阅读说明：本技术 移植有人肝细胞的非人脊椎动物及其制造方法 (Non-human vertebrate for transplantation of human hepatocytes and method for producing the same ) 是由 末水洋志 于 2019-12-12 设计创作，主要内容包括：本发明提供一种人肝细胞的增殖速度比目前存在的人肝脏移植非人脊椎动物更快,且人肝细胞的取代率、组织学及生理学上的人再造性也很高的非人脊椎动物及其制造方法。一种移植有人肝细胞的基因改造非人脊椎动物的制造方法,包括在体内存在人IL-6的状态下向对于人的免疫反应缺失或降低的非人脊椎动物中移植人肝细胞。(The present invention provides a non-human vertebrate which has a higher proliferation rate of human hepatocytes than that of a non-human vertebrate transplanted with a human liver, and which has a high substitution rate of human hepatocytes, a high histological and physiological human reproducibility, and a method for producing the same. A method for producing a genetically modified non-human vertebrate into which human hepatocytes have been transplanted, comprising transplanting human hepatocytes into a non-human vertebrate in which an immune response to a human is deficient or reduced in the presence of human IL-6 in vivo.)

1. A method for producing a genetically modified non-human vertebrate into which human hepatocytes have been transplanted, comprising transplanting human hepatocytes into a non-human vertebrate in which an immune response to a human is deficient or reduced in the presence of human IL-6 in vivo.

2. The manufacturing method according to claim 1,

non-human vertebrates in which the immune response to humans is absent or reduced are immunodeficient animals.

3. The manufacturing method according to claim 1 or 2,

liver injury is induced in the liver of a non-human vertebrate in which an immune response to the human is absent or reduced by a liver injury induction method, and transplanted human hepatocytes are allowed to survive in the liver of the non-human vertebrate instead of the hepatocytes of the non-human vertebrate.

4. The manufacturing method according to claim 3,

liver damage is induced by any one of the following methods:

(i) a method of inducing liver damage by maintaining thymidine kinase gene expression in the liver of a non-human vertebrate in which an immune response to the human is absent or reduced, and administering human hepatocytes to the non-human vertebrate;

(ii) a method of inducing liver damage by maintaining urokinase-type plasminogen activator gene expressible in the liver of a non-human vertebrate in which the immune response to the human is absent or reduced;

(iii) a method for inducing liver damage by deleting a fumarylacetoacetate hydrolase Fah gene of a non-human vertebrate in which an immune response to human is deleted or reduced;

(iv) a method for inducing liver damage by administering a compound selected from any one of the following (a) to (e):

(a) carbon tetrachloride

(b) Acetaminophen

(c) d-galactosamine

(d) Thioacetamide

(e) An anti-Fas antibody; and

(v) two, three or four of the above-mentioned (i) to (iv) are used in combination.

5. The manufacturing method according to claim 4,

liver damage is induced by maintaining thymidine kinase gene expression in the liver of a non-human vertebrate in which the immune response to the human is absent or reduced, and administering human hepatocytes with a suicide substrate to the non-human vertebrate.

6. The production method according to any one of claims 1 to 5,

human IL-6 is made to exist in vivo by introducing and expressing a human IL-6 gene into a non-human vertebrate.

7. The production method according to any one of claims 1 to 5,

human IL-6 is made present in vivo by administering human IL-6 to a non-human vertebrate.

8. The production method according to any one of claims 1 to 5,

human IL-6 is present in vivo by administering to a non-human vertebrate a cultured cell expressing a human IL-6 gene or a vector molecule expressing the gene and allowing expression thereof.

9. The production method according to any one of claims 4 to 8,

the non-human vertebrate is a TK-NOG mouse made by the following procedure:

(i) microinjecting human herpes simplex virus type 1-thymidine kinase HSV-tk gene into fertilized eggs of NOD/Shi mice; and

(ii) the NOD/Shi mice having the HSV-tk gene of human herpes simplex virus type 1 obtained in step (i) were combined with NOG, i.e., NOD/SCID/gammac^nullMice were mated.

10. The manufacturing method according to claim 9,

the TK-NOG mouse is selected from

NOD.Cg-Prkdc^scidIl2rg^tm1SugTg(Alb-UL23)7-2/ShiJic、

NOD.Cg-Prkdc^scidIl2rg^tm1SugTg(Ttr-UL23 mutant30)4-9/ShiJic、NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Ttr-UL23mutant30)5-2/Shijic mice and

NOD.Cg-Prkdc^scidIl2rg^tm1SugTg(Ttr-UL23 mutant30)10-15/ShiJic

mice represented by the genetic code in the group.

11. The manufacturing method according to claim 9 or 10, wherein the manufacturing method includes the steps of:

the mouse of claim 9 or 10, which is a TK-NOG mouse in which human IL-6 is present in vivo, is administered with a suicide substrate and hepatocytes isolated from a human, thereby damaging and replacing the mouse hepatocytes with human hepatocytes.

12. The manufacturing method according to claim 9 or 10,

the mouse according to claim 9 or 10, wherein the TK-NOG mouse in which human IL-6 exists in vivo is a TK-NOG-IL-6 mouse obtained by the following steps:

(i) microinjecting a DNA fragment containing the human IL-6cDNA into a fertilized egg made of a female NOD mouse and a male NOG mouse to obtain a human IL-6 transgenic first-construction mouse;

(ii) scid and IL2Rg were obtained by mating human IL-6 transgenic naive mice with NOG mice^nullA variant of (a); and

(iii) (iii) mating the NOG-IL-6 mice obtained in (ii) with TK-NOG mice.

13. The manufacturing method according to claim 12,

the obtained TK-NOG-IL-6 mice are selected from the group consisting of

N NOD.Cg-Prkdc^scidIl2rg^tm1SugTg((Alb-UL23)7-2,CMV-IL-6)/ShiJic、

NOD.Cg-Prkdc^scidIl2rg^tm1SugTg(Ttr-UL23 mutant30)4-9,CMV-IL-6/ShiJic、

NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Ttr-UL23mutant30)5-2, CMV-IL-6/Shijic and

NOD.Cg-Prkdc^scidIl2rg^tm1Sugtg (Ttr-UL23mutant30)10-15, CMV-IL-6/ShiJic.

14. The production method according to any one of claims 4 to 13,

suicide substrates are substances that are metabolized by thymidine kinase into toxic substances.

15. The manufacturing method according to claim 14,

the substance metabolized into a toxic substance is aciclovir or ganciclovir.

16. The production method according to any one of claims 1 to 15,

human hepatocytes are human liver cell lines.

17. The manufacturing method according to claim 16,

the human liver cell line is HepG2, Hep3B or HuH-7.

18. The production method according to any one of claims 1 to 15,

the human hepatocytes are primary cultured hepatocytes.

19. The production method according to any one of claims 4 to 18,

the human herpes simplex virus type 1-thymidine kinase HSV-tk gene is placed under the control of an albumin promoter, a thyroxine transporter promoter, a thyroxine binding globulin promoter, an LST-1 promoter, an alpha-fetoprotein promoter or an alpha-TTP promoter, so that the specific expression of the human herpes simplex virus type 1-thymidine kinase HSV-tk gene in the liver is kept.

20. A mouse transplanted with human hepatocytes, wherein 90% or more of the hepatocytes of the mouse are replaced with human hepatocytes to reconstruct a human liver having a three-dimensional structure of the human liver and a function of the human liver, and human IL-6 is present in the body.

21. The mouse of claim 20,

damaging hepatocytes of the mouse by any one of the following methods:

(i) inducing liver damage by maintaining thymidine kinase gene expression in the liver of a non-human vertebrate in which an immune response to the human is absent or reduced, and administering human hepatocytes to the non-human vertebrate with a suicide substrate;

(ii) inducing liver damage by maintaining urokinase-type plasminogen activator gene expressible in the liver of a non-human vertebrate in which the immune response to the human is absent or reduced;

(iii) inducing liver damage by deleting the fumarylacetoacetate hydrolase Fah gene of a non-human vertebrate that has a deleted or reduced immune response to the human;

(iv) inducing liver damage by administering any one of the following compounds (a) to (e):

(a) carbon tetrachloride

(b) Acetaminophen

(c) d-galactosamine

(d) Thioacetamide

(e) An anti-Fas antibody; and

(v) two, three or four of the above-mentioned (i) to (iv) are used in combination.

22. The mouse transplanted with human hepatocytes according to claim 21, wherein,

the specific expression of the HSV-tk gene in the liver can be maintained, more than 90% of the liver cells of a mouse are replaced by the liver cells of a human, so that the human liver is reconstructed, the liver has the three-dimensional structure of the human liver and the functions of the human liver, and the human IL-6 exists in the body.

23. The mouse of any one of claims 20 to 22,

the hepatobiliary system with human liver is constructed, has functional lobular structure of human liver, and can normally exert the function of excreting foreign body from liver.

24. The mouse of claim 22 or 23,

Cg-Prkdc obtained by the following steps^scidIl2rg^tm1SugTg((Alb-UL23)7-2,CMV-IL-6)/ShiJic：

(i) Microinjecting human herpes simplex virus type 1-thymidine kinase HSV-tk gene into fertilized eggs of NOD/Shi mice;

(ii) by combining the NOD/Shi mice having the human herpes simplex virus type 1 thymidine kinase (HSV-tk) gene obtained in step (i) with NOG, i.e., NOD/SCID/gammac^nullMating mice to make NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Alb-UL23)7-2/Shijic mice;

(iii) microinjecting a DNA fragment containing the human IL-6cDNA into a fertilized egg made of a female NOD mouse and a male NOG mouse to obtain a human IL-6 transgenic first-established mouse;

(iv) scid and IL2Rg were obtained by crossing human IL-6 transgenic naive and NOG mice^nullThe NOG-IL-6 mouse obtained in the step (3) of (1) is mated with the mouse of (ii).

25. The human hepatocyte-transplanted mouse according to any one of claims 20 to 24,

human hepatocytes are human hepatocyte cell lines.

26. The human hepatocyte-transplanted mouse according to claim 25,

the human liver cell line is HepG2, Hep3B or HuH-7.

27. The human hepatocyte-transplanted mouse according to any one of claims 20 to 24,

human hepatocytes are primary cultured hepatocytes.

28. The human hepatocyte-transplanted mouse according to any one of claims 20 to 27,

the human herpes simplex virus type 1-thymidine kinase HSV-tk gene is maintained to be specifically expressed in the liver by placing it under the control of an albumin promoter, a thyroxine transporter promoter, a thyroxine binding globulin promoter, a LST-1 promoter, an alpha-fetoprotein promoter, or an alpha-TTP promoter.

29. The human hepatocyte-transplanted mouse according to any one of claims 20 to 28,

further keeping the urokinase-type plasminogen activator gene specifically expressed in the liver.

30. The production method according to any one of claims 1 to 19,

the proliferation rate of the transplanted human hepatocytes after transplantation is at least 1.5-fold faster than when human IL-6 is not present in vivo.

31. The production method according to any one of claims 1 to 19,

further comprising administering a human anti-IL-6 antibody after transplantation of human hepatocytes.

Technical Field

The present invention relates to a non-human vertebrate into which human hepatocytes have been transplanted.

Background

In order to analyze infection with human specific hepatitis virus, metabolism of an agent to be administered, or proliferation of human liver, immune-tolerant mice retaining human hepatocytes are currently produced by many groups. Among them, in a mouse in which a human hepatocyte is transplanted while maintaining specific expression of an exogenous thymidine kinase gene in the liver, the hepatocyte of the mouse is replaced by the human hepatocyte, and the human hepatocyte replacement rate is good, reaching about 80% (see patent document 1).

Documents of the prior art

Patent document

Patent document 1: japanese patent No. 5073836

Disclosure of Invention

The present invention has an object to provide a non-human vertebrate which has a higher proliferation rate of human hepatocytes than that of a non-human vertebrate transplanted with a human liver, and which has a high substitution rate of human hepatocytes, a high histological and physiological human reproducibility, a method for producing the non-human vertebrate, a non-human vertebrate which is prepared by the effective action and which has a structure and a high physiological reconstruction of human hepatocytes to a higher degree, and a method for producing the non-human vertebrate.

The present inventors have made intensive studies on a method for producing a mouse transplanted with human hepatocytes, which has a higher proliferation rate of human hepatocytes than that of existing human liver transplanted mice and a high replacement rate of human hepatocytes.

The present inventors have found that the presence of human IL-6 in the transplantation of human hepatocytes into an immunodeficient mouse makes it possible to produce a mouse into which human hepatocytes have been transplanted with a high proliferation rate and a high substitution rate of human hepatocytes. Further, it was found that the mouse is a mouse model having a liver very similar to a human liver, and the liver of the mouse, in which the human hepatocytes are rapidly substituted and the substitution rate is high, has a higher degree of reconstruction of human liver tissue both histologically and physiologically, and the present invention was completed.

Namely, the present invention is as follows.

[1] A method for producing a genetically modified non-human vertebrate into which human hepatocytes have been transplanted, comprising transplanting human hepatocytes into a non-human vertebrate in which an immune response to a human is deficient or reduced in the presence of human IL-6 in vivo.

[2] The production method according to [1], wherein the non-human vertebrate in which an immune response to a human is absent or reduced is an immunodeficient animal.

[3] The production method according to [1] or [2], wherein the liver of the non-human vertebrate, in which the immune response to the human is deficient or reduced, is injured by a liver injury-inducing method, and the transplanted human hepatocytes are allowed to survive in the liver of the non-human vertebrate instead of the hepatocytes of the non-human vertebrate.

[4] The production method according to [3], wherein the liver injury is induced by any one of the following methods:

(i) inducing liver damage by maintaining thymidine kinase gene expression in the liver of a non-human vertebrate in which an immune response to the human is absent or reduced, and administering human hepatocytes to the non-human vertebrate with a suicide substrate;

(ii) inducing liver damage by maintaining urokinase-type plasminogen activator gene expressible in the liver of a non-human vertebrate in which the immune response to the human is absent or reduced;

(iii) inducing liver damage by deleting a fumarylacetoacetate hydrolase (Fah) gene from a non-human vertebrate that has a deleted or reduced immune response to the human;

(iv) inducing liver damage by administering any one of the following compounds (a) to (e):

(a) carbon tetrachloride

(b) Acetaminophen

(c) d-galactosamine

(d) Thioacetamide

(e) An anti-Fas antibody; and

(v) two, three or four of the above-mentioned (i) to (iv) are used in combination.

[5] The production method according to [4], wherein the liver injury is induced by maintaining thymidine kinase gene expression in the liver of a non-human vertebrate in which immune response to human is absent or reduced, and administering human hepatocytes and a suicide substrate to the non-human vertebrate.

[6] The production method according to any one of [1] to [5], wherein the human IL-6 is allowed to exist in vivo by introducing and expressing a human IL-6 gene into a non-human vertebrate.

[7] The production method according to any one of [1] to [5], wherein the human IL-6 is allowed to exist in vivo by administering the human IL-6 to a non-human vertebrate.

[8] The production method according to any one of [1] to [5], wherein the human IL-6 is caused to be present in vivo by administering to a non-human vertebrate and expressing cultured cells expressing the human IL-6 gene or a vector molecule expressing the gene.

[9] The process according to any one of [4] to [8], wherein the non-human vertebrate is a TK-NOG mouse produced by the following steps:

(i) microinjecting human herpes simplex virus type 1-thymidine kinase (HSV-tk) gene into fertilized egg of NOD/Shi mouse; and

(ii) the NOD/Shi mice having the human herpes simplex virus type 1 thymidine kinase (HSV-tk) gene obtained in step (i) and NOG (NOD/SCID/gammac)^null) Mice were mated.

[10]According to [9]]The method of (1), wherein the TK-NOG mouse is a mouse selected from the group consisting of NOD. Cg-Prkdc^scidIl2rg^tm1SugTg(Alb-UL23)7-2/ShiJic、NOD.Cg-Prkdc^scidIl2rg^tm1SugTg(Ttr-UL23 mutant30)4-9/ShiJic、NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Ttr-UL23mutant30)5-2/Shijic mice and NOD.Cg-Prkdc^scidIl2rg^tm1SugMice represented by the genetic code in the group consisting of Tg (Ttr-UL23mutant30) 10-15/ShiJic.

[11] The production method according to [9] or [10], wherein the production method comprises the steps of:

the mouse of [9] or [10], that is, the TK-NOG mouse in which human IL-6 is present in vivo, is administered with a suicide substrate and hepatocytes isolated from human, thereby damaging and replacing the mouse hepatocytes with human hepatocytes.

[12] The production method according to [9] or [10], wherein the mouse of [9] or [10], i.e., the TK-NOG mouse in which human IL-6 exists in vivo is a TK-NOG-IL-6 mouse obtained by the following steps:

(i) microinjecting a DNA fragment containing the human IL-6cDNA into a fertilized egg made of a female NOD mouse and a male NOG mouse to obtain a human IL-6 transgenic first-construction mouse;

(ii) scid and IL2Rg were obtained by mating human IL-6 transgenic naive mice with NOG mice^nullA variant of (a); and

(iii) (iii) mating the NOG-IL-6 mice obtained in (ii) with TK-NOG mice.

[13]According to [12]]The method of (1), wherein the TK-NOG-IL-6 mouse obtained is selected from the group consisting of N NOD^scidIl2rg^tm1SugTg((Alb-UL23)7-2,CMV-IL-6)/ShiJic、NOD.Cg-Prkdc^scidIl2rg^tm1SugTg(Ttr-UL23 mutant30)4-9,CMV-IL-6/ShiJic、NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Ttr-UL23mutant30)5-2, CMV-IL-6/Shijic and NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Ttr-UL23mutant30)10-15, CMV-IL-6/ShiJic.

[14] The process according to any one of [4] to [13], wherein the suicide substrate is a substance which is metabolized into a toxic substance by thymidine kinase.

[15] The production method according to [14], wherein the substance metabolized into a toxic substance is aciclovir or ganciclovir.

[16] The production method according to any one of [1] to [15], wherein the human hepatocyte is a human hepatocyte cell line.

[17] The process according to [16], wherein the human liver cell line is HepG2, Hep3B or HuH-7.

[18] The production method according to any one of [1] to [15], wherein the human hepatocytes are primary-cultured hepatocytes.

[19] The process according to any one of [4] to [18], wherein the specific expression of the human herpes simplex virus type 1-thymidine kinase (HSV-tk) gene in the liver is maintained by placing the human herpes simplex virus type 1-thymidine kinase (HSV-tk) gene under the control of an albumin promoter, a thyroxine transporter promoter, a thyroxine-binding globulin promoter, an LST-1 promoter, an alpha-fetoprotein promoter, or an alpha-TTP promoter.

[20] A mouse transplanted with human hepatocytes, wherein 90% or more of the hepatocytes of the mouse are replaced with human hepatocytes to reconstruct a human liver having a three-dimensional structure of the human liver and a function of the human liver, and human IL-6 is present in the body.

[21] The mouse according to [20], wherein hepatocytes of the mouse are injured by any one of the following methods:

(i) inducing liver damage by maintaining thymidine kinase gene expression in the liver of a non-human vertebrate in which an immune response to the human is absent or reduced, and administering human hepatocytes to the non-human vertebrate with a suicide substrate;

(ii) inducing liver damage by maintaining urokinase-type plasminogen activator gene expressible in the liver of a non-human vertebrate in which the immune response to the human is absent or reduced;

(iii) inducing liver damage by deleting a fumarylacetoacetate hydrolase (Fah) gene from a non-human vertebrate that has a deleted or reduced immune response to the human;

(iv) inducing liver damage by administering any one of the following compounds (a) to (e):

(a) carbon tetrachloride

(b) Acetaminophen

(c) d-galactosamine

(d) Thioacetamide

(e) An anti-Fas antibody; and

(v) two, three or four of the above-mentioned (i) to (iv) are used in combination.

[22] The mouse transplanted with human hepatocytes according to [21], wherein the human herpes simplex virus type 1-thymidine kinase (HSV-tk) gene is maintained to be specifically expressed in the liver, and 90% or more of the hepatocytes of the mouse are replaced with the human hepatocytes, thereby reconstructing a human liver having the three-dimensional structure and function of the human liver in which human IL-6 is present.

[23] According to any one of [20] to [22], a hepatobiliary system of a human liver is constructed, and the hepatobiliary system has a functional lobular structure of the human liver and normally exerts a function of excreting a foreign substance from the liver.

[24]According to [22]]Or [23]]The mouse of (1), wherein the mouse is a mouse obtained by the following process (NOD. Cg-Prkdc)^scidIl2rg^tm1SugTg((Alb-UL23)7-2,CMV-IL-6)/ShiJic)：

(i) Microinjecting human herpes simplex virus type 1-thymidine kinase (HSV-tk) gene into fertilized egg of NOD/Shi mouse;

(ii) by combining NOD/Shi mice having human herpes simplex virus type 1 thymidine kinase (HSV-tk) gene obtained in step (i) with NOG (NOD/SCID/gammac)^null) Mating mice to make NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Alb-UL23)7-2/Shijic mice;

(iii) microinjecting a DNA fragment containing the human IL-6cDNA into a fertilized egg made of a female NOD mouse and a male NOG mouse to obtain a human IL-6 transgenic first-established mouse; ,

(iv) scid and IL2Rg were obtained by crossing human IL-6 transgenic naive and NOG mice^nullThe NOG-IL-6 mouse obtained in the step (3) of (1) is mated with the mouse of (ii).

[25] A mouse into which human hepatocytes have been transplanted according to any one of [20] to [24], wherein the human hepatocytes are human hepatocyte cell lines.

[26] The human hepatocyte-transplanted mouse according to [25], wherein the human hepatocyte cell line is HepG2, Hep3B or HuH-7.

[27] A mouse into which human hepatocytes have been transplanted according to any one of [20] to [24], wherein the human hepatocytes are primary-cultured hepatocytes.

[28] The mouse transplanted with human hepatocytes according to any one of [20] to [27], wherein specific expression of a human herpes simplex virus type 1-thymidine kinase (HSV-tk) gene in the liver is maintained by placing the human herpes simplex virus type 1-thymidine kinase (HSV-tk) gene under the control of an albumin promoter, a thyroxine transporter promoter, a thyroxine-binding globulin promoter, an LST-1 promoter, an alpha-fetoprotein promoter, or an alpha-TTP promoter.

[29] A mouse transplanted with human hepatocytes according to any one of [20] to [28], wherein the urokinase-type plasminogen activator gene is further maintained to be specifically expressed in the liver.

[30] The production method according to any one of [1] to [19], wherein the proliferation rate of the transplanted human hepatocytes after transplantation is at least 1.5 times higher than that in the absence of human IL-6 in vivo.

[31] The production method according to any one of [1] to [19], which further comprises administering a human anti-IL-6 antibody after the transplantation of human hepatocytes.

The present specification includes the disclosure of japanese patent application No. 2018-234945 as the basis for priority of the present application.

When human hepatocytes are transplanted into a non-human vertebrate in which an immune response to human is deficient or decreased, the presence of human IL-6 makes it possible to produce a non-human vertebrate into which human hepatocytes have been transplanted with high human hepatocytes proliferation rate and high human hepatocyte replacement rate.

This indicates that human IL-6 has a significant effect of promoting the proliferation of human hepatocytes transplanted into a human body, and can be used as a method for proliferating human hepatocytes in a non-human animal body. The animal having human hepatocytes thus produced can be used as a source for providing hepatocytes for in vitro studies using human hepatocytes, in addition to drug metabolism and liver studies.

Drawings

FIG. 1 is a diagram showing the structure of a human interleukin 6 gene expression unit.

FIG. 2 is a diagram showing the structure of an HSV-tk mutant30(TKmut30) gene expression unit.

FIG. 3 is a graph showing the temporal change (average value) of the human albumin concentration in the serum of TK-NOG-IL-6 mice into which LHum17003 had been transplanted and TK-NOG mice.

FIG. 4 is a graph showing temporal changes in the presumed substitution index for each of TK-NOG-IL-6 mouse into which LHum17003 has been transplanted, and TK-NOG mouse.

FIG. 5 is a diagram showing the morphology of liver cells obtained by inoculating liver cells isolated and purified from TK-NOG-IL-6 mice after transplantation of human liver cells into a vessel coated with type 1 collagen and after 48 hours.

FIG. 6 is a graph showing the average values of the human albumin concentration in the serum of TK-NOG-IL-6 mice into which human hepatocytes HUM4122B were transplanted and TK-NOG mice measured with time.

FIG. 7 is a graph showing values obtained by measuring the presumed substitution index with time for each of TK-NOG-IL-6 mouse into which human hepatocytes HUM4122B have been transplanted and TK-NOG mouse.

FIG. 8 is a graph showing the results of staining (A: H & E staining, B: HLA staining) of TK-NOG-IL-6 mice and TK-NOG mice 2 weeks after LHum17003 donor cell transplantation.

FIG. 9 is a graph showing the results of staining (A: H & E staining, B: HLA staining) of TK-NOG-IL-6 mice and TK-NOG mice 3 weeks after LHum17003 donor cell transplantation.

FIG. 10 is a graph showing the results of staining (A: H & E staining, B: HLA staining) of TK-NOG-IL-6 mice and TK-NOG mice 4 weeks after LHum17003 donor cell transplantation.

FIG. 11 is a view showing the results of staining (A: H & E staining, B: HLA staining) of TK-NOG-IL-6 mice and TK-NOG mice 2 weeks after the transplantation of HUM4122B donor cells.

FIG. 12 is a graph showing the results of staining (A: H & E staining, B: HLA staining) of TK-NOG-IL-6 mice and TK-NOG mice 3 weeks after the HUM4122B donor cell transplantation.

FIG. 13 is a view showing the results of staining (A: H & E staining, B: HLA staining) of TK-NOG-IL-6 mice and TK-NOG mice 4 weeks after the HUM4122B donor cell transplantation.

FIG. 14 is a view showing liver tissue images (H & E staining and HLA staining) of TK-NOG-IL-6 mouse (A) and TK-NOG mouse (B) 8 weeks after transplantation.

FIG. 15 is a graph showing temporal changes in the concentration of human albumin in the serum of TKmut30-NOG-IL-6 mice and TKmut30-NOG mice in which human hepatocytes LHum17003 were transplanted.

FIG. 16 is a graph showing temporal changes in the presumed substitution index for each of TKmut30-NOG-IL-6 mice and TKmut30-NOG mice transplanted with human hepatocytes LHum 17003.

FIG. 17 is a diagram showing liver tissue images (H & E staining (A) and HLA staining (B)) of TKmut30-NOG-IL-6 mice 7 weeks after human hepatocyte LHum17003 transplantation.

FIG. 18 is a graph showing the results of measurement of the human albumin concentration in the serum of TKmut30-NOG-IL-6 mice and TK-NOG-IL-6 mice after human hepatocyte transplantation.

FIG. 19 is a graph showing the doubling time of human hepatocytes in TK-NOG-IL-6 mouse (A) and TK-NOG mouse (B).

FIG. 20 is a graph showing the results of measurement of the blood human albumin concentration (A) and cholinesterase activity (B) when an anti-IL-6 antibody is administered after transplantation of human hepatocytes into TK-NOG-IL-6 mice.

Detailed Description

The present invention will be described in detail below.

The present invention relates to a non-human vertebrate into which human hepatocytes have been transplanted and a method for producing the same.

The genetically modified vertebrate into which human hepatocytes are transplanted according to the present invention is a vertebrate to which human interleukin 6 (hereinafter referred to as human IL-6) is administered, or an animal in which human hepatocytes are transplanted into a vertebrate in which human IL-6 gene is maintained to be expressed in vivo and the human hepatocytes survive in the liver.

The vertebrate is not limited, and there may be mentioned: mouse, rat, rabbit, dog, cat, mini-pig, monkey, etc. The monkey includes marmoset monkey.

The animal to which the human hepatocytes have been transplanted to be used in the present invention is an animal from which human IL-6 and human hepatocytes are not eliminated by immunization, that is, an animal from which an immune response to a human is suppressed. Examples of such animals include animals whose immune function is reduced or lost and whose immune response to humans is inactivated, and for example, immunodeficient animals or immune-tolerant animals can be used.

An immunodeficient animal is an animal with reduced or absent immune function, and is an animal deficient in some or all of T cells, B cells, NK cells, dendritic cells, and macrophages. The immunodeficient animal can be prepared by irradiating the whole body with X-rays, or an animal with a genetically compromised immune function can be used. The immune-tolerant animal refers to an animal in which a specific immune response to a specific antigen is absent or suppressed, and in the present invention, the immune-tolerant animal is an animal in which immune tolerance is established for human IL-6 and human hepatocytes. As for the immunological tolerance against human IL-6 and human hepatocytes, the immunological tolerance is obtained by administering human IL-6 or human hepatocytes to an animal. To achieve immune tolerance, animals can be injected subcutaneously with human IL-6 or human hepatocytes, or administered orally.

In the present invention, an immunodeficient animal and an immune-tolerant animal are referred to as animals in which an immune response to a human is absent or reduced.

The animal species of the immunodeficient animal is not limited, and immunodeficient mice, immunodeficient rats, immunodeficient pigs, and the like can be suitably used.

As the immunodeficient mouse, there can be mentioned: nude mice, NOD/SCID mice, Rag2 knockout mice, mice after administration of asialo-GM1 antibody or TM β 1 to SCID mice, X-ray irradiated mice, and the like. Further, a knockout animal (hereinafter referred to as "dKO (double knockout) animal") obtained by adding an IL-2R γ knockout to these NOD/SCID mice or Rag2 knockout mice can also be used. For example, dKO mice (Rag 2KO, IL-2R) can be used^null). In the present invention, a dKO mouse having Balb/c as a genetic background is referred to as a Balb/c dKO mouse, and a mouse having NOD as a genetic background is referred to as an NOD dKO mouse. The genetic background of the mouse is not limited to this, and may be a strain of C57BL/6, C3H, DBA2 or IQI, a strain having SCID mutation and IL-2R γ knockout, or Rag2 knockout and IL-2R γ knockout mutation, or a strain having deletion of Jak3 protein responsible for signal transduction downstream of the common γ chain of IL-2 receptor and IL2R γ knockout^nullSimilarly, a knockout mouse obtained by adding a Jak3 knockout to a Rag2 knockout mouse, a knockout mouse combining SCID mutation and a Jak3 knockout mouse, and an inbred line, a non-inbred line, or a hybrid line (F1 hybrid) mouse obtained by crossing these mice may be used.

In addition, to exclude the miceAs a result of observation of the influence of immune cells such as NK cells, in addition to the administration of asialo-GM1 antibody to SCID mice as described above, other mice used in the present invention include genetically modified immunodeficient mice in which mutation is introduced into IL-2 receptor gamma chain gene to delete IL-2 receptor gamma chain and SCID mutation of genes involved in rearrangement of antigen receptor genes of T cells and B cells is located at two allelic positions. Examples of such mice include: NOG mice (NOD/SCID/gammac) mice derived from NOD/SCID mice and having a knockout of the same gamma chain of the IL-2 receptor^null(NOD/Shi-Scid, IL-2R γ KO mouse)), NSG mouse (NOD/Scid/IL2R γ)^null(NOD.Cg-Prkdc^scidIL2rgtm1Wjl/SzJ)), NCG mice (NOD-Prkdc)^em26Cd52IL2rg^em26Cd22NjuCrl), etc. In addition, genetically engineered immunodeficient mice NOJ (NOD/Scid/Jak 3) can also be used^null(NOD.Cg-Prkdc^scidJal3tm1card), in which Jak3 is deleted by introducing a mutation into the Jak3 gene, and SCID mutations of genes associated with rearrangement of antigen receptor genes of T cells and B cells are located at two allelic positions. Hereinafter, these animals in which the function of Prkdc gene and its gene product is deleted by scid mutation or the like and the normal function of IL2R γ gene product is lost by deletion and mutation of IL2R γ gene or loss of function of gene located downstream of signal transduction and its product are referred to as NOG mice ("NOG mouse" is a registered trademark), and can be used as hosts. Lymphocytes were not found in these mice, so NOG mice showed no NK activity and lost dendritic cell function. The preparation of NOG mice is described in WO 2002/043477. NSG mice are prepared in Ishikawa F.et al, Blood 106: 1565-.

The non-human vertebrate animal with a missing or reduced immune response, such as an immunodeficient animal used in the method of the present invention, is preferably an animal with liver damage by various liver damage-inducing methods. In this animal, human hepatocytes transplanted into the non-human vertebrate instead of hepatocytes of the non-human vertebrate that produced the liver injury survive in the liver of the non-human vertebrate.

The following methods can be mentioned as various methods for inducing liver injury.

(i) Liver damage is induced by maintaining thymidine kinase gene expression in the liver of a non-human vertebrate in which the immune response to the human is absent or reduced, and administering human hepatocytes with a suicide substrate to the non-human vertebrate.

(ii) Liver damage is induced by maintaining urokinase-type plasminogen activator gene expression in the liver of a non-human vertebrate in which the immune response to the human is absent or reduced.

(iii) Liver damage is induced by deletion of the fumarylacetoacetate hydrolase (Fah) gene in a non-human vertebrate in which the immune response to the human is absent or reduced.

(iv) Inducing liver damage by administering any one of the following compounds (a) to (e):

(a) carbon tetrachloride

(b) Acetaminophen

(c) d-galactosamine

(d) Thioacetamide

(e) An anti-Fas antibody.

Each method will be described in detail below.

(i) Method for inducing liver damage by maintaining thymidine kinase gene expression in the liver of a non-human vertebrate subject having a missing or reduced immune response to the human subject, and administering human hepatocytes together with a suicide substrate to the non-human vertebrate subject

The thymidine kinase gene is maintained in expression in the liver of a non-human vertebrate in this method.

Hereinafter, a case where the non-human vertebrate is a mouse will be described, but the mouse can be used for other non-human vertebrates. The immunodeficient mice used in the methods of the invention maintain thymidine kinase gene expression in the mouse liver. By "maintaining the thymidine kinase gene expressible in the liver of a mouse" is meant that the thymidine kinase gene described above is contained in and expressed in liver cells of a mouse. That is, a foreign thymidine kinase gene was introduced so as to be specifically expressed in mouse liver. The biological species from which the thymidine kinase gene is derived may be human or mammalian, or prokaryotic cells or viruses, and the biological species from which the thymidine kinase gene is derived is not limited, but is preferably human herpes simplex virus type 1-thymidine kinase (HSV-tk). Further, these thymidine kinase gene variants may be used. Examples of the variant include HSV-tk mutant clone # 30. HSV-tk mutant clone #30 is described in PNAS 1996, Vol93, pp 3525-3529. HSV-TK mutant clone #30 is referred to as TK mutant 30. The base sequence of the wild type HSV-TK is shown in SEQ ID NO. 1, the amino acid sequence is shown in SEQ ID NO. 2, the base sequence of TK mutant30 is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4. In the present invention, TK mutant30 and other variants are also included in the case of HSV-TK. Transgenes in which thymidine kinase is introduced by a suicide substrate that is metabolized by thymidine kinase to produce toxicity exhibit cytotoxicity only on cells having thymidine kinase activity, and therefore can be used as selective therapeutic agents that cause cell damage only on target cells. As such a suicide substrate, guanosine analogs can be used. More preferably, as these guanosine analogs, there can be mentioned: ganciclovir (ganciclovir; GCV), Valganciclovir (Valganciclovir; Val GCV), aciclovir (acyclovir) are commonly used as antiviral agents.

By "maintaining the thymidine kinase gene expressible in the liver of a mouse" is meant that the thymidine kinase gene described above is contained in and expressed in liver cells of a mouse.

In order to express the thymidine kinase gene of the present invention in the liver of a mouse, a regulatory gene that functions in the liver can be suitably used. As the regulatory gene, a regulatory gene of a gene encoding a protein that functions in hepatocytes is used. Herein, "regulatory gene" refers to a sequence acting on DNA that increases or decreases the transcription efficiency of a gene, and includes, but is not limited to, promoters, enhancers, upstream activating sequences, silencers, upstream inhibitory sequences, attenuators, and the like. The biological species from which the regulatory gene is derived is not limited to a specific biological species. In order that the thymidine kinase group may be appropriately expressed in the liver of a mouse, a regulatory gene derived from a mouse may be used.

The promoter is not particularly limited as long as it is a promoter that allows the thymidine kinase gene to be expressed in the liver. Examples can be given such as: albumin promoter, thyroxine transporter promoter, thyroxine-binding globulin promoter, organic anion transporter LST-1 promoter, alpha fetoprotein promoter, alpha-tocopherol transporter (alpha-TTP) gene promoter, etc.

The expression mechanism of a gene specifically expressed in the liver (promoter analysis of the gene) has been studied intensively. That is, it is known that a binding site for a transcription regulatory factor, a binding site for a liver-specific transcription factor existing in the liver in a large amount, and an upstream activating sequence responding to an extracellular stimulus such as a hormone are inserted into a narrow region 5' to the upstream of the transcription initiation site of a gene specifically expressed in the liver. Examples of such transcription factors include: HNF-1, HNF-3, HNF-4, HNF-6, C/EBP, DBP and the like. In the present invention, to maintain the thymidine kinase gene expressible in the liver of mice, the thymidine kinase gene is linked to an upstream activating sequence that functions in the liver of mice.

Therefore, it is possible to use an upstream activating sequence to which a liver-specific transcription factor potentially comprising a certain region such as the above-mentioned albumin promoter, thyroxine transporter promoter, thyroxine-binding globulin promoter, organic anion transporter LST-1 promoter, alpha-fetoprotein promoter, promoter region of alpha-tocopherol transporter (alpha-TTP) gene promoter region is bound by using the above-mentioned linkage. In addition, in other embodiments, liver-specific upstream activating sequences that have been identified can be integrated as exogenous genes by means of gene recombination.

It has also been shown that, as an enhancer for increasing the level of transcription from a promoter, it is present not only in the upstream activating sequence but also downstream of the transcription initiation point. For example, higher expression of the human angiotensinogen gene was seen in the liver in transgenic mice with a full-length 14kb human angiotensinogen gene comprising a5 'upstream 1.3kb and 3' downstream region. In addition, in HepG2 cells derived from human liver cancer, the presence of an enhancer was confirmed in a 3.8kb DNA fragment which is a downstream region of the transcription start point of the gene. In the present invention, such an enhancer can be suitably used in order to cause liver-specific expression of the thymidine kinase gene.

In the present invention, a 3' untranslated sequence including a tailing signal (poly a signal) may be used for liver-specific expression of the thymidine kinase gene.

In the present invention, to maintain the thymidine kinase gene expressible in the liver of mice, the thymidine kinase gene is linked to an upstream activating sequence that functions in the liver of mice. In addition, an enhancer or a 3' untranslated sequence containing a tailing signal, or the like can be linked. By inserting the thus-ligated genome into a vector containing a marker gene such as a drug resistance gene, an integration vector for preparing a gene expression unit for maintaining the thymidine kinase gene expressible in the liver of a host is prepared.

Transgenic mice into which the gene expression units prepared from the integration vector have been introduced can be prepared. For example, a transgenic mouse can be produced by a known method (Proc. Natl. Acad. Sci. USA (1980)77,7380-4).

Specifically, the prepared gene expression unit was introduced into totipotent cells of mice in order to maintain the expression of the target thymidine kinase gene in the liver of the host. Examples of totipotent cells into which genes are introduced include: fertilized eggs and early embryos, ES cells having differentiation pluripotency, cultured cells such as iPS cells, and the like. For example, by mating a female mouse to which an ovulation inducer has been administered with a normal male mouse, a fertilized egg into which the gene expression unit can be introduced can be appropriately recovered. Gene expression units are typically introduced into mouse zygotes by microinjection into the male pronuclei. After the fertilized egg cells are cultured in vitro, cells into which the gene expression unit is considered to be successfully introduced are selected. The selected cells were transplanted into the oviduct of a surrogate mother, and then transgenic chimeric mice were generated. As the surrogate mother, a female that forms a pseudopregnant state by mating with a male whose vas deferens has been cut is generally used.

By selecting an individual having an introduced gene integrated into somatic cells and germ cells from the obtained plurality of individuals, a transgenic mouse of interest can be produced.

As a mouse capable of maintaining the thymidine kinase gene expressed in its liver, there can be mentioned: a transgenic mouse produced by introducing a gene designed to maintain the expression of a target thymidine kinase gene in the liver of a host into a fertilized egg of the immunodeficient mouse. In addition, an immunodeficient mouse selected from offspring produced by mating the immunodeficient mouse with a transgenic mouse generated by introducing the designed gene into a fertilized egg of an inbred mouse of the immunodeficient mouse whose immune function is not deficient can be used as a mouse in which the characteristics of the immunodeficient mouse are maintained and the target thymidine kinase gene is expressed in the liver of the host. In the present invention, a mouse selected as a mouse which retains the properties of a NOG mouse and in which a target thymidine kinase gene is expressed in the liver of a host is referred to as a TK-NOG mouse. A method for producing TK-NOG mice is described in patent No. 507836. Examples of thymidine kinase genes retained by TK-NOG mice include: human herpes simplex virus type 1-thymidine kinase (HSV-tk) and variants thereof. Examples of the variant include the above-mentioned HSV-TK mutant clone #30(TK mutant 30). The mice that retained TKmutant30 were designated TKmut30-NOG mice. In the present invention, TKmut30-NOG mice are included in the case of what are called TK-NOG mice. TKmut30-NOG mice can be made by the same method as TK-NOG mice. That is, the expression unit for TK-NOG mice was prepared by replacing the Alb promoter used to prepare the expression unit with the thyroxine transporter promoter and the HSV-TK gene cDNA (from the start codon to the stop codon) with the HSV-TK mutant30 gene cDNA (from the start codon to the stop codon). As the genotype of TK-NOG mouse, there can be mentioned: Cg-Prkdc^scidIl2rg^tm1SugTg(Alb-UL23)7-2/SHiJic mice (TK-NOG), NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Ttr-UL23 mutant30)4-9/Shijic mice (TKmut30-4-9-NOG), NOD. Cg-Prkdc^scidIl2rg^tm1SugTg (Ttr-UL23mutant30)5-2/Shijic mice (TKmut30-5-2-NOG) and NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Ttr-UL23 mutant30)10-15/Shijic mice (TKmut 30-10-15-NOG).

Furthermore, an immunodeficient mouse selected from the offspring produced by the mating of the immunodeficient mouse and a transgenic mouse produced by introducing the above designed gene into a fertilized egg of an inbred mouse of the mouse, in which the immune function is partially deleted, as a mouse capable of maintaining the properties of the immunodeficient mouse and maintaining the expression of the target thymidine kinase gene in the liver of the host can be used. More specifically, preferred examples include: mice selected as mice that retain the properties of NOG mice and that can express the target thymidine kinase gene in the liver of the host, from offspring born by mating NOG mice with NOD/SCID transgenic mice produced by introducing the gene into fertilized eggs of the NOD/SCID mice, and by combining TK-NOG mice with Balb/cAdKO mice (RAG-2KO, IL-2R)^null) TK-Balb/cA dKO mice (HSV-Tk (+), SCID world, RAG-2KO, IL-2R) prepared by mating and repeatedly mating with Balb/cA dKO mice^null) Or by combining TK-NOG mice with NOD dKO mice (RAG-2KO, IL-2R)^null) TK-NOD dKO mice (HSV-Tk (+), SCID wild, RAG-2KO, IL-2R) prepared by mating and repeated mating with NOD dKO mice^null) By contacting TK-NOG mice with IQI/SCID, IL-2R^nullTK-IQI/NOG F1 mice (HSV-Tk (+), SCID, IL-2R) prepared by mouse mating^null) Then combined with IQI/SCID, IL-2R^nullTK-IQI SCID, IL-2R prepared by repeated mating of mice^nullMice, and the like.

By administering the guanosine analog to a mouse in which the thymidine kinase gene of the present invention is expressed in the liver, the guanosine analog is metabolized into a toxic substance in the liver cells of the mouse, and liver damage is caused to the mouse. Ganciclovir, which can be cited as a suitable example of the guanosine analog, is metabolized to ganciclovir-triphosphate in mouse hepatocytes to cause liver damage in mice. The method of administration of the guanosine analog to the mouse can be freely selected. For example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and application to the skin can be selected as appropriate. In addition to in vivo administration to mice by these common routes, in vivo administration to mice may also be carried out by mixing in the diet or in drinking water. The dosage of the guanosine analogue is not limited, and is 0.1 to 10mg/Kg of body weight, preferably 0.5 to 1.5mg/Kg of body weight. In addition, the composition can be added to drinking water at a concentration of 0.05-0.5 mg/mL, and the composition can be freely drunk by mice for 1 day to 1 week.

In addition to the above, as a method for causing liver damage in a mouse retaining the thymidine kinase gene of the present invention expressible in its liver, in addition to the administration of ganciclovir, there can be mentioned: the treatment with known liver injury-inducing substances such as carbon tetrachloride, D-galactosamine, pyrrolidine alkaloids, and 2-acetamidofluorene, and the surgical treatment such as surgical hepatectomy. In addition, in other embodiments, the mouse can also be made to develop liver damage by administering an anti-mouse Fas antibody to the mouse. The anti-mouse Fas antibody does not bind to the Fas antigen expressed in human hepatocytes but binds to the Fas antigen expressed in mouse hepatocytes, thereby specifically causing apoptosis in mouse hepatocytes.

(ii) Method for inducing liver damage by maintaining urokinase-type plasminogen activator gene expressible in liver of non-human vertebrate with missing or reduced immune response to human

Mice with NOG variants that maintain urokinase-type plasminogen activator gene expressible in their liver can also be used in place of the thymidine kinase gene described above. That is, a transgenic mouse can be used which is produced by introducing a gene expression unit prepared to maintain the urokinase-type plasminogen activator gene expressible in the liver of a host into totipotent cells of the mouse having NOG mutation. As the urokinase-type plasminogen activator gene, a polynucleotide encoding a mouse urokinase-type plasminogen activator can be used. Such a mouse is called a uPA-NOG mouse and can be prepared according to the description of Suemizu H.et al, Biochme Biophys Res Commun,377,248,2008.

Furthermore, mice with NOG variants that retain both thymidine kinase gene and urokinase-type plasminogen activator gene expressed in their liver can also be used. By appropriately selecting the period of liver damage by the action of thymidine kinase and the period of liver damage by the action of urokinase-type plasminogen activator, the survival of human hepatocytes after administration can be optimized.

The mouse can be obtained, for example, by: mice retaining thymidine kinase gene in their livers can be expressed, and mice having NOG mutation retaining urokinase-type plasminogen activator gene in their livers can be bred by appropriately combining them, thereby selecting mice having desired traits, i.e., progeny mice retaining both thymidine kinase gene and urokinase-type plasminogen activator gene in their livers and having NOG mutation. The present invention can also be produced, for example, by the following operations: a gene expression unit prepared for maintaining the expression of a urokinase-type plasminogen activator gene in the liver of a host is introduced into totipotent cells of a mouse in which a thymidine kinase gene is maintained in the liver of a host.

(iii) Method for inducing liver damage by deletion of fumarylacetoacetate hydrolase (Fah) gene in non-human vertebrate with absent or reduced immune response to human

Methods of inducing liver damage by deleting the Fah gene are described in Azuma h.et al, Nat Biotechnol,25,8, 2007. Faysal Elgilano et al, Am J Pathol,187,1,2017, describe the creation of Fah gene-deleted animals using pigs by CRISPR/Cas9 genome editing technology.

For example, human liver chimeric pigs can be easily made by deleting immunity-related genes using immunosuppressive agents, or by CRISPR/Cas9 genome editing techniques.

(iv) Method for inducing liver damage by administering any one of the following compounds (a) to (e)

(a) Carbon tetrachloride

(b) Acetaminophen

(c) d-galactosamine

(d) Thioacetamide

(e) An anti-Fas antibody.

The above-mentioned compounds are known as chemical substances that cause damage to the liver.

Administration of these chemicals to animals with reduced immunity to human hepatocytes (genetic immunodeficiency, acquired immune tolerance by sensitization during fetal or neonatal periods, or immunosuppressive agents) can also induce liver damage and can be made viable by transplantation of human hepatocytes.

The methods (i) to (iv) above may be carried out alone, or two, three or four methods may be used in combination.

The vertebrate used for producing the vertebrate into which the human hepatocytes have been transplanted according to the present invention may be any animal in which human IL-6 is present in the body of the aforementioned immunodeficient animal.

As a method for allowing human IL-6 to exist in vivo, the following methods can be mentioned:

1. introducing and expressing a human Il-6 gene into the immunodeficient animal in such a manner that the human Il-6 gene can be expressed; and

2. the immunodeficient animal is administered human IL-6.

The method of 1, may further include:

(1) mating an immunodeficient animal with a human IL-6 transgenic animal obtained by introducing a human IL-6 gene into the immunodeficient animal so that the human IL-6 gene can be expressed;

(2) administering cultured cells expressing human IL-6 gene to the above-mentioned immunodeficient animal to thereby cause the animal to have the cells; and

(3) the human IL-6 gene expression vector was inoculated to the above-mentioned immunodeficient animal.

The method (1) for mating an immunodeficient animal with a human IL-6 transgenic animal obtained by introducing a human IL-6 gene into the immunodeficient animal so that the human IL-6 gene can be expressed can be carried out, for example, by the following steps.

The human IL-6 gene expression unit may be introduced into a human IL-6 transgenic animal into which the human IL-6 gene is introduced so that the human IL-6 gene can be expressed. The human IL-6 gene expression unit includes a promoter, an enhancer, and further may include elements such as a tailing sequence. An example of a human IL-6 expression unit is shown in FIG. 1.

(i) Injecting a DNA fragment containing human IL-6cDNA into a fertilized egg of an immunodeficient animal

In this step, a vector into which a DNA fragment containing human IL-6cDNA has been introduced can be used. The injection of the DNA fragment may be performed by, for example, microinjection, electroporation, or the like.

(ii) (ii) culturing the DNA fragment obtained in (i) and injecting the DNA fragment into a fertilized egg to obtain a young newborn

The animals obtained in this procedure are called human IL-6 transgenic founder animals. Examples of the method for obtaining a newborn baby from a fertilized egg include the following methods: and culturing the fertilized eggs injected with the DNA fragments in vitro at 30-40 ℃ for 18-24 hours, transplanting the fertilized eggs into the uterus of a surrogate mother body, and implanting the fertilized eggs to obtain the newborn young.

(iii) (iii) a step of mating the founder animal obtained in (ii) with an immunodeficient animal

When the TK gene is introduced, animals having the TK gene introduced therein may be further bred.

The animals thus obtained were immunodeficient animals expressing human IL-6 in vivo.

For example, in the case of using TK-NOG mice as an immunodeficient animal, preparation is as follows.

(i) A DNA fragment containing human IL-6cDNA was microinjected into fertilized eggs prepared from female NOD mice and male NOG mice to obtain human IL-6 transgenic naive mice.

(ii) Scid and IL2Rg were obtained by mating human IL-6 transgenic naive mice with NOG mice^nullA variant of (1). This mouse was designated NOG-IL-6.

(iii) NOG-IL-6 mice were mated with TK-NOG mice. In the resulting miceMice bearing both the human IL-6 transgene and the HSVtk transgene are referred to as TK-NOG-IL-6 mice. Cg-Prkdc from, for example, NOD^scidIl2rg^tm1SugTg((Alb-UL23)7-2,CMV-IL-6)/ShiJic(TK-NOG-IL-6)、NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Ttr-UL23mutant30)4-9, CMV-IL-6/Shijic mice (TKmut30-4-9-NOG-IL-6), NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Ttr-UL23mutant30)5-2, CMV-IL-6/Shijic mice (TKmut30-5-2-NOG-IL6) and NOD.Cg-Prkdc^scidIl2rg^tm1SugTg (Ttr-UL23mutant30)10-15, CMV-IL-6/Shijic mice (TKmut30-10-15-NOG-IL 6).

By transplanting human hepatocytes into the thus obtained non-human vertebrate having human IL-6 in vivo, a non-human vertebrate into which human hepatocytes have been transplanted can be produced. In the case of a non-human vertebrate accompanied by genetic modification, it is referred to as a genetically modified non-human vertebrate.

As a method for transplanting human hepatocytes into a non-human vertebrate, a method of transplanting hepatocytes directly from the portal vein can be suitably employed in addition to a method of transplanting hepatocytes into a non-human vertebrate through the spleen. In addition, it may be implanted in the abdominal cavity or in a vein. The number of human hepatocytes to be transplanted at one time can be appropriately selected from 1 to 2,000,000 (2X 106) cells.

As the hepatocytes, normal hepatocytes or primary hepatocytes which are maintained (including culture, passage, and storage) in the presence of serum (e.g., fetal bovine serum) can be suitably used, and established hepatocyte cell lines are preferred. As the human hepatocytes to be transplanted, hepatocytes derived from any kind of cells may be used as long as they are cells in the human liver including human parenchymal hepatocytes. In the case of using normal hepatocytes, hepatocytes obtained from liver tissues of a subject can be used. As a method for collecting liver tissue, biopsy (biopsy) which is a known method may be mentioned in addition to resection at the time of surgery. Liver biopsy refers to a method of collecting liver tissue by directly penetrating a long and thin needle into the liver from the surface of the skin. Usually, the part punctured by the needle is the intercostal space in the lower part of the right chest. After confirming the safety of the puncture site with an ultrasonic examination device before the operation, the puncture site is sterilized. The skin is anesthetized to the surface of the liver, and the puncture needle is inserted after a small opening is cut in the skin of the puncture part. In addition, commercially available frozen human hepatocytes can also be used.

In the case of using primary hepatocytes, hepatocytes are appropriately separated from the collected liver or liver tissue by fractionating a cell suspension dispersed in ice-cold kirschner's fluid or the like by a known technique such as perfusion or osmosis by centrifugation or the like. Culture medium such as Williams' E supplemented with bovine serum at 5% CO₂The obtained hepatocytes were cultured at 37 ℃ for 24 hours in the presence of oxygen. In addition, cells obtained by culturing for about 1 week every 3 days with a medium such as ASF104 medium (ajinomotol) can also be used. The culture medium may be supplemented with a cell growth factor such as HGF and EGF, and the culture medium, three-dimensional culture, or the like may be appropriately changed.

When an established hepatocyte cell line is used, the type of hepatocyte is not particularly limited, and examples thereof include: SSP-25, RBE, HepG2, TGBC50TKB, HuH-6, HuH-7, ETK-1, Het-1A, PLC/PRF/5, Hep3B, SK-HEP-1, C3A, THLE-2, THLE-3, HepG2/2.2.1, SNU-398, SNU-449, SNU-182, SNU-475, SNU-387, SNU-423, FL62891, DMS153, and the like. These cells can be obtained from the American Type Culture Collection (ATCC) or the like. For example, Hep3B and HepG2 are registered with ATCC as accession numbers HB-8064 and HB-8065, respectively, and HuH-7 is registered with JCRB0403 accession number JCRB cell bank, national institute of research and development, institute of medicine and health, Nutrition.

In the case of using a non-human vertebrate animal in which an immune response to a human is absent or reduced, the animal may be induced to develop liver damage by a liver damage induction method. In the case of administration of human hepatocytes, human hepatocytes transplanted into the animal survive in the liver of the animal instead of hepatocytes of the animal after causing liver injury. A chimeric animal in which human hepatocytes account for a certain number or more can be produced by killing animal hepatocytes and proliferating human hepatocytes that survive in the liver of the animal. Examples of the chimeric animal include: an animal in which 50% or more, preferably 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 90% or more, particularly preferably 95% or more, 96% or more, or 97% or more of the animal liver is replaced with human hepatocytes and occupies the animal liver. This substitution rate is referred to as a chimerism rate.

In the case of using an immunodeficient animal into which the TK gene has been introduced or an animal having acquired immune tolerance to IL-6, a guanosine analogue or the like may be administered. The guanosine analog and the human hepatocytes may be administered to the animal simultaneously or separately. For example, a guanosine analog can be administered into the abdominal cavity of an animal or mixed in drinking water to be drunk, and then human hepatocytes can be transplanted from the spleen or vein.

Replacement of an animal hepatocyte with a human hepatocyte is referred to as human hepatocyte or liver reconstruction (repopulation), and an animal in which a liver is reconstituted by replacing an animal hepatocyte with a human hepatocyte is referred to as human hepatocyte reconstruction or human liver reconstruction chimeric animal. In the case where the animal is a mouse, it is referred to as a human hepatocyte reconstitution chimeric mouse or a human liver reconstitution chimeric mouse. In addition, a liver in which hepatocytes are replaced with human hepatocytes is called a humanized liver (humanized liver).

The present invention also includes human liver tissue reconstructed in the above-mentioned animals such as mice. The liver tissue has a three-dimensional structure or function of a human liver, and is reconstructed by substituting 75% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more, 96% or more, or 97% or more of hepatocytes of a non-human vertebrate with human hepatocytes. In addition, a hepatobiliary system of a human liver is constructed, which has a functional lobular structure of the human liver and normally exerts a function of excreting a foreign substance of the liver.

For example, in the case of comparing the case where human IL-6 is present with the case where human IL-6 is not present, that is, in the case of using TK-NOG-IL-6 mice, the case where TK-NOG mice are used in the absence of human IL-6, the proliferation of human hepatocytes is rapid, and the chimerism rate (survival rate) of human hepatocytes is also high. For example, since the number of human hepatocytes in a mouse correlates with the concentration of human albumin in the blood of the mouse, when the proliferation of human hepatocytes is compared using the concentration of human albumin in the blood of a mouse into which human hepatocytes have been transplanted as an index, the proliferation rate in a TK-NOG-IL-6 mouse is 2 times as high as that in a TK-NOG mouse at 2 weeks after transplantation, and the proliferation rate after 4 weeks after transplantation is 1.2 times or more, preferably 1.3 times or more, and more preferably 1.4 times or more as high as that in a TK-NOG mouse. In addition, in TK-NOG-IL-6 mice, the ratio of the number of mice with a chimerism ratio of more than 70% (substitution index 70%) to the number of mice transplanted with hepatocytes was 80% or more after the 4 th week of transplantation, while in TK-NOG mice, the ratio was 10% or less or 50% or less. When the ratio of the number of individual mice having a chimerism rate of 70% or more is used as an index, the chimerism rate (survival rate) of human hepatocytes in a TK-NOG-IL-6 mouse is 1.3 times or more, preferably 1.4 times or more, that of a TK-NOG mouse.

In addition, in the case of transplanting human hepatocytes into a genetically modified non-human vertebrate in the presence of human IL-6 in vivo, the doubling time of human hepatocytes in the non-human vertebrate is shorter than in the case of transplanting human hepatocytes into a genetically modified non-human vertebrate in the absence of IL-6.

For example, in the case of transplanting human hepatocytes into TK-NOG-IL-6 mice, the doubling time of human hepatocytes in the mice is shorter than when human hepatocytes are transplanted into TK-NOG mice in which IL-6 is not present.

The doubling time of human hepatocytes in the case of using a human liver chimeric mouse can be determined by the following method.

First, whole hepatocytes were recovered by collagenase perfusion of the liver of human liver chimeric mice. The number of recovered cells was counted by trypan blue staining. The HLA positive cell rate (human cell rate) was determined by flow cytometry. The number of recovered human cells was calculated by the number of recovered cells × HLA positivity. The doubling time can be calculated by the following equation.

The time (day) elapsed after the cell transplantation, day of isolation and recovery of hepatocytes, day of expansion rate (double) (number of cells recovered × HLA positivity rate) of the transplanted cells, number of cell divisions (double) ═ LOG (expansion rate, 2)

Doubling time (days) as the time elapsed after cell transplantation divided by the number of cell divisions

The doubling time of human hepatocytes in a mouse is at least 2.4 days (56.9 hours), preferably at least 3 days (72.0 hours) when human hepatocytes are transplanted into a TK-NOG-IL-6 mouse. The time for the human hepatocytes in the mouse to be multiplied when the human hepatocytes are transplanted into the TK-NOG-IL-6 mouse is at least 1.2 times, preferably at least 1.3 times, more preferably at least 1.4 times, and particularly preferably at least 1.5 times shorter than the time for the human hepatocytes to be multiplied in the mouse when the human hepatocytes are transplanted into the TK-NOG mouse. That is, the proliferation rate of human hepatocytes in a TK-NOG-IL-6 mouse is at least 1.2 times, preferably at least 1.3 times, more preferably at least 1.4 times, and particularly preferably at least 1.5 times faster than the proliferation rate of human hepatocytes in a mouse in which human hepatocytes are transplanted in a TK-NOG mouse.

As is clear from histology and immunohistochemistry, in TK-NOG-IL-6 mice, the proliferated foci disappeared after 4 weeks of transplantation and the hepatocytes were mostly replaced with human hepatocytes. The structure of the foci of proliferation of hepatocytes seen in TK-NOG mice is in a form after proliferation of cultured cells, whereas the structure of foci of proliferation of hepatocytes seen in TK-NOG-IL-6, and Tkmut30-NOG-IL-6 mice is significantly different from that of the form after proliferation of cultured cells, and has the following characteristics: the hepatic sinus structure is visible, and the hepatic lobule structure is close to the human lobule structure, and the structure of the interlobular artery and vein-interlobular bile duct called the portal triple is also well preserved. It is considered that, as described above, the high chimerization efficiency provided by the present invention, i.e., in other words, the faster destruction of mouse hepatocytes, exerts an unexpected effect of ultimately achieving histological reconstruction of human liver tissue, and further, the present invention can provide human liver reconstruction (repopulation) more efficiently in terms of the physiology of albumin production.

For example, a test substance is administered to a chimeric animal having human hepatocytes after about 60 days after transplantation of the human hepatocytes. Examples of the test substance include drug candidates. The drug candidate substance is a substance in the process of drug development, and substances required to predict drug interactions in the human body are appropriately listed. Such a substance may be a main component substance that is effective against the drug effect of the drug, or may be a composition containing a main component substance. The dose of the drug candidate substance varies depending on the kind of the disease to be treated, the kind of the substance composition, the administration route, and the like, and can be appropriately selected from the range of 0.1mg/kg body weight to 2000mg/kg body weight. The route of administration can be appropriately selected from oral administration, transdermal administration, subcutaneous administration, intravenous administration, intraperitoneal administration, and the like, depending on the type of the candidate drug substance and the dosage form to which the candidate drug substance is administered.

The degree of metabolism of a test substance such as a drug candidate in the liver of a chimeric animal and a metabolite can be obtained by appropriately measuring and identifying the concentration of the drug candidate in mouse plasma by a predetermined method in the technical field such as chromatography. The measurement can be performed by time-lapse measurement, which is measurement at one or more time points appropriately selected from the time of administration of the candidate substance to about 30 minutes to 24 hours.

Further, it is possible to determine whether the drug candidate is a substance that is easily metabolized by, for example, a CYP2D6, CYP2C9, or CYP2C 19-deficient person or a substance that is not easily metabolized, based on the concentration of the drug candidate in the plasma and the concentration of the substance metabolized by the drug metabolizing enzyme in the liver.

Human drug metabolizing enzymes are CYP1a1, CYP1a2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C10, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A3, CYP3a4, CYP3A5, CYP3a7, CYP4F1, CYP4a2, CYP4A3 and the like which are CYP group enzymes, and as the respective indicator compounds, for example, the indicator compound metabolized by CYP1a2 is caffeine, CYP2C9 is tolbutamide, CYP2D6 is dextromethorphan, CYP2C19 is omeprazole, CYP3a4 is erythromycin and the like.

After administering the pharmaceutical candidate substance to the chimeric animal of the present invention, a mixture of these indicator compounds is administered, and the concentration in the plasma of each compound is measured over time, whereby it can be determined whether the pharmaceutical candidate substance is a substance that promotes the activity of each enzyme or a substance that inhibits the activity of each enzyme. For example, when a drug candidate substance is administered, if the metabolism of caffeine is promoted and the concentration in plasma is decreased, it can be judged that the drug candidate substance specifically promotes the activity of CYP1a 2.

That is, the animal of the present invention can be used to evaluate how a drug administered to a human is metabolized in the liver of the human and what metabolites are produced, that is, to evaluate the plasma kinetics of the drug, identification of the metabolites, metabolic rate, and the like. From a metabolic point of view, it is possible to screen a medicine suitable for administration to a human and predict the amount of the medicine suitable for administration to the human. In addition, it can be used for research on proliferation and regeneration of human liver.

The candidate agent can be administered by any of a number of desired methods and/or methods suitable for agent delivery. For example, the candidate agent can be suitably administered by injection (e.g., intravenous injection, intramuscular injection, subcutaneous injection, or direct injection into a tissue for achieving a desired effect), oral administration, or any other preferred method. Generally, in vivo screening methods include: methods of delivering agents by various dosage forms and routes are provided for a plurality of animals receiving different amounts and concentrations of a candidate agent (from no administration to an amount of the agent that reaches an upper limit of the amount that can be well delivered to the animal). The drug may be administered alone, or may be administered in combination with two or more drugs, particularly when the combined administration of the drugs produces a synergistic effect.

As the candidate agent to be screened, there can be used: synthetic molecules, natural molecules, or recombinantly produced molecules (e.g., low molecular weight molecules; drugs; peptides; antibodies (including antigen-binding antibody fragments, e.g., fragments that confer passive immunity), or other immunotherapeutic drugs: endogenous factors (e.g., polypeptides and plant extracts) contained in eukaryotic or prokaryotic cells, etc.). Screening assays for agents with low toxicity to human cells are particularly important.

In addition, another object of the present invention is to provide a mouse model that can be used for searching, detecting, and identifying cells that can be used for cell transplantation therapy or the like to become a liver in the future, for example, hepatic stem cells. The number of generations required for the differentiation of hepatic stem cells into hepatocytes is not particularly limited, as long as hepatic stem cells will differentiate into hepatocytes in the future. That is, the present invention also provides a method for transplanting primary isolated cells obtained from human liver tissue into a mouse model via the splenomegaly vein, and searching for or further identifying cells that can be used in cell transplantation therapy or the like, such as human hepatic stem cells, into the liver in the future, from the liver tissue after survival. In addition, a cell population including cells that can be used as a liver in the future in cell transplantation therapy, such as human hepatic stem cells, can be collected from the liver tissue after survival. The present invention also provides a method for collecting a cell population containing cells that can be converted into liver cells in the future for use in cell transplantation therapy or the like, such as the hepatic stem cells. In place of transsplenomental portal vein transplantation, orthotopic transplantation may also be suitably used. Stem cells cultured in vitro under appropriate conditions and induced to differentiate into hepatocytes may also be suitably used for the present purpose in place of the primary isolated cells. Such methods of inducing differentiation into hepatocytes using stem cells are described, for example, in Gastroenterology (2009)136,990-999, and the like. In order to search for and identify hepatic stem cells from liver tissues using proliferation as an indicator, tissue immunostaining can be performed using an antigen that recognizes a marker for detecting hepatocytes and/or an antibody that recognizes proliferating cells. The marker for detecting hepatocytes can be appropriately selected from albumin, tyrosine aminotransferase (tyrosinase), glucose-6-phosphatase (glucose-6-phosphatase), Coagulation Factor (CF) VII, asialoglycoprotein receptor (asialoglycoprotein receptor), and Cytokeratin (cytokerin) 8/18. The cell growth marker can be appropriately selected from Ki-67 antigen, 5-bromo-2 '-deoxyuridine (5-bromo-2' -deoxyuridine) absorbability, PCNA, and the like. By using these markers, a cell population containing human hepatic stem cells can be obtained, hepatic cells expressing such markers can be suitably isolated using FACS Calibur (Becton Dickinson, japan) or the like, and the isolated hepatic stem cells can be suitably used for cell transplantation therapy or the like.

As described above, it is possible to confirm whether or not human hepatocytes transplanted to a genetically modified non-human vertebrate survive and proliferate using the concentration of human albumin in blood of the non-vertebrate as a human hepatocyte survival marker. In addition, almost no cholinesterase activity was observed in blood of mice and the like. Therefore, it is possible to confirm whether or not human hepatocytes transplanted into a non-human vertebrate survive and proliferate using the blood cholinesterase activity as a survival marker of human hepatocytes. In fact, in mice transplanted with human hepatocytes, the concentration of human albumin in blood is highly correlated with the blood cholinesterase activity.

Human livers constructed from genetically modified vertebrates in which human hepatocytes have been transplanted can also be removed and used for screening, and screening can be performed both in vivo (in vivo) and in vitro (in vitro) by using genetically modified vertebrates in which human hepatocytes have been transplanted and woven human livers.

When an anti-human IL-6antibody is further administered to a genetically modified non-human vertebrate into which human hepatocytes have been transplanted in the presence of human IL-6 in vivo, the human hepatocytes proliferate more rapidly in vivo than when not administered. That is, by administering an anti-human IL-6antibody to a genetically modified non-human vertebrate into which human hepatocytes have been transplanted, survival and proliferation of the transplanted human hepatocytes can be promoted. This is believed to be because anti-IL-6 antibodies act for a long time as a carrier protein for human IL-6 in non-human vertebrates in which human IL-6 is present, and protect it from degradation. The antibody may be a monoclonal antibody or a polyclonal antibody, preferably a polyclonal antibody. The amount of the anti-human IL-6antibody to be administered may be, for example, several μ g to several hundred μ g per individual. When the non-human vertebrate is a mouse, it is sufficient to administer 1. mu.g to 100. mu.g, preferably 2. mu.g to 20. mu.g, more preferably 2. mu.g to 10. mu.g, and particularly preferably 2. mu.g to 5. mu.g to an individual.

The present invention also encompasses a method for producing a genetically modified non-human vertebrate into which human hepatocytes have been transplanted, which comprises transplanting human hepatocytes into a non-human vertebrate in which an immune response to human is deficient or reduced in the presence of human IL-6 in vivo, and administering the human IL-6.

Examples

The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

[ example 1]

Proliferation of human liver cells in TK-NOG-IL-6 mice

An example is shown in which Human liver cells transplanted into a mouse can be significantly proliferated by using Human Interleukin 6(Human Interleukin-6)/TK-NOG double knockout transgenic mouse (TK-NOG-IL-6 mouse).

Human Interleukin 6(Human Interleukin-6: Human IL-6) gene expression units are described in the prior reports (PMID:29456539, Hanazawa A, Ito R, Katano I, Kawai K, Goto M, Suemizu H, Kawakami Y, Ito M, Takahashi T. (2018), Generation of Human immunological competent Cell sites in Human Interleukin-6Transgenic NOG Mice. front Immunol,9,152) (FIG. 1). That is, a DNA fragment containing Human IL-6cDNA whose expression is controlled by Human Cytomegalovirus (CMV) immediate early enhancer and promoter was microinjected into fertilized eggs prepared from female NOD mice and male NOG mice to obtain Human IL-6 transgenic naive mice. Scid and IL2Rg were obtained by mating human IL-6 transgenic naive mice with NOG mice^nullA variant of (1). The strain of the identified mice is represented by NOD^scidIl2rg^tm1SugTg (CMV-IL-6)/ShiJic is indicated by the gene notation, which is abbreviated as NOG-IL-6. Cg-Prkdc among the pups from the mating of NOG-IL-6 and TK-NOG mice, mice harboring both the Human Interleukin-6 transgene and the HSVtk transgene were derived from NOD.Cg-Prkdc^scidIl2rg^tm1SugTg ((Alb-UL23)7-2, CMV-IL-6)/ShiJic, which is designated by the genetic code, is abbreviated as TK-NOG-IL-6 mouse.

Induction of liver injury

Ganciclovir (GCV) sodium (Denosine-IV; Mitsubishi tannabe Pharma) dissolved in distilled water was administered to the mouse intraperitoneally once or twice every other day. In addition, Valganciclovir (ValGCV) (Valganciclovir Hydrochloride; Sigma-Ardrich, Merck) was orally administered in place of GCV dissolved in drinking water. The extent of liver damage was investigated by biochemical serum examination and pathological analysis. Heparin blood was collected 1 week after GCV or ValGCV administration, and after plasma isolation, clinical chemistry analysis (alanine aminotransferase; ALT) was performed by FUJI DRI-CHEM7000 (Fujifilm).

Transplantation of human hepatocytes

As the donor cells, commercially available frozen human hepatocytes (Biopredic, 31-year-old male LHum170003, Lonza, 35-year-old HUM4122B) were used. The transfer to TK-NOG-IL-6 and TK-NOG was carried out by the following method. Adult TK-NOG-IL-6 and TK-NOG receptor mice aged 6 to 8 weeks were administered 48 hours after administration of ValGCV solution (0.06 to 0.08mg/mL) as drinking water, and blood was collected 1 week after the start of administration to measure plasma ALT value. Individuals showing plasma ALT values of 600U/L or more were used as recipients for human hepatocyte transplantation. The number of human hepatocytes and their survival rate were calculated by trypan blue exclusion method using a hematocrit meter. Approximately 1X 10 suspended in 40. mu.L of William's medium E medium using a 29 gauge needle syringe (Myjector) for subcutaneous administration of insulin⁶One live hepatocyte was administered into the spleen.

Human albumin assay

A small amount of blood was collected from the fundus venous plexus every week using a polyethylene tube 1 week after human hepatocyte transplantation. Human albumin concentrations were determined using a human albumin ELISA quantification kit (Bethy Laboratories) diluted 5,000-250,000 fold in TBS (Tris buffered saline) containing 1% bovine serum albumin/0.05% Tween 20. The threshold concentration was 0.016 mg/mL.

Histology and immunohistochemistry

Formalin-fixed liver was embedded in paraffin to prepare a5 μm thick section, and hematoxylin was added&Eosin staining. The sections to be subjected to the immunohistochemical staining were placed in a target regenerative solution (0.1M citrate buffer, pH 6.0; 1mM EDTA, pH 9.0) and autoclaved for 10 minutes, followed by standing at room temperature for 20 minutes. Monoclonal mouse anti-human HLA-classI-A, B, C antibody (clone EMR 8-5; Hokudo) was used as the primary antibody. Antibodies (Histofine Simple Stain Mouse MAX PO (M); Nichirei Bioscience) and diaminobenzidine (DAB; Dojindo Laboratories) matrices (0.2M) were labeled with amino acid polymer/peroxidase complexg/mL 3,3' -diaminobenzidine tetrahydrochloride, 0.05M Tris-HCl, pH 7.6, and 0.005% H₂O₂) Mouse Ig was visualized for brightfield immunization group chemistry. Sections were counterstained with hematoxylin. The images were taken using an Axio Imager (Carl Zeiss) equipped with an axioCam HRm and an axioCam MRc5 CCD cameras (Carl Zeiss).

Separation and purification of human hepatocytes

Hepatocytes were isolated from TK-NOG-IL-6 mice 5 weeks after human hepatocyte transplantation by a two-step collagenase perfusion method. Briefly, a 27G winged needle was inserted into the inferior vena cava, secured with adhesive and then the portal vein was severed. Next, the liver was perfused with 1 Xliver perfusion medium (manufactured by Thermo Fisher Scientific Co.) at 37 ℃ for 7 minutes (6 mL/min). Next, the perfusion medium was replaced with 0.15% collagenase medium [360U/mL collagenase type IV (CLSS 4; Worthington Biochemical Corporation, Rickward, N.J.; 140U/mL collagenase type IV (C1889; Sigma-Aldrich)/mL, 0.6mg/mL CaCl₂10mM HEPES (pH7.4), and 10mg/mL gentamicin]The infusion was performed at 1.5 mL/min for 10 minutes. The livers were harvested, transferred to a petri dish containing 50mL of Phosphate Buffered Saline (PBS) containing 1% fetal bovine serum (FBS; Thermo Fisher Scientific), and shaken steadily to disperse the cells from the collagenase digested livers. The liver cells were filtered through a 100 μm nylon filter and centrifuged at 50 Xg for 4 minutes at 4 ℃. The cells were washed 2 times with ice-cold PBS 50mL containing 1% FBS. Dead cells were removed by density gradient centrifugation (60 × g, 7 min) with 27% Percoll (GE Healthcare, Buckinghamshire, UK), and washed 3 times at 50 × g for 4 min with ice-cold PBS 50mL containing 1% FBS. After 3 washes, cells were suspended in 44mM NaHCO containing 10% FBS₃1mM sodium pyruvate, and two antibiotics (50 units/mL penicillin G and 50. mu.g/mL streptomycin; Sigma-Aldrich) in Duchen's modified eagle medium (DMEM; Sigma-Aldrich)). The cell number and the survival rate of the prepared Liver cells (Hu-Liver cells) were determined by trypan blue exclusion test.

Flow cytometry analysis

The ratio of Human Leukocyte Antigen (HLA) -expressing human cells to mouse H-2 kd-expressing mouse cells to the total number of isolated and purified Hu-Liver cells was determined by flow cytometry analysis using BD FACSCAnto (BD Biosciences). Briefly, cells were stained with an anti-HLA mouse monoclonal antibody (clone G46-2.6; BD Biosciences) and an anti-mouse H-2kd mouse monoclonal antibody (clone SF 1-1.1; BD Biosciences), while cell survival was evaluated with propidium iodide (BD Biosciences). Data analysis used the BD FACSDiva software program (BD Biosciences) and the FlowJo program (Tree Star, San Carlos, Calif., USA).

The results are as follows.

Transplantation of human hepatocytes (LHum17003 donor cells) into TK-NOG-IL-6 mice

Human albumin in the serum of TK-NOG-IL-6 mice and TK-NOG mice was measured by ELISA every week from the second week of human hepatocyte transplantation to 5 weeks after transplantation. The results are shown in FIG. 3. Patent publication No. 5073836 shows that the ratio of human hepatocytes to human hepatocytes (presumed substitution index (RI)), that is, the number of human hepatocytes, in the liver of TK-NOG mice into which human hepatocytes have been transplanted is highly correlated with the concentration of human albumin in serum. In addition, Suemizu H.et al, Pest Management Science,74,1424,2018, indicate that in the liver of TK-NOG mice, the proportion of human hepatocyte substitution (presumed substitution index (RI)), i.e., the number of human hepatocytes, is also highly correlated with the butylcholinesterase (CHE) activity in serum. After the liver cells are transplanted for 1 week, the concentration of human albumin in blood of the TK-NOG-IL-6 mouse is obviously increased compared with that of the TK-NOG mouse, and after the liver cells are transplanted for 2 weeks, the concentration of the human albumin in blood of the TK-NOG-IL-6 mouse reaches 2 times that of the TK-NOG mouse. Then, at 3 weeks, 4 weeks, TK-NOG-IL-6 mice still showed significantly higher blood human albumin concentrations compared to TK-NOG mice. TK-NOG-IL-6 mice still had a higher tendency at 5 weeks post-transplantation, but the human albumin concentration in blood no longer had a significant difference, indicating that rapid proliferation of human hepatocytes reached plateau 5 weeks post-transplantation.

For the purpose of drug metabolism experiments, mice having human hepatocytes, which are assumed to have a substitution index of 70% or more, are generally used. The concentration of human albumin in blood for each individual of the mice transplanted with human hepatocytes is shown in fig. 4. In TK-NOG-IL-6 mice transplanted with human hepatocytes, the substitution index was over 70% in most of the mice 4 weeks after transplantation (10 out of 13, 76.9%), while in mice with human hepatocytes (TK-NOG mice), the substitution index was over 70% and very few (2 out of 17, 11.8%). At 5 weeks post-transplantation, 9 of 16 (56.2%) of TK-NOG mice had a substitution index of over 70%, while 8 of 9 (88.9%) of TK-NOG-IL-6 mice had a substitution index of over 70%. It is thus predicted that TK-NOG-IL-6 allows the proliferation of transplanted human hepatocytes to be early and allows mice having human hepatocytes with a substitution index of 70% or more to be obtained efficiently.

Transplantation of human hepatocytes (HUM4122B donor cells) into TK-NOG-IL-6 mice

Human albumin in the serum of TK-NOG-IL-6 mice and TK-NOG mice was measured by ELISA every week from the second week of human hepatocyte transplantation to 5 weeks after transplantation. The results are shown in FIG. 6. Patent publication No. 5073836 shows that the ratio of human hepatocytes to human hepatocytes (presumed substitution index (RI)), that is, the number of human hepatocytes, in the liver of TK-NOG mice into which human hepatocytes have been transplanted is highly correlated with the concentration of human albumin in serum. Before 5 weeks of human hepatocyte transplantation, if the blood human albumin concentration of the TK-NOG-IL-6 mouse and the TK-NOG mouse is compared, the TK-NOG-IL-6 mouse is always remarkably higher, and particularly the blood human albumin concentration reaches 3 times of that of the TK-NOG mouse after 3 weeks of human hepatocyte transplantation. Then, at 4 weeks, 5 weeks, TK-NOG-IL-6 mice still showed significantly higher blood human albumin concentrations compared to TK-NOG mice.

For the purpose of drug metabolism experiments, mice having human hepatocytes, which are assumed to have a substitution index of 70% or more, are generally used. The concentration of human albumin in blood for each individual of the mice transplanted with human hepatocytes is shown in fig. 7. Of TK-NOG-IL-6 mice transplanted with human hepatocytes, the substitution index of most of the mice 4 weeks after transplantation (5 out of 6, 83.3%) exceeded 70%, whereas none of the mice with human hepatocytes (TK-NOG mice) exceeded 70% (0 out of 4, 0.0%). At 5 weeks post-transplantation, none of the 3 TK-NOG mice (0.0%) had a substitution index exceeding 70%, while the 3 TK-NOG-IL-6 mice (100%) had a substitution index exceeding 70%. This indicates that TK-NOG-IL-6 has a high proliferation rate of human hepatocytes after transplantation, and TK-NOG mice have a high survival rate of cells with poor viability, and therefore, it is expected that a large number of cells can be used, and mice having human hepatocytes with a substitution index of 70% or more can be obtained with high efficiency.

Next, the histological and immunohistochemical results of the liver of TK-NOG-IL-6 mice obtained 2, 3 and 4 weeks after transplantation of human hepatocytes, respectively, are shown. The serial sections were stained with H & E and Huma Leucocyte Antigen (HLA), and the results are shown in FIGS. 8 to 13. FIG. 8 shows the staining results of TK-NOG-IL-6 mice and TK-NOG mice 2 weeks after LHum17003 donor cell transplantation, FIG. 9 shows the staining results 3 weeks after transplantation, and FIG. 10 shows the staining results 4 weeks after transplantation. Most human hepatocytes were present as large proliferating foci in host liver tissue 2 weeks after transplantation (fig. 8). After 3 weeks of transplantation, large pools of human hepatocytes coalesce, occupying more than half of the host liver tissue (fig. 9). Furthermore, the substitution index of human hepatocytes estimated from the serum human albumin concentration after 4 weeks of transplantation exceeded 70%, and in fact, large foci that were visible 3 weeks before transplantation disappeared, and the host liver tissue was largely replaced by human hepatocytes (fig. 10). FIG. 11 shows the staining results of TK-NOG-IL-6 mice and TK-NOG mice 2 weeks after transplantation of HUM4122B donor cells, FIG. 12 shows the staining results 3 weeks after transplantation, and FIG. 13 shows the staining results 4 weeks after transplantation. Most human hepatocytes were present as large proliferating foci in host liver tissue 2 weeks after transplantation (fig. 11). At 3 weeks of transplantation, large aggregates of human hepatocytes coalesced and occupied more than half of the host liver tissue (fig. 12). Furthermore, the substitution index of human hepatocytes estimated from the serum human albumin concentration after 4 weeks of transplantation exceeded 70%, and in fact, large foci that were visible 3 weeks before transplantation disappeared, and the host liver tissue was largely replaced by human hepatocytes (fig. 13). Liver tissue images of TK-NOG-IL-6 mice and TK-NOG mice 8 weeks after transplantation are shown in FIG. 14. When the liver tissue H & E of TK-NOG mouse is stained, the human liver cells proliferate without gaps, and a large amount of vacuoles are visible in the human liver cells, while when the liver tissue H & E of TK-NOG-IL-6 mouse is stained, the liver tissue H & E has the following characteristics: chordal structures are visible in the hepatic lobules, and inter-lobular pulsating flow, called portal triple, and inter-lobular vein, inter-lobular bile duct, also show very close to human architecture.

It was verified whether a mouse with human hepatocytes with a high substitution index, which can be made in a short time by using TK-NOG-IL-6 mouse, could be used as a source of hepatocyte supply for in vitro studies. That is, liver cells were isolated from 3 TK-NOG-IL-6 mice by a two-step collagenase perfusion method before 5 weeks after transplantation of rapid proliferation of human liver cells. Table 1 shows the information on the isolation and purification of hepatocytes from TK-NOG-IL-6 mice after human hepatocyte transplantation. The number of liver cells recovered was 3.9X 10 per average⁷The average survival rate showed a value as high as 90.8%. Although positive selection of human cells or negative selection of mouse cells was not performed, the human cell rate was up to 94.4% on average relative to the mouse cell contamination rate of 2.3% on average, and the human cell occupancy was absolutely superior. Inoculated into a vessel coated with type 1 collagen, showing the morphology of the liver cells after 48 hours (fig. 5). Polygonal cells are formed into a sheet in a shape of a paving stone, and many special binuclear cells are visible among hepatocytes, and exhibit a morphology similar to that of general human hepatocytes.

The number of cells transplanted to 1 was 0.1X 10⁷The number of cells recovered was about 4X 10⁷Thus, it is shown that the present invention is a breakthrough technique that can actually expand 40-fold human hepatocytes, which are absolutely no longer proliferating in vitro, within only 5 weeks. At present, in human clinical materials that are not regularly available, fresh human hepatocytes cannot be supplied as planned, but by using the animal produced by the present invention as a source for hepatocyte supply for in vitro studies, fresh human hepatocytes can be supplied as planned and as needed.

[ Table 1]

In addition, cases with high substitution index are addedIt was verified whether a mouse having human hepatocytes can be used as a source for hepatocyte provision in an in vitro study, wherein the mouse can be prepared in a short time by using a TK-NOG-IL-6 mouse. That is, liver cells were isolated from 43 TK-NOG-IL-6 mice by a two-step collagenase perfusion method 5 weeks after transplantation of rapidly proliferating human liver cells (Biopredic: 31-year-old male LHum170003, 5-year-old male LHum170003, Lonza: 30-year-old female HUM4119F, 0.9-year-old male HUM4282, 12-year-old female HUM 181001B). Table 2 shows the information (5 representative examples) obtained when purified hepatocytes were isolated from TK-NOG-IL-6 mice after human hepatocyte transplantation. The number of liver cells recovered from 43 cells was 11.7X 10 per average⁷The average survival rate showed a value as high as 87.8%. Although positive selection of human cells or negative selection of mouse cells was not performed, the human cell rate averaged up to 95.3% relative to the mouse cell contamination rate, which averaged 2.9%, and the human cells occupied an absolute preponderance.

The number of cells transplanted to 1 was 0.03X 10⁷The number of cells that can be recovered is about 11.6X 10⁷Thus, it is shown that the present invention is a breakthrough technique that can actually expand 370-fold human hepatocytes, which are absolutely unable to proliferate in vitro, within only 5 weeks.

[ Table 2]

Transplantation of human hepatocytes (LHum17003 donor cells) into TKmut30-NOG-IL-6 mice

Further, an example is shown in which Human liver cells transplanted into a mouse can be significantly proliferated by using a TKmut30-NOG mouse and a double knockout transgenic mouse of Human Interleukin 6(Human Interleukin-6) (TKmut-NOG-IL-6 mouse) in which a variant TKmut30 of the Human herpes simplex virus type 1-thymidine kinase (HSV-TK) gene is placed under the control of a thyroxine transporter promoter.

As an expression unit of a human herpes simplex virus type 1-thymidine kinase gene variant strain (HSV-TK mutant30) (FIG. 2), a mouse formazan-like gene variant was includedThe DNA fragment of HSV-TKmutant30 cDNA expressed by the promoter of the acinin transporter gene is injected into fertilized eggs made of female NOD mice and male NOG mice to obtain HSV-TKmutant30 transgenic first-building mice. Scid and IL2Rg were obtained by mating HSV-TKmutant30 transgenic naive mice with NOG mice^nullA variant of (1). The strain of the identified mice is represented by NOD^scidIl2rg^tm1SugTg (Ttr-UL23 (commonly referred to as tk) mutant30)/Shijic, which is abbreviated as TKmut 30-NOG. Cg-Prkdc of mice bearing both the Human Interleukin-6 transgene and the HSVtk mutant30 transgene in the young animals from TKmut30-NOG mice mating with NOG-IL-6 mice^scidIl2rg^tm1SugTg ((Ttr-UL23 mutant30), CMV-IL-6)/Shijic, which is designated by the genetic code and is abbreviated as TKmut30-NOG-IL-6 mouse.

Induction of liver injury

Valganciclovir (ValGCV) (Valganciclovir Hydrochloride; Sigma-Ardrich, Merck) dissolved in distilled water is added to drinking water at a concentration of 0.05-0.5 mg/mL and is orally administered by free drinking water for 24-72 hours. In addition, Ganciclovir (GCV) sodium (Denoside-IV; Mitsubishi Tanabe Pharma) was administered to the mouse intraperitoneal cavity in place of ValGCV either singly or 2 times every 1 day. The extent of liver damage was investigated by biochemical serum examination and pathological analysis. Heparin blood was collected 1 week after GCV or ValGCV administration, and after plasma isolation, clinical chemistry analysis (alanine aminotransferase; ALT) was performed by FUJI DRI-CHEM7000 (Fujifilm).

Transplantation of human hepatocytes

As donor cells, commercially available frozen human hepatocytes (Biopredic, 31-year-old male LHum170003) were used. The transfer to TKmut30-NOG-IL-6 and TKmut30-NOG was performed by the following method. Adult TKmut30-NOG-IL-6 and TKmut30-NOG receptor mice 6 to 8 weeks old were administered 72 hours while ValGCV solution (0.4mg/mL) was used as drinking water, and blood was collected 1 week after the start of administration to determine plasma ALT values. Individuals showing plasma ALT values of 600U/L or more were used as recipients for human hepatocyte transplantation. Human liver details were calculated by trypan blue exclusion and using a hematocrit meterThe number of cells and the survival rate thereof. Using a 29 gauge needle syringe (Myjector) for subcutaneous administration of insulin, approximately 1X 10 aliquots of insulin were suspended in 40. mu.L William's medium E medium⁶The live hepatocytes of (1) are administered into the spleen.

Human albumin assay

A small amount of blood was collected from the fundus venous plexus every week by using a polyethylene tube as appropriate 1 week after the transplantation of human hepatocytes. Human albumin concentrations were determined using a human albumin ELISA quantification kit (Bethy Laboratories) diluted 5,000-250,000 fold in TBS (Tris buffered saline) containing 1% bovine serum albumin/0.05% Tween 20. The threshold concentration was 0.016 mg/mL.

Human albumin was assayed in the serum of TKmut30-NOG-IL-6 mice by ELISA 4 weeks after human hepatocyte transplantation. In addition, human albumin was measured in the serum of TKmut30-NOG mice by ELISA 4.7 weeks after human hepatocytes were transplanted. The results are shown in FIG. 15. Patent publication No. 5073836 shows that the ratio of human hepatocyte replacement (presumed substitution index (RI)), that is, the number of human hepatocytes, is highly correlated with the concentration of human albumin in serum in the liver of TK-NOG mice into which human hepatocytes have been transplanted, and that the number of human hepatocytes is highly correlated with the concentration of human albumin in serum in TKmut30-NOG mice. At a time 5 weeks before human hepatocyte transplantation, when comparing the human albumin concentration in blood of TKmut30-NOG-IL-6 mice with TKmut-NOG mice, a significantly poor test could not be performed due to the smaller number of experiments, but showed as high viability as TK-NOG-IL-6 in TKmut30-NOG-IL-6 mice, reaching 5 times that of TKmut30-NOG mice.

For the purpose of drug metabolism experiments, mice with human hepatocytes, which are assumed to have a substitution index of 70% or more (fig. 16, 70% chimera), are generally used. The presumed substitution index for each individual of the mice transplanted with human hepatocytes is shown in fig. 16. Of the TKmut30-NOG-IL-6 mice transplanted with human hepatocytes, all the transplanted mice (2 out of 2, 100%) had a substitution index of more than 70% after 4.7 weeks of transplantation, while of the TKmut30-NOG mice not expressing human IL-6, no mice (0 out of 22, 0.0%) had a substitution index of more than 70%.

FIG. 17 shows the staining of TKmut30-NOG-IL-6 mice 7 weeks after LHum17003 donor cell transplantation. Host liver tissue was largely replaced by human hepatocytes (fig. 17). Furthermore, H & E staining, as well as TK-NOG-IL-6 mice, had the following characteristics: chordal structures are visible in the hepatic lobules, and the structure of the interlobular pulse, the interlobular vein, and the interlobular bile duct, known as the portal triad, also shows a very close approach to the human structure.

FIG. 18 shows the results of measurement of human albumin in the serum of TKmut30-NOG mouse expressing human IL-6 (TKmut30-NOG-IL-6) and TK-NOG mouse (TK-NOG-IL-6) by ELISA method every week from the second week after transplantation of human hepatocytes to 5 weeks after transplantation. It was also revealed that the same effect was obtained by the presence of human IL-6 in mice in which a human herpes simplex virus type 1-thymidine kinase gene variant (HSV-TK mutant30) was used as the human herpes simplex virus type 1-thymidine kinase gene and expression was controlled by the mouse thyroxine transporter gene promoter, as in TK-NOG mice. Note that, with regard to TKmut30-NOG mice, data were not obtained over 2, 3, and 4 weeks.

This short-time replacement by human hepatocytes is a phenomenon specific to mice in which human IL-6 is present in vivo, and is not seen in TK-NOG mice and TKmut30-NOG mice, which do not have human IL-6 Tg. This indicates that human IL-6 has a significant effect of promoting the proliferation of human hepatocytes transplanted into a human body, and can be used as a method for proliferating human hepatocytes in a non-human animal body. The animal having human hepatocytes thus produced can be used as a source for providing hepatocytes for in vitro studies using human hepatocytes as well as for drug metabolism and liver studies.

[ example 2]

Determination of the doubling time of human hepatocytes transplanted into TK-NOG-IL-6 mice

After proliferation of human hepatocytes transplanted to mice, the proliferation was stopped when the liver reached a certain size. Since the time elapsed after the cessation of cell proliferation is no longer related to the number of proliferating cells, it is necessary to obtain the minimum doubling time, i.e., the maximum proliferation rate of the cell, before the cell proliferation is stopped. Thus, at various elapsed days after human hepatocyte transplantation, hepatocytes were isolated from TK-NOG-IL-6 mice, TK-NOG mice by the above-described method, and the cell number and survival rate were determined by trypan blue exclusion test. HLA positive cell rate (human cell rate) was then determined by flow cytometry analysis. The doubling time of human hepatocytes transplanted into mice was determined by the following formula, and the minimum doubling time was determined from a graph obtained by plotting the elapsed time after cell transplantation on the horizontal axis and the doubling time on the vertical axis.

Doubling time (days) as the time elapsed after cell transplantation divided by the number of cell divisions

Day after cell transplantation for separating and recovering hepatocytes (day), day of performing hepatocyte transplantation

Cell division times LOG (amplification rate, 2)

Amplification rate (fold) — (number of recovered cells × HLA positivity ÷ number of transplanted cells) ÷ expansion rate

1. Materials and methods

(1) Separation and purification of human hepatocytes

Hepatocytes were isolated from TK-NOG-IL-6 mice and TK-NOG mice after the 7 th day of human hepatocyte transplantation by a two-step collagenase perfusion method. Briefly, a 27G winged needle was inserted into the inferior vena cava, secured with adhesive and then the portal vein was severed. Next, the liver was perfused with 1 Xliver perfusion medium (manufactured by Thermo Fisher Scientific Co.) at 37 ℃ for 7 minutes (6 mL/min). Next, the perfusion medium was replaced with 0.15% collagenase medium [360U/mL collagenase type IV (CLSS 4; Worthington Biochemical Corporation, Rickward, N.J.; 140U/mL collagenase type IV (C1889; Sigma-Aldrich)/mL, 0.6mg/mL CaCl₂10mM HEPES (pH7.4), and 10mg/mL gentamicin]The infusion was performed at 1.5 mL/min for 10 minutes. Livers were harvested, transferred to a petri dish containing 50mL Phosphate Buffered Saline (PBS) containing 1% fetal bovine serum (FBS; Thermo Fisher Scientific), and shaken steadily to disperse cells from the collagenase digested livers. The liver cells were filtered through a 100 μm nylon filter and centrifuged at 50 Xg for 4 minutes at 4 ℃. The cells were washed 2 times with ice-cold PBS 50mL containing 1% FBS. Cell determination of prepared Liver cells (Hu-Liver cells) by Trypan blue exclusion testCounting and survival rate. In the case of a survival rate of less than 70%, dead cells were removed by density gradient centrifugation (60 × g, 7 minutes) with 27% Percoll (GE Healthcare, Buckinghamshire, UK), and washed repeatedly 3 times at 50 × g for 4 minutes with ice-cold PBS 50mL containing 1% FBS. After 3 washes, cells were suspended in 44mM NaHCO containing 10% FBS₃1mM sodium pyruvate, and two antibiotics (50 units/mL penicillin G and 50. mu.g/mL streptomycin; Sigma-Aldrich) in Duchen's modified eagle medium (DMEM; Sigma-Aldrich)). The cell number and the survival rate of the prepared Liver cells (Hu-Liver cells) were determined by trypan blue exclusion test.

(2) Flow cytometry analysis

The ratio of Human Leukocyte Antigen (HLA) -expressing human cells to mouse H2 kd-expressing mouse cells to the total number of isolated and purified Hu-lever cells was determined by flow cytometry analysis using BD facscan (BD Biosciences). Briefly, cells were stained with an anti-HLA mouse monoclonal antibody (clone G46-2.6; BD Biosciences) and an anti-mouse H-2kd mouse monoclonal antibody (clone SF 1-1.1; BD Biosciences), while cell survival was evaluated with propidium iodide (BD Biosciences). Data analysis used the BD FACSDiva software program (BD Biosciences) and the FlowJo program (Tree Star, San Carlos, Calif., USA).

2. Results

FIG. 19A shows the results of measurement of the doubling time of human hepatocytes transplanted to TK-NOG-IL-6 mice, and FIG. 19B shows the results of measurement of the doubling time of human hepatocytes transplanted to TK-NOG mice.

The minimum doubling time of human hepatocytes in TK-NOG-IL-6 mice was 2.4 days, on the other hand, the minimum doubling time of human hepatocytes in TK-NOG mice was 3.6 days. From this, it was determined that human hepatocytes were proliferated 1.5 times faster by using TK-NOG-IL-6 mice.

Although in this example, an immunodeficient mouse was used as the non-human vertebrate, it is expected that the same effect of human IL-6 can be obtained as in the case of using an immunodeficient non-human vertebrate other than a mouse, considering the mechanism of survival of human hepatocytes.

[ example 3]

Effect of anti-IL-6 neutralizing antibodies on proliferation of human hepatocytes in TK-NOG-IL-6 mice

As shown in example 1, when human hepatocytes are proliferated in a non-human mammal, human IL-6 effectively functions and proliferation of transplanted human hepatocytes is accelerated in TK-NOG-IL-6 mice.

It has been reported that "an anti-cytokine antibody acts for a long period of time by acting as a carrier protein for cytokines in vivo and preventing degradation" (FD Finkelman et al, J Immunol,1993Aug 1,151(3),1993, pp.1235-44: May LT.et al, J Immunol,1993Sep 15,151(6), pp.3225-36), and it was investigated whether or not the effect of human IL-6 can be enhanced by administering a neutralizing antibody against human IL-6 after transplanting human hepatocytes into TK-NOG-IL-6 mice.

1. Materials and methods

(1) Induction of liver injury and transplantation of human hepatocytes

Ganciclovir was administered to TK-NOG-IL-6 mice as described in example 1, heparin blood was collected 1 week after administration, plasma was separated, and clinical chemistry analysis (alanine aminotransferase; ALT) was performed by FUJI DRI-CHEM7000(Fujifilm)

As donor cells, commercially available frozen human hepatocytes (Lonza, example HUM4282 for 11 months) were used. The donor cells were transplanted into TK-NOG-IL-6 by the following method. Adult TK-NOG-IL-6 receptor mice aged 6 to 8 weeks were administered 72 hours after the administration of ValGCV solution (0.27mg/mL) as drinking water, and blood was collected 1 week after the start of the administration to measure the plasma ALT value. Individuals showing plasma ALT values of 300U/L or more were used as recipients for human hepatocyte transplantation. The number of human hepatocytes and their survival rate were calculated by trypan blue exclusion method using a hematocrit meter. Using a 29 gauge needle syringe (Myjector) for subcutaneous administration of insulin, approximately 1X 10 aliquots of insulin were suspended in 40. mu.L William's medium E medium⁶One live hepatocyte was administered into the spleen.

(2) Grouping of administration group and non-administration group of anti-human IL-6antibody

ALT activity measured before human hepatocyte transplantation was ranked from higher individuals for cross-grouping (4 females per group) and no difference in the mean ALT activity between 2 groups was determined by the significant difference test (Mann-Whitney test). It was further determined that there was no significant difference in the average body weight before antibody administration between the 2 groups (Mann-Whitney test). The arbitrarily selected group was used as an antibody administration group.

(3) Dosing and blood sampling schedules for anti-human IL-6 antibodies

Anti-human IL-6Antibody (2. mu.g of Sino Biological, IL6/IL-6 neutraing Antibody, Clone mhk23, and 2. mu.g of Invitrogen IL-6Antibody, Monoclonal,505E9A12A3, 4. mu.g each, was administered once daily for 4 consecutive days (tuesday, wednesday, thursday, friday), 2 days after drug withdrawal, 5 days (Monday, tuesday, Wednesday, friday) subcutaneous administration in the same amount was performed for 5 days (Monday, Wednesday, friday) after drug withdrawal for 2 days (Saturday, friday), and 5 days (Monday, Wednesday, thursday, friday) subcutaneous administration in the same amount was performed for 2 days after drug withdrawal for 2 days (Saturday, friday) and once (15 times in total). Blood was collected every Tuesday from Tuesday 2 weeks after transplantation to 5 weeks after transplantation.

(4) Human albumin assay

A small amount of blood was collected from the fundus venous plexus using a polyethylene tube, and plasma was separated. Plasma was diluted 5,000 to 250,000 fold with TBS (Tris-buffered saline) containing 1% bovine serum albumin/0.05% Tween20, and human albumin concentration was determined using a human albumin ELISA quantification Kit (Bethy Laboratories).

(5) Cholinesterase activity assay

A small amount of blood was collected from the fundus venous plexus using a polyethylene tube, and plasma was separated. Clinical chemistry analysis (Cholinesterase; CHE) was performed on plasma by FUJI DRI-CHEM7000 (Fujifilm). Since there is almost no cholinesterase activity in mouse plasma, the detected cholinesterase activity is secreted by human hepatocytes, and it has been reported that this activity is highly correlated with human albumin concentration and can be used as a survival marker for human hepatocytes instead of human albumin (Suemizu et al 2018, Pest management Sci,74, 1424-1430).

2. Results

Fig. 20A shows the measurement results of the blood human albumin concentration, and fig. 20B shows the measurement results of cholinesterase activity.

As shown in fig. 20A, in the group to which the human anti-IL-6 antibody was administered, the amount of human albumin was higher than that in the group to which the antibody was not administered, from 2 weeks to 5 weeks after the transplantation of human hepatocytes. Wherein, the human albumin amount is higher value after 3 weeks and 4 weeks of human liver cell transplantation, and has a statistically significant difference. As shown in fig. 20B, cholinesterase was higher in the group to which the human anti-IL-6 antibody was administered, compared with the group to which no antibody was administered, and was statistically significantly inferior, throughout the period from 2 weeks after the transplantation of human hepatocytes to 5 weeks after the transplantation of human hepatocytes. It was thus determined that administration of human anti-IL-6 antibody promotes survival and proliferation of human hepatocytes after transplantation.

Industrial applicability

The non-human vertebrate of the present invention is a non-human vertebrate in which liver cells are replaced with human liver cells, and is a non-human animal having the structure or function of a human liver. The non-human animal of the present invention can be used for developing a medicine for treating a liver disease in a human and recovering human hepatocytes.

All publications, patents and patent applications cited in this specification are herein incorporated by reference as if fully set forth.

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Ala Arg Ser Arg Gly His Ser Asn Arg Arg Thr Ala Leu Arg Pro Arg

20 25 30

Arg Gln Gln Glu Ala Thr Glu Val Arg Leu Glu Gln Lys Met Pro Thr

35 40 45

Leu Leu Arg Val Tyr Ile Asp Gly Pro His Gly Met Gly Lys Thr Thr

50 55 60

Thr Thr Gln Leu Leu Val Ala Leu Gly Ser Arg Asp Asp Ile Val Tyr

65 70 75 80

Val Pro Glu Pro Met Thr Tyr Trp Gln Val Leu Gly Ala Ser Glu Thr

85 90 95

Ile Ala Asn Ile Tyr Thr Thr Gln His Arg Leu Asp Gln Gly Glu Ile

100 105 110

Ser Ala Gly Asp Ala Ala Val Val Met Thr Ser Ala Gln Ile Thr Met

115 120 125

Gly Met Pro Tyr Ala Val Thr Asp Ala Val Leu Ala Pro His Ile Gly

130 135 140

Gly Glu Ala Gly Ser Ser His Ala Pro Pro Pro Ala Leu Thr Leu Ile

145 150 155 160

Phe Asp Arg His Pro Ile Ala Ala Leu Leu Cys Tyr Pro Ala Ala Arg

165 170 175

Tyr Leu Met Gly Ser Met Thr Pro Gln Ala Val Leu Ala Phe Val Ala

180 185 190

Leu Ile Pro Pro Thr Leu Pro Gly Thr Asn Ile Val Leu Gly Ala Leu

195 200 205

Pro Glu Asp Arg His Ile Asp Arg Leu Ala Lys Arg Gln Arg Pro Gly

210 215 220

Glu Arg Leu Asp Leu Ala Met Leu Ala Ala Ile Arg Arg Val Tyr Gly

225 230 235 240

Leu Leu Ala Asn Thr Val Arg Tyr Leu Gln Gly Gly Gly Ser Trp Arg

245 250 255

Glu Asp Trp Gly Gln Leu Ser Gly Thr Ala Val Pro Pro Gln Gly Ala

260 265 270

Glu Pro Gln Ser Asn Ala Gly Pro Arg Pro His Ile Gly Asp Thr Leu

275 280 285

Phe Thr Leu Phe Arg Ala Pro Glu Leu Leu Ala Pro Asn Gly Asp Leu

290 295 300

Tyr Asn Val Phe Ala Trp Ala Leu Asp Val Leu Ala Lys Arg Leu Arg

305 310 315 320

Pro Met His Val Phe Ile Leu Asp Tyr Asp Gln Ser Pro Ala Gly Cys

325 330 335

Arg Asp Ala Leu Leu Gln Leu Thr Ser Gly Met Val Gln Thr His Val

340 345 350

Thr Thr Pro Gly Ser Ile Pro Thr Ile Cys Asp Leu Ala Arg Thr Phe

355 360 365

Ala Arg Glu Met Gly Glu Ala Asn

370 375