JP6923186B2 - Smooth muscle specific TRPV2 knockout non-human animal - Google Patents

Description

The present invention relates to non-human animals that do not cause medial thickening and hypertension when angiotensin II is administered.

Hypertension is classified into hypertension in which there is a clear causative disease such as kidney and adrenal gland and hypertension in which there is no clear causative disease. Hypertension without a causative disorder is called essential hypertension. About 90% or more of hypertension is said to be essential hypertension, and it is said that the number of patients in Japan exceeds about 7.5 million. Although many studies have been conducted on essential hypertension, the developmental process has not been elucidated.

Many model animals for hypertension are known and are used to evaluate various types of antihypertensive agents. Examples of hypertension model animals include a low / normal renin type hypertension model that depends on salt and water load, and a hypertension type hypertension model that involves activation of the renin-angiotensin system that does not depend on salt and water load. .. For example, Dahl SS / jrCtr rats are a salt-sensitive model of low / normal renin-type hypertension, and salt loading increases blood pressure with age. The hypertension model, known as the aldosterone / salt load-induced model, causes salt loading in rats with one kidney removed, followed by continuous administration of aldosterone, ^{which causes Na +} retention in the body, resulting in water. It is considered that hypertension is caused by the accumulation in the body. Stroke-prone spontaneous hypertension rats (SHRSP), a model of genetic hypertension, is a salt-insensitive anti-renin-type genetic hypertension model with activation of the renin-angiotensin system. In the 11-β-hydroxysteroid dehydrogenase2 (11-β-HSD2) -inhibited hypertension model, mineralocorticoid receptor stimulation by glucocorticoids occurs continuously, leading to a condition similar to hyperaldosteronism with severe hypertension.

It is known that Trp (transient receptor potential) forms a TRP superfamily consisting of 29 kinds of genes and 6 groups (TRPC, TRPV, TRPM, TRPA, TRPML and TRPP) in mammals. Both are non-specific cation channels that penetrate the plasma membrane six times and are polymodal receptors that act as tetramers. The existence of TRPV2 and its agonist 2APB is known, and there are reports on TRPV1, TRPV2, and TRPV3 measurement systems that utilize the electrophysiological properties of ^{Ca 2+ channels.} TRPV2 is a stretch-activated Ca ²⁺ channel that is present in the intracellular membrane system in normal tissues, but migrates to the cell membrane with muscle degenerative diseases such as muscular dystrophy and cardiomyopathy, and is activated and abnormal into the cell. It is said to contribute to the influx of Ca ²⁺ (J. Cell Biology; 161 (5): 957-967 (2003), Hum Mol Genet .; 18 (5): 824-834 (2009)). TRPV2 systemic knockout (KO) mice die during the embryonic period (Non-Patent Document 1).

There is a report confirming the function of TRPV2 in the heart by suppressing the expression of TRPV2 in a myocardial manner (Non-Patent Document 2). Here, the physiological importance of TRPV2 in maintaining the structure and function of the heart has been reported, but there is no description regarding angiotensin administration, and there is no description that directly indicates the relationship between TRPV2 and blood pressure.

The action of angiotensin II is known as one of the causes of hypertension. Angiotensin II thickens and contracts the media consisting of vascular smooth muscle cells, and plays an important role in the pathogenesis and progression of hypertension. Focusing on the action of angiotensin II, several model animals have been reported that do not cause medial hyperplasia / hypertension even when angiotensin II is administered. For example, angiotensin receptor KO mice (Non-Patent Document 3) and G protein-coupled receptor KO mice (Non-Patent Document 4) have been reported. The model animals shown in Non-Patent Documents 3 and 4 are merely KOs targeting angiotensin receptors and G protein-coupled receptors, not Ca ²⁺ channels. Although it is known that medial thickening caused by angiotensin II administration ^{is triggered by an increase in Ca 2+ in smooth muscle cells, the molecular substance of Ca 2+} channels is still unknown.

J. Neuteoscience; 31 (32): 11425-11436 (2011) Nature Comunications; NATURE COMMUNICATIONS | 5: 3932 | DOI: 10.1038 / ncomms4932 J. Biological Chemistry; 270: 18719-18722 (1995) Science Signaling; 9 (411): ra7.doi.1126 / scisignal.aac9187 (2016)

An object of the present invention is to provide a non-human animal that does not cause medial thickening and hypertension even when angiotensin II is administered.

^{The inventors of the present application focused on the function of the Ca 2+} channel in order to solve the above-mentioned problems, and as a result of intensive research, angiotensin II was developed according to a non-human animal in which the function of TRPV2 was specifically suppressed in smooth muscle. The present invention was completed by finding that administration does not cause medial thickening and hypertension.

That is, the present invention comprises the following.
1. 1. A non-human animal whose TRPV2 protein function is suppressed specifically in smooth muscle.
2. The non-human animal according to item 1 above, wherein the expression of all or part of the TRPV2 gene is suppressed in a smooth muscle-specific manner, and the function of the TRPV2 protein is suppressed in a smooth muscle-specific manner.
3. 3. The non-human animal according to item 1 or 2 above, wherein the non-human animal is a mouse that suppresses medial thickening.
4. The non-human animal according to item 1 or 2 above, wherein the non-human animal is a hypertension-suppressed mouse.
5. Smooth muscle-specific TRPV2 protein function is suppressed, including the step of suppressing smooth muscle-specific expression of all or part of TRPV2 using a vector equipped with a promoter capable of smooth muscle-specific expression. A method for producing a non-human animal.
6. The non-existence according to the above item 5, wherein all or a part of TRPV2 whose expression is suppressed includes at least the 63rd to 756th amino acid sequences among the amino acid sequences disclosed in NCBI Reference Sequence: NP_035836.2. Method for producing human animals.
7. The step of suppressing the expression of all or part of TRPV2 specifically in smooth muscle includes the step of knocking out (KO) all or part of the gene encoding TRPV2 using a Cre expression-dependent gene deficiency system. The production method according to 6.
8. 2. The gene encoding TRPV2 is described in item 7 above, wherein all or part of the gene encoding TRPV2 is a gene encoding at least the 63rd to 756th amino acid sequences among the amino acid sequences disclosed in NCBI Reference Sequence: NP_035836.2. How to make.
9. The production method according to any one of 5 to 8 above, wherein the promoter that can be specifically expressed in smooth muscle is the SM22α gene promoter.
10. A non-human animal produced by the production method according to any one of 5 to 9 above, wherein the function of the TRPV2 protein is suppressed in a smooth muscle-specific manner.
11. The non-human animal according to any one of the above items 1 to 4, which is produced by the production method according to any one of the above items 5 to 9.
12. The method for screening an antihypertensive agent using a non-human animal according to the above items 1 to 4, 10 or 11.

According to the non-human animal in which the function of TRPV2 is specifically suppressed in the smooth muscle of the present invention, medial thickening does not occur and blood pressure does not increase even when angiotensin II, which causes hypertension, is administered. rice field.

a. It is a figure which confirmed the production scheme of the smooth muscle-specific TRPV2 knockout (KO) mouse, and the expressed protein of the produced mouse media cells. b. It is a figure which shows the gene expression (genotyping) result in the genomic DNA of the prepared mouse. (Example 1) It is a figure which shows the composition of the gene for the production of a smooth muscle specific TRPV2 KO mouse. (Example 1) It is a photographic figure which confirmed the suppression of TRPV2 expression in the aorta of smooth muscle specific TRPV2 KO mouse. (Example 2) It is a result figure which confirmed the morphological change of the aorta of the smooth muscle specific TRPV2 KO mouse before and after the administration of angiotensin II. (Example 3) It is a result figure which confirmed the blood pressure change of the smooth muscle specific TRPV2 KO mouse before and after the administration of angiotensin II. (Example 4)

The present invention relates to non-human animals that do not cause medial thickening and hypertension upon administration of angiotensin II. The blood vessel wall has a three-layer structure of an intima, a media, and an adventitia from the luminal side, and the “media” in the present specification means the media of the blood vessel wall. The media is well-developed in the arteries and is mainly composed of elastic fibers and smooth muscle. There are three types of muscles: smooth muscle, striated muscle, and myocardium. Unlike striated muscle, smooth muscle refers to muscle without sarcomere (myomere). Smooth muscle is involved in the movement of internal organs and blood vessels such as blood vessels, gastrointestinal tract, bladder, and uterus. Unlike skeletal muscle, smooth muscle contraction is involuntary. In the present invention, the smooth muscle derived from the blood vessel wall is particularly preferable. According to the "non-human animal in which the function of the TRPV2 protein is specifically suppressed in smooth muscle" of the present invention, the medial thickening of the non-human animal is also suppressed by administration of angiotensin II. Then, administration of angiotensin II usually causes hypertension, but according to the non-human animal of the present invention, the onset of hypertension is suppressed.

The phrase "the function of the TRPV2 protein is specifically suppressed in smooth muscle" in the present invention means that the function of the TRPV2 protein is suppressed in smooth muscle as the word implies. The function of the TRPV2 protein specific to smooth muscle may be suppressed, for example, the expression of all or part of the TRPV2 gene may be suppressed in a smooth muscle specific manner. Suppression of partial expression of the TRPV2 gene means that the protein consisting of a partial sequence of TRPV2 expressed by suppressing the partial expression of the TRPV2 gene does not have to exert its function as a TRPV2 protein. The amino acid sequences that make up the TRPV2 protein are disclosed in NCBI Reference Sequence: NP_035836.2 (SEQ ID NO: 1). Among the amino acid sequences shown in SEQ ID NO: 1, the 63rd to 756th amino acid sequences are considered to be particularly important in order to exert the function as a TRPV2 protein. Therefore, in the present specification, "the expression of a part of the TRPV2 gene is suppressed" means that the expression of the gene encoding at least the 63rd to 756th amino acid sequences of the amino acid sequences shown in SEQ ID NO: 1 is expressed. It is preferable that it is suppressed.

The non-human animal in which the function of the TRPV2 protein is suppressed specifically for smooth muscle of the present invention can be produced by suppressing the expression of all or part of TRPV2 specifically for smooth muscle. Smooth muscle-specific expression suppression of all or part of TRPV2 is an expression protein in which all or part of the gene encoding TRPV2 is deficient and all or part of TRPV2 is deficient, and its function is suppressed. I just need to be there. Suppression of the expression of all or part of TRPV2 means that the gene encoding at least the 63rd to 756th amino acid sequences of the gene encoding TRPV2 is targeted, and the relevant portion may be knocked out (KO). As the method of gene KO, any method known or developed in the future can be applied. In the present invention, it is necessary that the gene is KOed specifically for smooth muscle. This is because it is fatal if the function of the expressed protein is suppressed systemically. Therefore, as a method for KOing a gene specifically for smooth muscle, for example, a DNA recombination enzyme Cre expression-dependent gene deficiency system can be used. Specifically, the following method can be used.

F1 animals for KO non-human animal production of all or part of the gene encoding TRPV2 are produced. Hereinafter, it is also referred to as a "target gene" as a gene for which "all or part of the gene encoding TRPV2" is desired to be deleted. A KO vector for deleting a target gene is called a "targeting vector". The targeting vector of the target gene can be constructed so as to facilitate screening of the recombinant after homologous recombination has occurred. For example, a selectable marker such as a drug resistance gene or a toxin gene may be linked to a targeting vector for positive-negative selection. As the selectable marker gene, for example, neomycin resistance gene (neo), hygromycin B phosphotransferase gene, ampicillin resistance gene, tetracycline resistance gene and the like can be used for positive selection, and for example, herpesvirus thymidine kinase gene (for negative selection). HSV-tk), diphtheria toxin A gene, etc. can be used. Homologous recombination is performed using the targeting vector prepared by the above method. Currently established gene targeting methods can use, for example, ES cells when producing KO non-human animals.

In order to cause homologous recombination, a targeting vector is introduced into, for example, ES cells. As a method for introducing the targeting vector into cells, for example, a method known per se, such as an electroporation method, a calcium phosphate method, a DEAE-dextran method, a liposome method, or any method developed in the future can be used.

Gene targeting using the Cre-loxP system, which is a recombinant system derived from bacteriophage P1, will be described. loxP is the target sequence for Cre recombinase. According to the Cre-loxP system, loxP and Cre can be used to delete a target gene at a specific site (organ). By deleting the target gene, the expression of the protein encoded by the target gene is suppressed, or even if it is expressed, the function of the protein is suppressed.

Cre is a DNA recombination enzyme that can recognize the sequence of 34 bases called loxP and cause recombination at this site. Create a non-human animal in which the target gene to be deleted is sandwiched between the loxP sequence and the loxP sequence (flox animal). In addition, a non-human animal into which a targeting vector in which the Cre gene is incorporated downstream of a tissue-specific promoter is introduced (Cre animal). A heterozygous non-human animal (F2) lacking the desired target gene can be produced by crossing a flox animal and a Cre animal, and a homozygous non-human animal can be produced with F3.

Specifically, first, a non-human animal in which all or part of the gene encoding TRPV2 is sandwiched between loxP as a target gene is prepared as a flox animal (TRPV2 ^{flox / flox} ). Separately, Cre animals that express Cre under the control of a promoter capable of expressing smooth muscle specifically are prepared. Heterozygous non-human animals (F2) are produced by crossing flox animals and Cre animals, and non-human animals lacking all or part of the gene encoding RPV2 at the site where the smooth muscle-specific promoter acts in F3. It can be made (see FIG. 1a). The promoter that can be specifically expressed in smooth muscle is not particularly limited, and examples thereof include a SM22α gene promoter, a smooth muscle actin promoter, and a smooth muscle myosin heavy chain promoter. Particularly preferred is the SM22α gene promoter.

The non-human animal of the present invention is a non-human animal in which the function of the TRPV2 protein is suppressed in a smooth muscle-specific manner. Examples of non-human animals in the present invention include mammals other than humans, and examples thereof include cows, monkeys, pigs, dogs, cats, guinea pigs, rabbits, rats, and mice. In particular, rodents such as guinea pigs, rats, and mice are preferable because they are easy to handle, and mice are particularly preferable. After that, it can be confirmed whether or not the target gene is targeted by PCR method, Southern blotting method or the like. In PCR, primers can be designed, for example, on the genomic DNA region outside the targeting vector and on the drug resistance gene of the targeting vector sequence. Southern blot analysis can be performed, for example, as follows. Hybridization of the DNA fragment obtained by designing one or two types of probes that recognize the genomic region not included in the targeting vector and treating the clone containing the target gene genome with an appropriate restriction enzyme and the above probe. By doing so, it is possible to confirm the presence or absence of disruption of the target gene.

From the non-human animals obtained as described above, a target gene-deficient heterozygous KO non-human animal can be bred. A gene-deficient homozygous KO non-human animal can be obtained by mating the target gene heterozygous KO non-human animals obtained as described above. Confirmation that a KO non-human animal has been obtained can be performed by extracting chromosomal DNA from the tissue and using, for example, Southern blotting or PCR. Furthermore, RNA can be extracted from tissues and the gene expression pattern can be analyzed by Northern blot analysis. Western blotting may be performed using an antibody against TRPV2, which is a gene of interest.

It is also possible to analyze the phenotypes of heterozygous KO non-human animals and homozygous KO non-human animals for established non-human animal strains. Phenotypic analysis is performed by macroscopic observation, internal observation by dissection, tissue section of each organ, observation by X-ray photography, observation of behavior and memory, blood test and serum biochemical test. The analysis period may be any period from the embryonic period to the adult period, and is not particularly limited.

The present invention also extends to a method for screening a therapeutic agent for hypertension using a non-human animal in which the function of the TRPV2 protein is suppressed in a smooth muscle-specific manner. The non-human animal of the present invention is a non-human animal that does not cause medial thickening and hypertension even when angiotensin II is administered. By administering the candidate compound to the non-human animal of the present invention and the wild-type non-human animal and comparing the phenotypes, the effect of the candidate compound can be confirmed and the mechanism of action of the candidate compound can be confirmed.

The best embodiments and specific examples for carrying out the invention show preferred embodiments of the present invention and are shown for illustration or explanation purposes, and the present invention is limited thereto. It's not something to do. It will be apparent to those skilled in the art that various modifications can be made based on the description herein within the intent and scope of the invention disclosed herein.

Hereinafter, the present invention will be specifically described with reference to Examples and Experimental Examples in order to deepen the understanding of the present invention, but it goes without saying that these do not limit the scope of the present invention.

(Example 1) Preparation of smooth muscle-specific TRPV2 knockout (KO) mouse In this example, smooth muscle-specific TRPV2 KO mouse (TRPV2 ^{flox / flox} : TRPV2-Cre ^+/- (cKO)) using the Cre-loxP system. )) Will be described. C57 / BL6J was used as the mouse, and the gene was introduced into ES cells by the electroporation method.

In this example, the target gene to be deleted is a gene encoding the amino acid sequence (SEQ ID NO: 1) disclosed in NCBI Reference Sequence: NP_035836.2. ^{A TRPV2 flox / flox} mouse (F1) was prepared by sandwiching the target gene between the loxP sequence and the loxP sequence. ^{In addition, SM22α-Cre +/-} mice (F1) were prepared by introducing a targeting vector in which the Cre gene was incorporated downstream of the SM22α gene promoter. The composition of genes such as the targeting vector related to KO of TRPV2 is as shown in FIG. TRPV2 ^{flox / flox} mice and SM22α-Cre ^+/- mice were crossed to produce heterozygous TRPV2 ^{flox / +} : SM22α-Cre ^+/- mice (F2). TRPV2 ^{flox / +} : SM22α-Cre ^+/- Mice further mated with ^{TRPV2 flox / flox mice, TRPV2 flox / +} , TRPV2 ^{flox / flox} , TRPV2 ^{flox / +} : SM22α-Cre ^+/- (HET) and TRPV2 ^{flox / flox} : SM22α-Cre ^+/- (cKO) F3 mice were produced (Fig. 1a).

Among the above-mentioned mice obtained, TRPV2 ^{flox / flox} : SM22α-Cre ^+/- (cKO) mice were used as smooth muscle-specific TRPV2 KO mice of the present invention, and TRPV2 ^{flox / flox} was used as a control. The results of gene expression (genotyping) in genomic DNA extracted from mouse tails are shown in FIG. 1b. Based on this, the genetic background of individual mice can be confirmed.

(Example 2) Confirmation of TRPV2 expression in aortic smooth muscle cells of smooth muscle-specific TRPV2 KO mice In the smooth muscle-specific TRPV2 KO mice of the present invention, TRPV2 expression was confirmed in aortic smooth muscle cells (intermediate membrane). From the mice that had completed genotyping in Example 1, the aorta was isolated, frozen sections were prepared, and immunostaining was performed with TRPV2 antibody. The TRPV2 antibody was visualized with a secondary antibody added by Alexa-Fluor 488.
As a result, in the control, expression of TRPV2 was observed in smooth muscle cells, but expression of TRPV2 was hardly observed in the smooth muscle-specific TRPV2 KO mouse of the present invention (Fig. 3).

(Example 3) Anti-middle film thickening by administration of angiotensin II Angiotensin II was administered subcutaneously at 2 mg / kg per day for 4 weeks to the smooth muscle-specific TRPV2 KO mice of the present invention by implanting an osmotic pump. In the control mice, the media was thickened to 150 mmHg or more, but in the smooth muscle-specific TRPV2 KO mice of the present invention, no thickening was observed, which was almost the same as before the administration of angiotensin II (Fig. 4).

(Example 4) Antihypertensive effect by administration of angiotensin II Angiotensin II was administered subcutaneously at 2 mg / kg per day for 4 weeks to the smooth muscle-specific TRPV2 KO mice of the present invention by implanting an osmotic pump. The mean blood pressure of the control mouse was about 150 mmHg, but that of the smooth muscle-specific TRPV2 KO mouse of the present invention was maintained at about 100 mmHg, which was almost the same as that before the administration of angiotensin II (Fig. 5).

As explained above, this book is composed of non-human animals in which the function of TRPV2 is specifically suppressed in smooth muscle, and more specifically, the expression of all or part of the gene encoding TRPV2 in a smooth muscle specific manner is suppressed. According to the non-human animal of the present invention, even when angiotensin II, which causes hypertension, was administered, medial thickening did not occur and blood pressure did not increase. Therefore, the use of the non-human animal of the present invention can contribute to the elucidation of the mechanism of action of essential hypertension and the development of a therapeutic agent for essential hypertension.

Claims (9)

A non-human animal in which the expression of all or part of the gene encoding the TRPV2 protein in a smooth muscle-specific manner is suppressed, and the function of the TRPV2 protein is suppressed in a smooth muscle-specific manner. , A non-human animal characterized in that medial thickening is suppressed even by administration of angiotensin II . The non-human animal according to claim 1, wherein the non-human animal has a property of not causing hypertension caused by angiotensin II even when angiotensin II is administered. A smooth muscle-specific TRPV2 protein that comprises the step of suppressing the expression of all or part of a smooth muscle-specific TRPV2 protein-encoding gene using a vector carrying a smooth muscle-specificly expressable promoter. function Ri Na is suppressed, a method for manufacturing a non-human animal characterized by having the property of tunica hypertrophy is suppressed by angiotensin II administration. The step of suppressing the expression of all or part of the gene encoding TRPV2 specifically in smooth muscle includes the step of knocking out all or part of the gene encoding TRPV2 using a Cre expression-dependent gene deletion system. The production method according to claim 3. Claim 3 or claim 3, wherein all or part of the gene encoding TRPV2 is a gene encoding at least the 63rd to 756th amino acid sequences among the amino acid sequences disclosed in NCBI Reference Sequence: NP_035836.2. The production method according to 4. The production method according to any one of claims 3 to 5 , wherein the promoter that can be specifically expressed in smooth muscle is the SM22α gene promoter. Produced by the manufacturing method according to any one of claims 3-6, Ri Na function of smooth muscle specific TRPV2 protein is suppressed, it has a property of tunica hypertrophy is suppressed by angiotensin II administration A non-human animal characterized by. The non-human animal according to any one of claims 1, 2 and 7 for elucidating the mechanism of action of essential hypertension or developing a therapeutic agent for essential hypertension. The method for screening an antihypertensive agent using a non-human animal according to any one of claims 1 , 2, 7 and 8.

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