Luminescence detection method, drug delivery evaluation method, adjuvant screening method, and luminescence detection kit

Abstract

To provide a luminescence detection method for detecting whether or not a substance such as a drug has reached a target area by detecting luminescence signals from a deep part inside the body producing a non-human animal capable of stably expressing modified luciferase over a long time.SOLUTION: A disclosed luminescence detection method is a method including administering luciferase luminescent substrate and siRNA that suppresses expression of a modified luciferase gene to a transgenic non-human animal carrying modified luciferase genes. The modified luciferase gene expresses modified luciferase that makes a luminescent substrate emit light with higher luminescence intensity than wild-type D-luciferase.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to a luminescence detection method, a drug delivery evaluation method, a screening method for auxiliary agents, and a luminescence detection kit, using a transgenic non-human animal into which a modified luciferase gene has been introduced. [Background technology]

[0002] The luminescence of fireflies, known as bioluminescence, is the result of a reaction between firefly luciferin (D-luciferin) as a luminescent substrate and firefly luciferase as a catalyst. Specifically, adenosine triphosphate (ATP) and magnesium ions (Mg 2+ In the presence of D-luciferin, the luminescent substrate is converted by luciferase to the luminescent substance oxyluciferin, producing light.

[0003] In recent years, there has been an increasing need in biological and medical research to dynamically capture life phenomena at the molecular level. For example, fluorescent probes such as fluorescent dyes and fluorescent proteins are used to investigate cellular functions such as intracellular signal transduction and gene expression. In addition, the above-mentioned firefly bioluminescence system has attracted attention and is being applied in various fields such as life science, biotechnology, medicine, and pharmacology.

[0004] For example, Patent Document 1 discloses a modified luciferase with improved substrate specificity for a luminescent substrate (e.g., acarmine) having a different emission wavelength from D-luciferin, and Patent Document 2 discloses a mutant luciferase that causes luciferin to emit light with high intensity.

[0005] Non-Patent Document 1 discloses an in vivo luminescence imaging system that utilizes the firefly luminescence system. In Non-Patent Document 1, the Akaluc gene, which is a modified luciferase gene, is expressed in vivo, and the substrate Akalumine is administered in vivo to detect luminescence signals from deep within the animal's body, and the construction of a non-invasive imaging system that can detect luminescence signals 100 to 1,000 times stronger than conventional systems is being considered. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 038248 [Patent Document 2] JP 2012-235756 A [Non-patent literature]

[0007] [Non-Patent Document 1] Iwano et al., Science 359,935-939(2018) Summary of the Invention [Problem to be solved by the invention]

[0008] Non-Patent Document 1 discloses a non-invasive imaging system capable of detecting luminescence signals from deep inside an animal's living body, but since the Akaluc gene is introduced using a virus, the expression of Akaluc (modified luciferase) in the living body is transient. Therefore, the period during which Akaluc is expressed in the living body of an animal is at most several months, making it difficult to observe life phenomena over a long period of time. In other words, Non-Patent Document 1 has a problem in that it is difficult to stably express Akaluc over a long period of time.

[0009] However, unlike other organs, the brain has a blood-brain barrier, and it is known that administered substances such as drugs have difficulty reaching the brain. For this reason, intranasal administration of drugs and the like is being considered, but in this case, it is necessary to establish a technique for confirming over time that the administered substances such as drugs have reached the brain.

[0010] Therefore, the inventors conducted research with the aim of producing a non-human animal capable of stably expressing modified luciferase over a long period of time, and establishing a method for detecting whether a substance such as a drug has reached the target site by detecting a luminescent signal from deep within the body (e.g., the brain). [Means for solving the problem]

[0011] As a result of intensive research to solve the above problems, the present inventors have succeeded in developing a luminescence detection method that makes it possible to detect whether a substance such as a drug has reached a target site by administering a luminescent substrate for luciferase and an siRNA that suppresses the expression of a modified luciferase gene to a transgenic non-human animal into which a modified luciferase gene has been introduced. Specifically, the present invention has the following configuration.

[0012] [1] A luminescence detection method comprising administering a luminescent substrate for luciferase and an siRNA that suppresses expression of the modified luciferase gene to a transgenic non-human animal into which a modified luciferase gene has been introduced, A luminescence detection method, wherein the modified luciferase gene expresses a modified luciferase that causes a luminescent substrate to emit light with a luminescence intensity higher than that of wild-type D-luciferase. [2] The luminescence detection method described in [1], wherein at least a portion of the luminescent substrate and siRNA are administered intranasally and reach the brain. [3] The luminescence detection method according to [1] or [2], further comprising administering an auxiliary agent. [4] The luminescence detection method according to [3], wherein the auxiliary agent is polyethyleneimine. [5] The luminescence detection method according to any one of [1] to [4], wherein the modified luciferase contains at least one amino acid substitution selected from the group consisting of T39A, E48Q, I51V, K68R, L86S, Q134R, I136V, Q147R, G175S, N229Y, I231N, F294C, F295L, N308S, H310R, H332R, S347N, I349V, L350M, D357R, A361S, D377V, S456G, N463Y, K524R, L526S, I540T and G545D in the amino acid sequence shown in SEQ ID NO:1. [6] The luminescent detection method according to any one of [1] to [5], wherein the modified luciferase contains at least one amino acid substitution selected from the group consisting of S347N and N229Y in the amino acid sequence set forth in SEQ ID NO:1. [7] The luminescence detection method according to any one of [1] to [6], wherein the modified luciferase has the amino acid sequence shown in SEQ ID NO:3. [8] The luminescence detection method according to any one of [1] to [7], wherein the modified luciferase is an artificial enzyme, Akaluc. [9] The luminescence detection method according to any one of [1] to [8], which is used for evaluating drug delivery.

[10] A method for evaluating drug delivery, which utilizes the luminescence detection method according to any one of [1] to [9].

[11] A method for screening an auxiliary agent, comprising administering a luminescent substrate for luciferase, an siRNA that suppresses expression of the modified luciferase gene, and an auxiliary agent to a transgenic non-human animal into which a modified luciferase gene has been introduced, The modified luciferase gene expresses a modified luciferase that causes a luminescent substrate to emit light with a luminescence intensity higher than that of wild-type D-luciferase.

[12] The method for screening an auxiliary agent described in

[11] , wherein the modified luciferase contains at least one amino acid substitution in the amino acid sequence shown in SEQ ID NO:1 selected from the group consisting of T39A, E48Q, I51V, K68R, L86S, Q134R, I136V, Q147R, G175S, N229Y, I231N, F294C, F295L, N308S, H310R, H332R, S347N, I349V, L350M, D357R, A361S, D377V, S456G, N463Y, K524R, L526S, I540T and G545D.

[13] The method for screening an auxiliary agent described in

[11] or

[12] , wherein the modified luciferase contains at least one amino acid substitution selected from the group consisting of S347N and N229Y in the amino acid sequence described in SEQ ID NO:1.

[14] The method for screening an auxiliary agent according to any one of

[11] to

[13] , wherein the modified luciferase has the amino acid sequence shown in SEQ ID NO:3.

[15] The method for screening an auxiliary agent according to any one of

[11] to

[14] , wherein the modified luciferase is an artificial enzyme, Akaluc.

[16] A transgenic non-human animal having a modified luciferase gene introduced therein. A luminescent substrate for luciferase; A luminescence detection kit comprising an siRNA that suppresses the expression of a modified luciferase gene, A luminescence detection kit in which the modified luciferase gene expresses a modified luciferase that causes a luminescent substrate to emit light with a higher luminescence intensity than that of wild-type D-luciferase.

[0013] The present invention may also have the following configuration. [A] A method for screening a drug, comprising administering a luminescent substrate for luciferase, an siRNA that suppresses the expression of the modified luciferase gene, and a drug to a transgenic non-human animal into which a modified luciferase gene has been introduced. [B] An siRNA used in the luminescent detection method according to any one of [1] to [9], which is shown in SEQ ID NO:9. Effect of the Invention

[0014] According to the present invention, by administering siRNA that suppresses the expression of modified luciferase gene to a non-human animal capable of stably expressing modified luciferase for a long period of time, it is possible to detect whether a substance such as a drug has reached a target site. By using the method of the present invention, it is possible to non-invasively detect luminescence while keeping a transgenic non-human animal into which a modified luciferase gene has been introduced alive, and therefore it is possible to evaluate the arrival of a substance such as a drug in real time. [Brief description of the drawings]

[0015] [Figure 1] Figure 1 shows the design of the expression vector used to generate transgenic non-human animals. The Venus-Akaluc gene is linked to the Thy1 promoter, so that Venus-Akaluc is expressed in mammalian brain neurons. After enzyme treatment with XhoI, the target gene was inserted, and linearization was performed with ApaIxEcoRI. [Diagram 2] Figure 2 shows the results of Western blot analysis to confirm the effect of suppressing the expression of modified luciferase gene for three types of Venus-Akaluc siRNA (VA-siRNA) that suppress the expression of modified luciferase gene. A decrease in the expression level of Akaluc was confirmed when VA-siRNA 1 (GCACUCUCAUCGACAAGUAdtdt) was used. [Diagram 3] Figure 3 shows the results of measuring the luminescence intensity of cultured cells transfected with a modified luciferase gene and siRNA (VA-siRNA 1) and adding a luminescence substrate to the medium. A decrease in the amount of luminescence was observed in the VA-siRNA 1 treatment group. [Figure 4]Figure 4 shows the results of PCR performed on DNA extracted from the tails of transgenic mice. + indicates a transgenic mouse (Akaluc mouse), and - indicates a normal mouse. [Diagram 5] FIG. 5 is a schematic diagram illustrating a scheme for intranasal administration of VA-siRNA 1 together with a brain delivery aid (polyethyleneimine) to Akaluc mice. [Figure 6] Figure 6 shows a comparison of the luminescence intensity before and after administration of VA-siRNA 1. A decrease in luminescence intensity was confirmed by administration of VA-siRNA 1 (lower panel). [Figure 7] Figure 7 shows a comparison of the luminescence intensity before and after administration of VA-siRNA 1 together with a brain delivery aid (polyethyleneimine). A decrease in luminescence intensity was confirmed by administering the aid together with VA-siRNA 1 (lower panel). [Figure 8] FIG. 8 is a graph quantifying the luminescence signals of FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention will be described in detail below. The following description of the constituent elements may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "~" means a range including the numerical values ​​before and after "~" as the lower and upper limits.

[0017] In the present specification, the 20 types of amino acid residues in the amino acid sequence may be expressed by one-letter abbreviations. In this case, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y, tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine ​​(Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E, asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P. In addition, in the amino acid sequence shown in the present specification, the left end is the N-terminus and the right end is the C-terminus.

[0018] (Luminescence detection method) The present invention relates to a luminescence detection method comprising administering a luminescent substrate for luciferase and an siRNA that suppresses the expression of the modified luciferase gene to a transgenic non-human animal into which a modified luciferase gene has been introduced. Luciferase is an oxidoreductase that catalyzes a chemical reaction (luminescence reaction) in which the luminescent substrate emits light. In the present invention, the modified luciferase gene expresses a modified luciferase that causes the luminescent substrate to emit light with a luminescence intensity higher than that of wild-type D-luciferase. In this embodiment, the luminescent substrate for luciferase and the siRNA that suppresses the expression of the modified luciferase gene are preferably administered intranasally, and at least a part of the luminescent substrate and the siRNA reaches the brain after intranasal administration.

[0019] In order to treat dementia, brain diseases, and the like, various pharmaceuticals are being developed. However, the brain has a brain barrier called the blood-brain barrier (BBB), and only a very small amount (1% or less) of administered drugs may reach the brain. For this reason, development of drugs that easily permeate the brain barrier and nasal administration of drugs to efficiently deliver administered drugs to the brain are being considered. In the present invention, in a transgenic non-human animal into which a modified luciferase gene has been introduced, the modified luciferase can be expressed in the brain of the transgenic non-human animal, so that in addition to a luminescent substrate and siRNA that suppresses the expression of the modified luciferase gene, a drug is administered nasally, and a luminescence signal from the brain is detected, thereby making it possible to detect whether or not the target drug has been delivered to the brain.

[0020] In a transgenic non-human animal in which modified luciferase is expressed in the brain, when a luminescent substrate is administered into the brain, a strong luminescent signal is obtained by the reaction of the modified luciferase and the luminescent substrate. However, since siRNA that suppresses the expression of modified luciferase gene suppresses the expression of modified luciferase, when siRNA is administered into the brain and reaches the brain, the luminescent signal is weakened. The luminescence detection method of this embodiment preferably includes, for example, a step (A) of administering a luminescent substrate of luciferase to a transgenic non-human animal into which a modified luciferase gene has been introduced and detecting a luminescent signal, and a step (B) of administering an siRNA that suppresses the expression of the modified luciferase gene after the step (A) and detecting a luminescent signal. A luminescent substrate of luciferase is administered to a transgenic non-human animal, a luminescent signal is detected, and then an siRNA that suppresses the expression of the modified luciferase gene is administered, the intensity of the luminescent signal is measured, and the difference in luminescence intensity is calculated, whereby the amount and efficiency of siRNA reaching the brain can be evaluated.

[0021] In this embodiment, for example, by administering a drug together with siRNA, the delivery efficiency of the drug can be evaluated in the same manner. In this case, the drug and siRNA may be linked and administered. By administering a drug together with siRNA, it becomes possible to detect the delivery status of the drug to the brain in real time. In this way, the luminescence detection method of the present invention can be used for drug delivery evaluation. The present invention may also relate to a drug delivery evaluation method using the above-mentioned luminescence detection method.

[0022] In this way, a luminescent substrate and an siRNA that suppresses the expression of the modified luciferase gene are administered to a transgenic non-human animal into which a modified luciferase gene has been introduced, and the luminescence intensity from the brain is measured in real time, thereby making it possible to evaluate the delivery of a drug in real time. By using the luminescence detection method of the present invention, it is also possible to perform drug delivery evaluation in real time.

[0023] In this embodiment, it is preferable to express the modified luciferase in the brain, but it can also be expressed in organs other than the brain (for example, the heart, lungs, liver, stomach, kidneys, etc.). For example, by linking a promoter that promotes expression in each organ to the modified luciferase gene, the modified luciferase can also be expressed in organs other than the brain. On the other hand, by linking a promoter that promotes brain-specific expression to the modified luciferase gene, a transgenic non-human animal that expresses the modified luciferase specifically in the brain can be obtained. In addition, by using a promoter that promotes expression in multiple organs, a transgenic non-human animal that expresses the modified luciferase in the whole body can also be obtained. By using a transgenic non-human animal that expresses the modified luciferase in organs other than the brain, it is also possible to evaluate drug delivery to organs other than the brain.

[0024] The luminescence detection method of the present invention is a non-invasive method, and by using such a method, luminescence can be detected in real time while maintaining the survival of a transgenic non-human animal. Therefore, it is also possible to measure the time it takes for a drug to reach a specific organ such as the brain. In addition, by detecting the difference in luminescence intensity before and after administration of siRNA, it is also possible to detect the amount and speed of the drug in real time.

[0025] <Transgenic non-human animals> A modified luciferase gene is introduced into the transgenic non-human animal used in the luminescence detection method of the present invention. In the transgenic non-human animal, since the modified luciferase gene is inserted into the genome, the transgenic non-human animal can stably express the modified luciferase for a long period of time. The modified luciferase can cause the luminescence substrate to emit light with a luminescence intensity higher than that of wild-type D-luciferase. Therefore, in the transgenic non-human animal in which the modified luciferase is expressed, it is possible to detect a luminescence signal from deep inside the living body. For example, it is also possible to detect a luminescence signal from inside the brain covered by the skull. In addition, in the present invention, since the modified luciferase gene is inserted into the genome, the modified luciferase can be expressed stably for a long period of time. Therefore, it is possible to detect a luminescence signal stably for a long period of time. By using such a transgenic non-human animal, a non-invasive imaging system that enables luminescence detection in real time for a long period of time can be constructed.

[0026] In transgenic non-human animals, the modified luciferase gene is also introduced into the germline, so that the modified luciferase gene is inherited to the next generation. Therefore, the luminescence signal from the modified luciferase and the luciferase luminescence substrate can be detected in the next generation of non-human animals, making it possible to construct a non-invasive imaging system that transcends generations.

[0027] Examples of transgenic non-human animals used in the luminescence detection method of the present invention include mammals other than humans, such as mice, rats, guinea pigs, rabbits, pigs, dogs, cats, monkeys, sheep, cows, and horses, and among these, rodents (rodents) such as mice, rats, and guinea pigs are preferred, and mice are more preferred.

[0028] <Modified luciferase gene / modified luciferase> In the transgenic non-human animal, a modified luciferase that causes a luminescent substrate to emit light with a luminescence intensity higher than that of wild-type D-luciferase is expressed. The modified luciferase has a mutation in the amino acid sequence compared to the wild-type D-luciferase. In this specification, the mutation refers to a substitution, deletion, or insertion. In the present invention, the mutation is preferably a substitution or deletion, and more preferably a substitution.

[0029] The modified luciferase preferably contains an amino acid sequence that shows 80% or more sequence identity with the amino acid sequence described in SEQ ID NO: 1 (wild-type D-luciferase derived from Common Eastern Firefly). The amino acid sequence identity between the modified luciferase and the wild-type D-luciferase is more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more. The amino acid sequence identity can be determined using a method well known to those skilled in the art, sequence analysis software, etc. For example, the blastp program of the BLAST algorithm and the fasta program of the FASTA algorithm can be mentioned. The sequence identity of the amino acid sequence here is a value expressed as a percentage, which is obtained by comparing the amino acid sequence of the modified luciferase with that of the wild-type D-luciferase, and expressing the frequency of the appearance of the same amino acid at the same site.

[0030] In the modified luciferase, the substrate specificity for the luminescent substrate acarmine is improved compared to the wild-type D-luciferase. The substrate specificity for the luminescent substrate can be determined by the intensity of the luminescence of the luminescent substrate, as described below.

[0031] The luminescence intensity at which the modified luciferase makes the luminescent substrate luminous is preferably at least twice, more preferably at least 5 times, even more preferably at least 10 times, even more preferably at least 100 times, even more preferably at least 500 times, and particularly preferably at least 1000 times. The upper limit of the luminescence intensity at which the modified luciferase makes the luminescent substrate luminous is not particularly limited, and the upper limit may be about 1 million times. When measuring the luminescence intensity, for example, the luminescence intensity at the maximum luminescence wavelength when the target luciferase is reacted with the luminescent substrate is measured. Then, the integrated value of the luminescence intensity measured for one minute can be calculated to obtain comparative data.

[0032] The modified luciferase preferably contains at least one amino acid substitution selected from the group consisting of T39A, E48Q, I51V, K68R, L86S, Q134R, I136V, Q147R, G175S, N229Y, I231N, F294C, F295L, N308S, H310R, H332R, S347N, I349V, L350M, D357R, A361S, D377V, S456G, N463Y, K524R, L526S, I540T and G545D in the amino acid sequence of the wild-type D-luciferase set forth in SEQ ID NO:1. In the present specification, an amino acid substitution is expressed by a combination of a one-letter abbreviation representing the amino acid residue before substitution, a number representing the position of the amino acid residue where the amino acid substitution occurs (the position from the N-terminus in a specific amino acid sequence), and a one-letter abbreviation representing the amino acid residue after substitution. For example, if threonine at position 39 is replaced by alanine, it is expressed as "T39A".

[0033] In particular, the modified luciferase preferably contains at least one amino acid substitution selected from the group consisting of S347N and N229Y.Moreover, the modified luciferase preferably contains amino acid substitutions at T39A, E48Q, I51V, K68R, L86S, Q134R, I136V, Q147R, G175S, N229Y, I231N, F294C, F295L, N308S, H310R, H332R, S347N, I349V, L350M, D357R, A361S, D377V, S456G, N463Y, K524R, L526S, I540T, and G545D.

[0034] The modified luciferase gene preferably has the nucleotide sequence shown in SEQ ID NO: 2, and the modified luciferase preferably has the amino acid sequence shown in SEQ ID NO: 3. That is, the modified luciferase is preferably the artificial enzyme Akaluc.

[0035] The modified luciferase gene is preferably linked to a promoter that promotes selective expression in brain nerve cells. Examples of promoters that promote selective expression in brain nerve cells include Thy1 promoter, synapsin-I promoter, synuclein 1 promoter, calcium / calmodulin-dependent kinase IIα (CAMKIIα) promoter, etc. Among them, it is particularly preferable that the promoter that promotes selective expression in brain nerve cells is Thy1 promoter.

[0036] <Method for producing transgenic non-human animals> The transgenic non-human animal used in the luminescence detection method of the present invention can be produced by a known production method. For example, a method of producing a chimera after introducing DNA into an embryonic stem cell (ES cell), a method of producing a chimera after introducing DNA into a pronucleus of a fertilized egg by microinjection, etc. can be used. The method of producing a chimera after introducing DNA into an embryonic stem cell (ES cell) will be described below.

[0037] (i) Construction of a targeting vector to be introduced into ES cells The modified luciferase gene (Venus-Akaluc portion) is obtained by a method such as PCR from a plasmid vector (for example, pcDNA3 Venus-Akaluc (provided by RIKEN BRC)) into which the modified luciferase gene has been previously inserted.

[0038] Then, a targeting vector is obtained by inserting the modified luciferase gene. When constructing a targeting vector, a tissue-specific plasmid may be selected. For example, a tissue-specific plasmid linked with a promoter that promotes selective expression in brain nerve cells may be used. Examples of promoters that promote selective expression in brain nerve cells include Thy1 promoter, synapsin-I promoter, synuclein 1 promoter, calcium / calmodulin-dependent kinase IIα (CAMKIIα) promoter, etc. Among them, it is particularly preferable that the promoter that promotes selective expression in brain nerve cells is Thy1 promoter.

[0039] The targeting vector into which the modified luciferase gene has been inserted may have a configuration in which, for example, the following fragments are linked together: Fragment 1) Thy1 promoter that expresses the target protein in brain neurons Fragment 2) Venus-Akaluc cDNA, a gene encoding modified luciferase Fragment 3) pUC18 sequence that can determine the presence or absence of genes incorporated into the plasmid Fragment 4) Ampicillin (amp) resistance gene (for positive selection)

[0040] (ii) Introduction of a targeting vector into ES cells It is preferable to use a targeting vector that has been purified as necessary. For example, the targeting vector may be linearized with an appropriate restriction enzyme and then separated for use. When the targeting vector is introduced into the ES cells of a non-human animal, the targeting vector is introduced into the ES cells by electroporation. Then, the ES cells into which the targeting vector has been introduced are microinjected into mouse blastocysts. At this time, it is preferable to inject at least one ES cell, usually about 10 to 15 ES cells, into the blastocoel of the blastocyst. The microinjected embryo is transplanted into the uterus of a pseudopregnant female mouse that will serve as a surrogate mother. The female mouse after transplantation is kept under normal breeding conditions and allowed to give birth to a baby mouse (chimeric mouse).

[0041] By mating the chimeric mice (founder mice) born from the foster parents with wild-type mice, transgenic mice expressing modified luciferase (heterozygous mice (+ / -)) can be obtained. Furthermore, homozygous mice (+ / +) can be obtained by mating these heterozygous mice (+ / -) with each other.

[0042] The selection of transgenic non-human animals can be carried out by detecting whether or not the modified luciferase gene is incorporated into the chromosomal DNA of the resulting offspring. The detection can be carried out according to a conventional method, for example, by extracting DNA from the tail pieces of the offspring, followed by PCR or Southern blotting to confirm germline transmission.

[0043] <sirna> The siRNA used in the luminescence detection method of the present invention is not particularly limited as long as it suppresses the expression of modified luciferase gene. For example, GCACUCUCAUCGACAAGUA dtdt can be exemplified as such an siRNA. Note that the present invention may relate to the siRNA used in the above-mentioned luminescence detection method.

[0044] The dosage of siRNA used in the luminescent detection method of the present invention is not particularly limited as long as it is an amount that can suppress the expression of the modified luciferase gene. For example, when administering to the transgenic mouse described above, it is preferable to administer it at 0.5 to 100.0 μg.

[0045] <Luminescent Substrate> Examples of the luminescent substrate include those described in paragraph 0108 of International Publication No. 2018 / 038248. For example, firefly luciferin (D-luciferin), acarmine TM , acarmine-OH, 6-acarmine, O-acarmine, monoene-NMe2, monoene-NH2, monoene-OH, biphenyl, etc. Adenosine triphosphate (ATP) and magnesium ion (Mg 2+ In the presence of , the luminescent substrate is converted to a luminescent substance by luciferase, resulting in luminescence. The color of the luminescence produced in the luminescence reaction depends on the luminescent substrate. For example, when the luminescent substrate is D-luciferin, the luminescence peak is at about 560 nm, and when the luminescent substrate is acarmine, the luminescence peak is at about 570 nm. TM In this case, the emission peak is 675 nm.

[0046] The amount of the luminescent substrate administered is not particularly limited as long as it is an amount at which luminescence can be detected by reacting the luminescent substrate with luciferase. For example, when administered to a transgenic mouse as described above, the amount can be 1 to 100 nmol.

[0047] <Adjuvants> In one embodiment of the luminescence detection method of the present invention, the method may further include a step of administering an auxiliary agent. In this specification, the auxiliary agent refers to an agent that promotes the delivery of siRNA or a drug that suppresses the expression of a modified luciferase gene to the brain. Specifically, by using the auxiliary agent, the amount of siRNA or a drug that reaches the brain is preferably 1.2 times or more, more preferably 1.5 times or more, and even more preferably 2.0 times or more, compared to the case where the auxiliary agent is not used. In one embodiment of the luminescence detection method of the present invention, the siRNA or a drug can be delivered to the brain more efficiently by administering the auxiliary agent together with the siRNA or a drug.

[0048] In this embodiment, an auxiliary agent that promotes the luminescent substrate reaching the brain may be included. By using the auxiliary agent, the amount of the luminescent substrate reaching the brain is preferably 1.2 times or more, more preferably 1.5 times or more, and even more preferably 2.0 times or more, compared to the case where the auxiliary agent is not used.

[0049] Examples of auxiliary agents include polymers, carbohydrates such as monosaccharides, disaccharides, oligosaccharides, and polysaccharides (e.g., pullulan), lipids, phospholipid compounds, amino acids, peptides, proteins, DNA compounds, and particles formed by combining these (such as extracellular vesicles).

[0050] Examples of the polymer include polyethyleneimine, polyethylene glycol, polylysine, etc. Among them, it is preferable to use polyethyleneimine as an auxiliary agent. By using polyethyleneimine as an auxiliary agent, the amount of siRNA that suppresses the expression of the modified luciferase gene that reaches the brain can be more effectively increased.

[0051] The preferred dosage of the adjuvant is not particularly limited, and is preferably adjusted appropriately depending on the type of adjuvant and the subject to be supported. For example, when administered to the transgenic mouse described above, the dosage of the adjuvant can be 10 to 1000 nmol.

[0052] Examples of carbohydrates include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as maltose, lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, pullulan, starch, cellulose, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myo-inositol, etc. In the present invention, it is preferable to use, as the adjuvant, disaccharides such as maltose, lactose, sucrose, trehalose, cellobiose, etc., it is more preferable to use at least one selected from maltose and trehalose, and it is even more preferable to use trehalose.

[0053] Examples of lipids include positively charged lipids such as 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), cholesteryl-3β-N-(dimethylaminoethyl)carbamate (DC-chol), and heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); neutral lipids such as cholesterol, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG2000), lecithin, phosphatidylethanolamine, and sphingomyelin; and negatively charged lipids such as phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine.

[0054] In this embodiment, various extracellular vesicles can be used instead of the auxiliary agent. Examples of the extracellular vesicles include exosomes. For example, by encapsulating a drug or the like in an extracellular vesicle covered with a lipid membrane, it is possible to deliver the drug to the brain more reliably.

[0055] (Screening Method) The present invention may also relate to a method for screening an auxiliary agent, comprising administering a luminescent substrate for luciferase, an siRNA that suppresses expression of the modified luciferase gene, and an auxiliary agent to a transgenic non-human animal into which a modified luciferase gene has been introduced. In the present invention, the modified luciferase gene expresses a modified luciferase that causes the luminescent substrate to emit light with a luminescence intensity higher than that of wild-type D-luciferase. The transgenic non-human animal, the luminescent substrate for luciferase, and the siRNA used in the screening method of the present invention may be those described above.

[0056] The screening method of this embodiment preferably includes, for example, a step (A) of administering a luminescent substrate for luciferase to a transgenic non-human animal into which a modified luciferase gene has been introduced and detecting a luminescent signal, and a step (B) of administering an siRNA that suppresses the expression of the modified luciferase gene and a target auxiliary agent and detecting the luminescent signal after the step (A). After administering the luminescent substrate for luciferase to the transgenic non-human animal and detecting the luminescent signal, an siRNA that suppresses the expression of the modified luciferase gene and an auxiliary agent are administered, the intensity of the luminescent signal is measured, and the difference in the luminescence intensity is calculated, whereby the ability of the auxiliary agent to promote delivery to the brain can be evaluated.

[0057] The screening method for auxiliary agents of the present invention makes it possible to screen auxiliary agents for more efficiently delivering siRNA or drugs into the brain, thereby making it possible to select appropriate auxiliary agents at low cost during the development of pharmaceuticals, etc.

[0058] The present invention may also relate to a method for screening a medicine (drug), comprising administering a luminescent substrate for luciferase, an siRNA that suppresses the expression of a modified luciferase gene, and a drug to the above-mentioned transgenic non-human animal. In this case, the drug and the siRNA may be linked and administered. In the method for screening a drug, for example, a drug targeting a molecule or tissue in the brain and an siRNA are simultaneously administered, whereby it is possible to analyze whether or not the drug has reached the brain and the amount of the drug that has reached the brain. This makes it possible to select a drug that can reach the brain and to analyze the dosage of the drug.

[0059] (Luminescence detection kit) The present invention may relate to a luminescence detection kit including a transgenic non-human animal into which a modified luciferase gene has been introduced, a luminescence substrate for luciferase, and an siRNA that suppresses the expression of the modified luciferase gene. In the present invention, the modified luciferase gene expresses a modified luciferase that causes the luminescence substrate to emit light with a luminescence intensity higher than that of wild-type D-luciferase. The transgenic non-human animal, the luminescence substrate for luciferase, and the siRNA used in the luminescence detection kit of the present invention may be those described above. In addition, the luminescence detection kit of the present invention may further include an auxiliary agent as described above. EXAMPLES

[0060] The features of the present invention will be described in more detail below with reference to examples. The materials, amounts, ratios, processing contents, processing procedures, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the specific examples shown below.

[0061] (Example 1: Luminescence observation of cultured cells) <Expression vector> The expression vector pThy1.2, provided by Friedrich Miescher Institute, Basel, Switzerland, was cleaved with the restriction enzymes described in Journal of Neuroscience Methods 71 ​​(1) 3-9 1997 and used.

[0062] The Venus-Akaluc portion was amplified by PCR from the plasmid gene pcDNA3 Venus-Akaluc (Non-Patent Document 1, provided by RIKEN BRC) encoding the target protein (Akaluc) using the following two primer sets. Forward: 5'-cctagaggatctcgagatggtgagcaagggcgag-3' (SEQ ID NO: 5) Reverse: 5'-gaggaaggacctcgagttacacggcgatcttgcc-3' (SEQ ID NO: 6) The amplified fragment was 2376 bp in length, with the last 3 bp being a stop codon, and the expressed protein contained 791 amino acids.

[0063] The cDNA of the Venus-Akaluc portion amplified by PCR was inserted into the XhoI site in the expression vector (Figure 1). The expression vector was confirmed by sequencing. In this way, the expression vector was constructed. The structure of the expression vector is as follows: 1) Thy1 promoter (SEQ ID NO: 4) for expressing a target protein in brain neurons 4118bp 2) Venus-Akaluc cDNA 2376bp, which is the gene encoding the protein to be expressed 3) pUC18 sequence (2363bp) that can determine the presence or absence of genes incorporated into the plasmid 4) Ampicillin (amp) resistance gene (for positive selection) 861bp

[0064] <Cell culture> HeLa cells (JCRB Cell Bank, Osaka, Japan) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and 1.0 x 10 5 Cells were seeded. 1.5 μg of pcDNA3 Venus-Akaluc was transfected using X-treamGeneHP DNA Transfection Reagent (06366236001; Roche, Basel, Switzerland), and 24 μL of three types of Venus-Akaluc siRNA (VA-siRNA) were simultaneously transfected using Lipofectamine RNAiMAX Transfection Reagent (13778030; Invitrogen, Waltham, MA, USA). The sequences of the siRNAs were VA-siRNA 1: GCACUCUCAUCGACAAGUAdtdt (SEQ ID NO: 9), VA-siRNA 2: CGAACGCUCUCAUCGACAAdtdt (SEQ ID NO: 10), and VA-siRNA 3: AGCUACCGAUCAUACGAAAdtdt (SEQ ID NO: 11) (Nippon Gene Co., Ltd, Tokyo, Japan). In SEQ ID NOs: 9 to 11, dt represents deoxythymidine. After 24 hours, the cells were washed with washing solution (1 x PBS(-); 137 mM NaCl, pH 7.4, 2.68 mM KCl, 20.4 mM Na2PO4, 1.76 mM KH2PO4) and 100 μL of sample buffer (75 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 25% glycerol, 5% β-mercaptoethanol) was added. The cells were sheared 20 times using a 23G needle and stored at -20℃ until use in Western blotting.

[0065] <Western Blot> The samples (sample buffer containing cells) were electrophoresed using a 5%-20% Tris-glycine gradient gel (ATTO, Tokyo, Japan) at a constant current (90 mA, 1 h). Then, the proteins were transferred to a polyvinylidene difluoride membrane (IPVH00010; EMD Millipore, Burlington, MA, USA) soaked in methanol at a constant voltage (90 V, 1 h) using a Mini Trans-Blot cell system (Bio-Rad, Hercules, CA, USA). After transfer, the membrane was blocked with blocking buffer (20 mM Tris-HCl, pH 7.5, 133 mM NaCl, 0.1% Tween 20 [TBS-T], 5% skim milk). Then, the primary antibody was dissolved in 1% skim milk buffer (TBS-T containing 1% skim milk) and placed on the membrane and incubated for 1 h. The primary antibodies used were anti-actin antibody (A5044; Sigma, St Louis, MO, USA, Rabbit; 1:5000) and anti-GFP antibody (A11122; Invitrogen, Waltham, MA, USA, Rabbit; 1:1000). After washing twice with 1% skim milk buffer, the secondary antibodies were dissolved in the same buffer and incubated for 1 h as for the primary antibodies. The secondary antibodies used were goat anti-mouse IgG (ab6789; Abcam, Cambridge, UK; 1:30000) and goat anti-rabbit IgG (ab6721; Abcam; 1:30000). After washing three times with TBS-T, protein detection was performed according to the ECL or ECL prime (Amersham Pharmacia Biotech, Amersham, UK) protocol.

[0066] <Result> Western blot analysis confirmed that the expression level of Akaluc was reduced when VA-siRNA 1 (GCACUCUCAUCGACAAGUAdtdt) was used among the three types of Venus-Akaluc siRNA (VA-siRNA) (Figure 2).

[0067] <Luminescence observation> 1.0 x 10 in a 35 mm dish 5 HeLa cells were seeded. 2.0 μg of pcDNA3 Venus-Akaluc was transfected using X-treamGeneHP DNA Transfection Reagent. 1 24 μL of VA-siRNA was simultaneously transfected using Lipofectamine RNAiMAX Transfection Reagent. After 24 hours, 3 mM AkaLumine n-hydrochloride (AkaLumine) was added as a luminescence substrate. TM 10 μL of 1,2-dichlorophenyl ether hydrochloride was added to the medium, and the luminescence intensity of the cultured cells was measured every 5 minutes for a total of 15 minutes using an IVIS LuminaXRMS seriesIII ver4.7 (Perkin Elmer, Waltham, MA, USA).

[0068] <Result> When the effect of VA-siRNA 1 was further examined using cultured cells, a decrease in luminescence intensity was observed in the VA-siRNA 1-treated group, consistent with the results of Western blot analysis (Fig. 3).

[0069] (Example 2: Luminescence observation using transgenic mice) (Creation of transgenic mice) <Construction of expression vector> The expression vector pThy1.2, kindly provided by the Friedrich Miescher Institute, Basel, Switzerland, was used after cleavage with the restriction enzymes described in Journal of Neuroscience Methods 71 ​​(1) 3-9 1997.

[0070] The Venus-Akaluc portion was amplified by PCR from the plasmid gene pcDNA3 Venus-Akaluc (Non-Patent Document 1, provided by RIKEN BRC) encoding the target protein (Akaluc) using the following two primer sets. Forward: 5'-cctagaggatctcgagatggtgagcaagggcgag-3' (SEQ ID NO: 5) Reverse: 5'-gaggaaggacctcgagttacacggcgatcttgcc-3' (SEQ ID NO: 6) The amplified fragment was 2376 bp in length, with the last 3 bp being a stop codon, and the expressed protein contained 791 amino acids.

[0071] The cDNA of the Venus-Akaluc portion amplified by PCR was inserted into the XhoI site in the expression vector (Figure 1). The expression vector was confirmed by sequencing. In this way, the expression vector was constructed. The composition of the expression vector is as follows. Akaluc mice were generated so that Venus-tagged Akaluc is expressed under the control of the Thy1 promoter, which is strongly activated in nerve cells. 1) Thy1 promoter (SEQ ID NO: 4) for expressing a target protein in brain neurons 4113bp 2) Venus-Akaluc cDNA 2376bp, which is the gene encoding the protein to be expressed 3) pUC18 sequence (2369bp) that can determine the presence or absence of genes incorporated into the plasmid 4) Ampicillin (amp) resistance gene (for positive selection) 861bp

[0072] <Creation of transgenic mice> The expression vector obtained above was linearized by enzyme digestion with ApaI and EcoRI, and dissolved in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA·Na2) to a concentration of 100 ng / μL. Then, ES cells were electroporated using a Gene Pulser (800 V and 3 mF at 0.4 cm electrode distance, Bio-Rad).

[0073] E14 cells derived from mouse blastocyst line 129 were used as the ES cell line (Hooper et al., Nature 326, 292-295 1987). Cell culture and cell bombardment were performed according to Itohara et al., Cell 72, 337-348 199. Chimeric mice were produced according to the method described in Nature 309, 255-256, 1984. ES cells were microinjected into C57BL / 6J blastocysts 3.5 days after mating. After injection, the embryos were transferred to the uterus of pseudopregnant ICR mice. The obtained chimeric mice were visually confirmed for coat color, and then further crossed with C57BL / 6J mice to produce heterozygous mice (transgenic mice). In this specification, the transgenic mice obtained by the above procedure are sometimes called brain visualization mice (Akaluc mice).

[0074] The genotypes of the transgenic mice were determined by PCR analysis of genomic DNA prepared from the tails using the following two primer sets: Forward: 5'- ggacacgctgaacttgtgg -3' (SEQ ID NO: 7) Reverse: 5'-ctggctgacctgtagctttcc-3' (SEQ ID NO: 8) The size of the PCR product was 162 bp (Figure 4).

[0075] Mice for brain visualization (Akaluc mice) were housed in a temperature- and humidity-controlled room with automatic lighting and a 12-h / 12-h light / dark cycle. Mice were housed 3–5 per cage and had free access to water and food.

[0076] <In vivo imaging> Akaluc mice were intranasally administered 9 μL of 0.5 mM Akalumine, and the results were measured every 3 minutes for a total of 30 minutes or every 5 minutes for a total of 25 minutes using IVIS LuminaXRMS seriesIII ver4.7. Next, VA-siRNA 1 and polyethyleneimine (101000040; polyplus, Illkirch, France) were incubated in 50 μL of 10% glucose solution for 30 minutes to prepare a suspension containing 8 μg, 16 μg, or 24 μg of VA-siRNA 1 at an N / P ratio of 7. 50 μL of this suspension was intranasally administered to Akaluc mice using a pipette. 24 hours after intranasal administration of the suspension, 9 μL of 0.5 mM Akalumine was intranasally administered, and the results were measured every 3 minutes for a total of 30 minutes or every 5 minutes for a total of 25 minutes using IVIS LuminaXRMS seriesIII ver4.7.

[0077] <Statistical analysis> Statistical analysis was performed using the Shapiro-Wilk test followed by t-test, with P < 0.05 considered statistically significant.

[0078] <Result> As shown in Figure 5, VA-siRNA 1 was administered intranasally together with a brain delivery aid (polyethyleneimine) to Akaluc mice administered Akalumine, a luminescent substrate, and the delivery efficiency of the brain delivery aid was calculated by comparing the amount of luminescence before and after siRNA treatment. To verify the in vivo effect of VA-siRNA 1, VA-siRNA 1 (8 μg, 16 μg, 24 μg) was administered intranasally to Akaluc mice using a pipette, and luminescence was measured every 5 minutes for a total of 25 minutes. A decrease in the amount of luminescence was observed after VA-siRNA 1 treatment at all concentrations (Figure 6).

[0079] Akaluc mice were intranasally administered 1-18 μg of VA-siRNA together with a brain delivery aid (polyethyleneimine), and the luminescence intensity was compared before and 30 minutes after siRNA treatment. A significant decrease in the luminescence intensity was observed after treatment (Figures 7 and 8).

[0080] In the present invention, we have succeeded in observing the delivery efficiency of the brain delivery aid in real time without sacrificing animals. In the future, we believe that the delivery efficiency to the brain can be further improved by using various extracellular vesicles instead of the brain delivery aid.

[0081] SEQ ID NO:1 (wild-type D-luciferase derived from Common eastern firefly) Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro 1 5 10 15 Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg 20 25 30 Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu 35 40 45 Val Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala 50 55 60 Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val 65 70 75 80 Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu 85 90 95 Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg 100 105 110 Glu Leu Leu Asn Ser Met Asn Ile Ser Gln Pro Thr Val Val Phe Val 115 120 125 Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro 130 135 140 Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly 145 150 155 160 Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe 165 170 175 Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile 180 185 190 Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val 195 200 205 Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp 210 215 220 Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val 225 230 235 240 Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 245 250 255 Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu 260 265 270 Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val 275 280 285 Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr 290 295 300 Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser 305 310 315 320 Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile 325 330 335 Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr 340 345 350 Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe 355 360 365 Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val 370 375 380 Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly 385 390 395 400 Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly 405 410 415 Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe 420 425 430 Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln 435 440 445 Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn Ile 450 455 460 Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu 465 470 475 480 Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys 485 490 495 Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu 500 505 510 Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly 515 520 525 Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys 530 535 540 Gly Gly Lys Ser Lys Leu 545 550

[0082] Sequence No. 2 (Modified Luciferase Gene) atggaagatg ccaaaaacat taagaagggc ccagcgccgt tctacccact cgaagacggg 60 accgccggcg agcagctgca caaagccatg aagcgctacg ccctggtgcc cggcgccatc 120 gcctttaccg acgcacatat tcaggtggac gttacctacg ccgagtactt cgagatgagc 180 gttcggctgg cagaagctat gaggcgctat gggctgaata caaaccatcg gatcgtggtg 240 tgcagcgaga atagctcgca gttcttcatg cccgtgttgg gtgccctgtt catcggtgtg 300 gctgtggccc cagctaacga catctacaac gagcgcgagc tgctgaacag catgggcatc 360 agccagccca ccgtcgtatt cgtgagcaag aaagggctgc gaaaggtcct caacgtgcaa 420 aagaagctac cgatcatacg aaagatcatc atcatggata gcaagaccga ctaccagggc 480 ttccaaagca tgtacacctt cgtgacttcc catttgccac ccagcttcaa cgagtacgac 540 ttcgtgcccg agagcttcga ccgggacaaa accatcgccc tgatcatgaa cagtagtggt 600 agtacaggat tacccaaggg cgtagcccta ccgcaccgca ccgcttgtgt ccgattcagt 660 catgcccgcg accccatctt cggctaccag aacatccccg acaccgctat cctcagcgtg 720 gtgccatttc accacggctt cggcatgttc accacgctgg gctacttgat ctgcggcttt 780 cgggtcgtgc tcatgtaccg cttcgaggag gagctattct tgcgcagctt gcaagactat 840 aagattcaat ctgccctgct ggtgcccaca ctatttagct gcctcgctaa gagcactctc 900 atcgacaagt acgacctaag cagcttgcgc gagatcgcca gcggcggggc gccgctcagc 960 aaagggtag gtgaggccgt ggccaaacgc ttccgcctac caggcatccg ccagggctat 1020 ggcctgacag aaaaaccaa cgccgtcatg atcaccccccg agggggaccg taagcctggc 1080 tcagtaggca aggtggtgcc cttcttcgag gctaaggtgg tagacttggt caccggtaag 1140 acactgggtg tgaaccagcg cggtgagctg tgcgtccgtg gccccatgat catgagcggc 1200 tacgttaaca accccgaggc tacgaacgct ctcatcgaca aggacggctg gctgcacagc 1260 ggcgacatcg cctactggga cgaggacgag cacttcttca tcgtggaccg gctgaagagc 1320 ctgatcaaat acaagggcta ccaggtagcc ccagccgaac tggagggcat cctgctgcaa 1380 cacccctaca tcttcgacgc cggagtcgcc ggcctgcccg acgacgatgc cggcgagctg 1440 cccgccgcag tcgtcgtgtt ggaacacggt aaaaccatga ccgagaaaga gatcgtggac 1500 tatgtggcca gccaggttac aaccgccaag aagctgcgcg gtggtgttgt gtttgtggat 1560 gaagtcccta gaggatcgac cggcaagtta gacgcccgca agatccgga gattctcact 1620 aaggccaaga aggacggcaa gatcgccgtg taa 1653

[0083] SEQ ID NO: 3 (Modified Luciferase) Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro 1 5 10 15 Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg 20 25 30 Tyr Ala Leu Val Pro Gly Ala Ile Ala Phe Thr Asp Ala His Ile Gln 35 40 45 Val Asp Val Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala 50 55 60 Glu Ala Met Arg Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val 65 70 75 80 Cys Ser Glu Asn Ser Ser Gln Phe Phe Met Pro Val Leu Gly Ala Leu 85 90 95 Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg 100 105 110 Glu Leu Leu Asn Ser Met Gly Ile Ser Gln Pro Thr Val Val Phe Val 115 120 125 Ser Lys Lys Gly Leu Arg Lys Val Leu Asn Val Gln Lys Lys Leu Pro 130 135 140 Ile Ile Arg Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly 145 150 155 160 Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Ser Phe 165 170 175 Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile 180 185 190 Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val 195 200 205 Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp 210 215 220 Pro Ile Phe Gly Tyr Gln Asn Ile Pro Asp Thr Ala Ile Leu Ser Val 225 230 235 240 Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 245 250 255 Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu 260 265 270 Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val 275 280 285 Pro Thr Leu Phe Ser Cys Leu Ala Lys Ser Thr Leu Ile Asp Lys Tyr 290 295 300 Asp Leu Ser Ser Leu Arg Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser 305 310 315 320 Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe Arg Leu Pro Gly Ile 325 330 335 Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Asn Ala Val Met Ile Thr 340 345 350 Pro Glu Gly Asp Arg Lys Pro Gly Ser Val Gly Lys Val Val Pro Phe 355 360 365 Phe Glu Ala Lys Val Val Asp Leu Val Thr Gly Lys Thr Leu Gly Val 370 375 380 Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly 385 390 395 400 Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly 405 410 415 Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe 420 425 430 Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln 435 440 445 Val Ala Pro Ala Glu Leu Glu Gly Ile Leu Leu Gln His Pro Tyr Ile 450 455 460 Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu 465 470 475 480 Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys 485,490,495 Glu Ile Val Asp Tyr Val Val Ser Gln Val Thr Thr Val Lys Lys Leu 500 505 510 Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Arg Gly Ser Thr Gly 515,520,525 Lys Leu Asp With Arg Lys And Arg Glu With Leu Thr Lys Ala Lys Lys 530 535 540 Asp Gly Lys Ile Ala Val 545,550

[0084] Movie 4(Thy1 is on the cover of Thy1 cagagaccgg gaaccaact agccttttaa aaacatagt acaggagcca gcagatggc 60 tcagtgggta aaggtgccta ccagcaagcc tgacagcctg agttcagtcc ccacgaacta 120 cgtggtagga gaggaccaac caacctgga aatctgttct gcaacacat gctcacacac 180 acacacaca atagtataaa caatttaaa ttcatttaa aaataatttg taaacaaat 240 cattagcaca ggttttagaa agagcctctt ggtgacatca agttgatgct gtagatgggg 300 tatcattcct gaggacccaa aaccgggtct cagcctttcc ccattctgag agttctctct 360 tttctcagcc actagctgaa gagtagagtg gctcagcact gggctcttga gttcccaagt 420 cctacaactg gtcagcctga ctactaacca gccatgaaga aacaagggt ggatggggctg 480 agtctgctgg gatgggagtg gagttagtaa gtggccatgg atgtaatgac cccagcaatg 540 ctggctagaa ggcatgcctc ctttccttgt ctggagacgg aacgggagg atcatcttgt 600 actcacagaa gggagaaacat tctagctggt tgggccaaaa tgtgcaagtt cacctggagg 660 tggtggtgca tgcttttaac tccagtactc aggaggcagg gccaggtgga tctctgtgag 720 ttcaagacca gcctgcacta tggagagagt tttgggacag ccagagttac agaaaaat 780 cctggtggaa aatctgaaag aaagagaga agaagaaag agaagaagga agaagaaag 840 aaagagtggc aggcaggcag ccaggagaa ggaaggaagg aaagaaggaa ggaaggaagg 900 aaagaaggaa aataggtgcg acttcaagat ccggagttac aagcagaatg cactgtttcc 960 ctaacagggc caagtgtttt gagtaactga aggtgggcat gatgcctggg aagcagaaac 1020 aagccaggca gatgcacccc ttgccttgct tccgaagggc tgcagtagca tggaaaacat 1080 ggaaaacaac caatccattc cctttgctga tataacaggc tccaaagcca aaacctgtca 1140 ctggaggctc aagagcagat ctccagccaa gaggcaaagg aatgggggaa gctggagggc 1200 ctccctctgg ttatccaggc ttctgaaggt tcaagcaaag aaagggttac aaccttaaaa 1260 ggagagcgtc ccggggtatg ggtagaagac tgctccaccc cgacccccag ggtccctaac 1320 cgtcttttcc ctgggcgagt cagcccaatc acaggactga gagtgcctct ttagtagcag 1380 caagccactt cggacaccca aatggaacac ctccagtcag ccctcgccga ccaccccacc 1440 ccctccatcc ttttccctca gcctccgatt ggctgaatct agagtccctc cctgctcccc 1500 cctctctccc cacccctggt gaaaactgcg ggcttcagcg ctgggtgcag caactggagg 1560 cgttggcgca ccaggaggag gctgcagcta ggggagtcca ggtgagagca ggccgacggg 1620 agggacccgc acatgcaagg accgccgcag ggcgaggatg caagccttcc ccagctacag 1680 ttttgggaaa ggataccagg gcgctcctat atggggcgc gggaactggg gaagaaaggt 1740 gctcccaggt cgaggtggga ggaaggca gtgcggggtc acgggctttc tccctgctaa 1800 cggacgcttt cgaagagtgg gtgccggagg agaaccatga ggaaggacat caaggacagc 1860 ctttggtccc caagctcaaa tcgctttagt ggtgcgaata gaggggagg gtgggtggca 1920 aactggaggg agtccccagc gggtgacctc gtggctggct gggtgcgggg caccgcaggt 1980 aagaaaaccg caatgttgcg ggaggggact gggtggcagg cgcgggggag gggaaagcta 2040 gaaaggatgc gaggagcgg agggggagg gagcgggaga atctcaactg gtagaggaag 2100 attaaaatga ggaatagca tcagggtggg gttagyyars cmrgscsksm sksarrgrrm 2160 gsrmrswwwg tytgkstgkg kgykkrsktr ggykggstrk gwrwkwsrkk ssgsssrmmr 2220 mamwsactgc tgcgcctctg ccaaatcacg ctacccctgt atctagttct gccaggcttc 2280 tccagcccca gccccaattc ttttctctag tgttccccct tccctcccct gaatctcaag 2340 cccacactcc ctcctccata acccactgtt atcaaatcta agtcatttgc cacccaacaa 2400 ccatcaggag gcggaagcag acgggaggag tttgagatca acttgggcta catcacgagt 2460 tccaggctca ccaaggcttc ttaaggagac cttgtctcta aaattaatta attaattaat 2520 taatagtccc ctttctctgc cacagaacct tgggatctgg ctcctggtcg cagctccccc 2580 cacccccaggc tgacattcac tgccatagcc catccggaaa tcctagtcta tttccccatg 2640 gatcttgaac tgcagagaga atggcagagt ggccccgccct gtgcaaagga tgttcctagc 2700 ctaggtggag ctcgcgaact cgcagactgt gcctctcttg ggcaaggaca ggctagacag 2760 cctgccggtg tgttgagcta gggcactgtg gggaaggcag agaacctgtg cagggcagca 2820 atgaacacag gaccagaaaa ctgcagccct aggaacactc aagagctggc catttgcaag 2880 catctctggc ctccgtgctt ctcactcatg tcccatgtct tatacaggcc tctgtggcac 2940 ctcgcttgcc tgatctcatc cctagccgtt aagctttctg catgacttat cacttggggc 3000 ataatgctgg atacctacca ttttcttag ccccatcaaa atcctatttg agtgtacggt 3060 tcggagaacc tcatttatcc ggtaaatgtc ttttactctg ctctcaggga gctgaggcag 3120 3180 aggtacatgc ctataatgcc accactcagg aaatggtggc agcttcgtga gtttgaggcc 3240 3300 ctatatgcta gggctgacct ggacattagg ggtcatcttc tgaacaaact agtgcttgag 3360 ggaggtattt ggggtttttg tttgtttaat ggatctgaat gagttccaga gactggctac 3420 acagcgatat gactgagctt aacaccccta aagcatacag tcagaccaat tagacaataa 3480 aaggtatgta tagcttacca aataaaaaa ttgtattttc aagagagtgt ctgtctgtgt 3540 agccctggct gttcttgaac tcactctgta gaccaggctg gcctggaaat ccatctgcct 3600 gcctctgcct ctctgcctct ctgcctctct gcctctctct ctgcctctct ctgcctctct 3660 ctgcccctct ctgcccctct ctgcccctct ctgccgccct ctgccttttg ccctctgccc 3720 tctgttctct ggcctctgcc ctctgccctc tggcctctgg cctctgcctc tgcctcttga 3780 gtgctgggaat caaaggtgtg agctctgtag gtcttaagtt ccagaagaa gtaatgaagt 3840 cacccagcag ggaggtgctc agggacagca cagacacaca cccaggacat aggctcccac 3900 ttccttggct ttctctgagt ggcaaaggac cttaggcagt gtcactccct aagagaaggg 3960 gataaagaga ggggctgagg tattcatcat gtgctccgtg gatctcaagc cctcaaggta 4020 aatggggacc cacctgtcct accagctggc tgacctgtag ctttccccac cacagaatcc 4080 aagtcggaac tcttggcacc tagaggatct cga 4113

[0085] SEQ ID NO:5 (primer) cctagaggat ctcgagatgg tgagcaaggg cgag

[0086] SEQ ID NO:6 (primer) gaggaaggac ctcgagttac acggcgatct tgcc

[0087] SEQ ID NO:7 (primer) ggacacgctgaacttgtgg

[0088] SEQ ID NO:8 (primer) ctggctgacc tgtagctttc c

[0089] SEQ ID NO: 9 (Venus-Akaluc siRNA 1 (VA-siRNA 1)) gcacucucaucgacaagua dtdt

[0090] SEQ ID NO: 10 (Venus-Akaluc siRNA 2 (VA-siRNA 2)) cgaacgcucucaucgacaa dtdt

[0091] SEQ ID NO: 11 (Venus-Akaluc siRNA 3 (VA-siRNA 3)) agcuaccgaucauacgaaa dtdt< / sirna>

Claims

1. A luminescence detection method comprising administering a luminescent substrate for luciferase and an siRNA that suppresses expression of the modified luciferase gene to a transgenic non-human animal into which a modified luciferase gene has been introduced, The modified luciferase gene expresses a modified luciferase that causes a luminescent substrate to emit light with a higher luminescence intensity than that of wild-type D-luciferase.

2. The luminescence detection method according to claim 1 , wherein the luminescent substrate and at least a portion of the siRNA are administered intranasally to reach the brain.

3. The luminescent detection method of claim 1 , further comprising administering an auxiliary agent.

4. The luminescent detection method according to claim 3 , wherein the auxiliary agent is polyethyleneimine.

5. The luminescence detection method according to claim 1, wherein the modified luciferase contains at least one amino acid substitution selected from the group consisting of T39A, E48Q, I51V, K68R, L86S, Q134R, I136V, Q147R, G175S, N229Y, I231N, F294C, F295L, N308S, H310R, H332R, S347N, I349V, L350M, D357R, A361S, D377V, S456G, N463Y, K524R, L526S, I540T and G545D in the amino acid sequence shown in SEQ ID NO:

1.

6. The luminescence detection method according to claim 1 , wherein the modified luciferase comprises at least one amino acid substitution selected from the group consisting of S347N and N229Y in the amino acid sequence set forth in SEQ ID NO:

1.

7. The luminescence detection method according to claim 1 , wherein the modified luciferase has the amino acid sequence shown in SEQ ID NO:

3.

8. The luminescence detection method according to claim 1 , wherein the modified luciferase is an artificial enzyme Akaluc.

9. The luminescence detection method according to claim 1 , which is used for evaluating drug delivery.

10. A drug delivery evaluation method utilizing the luminescence detection method according to any one of claims 1 to 9.

11. A method for screening an auxiliary agent, comprising administering to a transgenic non-human animal into which a modified luciferase gene has been introduced a luminescent substrate for luciferase, an siRNA that suppresses expression of the modified luciferase gene, and an auxiliary agent, The method for screening an auxiliary agent, wherein the modified luciferase gene expresses a modified luciferase that causes a luminescent substrate to emit light with a luminescence intensity higher than that of wild-type D-luciferase.

12. The method for screening an auxiliary agent according to claim 11, wherein the modified luciferase comprises at least one amino acid substitution selected from the group consisting of T39A, E48Q, I51V, K68R, L86S, Q134R, I136V, Q147R, G175S, N229Y, I231N, F294C, F295L, N308S, H310R, H332R, S347N, I349V, L350M, D357R, A361S, D377V, S456G, N463Y, K524R, L526S, I540T and G545D in the amino acid sequence shown in SEQ ID NO:

1.

13. The method for screening an auxiliary agent according to claim 11, wherein the modified luciferase contains at least one amino acid substitution selected from the group consisting of S347N and N229Y in the amino acid sequence set forth in SEQ ID NO:

1.

14. The method for screening an auxiliary agent according to claim 11 , wherein the modified luciferase has the amino acid sequence shown in SEQ ID NO:

3.

15. The method for screening an auxiliary agent according to claim 11, wherein the modified luciferase is an artificial enzyme Akaluc.

16. A transgenic non-human animal into which a modified luciferase gene has been introduced, A luminescent substrate for luciferase; and an siRNA that suppresses expression of the modified luciferase gene, The modified luciferase gene expresses a modified luciferase that causes a luminescent substrate to emit light with a higher luminescence intensity than that of wild-type D-luciferase.

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