Immunodeficient rodents capable of maintaining human red blood cells for extended periods.

Abstract

Providing immunodeficient rodents capable of maintaining human red blood cells in the bloodstream for extended periods. [Solution] An immunodeficient rodent in which three genes—the gene encoding complement C3, the clec4f gene, and the Axl gene—are knocked out.

Description

[Technical Field]

[0001] This invention relates to immunodeficient rodents capable of maintaining human red blood cells in the blood for extended periods. [Background technology]

[0002] Severely immunodeficient mice (NOG) are known as a highly superior immunodeficient mouse (Patent Document 1).

[0003] Severely immunodeficient mice (NOGs) are mice that can efficiently engraft normal human cells, and humanized NOG mice have been created by transplanting human cells and tissues into NOG mice. However, some cells are difficult to engraft. Human red blood cells are a prime example. Human red blood cells are essential for malaria infection research, and researchers need to transplant them on a daily basis. Furthermore, with the development of artificial blood and the progress of gene therapy using genome editing for thalassemia, a genetic disease affecting red blood cells, there has been a need to develop mice that can maintain human red blood cells for extended periods. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2002 / 043477 [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to provide an immunodeficient rodent animal capable of maintaining human red blood cells in the blood for an extended period. [Means for solving the problem]

[0006] The inventors of this invention have diligently researched the development of a rodent animal capable of maintaining human red blood cells over a long period of time. The inventors of the present invention have discovered that human red blood cells can be maintained in the blood for a long period of time in triple-deficient immunodeficient rodents in which three genes—the gene encoding complement C3, the clec4f gene encoding the CLEC4F protein which is involved in phagocytosis of red blood cells, and the Axl gene encoding the tyrosine kinase AXL protein—are knocked out, and have completed the present invention.

[0007] In other words, the present invention is as follows. [1] Immunodeficient rodents in which three genes are knocked out: the gene encoding complement C3, the clec4f gene, and the Axl gene. [2] The mouse, a rodent of [1]. [3] Immunodeficient rodents of [2], which are SCID mutant mice, RAG1 knockout mice, RAG2 knockout mice, or NOD / SCID mice. [4] NOG mouse, formally known as NOD.Cg-Prkdc scid Il2rg tm1Sug Mpl em1 An immunodeficient rodent represented as / Jic, [3]. [5] A method for producing an immunodeficient knockout rodent according to any of [1] to [4], comprising knocking out three genes: the gene encoding complement C3, the clec4f gene, and the Axl gene of the immunodeficient rodent. [6] An immunodeficient knockout rodent that maintains transplanted human red blood cells in the blood for an extended period, one of the following [1]-[4]. [7] An immunodeficient rodent from any of the categories [1] to [4] in which, when human red blood cells are transferred into the rodent, the percentage of human red blood cells in the rodent's blood 5 days after transfer is 15% or more. [8] Immunodeficient rodents of any of the following categories, in which human red blood cells are transferred into any of the following categories, and the survival rate of human red blood cells in the blood of the rodents 5 days after transfer is 60% or higher. [9] A method for producing a rodent in which human red blood cells are maintained in the blood for a long period of time, comprising transferring human red blood cells into any of the rodents described in [1] to [4].

[10] A method for producing immunodeficient rodents according to [9], wherein, when human red blood cells are transferred into any of the rodents [1] to [4], the percentage of human red blood cells in the blood of the rodents 5 days after transfer is 15% or more.

[11] A method for producing immunodeficient rodents according to [9], wherein, when human red blood cells are transferred into any of the rodents [1] to [4], the survival rate of human red blood cells in the blood of the rodents five days after transfer is 60% or more.

[12] [6] Use of rodents as model rodents for research on the treatment of malaria infections in rodents.

[13] [6] A rodent model of human malaria infection created by infecting the rodents described above with malaria.

[14] A method for screening drugs and vaccines for malaria infection using the rodent animal malaria infection model described in

[13] .

[15] [6] Use of rodents as model rodents for research into the treatment of thalassemia or sickle cell disease in rodents.

[16] [6] Use for the development of artificial red blood cells in rodents. [Effects of the Invention]

[0008] Triple knockout immunodeficiency mice (NOG-C3 / clec4f / Axl triple knockout (TKO) mice) in which the gene encoding complement C3, the clec4f gene encoding the CLEC4F protein (a molecule involved in phagocytosis of red blood cells), and the Axl gene encoding the tyrosine kinase AXL protein are knocked out are capable of maintaining human red blood cells for extended periods.

[0009] While malaria infection experiments using immunodeficient mice, such as NSG or NOG mice, require daily transplantation of human red blood cells, the triple-deficient mice described above significantly reduce the effort involved in these experiments and allow for tracking the fate of identical red blood cells, enabling a more accurate reproduction of the disease state.

Brief Description of the Drawings

[0010] [Figure 1] It is a figure showing the result of comparison of the mouse Clec4f gene sequence, and shows the gene sequence of wild type (WT) (NCBI, NM_016751.3) (upper row) and the gene sequence of genome-edited mouse (lower row). [Figure 2] It is a figure showing the result of comparison of the mouse Axl gene sequence, and shows the gene sequence of wild type (WT) (NCBI, NM_001190974.1) (upper row) and the gene sequence of genome-edited mouse (lower row). [Figure 3-1] It is a figure showing the extension of human red blood cell survival in NOG-C3 / Clec4f / Axl TKO mice by chimerism rate. In NOG-C3 / Clec4f / Axl TKO mice, the survival period of human red blood cells is significantly extended compared to NOG mice and NOG-C3 / Clec4f DKO mice. [Figure 3-2] It is a figure showing the extension of human red blood cell survival in NOG-C3 / Clec4f / Axl TKO mice by survival rate. The survival rate shows the attenuation of human red blood cells over time in mouse peripheral blood. [Figure 4-1] It is a figure showing the effect of macrophage depletion by clodronic acid. In NOG mice, the survival period of human red blood cells is significantly extended by macrophage depletion with clodronic acid, but in NOG / C3 / Clec4f / Axl TKO mice, the survival period of human red blood cells is significantly longer than that of clodronic acid-administered NOG mice even without administration of clodronic acid. [Figure 4-2] It is a figure showing the effect of macrophage depletion by clodronic acid by survival rate. It shows the attenuation of human red blood cells over time in mouse peripheral blood. [Figure 5] It is a figure showing the long-term survival maintenance of human red blood cells in NOG-C3 / Clec4f / Axl TKO mice. In NOG-C3 / Clec4f / Axl TKO mice, once the ratio of human red blood cells in peripheral blood exceeds 70%, without new blood transfer, a chimerism rate of 60% or more is maintained for the following 3 weeks. [Modes for carrying out the invention]

[0011] The present invention will be described in detail below.

[0012] 1. The present invention relates to a triple-deficient immunodeficient rodent in which three genes are knocked out: the gene encoding complement C3, the crec4f gene encoding the CLEC4F protein, a molecule involved in phagocytosis of red blood cells, and the Axl gene encoding the tyrosine kinase AXL protein. When human red blood cells are transplanted into this rodent, the human red blood cells are maintained in the blood of the rodent for a long period of time.

[0013] The inventors have clarified the involvement of complement C3 as a molecule involved in macrophage phagocytosis and have created NOG-C3 KO mice (Yamaguchi, Front. Immunol., 27 July 2021, Volume 12 - 2021 | https: / / doi.org / 10.3389 / fimmu.2021.671648).

[0014] The inventors further identified Clec4f as a molecule involved in the phagocytosis of human erythrocytes (Yamaguchi, International workshop on humanized mice (IWHM)6, poster presentation 2022). NOG-C3 / clec4f double knockout (DKO) mice were able to maintain human erythrocytes for a longer period compared to NOG-C3 KO mice.

[0015] We screened NOG-C3 / clec4f DKO mice to see if administering molecularly targeted drugs could prolong human erythrocyte engraftment, and identified the involvement of the Axl gene. When we generated NOG-C3 / clec4f / Axl triple knockout (TKO) mice, they were significantly better able to maintain human erythrocytes for longer periods compared to NOG-C3 / clec4f DKO mice.

[0016] Here, "knockout" (gene knock-out) is a genetic engineering technique that involves deleting all or part of a gene, thereby causing the gene to lose its function. When a gene loses its function, the protein it expresses becomes inactive. Therefore, an immunodeficient rodent in which the gene encoding complement C3 has been knocked out is an immunodeficient rodent in which part or all of the gene encoding complement C3 is deleted, resulting in a loss of function of the complement C3 gene. Similarly, an immunodeficient rodent in which the Clec4f gene has been knocked out is an immunodeficient rodent in which part or all of the Clec4f gene is deleted, resulting in a loss of function of the Clec4f gene. Furthermore, an immunodeficient rodent in which the Axl gene has been knocked out is an immunodeficient rodent in which part or all of the Axl gene is deleted, resulting in a loss of function of the Axl gene.

[0017] Immunodeficient rodents in which the gene encoding complement C3 is knocked out can be created by crossing a rodent in which the gene encoding complement C3 is knocked out with an immunodeficient rodent. Immunodeficient rodents in which the gene encoding complement C3 is knocked out can also be created using genome editing technologies such as Talen or CRISPR / CAS9. Similarly, immunodeficient rodents in which the Clef4f gene and the Axl gene are knocked out can be created. By crossing these immunodeficient rodents, it is possible to create immunodeficient rodents in which all three genes—the gene encoding complement C3, the Clef4f gene, and the Axl gene—are knocked out. Furthermore, by creating an immunodeficient rodent in which one of the three genes is knocked out, and then knocking out the other two genes using genome editing or homologous recombination, it is possible to create an immunodeficient rodent in which all three genes—the gene encoding complement C3, the Clec4f gene, and the Axl gene—are knocked out.

[0018] Preferably, an immunodeficient rodent in which the gene encoding complement C3 is knocked out can be created by crossing an immunodeficient rodent with a rodent in which the gene encoding complement C3 is knocked out, and then the Clec4f gene and Axl gene of the immunodeficient rodent can be knocked out by genome editing or homologous recombination.

[0019] Knockout of the gene encoding complement C3 can be performed by deleting all or part of the gene encoding complement C3 in rodents. For example, all or part of the gene encoding complement C3 in rodents can be deleted. By deleting all or part of the gene encoding complement C3 in rodents, the function of complement C3 in rodents is lost.

[0020] Clef4f gene knockout can be performed by deleting all or part of the Clef4f gene in rodents. For example, all or part of the Clef4f gene in rodents can be deleted. By deleting all or part of the Clef4f gene in rodents, the function of the CLEF4F protein is lost.

[0021] Axl gene knockout can be performed by deleting all or part of the rodent Axl gene. For example, all or part of the rodent Axl gene can be deleted. By deleting all or part of the rodent Axl gene, the function of the AXL protein is lost.

[0022] Sequence ID 1 shows the gene sequence of the wild-type gene encoding mouse Clef4f, and Sequence ID 2 shows the gene sequence of the gene encoding mouse Clef4f that has lost its function due to genome editing. The gene sequence in Sequence ID 2 is an example of a gene encoding mouse Clef4f that has lost its function due to genome editing, and is not limited to this gene sequence.

[0023] Furthermore, the gene sequence of the wild-type gene encoding mouse Axl is shown in Sequence ID No. 3, and the gene sequence of the gene encoding mouse Axl that has lost its function due to genome editing is shown in Sequence ID No. 4. The gene sequence in Sequence ID No. 2 is just one example of a gene encoding mouse Axl that has lost its function due to genome editing, and is not limited to this gene sequence.

[0024] In this invention, rodents are not limited, but examples include mice, rats, guinea pigs, hamsters, rabbits, nutrias, etc., with mice being preferred among these.

[0025] The rodents used in this invention, in which human red blood cells are maintained for a long period of time in the blood of the rodents, are rodents that do not eliminate human red blood cells by immunity, that is, rodents in which the immune response to humans has been inactivated. Examples of such animals include rodents with reduced or absent immune function and inactivated immune response to humans, for example, immunodeficient rodents can be used.

[0026] Immunodeficient rodents are animals with reduced or absent immune function, lacking some or all of T cells, B cells, NK cells, dendritic cells, and macrophages. Immunodeficient rodents can be created by whole-body radiation, or by using rodents that are genetically immune-deficient.

[0027] Examples of immunodeficient mice include nude mice, NOD / SCID mice, Rag1 knockout mice, Rag2 knockout mice, SCID mice administered with asialoGM1 antibody or TMβ1, and irradiated mice. Furthermore, knockout animals (hereinafter referred to as dKO (double knockout) animals) obtained by crossing these NOD / SCID mice, Rag1 knockout mice, or Rag2 knockout mice with IL-2Rγ knockout can also be used. For example, dKO mice (Rag2 KO, IL-2R nullcan be used. In the present invention, a dKO mouse with a Balb / c genetic background is referred to as a Balb / c dKO mouse, and a mouse with a NOD genetic background is referred to as a NOD dKO mouse. Moreover, the genetic background of the mouse is not limited to these, and it may also be C57BL / 6, C3H, DBA2, or an IQI strain, or a strain having SCID mutation and IL-2Rγ knockout, Rag1 knockout and IL-2Rγ knockout, or Rag2 knockout and IL-2Rγ knockout mutation. Also, the deficiency of Jak3 protein responsible for signal transduction downstream of the common γ chain of the IL-2 receptor is also IL2Rγ null Since the phenotype is the same as that of a Rag2 knockout mouse, a knockout mouse obtained by crossing a Rag2 knockout mouse with a Jak3 knockout, a knockout mouse obtained by crossing a Rag1 knockout mouse with a Jak3 knockout, a knockout mouse obtained by crossing a SCID mutation with a Jak3 knockout, or an inbred, non-inbred, or hybrid (F1 hybrid) mouse obtained by mating them may also be used.

[0028] Furthermore, in order to exclude the influence of immune cells such as NK cells observed in the mouse, in addition to the mode in which an asialo GM1 antibody is administered to SCID mice and used as described above, as another mouse used in the present invention, a gene-modified immunodeficient mouse in which a mutation is introduced into the IL-2 receptor γ chain gene, the IL-2 receptor γ chain is deficient, and the SCID mutation of the gene involved in the rearrangement of the antigen receptor genes of T cells and B cells is present at both allelic loci can be mentioned. Such a mouse is a NOG mouse (NOD / SCID / γc null (NOD / Shi-scid,IL-2RγKO mouse)), NSG mouse (NOD / Scid / IL2Rγ null (NOD.Cg-Prkdc scid IL2rg tm1Wjl / SzJ)), NCG mouse (NOD-Prkdc em26Cd52 IL2rg em26Cd22Examples include / NjuCrl). Furthermore, there are genetically modified immunodeficient mice (NOJ mice, NOD / Scid / Jak3) in which a mutation has been introduced into the Jak3 gene, resulting in a Jak3 deficiency, and SCID mutations in genes involved in the rearrangement of T cell and B cell antigen receptor genes are present at both allele loci. null (NOD.Cg-Prkdc scid Jak3 tm1card ) can also be used. Hereinafter, animals in which the function of the Prkdc gene and its gene product is lost due to scid mutations, etc., or in which the normal function of the IL2Rγ gene product is lost due to deletion or mutation of the IL2Rγ gene, or due to the function of genes downstream of signal transduction and their products, will be referred to as NOG mice ("NOG mouse" is a registered trademark) and can also be used as hosts. Since lymphocytes are not found in these mice, NOG mice do not show NK activity and also lack dendritic cell function. The method for producing NOG mice is described in WO2002 / 043477. The method for producing NSG mice is described in Ishikawa F. et al., Blood 106:1565-1573, 2005; the method for producing NCG mice is described in Zhou J. et al., Int J Biochem Cell Biol 46:49-55, 2014; and the method for producing NOJ mice is described in Okada S. et al., Int J Hematol 88:476-482, 2008.

[0029] Furthermore, W41 mutant mice can be used as immunodeficient mice in the present invention. The W41 mutant gene is a mouse in which the V831M mutation has been introduced into the c-kit gene. A method for producing W41 mutant mice is disclosed, for example, in Patent Document US9,668,463.

[0030] Furthermore, examples of immunodeficient mice used in the present invention include RAG1 knockout mice and RAG2 knockout mice.

[0031] Genome editing is a method of modifying target genes using site-specific nucleases. Genome editing methods include the ZFN (zinc finger nuclease) method (Urnov, Fyodor D. et al., Natur, Vol 435, 2 June 2005, pp.642-651), the TALEN (Tale nuclease) method (Mahfouz, Magdy M et al., PNAS February 8, 2011, 108(6), pp.2623-2628), CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 (Crispr Associated protein 9) (Jinek, Martin, et al., Science, Vol 337, 17 August 2012, pp.816-821), and CRISPR / Cas systems such as CRISPR / Cas3. These methods include methods using modified nucleases, such as those using modified Nicasse Cas. Of these, the method using the CRISPR / Cas9 system is preferred. In the CRISPR / Cas9 system, for example, a guide RNA (crRNA, tracrRNA) containing a sequence complementary to the target sequence of the complement C3 encoding gene, the clef4f gene, or the Axl gene of a rodent whose function is to be lost by cleavage is injected into a fertilized egg of an immunodeficient rodent gene. Within the fertilized egg, the complement C3 encoding gene, the clef4f gene, or the Axl gene of the rodent is deleted. The base length of the guide RNA is preferably 20 or more. Genome editing using CRISPR / Cas9 can be performed using commercially available CRISPR / Cas9 tools.

[0032] Homologous recombination is a phenomenon in which two DNA molecules in a cell undergo recombination via the same base sequence, and it is a method often used for recombination in organisms with large genomic DNA. A plasmid (called a targeting vector) is constructed by ligating other DNA to the sequence of the target gene region, with the sequence being divided in the middle. Specifically, a construct is made in which the DNA of a gene encoding complement C3, the clef4f gene, or the Axl gene, which does not function as other DNA, is sandwiched between homologous sequences of the upstream and downstream parts of a specific region in a rodent animal. This construct is then inserted into a known vector to create a targeting vector. This is then introduced into ES cells (embryonic stem cells) of an immunodeficient rodent animal. Through homologous recombination, exchange occurs between the DNA of the specific region in the rodent animal and the same sequence region on the targeting vector, and the sandwiched other DNA is incorporated into the rodent genome. In this way, ES cells in which the complement C3 encoding gene, Clec4f gene, or Axl gene is knocked out are established, and these ES cells are injected into rodent embryos or blastocysts. The chimeric embryos are then transplanted into the uterus of rodents that have been given a pseudopregnancy to create chimeric mice. By mating the resulting chimeric mice with the aforementioned immunodeficient rodents, individuals in which the complement C3 encoding gene, Clec4f gene, or Axl gene is knocked out can be obtained.

[0033] In this process, the vector used to construct the targeting vector can be one that is expressible and transformable in rodent ES cells, such as plasmids derived from E. coli, retroviruses, lentiviruses, adeno-associated viruses, and vaccinia viruses. The targeting vector can be introduced into ES cells by known methods such as electroporation, calcium phosphate coprecipitation, lipofection, microinjection, and particle gun. The vector may contain marker genes for selection, such as hygromycin resistance genes, neomycin resistance genes, and puromycin resistance genes. The marker genes may be removed after the selection of homologous recombinants, and can be removed using the Cre-loxP system or the Flp-frt system. Stem cells such as iPS cells from rodents can be used instead of ES cells.

[0034] Knockout rodents include both conventional knockout rodents and conditional knockout rodents. Conventional knockout rodents can be produced by homologous recombination and crossbreeding.

[0035] Deletions in the gene encoding complement C3, the Ckef4f gene, or the Axl gene in rodents can be investigated, for example, by isolating cells from rodents and performing expression analysis of the aforementioned genes.

[0036] 2. Characteristics of immunodeficient rodents in which the following three genes are knocked out: the gene encoding complement C3, the clec4f gene encoding the CLEC4F protein (a molecule involved in phagocytosis of red blood cells), and the Axl gene encoding the tyrosine kinase AXL protein. Immunodeficient rodents in which the three genes—the gene encoding complement C3 (created in step 1), the clec4f gene encoding the CLEC4F protein, a molecule involved in phagocytosis of red blood cells, and the Axl gene encoding the tyrosine kinase AXL protein—are knocked out have the following characteristics.

[0037] When human erythrocytes are transferred into the immunodeficient mice described above, the human erythrocytes are maintained in the blood of the immunodeficient mice without being destroyed. When human erythrocytes are introduced intravenously into immunodeficient mice, the cell density is increased to 5 × 10⁶ per mouse. 9 When prepared to a volume of 700 μl and transferred into 6-8 week old mice via the tail vein, the percentage of human red blood cells in the mouse blood (chimera rate) was 20% or more, preferably 25% or more, 3 days after transfer, and 15% or more, preferably 20% or more, 5 days after transfer. Furthermore, it was 7% or more, preferably 15% or more, 10 days after transfer, and 5% or more, 14, 15, 16, and 17 days after transfer.

[0038] Furthermore, the survival rate of human red blood cells is 70% or more, preferably 80% or more, 3 days after transplantation, and 60% or more, preferably 65% ​​or more, 5 days after transplantation. Moreover, it is 25% or more, preferably 30% or more, 10 days after transplantation, and 20% or more, 14, 15, 16, and 17 days after transplantation.

[0039] Furthermore, the half-life of the transplanted human erythrocytes is 5 days or more, preferably 7 days or more, more preferably 8 days or more, even more preferably 9 days or more, and even more preferably 10 days or more. When comparing the half-lives, the half-lives were 1.5 days, 3 days, 4 days, and 10 days for NOG mice, NOG-C3KO (NOG mice lacking only C3), NOG-C3 / clec4f DKO (NOG mice with double deletions of C3 and clec4f genes), and NOG-C3 / clec4f / Axl TKO (NOG mice with triple deletions of C3, clec4f genes, and Axl genes), respectively. A significant extension was observed in NOG-C3 / clec4f / Axl TKO (NOG mice with triple deletions of C3, clec4f genes, and Axl genes).

[0040] NOG-C3 / clec4f / Axl TKO mice with human erythrocytes (5x10) 9 If human erythrocytes are transplanted three times at one-week intervals, and the chimera rate (human erythrocytes / (human erythrocytes + mouse erythrocytes) × 100 (%)) is increased to 70-80%, the erythrocytes can be maintained for the following three weeks without additional transplantation. In other words, if human erythrocytes are transferred to the above-mentioned immunodeficient mice so that the proportion of human erythrocytes in the total blood cells of the mice is 70% or more, a chimera rate of 50% or more, preferably 60% or more, can be maintained for three weeks without further transfer.

[0041] Here, the percentage of human red blood cells in mouse blood (chimerism rate) and the survival rate of human red blood cells at each time point can be calculated using the following formula. The percentage of human red blood cells in mouse blood (chimerism rate) (%) = Human red blood cell count (cell count) / Total red blood cell count (human red blood cell count + mouse red blood cell count) × 100 Survival rate (%) = Percentage of human red blood cells (measurement day) / Percentage of human red blood cells (experiment start day) × 100

[0042] 3. Use of immunodeficient rodents in which three genes are knocked out: the gene encoding complement C3, the clec4f gene encoding the CLEC4F protein, a molecule involved in phagocytosis of red blood cells, and the Axl gene encoding the tyrosine kinase AXL protein. Immunodeficient rodents in which the gene encoding complement C3, the clec4f gene encoding the CLEC4F protein (a molecule involved in phagocytosis of red blood cells), and the Axl gene encoding the tyrosine kinase AXL protein are knocked out can be used for the following purposes.

[0043] (1) Use as a model animal for malaria infection Malaria is an infectious disease caused by the malaria parasite transmitted by the Anopheles mosquito, infecting more than 200 million people worldwide annually and causing 500,000 to 600,000 deaths. The life cycle of the malaria parasite is complex; after entering the human body, it infects liver cells, then migrates to the bloodstream and infects red blood cells. Therefore, developing a drug requires an infection model animal that can reproduce the liver stage, the red blood cell stage, or both stages. The parasite infects human red blood cells in a state called merozoites, forms a state called schizonts within the red blood cell, and ultimately destroys the human red blood cell while releasing a large amount of merozoites and sexual reproductive organs (gametocytes). Therefore, developing a drug that interrupts the parasite's life cycle at the red blood cell stage is considered important to prevent the spread and reinfection of red blood cells by merozoites and the migration of gametocytes to mosquitoes. The life cycle of the malaria parasite varies from 48 hours to 72 hours depending on the species of parasite, and developing a malaria drug requires experimental animals that can repeat this cycle several times. Malaria parasites exhibit high species specificity in infection, making infection experiments impossible in conventional laboratory animals. Therefore, infection experiments are conducted by transferring human red blood cells into immunodeficient mice, which are relatively easy to engraft with human cells and tissues. However, human red blood cells are eliminated from the mouse bloodstream very quickly, requiring daily engraftment. The triple-deficient immunodeficient rodent of this invention, in which the gene encoding complement C3, the clec4f gene encoding the CLEC4F protein (a molecule involved in red blood cell phagocytosis), and the Axl gene encoding the tyrosine kinase AXL protein are knocked out, is suitable for studying the red blood cell stages of malaria because it allows for long-term engraftment of human red blood cells. Furthermore, daily engraftment via the tail vein of rodents such as mice requires considerable effort in blood preparation and can lead to keratinization of the tail vein, making injection increasingly difficult. Such problems do not occur with the rodent of this invention. In other words, a triple-deficient immunodeficient rodent in which the gene encoding complement C3, the clec4f gene encoding the CLEC4F protein (a molecule involved in phagocytosis of red blood cells), and the Axl gene encoding the tyrosine kinase AXL protein are knocked out can be used as a malaria infection model animal that can reproduce human malaria infection.This model can be used as a powerful tool for screening therapeutic drugs and vaccines, and for developing treatments. For example, if the human red blood cells of the rodent animal of the present invention, which maintains human red blood cells in its blood, are infected with malaria, and a candidate therapeutic compound is administered to the rodent animal, and the malaria infection improves or is cured, it can be determined that the candidate compound can be used as a therapeutic agent for malaria. Alternatively, if a candidate vaccine compound is administered to the rodent animal of the present invention, which maintains human red blood cells in its blood, and then malaria parasites are administered to human red blood cells, and the human red blood cells are not infected with malaria, it can be determined that the candidate compound can be used as a vaccine for malaria.

[0044] (2) Development of artificial red blood cells Artificial red blood cells are being developed to replace human-derived red blood cells, which currently rely on blood donations. While particles consisting of hemoglobin molecules encapsulated in an artificial lipid bilayer are the mainstream, practical application of such lipid particles requires verification of their oxygen-carrying capacity, gas exchange capacity, stability in the blood, and safety for the body. In this mouse model, human red blood cells are maintained for a relatively long period, so artificial red blood cells are expected to maintain a similar engraftment period, enabling analysis of their functionality. Furthermore, while red blood cells express abundant glycans on their cell membranes, the roles of these glycans remain unclear. Since these glycans are absent in artificial red blood cells, a triple-deficient immunodeficient rodent animal, in which the gene encoding complement C3, the clec4f gene encoding the CLEC4F protein (a molecule involved in red blood cell phagocytosis), and the Axl gene encoding the tyrosine kinase AXL protein are knocked out, can be used to clarify the role of glycans in vivo.

[0045] (3) Use as an animal model for thalassemia and sickle cell disease Thalassemia and sickle cell disease are both genetic diseases that cause abnormalities in hemoglobin production. In severe cases, both conditions lead to hemolytic anemia, requiring treatment such as blood transfusions. Recent genome editing technology has enabled the development of gene therapy methods to target mutated genes in patient hematopoietic stem cells, and clinical trials have begun. The use of experimental animal models is necessary to examine the effectiveness of gene therapy and the normality of differentiation. By using these animals, it is possible to analyze whether the transplanted cells have the same function as normal human red blood cells through experiments such as transplanting hematopoietic stem cells after gene therapy, or differentiating them into red blood cells in vitro and transplanting them. Specifically, triple-deficient immunodeficient rodents in which the gene encoding complement C3, the clec4f gene encoding the CLEC4F protein (a molecule involved in phagocytosis of red blood cells), and the Axl gene encoding the tyrosine kinase AXL protein are knocked out can be used as animal models of thalassemia or sickle cell disease infection that can reproduce human thalassemia or sickle cell disease. This model can be used as a promising tool for developing treatment methods. [Examples]

[0046] The present invention will be specifically described by the following embodiments, but the present invention is not limited to these embodiments.

[0047] [Example 1] Preparation of mice (1) Generation of NOG-C3 deficient mice NOG-C3 knockout mice (NOG-C3KO mice) were created as previously reported (Yamaguchi, Front. Immunol., 27 July 2021, Volume 12 - 2021, https: / / doi.org / 10.3389 / fimmu.2021.671648).

[0048] (2) Disruption of the Clef4f gene The mouse Clec4f gene was disrupted using CRISPR / Cas9 genome editing technology with fertilized eggs derived from NOG-C3 KO mice. The guide RNA (gRNA) consisted of Clec4f gene-specific Crispr RNA (crRNA) and TransCrispr RNA (tracrRNA), and the sequence of the crRNA used is as follows. 5'-GGCCGAGCTGAACAGAGATATGG-3'(Sequence ID 5)

[0049] The above gRNA and recombinant Cas9 protein were introduced into mouse fertilized eggs using electroporation. These fertilized eggs were then transplanted into the uteruses of pseudopregnant mice.

[0050] A mouse strain was established with a 49-base pair deletion (nt 17-32) in the first exon of the Clec4f gene as a founder line (Figure 1). This mouse was crossed with NOG-C3 KO mice, and the resulting F1 mice were backcrossed with the founder mice to establish NOG-C3 / Clec4f double knockout (DKO) mice.

[0051] (3) Disruption of the mouse Axl gene The mouse Axl gene was disrupted using CRISPR / Cas9 genome editing technology with fertilized eggs derived from NOG-C3 / Clec4f DKO mice. The guide RNA (gRNA) consisted of Axl gene-specific Crispr RNA (crRNA) and TransCrispr RNA (tracrRNA), and the sequences of the crRNAs used are as follows. 5'-TCCCTGGGTTCCCCACAAACGGG-3'(Sequence No. 6)

[0052] The above gRNA and recombinant Cas9 protein were introduced into mouse fertilized eggs using electroporation. These fertilized eggs were then transplanted into the uteruses of pseudopregnant mice.

[0053] A mouse strain with a 16-base pair deletion (nt 88-104) in the second exon of the Axl gene was established as a founder line (Figure 2). This mouse was crossed with NOG-C3 / Clec4f DKO mice, and the resulting F1 mice were backcrossed with the founder mice to establish NOG-C3 / Clec4f / Axl triple knockout (TKO) mice.

[0054] [Example 2] Human red blood cell transfer experiment (1) Transfer of human red blood cells Peripheral blood was collected from healthy individuals using heparinized blood collection tubes, and plasma and red blood cells were separated by centrifugation (1700 rpm, room temperature, 5 minutes). After removing the plasma and intermediate layer (buffy coat), the precipitated human red blood cells were resuspended in 10 ml of phosphate buffer (PBS). The human red blood cells were again centrifuged, the supernatant and buffy coat were removed, and the cells were resuspended in PBS. The number of human red blood cells in this cell suspension was measured, and the cell density was determined to be 5 × 10⁶ per mouse. 9 The solution was prepared to a volume of 700 μl.

[0055] The above human erythrocytes were transferred via tail vein into 6-8 week old mice (NOG, NOG-C3 / Clec4f DKO mice, or NOG-C3 / Clec4f / Axl TKO mice). It has been suggested that the transferred human erythrocytes are phagocytosed by mouse macrophages. In fact, macrophages can be depleted in NOG mice by administering clodronate (Clo-lip). Therefore, in some experiments, mouse macrophages were removed beforehand by administering clodronate liposomes (Clo-lip, Xygieia Bioscience). Clo-lip was administered 7 days and 1 day before erythrocyte transplantation. The volume of Clo-lip was adjusted to 400 μL / kg of mouse body weight and suspended in 200 μL of physiological saline per mouse before being administered via tail vein.

[0056] For long-term survival experiments of human red blood cells, 5 x 10 units were used on the first day of the experiment. 9 Human red blood cells were transplanted into NOG-C3 / Clec4f / Axl TKO mice via the tail vein twice, at 6-hour intervals (5 x 10⁶ cells each time).9 , total 10 x 10 9 Using the same mice, 5 × 10⁶ mice were used on day 7 and day 14 after the start of the experiment. 9 Human erythrocytes were transplanted from the tail vein of mice. After confirming that the proportion of human erythrocytes in the total erythrocytes in the mouse blood reached 70% or more, the transplantation of human erythrocytes was terminated, and the proportion of human erythrocytes in the mouse blood was then analyzed over time.

[0057] (2) Measurement of human erythrocytes in mouse peripheral blood Human erythrocyte viability was analyzed using flow cytometry. Mouse peripheral blood (10 μl) was collected from the orbital venous plexus of mice anesthetized with isoflurane using heparin-coated capillaries. Blood was collected at 1 hour, 1, 2, 3, 4, 7, 10, 14, and 17 days after human erythrocyte transplantation. For long-term survival experiments, mouse peripheral blood was analyzed at 21, 28, and 34 days after administration of human red blood cells.

[0058] For flow cytometry analysis, mouse blood was diluted 10-fold with physiological saline after collection, and 5 μl of this diluted blood was used for antibody staining. Cells were stained with phycoerythrin (PE)-labeled anti-mouse TER119 antibody (Biolegend, Cat #116207) and allophycocyanin / Cyanine 7 (APC / Cy7)-labeled anti-human CD235a antibody (Glycophorin A, clone name HI264, Biolegend, Cat #349115) in the dark at room temperature for 30 minutes. The staining reaction was stopped by adding 100 μl of physiological saline, and 10 μl of this diluted blood was further diluted with 100 μl of physiological saline before detecting human erythrocytes using a FACS Fortessa X-20 flow cytometer (BD Biosciences).

[0059] The percentage of human red blood cells in mouse blood (chimerism rate) was calculated as follows: Human red blood cell count (cell count) / Total red blood cell count (human red blood cell count + mouse red blood cell count) × 100 (%).

[0060] The survival rate of human red blood cells at each time point was calculated as follows: Survival rate (%) = Percentage of human red blood cells (measurement day) / Percentage of human red blood cells (experiment start day) × 100.

[0061] Figures 3-1 and 3-2 show the results of the extension of human erythrocyte survival in NOG-C3 / Clec4f / Axl TKO mice. Figure 3-1 shows the chimera rate, and Figure 3-2 shows the survival rate. As shown in Figures 3-1 and 3-2, the survival period of human erythrocytes was significantly extended in NOG-C3 / Clec4f / Axl TKO mice compared to NOG mice and NOG-C3 / Clec4f DKO mice. Specifically, in NOG-C3 / clec4f / Axl TKO mice, human erythrocytes (5 × 10⁶) were significantly prolonged. 9 In an experiment in which human red blood cells were transplanted three times at one-week intervals, increasing the chimera rate (human red blood cells / (human red blood cells + mouse red blood cells) × 100 (%)) to 70-80%, it was possible to maintain the red blood cells for the following three weeks without additional transplantation.

[0062] When comparing the half-lives, NOG, NOG-C3KO, NOG-C3 / clec4f DKO, and NOG-C3 / clec4f / Axl TKO had half-lives of 1.5 days, 3 days, 4 days, and 10 days, respectively, showing a significant extension.

[0063] Figures 4-1 and 4-2 show the results of macrophage removal by clodronate. Figure 4-1 shows the chimera rate, and Figure 4-2 shows the survival rate. As shown in Figures 4-1 and 4-2, macrophage removal by clodronate significantly extended the survival period of human erythrocytes in NOG mice, but in NOG / C3 / Clec4f / Axl TKO mice, the survival period of human erythrocytes was significantly longer compared to clodronate-treated NOG mice, even without clodronate administration.

[0064] Figure 5 shows the long-term survival of human erythrocytes in NOG-C3 / Clec4f / Axl TKO mice. As shown in Figure 5, once the percentage of human erythrocytes in the peripheral blood of NOG-C3 / Clec4f / Axl TKO mice exceeded 70%, a chimeric rate of over 60% was maintained for the following three weeks without further blood transfusion. [Industrial applicability]

[0065] Triple-deficient immunodeficient rodents, in which the gene encoding complement C3, the clec4f gene encoding the CLEC4F protein (a molecule involved in phagocytosis of red blood cells), and the Axl gene encoding the tyrosine kinase AXL protein are knocked out, can maintain human red blood cells in their blood for extended periods and are useful for malaria research. [Sequence Listing Free Text]

[0066] Sequence IDs 2, 4, 5, and 6 are synthesized.

Claims

1. An immunodeficient rodent in which three genes—the gene encoding complement C3, the clec4f gene, and the Axl gene—have been knocked out.

2. The rodent animal according to claim 1, which is a mouse.

3. The immunodeficient rodent according to claim 2, which is a SCID mutant mouse, a RAG1 knockout mouse, a RAG2 knockout mouse, or a NOD / SCID mouse.

4. This is a NOG mouse, and its official strain name is NOD.Cg-Prkdc scid Il2rg tm1Sug Mpl em1 An immunodeficient rodent according to claim 3, represented as / Jic.

5. A method for producing an immunodeficient rodent according to any one of claims 1 to 4, comprising knocking out three genes: the gene encoding complement C3, the clec4f gene, and the Axl gene of the immunodeficient rodent.

6. An immunodeficient rodent animal according to any one of claims 1 to 4, which maintains transferred human red blood cells in the blood for a long period of time.

7. An immunodeficient rodent according to any one of claims 1 to 4, wherein, when human red blood cells are transferred to the rodent according to any one of claims 1 to 4, the percentage of human red blood cells in the blood of the rodent five days after transfer is 15% or more.

8. An immunodeficient rodent according to any one of claims 1 to 4, wherein, when human red blood cells are transferred to the rodent according to any one of claims 1 to 4, the survival rate of human red blood cells in the blood of the rodent five days after transfer is 60% or more.

9. A method for producing a rodent in which human red blood cells are maintained in the blood for a long period of time, comprising transferring human red blood cells into a rodent according to any one of claims 1 to 4.

10. A method for producing an immunodeficient rodent according to claim 9, wherein, when human red blood cells are transferred to a rodent according to any one of claims 1 to 4, the percentage of human red blood cells in the blood of the rodent five days after the transfer is 15% or more.

11. A method for producing an immunodeficient rodent according to claim 9, wherein, when human red blood cells are transferred to the rodent according to any one of claims 1 to 4, the survival rate of human red blood cells in the blood of the rodent five days after transfer is 60% or more.

12. Use as a model rodent for research on the treatment of malaria infection in rodents as described in claim 6.

13. A human malaria infection model rodent prepared by infecting the rodent described in claim 6 with malaria.

14. A method for screening therapeutic drugs and vaccines for malaria infection using a malaria infection model rodent animal as described in claim 13.

15. Use of the rodent described in claim 6 as a model rodent for research on the treatment of thalassemia or sickle cell disease.

16. Use for the development of artificial red blood cells in rodents as described in claim 6.

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