JP2019118334A - Mitochondrial dysfunctional genetically modified silkworm - Google Patents

Abstract

To develop and provide novel mitochondrial dysfunctional model organisms that are easy to maintenance lineage and to reproduce dysfunction in mitochondria as well as to detect recovery thereof.SOLUTION: A mitochondrial dysfunctional silkworm comprising a mitochondrial transcriptional factor A gene or a functional fragment thereof which is arranged under the control of a central or posterior silk gland specific promoter is produced and provided.SELECTED DRAWING: None

Description

  The present invention relates to a genetically modified silkworm that serves as a mitochondrial dysfunctional model, a method for producing the same, a method of screening a drug for treating mitochondrial disease using the genetically modified silkworm, and a method for evaluating the efficacy of a remedy for mitochondrial disease About.

  Mitochondria are organelles that play a central role in energy production in eukaryotic cells. Citric acid cycle (TCA cycle), an electron transfer system, and a site that produces ATP essential for cell survival by oxidative phosphorylation coupled to both, homeostasis of intracellular calcium ion concentration, production of active oxygen And control apoptosis. In addition, the mitochondria has an autoproliferative ability and retains its own DNA (mitochondrial DNA: mtDNA) independent of nuclear DNA, including a gene necessary for oxidative phosphorylation.

  Therefore, the energy production balance is disrupted when a qualitative or quantitative mutation of mtDNA or a slight abnormality occurs in a gene on nuclear DNA related to mitochondrial function. As a result, various cellular disorders caused by energy metabolism abnormalities caused by mitochondrial dysfunction are caused. For example, mitochondrial disease is one of the most common metabolic disorders caused by mitochondrial dysfunction, and is known to exhibit various symptoms among human diseases (Non-patent Document 1). In particular, in tissues such as muscles and nervous systems where energy demand is high, serious damage may occur (Non-patent Documents 2 and 3).

  For example, a disease called mitochondrial encephalomyopathy is caused by mutations in genes on nuclear DNA, such as mitochondrial DNA depletion syndrome-15 (MTDPS15), as well as deletion mutations in mtDNA. This results in a marked decrease in respiratory activity (4). In addition, nerve degeneration disease (non-patent document 5), depression (non-patent document 6), diabetes (non-patent document 7), malignant transformation of cancer (non-patent document 8), male infertility (non-patent document 9), etc. Can also develop various diseases. Therefore, pathological analysis and treatment drug development of diseases caused by these mitochondrial dysfunctions are considered urgent.

  In general, not only cultured cells but also the presence of experimental model animals in in vivo systems are indispensable for pathological analysis and screening of therapeutic agents. However, disease model animals associated with mitochondrial dysfunction are extremely limited. Because mtDNA is essential for cell energy production, individuals lacking mtDNA usually have embryonic lethality. Mutation of a protein gene that regulates mtDNA, which is present on nuclear DNA, can induce mitochondrial dysfunction, but heterozygosity causes a phenotype similar to that of wild-type, and homozygosity leads to embryonic lethality, so analysis is still difficult. Become. It is generally extremely difficult to construct a screening system using mutations in this manner, and therefore, effective mutant model animals related to mitochondrial dysfunction are hardly known.

  At present, attempts are being made to create various model mice for diseases related to mitochondrial dysfunction, and abnormal mitochondrial DNA is contained by the cybrid method in which mouse cytoplasm having mutant mitochondrial DNA is transferred to a normal mouse pronuclear embryo. "Mito mice" have been produced (Non-patent Document 9). Mitomouse is a very good model animal as a disease model of mitochondrial disease that can reproduce mitochondrial dysfunction. However, in the case of a mouse, it requires a large amount of cost for preparation and breeding, and there is a problem that it is difficult to adjust a large number of individuals having the same condition, because the content of mutated mtDNA varies depending on individuals. In addition, ethical issues are likely to arise with the use of mice in large scale validations, such as screening of active ingredients. In addition, in Japan, a partial revision (Law No. 68 of 2005) of the Act on the Protection and Management of Animals (Animal Protection and Management Act) was promulgated, and the Remediation (reduction of pain) prescribed so far In addition to the provisions on (1), provisions on Replacement (use of alternative method) and Reduction (reduction of the number of animals used) were included. As a result, Japan has to comply with the "3R principle" that is widely used and established internationally.

  As described above, in recent years, the use of experimental model animals has become difficult due to social trends. Therefore, there is a need to develop mitochondrial dysfunction models in new model organisms that replace mammalian models.

Scheibye-Knudsen et al., 2015. Trends Cell Biol 25: 158-170. Schaefer et al., 2008. Ann Neurol, 63: 35-39. Ylikallio and Suomalainen, 2012. Ann Med 44: 41-59. Stiles et al., 2016. Mol. Genet. Metab. 119: 91-99. Correia et al., 2012. Adv Exp Med Biol 724: 205-221. Kasahara et al., 2006. MoI Psychiatry 11: 577-593. Gorman et al., 2015. Ann Neurol 77: 753-759. Ohsawa et al., 2012. Nature 490: 547-551. Nakada et al., 2006. Proc Natl Acad Sci USA A. 103: 15148-151453.

  An object of the present invention is to develop and provide a novel mitochondrial dysfunction model organism that is easy to detect lineage maintenance, reproduction of functional abnormalities in mitochondria, and recovery thereof.

  Another object of the present invention is to provide a screening method for a therapeutic drug for mitochondrial disease and a method for evaluating the efficacy of a known therapeutic drug using the model organism.

  In order to solve the above problems, the present inventors focused on using insects as a new model animal instead of mammals such as mice. Insects such as silkworms can significantly reduce breeding space and maintenance costs compared to mice, and can produce a large number of individuals with the same symptoms in any generation. Furthermore, since the model animal is an insect, it is not necessary to consider measures, prevention, treatment and the like of zoonotic diseases. In addition, because it is not subject to the "Animal Protection Act", it is not necessary to follow the Act. Furthermore, when silkworms are used as model animals, silk glands possessed by silkworms can be said to be substitutes for brain and muscle tissue in terms of large energy demand. In addition, analysis is easy because the cell number of silk gland does not change from the juvenile stage. In addition, there is also a feature that the cell function can be evaluated by the size of the cell volume.

  By the way, as a method for inducing mitochondrial dysfunction, a method is known which overexpresses mitochondrial transcription factor A (Transcription Factor A, Mitochondrial (herein often referred to as "TFAM")). For example, systemic overexpression of TFAM in Drosophila melanogaster results in larval lethality, and overexpression of motor neurons specifically has been reported to result in abnormal climb behavior (Cagin et al., 2016. Proc. Natl. Acad. Sci. USA 112: E6000-9.). On the other hand, overexpression of TFAM in the whole body does not lead to lethality, and it has also been reported that the lifespan is prolonged reversely under oxidative stress conditions (Matsuda et al., 2013. BBRC 430: 717-721). Overexpression of TFAM has also been reported in mice. For example, in F1 individuals obtained by crossing transgenic mice capable of overexpressing TFAM with myocardial infarction mice, it has been reported that complementation of mtDNA reduction resulting from myocardial infarction improves symptoms by complementation of TFAM (Ikeuchi et al., 2005, Circulation 112: 683-690). In addition, Japanese Patent Application No. 2008-549398 has been filed for a therapeutic method for improving cell function by supplementation with TFAM. Thus, although overexpression of TFAM may also cause a decrease in mitochondrial function, on the other hand, positive effects may be obtained due to the protective action of mtDNA. This is due to the fact that the functions of TFAM are very versatile.

  The present inventors are able to cause symptoms similar to cell function decline due to mtDNA deletion locally in silk gland by overexpressing TFAM specifically in silk gland in silkworm including silkworm Found out. Since this condition is limited to silk glands and silk glands are not essential organs for survival, individuals will not be killed even if symptoms become serious. Therefore, it can be a new model animal for pathological analysis of mitochondrial diseases and screening of therapeutic agents. The present invention is based on the development result, and provides the following.

(1) A mitochondrial dysfunctional silkworm inducer comprising, as an active ingredient, a nucleic acid molecule encoding a gene encoding mitochondrial transcription factor A or a functional fragment thereof.
(2) A gene expression unit for producing a mitochondrial dysfunctional silkworm, which is a promoter of a gene encoding a protein specifically expressed in the central or posterior silk gland, and a mitochondrial transcription factor functionally linked downstream of the promoter Said gene expression unit comprising a nucleic acid molecule encoding a gene encoding A or a functional fragment thereof.
(3) The gene expression unit according to (2), further comprising an additional gene or a fragment thereof linked to the 3 'end of the gene encoding mitochondrial transcription factor A.
(4) The gene expression unit according to (2) or (3), wherein the protein specifically expressed in the posterior silk gland is fibroin H chain, fibroin L chain and p25 / FHX.
(5) The gene expression unit according to (2) or (3), wherein the protein specifically expressed in the middle silk gland is sericin.
(6) The gene expression unit according to any one of (2) to (5), wherein the mitochondrial transcription factor A is any of the following (a) to (d).
(A) A silkworm-derived mitochondrial transcription factor A comprising the amino acid sequence shown in SEQ ID NO: 1,
(B) a mitochondrial transcription factor A comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence described in (a),
(C) Mitochondrial transcription factor A consisting of an amino acid sequence having an amino acid identity of 90% or more to the amino acid sequence described in (a), or (d) another type ortholog of mitochondrial transcription factor A described in (a) Protein (7) The gene expression unit according to any one of (3) to (6), wherein the additional gene is a gene encoding a labeled protein.
(8) The gene expression unit comprises the first and second subunits, and the first subunit is functionally a promoter of a gene encoding a protein specifically expressed in the middle or rear silk gland and its 3 'end side The second subunit comprises a gene encoding a transcriptional regulatory factor bound thereto, and a second subunit comprising a target promoter of the transcriptional regulatory factor and a gene encoding a mitochondrial transcription factor A functionally linked to the 3 'end thereof or a functional fragment thereof The gene expression unit according to any one of (2) to (7), which comprises a coding nucleic acid molecule.
(9) A gene expression vector comprising the gene expression unit according to any one of (2) to (7).
(10) A gene expression vector comprising a second subunit constituting the gene expression unit according to (8).
(11) A mitochondrial dysfunction abnormality type recombinant silkworm comprising a gene expression unit for producing a mitochondrial dysfunction abnormality type silkworm according to any one of (2) to (8).
(12) The genetically modified silkworm according to (11), which is subordinate to (8), wherein the first subunit and the second subunit are present on different chromosomes.
(13) The recombinant silkworm according to (11) or (12), wherein the silkworm is a silkworm.
(14) A method for producing a mitochondrial dysfunction abnormality type recombinant silkworm, wherein an introducing step of introducing the gene expression vector according to (9) into silkworm and selecting an individual containing the gene expression vector after the introducing step Said method comprising a selection step.
(15) A method for producing a mitochondrial dysfunction abnormality type recombinant silkworm, comprising a first subunit silkworm strain comprising the first subunit according to (8) and a second subunit according to (8) Selecting an individual including the first and second subunits from the eggs obtained in the mating step of mating with the second subunit silkworm strain containing, the egg collecting step of collecting eggs from the silkworm after the mating step, and the egg collecting step Said method comprising a selection step.
(16) A screening method for a drug treating mitochondrial disease, which comprises administering a candidate drug to the mitochondrial dysfunction type recombinant silkworm according to any of (11) to (13), and after the administration step A confirmation step of confirming the shape of the silk gland in the genetically modified silkworm of the present invention, and a selection step of selecting the candidate drug as a treatment for mitochondrial disease when the silk gland shape of the genetically modified silkworm which has been contracted has recovered Including the above method.
(17) A method for evaluating a mitochondrial disease therapeutic agent, which comprises administering a known mitochondrial disease therapeutic agent to a mitochondrial dysfunction type recombinant silkworm according to any of (11) to (13), administration A confirmation step of confirming the shape of the silk gland in the genetically modified silkworm after the step; and, if the silk gland shape of the genetically modified silkworm which has been contracted has recovered, the agent for treating mitochondrial disease is for treatment of mitochondrial disease Said method comprising an evaluation step of evaluating as effective.

  The mitochondrial dysfunction type recombinant silkworm of the present invention is easy to detect lineage maintenance, reproduction of functional abnormality in mitochondria and detection of its recovery, and is not a target of application of the "animal protection management method". It is useful as a mitochondrial dysfunctional model organism.

FIG. 2 is a schematic view of a gene expression vector pBac [UAS-BmTFAM-GFP-SV40, 3 × P3-GFP] containing the second subunit of the present invention prepared in Example 1. It is a microscope image of the silk gland extracted from the mitochondrial dysfunction abnormality type | mold recombinant silkworm etc. of this invention. A and C are silk glands in a silkworm strain containing only the first subunit for control, and B and D are mitochondrial dysfunction type recombinant silkworms of the present invention containing the first and second subunits. It is a figure which shows a silk gland, respectively. A and B are stereomicrographs, and C and D are enlarged views of the posterior silk gland. The small images in A and B in the dashed white frame show the posterior silk gland, and the lower right images in C and D show fluorescence microscopic images in which the nucleus of the posterior silk gland tissue is stained with DAPI. It is a transmission electron microscope image of the silk gland extracted from the mitochondrial dysfunction abnormality type recombinant silkworm etc. of the present invention. The upper stages A and B show silk glands in a silkworm strain containing only the first subunit for control, and the lower stages C and D show the mitochondrial dysfunction type genetic recombination of the present invention containing the first and second subunits. It is a figure which shows the silk gland in a silkworm, respectively. In the figure, ER indicates rough endoplasmic reticulum, mt indicates mitochondria, and G indicates the Golgi body. It is a microscope image of the silk gland which shows the recovery effect of the posterior silk gland atrophy symptom in the mitochondrial dysfunction type recombinant silkworm by administration of a mitochondrial disease therapeutic candidate drug. A represents a control silk gland in which a solvent only was administered to a mitochondrial dysfunction type recombinant silkworm, and B represents a silk gland in which 5-ALA, which is a mitochondrial disease treatment candidate, was administered. The dashed frame corresponds to the posterior silk gland.

1. Mitochondrial dysfunction type silkworm production gene expression unit 1-1. SUMMARY A first aspect of the present invention relates to a gene expression unit (herein often referred to simply as "gene expression unit") for producing mitochondrial dysfunctional silkworm. The gene expression unit of the present invention comprises a promoter of a gene encoding a protein specifically expressed in the central or posterior silk gland and a nucleic acid molecule encoding a gene encoding mitochondrial transcription factor A or a functional fragment thereof By introducing into silkworms, genetically modified silkworms exhibiting symptoms of mitochondrial dysfunction can be produced.

1-2. Definitions The following terms frequently used in this specification are defined.

  As used herein, "silk thread" refers to a fibrous protein complex which is spun by a silkworm. Wild-type silk yarn is composed of fibroin which is a fiber component and sericin which coats fibroin.

  As used herein, "silkworm" is a generic term for insects having a silk gland and capable of spiting silk thread. Usually refers to species capable of spinning for nesting, nesting or transfer at the larval stage. Specifically, it is a species belonging to the order Lepidoptera, Hymenoptera, Amimeca-lowe, Tobichela. Preferably, it is a species belonging to the order Cichlid which can spit a large amount of silk thread. Belonging to the silkworm family (Bombycidae), the saturnidae (Saturnidae), the family Bivotaidae (Brahmaeidae), the family Obiga (Eupterotidae), the family of the long-tailed lizard family (Lasiocampidae), the family of the long-legged family (Psychidae), the family of long-eared moth (Archtiidae), the family noctidae (Noctuidae) Species are preferred as silkworms herein. As a silkworm of the present invention, a more preferable species is a species belonging to the family Bichidae or Phyllaceae, and examples include species belonging to the genera Bombyx, Samia, Antheraea, Saturnia, Attacus, Rhodinia. More specifically, silkworm, Bombyx mandarina, Shinjusan (Samia cynthia; including Erisan Samia cynthia ricini and a hybrid of Shinjusan and Erisan), Yamame-ga (Antheraea yamamai), Saxan (Antheraea pernyi), Himeyamamay ( Saturnia japonica), Actias gnoma, etc. Furthermore, in addition to industrial applicability, insects with limited mobility and / or insects rearable within enclosed spaces are particularly suitable as silkworms herein. For example, silkworm is a typical example.

  The “silk gland” is a tubular organ in which the salivary gland is changed, and has a function of producing, accumulating and secreting liquid silk. The silk glands exist in a pair mainly along the digestive tract of the larvae of the silkworm, and each silk gland is composed of three regions of front, middle and rear silk glands. In general, the posterior silk gland has a function of producing and secreting fibroin, and the middle silk gland has a function of producing and secreting sericin and accumulating in the lumen along with fibroin transferred from the posterior silk gland.

  In the present specification, "mitochondrial dysfunction" (herein often referred to as "mt dysfunction") refers to the fact that mitochondrial intrinsic function is impaired due to various causes. Although the cause of mt dysfunction is not limited, for example, mutation of a gene contained in nuclear DNA or abnormality of mtDNA may be mentioned. The abnormalities of mtDNA correspond to mutations (including substitution and deletion of bases) and depletion of mtDNA. mt dysfunction causes clinical symptoms such as mitochondrial disease.

  In the present specification, the term "mitochondrial dysfunctional silkworm" (herein often referred to as "mt dysfunctional silkworm") refers to a trait of host silkworm comprising a gene expression unit of this aspect in its cell. It means a transformant or its progeny. The mt dysfunctional silkworm will be described in detail in the third embodiment.

  As used herein, the term "gene expression unit" refers to a promoter and a nucleic acid molecule such as a gene arranged in a state capable of expression on the 3 'end side of the promoter as an essential component, and downstream nucleic acid molecules by activation of the promoter. An artificial gene expression unit configured to be over expressed.

1-3. Configuration The gene expression unit of the present invention comprises, as an essential component, a gene promoter that specifically expresses the middle or rear silk gland, and a nucleic acid molecule encoding a gene encoding mitochondrial transcription factor A or a functional fragment thereof The gene or a fragment thereof, a terminator and a 5 'UTR and a 3' UTR are included as optional components. Furthermore, when the gene expression unit is composed of two subunits of the first subunit and the second subunit described later, a gene encoding a transcriptional regulatory factor and a target promoter of the transcriptional regulatory factor are essential components As included. Each component will be specifically described below.

1-3-1. Component (1) Middle or Posterior Silk Gland Specific Promoter A "promoter" is a gene expression regulatory region capable of controlling the expression of a nucleic acid molecule such as a gene placed under its control. A gene promoter specifically expressed in the central or posterior silk gland, which is contained in the gene expression unit of the present invention (herein often referred to as "central or posterior silk gland specific promoter"), is a central or posterior silk gland It regulates the expression of a gene encoding a specifically expressed protein.

  As used herein, the term "protein specifically expressed in the central or posterior silk gland" refers to a protein that is expressed site-specifically in each silk gland in silkworm. The type is not limited. For example, if it is a protein specifically expressed in the middle silk gland, sericin will be mentioned, and if it is a protein specifically expressed in the posterior silk gland, fibroin H chain, fibroin L chain and p25 / FHX will be mentioned.

  Sericin (Ser) is a water-soluble paste-like protein produced by the central silk gland and coats fibroin which is a fiber component of silk thread as described above. In silkworm et al., Three variants of sericin 1 (Ser1), sericin 2 (Ser 2) and sericin 3 (Ser 3) are known, but all of them are produced in the middle silk gland and sericin in the present specification. Is not particularly limited.

  Fibroin H chain (Fib H), fibroin L chain (Fib L) and p25 / FHX (p25) are all proteins produced in the posterior silk gland and constitute the fiber component of silk thread. Usually, in silk thread, silk fiber is configured as a composite (silk fibroin elementary unit; SFEU composite) at a ratio of Fib H: Fib L: p 25 = 6: 1.

  The term "under the control of a promoter" refers to a region at the 3 'end of the promoter in the gene expression unit and under the control of the promoter. Thus, "arranged under the control of a promoter" means that genes and the like are linked directly to the 3 'end side of the promoter or indirectly with an intervening sequence such as a spacer interposed therebetween.

  In the present specification, a donor species from which a central or posterior silk gland-specific promoter is derived is a host cell having a gene expression unit of the present invention or in a host cell on the recipient side into which the gene expression unit of the present invention is introduced. There is no particular limitation in the cells as long as they are operable. The term "operable" as used herein means that it exerts a function as a promoter and can express a gene or the like placed downstream. Therefore, the promoter in the gene expression unit of the present invention is preferably a promoter from a species which belongs to the same taxonomical position as the host into which the gene expression unit of the present invention is introduced. Promoters derived from species belonging to the same family are more preferred, and promoters derived from species belonging to the same genus are more preferred. Most preferred are promoters from the same species as the host. For example, when the host into which the gene expression unit of the present invention is introduced is Bombyx mori, a lepidopteran insect, the above promoter used in the gene expression unit is preferably a lepidopteran-derived promoter, and belongs to the Bombyxidae It is more preferable if it is derived from a species, and it is further preferable if it is derived from a species belonging to the same Bombyx genus, such as Bombyx mandarina. In this case, the most preferable promoter is a promoter derived from homologous silkworms. However, in general, the base sequence of the central or posterior silk gland-specific promoter is evolutionarily highly conserved among silkworms, and therefore the silkworm which differs from the host silkworm in the promoter contained in the gene expression unit of the present invention It is known that even if they are derived, they can function in the cells of host silkworm (Sezutsu H., et al., 2009, Journal of Insect Biotechnology and Sericology, 78: 1-10). Therefore, as long as it is silkworm-derived, it does not necessarily have to be a species that belongs to the same eye, the same family, or the same genus as the host silkworm, or a promoter derived from the same species.

  As a specific example of the middle or rear silk gland-specific promoter contained in the gene expression unit of the present invention, if it is a middle silk gland-specific promoter, a promoter (BmSer1 promoter) consisting of the nucleotide sequence shown in SEQ ID NO: 3 And a promoter of B. coccicolysin 2 gene consisting of the base sequence shown in SEQ ID NO: 4 (BmSer2 promoter) and a promoter of B. caicosicin 3 gene consisting of the base sequence shown in SEQ ID NO: 5 (BmSer3 promoter). In addition, if it is a posterior silk gland-specific promoter, a promoter of B. fibronectin H gene consisting of a nucleotide sequence shown in SEQ ID NO: 6 (BmFib H promoter), a promoter of B. ca. Promoter of the silkworm p25 gene consisting of the nucleotide sequence shown in SEQ ID NO: 8 (Bmp 25 promoter), Promoter of the San Fib H gene consisting of the nucleotide sequence shown in SEQ ID NO: 9 (ApFib H promoter), and shown in SEQ ID NO: 10 The promoter (ApFib L promoter) of the San Fib L gene consisting of a nucleotide sequence can be mentioned.

(2) Nucleic acid molecule encoding mitochondrial transcription factor A gene or functional fragment thereof "Mitotochondrial transcription factor A" (TFAM) is a protein belonging to the high mobility group (HMG) family and binds to the transcription promoter region on mtDNA It functions as a transcription factor that promotes gene expression, and functions as a structural protein that binds non-specifically to mtDNA to maintain the structure and stability of mtDNA.

  "Mitochondrial transcription factor A gene (TFAM gene)" refers to a gene encoding TFAM. The present invention is based on the basic technology of overexpressing the exogenous TFAM gene in the mid or posterior silk gland of silkworm to cause symptoms similar to mt dysfunction in the central or posterior silk gland. Therefore, a nucleic acid molecule encoding the TFAM gene or a functional fragment thereof can be an active ingredient of the inducing agent for inducing mt dysfunctional silkworm of the present invention.

  There are no particular limitations on the species from which the TFAM gene is derived from the gene expression unit. TFAM has a unique structure including two HMG-box motifs (for example, HMG1 shown in SEQ ID NO: 18 and HMG2 shown in SEQ ID NO: 19 in the case of silkworm) in the amino acid sequence. However, the amino acid identity of the protein encoded by the TFAM ortholog (often referred to herein as "TFAM ortholog protein") in each species is not very high, not only between full length but also between each HMG-box motif. However, their functions are evolutionarily highly conserved, and it is known that each TFAM ortholog protein is compatible between species. For example, silkworm TFAM, an invertebrate, has only 26.5% full-length amino acid sequence identity to vertebrate human TFAM ortholog protein, but has functional compatibility with human TFAM ortholog protein (Sumitani) M., et al., 2016, Gene, 608: 103-113). Therefore, even a TFAM orthologue protein from a species taxonomically distant from the host organism into which the gene expression unit is introduced can function as a TFAM in the host cell, so any TFAM gene of any species is used be able to.

  However, in consideration of the expression efficiency of the TFAM gene and the functional efficiency of the expressed TFAM, a TFAM gene derived from a species which belongs to the same taxonomical position as the host into which the gene expression unit is introduced is preferable. The TFAM gene derived from a species belonging to the same family is more preferred, and the TFAM gene derived from a species belonging to the same genus is more preferred. Most preferred is the TFAM gene from the same species as the host. For example, if the host into which the gene expression unit is introduced is a silkworm, the above TFAM gene is preferably derived from an insect of the order Lepidoptera, more preferably derived from a species belonging to the family Bichidae and more preferably derived from a species belonging to the genus Bombyx Most preferred is the silkworm-derived TFAM gene (Bmtfam gene). Specifically, a WT-Bmtfam gene encoding a wild-type silkworm TFAM (WT-BmTFAM) consisting of the amino acid sequence shown in SEQ ID NO: 1, one or more amino acids are deleted, substituted or added in the wild-type BmTFAM And the MT-Bmtfam gene encoding a mutant BmTFAM (MT-BmTFAM) consisting of an amino acid sequence having an amino acid identity of 90% or more to the amino acid sequence of the wild type BmTFAM. Specific examples of the WT-Bmtfam gene encoded by WT-BmTFAM consisting of the amino acid sequence shown by SEQ ID NO: 1 include the WT-Bmtfam gene consisting of the nucleotide sequence shown by SEQ ID NO: 2 and degenerate variants thereof. It may also be another species TFAM ortholog of the WT-BmTFAM gene.

  As used herein, “functional fragment thereof” refers to a peptide fragment which is a part of TFAM and retains a function equal to or higher than that of TFAM. Also, "a nucleic acid molecule encoding a functional fragment thereof" refers to a TFAM gene fragment encoding the peptide fragment.

  The nucleic acid molecule encoding the TFAM gene or its functional fragment is an essential component whether the gene expression unit of the present invention is constituted by a single unit or by two subunits described later . When the gene expression unit is composed of a single unit, the nucleic acid molecule encoding the TFAM gene or its functional fragment is placed under the control region of the middle or rear silk gland specific promoter. On the other hand, when the gene expression unit is composed of the first and second two subunits, the nucleic acid molecule encoding the TFAM gene or its functional fragment is contained in the second subunit and is a target of a transcriptional regulatory factor described later It is placed under the promoter control region.

(3) Additional gene or fragment thereof In the present specification, the term "additional gene" refers directly to the 3 'end of the nucleic acid molecule encoding the TFAM gene or its functional fragment in the gene expression unit, or a spacer sequence, etc. Indirectly, the gene is linked, and encodes an additional protein. In the present specification, the type of added protein, the number of amino acids, and the presence or absence of activity are not particularly limited. Various proteins can be added proteins.

  When the gene expression unit contains an additional gene, a marker gene or the like encoding a marker protein is convenient. The “labeling gene” is a gene encoding a labeling protein, and the “labeling protein” is generally a poly that can confirm the presence or absence and the expression level of the labeling gene or the fusion gene thereof based on its activity. Refers to a peptide. Here, "based on activity" means based on the result of detection of activity.

  When the additional protein is a labeled protein, the type is not particularly limited. However, a host protein containing a gene expression unit, that is, a labeled protein which is less invasive to a transformant is preferable. For example, fluorescent proteins, luminescent proteins, dye synthesis proteins, external secretion proteins and the like can be mentioned.

  As used herein, "fluorescent protein" refers to a protein that emits fluorescence of a specific wavelength when irradiated with excitation light. It may be either natural or non-natural type. Also, the excitation wavelength and the fluorescence wavelength are not particularly limited. Specifically, for example, GFP (including derivatives such as EGFP and 3xP3-EGFP), CFP, RFP, DsRed (including derivatives such as 3xP3-DsRed), YFP, PE, PerCP, APC, etc. Do.

  As used herein, “luminoprotein” refers to a substrate protein that can emit light without the need for excitation light, or an enzyme that catalyzes the luminescence of that substrate protein. For example, luciferin or aequorin as a substrate protein, and luciferase as an enzyme correspond.

  As used herein, "dye-synthesized protein" refers to a protein involved in the biosynthesis of a dye. Usually, an enzyme corresponds. The "pigment" referred to herein is a low molecular weight compound or peptide capable of providing a pigment to a transformant, regardless of its type. Preferably, it is a pigment which appears as an external color of an individual. For example, melanin pigments (including dopamine melanin), omochrome pigments, or pteridine pigments are applicable.

  In the present specification, "externally secreted protein" is a protein secreted extracellularly or extracorporeally, and in addition to exocrine enzymes, fiber proteins such as fibroin and sericins are applicable. The exocrine enzymes include digestive enzymes as well as enzymes that contribute to the degradation or inactivation of drugs such as blasticidin and impart drug resistance to the host.

  When the gene expression unit contains an additional gene, in principle, the TFAM gene and the additional gene become a fusion gene (denoted as "TFAM-added gene"), behave as one gene, and receive the same expression control etc. . For example, if the additional gene is a GFP gene, it is a TFAM-GFP fusion gene. Further, the protein produced by the expression of the TFAM-added gene is also a fusion protein of TFAM and the added protein (TFAM-added protein), and the behavior such as expression and localization is basically the same. In the case of the TFAM-GFP fusion gene, it is a TFAM-GFP fusion protein produced by its expression. Therefore, if the additional gene is a marker gene, it is also possible to detect the presence or absence of the expression of the TFAM gene and the expression amount thereof via the expression of the marker gene.

(4) Terminator The terminator is composed of a nucleotide sequence that can terminate transcription of mRNA in the posterior silk gland cell of the host into which the mRNA expression vector is introduced.

(5) 5 'UTR and 3' UTR
The 5 'UTR and 3' UTR are polynucleotides consisting of untranslated regions which themselves do not encode proteins or fragments thereof or functional nucleic acids. In the gene expression unit of the present invention, the upstream (5 'end side) of the start codon of the mRNA coding region of the TFAM gene or the TFAM-added gene (herein, often collectively referred to as "TFAM gene etc.") And a nucleotide sequence located downstream (3 'end side) of the stop codon, and the 3' UTR can contain a poly A signal.

(6) Gene encoding transcriptional regulatory factor "gene encoding transcriptional regulatory factor" refers to a gene of transcriptional regulatory factor. This gene is an essential component in the first subunit when the gene expression unit is composed of the first and second two subunits. The term "transcription regulatory factor" as used herein refers to a protein factor capable of binding to a target promoter described later to activate the target promoter. For example, GAL4 protein which is a galactose metabolism activating protein of yeast, tTA which is a tetracycline regulatory transactivator, and a variant thereof can be mentioned.

(7) Transcription regulatory factor target promoter The "transcriptional regulatory factor (of) target promoter" refers to a promoter to which the transcriptional regulatory factor encoded by the first subunit binds. This promoter is an essential component in the second subunit when the gene expression unit is composed of the first and second subunits. The binding of the transcriptional regulatory factor can activate gene expression under its promoter regulatory region. As described above, in the second subunit of the gene expression unit of the present invention, since the nucleic acid molecule encoding the TFAM gene or its functional fragment is disposed under the control region, the expression of these genes can be activated.

  The transcriptional regulatory factor and its target promoter correspond to the transcriptional regulatory factor, and usually, when a transcriptional regulatory factor is determined, the target promoter is also necessarily defined. For example, when the transcription regulatory factor is GAL4 protein, UAS (Upstream Activating Sequence) is used.

1-3-2. Unit Structure The gene expression unit of the present invention may be composed of a single unit or two subunits. Each case will be described below.

(1) When composed of a single unit: When the gene expression unit is composed of a single unit, the gene expression unit is an essential component of the middle or rear silk gland specific promoter, and the TFAM gene or its function It may include all of the components, such as a nucleic acid molecule encoding a fragment, as well as optional components additional genes or fragments thereof, and a terminator. Therefore, in this case, it can function as a gene expression unit which can overexpress the TFAM gene etc. with only a single unit.

(2) When composed of two subunits When the gene expression unit is composed of two subunits, the first subunit and the second subunit, the essential components for overexpressing the TFAM gene etc. are It exists in each subunit. Therefore, it can function as one gene expression unit which can overexpress the TFAM gene etc., only when the two subunits are simultaneously present in silkworm cells. The configuration of each subunit is described below.

(First subunit)
The first subunit comprises a gene encoding the above-mentioned transcriptional regulatory element functionally linked to the central or posterior silk gland-specific promoter and the 3 'end of the promoter. At this time, the same or different two or more transcription regulatory factors may be included under the control of one promoter.

  As used herein, “functionally linked” refers to linking in such a way that expression of a gene or the like is possible by the control of an upstream promoter.

(Second subunit)
The second subunit comprises a target promoter of a transcriptional regulatory factor encoded by the first subunit and a TFAM gene functionally linked to the 3 'end of the target promoter.

  The gene expression unit of this configuration controls the expression of the TFAM gene etc. by activating the target promoter of the second subunit by the transcriptional regulatory factor expressed from the first subunit by activating the central or posterior silk gland expression promoter. It can.

2. Gene expression vector 2-1. Overview A second aspect of the present invention is a gene expression vector. The gene expression vector of the present invention contains the gene expression unit according to the first aspect, and by introducing it into the cells of the target host silkworm, it mimics mt dysfunction in the middle or rear silk gland of the host silkworm. It can induce symptoms.

2-2. Configuration As used herein, “gene expression vector” refers to a gene expression system including the gene expression unit according to the first aspect in the mother nucleus. The gene expression vector of the present invention comprises, as an optional component, a marker gene, transposon inverted terminal repeat sequence, an insulator and the like in addition to the mother nucleus and the gene expression unit which are essential components. Each of these will be described below.

(1) Mother Nucleus Various vectors can be used for the mother nucleus. For example, an autonomously replicable expression vector such as a plasmid or bacmid (Bacmid), a viral vector, or an expression vector capable of homologous or non-homologous recombination in the chromosome or a part of the chromosome into which it has been inserted in the host chromosome It can be mentioned. It can also be a shuttle vector that can replicate in other bacteria such as E. coli.

(2) Gene Expression Unit The gene expression unit contained in the gene expression vector is the gene expression unit described in the first aspect. Since the configuration of this gene expression unit is described in detail in the first embodiment, only the characteristic configuration of the gene expression vector will be described here.

  The gene expression unit contained in the gene expression vector may be either a single unit or a set of subunits consisting of the first and second two subunits.

  When the gene expression unit is a single unit, one or more gene expression units may be included in the gene expression vector. When multiple gene expression units are included, each gene expression unit may have the same configuration or a different configuration. For example, when the gene expression unit contains two gene expression units, one gene expression unit contains a middle silk gland specific promoter and the other gene expression unit contains a posterior silk gland specific promoter.

  When the gene expression unit is one set of subunits, each subunit forming one set is, in principle, contained in different gene expression vectors. The gene expression vector (first gene expression vector) containing the first subunit and the gene expression vector (second gene expression vector) containing the second subunit do not have to be in one-to-one correspondence. For example, the first expression vector may be in a one-to-two correspondence in combination with two or more different second gene expression vectors. Specifically, the first gene expression vector comprising the GAL4 gene comprises a second gene expression vector A comprising the UAS promoter and the TFAM gene linked to the 3 'end thereof, and the UAS promoter and the 3' end thereof It can be paired with any of the second gene expression vector B containing the linked TFAM-GFP fusion gene. Conversely, two or more first expression vectors may have a two-to-one correspondence in combination with one second gene expression vector. For example, the first gene expression vector A containing the middle silk gland promoter and the GAL4 gene, and the first gene expression vector B containing the posterior silk gland promoter and the GAL4 gene were all linked to the UAS promoter and its 3 'end. It can be paired with a second gene expression vector containing a TFAM-GFP fusion gene.

  When the gene expression vector contains a plurality of gene expression units, each gene expression unit may be a single unit, a pair of subunits, or a combination thereof. When the gene expression vector includes a plurality of subunits, the first subunit and the second subunit derived from each of a different pair of subunits can be contained in the same gene expression vector.

(3) Labeled Gene The labeled gene is a polynucleotide consisting of a nucleotide sequence encoding a labeled protein also called a selective marker, and is a selective component of a gene expression vector. The configuration of the marker gene and the marker protein is described in detail in the first embodiment as an example of the additional gene and the additional protein, and thus the specific description thereof is omitted here.

(4) Transposon Inverted Terminal Repeat Sequence The “inverted terminal repeat sequence” of “transposon” is a transposon-derived gene which is included when the gene expression vector of the present invention is inserted into the host genome by homologous recombination. It is an array. Usually, they are used in pairs, and the nucleotide sequences to be inserted in the genome are placed between them. Transposons differ depending on the species of host into which the gene expression vector is introduced. For example, if the host is silkworm, piggyBac, mariner, minos etc. can be used (Shimizu, K. et al., 2000, Insect Mol. Biol., 9, 277-281; Wang W. et al., 2000, Insect Mol Biol 9 (2): 145-55).

(5) Insulator An insulator is a base sequence that can stably control transcription of a gene sandwiched between the sequences without being affected by the chromatin of surrounding chromosomes. For example, cHS4 sequence of chicken and gypsy sequence of Drosophila can be mentioned.

2-3. The gene expression vector of this aspect is the middle of host silkworm or the middle part of host silkworm by introducing the gene expression unit of the first aspect etc. into the cells of host silkworm and expressing the TFAM gene etc. contained in the gene expression unit. Symptoms similar to mt dysfunction can be induced in the posterior silk gland.

3. Mitochondrial dysfunction type genetically modified silkworm 3-1. Overview A third aspect of the present invention is a mt dysfunctional type transgenic silkworm (herein often referred to simply as "genetically modified silkworm"). The transgenic silkworm of the present invention is characterized in that the cell comprises the gene expression unit according to the first aspect. The transgenic silkworm of the present invention can overexpress the TFAM gene in the central or posterior silk gland, and can exhibit symptoms similar to mt dysfunction specifically in the central or posterior silk gland. Thus, the mt dysfunctional type recombinant silkworm of the present invention can be used as a mitochondrial dysfunction model animal.

3-2. Configuration As used herein, “mt dysfunctional type recombinant silkworm” refers to a transformant of host silkworm comprising the gene expression unit according to the first aspect in a cell, or a progeny thereof. As used herein, the term "progeny" refers to an individual who is a progeny of the first generation of transformants and retains the gene expression unit of the first aspect in a cell. The gene expression unit contained in the cell may be in the state of the gene expression vector described in the second aspect. The number of generations of the progeny does not matter as long as the gene expression unit of the first aspect is retained.

  The transgenic silkworm of the present invention exhibits symptoms similar to mt dysfunction. The “condition similar to mt dysfunction” includes, for example, a state in which the cells can not be differentiated normally because the capacity of the cells does not increase due to the ability to synthesize ATP and the like. In the genetically modified silkworm of the present invention, symptoms similar to the mt dysfunction are limited to the middle silk gland or the posterior silk gland, and in principle, no abnormality is shown in other organs or cells.

  The host of the genetically modified silkworm of the present invention is a silkworm described in "1-2. Definition" of the first aspect. When the transgenic silkworm of the present invention is used as a mitochondrial dysfunction model animal, the organ in which mt dysfunction is induced is the middle and / or posterior silk gland. Therefore, a silkworm having a large silk gland which is easy to detect morphological abnormalities or the like and to observe the recovery of its symptoms caused by mt dysfunction is preferable. For example, species belonging to the above-mentioned silkworm family or to the family Phyllaceae are suitable. Among them, silkworm is particularly preferable as a transgenic silkworm of the present invention for the reason described in the above definition.

  When the transgenic silkworm comprises the gene expression unit according to the first aspect in the state of being contained in the gene expression vector according to the second aspect, the gene expression unit according to the first aspect is a cell in the silkworm cell. Or may be present stably and continuously, such as in the state of being inserted into the genome. It is usually preferred that the gene be inserted into the genome. When the gene expression vector according to the second aspect is inserted into the genome, it is preferable that the gene expression vector according to the second aspect contains transposon inverted terminal repeat sequences.

  The gene expression unit according to the second aspect, which is included in the transgenic silkworm, may be composed of a single unit or two subunits. Each case will be described below.

(1) In the case where the gene expression unit is composed of a single unit: When the gene expression unit covered by a transgenic silkworm is composed of the single unit, at least one gene expression unit is included in the cell It should just be. This is because a single unit can overexpress the TFAM gene etc. alone as described above.

  The transgenic silkworm of the present invention can contain two or more single units in cells. For example, a single unit containing the middle silk gland specific promoter and a single unit containing the posterior silk gland specific promoter are applicable. A transgenic silkworm that contains these two gene expression units can induce symptoms similar to mt dysfunction in both the central and posterior silk glands.

(2) In the case where the gene expression unit is composed of two subunits When the gene expression unit contained in the transgenic silkworm is composed of one set of subunits consisting of the first subunit and the second subunit In order to overexpress the TFAM gene etc. in the central and / or posterior silk gland, at least one set of subunits must be present in the cell. As described above, this is because, only when one set of subunits is present in silkworm cells, it functions as one gene expression unit capable of overexpressing the TFAM gene and the like.

  Therefore, a strain of silkworm comprising only the first subunit (often referred to herein as "first subunit silkworm strain") and a strain of silkworm comprising only the second subunit (herein described) Silkworm worm strains containing only one subunit, such as often described as “the second subunit silkworm worm strain”, do not fall under the mt dysfunction type recombinant silkworm of the present invention. However, since only one subunit does not cause mt dysfunction, these two lines are genetically stable and very convenient for line maintenance.

  Even when silkworms contain only one subunit, if the respective strains are the same strain and one is male and the other is female, hybrids can be obtained by crossing both strains as needed. The transgenic silkworm of the present invention having both subunits at generation (F1) or later can be obtained.

  The first subunit contains a central or posterior silk gland specific promoter and a gene of a transcription factor such as GAL4 linked to its 3 'end, and expresses the transcription factor by activation of the promoter. Therefore, the first subunit silkworm strain can be regarded as a highly versatile gene expression driver strain because it can induce the expression of any target gene under the control of the target promoter of the transcription factor.

  The second subunit contains a target promoter of a transcription factor to be expressed from the first subunit and a TFAM gene linked to the 3 'end side thereof, and the gene is overexpressed by activation of the promoter. Thus, the second subunit silkworm caterpillar lineage is a passable transgenic or subsequent progeny that has the induction potential of mt dysfunction of the middle and / or posterior silk glands. Therefore, it can be considered as a mt dysfunction type gene recombinant production line specialized for the production of the genetically modified silkworm of the present invention.

  When the first and second subunits are present on the genome of the host silkworm, each subunit may be present on the same genome or on different genomes. However, when mating the aforementioned first subunit silkworm series with the second subunit silkworm strain to obtain the genetically modified silkworm of the present invention, each subunit should in principle be present on different genomes Is desirable.

3-4. Effect According to the genetically modified silkworm of the present invention, the TFAM gene etc. is overexpressed only in a specific organ, ie, the middle and / or posterior silk glands, whereby the cell function by the deficiency of mitochondrial DNA in the silk glands is limited. It can exhibit symptoms similar to depression. The genetically modified silkworm of the present invention has the advantage that it does not cause individual lethality due to the aggravation of symptoms, which has been a problem of conventional mitochondrial dysfunction animal models.

4. Mitochondrial dysfunction type recombinant silkworm production method 4-1. Overview A fourth aspect of the present invention is a method for producing mt dysfunction type recombinant silkworm. According to the production method of the present invention, it is possible to produce a strain of the mt dysfunction type recombinant silkworm according to the third aspect from silkworm.

4-2. Method The method for producing the mt dysfunction type recombinant silkworm of the present invention (herein often referred to as “genetically modified silkworm”) comprises (1) a host of the gene expression vector of the second aspect There is a method of introducing into silkworm or (2) a method of crossing a first subunit silkworm strain and a second subunit silkworm strain. Each method will be described below.

(1) A method for introducing the gene expression vector of the second aspect into a host silkworm to produce it This method comprises, when the gene expression unit is composed of a single unit, a gene expression vector containing the gene expression unit as a host silkworm. Can be achieved by introducing The method comprises an introduction step as an essential step and a selection step as a selection step. Each step will be described below.

(Introduction process)
The "introduction step" is a step of introducing the gene expression vector of the second aspect comprising a gene expression unit constituted by a single unit into a host cell. The host into which the gene expression vector is introduced is the silkworm described in "1-2. Definition" of the first aspect. In particular, silkworm is preferable as a silkworm of the present invention. When the gene expression vector of the second aspect is introduced into silkworm, there is no particular limitation on the developmental stage of an individual or sex and sex, and any stage of embryo, larva, wing, or adult may be used. Preferably, it is an embryo stage at which higher effects can be expected.

  The introduction method may be carried out by methods known in the art depending on the type of gene expression vector to be introduced. For example, the host to be introduced is silkworm, and the exogenous gene expression vector is a plasmid having transposon inverted terminal repeat (Handler AM. Et al., 1998, Proc. Natl. Acad. Sci. USA 95: 7520-5). In the case of (1), the method of Tamura et al. (Tamura T. et al., 2000, Nature Biotechnology, 18, 81-84) can be used. For example, the gene expression vector of the second aspect may be microinjected into an early embryo of silkworm together with a helper vector having a DNA encoding a transposon transfer enzyme. Helper vectors include, for example, pHA3PIG.

(Selection process)
The “selection step” is a step of selecting, from the silkworm after the introduction step, a transformant containing the gene expression vector of the second aspect. In silkworm after the introduction step, a transformant carrying a gene expression vector and a non-transformant not carrying it exist. In this step, it is an object to discriminate them accurately and to select only a target transformant. When the gene expression vector contains a marker gene such as a fluorescent protein, selection may be performed based on the presence or absence of expression of the gene or the like. The obtained recombinant silkworm may be subjected to brother-sister mating or inbred as necessary to obtain a homozygote of the expression vector inserted into the chromosome.

  In the first subunit silkworm series and the second subunit silkworm series generation method, except that the gene expression vector to be introduced is a gene expression vector containing the first subunit or the second subunit, The basic method is the same as this method.

(2) Method of crossing the first subunit silkworm series and the second subunit silkworm strain This method is to be used in the case where the gene expression unit comprises one set of first and second subunits. In this method, a silkworm series comprising a unit, that is, a first subunit silkworm series and a second subunit silkworm strain are crossed to obtain a recombinant silkworm comprising both subunits. The method comprises a mating step as an essential step, and an egg collecting step, and a selection step as a selection step. Each step will be described below.

(Matching process)
The “breeding step” is a step of crossing the first subunit silkworm strain with the second subunit silkworm strain. Crossing may be performed by crossing two silkworm caterpillar lines based on a conventional method. It is preferable that the silkworm series having each subunit is homozygous in advance by conducting brother-sister mating or inbreeding.

(Aggregation process)
The “ovulation process” is a process of obtaining eggs from a female individual after the mating process. The method of collecting eggs may be carried out by methods known in the art. For example, when the silkworm is a silkworm, a female individual after mating may be separated (cut away) from a male individual, transferred to a laying board, and laid overnight in the dark at room temperature.

(Selection process)
The “selection step” of the present method is basically the same as the selection step in that the target transformant is selected by the activity of the protein encoded by the gene based on the expression of the marker gene. However, in the selection step of the present method, individuals having both subunits are selected based on the expression of the marker gene linked to each of the first subunit and the second subunit from F1 individuals obtained after the egg collecting step. It differs in the point.

5. Therapeutic agent screening method for mitochondrial disease 5-1. Overview A fifth aspect of the present invention is a method of screening for a mitochondrial disease therapeutic agent. The method of the present invention can select an effective drug for treating or preventing mitochondrial disease from candidate drugs.

5-2. Definitions The following terms used in this aspect and the sixth aspect described below are defined.
The term "mitochondrial disease" refers to a general term for conditions that cause clinical symptoms due to mitochondrial dysfunction. Generally, it is considered that mitochondrial dysfunction is caused by qualitative change such as mutation (deletion, substitution, addition, etc.) of mtDNA or genomic DNA, or quantitative change such as decrease of the amount of mtDNA. Many energy disorders due to reduced energy production, such as muscle weakness, myopathy, or skeletal muscle symptoms such as hypercreatine kinase (CK) emia, convulsions, migraine headache, ataxia, intellectual regression, or psychiatric symptoms Such central nervous symptoms, cardiac conduction disorders, cardiac symptoms such as cardiomyopathy, glomerular lesions, renal tubular dysfunction, or renal symptoms such as myoglobinuria, or blood symptoms such as anemia are observed. In the mt dysfunctional type recombinant silkworm of the present invention, the cells of the central and / or posterior silk glands do not undergo apoptosis but become growth arrested, and the volume does not increase. As a result, the silk glands The symptoms are severe. Such conditions in silkworms are referred to herein as "mitochondrial disease-like conditions".

  "Treatment" refers to the cure or amelioration of a disease. As used herein, "improvement" refers to alleviation or elimination of symptoms associated with a disease, and / or prevention or suppression of progression.

  A "therapeutic agent" is a drug intended to treat a disease and is an active ingredient in the treatment.

  A "mitochondrial disease therapeutic agent" is a drug that is an active ingredient in the treatment of mitochondrial disease. Low molecular weight compounds, peptides such as antibodies, and nucleic acid molecules such as miRNAs are relevant.

  The “mitochondrial disease therapeutic composition” refers to a composition containing, in addition to the active ingredient, a mitochondrial disease therapeutic drug, other components such as an excipient.

  "Candidate drug" refers to a drug that can be used to treat mitochondrial disease.

5-3. Method The method of the present invention comprises an administration step, a confirmation step, and a selection step as essential steps. Each step will be described below.

(Administration process)
The “administration step” is a step of administering a candidate agent to the mt dysfunctional type recombinant silkworm according to the third aspect.
The method of administering the candidate drug is not limited. It may be either oral or parenteral.

  When the administration method is oral administration, the target of administration is silkworm and it is necessary to be mixed with the feed and administered. Although the method of mixing is not limited, for example, when the silkworm is a silkworm, the candidate agent may be applied, sprayed, or dispersed on the surface of the mulberry leaf, or the mulberry leaf may be dipped in a drug solution containing the candidate agent. Alternatively, candidate drugs can be added to the artificial feed. Thus, the dosage form of the candidate agent may be either a solid, a granule, a powder, a powder or a solution.

  When the administration method is parenteral administration, systemic administration to silkworm or local administration can be mentioned. In the case of systemic administration, a solution containing a candidate drug may be injected subcutaneously or in the circulatory system of silkworm. When injected subcutaneously, the site does not matter. For example, it may be administered to the abdomen using a microcapillary or the like. In addition, in the case of topical administration, a solution containing a candidate drug may be injected by injection or the like directly into the silk gland where mitochondrial disease-like symptoms are observed.

  In addition, it can be administered in the form of a mixed composition, appropriately combined with an excipient, an emulsifying agent, a suspending agent, a surfactant, a stabilizer, a pH regulator and the like together with the candidate agent.

  A single dose (unit dose) and the number of times of administration may be appropriately determined according to conditions such as the type of silkworm, age, type of candidate drug and the like.

(Confirmation process)
The "confirmation step" is a step of confirming the shape of the silk gland in the genetically modified silkworm after the administration step.

  As described above, in the mt dysfunctional type recombinant silkworm according to the third aspect, the middle and / or rear silk glands are in a contracted state. After the administration step, the silk glands of the genetically modified silkworm are observed 1 to 7 days, 2 to 6 days, 3 to 5 days, or 4 days after the administration step, and the shape is confirmed without being fixed. Although the method of confirming the silk gland is not limited, it is convenient to carry out an open treatment and observe directly by visual observation. Extraction of the silk gland may be performed as necessary using tweezers or the like (Mori, Ed., New biology experiment with silkworm, see Sanshodo, 1970, pp. 249-255). The weight, volume, etc. of the silk gland can also be measured and quantified. The silk gland to be subjected to current confirmation is a silk gland in which the TFAM gene is overexpressed, that is, a silk gland in which a silk gland specific promoter contained in the gene expression vector described in the second embodiment is activated. Specifically, when the gene expression vector according to the second aspect contains a middle silk gland specific promoter, the shape of the middle silk gland, and when the rear silk gland specific promoter, the shape of the rear silk gland If both of them are included, the shapes of both silk glands may be confirmed. As a control, the shape of the silk gland of a wild-type silkworm or a silkworm strain containing a control gene expression vector not containing a TFAM gene may be confirmed.

(Selection process)
The “selection step” is a step of selecting a candidate drug used in the administration step as a mitochondrial disease therapeutic agent when the silk gland shape of the above-mentioned recombinant silkworm has recovered from the atrophy state.

  Recovery may be either complete recovery or partial recovery. Complete recovery means that the shape of the silk gland returns to the same level as the control wild-type silkworm.

  The criterion for recovery is the corresponding silk gland of the control transgenic silkworm that has not received the candidate agent. When the atrophy state recovers from the shape of the silk gland of the control genetically modified silkworm, if it is determined if it is quantified, if it is increased numerically, the candidate drug used in the administration step is the mitochondria It is selected as a treatment for diseases.

6. Evaluation method for mitochondrial disease therapeutic agent 6-1. Overview A sixth aspect of the present invention is a method of evaluating a mitochondrial disease therapeutic agent. According to the evaluation method of the present invention, the efficacy of known mitochondrial disease therapeutic agents can be reconfirmed or reevaluated.

6-2. Method The basic configuration of the method of the present invention is similar to the method of screening for a mitochondrial disease therapeutic agent according to the fifth aspect. For example, the administration step is basically the same except that the agent to be administered is a candidate agent in the fifth aspect, whereas it is a known mitochondrial therapeutic agent or a known therapeutic agent candidate in this aspect . Here, "known mitochondrial therapeutic drug" is a drug that has been approved as a pharmaceutical product under the drug-approach, and "therapeutic candidate drug" refers to a drug or the like in the clinical trial stage for receiving approval. For example, as described in the Examples below, aminolevulinic acid (5-ALA) is a mitochondrial disease treatment candidate drug in Phase III of clinical trial.

  In addition, the basic operation of the evaluation step of this embodiment is the same as the operation of the selection step in the screening method. When the silk gland shape has recovered after the confirmation step, in the fifth embodiment, the candidate drug is selected as a mitochondrial therapeutic agent, whereas in this step, the known mitochondrial therapeutic agent is effective for treating mitochondrial disease. There is only a difference to certify.

Example 1 Construction of Gene Expression Unit and Gene Expression Vector
(the purpose)
The gene expression unit for producing mt dysfunctional silkworm of the present invention and a gene expression vector containing the same are prepared.

(Method)
In this embodiment, silkworms are silkworms. First, the Bmtfam gene (SEQ ID NO: 2) encoding silkworm TFAM was cloned. A primer pair capable of specifically amplifying the ORF sequence of Bmtfam gene using as a template a cDNA library prepared from embryo-derived total mRNA of p50 strain, Bmtfam-for (5'-ATGACTACTTATACTCAATTACAGCG-3 ': SEQ ID NO: 11) and Bmtfam- It amplified by PCR using rev (5'-CTATTGTGAACTATCAACTTTTTTGG-3 ': sequence number 12). Takara EX taq (Takara-Bio) was used as a DNA polymerase for PCR. PCR conditions were performed under the conditions of a conventional method. The amplified product after PCR was cloned by the TA cloning method using pGEM-T vector (Novagen). The obtained vector was called "pGEM-BmTFAM".

  Next, in order to fuse the green fluorescent protein (gfp) gene to the 3 'end of the Bmtfam gene, GFP is located downstream of the CMV promoter of the mammalian cell expression vector pCMV-SPORT (Thermo Fisher Scientific). The vector into which the sequence was introduced was designated "pCMV-SPORT-GFP". Subsequently, using specific primers designed to add BglII and HindIII restriction sites to both ends of the Bmtfam gene, PCR was performed using pGEM-BmTFAM as a template and KOD plus DNA polymerase (Toyobo) It amplified. The amplified BmTFAM fragment was inserted into the BglII / HindIII site of pCMV-SPORT-GFP. Thus, pCMV-BmTFAM-GFP containing a TFAM-GFP fusion gene consisting of the nucleotide sequence shown in SEQ ID NO: 13 was obtained.

  A gene expression vector was constructed in which BmTFAM was expressed depending on GAL4 so that the gene expression unit of the present invention was composed of a set of subunits.

  First, the mother nucleus vector containing the second subunit includes the silkworm transposon vector pBac [UAS-SV40, 3 × P3-GFP] vector (Sakudoh, T., et al., 2007, Proc Natl Acad Sci USA, 104: 8941-8946) was used. The BmTFAM-GFP fragment was amplified by PCR using the above-described pCMV-BmTFAM-GFP as a template and KOD plus DNA polymerase (Toyobo). As a primer, a specific primer in which 15 residues homologous to the terminal sequence when the pBac [UAS-SV40, 3 × P3-GFP] vector was linearized with BInI was used. After the amplification reaction, the purified PCR amplified fragment, that is, the BnTF-digested pBac [UAS-SV40, 3 × P3-GFP] vector obtained by BnTFA-GFP fragment, the In-Fusion HD Cloning Kit (Clontech) at the 3 ′ end of the UAS sequence. Was inserted by site-specific recombination to obtain pBac [UAS-BmTFAM-GFP-SV40, 3 × P3-GFP] as a gene expression vector containing the second subunit. The conceptual diagram is shown in FIG. The attached protocol of In-Fusion HD Cloning Kit was followed about the method of primer design and the cloning method.

<Example 2: Production of mitochondrial dysfunction type recombinant silkworm>
(the purpose)
An mt dysfunction type recombinant silkworm of the present invention is produced.

(Method)
Silkworm (Bombyx mori) was reared at 25 ° C., with a light period of 12 hours, and a dark period of 12 hours, using an artificial feed (Silkmate; Nosan). The experiments included p50, w1-pnd, w1 and A3-GAL4 (Uchino, K., et al., 2006, J. Insect Biotechnol. Sericology, 75: 89-97), AyFib 431a (Sezutsu, H., et al. Sericology 78: 1-10; Tsubota, T., et al., 2014, G3 (Bethesda) 4: 1347-1357) were used.

  Production of recombinant silkworms was performed under the following conditions based on the method of Tamura et al. (2000, Nature Biotechnology, 18 81-84). First, pBac [UAS-BmTFAM-GFP-SV40, 3 × P3-GFP] constructed in Example 1, ie, a gene expression vector containing the second subunit, helper plasmid pHA3PIG and transposase mRNA at a final concentration of 0.4 μg / each. The injection solution was prepared by dissolving in injection buffer (0.5 mM phosphate buffer, KCl 5 mM, pH 7.0) to be μL, 0.1 μg / μL, and 0.2 μg / μL. The injection solution was microinjected into the embryo of the silkworm w1-pnd strain (white eye, white egg, non-dormant strain) within 6 hours after egg laying using a tungsten needle and a glass capillary. The embryos after injection were closed with a superglue (Konishi # 30523) and incubated in a humidified condition at 25 ° C. until hatching. The hatched larvae were reared and mated with brothers and sisters, or with the non-injected white-eye dormant line w1 for line maintenance. The obtained embryos were selected for the presence or absence of expression of 3xP3-GFP to obtain a second subunit silkworm strain comprising a gene expression vector having a second subunit.

  The number of genomic insertions of the gene expression vector containing the second subunit in the generated second subunit silkworm strain was confirmed by Southern blot. The Southern blot method followed the usual method. Moreover, the genomic insertion position of the gene expression vector containing 2nd subunit was confirmed by inverse PCR. Inverse PCR is based on the method of Tamura et al. (2000), about the left arm (L-arm) side primer pair of pBac [UAS-BmTFAM-GFP-SV40, 3 × P3-GFP] inserted into the genome For the right arm (R-arm) side, inv-Larm-F1 (5'-AAATCAGTGACACTTACCGCATT-3 ': SEQ ID NO: 14) and inv-Larm-R2 (5'-ACTATAACGACGCGCGTGAGTCAA-3': SEQ ID NO: 15) Used 30K6G1gal-invF2 (5'-AAGTACAAAAAACTTTTATGGCGC-3 ': SEQ ID NO: 16) and 30K6G1gal-invR (5'-CCTCGATATACAGACCGATAAACA-3': SEQ ID NO: 17).

  As a result, it was revealed that only one copy of the gene expression vector containing the second subunit was inserted into the genome, and that the position did not destroy the endogenous gene of the host silkworm (data not shown).

  On the other hand, in the first subunit silkworm series, an existing silkworm strain that expresses GAL4 specifically in the posterior silk gland, that is, an existing silkworm strain including the first subunit in which the GAL4 gene is disposed at the 3 'end of the posterior silk gland promoter, For example, the first subunit silkworm series (AyFib 431a) having the first subunit for the posterior silk gland gene expression unit established in Example 2 of Table 2014/104269 was used.

  Both lines are crossed, and F1 individual containing both subunits is selected by the presence or absence of fluorescence of 3xP3DsRed2 marker (marker for the first subunit) and 3xP3-GFP marker (marker for the second subunit), and is specific for posterior silk gland The mt dysfunctional recombinant silkworm of the present invention overexpressing BmTFAM was obtained.

  The mt functionally deficient type transgenic silkworms produced were reared at all temperatures of 25 to 27 ° C. using artificial feed (Silk mate original type 1 to 3 years S, Nippon Agro-industry). Artificial feed was changed every two to three days (Uchino K. et al., 2006, J Insect Biotechnol Sericol, 75: 89-97). At the stage immediately before the 5th day 6th vomiting, the final-instar larva was anesthetized on ice, and the dorsal side was incised and removed so as not to damage the silk gland with tweezers. It was observed with a stereomicroscope (Olympus SZX16) without fixing it. The excised tissue was immediately pre-fixed with glutaraldehyde solution, and then post-fixed with 1% osmium tetraoxide solution. Next, after dehydration with an ethanol series, an epoxy resin was permeated and replaced. The tissue was placed in an embedding plate with fresh epoxy resin, polymerized in a thermostat for 3 days, and embedded. A piece of tissue embedded in epoxy resin was attached to the microtome body, unnecessary portions were cut off, and a thin section was cut out. At the same time, thick-cut sections were made, stained with toluidine blue, and the orientation of the tissue was confirmed before trimming. Subsequently, ultra-thin slices were made from the finished texture using a diamond knife. Ultrathin slices were placed on a grid, electron stained, and observed with a transmission electron microscope (JEOL: JEM1010).

(result)
The results are shown in FIG. 2 and FIG.
As a result of overexpression of TFAM-GFP specifically in the posterior silk gland, as shown in FIG. 2B, the posterior silk gland in the dashed white frame does not increase in cell volume as compared to FIG. Stopped and contracted. However, cell death did not occur in the posterior silk gland. In addition, such TFAM-GFP overexpressing individuals become normal adults while having morphological abnormalities in the posterior silk gland. Thus, the mt dysfunctional type recombinant silkworm of the present invention can observe severe phenotypes over time without causing individual death.

  As shown in FIGS. 3C and 3D, overexpression of TFAM-GFP specifically in the posterior silk gland resulted in abnormal mitochondrial morphology as compared to FIGS. 3A and 3B in the control section. Specifically, the structure (criste) of the inner mitochondrial membrane is organized in layers in the mitochondria of the control section, whereas in the mitochondria of the tissue overexpressing TFAM-GFP, a large number of small bubbles are formed. It had become. In addition, as shown in FIG. 3C, mitochondria entrapped in an autophagosome-like membrane (arrow) were observed. As described above, such a TFAM-GFP overexpressing individual has morphological abnormalities in the posterior silk gland but does not kill it, so that it is possible to observe phenotypes for mitochondrial activity and morphology over time .

<Example 3: Efficacy evaluation of a mitochondrial disease treatment candidate drug using mt dysfunction type recombinant silkworm>
(the purpose)
It is verified that the mt dysfunctional recombinant silkworm of the present invention can be a model animal replacing mt dysfunctional mouse and the like, and that the efficacy evaluation of a mitochondrial disease therapeutic drug or a therapeutic drug is possible.

(Method)
100 μg of aminolevulinic acid (5-ALA) in NaCl solution, 10 μL per head, to the individual (n = 15) at age 5 on day 5 of the mt dysfunction type recombinant silkworm produced in Example 1; Were injected subcutaneously using a microcapillary. 5-ALA has been developed as an effective candidate drug for human mitochondrial diseases centered on cranial nerve symptoms, and is currently in Phase III clinical trials in Japan, and its efficacy and safety for mitochondrial diseases with long-term administration are verified It is done. Five days after administration, the silkworm was anaesthetized on ice in the same manner as in Example 2, and the dorsal side was incised and extracted with tweezers so as not to damage the silk gland.

(result)
The administration results of 5-ALA are shown in FIG. When 5-ALA was administered, 90% of the individuals showed morphological recovery of the posterior silk gland as shown in FIG. 4B. This result indicates that the mt dysfunctional recombinant silkworm of the present invention can function as a model animal for mitochondrial diseases of mammals, and the mitochondrial function is indicated by the shape recovery in the mammary gland of the mt dysfunctional recombinant silkworm silkworm It suggests that it is possible to screen therapeutic agents and evaluate the efficacy of therapeutic candidates.

Claims (15)

  A mitochondrial dysfunctional silkworm-inducing agent comprising, as an active ingredient, a nucleic acid molecule encoding a gene encoding mitochondrial transcription factor A or a functional fragment thereof. A gene expression unit for producing a mitochondrial dysfunctional silkworm,
A promoter of a gene encoding a protein specifically expressed in the central or posterior silk gland, and a nucleic acid molecule encoding a gene encoding mitochondrial transcription factor A functionally linked downstream of the promoter, or a functional fragment thereof Gene expression unit.   The gene expression unit according to claim 2, further comprising an additional gene or a fragment thereof linked to the 3 'end of the gene encoding mitochondrial transcription factor A.   The gene expression unit according to claim 2 or 3, wherein the protein specifically expressed in the middle or rear silk gland is a protein selected from the group consisting of sericin, fibroin H chain, fibroin L chain and p25 / FHX. The gene expression unit according to any one of claims 2 to 4, wherein the mitochondrial transcription factor A is any one of the following (a) to (d).
(A) A silkworm-derived mitochondrial transcription factor A comprising the amino acid sequence shown in SEQ ID NO: 1,
(B) a mitochondrial transcription factor A comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence described in (a),
(C) Mitochondrial transcription factor A consisting of an amino acid sequence having an amino acid identity of 90% or more to the amino acid sequence described in (a), or (d) another type ortholog of mitochondrial transcription factor A described in (a) protein   The gene expression unit according to any one of claims 3 to 5, wherein the additional gene is a gene encoding a marker protein. The gene expression unit comprises the first and second subunits,
The first subunit comprises a promoter of a gene encoding a protein specifically expressed in the central or posterior silk gland and a gene encoding a transcriptional regulatory factor functionally linked to its 3 'end side,
The second subunit comprises a target promoter of the transcriptional regulatory factor and a nucleic acid molecule encoding a gene encoding mitochondrial transcription factor A functionally linked to the 3 'end thereof or a functional fragment thereof.
The gene expression unit according to any one of claims 2 to 6.   A gene expression vector comprising the gene expression unit according to any one of claims 2 to 6.   A gene expression vector comprising a second subunit constituting the gene expression unit according to claim 7.   A mitochondrial dysfunction abnormality type recombinant silkworm comprising a mitochondrial expression abnormality type silkworm gene expression unit according to any one of claims 2 to 7.   The transgenic silkworm according to claim 10, which is subordinate to claim 7, wherein the first subunit and the second subunit are present on different chromosomes. A method for producing a mitochondrial dysfunction abnormality type transgenic silkworm, comprising
The method, comprising: introducing the gene expression vector of claim 8 into silkworm; and selecting the individual comprising the gene expression vector after the introducing step. A method for producing a mitochondrial dysfunction abnormality type transgenic silkworm, comprising
A mating step of crossing a first subunit silkworm strain comprising the first subunit according to claim 7 with a second subunit silkworm strain comprising the second subunit according to claim 7;
The method, comprising: an egg collecting step of collecting eggs from silkworms after the mating step; and a selecting step of selecting an individual including the first and second subunits from the eggs obtained in the egg collecting step. A method of screening a drug for treating mitochondrial disease, comprising
An administration step of administering a candidate agent to the mitochondrial dysfunction type recombinant silkworm according to claim 10 or 11;
A confirmation step of confirming the shape of the silk gland in the genetically modified silkworm after the administration step, and selecting the candidate drug as a treatment for mitochondrial disease when the silk gland shape of the genetically modified silkworm which has been contracted is recovered Said method comprising a selection step. It is a method of evaluating a mitochondrial disease therapeutic agent,
An administration step of administering a known mitochondrial disease therapeutic agent to the mitochondrial dysfunction type recombinant silkworm according to claim 10 or 11;
The confirmation step of confirming the shape of the silk gland in the genetically modified silkworm after the administration step, and when the silk gland shape of the genetically modified silkworm which has been contracted has been recovered, the agent for treating mitochondrial disease is a mitochondrial disease Said method comprising the evaluation step of evaluating as being effective for treatment.