be used. In the examples below, the human cytomegalovirus promoter, which constitutively exerts strong promoter activity in cells, is used, but the present invention is not limited to this. Promoters are not limited to virus-derived promoters, and any sequence that exhibits promoter activity in cells, preferably human cells, may be used, regardless of origin.

[0016] The gene sequence employed in the nucleic acid construct of the present invention is not particularly limited as long as it is a gene sequence that encodes a protein. Hereinafter, the protein encoded by this gene sequence may be referred to as protein A. In a first embodiment of the nucleic acid construct of the present invention, in cells with HR activity, a partial region of the mutant gene sequence (2) containing the cleavage site (if the mutant sequence (2) contains a stop codon upstream of the cleavage site, the stop codon and the cleavage site) is replaced by the sequence (3) through HR, thereby recreating the gene sequence encoding protein A and producing protein A. On the other hand, in cells without HR activity, the cleavage site of the mutant gene sequence (2) (if the mutant sequence (2) contains a stop codon, the stop codon and the cleavage site) is not replaced by the sequence (3), and protein A is not expressed. Therefore, whether cells into which a nucleic acid construct has been introduced have HR activity can be detected based on the expression level of protein A, preferably the expression level of protein A activity, or the activity of protein A can be utilized to selectively kill cancer cells with restored HR activity. The gene sequence encoding protein A restored by homologous recombination may be a sequence containing the full-length coding region of a naturally occurring gene, or it may be a sequence consisting of a portion of the coding region or a modified sequence. For example, of a naturally occurring gene sequence, only the region encoding the domain necessary for protein activity (e.g., a sequence encoding a protein fragment from which domains not necessary for protein activity itself, such as a domain necessary for membrane localization, have been removed) may be used as the gene sequence in the present invention. Furthermore, for fluorescent proteins and luminescent proteins, genes with various modifications are widely used commercially, and gene sequences encoding such non-natural proteins may also be used.

[0017] Protein A is preferably a protein whose expression level can be measured quickly and easily. For example, rather than measuring the amount of protein produced or accumulated, it is preferable to use as protein A a protein whose activity can be measured directly or indirectly using as an indicator, for example, a phenotypic change caused in cells by the activity of the protein. Examples of such proteins include proteins whose expression in cells can be detected by signals such as luminescence from a reaction with a substrate or fluorescence from the protein itself. Another example is a protein that has the effect of reducing cell viability, in which case protein activity can be measured indirectly as a reduction in cell viability.

[0018] The term "measuring the expression (expression level) of protein A" includes measuring the production or accumulation level of protein A and measuring the activity of protein A. As described above, in the present invention, it is preferable to measure the expression of protein A by directly or indirectly measuring the activity of protein A, i.e., to employ as protein A a protein whose activity can be measured directly or indirectly.

[0019] Preferred examples of protein A include proteins that have the effect of reducing cell viability and proteins whose expression in cells can be detected.

[0020] Specific examples of genes encoding proteins that have the effect of reducing cell viability include, but are not limited to, suicide genes, DNA damage-inducing genes, and DNA repair inhibitor genes.

[0021] Suicide genes include genes encoding proteins that are themselves toxic or damaging to cells, genes encoding proteins that react with other compounds to produce toxic substances, and genes encoding natural or non-natural proteins that exert cytotoxicity by inhibiting endogenous proteins through a dominant-negative effect. Specific examples of suicide genes include the diphtheria toxin A fragment gene (DT-A) (the DT-A protein itself is toxic to cells and kills them), the herpes virus-derived thymidine kinase gene (HSV-tk) (the HSV-tk protein reacts with ganciclovir or 5-iodo-2'-fluoro-2'deoxy-1-beta-D-arabino-furanosyl-uracil (FIAU) to convert it into a toxic substance that inhibits DNA synthesis; when this gene is used as a suicide gene, cells must be treated with ganciclovir or FIAU), and the p53 gene (overexpression of which induces cell growth cycle arrest or apoptosis, resulting in cell death). Further specific examples of suicide genes include natural nucleases such as restriction enzymes and meganucleases, and artificial nucleases such as ZFN, TALEN, and CRISPR systems (these are also specific examples of DNA damage-inducible genes). However, suicide genes are not limited to these specific examples.

[0022] Examples of genes encoding proteins whose intracellular expression can be detected include, but are not limited to, luminescent enzyme genes such as luciferase genes (including genes encoding secreted luciferase), fluorescent protein genes (genes encoding natural or artificial fluorescent proteins, including GFP, CFP, OFP, RFP, YFP, and modified versions thereof), cell surface antigen genes, secreted protein genes, membrane protein genes, etc. Luminescent enzyme genes and fluorescent protein genes are genes that encode proteins whose intracellular expression can be detected as a signal such as luminescence or fluorescence, and are examples of genes that can be preferably used in the present invention.

[0023] The sequence (2) is a mutant gene sequence having a structure in which a mutation (i.e., a cleavage site) has been introduced into the gene sequence encoding protein A. When the gene sequence encoding protein A employed in the sequence (2) is derived from a eukaryote, the base sequence of mRNA from which introns have been removed may be used. As long as it encodes protein A, it may be a naturally occurring gene sequence, or a sequence containing one or more bases different from the natural gene sequence (typically a silent mutation) at a site other than the mutation introduction site.

[0024] A stop codon may be included upstream of the cleavage site. In this case, the stop codon and cleavage site are introduced into the gene sequence encoding protein A so that they are arranged in the order of [stop codon]-[cleavage site] from the 5' side. It is not essential to include a stop codon upstream of the cleavage site; however, when the nucleic acid construct of the first embodiment is prepared as a circular construct and used in its circular form (introduced into cells or administered to patients) without being cleaved at the cleavage site to linearize it, it is desirable to include a stop codon upstream of the cleavage site to reliably stop the expression of active protein A from the gene sequence of (2) in cells lacking homologous recombination activity. The stop codon and cleavage site may be directly linked, or some base sequence may exist between them. However, it is preferable that the chain length of [stop codon]-[cleavage site] be approximately 50 bases or less, for example, approximately 30 bases or less.

[0025] Typical examples of cleavage sites include, but are not limited to, restriction enzyme recognition sites. In addition to cleavage by restriction enzymes, cleavage can also be achieved using genome editing techniques that cause DNA strand cleavage. A specific example is cleavage by the CRISPR / Cas system, i.e., cleavage by a complex of guide RNA and a Cas protein such as Cas9. When using this complex, a PAM sequence is introduced at an appropriate position downstream of the stop codon so that cleavage by the complex occurs at an appropriate position. In the case of Cas9 derived from Streptococcus pyogenes type II, which is the most commonly used known CRISPR / Cas system, the PAM sequence is 5'-NGG (N is A, T, G, or C). The complex cleaves the nucleic acid construct several bases upstream of the PAM sequence. In the case of cleavage by the CRISPR / Cas system, the PAM sequence and the actual cleavage site several bases upstream thereof constitute the cleavage site in the present invention. In addition, since (3) of the nucleic acid construct of the first embodiment has the structure described above and does not contain a cleavage site, it goes without saying that (3) will not be cleaved by a process that cleaves the nucleic acid construct at the cleavage site.

[0026] In the present invention, the unit of nucleic acid chain length is expressed as "base," but when the nucleic acid is a double-stranded nucleic acid, it means "base pair." Furthermore, the [cleavage site] or [stop codon]-[cleavage site] may be inserted into the gene sequence encoding protein A, or may be introduced by replacing a part of the gene sequence encoding protein A with the [cleavage site] or [stop codon]-[cleavage site]. The pCMV-DR-DTA constructs (versions 1 and 2), pCMV-DR-Nluc constructs (versions 1 and 2), and pCMV-DR-TK construct constructed in the Examples below are examples of the latter.

[0027] Hereinafter, in the mutant gene sequence (2), the upstream side of the [cleavage site] (when a stop codon is also included, [stop codon]-[cleavage site]) is referred to as the 5' region, and the downstream side is referred to as the 3' region. When used in a linearized state after cleavage at the cleavage site, a protein A fragment lacking the region downstream of the cleavage site is generated in cells in which HR does not occur. Furthermore, when the circular construct is used as is, translation stops in cells in which HR does not occur due to the introduced stop codon, and a protein A fragment consisting of the 5' region is generated. Therefore, the position of the [cleavage site] or [stop codon]-[cleavage site] in the mutant gene sequence is determined taking into consideration the size of the 5' region so that the 5' region fragment does not exhibit the activity of protein A. The size of the 5' region may be appropriately set depending on the type of protein A and is not particularly limited. However, the size of the 5' region can generally be less than half the total length of protein A, for example, less than 40 / 100, less than 30 / 100, or less than 25 / 100. There is no particular lower limit to the size of the 5'-side region, but it is usually set to a size of 50 bases or more.

[0028] The sequence (3) is a partial region in the mutant gene sequence (2), and is a base sequence consisting of a first homologous region and a second homologous region that can replace the partial region containing the cleavage site by homologous recombination, and is any of the following (i) to (iii): (i) A continuous subsequence in the gene sequence encoding protein A, which subsequence includes the upstream and downstream regions adjacent to the cleavage site in the mutant gene sequence of (2). (ii) A nucleotide sequence that is homologous to the partial sequence of (i) and encodes the same amino acid sequence as the partial sequence of (i). (iii) A contiguous partial sequence in a gene sequence that encodes a protein that has 80% or more sequence identity with Protein A and the same activity as Protein A, and that has homology with the partial sequence of (i).

[0029] The entire 5'-side region in the mutant gene sequence of (2), or a portion of its 3'-end (i.e., at least a portion of the 3'-end of the 5'-side region), corresponds to the upstream region adjacent to the cleavage site (or the stop codon and cleavage site) in the mutant gene sequence of (2) in the sequence of (3)(i) (hereinafter, for convenience, referred to as the "adjacent upstream region"). In the sequence of (i), this adjacent upstream region constitutes the first homologous region. At least a portion of the 3'-end of the 5'-side region in the mutant gene sequence of (2) and the adjacent upstream region of (3)(i) are referred to for convenience as the "first homologous region set." Furthermore, the entire 3'-side region or a portion of its 5'-end (i.e., at least a portion of the 5'-end of the 3'-side region) corresponds to the downstream region adjacent to the cleavage site (or the stop codon and cleavage site) in the mutant gene sequence of (2) in the sequence of (3)(i) (the "adjacent downstream region"). In the sequence of (i), this adjacent downstream region constitutes the second homologous region. At least a portion of the 5'-end of the 3'-side region in the mutant gene sequence of (2) and the adjacent downstream region of (3)(i) are conveniently referred to as the "second homologous region set." The length of each homologous region should be at least 20 bases, and may be, for example, 50 bases or more, 60 bases or more, 70 bases or more, or 80 bases or more. The upper limit of the length is not particularly limited, but is usually 10,000 bases or less, and may be, for example, 5,000 bases or less, 1,000 bases or less, 500 bases or less, or 300 bases or less.

[0030] The above (3) (i) is an embodiment in which the homology between the first set of homologous regions and the homology between the second set of homologous regions is 100%, and the corresponding sequences are completely identical.

[0031] The amino acid sequence encoded by (ii) of (3) above is identical to (a partial sequence of) the amino acid sequence of protein A, but at least one of the homology between the first set of homologous regions and the homology between the second set of homologous regions is less than 100%. It is widely known in the art that homologous recombination occurs when the corresponding sequences do not completely match but have a certain level of homology. The sequence of (ii) preferably has a homology of 90% or more with the partial sequence of (i), more preferably 95% or more, or 98% or more. For example, the homology between the first set of homologous regions is preferably 90% or more, 95% or more, or 98% or more, and the homology between the second set of homologous regions is preferably 90% or more, 95% or more, or 98% or more. In the sequence of (ii), the regions corresponding to the adjacent upstream region and the adjacent downstream region in (i), respectively, naturally constitute the first and second homologous regions.

[0032] (iii) of (3) above is a contiguous partial sequence in a gene sequence encoding a protein having 80% or more, for example 85% or more, 90% or more, 95% or more, or 98% or more, sequence identity with protein A and the same activity as protein A, and is a nucleotide sequence that is homologous to the partial sequence of (i). This is an embodiment in which at least one of the homology between the first set of homologous regions and the homology between the second set of homologous regions is less than 100%, and the encoded amino acid sequence does not match the amino acid sequence of protein A. Even if the encoded amino acid sequence is partially different from the amino acid sequence of protein A, it can be adopted as the sequence of (3) as long as it has the activity of protein A. As for (iii), a sequence that has 90% or more, for example 95% or more, or 98% or more homology with the partial sequence of (i), and the encoded amino acid sequence has 80% or more, for example 85% or more, 90% or more, 95% or more, or 98% or more sequence identity with the amino acid sequence encoded by the partial sequence of (i).

[0033] Conservative substitutions, i.e., substitutions with amino acids with similar chemical properties, are likely to preserve the properties and activity of proteins. Amino acids with similar side chains have similar chemical properties. Amino acids can be grouped based on side chain similarity, for example, into groups with aliphatic side chains (glycine, alanine, valine, leucine, isoleucine), aliphatic hydroxyl side chains (serine, threonine), amide-containing side chains (asparagine, glutamine), aromatic side chains (phenylalanine, tyrosine, tryptophan), basic side chains (arginine, lysine, histidine), acidic side chains (aspartic acid, glutamic acid), and sulfur-containing side chains (cysteine, methionine). Substitutions with other amino acids from the same group are conservative substitutions. A typical example of the amino acid sequence encoded by the base sequence of (iii) is an amino acid sequence in which conservative substitutions have been introduced into the amino acid sequence encoded by the partial sequence of (i), but is not limited to this.

[0034] The sequences (2) and (3) used in the nucleic acid construct of the first embodiment can be prepared by PCR amplification using appropriate primers from a known plasmid incorporating a gene encoding protein A, or from cultured cells or a cDNA library of an organism expressing protein A. A stop codon is required at the 3' end of the mutant gene sequence (2). The sequence (3) serves as a substrate for replacing a portion of the mutant gene sequence (2) by homologous recombination. Because this region is not translated before homologous recombination, it is not necessary to add a stop codon to the 3' end of the sequence (3). The examples below describe examples in which stop codons were introduced at the 3' ends of the iNluc and iDTA fragments corresponding to the sequence (3). While introduction of a stop codon, as in these examples, is permissible, it is essentially unnecessary. During PCR amplification, if desired, appropriate adapter sequences, etc., used in cloning the nucleic acid construct of the present invention, may be introduced into the two gene fragments in addition to a stop codon. The sequence (2) containing one or more bases different from the natural gene sequence at a site other than the mutation introduction site, and the sequences (ii) and (iii) of (3) can be prepared by methods such as in vitro mutagenesis or artificial gene synthesis. The poly(A) addition signal may be located downstream of the mutant gene sequence (2) (or between the mutant gene sequence (2) and the sequence (3) when the sequence (3) is located downstream of the mutant gene sequence (2)). However, the poly(A) addition signal is not an essential element in the nucleic acid construct of the present invention. mRNAs that function properly without poly(A), such as histone mRNAs, are known, and it is possible to omit the poly(A) addition signal from the nucleic acid construct of the present invention.

[0035] The nucleic acid construct of the first embodiment may be a plasmid vector into which (1) to (3) have been incorporated, a viral vector into which (1) to (3) have been incorporated, or may be in the form of a nucleic acid fragment not incorporated into a vector. As described above, the poly(A) addition signal may be located downstream of (2) (between (2) and (3) when (3) is located downstream of (2)). In either the case of a plasmid vector or a viral vector, the elements are arranged in the vector from the 5' end to the downstream end in the order [promoter region]-[mutant gene sequence of (2)]-[poly(A) addition signal]-[sequence of (3)], or [sequence of (3)]-[promoter region]-[mutant gene sequence of (2)]-[poly(A) addition signal]. In the case of a nucleic acid fragment not incorporated into a vector, the arrangement from the 5' end is the same as described above. When used as pharmaceuticals such as therapeutic agents and diagnostic agents, or as reagents such as assay reagents, the nucleic acid construct of the first embodiment using a plasmid vector may be used as is, in the form of a circular nucleic acid construct containing (2) and (3) in this order downstream of the promoter region, or a circular nucleic acid construct containing (3) upstream of the promoter region and (2) downstream, or may be used in a linear form by cleaving this circular nucleic acid construct at a cleavage site within the mutant gene sequence of (2). Nucleic acid constructs using viral vectors may be linear nucleic acid constructs containing (2) and (3) in this order downstream of the promoter region, or containing (3) upstream of the promoter region and (2) downstream. This linear nucleic acid construct may also be used without cleavage at the cleavage site, or may be cleaved before use. Nucleic acid constructs in the form of nucleic acid fragments not incorporated into vectors are typically linear and contain (2) and (3) in this order downstream of the promoter region, or contain (3) upstream of the promoter region and (2) downstream. Furthermore, the nucleic acid construct of the present invention incorporated into a vector may contain, in addition to (1) to (3), general elements depending on the type of vector, such as a replication origin, a translation origin, and a selectable marker gene for drug resistance or the like.

[0036] A second aspect of the nucleic acid construct of the present invention is a linear nucleic acid construct comprising the following (a) to (d), in the order (c), (d), (a), and (b) from upstream to downstream: (a) Promoter region. (b) The upstream region of a protein-coding gene sequence. (c) a downstream region of said gene sequence; (d) A base sequence consisting of a first homologous region and a second homologous region that can replace the upstream region and the downstream region by homologous recombination, which is any of the base sequences (d-1) to (d-3) below. (d-1) A continuous partial sequence in a gene sequence encoding the protein, the partial sequence including a region spanning the upstream region and the downstream region. (d-2) A base sequence that is homologous to a partial sequence of (d-1) and encodes the same amino acid sequence as the partial sequence. (d-3) A contiguous partial sequence in a gene sequence that encodes a protein having 80% or more sequence identity with the protein and the same activity as the protein, said partial sequence having homology with the partial sequence of (d-1).

[0037] (b) and (c) are elements corresponding to element (2) in the first embodiment, and are respectively composed of an upstream region and a downstream region of a gene sequence encoding the same protein (protein A). They may be composed of two fragments obtained by dividing the entire gene sequence, or may be composed of an upstream partial region including the 5' end and a downstream partial region including the 3' end, excluding the intermediate partial region of the gene sequence. In the latter case, even if the base sequences of (b) and (c) are joined, they will not match the entire gene sequence encoding protein A, and will result in a shorter base sequence lacking the intermediate partial region.

[0038] (d) is an element corresponding to (3) in the first aspect, and (d-1) to (d-3) correspond to (i) to (iii) of (3), respectively. When (b) and (c) are composed of fragments obtained by dividing the entire length of a gene sequence into two, (d-1) is a partial sequence of the gene sequence including the division point, with the region upstream of the division point constituting the first homologous region and the region downstream of the division point constituting the second homologous region. When (b) and (c) are composed of an upstream partial region including the 5' end and a downstream partial region including the 3' end, excluding a partial region in the middle of the gene sequence, (d-1) is a partial sequence of the gene sequence including the intermediate partial region, with the upstream region adjacent to the intermediate partial region constituting the first homologous region and the downstream region adjacent to the intermediate partial region constituting the second homologous region.

[0039] The nucleic acid construct of the second embodiment may or may not contain a poly(A) addition signal. If the poly(A) addition signal is (e), then when (e) is contained, it is sufficient that the elements are contained in the following order from upstream to downstream: (c), (e), (d), (a), and (b).

[0040] The above are the differences from the first embodiment, and other conditions (such as the gene sequence encoding protein A, protein A, the promoter region of (a), preferred examples of (d-2) and (d-3), and the size of the homologous region) are the same as those of the nucleic acid construct of the first embodiment.

[0041] In the nucleic acid construct of the second embodiment, in cells that have HR activity, at least a portion of the 3'-terminus of (b) and at least a portion of the 5'-terminus of (c) are replaced by (d) by HR, and the gene sequence encoding protein A is reproduced, thereby producing protein A. On the other hand, in cells that do not have HR activity, the above-mentioned replacement by (d) does not occur, and therefore protein A is not expressed. Therefore, the nucleic acid construct of the second embodiment can be used in the same way as in the first embodiment.

[0042] Hereinafter, when the term "nucleic acid construct of the present invention" is used simply, it encompasses both the first and second embodiments unless otherwise specified.

[0043] The nucleic acid construct of the present invention may be DNA or RNA. Generally, DNA is preferred for plasmid vector-type nucleic acid constructs. A typical example of a viral vector-type nucleic acid construct is a nucleic acid construct that is double-stranded DNA in the form of a viral expression plasmid, but viruses expressed from viral expression plasmids are also included in viral vector-type nucleic acid constructs. A nucleic acid construct that is a virus expressed from a viral expression plasmid is RNA if it is an RNA viral vector, and DNA if it is a DNA viral vector. Nucleic acid constructs not incorporated into a vector are usually DNA. Furthermore, the nucleic acid construct of the present invention may partially contain a nucleotide analog. Examples of nucleotide analogs include, but are not limited to, crosslinked nucleic acids such as LNA, ENA, and PNA, and modified bases such as Super T (registered trademark) and Super G (registered trademark).

[0044] The nucleic acid construct of the present invention can be used, for example, as a reagent for measuring homologous recombination activity. In this embodiment, a gene sequence encoding a protein whose expression in cells can be detected can be preferably used as the gene sequence encoding protein A. The measurement reagent may be a liquid reagent containing appropriate additives for stability of the nucleic acid construct, or a powdered reagent obtained by lyophilizing the nucleic acid construct. The nucleic acid construct of the present invention is introduced into test cells in which homologous recombination activity is to be measured and into cells with normal homologous recombination activity or cells not deficient in homologous recombination activity (HR-non-deficient control cells), and the expression of protein A is measured. The expression levels of protein A (protein A signals) between the test cells and the HR-non-deficient control cells can be compared. The nucleic acid construct may be introduced transiently. Furthermore, cells deficient in homologous recombination activity may be used as HR-deficient control cells.

[0045] The HR-non-deficient control cells are not particularly limited as long as they have normal homologous recombination activity. HR-related genes (ATM, CDK1, CDK12, MRE11, NBS1, Rad50, PLK1, RNF1, RNF8, RNF168, ARID1A, SMARCAD1, CHD1, CHD4, LEDGF, RBBP8 (CtIP), BLM, Exo1, DNA2, BRCA1, BRCA2, PALB2, BARD1, BRIP1, BAP1, Rad51B, Rad51C, Rad51D, Rad51AP1, Rad52, XRCC Any of the many known wild-type mammalian cell lines that do not have mutations in any of the following genes (e.g., 2, XRCC3, Rad51, Rad54L (Rad54), Rad54B, FBH1, WRN, SMARCAL1, ATRX, PTEN, MUS81, EME1, EME2, SLX1, SLX4, XPF, ERCC1, RPA1, RPA2, PRA3, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, LIG1, LIG3, POLD, HROB, MCM8, MCM9, HELQ, RAD21, RAD21L1) can be preferably used as HR-non-deficient control cells. Specific examples of known mammalian cell lines having normal homologous recombination activity include, for example, human cell lines such as Nalm-6 (a human pre-B cell leukemia cell-derived cell line) and HT1080 (a human fibrosarcoma cell line), which are also used in the Examples below, U2OS (a human osteosarcoma-derived cell line), HeLa (a human cervical carcinoma-derived cell line), HCT116 (a human colon adenocarcinoma-derived cell line), MCF-7 (a human breast adenocarcinoma-derived cell line), HAP1 (a human chronic myeloid leukemia-derived cell line), HEK293 (a human embryonic kidney-derived cell line), TIG-7 (a human lung-derived cell line), TIG-3 (a human lung-derived cell line), iPS cells (human induced pluripotent stem cells: established from normal human cells), and ES cells (human embryonic stem cells), but are not limited to these. Cell lines derived from human hereditary breast cancer and hereditary ovarian cancer have mutations in the HR-related genes BRCA1 and BRCA2, and many of them are deficient in homologous recombination activity, making them unsuitable as HR-non-deficient control cells.Whether or not a given cell line has normal homologous recombination activity (whether or not it is not deficient in homologous recombination activity) can be determined using the nucleic acid construct of the present invention, or by using conventionally known techniques. When determining whether or not a cell line has homologous recombination activity at or above the level of the specific example of the non-HR-deficient control cells described above, the cell can be determined to have normal homologous recombination activity (not deficient in homologous recombination activity).

[0046] As an additional positive control, a construct (positive control construct) in which a normal gene sequence encoding protein A is linked under the control of a promoter may be introduced into the test cells (and, if desired, the control cells). The lower the homologous recombination activity of the test cells, the lower the protein A expression in the test cells (the protein A signal detected from the test cells). Therefore, a lower protein A signal from the test cells compared to the protein A signal from HR-non-deficient control cells indicates that the test cells have lower homologous recombination activity.

[0047] HR-deficient control cells can be, for example, cell lines derived from homologous recombination-deficient cancers. Cell lines derived from homologous recombination-deficient cancers with mutations in the HR-related genes described above (e.g., cell lines derived from human hereditary breast cancer and hereditary ovarian cancer with mutations in the BRCA1 or BRCA2 gene, as examples of human cell lines) and cell lines created by introducing mutations into one or more HR-related genes, or methods for creating such cell lines, are known. Any of these cell lines can be used as HR-deficient control cells. Since gene knockout technology has also been established, HR-deficient cells from various animal species can be prepared by knocking out one or more HR-related genes in cultured mammalian cells with normal homologous recombination activity.Specific examples of known homologous recombination-deficient cell lines include, for example, HR-deficient human cell lines derived from Nalm-6 cells (Rad54B-deficient line, Rad54 / Rad54B double-deficient line) used in the Examples below, as well as HR-deficient human cell lines available from ATCC (American Type Culture Collection), such as HCC1395 (human breast cancer-derived cell line, BRCA1-deficient), HCC1599 (human breast cancer-derived cell line, BRCA2-deficient), HCC1937 (human breast cancer-derived cell line, BRCA1-deficient), MDA-MB-436 (human breast cancer-derived cell line, BRCA1-deficient), DoTc2-4510 (human cervical cancer-derived cell line, BRCA2-deficient), RL95-2 (human uterine cancer-derived cell line, BRCA2-deficient), AGS (human gastric cancer-derived cell line, CHD1-deficient), SNU-5 (human gastric cancer-derived cell line, CHD1-deficient); and Horizon Examples of HR-deficient human cell lines available from Discovery include genetically modified HAP1 cell lines (human chronic myeloid leukemia-derived cell lines) (ATRX-deficient line, CHD1-deficient line, CHD4-deficient line, EME1-deficient line, EME2-deficient line, FANCA-deficient line, FANCC-deficient line, FANCE-deficient line, FANCG-deficient line, FANCF-deficient line, LEDGF-deficient line, LIG3-deficient line, MUS81-deficient line, RAD51AP1-deficient line, RAD54L-deficient line, and RNF8-deficient line), genetically modified HCT116 cells (human colon adenocarcinoma-derived cell line) (ARID1A-deficient line, LIG3-deficient line, and PTEN-deficient line), and genetically modified DLD-1 cell lines (human colon adenocarcinoma-derived cell line) (BRCA2-deficient line).Specific examples of non-human animal cell lines include HR-deficient mice (frozen embryos) available from the NCI Mouse Repository (BRCA1-deficient mice, BRCA2-deficient mice); and HR-deficient canine Parks cells (MCM8-deficient) (Sunetra Das et al., Mol Cancer Ther. Author manuscript; available in PMC 2020 Feb 1. Published in final edited form as: Mol Cancer Ther. 2019 Aug; 18(8): 1460-1471. Published online 2019 Jun 7. doi: 10.1158 / 1535-7163.MCT-18-1346). However, as described above, HR-deficient cells can be prepared in various animal species by generating cells in which one or more HR-related genes are knocked out, and therefore the cell lines are not limited to these specific examples.

[0048] Furthermore, the measurement reagent of the present invention can also be provided as a kit for measuring homologous recombination activity by combining it with at least one item selected from a positive control plasmid (for example, a plasmid in which a normal protein A coding sequence is placed downstream of a promoter and which can constitutively express protein A), a negative control plasmid (a plasmid in which a gene sequence encoding protein A is not incorporated), HR-non-deficient control cells, HR-deficient control cells, an instruction manual, and, if protein A is an enzyme, an appropriate substrate substance.

[0049] The nucleic acid construct of the present invention can also be used, for example, as a screening system for drugs that affect homologous recombination activity. That is, the nucleic acid construct of the present invention can be used to identify candidate drugs that affect homologous recombination activity. A method for identifying candidate drugs that affect homologous recombination activity using the nucleic acid construct of the present invention includes introducing the nucleic acid construct of the present invention into cells with normal homologous recombination activity and then treating the cells with individual compounds from a group of compounds, or treating cells with normal homologous recombination activity with individual compounds from a group of compounds and then introducing the nucleic acid construct of the present invention into the cells; and measuring the expression of protein A in compound-treated cells transfected with the nucleic acid construct and in cells transfected with the nucleic acid construct but not treated with the compound. As described above, the measurement of protein A expression is preferably the measurement of protein A activity. In this embodiment, the gene sequence encoding protein A is preferably a gene sequence encoding a protein whose expression in cells can be detected, particularly a protein whose expression in cells can be detected as a signal. However, a gene sequence encoding a protein that reduces cell viability can also be used. The nucleic acid construct of the present invention can be cleaved, if desired, at the cleavage site provided within the sequence of (2), and then introduced into cells with normal homologous recombination activity (HR-normal cells, typically mammalian cells such as cultured human cells, are preferably used; details are the same as for the HR-non-deficient control cells described above). The cells are then treated with individual compounds from a compound library or other such compound group (the order of introduction of the nucleic acid construct and compound treatment can be reversed), and the expression of protein A can be measured to determine whether the compound treatment has caused a decrease or increase in the expression level of protein A, preferably the activity level of protein A (whether a decrease or increase in homologous recombination activity has occurred). If protein A is luciferase, changes in luciferase activity (luminescence reaction) can be measured. If protein A is a protein that has the effect of reducing cell viability, changes in cell viability can be determined.Whether or not the expression of protein A has decreased or increased can be evaluated by introducing a nucleic acid construct into cells with normal homologous recombination activity, measuring the expression of protein A without compound treatment, and comparing this measurement with the measured value to determine whether the expression level has decreased or increased. In addition to HR-normal cells, HR-deficient cells that are deficient in homologous recombination activity (e.g., the RAD54 / RAD54B double-deficient strain used in the Examples below; details are the same as for the HR-deficient control cells above) may also be used.

[0050] When protein A is a protein whose intracellular expression can be used as a signal to detect it, the compound can be selected as follows. If a lower signal for protein A is detected in compound-treated HR normal cells than in compound-untreated HR normal cells, the compound can be selected as a candidate HR inhibitor that inhibits HR activity. If the protein A signal rises earlier or a higher signal is detected in compound-treated HR normal cells compared to compound-untreated HR normal cells, the compound can be selected as a candidate HR promoter that promotes HR activity.

[0051] When protein A is a protein that has the effect of reducing cell viability, the compound can be selected as follows. If the decrease in cell viability is suppressed in compound-treated HR normal cells compared to compound-untreated HR normal cells (i.e., if the viability of compound-treated HR normal cells is higher than the viability of compound-untreated HR viable cells), the compound can be selected as a candidate HR inhibitor that inhibits HR activity. If the decrease in viability of compound-treated HR normal cells occurs more quickly than the decrease in viability of compound-untreated HR cells, or if the viability of compound-treated HR normal cells is lower than the viability of compound-untreated HR cells, the compound can be selected as a candidate HR promoter that promotes HR activity.

[0052] The nucleic acid construct of the present invention can also be used, for example, as a diagnostic agent for homologous recombination-deficient cancers. Homologous recombination deficiency has been reported in various cancers. Specifically, homologous recombination deficiency has been reported in breast cancer and ovarian cancer (including hereditary breast and ovarian cancer syndrome), as well as colorectal cancer, endometrial cancer, gastric cancer, esophageal cancer, pancreatic cancer, hepatobiliary cancer, prostate cancer, liver cancer, non-small cell lung cancer, and small cell lung cancer (Non-Patent Documents 6 to 9). It is expected that homologous recombination-deficient cancers will be discovered in various other cancers (including cancers of various animal species, including non-human animals) in the future. It has also been reported that molecular-targeted cancer therapies such as EGFR inhibitors and BRAF inhibitors reduce homologous recombination activity (Science 20 Dec 2019: Vol. 366, Issue 6472, pp. 1473-1480, DOI: 10.1126 / science.aav4474). Homologous recombination-deficient cancers in the present invention include various cancers, including the specific examples mentioned above.

[0053] When used as a diagnostic agent for homologous recombination-deficient cancer, the nucleic acid construct of the present invention is introduced into cancer cells of a cancer patient, and the expression of protein A is measured. As described above, the measurement of protein A expression is preferably a measurement of protein A activity. A gene sequence encoding protein A is preferably a gene sequence encoding a protein whose expression in cells can be detected, particularly a protein whose expression in cells can be detected as a signal. However, a gene sequence encoding a protein that reduces cell viability can also be used. When homologous recombination is not deficient in cells, homologous recombination occurs between a portion of the sequence (2) and the sequence (3), and a gene sequence capable of expressing protein A is reproduced in the cell, resulting in the expression of protein A. When protein A is a protein whose expression in cells can be detected as a signal, a protein A signal is detected, and when it is a protein that reduces cell viability, cell viability decreases. On the other hand, when homologous recombination is deficient in cells, a gene sequence capable of expressing protein A is not reproduced, and protein A is not expressed. When protein A is a protein whose expression within cells can be detected as a signal, the signal of protein A is not detected, and when protein A is a protein that has the effect of reducing cell viability, the reduction in cell viability does not occur.

[0054] In one embodiment of the diagnostic agent for homologous recombination-deficient cancer, the cancer cells are cells isolated from a cancer patient, and a nucleic acid construct is introduced into the cancer cells ex vivo. Among the usage modes described below, (1) and (3) are specific examples of this mode. In this mode, either a protein whose expression in cells can be detected or a protein that reduces cell viability can be used as protein A, with the former being more preferred. In this mode, the cancer patient includes patients with solid cancers and patients with blood cancers. Cancer tissue cells isolated from cancer patients by biopsy or surgery, or, in the case of leukemia, cancer cells in the blood collected from cancer patients, can be used. If desired, the cancer cells may be concentrated before introduction of the nucleic acid construct. The introduction of the nucleic acid construct may be transient. Whether or not cancer cells are deficient in homologous recombination activity can be determined by measuring the homologous recombination activity of the cancer cells in the same manner as in the method for using the reagent for measuring homologous recombination activity described above. In addition to the above-mentioned cells with normal homologous recombination activity, non-HR-deficient control cells can be used, such as non-cancer cells (non-cancer peripheral blood cells, etc.) collected from the same cancer patient. Furthermore, the above-mentioned homologous recombination-deficient cells can be used as HR-deficient control cells.

[0055] In this embodiment, it is preferable to introduce the nucleic acid construct into cancer cells isolated from a cancer patient and control cells (HR non-deficient control cells or HR-deficient control cells, or both), and compare the expression of protein A between the cancer cells and the control cells.

[0056] When protein A expression / activity is detected in cancer cells, for example, when the expression / activity level of protein A detected in cancer cells is higher than that of HR-deficient control cells, it is indicated that the cancer in the cancer patient is not a homologous recombination-deficient cancer. When protein A is a protein whose intracellular expression can be detected as a signal, when the protein A signal is detected in cancer cells, for example, when the protein A signal detected in cancer cells is higher than that of HR-deficient control cells, it is indicated that the cancer in the cancer patient is not a homologous recombination-deficient cancer. When protein A is a protein that has the effect of reducing cell viability, when the viability of cancer cells is reduced, for example, when the viability of cancer cells is lower than that of HR-deficient control cells, it is indicated that the cancer in the cancer patient is not a homologous recombination-deficient cancer. If no expression / activity of protein A is detected in cancer cells, or if protein A expression / activity is detected at a lower level than in HR-non-deficient control cells, this indicates that the cancer in the cancer patient is a homologous recombination-deficient cancer. If protein A is a protein whose expression in cells can be detected as a signal, if no protein A signal is detected in cancer cells, or if a signal lower than the protein A signal in the HR-non-deficient control is detected, this indicates that the cancer in the cancer patient is a homologous recombination-deficient cancer. If protein A is a protein that reduces cell viability, if the viability of cancer cells is not reduced, or is higher than the viability of HR-non-deficient control cells, this indicates that the cancer in the cancer patient is a homologous recombination-deficient cancer.

[0057] In another embodiment, protein A is a protein whose intracellular expression can be detected as a signal, and the introduction of the nucleic acid construct into cancer cells is carried out by administering the nucleic acid construct to a cancer patient, and whether a protein A signal is detected from the cancer lesion is examined. Among the usage modes described below, (2) is a specific example of this mode. In this mode, the cancer patient typically has a solid cancer. The nucleic acid construct is preferably administered locally into or near the tumor of the cancer patient. From the perspective of detecting a protein A signal from a cancer lesion in the body of a cancer patient, it is preferable to use a protein A that generates a signal without reacting with a substrate, such as a fluorescent protein. If a protein A signal is detected from the cancer lesion (e.g., if a signal of the same level as that detected in a non-cancerous site near the tumor is detected), it is indicated that the patient's cancer is not a homologous recombination-deficient cancer. If a protein A signal is not detected from the cancer lesion (e.g., if the signal from the cancer lesion is clearly lower than that from a non-cancerous site near the tumor), it is indicated that the patient's cancer may be a homologous recombination-deficient cancer. In this embodiment, for a patient diagnosed with a possible homologous recombination deficient cancer, cancer cells may be collected and transfected with a nucleic acid construct to confirm that the cancer is homologous recombination deficient.

[0058] In yet another embodiment, protein A is a secreted protein whose expression in cells can be detected (preferably as a signal), and the nucleic acid construct is introduced into cancer cells by administering the nucleic acid construct to a cancer patient, and the activity of protein A in blood isolated from the patient after administration of the nucleic acid construct is measured. Among the usage modes described below, (4) is a specific example of this mode. In this mode, the cancer patient typically has a solid cancer. The nucleic acid construct is preferably administered locally into or near the tumor of the cancer patient. If the patient's cancer is not deficient in homologous recombination, secreted protein A is expressed from the protein A gene reproduced in the cancer cells and secreted outside the cancer cells, allowing the activity of protein A to be detected using a blood sample from the cancer patient. Since the activity of protein A secreted into the blood is detected in vitro, proteins that generate a signal upon reaction with a substrate can also be preferably used, and a typical example is secreted luciferase. The gene sequence encoding a secreted protein such as secreted luciferase may be a known secreted protein coding sequence as is, or may be prepared by adding a sequence encoding a secretion signal to the protein coding sequence. A negative control sample can be, for example, a blood sample collected from a patient before administration of a nucleic acid construct. A positive control can be, for example, a culture supernatant obtained by introducing a nucleic acid construct into a cell line with normal homologous recombination activity (details are the same as for the HR-non-deficient control cells described above) and culturing the cell line. Detection of protein A activity in the blood indicates that the patient's cancer is not a homologous recombination-deficient cancer. Failure to detect protein A activity in the blood indicates that the patient's cancer may be a homologous recombination-deficient cancer. In this embodiment, cancer cells from a patient diagnosed with a possible homologous recombination-deficient cancer can be collected, transfected with a nucleic acid construct, and confirmed to be a homologous recombination-deficient cancer.

[0059] By using the diagnostic technique for homologous recombination deficient cancer according to the present invention, cancer patients diagnosed with homologous recombination deficient cancer can be preferably administered DNA-damaging anticancer drugs such as PARP inhibitors and platinum preparations for cancer treatment. Cancer patients diagnosed with cancer other than homologous recombination deficient cancer can be preferably administered anticancer drugs other than DNA-damaging anticancer drugs.

[0060] Examples of usage of the diagnostic agent for homologous recombination deficient cancer include, but are not limited to, the following.

[0061] (1) Cancer cells collected from a cancer patient are cultured and transfected with the nucleic acid construct of the present invention to examine whether a protein A signal can be detected (or whether a cytocidal effect of protein A is observed). If desired, non-cancerous cells (such as peripheral blood cells) collected from the same cancer patient may be used as a control. If protein A is luciferase, a luciferase substrate is added and the detection reaction is carried out. If protein A is a fluorescent protein, the fluorescent signal can be detected using a luminometer or the like. If a protein A signal is detected (especially if a signal comparable to that of control non-cancerous cells is detected), it can be determined that the patient's cancer is not deficient in homologous recombination. If no protein A signal is detected, or if a signal lower than that of control non-cancerous cells is detected, it can be determined that the patient's cancer is deficient in homologous recombination. Methods for measuring cell viability are well known in the art and can be easily measured using commercially available kits, etc. If the viability of cancer cells into which a nucleic acid construct has been introduced decreases (especially if it decreases to the same extent as that of control non-cancer cells), it can be determined that the patient's cancer is not deficient in homologous recombination. If no decrease in cancer cell viability is observed, or if the decrease in cell viability is more gradual than that of control non-cancer cells, it can be determined that the patient's cancer is deficient in homologous recombination.

[0062] (2) The nucleic acid construct of the present invention is administered to a patient, and whether a protein A signal is detected from the cancer lesion is examined. If a signal is detected, it can be determined that the patient's cancer is not deficient in homologous recombination, and if a signal is not detected, it can be determined that the patient's cancer may be deficient in homologous recombination. In this embodiment, a protein whose intracellular expression can be detected as a signal is used as protein A, but it is necessary to use a protein with sufficiently low biotoxicity. The nucleic acid construct is preferably administered to the patient by local administration into or near the tumor.

[0063] (3) Cancer cells present in the blood are isolated from the patient, concentrated as necessary, and transfected with the nucleic acid construct of the present invention to examine whether a signal from Protein A is detected (or whether a cytocidal effect by Protein A is observed) (in the case of leukemia).

[0064] (4) A nucleic acid construct employing a gene sequence encoding secreted luciferase as the gene sequence encoding protein A is administered locally into or near the patient's tumor, and then a blood sample is collected and examined for the presence or absence of a luciferase reaction. If the patient's cancer is not deficient in homologous recombination, secreted luciferase is produced from the luciferase gene replicated within the cancer cells and secreted extracellularly, allowing the luciferase reaction to be detected using the blood sample. If the patient's cancer is deficient in homologous recombination, secreted luciferase is not produced, and therefore no luciferase reaction is detected in the blood sample.

[0065] The nucleic acid construct of the present invention can also be used, for example, as a detection agent for homologous recombination-restored cancer cells. Homologous recombination-restored cancer refers to cancer that was initially deficient in homologous recombination but has regained homologous recombination activity as a result of therapeutic treatment or other measures. A typical example is cancer that has acquired resistance to treatment with a PARP inhibitor (PARP inhibitor-resistant cancer). The term homologous recombination-restored cancer also encompasses cancer in which BRCA1 deficiency has been restored (reversion mutation) and BRCA1 has been normalized. In this use mode, a gene sequence encoding a protein whose intracellular expression can be detected can be preferably used as the gene sequence encoding protein A. The specific use mode is similar to that of a diagnostic agent for homologous recombination-deficient cancer, but the target cancer patients may primarily be patients currently undergoing PARP inhibitor treatment or patients previously diagnosed with homologous recombination-deficient cancer. The nucleic acid construct of the present invention can be introduced into cancer cells of a patient using a method similar to that used for diagnosing homologous recombination-deficient cancer described above, and the expression of protein A, preferably protein A activity, can be measured. As with the method for diagnosing homologous recombination-deficient cancer, the method for detecting homologous recombination-restored cancer cells may also use control cells (HR-non-deficient control cells, HR-deficient control cells, or both). Detection of protein A expression / activity can be considered to indicate that homologous recombination-restored cancer cells have been detected. One possible method for using the detection agent is to measure homologous recombination activity in homologous recombination-deficient cancer cells before homologous recombination restoration using a homologous recombination activity assay kit according to the present invention, and then compare this measurement to determine whether homologous recombination has been restored. An increase in homologous recombination activity compared to previous measurements, i.e., an increase in the protein A expression / activity level compared to the protein A expression / activity level previously measured in cancer cells of the cancer patient using the nucleic acid construct of the present invention, indicates restoration of homologous recombination. For patients diagnosed with homologous recombination-restored cancer, possible measures include discontinuing administration of PARP inhibitors and switching to treatment with another anticancer drug (for patients currently undergoing PARP inhibitor treatment).One option for other anticancer drugs is the therapeutic agent for homologous recombination-restoring cancer of the present invention, which will be described later. For patients diagnosed with cancer other than homologous recombination-restoring cancer, treatment with the same anticancer drug may be continued or switched to treatment with another anticancer drug.

[0066] The nucleic acid construct of the present invention can also be used, for example, as a companion diagnostic agent for predicting the effectiveness of anticancer drugs against homologous recombination-deficient cancers. Examples of anticancer drugs include DNA-damaging anticancer drugs, including PARP inhibitors. Specific examples of use as a companion diagnostic agent are the same as those for the diagnostic agent for homologous recombination-deficient cancers described above. A gene sequence encoding a protein whose intracellular expression can be detected can preferably be used as the gene sequence encoding protein A, but a gene sequence encoding a protein that has the effect of reducing cell viability can also be used. When a patient is diagnosed with homologous recombination-deficient cancer, the patient can be determined to be one for whom a DNA-damaging anticancer drug, such as a PARP inhibitor, is likely to produce the desired anticancer effect. Therefore, DNA-damaging anticancer drugs can be preferably administered to such cancer patients.

[0067] DNA-damaging anticancer drugs include inhibitors of proteins that are involved in synthetic lethality (or synthetic growth retardation) with homologous recombination, and drugs that induce DNA damage that is repaired by homologous recombination. Examples of the former include inhibitors of proteins such as PARP, PolQ, and Rad52, while examples of the latter include, but are not limited to, camptothecin, cisplatin, and PARP inhibitors.

[0068] Specific examples of PARP inhibitors include, but are not limited to, olaparib, rucaparib, niraparib, veliparib, talazoparib, PJ34 (Visochek et al., Oncotarget, 2019, Vol. 10, (No. 58), pp: 6269-6282), NU1025 (CAS 90417-38-2), and the like.

[0069] PolQ inhibitors can be obtained, for example, by the method described in JP 2017-201978 A. PolQ inhibitors include various PolQ inhibitors that will be discovered in the future by this method or by other methods.

[0070] Examples of Rad52 inhibitors include, but are not limited to, 6-hydroxyDL-dopa, AICAR (CAS 2627-69-2), AICAR 50 phosphate (ZMP), 6-hydroxyDL-dopa (CAS 21373-30-8), D-103, D-G23, (-)-epigallocatechin, EGC (CAS 490-46-0), and NP-004255 (CAS 23094-69-1) (Hengel SR et al. Cell Chem Biol. 2017 Sep 21;24(9):1101-1119.).

[0071] Further examples of DNA-damaging anticancer drugs include DDR-related inhibitors that target synthetic lethality (see, e.g., Gourley C et al. J Clin Oncol. 2019 Sep 1;37(25):2257-2269, Gourley C et al. J Clin Oncol. 2019 Sep 1;37(25):2257-2269, and Ashworth A and Lord CJ. Nat Rev Clin Oncol. 2018 Sep;15(9):564-576). Specific examples include, but are not limited to, CBP-501, Prexasertib, GDC-0575, and SRA-737, which target CHK1 / 2; AZD-1775, which targets WEE1; AZD-6738, M-4344, and M6620 (VX-970), which target ATR; CC-115, LY-3023414, AsiDNA, and M-3814, which target DNA-PK; and AZD-0156, which targets ATM (Gourley C et al., 2019 (supra)).

[0072] Specific examples of DNA-damaging anticancer agents include platinum drugs (DNA synthesis inhibitors) such as cisplatin, carboplatin, and oxaliplatin; pyrimidine drugs (DNA synthesis inhibitors) such as fluorouracil and gemcitabine; camptothecin drugs (DNA synthesis inhibitors) such as irinotecan and topotecan; epipodophyllotoxin drugs (DNA synthesis inhibitors) such as etoposide; anthracycline drugs (DNA synthesis inhibitors) such as doxorubicin, epirubicin, and pirarubicin; alkylating agents (DNA synthesis inhibitors) such as cyclophosphamide and ifosfamide; and vinca alkaloid drugs (cell division inhibitors) such as vinblastine, vincristine, vindesine, and vinorelbine, as well as taxane drugs (apoptosis inducers) such as paclitaxel and docetaxel. However, DNA-damaging anticancer agents are not limited to the above examples.

[0073] The nucleic acid construct of the present invention can also be used, for example, as a therapeutic agent for homologous recombination-restoring cancer. The definition of homologous recombination-restoring cancer is as described above, and includes cancers that have acquired resistance to treatment with a PARP inhibitor (PARP inhibitor-resistant cancer) and cancers in which BRCA1 deficiency has been restored (reversion mutation) and BRCA1 has been normalized. In this use mode, the gene sequence encoding protein A is a gene sequence encoding a protein that has the effect of reducing cell viability. The nucleic acid construct of the present invention can kill cancer cells with homologous recombination activity through the action of protein A, making it possible to treat homologous recombination-restoring cancers, such as PARP inhibitor-resistant cancers, for which there is currently no treatment.

[0074] The nucleic acid constructs of the present invention can also be used, for example, to determine or predict whether a genetic mutation (particularly a mutation in an HR-related gene) identified in a cancer patient is a pathogenic mutation that impairs homologous recombination activity.

[0075] When a mutation in a gene X (preferably an HR-related gene) is identified in the cancer cells of a cancer patient, an expression vector expressing mutant gene X having the same mutation is first constructed. Mutant gene X can be obtained by extracting mRNA from cancer cells collected from the cancer patient and performing reverse transcription PCR using a primer set targeting gene X. In addition, an expression vector expressing wild-type gene X without mutation is also prepared. Wild-type gene X can be prepared from a known cell line with normal homologous recombination activity (see the above-mentioned HR-non-deficient control cells for details) or from the patient's non-cancerous cells. If an expression vector expressing wild-type gene X is already known, the known expression vector can be used without the need for newly constructing one.

[0076] Next, a mutant gene X expression vector and a wild-type gene X expression vector are introduced into the gene X-deficient cells, and then the nucleic acid construct of the present invention is introduced. The introduction of each expression vector and the introduction of the nucleic acid construct can be performed in any order. Gene X-deficient cells can be prepared by knocking out gene X in cells with normal homologous recombination activity. Techniques for knocking out specific genes have been established, and those skilled in the art can easily prepare gene X knockout cells using well-known techniques. Alternatively, if there is a known cell line known to be completely deficient in the function of gene X, such a known cell line may be used.

[0077] Next, the expression of protein A is measured in cells into which each expression vector and nucleic acid construct has been introduced. Protein A expression may also be measured in gene X-deficient cells into which an empty vector without an insert has been introduced, or in gene X-deficient cells into which no vector has been introduced and into which the nucleic acid construct of the present invention has been introduced. As described above, measuring the expression of protein A preferably means measuring the activity of protein A. Based on the expression level of protein A, it can be determined whether the mutation in gene X is a pathogenic mutation (whether the mutation results in the loss of homologous recombination activity). This determination can be made using the same criteria as in the above-mentioned technique for measuring homologous recombination activity. If the expression level / activity level of protein A in cells into which mutant gene X has been introduced is lower than the expression level / activity level of protein A in cells into which wild-type gene X has been introduced, it is indicated that the mutation in gene X is a pathogenic mutation that impairs homologous recombination activity.

[0078] In this pathogenic mutation prediction technique, a protein whose intracellular expression can be preferably detected as a signal can be preferably used as protein A, but it is also possible to use a protein that has the effect of reducing cell viability.

[0079] When the agent of the present invention is administered to a patient, the administration route may be oral or parenteral, although parenteral administration, such as intravenous, intraarterial, subcutaneous, or intramuscular administration, is generally preferred. Systemic administration may be used, or administration may be intratumor or peritumor, or administration may be to tumor-draining lymph nodes. However, in the case of a therapeutic agent for cancers that are restored by homologous recombination, local administration into or near a tumor is preferred, since systemic administration may also have a cytocidal effect on normal non-cancer cells that possess homologous recombination activity. The dosage is not particularly limited, but may be approximately 1 pg to 10 g, for example, approximately 0.01 mg to 100 mg, of nucleic acid construct per day per patient. Daily administration may be a single dose or may be divided into several doses. Administration may be daily, or every few days or weeks.

[0080] The dosage form of the agent of the present invention to be administered to a patient is not particularly limited, and can be formulated by appropriately mixing additives such as pharmaceutically acceptable carriers, diluents, and excipients with the nucleic acid construct of the present invention depending on the administration route. Examples of dosage forms include parenteral preparations such as drip infusions, injections, suppositories, and inhalants, and oral preparations such as tablets, capsules, granules, powders, and syrups. Formulation methods and usable additives are well known in the field of pharmaceutical formulations, and any method and additive can be used.

[0081] In the case of diagnostic agents, companion diagnostic agents, and detection agents used in vitro, the amount used when treating cells may be, for example, about 10 pg to 1 mg, e.g., about 0.1 ng to 100 μg, of nucleic acid construct per 1 million cells. The dosage form may be a liquid agent to which additives useful for the stability of the nucleic acid construct are added as desired, or a powdered agent obtained by lyophilizing the nucleic acid construct.

[0082] When the nucleic acid construct of the present invention is used as a therapeutic agent, diagnostic agent, or the like, the target cancer patients include cancer patients of various mammals such as humans, dogs, cats, ferrets, hamsters, mice, rats, horses, pigs, etc. The nucleic acid construct of the present invention can be applied to various animal species without changing its configuration, except for changing the origin of the promoter as necessary. [Example]

[0083] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0084] A. How to prepare vectors (DNA constructs) A-1. Construction of pCMV-DR-DTA construct (version 1) The two DT-A gene fragments used in pCMV-DR-DTA were amplified by PCR using pMC1DT-ApA (KURABO, Osaka, Japan) as a template. The 588-bp SceDTA fragment (SEQ ID NO: 15) was obtained by replacing positions 133 to 153 in the DT-A gene ORF sequence (SEQ ID NO: 13) with a stop codon (TGA) plus an I-SceI recognition sequence. The 357-bp iDTA fragment (SEQ ID NO: 18) consisted of the region from positions 1 to 357 in the DT-A gene ORF sequence, including the replaced region. PrimeSTAR HS (trade name) DNA Polymerase (Takara Bio) was used as the polymerase for PCR. The primers used are listed in Table 1 below. The SceDTA fragment was amplified separately into two fragments: the 5'SceDTA fragment and the 3'SceDTA fragment. The 5'SceDTA fragment was amplified using DTA-Fw and Sce-5'DTA-Rv, resulting in a DNA fragment with a sequence for In-Fusion reaction added to the 5' end of the 5'SceDTA fragment and a stop codon (TGA) + I-SceI recognition sequence added to the 3' end. The 3'SceDTA fragment was amplified using Sce-3'DTA-Fw and DTA-Rv, resulting in a DNA fragment with an I-SceI recognition sequence added to the 5' end of the 3'SceDTA fragment and a stop codon (TGA) and sequences for In-Fusion reaction added to the 3' end. The iDTA fragment was amplified using iDTA-Fw and iDTA-Rv, resulting in a DNA fragment with a stop codon (TGA) ligated to the 3' end of the iDTA fragment and sequences for In-Fusion reaction added to both ends. Next, pIRES (Takara Bio, Figure 2) was digested with NheI and XbaI to remove the IRES region and recover a fragment containing the CMV promoter and poly(A) addition signal. Using the In-Fusion HD Cloning Kit (Takara Bio), the 5'SceDTA fragment and 3'SceDTA fragment were inserted downstream of the CMV promoter (the site where the IRES region had been located), yielding pCMV-Sce-DTA, which is a CMV promoter-SceDTA fragment-poly(A) addition signal ligation.Finally, pCMV-Sce-DTA was digested with ClaI, and the iDTA fragment was inserted downstream of the poly(A) addition signal using the In-Fusion HD Cloning Kit (Takara Bio). This resulted in pCMV-DR-DTA, which contained the CMV promoter, SceDTA fragment, poly(A) addition signal, and iDTA fragment. The resulting plasmid was purified using a Qiagen Plasmid Plus Midi Kit (Qiagen KK) and linearized by digestion with I-SceI (New England Biolabs, Ipswich, MA, USA) before transfection.

[0085] A-2. Construction of pCMV-DR-Nluc construct (version 1) The two Nluc gene fragments used in pCMV-DR-Nluc were a 513-bp SceNluc fragment (SEQ ID NO: 22), in which the region from positions 223 to 243 in the Nluc gene sequence (SEQ ID NO: 20) was replaced with a stop codon (TGA) plus an I-SceI recognition sequence, and a 459-bp iNluc fragment (SEQ ID NO: 25), consisting of the region from positions 16 to 474 in the Nluc gene sequence including the replaced region, were amplified by PCR using pNL1.1[Nluc] Vector (Promega, Madison, WI, USA) as a template. PrimeSTAR HS (trade name) DNA Polymerase (Takara Bio) was used as the polymerase for PCR. The primers used are listed in Table 1 below. The SceNluc fragment was amplified separately into two fragments: the 5'SceNluc fragment and the 3'SceNluc fragment. The 5'SceNluc fragment was amplified using Nluc-Fw (SEQ ID NO: 7) and Sce-5'Nluc-Rv (SEQ ID NO: 8), resulting in a DNA fragment with a structure in which a sequence for the In-Fusion reaction was added to the 5' end of the 5'SceNluc fragment and a stop codon (TGA) + I-SceI recognition sequence was added to the 3' end. The 3'SceNluc fragment was amplified using Sce-3'Nluc-Fw (SEQ ID NO: 9) and Nluc-Rv (SEQ ID NO: 10), resulting in a DNA fragment with a structure in which an I-SceI recognition sequence was added to the 5' end of the 3'SceNluc fragment and a stop codon (TAA) and a sequence for the In-Fusion reaction were added to the 3' end. The iNluc fragment was amplified using iNluc-Fw (SEQ ID NO: 11) and iNluc-Rv (SEQ ID NO: 12) to amplify a DNA fragment containing a stop codon (TGA) at the 3' end of the iNluc fragment and sequences for the In-Fusion reaction added to both ends. Next, pIRES (Takara Bio, Figure 2) was digested with NheI and XbaI to remove the IRES region and recover a fragment containing the CMV promoter and poly(A) addition signal.Using the In-Fusion HD Cloning Kit (Takara Bio), the 5'SceNluc and 3'SceNluc fragments were inserted downstream of the CMV promoter (the site where the IRES region was located), yielding pCMV-Sce-Nluc, which contains the CMV promoter, SceNluc fragment, and poly(A) addition signal. Finally, pCMV-Sce-Nluc was digested with BamHI, and the iNluc fragment was inserted downstream of the poly(A) sequence using the In-Fusion HD Cloning Kit (Takara Bio). This yielded pCMV-DR-Nluc, which contains the CMV promoter, SceNluc fragment, poly(A) addition signal, and iNluc fragment. The resulting plasmid was purified using the Qiagen Plasmid Plus Midi Kit (Qiagen KK) and linearized by digestion with I-SceI (New England Biolabs, Ipswich, MA, USA) before transfection.

[0086] A-3. Construction of pCMV-DR-Nluc-v2 construct A modified version of the pCMV-DR-Nluc construct (version 1) was constructed, in which the iNluc fragment was located upstream of the promoter instead of at the most downstream position.

[0087] The two Nluc gene fragments used in pCMV-DR-Nluc-v2 (the 513-bp SceNluc fragment (SEQ ID NO: 22) and the 459-bp iNluc fragment (SEQ ID NO: 25)) were amplified from pNL1.1[Nluc] Vector as described in A-2. However, for amplification of the iNluc fragment, iNluc-Fw2 (SEQ ID NO: 27) and iNluc-Rv2 (SEQ ID NO: 28) were used instead of iNluc-Fw and iNluc-Rv, which had modified sequences added for the In-Fusion reaction. pIRES (Takara Bio, Figure 2) was digested with NheI and XbaI to remove the IRES region, and a fragment containing the CMV promoter and poly(A) addition signal was recovered. Using the In-Fusion HD Cloning Kit (Takara Bio), the 5'SceNluc and 3'SceNluc fragments were inserted downstream of the CMV promoter (where the IRES region was located) to obtain pCMV-Sce-Nluc, which contains the CMV promoter-SceNluc fragment-poly(A) addition signal ligation. Finally, pCMV-Sce-Nluc was digested with BglII, and the iNluc fragment was inserted upstream of the CMV promoter using the In-Fusion HD Cloning Kit (Takara Bio). This resulted in pCMV-DR-Nluc-v2, which contains the iNluc fragment-CMV promoter-SceNluc fragment-poly(A) addition signal ligation. The resulting plasmid was purified using the Qiagen Plasmid Plus Midi Kit (Qiagen KK, Tokyo, Japan) and linearized by digestion with I-SceI (New England Biolabs, Ipswich, MA, USA) before transfection.

[0088] A-4. Construction of pCMV-DR-DTA-v2 construct A modified version of the pCMV-DR-DTA construct (version 1) was constructed, in which the iDTA fragment was located upstream of the promoter instead of at the most downstream position.

[0089] The two DT-A gene fragments used in pCMV-DR-DTA-v2 (a 588-bp SceDTA fragment (SEQ ID NO: 15) and a 357-bp iDTA fragment (SEQ ID NO: 18)) were amplified from pMC1DT-ApA in the same manner as in A-1. However, for amplification of the iDTA fragment, iDTA-Fw2 (SEQ ID NO: 29) and iDTA-Rv2 (SEQ ID NO: 30), which had modified sequences added for the In-Fusion reaction, were used instead of iDTA-Fw and iDTA-Rv. pIRES (Takara Bio, Figure 2) was digested with NheI and XbaI to remove the IRES region, and a fragment containing the CMV promoter and poly(A) addition signal was recovered. Using the In-Fusion HD Cloning Kit (Takara Bio), the 5'SceDTA and 3'SceDTA fragments were inserted downstream of the CMV promoter (the site where the IRES region was located), resulting in pCMV-Sce-DTA, which contained a CMV promoter-SceDTA fragment-poly(A) addition signal ligation. Finally, pCMV-Sce-DTA was digested with BglII, and the iDTA fragment was inserted upstream of the CMV promoter using the In-Fusion HD Cloning Kit (Takara Bio). This resulted in pCMV-DR-DTA-v2, which contained a CMV promoter-SceDTA fragment-poly(A) addition signal ligation. The resulting plasmid was purified using the Qiagen Plasmid Plus Midi Kit (Qiagen KK) and linearized by digestion with I-SceI (New England Biolabs) before transfection.

[0090] A-5. Construction of pCMV-DR-TK construct A construct was constructed using the HSV-TK gene as the suicide gene. The two HSV-TK gene fragments used in pCMV-DR-TK were a 1,128-bp SceTK fragment (SEQ ID NO: 39) in which the region from positions 499 to 519 in the HSV-TK gene ORF sequence (SEQ ID NO: 37) was replaced with a "stop codon (TGA) + I-SceI recognition sequence," and a 1,063-bp iTK fragment (SEQ ID NO: 42) consisting of the region from positions 27 to 1,089 in the HSV-TK gene ORF sequence containing the replaced region. These fragments were obtained by PCR amplification using the HSV-TK gene fragment (GenBank: V00470.1, Kobayashi et al. (2001) Decreased topoisomerase IIalpha expression confers increased resistance to ICRF-193 as well as VP-16 in mouse embryonic stem cells. Cancer Lett. 166(1): 71-77) as a template. PrimeSTAR HS (trade name) DNA Polymerase (Takara Bio) was used as the polymerase for PCR. The primers used are shown in Table 1 below. The SceTK fragment was amplified by dividing it into two fragments: the 5'SceTK fragment and the 3'SceTK fragment containing a poly(A) addition signal. Sce-5'TK-Fw (SEQ ID NO: 31) and Sce-5'TK-Rv (SEQ ID NO: 32) were used to amplify the 5'SceTK fragment, and a DNA fragment with a sequence for the In-Fusion reaction added to the 5' end of the 5'SceTK fragment and a stop codon (TGA) added to the 3' end was amplified. The 3'SceTK fragment was amplified using Sce-3'TK-Fw (SEQ ID NO: 33) and Sce-3'TK-Rv (SEQ ID NO: 34), resulting in a DNA fragment with an I-SceI recognition sequence added to the 5' end of the 3'SceTK fragment and a sequence for In-Fusion reaction added to the 3' end. The iTK fragment was amplified using iTK-Fw (SEQ ID NO: 35) and iTK-Rv (SEQ ID NO: 36), resulting in a DNA fragment with a sequence for In-Fusion reaction added to both ends. Next, pIRES (Takara Bio, Figure 2) was digested with NheI and BamHI, and a fragment containing the CMV promoter was recovered.Using the In-Fusion HD Cloning Kit (Takara Bio), the 5'SceTK fragment and the 3'SceTK fragment were inserted downstream of the CMV promoter to obtain pCMV-Sce-TK, which contains the CMV promoter-SceTK fragment-poly(A) addition signal ligation. Finally, pCMV-Sce-TK was digested with BglII, and the iTK fragment was inserted upstream of the CMV promoter using the In-Fusion HD Cloning Kit (Takara Bio). This resulted in pCMV-DR-TK, which contains the iTK fragment-CMV promoter-SceTK fragment-poly(A) addition signal ligation. The resulting plasmid was purified using the Qiagen Plasmid Plus Midi Kit (Qiagen KK) and linearized by digestion with I-SceI (New England Biolabs) before transfection.

[0091] [Table 1]

[0092] B. Experiment 1 (Experiment using Version 1 construct) B-1. Evaluation of cell killing effect in human cells and luciferase assay method The human pre-B cell line Nalm-6 and its derivatives were cultured at 37°C in a 5% CO2 incubator (So et al. (2004) Genetic interactions between BLM and DNA ligase IV in human cells. J. Biol. Chem. 279: 55433-55442).

[0093] RAD54 / RAD54B double-deficient cells were obtained as previously reported (Saito S, Kurosawa A, Adachi N. Mechanistic basis for increased human gene targeting by promoterless vectors—roles of homology arms and Rad54 paralogs. FEBS J. 2017 Sep;284(17):2748-2763. doi: 10.1111 / febs.14137.).

[0094] Gene transfer into Nalm-6 cells and their derivatives was performed by electroporation as previously reported (Saito S, Maeda R, Adachi N. Dual loss of human POLQ and LIG4 abolishes random integration. Nat Commun. 2017 Jul 11;8:16112. doi: 10.1038 / ncomms16112.). 2 x 10 6 Each cell was transfected with each construct (1 μg), and the cell-killing effect was examined by one of the following methods. (1) Growth inhibition test After transfection, cells were cultured at 1 x 10 5 The cells were seeded at 1000 cells / ml into a 24-well dish, and cell viability was measured every 24 hours using CellTiter-Glo (CellTiter-Glo® Luminescent Cell Viability Assay, Promega). (2) Colony formation method 24 hours after gene transfer, 200 or 5,000 cells were seeded into a 60 mm dish, colony formation was allowed to occur for 17 days, and the number of colonies was counted.

[0095] Luciferase assays were performed as follows: 2 x 10 6 Each construct (1 μg) was transfected into 5 x 10 cells. 5The cells were seeded into a 12-well dish at 100 cells / ml, and luciferase activity (RLU value) was measured over time using the Nano-Glo Luciferase Assay System (Promega).

[0096] B-2. Results Figure 3 shows an example of the results of a luciferase assay using the version 1 DR-Nluc construct. In wild-type Nalm-6 cells transfected with pCMV-DR-Nluc, luciferase activity increased 2 hours after transfection. In contrast, in a RAD54 / RAD54B double-deficient strain (cells that have lost the ability to homologous recombination), luciferase activity barely increased even 24 hours after transfection with pCMV-DR-Nluc. The RLU / cell value in the homologous recombination-deficient cells after 24 hours was less than 1 / 100 of that of the wild-type strain. Note that pCMV-Nluc in this figure represents the results of cells transfected with a construct expressing the normal Nluc gene under the control of the CMV promoter. It is believed that transfection of the pCMV-DR-Nluc construct into cells generates the normal Nluc gene via extrachromosomal homologous recombination, resulting in transient expression of the Nluc protein.

[0097] Examples of experimental results using the version 1 DR-DTA construct are shown in Figure 4 (colony formation assay) and Figure 5 (growth inhibition test). The viability of cells transfected with pCMV-DR-DTA was approximately 1 / 100 of that of cells transfected with pCMV-Nluc (negative control) (Figure 5). pCMV-DTA in Figures 4 and 5 represents the results of cells transfected with a construct expressing the normal DT-A gene under the control of the CMV promoter. These results confirmed the cell-killing effect of pCMV-DR-DTA. These results suggest that the use of a DNA construct based on a suicide gene (DT-A gene) can selectively kill cells with normal homologous recombination ability.

[0098] C. Experiment 2 C-1. Method <Cells and gene transfer> The human pre-B cell line Nalm-6 and its derivatives were cultured at 37°C in a 5% CO2 incubator (So et al. (2004) Genetic interactions between BLM and DNA ligase IV in human cells. J. Biol. Chem. 279: 55433-55442). Nalm-6 cells were transfected by electroporation as previously described (Saito et al. (2017) Dual loss of human POLQ and LIG4 abolishes random integration. Nat. Commun. 8: 16112). The human fibrosarcoma cell line HT1080 was cultured at 37°C in a 5% CO2 incubator (Saito et al. (2015) Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection. BMC Res. Notes 8, 278). The human breast cancer cell lines MDA-MB-436 (ATCC, Manassas, VA, USA) and HCC1937 (ATCC) were cultured at 37°C in a 5% CO2 incubator. The medium used was Eagle's MEM (Nissui Pharmaceutical, Tokyo, Japan) supplemented with calf serum (Global Life Science Technologies Japan, Tokyo, Japan; 50 ml added per 500 ml of Eagle's MEM), L(+)-glutamine (Fujifilm Wako Pure Chemical Industries, Osaka, Japan; added to a final concentration of 2 mM), MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Osaka, Japan; added to a final concentration of 1 mM), 100 mmol / L sodium pyruvate solution (Fujifilm Wako Pure Chemical Industries, St. Louis, MO, USA; added to a final concentration of 0.15 μM), and 2-mercaptoethanol (Fujifilm Wako Pure Chemical Industries, St. Louis, MO, USA; added to a final concentration of 50 μM). Transfection of HT1080, MDA-MB-436, and HCC1936 cells was performed with jetPEI (Polyplus-Transfection, Illkirch, France) according to the manufacturer's protocol.

[0099] <Evaluation of cell killing effect> 2x 10 6 Each construct (1 μg) was transfected into Nalm-6 cells, and the cell-killing effect was examined by either a growth inhibition test or a colony formation assay. (1) Growth inhibition test method 1 x 10 cells after transfection 5The cells were seeded into a 24-well dish at 1000 cells / ml, and 24 hours later, ganciclovir (Fujifilm Wako Pure Chemical Industries, Ltd.) was added at a final concentration of 500 nM. Cell viability was measured every 48 hours using CellTiter-Glo (CellTiter-Glo® Luminescent Cell Viability Assay, Promega). (2) Colony formation method Twenty-four hours after transfection, 200 or 5,000 cells were seeded onto a 60 mm dish to allow colony formation. For pCMV-DR-TK transfection, 200 or 5,000 transfected cells were seeded onto agar medium containing 500 nM ganciclovir. After 17 days of culture, the number of colonies that grew was counted, and the survival rate was calculated.

[0100] <Luciferase assay> Luciferase assays in Nalm-6 cells and their derivatives were performed as follows: 2 x 10 6 Each construct (1 μg) was transfected into 5 x 10 Nalm-6 cells. 5 The cells were seeded at 1000 cells / ml into a 24-well dish, and luciferase activity (RLU value) was measured 24 hours later using the Nano-Glo Luciferase Assay System (Promega). Luciferase assays in HT1080 cells, MDA-MB-436 cells, and HCC1937 cells were performed as follows: 5 x 10 4 Cells were seeded into 24-well dishes, cultured overnight, and then transfected with 1 μg of each construct. 24 hours after transfection, the cells were counted and luciferase activity (RLU) was measured using the Nano-Glo Luciferase Assay System (Promega).

[0101] C-2. Results An example of the results of luciferase assays performed on HT1080, MDA-MB-436, and HCC1937 cells transfected with the version 1 DR-Nluc construct is shown in Figure 6. The RLU values ​​of BRCA1-deficient cells (HCC1937 and MDA-MB-436, cells deficient in homologous recombination) transiently transfected with pCMV-DR-Nluc were less than 1 / 1000 of those of BRCA1-normal cells (HT1080, with normal homologous recombination activity) transfected with pCMV-DR-Nluc.

[0102] The differences between versions 1 and 2 of the DR-Nluc construct are illustrated in Figure 7, and an example of the results of a luciferase assay using the DR-Nluc-v2 construct is shown in Figure 8. Changing the position of iNluc did not change the RLU value of HR-dependently expressed luciferase, confirming that the position of iNluc does not affect HR frequency.

[0103] An example of the results of examining the cell-killing effect of the DR-DTA-v2 construct by colony formation assay is shown in Figure 9, along with the experimental results using the version 1 DR-DTA construct (the results for cells transfected with the positive control pCMV-DTA are omitted). Changing the position of the iDTA did not affect the cell-killing effect on cells with normal homologous recombination ability, and it was confirmed that the same cell-killing effect as version 1 was achieved.

[0104] An explanatory diagram of the DR-TK construct is shown in Figure 10. An example of the results of a growth inhibition test to examine the cell-killing effect of the DR-TK construct is shown in Figure 11. The cell viability 6 days after gene transfer is shown as a relative value, with the value without ganciclovir set at 100. As with the use of the DT-A gene, it was confirmed that the use of HSV-TK as the suicide gene was also effective.

[0105] D. Experiment 3 D-1. Construction of pCMV-DR-SecNluc construct To create a vector expressing the Nluc gene with a secretory signal added, a secretory signal sequence derived from human interleukin 6 (IL6) was synthesized by artificial gene synthesis (pIL6-Nluc). The artificially synthesized pIL6-Nluc was digested with SmaI and EcoNI, and a fragment containing the secretory signal sequence was recovered. The recovered fragment containing the secretory signal sequence was mixed with a DNA fragment obtained by digesting pCMV-Nluc with SmaI and EcoNI, and ligated using a DNA Ligation Kit.<Mighty Mix> Using a recombinant vector (Takara Bio), we inserted a fragment containing a secretory signal sequence upstream of the Nluc gene to obtain pCMV-SecNluc, which is a CMV promoter-secretory signal-Nluc gene-poly(A) addition signal ligation. The nucleotide sequence of the secretory signal-Nluc gene portion (SecNluc gene) is shown in SEQ ID NO: 44 (positions 1 to 87 of the DNA sequence encode the secretory signal), and the amino acid sequence encoded by this is shown in SEQ ID NO: 45 (amino acids 1 to 29 of the secretory signal sequence).

[0106] To add a secretory signal sequence to the [SceNluc fragment] portion (SceNluc gene) of pCMV-Sce-Nluc prepared in A-2 above, pCMV-SecNluc was digested with XhoI and EcoNI, and a fragment containing the secretory signal sequence was recovered. Next, pCMV-DR-Nluc was digested with XhoI and EcoNI, and a fragment containing the CMV promoter was recovered. The recovered fragment containing the secretory signal sequence and the fragment containing the CMV promoter were mixed and ligated using a DNA Ligation Kit.<Mighty Mix> Using a PCR product (Takara Bio), a fragment containing the secretory signal sequence was inserted upstream of the SceNluc gene to obtain pCMV-Sce-SecNluc, which is a CMV promoter-secretory signal-SceNluc fragment-poly(A) addition signal ligation. Finally, pCMV-Sce-SecNluc was digested with BamHI, and the iNluc fragment prepared in A-2 above was inserted downstream of the poly(A) addition signal using an In-Fusion HD Cloning Kit (Takara Bio), to obtain pCMV-DR-SecNluc, which is a CMV promoter-secretory signal-SceNluc fragment-poly(A) addition signal ligation (Figure 12). The nucleotide sequence of the [secretion signal]-[SceNluc fragment] portion (SceSecNluc gene) is shown in SEQ ID NO: 46 (positions 1 to 87 of the DNA sequence encode the secretion signal), and the amino acid sequence encoded thereby is shown in SEQ ID NO: 47 (amino acids 1 to 29 are the secretion signal sequence) and SEQ ID NO: 48. The constructed plasmid was purified using a Qiagen Plasmid Plus Midi Kit (Qiagen KK) and linearized by cleavage with I-SceI (New England Biolabs, Ipswich, MA, USA) before use in transfection.

[0107] D-2. Cells, gene transfection, and luciferase assay D-2-1. Cells and gene transfer Human colon cancer-derived cell line HCT116 (Horizon Discovery, Cambridge, UK) and MLH1+ HCT116 cells (Horizon Discovery) were cultured at 37°C in a 5% CO2 incubator. The culture medium used was Dulbecco's modified Eagle's medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with calf serum (Global Life Science Technologies Japan, Tokyo, Japan; 50 ml added per 500 ml of Eagle's MEM) and L(+)-glutamine (Fujifilm Wako Pure Chemical Industries, Osaka, Japan; added to a final concentration of 2 mM). Transfection was performed using jetPEI (Polyplus-transfection, Illkirch, France) according to the manufacturer's protocol.

[0108] D-2-2. Luciferase assay Luciferase assays in HCT116 cells and MLH1+ HCT116 cells were performed as follows: 5 x 10 4 Cells were seeded into 24-well dishes, cultured overnight, and transfected with 1 μg of each construct. 24 hours after transfection, luciferase activity (RLU) was measured using 50 μl of the cell culture supernatant using the Nano-Glo Luciferase Assay System (Promega).

[0109] D-3. Results The results of measuring luciferase activity using the culture supernatant are shown in Figure 13. Similar results were obtained as with the non-secreted luciferase construct. The use of secreted luciferase eliminates the need for cell lysis and allows for convenient assays using the culture supernatant.

Claims

1. A nucleic acid construct comprising the following (1) to (3): (1) Promoter region. (2) A mutant gene sequence that has a cleavage site within the gene sequence that codes for a protein. (3) A partial region in the mutant gene sequence of (2), which is a base sequence consisting of a first homologous region and a second homologous region that can replace the partial region containing the cleavage site by homologous recombination, and which is any of the following base sequences (i) to (iii): (i) A continuous subsequence in the gene sequence encoding the protein, which subsequence includes the upstream and downstream regions adjacent to the cleavage site in the mutant gene sequence of (2). (ii) A nucleotide sequence that is homologous to the partial sequence of (i) and encodes the same amino acid sequence as the partial sequence. (iii) A contiguous partial sequence in a gene sequence encoding a protein having 80% or more sequence identity with the protein and the same activity as the protein, said partial sequence having homology with the partial sequence of (i).

2. The nucleic acid construct according to claim 1, wherein (2) comprises a stop codon upstream of the cleavage site.

3. The nucleic acid construct according to claim 1, wherein the sequence (3) is a continuous subsequence in the gene sequence encoding the protein, and is a subsequence including the upstream and downstream regions adjacent to the cleavage site in the mutant gene sequence of (2).

4. 3. The nucleic acid construct according to claim 2, wherein the sequence (3) is a continuous subsequence in the gene sequence encoding the protein, and is a subsequence including the upstream and downstream regions adjacent to the stop codon and cleavage site in the mutant gene sequence of (2).

5. The nucleic acid construct according to any one of claims 1 to 4, wherein the sequences of (ii) and (iii) have 90% or more homology with the partial sequence of (i).

6. 6. The nucleic acid construct according to claim 1, wherein in the sequence (3), the first homologous region and the second homologous region each have a chain length of at least 20 bases.

7. 7. The nucleic acid construct according to claim 1, which comprises a poly(A) addition signal between the mutant gene sequence (2) and the sequence (3).

8. The nucleic acid construct according to any one of claims 1 to 7, wherein the cleavage site is a restriction enzyme recognition site.

9. The nucleic acid construct according to any one of claims 1 to 8, wherein the nucleic acid construct is a circular nucleic acid construct comprising (2) and (3) in this order downstream of (1), or a linear nucleic acid construct obtained by cleaving the circular nucleic acid construct at the cleavage site, or a linear nucleic acid construct comprising (2) and (3) in this order downstream of (1).

10. The nucleic acid construct according to any one of claims 1 to 8, wherein the nucleic acid construct is a circular nucleic acid construct comprising (2) downstream of (1) and (3) upstream of (1), or a linear nucleic acid construct obtained by cleaving the circular nucleic acid construct at the cleavage site, or a linear nucleic acid construct comprising (2) downstream of (1) and (3) upstream of (1).

11. The nucleic acid construct according to any one of claims 1 to 10, wherein the gene sequence encodes a protein that has the effect of reducing cell viability, or a protein whose expression in a cell can be detected.

12. The nucleic acid construct according to claim 11, wherein the gene sequence is a sequence of a suicide gene, a DNA damage-inducing gene, a DNA repair inhibitor gene, a luciferase gene, a fluorescent protein gene, a cell surface antigen gene, a secreted protein gene, or a membrane protein gene.

13. A linear nucleic acid construct comprising the following (a) to (d) in the order (c), (d), (a), and (b) from upstream to downstream: (a) Promoter region. (b) The upstream region of a protein-coding gene sequence. (c) a downstream region of said gene sequence; (d) A base sequence consisting of a first homologous region and a second homologous region that can replace the upstream region and the downstream region by homologous recombination, which is any of the base sequences (d-1) to (d-3) below. (d-1) A continuous partial sequence in a gene sequence encoding the protein, the partial sequence including a region spanning the upstream region and the downstream region. (d-2) A base sequence that is homologous to a partial sequence of (d-2) and encodes the same amino acid sequence as the partial sequence. (d-3) A contiguous partial sequence in a gene sequence encoding a protein having 80% or more sequence identity with the protein and the same activity as the protein, said partial sequence having homology with the partial sequence of (d-1).

14. A reagent for measuring homologous recombination activity, comprising the nucleic acid construct according to any one of claims 1 to 13, wherein the gene sequence is a gene sequence encoding a protein whose expression in a cell can be detected.

15. A kit for measuring homologous recombination activity, comprising the reagent according to claim 14.

16. A screening system for drugs that affect homologous recombination activity, comprising the nucleic acid construct according to any one of claims 1 to 13.

17. A diagnostic agent for homologous recombination deficient cancer, comprising the nucleic acid construct according to any one of claims 1 to 13.

18. A detection agent for detecting homologous recombination restored cancer cells, comprising the nucleic acid construct according to any one of claims 1 to 13, wherein the gene sequence is a gene sequence encoding a protein whose expression in the cell can be detected.

19. The detection agent according to claim 18, wherein the homologous recombination restored cancer cells are PARP inhibitor-resistant cancer cells.

20. A companion diagnostic agent for predicting the effect of an anticancer drug on a homologous recombination-deficient cancer, comprising the nucleic acid construct according to any one of claims 1 to 13.

21. The companion diagnostic according to claim 20, wherein the anticancer agent is a DNA-damaging anticancer agent.

22. The companion diagnostic agent of claim 21, wherein the DNA-damaging anticancer agent is a PARP inhibitor.

23. A therapeutic agent for homologous recombination-restoring cancer, comprising the nucleic acid construct according to any one of claims 1 to 13, wherein the gene sequence is a gene sequence encoding a protein having the effect of reducing cell viability.

24. The therapeutic agent according to claim 23, wherein the homologous recombination restored cancer is a PARP inhibitor resistant cancer.

25. 14. The nucleic acid construct according to any one of claims 1 to 13, wherein the gene sequence encodes a protein whose expression in cells can be detected, and the nucleic acid construct is introduced into test cells in which homologous recombination activity is to be measured and into HR-non-deficient control cells having normal homologous recombination activity; measuring the expression level of the protein in the test cells and in non-HR deficient control cells; and Comparing the expression levels in the test cells with those in HR-non-deficient control cells 10. A method for measuring homologous recombination activity in a test cell, comprising:

26. Introduction of the nucleic acid construct according to any one of claims 1 to 13 into a cell having normal homologous recombination activity, followed by treatment of the cell with an individual compound from the group of compounds, or treatment of a cell having normal homologous recombination activity with an individual compound from the group of compounds, followed by introduction of the nucleic acid construct according to any one of claims 1 to 13 into the cell; and measuring the expression of said protein A method for identifying candidate drugs that affect homologous recombination activity, comprising:

27. The method according to claim 26, wherein the protein is a protein whose expression in cells can be detected as a signal, and the method comprises selecting the compound as a candidate inhibitor that inhibits homologous recombination activity when a lower protein signal is detected in compound-treated cells than in compound-untreated cells, or selecting the compound as a candidate promoter that promotes homologous recombination activity when a faster rise in the protein signal or a higher signal is detected in compound-treated cells than in compound-untreated cells.

28. The method according to claim 26, wherein the protein is a protein having the effect of reducing cell viability, and the method comprises selecting the compound as a candidate inhibitor that inhibits homologous recombination activity when the reduction in cell viability is suppressed in compound-treated cells compared to compound-untreated cells, or selecting the compound as a candidate promoter that promotes homologous recombination activity when the reduction in viability of compound-treated cells occurs more quickly than the reduction in viability of compound-untreated cells.

29. Introducing the nucleic acid construct according to any one of claims 1 to 13 into cancer cells of a cancer patient; and measuring the expression of said protein A method for diagnosing homologous recombination deficient cancer, comprising:

30. 30. The method of claim 29, wherein the cancer cells are cells isolated from the cancer patient, and the introduction of the nucleic acid construct into the cancer cells is performed ex vivo.

31. The method according to claim 29, wherein the protein is a protein whose expression within a cell can be detected as a signal, the introduction of the nucleic acid construct into the cancer cells is carried out by administering the nucleic acid construct to the cancer patient, and whether a signal of the protein can be detected from the cancer lesion is examined.

32. The method according to claim 29, wherein the protein is a secreted protein whose expression in cells can be detected, the introduction of the nucleic acid construct into the cancer cells is carried out by administering the nucleic acid construct to the cancer patient, and the activity of the protein in blood isolated from the patient after administration of the nucleic acid construct is measured.

33. Introducing the nucleic acid construct according to any one of claims 1 to 13 into cancer cells of a cancer patient who is currently undergoing PARP inhibitor treatment or who has previously been diagnosed with a homologous recombination deficient cancer; and measuring the expression of said protein A method for detecting homologous recombination-recovered cancer cells, comprising:

34. 34. The method of claim 33, wherein the cancer cells are cells isolated from the patient and the introduction of the nucleic acid construct into the cancer cells is performed ex vivo.

35. The method described in claim 33, wherein the protein is a protein whose expression in cells can be detected as a signal, the introduction of the nucleic acid construct into the cancer cells is carried out by administering the nucleic acid construct to the patient, and it is examined whether a signal of the protein can be detected from the cancer lesion.

36. Introducing the nucleic acid construct according to any one of claims 1 to 13 into cancer cells of a cancer patient; and measuring the expression of said protein A method for predicting the efficacy of an anticancer drug against a homologous recombination-deficient cancer, comprising:

37. 37. The method of claim 36, wherein the anticancer agent is a DNA damaging anticancer agent.

38. 38. The method of claim 37, wherein the DNA damaging anticancer agent is a PARP inhibitor.

39. The method according to any one of claims 36 to 38, wherein the cancer cells are cells isolated from the patient, and the introduction of the nucleic acid construct into the cancer cells is carried out ex vivo.

40. The method according to any one of claims 36 to 38, wherein the protein is a protein whose expression in cells can be detected as a signal, the introduction of the nucleic acid construct into the cancer cells is carried out by administering the nucleic acid construct to the patient, and whether a signal of the protein is detected in a cancer lesion is examined.

41. The method according to any one of claims 36 to 38, wherein the protein is a secreted protein whose expression in cells can be detected, the introduction of the nucleic acid construct into the cancer cells is carried out by administering the nucleic acid construct to the cancer patient, and the activity of the protein in blood isolated from the patient after administration of the nucleic acid construct is measured.

42. A method for treating homologous recombination-restoring cancer, comprising administering the nucleic acid construct according to any one of claims 1 to 13, wherein the gene sequence is a gene sequence encoding a protein that has the effect of reducing cell viability, to a patient having homologous recombination-restoring cancer.

43. 43. The method of claim 42, wherein the nucleic acid construct is administered locally to the patient in or near a tumor.

44. The method of claim 42 or 43, wherein the homologous recombination restored cancer is a PARP inhibitor resistant cancer.

45. A method for predicting whether a genetic mutation identified in a cancer patient is a pathogenic mutation that impairs homologous recombination activity, comprising: constructing an expression vector that expresses a mutant gene having the same mutation as the mutation; preparing an expression vector that expresses a wild-type gene that does not have the mutation; introducing a mutant gene expression vector and a wild-type gene expression vector into the gene-deficient cell, and also introducing the nucleic acid construct according to any one of claims 1 to 11; measuring the expression of the protein in cells into which any of the expression vectors and nucleic acid constructs has been introduced; wherein, when the expression level of the protein in a cell into which a mutant gene expression vector has been introduced is lower than the expression level of the protein in a cell into which a wild-type gene expression vector has been introduced, it is indicated that the mutation is a pathogenic mutation that impairs homologous recombination activity.

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