JP2009079042A - Recombinant vector for treatment of nephrogenic diabetes insipidus, and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new therapeutic method for nephrogenic diabetes insipidus. <P>SOLUTION: A Sendai virus vector is used as the recombinant vector for treatment of nephrogenic diabetes insipidus and retaining an aquaporin-2 (AQP2) gene, AQP2 of a water channel is selected as a gene essentially acting in the final stage of water re-absorption, and the therapeutic effects of introduction thereof are examined on animal models. When inversely injecting the vector into the kidney through the ureter to a nephrogenic diabetes insipidus animal model of the AQP2 aberrant form (no particular phosphorylation caused by V2R), the AQP2 derived from the vector is successfully expressed in the collecting tube epithelial cells and, thereby, the therapeutic effect of about 40% urine volume decrease is expressed. On the one hand, the same therapeutic effects is recognized when simian immunodeficiency virus (SIV) vector including the AQP2 gene is used. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a recombinant vector useful for the treatment of nephrogenic diabetes insipidus and its use.

  The neuropeptide arginine vasopressin (AVP, also known as antidiuretic hormone) plays a central role in maintaining body fluid homeostasis. AVP secreted into the circulating blood from the posterior pituitary gland binds to the vasopressin receptor (V2R) on the cell membrane surface of the renal medullary collecting duct cells on the vascular cavity side. As a result, intracellular cAMP concentration increases, A kinase (PKA) is activated, and activated PKA phosphorylates aquaporin 2 (AQP2). As a result, AQP2 vesicles enter the plasma membrane on the tubular lumen side. A series of reactions to move is triggered.

  AQP2 is usually present on the membrane surface of intracellular endosomes. In addition to AQP2, PKA, PKCδ, PP2B, AKAP, VAMP2 required for membrane fusion, and calcium receptor are present on the surface of the AQP2 vesicle membrane. When the intracellular cAMP concentration increases, PKA of AQP2 vesicles is activated and the 256th serine residue of AQP2 is phosphorylated. This reaction is essential for membrane transport of AQP2. Subsequent to phosphorylation, VAMP2 binds to syntaxin 4 (syn4) and AQP2 vesicles fuse to the cell membrane. As a result, AQP2, which is a water channel, is exposed on the cell surface, and water molecules flow into the cell by osmotic pressure from the tubular lumen. Water molecules that have flowed into the cells are further moved to the vascular cavity side by AQP3 / 4, which is another water channel present in the cell membrane on the vascular cavity side, and the reabsorption of water from the original urine is completed (Reference 1). .

  Nephrogenic diabetes insipidus (NDI) is a tubular disease caused by dysfunction of the collecting duct, which makes it impossible to concentrate urine in response to AVP. This improper reabsorption of water causes a characteristic symptom of dilute polyuria, and patients with nephrogenic diabetes insipidus always complain of thirst and are at risk of dehydration throughout their lives. . There are two types of nephrogenic diabetes insipidus, congenital and acquired. Congenital nephrogenic diabetes insipidus is further divided into two types, ie, a concomitant recessive genotype due to mutations in the V2R gene (hereinafter referred to as “V2R abnormal nephrogenic diabetes insipidus”) and autosomal recessive inheritance due to mutations in the AQP2 gene Type (hereinafter referred to as “APQ2 abnormal type renal diabetes insipidus”) (reference documents 2, 3). The prevalence is 9 for V2R abnormal renal diabetes insipidus and 1 for APQ2 abnormal renal diabetes insipidus, and the number of patients affected by the former is overwhelmingly larger. Acquired nephrogenic diabetes insipidus is caused by certain drugs such as lithium (Li). Currently, there are only limited treatments for congenital nephrogenic diabetes insipidus. For example, control therapy such as a salt-restricted diet, thiazide alone, or a combination of thiazide and a prostaglandin inhibitor is performed. However, these therapies are not sufficient, and the development and establishment of new therapies have been eagerly desired.

For patients with nephrogenic diabetes insipidus, the only way to avoid severe dehydration is to drink enough water. If hydration is not possible, there is a risk of falling into an unconscious state. Under anesthesia, patients with renal diabetes insipidus do not feel thirsty and cannot drink water, so there is a risk in surgical operations for patients with renal diabetes insipidus.
Due to polyuria that cannot be treated, it is difficult to maintain an appropriate water content and electrolyte balance (Reference 4). In Japan, a support group for patients with renal diabetes insipidus was formed, triggered by an accident in which a patient with renal diabetes insipidus who had undergone surgery after a traffic accident fell into a fatal state.

Since kidney tissue has a low mitotic index and a complex structure consisting of glomeruli, tubules, vasculature and stromal tissue, gene transfer into it is difficult. Recently, adenovirus (reference 5), lentivirus (reference 6), liposome (reference 7), electroporation (reference 8), ultrasound (reference) 9) etc. were attempted. However, these methods have been shown to target kidney glomeruli or tubules that are not involved in water reabsorption and have not been shown to successfully treat functional protein deficiency in the kidney in vivo. It cannot be said that it can be applied to patients with insipidus. The only successful example of reports on gene therapy targeting the kidney is that the expression of pathogenic genes (such as inflammatory cytokines) in model animals of glomerulonephritis was suppressed by short reverse complementary nucleotides. It is a report (references 10-12). Patent Document 1 discloses a technique relating to the preparation of a recombinant Sendai virus vector that is promising as a vector for gene therapy. Patent Document 2 describes a method for introducing a gene into the kidney using a recombinant Sendai virus. However, there is no mention of application to nephrogenic diabetes insipidus.
WO 97/16539 pamphlet International Publication No. 02/00264 Pamphlet

  Under the above background, an object of the present invention is to create a new treatment for renal diabetes insipidus.

  In gene therapy for nephrogenic diabetes insipidus, it is necessary to compensate for the function lost by mutations in molecules (V2R or AQP2) involved in water reabsorption. The present inventors selected AQP2 of the water channel as a therapeutic gene because it plays a central role in the final stage of water reabsorption, and decided to examine the therapeutic effect of the introduction in an animal model. First, a recombinant Sendai virus vector carrying the AQP2 gene was prepared. A lithium-induced AQP2 abnormal renal diabetes insipidus animal model (V2R phosphorylation was not abnormal) was injected into the kidney retrogradely from the ureter, and the vector-derived AQP2 was collected into the collecting tube. It was successfully expressed in epithelial cells, and as a result, there was a therapeutic effect that urine volume was reduced by about 40%. On the other hand, the same therapeutic effect was observed when a simian immunodeficiency virus (SIV) vector carrying the AQP2 gene was used.

On the other hand, through oocyte swelling assay, it was revealed that AQP2 overexpression increases without AVP stimulation, and congenital nephrogenic diabetes insipidus (ie, V2R abnormalities) caused by mutations in the V2R gene It was suggested that the introduction of AQP2 gene would be an effective treatment strategy for type diabetic diabetes insipidus. In view of this finding, when the above recombinant vector was administered to a V2R abnormal type renal diabetes insipidus animal model induced by a V2R selective inhibitor, the therapeutic effect was equivalent to that of an AQP2 abnormal type nephrogenic diabetes insipidus animal model. The therapeutic effect was recognized. This was presumed to be the result of membrane transport due to phosphorylation of a part of the overexpressed vector-derived AQP2 by a weak PKA activation mechanism other than V2R. Based on common sense so far, in the case of V2R abnormal renal diabetes insipidus, in which AQP2 phosphorylation does not occur due to V2R disorder, even if AQP2 is overexpressed, movement of AQP2 into the tubular lumen occurs. It was initially expected that no therapeutic effect would be obtained. The above findings by the inventors overturn this expectation and are surprising, and considering the prevalence of V2R abnormal type renal diabetes insipidus, the scope of clinical application of the therapeutic means of introducing AQP2 is greatly increased. It shows that it spreads, and its significance is extremely large.
As described above, introduction of AQP2 gene (forced expression) is a promising therapeutic strategy for diabetes insipidus, and its application target is not limited to AQP2 abnormal type nephrogenic diabetes insipidus (V2R abnormal type nephrogenic diabetes insipidus) And that the Sendai virus vector and the simian immunodeficiency virus vector are effective as a carrier for introducing the AQP2 gene into the renal medullary collecting duct epithelial cells, and the gene As an introductory method, it became clear that the method of injecting into the kidney retrogradely from the ureter was effective.
The present invention has been completed based on these findings, and provides a recombinant vector for the treatment of nephrogenic diabetes insipidus and uses thereof as listed below.
[1] A recombinant vector for treating renal diabetes insipidus, which retains the aquaporin 2 gene.
[2] The recombinant vector according to [1], wherein the vector is a Sendai virus vector or a simian immunodeficiency virus vector.
[3] The recombinant vector according to [2], wherein the Sendai virus vector does not contain one or more genes selected from the group consisting of F gene, HN gene and M gene.
[4] The recombinant vector according to any one of [1] to [3], wherein the aquaporin 2 gene is a human aquaporin 2 gene.
[5] The recombinant vector according to any one of [1] to [4], wherein the renal diabetes insipidus is congenital nephrogenic diabetes insipidus caused by a mutation in a vasopressin receptor gene.
[6] The recombinant vector according to any one of [1] to [5], which is retrogradely injected from the ureter when applied.
[7] A composition for treating renal diabetes insipidus comprising the recombinant vector according to any one of [1] to [6] and a pharmaceutically acceptable medium.
[8] Use of the aquaporin 2 gene for producing a recombinant vector for the treatment of nephrogenic diabetes insipidus.
[9] A method for treating renal diabetes insipidus comprising the step of forcibly expressing the aquaporin 2 gene in renal medullary collecting duct epithelial cells.
[10] The method according to [9], wherein the aquaporin 2 gene is forcibly expressed in the renal medullary collecting duct epithelial cell by delivering a recombinant vector carrying the aquaporin 2 gene to the renal medullary collecting duct epithelial cell. The described treatment.
[11] A recombinant vector carrying the aquaporin 2 gene is retrogradely injected from the ureter to deliver the recombinant vector to the renal medullary collecting duct epithelial cell, and the aquaporin 2 gene is transferred into the renal medullary collecting duct epithelial cell. The treatment according to [9], wherein the treatment is forcibly expressed.

  The first aspect of the present invention relates to a recombinant vector useful for the treatment of nephrogenic diabetes insipidus. “Recombinant vector” refers to a carrier (vector) for transporting a target gene to a target cell and constructed by a genetic engineering technique (gene manipulation). The type of vector is not particularly limited as long as the aquaporin 2 gene can be introduced into the target cell and expressed in the target cell. The “vector” herein includes viral vectors and non-viral vectors. The gene transfer method using a virus vector skillfully utilizes the phenomenon that a virus infects cells, and high gene transfer efficiency can be obtained. Adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, herpes virus vectors, Sendai virus vectors (see, for example, WO 97/16539 pamphlet, WO 02/000264 pamphlet) Simian immunodeficiency virus (SIV) vector (for example, WO 2002/101057 pamphlet; Nakajima T, Nakamaru K, Ido E, Terao K, Hayami M, Hasegawa M. Development of novel simian immunodeficiency virus vectors carrying a dual gene expression system. Hum Gene Ther 11, 1863-1874, 2000 .; Ikeda Y, Goto Y, Yonemitsu Y, Miyazaki M, Sakamoto T, Ishibashi T, Ueda Y, Hasegawa M, Tobimatsu S, Sueishi K. Simian Immunodeficiency Virus -Based Lentiviral Vector For Retinal Gene Transfer: a preclinical safety study in adult rats. ene Therapy 10: 1161-1169, 2003) has been developed.

  Non-viral vectors include liposomes, positively charged liposomes (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84: 7413-7417, 1987), HVJ (Hemagglutinating virus of Japan) -Liposome (Dzau, VJ, Mann, M., Morishita, R. et al., Proc. Natl. Acad. Sci., 93: 11421-11425, 1996, Kaneda, Y., Saeki, Y. & Morishita , R., Molecular Med. Today, 5: 298-303, 1999). The recombinant vector of the present invention may be constructed as such a non-viral vector.

  In adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors, a foreign gene incorporated into the vector is incorporated into the host chromosome, and stable and long-term expression can be expected. Retroviral vectors are not suitable for gene transfer into non-dividing cells because cell division is required for integration of the viral genome into the host chromosome. On the other hand, lentivirus vectors and adeno-associated virus vectors cause integration of foreign genes into the host chromosome after infection even in non-dividing cells. Therefore, these vectors are effective for stably and long-term expressing foreign genes in non-dividing cells.

  Sendai virus vectors have many advantages such as (1) low toxicity, (2) high transgene expression efficiency, and (3) excellent safety because the transgene is not integrated into the host chromosome.

“Monkey immunodeficiency virus (SIV) vector” refers to a vector in which a sequence essential for function as a viral vector among nucleic acid molecules in a virus particle is a sequence based on the SIV genome (International Publication WO2006 / 090689, International Publication) WO2006 / 090697, International Publication WO2007 / 049752). “Sequence essential for the function as a viral vector” means, in order from 5 ′ side, 5 ′ LTR R region, U5 region, packaging signal (φ), RRE, U3 region other than 3 ′ LTR promoter region, R region sequence. The SIV vector may be modified as long as the above definition is applied.For example, as long as the “sequence essential for the function as a viral vector” is derived from SIV, other SIV-derived sequences or SIV Sequences derived from other than may be included. Examples of sequences that can be suitably included include cPPT (central polypurine tract), internal promoter (CMV), internal promoter (CAG), and WPRE (woodchuck hepatitis virus posttranscriptional regulatory element).
The simian immunodeficiency virus vector is a chromosomally integrated vector. The simian immunodeficiency virus vector is characterized in that it does not activate surrounding genes even when it is integrated into a chromosome, and can express two therapeutic genes simultaneously and in a desired ratio.

  Each viral vector can be prepared according to a previously reported method or using a commercially available dedicated kit. For example, an adenovirus vector can be prepared by the COS-TPC method or full-length DNA introduction method. The COS-TPC method is a homologous recombination that occurs in 293 cells by co-transfecting a recombinant cosmid incorporating the target cDNA or expression cassette and a parent virus DNA-terminal protein complex (DNA-TPC) into 293 cells. (Mayake, S., Makimura, M., Kanegae, Y., Harada, S., Takamori, K., Tokuda, C., and Saito, I.) (1996) Proc. Natl. Acad. Sci. USA, 93, 1320.). On the other hand, the full-length DNA introduction method is a method for producing a recombinant adenovirus by subjecting a recombinant cosmid inserted with a target gene to restriction digestion and then transfecting 293 cells (Miho Terashima, Koki Kondo). Hiromi Kanegae, Izumi Saito (2003) Experimental Medicine 21 (7) 931.). The COS-TPC method can be performed using Adenovirus Expression Vector Kit (Dual Version) (Takara Bio Inc.) and Adenovirus genome DNA-TPC (Takara Bio Inc.). The full-length DNA introduction method can be performed using Adenovirus Expression Vector Kit (Dual Version) (Takara Bio Inc.).

  On the other hand, a retroviral vector can be prepared by the following procedure. First, a virus genome (gag, pol, env gene) other than the packaging signal sequence between LTRs (Long Terminal Repeat) existing at both ends of the virus genome is removed, and a target gene is inserted therein. The viral DNA thus constructed is introduced into a packaging cell that constitutively expresses the gag, pol, and env genes. Thereby, only the vector RNA having the packaging signal sequence is incorporated into the viral particle, and a retroviral vector is produced.

  Developed by improving or specificizing fiber protein by modifying adeno vectors (specific infection vectors) and gutted vectors (helper-dependent vectors) that can be expected to improve the expression efficiency of target genes. Has been. The recombinant vector of the present invention may be constructed as such a viral vector.

  Sendai virus vectors have various advantages as described above. Therefore, in a preferred embodiment of the present invention, a recombinant vector is constructed using a Sendai virus vector. As used herein, “Sendai virus vector” refers to a carrier derived from Sendai virus and capable of introducing a desired gene into a target cell. By constructing a Sendai virus vector so as to retain the aquaporin 2 gene, the recombinant Sendai virus vector of the present invention can be obtained. The recombinant Sendai virus vector of the present invention may be in the form of ribonucleoprotein (RNP). Moreover, the form of the virus particle which has infectivity may be sufficient. Here, “infectivity” refers to the ability to introduce a nucleic acid or the like held by a cell into the cell by its ability to adhere to a cell / membrane fusion ability.

  The genome of the native Sendai virus follows the 3 'short leader region, followed by the nucleocapsid (N) gene, phospho (P) gene, matrix (M) gene, fusion (F) gene, hemagglutinin-neuraminidase (HN) gene, And the large (L) gene and the short 5 'trailer region in this order.

  As long as the reconstituted recombinant Sendai virus vector infects the target cell and the aquaporin 2 gene carried by the vector can be expressed in the target cell, the Sendai virus gene as a starting material for producing the recombinant Sendai virus vector Some may be deleted or modified. For example, incomplete viruses such as DI particles (J. Virol. 68: 8413-8417, 1994) can be used.

The recombinant Sendai virus vector preferably has infectivity but lacks transmission ability. In order to make the transmission power lack, it is preferable to delete at least one of the F gene, the HN gene, and the M gene. Such vectors include, for example, the Sendai virus Z strain gene lacking the F gene. As further examples, pSeV18 ⁺ b (+) (Yu, D. et al., Genes to Cells 2: 457-466, 1997) or pSeV (+) (Kato, A. et al., EMBO J. 16: 578-587, 1997) ).

  Recombinant Sendai virus vectors carrying the aquaporin 2 gene are described, for example, by Kato, A. et al. (1997, EMBO J. 16: 578-587) and Yu, D. et al. (1997, Genes Cells 2: 457-466). Based on the description, it can be constructed as follows. First, a DNA sample containing the cDNA base sequence of the aquaporin 2 gene is prepared. It is preferable that the DNA sample can be confirmed as a single plasmid by electrophoresis at a concentration of 25 ng / μl or more. However, if the cDNA sequence contains a NotI recognition site, it is removed in advance. In order to amplify and recover a desired gene fragment from this sample, a NotI restriction enzyme cleavage site sequence; a transcription termination sequence (E), an intervening sequence (I), and a transcription initiation sequence (S) sequence described below; and one of aquaporin 2 As a primer pair including a partial sequence, a forward-side synthetic DNA sequence and a reverse-side synthetic DNA sequence (antisense strand) are prepared.

  For the synthetic DNA sequence on the forward side, select any 2 or more oligo DNAs from the 5 'side, preferably 4 bases that do not contain sequences derived from the NotI recognition site of GCG or GCC, more preferably ACTT, and the 3' side. The NotI recognition site gcggccgc is added to, and any 9 bases as a spacer sequence or a number obtained by adding a multiple of 6 to 9 is added to the 3 ′ side thereof. Further, a sequence corresponding to 25 bases of ORF including the start codons ATG to ATG of the desired cDNA is added to the 3 ′ side. However, the vicinity of 25 bases is selected from the cDNA so that the 3 ′ end of the forward synthetic oligo DNA is either G or C.

  The reverse-side synthetic DNA sequence is selected from the 5 'side to any two or more oligo DNAs, preferably 4 bases that do not contain sequences derived from GCG and GCC NotI recognition sites, more preferably ATCC, and the 3' side. Is added with a NotI recognition site gcggccgc, and an oligo DNA of an inserted fragment for adjusting the length is added to the 3 ′ side thereof. The length of this oligo DNA, including the NotI recognition site gcggccgc, is designed so that the total number of complementary strand bases of cDNA and the number of EIS bases of Sendai virus genome derived from Sendai virus described later is a multiple of 6 (so-called "Rule of six"; Kolakofski, D. et al., J. Virol. 72: 891-899, 1998, Calain, P. and Roux, L., J. Virol. 67: 4822-4830, 1993 ). Complementary strand sequence of Sendai virus S sequence, preferably 5'-CTTTCACCCT-3 ', I sequence, preferably 5'-AAG-3', and E sequence on the 3 'side of the insert, preferably 5'-TTTTTCTTACTACGG-3 ', reverse-side synthetic oligo DNA by adding a complementary sequence such that the end corresponding to 25 bases is either G or C, counting backward from the start codon of the desired cDNA sequence Prepare the 3 'end of.

For PCR, a usual method using ExTaq polymerase (Takara Shuzo) can be used. Vent polymerase (NEB) is preferably used. The amplified fragment of interest is digested with NotI and then inserted into the NotI site of the plasmid vector pBluescript. The base sequence of the obtained PCR product is confirmed with a sequencer, and a plasmid with the correct sequence is selected. This was cut with NotI and Sendai virus genomic cDNA plasmid, such as pSeV18 ⁺ b (+) (Yu, D. et al., Genes to Cells 2: 457-466, 1997) or pSeV (+) (Kato, A. et al. , EMBO J. 16: 578-587, 1997), to obtain a recombinant Sendai virus cDNA in which exogenous cDNA is incorporated. It is also possible to obtain a recombinant Sendai virus cDNA by directly inserting into the NotI site without using the plasmid vector pBluescript.

  A recombinant Sendai virus vector can be obtained by transcribing the recombinant Sendai virus cDNA produced as described above in vitro or in a cell to reconstitute the virus. Reconstitution of virus from cDNA can be performed using a known method (see, for example, WO 97/16538 pamphlet and WO 97/16539 pamphlet).

Reconstitution from cDNA can be performed, for example, as follows. First, a monkey kidney-derived cell line using 6% fetal bovine serum (FCS) and minimal essential medium (MEM) containing antibiotics (100 units / ml penicillin G and 100 μg / ml streptomycin) on a 6-well plastic plate Culture LLCMK2 until it is 70-80% confluent (1 × 10 ⁶ cells). Recombinant vaccinia virus vTF7-3 inactivated by UV irradiation expressing T7 polymerase (Fuerst, TR et al., Proc. Natl. Acad. Sci. USA 83: 8122-8126, 1986, Kato, A. et al. , Genes Cells 1: 569-579, 1996) are infected with 2 PFU / cell. One hour after infection, 2-60 μg, more preferably 3-5 μg of the above recombinant Sendai virus cDNA is transformed into a plasmid (0.5-24 μg of pGEM expressing a viral protein acting in trans essential for the generation of the full-length Sendai virus genome. -N, 0.25-12 μg pGEM-P, and 0.5-24 μg pGEM-L, more preferably 1 μg pGEM-N, 0.5 μg pGEM-P, and 1 μg pGEM-L) (Kato, A. et al., Genes Cells 1: 569-579, 1996) and transfection by a transfection method such as a lipofection method using Superfect (QIAGEN). Transfected cells are treated with serum-free MEM containing 100 μg / ml rifampicin (Sigma) and cytosine arabinoside (AraC), more preferably 40 μg / ml cytosine arabinoside (AraC) (Sigma). Incubate and set the optimal concentration of drug to minimize cytotoxicity by vaccinia virus and maximize virus recovery (Kato, A. et al., Genes Cells 1: 569-579, 1996). Forty-eight hours after transfection, the cells are collected, and freeze-thaw is repeated three times to disrupt the cells. Thereafter, the cells are inoculated into the chorioallantoic membrane of 10-day-old embryonated chicken eggs. After 3 days, chorioallantoic fluid is collected and the virus titer is determined by measuring hemagglutination activity (HA). HA can be determined by the “endo-point dilution method” (Kato, A. et al., Genes Cells 1: 569-579, 1996). For samples for which HA has not been confirmed, further inoculate the eggs of the growing chicken. The titer of the collected Sendai virus is usually 10 ⁸ to 10 ⁹ PFU / ml, and the vaccinia virus vTF7-3 contained together is usually 10 ³ to 10 ⁴ PFU / ml or less. The sample is diluted 10 ⁶ times and reamplified with chicken eggs to remove vaccinia virus. The recombinant virus obtained by the passage in the second or third breeding chicken egg is preserved to obtain a recombinant Sendai virus vector in which the desired cDNA is incorporated. The plaque-forming ability of a stored virus generally has a titer of 10 ⁹ PFU / ml or 10,240 HA unit / ml, and it is considered that the titer is maintained when the virus is stored at −80 ° C.

  As long as the recombinant Sendai virus cDNA is reconstituted within the cell, the host cell used for reconstitution is not particularly limited. In addition to LLCMK2 cells and human-derived cells, cultured cells such as monkey kidney-derived CV-1 cells and hamster kidney-derived BHK cells can be used as the cell line used as the host.

  The reconstituted recombinant Sendai virus may have an adhesion factor, a ligand, a receptor, etc. bound to the envelope surface so that it can adhere to specific cells.

The recombinant vector of the present invention holds the aquaporin 2 gene as a transgene. Usually, the species of the aquaporin 2 gene is selected according to the species to be treated. For example, when a human is a treatment target, the human aquaporin 2 gene may be selected. The human aquaporin 2 gene sequence and other information are registered in a public database and can be easily obtained. For example, the human aquaporin 2 gene is registered with GeneID: 359 in the NCBI (National Center for Biotechnology Information) database. Similarly, the sequence of the human aquaporin 2 gene is registered under ACCESSION: NM_000486 (DEFINITION Homo sapiens aquaporin 2 (collecting duct) (AQP2), mRNA.), And the amino acid sequence of human aquaporin 2 is ACCESSION: NP_000477 (DEFINITION aquaporin 2 [Homo sapiens].). In addition to natural genes, genes modified by deletion, substitution, insertion, addition, etc. of part of the nucleic acid sequence (however, it is necessary to encode a protein having the same function as the protein encoded by the natural gene) Can also be used.
Examples of the sequence of the aquaporin 2 gene retained by the recombinant vector of the present invention are shown in SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing.

By combining the recombinant vector of the present invention with a pharmaceutically acceptable medium, a composition for treating nephrogenic diabetes insipidus can be constructed. “Pharmaceutically acceptable vehicle” refers to advantages or benefits relating to administration or storage of a recombinant vector without substantially affecting the efficacy of the recombinant vector (ie, therapeutic effect on nephrogenic diabetes insipidus). The substance to bring. Examples of the “pharmaceutically acceptable medium” include deionized water, ultrapure water, physiological saline, phosphate buffered saline (PBS), and 5% dextrose aqueous solution. The composition of the present invention may contain other components such as a suspending agent, a soothing agent, a stabilizer (such as albumin or (Prionex (registered trademark), Penta Farm Japan)), a preservative, and an antiseptic. . The composition of the present invention can also be prepared as a lyophilized product.
When the recombinant vector of the present invention is in the form of a viral vector, it is preferable to use a biocompatible polyol (for example, poloxamer 407) in combination. The use of polyols can increase the transduction rate of viral vectors 10 to 100 times (March et al., Human Gene Therapy 6: 41-53, 1995). Therefore, if a polyol is used in combination, the dose of the viral vector can be kept low. In addition, you may decide to use a polyol as one component of the composition of this invention, or to prepare a polyol (or composition containing it) separately from the composition of this invention. In the latter case, when the composition of the present invention is administered, polyol (or a composition containing the same) is administered together.

  The recombinant vector of the present invention is administered to a subject (patient) suffering from nephrogenic diabetes insipidus for the purpose of treating nephrogenic diabetes insipidus. It is also possible to administer the recombinant vector of the present invention to a patient (potential patient) before being diagnosed with nephrogenic diabetes insipidus. In other words, the present invention may be used for preventive medical measures.

“Renal diabetes insipidus” is a tubular disease caused by dysfunction of the collecting duct, which makes it impossible to concentrate urine in response to arginine vasopressin (AVP). Renal diabetes insipidus is broadly classified into congenital nephrogenic diabetes insipidus caused by a specific gene mutation and acquired nephrogenic diabetes insipidus caused by drug use. Congenital nephrogenic diabetes insipidus is further divided into two types depending on the causative gene: concomitant recessive genotype due to mutation of V2R gene (V2R abnormal nephrogenic diabetes insipidus) and autosomal recessive genotype due to mutation of AQP2 gene Classified as (APQ2 Abnormal Renal Insipidus). The recombinant vector of the present invention is effective for both V2R abnormal nephrogenic diabetes insipidus and APQ2 abnormal nephrogenic diabetes insipidus.
Subjects to which the recombinant vector of the present invention is administered include humans or non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, Goats, sheep, dogs, cats, etc.). Preferably, the recombinant vector of the present invention is used for humans.

The administration method of the recombinant vector of the present invention is not particularly limited as long as the recombinant vector can be delivered to the target medullary collecting duct epithelial cells. In selecting the administration method, the form of the recombinant vector is considered. For example, if it is in the form of a viral vector, it is administered so that efficient infection to the target cells is achieved.
Although the recombinant vector of the present invention is administered to a subject by systemic administration or local administration, it is preferable to adopt the latter administration method in order to achieve efficient delivery and reduce side effects. For example, the recombinant vector may be delivered by local administration such as intrarenal, intraureteral, intraperitoneal, or intraparenchymal injection (Tomita, N. et al., Biochem. Biophys. Res. Commun. 186). : 129-134, 1992; Isaka, Y et., J. Clin. Invest. 92: 2597-2601; Arai, M. et al., Biochem. Biophys. Res. Commun. 206: 525-532, 1995; Heikkila , P. et al., Gene Ther. 3: 21-27, 1996; Rappaport, J. et al., Kidney Int. 47: 1462-1469, 1995; Oberbauer, R. et al., Kidney Int. 48: 1226-1232, 1995; Haller, H. et al., Kidney Int. 50: 473-480, 1996; Lien, Y. et al., Exp. Nephrol. 5: 132-136; Moullier, P. et al. , Kidney Int. 45: 1220-1225, 1994; Bosch, R. et al., Exp. Nephrol. 1: 49-54, 1993).

Among them, it is preferable to employ a method of injecting the recombinant vector retrogradely from the ureter. According to this method, it is possible to efficiently introduce a gene into the renal medullary collecting duct epithelial cell by a relatively simple operation. Therefore, it is possible to increase the therapeutic effect while reducing the burden on the patient. As shown in the examples below, according to the method of injecting the recombinant vector retrogradely from the ureter, the recombinant vector is efficiently delivered to the renal medullary collecting duct epithelial cells, and a good therapeutic effect is obtained. It was actually confirmed to play.
In order to inject the recombinant vector retrogradely from the ureter, a syringe or catheter may be inserted into the ureter and the recombinant vector injected into the kidney. In addition, it is good to clamp a ureter for several minutes (for example, about 3 to 10 minutes) after injection | pouring of a recombinant vector.

The dosage of the recombinant vector of the present invention is set so that a therapeutically effective amount of the aquaporin 2 gene is introduced into the renal medullary collecting duct cells. “Therapeutically effective amount” refers to an amount capable of producing a therapeutic effect (amelioration of symptoms, cure, etc.) by the aquaporin 2 gene. The dose of the recombinant vector of the present invention generally varies depending on the form of the recombinant vector, the route of administration, the symptom of the subject to be treated, age, sex, body weight, etc., but those skilled in the art will appropriately set an appropriate dose. Is possible. The dose of the recombinant vector may be set so that the amount of aquaporin 2 gene administered to an adult (body weight of about 60 kg) per administration is, for example, 40 to 400 μg, preferably 200 to 400 μg. In addition, when the recombinant vector of the present invention is constructed using a viral vector, the amount of viral vector administered per time for an adult (body weight of about 60 kg) is, for example, 2.0 × 10 ^{8 to} 2.0. The dose of the recombinant vector may be set to x 10 ⁹ CIU (cell-infectious units) / ml, preferably 1.0 x 10 ^{9 to} 2.0 x 10 ⁹ CIU / ml.
As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the symptom, sex, age, disease state, etc. of the subject (recipient) can be considered.

1. Effect of APQ2 gene transfer on APQ2 abnormal renal diabetes insipidus model
The effect of AQP2 gene transfer on APQ2 abnormal renal diabetes insipidus was examined using an APQ2 abnormal renal diabetes insipidus model induced by Li (Li phagocytic diabetes insipidus rat).

(1) Materials and Methods (a) Construction of human aquaporin 2 (AQP2) cDNA containing FLAG Normal human kidney tissue surrounding kidney cancer was collected from a surgically excised sample. The experimental protocol was approved by the Ethics Committee of Nagoya University Graduate School of Medicine (protocol number 05-76). Total RNA was prepared from the above tissue using Trizol reagent (Gibco). A first strand cDNA library was synthesized from human kidney RNA according to the protocol attached to the product.
Primer set specific to human aquaporin 2 (AQP2) gene (sense primer (including FLAG epitope tag): 5'-CTACCATGGACTACAAAGACGATGACGACAAGGAATTCATGTGGGAGCTCCGCTCCATAGCCTTC-3 '(SEQ ID NO: 1), antisense primer: 5'-CCTCTAGACTCGAGCGGCCGCC-3' ( Following amplification by PCR using SEQ ID NO: 2)), the PCR amplification product was subcloned into a pcDNA3.1 / V5-His-TOPO vector (Invitrogen). The sequence of the construct was examined by DNA sequencing service provider (Macrogen), and it was confirmed that the sequence of human aquaporin 2 (SEQ ID NO: 5) was included.

(B) Vector construction (AQP2-SeV / ΔF, GFP-SeV / ΔF)
A human AQP2 expression vector (AQP2-SeV / ΔF) and a GFP expression vector (GFP-SeV / ΔF) (FIG. 1A) containing a FLAG tag were constructed by a previously reported method (reference document 13). Briefly, a primer containing a FLAG sequence was synthesized, and human AQP2 cDNA was amplified by PCR to produce an AQP2 cDNA fragment with a FLAG sequence added to the N-terminus. Similarly, the GFP sequence was amplified by PCR. These DNA fragments were joined to an expression vector SeV / ΔF cleaved at a predetermined location using a restriction enzyme to obtain each expression vector (FIG. 1A).

On the other hand, two SIV pseudo-type human AQP2 expression vectors (SIV-FLAG-hAQP2-VSVG, SIV-FLAG-hAQP2-F / HN) and corresponding control vectors (SIV-EGFP-VSVG, SIV-EGFP-F / HN) was produced (FIG. 1B).
“SIV-FLAG-hAQP2-VSVG” is pseudotyped with VSV-G protein, which is a surface glycoprotein of vesicular stomatitis virus (VSV) (International Publication WO2002 / 101057, International Publication WO2006 / 090689, International Publication WO2006 / [090697] is a SIV vector containing human-derived AQP2 to which FLAG has been added.
"SIV-FLAG-hAQP2-F / HN" was produced by pseudotyping with Sendai virus envelope glycoprotein F and HN proteins (International Publication WO2001 / 92508, International Publication WO2007 / 049752), An SIV vector containing human-derived AQP2 to which FLAG is added.
“SIV-EGFP-VSVG” refers to an SIV vector containing a marker gene EGFP, which is prepared by pseudotyping with VSV-G protein, which is a surface glycoprotein of vesicular stomatitis virus (VSV).
“SIV-EGFP-F / HN” is a SIV containing the marker gene EGFP produced by pseudotyping with Sendai virus envelope glycoprotein F and HN proteins (International Publication WO2007 / 049752) A vector.
In addition, although the form shown in a present Example is a form using the human origin AQP2 (FLAG-hAQP2) to which the flag was added, it is thought that AQP2 to which FLAG is not added shows the same effect, for example.

Transfection method using the above four SIV vectors ("SIV-FLAG-hAQP2-VSVG", "SIV-FLAG-hAQP2-F / HN", "SIV-EGFP-VSVG", "SIV-EGFP-F / HN") (Masanori Kobayashi et al., J. Virol. 2003 77: 2607-2614).
The following mixture (FIG. 1B) was administered (transfected) to 293T / 17 cells (ATCC (URL: http://www.atcc.org/) CRL-11268) seeded in a 15 cm culture dish.
<Mixture for making “SIV-FLAG-hAQP2-VSVG”>
A mixture containing 10 μg of the vector described in FIG. 1B (2), 3 μg of the vector described in FIG. 1B (3), and 2 μg of the plasmid described in FIG. 1B (6) <mixture for producing “SIV-FLAG-hAQP2-F / HN” >
A mixture containing 10 μg of the vector described in FIG. 1B (2), 3 μg of the vector described in FIG. 1B (3), 2 μg of the plasmid described in FIG. 1B (4), and 2 μg of the plasmid described in FIG. 1B (5) <“SIV-EGFP-VSVG” Mixture for making
A mixture containing 10 μg of the vector described in FIG. 1B (1), 3 μg of the vector described in FIG. 1B (3), and 2 μg of the plasmid described in FIG. 1B (6) <mixture for producing “SIV-EGFP-F / HN”>
A mixture containing 10 μg of the vector described in FIG. 1B (1), 3 μg of the vector described in FIG. 1B (3), 2 μg of the plasmid described in FIG. 1B (4), and 2 μg of the plasmid described in FIG. 1B (5)

  After the administration of the above mixture, the cells were cultured for 2 days. After culture, the supernatant is recovered, and SIV vectors (VSVG-SIV-EGFP, F / HN-SIV-EGFP, VSVG-SIV-FLAG-hAQP2, F / HN-SIV-FLAG-hAQP2) are purified from the supernatant. did.

(C) Animal and surgical procedures
Since introduction of mutations into the V2R gene or AQP2 gene causes embryonic lethality, we decided to study using an animal model of nephrogenic diabetes insipidus induced by Li. Li causes severe renal diabetes insipidus mainly through deficiency of AQP2 in the collecting duct. Therefore, this drug induction model is preferable as an animal model for evaluating the therapeutic effect of AQP2 gene introduction (Reference Document 14).
Male SD (Sprague-Dawley) rats (10 weeks old, 250-300 g body weight, Chubu Scientific Materials Co., Ltd.) are metabolized one by one under certain conditions (23.0 ± 0.5 ° C, light period: 9 am to 9 pm) Raised in cages. All animal experiments were approved by the Ethics Committee of Nagoya University Graduate School of Medicine (Protocol No. 05-76). On the 14th day, the right renal artery and renal vein and ureter were tied and the right kidney was removed under sufficient anesthesia by intraperitoneal administration of pentobarbital (50 mg / kg). After the operation, lithium chloride was mixed with food (Li food) and reared. In accordance with the previous report (Reference 15), the lithium chloride concentration was 40 mg / kg (per dry weight of food) in the first week, and the lithium chloride concentration was 60 mg / kg (per dry weight of food) in the second week. ). Breeding in metabolic cages 3 days before gene transfer, urine volume, water consumption, food intake and body weight were monitored. Ten days after starting feeding on the Li diet, the rats showed polyuria and polyposis symptoms typical of drug-induced nephrogenic diabetes insipidus (Li-phagocytic diabetes insipidus rats). On the other hand, immunostaining for AQP2 was completely suppressed in the kidney (FIG. 13c).
On day 0, the rat was anesthetized and a longitudinal incision was made in the abdomen. A 30-gauge injection needle (BD Bioscience) was punctured into the left ureter, and 200 μl of AQP2-SeV / ΔF vector (1.4 × 10 ⁹ CIU / ml) or control GFP-SeV / ΔF vector (1.4 with phosphate buffer) × 10 ⁹ CIU / ml) was injected retrogradely from the left ureter toward the renal pelvis. After injection, the left ureter was clamped for 5 minutes. After spraying antibiotics into the abdominal cavity, the abdominal wall and skin were sutured.
On the other hand, a similar experiment was performed using an SIV pseudo-type human AQP2 expression vector. In addition, 0.2 ml of each vector having the following virus titer was injected per rat.
SIV-EGFP-VSVG: 2.8x10 ⁹ TU / ml
SIV-FLAG-hAQP2-VSVG: 2.3x10 ⁹ TU / ml
SIV-EGFP-F / HN: 3.2x10 ⁸ TU / ml
SIV-FLAG-hAQP2-F / HN: 1.7x10 ⁷ TU / ml

(D) Immunohistochemistry For immunohistochemical analysis, isolated kidney tissue sections were fixed with 4% formaldehyde and then embedded in paraffin. 10 μm thick sections were deparaffinized with xylene and then hydrated. In order to suppress endogenous peroxidase activity, the sections were treated with methanol containing 0.3% H ₂ O ₂ for 30 minutes. After the sample prepared in this way was blocked with bovine serum albumin or normal horse serum, anti-AQP2 antibody (Sigma) or affinity purified anti-FLAG antibody (Sigma) was added as a primary antibody and incubated overnight. Next, the sample was incubated with a predetermined secondary antibody (anti-rabbit or anti-mouse IgG) for 2 hours. The sample after this reaction was visualized with Vectastain ABC Kit (Vector Laboratories) (substrate was diaminobenzidine) and fixed. After rinsing, it was stained with Meyer's hematoxylin (Sigma). After hydration, it was sampled with Permount (Fisher Scientific).

(E) Western blot Kidney samples were lysed with Laemmli buffer. For standardization, the total protein amount was measured using a commercially available protein assay system (BD Bioscience). Urine samples were concentrated by dialysis and normalized (urine volume / day). After boiling for 5 minutes, the sample was subjected to 15% SDS-PAGE and subsequently transferred to Immobilon-P membrane (Millipore). After blocking with Tris buffer containing 5% skim milk and 0.1% TWEEN for 1 hour, the transferred membrane was treated with anti-AQP2 antibody (Sigma) or anti-FLAG antibody (Sigma). The treated membrane was incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody, and then visualized by a chemiluminescence method using ECL-Plus (Amersham Pharmacia Biotech).

(F) Statistical analysis The experimental results were expressed as mean ± standard error. Statistical significance of the differences between the two groups was assessed by Student's t-test. There was a significant difference when P <0.05.

(2) Results APQ2 gene was used as a transgene because it plays a central role in the final stage of water reabsorption. As the carrier, we decided to use recombinant Sendai virus (SeV). This is because SeV can infect most mammalian cells and replicate its genome. In addition, there is no risk of SeV integration into the host genome because its replication is localized in the cytoplasm and occurs independently of nuclear function. Therefore, SeV vectors do not show any genotoxicity. The SeV vector loses its transmission ability by deleting the fusion protein (F) gene, which is a glycoprotein inserted into the viral envelope and is essential for the vector genome to reach the cytoplasm through cell fusion (references) 13). Sendai virus vectors lacking the F gene and trans-fed with the envelope F protein show high transfer efficiency and transient infection.

For treatment of nephrogenic diabetes insipidus, a recombinant Sendai virus vector (APQ2-SeV / ΔF) carrying the human AQP2 gene was prepared (FIG. 1A). A FLAG epitope tag was fused to the N-terminus of the human APQ2 gene for use in later evaluation (FIG. 12). The titer of AQP2-SeV / ΔF was 1.4 × 10 ⁹ CIU / ml.

  As the administration route, retrograde injection via the ureter was adopted so that the vector penetrated into the renal medullary collecting duct via the renal pelvis. Although administration through the renal artery or renal vein is possible, the administration route was not examined in consideration of the risk of the vector penetrating throughout the body. The administration method for retrograde injection is particularly invasive to humans because it is minimally invasive and can be performed using a ureterocystoscope, which is a routine procedure.

  Prior to the evaluation of the therapeutic effect by forced expression of AQP2, the control GFP-SeV / ΔF vector was retrogradely injected into the renal pelvis of control rats and Li-eating rats. In cross-sections of the inner medulla of rats treated in this way, more than 50% of the collecting ducts expressed GFP (FIG. 13a). GFP expression was confined to the epithelium of the collecting duct, and GFP expression was not observed in tubule cells. In addition, GFP expression was not observed in other parts of nephron, outer medulla, and renal cortex. From these results, it was shown that efficient expression of the transgene was achieved and that the expression was limited to the region in direct contact with the vector. In addition, expression of GFP was not observed in other tissues (liver, spleen, heart, lung, intestine, etc.) (results not shown).

  Based on the above results, the therapeutic effect of the injection of the AQP2-SeV / ΔF vector was examined using the GFP-SeV / ΔF vector as a control. On the 4th to 8th days, the rats injected with the AQP2-SeV / ΔF vector showed a significant decrease (about 40% decrease) in urine volume compared to the control (FIG. 3). Moreover, the amount of drinking water also showed the same tendency (FIG. 4). The amount of food intake and body weight were not different from the control (FIGS. 5 and 6).

  The urine osmotic pressure on day 5 of rats injected with the AQP2-SeV / ΔF vector was significantly higher than that of the control (FIG. 7).

  As a result of immunostaining the kidney tissue on day 5 after the treatment, many AQP2-positive and FLAG-positive cells were observed in the collecting tube of the rat injected with the AQP2-SeV / ΔF vector (FIGS. 8 and 9). These results coincided with the results of Western blotting using renal lysates and urine samples on day 5 (FIGS. 10 and 11). The expression level of endogenous AQP2 was significantly reduced by ingestion of Li and recovered by injection of AQP2-SeV / ΔF vector (FIGS. 10 and 11). In the rats injected with the AQP2-SeV / ΔF vector, the AQP2 positive band slightly shifted upward was also FLAG positive (FIGS. 10 and 11). This suggests that the band is derived from the AQP2-SeV / ΔF vector. These results show that forced AQP2 expression with the AQP2-SeV / ΔF vector compensates for water reabsorption in the renal medulla of Li-induced diabetes insipidus rats, thereby concentrating urine, reducing urine output, and food intake It strongly suggests that the decrease in To the best of our knowledge, this is the first successful case where the function of a defective gene can be restored by gene therapy targeting the kidney collecting duct.

The results of experiments using SIV pseudo-type vectors are shown in FIGS. The therapeutic vector SIV-FLAG-hAQP2-VSVG or the control vector SIV-EGFP-VSVG was administered retrogradely to 2 mice in each group of Li-induced diabetes insipidus model rats, reared in a metabolic cage, and urine volume was measured. The change in urine volume was evaluated based on the average of urine volume on the 2nd day before injection (100%). When the therapeutic vector was administered, the urine volume decreased from 40% to 60% of the control group from the 4th day, showing a significant therapeutic effect (FIG. 19). Moreover, as a result of measuring the amount of drinking water, when the therapeutic vector was administered, the amount of drinking water decreased from 40% to 60% of the control group from the 4th day, and showed a significant therapeutic effect (FIG. 20). In addition, the change of the amount of drinking water was evaluated on the basis of the average of the amount of drinking water on the 2nd before injection (100%).
On the other hand, the therapeutic vector SIV-FLAG-hAQP2-F / HN or the control vector SIV-EGFP-F / HN was administered ureterally retrogradely to each group of 2 Li-induced diabetes insipidus model rats, reared in normal cages, The amount of drinking water was measured. Changes in the amount of drinking water were evaluated based on the average amount of drinking water on the 2nd day before injection (100%). In the treatment vector, the amount of drinking water decreased from 40% to 60% of the control group from around 4th, and showed a significant therapeutic effect.
As described above, a clear therapeutic effect was also observed when the SIV pseudo-type therapeutic vector was used. This confirms that the water reabsorption function is supplemented by forced expression of the AQP2 gene, and as a result, a therapeutic effect is exerted.

  The technique adopted in the above experiments is promising as a means of repairing the fatal imbalance of fluid homeostasis in patients with renal diabetes insipidus. In particular, it can be said that this is an effective means for avoiding the risk of nephrogenic diabetes insipidus patients becoming fatal during the operation.

2. Oocyte Swelling Assay (1) Materials and Methods (a) Construction and Transfection of Expression Construct (FLAG-hAQP2) Capped human aquaporin 2 (hAPQ2) cRNA was transformed into pXβG plasmid (Tohoku University Graduate School of Engineering) using T3 RNA polymerase. The expression construct pXβG-ev1 was obtained. 10 ng of the synthetic cRNA was injected into Xenopus oocytes from which follicles had been removed (test group). Distilled water was injected into the control group instead of the synthetic cRNA. The oocyte after the injection operation was incubated at 18 ° C. for 3 days in a modified Barth's solution of 200 mOsm / l.

(B) Measurement of water permeability In order to measure water permeability, an oocyte swelling assay was performed. First, the oocyte after the introduction operation was transferred into a modified Barth's solution diluted to 70 mOsm / l with distilled water. The change in oocyte volume over time at room temperature was measured with a video microscope. The relative volume (V / V ₀ ) was calculated from the volume at the start of measurement (A ₀ ) and the volume after the predetermined time (A _t ) (relative volume (V / V ₀ ) = (A ₀ / A _t ) ^{3 / 2} ). Initial gradient (d (V / V ₀ ) / dt), initial oocyte volume (V ₀ = 9 × 10 ^-4 cm ³ ), initial oocyte area (S = 0.045 cm ² ), and water Permeability coefficient according to the formula using molar volume (V _w = 18 cm ³ / mol): Pf = (V ₀ × d (V / V ₀ ) / dt) / (S × V _w × (Osm _in -Osm _out )) (Pf) was calculated.

(2) Results
In the oocytes into which the hAQP2 gene was introduced, rapid swelling was observed (upper part of FIG. 14a). When the hydraulic conductivity was compared with the control group, a significant difference (p <0.0001) was observed (FIG. 14b). Thus, it was shown that water permeability is enhanced by overexpression of AQP2 even without stimulation by AVP. This result suggests that the AQP2-SeV / ΔF vector can also be applied to nephrogenic diabetes insipidus (V2R abnormal type nephrogenic diabetes insipidus) caused by mutation of the V2R gene.

3. Effect of APQ2 gene introduction on V2R abnormal type renal diabetes insipidus model As a result of egg cell swelling assay, it was suggested that introduction of AQP2 gene is also effective for V2R abnormal type renal diabetes insipidus. Therefore, V2R abnormal type renal diabetes insipidus induced by OPC-31260 (Mozabaptan hydrochloride, Otsuka Pharmaceutical Co., Ltd.), a V2R selective inhibitor, was introduced to the effect of AQP2 gene transfer on V2R abnormal type nephrogenic diabetes insipidus. We decided to investigate using a model (OPC food nephrogenic diabetes insipidus rat).

(1) Material and method (a) Expression vector AQP2 expression vector (AQP2-SeV / ΔF) and GFP expression vector (GFP-SeV / ΔF) prepared by the above method (see 1. (1) (b)) It was used.

(B) Animal and surgical treatment, statistical analysis Except for using a diet mixed with OPC-31260 (0.05%) instead of using a diet mixed with lithium chloride, “APQ2 abnormal type renal diabetes insipidus model” By the same procedure as the experiment on “the effect of introducing APQ2 gene”, a V2R abnormal type renal diabetes insipidus model (OPC phagocytic diabetes insipidus rat) induced by OPC-31260 was obtained (above 1. (1). (See (c)). The injection operation of the recombinant Sendai virus vector and other treatments were the same. The experimental results were expressed as mean ± standard error. Statistical significance of the differences between the two groups was assessed by Student's t-test. There was a significant difference when P <0.05.

(2) Results
Using the GFP-SeV / ΔF vector as a control, the physiological effect of the injection of the AQP2-SeV / ΔF vector was examined. On the 4th to 8th days, the rats injected with the AQP2-SeV / ΔF vector showed a significant decrease (approximately 40% decrease) in urine volume compared to the control (FIG. 15). Moreover, the amount of drinking water also showed the same tendency (FIG. 16). The amount of food intake and body weight were not different from the controls (FIGS. 17 and 18).

  As described above, even in the V2R abnormal type nephrogenic diabetes insipidus model induced by the V2R selective inhibitor, a therapeutic effect equivalent to the therapeutic effect on the AQP2 abnormal type nephrogenic diabetes insipidus model was recognized. This was presumably because part of the overexpressed vector-derived AQP2 was phosphorylated by a weak PKA activation mechanism other than V2R, resulting in membrane transport. In V2R abnormal nephrogenic diabetes insipidus, in which AQP2 phosphorylation does not occur due to V2R injury, it was initially expected that AQP2 would not migrate to the tubular lumen even if AQP2 was overexpressed, Contrary to this expectation, the surprising finding that introduction of AQP2 is effective for V2R abnormal renal diabetes insipidus was obtained.

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  The recombinant vector and composition of the present invention are used for the treatment of nephrogenic diabetes insipidus. Currently, there is no effective drug treatment for polyuria of nephrogenic diabetes insipidus. Therefore, the patient is constantly showing polyuria, and in order to prevent dehydration symptoms, it is necessary to constantly replenish water. In particular, in general anesthesia during surgery, water control is very difficult and may have serious outcomes. If the recombinant vector or composition of the present invention is used, AQP2 can be transiently expressed locally in the kidney, and perioperative polyuria can be suppressed, and safe body fluid management can be achieved.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Composition of recombinant Sendai virus vectors AQP2-SeV / ΔF and GFP-SeV / ΔF (bottom). The upper row shows the structure of the wild type Sev vector. The human aquaporin 2 (hAQP2) gene or GFP gene fused with the FLAG epitope tag at the N-terminus was introduced into the 3 ′ vicinity region of the SeV / ΔF vector lacking the propagation ability due to the deletion of the F gene. NP represents a nucleocapsid protein gene, P represents a phosphoprotein gene, M represents a matrix protein gene, HN represents a hemagglutinin neuraminidase gene, and L represents a large protein gene. The structure of the material used to construct the SIV vector used in the experiment was shown. (1) In order from the 5 ′ side, the gene encoding the promoter (CMV), the gene encoding the R region of the 5 ′ LTR, the gene encoding the U5 region, the gene encoding the packaging signal (φ), and the RRE encoding , Gene encoding cPPT, gene encoding internal promoter (CMV), gene encoding EGFP, gene encoding WPRE, gene encoding U3 region other than 3'LTR promoter region, encoding R region A vector containing the gene to be (2) In order from the 5 ′ side, a gene encoding a promoter (CMV), a gene encoding the R region of 5′LTR, a gene encoding the U5 region, a gene encoding RRE, a gene encoding cPPT, an internal promoter This is a vector containing a gene encoding (CMV), a gene encoding FLAG-hAQP2, a gene encoding WPRE, a gene encoding a U3 region other than the 3′LTR promoter region, and a gene encoding an R region. (3) A gene containing a gene encoding a promoter (CAG), a gene encoding gag, a gene encoding pol, a gene encoding Rev, a gene encoding RRE, and a polyA signal in this order from the 5 ′ side. . (4) A gene containing a gene encoding a promoter (CAG), a gene encoding an F protein, and a polyA signal in this order from the 5 'side. (5) A plasmid containing a gene encoding a promoter (CAG), a gene encoding an HN protein, and a polyA signal in this order from the 5 'side. (6) A plasmid containing a gene encoding a promoter (CAG), a gene encoding VSVG protein, and a polyA signal in this order from the 5 'side. Route of administration of recombinant Sendai virus vector. The recombinant Sendai virus vector was retrogradely injected into the ureter (i). The injected recombinant viral vector reaches the renal pelvis (ii) and penetrates the collecting duct in the renal medulla (iii) covered by the renal cortex (iv). The therapeutic effect by retrograde administration of AQP2-SeV / ΔF via the ureter. GFP-SeV / ΔF (◯) or AQP2-SeV / ΔF (●) was administered to Li-phagocytic diabetes insipidus rats (day 0), and urine volume was measured over time. The measurement result was expressed as an average value ± standard error. The amount of urine in rats administered with AQP2-SeV / ΔF is significantly lower over several days than in rats administered with GFP-SeV / ΔF. * P <0.05 (t test) The therapeutic effect by retrograde administration of AQP2-SeV / ΔF via the ureter. GFP-SeV / ΔF (◯) or AQP2-SeV / ΔF (●) was administered to Li-phagocytic diabetes insipidus rats (day 0), and the amount of water consumed was measured over time. The measurement result was expressed as an average value ± standard error. The amount of water consumed by rats administered with AQP2-SeV / ΔF is significantly lower over several days than that of rats administered with GFP-SeV / ΔF. * P <0.05 (t test) The therapeutic effect by retrograde administration of AQP2-SeV / ΔF via the ureter. GFP-SeV / ΔF (◯) or AQP2-SeV / ΔF (●) was administered to Li-nephrogenic diabetes insipidus rats (day 0), and the amount of food intake was measured over time. The measurement result was expressed as an average value ± standard error. There is no significant difference in food intake between rats administered with AQP2-SeV / ΔF and rats administered with GFP-SeV / ΔF. The therapeutic effect by retrograde administration of AQP2-SeV / ΔF via the ureter. GFP-SeV / ΔF (◯) or AQP2-SeV / ΔF (●) was administered to Li-phagocytic diabetes insipidus rats (day 0), and body weight was measured over time. The measurement result was expressed as an average value ± standard error. There is no significant difference in body weight between rats administered with AQP2-SeV / ΔF and rats administered with GFP-SeV / ΔF. The therapeutic effect by retrograde administration of AQP2-SeV / ΔF via the ureter. The urine osmotic pressure (mean ± standard error, n = 5) on day 5 of rats administered with AQP2-SeV / ΔF is significantly higher than that of rats administered with GFP-SeV / ΔF (control). * P = 0.0109 (t-test) Immunohistochemical staining on day 5 after administration of recombinant Sendai virus vector. Anti-APQ2 antibody was used for staining. Stained image of rats administered with GFP-SeV / ΔF (left) and stained image of rats administered with AQP2-SeV / ΔF (right). It can be seen that AQP2 is expressed in rats administered with AQP2-SeV / ΔF. The scale bar is 200 μm. Immunohistochemical staining on day 5 after administration of recombinant Sendai virus vector. Anti-FLAG antibody was used for staining. Stained image of rats administered with GFP-SeV / ΔF (left) and stained image of rats administered with AQP2-SeV / ΔF (right). It can be seen that FLAG is expressed in rats administered with AQP2-SeV / ΔF. The scale bar is 200 μm. Western blot analysis of kidney lysates. Lane 1: control SD rat, lane 2: Li phagocytic diabetes insipidus rat, lane 3: Li phagocytic diabetes insipidus rat administered with GFP-SeV / ΔF on the 5th day, lane 4: 5th day Rats of Li diet administered AQP2-SeV / ΔF. It can be seen that the expression of APQ2 was suppressed in the rats with Li pneumoniae diabetes insipidus, and APQ2 was re-expressed in the Li pyeloneuria diabetes mellitus rat to which AQP2-SeV / ΔF was administered. In phagocytic diabetes insipidus rats administered with AQP2-SeV / ΔF, FLAG expression is also observed. Western blot analysis of urine samples. Lane 1: control SD rat, lane 2: Li phagocytic diabetes insipidus rat, lane 3: Li phagocytic diabetes insipidus rat administered with GFP-SeV / ΔF on day 5, lane 4: day 5 Li phagocytic diabetes insipidus rats administered with AQP2-SeV / ΔF. It can be seen that the expression of APQ2 was suppressed in the rats with Li pneumoniae diabetes insipidus, and APQ2 was re-expressed in the Li pyeloneuria diabetes mellitus rat to which AQP2-SeV / ΔF was administered. In phagocytic diabetes insipidus rats administered with AQP2-SeV / ΔF, FLAG expression is also observed. The deduced amino acid sequence of human AQP2 (hAQP2) with FLAG added. The FLAG epitope (SEQ ID NO: 3) is shown in italics. The sequence of human AQP2 (SEQ ID NO: 4) is underlined. Immunohistochemistry of the medullary lining of rats after feeding with Li diet or after feeding and gene transfer with Li diet. At least three independent experiments were performed and representative stained images were shown. Paraffin-embedded sections were stained with anti-GFP antibody (a), anti-FLAG antibody (b), or anti-AQP2 antibody. Normal: reared on normal diet, Li diet: 14 days after start of Li diet, dissect rat, Li-fed GFP-SeV / ΔF: Li-fed rat was transfected with GFP-GFP-SeV / ΔF, 4 days later Dissect, Li-fed AQP2-SeV / ΔF: Li-fed rats were transfected with AQP2-SeV / ΔF and dissected four days later. Effect by transfecting oA cells with hAQP2 cRNA. (a) Results of oocyte swelling assay. (b) Comparison of hydraulic conductivity (Pf). Scale bar is 1mm. Therapeutic effect of AQP2-SeV / ΔF for V2R abnormal renal diabetes insipidus. GFP-SeV / ΔF (■) or AQP2-SeV / ΔF (●) was administered to OPC pneumonia diabetes insipidus rats (day 0), and urine volume was measured over time. The measurement result was expressed as an average value ± standard error. The amount of urine in rats administered with AQP2-SeV / ΔF is significantly lower over several days than in rats administered with GFP-SeV / ΔF. * P <0.05 (t test) Therapeutic effect of AQP2-SeV / ΔF for V2R abnormal renal diabetes insipidus. GFP-SeV / ΔF (■) or AQP2-SeV / ΔF (●) was administered to OPC pneumonia diabetes insipidus rats (day 0), and the amount of water consumed was measured over time. The measurement result was expressed as an average value ± standard error. The amount of water consumed by rats administered with AQP2-SeV / ΔF is significantly lower over several days than that of rats administered with GFP-SeV / ΔF. * P <0.05 (t test) Therapeutic effect of AQP2-SeV / ΔF for V2R abnormal renal diabetes insipidus. GFP-SeV / ΔF (◯) or AQP2-SeV / ΔF (●) was administered to OPC-eating nephrogenic diabetes rats (day 0), and the amount of food intake was measured over time. The measurement result was expressed as an average value ± standard error. There is no significant difference in food intake between rats administered with AQP2-SeV / ΔF and rats administered with GFP-SeV / ΔF. Therapeutic effect of AQP2-SeV / ΔF for V2R abnormal renal diabetes insipidus. GFP-SeV / ΔF (◯) or AQP2-SeV / ΔF (●) was administered to OPC pneumonia diabetes insipidus rats (day 0), and body weight was measured over time. The measurement result was expressed as an average value ± standard error. There is no significant difference in body weight between rats administered with AQP2-SeV / ΔF and rats administered with GFP-SeV / ΔF. The therapeutic effect by retrograde administration of SIV-FLAG-hAQP2-VSVG via the ureter. SIV-EGFP-VSVG (◯, □) or SIV-FLAG-hAQP2-VSVG (●, □) was administered to Li pyelic diabetes insipidus rats (day 0), and urine volume was measured over time. The therapeutic effect by retrograde administration of SIV-FLAG-hAQP2-VSVG via the ureter. SIV-EGFP-VSVG (◯, □) or SIV-FLAG-hAQP2-VSVG (●, ■) was administered to Li pyeloneuria diabetic rats (day 0), and the amount of water consumed was measured over time. The therapeutic effect by retrograde administration of SIV-FLAG-hAQP2-F / HN via the ureter. SIV-EGFP-F / HN (◯) or SIV-FLAG-hAQP2-F / HN (●) was administered to Li pyelic diabetes insipidus rats (day 0), and the amount of water consumed was measured over time.

Claims (11)

  A recombinant vector for the treatment of renal diabetes insipidus, which retains the aquaporin 2 gene.   The recombinant vector according to claim 1, wherein the vector is a Sendai virus vector or a simian immunodeficiency virus vector.   The recombinant vector according to claim 2, wherein the Sendai virus vector does not contain one or more genes selected from the group consisting of F gene, HN gene and M gene.   The recombinant vector according to any one of claims 1 to 3, wherein the aquaporin 2 gene is a human aquaporin 2 gene.   The recombinant vector according to any one of claims 1 to 4, wherein the nephrogenic diabetes insipidus is congenital nephrogenic diabetes insipidus resulting from a mutation in a vasopressin receptor gene.   The recombinant vector according to any one of claims 1 to 5, wherein the recombinant vector is injected retrogradely from the ureter when applied.   A composition for treating renal diabetes insipidus comprising the recombinant vector according to any one of claims 1 to 6 and a pharmaceutically acceptable medium.   Use of aquaporin 2 gene to produce a recombinant vector for the treatment of nephrogenic diabetes insipidus.   A method for treating renal diabetes insipidus comprising the step of forcibly expressing aquaporin 2 gene in renal medullary collecting duct epithelial cells.   The treatment according to claim 9, wherein the aquaporin 2 gene is forcibly expressed in the renal medullary collecting duct epithelial cell by delivering a recombinant vector carrying the aquaporin 2 gene to the renal medullary collecting duct epithelial cell. Law.   A recombinant vector carrying the aquaporin 2 gene is retrogradely injected from the urinary tract to deliver the recombinant vector to the renal medullary collecting duct epithelial cell, and the aquaporin 2 gene is forcibly expressed in the renal medullary collecting duct epithelial cell The method according to claim 9, wherein: