WO2019045049A1 - Renal tubule cell-specific expression vector - Google Patents

Renal tubule cell-specific expression vector Download PDF

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WO2019045049A1
WO2019045049A1 PCT/JP2018/032360 JP2018032360W WO2019045049A1 WO 2019045049 A1 WO2019045049 A1 WO 2019045049A1 JP 2018032360 W JP2018032360 W JP 2018032360W WO 2019045049 A1 WO2019045049 A1 WO 2019045049A1
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vector
gene
egfp
promoter
renal
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Japanese (ja)
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内田　俊也
秀美代 渡邉
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学校法人帝京大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/86—Viral vectors

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  • the present invention relates to renal tubular cell-specific expression vectors.
  • Priority is claimed on Japanese Patent Application No. 2017-168924, filed September 1, 2017, the content of which is incorporated herein by reference.
  • Renal tubules are tissues from the glomeruli in the kidney to the collecting ducts. In the process of raw urine passing through the renal tubule, it is reabsorbed and secreted. In renal tubules, there are sites called proximal tubule, Henreloop, thick Henle hook, distal tubule, collecting duct, etc. sequentially from the glomerulus side, and the shape and function are different for each site ing.
  • Barter's syndrome for example, is a disease characterized by dysfunction of the thick Henle's foot, which mainly affects children.
  • the symptoms of Barter syndrome include hypokalemia, metabolic alkalosis, blood pressure reduction and the like.
  • One of the causes of Barter syndrome is known to be an increase in function of a calcium-sensing receptor (CaSR) gene mutation.
  • CaSR calcium-sensing receptor
  • hereditary tubular diseases including Barter's syndrome
  • fundamental gene therapy has not been performed, but only symptomatic treatment has been performed.
  • the QOL of patients is extremely low, and the effects on complications due to complications on the kidney and the whole body are great, and an immediate solution is required.
  • Non-Patent Document 1 describes that a promoter of a sodium-potassium 2-chloride cotransporter (Nkcc2) gene, which is specifically expressed in the thick Henle's leg of the renal tubule, is cloned and analyzed.
  • Nkcc2 sodium-potassium 2-chloride cotransporter
  • An object of the present invention is to provide a vector for expressing a gene specifically in renal tubular cells.
  • a vector for expressing the gene specific to renal tubular cells which comprises a viral vector having a renal tubular cell specific promoter and a desired gene linked downstream of the promoter.
  • the promoter is sodium-dependent phosphate transporter type 2a (NPT2a), sodium-potassium-2-chloride cotransporter (NKCC2), aquaporin 2 (AQP2), sodium-chloride cotransporter (NCC), epithelial sodium-channel (ENaC)
  • NTT2a sodium-dependent phosphate transporter type 2a
  • NKCC2 sodium-potassium-2-chloride cotransporter
  • AQP2 aquaporin 2
  • NCC sodium-chloride cotransporter
  • ENaC epithelial sodium-channel
  • [3] The vector according to [1] or [2], wherein the gene is a gene selected from the group consisting of NPT2a, NKCC2, AQP2, NCC, and ENaC.
  • [4] The vector according to any one of [1] to [3], which is administered by bolus injection into a renal artery of a kidney whose blood flow has been blocked.
  • [5] The vector according to any one of [1] to [4], which is for gene therapy of hereditary tubular disease.
  • FIG. (A) and (b) is a photograph which shows the result of the immunohistochemical analysis of the kidney of the rat which introduce
  • FIG. (C) and (d) are photographs showing the results of immunohistochemical analysis of rat kidneys into which the NKCC2 pvu-EGFP adenoviral vector has been introduced in Experimental Example 4.
  • (E) and (f) are photographs showing the results of immunohistochemical analysis of rat kidneys into which the NKCC2-EGFP adenoviral vector has been introduced in Experimental Example 4.
  • FIG. (A) to (d) are photographs showing the results of immunostaining of the kidneys of rats into which the NKCC2DI-EGFP adenoviral vector has been introduced in Experimental Example 4 and observation with a confocal laser microscope.
  • (A) and (b) is a photograph which shows the result of the immunohistochemical analysis of the kidney of the rat which introduce
  • FIG. (A) to (d) are photographs showing the results of immunostaining of the kidney of a rat into which the NPT2a-EGFP adenoviral vector has been introduced in Experimental Example 5 and observation with a confocal laser microscope.
  • FIG. (A) is a photograph showing the result of detecting the nucleus
  • (b) is a photograph showing the result of detecting the EGFP
  • (c) is a photograph showing the result of detecting the endogenous NHE3 protein
  • (d) is a photograph in which (a) to (c) are synthesized.
  • (A) and (b) is a photograph which shows the result of the immunohistochemical analysis of the kidney of the rat which introduce
  • FIG. (A) to (d) are photographs showing the results of immunostaining of rat kidneys into which the AQP2-EGFP adenoviral vector has been introduced in Experimental Example 6 and observation with a confocal laser microscope.
  • (A) is a photograph showing the result of detecting the nucleus
  • (b) is a photograph showing the result of detecting the EGFP
  • (c) is a photograph showing the result of detecting the endogenous AQP2 protein
  • (d) is a photograph in which (a) to (c) are synthesized.
  • the present invention specifically expresses renal tubule cells specifically comprising a viral vector having a renal tubular cell specific promoter and a desired gene linked downstream of the promoter.
  • a viral vector having a renal tubular cell specific promoter and a desired gene linked downstream of the promoter.
  • the inventors succeeded in expressing a desired gene in renal tubular cells specifically in vivo by using the vector of the present embodiment. This is the first example of successful expression of a desired gene specifically in renal tubular cells.
  • the vector of this embodiment can be suitably used for the purpose of expressing a desired gene specifically in renal tubular cells in humans or animals.
  • renal tubular cell specific promoters include promoters of genes such as NPT2a, NKCC2, AQP2, NCC, and ENaC.
  • NPT2a is a gene also called SLC34A1, which is a gene specifically expressed in the proximal tubule.
  • NKCC2 is a gene also called SLC12A1, which is a gene specifically expressed in thick Henle's hook.
  • AQP2 is a gene specifically expressed in the collecting duct or distal tubule.
  • the renal tubular cell-specific promoter is preferably a promoter derived from a target species into which the vector of the present embodiment is to be introduced. However, it is also possible to express the desired gene using promoters from different species.
  • the nucleotide sequence of the renal tubular cell-specific promoter is preferably long. Moreover, it is preferable to include a base as close as possible to the translation initiation codon (ATG) or to include a translation initiation codon.
  • the length of a renal tubular cell specific promoter may be, for example, 2 kbp or more, for example, 3 kbp or more, for example, 4 kbp or more, for example, 5 kbp or more, for example, 6 kbp It may be more than.
  • the desired gene includes a gene transcriptionally controlled by the renal tubular cell-specific promoter described above. Specifically, genes such as NPT2a, NKCC2, AQP2, NCC, ENaC, etc. may be used.
  • the desired gene is preferably a gene from a species to which the vector of the present embodiment is to be introduced.
  • the desired gene is preferably a gene having no mutation causing disease. This makes it possible to use the vector of the present embodiment for gene therapy of hereditary tubular disease. That is, the vector of the present embodiment may be for gene therapy of hereditary tubular disease.
  • the desired gene is preferably a gene transcriptionally regulated by the native renal tubular cell-specific promoter.
  • the combination of renal tubular cell specific promoter and desired gene may be different from the original combination.
  • the virus vector is not particularly limited as long as it is generally used for gene transfer, and examples thereof include adenovirus vector, adeno-associated virus vector, lentivirus vector, retrovirus vector and the like.
  • the viral vector may be commercially available.
  • adenoviral vectors can be suitably used as viral vectors. It is preferable to use a viral vector which can not self-replicate from the viewpoint of viral propagation.
  • adenoviral vectors which can not replicate autonomously include those in which the E1A and E3 regions have been artificially deleted, adenochimeric viruses, modified adenovirus vectors and the like.
  • packaging cells having the corresponding deleted region.
  • packaging cells include HEK293 cells having E1A and E3 regions.
  • the vector of this embodiment is administered by bolus injection into the renal artery of the kidney which has blocked the blood flow.
  • Bolus injection is rapid injection.
  • the bolus injection is not particularly limited as long as the pressure is such that the kidney tissue of the patient is not injured.
  • injection may be performed in about 2 to 3 minutes per 1 ml of virus vector solution.
  • the blocking of the blood flow can be done by clamping the blood vessel with a clip. After blocking the renal artery, a bolus injection of viral vector is performed. In addition, after blocking the renal artery, it is preferable to infuse isotonic fluid such as physiological saline and wash out the blood in the kidney to increase infection efficiency more preferably. Furthermore, after injecting the viral vector, the infection efficiency of the viral vector can be further improved by blocking the renal vein and maintaining it for several minutes. Here, as a time for blocking the renal vein, for example, about 5 to 10 minutes can be mentioned.
  • the vector of the present embodiment may be formulated in the form of a pharmaceutical composition comprising the above-described viral vector and a pharmaceutically acceptable solvent.
  • a pharmaceutically acceptable solvent those used for the production of general pharmaceutical compositions can be appropriately used.
  • the present invention comprises administering to a patient in need of treatment an effective amount of a viral vector having a renal tubular cell specific promoter and a desired gene linked downstream of said promoter.
  • a method of treating hereditary tubular disease comprises administering to a patient in need of treatment an effective amount of a viral vector having a renal tubular cell specific promoter and a desired gene linked downstream of said promoter.
  • the present invention provides a vector for the treatment of hereditary tubular disease, which comprises a renal tubular cell-specific promoter and a desired gene linked downstream of said promoter. provide.
  • the present invention is the use of a vector for producing a therapeutic agent for hereditary tubular disease, wherein the vector is linked to a renal tubular cell specific promoter and downstream of the promoter.
  • the use is provided, which is a viral vector having the desired gene.
  • the above promoter may be a promoter of a gene selected from the group consisting of NPT2a, NKCC2, AQP2, NCC and ENaC.
  • the above gene may be a gene selected from the group consisting of NPT2a, NKCC2, AQP2, NCC and ENaC.
  • these genes are preferably genes having no mutation causing disease.
  • the above-mentioned vector is administered by bolus injection into the renal artery of the kidney which has blocked the blood flow.
  • accession number ENSMUSG00000027202 was used as the nucleotide sequence data of the 5 'side of the Npt2a gene.
  • base sequence data of accession number ENSRNOG00000015262 was used as base sequence data of the 5 'side of Nkcc2 gene.
  • base sequence data of accession number ENSRNOG00000000297 was used as base sequence data of the 5 'side of Aqp2 gene.
  • Npt2a gene Rat genomic DNA was extracted from the blood of Wistar rats, and the promoter of the Npt2a gene was cloned by PCR. In order to make the promoter region to be cloned as long as possible, a region including a region as close as possible to the translation initiation codon was obtained. The cloned promoter was in the region from -3379 to +630. Here, -1 corresponds to the base immediately before the translation initiation codon (ATG) of the Npt2a gene, and so forth.
  • Nkcc2 gene ⁇ Each promoter described in Table 1 below was used.
  • pNKCC2DI promoter and pNKCC2pvu promoter were distributed by Dr. Peter Igarashi, University of Texas Southwest Medical Center.
  • the pNKCC2 promoter was prepared by PCR of rat genomic DNA.
  • Promoter of Aqp2 gene >> Rat genomic DNA was extracted from the blood of Wistar rats, and the promoter of the Aqp2 gene was cloned by PCR. The cloned promoter was in the region of -5338 to +930.
  • Each prepared adenovirus vector was used by selecting a single clone correctly.
  • the nucleotide sequence of each adenoviral vector was confirmed by restriction enzyme cleavage and sequence analysis of the nucleotide sequence.
  • Each adenovirus vector was used to infect HEK 293 cells, a cell line derived from human embryonic kidney cells, to prepare adenovirus.
  • the titer of the obtained adenovirus was 10 13 pfu / mL or more.
  • the blood flow was interrupted by clamping with a clip between the rat's abdominal aorta and the left renal artery. Subsequently, 1 mL of saline was infused over 2 minutes into the left renal artery. Thereby, the blood in the kidney was washed away. Subsequently, 1 mL of adenoviral vector solution was infused into the left renal artery over 2 minutes. Subsequently, the renal vein was clamped with a clip to block blood flow and maintained for 10 minutes. As a result, the adenoviral vector remained in the kidney and efficiently infected the kidney cells. After that, the clip was removed and blood flow was resumed.
  • Rat renal artery was infused slowly with 1 mL of adenovirus vector solution for 16 hours.
  • adenoviral vector solution 1 mL of adenoviral vector solution.
  • adenoviral vectors were expected to backflow to the ureter and infect kidney cells.
  • Rat kidneys were directly injected with 1 mL of adenovirus vector solution. The injections were divided into 10 parts and injected in all directions.
  • kidneys were removed from each rat and fixed with 4% paraformaldehyde for 2 days. Subsequently, each kidney sample was embedded in paraffin to prepare a sliced section.
  • each tissue section was stained with anti-EGFP antibody, anti-NKCC2 antibody, anti-AQP2 antibody, anti-sodium-hydrogen exchanger 3 (NHE3; SLC9A3P2) antibody.
  • the anti-NHE3 antibody was used instead of the anti-NPT2a antibody because the anti-NPT2a antibody was not available.
  • the region where EGFP was expressed was limited to the site where the adenoviral vector was injected.
  • the structure of the renal parenchyma was damaged by repeated puncture and fluid injection.
  • adenoviral vectors were administered by bolus administration to the renal arteries.
  • Example 4 (examination of NKCC2 promoter)
  • the inventors administered the three types of adenoviral vectors, NKCC2DI-EGFP, NKCC2pvu-EGFP and NKCC2-EGFP, prepared in Experimental Example 2 to rats by bolus administration to the renal artery. Subsequently, 4 days after administration of the adenoviral vector, kidneys were removed from each rat, and tissue sections were prepared in the same manner as in Experimental Example 3.
  • FIGS. 1 (a) and (b) are photographs showing the results of introducing the NKCC2DI-EGFP adenoviral vector.
  • FIG. 1 (a) is a photograph showing the results of staining with anti-EGFP antibody
  • FIG. 1 (b) is a staining of another tissue section adjacent to the tissue section of FIG. 1 (a) with anti-NKCC2 antibody
  • Each scale bar shows 1 cm.
  • FIG.1 (c) and (d) are photographs which show the result of introduce
  • Fig. 1 (c) is a photograph showing the results of staining with anti-EGFP antibody
  • Fig. 1 (d) is another tissue section adjacent to the tissue section of Fig. 1 (c) stained with anti-NKCC2 antibody
  • Each scale bar shows 1 cm.
  • FIG. 1 (e) and (f) is a photograph which shows the result of introduce
  • FIG. 1 (e) is a photograph showing the results of staining with anti-EGFP antibody
  • FIG. 1 (f) is a staining of another tissue section adjacent to the tissue section of FIG. 1 (e) with anti-NKCC2 antibody, It is a photograph which shows the result of having detected sexual NKCC2 protein. Each scale bar shows 1 cm.
  • EGFP was expressed in a broader region than the endogenous NKCC2 protein.
  • FIGS. 2 (a) to 2 (d) are photographs showing the results of observation of the same field of view with a confocal laser microscope.
  • FIG. 2 (a) is a photograph showing the result of detecting the nucleus
  • FIG. 2 (b) is a photograph showing the result of detecting the EGFP
  • FIG. 2 (c) is a result of detecting the endogenous NKCC2 protein
  • FIG. 2 (d) is a photograph showing a composite of FIGS. 2 (a) to 2 (c). Scale bars all show 50 ⁇ m.
  • Non-Patent Document 1 it is reported that the pNKCC2 pvu promoter showed the highest promoter activity and the pNKCC2 DI promoter showed the lowest promoter activity in in vitro studies using primary culture cells of renal tubules.
  • the promoter is preferably as long as possible.
  • FIGS. 3 (a) and 3 (b) are photographs showing the results of introducing the NPT2a-EGFP adenoviral vector.
  • FIG. 3 (a) is a photograph showing the results of staining with anti-EGFP antibody
  • FIG. 3 (b) is a staining of another tissue section adjacent to the tissue section of FIG. 3 (a) with anti-NHE3 antibody
  • anti-NHE3 antibody was used instead of anti-NPT2a antibody because anti-NPT2a antibody was not available.
  • Each scale bar shows 1 cm.
  • FIGS. 4 (a) to 4 (d) are photographs showing the results of observation of the same field of view with a confocal laser microscope.
  • FIG. 4 (a) is a photograph showing the result of detecting the nucleus
  • FIG. 4 (b) is a photograph showing the result of detecting the EGFP
  • FIG. 4 (c) is a result of detecting the endogenous NHE3 protein
  • FIG. 4 (d) is a photograph showing a composite of FIGS. 4 (a) to 4 (c).
  • NPT2a-EGFP adenoviral vector makes it possible to express EGFP with an expression pattern close to that of the endogenous NKCC2 protein.
  • EGFP can be expressed in the same cells as cells expressing endogenous NHE3 protein.
  • FIGS. 5 (a) and (b) are photographs showing the results of introducing the AQP2-EGFP adenoviral vector.
  • Fig. 5 (a) is a photograph showing the results of staining with anti-EGFP antibody
  • Fig. 5 (b) is another tissue section adjacent to the tissue section of Fig. 3 (a) stained with anti-AQP2 antibody
  • Each scale bar shows 1 cm.
  • FIGS. 6 (a) to 6 (d) are photographs showing the results of observation of the same field of view with a confocal laser microscope.
  • FIG. 6 (a) is a photograph showing the result of detecting the nucleus
  • FIG. 6 (b) is a photograph showing the result of detecting the EGFP
  • FIG. 6 (c) is a result of detecting the endogenous AQP2 protein
  • FIG. 6 (d) is a photograph showing a composite of FIGS. 6 (a) to 6 (c). Scale bars all show 50 ⁇ m.
  • AQP2-EGFP adenoviral vector can express EGFP with an expression pattern close to that of endogenous AQP2 protein.
  • EGFP can be expressed in the same cells as cells expressing endogenous AQP2 protein.

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Abstract

A vector for causing a desired gene linked downstream of a renal tubule cell-specific promotor to be expressed specifically in renal tubule cells, the vector comprising a viral vector having the renal tubule cell-specific promotor and the desired gene.

Description

腎尿細管細胞特異的発現ベクターRenal tubular cell specific expression vector
　本発明は、腎尿細管細胞特異的発現ベクターに関する。本願は、２０１７年９月１日に、日本に出願された特願２０１７－１６８９２４号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to renal tubular cell-specific expression vectors. Priority is claimed on Japanese Patent Application No. 2017-168924, filed September 1, 2017, the content of which is incorporated herein by reference.
　腎尿細管は、腎臓における糸球体から集合管に至るまでの組織である。腎尿細管を原尿が通る過程で再吸収・分泌等を受ける。腎尿細管には、糸球体側から順に、近位尿細管、ヘンレループ、太いヘンレ係蹄脚、遠位尿細管、集合管等と呼ばれる部位が存在し、個々の部位ごとに形状・機能が異なっている。 Renal tubules are tissues from the glomeruli in the kidney to the collecting ducts. In the process of raw urine passing through the renal tubule, it is reabsorbed and secreted. In renal tubules, there are sites called proximal tubule, Henreloop, thick Henle hook, distal tubule, collecting duct, etc. sequentially from the glomerulus side, and the shape and function are different for each site ing.
　遺伝性尿細管疾患には、バーター症候群、ギテルマン症候群、リドル症候群等が知られている。例えば、バーター症候群は、太いヘンレ係蹄脚の機能不全を特徴とする疾患であり、主に小児が発症する。バーター症候群の症状としては、低カリウム血症、代謝性アルカローシス、血圧低下等が挙げられる。バーター症候群の原因の１つとして、カルシウム感知受容体（ｃａｌｃｉｕｍ－ｓｅｎｓｉｎｇ　ｒｅｃｅｐｔｏｒ、ＣａＳＲ）の遺伝子変異による機能亢進が知られている。 Known hereditary tubular diseases include Barter's syndrome, Gittermann's syndrome, Liddle's syndrome and the like. Barter's syndrome, for example, is a disease characterized by dysfunction of the thick Henle's foot, which mainly affects children. The symptoms of Barter syndrome include hypokalemia, metabolic alkalosis, blood pressure reduction and the like. One of the causes of Barter syndrome is known to be an increase in function of a calcium-sensing receptor (CaSR) gene mutation.
　現在、バーター症候群を始めとする遺伝性尿細管疾患には、根本的な遺伝子治療はなされておらず、対症療法を行っているに過ぎない。このため、患者のＱＯＬは著しく低く、合併症による腎及び全身に及ぼす影響は多大であり、早急な解決が求められている。このため、腎尿細管細胞特異的に遺伝子を発現させる技術の開発が必要である。 At present, for hereditary tubular diseases including Barter's syndrome, fundamental gene therapy has not been performed, but only symptomatic treatment has been performed. For this reason, the QOL of patients is extremely low, and the effects on complications due to complications on the kidney and the whole body are great, and an immediate solution is required. For this reason, it is necessary to develop a technique for expressing a gene specifically in renal tubular cells.
　例えば、非特許文献１には、腎尿細管の太いヘンレ係蹄脚特異的に発現するｓｏｄｉｕｍ－ｐｏｔａｓｓｉｕｍ－２－ｃｈｌｏｒｉｄｅ　ｃｏｔｒａｎｓｐｏｒｔｅｒ（Ｎｋｃｃ２）遺伝子のプロモーターをクローニングして解析したことが記載されている。 For example, Non-Patent Document 1 describes that a promoter of a sodium-potassium 2-chloride cotransporter (Nkcc2) gene, which is specifically expressed in the thick Henle's leg of the renal tubule, is cloned and analyzed.
　本発明は、腎尿細管細胞特異的に遺伝子を発現させるためのベクターを提供することを目的とする。 An object of the present invention is to provide a vector for expressing a gene specifically in renal tubular cells.
　本発明は以下の態様を含む。
［１］腎尿細管細胞特異的プロモーターと、前記プロモーターの下流に連結された所望の遺伝子とを有するウイルスベクターからなる、腎尿細管細胞特異的に前記遺伝子を発現させるためのベクター。
［２］前記プロモーターが、ｓｏｄｉｕｍ－ｄｅｐｅｎｄｅｎｔ　ｐｈｏｓｐｈａｔｅ　ｔｒａｎｓｐｏｒｔｅｒ　ｔｙｐｅ　２ａ（ＮＰＴ２ａ）、ｓｏｄｉｕｍ－ｐｏｔａｓｓｉｕｍ－２－ｃｈｌｏｒｉｄｅ　ｃｏｔｒａｎｓｐｏｒｔｅｒ（ＮＫＣＣ２）、ａｑｕａｐｏｒｉｎ２（ＡＱＰ２）、ｓｏｄｉｕｍ－ｃｈｌｏｒｉｄｅ　ｃｏｔｒａｎｓｐｏｒｔｅｒ（ＮＣＣ）、ｅｐｉｔｈｅｌｉａｌ　ｓｏｄｉｕｍ－ｃｈａｎｎｅｌ（ＥＮａＣ）等からなる群より選択される遺伝子のプロモーターである、［１］に記載のベクター。
［３］前記遺伝子が、ＮＰＴ２ａ、ＮＫＣＣ２、ＡＱＰ２、ＮＣＣ、及びＥＮａＣからなる群より選択される遺伝子である、［１］又は［２］に記載のベクター。
［４］血流を遮断した腎臓の腎動脈へのボーラス注入により投与される、［１］～［３］のいずれかに記載のベクター。
［５］遺伝性尿細管疾患の遺伝子治療用である、［１］～［４］のいずれかに記載のベクター。 The present invention includes the following aspects.
[1] A vector for expressing the gene specific to renal tubular cells, which comprises a viral vector having a renal tubular cell specific promoter and a desired gene linked downstream of the promoter.
[2] The promoter is sodium-dependent phosphate transporter type 2a (NPT2a), sodium-potassium-2-chloride cotransporter (NKCC2), aquaporin 2 (AQP2), sodium-chloride cotransporter (NCC), epithelial sodium-channel (ENaC) The vector according to [1], which is a promoter of a gene selected from the group consisting of and the like.
[3] The vector according to [1] or [2], wherein the gene is a gene selected from the group consisting of NPT2a, NKCC2, AQP2, NCC, and ENaC.
[4] The vector according to any one of [1] to [3], which is administered by bolus injection into a renal artery of a kidney whose blood flow has been blocked.
[5] The vector according to any one of [1] to [4], which is for gene therapy of hereditary tubular disease.
　本発明によれば、腎尿細管細胞特異的に遺伝子を発現させるためのベクターを提供することができる。 According to the present invention, it is possible to provide a vector for expressing a gene specifically in renal tubular cells.
（ａ）及び（ｂ）は、実験例４において、ＮＫＣＣ２ＤＩ－ＥＧＦＰアデノウイルスベクターを導入したラットの腎臓の免疫組織化学解析の結果を示す写真である。（ｃ）及び（ｄ）は、実験例４において、ＮＫＣＣ２ｐｖｕ－ＥＧＦＰアデノウイルスベクターを導入したラットの腎臓の免疫組織化学解析の結果を示す写真である。（ｅ）及び（ｆ）は、実験例４において、ＮＫＣＣ２－ＥＧＦＰアデノウイルスベクターを導入したラットの腎臓の免疫組織化学解析の結果を示す写真である。(A) and (b) is a photograph which shows the result of the immunohistochemical analysis of the kidney of the rat which introduce | transduced the NKCC2DI-EGFP adenovirus vector in Experimental example 4. FIG. (C) and (d) are photographs showing the results of immunohistochemical analysis of rat kidneys into which the NKCC2 pvu-EGFP adenoviral vector has been introduced in Experimental Example 4. (E) and (f) are photographs showing the results of immunohistochemical analysis of rat kidneys into which the NKCC2-EGFP adenoviral vector has been introduced in Experimental Example 4. （ａ）～（ｄ）は、実験例４において、ＮＫＣＣ２ＤＩ－ＥＧＦＰアデノウイルスベクターを導入したラットの腎臓を免疫染色し、共焦点レーザー顕微鏡で観察した結果を示す写真である。(A) to (d) are photographs showing the results of immunostaining of the kidneys of rats into which the NKCC2DI-EGFP adenoviral vector has been introduced in Experimental Example 4 and observation with a confocal laser microscope. （ａ）及び（ｂ）は、実験例５において、ＮＰＴ２ａ－ＥＧＦＰアデノウイルスベクターを導入したラットの腎臓の免疫組織化学解析の結果を示す写真である。(A) and (b) is a photograph which shows the result of the immunohistochemical analysis of the kidney of the rat which introduce | transduced NPT2a-EGFP adenoviral vector in Experimental example 5. FIG. （ａ）～（ｄ）は、実験例５において、ＮＰＴ２ａ－ＥＧＦＰアデノウイルスベクターを導入したラットの腎臓を免疫染色し、共焦点レーザー顕微鏡で観察した結果を示す写真である。（ａ）は、核を検出した結果を示す写真であり、（ｂ）は、ＥＧＦＰを検出した結果を示す写真であり、（ｃ）は、内在性のＮＨＥ３タンパク質を検出した結果を示す写真であり、（ｄ）は、（ａ）～（ｃ）を合成した写真である。(A) to (d) are photographs showing the results of immunostaining of the kidney of a rat into which the NPT2a-EGFP adenoviral vector has been introduced in Experimental Example 5 and observation with a confocal laser microscope. (A) is a photograph showing the result of detecting the nucleus, (b) is a photograph showing the result of detecting the EGFP, and (c) is a photograph showing the result of detecting the endogenous NHE3 protein And (d) is a photograph in which (a) to (c) are synthesized. （ａ）及び（ｂ）は、実験例６において、ＡＱＰ２－ＥＧＦＰアデノウイルスベクターを導入したラットの腎臓の免疫組織化学解析の結果を示す写真である。(A) and (b) is a photograph which shows the result of the immunohistochemical analysis of the kidney of the rat which introduce | transduced AQP2-EGFP adenovirus vector in Experimental example 6. FIG. （ａ）～（ｄ）は、実験例６において、ＡＱＰ２－ＥＧＦＰアデノウイルスベクターを導入したラットの腎臓を免疫染色し、共焦点レーザー顕微鏡で観察した結果を示す写真である。（ａ）は、核を検出した結果を示す写真であり、（ｂ）は、ＥＧＦＰを検出した結果を示す写真であり、（ｃ）は、内在性のＡＱＰ２タンパク質を検出した結果を示す写真であり、（ｄ）は、（ａ）～（ｃ）を合成した写真である。(A) to (d) are photographs showing the results of immunostaining of rat kidneys into which the AQP2-EGFP adenoviral vector has been introduced in Experimental Example 6 and observation with a confocal laser microscope. (A) is a photograph showing the result of detecting the nucleus, (b) is a photograph showing the result of detecting the EGFP, and (c) is a photograph showing the result of detecting the endogenous AQP2 protein And (d) is a photograph in which (a) to (c) are synthesized.
［腎尿細管細胞特異的発現ベクター］
　１実施形態において、本発明は、腎尿細管細胞特異的プロモーターと、前記プロモーターの下流に連結された所望の遺伝子とを有するウイルスベクターからなる、腎尿細管細胞特異的に前記遺伝子を発現させるためのベクターを提供する。 [Renal tubular cell specific expression vector]
In one embodiment, the present invention specifically expresses renal tubule cells specifically comprising a viral vector having a renal tubular cell specific promoter and a desired gene linked downstream of the promoter. Provide a vector of
　実施例において後述するように、発明者らは、本実施形態のベクターを用いることにより、インビボで腎尿細管細胞特異的に所望の遺伝子を発現させることに成功した。これは、腎尿細管細胞特異的に所望の遺伝子を発現させることに成功した初めての例である。 As described later in the examples, the inventors succeeded in expressing a desired gene in renal tubular cells specifically in vivo by using the vector of the present embodiment. This is the first example of successful expression of a desired gene specifically in renal tubular cells.
　したがって、本実施形態のベクターは、ヒト又は動物において、腎尿細管細胞特異的に所望の遺伝子を発現させる用途に好適に用いることができる。 Therefore, the vector of this embodiment can be suitably used for the purpose of expressing a desired gene specifically in renal tubular cells in humans or animals.
　本実施形態のベクターにおいて、腎尿細管細胞特異的プロモーターとしては、例えば、ＮＰＴ２ａ、ＮＫＣＣ２、ＡＱＰ２、ＮＣＣ、ＥＮａＣ等の遺伝子のプロモーターが挙げられる。 In the vector of the present embodiment, examples of renal tubular cell specific promoters include promoters of genes such as NPT2a, NKCC2, AQP2, NCC, and ENaC.
　ＮＰＴ２ａは、ＳＬＣ３４Ａ１とも呼ばれる遺伝子であり、近位尿細管特異的に発現する遺伝子である。また、ＮＫＣＣ２は、ＳＬＣ１２Ａ１とも呼ばれる遺伝子であり、太いヘンレ係蹄脚特異的に発現する遺伝子である。また、ＡＱＰ２は、集合管又は遠位尿細管に特異的に発現する遺伝子である。 NPT2a is a gene also called SLC34A1, which is a gene specifically expressed in the proximal tubule. In addition, NKCC2 is a gene also called SLC12A1, which is a gene specifically expressed in thick Henle's hook. In addition, AQP2 is a gene specifically expressed in the collecting duct or distal tubule.
　腎尿細管細胞特異的プロモーターは、本実施形態のベクターを導入する対象の種由来のプロモーターであることが好ましい。しかしながら、異なる種由来のプロモーターを使用しても所望の遺伝子を発現させることは可能である。 The renal tubular cell-specific promoter is preferably a promoter derived from a target species into which the vector of the present embodiment is to be introduced. However, it is also possible to express the desired gene using promoters from different species.
　実施例において後述するように、腎尿細管細胞特異的プロモーターの塩基配列は長いことが好ましい。また、翻訳開始コドン（ＡＴＧ）にできるだけ近接した塩基を含むか、翻訳開始コドンを含むことが好ましい。腎尿細管細胞特異的プロモーターの長さは、例えば２ｋｂｐ以上であってもよく、例えば３ｋｂｐ以上であってもよく、例えば４ｋｂｐ以上であってもよく、例えば５ｋｂｐ以上であってもよく、例えば６ｋｂｐ以上であってもよい。 As described later in the Examples, the nucleotide sequence of the renal tubular cell-specific promoter is preferably long. Moreover, it is preferable to include a base as close as possible to the translation initiation codon (ATG) or to include a translation initiation codon. The length of a renal tubular cell specific promoter may be, for example, 2 kbp or more, for example, 3 kbp or more, for example, 4 kbp or more, for example, 5 kbp or more, for example, 6 kbp It may be more than.
　本実施形態のベクターにおいて、所望の遺伝子は、上記の腎尿細管細胞特異的プロモーターにより転写制御されている遺伝子が挙げられる。具体的には、ＮＰＴ２ａ、ＮＫＣＣ２、ＡＱＰ２、ＮＣＣ、ＥＮａＣ等の遺伝子であってもよい。所望の遺伝子は、本実施形態のベクターを導入する対象の種由来の遺伝子であることが好ましい。 In the vector of the present embodiment, the desired gene includes a gene transcriptionally controlled by the renal tubular cell-specific promoter described above. Specifically, genes such as NPT2a, NKCC2, AQP2, NCC, ENaC, etc. may be used. The desired gene is preferably a gene from a species to which the vector of the present embodiment is to be introduced.
　所望の遺伝子は、疾患の原因となる変異を有しない遺伝子であることが好ましい。これにより、本実施形態のベクターを遺伝性尿細管疾患の遺伝子治療に用いることが可能になる。すなわち、本実施形態のベクターは、遺伝性尿細管疾患の遺伝子治療用であってもよい。 The desired gene is preferably a gene having no mutation causing disease. This makes it possible to use the vector of the present embodiment for gene therapy of hereditary tubular disease. That is, the vector of the present embodiment may be for gene therapy of hereditary tubular disease.
　所望の遺伝子は、本来の腎尿細管細胞特異的プロモーターにより転写制御されている遺伝子であることが好ましい。しかしながら、腎尿細管細胞特異的プロモーターと、所望の遺伝子の組み合わせは、本来の組み合わせと異なっていてもよい。 The desired gene is preferably a gene transcriptionally regulated by the native renal tubular cell-specific promoter. However, the combination of renal tubular cell specific promoter and desired gene may be different from the original combination.
　ウイルスベクターとしては、一般に遺伝子導入に用いられるものであれば特に限定されず使用することができ、例えば、アデノウイルスベクター、アデノ随伴ウイルスベクター、レンチウイルスベクター、レトロウイルスベクター等が挙げられる。ウイルスベクターは市販されているものであってもよい。 The virus vector is not particularly limited as long as it is generally used for gene transfer, and examples thereof include adenovirus vector, adeno-associated virus vector, lentivirus vector, retrovirus vector and the like. The viral vector may be commercially available.
　ウイルスベクターとしては、なかでも、アデノウイルスベクターを好適に用いることができる。ウイルスベクターは、ウイルス増殖の観点から自己複製できないものを使用することが好ましい。 Among them, adenoviral vectors can be suitably used as viral vectors. It is preferable to use a viral vector which can not self-replicate from the viewpoint of viral propagation.
　自己複製できないアデノウイルスベクターとしては、例えば人為的にＥ１Ａ及びＥ３領域を欠失させたもの、アデノキメラウイルス、改良型アデノウイルスベクター等が挙げられる。 Examples of adenoviral vectors which can not replicate autonomously include those in which the E1A and E3 regions have been artificially deleted, adenochimeric viruses, modified adenovirus vectors and the like.
　自己複製ができないアデノウイルスベクターを使用する場合、相当する欠失領域を有するパッケージング細胞を用いて相同組換えを行なう必要がある。パッケージング細胞としては、Ｅ１ＡおよびＥ３領域を有するＨＥＫ２９３細胞等が挙げられる。 When using an adenoviral vector which can not replicate itself, it is necessary to carry out homologous recombination using packaging cells having the corresponding deleted region. Examples of packaging cells include HEK293 cells having E1A and E3 regions.
　本実施形態のベクターは、血流を遮断した腎臓の腎動脈へのボーラス注入により投与されるものであることが好ましい。ボーラス注入とは急速注入のことである。ボーラス注入は、患者の腎組織に傷害を与えない程度の圧力となるように行えば特に限定されず、例えば、ウイルスベクター液１ｍＬあたり２～３分程度で注入することが挙げられる。 It is preferable that the vector of this embodiment is administered by bolus injection into the renal artery of the kidney which has blocked the blood flow. Bolus injection is rapid injection. The bolus injection is not particularly limited as long as the pressure is such that the kidney tissue of the patient is not injured. For example, injection may be performed in about 2 to 3 minutes per 1 ml of virus vector solution.
　実施例において後述するように、発明者らが様々な投与ルートを検討した結果、血流を遮断した腎臓の腎動脈へのボーラス注入により本実施形態のベクターを投与することが、腎組織への傷害も少なく、所望の遺伝子の発現効率も高いことが明らかとなった。 As described later in the examples, as a result of the inventors studying various administration routes, administration of the vector of the present embodiment by bolus injection into the renal artery of the kidney whose blood flow has been blocked is effective for renal tissue. It was revealed that the injury was less and the expression efficiency of the desired gene was also high.
　血流の遮断はクリップで血管をクランプすることにより行うことができる。腎動脈を遮断後、ウイルスベクターをボーラス注入する。また、腎動脈を遮断後、生理食塩水等の等張液を注入して腎臓内の血液を洗い流すとより感染効率が高まり好ましい。更に、ウイルスベクターを注入後、腎静脈を遮断して数分間維持することにより、ウイルスベクターの感染効率を更に向上させることができる。ここで、腎静脈を遮断する時間としては、例えば、５～１０分程度が挙げられる。 The blocking of the blood flow can be done by clamping the blood vessel with a clip. After blocking the renal artery, a bolus injection of viral vector is performed. In addition, after blocking the renal artery, it is preferable to infuse isotonic fluid such as physiological saline and wash out the blood in the kidney to increase infection efficiency more preferably. Furthermore, after injecting the viral vector, the infection efficiency of the viral vector can be further improved by blocking the renal vein and maintaining it for several minutes. Here, as a time for blocking the renal vein, for example, about 5 to 10 minutes can be mentioned.
　本実施形態のベクターは、上述したウイルスベクターと、薬学的に許容される溶媒とを含む医薬組成物の形態に製剤化されていてもよい。薬学的に許容される溶媒としては、一般的な医薬組成物の製造に用いられるものを適宜使用することができる。 The vector of the present embodiment may be formulated in the form of a pharmaceutical composition comprising the above-described viral vector and a pharmaceutically acceptable solvent. As the pharmaceutically acceptable solvent, those used for the production of general pharmaceutical compositions can be appropriately used.
［その他の実施形態］
　１実施形態において、本発明は、腎尿細管細胞特異的プロモーターと、前記プロモーターの下流に連結された所望の遺伝子とを有するウイルスベクターの有効量を、治療を必要とする患者に投与することを含む遺伝性尿細管疾患の治療方法を提供する。 Other Embodiments
In one embodiment, the present invention comprises administering to a patient in need of treatment an effective amount of a viral vector having a renal tubular cell specific promoter and a desired gene linked downstream of said promoter. Provided is a method of treating hereditary tubular disease.
　１実施形態において、本発明は、遺伝性尿細管疾患の治療のためのベクターであって、腎尿細管細胞特異的プロモーターと、前記プロモーターの下流に連結された所望の遺伝子とを有するウイルスベクターを提供する。 In one embodiment, the present invention provides a vector for the treatment of hereditary tubular disease, which comprises a renal tubular cell-specific promoter and a desired gene linked downstream of said promoter. provide.
　１実施形態において、本発明は、遺伝性尿細管疾患の治療薬を製造するためのベクターの使用であって、前記ベクターは、腎尿細管細胞特異的プロモーターと、前記プロモーターの下流に連結された所望の遺伝子とを有するウイルスベクターである、使用を提供する。 In one embodiment, the present invention is the use of a vector for producing a therapeutic agent for hereditary tubular disease, wherein the vector is linked to a renal tubular cell specific promoter and downstream of the promoter. The use is provided, which is a viral vector having the desired gene.
　上記各実施形態において、上記のプロモーターは、ＮＰＴ２ａ、ＮＫＣＣ２、ＡＱＰ２、ＮＣＣ及びＥＮａＣからなる群より選択される遺伝子のプロモーターであってもよい。 In each of the above embodiments, the above promoter may be a promoter of a gene selected from the group consisting of NPT2a, NKCC2, AQP2, NCC and ENaC.
　また、上記の遺伝子は、ＮＰＴ２ａ、ＮＫＣＣ２、ＡＱＰ２、ＮＣＣ及びＥＮａＣからなる群より選択される遺伝子であってもよい。ここで、これらの遺伝子は、疾患の原因となる変異を有しない遺伝子であることが好ましい。 In addition, the above gene may be a gene selected from the group consisting of NPT2a, NKCC2, AQP2, NCC and ENaC. Here, these genes are preferably genes having no mutation causing disease.
　また、上記のベクターは、血流を遮断した腎臓の腎動脈へのボーラス注入により投与するものであることが好ましい。 In addition, it is preferable that the above-mentioned vector is administered by bolus injection into the renal artery of the kidney which has blocked the blood flow.
　次に実施例を示して本発明を更に詳細に説明するが、本発明は以下の実施例に限定されるものではない。 EXAMPLES The present invention will next be described in more detail by way of examples, which should not be construed as limiting the invention thereto.
［実験例１］（プロモーターのクローニング）
　ウイスターラットのゲノムＤＮＡから、Ｎｐｔ２ａ遺伝子、Ｎｋｃｃ２遺伝子及びＡｑｐ２遺伝子のプロモーターをクローニングして使用した。クローニングするプロモーター領域としては、ヒト、マウス、ラットの塩基配列データを比較し、相同性の高い領域を選択した。 [Experimental Example 1] (Cloning of Promoter)
The promoters of Npt2a gene, Nkcc2 gene and Aqp2 gene were cloned and used from Genomic DNA of Wistar rat. As promoter regions to be cloned, nucleotide sequence data of human, mouse and rat were compared, and regions with high homology were selected.
　Ｎｐｔ２ａ遺伝子の５’側上流の塩基配列データとして、アクセッション番号ＥＮＳＭＵＳＧ００００００２７２０２の塩基配列データを使用した。また、Ｎｋｃｃ２遺伝子の５’側上流の塩基配列データとして、アクセッション番号ＥＮＳＲＮＯＧ００００００１５２６２の塩基配列データを使用した。また、Ａｑｐ２遺伝子の５’側上流の塩基配列データとして、アクセッション番号ＥＮＳＲＮＯＧ００００００００２９７の塩基配列データを使用した。 The nucleotide sequence data of accession number ENSMUSG00000027202 was used as the nucleotide sequence data of the 5 'side of the Npt2a gene. Moreover, the base sequence data of accession number ENSRNOG00000015262 was used as base sequence data of the 5 'side of Nkcc2 gene. Moreover, the base sequence data of accession number ENSRNOG00000000297 was used as base sequence data of the 5 'side of Aqp2 gene.
《Ｎｐｔ２ａ遺伝子のプロモーター》
　ウイスターラットの血液からラットゲノムＤＮＡを抽出し、ＰＣＲによりＮｐｔ２ａ遺伝子のプロモーターをクローニングした。クローニングするプロモーター領域ができるだけ長くなるように、翻訳開始コドンにできるだけ近接した領域を含む領域を取得した。クローニングしたプロモーターは－３３７９～＋６３０の領域であった。ここで、－１がＮｐｔ２ａ遺伝子の翻訳開始コドン（ＡＴＧ）の直前の塩基に対応し、以下同様である。 プ ロ モ ー タ ー Promoter of Npt2a gene》
Rat genomic DNA was extracted from the blood of Wistar rats, and the promoter of the Npt2a gene was cloned by PCR. In order to make the promoter region to be cloned as long as possible, a region including a region as close as possible to the translation initiation codon was obtained. The cloned promoter was in the region from -3379 to +630. Here, -1 corresponds to the base immediately before the translation initiation codon (ATG) of the Npt2a gene, and so forth.
《Ｎｋｃｃ２遺伝子のプロモーター》
　下記表１に記載の各プロモーターを使用した。表１中、ｐＮＫＣＣ２ＤＩプロモーター及びｐＮＫＣＣ２ｐｖｕプロモーターはテキサス大学南西医療センターのピーター・イガラシ博士より分与された。ｐＮＫＣＣ２プロモーターはラットゲノムＤＮＡのＰＣＲにより調製した。 プ ロ モ ー タ ー Promoter of Nkcc2 gene》
Each promoter described in Table 1 below was used. In Table 1, pNKCC2DI promoter and pNKCC2pvu promoter were distributed by Dr. Peter Igarashi, University of Texas Southwest Medical Center. The pNKCC2 promoter was prepared by PCR of rat genomic DNA.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
《Ａｑｐ２遺伝子のプロモーター》
　ウイスターラットの血液からラットゲノムＤＮＡを抽出し、ＰＣＲによりＡｑｐ２遺伝子のプロモーターをクローニングした。クローニングしたプロモーターは－５３３８～＋９３０の領域であった。 << Promoter of Aqp2 gene >>
Rat genomic DNA was extracted from the blood of Wistar rats, and the promoter of the Aqp2 gene was cloned by PCR. The cloned promoter was in the region of -5338 to +930.
［実験例２］（アデノウイルスベクターの調製）
　実験例１でクローニングした各プロモーターは、緑色蛍光タンパク質発現ベクターであるｐＥＧＦＰＮ１（クロンテック社）にサブクローニングし、各プロモーターの下流に緑色蛍光タンパク質（ＥＧＦＰ）遺伝子を連結した。続いて、各プロモーター及びその下流に連結されたＥＧＦＰ遺伝子をアデノウイルスベクターであるｐＡｘＣｗｉｔ（理化学研究所バイオリソースセンター）にそれぞれサブクローニングした。下記表２に、調製したアデノウイルスベクター名及びその内容を記載した。 [Experimental Example 2] (Preparation of Adenoviral Vector)
Each promoter cloned in Experimental Example 1 was subcloned into a green fluorescent protein expression vector pEGFPN1 (Clontech), and a green fluorescent protein (EGFP) gene was linked downstream of each promoter. Subsequently, each promoter and the EGFP gene linked downstream thereof were subcloned respectively into an adenovirus vector pAxCwit (RIKEN BioResource Center). The following Table 2 describes the prepared adenovirus vector name and its contents.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　調製した各アデノウイルスベクターは、正確に単一クローンを選択して使用した。また、各アデノウイルスベクターの塩基配列は、制限酵素による切断及び塩基配列のシークエンス解析により確認した。 Each prepared adenovirus vector was used by selecting a single clone correctly. In addition, the nucleotide sequence of each adenoviral vector was confirmed by restriction enzyme cleavage and sequence analysis of the nucleotide sequence.
　各アデノウイルスベクターを、ヒト胎児腎細胞由来の細胞株であるＨＥＫ２９３細胞に感染させ、アデノウイルスを調製した。得られたアデノウイルスの力価は１０１３ｐｆｕ／ｍＬ以上であった。 Each adenovirus vector was used to infect HEK 293 cells, a cell line derived from human embryonic kidney cells, to prepare adenovirus. The titer of the obtained adenovirus was 10 13 pfu / mL or more.
［実験例３］（投与ルートの検討）
　アデノウイルスベクターを静脈投与により生体に投与した場合、９５％以上が肝臓にトラップされてしまう。そこで、アデノウイルスベクターを腎臓に特異的に感染させるための投与ルートの検討を行った。具体的には、８～１０週齢のラットを用いて、以下の投与ルートによりアデノウイルスベクターを投与し検討した。実験は、動物実験委員会の承認を得て行い、４３匹のラットを本実験に使用した。 [Experimental example 3] (examination of administration route)
When an adenovirus vector is administered to a living body by intravenous administration, 95% or more is trapped in the liver. Therefore, the administration route for specifically infecting the kidney with an adenoviral vector was examined. Specifically, adenoviral vectors were administered and examined by the following administration route using 8- to 10-week-old rats. The experiment was conducted with the approval of the Animal Experiment Committee, and 43 rats were used for this experiment.
《腎動脈へのボーラス投与》
　まず、ラットの腹部大動脈と左腎動脈の間をクリップでクランプして血流を遮断した。続いて、１ｍＬの生理食塩水を２分間かけて左腎動脈に注入した。これにより、腎臓内の血液が洗い流された。続いて、１ｍＬのアデノウイルスベクター液を２分間かけて左腎動脈に注入した。続いて、腎静脈をクリップでクランプして血流を遮断し１０分間維持した。これにより、アデノウイルスベクターが腎臓内に留まり、腎細胞に効率よく感染した。その後、クリップをはずし、血流を再開させた。 Bolus administration to the renal arteries
First, the blood flow was interrupted by clamping with a clip between the rat's abdominal aorta and the left renal artery. Subsequently, 1 mL of saline was infused over 2 minutes into the left renal artery. Thereby, the blood in the kidney was washed away. Subsequently, 1 mL of adenoviral vector solution was infused into the left renal artery over 2 minutes. Subsequently, the renal vein was clamped with a clip to block blood flow and maintained for 10 minutes. As a result, the adenoviral vector remained in the kidney and efficiently infected the kidney cells. After that, the clip was removed and blood flow was resumed.
《腎動脈への緩徐な投与》
　ラットの腎動脈にアデノウイルスベクター液１ｍＬを１６時間かけて緩徐に注入した。 Slow Administration to the Renal Artery
Rat renal artery was infused slowly with 1 mL of adenovirus vector solution for 16 hours.
《膀胱への投与》
　ラットの膀胱にアデノウイルスベクター液１ｍＬを注入した。その結果、アデノウイルスベクターは尿管を逆流して腎細胞に感染することが期待された。 Administration to the bladder
The rat bladder was infused with 1 mL of adenoviral vector solution. As a result, adenoviral vectors were expected to backflow to the ureter and infect kidney cells.
《腎実質への投与》
　ラットの腎臓にアデノウイルスベクター液１ｍＬを直接注射した。注射は１０回に分けて行い、あらゆる方向に向けて注射した。 Administration to the renal parenchyma
Rat kidneys were directly injected with 1 mL of adenovirus vector solution. The injections were divided into 10 parts and injected in all directions.
《免疫組織化学解析》
　アデノウイルスベクターの投与から４日後に各ラットから腎臓を摘出し、４％パラホルムアルデヒドで２日間固定した。続いて、各腎臓標本をパラフィン包埋して薄切切片を作製した。 <Immunohistochemical analysis>
Four days after administration of the adenoviral vector, kidneys were removed from each rat and fixed with 4% paraformaldehyde for 2 days. Subsequently, each kidney sample was embedded in paraffin to prepare a sliced section.
　続いて、各組織切片を抗ＥＧＦＰ抗体、抗ＮＫＣＣ２抗体、抗ＡＱＰ２抗体、抗ｓｏｄｉｕｍ－ｈｙｄｒｏｇｅｎ　ｅｘｃｈａｎｇｅｒ３（ＮＨＥ３；ＳＬＣ９Ａ３Ｐ２）抗体で染色した。抗ＮＨＥ３抗体は、抗ＮＰＴ２ａ抗体が入手できなかったため、抗ＮＰＴ２ａ抗体の代わりに使用した。 Subsequently, each tissue section was stained with anti-EGFP antibody, anti-NKCC2 antibody, anti-AQP2 antibody, anti-sodium-hydrogen exchanger 3 (NHE3; SLC9A3P2) antibody. The anti-NHE3 antibody was used instead of the anti-NPT2a antibody because the anti-NPT2a antibody was not available.
　その結果、腎動脈への緩徐な投与では、ＥＧＦＰの発現が検出できなかった。この投与方法では、血液の存在により、アデノウイルスの感染率が低かったものと考えられた。 As a result, expression of EGFP could not be detected by slow administration to the renal artery. In this administration method, it was considered that the infection rate of adenovirus was low due to the presence of blood.
　また、膀胱への投与では、鮮明なＥＧＦＰの発現が検出された。しかしながら、近位尿細管の微絨毛が消失していた。これはアデノウイルスベクターの尿細管への逆流の圧力が高すぎたためであると考えられた。膀胱への投与では、腎臓の構造を破壊しただけでなく、尿管へのアデノウイルスの感染のリスクも考えられた。 In addition, in the case of administration to the bladder, clear expression of EGFP was detected. However, the microvilli in the proximal tubule have disappeared. This was considered to be due to the pressure for backflow of the adenoviral vector into the renal tubule being too high. The administration to the bladder not only destroyed the structure of the kidney, but also considered the risk of adenovirus infection to the ureter.
　また、腎実質への投与では、ＥＧＦＰが発現した領域は、アデノウイルスベクターを注射した部位に限定されていた。また、繰り返された穿刺及び液注入により腎実質の構造が傷害を受けていた。 In addition, in the administration to the renal parenchyma, the region where EGFP was expressed was limited to the site where the adenoviral vector was injected. In addition, the structure of the renal parenchyma was damaged by repeated puncture and fluid injection.
　これに対し、顕微鏡観察の結果、アデノウイルスベクターの腎動脈へのボーラス投与による腎組織構造への目立った傷害は認められなかった。また、血清及び尿の解析結果から、アデノウイルスベクターの腎動脈へのボーラス投与による、腎臓及び肝臓の機能の変化も認められなかった。 On the other hand, as a result of microscopic observation, no remarkable damage to the renal tissue structure was observed by the bolus administration of the adenoviral vector to the renal artery. Further, from the analysis results of serum and urine, no change in function of kidney and liver was observed by bolus administration of adenoviral vector to renal artery.
　また、後述するように、アデノウイルスベクターの腎動脈へのボーラス投与により、最も高い遺伝子発現効率が得られることが明らかとなった。そこで、以降の実験では、腎動脈へのボーラス投与によりアデノウイルスベクターを投与した。 In addition, as described later, it was revealed that bolus administration of an adenoviral vector to the renal artery provides the highest gene expression efficiency. Therefore, in subsequent experiments, adenoviral vectors were administered by bolus administration to the renal arteries.
［実験例４］（ＮＫＣＣ２プロモーターの検討）
　発明者らは、実験例２で調製した、ＮＫＣＣ２ＤＩ－ＥＧＦＰ、ＮＫＣＣ２ｐｖｕ－ＥＧＦＰ及びＮＫＣＣ２－ＥＧＦＰの３種類のアデノウイルスベクターを腎動脈へのボーラス投与によりラットに投与した。続いて、アデノウイルスベクターの投与から４日後に各ラットから腎臓を摘出し、実験例３と同様にして組織切片を作製した。 [Experimental example 4] (examination of NKCC2 promoter)
The inventors administered the three types of adenoviral vectors, NKCC2DI-EGFP, NKCC2pvu-EGFP and NKCC2-EGFP, prepared in Experimental Example 2 to rats by bolus administration to the renal artery. Subsequently, 4 days after administration of the adenoviral vector, kidneys were removed from each rat, and tissue sections were prepared in the same manner as in Experimental Example 3.
《免疫組織化学解析》
　続いて、作製した各組織切片を免疫組織化学解析により解析した。図１（ａ）及び（ｂ）は、ＮＫＣＣ２ＤＩ－ＥＧＦＰアデノウイルスベクターを導入した結果を示す写真である。図１（ａ）は抗ＥＧＦＰ抗体で染色した結果を示す写真であり、図１（ｂ）は、図１（ａ）の組織切片に隣接する別の組織切片を抗ＮＫＣＣ２抗体で染色し、内在性のＮＫＣＣ２タンパク質を検出した結果を示す写真である。スケールバーは、いずれも１ｃｍを示す。 <Immunohistochemical analysis>
Subsequently, each tissue section prepared was analyzed by immunohistochemical analysis. FIGS. 1 (a) and (b) are photographs showing the results of introducing the NKCC2DI-EGFP adenoviral vector. FIG. 1 (a) is a photograph showing the results of staining with anti-EGFP antibody, and FIG. 1 (b) is a staining of another tissue section adjacent to the tissue section of FIG. 1 (a) with anti-NKCC2 antibody, It is a photograph which shows the result of having detected sexual NKCC2 protein. Each scale bar shows 1 cm.
　また、図１（ｃ）及び（ｄ）は、ＮＫＣＣ２ｐｖｕ－ＥＧＦＰアデノウイルスベクターを導入した結果を示す写真である。図１（ｃ）は抗ＥＧＦＰ抗体で染色した結果を示す写真であり、図１（ｄ）は、図１（ｃ）の組織切片に隣接する別の組織切片を抗ＮＫＣＣ２抗体で染色し、内在性のＮＫＣＣ２タンパク質を検出した結果を示す写真である。スケールバーは、いずれも１ｃｍを示す。 Moreover, FIG.1 (c) and (d) are photographs which show the result of introduce | transducing NKCC2pvu-EGFP adenoviral vector. Fig. 1 (c) is a photograph showing the results of staining with anti-EGFP antibody, and Fig. 1 (d) is another tissue section adjacent to the tissue section of Fig. 1 (c) stained with anti-NKCC2 antibody, It is a photograph which shows the result of having detected sexual NKCC2 protein. Each scale bar shows 1 cm.
　また、図１（ｅ）及び（ｆ）は、ＮＫＣＣ２－ＥＧＦＰアデノウイルスベクターを導入した結果を示す写真である。図１（ｅ）は抗ＥＧＦＰ抗体で染色した結果を示す写真であり、図１（ｆ）は、図１（ｅ）の組織切片に隣接する別の組織切片を抗ＮＫＣＣ２抗体で染色し、内在性のＮＫＣＣ２タンパク質を検出した結果を示す写真である。スケールバーは、いずれも１ｃｍを示す。 Moreover, FIG. 1 (e) and (f) is a photograph which shows the result of introduce | transducing a NKCC2-EGFP adenoviral vector. FIG. 1 (e) is a photograph showing the results of staining with anti-EGFP antibody, and FIG. 1 (f) is a staining of another tissue section adjacent to the tissue section of FIG. 1 (e) with anti-NKCC2 antibody, It is a photograph which shows the result of having detected sexual NKCC2 protein. Each scale bar shows 1 cm.
　その結果、ＮＫＣＣ２ＤＩ－ＥＧＦＰアデノウイルスベクターを導入した場合に、ＥＧＦＰの発現パターンが内在性のＮＫＣＣ２タンパク質の発現パターンに最も近くなることが明らかとなった。 As a result, when NKCC2DI-EGFP adenoviral vector was introduced, it became clear that the expression pattern of EGFP was the closest to the expression pattern of endogenous NKCC2 protein.
　これに対し、ＮＫＣＣ２ｐｖｕ－ＥＧＦＰアデノウイルスベクターを導入した場合、及びＮＫＣＣ２－ＥＧＦＰアデノウイルスベクターを導入した場合では、ＥＧＦＰが内在性のＮＫＣＣ２タンパク質よりも広範囲の領域で発現されていた。 On the other hand, when the NKCC2 pvu-EGFP adenoviral vector was introduced and when the NKCC2-EGFP adenoviral vector was introduced, EGFP was expressed in a broader region than the endogenous NKCC2 protein.
《共焦点レーザー顕微鏡》
　続いて、共焦点レーザー顕微鏡を使用して、ＮＫＣＣ２ＤＩ－ＥＧＦＰアデノウイルスベクターを導入した場合のＥＧＦＰ及び内在性のＮＫＣＣ２タンパク質の発現を、より高解像度で検討した。組織切片の免疫染色には、実験例３で用いたものと同じ抗体を使用し、ＥＧＦＰ及び内在性のＮＫＣＣ２タンパク質を二重染色した。また、ヘキスト３３３４２を用いて核を染色した。 Confocal laser microscope
Subsequently, using confocal laser microscopy, the expression of EGFP and endogenous NKCC2 protein when NKCC2DI-EGFP adenoviral vector was introduced was examined at higher resolution. For immunostaining of tissue sections, the same antibody as that used in Experimental Example 3 was used to double-stain EGFP and endogenous NKCC2 protein. The nuclei were also stained using Hoechst 33342.
　図２（ａ）～（ｄ）は、同一視野をそれぞれ共焦点レーザー顕微鏡で観察した結果を示す写真である。図２（ａ）は核を検出した結果を示す写真であり、図２（ｂ）はＥＧＦＰを検出した結果を示す写真であり、図２（ｃ）は内在性のＮＫＣＣ２タンパク質を検出した結果を示す写真であり、図２（ｄ）は図２（ａ）～（ｃ）を合成した写真である。スケールバーは、いずれも５０μｍを示す。 FIGS. 2 (a) to 2 (d) are photographs showing the results of observation of the same field of view with a confocal laser microscope. FIG. 2 (a) is a photograph showing the result of detecting the nucleus, FIG. 2 (b) is a photograph showing the result of detecting the EGFP, and FIG. 2 (c) is a result of detecting the endogenous NKCC2 protein FIG. 2 (d) is a photograph showing a composite of FIGS. 2 (a) to 2 (c). Scale bars all show 50 μm.
　その結果、ＮＫＣＣ２ＤＩ－ＥＧＦＰアデノウイルスベクターの導入により発現したＥＧＦＰ及びＮＫＣＣ２タンパク質は、いずれも太いヘンレ係蹄脚に発現していることが確認された。すなわち、太いヘンレ係蹄脚特異的に所望の遺伝子を発現させることができることが明らかとなった。 As a result, it was confirmed that the EGFP and NKCC2 proteins expressed by the introduction of the NKCC2DI-EGFP adenoviral vector were both expressed in thick Henle-stitched legs. That is, it has become clear that the desired gene can be expressed specifically in thick Henle's wing.
　以上の結果から、ＮＫＣＣ２ＤＩ－ＥＧＦＰアデノウイルスベクターの導入により、内在性のＮＫＣＣ２タンパク質に近い発現パターンでＥＧＦＰを発現させることができることが明らかとなった。 From the above results, it has become clear that the introduction of the NKCC2DI-EGFP adenoviral vector makes it possible to express EGFP with an expression pattern close to that of the endogenous NKCC2 protein.
　非特許文献１によれば、尿細管の初代培養細胞を使用したインビトロの検討において、ｐＮＫＣＣ２ｐｖｕプロモーターが最も高いプロモーター活性を示し、ｐＮＫＣＣ２ＤＩプロモーターが最も低いプロモーター活性を示したことが報告されている。 According to Non-Patent Document 1, it is reported that the pNKCC2 pvu promoter showed the highest promoter activity and the pNKCC2 DI promoter showed the lowest promoter activity in in vitro studies using primary culture cells of renal tubules.
　これに対し、インビボにおいて、内在性のＮＫＣＣ２タンパク質に近い発現パターンで遺伝子を発現させるためには、ｐＮＫＣＣ２ＤＩプロモーターを用いることが最もよいことが明らかとなった。すなわち、プロモーターはできるだけ長いことが好ましいことが明らかとなった。 On the other hand, in order to express the gene in an expression pattern close to the endogenous NKCC2 protein in vivo, it was revealed that it is best to use the pNKCC2DI promoter. That is, it has become clear that the promoter is preferably as long as possible.
［実験例５］（ＮＰＴ２ａプロモーターの検討）
　発明者らは、実験例２で調製した、ＮＰＴ２ａ－ＥＧＦＰアデノウイルスベクターを腎動脈へのボーラス投与によりラットに投与した。続いて、アデノウイルスベクターの投与から４日後に各ラットから腎臓を摘出し、実験例３と同様にして組織切片を作製した。 [Experimental example 5] (examination of NPT2a promoter)
The inventors administered the NPT2a-EGFP adenoviral vector prepared in Experimental Example 2 to rats by bolus administration to the renal artery. Subsequently, 4 days after administration of the adenoviral vector, kidneys were removed from each rat, and tissue sections were prepared in the same manner as in Experimental Example 3.
《免疫組織化学解析》
　続いて、作製した各組織切片を免疫組織化学解析により解析した。図３（ａ）及び（ｂ）は、ＮＰＴ２ａ－ＥＧＦＰアデノウイルスベクターを導入した結果を示す写真である。図３（ａ）は抗ＥＧＦＰ抗体で染色した結果を示す写真であり、図３（ｂ）は、図３（ａ）の組織切片に隣接する別の組織切片を抗ＮＨＥ３抗体で染色し、内在性のＮＨＥ３タンパク質を検出した結果を示す写真である。上述したように、抗ＮＨＥ３抗体は、抗ＮＰＴ２ａ抗体が入手できなかったため、抗ＮＰＴ２ａ抗体の代わりに使用した。スケールバーは、いずれも１ｃｍを示す。 <Immunohistochemical analysis>
Subsequently, each tissue section prepared was analyzed by immunohistochemical analysis. FIGS. 3 (a) and 3 (b) are photographs showing the results of introducing the NPT2a-EGFP adenoviral vector. FIG. 3 (a) is a photograph showing the results of staining with anti-EGFP antibody, and FIG. 3 (b) is a staining of another tissue section adjacent to the tissue section of FIG. 3 (a) with anti-NHE3 antibody, It is a photograph which shows the result of having detected sex NHE3 protein. As mentioned above, anti-NHE3 antibody was used instead of anti-NPT2a antibody because anti-NPT2a antibody was not available. Each scale bar shows 1 cm.
　その結果、ＮＰＴ２ａ－ＥＧＦＰアデノウイルスベクターの導入によるＥＧＦＰの発現パターンと、内在性のＮＨＥ３タンパク質の発現パターンは近いことが明らかとなった。 As a result, it was revealed that the expression pattern of EGFP by the introduction of the NPT2a-EGFP adenoviral vector and the expression pattern of the endogenous NHE3 protein are similar.
《共焦点レーザー顕微鏡》
　続いて、共焦点レーザー顕微鏡を使用して、ＥＧＦＰ及び内在性のＮＫＣＣ２タンパク質の発現を、より高解像度で検討した。組織切片の免疫染色には、実験例３で用いたものと同じ抗体を使用し、ＥＧＦＰ及び内在性のＮＨＥ３タンパク質を二重染色した。また、ヘキスト３３３４２を用いて核を染色した。スケールバーは、いずれも５０μｍを示す。 Confocal laser microscope
Subsequently, expression of EGFP and endogenous NKCC2 protein was examined at higher resolution using confocal laser microscopy. For immunostaining of tissue sections, the same antibody as that used in Experimental Example 3 was used to double-stain EGFP and endogenous NHE3 protein. The nuclei were also stained using Hoechst 33342. Scale bars all show 50 μm.
　図４（ａ）～（ｄ）は、同一視野をそれぞれ共焦点レーザー顕微鏡で観察した結果を示す写真である。図４（ａ）は核を検出した結果を示す写真であり、図４（ｂ）はＥＧＦＰを検出した結果を示す写真であり、図４（ｃ）は内在性のＮＨＥ３タンパク質を検出した結果を示す写真であり、図４（ｄ）は図４（ａ）～（ｃ）を合成した写真である。 FIGS. 4 (a) to 4 (d) are photographs showing the results of observation of the same field of view with a confocal laser microscope. FIG. 4 (a) is a photograph showing the result of detecting the nucleus, FIG. 4 (b) is a photograph showing the result of detecting the EGFP, and FIG. 4 (c) is a result of detecting the endogenous NHE3 protein FIG. 4 (d) is a photograph showing a composite of FIGS. 4 (a) to 4 (c).
　その結果、ＮＰＴ２ａ－ＥＧＦＰアデノウイルスベクターの導入により発現したＥＧＦＰ及びＮＨＥ３タンパク質は、いずれも近位尿細管細胞に発現していることが確認された。すなわち、近位尿細管特異的に所望の遺伝子を発現させることができることが明らかとなった。しかしながら、ＥＧＦＰは細胞質に局在し、ＮＨＥ３タンパク質は同一の細胞の細胞膜に局在していることが明らかとなった。ＥＧＦＰとＮＨＥ３タンパク質の局在の違いは、膜局在シグナルの有無によるものと考えられた。 As a result, it was confirmed that both EGFP and NHE3 proteins expressed by the introduction of the NPT2a-EGFP adenoviral vector were expressed in proximal tubular cells. That is, it became clear that the desired gene can be expressed in a proximal tubule-specific manner. However, it was revealed that EGFP is localized in the cytoplasm, and the NHE3 protein is localized in the cell membrane of the same cell. The difference in the localization of EGFP and NHE3 protein was considered to be due to the presence or absence of the membrane localization signal.
　以上の結果から、ＮＰＴ２ａ－ＥＧＦＰアデノウイルスベクターの導入により、内在性のＮＫＣＣ２タンパク質に近い発現パターンでＥＧＦＰを発現させることができることが明らかとなった。また、内在性のＮＨＥ３タンパク質を発現する細胞と同一の細胞にＥＧＦＰを発現させることができることが明らかとなった。 From the above results, it became clear that the introduction of the NPT2a-EGFP adenoviral vector makes it possible to express EGFP with an expression pattern close to that of the endogenous NKCC2 protein. In addition, it became clear that EGFP can be expressed in the same cells as cells expressing endogenous NHE3 protein.
［実験例６］（ＡＱＰ２プロモーターの検討）
　発明者らは、実験例２で調製した、ＡＱＰ２－ＥＧＦＰアデノウイルスベクターを腎動脈へのボーラス投与によりラットに投与した。続いて、アデノウイルスベクターの投与から４日後に各ラットから腎臓を摘出し、実験例３と同様にして組織切片を作製した。 [Experimental example 6] (examination of AQP2 promoter)
The inventors administered AQP2-EGFP adenoviral vector prepared in Experimental Example 2 to rats by bolus administration to the renal artery. Subsequently, 4 days after administration of the adenoviral vector, kidneys were removed from each rat, and tissue sections were prepared in the same manner as in Experimental Example 3.
《免疫組織化学解析》
　続いて、作製した各組織切片を免疫組織化学解析により解析した。図５（ａ）及び（ｂ）は、ＡＱＰ２－ＥＧＦＰアデノウイルスベクターを導入した結果を示す写真である。図５（ａ）は抗ＥＧＦＰ抗体で染色した結果を示す写真であり、図５（ｂ）は、図３（ａ）の組織切片に隣接する別の組織切片を抗ＡＱＰ２抗体で染色し、内在性のＡＱＰ２タンパク質を検出した結果を示す写真である。スケールバーは、いずれも１ｃｍを示す。 <Immunohistochemical analysis>
Subsequently, each tissue section prepared was analyzed by immunohistochemical analysis. FIGS. 5 (a) and (b) are photographs showing the results of introducing the AQP2-EGFP adenoviral vector. Fig. 5 (a) is a photograph showing the results of staining with anti-EGFP antibody, and Fig. 5 (b) is another tissue section adjacent to the tissue section of Fig. 3 (a) stained with anti-AQP2 antibody, It is a photograph which shows the result of having detected sex AQP2 protein. Each scale bar shows 1 cm.
　その結果、ＡＱＰ２－ＥＧＦＰアデノウイルスベクターの導入によるＥＧＦＰの発現パターンと、内在性のＡＱＰ２タンパク質の発現パターンは近いことが明らかとなった。 As a result, it was revealed that the expression pattern of EGFP by the introduction of the AQP2-EGFP adenoviral vector and the expression pattern of the endogenous AQP2 protein were close.
《共焦点レーザー顕微鏡》
　続いて、共焦点レーザー顕微鏡を使用して、ＥＧＦＰ及び内在性のＡＱＰ２タンパク質の発現を、より高解像度で検討した。組織切片の免疫染色には、実験例３で用いたものと同じ抗体を使用し、ＥＧＦＰ及び内在性のＡＱＰ２タンパク質を二重染色した。また、ヘキスト３３３４２を用いて核を染色した。 Confocal laser microscope
Subsequently, expression of EGFP and endogenous AQP2 protein was examined at higher resolution using confocal laser microscopy. For immunostaining of tissue sections, the same antibody as used in Experimental Example 3 was used to double-stain EGFP and endogenous AQP2 protein. The nuclei were also stained using Hoechst 33342.
　図６（ａ）～（ｄ）は、同一視野をそれぞれ共焦点レーザー顕微鏡で観察した結果を示す写真である。図６（ａ）は核を検出した結果を示す写真であり、図６（ｂ）はＥＧＦＰを検出した結果を示す写真であり、図６（ｃ）は内在性のＡＱＰ２タンパク質を検出した結果を示す写真であり、図６（ｄ）は図６（ａ）～（ｃ）を合成した写真である。スケールバーは、いずれも５０μｍを示す。 6 (a) to 6 (d) are photographs showing the results of observation of the same field of view with a confocal laser microscope. FIG. 6 (a) is a photograph showing the result of detecting the nucleus, FIG. 6 (b) is a photograph showing the result of detecting the EGFP, and FIG. 6 (c) is a result of detecting the endogenous AQP2 protein FIG. 6 (d) is a photograph showing a composite of FIGS. 6 (a) to 6 (c). Scale bars all show 50 μm.
　その結果、ＡＱＰ２－ＥＧＦＰアデノウイルスベクターの導入により発現したＥＧＦＰ及びＡＱＰ２タンパク質は、いずれも集合管細胞に発現していることが確認された。すなわち、集合管特異的に所望の遺伝子を発現させることができることが明らかとなった。しかしながら、ＥＧＦＰは細胞質に局在し、ＡＱＰ２タンパク質は同一の細胞の細胞膜に局在していることが明らかとなった。ＥＧＦＰとＡＱＰ２タンパク質の局在の違いは、膜局在シグナルの有無によるものと考えられた。 As a result, it was confirmed that both EGFP and AQP2 proteins expressed by the introduction of the AQP2-EGFP adenoviral vector were expressed in collecting duct cells. That is, it has become clear that the desired gene can be expressed specifically in the collecting duct. However, it was revealed that EGFP is localized in the cytoplasm and AQP2 protein is localized in the cell membrane of the same cell. The difference in the localization of EGFP and AQP2 protein was considered to be due to the presence or absence of a membrane localization signal.
　以上の結果から、ＡＱＰ２－ＥＧＦＰアデノウイルスベクターの導入により、内在性のＡＱＰ２タンパク質に近い発現パターンでＥＧＦＰを発現させることができることが明らかとなった。また、内在性のＡＱＰ２タンパク質を発現する細胞と同一の細胞にＥＧＦＰを発現させることができることが明らかとなった。 From the above results, it became clear that the introduction of AQP2-EGFP adenoviral vector can express EGFP with an expression pattern close to that of endogenous AQP2 protein. In addition, it became clear that EGFP can be expressed in the same cells as cells expressing endogenous AQP2 protein.
　本発明によれば、腎尿細管細胞特異的に遺伝子を発現させるためのベクターを提供することができる。 According to the present invention, it is possible to provide a vector for expressing a gene specifically in renal tubular cells.

Claims (5)

  1. 　腎尿細管細胞特異的プロモーターと、前記プロモーターの下流に連結された所望の遺伝子とを有するウイルスベクターからなる、腎尿細管細胞特異的に前記遺伝子を発現させるためのベクター。 A vector for expressing a gene in renal tubular cells specifically, which comprises a viral vector having a renal tubular cell specific promoter and a desired gene linked downstream of the promoter.
  2. 　前記プロモーターが、ｓｏｄｉｕｍ－ｄｅｐｅｎｄｅｎｔ　ｐｈｏｓｐｈａｔｅ　ｔｒａｎｓｐｏｒｔｅｒ　ｔｙｐｅ　２ａ（ＮＰＴ２ａ）、ｓｏｄｉｕｍ－ｐｏｔａｓｓｉｕｍ－２－ｃｈｌｏｒｉｄｅ　ｃｏｔｒａｎｓｐｏｒｔｅｒ（ＮＫＣＣ２）、ａｑｕａｐｏｒｉｎ２（ＡＱＰ２）、ｓｏｄｉｕｍ－ｃｈｌｏｒｉｄｅ　ｃｏｔｒａｎｓｐｏｒｔｅｒ（ＮＣＣ）及びｅｐｉｔｈｅｌｉａｌ　ｓｏｄｉｕｍ－ｃｈａｎｎｅｌ（ＥＮａＣ）からなる群より選択される遺伝子のプロモーターである、請求項１に記載のベクター。 The promoter comprises a group consisting of sodium-dependent phosphate transporter type 2a (NPT2a), sodium-potassium-2-chloride cotransporter (NKCC2), aquaporin 2 (AQP2), sodium-chloride cotransporter (NCC) and epithelial sodium-channel (ENaC) The vector according to claim 1, which is a promoter of a gene selected from
  3. 　前記遺伝子が、ＮＰＴ２ａ、ＮＫＣＣ２、ＡＱＰ２、ＮＣＣ及びＥＮａＣからなる群より選択される遺伝子である、請求項１又は２に記載のベクター。 The vector according to claim 1 or 2, wherein the gene is a gene selected from the group consisting of NPT2a, NKCC2, AQP2, NCC and ENaC.
  4. 　血流を遮断した腎臓の腎動脈へのボーラス注入により投与される、請求項１～３のいずれか一項に記載のベクター。 The vector according to any one of claims 1 to 3, which is administered by bolus injection into the renal artery of the kidney whose blood flow has been blocked.
  5. 　遺伝性尿細管疾患の遺伝子治療用である、請求項１～４のいずれか一項に記載のベクター。 The vector according to any one of claims 1 to 4, which is for gene therapy of hereditary tubular disease.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004111238A1 (en) * 2003-06-16 2004-12-23 Tohoku Techno Arch Co., Ltd Transcription regulatory sequence
JP2008175630A (en) * 2007-01-17 2008-07-31 Univ Of Miyazaki Diagnostic method of acute renal failure
JP2010506563A (en) * 2006-10-12 2010-03-04 エシコン・インコーポレイテッド Kidney-derived cells and their use in tissue repair and regeneration
JP2014528076A (en) * 2011-09-22 2014-10-23 ウニベルシダ デ ロス アンデス Method for monitoring, diagnosing and / or prognosing acute kidney injury in the early stage

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020184655A1 (en) * 1997-12-01 2002-12-05 Henryk Lubon Methods for the degradation and detoxification of organic material using urine produced by transgenic animals and related transgenic animals and proteins
WO2000015772A2 (en) * 1998-09-16 2000-03-23 Nexia Biotechnologies, Inc. Recombinant protein production in urine
AU1621900A (en) * 1998-11-13 2000-06-05 New York University Transgenic animals as urinary bioreactors for the production of protein in the urine, recombinant dna construct for kidney-specific expression, and method of using same
US9388427B2 (en) * 2002-12-02 2016-07-12 Biovec, Llc In vivo and ex vivo gene transfer into renal tissue using gutless adenovirus vectors
JP2010110322A (en) * 2008-10-10 2010-05-20 Tohoku Univ Human oatp-r gene introduced model nonhuman animal

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004111238A1 (en) * 2003-06-16 2004-12-23 Tohoku Techno Arch Co., Ltd Transcription regulatory sequence
JP2010506563A (en) * 2006-10-12 2010-03-04 エシコン・インコーポレイテッド Kidney-derived cells and their use in tissue repair and regeneration
JP2008175630A (en) * 2007-01-17 2008-07-31 Univ Of Miyazaki Diagnostic method of acute renal failure
JP2014528076A (en) * 2011-09-22 2014-10-23 ウニベルシダ デ ロス アンデス Method for monitoring, diagnosing and / or prognosing acute kidney injury in the early stage

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
IGARASHI P. ET AL.: "Cloning and Kidney Cell -specific Activity of the Promoter of the Murine Renal Na-K-Cl cotransporter Gene", J. BIOL. CHEM., vol. 271, no. 16, 1996, pages 9666 - 9674, ISSN: 0021-9258 *
OBERMULLER N. ET AL.: "Expression of the Na-K-2Cl Cotransporter by Macula Densa and Thick Ascending Limb Cells of Rat and Rabbit Nephron", J. CLIN. INVEST., vol. 98, no. 3, 1996, pages 635 - 640, XP055578946, ISSN: 0021-9738 *
WATANABE S. ET AL.: "Targeting gene expression to specific cells of kidney tubules in vivo, using adenoviral promoter fragments", PLOS ONE, vol. 12, no. 3, 2 March 2017 (2017-03-02), pages 1 - 14, XP055578926, ISSN: 1932-6203 *
WEINMAN E.J. ET AL.: "The role of NHERF-1 in the regulation of renal proximal tubule sodium-hydrogen exchanger 3 and sodium-dependent phosphate cotransporter 2a", J. PHYSIOL., vol. 567, no. 1, 2005, pages 27 - 32, XP055578944, ISSN: 1469-7793 *

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