WO2010010887A1 - Promoteur d'expression tissulaire - Google Patents

Promoteur d'expression tissulaire Download PDF

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WO2010010887A1
WO2010010887A1 PCT/JP2009/063088 JP2009063088W WO2010010887A1 WO 2010010887 A1 WO2010010887 A1 WO 2010010887A1 JP 2009063088 W JP2009063088 W JP 2009063088W WO 2010010887 A1 WO2010010887 A1 WO 2010010887A1
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dna
seq
base sequence
promoter
adipose tissue
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Japanese (ja)
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真一郎 堀
和彦 前川
鈴木　稔
五紀 大島
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塩野義製薬株式会社
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Publication of WO2010010887A1 publication Critical patent/WO2010010887A1/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the present invention relates to a novel promoter useful for specific expression in adipose tissue and / or pancreatic tissue and use thereof.
  • adipocyte P2 promoter (hereinafter also referred to as aP2 promoter) region is known to specifically express a gene connected downstream thereof in adipose tissue (cell), and a fusion gene with this promoter region.
  • MCP-1 monocytochemotractant protein-1
  • CDO cyste dioxidase
  • taurine synthesis can be promoted in the adipose tissue of transgenic mice prepared by introducing a gene in which this enzyme is fused with the aP2 promoter. It has been.
  • CDO is overexpressed 2 to 3 times that of normal mice, and is released as a research tool for the association of taurine with obesity and metabolic syndrome.
  • a sequence of about 520 bp (SEQ ID NO: 21) located at the most upstream of the aP2 promoter (SEQ ID NO: 24) derived from a mouse of about 5.4 kb has been shown to be adipose tissue-specific expression in vivo by experiments with transgenic mice. It has been reported to be important. However, as a result of comparing the in vivo expression intensity with the case where the full length of the promoter is used, the activity is greatly reduced (Non-patent Document 1), and only this region is used as a promoter for adipose tissue-specific expression. Even if it was used, its expression was weak, so that a practical transgenic mouse could not be produced.
  • transgenic mice can be produced with very high efficiency by introducing genes into fertilized eggs using a lentiviral vector, compared to the conventional microinjection method.
  • aP2 gene promoter has a sequence length of about 5.4 kb, and when incorporated into a lentiviral vector, its large size becomes a barrier, making it difficult to produce a transgenic mouse using this promoter. there were.
  • the reason is that, due to the large length of the promoter sequence, the packaging efficiency of the virus is reduced, and it is not possible to prepare a virus having the titer necessary for the production of a transgenic mouse.
  • the same reason can be considered for other viral vectors used for gene therapy such as adenovirus and AAV.
  • An object of the present invention is to provide an element capable of specific expression in adipose tissue and / or pancreatic tissue and having high introduction efficiency.
  • a partial region in the sequence derived from the mouse aP2 gene promoter is important for promoting expression in adipose tissue, Even when a transgenic mouse is produced using a truncated promoter containing this region, it has a specific activity and sufficient expression level in adipose tissue and pancreatic tissue, and human-derived sequences are similar to those in mice. It has been found that it has activity, and the present invention has been completed.
  • the present invention (1) an isolated DNA having at least the base sequence shown in SEQ ID NO: 22 and having specific promoter activity in adipose tissue and / or pancreas, (2) (a) DNA having at least one base substitution, deletion, insertion or addition in the base sequence shown in SEQ ID NO: 22; (b) complementary DNA consisting of the base sequence shown in SEQ ID NO: 22 A DNA selected from the group consisting of DNA that can hybridize with the strand under stringent conditions, and (c) a DNA having a sequence identity of at least 90% to the DNA consisting of the base sequence represented by SEQ ID NO: 22 Isolated DNA having specific promoter activity in adipose and / or pancreatic tissue, (3) The isolated DNA according to (1) or (2), which is further DNA having the base sequence represented by SEQ ID NO: 21; (4) The isolated DNA according to any one of (1) to (3), which is further DNA having the base sequence represented by SEQ ID NO: 23, (5) an isolated DNA having at least the base sequence shown in SEQ ID NO: 28 and
  • transgenic mouse capable of specifically expressing a target gene in adipose tissue and / or pancreatic tissue, and to perform gene therapy targeting these tissues. Become.
  • FIG. 1 is a schematic diagram of a reporter assay construct for the aP2 promoter-derived sequence prepared this time.
  • FIG. 2 is a graph showing the luciferase activity when co-expressing a reporter assay construct of an aP2 promoter-derived sequence and a pRL-Tk vector, which is a vector expressing Renilla luciferase as an internal control reporter. The horizontal axis shows the relative value of the emission intensity when other constructs are used with the emission intensity when aP2-11-luc is introduced at 293 being 1.
  • FIG. 3 is a graph showing the luciferase activity when reporter vectors of human and mouse aP2 promoter-derived sequences are coexpressed with the pRL-Tk vector, respectively.
  • the abscissa represents the relative value of the luminescence intensity when other constructs are used with the luminescence intensity when aP2-11 is introduced into undifferentiated 3T3-L1 being 1.
  • the “promoter” includes an expression regulatory region capable of associating with RNA polymerase in a cell, and initiates transcription of a coding sequence existing downstream (toward the 3 ′ end) of the region. It includes a region having a function to be performed. Furthermore, the “promoter” refers to a region including a so-called enhancer region responsible for tissue-specific expression regulation.
  • adipose tissue and / or pancreatic tissue means that expression in adipose tissue and / or pancreatic tissue is clearly higher compared to other tissues.
  • the adipose tissue is not particularly limited as long as it is a mammalian adipose tissue, such as subcutaneous adipose tissue, visceral adipose tissue, white adipose tissue, brown adipose tissue, perigenital adipose tissue, mesenteric adipose tissue, scapula Contains brown adipose tissue.
  • the DNA of the present invention is an DNA having at least the base sequence shown in SEQ ID NO: 22 and having a specific promoter activity in adipose tissue and / or pancreatic tissue. DNA. Since the DNA of the present invention has the base sequence shown in SEQ ID NO: 22, it has an excellent property that it can be expressed specifically in adipose tissue and / or pancreatic tissue even in vivo.
  • the base sequence shown in SEQ ID NO: 22 is a sequence of the region consisting of positions 1662 to 2357 in the mouse-derived adipocyte P2 gene promoter.
  • the inventors have now found that these sequences play an important role in the specific expression of adipose and / or pancreatic tissue in vivo. In general, gene expression trends in vitro often differ greatly from actual gene expression trends in vivo.
  • the promoter activity of the DNA of the present invention is, for example, the following steps I to III: I a sequence in which a reporter gene (for example, luciferase gene, alkaline phosphatase gene, etc.) is operably linked under the control of the DNA to be tested.
  • a step of preparing a plasmid for activity measurement retaining II, a step of transforming appropriate adipose tissue cells (or cells of pancreatic tissue) and control cells with the activity measurement plasmid of I above, and III This includes a step of detecting the presence or absence of expression of a reporter gene in the obtained cells and measuring the expression intensity, and can be determined by a reporter gene assay or the like.
  • the DNA is an indicator that it is a promoter specific to adipose tissue (pancreatic tissue) It becomes.
  • the amount of the expression product of the reporter gene is an indicator of the strength of the promoter activity.
  • the DNA of the present invention is a DNA having a substitution, deletion, insertion or addition of at least one base, specifically, one or more bases in the base sequence shown in SEQ ID NO: 22, and It may be an isolated DNA having specific promoter activity in adipose tissue and / or pancreatic tissue.
  • the number of substitutions, deletions, insertions or additions (hereinafter may be simply referred to as “mutation”) and the position of introduction are determined by performing a reporter gene assay comprising the steps I to III above and / or Any promoter that is determined to be a pancreatic tissue-specific promoter may be used.
  • mutation substitutions, deletions, insertions or additions are included.
  • Artificial mutation can be introduced by a conventional site-specific mutation introduction method.
  • methods for introducing site-specific mutation include a method using amber mutation (gapped duplex method, Nucleic Acids Research, 12, 9441-9456 (1984)) and a PCR method using amber mutation (international publication). 98/02535) or the like can be used.
  • the DNA of the present invention has a specific promoter activity in adipose tissue and / or pancreatic tissue, it hybridizes under stringent conditions with a complementary strand of DNA consisting of the base sequence shown in SEQ ID NO: 22.
  • Isolated DNA having specific promoter activity in adipose tissue and / or pancreatic tissue is also encompassed by the present invention.
  • Such DNA is not particularly limited as long as it is determined to be a promoter specific to adipose tissue and / or pancreatic tissue by performing a reporter gene assay including the steps I to III.
  • hybridization operation can be performed, for example, according to the method described in Molecular Cloning: A Laboratory Manual Second Edition (issued by Cold Spring Harbor Laboratory, 1989).
  • stringent conditions include hybridization conditions described in the above-mentioned books and the like. Specifically, 10% dextran sulfate, 1M NaCl, 1% SDS, 100 mg / ml salmon sperm DNA, 1 After performing a hybridization reaction under the conditions of ⁇ 10 6 cpm / ml 32 P-labeled probe (SEQ ID NO: 22), 2 ⁇ SSC, 0.1% SDS, twice at room temperature, 0.1 ⁇ SSC, 0. The conditions are such that washing is performed twice at 1% SDS and 60 ° C.
  • the DNA of the present invention includes DNA having at least 90% sequence identity to the DNA consisting of the base sequence shown in SEQ ID NO: 22, preferably 95% or more, more preferably 98% or more. Also included is isolated DNA having specific enhancer activity in adipose tissue and / or pancreatic tissue. If DNA having such sequence identity is determined to be a promoter specific to adipose tissue and / or pancreatic tissue by performing a reporter gene assay including the steps I to III described above, for example. Good.
  • sequence identity can be determined using a homology search program (for example, BLAST etc.) commonly used in this field by default.
  • sequence identity of nucleotide sequences is calculated by aligning the two types of nucleotide sequences to be compared and dividing the number of nucleotide sequences matched by the alignment by the total number of reference nucleotide sequences. It is the number which showed the ratio which was done in%. Note that the gap generated by the alignment is calculated as a mismatch.
  • the base sequence shown in SEQ ID NO: 21 is further included.
  • This DNA is operably linked, but it is generally desirable that the DNA is linked so that SEQ ID NO: 21 is upstream.
  • the base sequence shown in SEQ ID NO: 21 is a sequence of a region consisting of about 520 bases at the most upstream of the mouse-derived adipocyte P2 gene promoter. This region is generally called an “enhancer” and has a function of increasing the transfer speed. It has also been reported to have an important role in adipose tissue-specific expression (Journal of Cellular Biochemistry, Vol. 49, 1992, pages 219-224).
  • the base sequence shown in SEQ ID NO: 23 is a sequence of about 200 bp in the region from 5359 to 5548 in the mouse-derived adipocyte P2 gene promoter (aP2 promoter).
  • This region is called a proximal-promoter and includes a transcription factor binding region such as AP-1 or C / EBP (CCAAT / enhancer binding protein).
  • AP-1 or C / EBP CCAAT / enhancer binding protein
  • This region plays an important role in adipose differentiation, it has been reported that it is not an essential region for adipose tissue-specific expression in vivo such as in transgenic animals. (Proc. Natl. Acad. Sci. USA, Vol. 87, pp 9590-9594, 1990).
  • This downstream part contains sequences called TATA box and CAAT box, and it is known to lead RNA polymerase to its normal start site and to promote transcription.
  • the DNA of the present invention provides an expression vector capable of expressing a foreign gene specifically in adipose tissue and / or pancreatic tissue.
  • expression vector refers to a vector comprising a sequence of a foreign gene that encodes at least a portion of a gene product that can be transcribed.
  • the sequence of the foreign gene is optionally translated into a protein, polypeptide or peptide. In other cases, these sequences are not translated.
  • siRNA that specifically cleaves mRNA using RNA interference action such as miRNA (microRNA) and shRNA (short hairpin structure RNA).
  • miRNA miRNA
  • shRNA short hairpin structure RNA
  • the expression vector of the present invention can be obtained by inserting the DNA of the present invention into an appropriate vector.
  • the vector used in the present invention include plasmids, phages, cosmids, viruses (bacteriophages, animal viruses and plant viruses) and artificial chromosomes (for example, YAC).
  • the expression vector of the present invention is characterized in that the DNA of the present invention is operably linked upstream of the foreign gene sequence in the transcription direction, and is specific to adipose tissue and / or pancreatic tissue. In which foreign genes can be expressed.
  • a preferred embodiment is an expression vector in which a foreign gene is operably linked downstream of DNA having the base sequence shown in SEQ ID NO: 22 and the base sequence shown in SEQ ID NO: 21.
  • an expression in which a foreign gene is operably linked downstream of a DNA having the base sequence shown in SEQ ID NO: 22, the base sequence shown in SEQ ID NO: 21, and the base sequence shown in SEQ ID NO: 23 It is a vector.
  • the expression vector of the present invention can itself be a very useful therapeutic tool for diseases in adipose tissue and / or pancreatic tissue.
  • the transgenic non-human animal having the expression vector of the present invention and cells derived therefrom are adipose tissues (or fat cells) such as anti-obesity drugs, diabetes drugs, hyperlipidemia drugs, hypertension drugs, It can be a tool for searching for drugs that improve diseases associated with pancreatic tissue (or pancreatic cells) (such as metabolic syndrome-related diseases).
  • the base sequences shown in SEQ ID NOs: 21 to 23 are all derived from mice, but the present inventors have found a base sequence having the same action from a human-derived gene. Specifically, the base sequence represented by SEQ ID NO: 21 is the base sequence represented by SEQ ID NO: 27, the base sequence represented by SEQ ID NO: 22 is the base sequence represented by SEQ ID NO: 28, and the base sequence represented by SEQ ID NO: 23. Since the sequence corresponds to the base sequence represented by SEQ ID NO: 29, it is within the common sense of those skilled in the art to obtain the same effect by replacing these sequences.
  • Examples of the foreign gene include (A) a gene encoding a protein for treating the target disease, (B) a nucleic acid for treating the target disease, and (C) a marker gene.
  • target disease examples include diseases that can be treated by expressing a foreign gene specifically in adipose tissue and / or pancreatic tissue. Specific examples include adipose tissue atrophy, type II diabetes, hyperlipidemia, obesity and the like.
  • proteins for treating a target disease include insulin, leptin, and glucose transporter.
  • Nucleic acids for treating the target disease include synthetic or non-naturally occurring forms of nucleic acids, such as phosphorothioate antisense oligonucleotides, aptamers, siRNAs, or double stranded RNAi, and nucleic acids well known in the art Other forms of nucleic acids such as the chemical derivatives of can also be used.
  • nucleic acids such as the chemical derivatives of can also be used.
  • Various nucleotide sequences can be used for designing siRNA and other interference sequences. For example, siDirect (http://design.RNAi.jp/) can be used for sequence design.
  • marker gene examples include ⁇ -galactosidase gene, alkaline phosphatase gene, chloramphenicol acetyltransferase gene, growth hormone gene, luciferase gene, green fluorescent protein gene and derivatives thereof.
  • the “derivative” includes an artificially produced mutant.
  • the foreign gene when the foreign gene is “the gene encoding a protein for treating the target disease” or “the nucleic acid for treating the target disease”, it is specific to adipose tissue and / or pancreatic tissue. It can be used as an active ingredient of a therapeutic agent. Such therapeutic agents are also included in the present invention.
  • the therapeutic agent of the present invention is characterized by containing the expression vector as an active ingredient, and encodes a foreign gene specifically for a fat tissue and / or pancreatic tissue, specifically a protein for treating a target disease. Or a nucleic acid for treating a target disease. Since such a therapeutic agent contains the expression vector as an active ingredient, it has an excellent property of exhibiting high specificity for adipose tissue and / or pancreatic tissue.
  • the therapeutic agent of the present invention may appropriately contain components for maintaining the expression vector, which is an active ingredient, in a stable state, for example, buffer components, degradation protective agents (for example, inhibitors of nucleolytic enzymes, etc.) and the like. Good.
  • the gene expression agent of the present invention may further contain a drug suitable for introduction into cells and / or tissues.
  • the amount of the expression vector in the therapeutic agent of the present invention can be appropriately set depending on the purpose of use. For example, when used in the therapeutic agent described later, it can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient.
  • the expression vector amount is 0.0001 to 100 mg, preferably 0.001 to 10 mg. It is desirable. It is desirable to administer such a dose once every few days to several months.
  • the therapeutic agent of the present invention can be introduced into cells by, for example, a lipofection method, a phosphate-calcium coprecipitation method; a DEAE-dextran method; a direct injection method using a micro glass tube.
  • Examples of the method for introducing the therapeutic agent of the present invention into a tissue include introduction method using encapsulated liposome, introduction method using electrostatic liposome, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), particle gun Examples thereof include a method of transferring an active ingredient together with a carrier (metal particles) into a cell, a method of introduction with a positively charged polymer, and the like.
  • the therapeutic agent of the present invention since it has high specificity for adipose tissue and pancreatic tissue, diseases that can be treated by expressing foreign genes specifically for adipose tissue and / or pancreatic tissue (for example, , Obesity, type II diabetes, adipose tissue atrophy, pancreatic cancer, etc.), and can exhibit a symptom reduction or curative effect. Therefore, at the same time, it has an excellent property that it becomes possible to reduce the influence of the therapeutic agent on tissues other than adipose tissue and pancreatic tissue.
  • diseases that can be treated by expressing foreign genes specifically for adipose tissue and / or pancreatic tissue for example, Obesity, type II diabetes, adipose tissue atrophy, pancreatic cancer, etc.
  • Examples of the “disease that can be treated by expressing a foreign gene specifically in adipose tissue and / or pancreatic tissue” include the same as the “target disease”.
  • the therapeutic agent of the present invention is administered, for example, intravenously, artery, subcutaneously, intradermally, intramuscularly, or directly locally to the subject adipose tissue and / or pancreatic tissue itself. be able to.
  • various preparation forms for example, a liquid agent etc.
  • the injection can be prepared by a conventional method, for example, after dissolving in an appropriate solvent (buffer solution such as PBS, physiological saline, sterilized water, etc.)
  • buffer solution such as PBS, physiological saline, sterilized water, etc.
  • it can be prepared by sterilizing by filtration with a filter or the like and then filling into an aseptic container.
  • a conventional carrier or the like may be added to the injection as necessary.
  • Liposomes such as HVJ-liposomes can be in the form of liposome preparations such as suspensions, freezing agents, and centrifugal concentrated freezing agents.
  • a sustained-release preparation (mini-pellet preparation or the like) can be prepared and implanted near the affected area, or can be gradually and gradually administered to the affected area using an osmotic pump or the like.
  • the dosage form of the therapeutic agent of the present invention is described in detail, for example, in the experiment manual, etc., its preparation method, administration method, etc. (separate volume experimental medicine, basic technology of acupuncture gene therapy, Yodosha, 1996, separate volume experiment) Medicine, Acupuncture Gene Introduction & Expression Analysis Experimental Method, Yodosha, 1997).
  • experiment manual etc.
  • administration method etc.
  • the therapeutic agent of the present invention can be introduced into cells or tissues by the following technique.
  • Examples of the method for introducing a gene into a cell include a lipofection method, a phosphate-calcium coprecipitation method, and a direct injection method using a micro glass tube.
  • Gene transfer methods to tissues include gene transfer method using encapsulated liposomes, gene transfer method using electrostatic liposomes, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), receptor-mediated gene transfer And a method of transferring an active ingredient together with a carrier (metal particles) with a particle gun, a direct introduction method of naked-DNA, an introduction method using a positively charged polymer, and the like.
  • a viral vector When a viral vector is used in the construction of the expression vector of the present invention, examples of such a viral vector include recombinant adenovirus, retrovirus and the like. More specifically, for example, lentivirus, detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, shinbis virus, Sendai virus, SV40, immunodeficiency virus ( HIV) and other DNA viruses or RNA viruses can be introduced into a cell by introducing the DNA of the present invention and a foreign gene operably linked to the DNA, and infecting the resulting expression vector. Is possible. Of the viral vectors, it is desirable to use a lentivirus to infect fertilized eggs.
  • Lentiviruses are a subgroup of retroviruses, various primate viruses such as human immunodeficiency viruses HIV-1 and HIV-2, simian immunodeficiency virus (SIV), and non-primate viruses (eg, maedi bisna virus ( MW), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), goat arthritis encephalitis virus (CAEV) and bovine immunodeficiency virus (BIV)).
  • various primate viruses such as human immunodeficiency viruses HIV-1 and HIV-2, simian immunodeficiency virus (SIV), and non-primate viruses (eg, maedi bisna virus ( MW), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), goat arthritis encephalitis virus (CAEV) and bovine immunodeficiency virus (BIV)).
  • the method for introducing the therapeutic agent of the present invention is the same as described above.
  • the dose can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient.
  • the amount of the expression vector contained in the therapeutic agent is 0.0001 to 100 mg, preferably 0.001 to 10 mg. It is desirable to be. It is desirable to administer such a dose once every few days to several months.
  • a transgenic non-human mammal carrying such an expression vector is provided.
  • the transgenic non-human mammal of the present invention is a transgenic non-human mammal having the expression vector, wherein a foreign gene in the expression vector is expressed specifically in adipose tissue and / or pancreatic tissue.
  • Transgenic non-human mammals that retain an expression vector containing a marker gene as a foreign gene retain a specific promoter in adipose tissue or pancreatic tissue. Using the presence / absence and strength as an index, it has an excellent property that it can easily screen for therapeutic agents for metabolic syndrome-related diseases including diabetes drugs and anti-obesity drugs.
  • a transgenic non-human mammal having an expression vector containing a gene encoding a protein for treating the target disease or a nucleic acid such as miRNA or shRNA as a foreign gene is used in adipose tissue and / or pancreatic tissue. Since a specific promoter is retained, the therapeutic effect of the protein or the nucleic acid when expressed in these tissues can be evaluated.
  • the transgenic non-human mammal of the present invention is used to examine the influence of a specific gene on these tissues in the living body. For example, if there are genes with different expression levels in adipose tissue between obese patients and healthy individuals, the effects can be examined in vivo by expressing proteins encoding these in adipose tissue.
  • the therapeutic effect of a nucleic acid such as miRNA expressed specifically in adipose tissue can be examined.
  • the expression vector may be incorporated and retained in the chromosome of the mammal.
  • non-human mammal examples include mammals other than humans, for example, mammals such as mice, rats, rabbit pigs, dogs, sheep and goats. Above all, there is a long history of use in medical research and accumulation of data, and there is a track record that many pathological models have been created so far, and the production technology of genetically modified animals has been established. Therefore, rodents represented by mice, rats and the like are preferable, and mice are particularly preferable.
  • the transgenic non-human mammal of the present invention can be produced by a known method.
  • it can be produced by a method including the following steps (a) to (c), for example.
  • (A) a step of introducing a vector or the like into a fertilized egg and transplanting the transgenic fertilized egg into a foster animal;
  • (B) selecting a transgenic animal from offspring born from the animal; and
  • (c) establishing a line from the selected animal (founder).
  • step (a) When introducing the vector of step (a) into a fertilized egg, a microinjection method (manual operation manual for mouse embryo (Modern Publishing, 1989), molecular biology protocol (Nanedo, 1994)) or lentivirus
  • a microinjection method manual operation manual for mouse embryo (Modern Publishing, 1989), molecular biology protocol (Nanedo, 1994)
  • lentivirus The lentivirus method for infecting fertilized eggs (Lois C et al., Science, 295, 2002, pages 868-872) is used.
  • transgenic non-human mammals In general, however, it is desirable to produce transgenic non-human mammals by the lentiviral method. This is because in the microinjection method, several tens to several hundreds of transgenes are inserted into one unspecified chromosome location and inserted, and depending on the insertion location, an individual with no transgene expression is born. Therefore, it takes a period of 1 to 2 years after selection of useful strains after production of mice and production of mice for use in experiments. On the other hand, the lentivirus method has a short production period and high production efficiency of transgenic mice, and can obtain the number of mice used for experiments in the first-generation offspring.
  • the transgene is inserted one copy at a different location on the genome, a plurality of mice showing various expression levels can be obtained at a time.
  • the promoter of the present invention is characterized in that its sequence length is shorter than that of the natural type, the packaging efficiency of the lentiviral vector is improved, and it becomes possible to prepare a high titer virus. Therefore, it is possible to produce a transgenic non-human mammal by the lentivirus method with high efficiency.
  • a transgenic non-human mammal carrying an expression vector containing a marker gene or a cell derived from the transgenic non-human mammal is subjected to treatment.
  • a method for evaluating the expression level of a marker gene in animal tissues or cells after administration of a test substance can be mentioned.
  • a test substance to the transgenic non-human mammal of the present invention to suppress the expression of a marker gene is evaluated as an index for reducing adipocytes in each tissue in vivo. be able to.
  • the test substance may be any known substance or novel substance, for example, a nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound library prepared using combinatorial chemistry technology And random peptide libraries prepared by solid phase synthesis or phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.
  • a nucleic acid carbohydrate, lipid, protein, peptide, low molecular organic compound
  • compound library prepared using combinatorial chemistry technology
  • random peptide libraries prepared by solid phase synthesis or phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.
  • the mixture of 2 or more types of these compounds can also be provided as a sample.
  • an amplified fragment into which BglII and HindIII restriction enzyme sites were introduced was obtained.
  • a promoter region at about 200 bp from the transcription start point was used at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds using a primer consisting of the base sequence of SEQ ID NO: 3 and a primer consisting of the base sequence of SEQ ID NO: 4.
  • a fragment of the enhancer region was treated with BglII and HindIII, and inserted into the BglII and HindIII sites of pEGFP-N1 (Clontech) to obtain a ligated plasmid.
  • This plasmid was treated with BglII and NotI and inserted into the BamHI and NotI sites of pENTR4 (Invitrogen) to obtain a ligated plasmid.
  • This plasmid was treated with KpnI and BamHI, and a promoter fragment previously introduced with a KpnI and BamHI restriction enzyme site was ligated to prepare aP2-0-EGFP / pENTR.
  • This DNA includes a sequence of about 200 bp of a region consisting of about 520 bases at the most upstream and a region consisting of positions 5359-5548 of the full length of the aP2 promoter shown in SEQ ID NO: 24.
  • aP2-0-EGFP / pENTR was treated with SalI to remove the fragment containing the enhancer region, and then ligated with itself to prepare aP2-11-EGFP / pENTR.
  • This DNA includes a sequence of about 200 bp in the region consisting of positions 5359-5548 of the full length aP2 promoter shown in SEQ ID NO: 24.
  • the following primer pair (primer consisting of the base sequence of SEQ ID NO: 5 and primer consisting of the base sequence of SEQ ID NO: 6), (primer consisting of the base sequence of SEQ ID NO: 7) , And a primer consisting of the base sequence of SEQ ID NO: 8), (a primer consisting of the base sequence of SEQ ID NO: 9 and a primer consisting of the base sequence of SEQ ID NO: 10), (a primer consisting of the base sequence of SEQ ID NO: 11, and SEQ ID NO: A primer comprising 12 nucleotide sequences), (SEQ ID NO: 13) A primer consisting of a base sequence and a primer consisting of a base sequence of SEQ ID NO: 14) (a primer consisting of a base sequence of SEQ ID NO: 15 and a primer consisting of a base sequence of SEQ ID NO: 16), (consisting of a base sequence of SEQ ID
  • DNAs include the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 23 in common, and are located at positions 481 to 1137 in the full length of the aP2 promoter shown in SEQ ID NO: 24, respectively. It includes an array of regions consisting of 1069 to 1780, 1662 to 2357, 2296 to 2988, 2908 to 3558, 3496 to 4199, 4122 to 4823, and 4728 to 5380.
  • the vector aP2 promoter / pBluescript II containing the entire length of the aP2 promoter region of about 5.5 kb was used as a template, and the above primer pairs were used under the conditions of repeating 94 ° C. for 30 seconds, 55 ° C.
  • This DNA includes the nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 23, and includes the sequence of the region consisting of positions 481 to 5380 in the full length of the aP2 promoter shown in SEQ ID NO: 24. It is out.
  • This fragment was treated with AgeI and NotI, and aP2-0-EGFP / pENTR, aP2-1-EGFP / pENTR, aP2-2-EGFP / pENTR, aP2-3-EGFP / pENTR, aP2-4-EGFP / pENTR, AgeI of aP2-5-EGFP / pENTR, aP2-6-EGFP / pENTR, aP2-7-EGFP / pENTR, aP2-8-EGFP / pENTR, aP2-11-EGFP / pENTR, aP2-12-EGFP / pENTR Plasmids inserted into NotI sites to convert EGFP into luciferase gene, aP2-0-luc / pENTR, aP2-1-luc / pENTR, aP2-2-luc / pENTR, aP2-3-luc / pENTR, aP
  • the pCDH- ⁇ CMV-GW vector was obtained by introduction into the NheI and SalI sites of pCDH-MCS1- ⁇ CMV.
  • aP2-0-EGFP / pENTR aP2-3-EGFP / pENTR, aP2-5-EGFP / pENTR, and aP2-12-EGFP / pENTR with LR clonase II enzyme mix (Invitrogen)
  • AP2-0-EGFP / pCDH, aP2-3-EGFP / pCDH, aP2-5-EGFP / pCDH, aP2-12-EGFP / pCDH were obtained.
  • aP2-8-luc was introduced, the transcriptional activity of 2 times or more was obtained in 3T3-L1 adipose differentiated cells, but the specific activity differed by about 6 to 14 times.
  • an approximately 520 bp sequence located at the most upstream of the aP2 promoter shown in SEQ ID NO: 21 is necessary for specific expression in adipocytes, but other regions are also expected to affect the promoter activity. It was.
  • aP2-12-luc contained a sequence of about 520 bp located at the most upstream of the aP2 promoter, it was less active in adipocytes than other constructs.
  • transgenic mice using lentivirus and expression in each tissue Using DMEM containing 10% FCS (fetal calf serum), 293TN cells (System Biosciences) under conditions of 5% CO 2 and 37 ° C. Cultured. For transfection, FuGene6 (Roche) was used and the attached protocol was followed. Among the vectors that were found to be specific in adipocytes from the results of Example 2, the vectors used were aP2-0-EGFP / pCDH, 3T3-L1 undifferentiated cells with the smallest promoter size, and relatively active in 293 cells.
  • aP2-3-EGFP / pCDH, aP2-5-EGFP / pCDH, and aP2-12-EGFP / pCDH containing the full length of the aP2 promoter were selected and co-introduced with pPACKH1 (SBI). 72 hours after the transfection, the medium was collected, concentrated by ultracentrifugation, and purified by sucrose to prepare a lentivirus solution for producing a transgenic mouse. Virus titer was measured using p24 Antigen ELIZA kit (ZeptoMetrix).
  • Superovulation was induced by administering 5 IU of PSG (pregnant horse serum gonadotropin) to BDF1 female mice and 48 I HCG (placental gonadotropin) 48 hours later, and mated with BDF1 male mice.
  • PSG pregnant horse serum gonadotropin
  • I HCG placental gonadotropin
  • the oviducts of female mice whose vaginal plug was confirmed the day after mating were collected approximately 40 hours after mating and perfused with 0.4% BSA solution.
  • the recovered 2-cell embryo was cultured in kSOM medium (Arc Resource) for a short period, and then treated with acidic Tyrode (Arc Resource) for about 1 to 2 minutes to remove the embryo transparency.
  • Embryos from which the zona pellucida was successfully removed were washed in kSOM medium and cultured in the same medium until viral infection. Viral infection of embryos is performed by culturing embryos from which the zona pellucida has been removed for 2 days with a drop (6 ⁇ l / spot) of kSOM medium diluted to contain about 150 pg / ⁇ l of viral particles or 1500 pg / ⁇ l. It was. All embryos were cultured under conditions of 5% CO 2 and 37 ° C.
  • Genomic DNA was purified from the tail of a litter collected at 4 weeks of age using DNeasy Blood & Tissue Kit (QIAGEN). Using purified DNA as a template, PCR was performed under the conditions of 95 ° C 15 seconds, 57 ° C 15 seconds, 72 ° C 30 seconds 40 times using a primer consisting of the base sequence of SEQ ID NO: 25 and a primer consisting of the base sequence of SEQ ID NO: 26.
  • the transgene was detected by performing agarose gel electrophoresis, and the genotype of the transgenic mouse was determined.
  • a portion of perigenital fat was biopsyed from the transgenic mice and observed for EGFP fluorescence under a fluorescent stereomicroscope. Individuals in which EGFP fluorescence was observed were necropsied, and EGFP fluorescence was observed in major organs and 4 types of adipose tissue under a fluorescent stereomicroscope. The results are shown in Table 1 below.
  • the efficiency of producing the transgenic mouse is reduced to 1/10 or less compared to the case of using other truncated promoters.
  • no fluorescence was observed in any tissue. This is thought to be due to a decrease in virus packaging efficiency.
  • fluorescence was confirmed in the adipose tissue, but its intensity was weak, and it was similar in the pancreas.
  • primer pair primer consisting of the base sequence of SEQ ID NO: 30 and primer consisting of the base sequence of SEQ ID NO: 31
  • primer consisting of the base sequence of SEQ ID NO: 33 primer consisting of the base sequence of SEQ ID NO: 34 and a primer consisting of the base sequence of SEQ ID NO: 35
  • NheI, NheI and XhoI, and BglII and HindIII restriction enzyme sites were obtained.
  • the amplified fragment containing SEQ ID NO: 29 was treated with BglII and HindIII, and ligated to the BglII and HindIII sites of pGL3-basic (Promega) to obtain human aP2-11 / pGL3.
  • this plasmid was treated with SacI and NheI, and the amplified fragment containing SEQ ID NO: 27 was ligated with the fragment treated with SacI and NheI to prepare human aP2-0 / pGL3.
  • this plasmid was treated with NheI and XhoI, and the amplified fragment containing SEQ ID NO: 28 was ligated with the fragment treated with NheI and XhoI to prepare human aP2-3 / pGL3.
  • 3T3-L1 adipose differentiated cells Reaction of each promoter in cultured cells Using a transient assay in 3T3-L1 adipose differentiated cells, the transcriptional activity of each construct derived from the human aP2 promoter in adipocytes was examined. For comparison, 3T3-L1 cells, HeLa cells, and 293 cells that were not induced to differentiate were used. Using DMEM containing 10% FCS (fetal calf serum), 3T3-L1 cells are cultured under conditions of 5% CO 2 and 37 ° C. After 2 days of confluence, differentiation induction is performed in a medium containing insulin, dexamethasone and IBMX. Started. Transfection was performed by electroporation.
  • FCS fetal calf serum
  • This figure shows the relative value of the luminescence intensity when other constructs were used with the luminescence intensity when aP2-11 was introduced into undifferentiated 3T3-L1 cells (indicated as Luciferase activity in the figure) as 1. It was.

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Abstract

Les promoteurs classiques ont de longues séquences, et par conséquent, ils ne peuvent pas être introduits dans les organismes vivants avec une efficacité élevée. Ainsi, l'invention concerne un nouveau promoteur. Le promoteur a une activité de promoteur spécifique d'un tissu adipeux et/ou d'un tissu pancréatique.
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JP2021007395A (ja) * 2020-09-25 2021-01-28 ウニベルシダッド アウトノマ デ バルセロナ 脂肪組織に形質導入するのに有用なアデノ随伴ウイルスベクター
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US11001857B2 (en) 2010-07-12 2021-05-11 Universitat Autonoma De Barcelona Gene therapy composition for use in diabetes treatment
US11033638B2 (en) 2015-01-07 2021-06-15 Universität Autonoma De Barcelona Single-vector gene construct comprising insulin and glucokinase genes

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Cited By (13)

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US11001857B2 (en) 2010-07-12 2021-05-11 Universitat Autonoma De Barcelona Gene therapy composition for use in diabetes treatment
US10711281B2 (en) 2012-08-02 2020-07-14 Universitat Autónoma De Barcelona Adeno-associated viral (AAV) vectors useful for transducing adipose tissue
JP2016525361A (ja) * 2012-08-02 2016-08-25 ウニベルシダッド アウトノマ デ バルセロナ 脂肪組織に形質導入するのに有用なアデノ随伴ウイルスベクター
US20160319303A1 (en) * 2012-08-02 2016-11-03 Universitat Autònoma De Barcelona Adeno-associated viral (aav) vectors useful for trasducing adipose tissue
EP2692868A1 (fr) * 2012-08-02 2014-02-05 Universitat Autònoma De Barcelona Vecteurs viraux adéno-associés (AAV) utiles pour la transduction de tissu adipeux
WO2014020149A1 (fr) * 2012-08-02 2014-02-06 Universitat Autonoma De Barcelona Vecteurs viraux adéno-associés (aav) utiles pour la transduction du tissu adipeux
US11629361B2 (en) 2012-08-02 2023-04-18 Universitat Autònoma De Barcelona Adeno-associated viral (AAV) vectors useful for transducing adipose tissue
CN105916990A (zh) * 2013-08-02 2016-08-31 巴塞罗那自治大学 用于转导脂肪组织的腺相关病毒载体
US10973931B2 (en) 2014-09-16 2021-04-13 Universitat Autònoma De Barcelona Adeno-associated viral vectors for the gene therapy of metabolic diseases
US11033638B2 (en) 2015-01-07 2021-06-15 Universität Autonoma De Barcelona Single-vector gene construct comprising insulin and glucokinase genes
JP2018198611A (ja) * 2018-08-03 2018-12-20 ウニベルシダッド アウトノマ デ バルセロナ 脂肪組織に形質導入するのに有用なアデノ随伴ウイルスベクター
JP2021007395A (ja) * 2020-09-25 2021-01-28 ウニベルシダッド アウトノマ デ バルセロナ 脂肪組織に形質導入するのに有用なアデノ随伴ウイルスベクター
JP7121086B2 (ja) 2020-09-25 2022-08-17 ウニベルシダッド アウトノマ デ バルセロナ 脂肪組織に形質導入するのに有用なアデノ随伴ウイルスベクター

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