Maintenance of programmed chronic liver injury in Fah gene-deficient animals and its application in the generation of xenogeneic liver models

Abstract

The present invention provides the maintenance of programmed chronic liver injury in Fah gene-deficient animals and its application in the generation of xenogeneic liver models. The present invention provides a novel mechanism for the maintenance of programmed chronic liver injury in Fah gene-deficient animals and the generation of xenogeneic liver models. P ) method, which can enable long-term survival of animal models in a state of chronic liver injury and can achieve a high xenogeneic replacement rate after xenogeneic hepatocyte transplantation.

Description

Detailed description of the invention

[0001] Technical field The present invention belongs to the field of biotechnology and animal model production, and more specifically, relates to a method for maintaining programmed chronic liver injury in Fah gene-deficient animals and its application in the production of heterologous liver models.

[0002] Background technology The liver is the primary site of drug metabolism and removal in the human body, and also a host for many specific pathogens. Due to interspecies differences, these human liver-specific functions and characteristics are difficult to effectively simulate in other animals. Currently, the humanization of the liver has been successfully achieved in mouse models through the transplantation of human primary hepatocytes (PHH) (M Dandri et al., Hepatology. 33(4): 981-988. 2001; DF Mercer et al., Nat Med. 7(8): 927-933. 2001; C Tateno et al., Am J Pathol. 165(3): 901-912. 2004), and is used to accurately predict human liver-specific drug metabolism and pathogen infection processes. Furthermore, humanized animal models can also produce functional human hepatocytes for use in large quantities and diverse applications as bioreactors, such as "artificial liver systems" that require the support of a large number of hepatocytes (H Azuma et al., Nat Biotechnol. 25(8):903-910. 2007; E Michailidis et al., Proc Natl Acad Sci US A. 117(3):1678-1688. 2020). The results of research and development over the past decade have successfully led to the efficient and stable production of humanized liver models in mouse models (H Azuma et al., Nat Biotechnol. 25(8):903-910. 2007; C Tateno et al., PLoS One. 10(11):e0142145. 2015; EM Wilson et al., Stem Cell Res. 13(3 Pt A):404-412. 2014; M Hasegawa et al., Biochem Biophys Res Commun. 405(3):405-410. 2011; KD Bissig et al., J Clin Invest. 120(3):924-930. 2010; ML Washburn et al., Gastroenterology. 140(4):1334-1344. 2011). However, the size of mice significantly limits the amount of biological samples that can be provided, such as blood and bile necessary for pharmaceutical analysis, and hepatocytes necessary for regenerative medicine research (K Yoshizato et al., Expert Opin Drug Metab Toxicol. 9(11):1419-1435. 2013).

[0003] Rats are at least 10 times larger than mice and are physiologically and pathologically closer to humans (PM Iannaccone et al., Dis Model Mech. 2(5-6):206-210. 2009). In particular, for drug metabolism and toxicity, the US FDA guidelines also recommend preclinical studies using rats (HJ Jacob et al., Nat Rev Genet. 3(1):33-42. 2002). Furthermore, theoretically, after rats have achieved complete humanized liver replacement, 10 9 We can provide the above-mentioned human liver cells. Therefore, constructing a humanized liver on a rat model is of greater significance than on a mouse, especially in the fields of drug development and regenerative medicine research. However, the development of humanized livers on rats is greatly hindered by the lack of rat models that can effectively support xenotransplantation of human-derived liver cells. At the same time, there is currently a lack of practical experience in humanizing livers in large animals other than mice.

[0004] The inventors previously used fumarylacetase (Fah) gene-deficient linked severe immunodeficiency FRG (Fah) to construct a humanized liver. - / - Rag 2- / - IL2rg - / - We participated in the construction of a rat model (currently under substantive examination for a Chinese patent, application number: 20180621840.8) and confirmed that this rat model has the potential for constructing a humanized liver. However, for the long-term maintenance of transplanted rats, it is necessary to explore more effective liver injury control models to achieve stable and efficient levels of humanized replacement.

[0005] Content of the invention The object of the present invention is to provide a method for maintaining nonacute lethal, persistent chronic liver injury in Fah gene-deficient rats, and to achieve stable amplification of human hepatocytes in the rat body by this method, ultimately obtaining a heterologous liver with a high replacement rate.

[0006] The object of the present invention is to provide a system or kit used for breeding a Fah gene-deficient rat model and maintaining its chronic liver injury.

[0007] In a first aspect of the present invention, Fah gene deletion (Fah - / - ) Provides a method for maintaining chronic liver injury in an animal model, the method comprising administering nitisinone (NTBC) to a Fah gene-deficient animal model in the following program: (1) Administer a low dose of nitisinone daily for 3 to 12 days, the low dose being 0.005 to 0.1 mg / kg animal body weight / day; (2) Administer a high dose of nitisinone daily for 2 to 6 days, the high dose being 0.25 to 1.5 mg / kg animal body weight / day; (3) Repeat the cycle of steps (1) and (2).

[0008] In a first aspect of the present invention, a method is provided for producing a xenotransplant animal model, wherein the animal model has a xenotransplant liver, and the method comprises: (1) administering a low dose of nitisinone daily for 3 to 12 days, the low dose being 0.005 to 0.1 mg / kg animal body weight / day; (2) administering a high dose of nitisinone daily for 2 to 6 days, the high dose being 0.25 to 1.5 mg / kg animal body weight / day; (3) performing a cycle of steps (1) and (2); and transplanting xenotransplanted hepatocytes into the animal 3 to 30 days after the start of the program.

[0009] In a preferred example of the present invention, prior to performing the above program, the following is further included: pre-treating the animals with retrorsine; preferably, the amount of retrorsine used is 10 to 50 mg / kg of animal body weight; more preferably, the amount of retrorsine used is 20 to 40 mg / kg of animal body weight; even more preferably, the amount of retrorsine used is 25 to 35 mg / kg of animal body weight.

[0010] In another preferred example of the present invention, step (1) involves administering a low dose of nitisinone daily for 4 to 10 days; preferably, a low dose of nitisinone daily for 5, 6, 7, 8, or 9 days.

[0011] In another preferred example of the present invention, in step (1), the above low dose is 0.008-0.08 mg / kg animal body weight / day; preferably, the above low dose is 0.01-0.05 mg / kg animal body weight / day; for example, 0.015-0.04 mg / kg animal body weight / day, or 0.015-0.035 mg / kg animal body weight / day; more specifically, for example, 0.02 mg / kg animal body weight / day, 0.03 mg / kg animal body weight / day.

[0012] In another preferred example of the present invention, step (2) involves administering a high dose of nitisinone daily for 3 to 5 days.

[0013] In another preferred example of the present invention, in step (2), the above high dose is 0.3 to 1.2 mg / kg animal body weight / day; preferably, the above high dose is 0.35 to 1 mg / kg animal body weight / day; for example, 0.35 to 0.85 mg / kg animal body weight / day; more specifically, for example, 0.4, 0.5, 0.6, 0.7, 0.8 mg / kg animal body weight / day.

[0014] In another preferred example of the present invention, the animal is a mammal having a body size of twice that of a mouse (e.g., 2 to 2000 times, more specifically, 5, 10, 15, 20, 50, 100, 200, 500 times); preferably, the animal is selected from the following group: rat, rabbit, monkey, pig, guinea pig, dog; preferably, the animal is an animal in which the interleukin-2 receptor γ gene and recombinant activator gene 2 are disrupted (Rag2). - / - IL2rg - / - )

[0015] In another preferred example of the present invention, the above-mentioned heterologous hepatocytes include, but are not limited to: human-derived, mouse-derived, pig-derived, monkey-derived or dog-derived hepatocytes belonging to a species different from the animal model; preferably, including, but not limited to: (a) primary hepatocytes; (b) hepatic stem / progenitor cells, hepatocytes or hepatocyte-like cells, endoderm cells, etc. induced and differentiated from induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs); (c) hepatic stem / progenitor cells, hepatocytes or hepatocyte-like cells differentiated from the induction of endoderm and other germ layer stem cells; (d) hepatic stem / progenitor cells, hepatocytes or hepatocyte-like cells directly differentiated and converted from adult or fetal stem / progenitor cells or hepatocytes and other somatic cells; (f) hepatic stem / progenitor cells, hepatocytes or hepatocyte-like cells after in vitro induced proliferation of cells derived from (a)-(d); (g) hepatic organoids constructed from cells derived from (a)-(f).

[0016] In another preferred example of the present invention, the cycle of (3) is carried out until the animal model is used or until the end of the lifespan of the animal model.

[0017] In another preferred example of the present invention, the above-mentioned heterologous liver is a liver with a high heterologous chimerism rate.

[0018] In another preferred example of the present invention, the above-mentioned high heterologous chimerism rate (heterologous substitution rate) refers to a heterologous chimerism rate of more than 25%, preferably more than 28%, more preferably more than 30%. At the same time, with the extension of the culture time of the recipient animal model, the chimerism rate increases.

[0019] In another preferred example of the present invention, the above-mentioned heterologous hepatocytes are separated from the liver of an organ donor, separated from surgical resection specimens, or derived from induced pluripotent stem cells, embryonic stem cells, embryonic tissues and their cells, amniotic epithelial cells, monocytes or amniotic fluid cells.

[0020] In another preferred example of the present invention, the heterologous hepatocytes transplanted into the above-mentioned animals are the separated heterologous hepatocytes.

[0021] In another preferred example of the present invention, the above-mentioned nitisinone is administered into drinking water.

[0022] In another preferred example of the present invention, the above-mentioned nitisinone is administered in food.

[0023] In another aspect of the present invention, a system is provided for raising a model of Fah gene-deficient animals and maintaining its chronic liver injury, comprising: a dosing component or module 1 for administering a low dose of nitisinone to an animal daily for 3 to 12 days, wherein the low dose is set to 0.005 to 0.1 mg / kg animal body weight / day; and a dosing component or module 2 for administering a high dose of nitisinone to an animal daily for 2 to 6 days, wherein the high dose is set to 0.25 to 1.5 mg / kg animal body weight / day.

[0024] In a preferred example of the present invention, the system used for breeding the above-mentioned Fah gene-deficient animal model and maintaining its chronic liver injury includes a mechanical device or a computer system.

[0025] In another preferred example of the present invention, the apparatus described above is used to cultivate a xenograft animal model having a xenografted liver.

[0026] In another preferred example of the present invention, the apparatus further includes a dosing component or module 3 capable of administering retrorucin to an animal.

[0027] In another preferred example of the present invention, the apparatus described above is configured to administer 10 to 50 mg / kg of animal body weight of retrorucin.

[0028] In another preferred example of the present invention, the apparatus outputs the aforementioned amount of drug from a computer program and transfers it to an animal via a mechanical device.

[0029] In another aspect of the present invention, a kit is provided for use in raising a Fah gene-deficient animal model and maintaining its chronic liver injury, comprising: Container group 1: a container and a low dose of nitisinone contained in the container, the low dose being 0.005 to 0.1 mg / kg animal body weight / day; preferably, the number of containers in container group 1 is 3 to 12; and Container group 2: a container and a high dose of nitisinone contained therein, the high dose being 0.25 to 1.5 mg / kg animal body weight / day; preferably, the number of containers in container group 2 is 2 to 6.

[0030] In a preferred example of the present invention, the kit further comprises a container 3 and retrorucin contained therein; preferably, the retrorucin is 1.2 to 10 mg.

[0031] In a preferred example of the present invention, the above-described container groups 1 and 2, and preferably further including container group 3, are used for one complete dosing cycle (one "low-dose-high-dose" cycle) for one animal.

[0032] In another preferred example of the present invention, the above-described container groups 1 and 2, and preferably further comprising container group 3, are used for two or more complete administration cycles for one animal, or for one, two or more administration cycles for multiple animals.

[0033] In another preferred example of the present invention, a kit group is employed for subsequent animal rearing, the kit group comprising 2 to 200 kits, for example, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, or 150 kits.

[0034] In another aspect of the present invention, the application of low-dose nitisinone, high-dose nitisinone, and letrolsin for use in maintaining chronic liver injury in Fah gene-deficient animal models; or for preparing dose groups or kit groups for maintaining chronic liver injury in Fah gene-deficient animal models; wherein the low dose is 0.005 to 0.1 mg / kg animal body weight / day, and the high dose is 0.25 to 1.5 mg / kg animal body weight / day.

[0035] Another aspect of the present invention provides the preparation of a xenograft animal model having a xenografted liver; or the application of low-dose nitisinone, high-dose nitisinone, and letrolsin for use in the preparation of a dosage group or kit group for preparing a xenograft animal model; wherein the low dose is 0.005 to 0.1 mg / kg animal body weight / day, and the high dose is 0.25 to 1.5 mg / kg animal body weight / day.

[0036] Based on the information disclosed herein, other aspects of the present invention will be obvious to those skilled in the art. [Brief explanation of the drawing]

[0037] [Figure 1] Figure 1. NTBC-mediated liver injury in FRG rats. A. Diagram of FRG rat states under NTBC supply (w / ) and removal (w / o) conditions. B. Serum ALT and AST levels in healthy FRG rats under NTBC supply and dying rats under NTBC removal. ****P < 0.0001. C. Staining results for HE and Sirius scarlet in liver tissue of FRG rats under NTBC supply and removal conditions. Scale bar: 100 μm. D. Survival curves of FRG rats under NTBC supply and removal conditions. [Figure 2]Figure 2. Construction of a rat chronic liver injury model. A. Survival curves of FRG rats under different NTBC concentration conditions. ***P < 0.001,****P < 0.0001; ns, no statistically significant difference. B. AST, ALT levels and body weight changes in rats under 4% and 10% NTBC conditions. *P < 0.05,**P < 0.01,***P < 0.001,****P < 0.0001. C. Schematic diagram of the control design for NTBC programming with fixed cycle period. D. Survival curves of FRG rats under three different NTBC programming controls (5+4, 7+4, and 9+4). E. AST and ALT levels in rats under 5+4 and 7+4 NTBC programming controls. **P < 0.01,****P < 0.0001; ns, no statistically significant difference. F. Degree of fibrosis in rat liver tissue under 5+4 and 7+4 NTBC programmed control, as indicated by Sirius scarlet staining. Scale bar: 100 μm. G. Afp gene expression levels in rat liver tissue under 5+4 and 7+4 NTBC programmed control. ****P < 0.0001; ns, no statistically significant difference. [Figure 3] Figure 3. Hepatocyte repair and regeneration in transplanted rats pre-treated with retrorsine (RS). A. Ki67 staining results of rat liver tissue pre-treated (with) and untreated (without) RS. Scale bar: 100 μm. B. Statistical analysis of the proportion of proliferative cells in rat liver tissue pre-treated (with) and untreated (without) RS. *P < 0.05. C. Human albumin secretion levels in rats pre-treated (with) and untreated (without) RS within 4 weeks after PHH transplantation. *P < 0.05. [Figure 4] Figure 4. Construction of a rat chronic liver injury model. A. Illustration of NTBCP and NTBC△BW liver injury control methods. B. Live curves of FRG rats after PHH transplantation under NTBCP and NTBC△BW liver injury control. C. Time course of human albumin concentration in rat serum after transplantation. D. Changes in the mating rate of human-derived cells in FRG rat liver at 4 days, 1 month, and 7 months after transplantation. E. Staining results of human-derived cell nuclei in rat liver lobes (middle lobe, left lobe, and right lobe) in slices 7 months after transplantation. Scale bar: 2 mm. [Figure 5] Figure 5. Identification of humanized liver cells. A. Human hepatocytes were identified using the restaining results for hALB, hAAT, and hNuclei. Scale bar: 100 μm. B. FAH and HE staining on serial slices of humanized liver. R: rat hepatocytes; H: human hepatocytes. Scale bar: 100 μm. C. FAH and PAS staining on serial slices of humanized liver. Scale bar: 100 μm. D. Restaining results for CK19 and hNuclei on humanized liver slices. Scale bar: 1 mm (magnified view: 100 μm). E. Restaining results for Ki67 and hALB at 4 days, 1 month, and 7 months after transplantation. Scale bar: 100 μm. F. Percentage of Ki67+ proliferating cells in hALB+ human hepatocytes at different time points after transplantation. [Figure 6] Figure 6. Examination of tumorigenicity in humanized liver. A. Liver weight ratio between FRG rats transplanted with PHH and those that were not. ns, no statistically significant difference. B. hAFP gene expression in FRG rat liver tissue transplanted with PHH and those that were not. HepG2 hepatocellular carcinoma cell line is the positive control. ****P < 0.0001; ns, no statistically significant difference; UD, not detected. C. Results of AFP protein immunostaining in FRG rat liver tissue transplanted with PHH. hGAPDH is the internal standard for human-derived cells. Scale bar: 100 μm. [Figure 7] Figure 7. Gene and protein expression related to metabolism in humanized liver. A. Expression of mature hepatocyte markers, phase I and II metabolic enzymes, and transporter-related genes was compared in humanized liver tissue and donor PHH by qPCR analysis. PHH, human primary hepatocytes. B. Immunostaining results for ALB, FAH, CYP3A4, CYP1A2, and ARG1. CV: central vein, PV: portal vein; scale bar: 100 μm. C. Immunostaining results for FAH, UGT2B7, and MRP2. Scale bar: 100 μm. [Figure 8]Figure 8, UGT2B7 drug metabolism in humanized liver. A. Schematic diagram of the UGT2B7 metabolism test of AZT drug. After orally administering AZT (15 mg / kg) to rats, sampling and testing were performed at the designated time points. B. Time-dependent changes in the concentrations of AZT and AZT-5'-glucoside in the liver humanized rats and the control group. ***P < 0.001; ns, no statistical difference. C. AUC values of the AZT-5'-glucoside / AZT ratio in the liver humanized rats and the control group. **P < 0.01. D. Correlation analysis of the AUC value (AZT-5'-glucoside / AZT) and the human albumin secretion amount in liver humanized rats.

[0038] Specific embodiments In this field, with respect to the technical defect that it is difficult to obtain an animal model with a heterologous liver that can maintain survival for a long time and has a high engraftment rate, as a result of the inventor's intensive study, it was found that the cycle of nitisinone (2-(2-nitro-4-trifluoromethyl-benzoyl)-1,3 cyclohexanedione, NTBC), whose cycle period is fixed, was programmed control (Programmed NTBC controlling, NTBC P ) The plan was clarified. The plan of the present invention can realize that the animal survives for a long time in a state of chronic liver injury, and can realize a high heterologous replacement rate after transplantation of heterologous hepatocytes.

[0039] As used in the present invention, the above-mentioned "fumarylacetoacetase gene deficiency (Fah - / - )", "recombinant activation gene 2 deficiency (Rag2 - / - )" or "interleukin 2 receptor γ gene deficiency (IL2rg - / - )" means that Fah, Rag2 or IL2rg is disrupted by a method including knockout, gene editing, homologous recombination or site-directed mutagenesis. In some embodiments of the present invention, the above-mentioned "deficiency" further includes a significant decrease in gene / protein expression, for example, a decrease in gene / protein expression of about 80%, about 90%, about 95% or about 99%.

[0040] For use in the present invention, the above-mentioned animal is a mammal. Preferably, the above-mentioned animal is a mammal having a body size of twice (e.g., 2 to 2000 times) or more that of a mouse; preferably, the above-mentioned animal is an animal selected from the following group: rats, rabbits, pigs, guinea pigs, dogs, and non-human primates such as monkeys and orangutans; preferably, the above-mentioned animal is a Fa - / - Rag2 - / - IL2rg - / - It is an animal. Most preferably, the above animal is a rat.

[0041] As used in this invention, the term "heterogeneous" above refers to a relationship between two types of hepatocytes originating from different species, or between cells from different sources and animal receptors. For example, if a combination of cells and an animal receptor is not typically found in nature, then the cells are heterogeneous with respect to that animal receptor. The term "heterogeneous" above is also referred to as "exogenous." The term "heterogeneous" above refers to the process by which heterogeneous cells are implanted in the liver of a receptor animal, grow, amplify, and function.

[0042] As used in the present invention, the above-mentioned "heterogeneous interlocking rate" refers to the proportion of heterogeneous cells implanted, grown, and proliferated in the liver of a receptor animal in the total number of hepatocytes of that receptor animal.

[0043] Creating animal models The FAH enzyme is a crucial enzyme in the hepatic tyrosine metabolic pathway. In animal models, deficiency of the Fah gene leads to the accumulation of butadiyl, a tyrosine metabolic intermediate, which, when reached a certain level, can cause hepatocyte death. Supplementing daily drinking water with NTBC has the effect of suppressing the tyrosine metabolic pathway, preventing the accumulation of toxic metabolic intermediates, and relieving liver damage. In this field, a strategy of controlling NTBC elimination / recovery cycles based on stepwise elimination or body weight changes has been applied to human-derived hepatocyte transplantation in Fah-deficient mice. However, in previous studies, the inventors found that this control strategy could only support a low percentage (less than 10%) of human-derived hepatocyte chimeras in Fah-deficient rats and could not be applied to the construction of highly humanized livers. The inventors discovered that Fah-deficient rats are more sensitive to liver damage caused by NTBC removal than mice with the same gene deficiency; they also exhibit a serious delay in weight indicators, making timely rescue impossible, and they have an extremely high mortality rate, resulting in an insufficient window for rescue after mature hepatocyte transplantation; therefore, a stable and efficient humanized replacement rate cannot be obtained. To address this, the inventors have strived to find a way to solve this problem.

[0044] After thorough research and analysis, the inventors have disclosed a novel method that is easy to operate, highly controllable, non-acutely lethal, and capable of sustaining chronic liver injury. The method described in the present invention avoids premature death in model rats after xenogeneic hepatocyte transplantation, creates a favorable intrahepatic environment and sufficient amplification window for the implantation and survival of xenogeneic hepatocytes / hepatoid cells in FRG rats, and can construct a xenogeneic liver with a high survival rate and high replacement rate. This method can also be applied to xenogeneic hepatocyte transplantation in models of Fah-deficient large animals other than rats, such as rabbits, pigs, guinea pigs, dogs, and non-human primates such as monkeys.

[0045] A new discovery by the inventors provides a method for maintaining chronic liver injury in an animal model of Fah gene deficiency (NTBC PThe above method provides a Fah gene-deficient animal model and includes administering NTBC to the Fah gene-deficient animal model in the following program: (1) administer a low dose of NTBC daily for 3 to 12 days, the low dose being 0.005 to 0.1 mg / kg animal body weight / day; (2) administer a high dose of NTBC daily for 2 to 6 days, the high dose being 0.25 to 1.5 mg / kg animal body weight / day; (3) repeat the cycle of steps (1) and (2). Depending on the actual needs, the above program may be continued until the animal model is used or until the animal model reaches the end of its lifespan.

[0046] Considering individual differences between different animals, and given that there are certain fluctuations in daily NTBC intake when administered orally, the low and high doses of the present invention are calculated based on the average intake of individual animals. Specifically, for the low dose stage, when administering an average of 0.005 to 0.1 mg / kg of animal body weight NTBC per day, if 5 mg / L NTBC is considered 100% NTBC, the corresponding NTBC usage concentration may be 0.665 to 13.33%. Specifically, for the high dose stage, when administering an average of 0.25 to 1.5 mg / kg of animal body weight NTBC per day, if 5 mg / L NTBC is considered 100% NTBC, the corresponding NTBC usage concentration may be 33.33% to 200%.

[0047] Fah gene-deficient animals can have their liver damage controlled by artificially controlling the NTBC supply pattern, thereby creating conditions for Fah wild-type allogeneic hepatocyte transplantation or xenogeneic hepatocyte transplantation. Therefore, based on maintaining chronic liver damage in the above-mentioned Fah gene-deficient animal model, the present invention further provides a method for creating a xenogeneic liver transplant animal model, comprising: carrying out the steps of (1) and (2) above; and transplanting human primary hepatocytes into the animal on days 3 to 30 of the above program (e.g., days 4, 5, 6, 8, 10, 12, 15, 18, 20, 22, 25, and 28).

[0048] In a preferred method of the present invention, the above-mentioned Fah gene-deficient animal is Fah- / - Rag2 - / - IL2rg - / - These are animals. Fah is an enzyme with 419 amino acids, a gene length of 22586 bp, 14 exons and 13 introns, and its GenBank registry number is NM_017181.2. Rag2 is a protein with 527 amino acids, a gene length of 8297 bp, 3 exons and 2 introns, and its GenBank registry number is NM_001100528.1. IL2rg is a protein with 368 amino acids, a gene length of 7281 bp, 12 exons and 11 introns, and its GenBank registry number is NM_080889.1. Fah gene deficiency can induce liver damage in animals, Rag2 gene knockout can cause T and B cell deficiency in animals, and IL2rg gene knockout can cause NK cell deficiency and T and B cell reduction in animals. Individual and combined knockouts of Rag 2 and IL 2 rg can both produce immunodeficient animals, but simultaneous knockout of Rag 2 and IL 2 rg results in the most severe immunodeficiency and is suitable for human hepatocyte transplantation.

[0049] In this invention, NTBC can be administered to animals in various ways. Some preferred methods employ oral administration, including mixing NTBC with drinking water or mixing NTBC with the animal's food.

[0050] In the present invention, the above-mentioned heterologous hepatocytes include: (a) primary hepatocytes; (b) hepatic stem / ancestral cells, hepatocytes or hepatic sac cells, endodermal cells, etc., derived and differentiated from induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs); (c) hepatic stem / ancestral cells, hepatocytes or hepatic sac cells, derived from the induction of endoderm and other germinal stem cells; (d) hepatic stem / ancestral cells, hepatocytes or hepatic sac cells, derived directly from adult or fetal stem / ancestral cells or hepatocytes and other somatic cells; (f) hepatic stem / ancestral cells, hepatocytes or hepatic sac cells, derived from cells derived from (a) to (d) after in vitro induced proliferation; and (g) liver-like organs constructed from cells derived from (a) to (f). Heterogeneous primary hepatocytes may be isolated from the liver of an organ donor, isolated from surgically resected tissue, or derived from induced pluripotent stem cells, embryonic stem cells, embryonic tissue and its cells, amniotic epithelial cells, mononuclear cells, or amniotic fluid cells.

[0051] In the present invention, xenogeneic hepatocytes for transplantation into receptor animals can be isolated from liver tissue by any method known in the art. Liver tissue can be mechanically or enzymatically digested to provide a single-cell suspension, or a complete liver tissue block can be used. For example, hepatocytes can be separated from donor tissue by normal collagen perfusion followed by slow centrifugation; the hepatocytes can then be purified by screen filtration, followed by density gradient centrifugation. As a different option, other methods for concentrating hepatocytes can also be used, such as flow cytometer separators, bead separation, density gradient centrifugation, or other methods known in the art. Proliferated human hepatocytes can be collected from the liver of receptor animals using similar hepatocyte isolation methods. In addition to obtaining xenogeneic hepatocytes from organ donors or hepatic resections, the cells used for transplantation may be human hepatocytes that are implanted and proliferated after transplantation into receptor animals, or hepatocyte precursor cells, ancestral cells, and other hepatocyte-like cells that have been proliferated in vitro.

[0052] A preferred method of the present invention is the program (NTBC P The procedure further includes pre-treating the animals with retrorsine (RS) before proceeding. NTBC PBefore developing the program, the inventors also attempted to treat animals using a combination of retrorucin and partial hepatectomy. However, the animal model used had limitations in that it could not control liver damage, resulting in a detected human albumin secretion level of only 90 μg / ml. In humanized animals, the human drug metabolism process can only be simulated relatively accurately if the humanization fit rate exceeds 30% or if there is an albumin secretion level of 1 mg / ml. Therefore, the inventors used Fah-deficient and immunodeficient rats as a model and developed an optimized NTBC P By developing a program that significantly improves the humanized implantation rate to over 30% at 7 months after transplantation, and can achieve albumin secretion levels exceeding 2 mg / ml, this program can significantly increase the implantation success rate.

[0053] In the present invention, the amount of retrorusin used is 10-50 mg / kg animal body weight; more preferably, the amount of retrorusin used is 20-40 mg / kg animal body weight; even more preferably, the amount of retrorusin used is 25-35 mg / kg animal body weight. Demonstration of specific examples of the present invention shows that the percentage of Ki67-positive cells in the liver tissue of animals pre-treated with RS is significantly lower. Human albumin (hAlbumin, ALB) secretion levels are an important indicator for evaluating the level of in vivo mating and proliferation of xenogeneic hepatocytes. By comparison, the human albumin secretion level after transplantation of xenogeneic hepatocytes is significantly faster in animals pre-treated with RS, indicating that the combined application of RS pre-treatment and NTBC programmed control further promotes in vivo mating and proliferation of xenogeneic hepatocytes.

[0054] A specific embodiment of the present invention provides a preferred approach, which includes: intraperitoneal injection of 30 mg / kg of animal body weight of retrorucin into Fah-deficient rats two weeks prior to human liver / hepatoid cell transplantation; one week prior to transplantation, the NTBC concentration in the daily drinking water is reduced from a normal maintenance concentration (5-8 mg / L) to a low concentration (0.2 mg / L), NTBC programmed control is initiated, and NTBC is added to the daily drinking water in the manner of 0.2 mg / L for 7 days + 5 mg / L for 4 days (1 cycle is 11 days).

[0055] By employing the method of the present invention, heterologous livers having a high heterologous interlocking rate can be obtained, which may be greater than 25%, preferably greater than 28%, and more preferably greater than 32%; and this interlocking rate can be further increased by increasing the culture time of the animals.

[0056] The technical solution of the present invention has very remarkable technical effects and includes, but is not limited to, the following: (1) It can maintain a long-term chronic liver injury state in Fah-deficient animals, achieve non-acute lethal sustainable chronic liver injury, avoid premature death of animal models after xenogeneic liver / hepatoid cell transplantation, and achieve a high animal survival rate; (2) It can create an intrahepatic environment favorable for the implantation and survival of xenogeneic liver / hepatoid cells in animal models, provide a sufficiently long in vivo amplification window, and construct a xenogeneic liver with a high survival rate and high replacement rate; (3) It has convenient operation and management methods, is highly controllable, and enables the development of automated animal model production equipment or computer systems, thereby realizing the production of large-scale animal models.

[0057] application The xenotransplant animal models disclosed in this invention can be used for a wide variety of research and therapeutic purposes, and include, but are not limited to, the following applications:

[0058] Animal Chronic Liver Injury Model: This invention allows for the stable induction of chronic liver injury in Fah-deficient animals, enabling the construction of chronic liver disease models primarily representing liver fibrosis and cirrhosis, which can be used for disease mechanism and drug research.

[0059] Preclinical Drug Testing: The advanced heterologous liver animal model constructed according to the present invention is expected to be applicable to preclinical drug metabolism, toxicity and efficacy testing, and experiments on in vivo human hepatitis virus infection. It is not only physiologically similar to the human body, but also supports long-term sampling, testing, and follow-up, which is advantageous for promoting the research and development of new drugs. The animal model of the present invention can be used in the study of various liver diseases, including HCC, liver cancer, and cirrhosis. The animal model of the present invention can be used, for example, as a model of liver disease caused by exposure to toxins, infectious diseases or malignant tumors, or genetic defects. Examples of hereditary liver diseases suitable for drug research using the animal model of the present invention include, but are not limited to, hypercholesterolemia, hypertriglyceridemia, hyperoxaluria, phenylpyruvinuria, glycogen storage disorders, and several congenital metabolic defects. The model system of the present invention can be used to better understand specific liver diseases and to identify drugs that can inhibit, delay, or reverse disease progression. Alternatively, the animal model constructed according to the present invention can be used as a model of liver disease caused by toxins. The animal models constructed in this invention can be used to screen the ability of candidate vaccines to prevent or mitigate infection by hepatopathogenic pathogens. In this invention, the type of candidate drug for drug testing is not particularly limited and can be obtained from a variety of sources, including synthetic or natural compound libraries. For example, there are various methods for random and oriented synthesis of multiple organic compounds and biomolecules, including the expression of random oligonucleotides and oligopeptides, or natural compound libraries can be obtained or readily produced in the form of bacterial, fungal, plant, and animal extracts. Furthermore, libraries and compounds produced by natural or synthetic methods can be readily modified by conventional chemical, physical, and biochemical methods and used to produce complex libraries. Known pharmacological agents can be oriented or randomly chemically modified (e.g., acylation, alkylation, esterification, amidation, etc.) to produce structural analogs.

[0060] Xenogeneic (Human-Derived) Hepatocyte Amplification and Transplantation Therapy: The method of the present invention allows for the collection of xenogeneic (e.g., human) hepatocytes from receptor animals, which are grown in receptor animals, as a source of human hepatocytes for liver reconstruction in subjects requiring liver reconstruction therapy. The present invention can support the significant proliferation of xenogeneic hepatocytes / hepatoid cells in Fah-deficient animals, developing alternative techniques for organ transplantation and providing important support for future clinical regenerative transplantation therapies. Introducing hepatocytes and reconstructing liver tissue in patients is a potential therapeutic option for patients with acute liver failure and can also be used as a preliminary treatment before liver transplantation. Hepatocyte reconstruction can be used, for example, to introduce genetically modified hepatocytes for gene therapy, or to replace hepatocyte loss due to disease, physical or chemical damage, or tumor. Human hepatocytes can be collected from receptor animals using many techniques known in the art; human hepatocytes collected from receptor animals can be separated from non-human cells or other impurities (e.g., tissue or cell debris) using any of the techniques known in the art. The present invention is expected to solve the severe problem of liver organ and hepatocyte transplant donor shortages.

[0061] Gene Therapy Research: Hepatocytes grown and collected in the animal model constructed according to the present invention can be used to evaluate changes in human hepatocyte gene expression by any pharmaceutical compound (e.g., small molecules, biological products, environmental agents, and biotoxins or gene delivery systems). It can also be applied to the study of gene therapy schemes and vectors. For example, parameters such as the transfer efficiency of gene delivery vectors (including viral and non-viral vectors); the integration frequency and positioning of genetic material (integration site analysis); the functionality of genetic material (gene expression levels, gene knockout efficiency); and the side effects of genetic material (in vivo human hepatocyte gene expression or protein group analysis) can be evaluated. For example, the use of transfected hepatocytes in gene therapy for patients with familial hypercholesterolemia has been reported.

[0062] Personalized Liver Disease Models and Precision Medicine: Using patient-derived iPSC-derived liver-like cells, we construct personalized animal models of liver disease and perform gene editing therapy for liver diseases ranging from hepatic fibrosis, cirrhosis, HCC, ICC, and CCC to viral and metabolic genetic defects such as NASH, HBV, HT1, and AAT. This promotes the research and development of personalized targeted drugs and cutting-edge novel therapeutic strategies.

[0063] In Vitro Testing: Xenogeneic liver / hepatoid cells can be obtained by growing them in an animal body and then recovering them via liver perfusion. Because they do not undergo in vitro culture, xenogeneic liver / hepatoid cells grown in vivo can maintain maximum similarity in characteristics and function to those transplanted from primitive cells. When used in relevant in vitro tests such as drug testing and disease simulation, the stability and reproducibility of results can be guaranteed.

[0064] Furthermore, using a method similar to that of the present invention, it is possible to determine NTBC control concentrations and maintenance times suitable for specific animal species, establish NTBC programmed control with fixed cycle periods, create targeted control of chronic liver injury, and achieve sustained and stable interlocking and proliferation in the body after xenogeneic liver / hepatoid cell transplantation.

[0065] Animal model making system / kit Based on the disclosed novel method, the present invention further provides a system for breeding and maintaining a Fah gene-deficient animal model. In the present invention, the system may be a mechanical device, preferably an automated mechanical device; it may also be a computer system comprising various modules for carrying out the method of the present invention.

[0066] As one preferred form of the present invention, a machine is provided, preferably an automated machine, and comprises: Dosage component 1, which administers a low dose of NTBC to an animal daily for 3 to 12 days, wherein the low dose is set to 2 to 10%; and Dosage component 2, which administers a high dose of NTBC to an animal daily for 2 to 6 days, wherein the low dose is set to 70 to 100%. Preferably further comprising a Dosage component 3, which can administer (e.g., by injection) letrolusin to the animal. The three types of Dosage components described above can be organically configured to form one whole, which can be organically integrated with an animal housing device, thereby providing a convenient and automated animal production system.

[0067] One preferred configuration of the present invention provides a computer-controlled system comprising a dosing module 1 including a control program, which instructs a downstream system (e.g., a mechanical nozzle) operably connected to (operated by) it to administer a low dose of NTBC to an animal daily for 3 to 12 days, wherein the low dose is 2 to 10%; further comprising a dosing module 2 including a control program, which instructs a downstream system (e.g., a mechanical nozzle) operably connected to (operated by) it to administer a high dose of NTBC to an animal daily for 2 to 6 days, wherein the high dose is 70 to 100%; preferably further comprising a dosing module 3 including a control program, which instructs a downstream system (e.g., a mechanical nozzle) operably connected to (operated by) it to administer letrolusin to an animal.

[0068] Based on the disclosed novel method, the present invention further comprises a kit (preferably one cycle) for breeding a Fah gene-deficient animal model and maintaining its chronic liver injury, comprising: Container group 1: a container and a low dose of nitisinone contained in the container, the low dose being 0.005 to 0.1 mg / kg animal body weight / day; preferably, the number of containers in container group 1 is 3 to 12 (preferably one cycle); and Container group 2: a container and a high dose of nitisinone contained therein, the high dose being 0.25 to 1.5 mg / kg animal body weight / day; preferably, the number of containers in container group 2 is 2 to 6 (preferably one cycle). Preferably, the above kit further comprises a container 3 and letrolusin contained therein. Preferably, the above container 3 may be a syringe. Multiple kits can be set up and applied to multiple rounds of NTBC cycles. Furthermore, in actual operation, a person skilled in the art can easily add a specific amount of NTBC to the container according to the animal's weight.

[0069] According to the overall disclosure and examples of embodiments of the present invention, those skilled in the art can easily design or assemble the invention. [Examples]

[0070] The present invention will be further described below with reference to specific examples. It should be understood that these examples are not intended to limit the scope of the present invention, but are merely illustrative. The experimental methods in the following examples, which do not specify particular conditions, are usually carried out according to the usual conditions described in J. Sambrook et al., Guide to Molecular Cloning, Third Edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0071] Materials and methods 1, animals Fah - / - Rag2 - / - IL2rg- / - The rats (FRG rats) are kept at the Experimental Animal Center of Jiangsu University.

[0072] 2, cells Table 1 shows the origin information of the primary hepatocytes used for transplantation in the examples. [Table 1] All of the above were purchased from BioreclamationIVT, USA.

[0073] 3. Reagents (1) RNA reverse transcription I used the Revert Aid RT kit (Thermo Fisher Scientific). (2) Quantitative qPCR The THUNDERBIRD SYBR qPCR Mix kit (Toyobo, Japan) was used. (3) Measurement of human albumin secretion The Human Albumin ELISA Quantitation Set (Bethyl Laboratory, USA) was used. (4) HE and immunohistochemistry 4% Paraformaldehyde (Wako); Methanol (Wako); Acetone (Wako); Twain-20 (Wako); Citric Acid (Wako); Hydrogen Peroxide (Wako); Goat Serum (Thermo Fisher Scientific); Donkey Serum (Jackson Lab, USA); Hematoxylin (Wako); Ibe (Wako); ABC Kit (Vector Laboratories, USA); Opal TM 4-Color Manual IHC Kit (PerkinElmer, USA); 4',6-Diamino-2-phenylindole (DAPI, Thermo Fisher Scientific); FA Cerebralizer (VMRD; USA). (5)Periodic acid-Schiff (PAS) staining The Periodic-Acid-Schiff kit (Shanghai Yuanye Biotechnology Co., Ltd.) was used. (6) UGT2B7 metabolic test Reference substance: 1.0 mg / mL zidovudine (zidovudine solution) (AZT, Sigma), 3'-azido 3'-deoxythioside β-D-glucoside (AZT-5'-glucaldehyde acid, Toronto Research Chemicals, Canada).

[0074] 4. Primer The primers used in the examples are shown in Table 2. [Table 2]

[0075] 5, Antibodies The antibodies used in the examples are shown in Table 3. [Table 3-1] [Table 3-2]

[0076] 6, HE staining Rat livers were fixed overnight with 4% PFA, dehydrated, embedded, and cut into 3 μm thick slices. (1) Dewax with xylene three times, for 5 minutes each time. (2) Gradient condensation: 100% ethanol, 100% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 50% ethanol, 5 minutes each. Immersion in deionized water for 1 minute. (3) Antigen repair (this step is adopted according to antibody characteristics): A sodium citrate antigen repair solution with pH=6.0 was heated at 95°C for 30 minutes, and then allowed to cool naturally to room temperature. Washed with deionized water for 15 minutes. (4) Re-stained with sappanwood extract for 20 minutes. Washed with tap water for 30 minutes. (5) Gradient condensation: 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol, 2 minutes each. (6) After treating with xylene for 15-30 minutes, the neutral resin-sealed sheets were stored.

[0077] 7, Immunohistochemistry (1) The dewaxing and condensation step is the same as described above. (2) A circle was drawn around the sample with an ImmunoPen, and one drop of 3% H2O2 was added to seal in the endogenous peroxidase. The sample was then treated at room temperature for 15 minutes. (3) Wash with PBS twice, for 5 minutes each time. (4) Block with 1:20 normal horse serum (dissolved in 1% BSA-PBS), incubate at room temperature for 20 minutes, and wash three times with PBS. (5) The primary antibody was added, the slices were placed in a moist box, sealed, and left overnight at 4°C. (6) Wash with PBS three times, for 5 minutes each time. (7) The secondary antibody was added and incubated at room temperature for 20 minutes. The mixture was washed with PBS three times for 5 minutes each time. (8) Add the AB solutions from the ABC kit (A and B solutions diluted 1:100) and incubate at room temperature for 30 minutes. Wash with PBS three times for 5 minutes each time. (9) DAB developed color, and the color development time was adjusted for each antibody. (10) Re-stained with sappanwood extract for 20 minutes. Washed with tap water for 30 minutes. (11) Gradient condensation: 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol, 2 minutes each. (12) After treating with xylene for 15-30 minutes, store the neutral resin-sealed sheet.

[0078] 8. Immunofluorescence staining (1) Frozen slices (7 μm) were fixed in 4% PFA at room temperature for 10 minutes, or fixed in methanol:acetone (1:1) at -30°C for 30 minutes. (2) Washed three times with 0.1% tween-PBS for 5 minutes each time. (3) Ten percent of goat or donkey serum was blocked at room temperature for 60 minutes. (4) The primary antibody was incubated overnight at 4°C. (5) Washed three times with 0.1% tween-PBS for 5 minutes each time. (6) A secondary antibody conjugated with fluorescein was incubated at room temperature for 60 minutes. (7) Wash with PBS three times, for 5 minutes each time. (8) DAPI was added, the slices were sealed with a sealant, and stored.

[0079] 9. Quantitative qPCR Following the instructions for the Revert Aid RT kit, 1 μg RNA was reverse transcribed into cDNA. The cDNA product was amplified using a quantitative PCR machine and quantitatively analyzed according to the instructions for the THUNDERBIRD SYBR qPCR Mix kit. ACTB was used as the internal standard for target gene relative expression levels. All data were repeated at least twice.

[0080] 10. Liver function tests The levels of glutamate aminotransferase (ALT) and glutamate aminotransferase (AST) in rat serum were measured according to the operating instructions for the FUJI DRI-CHEM 7000 V biochemical analyzer (Fujifilm, Japan).

[0081] 11. Measurement of human albumin secretion 100 μl of rat peripheral blood was collected from the tail vein, and after coagulation at room temperature, it was centrifuged at 400 × g for 10 minutes. The supernatant was then collected to obtain serum. The secretion level test for human albumin was performed according to the instructions for use of the Human Albumin ELISA Quantification Set kit. Serum was diluted 10 to 100,000 times based on the actual concentration, and the calculated values ​​were brought within the linear range of the standard curve to obtain accurate values.

[0082] 12. Primary hepatocyte transplantation and programmed NTBC cycle (1) Pre-transplant preparation: Female FRG rats aged 7-8 weeks were intraperitoneally injected with 30 mg / kg of retrorsine two weeks prior to transplantation. One week prior to transplantation, the NTBC concentration in the daily drinking water was reduced from a normal maintenance concentration (5-8 mg / L) to a low concentration (0.2 mg / L). A programmed NTBC cycle was then initiated, and daily NTBC drinking water was provided using the method of 0.2 mg / L for 7 days + 5 mg / L for 4 days (11 days per cycle). In conventional feeding, FRG rats are supplied with 5-8 mg / L daily. In this invention, this minimum dose of 5 mg / L NTBC is considered 100% NTBC, and the rat's body weight (250-300 g) and drinking water volume (25-50 ml / day) are used for calculations. 4% NTBC: Equivalent to an average of 0.03 mg / kg / day; 100% NTBC: Equivalent to an average of 0.75 mg / kg / day. (2) The transplantation process was carried out under anesthesia using isoflurane. The rats were fixed with their abdomens facing upwards, their hair was removed, and they were then cleaned and disinfected with iodine liquor and alcohol. (3) The epidermis and muscle layer were cut, and the hepatic portal vein was located. Using a 26 G needle, 2 × 10 6 Human primary hepatocytes (resuspended in 800 μl DMEM + 10% FBS) were slowly injected. After the injection was complete, the needle was withdrawn after a few seconds, and the needle tip was quickly pressed with a sterile cotton swab. (4) After pressing for about one minute and observing that there was no blood flow, the muscle and epidermis were sutured together and the wound was disinfected with an alcohol swab.

[0083] 13. In vivo UGT2B7 metabolism test FRG rats in the liver-humanized group and the non-control group were orally administered 15 mg / kg of zidovudine (Wako) via gastric irrigation, and peripheral blood was collected at 0, 0.5, 1, 2, 4, and 8 hours later. 30 μl of plasma was diluted 2-fold with PBS, and the substrate and metabolite content was measured using LC-MS / MS (Agilent 1200 HPLC and ABI 4000 mass-spectrometer).

[0084] Example 1: FRG rats produced lethal liver injury after NTBC removal. Long-term follow-up observations by the inventors showed that FRG rats remained healthy for at least 1.5 years with 100% NTBC (FRG rats can be routinely supplied with 5-8 mg / L, and in this invention, this minimum dose of 5 mg / L is considered 100%), which provided a sufficient time window (Figure 1 A and D) for the construction of a humanized liver. Upon removal of NTBC, the rats rapidly suffered liver damage. Necrosis and fibrosis appeared in the liver tissue (Figure 1 C), accompanied by significantly elevated ALT and AST levels (Figure 1 B). All rats died within 4 weeks of NTBC removal, with a median survival time of 9.6 days (Figure 1 D). The results showed that NTBC removal caused severe liver damage and death in FRG rats. This situation observed in rats differs from that observed in other animals such as mice, which maintain a longer survival period and higher levels of liver damage after NTBC removal. Therefore, some existing NTBC applications in the art are not applicable to rats.

[0085] Example 2: NTBC programmed control that induces chronic liver injury was established. To improve the survival rate of FRG rats under liver injury conditions and ensure the stability of the long-term humanization process, the inventors conducted in-depth research and multifaceted analyses of rat liver injury and its survival status, seeking the optimal conditions for increasing the survival rate under liver injury conditions. The optimal NTBC maintenance concentration was determined by comparing the survival rates of rats at different NTBC concentrations (0%, 1%, 4%, 10%, 100%). The results showed that when the NTBC concentration exceeded the 4% maintenance dose (5 mg / L), the median survival time exceeded 3 weeks (22 days), effectively preventing sudden death in rats (Figure 2A). Furthermore, comparing rat liver function and body weight under 4% and 10% NTBC conditions, the 4% condition maintained high ALT and AST levels while significantly suppressing rat body weight gain (Figure 2B), achieving an effect of inducing liver damage while maintaining survival. Because the humanization process requires a relatively long cycle, in order to further improve the long-term survival rate of rats under liver injury, the inventors designed a series of NTBC administration schemes called Programmed NTBC Control (NTBCP), which include: maintaining 4% NTBC conditions after 5, 7, and 9 days, restoring 100% NTBC for 4 days, and mitigating accumulated liver injury (Figure 2C).

[0086] The results show that FRG rats can maintain a high survival rate within 2 months under control with 5+4 NTBC programming (5 days 4% NTBC + 4 days 100% NTBC cycle) and 7+4 NTBC programming (7 days 4% NTBC + 4 days 100% NTBC cycle) (Figure 2 D). Furthermore, comparing the two modes, 5+4 and 7+4, the inventors found that under 7+4 control, significantly higher ALT and AST levels (Figure 2 E) were observed, and chronic liver damage could be better induced; the degree of liver tissue fibrosis (Figure 2 F) was observed, and the degree of fibrosis was more pronounced under 7+4 control. At the same time, the inventors demonstrated that the long-term effects of NTBC programmed control do not lead to abnormal upregulation of the specific oncogene Afp in rat liver tissue (Figure 2 G), thus proving that this strategy is safe. Based on these results, the inventors have concluded that 7+4 NTBC can sustainably induce chronic liver injury. P We established a control strategy and applied it to the subsequent creation of a humanized rat liver model.

[0087] Example 3: Combined application of retrorsine pretreatment and NTBC programmed control The inventors discovered that, in the NTBC programmed control process (7+4: a cycle of 4% NTBC for 7 days + 100% NTBC for 4 days), the mating and proliferation levels of human primary hepatocytes (PHH) in humanized rat models were still not ideal after transplantation. To achieve further optimization, the inventors conducted extensive analysis and experiments with a wide variety of target drugs and discovered that pretreatment with retrorsine (RS) has a promoting effect. The inventors placed retrorsine (RS) pre-treated (w / RS) and untreated (w / o RS) FRG rat hepatocytes and observed PHH mating and proliferation levels in the rat model. Two months after NTBC programmed control in Example 2 described above, the proportion of Ki 67-positive cells in rat liver tissue pretreated with RS was found to be significantly lower (Figure 3 A and B). Human albumin (hAlbumin, ALB) secretion levels are an important indicator for evaluating the in vivo mating and proliferation levels of human-derived hepatocytes. By comparison, human albumin secretion levels after transplantation of human primary hepatocytes (PHH) were significantly faster in rats pretreated with RS (Figure 3C), indicating that the combined application of RS pretreatment and NTBC programmed control further promotes the mating and proliferation of PHH in vivo.

[0088] Example 4: Humanization of rat liver under chronic liver injury As described above, human primary hepatocytes were transplanted into rats, and the NTBC programmed control NTBC with fixed cycle period was developed. P (7+4: a cycle of 7 days 4% NTBC + 4 days 100% NTBC; and retrolucin pretreatment) and NTBC using 10% body weight change as an indicator. △BW Survival curves of PHH-transplanted FRG rats when comparing conditions (NTBC removal, administration of 100% NTBC when rat body weight decreases by 10%, and removal of NTBC when body weight returns to pre-removal level, and so on, repeating the cycle) (Figure 4 A).

[0089] As a result, the transplanted rats were NTBC PUnder control conditions, they exhibited a higher survival rate, with 6 / 7 of the rats able to survive for at least 20 weeks (Figure 4B). NTBC P Under controlled conditions, with increasing time, the level of human albumin secretion in surviving rats steadily increases, reaching a maximum of 2.2 mg / ml (mean 1.7 ± 0.3 mg / ml) after 7 months (Figure 4 C). The humanized interlocking rate of rat liver can be calculated by immunostaining with the human-specific marker hNuclei. As shown in Figure 4D, NTBC P Under control conditions, the mating rate of transplanted human-derived cells continued to improve, reaching 31±4% after 7 months (Figure 4 E).

[0090] Example 5: Identification of the cellular composition of humanized liver NTBC by NTBC programming as described above P Under the (7+4: 7-day 4% NTBC + 4-day 100% NTBC cycle control; and retrorucin pretreatment) method, the characteristics of interlocked human-derived cells were obtained and identified 7 months after PHH transplantation in FRG rats. For human hepatocyte-specific antigens, the inventors restained and observed the cells using hALB, hAAT, and hNuclei antibodies. As a result, all hNuclei-positive human-derived cells expressed hALB and hAAT, indicating that these cells were mature hepatocytes (Figure 5A). Furthermore, human hepatocytes, represented by FAH positivity, showed a whiter color on HE staining compared to the surrounding rat hepatocyte region (Figure 5 B), clearly distinguishing their origin from two different species. Simultaneously, human hepatocytes also exhibited stronger glycogen storage capacity (Figure 5 C). Histological observations confirmed that the interposition of human-derived hepatocytes did not affect the existing liver structure in rats (Figure 5 B). These results were consistent with relevant reports in humanized mouse models. Simultaneously, CK 19 and hNuclei antibody restaining results showed no CK 19-positive human-derived cells, indicating that the transplanted human primary hepatocytes had not transformed into bile duct epithelial cells in the rat (Figure 5 D). Analysis of the proportion of Ki 67-positive cells in hALB-positive hepatocytes revealed a gradual decrease in the proliferative capacity of human hepatocytes over time (Figure 5 E). However, even 7 months after transplantation, the proportion remained above 10% (Figure 5 F), and NTBC P This demonstrates that it is possible to induce long-term in vivo proliferation potential in primary human hepatocytes.

[0091] Example 6: Detection of tumorigenicity in humanized liver NTBC by NTBC programming as described above P Under the (7+4: 7-day 4% NTBC + 4-day 100% NTBC cycle control; and retrolucin pretreatment) method, humanized livers were obtained 7 months after PHH transplantation in FRG rats, and the liver body weight ratio was calculated. As a result, no significant improvement was observed in the liver-to-body weight ratio compared to the untransplanted control (Figure 6A). Histologically, no tumor formation was observed. Furthermore, genetic and protein-level analysis of AFP, a major marker for liver cancer, revealed that no abnormal upregulation of AFP occurred 7 months after human hepatocytes were implanted in rat livers (Figure 6B and C). The above results are NTBC P Under these conditions, the humanization process of the liver did not induce malignant lesions.

[0092] Example 7: The humanized liver showed expression of metabolism-related genes and proteins similar to those of the human liver. NTBC by NTBC programming as described above P Under the (7+4: 7-day 4% NTBC + 4-day 100% NTBC cycle control; and retrolucin pretreatment) method, the function of the humanized liver in FRG rats was evaluated 7 months after PHH transplantation. The inventors first performed QPCR analysis on the expression status of metabolism-related genes using primers that specifically identify human genes (Table 2). The results showed that the humanized liver not only expresses mature hepatocyte marker-correlated genes (ALB, AAT, G6PC, Fah), but also expresses phase I metabolic enzymes (CYP2A6, CYP2E1, CYP3A4, CYP3A7 and CYP7A1), phase II metabolic enzymes (UGT2B7), and transporter-correlated genes (SLC22A1 and SLCO1B1) (Figure 7A). More importantly, the expression levels were close to those of PHH before transplantation. The orderly distribution of metabolic enzymes in liver tissue is an important structural feature of the liver. Immunostaining of randomly selected hepatic metabolic enzymes revealed that glutamine synthase (GS) is specifically distributed in hepatic parenchymal cells near the central vein (Figure 7B), and the phase I metabolic enzymes cytochrome P450 3A4 (CYP3A4) and 1A2 (CYP1A2) are also concentrated around the central vein. In contrast, alliinase (ARG1) is mainly concentrated near the hepatic portal vein (Figure 7B). Furthermore, the phase II enzyme uridine diphosphate glucosealdehyde transtransferase 2B79 (UGT2B7) and the transporter multidrug resistance-associated protein 2 (MRP2) were uniformly distributed throughout the hepatic lobule (Figure 7C). Importantly, the distribution of these metabolic enzymes in the humanized liver is consistent with the distribution rules in the human body. The results above indicate that, regardless of the expression characteristics of metabolism-related genes and enzymes, the humanized liver showed extremely high similarity to the human liver.

[0093] Example 8: Humanized liver possesses human-specific drug metabolism characteristics. The human UGT2B7 enzyme is known to be involved in the metabolism of approximately 35% of clinical drugs and is of significant research importance. The inventors investigated whether humanized rat livers could exhibit human-specific metabolic characteristics using zidovudine (AZT), a UGT2B7 metabolite. In humans, 75% of AZT is metabolized to AZT-5'-glucuronide, but in rats, the conversion rate was only 10%. Figure 8A shows the test flow. As a result, after oral administration of AZT, AZT-5'-glucuronaldehyde in liver-humanized rats (approximately 30% interfacing rate) was significantly higher than in the control group (Figure 8 B). The AUC (areas under the curves) values ​​for AZT-5'-glucuronaldehyde / AZT were 39% ± 16% and 6% ± 3% in liver-humanized and control rats, respectively (Figure 8 C). Furthermore, a comparison of the correlation between human albumin secretion and AUC values ​​(AZT-5'-glucuronaldehyde / AZT) also showed a positive correlation between the liver humanization rate and the human-type metabolic level of AZT (Figure 8 D). Based on these results, the humanized liver rats exhibit drug metabolism characteristics unique to humans and possess long-term survival capabilities.

[0094] Example 9, Kit / set for the production of humanized animal livers In this embodiment, a kit used for the preparation of a humanized rat liver is provided, which includes: Container group 1: A container containing a low dose of nitisinone; the low dose is 0.03 mg; the number of containers in container group 1 is 7; Container group 2: A container containing a high dose of nitisinone; the high dose is 0.75 mg; the number of containers in container group 2 is 4; Container group 3: A container containing 4 mg of retrorucin; the number of containers in container group 1 is 1; The kit was used for a single (11-day) administration and for the retrorucin pretreatment of rats. After integrating multiple kits, it can be used for administration over multiple cycles in rats, including simultaneous administration to multiple rats. In sustained animal rearing, the kit set is used, for example, and the kit set contains 2 to 200 kits. In a kit set consisting of multiple kits, retrorucin is supplied in only one kit.

[0095] All references cited in this application are cited and referred to in this application so as to be cited alone by reference. It should be understood that, based on the above disclosure of the present invention, those skilled in the art may make various changes or modifications to the invention, and these equivalent forms are also included within the scope defined in the claims attached to this application.

Claims

1. A method for maintaining chronic liver injury in a Fah gene-deficient animal model, the method comprising administering nitisinone to the Fah gene-deficient animal model according to the following program: (1) administering a low dose of nitisinone daily for 3 to 12 consecutive days, the low dose being 0.005 to 0.1 mg / kg animal body weight / day; (2) administering a high dose of nitisinone daily for 2 to 6 consecutive days, the high dose being 0.25 to 1.5 mg / kg animal body weight / day; (3) Cycle steps (1) and (2).

2. A method for producing a xenogeneic hepatocyte transplant animal model, wherein the animal model has a xenogeneic liver, and the method includes: (1) administering a low dose of nitisinone daily for 3 to 12 consecutive days, the low dose being 0.005 to 0.1 mg / kg animal body weight / day; (2) administering a high dose of nitisinone daily for 2 to 6 consecutive days, the high dose being 0.25 to 1.5 mg / kg animal body weight / day; (3) cycle steps (1) and (2); Additionally, the animals are transplanted with xenogeneic hepatocytes 3 to 30 days after the start of the above program.

3. 3. The method according to claim 1 or 2, further comprising pretreating the animal with retrorsine before carrying out the program.

4. The method of claim 3, wherein the amount of retrorsine used is 10 to 50 mg / kg of animal body weight.

5. Step (1) involves administering low doses of nitisinone daily for 4 to 10 consecutive days; or In step (1), the low dose is 0.008-0.08 mg / kg animal body weight / day; 3. The method according to claim 1 or 2.

6. In step (1), a low dose of nitisinone is administered daily for 5, 6, 7, 8, or 9 consecutive days; or In step (1), the low dose is 0.01 to 0.05 mg / kg animal body weight / day; 6. The method of claim 5.

7. Step (2) involves administering high doses of nitisinone daily for 3 to 5 consecutive days; or In step (2), the high dose is 0.3 to 1.2 mg / kg animal body weight / day; 3. The method according to claim 1 or 2.

8. In step (2), the high dose is 0.35 to 1 mg / kg animal body weight / day; 8. The method of claim 7.

9. 3. The method according to claim 1 or 2, wherein the animal is a mammal having a body size at least twice that of a mouse.

10. The method according to claim 9, wherein the animal is an animal selected from the following group: rat, rabbit, monkey, pig, guinea pig, dog.

11. The method described in claim 9, characterized in that the above animal is an animal in which the interleukin-2 receptor γ gene and recombination activating gene 2 have been disrupted.

12. 3. The method of claim 2, wherein the heterologous hepatocytes comprise hepatocytes of human, murine, porcine, simian or canine origin belonging to a species different from the animal model.

13. The method of claim 12, wherein the heterologous hepatocytes comprise: (a) Primary hepatocytes; (b) Liver stem / progenitor cells, hepatocytes or hepatocytes, and endoderm cells induced and differentiated from induced pluripotent stem cells and embryonic stem cells; (c) hepatic stem / progenitor cells, hepatocytes, or hepatocytes differentiated from endoderm and other germ layer stem cells; (d) hepatic stem / progenitor cells, hepatocytes, or hepatocytes directly transdifferentiated from adult or fetal stem / progenitor cells or hepatocytes and other somatic cells; (f) hepatic stem / progenitor cells, hepatocytes, or hepatocytes after in vitro induced expansion of cells derived from (a) to (d); (g) Liver organoid constructed from cells derived from (a) to (f).

14. a dosing component or module 1 configured such that animals receive a low dose of nitisinone daily for 3 to 12 consecutive days, said low dose being 0.005 to 0.1 mg / kg animal body weight / day; Administration component or module 2 is configured such that administration component or module 1 is followed by daily administration of a high dose of nitisinone to the animals for 2-6 consecutive days, the high dose being 0.25-1.5 mg / kg animal body weight / day. A system used for cultivating a Fah gene-deficient animal model and maintaining chronic liver injury thereof, comprising:

15. 15. The system of claim 14, further comprising: an administration component or module 3 capable of administering retrorsine to an animal; said administration component or module 3 arranged to administer 10-50 mg / kg animal body weight of retrorsine;

16. Container Group 1: A container and a low dose of nitisinone contained in the container; the low dose is 0.005 to 0.1 mg / kg animal body weight / day; and Container Group 2: A container containing a high dose of nitisinone, the high dose being 0.25-1.5 mg / kg animal body weight / day; A kit used for cultivating a Fah gene-deficient animal model and maintaining chronic liver injury thereof, comprising: The kit is used for one cycle of administration.

17. The number of containers in the container group 1 is 3 to 12, The kit according to claim 16, wherein the number of containers in container group 2 is 2 to 6.

18. 18. The kit of claim 16 or 17, further comprising: The container 3 includes retrorsine contained therein; the retrorsine is 1.2 to 10 mg.

19. A kit set comprising the kit according to any one of claims 16 to 18, comprising 2 to 200 of said kits; Kit set for use in sustainable animal breeding.

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