Genetically modified non-human animals that express human EPO

Abstract

To provide a gene-modified non-human animal which exerts a human EPO from an animal genome, and a method for creating the non-human animal.SOLUTION: There is provided a mouse for exerting human EPO protein (hEPO) which is created by: a process for making a nucleic acid sequence including a coding sequence or its segment which is functionally connected to an EPO promoter sequence contact with a mouse pluripotent stem cell, being a process for forming a cassette in which sequences in which the coding sequence and the promoter sequence are homologous with an endogenous mouse EPO gene locus adjacently contact with each other; a process for cultivating the pluripotent stem cell under a condition that the integration of the nucleic acid sequence to a mouse genome at the endogenous mouse EPO gene locus caused by a homologous recombination is promoted; and a process for creating a mouse from the mouse pluripotent stem cell including the nucleic acid sequence for coding the hEPO.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 000,460, filed on 19 May 2014, whose entire disclosure is incorporated herein by reference.

[0002] Field of Invention This invention relates to the field of genetically modified non-human animals. [Background technology]

[0003] introduction Genetically modified mice, genetically modified and engrafted mice, and their use in modeling human diseases are well known in the art. However, to date, the generation of genetically modified mice that model human infections by pathogens targeting human erythrocyte lineage cells has been largely unsuccessful. Such pathogens, such as protozoa of the genera Plasmodium, Babesia, and Theileria, can cause life-threatening diseases in humans.

[0004] For example, protozoa of the genus Plasmodium cause malaria. In 2010, a total of 106 countries worldwide were considered to be endemic to malaria, and an estimated 3.3 billion people were at risk of developing the disease. The global disease burden in 2010 was estimated at 216 million cases, with an estimated 655,000 deaths, 86% of which were children under the age of five. Currently, drugs and vaccines for the prevention and treatment of malaria remain extremely limited. Furthermore, the emergence of parasitic resistance to commonly used malaria treatments presents a persistent challenge. Therefore, the development of new drugs and vaccines for the control and treatment of pathogens that target human red blood cells is urgently needed.

[0005] Since many of these pathogens do not infect the red blood cells of laboratory rodents, in vivo studies have traditionally focused on malaria caused by the rodent parasite Plasmodium berghei ANKA, or on NOD / SCID mice and NOD / SCID / IL2rg mice, which are acutely engrafted by daily infusions of a large number of human red blood cells and simultaneously or subsequently infected by infusions of infected red blood cells. nullResearch has been limited to (NSG) mice or BXN mice (Angulo-Barturen et al. (2008) A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes. PLoS One 3:e2252 (Non-patent Literature 1); Jimenez-Diaz et al. (2009) Improved murine model of malaria using Plasmodium falciparum competent strains and non-myelodepleted NOD-scid IL2Rgnull mice engrafted with human erythrocytes. Antimicrob Agents Chemother 53:4533-4536 (Non-patent Literature 2); Badell et al. (2000) Human malaria in immunocompromised mice: an in vivo model to study defense mechanisms against Plasmodium falciparum. JEM 192(11):1653-1660 (Non-patent Literature 3); Moreno et al. (2006) The course of infections and pathology in immunomodulated NOD / LtSz-SCID mice inoculated with Plasmodium falciparum laboratory lines and clinical isolates. Int.J.Parasitol.36:361-369 (Non-patent Literature 4).To study the effects of malaria parasites and other pathogens on humans, and to test vaccines and drugs for their efficacy in preventing infection by these and other pathogens and in treating infected humans, it would be useful to have non-human animals, such as genetically modified mice, that are susceptible to such pathogens or that better support human erythrocytes acutely transplanted into the animals before infection, as is done in traditional rodent models. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Angulo-Barturen et al. (2008) A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes. PLoS One 3:e2252 [Non-Patent Document 2] Jimenez-Diaz et al. (2009) Improved murine model of malaria using Plasmodium falciparum competent strains and non-myelodepleted NOD-scid IL2Rgnull mice engrafted with human erythrocytes. Antimicrob Agents Chemother 53:4533-4536 [Non-Patent Document 3] Badell et al. (2000) Human malaria in immunocompromised mice: an in vivo model to study defense mechanisms against Plasmodium falciparum. JEM 192(11):1653-1660 [Non-Patent Document 4] Moreno et al. (2006) The course of infections and pathology in immunomodulated NOD / LtSz-SCID mice inoculated with Plasmodium falciparum laboratory lines and clinical isolates. Int.J.Parasitol.36:361-369 [Overview of the project]

[0007] overview Genetically modified non-human animals expressing human EPO from an animal genome are provided. Methods for producing non-human animals expressing human EPO from a non-human animal genome, and methods for using non-human animals expressing human EPO from a non-human animal genome are also provided. These animals and methods have many applications in the art, including, for example, modeling human erythropoiesis and erythrocyte function; modeling human pathogen infection of erythrocytes; in vivo screening for agents that modulate erythropoiesis and / or erythrocyte function in, for example, healthy or diseased states; in vivo screening for agents that are toxic to erythrocytes or erythrocyte progenitor cells; in vivo screening for agents that prevent, mitigate, or reverse the toxic effects of toxic agents to erythrocytes or erythrocyte progenitor cells; and in vivo screening of individual-derived erythrocytes or erythrocyte progenitor cells to predict individual responsiveness to disease treatment.

[0008] In some aspects of the present invention, a genetically modified non-human animal is provided that expresses human EPO from the genome of a non-human animal. In other words, the non-human animal contains a nucleic acid sequence that encodes the human EPO protein.

[0009] In some embodiments, the nucleic acid sequence encoding the human EPO protein is functionally ligated to the EPO gene promoter. In some embodiments, the EPO gene promoter is the human EPO promoter. In other embodiments, the EPO promoter is endogenous, i.e., non-human EPO promoter. In certain such embodiments, the endogenous EPO promoter is located at the non-human animal EPO locus. In other words, in certain embodiments, the nucleic acid sequence encoding the human EPO protein is functionally ligated to the non-human animal EPO promoter at the non-human animal EPO locus. In some such embodiments, the functional ligation results in a null mutation of the non-human EPO gene at the non-human EPO locus.

[0010] In some embodiments, the non-human animal of the present invention is heterozygous for alleles containing nucleic acid sequences encoding human EPO protein. In other embodiments, the non-human animal is homozygous for alleles containing nucleic acid sequences encoding human EPO protein.

[0011] In some embodiments, the nucleic acid sequence encoding the human EPO protein includes coding and non-coding sequences of the human EPO genome. In other embodiments, the nucleic acid sequence encoding the human EPO protein includes the human EPO cDNA sequence.

[0012] In some embodiments, the non-human animal of the present invention expresses one or more additional human proteins selected from the group consisting of M-CSF protein encoded by nucleic acid under the regulation of an M-csf promoter, IL-3 protein encoded by nucleic acid under the regulation of an Il-3 promoter, GM-CSF protein encoded by nucleic acid under the regulation of a Gm-csf promoter, TPO protein encoded by nucleic acid under the regulation of a TPO promoter, and Sirpa protein encoded by nucleic acid under the regulation of a Sirpa promoter. In some such embodiments, the promoter is an endogenous non-human animal promoter located at the corresponding non-human animal locus, and the non-human animal is heterozygous null for the non-human gene. In other embodiments, the promoter is an endogenous non-human animal promoter located at the corresponding non-human animal locus, and the non-human animal is homozygous null for the non-human gene. In certain embodiments, non-human animals express human proteins, e.g., humanized proteins, selected from the group consisting of human TPO, e.g., humanized TPO; human IL-3, e.g., humanized IL-3; human GM-CSF, e.g., humanized GM-CSF; human EPO, e.g., humanized EPO; and human Sirpa, e.g., humanized Sirpa; and combinations thereof.

[0013] In some embodiments, the genetically modified non-human animals of the present invention are mice having a genome containing nucleic acids encoding human proteins, such as humanized proteins, e.g., human EPO, e.g., humanized EPO; human Sirpa, e.g., humanized Sirpa; human IL-3, e.g., humanized IL-3; human GM-CSF, e.g., humanized GM-CSF; human M-CSF, e.g., humanized M-CSF; human TPO, e.g., humanized TPO; human IL-6, e.g., humanized IL-6, etc., functionally linked to corresponding non-human animal promoters, e.g., Sirpa promoter, IL-3 promoter, GM-CSF promoter, M-CSF promoter, TPO promoter, or IL-6 promoter, respectively, and expressing encoded human proteins and native mouse proteins. In other embodiments, the genetically modified non-human animal of the present invention is a mouse having a genome containing nucleic acids encoding human proteins, e.g., humanized proteins, e.g., human EPO, e.g., humanized EPO; human Sirpa, e.g., humanized Sirpa; human IL-3, e.g., humanized IL-3; human GM-CSF, e.g., humanized GM-CSF; human M-CSF, e.g., humanized M-CSF; human TPO, e.g., humanized TPO, functionally linked to the corresponding non-human animal promoters, e.g., Sirpa promoter, IL-3 promoter, IL-3 promoter, IL-3 promoter, e.g., humanized Sirpa; human IL-3, e.g., humanized IL-3; human GM-CSF, e.g., humanized GM-CSF; human M-CSF, e.g., humanized M-CSF; human TPO, e.g., humanized TPO, functionally linked to the corresponding non-human animal promoters, e.g., Sirpa promoter, IL-3 promoter, IL-3 promoter, GM-CSF promoter, M-CSF promoter, or TPO promoter, e.g., humanized EPO; human Sirpa, e.g., humanized Sirpa; human IL-3, e.g., humanized IL-3; human GM-CSF, e.g., humanized GM-CSF; human M-CSF, e.g., humanized M-CSF; human TPO, e.g., humanized TPO; expressing the encoded human proteins and not expressing native mouse proteins. Thus, in some embodiments, the genetically modified non-human animal is a mouse, and the mouse is heterozygous for some or all of the human genes, e.g., humanized genes, disclosed herein. In some embodiments, the genetically modified non-human animal is a mouse, and the mouse is homozygous for some or all of the human genes disclosed herein, e.g., humanization genes.

[0014] In some embodiments, the non-human animals of the present invention are immunodeficient with respect to their endogenous immune systems. In some such embodiments, the immunodeficiency is caused by a deficiency of one or both Rag2 and IL2rg.

[0015] In some embodiments, the genetically modified immunodeficient non-human animals of the present invention further include the engraftment of human hematopoietic cells. In some such embodiments, the human hematopoietic cells include one or more cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human myeloid progenitor cells, human erythrocyte progenitor cells, human myeloid cells, human dendritic cells, human monocytes, human granulocytes, human erythrocytes, human neutrophils, human mast cells, human thymocytes, and human B lymphocytes.

[0016] As demonstrated in the examples herein, genetically modified immunodeficient non-human animals engrafted with human hematopoietic cells, containing a nucleic acid sequence encoding a human EPO protein functionally linked to the EPO promoter at an endogenous locus, exhibit high levels of human erythrogenesis in the bone marrow and a 2- to 5-fold increase in human erythrocyte lineages in the bone marrow compared to control mice that do not express human EPO. In some embodiments, genetically modified immunodeficient non-human animals engrafted with human hematopoietic cells, containing a nucleic acid sequence encoding a human EPO protein functionally linked to the EPO promoter at an endogenous locus, exhibit high levels of human erythrogenesis in the bone marrow and a roughly 2- to 10-fold increase in human erythrocyte lineages in the bone marrow compared to control mice that do not express human EPO, for example, a roughly 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold increase in human erythrocyte lineages in the bone marrow compared to control mice that do not express human EPO. Accordingly, in some embodiments, the engrafted genetically modified immunodeficient animal of the present invention contains bone marrow in which 20% or more of the erythrocytes (CD235+) are human erythrocytes. In some embodiments, the engrafted genetically modified immunodeficient animal of the present invention contains bone marrow in which about 10% or more of the erythrocytes (CD235+), for example, about 20% or more, about 30% or more, about 40% or more, or about 50% or more are human erythrocytes.

[0017] In some such embodiments, the genetically modified animal of the invention comprises a nucleic acid encoding a human EPO protein operably linked to a non-human animal EPO promoter at the non-human animal EPO locus, a nucleic acid encoding a human TPO protein operably linked to a non-human animal TPO promoter at the non-human animal TPO locus, a nucleic acid encoding a human Il-3 protein operably linked to a non-human animal Il-3 promoter at the non-human animal Il-3 locus, a nucleic acid encoding a human GM-CSF protein operably linked to a GM-CSF promoter at the non-human animal GM-CSF locus, and a nucleic acid encoding a human M-CSF protein operably linked to a non-human animal M-CSF promoter at the non-human animal M-CSF locus, in an immunodeficient mouse, such as Rag2 - / - IL2rg y / - Tpo h / h Mcsf h / h Il3 h / h Gmcsf h / h Epo h / h (「MITER-G」) mice.

[0018] In some such embodiments, the genetically modified animal of the invention comprises a nucleic acid encoding a human EPO protein operably linked to a non-human animal EPO promoter at the non-human animal EPO locus, a nucleic acid encoding a human TPO protein operably linked to a non-human animal TPO promoter at the non-human animal TPO locus, a nucleic acid encoding a human Il-3 protein operably linked to a non-human animal Il-3 promoter at the non-human animal Il-3 locus, a nucleic acid encoding a human GM-CSF protein operably linked to a GM-CSF promoter at the non-human animal GM-CSF locus, and a nucleic acid encoding a human M-CSF protein operably linked to a non-human animal M-CSF promoter at the non-human animal M-CSF locus, and a nucleic acid encoding a human SIRPa protein operably linked to a non-human animal SIRPa promoter randomly integrated into the non-human animal genome, in an immunodeficient mouse, such as Rag2 - / - IL2rgy / - TPO h / h Mcsf h / h Il3 h / h Gmcsf h / h Epo h / h hSIRPα + This is a ("MISTER-G") mouse. In other such embodiments, a nucleic acid encoding a human SIRPa protein, for example, a humanized SIRPa protein, is functionally linked to a non-human SIRPa promoter at a non-human animal locus (e.g., Rag2). - / - IL2rg y / - TPO h / h Mcsf h / h Il3 h / h Gmcsf h / h Epo h / h SIRPα h / h ("SupER-G" mouse).

[0019] In some such embodiments, the genetically modified animal of the present invention comprises an immunodeficient mouse, for example, Rag2, comprising: one allele of a nucleic acid encoding a human EPO protein functionally linked to the non-human EPO promoter at the non-human EPO locus (i.e., the mouse is heterozygous for human EPO); a nucleic acid encoding a human SIRPa protein functionally linked to the non-human SIRPa promoter at the non-human SIRPa locus, e.g., a nucleic acid encoding a humanized SIRPa protein; a nucleic acid encoding a human TPO protein functionally linked to the non-human TPO promoter at the non-human TPO locus; a nucleic acid encoding a human Il-3 protein functionally linked to the non-human Il-3 promoter at the non-human Il-3 locus; and a nucleic acid encoding a human GM-CSF protein functionally linked to the GM-CSF promoter at the non-human GM-CSF locus. - / - IL2rg y / - TPO h / h Il3 h / h Gm-csf h / h Epo h / m SIRPα h / h This is a ("TIES") mouse.

[0020] In some embodiments, genetically modified immunodeficient non-human animals engrafted with human hematopoietic cells, containing a nucleic acid sequence encoding the human EPO protein functionally linked to the EPO promoter at an endogenous locus, may exhibit better survival and human erythrocyte engraftment when containing only one copy of the EPO protein-encoding nucleic acid sequence and when containing endogenous M-CSF. This is because high levels of human myeloid cell engraftment supported by human M-CSF in knock-in lead to the destruction of mouse erythrocytes, which in turn leads to anemia and death in the engrafted mice. Furthermore, heterozygosity for the human EPO allele improves reproductive capacity, developmental ability, and survival rate compared to mice that are homozygous for human EPO and null for mouse EPO. Therefore, in some embodiments, the engrafted genetically modified immunocompromised animals of the present invention may constitutively express EPO in mice, e.g., transgenic mice, or mice containing two copies of human EPO, e.g., EPO h / h It shows a more improved survival rate. In some such embodiments, genetically modified immunodeficient non-human animals are TIES mice (Rag2 - / - IL2rg y / - TPO h / h Il3 h / h Gm-csf h / h Epo h / m SIRPα h / h )

[0021] In some embodiments, genetically modified immunodeficient non-human animals with engrafted human hematopoietic cells injected with clodronate liposomes show a 1,000-fold increase in the number of human erythrocytes (CD235+) in peripheral blood compared to animals without injection. In some embodiments, genetically modified immunodeficient non-human animals with engrafted human hematopoietic cells injected with clodronate liposomes show an increase of approximately 10-fold, 50-fold, 100-fold, 500-fold, or 1,000-fold or more in the number of human erythrocytes (CD235+) in peripheral blood compared to animals without injection. Of these human erythrocytes, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more may be reticulocytes (erythrocyte progenitor cells, CD71+). Accordingly, in some embodiments, the engrafted genetically modified immunodeficient animal of the present invention contains peripheral blood in which 1% or more of the erythrocytes (CD235+), for example 5% or more or 10% or more, are human erythrocytes, and 10% or more of these human erythrocytes, for example 20% or more, 30% or more, 40% or more, or 50% or more, are human reticulocytes (CD71+). In some such embodiments, the engrafted genetically modified immunodeficient non-human animal of the present invention is a MISTER-G mouse, a SupER-G mouse, or a TIES mouse.

[0022] In some embodiments, the non-human animal further comprises infection with a pathogen that targets human cells of the erythrocyte lineage. In some such embodiments, the pathogen is selected from species of the genera Plasmodium, Babesia, and Theileria. In some embodiments, the infection is produced by injecting the parasite into the non-human animal. In some embodiments, the infection is produced by injecting infected human erythrocytes into the non-human animal. In some embodiments, the infection is produced by injecting infected human erythrocytes and healthy human erythrocytes into the non-human animal.

[0023] In some aspects of the present invention, a method is provided for identifying agents that inhibit infection by pathogens targeting human cells of the erythrocyte lineage.

[0024] In some embodiments, the method includes administering a candidate drug to a genetically modified non-human animal that includes a nucleic acid sequence encoding a human EPO protein functionally linked to an EPO gene promoter, one or more gene mutations causing immunodeficiency in the non-human animal, engraftment of human hematopoietic cells, and infection with a pathogen targeting human cells of the erythrocyte lineage; and determining whether the drug reduces the amount of pathogen in the non-human animal infected with the pathogen.

[0025] In some embodiments, the method includes the steps of: contacting a genetically modified immunodeficient non-human animal in which human hematopoietic cells have been engrafted, comprising a nucleic acid sequence encoding a human EPO protein functionally linked to an EPO gene promoter, with clodronate; administering a candidate drug to the non-human animal that has been contacted with clodronate; injecting parasitized reticulocytes or erythrocytes into the genetically modified non-human animal; and determining whether the drug prevents infection of human reticulocytes and / or erythrocytes in the non-human animal.

[0026] In some embodiments, the pathogen is selected from species of the genera Plasmodium, Babesia, and Theileria. In some such embodiments, the pathogen is selected from Plasmodium falciparum and Plasmodium vivax. In some embodiments, the non-human animal is a mammal. In some such embodiments, the mammal is a rodent. In certain such embodiments, the rodent is a mouse.

[0027] In some aspects of the present invention, a method for producing mice expressing human EPO protein is provided. In some embodiments, the method includes contacting mouse pluripotent stem cells with a nucleic acid sequence comprising a coding sequence or fragment thereof of human EPO protein functionally linked to an EPO promoter sequence, wherein the coding sequence and the EPO promoter sequence form a cassette in which sequences homologous to an endogenous mouse EPO locus are adjacent; culturing the pluripotent stem cells under conditions that promote the incorporation of the nucleic acid sequence into the mouse genome at the endogenous mouse EPO locus by homologous recombination; and producing mice from mouse pluripotent stem cells comprising a nucleic acid sequence encoding human EPO protein.

[0028] In some embodiments, mouse pluripotent stem cells are embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. In some embodiments, mouse pluripotent stem cells lack Rag2 and / or IL2rg. In some embodiments, the EPO promoter sequence is the sequence of the human EPO promoter. In other embodiments, the EPO promoter sequence is the sequence of the endogenous non-human EPO promoter. In some embodiments, incorporation results in the substitution of a non-human EPO gene at the non-human EPO locus. In some embodiments, the nucleic acid sequence encoding the human EPO protein includes coding and non-coding sequences of the human EPO genome. In some embodiments, the nucleic acid sequence encoding the human EPO protein includes the human EPO cDNA sequence.

[0029] In some aspects of the present invention, a method is provided for producing mice that express human EPO protein and include a human hematopoietic lineage. In some embodiments, the method includes transplanting a population of cells, including human hematopoietic progenitor cells, into a genetically modified immunodeficient mouse produced by the method of the present disclosure. In some embodiments, the transplantation includes tail vein injection, fetal liver injection, or posterior orbital injection. In some embodiments, the genetically modified mouse is irradiated to a sublethal dose prior to transplantation. In some embodiments, the human hematopoietic progenitor cells to be transplanted are CD34+ cells. In some embodiments, the human hematopoietic progenitor cells are derived from fetal liver, adult bone marrow, or umbilical cord blood.

[0030] In some aspects of the present invention, a method is provided for producing mice infected with human pathogens that target human cells of the erythrocyte lineage. In some embodiments, the method comprises the steps of producing mice expressing human EPO protein and including a human hematopoietic lineage according to the method of the present disclosure, injecting clodronate into the engrafted mice, and injecting parasitized human erythrocytes (PRBCs) into the clodronate-injected mice. In some embodiments, the method further comprises the step of injecting healthy human erythrocytes into the mice. In some embodiments, the parasites are selected from species of the genera Plasmodium, Babesia, and Theileria. In certain embodiments, the malaria parasites are selected from Plasmodium falciparum and Plasmodium vivax. [Brief explanation of the drawing]

[0031] This invention will be best understood from the following detailed description when referenced with the accompanying drawings. Following common practice, it should be emphasized that the various features in the drawings are not to a constant scale. Conversely, the dimensions of the various features have been arbitrarily enlarged or reduced for clarity. The drawings include the following figures:

[0032] [Figure 1]This provides protein alignment of mouse EPO (SEQ ID NO:2) with human EPO (SEQ ID NO:4). Underlined residues are conserved across species. [Figure 2] This provides schematic diagrams of the wild-type mouse EPO locus before and after knock-in of the nucleic acid sequence encoding human EPO. [Figure 3] A schematic diagram of the human EPO knock-in allele is provided. [Figure 4] This provides schematic diagrams of the wild-type mouse Sirpa locus before (top) and after (bottom) knock-in of the nucleic acid sequence encoding humanized Sirpa. [Figure 5] Panels A and B show the frequency of human erythrocytes in mice with HSC engraftment. (Panel A) Engraftment of human CD235a+ erythrocytes in the bone marrow 6–8 weeks after HSC engraftment in Rag2- / -Il2rg- / -Tpoh / hIL3h / hGmcsfh / hMcsfh / h mice ("MITRG mice") containing the indicated combination of human EPO expressed from the mouse EPO locus ("hEPO+") and / or human SIRPa expressed as a random integration into the mouse genome ("hSIRPa+"). (Panel B) Frequency of human CD235a+ erythrocytes in peripheral blood 6-8 weeks after HSC engraftment in Rag2- / -Il2rgnullTpoh / hMcsfh / hIl3h / hGmcsfh / hEpoh / hSIRPαh / h ("SupER-G") mice versus Rag2- / -Il2rgnullTpoh / hIl3h / hGmcsfh / hSirpah / h ("RGSKI-TI") control mice, in the presence or absence of hEPO. [Figure 6]Panels A and B demonstrate that clodronate treatment increases circulating human erythrocytes and reticulocytes in mice with engrafted hematocrit (HSCs). Seven weeks after HSC engraftment, SupER-G mice were treated with daily post-orbital injections of 50 μl of clodronate liposomes for 3–5 consecutive days. The frequency of human CD235+ cells (erythrocytes and reticulocytes) (Panel A) and CD235+ / CD71+ cells (reticulocytes) (Panel B) in peripheral blood was measured by FACS. Panel B: Three different mice after clodronate treatment. [Figure 7] Panels A-C illustrate the susceptibility of human RBCs produced from engrafted mice to infection by Plasmodium falciparum. Fetal liver HSCs or adult HSCs were engrafted into SupER-G mice. Seven weeks after engraftment, the mice were treated with daily post-orbital injections of 50 μl of clodronate liposomes for three consecutive days, after which blood was collected. The blood samples were then cultured with purified Plasmodium falciparum 3D7 erythrocyte-stage RBCs (99% purity). Fresh human RBCs were added to the culture 48 hours after infection, and the infection culture was maintained for a further 10 days. Giemsa staining and quantitative PCR were performed to quantify parasitemia. Human RBC control: Human RBCs; Mouse RBC control: RBCs from non-engrafted mice; Mouse RBC supplementation control: RBCs from non-engrafted mice supplemented with 0.1% hRBCs. In Panel A, red represents anti-human Band 3; blue represents Hoechst. In Panel C, the descriptions listed from top to bottom correspond to the x-axis of the bar graph from left to right. [Figure 8] This shows the disruption of human RBCs in mouse peripheral blood in the absence of clodronate. Mice that had not undergone engraftment were treated with either clodronate or PBS. In the case of clodronate treatment, mice received daily post-orbital injections of 50 μl of clodronate for three consecutive days. In the case of PBS treatment, only 500 μl of PBS was delivered one hour before human RBC infusion. Human RBCs were infused into pre-treated mice, and peripheral blood was collected at the indicated time points. Curves for clodronate and PBS are shown. [Modes for carrying out the invention]

[0033] Detailed explanation Genetically modified non-human animals expressing human EPO from an animal genome are provided. Methods for producing non-human animals expressing human EPO from a non-human animal genome, and methods for using non-human animals expressing human EPO from a non-human animal genome are also provided. These animals and methods have many applications in the art, including, for example, modeling human erythropoiesis and erythrocyte function; modeling human pathogen infection of erythrocytes; in vivo screening for agents that modulate erythropoiesis and / or erythrocyte function in, for example, healthy or diseased states; in vivo screening for agents that are toxic to erythrocytes or erythrocyte progenitor cells; in vivo screening for agents that prevent, mitigate, or reverse the toxic effects of toxic agents on erythrocytes or erythrocyte progenitor cells; and in vivo screening of individual-derived erythrocytes or erythrocyte progenitor cells to predict individual responsiveness to disease treatment. These and other objectives, advantages, and features of the present invention will become apparent to those skilled in the art by referring to the details of the compositions and methods described below.

[0034] Before describing the methods and compositions of the present invention, it should be understood that the present invention is not limited to the specific methods or compositions described and is therefore naturally subject to change. Since the scope of the present invention is limited only by the appended claims, it should also be understood that the terminology used herein is for describing specific embodiments only, and not for limiting them. The present invention is described by the patented claims, not by the specific embodiments described.

[0035] Where a range of values ​​is provided, unless the context clearly indicates otherwise, each value that falls between the upper and lower limits of that range is also specifically disclosed to the extent of one-tenth of the lower limit. Smaller ranges between a specified value or a value that falls within a specified range and other specified values ​​or values ​​that fall within a specified range are each included in the present invention. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, and each range that includes one of the limit values, does not include either, or includes both is also included in the present invention and follows the specifically excluded limit values ​​in the specified range. Where a specified range includes one or both limit values, ranges that exclude one or both of the limit values ​​that they include are also included in the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or trial of the invention, but several possible preferred methods and materials are described below. All publications referenced herein are incorporated herein by reference to disclose and explain methods and / or materials relating to citations of those publications. In the event of any conflict, it is understood that this disclosure shall prevail over the disclosures of the incorporated publications.

[0037] As will be obvious to those skilled in the art by reference to this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that may be readily separated from or combined with any of the features of several other embodiments without departing from the scope or spirit of the invention. The described methods may be carried out in the order of the described events, or in any other order that is logically possible.

[0038] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless the context explicitly indicates otherwise. Thus, for example, “a cell” includes multiple such cells, and “the peptide” includes one or more peptides and their equivalents, such as polypeptides known to those skilled in the art, and so on.

[0039] The publications described herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the present invention does not qualify as prior to such publications for the sake of prior art. Furthermore, the publication dates provided may differ from the actual publication dates and should be independently verified.

[0040] Genetically modified non-human animals In one aspect of the present invention, a non-human animal is provided that has been genetically modified to express one or more human proteins from its genome. In several aspects of the present invention, the human protein is human erythropoietin (hEPO) protein (SEQ ID NO: 4). In other words, the genetically modified non-human animal contains a nucleic acid sequence in its genome that codes for human EPO (hEPO) protein. For example, the non-human animal may contain a nucleic acid sequence in its genome that includes the coding and non-coding sequences of the human EPO genome, for example, the sequence or fraction thereof of nucleotides 100318423 to 100321323 of chromosome 7. Alternatively, the non-human animal may contain a nucleic acid sequence in its genome that includes the human EPO cDNA sequence (SEQ ID NO: 3) or a fraction thereof. In some cases, the non-human animal is further genetically modified to express one or more additional human proteins from the genome of the non-human animal.In some such embodiments, these one or more additional human proteins are human proteins that promote the development and / or function of human hematopoietic cells, e.g., human signal regulatory protein α (hSIRPa) protein (NCBI Gene ID: 140885, GenBank accession numbers NM_080792.2, NM_001040022.1, NM_001040023.1), human interleukin 3 (hIL-3) protein (NCBI Gene ID: 3562, GenBank accession number NM_000588.3), human colony-stimulating factor 2 (granulocyte-macrophage) (hGM-CSF) protein (NCBI Gene ID: 1437, GenBank accession number NM_000758.3), human colony-stimulating factor 1 (macrophage) (hM-CSF) protein (NCBI Gene ID:1435, GenBank accession numbers NM_000757.5, NM_172210.2, NM_172211.3, and NM_172212.2), human thrombopoietin (hTPO) protein (NCBI Gene ID:7066, GenBank accession numbers NM_000460.3, NM_001177597.2, NM_001177598.2, NM_001289997.1, NM_001290003.1, NM_001290022.1, NM_001290026.1, NM_001290027.1, NM_001290027.1), human interleukin-6 (hIL6) protein (NCBI Gene ID:3569, GenBank accession number NM_000600.3, etc.

[0041] Those skilled in the art will recognize that the terms “human nucleic acids” and “human proteins” encompass not only “wild-type” or “native” human nucleic acids and human proteins, but also variants of wild-type human nucleic acids and human proteins. As used herein, the term “variant” defines either an isolated, naturally occurring genetic variant of a human polypeptide or human nucleic acid sequence, or a variant prepared by recombination of a human polypeptide or human nucleic acid sequence, each containing one or more mutations compared to the corresponding wild-type human nucleic acid or human polypeptide sequence. For example, such mutations may be one or more amino acid substitutions, additions, and / or deletions. The term “variant” also includes human homologs and orthologues. In some embodiments, the variant polypeptides of the present invention have 70% or more identity with wild-type human polypeptides, for example, 75%, 80%, or 85% or more identity, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with wild-type human polypeptides.

[0042] The percentage of identity between two sequences can be determined, for example, by sequence alignment using any convenient technique in the art, such as publicly available software. Mutations can be introduced using standard molecular biological techniques such as site-directed mutagenesis, PCR-mediated mutagenesis, and directional evolution. Those skilled in the art will recognize that one or more nucleic acid substitutions can be introduced without altering the amino acid sequence, and that one or more amino acid mutations can be introduced without altering the functional properties of a human protein.

[0043] Conservative amino acid substitutions can be created in human proteins to produce human protein variants. A conservative amino acid substitution is a substitution of one amino acid with another amino acid having similar characteristics, as recognized in the art. For example, each amino acid may be described as having one or more of the following characteristics: positively charged, negatively charged, aliphatic, aromatic, polar, hydrophobic, and hydrophilic. A conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic with another amino acid having the same characteristic. Acidic amino acids include aspartic acid and glutamic acid; basic amino acids include histidine, lysine, and arginine; aliphatic amino acids include isoleucine, leucine, and valine; aromatic amino acids include phenylalanine, glycine, tyrosine, and tryptophan; polar amino acids include aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, and tyrosine; hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, and tryptophan; conservative substitutions include substitutions between amino acids within each group. Amino acids can also be described in terms of their relative size, with alanine, cysteine, aspartic acid, glycine, asparagine, proline, threonine, serine, and valine all typically considered small.

[0044] Human variants may include, but are not limited to, synthetic amino acid analogs, amino acid derivatives, and / or non-standard amino acids, including, but are not limited to, α-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxyphenylalanine, diencholic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, methylhistidine, and ornithine.

[0045] Human variants are thought to be encoded by nucleic acids that have a high degree of identity with the nucleic acids encoding wild-type humans. The complementary strand of the nucleic acid encoding the human variant specifically hybridizes with the nucleic acid encoding the wild-type human protein under high stringency conditions. In one embodiment, the nucleic acid encoding the human variant may be isolated using well-known methodologies, or may be generated by recombination or synthesis.

[0046] For example, with respect to “human EPO protein (hEPO),” “human SIRPa protein (hSIRPa),” “human IL-3 protein (hIL-3),” “human GM-CSF protein (hGM-CSF),” “human M-CSF protein (hM-CSF),” “human TPO protein (hTPO),” and “human IL-6 protein (hIL-6),” the term “human protein” includes, as used herein, “wild-type” or “native” human proteins and their “variants,” as well as fusion proteins, i.e., chimeric proteins, that contain one or more fragments of wild-type human proteins (or their variants) and retain one or more functions of wild-type human proteins, such as one or more signaling and / or receptor functions. For example, a fusion protein containing one or more fragments of wild-type human proteins (or their variants) in combination with one or more non-human peptides or polypeptides may also be referred to herein as a “humanized protein.” Therefore, for example, a protein containing the amino acid sequence of the extracellular domain of a wild-type human SIRPα protein fused with the signaling domain of a wild-type mouse SIRPα protein is included in the term "human SIRPα protein."

[0047] Therefore, a nucleic acid sequence encoding a human protein is a polynucleotide containing the coding sequence for a human protein, e.g., a wild-type human protein, a variant of a wild-type human protein that retains one or more functions of a wild-type human protein, e.g., one or more signaling and / or receptor functions, a fragment of a wild-type human protein (or its variant), or one or more fragments of a wild-type human protein (or its variant), and a fusion protein, e.g., a chimeric protein, that retains one or more functions of a wild-type human protein, e.g., one or more signaling and / or receptor functions.

[0048] Typically, in the genetically modified animals of this disclosure, nucleic acids encoding human proteins, such as hEPO protein, hSIRPa protein, hIL-3 protein, hGM-CSF protein, hM-CSF protein, hTPO protein, hIL-6 protein, etc., are functionally linked to one or more DNA regulatory elements. These DNA regulatory elements include transcriptional and translational regulatory sequences such as promoters, enhancers, polyadenylation signals, and terminators, which provide and / or control the expression of coding sequences in host cells. For example, a “promoter” or “promoter sequence” refers to a DNA regulatory region that can bind to RNA polymerase in a cell and initiate the transcription of a downstream (3' direction) coding sequence. The promoter sequence is adjacent to the transcription initiation site at its 3' end and extends upstream (5' direction) to contain the minimum number of bases or elements necessary to initiate transcription at a detectable level beyond the background. Within the promoter sequence, in addition to the transcription initiation site, a protein-binding domain responsible for binding to RNA polymerase may also be found. Eukaryotic promoters will often, though not always, contain "TATA" and "CAT" boxes. Of particular importance to this disclosure are DNA regulatory elements, such as promoters, that promote the transcription of human proteins in the same spatial and temporal expression patterns observed in the corresponding endogenous proteins, i.e., in the same cells and tissues and at the same time point.

[0049] In some embodiments, for example, when a human promoter promotes the precise spatial and temporal expression of a human protein in a non-human animal, the nucleic acid sequence encoding the human protein in the non-human animal according to the present invention is functionally ligated to the human promoter for that gene. Alternatively, the nucleic acid sequence encoding the human protein in the non-human animal according to the present invention is functionally ligated to the non-human animal promoter for the corresponding non-human animal gene. Thus, for example, with respect to a non-human animal expressing an hEPO protein, in some embodiments, the nucleic acid encoding the hEPO protein is functionally ligated to the human EPO promoter. In other cases, the nucleic acid encoding the human EPO protein is functionally ligated to the non-human EPO promoter. In yet another case, the nucleic acid encoding the human EPO protein is functionally ligated to the endogenous non-human EPO promoter.

[0050] In some cases, human proteins are expressed from the corresponding gene locus in non-human animals. In certain cases, the nucleic acid sequence encoding the corresponding non-human animal protein is replaced with the nucleic acid sequence encoding the human protein. In other cases, human proteins are expressed from genomic sites in non-human animals other than the gene locus for the corresponding non-human gene. Thus, for example, with respect to a non-human animal expressing the hEPO protein, in some embodiments, the hEPO protein is expressed from the EPO gene locus in the non-human animal genome. In certain embodiments, the non-human animal includes the substitution of the nucleic acid sequence encoding endogenous EPO with the nucleic acid sequence encoding the hEPO protein. In other embodiments, hEPO is expressed from genomic sites in non-human animals other than the non-human animal EPO gene locus.

[0051] In some cases, genetically modified non-human animals contain one copy of a nucleic acid sequence encoding a human protein. For example, a non-human animal may be heterozygous for a nucleic acid sequence encoding a human protein, i.e., one allele at a locus may be genetically modified and the other allele may be an endogenous allele. In other cases, a genetically modified non-human animal contains two copies of a nucleic acid sequence encoding a human protein. For example, a non-human animal may be homozygous for a nucleic acid sequence encoding a human protein, i.e., both alleles at a locus in a diploid genome may be genetically modified to encode a human protein, for example, both alleles may involve the substitution of an endogenous protein-encoding nucleic acid sequence with a human protein-encoding nucleic acid sequence. Therefore, for example, with respect to a non-human animal expressing the hEPO protein, as described above, the nucleic acid sequence encoding hEPO may be incorporated into the non-human animal EPO locus. In some such embodiments, the genetically modified non-human animal is heterozygous for the nucleic acid sequence encoding the hEPO protein, i.e., the genetically modified non-human animal contains one allele containing the nucleic acid encoding hEPO and one allele encoding endogenous EPO. In other words, the animal is heterozygous for EPO h / m It is an animal. Here, "h" represents the allele containing the human sequence, and "m" represents the endogenous allele. In another such embodiment, the genetically modified non-human animal is homozygous for the nucleic acid sequence encoding the hEPO protein, i.e., both alleles for the locus in the diploid genome will contain the nucleic acid sequence encoding the hEPO protein. In other words, the animal is EPO h / h It is an animal.

[0052] In some cases, non-human animals also express corresponding non-human animal proteins. For example, a nucleic acid sequence encoding a human protein, e.g., hEPO, may be located at a site in the animal genome other than the locus for the non-human animal gene, e.g., the mEPO locus. As a second example, a nucleic acid sequence encoding a human protein, e.g., hEPO, may be located at the corresponding animal locus, e.g., the mEPO locus, and may be incorporated into the animal locus in a manner that allows for the continued expression of the animal coding sequence, for example, by inserting the human coding sequence upstream or downstream of the animal coding sequence, and a 2A peptide sequence or IRES sequence may be included between the two coding sequences. As a third example, a nucleic acid sequence encoding a human protein, e.g., hEPO, may be located at the corresponding animal locus, e.g., the mEPO locus, in a manner that disrupts the expression of the animal coding sequence, e.g., as a substitution of some or all of the animal coding sequence, but the non-human animal is crossbred to be heterozygous for the insertion allele, i.e., the "knock-in" allele, i.e., to retain one knock-in allele and one wild-type allele. In other cases, non-human animals do not express the corresponding non-human animal proteins. For example, a human protein, such as the nucleic acid sequence encoding hEPO, may be located at the corresponding animal locus, such as the mEPO locus, by substituting a non-human animal coding sequence, for example, as a substitution of some or all of the animal coding sequence, in a manner that disrupts the expression of the animal coding sequence, and the animal is homozygous for the insertion allele, i.e., the "knock-in" allele.

[0053] Any non-human mammal may be genetically modified in accordance with this disclosure. Non-limiting examples include experimental animals, domesticated animals, livestock, etc., species such as mice, rodents, dogs, cats, pigs, horses, cattle, sheep, and non-human primates; for example, mice, rats, rabbits, hamsters, guinea pigs, cattle, pigs, sheep, goats, and other transgenic animal species known in the art, specifically mammalian species. In other embodiments, non-human animals may be birds, such as chickens, turkeys, quail, pheasants, or francolins (Galliformes); for example, ducks, geese, or swans (Anseriformes); or for example, pigeons or doves (Columbiformes). In various embodiments, the genetically modified animals of the present invention are mice, rats, or rabbits.

[0054] In one embodiment, the non-human animal is a mammal. In some such embodiments, the non-human animal is, for example, a small mammal of the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, genetically modified animals include the families Calomyscidae (e.g., kangaroo hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, white-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rats). The genetically modified rodents are derived from families selected from mice, bamboo rats, and zokors. Specifically, the genetically modified rodents are selected from typical mice and rats (Muridae), gerbils, spiny mice, and maned mice.

[0055] In one embodiment, the genetically modified non-human animal of the present invention is a rat. In one such embodiment, the rat is selected from Wistar rat, LEA strain, Sprague Dawley strain, Fischer strain, F344, F6, and Dark Agouti. In another embodiment, the rat strain is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0056] In another embodiment, the genetically modified non-human animal of the present invention is a mouse, for example, a mouse of the C57BL lineage (e.g., C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, C57BL / Ola) Etc.); 129 strains of mice (e.g., 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2); BALB strain mice; e.g., BALB / c, etc. See, for example, Festing et al. (1999) Mammalian Genome 10:836 and Auerbach et al (2000) Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines. In one embodiment, the genetically modified mouse is a hybrid of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In another embodiment, the mouse is a hybrid of the aforementioned 129 strain or a hybrid of the aforementioned BL / 6 strain. In yet another embodiment, the mouse is a hybrid of the BALB strain and another aforementioned strain.

[0057] In some embodiments, the genetically modified non-human animals of the present invention are also immunodeficient. "Immunodeficiency" includes a deficiency in one or more aspects of the animal's native or endogenous immune system, for example, the animal lacks one or more types of functional host immune cells, such as the number and / or function of non-human B cells, the number and / or function of non-human T cells, the number and / or function of non-human NK cells, etc.

[0058] One method for achieving immunodeficiency in animals according to the present invention is irradiation to a sublethal dose. Alternatively, immunodeficiency may be achieved by any of a number of gene mutations known in the art, which may be crossed alone or in combination with the genetically modified non-human animals of the present disclosure, or may be used as the source of stem cells into which the genetic modifications of the present disclosure may be introduced. Non-limiting examples include X-linked SCID associated with the IL2RG gene mutation and characterized by the lymphocyte phenotype T(-)B(+)NK(-); autosomal recessive SCID associated with the Jak3 gene mutation and characterized by the lymphocyte phenotype T(-)B(+)NK(-); ADA gene mutation characterized by the lymphocyte phenotype T(-)B(-)NK(-); IL-7Rα chain mutation characterized by the lymphocyte phenotype T(-)B(+)NK(+); lymph CD3δ or ε mutations characterized by a globular phenotype T(-)B(+)NK(+); RAG1 and RAG2 mutations characterized by a lymphocyte phenotype T(-)B(-)NK(+); Artemis gene mutations characterized by a lymphocyte phenotype T(-)B(-)NK(+); CD45 gene mutations characterized by a lymphocyte phenotype T(-)B(+)NK(+); and Prkdc mutations characterized by a lymphocyte phenotype T(-)B(-). scid Mutations are included. Therefore, in some embodiments, genetically modified immunodeficient non-human animals have IL2 receptor γ chain (Il2rγ y / -The animal models have one or more deficiencies selected from among ) deficiency, Jak3 deficiency, ADA deficiency, IL7R deficiency, CD3 deficiency, RAG1 and / or RAG2 deficiency, Artemis deficiency, CD45 deficiency, and Prkdc deficiency. These and other immunodeficiency animal models are known to those skilled in the art, and any of them may be used to generate the immunodeficient animals of this disclosure.

[0059] In some embodiments, the genetically modified non-human animals according to the present invention have a use as recipients of human hematopoietic cells, capable of developing human immune cells from engrafted human hematopoietic cells. Therefore, in some aspects of the present invention, the genetically modified animals of the present invention are genetically modified immunodeficient non-human animals in which human hematopoietic cells have been engrafted.

[0060] Any origin of human hematopoietic cells, human hematopoietic stem cells (HSCs), and / or hematopoietic stem primordial cells (HSPCs) known in the art or described herein may be transplanted into genetically modified immunodeficient non-human animals of this disclosure. One suitable origin of human hematopoietic cells known in the art is human umbilical cord blood cells, specifically CD34-positive (CD34) + ) are cells. Another origin of human hematopoietic cells is the human fetal liver. Another origin is the human bone marrow. For example, induced pluripotent stem cells (iPSCs) and induced hematopoietic stem cells (iHSCs) produced by dedifferentiation of somatic cells by methods known in the art are also included. Methods for transplanting human cells into non-human animals are well described in the art and elsewhere in this specification, and any of these can be used by those skilled in the art to reach the engrafted genetically modified non-human animals of the present invention.

[0061] Transplanted human hematopoietic cells produce one or more types of engrafted human cells selected from human CD34-positive cells, human hematopoietic stem cells, human hematopoietic cells, myeloid progenitor cells, erythrocyte progenitor cells, myeloid cells, dendritic cells, monocytes, neutrophils, mast cells, erythrocytes, and combinations thereof in genetically modified non-human animals. In one embodiment, the human cells are present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after engraftment. In a specific embodiment, the human cells include cells of the erythrocyte lineage.

[0062] In several embodiments, transplanted human hematopoietic cells give rise to an engrafted human hematopoietic lymphoid system in a genetically modified non-human animal, comprising human hematopoietic stem cell progenitor cells, human myeloid progenitor cells, human myeloid cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human erythrocytes, human thymocytes, human T cells, human B cells, and human platelets. In one embodiment, the human hematopoietic lymphoid system is present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after engraftment. In a specific embodiment, the human hematopoietic lymphoid system comprises cells of the erythrocyte lineage.

[0063] The erythrocyte lineage includes red blood cells and cells that produce red blood cells. "Red blood cells" include mature red blood cells, also called red cells or red corpuscles. Cells that produce red blood cells include erythrocyte primordial cells, i.e., proliferative pluripotent cells, and erythrocyte progenitor cells, i.e., proliferative or non-proliferative cells destined to become red blood cells.

[0064] Red blood cells are the primary cellular components of circulating blood, and their main function is to transport oxygen. The number of red blood cells per cubic millimeter of blood is generally maintained at 4.5 to 5.5 million in men and 4.2 to 4.8 million in women. This number varies depending on age, activity level, and environmental conditions. For example, above 10,000 feet of altitude, it is typically around 8 million / mm³. 3An increase in the red blood cell count can occur. Red blood cells normally survive for 110-120 days, after which they are removed from the bloodstream and broken down by the reticuloendothelial system. New red blood cells are produced at a rate slightly more than 1% per day; therefore, a constant level is generally maintained. Acute blood loss, hemolytic anemia, or chronic oxygen deficiency can cause a significant increase in red blood cell production.

[0065] Red blood cells originate from hematopoietic stem cells in the bone marrow of long bones and include myeloid common origin cells (CD123+,CD34+,c-kit+,Flt3+); megakaryocyte / erythroblast origin cells (CD34+,CD38+,CD45RA-); proerythroblasts (normally also called pro-northerythroblasts, abnormally also called pro-megaloblasts; large CD71+,EpoR+,c-kit+,Ter119+ origin cells); and basophilic erythroblasts (cytoplasm is basophilic, the nucleus is large and has agglutinating chromatin, and the nucleolus is small). Erythrocytes develop through a series of cellular stages, including: polychromatic erythroblasts (also called intermediate normoblasts, showing increased nuclear chromatin aggregation, with the cytoplasm beginning to acquire hemoglobin and exhibiting an eosinophilic hue); orthochromatic normoblasts (the final stage before nuclear loss, with a small nucleus, eventually becoming a homogeneous, unstructured mass of blue to black); and reticulocytes (circulating CD235+, CD71+ cells; cells characterized by a reticulate pattern of threads and particles in the former nuclear region).

[0066] Mature erythrocytes appear in peripheral smears as biconcave, circular or oval discs approximately 6–8 μm in diameter. They contain hemoglobin, have a pale blue zone in the center due to their biconcave shape, and can be readily identified by flow cytometry or immunohistochemistry-based methods due to elevated expression of cell surface markers CD235 and CD59 compared to non-erythrocytes.

[0067] For example, as demonstrated in Figure 5, Panel A and Figure 5, Panel B of this disclosure, expression of hEPO under the regulation of the EPO promoter from the genome of engrafted non-human animals of this disclosure increases the number of human erythrocyte lineage cells (CD235a+) in the bone marrow by an average of approximately twofold (e.g., from approximately 11% to approximately 22%). Expression of human Sirpa enhances this effect, resulting in an average increase of more than threefold (i.e., from approximately 11% to approximately 33%) in the presentation of human CD235a+ cells in the bone marrow compared to animals that do not express either human EPO or human Sirpa (Figure 5, Panel A). Therefore, engrafted, immunodeficient, genetically modified non-human animals expressing hEPO of this disclosure have applications in the study of human erythropoiesis and in the development of drugs that modulate (e.g., promote or inhibit) human erythropoiesis.

[0068] Furthermore, as demonstrated, for example in Figure 6, clodronate treatment of animals expressing hEPO with engrafted human HSCs increases the number of CD235+ erythrocytes (including CD71+ reticulocytes, Figure 6, panel B) in the peripheral blood of these animals 1,000-fold compared to untreated controls, i.e., to approximately 1% of all peripheral erythrocytes. Importantly, as demonstrated, for example in Figure 7, in animals expressing human EPO with engrafted HSCs, the human erythrocytes produced at these engraftment levels are susceptible to infection by Plasmodium falciparum. Therefore, the genetically modified non-human animals expressing hEPO described herein have specific applications in generating animal models of infection by parasites that target human cells of the erythrocyte lineage, such as pathogens that cause malaria or malaria-like diseases.

[0069] Accordingly, in some aspects of the present invention, the genetically modified animals of the present invention are non-human animals in which human hematopoietic cells have been engrafted and which are subject to infection by human pathogens. Of particular interest in these embodiments are human pathogens that target human cells of the erythrocyte lineage. Non-limiting examples of such pathogens include protozoa such as Plasmodium, Babesia, and Theileria. As will be described in more detail below, the genetically modified non-human animals of the present invention in which human hematopoietic cells have been engrafted can be infected with human pathogens using any suitable method known in the art or described herein for infecting animals with pathogens of interest. Animals thus infected will typically exhibit signs of parasitemia, including altered morphology by Giemsa-stained blood smears, as well as a severe decrease in total red blood cell concentration (e.g., 50%) and anemia.

[0070] Method for producing genetically modified mice according to the present invention In some aspects of the present invention, a method for producing a non-human animal of the present invention is provided. In carrying out the method of the present invention, for example, a non-human animal is produced that includes a nucleic acid sequence encoding an EPO promoter, for example, an hEPO protein functionally linked to a non-human animal EPO promoter, at the EPO locus of the non-human animal genome.

[0071] The generation of non-human animals containing a nucleic acid sequence encoding an hEPO protein functionally linked to an EPO promoter can be achieved, for example, by using any convenient method for producing genetically modified animals, such as those known in the art or described herein.

[0072] For example, nucleic acids encoding hEPO proteins can be incorporated into recombinant vectors in a form suitable for insertion into the genome of host cells and expression of human proteins in non-human host cells. In various embodiments, recombinant vectors contain one or more regulatory sequences functionally linked to nucleic acids encoding human proteins in a manner that enables transcription of the nucleic acid into mRNA and translation of the mRNA into human proteins, as described above. It will be understood that the design of the vector may depend on factors such as the selection of host cells to be transfected and / or the amount of human protein to be expressed.

[0073] Next, in order to create genetically modified animals that express human genes, one of various methods can be used to introduce human nucleic acid sequences into animal cells. Such techniques are well known in the art and include, but are not limited to, pronuclear microinjection, embryonic stem cell transformation, homologous recombination, and knock-in techniques. Methods for generating genetically modified animals that can be used include those described in Sundberg and Ichiki (2006, Genetically Engineered Mice Handbook, CRC Press), Hofker and van Deursen (2002, Genetically modified Mouse Methods and Protocols, Humana Press), Joyner (2000, Gene Targeting: A Practical Approach, Oxford University Press), Turksen (2002, Embryonic stem cells: Methods and Protocols in Methods Mol Biol., Humana Press), Meyer et al. (2010, Proc. Nat. Acad. Sci. USA 107:15022-15026), and Gibson (2004, A Primer Of Genome Science 2 ndThis includes, but is not limited to, the works described in Sunderland, Massachusetts: Sinauer (ed.), U.S. Patent No. 6,586,251, Rathinam et al. (2011, Blood 118:3119-28), Willinger et al. (2011, Proc Natl Acad Sci USA, 108:2390-2395), Rongvaux et al. (2011, Proc Natl Acad Sci USA, 108:2378-83), and Valenzuela et al. (2003, Nat Biot 21:652-659).

[0074] For example, the genetically modified animals of the present invention can be produced by introducing nucleic acids encoding human proteins into oocytes, for example, by microinjection, and developing the oocytes in female rearing animals. In a preferred embodiment, the expression product is injected into fertilized oocytes. Fertilized oocytes can be collected from superovulating females the day after mating and injected with the expression construct. The injected oocytes are cultured overnight or directly transferred into the fallopian tubes of pseudopregnant females 0.5 days after mating. Methods for superovulation, oocyte collection, expression construct injection, and embryo transfer are known in the art and are described in *Manipulating the Mouse Embryo* (2002, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press). The presence of the introduced nucleic acids can be evaluated in the derived animals by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing, etc.) or protein analysis (e.g., ELISA, Western blotting, etc.).

[0075] As another example, constructs containing nucleic acid sequences encoding human proteins can be transfected into stem cells (e.g., ES cells or iPS cells) using well-known methods such as electroporation, calcium phosphate precipitation, and lipofection. The presence of the introduced nucleic acid can then be evaluated in the cells by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing) or protein analysis (e.g., ELISA, Western blotting). Cells determined to have incorporated the expression construct can then be microinjected into preimplantation embryos. For a detailed description of known methods in the art that are useful for the compositions and methods of the present invention, see Nagy et al. (2002, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press), Nagy et al. (1990, Development 110:815-821), U.S. Patent No. 7,576,259, U.S. Patent No. 7,659,442, U.S. Patent No. 7,294,754, and Kraus et al. (2010, Genesis 48:394-399).

[0076] Furthermore, as described in some of the examples below, nucleic acid constructs can be constructed using VELOCIGENE® genetic engineering technology (see, for example, Valenzuela et al. (2003), High throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-59 and U.S. Patent No. 6,586,251), introduced into stem cells (e.g., ES cells), and precisely targeted clones can be determined using allele loss assays and allele gain assays (Valenzuela et al. (above)); precisely targeted ES cells can be determined using the VELOCIMOUSE® method (see, for example, U.S. Patent No. 7,294,754 and Poueymirou et al. 2007), which enables F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analysis. Using analyses (see Nature Biotech. 25(1):91-99), they can be used as donor ES cells for introduction into 8-cell stage mouse embryos.

[0077] A genetically modified primary animal can be crossbred with additional animals that carry the genetic modification. A genetically modified animal carrying the nucleic acid encoding the human protein of this disclosure can be further crossbred with other genetically modified animals that carry other genetic modifications, such as hSIRPa knock-in mice, hIL-3 knock-in mice, hGM-CSF knock-in mice, hM-CSF knock-in mice, hTPO knock-in mice, hIL-6 knock-in mice, etc., or with knockout animals, such as non-human animals that lack one or more proteins and do not express one or more of those genes, such as Rag2-deficient animals or Il2rg-deficient animals.

[0078] In another embodiment, stem cells, such as ES cells, can be generated to include several genetic modifications, for example, humanization or gene deletion as described herein, and such stem cells can be introduced into embryos to generate genetically modified animals having several genetic modifications.

[0079] As described above, in some embodiments, the genetically modified non-human animals of the present invention are immunodeficient animals. A genetically modified non-human animal that is immunodeficient and contains one or more human proteins, e.g., hEPO, hSIRPa, hIL-3, hGM-CSF, hM-CSF, and / or hTPO, can be produced using any convenient method for producing genetically modified animals, such as those known in the art or described herein. For example, the production of a genetically modified immunodeficient animal can be achieved by introducing a nucleic acid encoding a human protein into an oocyte or stem cell containing a mutated SCID gene allele that would result in immunodeficiency when homozygous, as described above and in the examples herein. Then, for example, a mouse having the modified oocyte or ES cell is produced using a method described herein or known in the art, and mated to produce an immunodeficient mouse containing the desired genetic modification. As another example, genetically modified non-human animals can be generated in an immunocompetent background, crossed with animals containing mutant alleles that would result in immunodeficiency when hemizygous or homozygous, and the offspring can be mated to create immunodeficient animals that express at least one human protein of interest.

[0080] In several embodiments, genetically modified mice are treated to eliminate any endogenous hematopoietic cells that may be present in the mice. In one embodiment, the treatment includes the step of irradiating the genetically modified mice. In a specific embodiment, the genetically modified neonatal mice are irradiated with a dose below lethal. In a specific embodiment, the neonatal mice are irradiated with 200 cGy twice, at 4-hour intervals.

[0081] Various aspects of the present invention provide genetically modified animals in which substantially all cells contain human nucleic acids, and genetically modified animals in which some but not all cells contain human nucleic acids. In some cases, for example, in target-specific recombination, one copy of human nucleic acid will be incorporated into the genome of the genetically modified animal. In other cases, for example, in random integration, multiple copies of human nucleic acid, adjacent or distant from each other, may be incorporated into the genome of the genetically modified animal.

[0082] Accordingly, in some embodiments, the genetically modified non-human animal of the present invention may be an immunodeficient animal comprising a genome containing nucleic acids encoding a human polypeptide functionally linked to a corresponding non-human animal promoter, and expressing the encoded human polypeptide. In other words, the genetically modified immunodeficient non-human animal of the present invention comprises a genome containing nucleic acids encoding at least one human polypeptide functionally linked to a corresponding non-human promoter and polyadenylation signal, and expresses the encoded human polypeptide.

[0083] As described above, in some embodiments, the genetically modified non-human animals of the present invention are engrafted with human hematopoietic cells. Any origin of human hematopoietic cells, human hematopoietic stem cells (HSCs), and / or human hematopoietic stem cells (HSPCs), such as umbilical cord blood, fetal liver, bone marrow, iPSCs, etc., as known in the art or described herein, can be transplanted into the genetically modified non-human animals of this disclosure. In one embodiment, the hematopoietic cells are selected from human umbilical cord blood cells and human fetal liver cells. The amount of cells to be transplanted may be well understood by those skilled in the art or may be determined empirically. In one embodiment, engraftment is about 1-2 × 10⁻⁶ 5The cells are human CD34+ cells. The cells can be transplanted into a non-human animal host of the present invention using any convenient technique known in the art, such as tail vein injection, postorbital injection, or injection into the neonatal liver. The cells can be transplanted into the host in any convenient buffer solution, such as PBS, Dulbecco's modified medium, Iskov's modified medium, etc. In some cases, the animals can be irradiated before engraftment, for example, as described above, to improve immunodeficiency.

[0084] Human hematopoietic cells that have engrafted in this manner give rise to one or more types of human cells selected from CD34+ cells, hematopoietic stem cells, hematopoietic cells, myeloid progenitor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, thymocytes, T cells, B cells, platelets, erythrocytes, and combinations thereof. In one embodiment, the human cells are present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after engraftment. For example, one of numerous assays, including flow cytometry assays, blood smears, and immunohistochemistry, can be performed on various human hematopoietic cells of interest to confirm successful engraftment.

[0085] As described above, in some embodiments, the genetically modified non-human animals of the present invention, in which human hematopoietic cells have been engrafted, are infected with human pathogens. Of particular interest in these embodiments are human pathogens that target human cells of the erythrocyte lineage. Non-limiting examples of such pathogens include protozoa of the genera Plasmodium, Babesia, and Theileria. In some embodiments, the pathogen strains used are naturally occurring strains. In certain embodiments, the strains used are selected in vivo for their ability to proliferate responsibly in immunodeficient mice in which human erythrocytes have been engrafted, e.g., Plasmodium falciparum strain Pf3D7. 0087 / N9 Or Pf3D7 0087 / N5 That is the case.

[0086] The engrafted immunodeficient animals of the present invention can be infected using any method known in the art for infecting animals with pathogens that target cells of the erythrocyte lineage. For example, the engrafted immunodeficient animals of the present invention can be intraperitoneally inoculated with parasitic erythrocytes (PRBCs). See, for example, Badell et al. (2000) Human malaria in immunocompromised mice: an in vivo model for studying defense mechanisms against Plasmodium falciparum JEM 192(11):1653-1660; and Moreno et al. (2006) Course of infection and pathology in immunomodulatory NOD / LtSz-SCID mice inoculated with experimental strains and clinical isolates of Plasmodium falciparum Int.J.Parasitol.36:361-369. As another example, the engrafted immunodeficient animals of the present invention can be intravascularly inoculated with parasitic erythrocytes. See, for example, Angulo-Barturen et al. (2008) Mouse model of Plasmodium falciparum malaria by in vivo selection of competent strains in bone marrow-non-depleted mice with engrafted human erythrocytes PLoS One 3:e2252; and Jimenez-Diaz et al. (2009) Improved mouse model of malaria using competent strains of Plasmodium falciparum and bone marrow-non-depleted NOD-scid IL2Rg null mice with engrafted human erythrocytes Antimicrob Agents Chemother 53:4533-4536. In some embodiments, infection is produced by injecting the parasite into a non-human animal, i.e., not related to erythrocytes. In some embodiments of the present invention, the engrafted immunodeficient animal undergoes in vivo phagocytic depletion of cells pre-infection and / or during infection.In such embodiments, any chemotherapeutic agent that selectively depletes host phagocytic cells, such as clodronate as described in the examples herein; dichloromethylene diphosphate as described in Badell et al. (above) and Moreno et al. (above); or a monoclonal antibody specific to polymorphonuclear neutrophils as described in Badell et al. (above) and Moreno et al. (above), such as NIMP-R14, can be administered to the animals of the present invention.

[0087] The percentage of parasitemia in the infected genetically modified non-human animals of this disclosure may be assessed by any convenient method in the art, for example, by microscopy from Giemsa-stained blood smears three days after injection; or, for example, by flow cytometry in or without TER-119 mAb, by measuring the luminescence of nucleic acid dye YOYO-1 or cell-permeable dye SYTO-16. See, for example, Jimenez-Diaz et al. Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence. Cytometry A 2009, 75:225-235.

[0088] Use of genetically modified mice of the present invention The ability to study human tissues in a mouse in vivo environment has opened up a range of potential research avenues. Major limitations have hindered the application of this approach, one of the most significant of which is the inability of mouse factors to support human cells. Indeed, in the immune system, many essential factors required for the development and function of human immune cells are species-specific and cannot be effectively provided by mice. Therefore, we decided to employ a strategy of substituting mouse genes with human counterparts to enable better development and function of human cells, and potentially prevent that of corresponding mouse cells. By applying this concept to the human cytokine EPO, we demonstrate herein that substitution of the nucleic acid sequence encoding the mouse EPO protein with the nucleic acid sequence encoding the human EPO protein improves the development and function of erythrocyte lineage cells in the engrafted human immune system in mice.

[0089] For example, the examples demonstrate, for instance, in Figure 5, that the expression of hEPO from nucleic acid sequences under the regulation of the EPO promoter in the genome of non-human animals increases the number of human cells (CD235a+) in the erythrocyte lineage developing in the bone marrow of animals engrafted with human HSCs by approximately twofold (i.e., from approximately 11% to approximately 22%). Expression of human Sirpa enhances this effect, resulting in a total threefold increase in the presentation of human CD235a+ cells in the bone marrow (i.e., from approximately 11% to approximately 33%) compared to animals that do not express either human EPO or human Sirpa (Figure 4a). Furthermore, as demonstrated, for example, in Figure 6, clodronate treatment of animals engrafted with human HSCs expressing human EPO increases the number of CD235+ erythrocytes (including CD71+ reticulocytes, Figure 6, panel B) in the peripheral blood of these animals by 1000-fold compared to untreated controls, i.e., to approximately 1% of total erythrocytes. Importantly, as demonstrated, for example in Figure 7, human erythrocytes produced at these engraftment levels in animals with engrafted HSCs expressing human EPO are susceptible to infection by Plasmodium species such as Plasmodium falciparum. Therefore, genetically modified non-human animals expressing human EPO, as described herein, have specific applications in generating animals that are susceptible to Plasmodium falciparum or that would better support human erythrocytes acutely transplanted into animals before infection in current rodent models.

[0090] Accordingly, the genetically modified non-human animals of this disclosure have many applications in the art. For example, the engrafted genetically modified animals of this disclosure are useful for studying human erythropoiesis and the function of human erythrocytes. As another example, the engrafted genetically modified mice of this disclosure provide a useful system for screening candidate drugs for desired in vivo activity, for example, to identify drugs that can modulate (i.e., promote or inhibit) human erythropoiesis and / or the function of human erythrocytes in a healthy or diseased state, for example, as cancer cells, during pathogen infection, for example, to identify novel therapeutic drugs; or as yet another example, to identify drugs that are toxic to human erythrocytes and to identify drugs that prevent, mitigate, or reverse the toxic effects of toxic drugs on human erythrocytes. As yet another example, the engrafted genetically modified animals of this disclosure provide a useful system for predicting an individual's responsiveness to disease treatment by, for example, providing an in vivo platform for screening the responsiveness of an individual's immune system to drugs, such as therapeutic agents, for predicting the individual's responsiveness to drugs.

[0091] As one non-limiting example, the engrafted genetically modified mice of this disclosure have applications in generating mouse models of pathogen infections caused by parasites that target human erythrocytes, such as Plasmodium malariae, Babesia, and Theileria. Such mouse models of infections would be useful, for example, in studies to better understand the progression of infection in humans, and in drug discovery to identify candidate drugs that protect against or treat infections caused by such parasites.

[0092] Plasmodium parasites are the cause of malaria in humans. Malaria begins with a bite from an infected Anopheles mosquito, which introduces the parasites into the circulatory system through its saliva, eventually reaching the liver, where the parasites mature and reproduce. The parasites then enter the bloodstream and infect cells of various stages of erythrocyte maturation.

[0093] Five species of malaria parasites can infect and transmit humans. The majority of deaths are caused by Plasmodium falciparum, while Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae cause generally milder forms of malaria that are rarely fatal. This is thought to be at least partly due to the type of cells targeted by each species: Plasmodium falciparum grows in red blood cells (RBCs) of all maturity stages, while Plasmodium vivax, for example, is limited to growth in reticulocytes, which represent only about 1-2% of all peripheral RBCs. Furthermore, Plasmodium falciparum causes severe malaria through a characteristic not shared by other human malaria species: sequestration. Within the 48-hour asexual red blood cell cycle, the mature form alters the surface properties of infected red blood cells, causing them to adhere to blood vessels (a process called cell adhesion). This can lead to obstruction of the microcirculation system and subsequent dysfunction of multiple organs.

[0094] Symptoms of malaria include fever, chills, headache, sweating, fatigue, anemia, nausea, dry (non-sputum) cough, muscle pain and / or back pain, and splenomegaly. Other symptoms and complications associated with malaria include brain infection (encephalitis), hemolytic anemia, renal failure, hepatic failure, meningitis, pulmonary edema, and splenic hemorrhage. Generally, individuals at risk of developing malaria may begin showing symptoms seven days or more after infection, for example, 9–14 days after a primary infection with Plasmodium falciparum, 12–18 days after a primary infection with Plasmodium vivax or Plasmodium ovale, 18–40 days after a primary infection with Plasmodium quartanum, or 11–12 days after a primary infection with Plasmodium bivitae (P. knowlesi). Antimalarial agents used in the art to treat or prevent malaria include chloroquine, quinidine, doxycycline, tetracycline, clindamycin, atovaquone + proguanil (Malarone), mefloquine, artesunate, and pyrimethamine + sulfadoxine (Fansidar).

[0095] Methods for determining whether a subject is infected with malaria parasites are well known in the art and include, for example, microscopic examination of blood using blood films, antigen-based rapid diagnostic tests (RDTs), such as immunochromatography-based RDTs, and detection of parasitic DNA by polymerase chain reaction (PCR). Any convenient method may be used to determine whether the human red blood cells of a subject are infected with the pathogen.

[0096] Another example of a pathogen of interest is the protozoa of the genus Babesia. Babesia infection results in a malaria-like illness called babesiosis. Babesiosis is an insect-borne disease commonly transmitted by the tick Ixodes scapularis. The disease is typically caused by B. microti in humans, B. canis rossi and B. canis canis in dogs, B. bovis in cows, and B. bigemina in cattle. Babesia microti, which infects humans, uses the same tick vector as Lyme disease and ehrlichiosis, and may occur in conjunction with these other diseases. The protozoa can also be transmitted through blood transfusions.

[0097] In humans, babesiosis can be asymptomatic or present with symptoms ranging from mild fever and diarrhea to high fever, chills, and severe anemia. In severe cases, organ failure, including respiratory distress syndrome, can occur. Severe cases most often occur in people who have undergone splenectomy or those with immunodeficiency, such as HIV / AIDS patients. Treatment typically involves a dual therapy regimen of quinine and clindamycin or atovaquone and azithromycin. If babesiosis is considered life-threatening, exchange transfusion is performed, which involves removing infected red blood cells and replacing them with uninfected ones.

[0098] A definitive diagnosis of babesiosis is made by identifying the parasite in a Giemsa-stained thin blood smear. The parasite appears in red blood cells as merozoite pairs that form a "maltacross" in humans or a "pear" in animals. Other diagnostic methods include PCR of peripheral blood and serological testing for antibodies (IgG, IgM) against babesiosis.

[0099] Another malaria-like disease, theyleriosis, is caused by protozoa of the genus Theyleria. Theyleriosis is caused in humans by T. microti; in horses by T. equi ("equine pyloriplasmosis"); in sheep and goats by T. lestoquardi; and in cattle, African buffalo, water buffalo, and waterbucks by T. annulata ("tropical theyleriosis," also known as "Mediterranean theyleriosis") or T. parva ("East Coast fever," also known as "Corridor disease"). Theyleriosis is transmitted to hosts by various tick species, including Ixodes ventricosus, Rhipicephalus, Dermacentor, Haemaphysalis, and Hyalomma. These organisms reproduce on ticks as their life stages progress, mature after the tick attaches to a host, and then invade the host's saliva. Generally, ticks become infectious only after several days of attachment. However, in high ambient temperatures, infectious sporozoites can develop on ticks on the ground and invade the host within a few hours of attachment.

[0100] Thelerii infection in humans typically presents with fever and hemolysis. Definitive diagnosis of Thelerii infection is made by identifying the parasite in a Giemsa-stained thin blood smear.

[0101] The engrafted genetically modified animals of this disclosure have use in screening candidate drugs for identifying drugs that prevent (e.g., vaccines) or treat infections caused by malaria parasites, babesiosis, Theileria, and other parasites that target human red blood cells. The terms “treatment,” “to treat,” etc., are used herein to generally include obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. “Treatment,” as used herein, includes any treatment of a disease in a mammal and includes: (a) preventing the onset of the disease in a subject that may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., preventing its development; or (c) reducing the disease, i.e., causing the disease to regress. Important candidate antiparasitic drugs include those that can be administered before, during, or after a parasitic infection and, when administered in an effective dose, inhibit the effects of the parasite on the individual (i.e., host) by, for example, killing the parasite or parasite-infected cells, preventing the transmission of the parasite, or preventing the production or action of drugs (i.e., toxins) produced by the parasite that are toxic to the individual. The terms “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject, specifically humans, for which diagnosis, treatment, or therapy is desired.

[0102] In a screening assay for drugs with biological activity, the human hematopoietic cells of this disclosure are engrafted in genetically modified non-human animals, such as engrafted Rag2 cells. - / - Il2rg null Epo h / m Mouse, Rag2 that has been engrafted - / - Il2rg null TPO h / h Il3 / Gmcsf h / h Epo h / m SIRPα h / h("TIES") Mouse, Rag2 that has been engrafted - / - IL2rg y / - TPO h / h Mcsf h / h Il3 h / h Gmcsf h / h Epo h / h hSIRPα+ ("MISTER-G") mice, Rag2 mice with successful engraftment - / - Il2rg null TPO h / h Mcsf h / h Il3 / Gmcsf h / h Epo h / h The effects of a candidate drug of interest are assessed by exposing SIRPα-tg+ ("SupER-G") mice or the like to the candidate drug and monitoring one or more output parameters. These output parameters may reflect cell viability, e.g., the total number of hematopoietic cells or the number of cells of a particular hematopoietic cell type, or the apoptotic state of cells, e.g., the amount of DNA fragmentation, the amount of cell vesicle formation, the amount of phosphatidylserine on the cell surface, etc., by methods well known in the art. Alternatively, the output parameters may reflect the differentiation potential of cells, e.g., the proportion of differentiated cells and differentiated cell types. Alternatively, the output parameters may reflect cell function, e.g., cytokines and chemokines produced by cells, antibodies produced by cells (e.g., quantity or type), the ability of cells to hom to the challenge site and extravasate, the ability of cells to modulate, i.e., promote or suppress the activity of other cells in vitro or in vivo, the ability to take up hemoglobin, etc. Furthermore, other parameters may reflect infection in animals, such as the effect of drugs on pathogen infection, or, for example, the titer of pathogens in mice, relevant to the research being conducted.

[0103] Parameters are quantifiable components of cells, particularly those that can be accurately measured, as is desirable in high-throughput systems. Parameters may include cell surface determinants, receptors, proteins or their structural or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids such as mRNA and DNA, or cellular components or cell products, or parts derived from such cellular components, or combinations thereof. While most parameters are expected to provide quantitative readouts, in some cases semi-quantitative or qualitative results may be acceptable. Readouts may include a single determination value, or they may include a mean, median, or variance, etc. Characteristically, a series of parameter readout values ​​are expected to be obtained for each parameter from a large number of identical assays. Variables are expected, and a series of values ​​for each of the set of test parameters are expected to be obtained using standard statistical methods, along with common statistical methods used to provide single values.

[0104] Candidate drugs of interest for screening include a large number of chemical classes, known and unknown compounds primarily comprising organic molecules, and may include organometallic molecules, inorganic molecules, gene sequences, vaccines, other drugs presumed to have antibiotic or antimicrobial properties, peptides, polypeptides, antibodies, and drugs that are pharmaceuticals approved for use in humans. A key aspect of the present invention is the evaluation of candidate drugs, including, for example, toxicity tests.

[0105] Candidate drugs include organic molecules containing structural interactions, specifically functional groups necessary for hydrogen bonding, typically at least one amine, carbonyl, hydroxyl, or carboxyl group, and often at least two functional chemical groups. Candidate drugs often include cyclic carbon structures, heterocyclic structures, and / or aromatic or polycyclic aromatic structures substituted with one or more of the above functional groups. Candidate drugs are also found in biomolecules, including peptides, polynucleotides, sugars, fatty acids, steroids, purines, pyrimidines, their derivatives, structural analogs, or combinations. These include drugs with pharmacological activity, molecules with genetic activity, etc. Compounds of interest include chemotherapeutic agents, hormones, or hormone antagonists, etc. Examples of pharmaceutical agents suitable for the present invention are described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth edition. Toxins, as well as biological and chemical weapons agents, are also included. For example, see Somani, SM (Ed.), "Chemical Warfare Agents," Academic Press, New York, 1992.

[0106] Candidate drugs of interest for screening include nucleic acids, such as nucleic acids encoding siRNA, shRNA, antisense molecules, or miRNA, or nucleic acids encoding polypeptides. Many vectors useful for transferring nucleic acids into target cells are available. The vector may be maintained as an episome, for example, as a viral vector such as a plasmid, minicircle DNA, cytomegalovirus, or adenovirus, or it may be incorporated into the target cell genome through homologous recombination or random incorporation (e.g., retroviral vectors such as MMLV, HIV-1, ALV). The vector may be directly delivered to the cells of the present invention. In other words, pluripotent cells are brought into contact with a vector containing the nucleic acid of interest so that the vector is taken up by the cells.

[0107] Methods for contacting cells, such as cultured cells or cells in a mouse body, with nucleic acid vectors, such as electroporation, calcium chloride transfection, and lipofection, are well known in the art. Alternatively, the nucleic acid of interest may be delivered to cells via a virus. In other words, cells are brought into contact with viral particles containing the nucleic acid of interest. Retroviruses, such as lentiviruses, are particularly suitable for the methods of the present invention. Commonly used retroviral vectors are "defective," meaning they cannot produce the viral proteins necessary for productive infection. Instead, vector replication requires proliferation in a packaging cell line. To generate viral particles containing the nucleic acid of interest, the retroviral nucleic acid containing that nucleic acid is packaged into a viral capsid by a packaging cell line. Different packaging cell lines provide different envelope proteins to be incorporated into the capsid, and these envelope proteins determine the specificity of the viral particles for the cells. There are at least three types of envelope proteins: homotropic, bitropic, and heterotropic. Retroviruses packaged by allotropic envelope proteins, such as MMLV, can infect most mouse and rat cell types and are produced using allotropic packaging cell lines such as BOSC23 (Pear et al. (1993) PNAS 90:8392-8396). Retroviruses possessing bitropic envelope proteins, such as 4070A (Danos et al. (above)), can infect most mammalian cell types, including human, dog, and mouse, and are produced using bitropic packaging cell lines such as PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); and GRIP (Danos et al. (1988) PNAS 85:6460-6464).Retroviruses packaged by heteromorphic envelope proteins, such as AKR env, can infect most mammalian cell types other than mouse cells. Appropriate packaging cell lines may be used to ensure that the cells of interest, in some cases engrafted cells, and in other cases the host, i.e., the cells of a genetically modified animal, are targeted by the packaged viral particles.

[0108] Vectors used to deliver nucleic acids of interest to the cells of the present invention typically contain a suitable promoter to activate the expression, i.e., transcription, of the nucleic acid of interest. This may include ubiquitous promoters, such as the CMV-β-actin promoter, or inducible promoters that are active in a specific cell population or respond to the presence of a drug such as tetracycline. Transcriptional activation means that transcription will increase at least about 10-fold, at least about 100-fold, and more commonly, at least about 1000-fold, above the basal level in the target cells. Furthermore, vectors used to deliver reprogramming factors to the cells of the present invention may contain genes that must be subsequently removed using a recombinase system such as Cre / Lox, or genes that enable selective toxicity, such as herpesvirus TK, bcl-xs, etc., thereby disrupting the cells expressing them.

[0109] Polypeptides are also included among the candidate drugs of interest for screening. Such polypeptides may optionally be fused with polypeptide domains that increase the solubility of the product. The domains may be linked to the polypeptide through distinct protease cleavage sites, e.g., TEV sequences cleaved by TEV proteases. The linker may contain one or more flexible sequences, e.g., 1 to 10 glycine residues. In some embodiments, cleavage of the fusion protein is carried out in a buffer that maintains the solubility of the product, e.g., in the presence of 0.5–2 M urea, or in the presence of polypeptides and / or polynucleotides that increase solubility. Domains of interest include endosomal lysis domains, e.g., influenza HA domains; and other polypeptides that assist in production, e.g., IF2 domains, GST domains, GRPE domains, etc. Furthermore, or alternatively, such polypeptides may be formulated for improved stability. For example, the peptide may be PEGylated, in which case the polyethyleneoxy group provides an enhanced lifespan in the bloodstream. Polypeptides may be fused with other polypeptides to provide additional functionality, for example, to increase in vivo stability. Generally, such fusion partners are stable plasma proteins that can extend the in vivo plasma half-life of the polypeptide when present as a fusion, and specifically, such stable plasma proteins are immunoglobulin constant domains. In most cases where stable plasma proteins are commonly found as immunoglobulins or lipoproteins, identical or different polypeptide chains are typically disulfide-bonded and / or non-covalently bonded to form an assembled polychain polypeptide, the fusions herein containing polypeptides will also be prepared and utilized as polymers having substantially the same structure as the stable plasma protein precursors. These polymers will be homogeneous with respect to the polypeptide agents they contain, or they may contain multiple polypeptide agents.

[0110] Candidate polypeptide agents may be produced from eukaryotic cells or prokaryotic cells. They may be further processed by unfolding, e.g., thermal denaturation, DTT reduction, etc., and further refolded using methods known in the art. Modifications of interest that do not alter the primary sequence include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Glycosylation modifications are also included, such as those produced by exposing polypeptides to enzymes that affect glycosylation, such as mammalian glycosylationases or deglycosylases, thereby altering the glycosylation pattern of polypeptides, for example, during synthesis and processing, or in further processing steps. Sequences having phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine, are also included. Polypeptides may be modified using conventional molecular biological techniques and synthetic chemistry to improve resistance to proteolysis, optimize solubility, or make them more suitable as therapeutic agents. Analogues of such polypeptides include those containing residues other than naturally occurring L-amino acids, such as D-amino acids or synthetic amino acids that do not exist in nature. D-amino acids can be used in place of some or all of the amino acid residues.

[0111] Candidate polypeptide agents can be prepared by in vitro synthesis using conventional methods known in the art. Various commercially available synthesis equipment, such as automated synthesis equipment from Applied Biosystems, Inc., Beckman, etc., are available. By using synthesis equipment, naturally occurring amino acids can be substituted with non-natural amino acids. The specific sequence and mode of preparation are considered to be determined by convenience, cost-effectiveness, required purity, etc. Alternatively, candidate polypeptide agents may be isolated and purified according to conventional recombinant synthesis methods. Lysates of the expression host can be prepared and purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In most cases, the composition used is considered to contain at least 20% by weight, more generally at least about 75% by weight, preferably at least about 95% by weight, and generally at least about 99.5% by weight for therapeutic purposes, in relation to contaminants associated with the preparation and purification methods of the product. Generally, the percentages are considered to be based on total protein.

[0112] In some cases, the candidate polypeptide drugs being screened are antibodies. The terms “antibody” or “antibody moiety” include any polypeptide chain that contains a molecular structure having a specific form that fits to and recognizes an epitope, and in which one or more non-covalent bonding interactions stabilize the complex between the molecular structure and the epitope. The specific or selective fit of a given structure to its specific epitope is sometimes referred to as a “lock and key” fit. Prototype antibody molecules are immunoglobulins, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all origins, e.g., humans, rodents, rabbits, cattle, sheep, pigs, dogs, other mammals, chickens, and other birds, are considered “antibodies.” The antibodies used in this invention may be either polyclonal or monoclonal antibodies. Antibodies are typically provided in the culture medium in which cells are cultured. Antibody production and screening are described in more detail below.

[0113] Candidate drugs can be obtained from diverse sources, including libraries of synthetic or natural compounds. For example, numerous methods, including the expression of randomized oligonucleotides and oligopeptides, are available for the random and specific synthesis of diverse organic compounds, including biomolecules. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily prepared. Furthermore, naturally occurring or synthetically prepared libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means and used to prepare combinatorial libraries. Known pharmacological agents can be subjected to specific or random chemical modifications, such as acylation, alkylation, esterification, and amidation, to produce structural analogues.

[0114] Candidate drugs are screened for biological activity by administering the drug to at least one, and generally more, samples, sometimes along with samples lacking the drug. Changes in parameters in response to the drug are measured, and the results are evaluated by comparison with reference samples, such as cultures in and without the drug, or cultures obtained with other drugs. When screening is performed to identify candidate drugs that may prevent, mitigate, or reverse the effects of a pathogen, the screening is typically performed in the presence of the pathogen, in which case the pathogen is added at the most appropriate time for the result to be determined. For example, in cases where the protective / preventive ability of a candidate drug is being tested, the candidate drug may be added before the pathogen, simultaneously with the pathogen, or after infection by the pathogen. In another example, when the ability of a candidate drug to reverse the effects of a pathogen is being tested, the pathogen may be added after treatment with the pathogen. As described above, in some cases, the "sample" is a genetically modified non-human animal on which cells have been engrafted. For example, the candidate drug is provided to an immunodeficient animal, such as a mouse, which contains nucleic acids encoding human EPO functionally linked to the EPO promoter, on which human hematopoietic cells have been engrafted. In some cases, the sample is the human hematopoietic cells to be engrafted, i.e., the candidate drug is provided to cells, such as reticulocytes or erythrocytes, before engraftment into an immunodeficient, genetically modified animal.

[0115] When candidate drugs are administered directly to engrafted genetically modified animals, the drugs may be administered by any of the many methods known in the art for the administration of peptides, small molecules, and nucleic acids to mice. For example, drugs may be administered orally, mucosally, topically, intradermally, or by injection, such as intraperitoneal injection, subcutaneous injection, intramuscular injection, intravenous injection, or intracranial injection. The drugs may be administered in a buffer or incorporated into any of a variety of formulations, for example, in combination with a suitable pharmaceutically acceptable medium. A “pharmaceutically acceptable medium” may be a medium approved by a federal or state regulatory authority for use in mammals such as humans, or listed in the US Pharmacopeia or other generally recognized pharmacopoeias. The term “medium” refers to a diluent, adjuvant, excipient, or carrier for formulating the compound of the present invention for administration to mammals. Such pharmaceutical media may include lipids, such as liposomes, such as liposomal dendrimers; water and liquids such as oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.; and saline solutions; as well as gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Furthermore, adjuvants, stabilizers, thickeners, lubricants, and colorants may be used. Pharmaceutical compositions can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. The drug may be systemic after administration, or topical by local administration, intramural administration, or the use of implants that act to retain the active dose at the implantation site. The active drug may be formulated for immediate action or for sustained release. For certain conditions, specifically those affecting the central nervous system, it may be necessary to formulate drugs that can cross the blood-brain barrier (BBB).One strategy for drug delivery across the blood-brain barrier (BBB) ​​requires disrupting the BBB biochemically, either by osmotic means such as mannitol or leukotrienes, or by the use of vasoactive substances such as bradykinin. When the composition is administered by intravascular injection, the BBB disruptor may be administered co-administered with the drug. Other strategies for crossing the BBB may require the use of endogenous transport systems, including transcellular transport mediated by caveolin 1, transporters mediated by carriers such as glucose and amino acid carriers, transcellular transport mediated by receptors for insulin or transferrin, and active efflux transporters such as p-glycoprotein. To facilitate transport across the endothelial wall of blood vessels, the active transport portion may be conjugated to the therapeutic compound for use in the present invention. Alternatively, drug delivery of the drug after the blood-brain barrier may be by local delivery, e.g., subarachnoid delivery, e.g., via an Ommaya reservoir (see, e.g., U.S. Patents 5,222,982 and 5385582 incorporated herein by reference); e.g., intravitreous or intracranial delivery, e.g., by bolus injection, e.g., with a syringe; e.g., continuous injection, e.g., by cannula insertion, e.g., by convection (see, e.g., U.S. Application No. 20070254842 incorporated herein by reference); or by implantation of a device to which the drug is reversibly attached (see, e.g., U.S. Applications No. 20080081064 and 20090196903 incorporated herein by reference).

[0116] When providing drugs to cells before engraftment, it is convenient to add the drug to the culture medium of the cells in solution or in an easily soluble form. The drug may be added through a flow-through system as an intermittent or continuous flow, or a bolus of the compound may be added to the static solution in a single or gradually increasing amount. In the flow-through system, two solutions are used: one is a physiologically neutral solution, and the other is the same solution to which the test compound has been added. The first solution is passed over the cells, followed by the second solution. In the single-solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentration of the components of the culture medium should not change significantly due to the addition of the bolus or between the two solutions in the flow-through method.

[0117] To obtain differential responses to various concentrations, multiple assays using different drug concentrations can be performed in parallel. As is well known in the art, determining the effective concentration of a drug typically involves using a series of concentrations resulting from dilutions of 1:10 or other logarithmic scales. If necessary, a second dilution series can be used to further refine the concentrations. Typically, one of these concentrations serves as a negative control, i.e., zero concentration, or a concentration of the drug below the detection level, or a concentration of the drug that does not produce a detectable change in phenotype.

[0118] Analysis of the cellular response to candidate drugs in genetically modified animals that have engrafted can be performed at any point in time after drug treatment. For example, cells may be analyzed 1 day, 2 days, or 3 days after contact with the candidate drug, sometimes 4 days, 5 days, or 6 days, sometimes 8 days, 9 days, or 10 days, sometimes 14 days, sometimes 21 days, sometimes 28 days, and sometimes more than a month later, for example, 2 months, 4 months, 6 months, or later. In some embodiments, the analysis involves analysis at multiple time points. The selection of time points for analysis is considered to be based on the type of analysis to be performed, as will be readily understood by those skilled in the art.

[0119] The analysis may include the measurement of any of the parameters described herein or known in the art for determining cell viability, cell proliferation, cell identity, cell morphology, and cell function, specifically those relating to immune cells. For example, flow cytometry can be used to determine the total number of hematopoietic cells or the number of cells of a particular hematopoietic cell type. Histochemistry or immunohistochemistry, such as terminal deoxynucleotide transferase dUTP nick-end labeling (TUNEL) to measure DNA fragmentation or immunohistochemistry to detect the binding of annexin V to phosphatidylserine on the cell surface, can be performed to determine the apoptotic state of cells. For example, flow cytometry can also be used to assess the proportion of differentiated cells and differentiated cell types to determine the viability and / or differentiation potential of hematopoietic cells in the presence of a drug. For example, ELISA, Western blotting, and Northern blotting can be performed to determine the levels of cytokines, chemokines, immunoglobulins, etc., expressed in genetically modified mice that have undergone engraftment, for example, to assess the function of engrafted cells or to assess the viability of erythrocytes. In vivo assays testing the function of immune cells, as well as assays related to specific diseases or disorders of interest, such as anemia, including sickle cell anemia, may also be performed. See, for example, Current Protocols in Immunology (Richard Coico, ed. John Wiley & Sons, Inc. 2012) and Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997) (these disclosures are incorporated herein by reference).

[0120] Therefore, for example, human EPO mice in which human reticulocytes and / or erythrocytes have engrafted, e.g., Rag2 - / - IL2rg - / - EPO m / hA method is provided for determining the effect of a drug on pathogen-infectable or infected erythrocytes, comprising the steps of: administering the drug to mice; measuring parameters of the viability of engrafted cells over time in the presence of the drug; and comparing the measurement with that of engrafted human EPO mice that have not been exposed to the drug. The drug is determined to be antipathogenic if, after a single dose or two or more doses of the drug over a selected period, it reduces the infection and / or death of human erythrocytes in the peripheral blood of mice by at least 20%, 30%, 40%, or more, and in some cases by 50%, 60%, 70%, or more, for example, 80%, 90%, or 100%, i.e., to an undetectable level. In specific embodiments, the administration of the drug or combination of drugs is at least 3 days, at least 1 week, at least 10 days after engraftment of human hematopoietic cells, for example, 2 weeks, 3 weeks, or 4 weeks after engraftment of human hematopoietic cells, for example, 6 weeks, 8 weeks, 10 weeks, or thereafter.

[0121] Other examples of the mouse applications of the present invention are provided elsewhere in this specification. Additional applications of the genetically modified and engrafted mice described herein will become apparent to those skilled in the art by reference to this disclosure.

[0122] Examples of non-limiting aspects of this disclosure Aspects including the aspects of the subject matter of the present invention described above may be useful on their own or in combination with one or more other aspects or aspects. Without limiting the foregoing description, certain non-limiting aspects of this disclosure, numbered 1 to 58, are provided below. Each of the individually numbered aspects may be used on its own or in combination with any of the preceding or following individually numbered aspects, as will be obvious to those skilled in the art by reference to this disclosure. This is to provide support for all such combinations of aspects, and is not limited to the combinations of aspects expressly provided below: 1. A genetically modified non-human animal containing a nucleic acid sequence encoding human EPO protein (hEPO) functionally linked to the EPO gene promoter. 2. A non-human animal as described in 1, wherein the EPO gene promoter is an endogenous non-human EPO promoter. 3. The non-human animal described in 1, wherein the functional linkage is an endogenous non-human EPO promoter at the non-human EPO locus. 4. The non-human animal described in 3, wherein the functional linkage results in a null mutation of the non-human EPO gene at the non-human EPO locus. 5. A non-human animal as described in 4, which is heterozygous for alleles containing the nucleic acid sequence encoding hEPO. 6. A non-human animal as described in 4, which is homozygous for an allele containing the nucleic acid sequence encoding hEPO. 7. A non-human animal as described in any one of items 1 to 6, wherein the nucleic acid sequence encoding hEPO includes the coding and non-coding sequences of the human EPO genome. 8. A non-human animal as described in any one of items 1 to 6, wherein the nucleic acid sequence encoding hEPO includes a human EPO cDNA sequence. 9. Nucleic acid-encoded hM-CSF protein under the regulation of the M-CSF promoter; The nucleotide-encoded hIL-3 protein under the regulation of the Il-3 promoter; hGM-CSF protein encoded by nucleic acids under the regulation of the Gm-CSF promoter; hTPO proteins encoded by nucleic acids under the regulation of the TPO promoter; and The hSirpa protein encoded by nucleic acids under the regulation of the Sirpa promoter. A non-human animal according to any one of items 1 to 8, which expresses one or more additional human proteins selected from the group consisting of the above. 10. The non-human animal described in 9, wherein the promoter is an endogenous non-human animal promoter located at the corresponding non-human animal locus, and the non-human animal is heterozygous null for the non-human gene. 11. The non-human animal described in 9, wherein the promoter is an endogenous non-human animal promoter located at the corresponding non-human animal locus, and the non-human animal is homozygous null for the non-human gene. 12. A non-human animal as described in 9, wherein the human protein comprises at least hTPO, hIL3, hGM-CSF, and hSirpa. 13. An immunodeficient non-human animal as described in any one of items 1-12. 14. Non-human animals as described in 13, in which immunodeficiency is caused by a deficiency of one or both Rag2 and Il2rg. 15. A non-human animal that is a mammal, as described in any one of items 1-14. 16. Non-human animals, as described in 15, whose mammals are rodents. 17. Non-human animals described in 16, in which rodents are mice. 18. A non-human animal as described in any one of items 1 to 17, further comprising the engraftment of human hematopoietic cells. 19. A non-human animal as described in 18, wherein the human hematopoietic cells include one or more cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human myeloid progenitor cells, human erythrocyte progenitor cells, human myeloid cells, human dendritic cells, human monocytes, human granulocytes, human erythrocytes, human neutrophils, human mast cells, human thymocytes, and human B lymphocytes. 20. A non-human animal as described in 19, containing clodronate. 21. Non-human animals as described in 20, further comprising infection by a pathogen that targets human cells of the erythrocyte lineage. 22. A non-human animal as described in 21, wherein the pathogen is selected from species of the genus Plasmodium, Babesia, and Theileri. 23. A method for identifying drugs that inhibit infection by pathogens targeting human cells of the erythrocyte lineage, comprising the following steps: (a) A step of administering a candidate drug to a genetically modified non-human animal, wherein the animal is: (i) A nucleic acid sequence encoding human EPO protein (hEPO) functionally linked to the EPO gene promoter; (ii) One or more gene mutations that result in immunodeficiency in non-human animals; (iii) Engraftment of human hematopoietic cells; and (iv) Infection caused by pathogens that target human cells of the red blood cell lineage Processes including, (b) A step of determining whether the drug reduces the amount of pathogen in a non-human animal infected with the pathogen. 24. A method for identifying drugs that prevent infection by pathogens targeting human cells of the erythrocyte lineage, comprising the following steps: (a) A step of contacting a genetically modified non-human animal with clodronate, wherein the non-human animal is: (i) A nucleic acid sequence encoding human EPO protein (hEPO) functionally linked to the EPO gene promoter; (ii) One or more gene mutations that result in immunodeficiency in non-human animals; and (iii) Engraftment of human hematopoietic cells Processes including (b) A process of administering a candidate drug to a genetically modified non-human animal, (c) The process of injecting parasitized reticulocytes or red blood cells into a genetically modified non-human animal, and (d) A step of determining whether the drug prevents infection of human reticulocytes and / or red blood cells in non-human animals. 25. The method according to 23 or 24, wherein the EPO gene promoter is an endogenous non-human promoter. 26. The method according to 23 or 24, wherein the functional linkage is made to an endogenous non-human EPO promoter at a non-human animal EPO locus. 27. The method according to 26, wherein the functional linkage results in a null mutation of a non-human EPO gene at a non-human EPO locus. 28. The method according to 27, wherein a non-human animal is heterozygous for an allele containing a nucleic acid sequence encoding hEPO. 29. The method according to 27, wherein a non-human animal is homozygous for an allele containing a nucleic acid sequence encoding hEPO. 30. The method according to any one of items 23 to 29, wherein the nucleic acid sequence encoding hEPO includes coding and non-coding sequences of the human EPO genome. 31. The method according to any one of items 23 to 29, wherein the nucleic acid sequence encoding hEPO includes a human EPO cDNA sequence. 32. Non-human animals, The hM-CSF protein encoded by nucleic acids under the regulation of the M-CSF promoter; The nucleotide-encoded hIL-3 protein under the regulation of the Il-3 promoter; hGM-CSF protein encoded by nucleic acids under the regulation of the Gm-CSF promoter; hTPO proteins encoded by nucleic acids under the regulation of the TPO promoter; and The hSirpa protein encoded by nucleic acids under the regulation of the Sirpa promoter. The method according to any one of items 23 to 31, wherein one or more additional human proteins selected from the group consisting of the above are expressed. 33. The method according to 32, wherein the promoter is an endogenous non-human animal promoter located at the corresponding non-human animal locus, and the non-human animal is heterozygous null for the non-human gene. 34. The method according to 33, wherein the promoter is an endogenous non-human animal promoter located at the corresponding non-human animal locus, and the non-human animal is homozygous null for the non-human gene. 35. The method according to any one of items 23 to 34, wherein the non-human animal lacks one or both of Rag2 and Il2rg. 36. The method according to any one of items 23 to 35, wherein the human hematopoietic cells include one or more cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human myeloid progenitor cells, human erythrocyte progenitor cells, human myeloid cells, human dendritic cells, human monocytes, human granulocytes, human erythrocytes, human neutrophils, human mast cells, human thymocytes, and human B lymphocytes. 37. The method according to any one of items 23 to 36, wherein the pathogen is selected from species of the genera Plasmodium, Babesia, and Theileria. 38. The method according to 37, wherein the pathogen is a species of the genus Plasmodium, and the species of Plasmodium is selected from Plasmodium falciparum and Plasmodium vivax. 39. The method described in any one of items 23 to 38, wherein the non-human animal is a mammal. 40. The method described in 39, which states that mammals are rodents. 41. The method described in 40, wherein the rodent is a mouse. 42. A method for producing mice that express human EPO protein (hEPO), comprising the following steps: A step of contacting mouse pluripotent stem cells with a nucleic acid sequence containing a coding sequence or fragment of hEPO functionally linked to an EPO promoter sequence, wherein the coding sequence and the EPO promoter sequence form a cassette in which sequences homologous to the endogenous mouse EPO locus are adjacent; A step of culturing pluripotent stem cells under conditions that promote the incorporation of nucleic acid sequences into the mouse genome at the endogenous mouse EPO gene locus by homologous recombination; and A process for generating mice from mouse pluripotent stem cells containing a nucleic acid sequence encoding hEPO. 43. The method according to 42, wherein the mouse pluripotent stem cells are ES cells or iPS cells. 44. The method according to 42 or 43, wherein mouse pluripotent stem cells lack Rag2 and / or IL2rg. 45. The method according to any one of items 42 to 44, wherein the EPO promoter sequence is the sequence of a human EPO promoter. 46. ​​The method according to any one of items 42 to 44, wherein the EPO promoter sequence is the sequence of an endogenous non-human EPO promoter. 47. The method according to any one of items 42-46, wherein the integration results in the substitution of a non-human EPO gene at a non-human EPO locus. 48. The method according to any one of items 42 to 47, wherein the nucleic acid sequence encoding hEPO includes coding and non-coding sequences of the human EPO genome. 49. The method according to any one of items 42 to 48, wherein the nucleic acid sequence encoding hEPO includes a human EPO cDNA sequence. 50. A method for producing mice that express human EPO protein (hEPO) and include human hematopoietic lineage, comprising the following steps: A step of transplanting a population of cells, including human hematopoietic primordial cells, into a genetically modified mouse produced by any one of the methods described in items 42 to 49. 51. The method according to 50, wherein the transplantation step includes tail vein injection, fetal liver injection, or posterior orbital injection. 52. The method according to 50 or 51, wherein genetically modified mice are irradiated to a non-lethal dose before transplantation. 53. The method according to any one of items 50-52, wherein the human hematopoietic cells are CD34+ cells. 54. The method according to any one of items 50 to 53, wherein the human hematopoietic organ cells are derived from fetal liver, adult bone marrow, or umbilical cord blood. 55. A method for producing mice infected with human pathogens that target human erythrocytes, comprising the following steps: A step of producing a mouse that expresses human EPO protein (hEPO) and includes a human hematopoietic line, according to the method described in any one of items 50 to 54; The process of injecting clodronate into mice; and The process of injecting parasitized human red blood cells (PRBCs) into mice. 56. The method of 55, further comprising the step of injecting healthy human red blood cells into a mouse. 57. The method of 55, wherein the parasite is selected from species of the genera Plasmodium, Babesia, and Theileria. 58. The method according to 57, wherein the parasite is a species of the genus Plasmodium, and the species of Plasmodium is selected from Plasmodium falciparum and Plasmodium vivax. [Examples]

[0123] The following examples are provided to the art to provide a complete disclosure and explanation of the methods for preparing and using the present invention, and are not intended to limit the scope of what the inventors consider to be the present invention, nor to represent that the following experiments are all or only experiments in which they were performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), but some experimental errors and deviations should be taken into consideration. Unless otherwise indicated, parts are by weight, molecular weights are weight-average molecular weights, temperatures are in degrees Celsius, and pressures are atmospheric pressure or approximately atmospheric pressure.

[0124] General methods in molecular and cellular biochemistry are described in: Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons). This information may be found in standard textbooks such as the 1998 edition, and their disclosures are incorporated herein by reference. The reagents, cloning vectors, and kits for genetic manipulation mentioned herein are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0125] Example 1 Generation of human EPO knock-in mice The coding sequence of the mouse erythropoietin locus (RefSeq transcript cDNA (SEQ ID NO:1) and encoded protein (SEQ ID NO:2) in mouse NCBI Gene ID:13856;MGI:95407;NM_007942.2) was replaced with a coding sequence derived from the human erythropoietin locus (RefSeq cDNA transcript (SEQ ID NO:3) and encoded protein (SEQ ID NO:4) in human NCBI Gene ID:2056;HGNC:3415;NM_000799.2).

[0126] [Table 1]

[0127] Specifically, the mouse genome region GRCm38:ch5:137482017:137485745 (minus strand) was deleted, and a human genome sequence derived from GRCh37:ch7:100318604:100321567 (plus strand) was inserted in its place. This resulted in the substitution of the entire coding region (coding exons 1-5) of the mouse Epo gene with the human uncoding region (exons 1-5 + 3') of the human EPO gene. In total, 3729 nt of mouse sequence were replaced with 2964 nt of human sequence.

[0128] In short, a targeting construct for replacing the mouse EPO gene with the human EPO gene in a single targeting step was constructed using VELOCIGENE® genetic engineering technology (see Valenzuela et al. (2003) (see above) and U.S. Patent No. 6,586,251). Mouse and human EPO DNA were obtained from bacterial artificial chromosomes bMQ-386K4 and RP11-797M3, respectively. A linearized targeting construct by PspXI, generated by gap repair cloning, was constructed using Rag2. This construct contains upstream and downstream homology arms of mouse EPO adjacent to a neo-selection cassette into which a 2964nt human EPO sequence + loxP extending from ATG in exon 1 to the stop codon in exon 5 (i.e., including the 3' downstream sequence) has been introduced. - / - IL2rg Y / - ES cells were electroporated (Figures 2 and 3). The junction between the mouse EPO 5' untranslated region (UTR) and exon 1 of human EPO was provided as SEQ ID NO:5. The last mouse nucleotide before the first nucleotide of the human gene is "G" (indicated in parentheses) in the non-bold portion of SEQ ID NO:5 in Table 2 below, and the first nucleotide of the human sequence is "A" (indicated in parentheses) in the bold portion of SEQ ID NO:5 in Table 2 below.

[0129] [Table 2]

[0130] The junction between the human 3'UTR and the 5' end of the selected cassette was provided as SEQ ID NO:6, where the last nucleotide of the human sequence was "C" (shown in single parentheses) in the bold portion of SEQ ID N:6 in Table 3 below, and the first nucleotide of the selected cassette sequence was "C" (shown in double parentheses) in the non-bold portion of SEQ ID NO:6 in Table 3 below; the downstream junction region also contained a loxP site at the 3' end for neocassette removal driven by a ubiquitin promoter into which loxP was introduced.

[0131] [Table 3]

[0132] The junction between the 3' end of the selection cassette and the mouse genome is provided as SEQ ID NO:7, and as shown in Table 4 below, "C" indicated by a single arc is the last nucleotide of the neocassette, and "G" indicated by double brackets is the first nucleotide of the mouse genome after the cassette.

[0133] [Table 4]

[0134] Precisely targeted hEPO ES cell clones were identified by a native allele loss assay (LONA) (Valenzuela et al. (2003) (see above)), which determines the number of copies of the native, unmodified EPO gene by performing two TaqMan® quantitative polymerase chain reaction (qPCR) assays specific to the sequence in the mouse EPO gene targeted for deletion. The qPCR assay included the following primer-probe set (written from 5' to 3'): Upstream forward primer TIFF0007833059000005.tif5128; Upstream reverse primer TIFF0007833059000006.tif5128; Upstream probe TIFF0007833059000007.tif5131; Downstream forward primer TIFF0007833059000008.tif5128; Downstream reverse primer TIFF0007833059000009.tif5128; Downstream probe TIFF0007833059000010.tif5129. FAM refers to a 5-carboxyfluorescein fluorescent probe, and BHQ refers to a black hole quencher type fluorescent quencher (Biosearch Technologies). Purified DNA from ES cell clones incorporating a targeting vector into their genomes was mixed with TaqMan® Gene Expression Master Mix (Life Technologies) in a 384-well PCR plate (MicroAmp® Optical 384-Well Reaction Plate, Life Technologies) according to the manufacturer's instructions. Fluorescence data was collected during PCR, and the sample was subjected to cycling in an Applied Biosystems Prism 7900HT to determine the threshold cycle number (Ct), which is the fractional PCR cycle at which the accumulated fluorescence reaches a predetermined threshold. Two types of qPCR were performed for each DNA sample: upstream and downstream EPO-specific qPCR and qPCR for a non-targeted reference gene. The difference in Ct values ​​(ΔCt) between each EPO-specific qPCR and each reference gene qPCR was calculated. Then, for all assayed samples, the difference between each ΔCt and the median ΔCt was calculated to obtain the ΔΔCt value for each sample. The copy number of the EPO gene in each sample was calculated using the following formula: copy number = 2·2 -ΔΔCtA precisely targeted clone that has lost one of its native copies will have an EPO gene copy number equal to 1. Confirmation that the human EPO gene sequence replaced the deleted mouse EPO gene sequence in the humanized allele was confirmed by a TaqMan® qPCR assay including the following primer-probe set (written from 5' to 3'): Human forward-direction primer TIFF0007833059000011.tif5128; Human reverse primer TIFF0007833059000012.tif5128, and human probe TIFF0007833059000013.tif5128.

[0135] Precisely targeted ES cells were electroporated with a transient Cre expression vector to remove the drug selection cassette. Precisely targeted ES cells were identified as described above and introduced into preimplantation embryos using techniques known in the art to generate mice containing human EPO and lacking Rag2 and Il2rg. Human EPO knock-in (KI) mice were then backcrossed to generate mice lacking Rag2 and Il2rg and expressing human EPO.

[0136] Example 2 Generation of human SIRPα mice In relation to some of the examples described herein, genetically modified mice containing nucleic acid sequences encoding human SIRPα randomly incorporated into the genome of genetically modified mice were prepared as described in U.S. Patent Application Publication No. 2013-0340105 (this disclosure is incorporated herein by reference).

[0137] For some of the examples described herein, human SIRPα knock-in mice were prepared as follows. It is known that human SIRPα exists in at least 10 alleles. In this specific example, human SIRPα variant 1 is used to humanize the endogenous SIRPα gene in mice.

[0138] A targeting vector for humanizing the extracellular region of the SIRP (e.g., SIRPα) gene was constructed using VELOCIGENE® technology (see, for example, U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) (see above)).

[0139] In short, mouse bacterial artificial chromosome (BAC) clone bMQ-261H14 was modified using human BAC clone CTD-3035H21 to delete the sequence containing exons 2-4 of the endogenous SIRPα gene and insert exons 2-4 of the human SIRPα gene. In BAC clone bMQ-261H14, the genomic DNA (approximately 8555 bp) corresponding to exons 2-4 of the endogenous SIRPα gene was replaced with a DNA fragment of approximately 8581 bp containing exons 2-4 of the human SIRPα gene derived from BAC clone CTD-3035H21. Sequence analysis of the human SIRPα allele contained in BAC clone CTD-3035H21 revealed that the allele corresponds to human variant 1. A neomycin cassette adjacent to the loxP region was attached to the end of an approximately 8581 bp human DNA fragment containing exons 2-4 of the human SIRPα gene (Figure 4).

[0140] To construct the final targeting vector for humanizing the endogenous SIRPα gene, the upstream and downstream homology arms located at the 5' and 3' positions of exon 2 and 4 of the endogenous SIRPα gene were obtained from mouse BAC DNA and added to the approximately 8581 bp human fragment-neomycin cassette. The targeting insertion of the targeting vector positioned the neomycin cassette in the fifth intron of the mouse SIRPα gene between exon 4 and exon 5. The targeting vector was linearized by digestion with SwaI and then used in homologous recombination in bacterial cells to achieve targeted substitution of exons 2-4 of the mouse SIRPα gene with exons 2-4 of the human SIRPα gene (Figure 4).

[0141] To create modified ES cells, which included the substitution of exons 2-4 of the endogenous mouse SIRPα gene with a genomic fragment containing exons 2-4 of the human SIRPα gene, the targeted BAC DNA (mentioned above) was used to electroporate mouse ES cells. Positive ES cells containing the genomic fragment containing exons 2-4 of the human SIRPα gene were identified by quantitative PCR using the TAQMAN® probe (Lie and Petropoulos, 1998. Curr. Opin. Biotechnology 9:43-48). The nucleotide sequence at the upstream insertion site included the following, which represents the endogenous mouse sequence upstream of the insertion site (contained in parentheses) that was continuously linked to the human SIRPα genomic sequence present at the insertion site. The nucleotide sequence at the downstream insertion site of the 5' end of the TIFF0007833059000014.tif27160 neomycin cassette contained the following, which represents a human SIRPα genome sequence that is continuous with the cassette sequence downstream of the insertion site (contained in parentheses, with the loxP sequence italicized): The nucleotide sequence at the downstream insertion site of the 3' end of the TIFF0007833059000015.tif27161 neomycin cassette contained the following cassette sequence, which is contiguous with the mouse genome sequence at 3' of exon 4 of the endogenous SIRPα gene (contained in parentheses): Next, to generate littermates containing the insertion of exons 2-4 of the human SIRPα gene into the endogenous SIRPα gene of the mouse, positive ES cell clones were used for implantation into female mice using the VELOCIMOUSE® method (e.g., U.S. Patent No. 7,294,754 and Poueymirou et al. 2007 Nature Biotech. 25(1):91-99 (see above) for F0 generation mice essentially entirely derived from ES cells targeted by a donor gene that enables direct phenotypic analysis).

[0142] The targeted ES cells described above were used as donor ES cells and introduced into 8-cell stage mouse embryos using the VELOCIMOUSE® method (described above). Mice retaining the humanization of exons 2-4 of the endogenous SIRPα gene were identified by genotyping using a modified allele assay (Valenzuela et al. (2003) (described above)) that detects the presence of the human SIRPα gene sequence.

[0143] For example, to remove a neomycin cassette into which loxP has been introduced by an unremoved targeting vector during the ES cell stage or in the embryo, mice carrying a humanized SIRPα gene construct (i.e., containing human SIRPα exons 2-4 in the mouse SIRPα gene) can be crossed with a Cre deleter mouse line (see, for example, International Patent Application Publication No. WO 2009 / 114400). Optionally, the neomycin cassette is retained in the mouse.

[0144] Example 3 Generation of compound knock-in mice Human EPO knock-in mice were crossed with mice expressing other human genes of interest, either as random integrations into the mouse genome or as knock-ins, i.e., from the corresponding mouse locus. For example, Rag2 - / - IL-2rg Y / - hEPO KI mice were used to create mice expressing human TPO from the mouse TPO locus (Rongvaux et al., 2011, Proc Natl Acad Sci USA, 108(6):2378-2383), mice expressing human IL-3 and human GM-CSF from the mouse IL-3 / GM-CSF locus (Willinger et al., 2011, Proc Natl Acad Sci USA, 108(6):2390-2395), mice expressing human M-CSF from the mouse M-CSF locus (Rathinam et al., 2011, Blood, 118(11):3119-3128), and / or as randomly incorporated (Strowig et al., 2011, Proc Natl Acad Sci (USA, 108(32):13218-13223) or mice expressing human SIRPa expressed from the aforementioned mouse locus, are crossed to produce mice (Rag2) that express these combinations of human proteins. - / - Il2rg null hSIRPa h / TPO h / h Mcsf h / h Il3 / Gmcsf h / h EPOh / h Genetically modified mice expressing one or more of the following: human TPO, human IL-3, human GM-CSF, human M-CSF, and human SIRPa are described in more detail in U.S. Patent Nos. 8,541,646 and 8,847,004; U.S. Patent Application Publication No. 2014 / 0134662; and PCT International Publication No. WO / 2014 / 039782 (each of these disclosures is incorporated herein by reference).

[0145] Example 4 Development of humanized mouse models of Plasmodium falciparum and Plasmodium vivax during the erythrocyte stage. This study demonstrates that genetic humanization of a mouse host by providing growth factors with limited cross-reactivity from mouse to human can successfully enhance human cell engraftment, specifically erythrocyte production, in mice engrafted with human hematopoietic stem cells (HSCs).

[0146] MITERG mice, which express human EPO from the genome (to enhance terminal erythropoiesis) and also express human TPO, MCSF, and IL-3 (to enhance HSC maintenance and early erythropoiesis), feature higher levels of human erythropoiesis in the bone marrow of mice with engrafted human HSCs, equivalent to mouse erythropoiesis in the same animal, compared to mice that do not express hEPO (Figure 5A, comparing "hSIRPa-,hEPO+" (i.e., "MITERG mice") with "hSIRPa-,hEPO-" (i.e., "MITRG mice"). However, these mice lack significant levels of circulating human erythrocytes (data not shown).

[0147] We hypothesized that the low levels of peripheral circulating human erythrocytes are due to the destruction of peripheral human RBCs (erythrocyte phagocytosis) by mouse macrophages. We investigated the role of SIRPa in this process by introducing hSIRPa into mice that express hSIRPa as a randomly incorporated transgene from a locus within the mouse genome other than the mSIRPa locus, i.e., hSIRPa-tg(Rag2) - / - Il2rgnull Tpo h / h Mcsf h / h Il3 h / h Gmcsf h / h Epo h / h SIRPα-tg+, that is, "MISTER-G mouse") and knock-in (KI) from the mSIRPa locus, that is, hSIRPa-expressing mice as hSIRPa KI (Rag2 - / - Il2rg null Tpo h / h Mcsf h / h Il3 h / h Gmcsf h / h Epo h / h SIRPα h / h , that is, "SupER-G mouse") were evaluated by generating.

[0148] For example, the expression of human SIRPa as a randomly integrated transgene in MISTER-G mice promoted a further increase in the number of human erythroid cells in the bone marrow of mice engrafted with human HSCs expressing human EPO (Figure 5, panel A, compare "hSIRPa+, hEPO+" with "hSIRPa-, hEPO+"). The introduction of human SIRPa significantly improved the survival of most hematopoietic cells in the periphery, and the frequency of human CD45 + cells, including lymphoid and myeloid cells in peripheral blood, increased at least 10-fold compared to that observed in mice not expressing hSIRPa. However, hSIRPa KI had little effect on the engraftment level of human RBCs in peripheral blood (Figure 5, panel B).

[0149] Because human SIRPα knock-in was insufficient to increase human erythrocytes in peripheral blood, clodronate liposomes were used to deplete macrophages, specifically splenic red medullary macrophages and hepatic Kupffer cells. Following depletion of tissue-resident macrophages with clodronate liposomes, a dramatic increase in circulating human erythrocytes to 1% of total circulating erythrocytes was observed in SuPER-G mice (Figure 6, Panel A). Furthermore, most peripheral human erythrocytes after clodronate treatment were reticulocytes (CD71). + ) was the result (Figure 6, Panel B). This is an exciting observation as it suggests that these mice would be good candidates to support Plasmodium vivax infection that preferentially infects reticulocytes.

[0150] Infection of red blood cells is an essential part of the life cycle of Plasmodium species. However, the required frequency of human RBCs for successful in vivo infection by different Plasmodium species has not been established. The only in vivo models currently available for Plasmodium falciparum are based on the transfer of human RBCs into immunodeficient lines such as NOD / SCID or NSG. In these mice, infection by merozoites can only be achieved by IV infusion of infected red blood cells after daily infusion of a large number of human red blood cells. At the time of infection, human RBCs constitute approximately half of the total red blood cell count in these animals.

[0151] As demonstrated above, SupER-G mice with engrafted human hematopoietic stem cells (HSCs) developed human erythrocytes in the bone marrow (Figure 5B), and (ii) clodronate treatment increased the frequency of peripheral human erythrocytes in engrafted SupER-G mice (Figure 6). To determine whether engrafted and clodronate-treated SupER-G mice contained a sufficient number of human erythrocytes peripherally to sustain a successful malaria parasite infection in vivo, peripheral blood was collected from fetal livers or from adult HSC-engrafted SupER-G mice, and the blood was cultured in vitro with blood infected with Plasmodium falciparum strain 3D7. Amplification due to subsequent reinfection with added human RBCs was expected to occur only if human RBCs collected from engrafted SuPER-G mice underwent a complete infection cycle with Plasmodium falciparum. To facilitate multiple parasitic replications, fresh human erythrocytes were added to the infection culture 48 hours later. Twelve days after infection, advanced stages of parasitic infection and amplification of merozoites from schizonts were observed in all blood samples from engrafted SuPER-G mice ("Engrafted Mouse RBCs, Adult 1", "Engrafted Mouse RBCs, Adult 2", and "Engrafted Mouse RBCs, Fetal Liver"), but not in non-engrafted control mice or control mice acutely engrafted with 0.1% hRBCs by injection ("Added Controls") (Figure 7A and unshown data). An exponential increase in parasitemia was observed by Giemsa staining and quantitative PCR (Figure 7B). This indicates that engrafted SupER-G treated with clodronate produces a sufficient number of human red blood cells to sustain infection by Plasmodium falciparum. Therefore, it is expected that in vivo infection with merozoites of Plasmodium falciparum and Plasmodium vivax in engrafted mice treated with clodronate will be successful.

[0152] Example 5 TIES mouse (Rag2) - / - Il2rg null TPO h / h Il3 / Gmcsf h / h Epoh / m SIRPα h / h ) Due to low reproductive capacity, developmental dysfunction, and high mortality rate, EPO h / h Mice possessing this gene are not ideal for infection research. Instead, mice heterozygous for the hEPO gene (i.e., Epo) are used. h / m For example, TIES mice possess the full ability to produce erythropoietin (EPO) and support all stages of erythropogenesis. High levels of human myeloid cell engraftment supported by human macrophage colony-stimulating factor (M-CSF) knock-in cause destruction of mouse erythrocytes, leading to anemia and death in engrafted mice. Therefore, by retaining the mouse M-CSF gene at the mouse locus rather than replacing it with human M-CSF, a further improvement in survival time from 8-10 weeks to 4 months was achieved.

[0153] Similar to SupER-G mice, TIES mice support human erythrogenesis and maintain a frequency of 1% human erythrocytes in peripheral blood after administration of clodronate. Furthermore, the majority of peripheral human erythrocytes after clodronate treatment were reticulocytes (CD71+). This suggests that these mice are good candidates for supporting Plasmodium vivax infection.

[0154] Engrafted TIES mice were shown to maintain a frequency of 1% human erythrocytes in their peripheral blood after administration of clodronate. Furthermore, the ability of TIES mice to support infusion of human RBCs was determined. As shown in Figure 8, clodronate treatment stabilized the infused population to over 20% of the total peripheral cell population. These levels, achieved approximately 4 hours post-infusion, persisted for at least 12 hours post-infusion. These RBC frequencies are expected to be sufficient to support in vivo infection with different species of malaria parasites.

[0155] The above is merely illustrative of the principles of the present invention. Those skilled in the art will recognize that various arrangements embodying the principles of the present invention and falling within its essence and scope may be devised, although these are not expressly described or shown herein. Furthermore, all language used to describe examples and conditions herein is primarily intended to help the reader understand the principles of the present invention and the concepts proposed by the inventors to advance the art, and should be interpreted without limitation to such specifically described examples and conditions. Moreover, all descriptions herein describing the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, shall encompass both their structural and functional equivalents. Furthermore, such equivalents shall include both currently known equivalents and those to be developed in the future, i.e., any elements to be developed that perform the same function regardless of their structure. Accordingly, the scope of the present invention is not limited to the exemplary embodiments shown and described herein. Rather, the scope and essence of the present invention are embodied in the appended claims.

[0156] Sequence information SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. Yale Plaza Institute for Research in Biomedicine (IRB) <120> GENETICALLY MODIFIED NON-HUMAN ANIMALS EXPRESSING HUMAN EPO <150> US 62 / 000,460 <151> 2014-05-19 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 715 <212> DNA <213> Mus musculus <400> 1 gatgaagact tgcagcgtgg acactggccc agccccgggt cgctaaggag ctccggcagc 60 taggcgcgga gatggggtg cccgaacgtc ccaccctgct gctttactc tccttgctac 120 tgattcctct gggcctccca gtcctctgtg ctcccccacg cctcatctgc gacagtcgag 180 ttctggagag gtacatctta gaggccaagg aggcagaaaa tgtcacgatg ggttgtgcag 240 aaggtcccag actgagtgaa aatattacag tcccagatac caaagtcaac ttctatgctt 300 ggaaaagaat ggaggtggaa gaacaggcca tagaagtttg gcaaggcctg tccctgctct 360 cagaagccat cctgcaggcc caggccctgc tagccaattc ctcccagcca ccagagaccc 420 ttcagcttca tatagacaaa gccatcagtg gtctacgtag cctcacttca ctgcttcggg 480 tactgggagc tcagaaggaa ttgatgtcgc ctccagatac caccccacct gctccactcc 540 gaacactcac agtggatact ttctgcaagc tcttccgggt ctacgccaac ttcctccggg 600 ggaaactgaa gctgtacacg ggagaggtct gcaggagagg ggacaggtga catgctgctg 660 ccaccgtggt ggaccgacga acttgctccc cgtcactgtg tcatgccaac cctcc 715 <210> 2 <211> 192 <212> PRT <213> Mus musculus <400> 2 Met Gly Val Pro Glu Arg Pro Thr Leu Leu Leu Leu Leu Ser Leu Leu Leu 1 5 10 15 Leu Ile Pro Leu Gly Leu Pro Val Leu Cys Ala Pro Pro Arg Leu Ile 20 25 30 Cys Asp Ser Arg Val Leu Glu Arg Tyr Ile Leu Glu Ala Lys Glu Ala 35 40 45 Glu Asn Val Thr Met Gly Cys Ala Glu Gly Pro Arg Leu Ser Glu Asn 50 55 60 Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met 65 70 75 80 Glu Val Glu Glu Gln Ala Ile Glu Val Trp Gln Gly Leu Ser Leu Leu 85 90 95 Ser Glu Ala Ile Leu Gln Ala Gln Ala Leu Leu Ala Asn Ser Ser Gln 100 105 110 Pro Pro Glu Thr Leu Gln Leu His Ile Asp Lys Ala Ile Ser Gly Leu 115 120 125 Arg Ser Leu Thr Ser Leu Leu Arg Val Leu Gly Ala Gln Lys Glu Leu 130 135 140 Met Ser Pro Pro Asp Thr Thr Pro Pro Ala Pro Leu Arg Thr Leu Thr 145 150 155 160 Val Asp Thr Phe Cys Lys Leu Phe Arg Val Tyr Ala Asn Phe Leu Arg 165 170 175 Gly Lys Leu Lys Leu Tyr Thr Gly Glu Val Cys Arg Arg Gly Asp Arg 180 185 190 <210> 3 <211> 1340 <212> DNA <213> homo sapiens <400> 3 cccggagccg gaccggggcc accgcgcccg ctctgctccg acaccgcgcc ccctggacag ccgccctctc ctccaggccc gtggggctgg ccctgcaccg ccgagcttcc cgggatgagg 120 gcccccggtg tggtcacccg gcgcgcccca ggtcgctgag ggaccccggc caggcgcgga 180 gatggggggtg cacgaatgtc ctgcctggct gtggcttctc ctgtccctgc tgtcgctccc 240 tctgggcctc ccagtcctgg gcgccccacc acgcctcatc tgtgacagcc gagtcctgga 300 360. gaggtacctc ttggaggcca aggaggccga gaatcacg acgggctgtg ctgaacactg cagcttgaat gagaatatca ctgtcccaga caccaaagtt aatttctatg cctggaagag 420 gatggaggtc gggcagcagg ccgtagaagt ctggcagggc ctggccctgc tgtcggaagc 480 tgtcctgcgg ggccaggccc tgttggtcaa ctcttcccag ccgtgggagc ccctgcagct 540 gcatgtggat aaagccgtca gtggccttcg cagcctcacc actctgcttc gggctctggg 600 agcccagaag gaagccatct cccctccaga tgcggcctca gctgctccac tccgaacaat 660 cactgctgac actttccgca aactcttccg agtctactcc aatttcctcc ggggaaagct 720 gaagctgtac acaggggagg cctgcaggac aggggacaga tgaccaggtg tgtccacctg 780 ggcatatcca ccacctccct caccaacatt gcttgtgcca caccctcccc cgccactcct 840 gaaccccgtc gaggggctct cagctcagcg ccagcctgtc ccatggacac tccagtgcca 900 gcaatgacat ctcaggggcc agaggaactg tccagagagc aactctgaga tctaaggatg 960 tcacagggcc aacttgaggg cccagagcag gaagcattca gagagcagct ttaaactcag 1020 ggacagagcc atgctgggaa gacgcctgag ctcactcggc accctgcaaa atttgatgcc 1080 aggacacgct ttggaggcga tttacctgtt ttcgcaccta ccatcaggga caggatgacc 1140 tggagaactt aggtggcaag ctgtgacttc tccaggtctc acgggcatgg gcactccctt 1200 ggtggcaaga gcccccttga caccggggtg gtgggaacca tgaagacagg atgggggctg 1260 gcctctggct ctcatggggt ccaagttttg tgtattcttc aacctcattg acaagaactg 1320 aaaaccaccaa aaaaaaaaaa 1340 <210> 4 <211> 193 <212> PRT <213> homo sapiens <400> 4 Put Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg <210> 5 <211> 226 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide sequence <220> <221> misc_feature <222> (1)..(115) <223> mouse sequence <220> <221> misc_feature <222> (116)..(128) <223> human Exon 1 <220> <221> misc_feature <222> (116)..(226) <223> human sequence <400> 5 tcttccaggc tagtggggtg atctggccct acagaacttc caaggatgaa gacttgcagc 60 gtggacactg gcccagcccc gggtcgctaa ggagctccgg cagctaggcg cggagatggg 120 ggtgcacggt gagtactcgc gggctgggcg ctcccgcccg cccgggtccc tgtttgagcg 180 gggatttagc gccccggcta ttggccagga ggtggctggg ttcaag 226 <210> 6 <211> 1054 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <220> <221> misc_feature <222> (1)..(115) <223> human Exon 5 <220> <221> misc_feature <222> (1)..(926) <223> human sequence <220> <221> misc_feature <222> (927)..(1054) <223> 5' end of selection cassette <400> 6 actccgaaca atcactgctg acactttccg caaactcttc cgagtctact ccaatttcct 60 ccggggaaag ctgaagctgt acacagggga ggcctgcagg acaggggaca gatgaccagg 120 tgtgtccacc tgggcatatc caccacctcc ctcaccaaca ttgcttgtgc cacaccctcc 180 cccgccactc ctgaaccccg tcgaggggct ctcagctcag cgccagcctg tcccatggac 240 actccagtgc cagcaatgac atctcagggg ccagaggaac tgtccagaga gcaactctga 300 gatctaagga tgtcacaggg ccaacttgag ggcccagagc aggaagcatt cagagagcag 360 ctttaaactc agggacagag ccatgctggg aagacgcctg agctcactcg gcaccctgca 420 aaatttgatg ccaggacacg ctttggaggc gatttacctg ttttcgcacc taccatcagg 480 gacaggatga cctggagaac ttaggtggca agctgtgact tctccaggtc tcacgggcat 540 gggcactccc ttggtggcaa gagccccctt gacaccgggg tggtgggaac catgaagaca 600 ggatgggggc tggcctctgg ctctcatggg gtccaagttt tgtgtattct tcaacctcat 660 tgacaagaac tgaaaccacc aatatgactc ttggcttttc tgttttctgg gaacctccaa 720 atcccctggc tctgtcccac tcctggcagc agtgcagcag gtccaggtcc gggaaacgag 780 gggtggaggg ggctgggccc tacgtgctgt ctcacacagc ctgtctgacc tctcgaccct 840 accgggcctg aggccacaag ctctgcctac gctggtcaat aaggtgtctc cattcaaggc 900 ctcaccgcag taaggcagct gccaacctcg agataacttc gtataatgta tgctatacga 960 agttatatgc atggcctccg cgccgggttt tggcgcctcc cgcgggcgcc cccctcctca 1020 cggcgagcgc tgccacgtca gacgaagggc gcag 1054 <210> 7 <211> 243 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <220> <221> misc_feature <222> (1)..(121) <223> 3' end of selection cassette <220> <221> misc_feature <222> (122)..(243) <223> mouse sequence <400> 7 gcctctgttc cacatacact tcattctcag tattgttttg ccaagttcta attccatcag 60 acctcgacct gcagccccta gataacttcg tataatgtat gctatacgaa gttatgctag 120 cgccaacccg ctaggacaag tgctgagtga gctggggcca ccgtttgagg aaacaggagc 180 cagtacagag gggttcccct ttaggggttg gtggcaatgg gcgaccctgg ttaatggatc 240 att 243 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <400> 8 catctgcgac agtcgagttc 20 <210> 9 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <400> 9 ccagggagct taccgtgac 19 <210> 10 <211> 27 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <400> 10 aggtacatct tagaggccaa ggaggca 27 <210> 11 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <400> 11 acagccgagt cctggagag 19 <210> 12 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <400> 12 aagccctgag cgtgagttc 19 <210> 13 <211> 26 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <400> 13 aggccaagga ggccgagaat atcacg 26 <210> 14 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <400> 14 gagccctgca ctggacaac 19 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <400> 15 tcccatgaac gctgagagtc 20 <210> 16 <211> 28 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <400> 16 agggtcaagg agccatagac agaatggc 28 <210> 17 <211> 200 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <220> <221> misc_feature <222> (1)..(100) <223> mouse sequence <220> <221> misc_feature <222> (101)..(200) <223> human sequence <400> 17 agctctccta ccactagact gctgagaccc gctgctctgc tcaggactcg atttccagta 60 cacaatctcc ctctttgaaa agtaccacac atcctggggt gctcttgcat ttgtgtgaca 120 ctttgctagc caggctcagt cctgggttcc aggtggggac tcaaacacac tggcacgagt 180 ctacattgga tattcttggt 200 <210> 18 <211> 199 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <220> <221> misc_feature <222> (1)..(100) <223> 5' end of neomycin cassette <220> <221> misc_feature <222> (101)..(199) <223> human sequence <220> <221> misc_feature <222> (106)..(139) <223> LoxP <400> 18 gctccccatt cctcactggc ccagcccctc ttccctactc tttctagccc ctgcctcatc 60 tccctggctg ccattgggag cctgccccac tggaagccag tcgagataac ttcgtataat 120 gtatgctata cgaagttata tgcatggcct ccgcgccggg ttttggcgcc tcccgcgggc 180 gcccccctcc tcacggcga 199 <210> 19 <211> 200 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide sequence <220> <221> misc_feature <222> (1)..(100) <223> 3' neomycin cassette <220> <221> misc_feature <222> (101)..(200) <223> mouse Exon 4 <400> 19 cattctcagt attgttttgc caagttctaa ttccatcaga cctcgacctg cagcccctag 60 ataacttcgt ataatgtatg ctatacgaag ttatgctagc tgtctcatag aggctggcga 120 tctggctcag ggacagccag tactgcaaag agtatccttg ttcatacctt ctcctagtgg 180 ccatctccct gggacagtca 200

Claims

1. Rodent embryonic stem (ES) cells, including the following: An EPO knock-in allele comprising a nucleic acid sequence encoding a human erythropoietin (hEPO) protein, functionally linked to the endogenous rodent EPO gene promoter at the rodent erythropoietin (EPO) locus of the genome of a rodent ES cell, wherein the EPO knock-in allele comprises at least several substitutions of the endogenous rodent EPO coding sequence, and the encoded hEPO protein has wild-type human EPO signaling function; A GM-CSF knock-in allele comprising a nucleic acid sequence encoding human granulocyte-macrophage colony-stimulating factor 2 (hGM-CSF) protein, functionally linked to the endogenous rodent GM-CSF gene promoter at the rodent granulocyte-macrophage colony-stimulating factor 2 (GM-CSF) locus in the genome of rodent ES cells, wherein the GM-CSF knock-in allele comprises at least several substitutions of the endogenous rodent GM-CSF coding sequence, and the rodent is homozygous for the GM-CSF knock-in allele (GM-CSF h / h ), and the encoded hGM-CSF protein has wild-type human GM-CSF signaling function, and is the GM-CSF knock-in allele; An IL-3 knock-in allele comprising a nucleic acid sequence encoding human interleukin-3 (hIL-3) protein, functionally linked to the endogenous rodent IL-3 gene promoter at the rodent interleukin-3 (IL-3) locus in the genome of rodent ES cells, wherein the IL-3 knock-in allele comprises at least several substitutions of the endogenous rodent IL-3 coding sequence, and the rodent is homozygous for the IL-3 knock-in allele (IL-3 h / h ), and the encoded hIL-3 protein has wild-type human IL-3 signaling function, and the IL-3 knock-in allele; A TPO knock-in allele comprising a nucleic acid sequence encoding human thrombopoietin (hTPO) protein, functionally linked to the endogenous rodent TPO gene promoter at the rodent thrombopoietin (TPO) locus in the genome of rodent ES cells, wherein the TPO knock-in allele comprises at least several substitutions of the endogenous rodent TPO coding sequence, and the rodent is homozygous for the TPO knock-in allele (TPO h / h ), and the encoded hTPO protein has wild-type human TPO signaling function, the TPO knock-in allele.

2. The aforementioned EPO knock-in allele is heterozygous (EPO h / m ), rodent ES cells as described in claim 1.

3. The aforementioned EPO knock-in allele is homozygous (EPO h / h ), rodent ES cells as described in claim 1.

4. The rodent ES cell according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding the hEPO protein includes a coding sequence and a non-coding sequence of the human EPO genome.

5. The rodent ES cell according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding the hEPO protein includes a human EPO cDNA sequence.

6. An M-CSF knock-in allele comprising a nucleic acid sequence encoding human macrophage colony-stimulating factor (hM-CSF) protein, functionally linked to the endogenous rodent M-CSF gene promoter at the rodent macrophage colony-stimulating factor (M-CSF) locus in the genome of rodent ES cells, wherein the M-CSF knock-in allele comprises at least several substitutions of the endogenous rodent M-CSF coding sequence, and the rodent ES cells are homozygous for the M-CSF knock-in allele (M-CSF h / h ), and the encoded hM-CSF protein has wild-type human M-CSF signaling function, and the M-CSF knock-in allele A rodent ES cell according to any one of claims 1 to 5, further comprising:

7. Endogenous rodent M-CSF is homozygous (M-CSF m / m ), rodent ES cells according to any one of claims 1 to 5.

8. A SIRPα knock-in allele comprising a nucleic acid sequence encoding a humanized signal regulatory protein α (hSirpα) protein, functionally linked to the endogenous rodent SIRPα gene promoter at the rodent signal regulatory protein α (SIRPα) locus in the genome of rodent ES cells, wherein the SIRPα knock-in allele comprises a partial substitution of the endogenous rodent SIRPα coding sequence, and the rodent ES cells are homozygous for the SIRPα knock-in allele (SIRPα h / h The SIRPα knock-in allele is characterized in that the encoded hSIRPα protein comprises the extracellular domain of the wild-type human SIRPα protein and the signaling domain of the endogenous rodent SIRPα protein, and the encoded hSIRPα protein has wild-type human SIRPα receptor function. A rodent ES cell according to any one of claims 1 to 7, further comprising:

9. Rodent ES cells according to any one of claims 1 to 7, further comprising a SIRPα transgene encoding the human Sirpα (hSirpα) protein.

10. Rodent ES cells according to any one of claims 1 to 9, comprising a deficiency of either or both Rag2 and IL2rg.

11. Rag2 null and IL2rg null The rodent ES cell according to claim 10, which is

12. A rodent ES cell according to any one of claims 1 to 11, which is a mouse ES cell.

13. A rodent embryo comprising rodent ES cells according to any one of claims 1 to 11.

14. A mouse embryo comprising mouse ES cells as described in claim 12.

15. A method for producing a genetically modified rodent, comprising the step of producing a genetically modified rodent from a rodent embryo as described in claim 13.

16. A method for producing a genetically modified mouse, comprising the step of producing a genetically modified mouse from a mouse embryo as described in claim 14.

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