Non-human animal modified in gene for exerting 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 May 19, 2014, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to the field of genetically modified non-human animals. [Background technology]

[0003] introduction Genetically modified mice, modified and engrafted mice, and their use in modeling human disease are known in the art.However, to date, the generation of genetically modified mice that model human infection with pathogens that target cells of the human erythroid lineage 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 around the world were considered malaria endemic, with an estimated 3.3 billion people at risk of developing the disease. The worldwide disease burden in 2010 was estimated at 216 million cases and 655,000 deaths, of which 86% were children under the age of five. Currently, drugs and vaccines for the prevention and treatment of malaria are still very limited. Furthermore, parasite resistance to commonly used antimalarial drugs is emerging and presents a constant challenge. Thus, there is an urgent need to develop new drugs and vaccines for the control and treatment of pathogens that target human red blood cells.

[0005] Because many of these pathogens do not infect laboratory rodent erythrocytes, in vivo studies have traditionally consisted of studies of malaria caused by the rodent parasite Plasmodium berghei ANKA, or of NOD / SCID mice, which are acutely engrafted by daily injection of large numbers of human erythrocytes and simultaneously or subsequently infected by injection of the parasitized erythrocytes, such as NOD / SCID / IL2rg mice. nullStudies on Plasmodium falciparum malaria have been limited to studies on non-myelodepleted (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 Document 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 Document 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 Document 3); Moreno et al. (2006) The course of infection 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 Document 4).To study the effects of malaria parasites and other pathogens on humans and to test vaccines and drugs for efficacy in preventing infection by these and other pathogens and treating infected humans, it would be useful to have non-human animals, such as genetically modified mice, that are susceptible to infection by such pathogens or that better support human red blood cells that are acutely transplanted into the animal prior to 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 Summary of the Invention

[0007] overview Provided is a genetically modified non-human animal that expresses human EPO from animal genome.Also provided is a method for making a non-human animal that expresses human EPO from non-human animal genome, and a method for using a non-human animal that expresses human EPO from non-human animal genome.These animals and methods have many applications in the art, including, for example, modeling human erythropoiesis and erythrocyte function; modeling human pathogen infection of erythrocyte; in vivo screening for agents that modulate erythropoiesis and / or erythrocyte function, for example, in healthy or diseased conditions; in vivo screening for agents that are toxic to erythrocytes or erythrocyte progenitor cells; in vivo screening for agents that prevent, alleviate or reverse the toxic effect of toxic agents on erythrocytes or erythrocyte progenitor cells; in vivo screening of erythrocytes or erythrocyte progenitor cells from individuals to predict the responsiveness of individuals to disease treatment.

[0008] In some aspects of the invention, genetically modified non-human animals are provided that express human EPO from the genome of the non-human animal, in other words, the non-human animal comprises 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 linked to the EPO gene promoter. In some embodiments, the EPO gene promoter is a human EPO promoter. In other embodiments, the EPO promoter is an endogenous, i.e., non-human, EPO promoter. In some such embodiments, the endogenous EPO promoter is at the non-human animal EPO locus. In other words, in some embodiments, the nucleic acid sequence encoding the human EPO protein is functionally linked to the non-human animal EPO promoter at the non-human animal EPO locus. In some such embodiments, the functional linkage results in a null mutation of the non-human EPO gene at the non-human EPO locus.

[0010] In some embodiments, the non-human animals of the invention are heterozygous for an allele that includes a nucleic acid sequence that encodes a human EPO protein. In other embodiments, the non-human animals are homozygous for an allele that includes a nucleic acid sequence that encodes a human EPO protein.

[0011] In some embodiments, the nucleic acid sequence encoding the human EPO protein comprises coding and non-coding sequences of the human EPO genome. In other embodiments, the nucleic acid sequence encoding the human EPO protein comprises a 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 a nucleic acid under the regulation of the M-csf promoter, IL-3 protein encoded by a nucleic acid under the regulation of the Il-3 promoter, GM-CSF protein encoded by a nucleic acid under the regulation of the Gm-csf promoter, TPO protein encoded by a nucleic acid under the regulation of the TPO promoter, and Sirpa protein encoded by a nucleic acid under the regulation of the Sirpa promoter. In some such embodiments, the promoter is an endogenous non-human animal promoter 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 at the corresponding non-human animal locus, and the non-human animal is homozygous null for the non-human gene. In certain embodiments, the non-human animal expresses a human protein, e.g., a humanized protein, 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 animal of the present invention is a mouse having a genome comprising a nucleic acid sequence encoding a human protein, e.g., a humanized protein, such as 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., operably linked to a corresponding non-human animal promoter, e.g., a Sirpa promoter, an IL-3 promoter, a GM-CSF promoter, an M-CSF promoter, a TPO promoter, or an IL-6 promoter, respectively, and expressing the encoded human protein and the native mouse protein. In other embodiments, the genetically modified non-human animal of the present invention is a mouse having a genome comprising a nucleic acid encoding a human protein, e.g., a humanized protein, functionally linked to a corresponding non-human animal promoter, e.g., a Sirpa promoter, an IL-3 promoter, a GM-CSF promoter, an M-CSF promoter, or a TPO promoter, respectively, such as a human EPO, e.g., a humanized EPO; a human Sirpa, e.g., a humanized Sirpa; a human IL-3, e.g., a humanized IL-3; a human GM-CSF, e.g., a humanized GM-CSF; a human M-CSF, e.g., a humanized M-CSF; a human TPO, e.g., a humanized TPO, and expressing the encoded human protein and not expressing a native mouse protein. 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., the 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, e.g., humanized genes, disclosed herein.

[0014] In some embodiments, the non-human animal of the invention is immunodeficient with respect to the innate immune system. In some such embodiments, the immunodeficiency is caused by a deficiency in one or both of Rag2 and IL2rg.

[0015] In some embodiments, the genetically modified immunodeficient non-human animals of the invention further comprise engraftment of human hematopoietic cells. In some such embodiments, the human hematopoietic cells comprise one or more cells selected from the group consisting of human CD34 positive cells, human hematopoietic stem cells, human myeloid progenitor cells, human erythroid 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 comprising a nucleic acid sequence encoding a human EPO protein operably linked to an EPO promoter at an endogenous locus exhibit high levels of human erythropoiesis in the bone marrow and a 2- to 5-fold increase in human cells of the erythroid lineage 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 comprising a nucleic acid sequence encoding a human EPO protein operably linked to an EPO promoter at an endogenous locus exhibit high levels of human erythropoiesis in the bone marrow and about a 2- to about 10-fold increase in human cells of the erythroid lineage in the bone marrow compared to control mice that do not express human EPO, for example, about a 2-fold, about a 3-fold, about a 4-fold, about a 5-fold, about a 6-fold, about a 7-fold, about a 8-fold, about a 9-fold, or about a 10-fold increase in human cells of the erythroid lineage in the bone marrow compared to control mice that do not express human EPO. Thus, in some embodiments, the engrafted genetically modified immunodeficient animals of the invention comprise bone marrow in which 20% or more of the red blood cells (CD235+) are human red blood cells. In some embodiments, the engrafted genetically modified immunodeficient animals of the invention comprise bone marrow in which about 10% or more of the red blood cells (CD235+) are human red blood cells, e.g., about 20% or more, about 30% or more, about 40% or more, or about 50% or more.

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

[0018] In some such embodiments, the genetically modified animal of the invention is an immunodeficient mouse, e.g., a Rag2 mouse, comprising a nucleic acid encoding a human EPO protein operably linked to a non-human animal EPO promoter at a non-human animal EPO locus, a nucleic acid encoding a human TPO protein operably linked to a non-human animal TPO promoter at a 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 a non-human animal Il-3 locus, a nucleic acid encoding a human GM-CSF protein operably linked to a GM-CSF promoter at a 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 a 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, e.g., a Rag2 mouse. - / - IL2rgy / - Tpo h / h Mcsf h / h Il3 h / h Gmcsf h / h Epo h / h hSIRPα + ("MISTER-G") mice. In other such embodiments, a nucleic acid encoding a human SIRPa protein, e.g., a humanized SIRPa protein, is operably linked to a non-human animal 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") mice).

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

[0020] In some embodiments, genetically modified immunodeficient non-human animals engrafted with human hematopoietic cells, comprising a nucleic acid sequence encoding a human EPO protein functionally linked to an EPO promoter at an endogenous locus, may exhibit better survival and engraftment of human red blood cells when they contain only one copy of the nucleic acid sequence encoding the EPO protein and when they contain endogenous M-csf. This is because high levels of human myeloid cell engraftment supported by human M-CSF in knock-in leads to destruction of mouse red blood cells, which in turn leads to anemia and death of the engrafted mice. Furthermore, heterozygosity for the human EPO allele improves fertility, developmental potential, and survival rate over mice homozygous for human EPO and null for mouse EPO. Thus, in some embodiments, the engrafted genetically modified susceptible animals of the present invention are mice that constitutively express EPO, e.g., transgenic mice, or mice that contain two copies of human EPO, e.g., EPO allele. h / h In some such embodiments, the genetically modified immunodeficient non-human animal is a TIES mouse (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 engrafted with human hematopoietic cells injected with clodronate liposomes exhibit a 1000-fold increase in the number of human red blood cells (CD235+) in the peripheral blood compared to non-injected animals. In some embodiments, genetically modified immunodeficient non-human animals engrafted with human hematopoietic cells injected with clodronate liposomes exhibit an increase in the number of human red blood cells (CD235+) in the peripheral blood of about 10-fold or more, about 50-fold or more, about 100-fold or more, about 500-fold or more, or about 1000-fold or more, compared to non-injected animals. Of these human red blood cells, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more can be reticulocytes (red blood cell precursor cells, CD71+). Thus, in some embodiments, the engrafted genetically modified immunodeficient animal of the invention comprises peripheral blood in which 1% or more, e.g., 5% or more or 10% or more of the red blood cells (CD235+) are human red blood cells, and 10% or more, e.g., 20% or more, 30% or more, 40% or more, or 50% or more of these human red blood cells are human reticulocytes (CD71+). In some such embodiments, the engrafted genetically modified immunodeficient non-human animal of the invention is a MISTER-G mouse, a SupER-G mouse, or a TIES mouse.

[0022] In some embodiments, the non-human animal further comprises an infection with a pathogen that targets human cells of the erythroid lineage. In some such embodiments, the pathogen is selected from Plasmodium species, Babesia species, and Theileria species. In some embodiments, the infection is created by injecting the parasite into the non-human animal. In some embodiments, the infection is created by injecting parasitized human red blood cells into the non-human animal. In some embodiments, the infection is created by injecting parasitized human red blood cells and healthy human red blood cells into the non-human animal.

[0023] In some aspects of the invention, methods are provided for identifying agents that inhibit infection by pathogens that target human cells of the erythroid lineage.

[0024] In some embodiments, the method includes administering a candidate agent to a genetically modified non-human animal comprising a nucleic acid sequence encoding a human EPO protein operably linked to an EPO gene promoter, one or more genetic mutations that result in immune deficiency in the non-human animal, engraftment of human hematopoietic cells, and infection with a pathogen that targets human cells of the erythroid lineage; and determining whether the agent reduces the amount of the 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 engrafted with human hematopoietic cells, the human hematopoietic cells comprising a nucleic acid sequence encoding a human EPO protein operably linked to an EPO gene promoter, with clodronic acid; administering a candidate agent to the non-human animal contacted with clodronic acid; injecting parasitized reticulocytes or red blood cells into the genetically modified non-human animal; and determining whether the agent prevents infection of human reticulocytes and / or red blood cells in the non-human animal.

[0026] In some embodiments, the pathogen is selected from Plasmodium spp., Babesia spp., and Theileria spp. In some such embodiments, the pathogen is selected from P. falciparum and P. 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, the method of producing mouse expressing human EPO protein is provided.In some embodiments, the method includes the following steps: contact mouse pluripotent stem cell with the nucleic acid sequence comprising the coding sequence of human EPO protein or its fragment functionally linked to EPO promoter sequence, and the coding sequence and EPO promoter sequence form a cassette flanked by the sequence that is homologous to endogenous mouse EPO locus; culture the pluripotent stem cell under the condition that promotes the integration of the nucleic acid sequence into mouse genome at endogenous mouse EPO locus by homologous recombination; and produce mouse from the mouse pluripotent stem cell that comprises the nucleic acid sequence that codes human EPO protein.

[0028] In some embodiments, the mouse pluripotent stem cell is an embryonic stem (ES) cell or an induced pluripotent stem (iPS) cell. In some embodiments, the mouse pluripotent stem cell lacks Rag2 and / or IL2rg. In some embodiments, the EPO promoter sequence is a human EPO promoter sequence. In other embodiments, the EPO promoter sequence is an endogenous non-human EPO promoter sequence. In some embodiments, the integration results in the replacement of the non-human EPO gene in the non-human EPO locus. In some embodiments, the nucleic acid sequence encoding the human EPO protein comprises coding and non-coding sequences of the human EPO genome. In some embodiments, the nucleic acid sequence encoding the human EPO protein comprises a human EPO cDNA sequence.

[0029] In some aspects of the present invention, a method for producing a mouse that expresses human EPO protein and comprises human hematopoietic system is provided.In some embodiments, the method comprises transplanting a population of cells that comprises human hematopoietic progenitor cells into the genetically modified immunodeficient mouse produced by the method of the present disclosure.In some embodiments, the transplantation comprises tail vein injection, fetal liver injection, or retro-orbital injection.In some embodiments, the genetically modified mouse is sublethally irradiated before transplantation.In some embodiments, the transplanted human hematopoietic progenitor cells 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 a mouse infected with a human pathogen that targets human cells of the erythroid lineage.In some embodiments, the method includes producing a mouse that expresses human EPO protein and includes a human hematopoietic system according to the method of the present disclosure, injecting clodronate into the engrafted mouse, and injecting parasitized human red blood cells (PRBCs) into the clodronate-injected mouse.In some embodiments, the method further includes injecting healthy human red blood cells into the mouse.In some embodiments, the parasite is selected from Plasmodium species, Babesia species, and Theileria species.In certain embodiments, the malaria parasite is selected from Plasmodium falciparum and Plasmodium vivax. [Brief description of the drawings]

[0031] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings, in which: It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, dimensions of the various features have been arbitrarily increased or reduced for clarity. The drawings include the following figures:

[0032] [Figure 1]1 provides a protein alignment of mouse Epo (SEQ ID NO:2) with human EPO (SEQ ID NO:4). Underlined residues are those that are conserved between species. [Diagram 2] 1 provides a schematic diagram of the wild-type mouse EPO locus before and after knock-in of a nucleic acid sequence encoding human EPO. [Diagram 3] 1 provides a schematic diagram of the human EPO knock-in allele. [Figure 4] 1 provides a schematic diagram of the wild-type mouse Sirpa locus before (top) and after (bottom) knock-in of the nucleic acid sequence encoding humanized Sirpa. [Diagram 5] Panels A and B show the frequency of human erythroid cells in mice engrafted with HSCs. (Panel A) Human CD235a+ erythroid engraftment in bone marrow 6-8 weeks after HSC engraftment into Rag2- / -Il2rg- / -Tpoh / hIL3h / hGmcsfh / hMcsfh / h mice ("MITRG mice") containing the indicated combinations of human EPO expressed from the mouse EPO locus ("hEPO+") and / or human SIRPa expressed as random integraters into the mouse genome ("hSIRPa+"). (Panel B) Frequency of human CD235a+ erythrocytes in peripheral blood in the presence versus absence of hEPO 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. [Figure 6]Panels A and B demonstrate that clodronate treatment increases circulating human red blood cells and reticulocytes in HSC-engrafted mice. Seven weeks after HSC engraftment, SupER-G mice were treated by daily retro-orbital injection of 50 μl of clodronate liposomes for 3-5 consecutive days. The frequency of human CD235+ cells (red blood cells and reticulocytes) (panel A) and CD235+ / CD71+ cells (reticulocytes) (panel B) in peripheral blood was measured by FACS. Panel B: Three different mice after treatment with clodronate. [Figure 7] Panels A-C illustrate how human RBCs produced from engrafted mice are susceptible to infection with P. falciparum. SupER-G mice were engrafted with fetal liver HSCs or adult HSCs. Seven weeks after engraftment, mice were treated with daily retro-orbital injections of 50 μl clodronate liposomes for three consecutive days, after which blood was collected. Blood samples were then cultured with RBCs (99% pure) infected with purified P. falciparum 3D7 erythrocytic stage parasites. Fresh human RBCs were added to the cultures 48 hours after infection, and infected cultures were maintained for an additional 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-loaded control: RBCs from non-engrafted mice loaded with 0.1% hRBCs. In (Panel A), red: anti-human Band3; blue: Hoechst. In Panel C, legends listed top to bottom correspond to the bar graph x-axis from left to right. [Figure 8] Figure 1 shows the destruction of human RBCs in mouse peripheral blood in the absence of clodronic acid. Non-engrafted mice were treated with clodronic acid or PBS. For clodronic acid treatment, mice received daily retro-orbital injections of 50 μl clodronic acid for three consecutive days. For PBS treatment, 500 μl PBS alone was delivered 1 hour prior to human RBC infusion. Human RBCs were infused into pre-treated mice and peripheral blood was collected at the indicated time points. Curves for clodronic acid and PBS are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description Genetically modified non-human animals are provided that express human EPO from animal genome. Methods for making non-human animals that express human EPO from non-human animal genome and methods for using non-human animals that express human EPO from 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, for example, in healthy or diseased conditions; in vivo screening for agents that are toxic to erythrocytes or erythrocyte progenitor cells; in vivo screening for agents that prevent, alleviate or reverse the toxic effects of toxic agents on erythrocytes or erythrocyte progenitor cells; in vivo screening of erythrocytes or erythrocyte progenitor cells from individuals to predict the responsiveness of individuals to disease treatment. These and other objects, advantages and features of the present invention will be apparent to those skilled in the art by referring to the details of the compositions and methods described more fully below.

[0034] Before the method and composition of the present invention are described, it should be understood that the present invention is not limited to the specific method or composition described, and therefore, of course, may vary.It should also be understood that the terminology used herein is only for describing specific embodiments, and not for the purpose of limitation, since the scope of the present invention is limited only by the appended claims.The present invention is not limited to the specific embodiments described, but is described by the allowed claims.

[0035] Where a range of values ​​is provided, it is understood that each value between the upper and lower limit of that range is also specifically disclosed, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each smaller range between a stated value or value falling within a stated range and another stated value or value falling within a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the range, and each range in which one, both or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included within the invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Any method and material similar or equivalent to those described herein can be used in carrying out or testing this invention, but some possible preferred methods and materials are described below.All publications mentioned herein are incorporated by reference to disclose and describe the method and / or material related to the publication citation.In the event of discrepancy, it is understood that the present disclosure takes precedence over the disclosure of the incorporated publication.

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

[0038] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, such as polypeptides known to those skilled in the art, and so forth.

[0039] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which must be independently confirmed.

[0040] Genetically modified non-human animals In one aspect of the invention, a non-human animal is provided that is genetically modified to express one or more human proteins from its genome. In some aspects of the invention, the human protein is human erythropoietin (hEPO) protein (SEQ ID NO:4). In other words, the genetically modified non-human animal comprises a nucleic acid sequence in its genome that encodes a human EPO (hEPO) protein. For example, the non-human animal may comprise a nucleic acid sequence in its genome that comprises coding and non-coding sequences of the human EPO genome, such as the sequence of chromosome 7 nucleotides 100318423-100321323, or a fraction thereof. Alternatively, the non-human animal may comprise a nucleic acid sequence in its genome that comprises a 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, the 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 alpha (hSIRPa) protein (NCBI Gene ID: 140885, GenBank Accession Nos. NM_080792.2, NM_001040022.1, NM_001040023.1), human interleukin 3 (hIL-3) protein (NCBI Gene ID: 3562, GenBank Accession No. NM_000588.3), human colony stimulating factor 2 (granulocyte macrophage) (hGM-CSF) protein (NCBI Gene ID: 1437, GenBank Accession No. NM_000758.3), human colony stimulating factor 1 (macrophage) (hM-CSF) protein (NCBI Gene ID: 1437, GenBank Accession No. NM_000758.3), human IL-1 (IL-2 ... 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: ID: 3569, GenBank accession number NM_000600.3).

[0041] Those skilled in the art will recognize that the terms "human nucleic acid" and "human protein" encompass "wild-type" or "native" human nucleic acid and human protein as well as variants of wild-type human nucleic acid and human protein. 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 recombinantly prepared variant of a human polypeptide or human nucleic acid sequence, each of which contains 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 orthologs. In some embodiments, variant polypeptides of the invention have 70% or more identity to a wild-type human polypeptide, e.g., 75%, 80%, or 85% or more identity, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a wild-type human polypeptide.

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

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

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

[0045] It is believed that the human variant is encoded by a nucleic acid that has a high degree of identity with the nucleic acid encoding the wild-type human. 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 some embodiments, the nucleic acid encoding the human variant may be isolated or produced recombinantly or synthetically using well-known methodologies.

[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" as used herein includes "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 the wild-type human protein (or variants thereof) and retain one or more functions of the wild-type human protein, e.g., one or more signaling functions and / or receptor functions. For example, a fusion protein that contains one or more fragments of the wild-type human protein (or variants thereof) in combination with one or more non-human peptides or polypeptides may also be referred to herein as a "humanized protein". Thus, for example, a protein comprising the amino acid sequence of the extracellular domain of a wild-type human SIRPα protein fused to the signaling domain of a wild-type mouse SIRPα protein is encompassed by the term "human SIRPα protein."

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

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

[0049] In some embodiments, the nucleic acid sequence encoding a human protein in the non-human animal of the present invention is operably linked to a human promoter for that gene, for example, when the human promoter drives the correct spatial and temporal expression of the human protein in the non-human animal. Alternatively, the nucleic acid sequence encoding a human protein in the non-human animal of the present invention is operably linked to a non-human animal promoter for the corresponding non-human animal gene. Thus, for example, with respect to a non-human animal expressing hEPO protein, in some embodiments, the nucleic acid encoding the hEPO protein is operably linked to a human EPO promoter. In other cases, the nucleic acid encoding the human EPO protein is operably linked to a non-human EPO promoter. In still other cases, the nucleic acid encoding the human EPO protein is operably linked to an endogenous non-human EPO promoter.

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

[0051] In some cases, the genetically modified non-human animal comprises one copy of the nucleic acid sequence encoding a human protein. For example, the non-human animal may be heterozygous for the nucleic acid sequence encoding a human protein, i.e., one allele at the locus is genetically modified and the other allele is an endogenous allele. In other cases, the genetically modified non-human animal comprises two copies of the nucleic acid sequence encoding a human protein. For example, the non-human animal may be homozygous for the nucleic acid sequence encoding a human protein, i.e., both alleles at the locus in the diploid genome may be genetically modified to encode a human protein, e.g., both alleles may comprise a substitution of the nucleic acid sequence encoding an endogenous protein with the nucleic acid sequence encoding a human protein. Thus, for example, for a non-human animal expressing hEPO protein, as described above, the nucleic acid sequence encoding hEPO may be integrated 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 that contains a nucleic acid encoding hEPO and one allele that encodes endogenous EPO. h / m In other such embodiments, 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 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. h / h It is an animal.

[0052] In some cases, the non-human animal also expresses the corresponding non-human animal protein. For example, the nucleic acid sequence encoding a human protein, e.g., hEPO, can be located at a site in the genome of the animal other than the locus for the non-human animal gene, e.g., the mEPO locus. As a second example, the nucleic acid sequence encoding a human protein, e.g., hEPO, can be located at the corresponding animal locus, e.g., the mEPO locus, and integrated into the animal locus in a manner that allows continued expression of the animal coding sequence, e.g., the human coding sequence can be inserted upstream or downstream of the animal coding sequence, and a 2A peptide sequence or an IRES sequence can be included between the two coding sequences. As a third example, the nucleic acid sequence encoding a human protein, e.g., hEPO, can 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 replacement of some or all of the animal coding sequence, but the non-human animal is crossed to be heterozygous for the inserted allele, i.e., the "knock-in" allele, i.e., to carry one knock-in allele and one wild-type allele. In other cases, the non-human animal does not express the corresponding non-human animal protein. For example, a nucleic acid sequence encoding a human protein, e.g., hEPO, may be located at a corresponding animal locus, e.g., the mEPO locus, in a manner that disrupts expression of the animal coding sequence, e.g., by replacing the non-human animal coding sequence, e.g., as a replacement of some or all 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 can be genetically modified according to the present disclosure. Non-limiting examples include laboratory animals, farm animals, livestock, etc., such as mice, rodents, dogs, cats, pigs, horses, cows, sheep, non-human primates, etc.; such as mice, rats, rabbits, hamsters, guinea pigs, cows, pigs, sheep, goats, and other transgenic animal species, particularly mammalian species, as known in the art. In other embodiments, the non-human animal can be a bird, such as Galliformes, such as chicken, turkey, quail, pheasant, or kokanee; anseriformes, such as duck, goose, or swan, or Columbiformes, such as pigeon or dove. In various embodiments, the genetically modified animal of the present invention is a mouse, rat, or rabbit.

[0054] In one embodiment, the non-human animal is a mammal. In some such embodiments, the non-human animal is a small mammal, for example, of the superfamily Dipodoidea or superfamily Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from mouse, rat, and hamster. In one embodiment, the rodent is selected from the superfamily Muridea. In one embodiment, the genetically modified animal is selected from the group consisting of Calomyscidae (e.g., mouse-like 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). In a specific embodiment, the genetically modified rodent is selected from the family of 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 rats, LEA strains, Sprague Dawley strains, Fischer strains, 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 invention is a mouse, e.g., a mouse of the C57BL strain (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 strain 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, e.g., Festing et al. (1999) Mammalian Genome 10:836, see also 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 mix of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In another embodiment, the mouse is a mix of the aforementioned 129 strain or the aforementioned BL / 6 strain. In yet another embodiment, the mouse is a mix of the BALB strain and another of the aforementioned strains.

[0057] In some embodiments, the genetically modified non-human animals of the invention are also immunodeficient. "Immune deficient" includes deficiencies in one or more aspects of the animal's native or endogenous immune system, e.g., the animal lacks one or more types of functional host immune cells, e.g., 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 way to achieve immune deficiency in the animals of the present invention is sublethal irradiation. Alternatively, immune deficiency can be achieved by any of a number of genetic 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 used as a source of stem cells into which the genetic modifications of the present disclosure can be introduced. Non-limiting examples include X-linked SCID associated with IL2RG gene mutations and characterized by lymphocyte phenotype T(-)B(+)NK(-); autosomal recessive SCID associated with Jak3 gene mutations and characterized by lymphocyte phenotype T(-)B(+)NK(-); ADA gene mutations characterized by lymphocyte phenotype T(-)B(-)NK(-); IL-7R alpha chain mutations characterized by lymphocyte phenotype T(-)B(+)NK(+); lymphocyte The lymphocyte phenotype T(-)B(+)NK(+) is characterized by mutations in CD3δ or ε; the lymphocyte phenotype T(-)B(-)NK(+) is characterized by mutations in RAG1 and RAG2; the lymphocyte phenotype T(-)B(-)NK(+) is characterized by mutations in the Artemis gene; the lymphocyte phenotype T(-)B(-)NK(+) is characterized by mutations in the CD45 gene; and the lymphocyte phenotype T(-)B(-) is characterized by mutations in the Prkdc gene. scid Thus, in some embodiments, the genetically modified immunodeficient non-human animal includes an IL2 receptor gamma chain (IL2rγ y / -) deficiency, Jak3 deficiency, ADA deficiency, IL7R deficiency, CD3 deficiency, RAG1 and / or RAG2 deficiency, Artemis deficiency, CD45 deficiency, and Prkdc deficiency. These and other animal models of immunodeficiency are known to those of skill in the art, any of which may be used to generate the immunodeficient animals of the present disclosure.

[0059] In some embodiments, the genetically modified non-human animals according to the invention have utility as recipients of human hematopoietic cells capable of developing human immune cells from the engrafted human hematopoietic cells. Thus, in some aspects of the invention, the genetically modified animals of the invention are genetically modified immunodeficient non-human animals engrafted with human hematopoietic cells.

[0060] Any source of human hematopoietic cells, human hematopoietic stem cells (HSCs), and / or hematopoietic stem progenitor cells (HSPCs) as known in the art or described herein may be transplanted into the genetically modified immunodeficient non-human animals of the present disclosure. One suitable source of human hematopoietic cells known in the art is human umbilical cord blood cells, specifically CD34 positive (CD34 + ) cells. Another source of human hematopoietic cells is human fetal liver. Another source is human bone marrow. Also included are induced pluripotent stem cells (iPSCs) and induced hematopoietic stem cells (iHSCs) produced by dedifferentiation of somatic cells, for example, by methods known in the art. Methods for transplantation of human cells into non-human animals are well described in the art and elsewhere herein, any of which may be utilized by the skilled artisan to arrive at the engrafted genetically modified non-human animals of the present invention.

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

[0062] In some embodiments, the transplanted human hematopoietic cells give rise to an engrafted human hematolymphoid system in the genetically modified non-human animal, comprising human hematopoietic stem and 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 hematolymphoid system is present at 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after engraftment. In a specific embodiment, the human hematolymphoid system comprises cells of the erythroid lineage.

[0063] Cells of the erythroid lineage include red blood cells and cells that give rise to red blood cells. "Erythrocytes" includes mature red blood cells, also called red cells or red corpuscles. Cells that give rise to red blood cells include erythroid progenitor cells, i.e., proliferative pluripotent cells, and erythroid precursor cells, i.e., proliferative or non-proliferative cells that are destined to become red blood cells.

[0064] Red blood cells are the main cellular element of circulating blood and their primary 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. It varies with age, activity, and environmental conditions. For example, at altitudes above 10,000 feet, the number is normally 8 million / mm 3An increase to levels of erythropoiesis can occur. Red blood cells normally survive for 110 to 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 of slightly more than 1% per day; thus, a constant level is generally maintained. Acute blood loss, hemolytic anemia, or chronic oxygen deprivation can cause a large increase in erythropoiesis.

[0065] Red blood cells arise from hematopoietic stem cells in the bone marrow of long bones and are classified into common myeloid progenitor cells (CD123+, CD34+, c-kit+, Flt3+); megakaryocyte-erythroid progenitor cells (CD34+, CD38+, CD45RA-); proerythroblasts (normally also called pronormoblasts, abnormally also called promegaloblasts; large CD71+, EpoR+, c-kit+, Ter119+ progenitor cells); and basophilic erythroblasts (basophilic cytoplasm, large nuclei with clumped chromatin, and absent nucleoli). They develop into erythrocytes through successive cell stages, including polychromatic erythroblasts (also called intermediate normoblasts, which show increased clumping of nuclear chromatin and whose cytoplasm begins to acquire hemoglobin, taking on an acidophilic hue); normochromatic normoblasts (the final stage before nuclear loss, in which the nucleus is small and eventually becomes a homogenous, structureless mass of blue-black color); and reticulocytes (circulating CD235+,CD71+ cells; the cells are characterized by a mesh-like pattern of threads and particles at the site of the former nucleus).

[0066] Mature erythrocytes appear in peripheral smears as biconcave round or ovoid disks approximately 6-8 μm in diameter. They contain hemoglobin, have a central pale zone due to the biconcave nature of the cells, and can be easily identified by flow cytometry or immunohistochemistry-based methods by elevated expression of the cell surface markers CD235 and CD59 compared to non-erythroid cells.

[0067] For example, as demonstrated in Figure 5, Panel A and Figure 5, Panel B of the present disclosure, expression of hEPO under the control of the EPO promoter from the genome of the engrafted non-human animal of the present disclosure increases the number of human cells of erythroid lineage (CD235a+) in the bone marrow by about 2-fold on average (e.g., from about 11% to about 22%). Expression of human Sirpa enhances this effect, resulting in an average increase of more than 3-fold (i.e., from about 11% to about 33%) in the representation of human CD235a+ cells in the bone marrow compared to animals that do not express human EPO or human Sirpa (Figure 5, Panel A). Thus, the engrafted hEPO-expressing immunodeficient genetically modified non-human animal of the present disclosure has applications in the study of human erythropoiesis and the development of drugs that modulate (e.g., promote or suppress) human erythropoiesis.

[0068] Moreover, as demonstrated, for example, in Figure 6, clodronate treatment of hEPO-expressing animals engrafted with human HSCs increases the number of CD235+ erythroid cells (including CD71+ reticulocytes, Figure 6, Panel B) in the peripheral blood of these animals by 1000-fold, or to about 1% of total peripheral erythrocytes, compared to untreated controls. Importantly, as demonstrated, for example, in Figure 7, human EPO-expressing animals engrafted with HSCs, the human erythrocytes produced at these engraftment levels are susceptible to infection with Plasmodium falciparum. Thus, the genetically modified non-human animals expressing hEPO described herein have particular use in generating animal models of infection with parasites that target human cells of the erythroid lineage, such as pathogens that result in malaria or malaria-like disease.

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

[0070] Method for Producing Genetically Modified Mice of the Present Invention In some aspects of the present invention, a method for producing the non-human animal of the present disclosure is provided.In carrying out the method of the present invention, a non-human animal is generated that comprises, for example, a nucleic acid sequence encoding a hEPO protein operably linked to an EPO promoter, for example, a non-human animal EPO promoter, in the EPO locus of the non-human animal genome.

[0071] The generation of non-human animals comprising a nucleic acid sequence encoding a hEPO protein operably linked to an EPO promoter can be accomplished using any convenient method of making genetically modified animals, for example, as known in the art or described herein.

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

[0073] Any of a variety of methods can then be used to introduce human nucleic acid sequences into animal cells to generate genetically modified animals that express human genes. Such techniques are well known in the art and include, but are not limited to, pronuclear microinjection, transformation of embryonic stem cells, homologous recombination, and knock-in techniques. Methods for generating genetically modified animals that may be used include those described by 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 nded. Sunderland, Massachusetts: Sinauer), U.S. Pat. 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 a nucleic acid encoding a human protein into an oocyte, e.g., by microinjection, and allowing the oocyte to develop in a female foster animal. In a preferred embodiment, the expression is injected into a fertilized oocyte. Fertilized oocytes can be collected from superovulated females the day after mating and injected with the expression construct. The injected oocytes are cultured overnight or transferred directly into the oviducts 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 described in Manipulating the Mouse Embryo (2002, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press). Derived animals can be evaluated for the presence of the introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or protein analysis (e.g., ELISA, Western blot, etc.).

[0075] As another example, constructs containing nucleic acid sequences encoding human proteins can be transfected into stem cells (e.g., ES or iPS cells) using well-known methods such as electroporation, calcium phosphate precipitation, and lipofection. The cells can be evaluated for the presence of the introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or protein analysis (e.g., ELISA, Western blot, etc.). Cells that are determined to have incorporated the expression construct can then be microinjected into preimplantation embryos. For detailed descriptions of methods known in the art useful for the compositions and methods of the 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] Additionally, as described in some of the Examples below, nucleic acid constructs can be constructed using VELOCIGENE® genetic engineering technology (see, e.g., 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. Pat. No. 6,586,251) and introduced into stem cells (e.g., ES cells) and precisely targeted clones can be determined using allelic loss and allelic gain assays (Valenzuela et al., supra); precisely targeted ES cells can be isolated using the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. 2007, F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analysis). In one embodiment, the ES cells can be used as donor ES cells for introduction into 8-cell mouse embryos using the previously described analyses (see Nature Biotech. 25(1):91-99).

[0077] The genetically modified founder animal can be crossed with additional animals carrying genetic modifications. The genetically modified animal carrying the nucleic acid encoding the human protein of the present disclosure can be further crossed with other genetically modified animals carrying 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 crossed with knock-out animals, such as non-human animals that lack one or more proteins, e.g., do not express one or more of its genes, such as Rag2-deficient animals, Il2rg-deficient animals.

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

[0079] As mentioned above, in some embodiments, the genetically modified non-human animal of the present invention is an immunodeficient animal. A genetically modified non-human animal that is immunodeficient and contains one or more human proteins, such as hEPO, hSIRPa, hIL-3, hGM-CSF, hM-CSF, and / or hTPO, can be generated using any convenient method for generating genetically modified animals, such as known in the art or described herein. For example, the generation of a genetically modified immunodeficient animal can be achieved by introducing a nucleic acid encoding a human protein into an oocyte or stem cell that contains a mutant SCID gene allele that will result in immunodeficiency when homozygous, as described in more detail herein above and in the examples. Mice with modified oocytes or ES cells are then generated, for example, using methods described herein and known in the art, and mated to generate an immunodeficient mouse that contains the desired genetic modification. As another example, genetically modified non-human animals can be generated in an immunocompetent background and bred with animals containing mutant gene alleles that would result in immunodeficiency when hemizygous or homozygous, and the progeny mated to produce immunodeficient animals that express at least one human protein of interest.

[0080] In some embodiments, the genetically modified mouse is treated to eliminate endogenous hematopoietic cells that may be present in the mouse.In one embodiment, the treatment comprises irradiating the genetically modified mouse.In a specific embodiment, the genetically modified newborn mouse is sublethally irradiated.In a specific embodiment, the newborn mouse is irradiated twice with 200cGy, 4 hours apart.

[0081] Various aspects of the present invention provide genetically modified animals that contain human nucleic acid in substantially all cells, and genetically modified animals that contain human nucleic acid in some but not all cells.In some cases, such as in target-specific recombination, one copy of human nucleic acid will be integrated into the genome of genetically modified animals.In other cases, such as in random integration, multiple copies of human nucleic acid that are adjacent or separate from each other can be integrated into the genome of genetically modified animals.

[0082] Thus, in some embodiments, the genetically modified non-human animals of the invention can be immunodeficient animals that comprise a genome comprising a nucleic acid encoding a human polypeptide operably linked to a corresponding non-human animal promoter and that express the encoded human polypeptide. In other words, the genetically modified immunodeficient non-human animals of the invention comprise a genome comprising a nucleic acid encoding at least one human polypeptide operably linked to a corresponding non-human promoter and polyadenylation signal and that express the encoded human polypeptide.

[0083] As mentioned above, in some embodiments, the genetically modified non-human animals of the present invention are engrafted with human hematopoietic cells. Any source of human hematopoietic cells, human hematopoietic stem cells (HSCs), and / or human hematopoietic stem progenitor 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 the present 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 is well understood by one of skill in the art or can be empirically determined. In one embodiment, engraftment is at least about 1-2×10 5with human CD34+ cells. The cells may be transplanted into the host non-human animal of the invention using any convenient technique known in the art, such as tail vein injection, retro-orbital injection, injection into the neonatal liver, etc. The cells may be transplanted into the host in any convenient buffer solution, such as PBS, Dulbecco's modified medium, Iscove's modified medium, etc. In some cases, animals may be irradiated prior to engraftment to ameliorate immune deficiencies, for example, as described above.

[0084] The engrafted human hematopoietic cells produce one or more 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. Any of a number of assays, including, for example, flow cytometry assays, blood smears, and immunohistochemistry for various human hematopoietic cells of interest, can be performed to confirm successful engraftment.

[0085] As mentioned above, in some embodiments, the genetically modified non-human animals of the present invention engrafted with human hematopoietic cells are infected with a human pathogen. Of particular interest in these embodiments are human pathogens that target human cells of the erythroid lineage. Non-limiting examples of such pathogens include protozoa such as Plasmodium, Babesia, and Theileria. In some embodiments, the strain of the pathogen used is a naturally occurring strain. In certain embodiments, the strain used is one that has been selected in vivo for its ability to grow reproductively in immunodeficient mice engrafted with human erythrocytes, such as the Plasmodium falciparum strain Pf3D7. 0087 / N9 or Pf3D7 0087 / N5 It is.

[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 erythroid lineage. For example, the engrafted immunodeficient animals of the present invention can be inoculated intraperitoneally with parasitized 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) Infection and pathology course in immunomodulated NOD / LtSz-SCID mice inoculated with Plasmodium falciparum laboratory strains and clinical isolates Int. J. Parasitol. 36:361-369. As another example, the engrafted immunodeficient animals of the present invention can be inoculated intravascularly with parasitized erythrocytes. See, for example, Angulo-Barturen et al. (2008) A mouse model of Plasmodium falciparum malaria by in vivo selection of competent strains in bone marrow-depleted mice engrafted with human erythrocytes PLoS One 3:e2252; and Jimenez-Diaz et al. (2009) An improved mouse model of malaria using Plasmodium falciparum competent strains and bone marrow-depleted NOD-scid IL2Rg null mice engrafted with human erythrocytes Antimicrob Agents Chemother 53:4533-4536. In some embodiments, the infection is created by injecting the parasite into the non-human animal, i.e., not associated with red blood cells. In some embodiments, the engrafted immunodeficient animals of the invention are subjected to in vivo chemical depletion of phagocytes prior to and / or during infection.In such embodiments, any chemotherapeutic agent that selectively depletes the host's phagocytes can be administered to the animals of the invention, including, for example, clodronic acid as described in the Examples herein; dichloromethylene diphosphate as described in Badell et al. (supra) and Moreno et al. (supra); monoclonal antibodies specific for polymorphonuclear neutrophils, such as NIMP-R14, as described in Badell et al. (supra) and Moreno et al. (supra).

[0087] Percent parasitemia in the infected genetically modified non-human animals of the present disclosure can be assessed by any convenient method in the art, e.g., by microscopy from Giemsa stained blood smears 3 days after injection; or by flow cytometry, e.g., by measuring the emission of the nucleic acid dye YOYO-1 or the cell permeable dye SYTO-16, e.g., in the presence or absence of TER-119 mAb. See, e.g., 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 the genetically modified mouse of the present invention The ability to study human tissues in the in vivo environment of mice has opened a series of possible research avenues. Major limitations have hindered the application of the approach, and one of the most important deficiencies among these has been the inability of mouse factors to support human cells. Indeed, many essential factors required for the development and function of human immune cells in the immune system are species-specific and cannot be effectively provided by mice. Therefore, it was decided to take a strategy of replacing mouse genes with human counterparts to allow better development and function of human cells and potentially not that of the corresponding mouse cells. By applying this concept to the human cytokine EPO, it is shown herein that the replacement 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 cells of the erythroid lineage in the engrafted human immune system in mice.

[0089] For example, the Examples demonstrate, for example, in FIG. 5, that expression of hEPO from a nucleic acid sequence under the regulation of the EPO promoter in the genome of a non-human animal increases the number of human cells of erythroid lineage (CD235a+) that develop in the bone marrow of animals engrafted with human HSCs by approximately 2-fold (i.e., from approximately 11% to approximately 22%). Expression of human Sirpa enhances this effect, resulting in a total of 3-fold increase in the representation of human CD235a+ cells in the bone marrow (i.e., from approximately 11% to approximately 33%) compared to animals that do not express human EPO or human Sirpa (FIG. 4a). Furthermore, as demonstrated, for example, in FIG. 6, clodronate treatment of animals engrafted with human HSCs expressing human EPO increases the number of CD235+ erythroid cells (including CD71+ reticulocytes, FIG. 6, panel B) in the peripheral blood of these animals by 1000-fold, i.e., to approximately 1% of total erythrocytes, compared to untreated controls. Importantly, human red blood cells produced at these engraftment levels in animals engrafted with HSCs expressing human EPO are susceptible to infection with Plasmodium species such as Plasmodium falciparum, as demonstrated, for example, in Figure 7. Thus, genetically modified non-human animals expressing human EPO as described herein have particular use in generating animals that are susceptible to infection with malaria parasites or that will better support human red blood cells acutely transplanted into the animal prior to infection in current rodent models.

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

[0091] As one non-limiting example, the engrafted genetically modified mice of the present disclosure have use in generating mouse models of pathogen infection with parasites that target human red blood cells, e.g., Plasmodium, Babesia, Theileria, etc. Such mouse models of infection would be useful, e.g., in research to better understand the progression of infection in humans, as well as in drug discovery, e.g., to identify candidate agents that protect against or treat infection with such parasites.

[0092] Protozoa of the genus Plasmodium are the cause of malaria in humans. Malaria begins with the bite of an infected Anopheles mosquito, which introduces the parasites via saliva into the circulatory system and ultimately to the liver, where they mature and reproduce. The parasites then enter the bloodstream and infect cells of the erythroid lineage at various stages of maturation.

[0093] Five species of malaria parasites can infect and be transmitted by humans. The majority of deaths are caused by P. falciparum, while P. vivax, P. ovale, and P. malariae cause generally milder forms of malaria that are rarely fatal. This is thought to be due at least in part to the type of cells targeted by each species: P. falciparum grows in red blood cells (RBCs) of all maturity levels, whereas P. vivax, for example, is restricted to growth in reticulocytes, which represent only approximately 1%-2% of all RBCs in the periphery. Furthermore, P. falciparum causes severe malaria through a distinctive property not shared by other human malaria, namely vascular sequestration. Within a 48-hour asexual erythrocytic phase cycle, mature forms change the surface properties of infected red blood cells, causing their attachment to blood vessels (a process called cytoadhesion). This leads to blockage of the microcirculatory system and to dysfunction of multiple organs.

[0094] Symptoms of malaria include fever, chills, headache, sweats, fatigue, anemia, nausea, dry (non-productive) cough, muscle and / or back pain, and splenomegaly. Other symptoms and complications associated with malaria include brain infection (encephalitis), hemolytic anemia, kidney failure, liver failure, meningitis, pulmonary edema, and bleeding from the spleen. In general, individuals at risk of developing malaria will begin to show symptoms after 7 days of infection, e.g., 9-14 days after initial infection with P. falciparum, 12-18 days after initial infection with P. vivax or P. ovale, 18-40 days after initial infection with P. vivax, or 11-12 days after initial infection with P. knowlesi. Antimalarial drugs used in the art to treat or prevent malaria include chloroquine, quinidine, doxycycline, tetracycline, clindamycin, atovaquone plus proguanil (Malarone), mefloquine, artesunate, and pyrimethamine plus sulfadoxine (Fansidar).

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

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

[0097] In humans, babesiosis may be asymptomatic or may be characterized by symptoms ranging from mild fever and diarrhea to high fever, shaking chills, and severe anemia. In severe cases, organ failure, including respiratory distress syndrome, may occur. Severe cases occur most often in people who have undergone splenectomy or who have a compromised immune system, such as HIV / AIDS patients. Treatment typically involves a two-drug regimen of quinine and clindamycin or atovaquone and azithromycin. If babesiosis is considered life-threatening, exchange transfusion is performed, in which infected red blood cells are removed and replaced with uninfected red blood cells.

[0098] Definitive diagnosis of Babesia infection is by identification of the parasite in a Giemsa-stained thin blood smear. The parasite appears in red blood cells as pairs of merozoites that form a "Maltese cross formation" in humans or a "double pear" in animals. Other diagnostic methods include PCR of peripheral blood and serological testing for antibodies (IgG, IgM) to Babesia.

[0099] Yet another malaria-like disease, Theileriosis, is caused by protozoa of the Theileria genus. Theileriosis is caused in humans by T. microti; in horses by T. equi ("equine piroplasmosis"); in sheep and goats by T. lestoquardi; in cattle, African buffalo, water buffalo, and waterbuck by T. annulata ("tropical theileriosis" also known as "Mediterranean theileriosis") or T. parva ("East Coast fever" also known as "Corridor disease"). Theileriosis is transmitted to the host by various tick species, including Ixodes punctatus, Rhipicephalus, Dermacentor, Haemaphysalis, and Hyalomma. These organisms reproduce in ticks as they progress through life stages, mature and invade the saliva after the tick has attached to the host. Typically, ticks become infective only after several days of attachment. However, if the environmental temperature is high, infective sporozoites can develop in ticks on the ground and invade the host within hours of attachment.

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

[0101] The engrafted genetically modified animals of the present disclosure have applications in screening candidate drugs to identify those that prevent (e.g., vaccines) or treat infection by Plasmodium, Babesia, Theileria, and other parasites that target human red blood cells. The terms "treatment," "treat," and the like are used generally herein to include obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it is a complete or partial prevention of a disease or its symptoms, and / or it may be therapeutic, in that it is a partial or complete cure of a disease and / or adverse effects caused by a disease. "Treatment," as used herein, includes any treatment of a disease in a mammal, including: (a) preventing the onset of a disease in a subject who 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) relieving the disease, i.e., causing regression of the disease. Important candidate agents as anti-parasitic therapeutics include those that may be administered before, during, or after a parasitic infection, and that when administered in an effective amount inhibit the effects of the parasite in an individual (i.e., host), for example, by killing the parasite or cells infected by the parasite, preventing transmission of the parasite, preventing the production or action of agents produced by the parasite that are toxic to the individual (i.e., toxins), etc. The terms "individual," "subject," "host," and "patient" are used interchangeably herein and include any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.

[0102] In a screening assay for biologically active agents, a genetically modified non-human animal engrafted with the human hematopoietic cells of the present disclosure, e.g., an engrafted Rag2 - / - Il2rg null Epo h / m Mice, engrafted Rag2 - / - Il2rg null Tpo h / h Il3 / Gmcsf h / h Epo h / m SIRPα h / h("TIES") mice, engrafted with Rag2 - / - IL2rg y / - Tpo h / h Mcsf h / h Il3 h / h Gmcsf h / h Epo h / h hSIRPα+ ("MISTER-G") mice, engrafted with Rag2 - / - Il2rg null Tpo h / h Mcsf h / h Il3 / Gmcsf h / h Epo h / h SIRPα-tg+ ("SupER-G") mice, etc. are contacted with a candidate agent of interest, and the effect of the candidate agent is assessed by monitoring one or more output parameters. These output parameters can reflect the viability of cells, for example, the total number of hematopoietic cells or the number of cells of a particular hematopoietic cell type, or the apoptotic state of cells, for example, the amount of DNA fragmentation, the amount of cell blebbing, the amount of phosphatidylserine on the cell surface, etc., by methods well known in the art. Alternatively or in addition, the output parameters can reflect the differentiation potential of cells, for example, the proportion of differentiated cells and differentiated cell types. Alternatively or in addition, the output parameters can reflect the function of cells, for example, the cytokines and chemokines produced by cells, the antibodies (for example, the amount or type) produced by cells, the ability of cells to home and extravasate to the site of challenge, 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. Still other parameters may reflect infection in animals, for example the effect of an agent on a pathogen infection, for example the titer of a pathogen in mice, etc., relevant to the study being conducted.

[0103] A parameter is a quantifiable component of a cell, particularly one that can be accurately measured, as is desirable in high-throughput systems. A parameter may be a cellular component or product, including cell surface determinants, receptors, proteins or their conformational or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids, such as mRNA, DNA, etc., or a portion derived from such a cellular component, or a combination thereof. Most parameters will provide a quantitative readout, but in some cases semi-quantitative or qualitative results will be acceptable. The readout may include a single determined value, or may include a mean, median, or variance, etc. Characteristically, a series of parameter readout values ​​will be obtained for each parameter from multiple identical assays. A series of values ​​will be obtained for each of a set of test parameters that are expected to be variable and are expected to be obtained using standard statistical methods with common statistical methods used to provide a single value.

[0104] Candidate agents of interest for screening include known and unknown compounds encompassing numerous chemical classes, primarily organic molecules, and may include organometallic molecules, inorganic molecules, genetic sequences, vaccines, antibiotics or other agents suspected of having antimicrobial properties, peptides, polypeptides, antibodies, agents that are pharmaceuticals approved for use in humans, etc. An important aspect of the present invention is the evaluation of candidate drugs, including, for example, toxicity testing, etc.

[0105] Candidate agents include organic molecules that contain functional groups necessary for structural interactions, specifically hydrogen bonding, typically at least one amine, carbonyl, hydroxyl, or carboxyl group, and often at least two functional chemical groups. Candidate agents often contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Included are pharmacologically active drugs, genetically active molecules, and the like. Compounds of interest include chemotherapeutic agents, hormones, or hormone antagonists, and the like. Examples of pharmaceutical agents suitable for the present invention are those described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth edition. Also included are toxins and biological and chemical warfare agents. See, e.g., Somani, SM (Ed.), "Chemical Warfare Agents," Academic Press, New York, 1992.

[0106] Candidate agents of interest for screening also include nucleic acids, such as siRNA, shRNA, antisense molecules, or miRNA-encoding nucleic acids, or nucleic acids that encode polypeptides. Many vectors are available that are useful for transferring nucleic acids into target cells. Vectors can be maintained as episomes, such as plasmids, minicircle DNA, virus-derived vectors, such as cytomegalovirus or adenovirus, or can be integrated into target cell genomes through homologous recombination or random integration (e.g., retrovirus-derived vectors, such as MMLV, HIV-1, ALV, etc.). Vectors can be directly provided to the cells of the present invention. In other words, pluripotent cells are contacted with vectors that contain the nucleic acid of interest, so that the vectors are taken up by the cells.

[0107] Methods for contacting cells, e.g., cultured cells or cells in mice, with nucleic acid vectors, such as electroporation, calcium chloride transfection, and lipofection, are well known in the art. Alternatively, the nucleic acid of interest can be provided to the cells via a virus. In other words, the cells are contacted with viral particles containing the nucleic acid of interest. Retroviruses, e.g., lentiviruses, are particularly suitable for the method of the present invention. Commonly used retroviral vectors are "defective", i.e., unable to produce viral proteins required for productive infection. Instead, vector replication requires growth in a packaging cell line. To generate viral particles containing the nucleic acid of interest, the retroviral nucleic acid containing the nucleic acid is packaged into a viral capsid by a packaging cell line. Different packaging cell lines provide different envelope proteins that are incorporated into the capsid, and this envelope protein determines the specificity of the viral particle for the cell. There are at least three types of envelope proteins: ecotropic, amphotropic, and xenotropic. Retroviruses packaged with ecotropic envelope proteins, such as MMLV, can infect most mouse and rat cell types and are generated by using ecotropic packaging cell lines such as BOSC23 (Pear et al. (1993) PNAS 90:8392-8396). Retroviruses carrying amphotropic envelope proteins, such as 4070A (Danos et al., supra), can infect most mammalian cell types, including human, dog, and mouse, and are generated by using amphotropic 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); GRIP (Danos et al. (1988) PNAS 85:6460-6464).Retroviruses packaged with xenotropic envelope proteins, such as AKR env, can infect most mammalian cell types except mouse cells. An appropriate packaging cell line can be used to ensure that the cells of interest, in some cases the engrafted cells, and in some cases the cells of the host, i.e., genetically modified animal, are targeted by the packaged viral particles.

[0108] The vectors used to provide the nucleic acid of interest to the cells of the invention will typically contain a suitable promoter to activate expression, i.e., transcription, of the nucleic acid of interest. This may include ubiquitously acting promoters, such as the CMV-β actin promoter, or inducible promoters, such as promoters that are active in a particular cell population or that respond to the presence of drugs, such as tetracycline. Transcriptional activation means that transcription will be increased at least about 10-fold, at least about 100-fold, and more commonly at least about 1000-fold over basal levels in the target cells. Additionally, the vectors used to provide the reprogramming factors to the cells of the invention may contain genes that must be subsequently removed, e.g., using a recombinase system such as Cre / Lox, or cells expressing them must be destroyed, e.g., by including genes that allow for selective toxicity, such as herpes virus TK, bcl-xs, etc.

[0109] Candidate agents of interest for screening also include polypeptides. Such polypeptides may optionally be fused to a polypeptide domain that increases the solubility of the product. The domain may be linked to the polypeptide through a distinct protease cleavage site, e.g., a TEV sequence that is cleaved by the TEV protease. The linker may contain one or more flexible sequences, e.g., 1-10 glycine residues. In some embodiments, cleavage of the fusion protein is performed in a buffer that maintains the solubility of the product, e.g., in the presence of 0.5-2M urea, in the presence of polypeptides and / or polynucleotides that increase solubility, etc. Domains of interest include endosomolytic domains, e.g., influenza HA domains; and other polypeptides that aid in production, e.g., IF2 domains, GST domains, GRPE domains, etc. Additionally or alternatively, such polypeptides may be formulated for improved stability. For example, the peptides may be PEGylated, where the polyethyleneoxy groups provide enhanced longevity in the bloodstream. A polypeptide may be fused to another polypeptide to provide additional functionality, e.g., to increase in vivo stability. Typically, such a fusion partner is a stable plasma protein that can, e.g., extend the in vivo plasma half-life of the polypeptide when present as a fusion; specifically, such a stable plasma protein is an immunoglobulin constant domain. In most cases where stable plasma proteins are usually found as multimeric forms, e.g., immunoglobulins or lipoproteins, in which identical or different polypeptide chains are usually disulfide-bonded and / or non-covalently linked to form an assembled multi-chain polypeptide, the fusions herein containing the polypeptides will also be made and utilized as multimers with substantially the same structure as the stable plasma protein precursor. These multimers will be homogeneous with respect to the polypeptide drug they contain. Alternatively, they may contain multiple types of polypeptide drugs.

[0110] Candidate polypeptide agents may be produced from eukaryotic cells or by prokaryotic cells. They may be further processed by unfolding, e.g., heat denaturation, DTT reduction, etc., and may be further refolded using methods known in the art. Modifications of interest that do not modify the primary sequence include chemical derivatization of the polypeptide, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are glycosylation modifications, e.g., those made by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes, to modify the glycosylation pattern of the polypeptide, e.g., during synthesis and processing, or in further processing steps. Sequences with phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine, are also included. Polypeptides may be modified using conventional molecular biology techniques and synthetic chemistry to improve resistance to proteolysis, optimize solubility properties, or make them more suitable as therapeutic agents. Analogs of such polypeptides include those that contain residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids, which can be substituted for some or all of the amino acid residues.

[0111] Candidate polypeptide agents may be prepared by in vitro synthesis using conventional methods as known in the art. A variety of commercially available synthesizers are available, such as automated synthesizers from Applied Biosystems, Inc., Beckman, and others. By using synthesizers, naturally occurring amino acids can be substituted with unnatural amino acids. The particular sequence and mode of preparation will be determined by convenience, economics, the required purity, and the like. Alternatively, candidate polypeptide agents may be isolated and purified according to conventional methods of recombinant synthesis. A lysate of the expression host may be prepared and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In most cases, the composition used will comprise at least 20% by weight, more typically at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes generally at least about 99.5% by weight of the desired product, relative to contaminants associated with the method of preparation of the product and its purification. Generally, the percentages will be based on total protein.

[0112] In some cases, the candidate polypeptide agent being screened is an antibody. The term "antibody" or "antibody portion" is intended to include any polypeptide chain that contains a molecular structure with a special shape that fits and recognizes an epitope, and one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The specific or selective fit of a given structure with its specific epitope is sometimes referred to as a "lock and key" fit. The prototypical antibody molecule is an immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g., human, rodent, rabbit, bovine, ovine, porcine, canine, other mammals, chicken, other birds, etc., are considered to be "antibodies." The antibodies utilized in the present invention can be either polyclonal or monoclonal. The antibodies are typically provided in the medium in which the cells are cultured. The production and screening of antibodies is described in more detail below.

[0113] Candidate agents can be obtained from a variety of sources, including libraries of synthetic or natural compounds. Numerous means are available for random and specific synthesis of a variety of organic compounds, including biomolecules, including, for example, expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or easily produced. In addition, natural or synthetically produced libraries and compounds can be easily modified through conventional chemical, physical, and biochemical means and used to produce combinatorial libraries. Known pharmacological agents can be subjected to specific or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to produce structural analogs.

[0114] Candidate agents are screened for biological activity by administering the agent to at least one, and generally multiple, samples, sometimes together with a sample lacking the agent. Changes in parameters in response to the agent are measured, and the results are evaluated by comparison with reference samples, such as cultures in the presence and absence of the agent, cultures obtained with other agents, etc. In cases where screening is performed to identify candidate agents that will prevent, mitigate, or reverse the effects of a pathogen, the screening is typically performed in the presence of a pathogenic agent, in which case the pathogenic agent is added at the most appropriate time for the results to be determined. For example, in cases where the protective / preventive ability of a candidate agent is tested, the candidate agent can be added before the pathogen, simultaneously with the pathogenic agent, or after infection by the pathogenic agent. As another example, in cases where the ability of a candidate agent to reverse the effects of a pathogen is tested, the pathogenic agent can be added after treatment with the pathogen. As noted above, in some cases, the "sample" is a genetically modified non-human animal into which the cells are engrafted, e.g., a candidate agent is provided to an immunodeficient animal, e.g., a mouse, into which human hematopoietic cells are engrafted, the candidate agent comprising a nucleic acid encoding human EPO operably linked to an EPO promoter. In some cases, the sample is the human hematopoietic cells to be engrafted, i.e., the candidate agent is provided to the cells, e.g., reticulocytes, red blood cells, etc., prior to engraftment into the immunodeficient, genetically modified animal.

[0115] When a candidate agent is administered directly to the engrafted genetically modified animal, the agent can be administered by any of the numerous methods known in the art for administration of peptides, small molecules, and nucleic acids to mice. For example, the agent can be administered orally, mucosally, topically, intradermally, or by injection, such as intraperitoneally, subcutaneously, intramuscularly, intravenously, or intracranially. The agent can be administered in a buffer or incorporated into any of a variety of formulations, for example, by combination with a suitable pharmacopoeia. A "pharmaceutical acceptable vehicle" can be a vehicle approved by a federal or state regulatory agency for use in mammals, such as humans, or listed in the US Pharmacopeia or other generally recognized pharmacopeia. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier for formulating the compound of the present invention for administration to a mammal. Such pharmaceutical vehicles may be lipids, e.g., liposomes, e.g., liposomal dendrimers; water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., liquids, such as saline; gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Additionally, auxiliary substances, stabilizers, thickeners, lubricants, and colorants may be used. The pharmaceutical compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injectables, inhalants, gels, microspheres, and aerosols. The agents may be systemic after administration or localized by use of topical administration, intramural administration, or the use of implants that act to retain the active dose at the site of implantation. The active agents may be formulated for immediate activity or for sustained release. For some conditions, particularly central nervous system conditions, it may be necessary to formulate an agent that crosses the blood-brain barrier (BBB).One strategy for drug delivery through the blood-brain barrier (BBB) ​​involves disrupting the BBB by osmotic means such as mannitol or leukotrienes, or biochemically by the use of vasoactive substances such as bradykinin. When the composition is administered by intravascular infusion, a BBB disrupting agent can be co-administered with the drug. Other strategies to cross the BBB may involve the use of endogenous transport systems, including caveolin-1 mediated transcytosis, carrier-mediated transporters such as carriers for glucose and amino acids, receptor-mediated transcytosis for insulin or transferrin, and active efflux transporters such as p-glycoprotein. To facilitate transport across the endothelial wall of blood vessels, active transport moieties may be conjugated to the therapeutic compounds for use in the present invention. Alternatively, drug delivery of the agent post-BBB can be by local delivery, e.g., intrathecal delivery, e.g., through an Ommaya reservoir (see, e.g., U.S. Patent Nos. 5,222,982 and 5,385,582, which are incorporated herein by reference); by bolus injection, e.g., intravitreally or intracranially, e.g., by a syringe; by continuous infusion, e.g., by cannulation, e.g., by convection (see, e.g., U.S. Application No. 20070254842, which is incorporated herein by reference); or by implantation of a device to which the agent is reversibly attached (see, e.g., U.S. Application Nos. 20080081064 and 20090196903, which are incorporated herein by reference).

[0116] When providing drugs to cells prior to engraftment, the drugs can conveniently be added to the culture medium of cultured cells in a solution or in a readily soluble form. Drugs can be added through a flow-through system as an intermittent or continuous stream, or a single or incremental bolus of compound can be added to a stationary solution. In a flow-through system, two liquids are used, one a physiologically neutral solution and the other an identical solution to which the test compound is added. The first liquid is passed over the cells, followed by the second liquid. In a 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 with 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 carried out in parallel.As known in the art, the determination of the effective concentration of a drug typically uses a series of concentrations resulting from 1:10 or other logarithmic scale dilutions.If necessary, the concentration can be further refined by a second dilution series.Typically, one of these concentrations serves as a negative control, i.e., zero concentration, or below the level of detection of the drug, or at or below the concentration of the drug that does not cause detectable changes in phenotype.

[0118] The analysis of the response of cells to the candidate drug in the engrafted genetically modified animal can be carried out at any time after the treatment with the drug.For example, the cells can be analyzed 1, 2, or 3 days after contact with the candidate drug, sometimes 4, 5, or 6 days after, sometimes 8, 9, or 10 days after, sometimes 14 days after, sometimes 21 days after, sometimes 28 days after, sometimes more than one month after, for example, 2 months after, 4 months after, 6 months after, or later.In some embodiments, the analysis includes the analysis at multiple time points.The selection of the time point for analysis will be based on the type of analysis to be carried out, as will be easily understood by those skilled in the art.

[0119] The analysis may include any of the parameters described herein or known in the art for measuring cell viability, cell proliferation, cell identity, cell morphology, and cell function, and may specifically relate 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 deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to measure DNA fragmentation or immunohistochemistry to detect Annexin V binding to phosphatidylserine on the cell surface, can be performed to determine the apoptotic state of cells. For example, flow cytometry can be used to assess the percentage of differentiated cells and differentiated cell types, to determine the viability and / or differentiation capacity of hematopoietic cells in the presence of a drug. For example, ELISA, Western, and Northern blots can be performed to determine the levels of cytokines, chemokines, immunoglobulins, etc. expressed in the engrafted genetically modified mice, to assess the function of engrafted cells, to assess the survival of red blood cells, etc. In vivo assays testing immune cell function and assays related to a particular disease or disorder of interest, such as anemia, e.g., sickle cell anemia, etc., may also be performed. See, e.g., Current Protocols in Immunology (Richard Coico, ed. John Wiley & Sons, Inc. 2012) and Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), the disclosures of which are incorporated herein by reference.

[0120] Thus, for example, human EPO mice engrafted with human reticulocytes and / or erythrocytes, e.g., Rag2 - / - IL2rg - / - EPO m / hMethods are provided for determining the effect of an agent on pathogen-infectable or infected red blood cells, comprising administering the agent to a mouse; measuring a parameter of viability of the engrafted cells over time in the presence of the agent; and comparing the measurements to measurements from engrafted human EPO mice not exposed to the agent. An agent is determined to be anti-pathogenic if it reduces infection and / or death of human red blood cells in the peripheral blood of the mouse by at least 20%, 30%, 40% or more, in some cases 50%, 60%, 70% or more, for example 80%, 90% or 100%, i.e., to an undetectable amount, after a single administration or administration of two or more agents over a selected period of time. In specific embodiments, administration of the drug or drug combination is at least 3 days, at least 1 week, at least 10 days, e.g., 2 weeks, 3 weeks, or 4 weeks after engraftment of the human hematopoietic cells, e.g., 6 weeks, 8 weeks, 10 weeks, or more after engraftment of the human hematopoietic cells.

[0121] Other examples of uses of the mice of the invention are provided elsewhere herein. Additional applications of the genetically modified and engrafted mice described in this disclosure will become apparent to those of skill in the art upon reference to this disclosure.

[0122] Examples of Non-Limiting Aspects of the Disclosure Aspects including the above-described aspects of the present inventive subject matter may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the present disclosure, numbered 1-58, are provided below. Each of the individually numbered aspects may be used or combined with any of the individually numbered aspects preceding or following it, as will be apparent to one of skill in the art upon reference to the present disclosure. This is intended 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 comprising a nucleic acid sequence encoding the human EPO protein (hEPO) operably linked to an EPO gene promoter. 2. The non-human animal 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 with an endogenous non-human EPO promoter in the non-human animal 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. The non-human animal of 4, which is heterozygous for an allele comprising a nucleic acid sequence encoding hEPO. 6. The non-human animal of 4, which is homozygous for an allele comprising a nucleic acid sequence encoding hEPO. 7. A non-human animal described in any one of 1 to 6, wherein the nucleic acid sequence encoding hEPO comprises coding and non-coding sequences of the human EPO genome. 8. The non-human animal described in any one of 1 to 6, wherein the nucleic acid sequence encoding hEPO comprises a human EPO cDNA sequence. 9. A hM-CSF protein encoded by a nucleic acid under the control of the M-csf promoter; a hIL-3 protein encoded by a nucleic acid under the regulation of an Il-3 promoter; hGM-CSF protein encoded by a nucleic acid under the regulation of the Gm-csf promoter; an hTPO protein encoded by a nucleic acid under the control of a TPO promoter; and hSirpa protein encoded by a nucleic acid under the control of the Sirpa promoter 9. The non-human animal according to any one of 1 to 8, which expresses one or more additional human proteins selected from the group consisting of: 10. The non-human animal according to 9, wherein the promoter is an endogenous non-human animal promoter 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 of 9, wherein the promoter is an endogenous non-human animal promoter at the corresponding non-human animal locus, and the non-human animal is homozygous null for the non-human gene. 12. The non-human animal according to 9, wherein the human proteins include at least hTPO, hIL3, hGM-CSF, and hSirpa. 13. A non-human animal described in any one of 1 to 12, which is immunodeficient. 14. The non-human animal according to 13, wherein the immune deficiency is caused by a deficiency of one or both of Rag2 and Il2rg. 15. A non-human animal described in any one of 1 to 14, which is a mammal. 16. The non-human animal according to claim 15, wherein the mammal is a rodent. 17. The non-human animal according to claim 16, wherein the rodent is a mouse. 18. The non-human animal described in any one of 1 to 17, further comprising engraftment of human hematopoietic cells. 19. The non-human animal described in 18, wherein the human hematopoietic cells include one or more types of cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human myeloid progenitor cells, human erythroid 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. The non-human animal according to 19, comprising clodronic acid. 21. The non-human animal of claim 20, further comprising an infection with a pathogen that targets human cells of the erythroid lineage. 22. The non-human animal according to 21, wherein the pathogen is selected from Plasmodium species, Babesia species, and Theileri species. 23. A method for identifying an agent that inhibits infection by a pathogen that targets human cells of the erythroid lineage, comprising the steps of: (a) administering a candidate agent to a genetically modified non-human animal, wherein the animal: (i) a nucleic acid sequence encoding a human EPO protein (hEPO) operably linked to an EPO gene promoter; (ii) one or more genetic mutations that result in immunodeficiency in the non-human animal; (iii) engraftment of human hematopoietic cells; and (iv) Infection by pathogens that target human cells of the erythroid lineage A process comprising: (b) determining whether the agent reduces the amount of the pathogen in a non-human animal infected with the pathogen. 24. A method for identifying an agent that prevents infection by a pathogen that targets human cells of the erythroid lineage, comprising the steps of: (a) contacting a genetically modified non-human animal with clodronic acid, wherein the non-human animal: (i) a nucleic acid sequence encoding a human EPO protein (hEPO) operably linked to an EPO gene promoter; (ii) one or more genetic mutations that result in immunodeficiency in a non-human animal; and (iii) Engraftment of human hematopoietic cells A process comprising: (b) administering the candidate agent to the genetically modified non-human animal; (c) injecting the parasitized reticulocytes or red blood cells into the genetically modified non-human animal; and (d) determining whether the agent prevents infection of the non-human animal with human reticulocytes and / or red blood cells. 25. The method of claim 23 or 24, wherein the EPO gene promoter is an endogenous non-human promoter. 26. The method of claim 23 or 24, wherein the functional linkage is with an endogenous non-human EPO promoter in a non-human animal EPO locus. 27. The method of claim 26, wherein the functional linkage results in a null mutation of the non-human EPO gene at the non-human EPO locus. 28. The method of claim 27, wherein the non-human animal is heterozygous for an allele comprising a nucleic acid sequence encoding hEPO. 29. The method of claim 27, wherein the non-human animal is homozygous for an allele comprising a nucleic acid sequence encoding hEPO. 30. The method of any one of 23 to 29, wherein the nucleic acid sequence encoding hEPO comprises coding and non-coding sequences of the human EPO genome. 31. The method of any one of 23 to 29, wherein the nucleic acid sequence encoding hEPO comprises a human EPO cDNA sequence. 32. Non-human animals: a hM-CSF protein encoded by a nucleic acid under the regulation of the M-csf promoter; a hIL-3 protein encoded by a nucleic acid under the regulation of an Il-3 promoter; hGM-CSF protein encoded by a nucleic acid under the regulation of the Gm-csf promoter; an hTPO protein encoded by a nucleic acid under the control of a TPO promoter; and hSirpa protein encoded by a nucleic acid under the control of the Sirpa promoter 32. The method according to any one of 23 to 31, wherein the method expresses one or more additional human proteins selected from the group consisting of: 33. The method of 32, wherein the promoter is an endogenous non-human animal promoter at the corresponding non-human animal locus, and the non-human animal is heterozygous null for the non-human gene. 34. The method of 33, wherein the promoter is an endogenous non-human animal promoter at a corresponding non-human animal locus, and the non-human animal is homozygous null for the non-human gene. 35. The method of any one of 23 to 34, wherein the non-human animal lacks one or both of Rag2 and Il2rg. 36. The method according to any one of 23 to 35, wherein the human hematopoietic cells comprise one or more types of cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human myeloid progenitor cells, human erythroid 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 of any one of 23 to 36, wherein the pathogen is selected from Plasmodium species, Babesia species, and Theileria species. 38. The method of claim 37, wherein the pathogen is a Plasmodium species, and the Plasmodium species is selected from P. falciparum and P. vivax. 39. The method according to any one of 23 to 38, wherein the non-human animal is a mammal. 40. The method according to claim 39, wherein the mammal is a rodent. 41. The method according to claim 40, wherein the rodent is a mouse. 42. A method for producing a mouse expressing human EPO protein (hEPO), comprising the steps of: contacting mouse pluripotent stem cells with a nucleic acid sequence comprising a coding sequence for hEPO, or a fragment thereof, operably linked to an EPO promoter sequence, wherein the coding sequence and the EPO promoter sequence form a cassette flanked by sequences that are homologous to the endogenous mouse EPO locus; Culturing the pluripotent stem cells under conditions that promote integration of the nucleic acid sequence into the mouse genome at the endogenous mouse EPO locus by homologous recombination; and Producing a mouse from mouse pluripotent stem cells comprising a nucleic acid sequence encoding hEPO. 43. The method according to claim 42, wherein the mouse pluripotent stem cells are ES cells or iPS cells. 44. The method of claim 42 or 43, wherein the mouse pluripotent stem cells lack Rag2 and / or IL2rg. 45. The method of any one of 42 to 44, wherein the EPO promoter sequence is a human EPO promoter sequence. 46. ​​The method of any one of 42 to 44, wherein the EPO promoter sequence is the sequence of an endogenous non-human EPO promoter. 47. A method according to any one of claims 42 to 46, wherein the integration results in replacement of the non-human EPO gene at the non-human EPO locus. 48. The method of any one of 42 to 47, wherein the nucleic acid sequence encoding hEPO comprises coding and non-coding sequences of the human EPO genome. 49. The method of any one of 42 to 48, wherein the nucleic acid sequence encoding hEPO comprises a human EPO cDNA sequence. 50. A method for producing a mouse that expresses human EPO protein (hEPO) and contains a human hematopoietic system, comprising the steps of: A step of transplanting a population of cells containing human hematopoietic progenitor cells into a genetically modified mouse produced by the method according to any one of 42 to 49. 51. The method of claim 50, wherein the transplanting step comprises tail vein injection, fetal liver injection, or retro-orbital injection. 52. The method according to claim 50 or 51, wherein the genetically modified mouse is irradiated at a sublethal dose before transplantation. 53. A method according to any one of claims 50 to 52, wherein the human hematopoietic progenitor cells are CD34+ cells. 54. A method according to any one of claims 50 to 53, wherein the human hematopoietic progenitor cells are derived from fetal liver, adult bone marrow, or umbilical cord blood. 55. A method for producing a mouse infected with a human pathogen that targets human cells of the erythroid lineage, comprising the steps of: Producing a mouse that expresses human EPO protein (hEPO) and contains a human hematopoietic system according to any one of the methods described in 50 to 54; injecting clodronic acid into the mouse; and Injecting parasitized human red blood cells (PRBCs) into mice. 56. The method of claim 55, further comprising the step of injecting healthy human red blood cells into the mouse. 57. The method of claim 55, wherein the parasite is selected from Plasmodium species, Babesia species, and Theileria species. 58. The method of claim 57, wherein the parasite is a Plasmodium species, and the Plasmodium species is selected from Plasmodium falciparum and Plasmodium vivax. EXAMPLES

[0123] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as the invention, nor are they intended to represent that the experiments described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0124] General methods in molecular and cellular biochemistry are described in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaR Bor 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 Genetic engineering techniques can be found in standard textbooks such as Thermologous Genetics and Its Applications (1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial sources 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 (mouse NCBI Gene ID: 13856; MGI: 95407; RefSeq transcript cDNA (SEQ ID NO: 1) in NM_007942.2 and encoded protein (SEQ ID NO: 2) in NP_031968) was replaced with the coding sequence from the human erythropoietin locus (human NCBI Gene ID: 2056; HGNC: 3415; RefSeq cDNA transcript (SEQ ID NO: 3) in NM_000799.2 and encoded protein (SEQ ID NO: 4) in NP_000790).

[0126] [Table 1]

[0127] Specifically, the mouse genomic region at GRCm38:ch5:137482017:137485745 (negative strand) was deleted and human genomic sequence from GRCh37:ch7:100318604:100321567 (positive strand) was inserted in its place. This resulted in the replacement of coding exons 1–5 (the entire coding region) of the mouse Epo gene with exons 1–5 + 3' human untranslated region of the human EPO gene. In total, 3729 nt of mouse sequence was replaced with 2964 nt of human sequence.

[0128] Briefly, 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) supra and U.S. Pat. No. 6,586,251). Mouse and human EPO DNA were obtained from bacterial artificial chromosomes bMQ-386K4 and RP11-797M3, respectively. A PspXI-linearized targeting construct generated by gap repair cloning containing 2964 nt of human EPO sequence spanning from the ATG in exon 1 to the stop codon in exon 5 (i.e., including 3′ downstream sequence) plus mouse EPO upstream and downstream homology arms flanking a loxP-introduced neo selection cassette was inserted into Rag2. - / - IL2rg Y / - The mouse EPO 5' untranslated region (UTR) and exon 1 of human EPO are provided as SEQ ID NO:5, with the last mouse nucleotide before the first nucleotide of the human gene being a "G" (shown in brackets) in the non-bolded portion of SEQ ID NO:5 in Table 2 below, and the first nucleotide of the human sequence being an "A" (shown in brackets) in the bolded 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 selection cassette is provided as SEQ ID NO:6, with the last nucleotide of the human sequence being "C" (indicated by a single bracket) in the bolded portion of SEQ ID NO:6 in Table 3 below, and the first nucleotide of the selection cassette sequence being "C" (indicated by a double bracket) in the non-bolded portion of SEQ ID NO:6 in Table 3 below; the downstream junction region also contained a loxP site at the 3' end for removal of the neo cassette driven by a loxP-introduced ubiquitin promoter.

[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 shown in Table 4 below, where the "C" indicated in single brackets is the last nucleotide of the neo cassette and the first nucleotide of the mouse genome after the cassette is a "G" indicated in double brackets.

[0133] [Table 4]

[0134] Correctly targeted hEPO ES cell clones were identified by loss of native allele assay (LONA) (Valenzuela et al. (2003) supra), which determines the number of copies of the native, unmodified EPO gene by two TaqMan™ quantitative polymerase chain reactions (qPCR) specific for sequences in the mouse EPO gene targeted for deletion. The qPCR assay contained the following primer-probe set (written 5' to 3'): Upstream forward primer TIFF2025061185000006.tif5128; Upstream reverse primer TIFF2025061185000007.tif5128; upstream probe TIFF2025061185000008.tif5131; downstream forward primer TIFF2025061185000009.tif5128; downstream reverse primer TIFF2025061185000010.tif5128; downstream probe TIFF2025061185000011.tif5129. FAM refers to 5-carboxyfluorescein fluorescent probe, and BHQ refers to black hole quencher type fluorescent quencher (Biosearch Technologies). DNA purified from ES cell clones that had taken up and integrated the targeting vector into their genome was combined with TaqMan™ Gene Expression Master Mix (Life Technologies) according to the manufacturer's instructions in a 384-well PCR plate (MicroAmp™ Optical 384-Well Reaction Plate, Life Technologies) and subjected to cycling in an Applied Biosystems Prism 7900HT, which collected fluorescence data during PCR and determined the threshold cycle number (Ct), the fractional PCR cycle when the accumulated fluorescence reaches a pre-determined threshold. Upstream and downstream EPO-specific qPCRs as well as two qPCRs for a non-targeted reference gene were performed for each DNA sample. The difference in Ct values ​​(ΔCt) between each EPO-specific qPCR and each reference gene qPCR was calculated, and then the difference between each ΔCt and the median ΔCt for all samples assayed was calculated to obtain the ΔΔCt value for each sample. The copy number of the EPO gene in each sample was calculated from the following formula: copy number = 2 2 -ΔΔCtCorrectly targeted clones that have lost one of the 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 containing the following primer-probe set (written 5' to 3'): Human forward primer TIFF2025061185000012.tif5128; Human reverse primer TIFF2025061185000013.tif5128, and human probes TIFF2025061185000014.tif5128.

[0135] The correctly targeted ES cells were electroporated with a transient Cre expression vector to remove the drug selection cassette.The correctly targeted ES cells were identified as above and introduced into preimplantation embryos using techniques known in the art to generate mice that contain human EPO and lack Rag2 and Il2rg.The human EPO knock-in (KI) mice were then backcrossed to generate mice that lack Rag2 and Il2rg and express human EPO.

[0136] Example 2 Generation of human SIRPα mice For some of the examples described herein, genetically modified mice containing a nucleic acid sequence encoding human SIRPα randomly integrated into the genome of the genetically modified mice were prepared as described in U.S. Patent Application Publication No. 2013-0340105, the disclosure of which is incorporated herein by reference.

[0137] For some of the examples described herein, human SIRPα knock-in mice were prepared as follows. Human SIRPα is known to exist in at least 10 allelic forms. In this specific example, human SIRPα variant 1 is utilized to humanize the endogenous SIRPα gene in mice.

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

[0139] Briefly, mouse bacterial artificial chromosome (BAC) clone bMQ-261H14 was modified to delete sequences containing exons 2-4 of the endogenous SIRPα gene and to insert exons 2-4 of the human SIRPα gene using human BAC clone CTD-3035H21. In BAC clone bMQ-261H14, genomic DNA (approximately 8555 bp) corresponding to exons 2-4 of the endogenous SIRPα gene was replaced with an approximately 8581 bp DNA fragment containing exons 2-4 of the human SIRPα gene 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 flanked by loxP sites was added to the ends of an approximately 8581 bp human DNA fragment containing exons 2 to 4 of the human SIRPα gene (Figure 4).

[0140] To generate the final targeting vector for humanization of the endogenous SIRPα gene, which contained, from 5' to 3', a 5' homology arm containing 19 kb of mouse DNA 5' of exon 2 of the endogenous SIRPα gene, an approximately 8581 bp DNA fragment containing exons 2-4 of the human SIRPα gene, a neomycin cassette flanked by loxP sites, and a 3' homology arm containing 21 kb of mouse DNA 3' of exon 4 of the endogenous SIRPα gene, the upstream and downstream homology arms located 5' and 3' of exons 2 and 4, respectively, were obtained from mouse BAC DNA and added to the approximately 8581 bp human fragment-neomycin cassette. The targeted 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 replacement of exons 2-4 of the mouse SIRPα gene with exons 2-4 of the human SIRPα gene (Figure 4).

[0141] The targeted BAC DNA (described above) was used to electroporate mouse ES cells to generate modified ES cells containing a replacement of exons 2-4 of the endogenous mouse SIRPα gene with a genomic fragment containing exons 2-4 of the human SIRPα gene. Positive ES cells containing a genomic fragment containing exons 2-4 of the human SIRPα gene were identified by quantitative PCR using a TAQMAN™ probe (Lie and Petropoulos, 1998. Curr. Opin. Biotechnology 9:43-48). The nucleotide sequence spanning the upstream insertion point included the following, which shows the endogenous mouse sequence upstream of the insertion point (contained within brackets) consecutively linked to the human SIRPα genomic sequence present at the insertion point: TIFF2025061185000015.tif27160 The nucleotide sequence spanning the downstream insertion point at the 5' end of the neomycin cassette included the following, which shows human SIRPα genomic sequence contiguous with the cassette sequence downstream of the insertion point (contained in brackets, with the loxP sequence in italics): TIFF2025061185000016.tif27161 The nucleotide sequence spanning the downstream insertion point at the 3' end of the neomycin cassette included the following, which shows the cassette sequence contiguous with mouse genomic sequence 3' of exon 4 of the endogenous SIRPα gene (contained in brackets): TIFF2025061185000017.tif27160 Positive ES cell clones were then used for implantation into female mice using the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. 2007 F0 generation mice essentially entirely derived from donor gene-targeted ES cells allowing direct phenotypic analysis Nature Biotech. 25(1):91-99, supra) to generate littermates containing an insertion of exons 2-4 of the human SIRPα gene into the mouse endogenous SIRPα gene.

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

[0143] For example, 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, e.g., International Patent Application Publication No. WO 2009 / 114400) to remove any loxP-transduced neomycin cassette introduced by the unremoved targeting vector at the ES cell stage or in the embryo. Optionally, the neomycin cassette is retained in the mice.

[0144] Example 3 Generation of compound knock-in mice The human EPO knock-in mice were bred 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 generated as 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 random integrants (Strowig et al., 2011, Proc Natl Acad Sci USA, 108(6):2378-2383). USA, 108(32):13218-13223) or mice expressing human SIRPa expressed from the mouse locus, to generate mice expressing a combination of these human proteins (Rag2 - / - Il2rg null hSIRPa h / Tpo h / h Mcsf h / h Il3 / Gmcsf h / h EPOh / h Genetically modified mice expressing one or more of human TPO, human IL-3, human GM-CSF, human M-CSF, and human SIRPa are described in more detail in U.S. Pat. Nos. 8,541,646 and 8,847,004; U.S. Patent Application Publication No. 2014 / 0134662; and PCT International Publication No. WO / 2014 / 039782, the disclosures of each of which are incorporated herein by reference.

[0145] Example 4 Development of humanized mouse models of Plasmodium falciparum and Plasmodium vivax erythrocytic stages It is demonstrated herein that genetic humanization of a mouse host by providing growth factors with limited mouse-to-human cross-reactivity can successfully enhance human cell engraftment in general, and erythropoiesis in particular, in mice engrafted with human hematopoietic stem cells (HSCs).

[0146] MITERG mice, which genomically express human EPO (to enhance definitive 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 engrafted with human HSCs than mice that do not express hEPO, equivalent in fact to mouse erythropoiesis in the same animals (Figure 5A, compare "hSIRPa-,hEPO+" (i.e., "MITERG mice") with "hSIRPa-,hEPO-" (i.e., "MITRG mice"). However, these mice lack significant levels of circulating human erythroid cells (data not shown).

[0147] We hypothesized that the low levels of peripheral circulating human red blood cells result from destruction of peripheral human RBCs (erythrophagocytosis) by mouse macrophages. The role of SIRPa in this process was examined by using mice in which hSIRPa is expressed from a locus in the mouse genome other than the mSIRPa locus, i.e., as a randomly integrated transgene, i.e., hSIRPa-tg(Rag2 - / - Il2rgnull Tpo h / h Mcsf h / h Il3 h / h Gmcsf h / h Epo h / h SIRPα-tg+, i.e., “MISTER-G mice”) and mice in which hSIRPa is expressed as a knock-in (KI) from the mSIRPa locus, i.e., as hSIRPa KI (Rag2 - / - Il2rg null Tpo h / h Mcsf h / h Il3 h / h Gmcsf h / h Epo h / h SIRPα h / h , i.e., "SupER-G mice."

[0148] For example, 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+"). Introduction of human SIRPa significantly improved the survival of most hematopoietic cells in the periphery and increased the number of human CD45+ cells, including lymphoid and myeloid cells in the peripheral blood. + The frequency of cells was increased at least 10-fold over that observed in mice not expressing hSIRPa. However, hSIRPa KI had little effect on the level of human RBC engraftment in peripheral blood (Figure 5, panel B).

[0149] Because human SIRPα knock-in was not sufficient to increase human red blood cells in peripheral blood, clodronate liposomes were used to deplete macrophages, specifically red pulp macrophages in the spleen and Kupffer cells in the liver. After depletion of tissue-resident macrophages with clodronate liposomes, a dramatic increase in circulating human cells of the erythroid lineage to 1% of the total circulating red blood cells was observed in SuPER-G mice (Figure 6, Panel A). Furthermore, the majority of human red blood cells in the periphery after clodronate treatment were reticulocytes (CD71 + ) (Figure 6, Panel B). This is an exciting observation because it indicates that these mice would be good candidates for supporting P. vivax infection, which 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 necessary for successful in vivo infection by different Plasmodium species has not been established. The only in vivo model for P. falciparum currently available is based on the transfer of human RBCs into immunodeficient strains such as NOD / scid or NSG. In these mice, infection with merozoites can be achieved by iv injection of infected RBCs only after daily injection of large numbers of human RBCs. At the time of infection, human RBCs constitute approximately half of the total RBC number in these animals.

[0151] As demonstrated above, SupER-G mice engrafted with human hematopoietic stem cells (HSCs) developed human erythroid cells in the bone marrow (Figure 5B), and (ii) clodronate treatment increased the frequency of peripheral human erythroid cells in engrafted SupER-G mice (Figure 6). To determine whether engrafted and clodronate-treated SupER-G mice contained sufficient numbers of human erythroid cells in the periphery to sustain successful Plasmodium infection in vivo, peripheral blood was collected from SupER-G mice engrafted with fetal liver or adult HSCs and the blood was cultured in vitro with blood infected with P. falciparum strain 3D7. Fresh human RBCs were added to the infected cultures after 48 h to facilitate multiple rounds of parasite replication, anticipating that amplification by subsequent reinfection with added human RBCs would occur only if the human RBCs harvested from HSC-engrafted SupER-G mice had undergone a complete P. falciparum infection cycle. 12 days after infection, advanced stages of parasite 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 from non-engrafted control mice or control mice acutely engrafted with 0.1% hRBCs by injection ("Addition Control") (Figure 7A and data not shown). An exponential increase in parasitemia was observed by Giemsa staining and quantitative PCR (Figure 7B). This indicates that clodronate-treated engrafted SupER-G produce sufficient numbers of human erythrocytes to sustain infection with P. falciparum, predicting that clodronate-treated engrafted mice will be successfully infected in vivo with P. falciparum and P. vivax merozoites.

[0152] Example 5 TIES mice (Rag2 - / - Il2rg null Tpo h / h Il3 / Gmcsf h / h Epoh / m SIRPα h / h ) Due to low reproductive success, developmental failure, and high mortality, Epo h / h Mice bearing hEPO are not ideal for infection studies. Instead, mice heterozygous for the hEPO gene (i.e., Epo h / m (e.g., TIES mice) have a full capacity to produce erythropoietin (EPO) and support all stages of erythropoiesis. Because high levels of human myeloid cell engraftment supported by human macrophage colony-stimulating factor (M-CSF) knock-in cause destruction of mouse red blood cells resulting in anemia and death of the engrafted mice, further improvement in survival time from 8-10 weeks to 4 months was achieved by retaining the mouse M-CSF gene in the mouse locus rather than replacing it with human M-CSF.

[0153] Similar to SupER-G mice, TIES mice support human erythropoiesis and maintain a frequency of 1% human red blood cells in the peripheral blood when administered clodronate. Furthermore, the majority of human red blood cells in the periphery after clodronate treatment were reticulocytes (CD71+), suggesting that these mice would be good candidates for supporting P. vivax infection.

[0154] It was demonstrated that engrafted TIES mice were able to maintain a frequency of 1% human red blood cells in the peripheral blood when administered clodronate. Furthermore, the ability of TIES mice to support transfusion of human RBCs was determined. As shown in Figure 8, clodronate treatment stabilized the transfused population to above 20% of the total population of cells in the periphery. These levels, achieved approximately 4 hours after transfusion, were sustained for at least 12 hours after transfusion. It is expected that these RBC frequencies will be sufficient to support in vivo infection with different species of malaria parasites.

[0155] The foregoing merely illustrates the principles of the present invention. It will be recognized that those skilled in the art will be able to devise various arrangements that embody the principles of the present invention and are within its spirit and scope, although not expressly described or shown herein. Furthermore, all language expressing examples and conditions described herein is intended to be interpreted primarily to aid the reader in understanding the principles of the present invention and the concepts proposed by the inventors to advance the art, and without being limited to such specifically described examples and conditions. Furthermore, all statements herein that recite principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

[0156] Sequence information SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. Yale University 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 gcttttactc 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: a human erythropoietin (hEPO) knock-in allele comprising a nucleic acid sequence encoding a human erythropoietin (hEPO) protein operably linked to an endogenous rodent EPO gene promoter at the rodent erythropoietin (EPO) locus in the genome of a rodent ES cell, wherein the EPO knock-in allele comprises at least some substitutions of the endogenous rodent EPO coding sequence and the encoded hEPO protein has wild-type human EPO signaling function; a human granulocyte-macrophage colony-stimulating factor 2 (hGM-CSF) knock-in allele comprising a nucleic acid sequence encoding a human granulocyte-macrophage colony-stimulating factor 2 (hGM-CSF) protein operably linked to an endogenous rodent GM-CSF gene promoter at a rodent GM-CSF locus in the genome of a rodent ES cell, wherein the GM-CSF knock-in allele comprises at least some 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 GM-CSF knock-in allele, wherein the encoded hGM-CSF protein has wild-type human GM-CSF signaling function; a rodent ES cell genome comprising a human interleukin-3 (hIL-3) knock-in allele, the human interleukin-3 (hIL-3) protein being operably linked to an endogenous rodent IL-3 gene promoter at the rodent interleukin-3 (IL-3) locus, the IL-3 knock-in allele comprising at least some substitutions of the endogenous rodent IL-3 coding sequence, the rodent being homozygous for the IL-3 knock-in allele (IL-3 h / h IL-3 knock-in allele), wherein the encoded hIL-3 protein has wild-type human IL-3 signaling function; and a human thrombopoietin (hTPO) knock-in allele comprising a nucleic acid sequence encoding a human thrombopoietin (hTPO) protein operably linked to an endogenous rodent TPO gene promoter at a rodent thrombopoietin (TPO) locus in the genome of a rodent ES cell, wherein the TPO knock-in allele comprises at least some substitution of the endogenous rodent TPO coding sequence, and the rodent is homozygous for the TPO knock-in allele (TPO h / h ), and wherein the encoded hTPO protein has wild-type human TPO signaling function.

2. Heterozygous for the EPO knock-in allele (EPO h / m ), the rodent ES cell of claim 1.

3. homozygous for the EPO knock-in allele (EPO h / h ), the rodent ES cell of claim 1.

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

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

6. a rodent macrophage colony-stimulating factor (M-CSF) knock-in allele comprising a nucleic acid sequence encoding a human macrophage colony-stimulating factor (hM-CSF) protein operably linked to an endogenous rodent M-CSF gene promoter at the rodent macrophage colony-stimulating factor (M-CSF) locus in the genome of a rodent ES cell, wherein the M-CSF knock-in allele comprises at least some substitution of the endogenous rodent M-CSF coding sequence, and the rodent ES cell is 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. The rodent ES cell of any one of claims 1 to 5, further comprising:

7. Homozygous for endogenous rodent M-CSF (M-CSF m / m ) A rodent ES cell according to any one of claims 1 to 5.

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

9. 8. The rodent ES cell of any one of claims 1 to 7, further comprising a SIRPα transgene encoding human Sirpα (hSirpα) protein.

10. 10. The rodent ES cell of claim 1, which comprises a deficiency of one or both of Rag2 and IL2rg.

11. Rag2 null and IL2rg null The rodent ES cell of claim 10,

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

13. A rodent embryo comprising the rodent ES cell of any one of claims 1 to 11.

14. A mouse embryo comprising the mouse ES cell of claim 12.

15. 14. A method of producing a genetically modified rodent, comprising producing the genetically modified rodent from the rodent embryo of claim 13.

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

17. A targeting construct for targeting the mouse EPO gene, comprising: （a） (i) a nucleotide sequence encoding a mouse EPO protein; or (ii) a nucleotide sequence that is complementary to a nucleotide sequence encoding a mouse EPO protein; upstream and downstream targeting arms that are complementary or substantially complementary to the upstream and downstream nucleotide sequences of either (b) a nucleotide sequence encoding a biologically active human EPO protein, or the complement of a nucleotide sequence encoding a biologically active human EPO protein; and (c) Marker and / or selection cassette The targeting construct comprising:

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