Genetically modified animal models and their use for modeling the human immune system

Abstract

(i) Rag1 and / or Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, and optionally a Fah gene knockout, and / or expressing one or more human or humanized polypeptides, genetically modified cells and genetically modified non-human animals (e.g., rats and mice) are provided herein. Methods and compositions for making and using such genetically modified cells and non-human animals are also provided.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 390,513, filed Jul. 19, 2022, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background For example, the use of genetically modified cells, genetically modified mice containing them, as well as modified and engrafted mice, and their use in modeling human diseases for the purpose of drug testing are known in the art. The use of genetically modified mice for modeling the human immune system (HIS) has been reported (Manz (2007) Immunity, 26:537-541). For example, HIS mice generated by transplanting human hematopoietic stem cells and progenitor cells into severely immunodeficient mouse strains (such as Rag2 KO Il2rg KO mice) have been reported. Multilineage hematopoietic development has been observed in these HIS mice, but they are known to lack human red blood cells (hRBC) in the peripheral blood. It has been found that human RBC precursors can develop in the bone marrow of such mice but cannot survive peripherally. This has hindered the use of these HIS models in modeling diseases or infections related to human red blood cells and testing the effects of therapeutic agents on human red blood cells.

[0003] Therefore, there is a need for genetically modified mice that can support the maintenance and propagation of human red blood cells, as well as for engraftable mice that can model or approximate certain aspects of human red blood cells.

Summary of the Invention

Means for Solving the Problems

[0004] Summary The present disclosure is, in part, based on the discovery that human erythrocytes in peripheral blood are restored by knocking out the Hmox-1 gene in immunodeficient mice engrafted with human hematopoietic cells (e.g., mice having Rag1 and / or Rag2 gene knockouts and an IL2rg gene knockout).

[0005] Accordingly, in one aspect, the present disclosure relates to a genetically modified non-human animal (e.g., a rodent) comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, the genetically modified non-human animal comprises a homozygous null mutation in the Rag1 gene. In some embodiments, the null mutation in the Hmox-1 gene is at least a deletion of the exon corresponding to mouse Hmox-1 exons 3-5. In some embodiments, the null mutation in the Hmox-1 gene is a deletion of the entire Hmox-1 endogenous coding sequence.

[0006] In some embodiments, the genetically modified non-human animal comprises a homozygous null mutation in the Fah gene. In some embodiments, the homozygous null mutation in the Fah gene comprises an insertion, deletion, and / or substitution in the endogenous Fah gene.

[0007] In some embodiments, the genetically modified non-human animal expresses a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter. In some embodiments, the genetically modified non-human animal comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, the Sirpa gene being operably linked to a Sirpa promoter. In some embodiments, the Sirpa gene comprises exons 2-4 of the human SIRPA gene. In some embodiments, the genetically modified non-human animal expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide. In some embodiments, the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide and / or the non-human animal Sirpa gene is an endogenous non-human animal gene. In some embodiments, the genetically modified non-human animal expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0008] In some embodiments, the genetically modified non-human animal further expresses one or more human or humanized proteins selected from the group consisting of a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0009] In some embodiments, at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter. In some embodiments, all promoters operably linked to a nucleic acid encoding a human or humanized protein are endogenous non-human animal promoters. In some embodiments, the endogenous non-human animal promoter is at the locus of the corresponding non-human animal gene.

[0010] In some embodiments, the genetically modified non-human animal described herein comprises a null mutation in at least one corresponding non-human animal gene at the locus of the corresponding non-human animal gene.

[0011] In some embodiments, the genetically modified non-human animal is heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein. In some embodiments, the genetically modified non-human animal is homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0012] In some embodiments, the at least one nucleic acid comprises genomic coding and non-coding sequences for a human or humanized protein. In some embodiments, the at least one nucleic acid comprises a cDNA sequence for a human or humanized protein.

[0013] In some embodiments, the genetically modified non-human animal expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

[0014] In some embodiments, the genetically modified non-human animal expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter. In some embodiments, the genetically modified non-human animal expresses a humanized CD47 protein, and the humanized CD47 protein comprises the extracellular portion of the human CD47 protein and the intracellular portion of the endogenous non-human animal CD47 protein.

[0015] In some embodiments, the genetically modified non-human animal expresses: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0016] In some embodiments, the genetically modified non-human animal expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0017] In certain aspects, the genetically modified non-human animals described herein further comprise engraftment of human hematopoietic cells. In some 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 hematopoietic progenitor cells, human erythroid progenitor cells, and human erythrocytes. In some embodiments, the animal comprises human cells of the erythroid lineage.

[0018] In some embodiments, the non-human animal further comprises infection by a pathogen that targets human cells of the erythroid lineage. In some embodiments, the animal comprises an inactivated endogenous Fah gene and further comprises transplanted human hepatocytes. In some embodiments, the pathogen can cause malaria in humans. Such pathogens can be selected from Plasmodium sp., Babesia sp., and Theileri sp.

[0019] In some embodiments, the engrafted human hematopoietic cells give rise to abnormal human cells of the erythroid lineage. In some embodiments, the engrafted human hematopoietic cells comprise a mutation in the β-globin gene that results in sickle cell disease.

[0020] In some embodiments, the genetically modified non-human animal is a mammal. In some embodiments, the mammal is a rodent, such as a rat or a mouse. In some embodiments, the rodent is a mouse.

[0021] In certain aspects, a method for identifying an agent that inhibits infection by a pathogen targeting human cells of the erythroid lineage, comprising: a. administering the agent to a genetically modified non-human animal, wherein the genetically modified non-human animal has: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; iii. engraftment of human hematopoietic cells; and iv. infection by a pathogen targeting human cells of the erythroid lineage, and b. determining whether the agent reduces the amount of the pathogen and / or inhibits the activity of the pathogen in the non-human animal infected with the pathogen.

[0022] In certain aspects, a method for identifying an agent that prevents infection by a pathogen targeting human cells of the erythroid lineage, comprising: a. administering the agent to a genetically modified non-human animal, wherein the genetically modified non-human animal has: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. engraftment of human hematopoietic cells, b. injecting parasitized reticulocytes or erythrocytes into the genetically modified non-human animal, and c. determining whether the agent prevents infection of human reticulocytes and / or erythrocytes in the non-human animal. In some embodiments, the pathogen can cause malaria in humans. Such pathogens can be selected from Plasmodium sp., Babesia sp., and Theileri sp.

[0023] In certain embodiments, a method for identifying an agent for treating sickle cell disease, the method comprising: a. administering an agent to a genetically modified non-human animal, wherein the genetically modified non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. engraftment of human hematopoietic cells comprising a mutation in the β-globin gene that causes sickle cell disease; and b. determining whether the agent prevents or reduces sickling of red blood cells in the non-human animal.

[0024] In certain embodiments, provided herein is a method for assessing the therapeutic efficacy of a drug candidate that targets human red blood cells, the method comprising: a. administering the drug candidate to a genetically modified non-human animal, wherein the genetically modified non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. engraftment of human hematopoietic progenitor cells; and b. monitoring human red blood cells in the non-human animal to assess the therapeutic efficacy of the drug candidate.

[0025] In some embodiments, human red blood cells are monitored to determine whether the number of human red blood cells in the non-human animal is reduced by the drug candidate. In some embodiments, the drug candidate is a chemotherapeutic agent or an anti-malarial agent. In some embodiments, human red blood cells are monitored to assess whether the drug candidate induces aggregation of red blood cells. In some embodiments, the drug candidate is a modulator (e.g., an antibody) of the human CD47 protein.

[0026] In certain embodiments, provided herein is a method of identifying an agent that reduces the toxicity of a toxic agent to human red blood cells, the method comprising: a. administering the agent and the toxic agent to a genetically modified non-human animal, wherein the genetically modified non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. engraftment of human hematopoietic progenitor cells; and b. determining whether the agent reduces the toxicity of the toxic agent to human red blood cells in the non-human animal. In some embodiments, the agent and the toxic agent are administered to the non-human animal together or sequentially. In some embodiments, the toxicity is on-target toxicity or off-target toxicity.

[0027] In certain embodiments, provided herein are genetically modified non-human animal cells comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

[0028] In some embodiments, the genetically modified non-human animal cells comprise a homozygous null mutation in the Rag1 gene. In some embodiments, the null mutation is at least a deletion of the exon corresponding to mouse Hmox-1 exons 3-5. In some embodiments, the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0029] In some embodiments, the genetically modified non-human animal cells comprise a homozygous null mutation in the Fah gene. In some embodiments, the homozygous null mutation in the Fah gene comprises an insertion, deletion, and / or substitution in the endogenous Fah gene.

[0030] In some embodiments, the genetically modified non-human animal cell expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter. In some embodiments, the genetically modified non-human animal cell comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, the Sirpa gene being operably linked to a Sirpa promoter. In some embodiments, the Sirpa gene comprises exons 2-4 of the human SIRPA gene. In some embodiments, the genetically modified non-human animal cell expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide. In some embodiments, the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide and / or the non-human animal Sirpa gene is an endogenous non-human animal gene. In some embodiments, the genetically modified non-human animal cell expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0031] In some embodiments, the genetically modified non-human animal cell further expresses one or more human or humanized proteins selected from the group consisting of a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0032] In some embodiments, at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter. In some embodiments, all promoters operably linked to a nucleic acid encoding a human or humanized protein are endogenous non-human animal promoters. In some embodiments, the endogenous non-human animal promoter is at the locus of the corresponding non-human animal gene.

[0033] In some embodiments, the genetically modified non-human animal cell comprises a null mutation in at least one corresponding non-human animal gene at the locus of the corresponding non-human animal gene. In some embodiments, the genetically modified non-human animal cell is heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein. In some embodiments, the genetically modified non-human animal cell is homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0034] In some embodiments, the genetically modified non-human animal cell expresses human M-CSF protein encoded by a nucleic acid operably linked to the M-CSF promoter.

[0035] In some embodiments, the genetically modified non-human animal cell expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to the CD47 promoter.

[0036] In some embodiments, the genetically modified non-human animal cell expresses a humanized CD47 protein, and the humanized CD47 protein comprises the extracellular portion of the human CD47 protein and the intracellular portion of the endogenous non-human animal CD47 protein.

[0037] In some embodiments, the genetically modified non-human animal cells express: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0038] In some embodiments, the genetically modified non-human animal cells express a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0039] In some embodiments, the genetically modified non-human animal cells are mammalian cells. In some embodiments, the mammalian cells are rodent cells, such as rat cells or mouse cells. In some embodiments, the rodent cells are mouse cells.

[0040] In some embodiments, the genetically modified non-human animal cells are non-human animal embryonic stem (ES) cells.

[0041] In certain aspects, provided herein is a method of generating non-human animal embryonic stem cells, the method comprising genetically engineering non-human animal embryonic stem cells such that the non-human animal embryonic stem cells have a genome comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the Il2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

[0042] In certain aspects, provided herein is a non-human animal embryo comprising the non-human animal embryonic stem cells described herein, or non-human animal embryonic stem cells generated according to the methods described herein.

[0043] In certain embodiments, a method of generating a non-human animal having in its genome (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, the method comprising: (a) obtaining a non-human animal embryonic stem cell described herein, or a non-human animal embryonic stem cell generated according to the methods described herein; and (b) using the non-human animal embryonic cell of (a) to generate a non-human animal, is provided herein.

[0044] In certain embodiments, a method of generating a non-human animal having in its genome (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, the method comprising modifying the genome of the non-human animal such that it comprises (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, is provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0045]

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Mode for Carrying Out the Invention

[0055] Detailed Description General The present disclosure relates to genetically modified non-human animals (e.g., mice or rats) that comprise (i) a Rag1 and / or Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, a genetically modified non-human animal (e.g., a mouse) is provided that comprises (i) a Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, a genetically modified non-human animal (e.g., a rat) is provided that comprises (i) a Rag1 gene knockout; (ii) a Rag2 gene knockout; (iii) an IL2rg gene knockout; and (iv) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, the genetically modified non-human animal expresses a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter. In some embodiments, the genetically modified non-human animal further comprises inactivation (e.g., deletion) of the fumarylacetoacetase (Fah) gene. In some embodiments, the genetically modified non-human animal further expresses one or more human or humanized proteins selected from the group consisting of a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.In certain embodiments, at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter. In other embodiments, the genetically modified animal expresses a human or humanized nucleic acid from native human promoters and native regulatory elements. One of ordinary skill in the art will understand that the genetically modified animals include genetically modified animals that express at least one human or humanized nucleic acid from any promoter. Examples of promoters useful in the present invention include, but are not limited to, DNA pol II promoter, PGK promoter, ubiquitin promoter, albumin promoter, globin promoter, ovalbumin promoter, SV40 early promoter, Rous sarcoma virus (RSV) promoter, retroviral LTR, and lentiviral LTR. Promoter and enhancer expression systems useful in the present disclosure also include inducible and / or tissue-specific expression systems.

[0056] Rodents having components of the human immune system, such as mice and rats (e.g., HIS mice and rats), are quite promising for studying the human immune system in vivo, testing human vaccines, and testing and developing drugs for treating human diseases and disorders. HIS mice are generated by transplanting human hematopoietic stem cells and progenitor cells (such as CD34+ HSC) into severely immunodeficient mouse strains (such as recombinant activation gene 2 (Rag2) knockout (KO) and interleukin 2 receptor gamma (IL2rg) KO mice). Compared to non-human primates, HIS mice have the advantage of being a small animal model, i.e., they allow for more diverse experiments, are more accessible to the research community, and are more ethically acceptable than conducting experiments using human subjects. Most importantly, experimental findings from HIS mice may be more relevant and applicable to humans. Similar rat models providing such advantages are also described and contemplated herein.

[0057] The HIS mouse develops human immune cells, such as B and T cells, but human red blood cells (hRBCs) do not develop in the peripheral blood of the HIS mouse model. Human RBC precursors can develop in the bone marrow of such mice, but cannot survive peripherally. Therefore, current HIS mice are not fully suitable for modeling diseases or infections related to human red blood cells (hRBCs) or for testing the effects of therapeutic agents on human red blood cells.

[0058] To overcome these limitations, the present disclosure provides an Hmox-1 deficient HIS mouse model. The injected human RBCs are rapidly cleared from the mouse, and their destruction is mainly thought to be due to phagocytosis by mouse macrophages. This idea is supported by the fact that elimination of macrophages by in vivo clodronate liposome treatment extends the survival of human RBCs. However, this approach requires repeated injections, which is not feasible due to mouse toxicity. Hmox-1 deficiency in mice results in the loss of erythrophagocytic macrophages, i.e., macrophages that eat red blood cells. This is because the Hmox-1 enzyme is required for macrophages to break down ingested RBCs and then recycle heme. Loss of this enzyme increases toxic heme in erythrophagocytic macrophages and leads to their death. Therefore, Hmox-1 KO in the HIS model enables the survival of human RBCs upon HSC engraftment. Hmox-1 deficient hSIRPA Rag2 - / - IL-2Rγ - / - It has been demonstrated herein that the mice show human RBCs in the peripheral blood, in contrast to Hmox-1 competent control mice to which the HSC donor is matched. This new model provides a useful tool for studying human RBC biology and a useful model for developing treatments for RBC diseases.

[0059] The genetically modified non-human animals provided herein are useful in, 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 states; in vivo screening for agents that are toxic to erythrocytes or erythrocyte precursors; in vivo screening for agents that prevent, mitigate or reverse the toxic effects of toxic agents on erythrocytes or erythrocyte precursors; and in vivo screening of erythrocytes or erythrocyte precursors from an individual to predict the responsiveness of the individual to disease treatment, among many uses in the art. These and other objects, advantages and features of the present invention will become apparent to those skilled in the art upon reading the following detailed description of the compositions and methods. Definitions

[0060] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0061] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that contain both an amino functional group and an acid functional group and can be included in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids, analogs, derivatives, and homologs thereof; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing.

[0062] The "coding region" of a gene includes the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene that are homologous or complementary, respectively, to the coding region of the mRNA molecule produced by transcription of the gene. The "coding region" of an mRNA molecule also includes the nucleotide residues of the mRNA molecule that match the anticodon region of a transfer RNA molecule or encode a stop codon during translation of the mRNA molecule. The coding region may thus include nucleotide residues that contain codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0063] As used herein, the terms "endogenous gene" or "endogenous gene segment" refer to a gene or gene segment found in a parental or reference organism prior to the introduction of a disruption, deletion, replacement, alteration or modification described herein. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is an engineered organism. In some embodiments, the reference organism is an organism mated in the laboratory (regardless of whether it is wild-type or engineered).

[0064] As used herein, the term "chimeric" refers to a nucleic acid or protein that contains portions derived from different species, i.e., having different nucleotide or amino acid sequences. In some embodiments, a "chimeric" nucleic acid or protein described herein contains nucleotide or amino acid sequences derived from both non-human and human origins. In such embodiments, the "chimeric" nucleic acid or protein may also be referred to as a "humanized" nucleic acid or protein.

[0065] As used herein, the term "corresponding to" refers to an exon encoding the same or a homologous functional domain or portion of a protein. For example, "an exon corresponding to mouse Hmox-1 exons 3-5" refers to an exon from a genetically modified non-human animal that encodes the same or a homologous functional domain or portion of the protein encoded by mouse exons 3-5. These can be exons 3-5 of the genetically modified non-human animal, or other exons resulting from differences in exon composition among different non-human animal species.

[0066] The term "humanized" is used herein in accordance with its meaning as understood in the art to refer to a nucleic acid or protein of non-human origin that has been engineered such that its structure (i.e., nucleotide or amino acid sequence) has a structure and function similar to that of a true human nucleic acid or protein. For example, humanizing can involve selecting amino acid substitutions to create a non-human sequence that is more similar to a human sequence. Humanizing can also involve grafting at least a portion of a non-human protein onto a human protein. By way of example, in the case of a membrane receptor, a "humanized" gene can encode a polypeptide having an extracellular portion with an amino acid sequence such as that of the human extracellular portion and the remaining sequence such as that of a non-human (e.g., mouse) polypeptide. In some embodiments, the humanized gene comprises at least a portion of the DNA sequence of a human gene. In some embodiments, a humanized protein comprises a sequence having a portion found in a human protein. The term "human" is recognized in the art and refers to a nucleic acid or protein whose structure (i.e., nucleotide or amino acid sequence) is derived entirely from human origin.

[0067] As used herein, the term "locus" refers to a position on a chromosome that contains a series of related genetic elements (e.g., genes, gene segments, regulatory elements). A locus can be endogenous or non-endogenous. The term "endogenous locus" refers to the position on a chromosome where a particular genetic element is found in nature. In some embodiments, the endogenous locus has a sequence found in nature. In some embodiments, the endogenous locus is a wild-type locus. In some embodiments, the endogenous locus is an engineered locus.

[0068] As used herein, the phrase "non-human animal" refers to any vertebrate organism that is not human. In some embodiments, the non-human animal is a cyclostome, bony fish, cartilaginous fish (e.g., sharks or rays), amphibian, reptile, mammal, and bird. In some embodiments, the non-human mammal is a primate, goat, sheep, pig, dog, cow, or rodent. In some embodiments, the non-human animal is a rodent, e.g., a rat or a mouse.

[0069] As used herein, the phrase "operably linked" refers to a juxtaposition wherein the components described are in a relationship that enables them to function in their intended manner. A coding sequence that is "operably linked" to a control sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. "Operably linked" arrays include both expression control sequences adjacent to the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" as used herein refers to a polynucleotide sequence necessary to affect the expression and processing of the coding sequence to which it is ligated. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending on the host organism. For example, in prokaryotes, such control sequences generally include a promoter, ribosome binding site, and transcription termination sequence, while in eukaryotes, such control sequences typically include a promoter and transcription termination sequence. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0070] The terms "polynucleotide" and "nucleic acid" are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. When present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. A polynucleotide can be further modified, for example, by conjugation with a labeled component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0071] The term "polypeptide" as used herein refers to any polymer chain of amino acids. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, the polypeptide has an amino acid sequence that has been engineered in that it is designed and / or produced by the activity of a human hand.

[0072] As used herein, the term "promoter" includes a DNA sequence operably linked to a transcribed nucleic acid sequence, such as a nucleic acid sequence encoding a desired molecule. A promoter is generally located upstream of the transcribed nucleic acid sequence and provides a site for specific binding by RNA polymerase and other transcription factors. In a specific embodiment, a promoter is generally located upstream of the transcribed nucleic acid sequence to produce a desired molecule and provides a site for specific binding by RNA polymerase and other transcription factors. The phrase "endogenous promoter" refers to a promoter that is native and associated with an endogenous gene, for example, in a wild-type organism.

[0073] As used herein, the term "recombinant" refers to a polypeptide that has been designed, engineered, prepared, expressed, made, or isolated by recombinant means (e.g., a signal regulatory protein as described herein), e.g., a polypeptide expressed using a recombinant expression vector transfected into a host cell, a polypeptide isolated from a recombinant combinatorial human polypeptide library (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), an antibody isolated from an animal (e.g., a mouse) transgenic for a human immunoglobulin gene (see, e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L. L. (2002) Current Opinion in Biotechnology 13:593-597, Little M. et al (2000) Immunology Today 21:364-370), or a polypeptide prepared, expressed, made, or isolated by any other means that involves splicing of selected sequence elements to each other. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements are obtained, for example, by mutagenesis (e.g., in vivo or in vitro) of known sequence elements of natural or synthetic origin.For example, in some embodiments, the recombinant polypeptide is composed of sequences found in the genome of the source organism of interest (e.g., human, mouse, etc.). In some embodiments, the recombinant polypeptide has an amino acid sequence obtained from mutagenesis (e.g., in vitro or in vivo, e.g., in a non-human animal), such that the amino acid sequence of the recombinant polypeptide is a sequence that is related to, but may not be naturally present in the genome of the non-human animal in vivo, where the polypeptide sequence originated.

[0074] The term "replacement" is used herein to refer to a process through which a "replaced" nucleic acid sequence (e.g., a gene) found at a host locus (e.g., in the genome) is removed from that locus and a different "replacement" nucleic acid is placed in its place. In some embodiments, the replaced nucleic acid sequence and the replacement nucleic acid sequence are equivalent to each other in that, for example, they are homologous to each other and / or contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.). In some embodiments, the replaced nucleic acid sequence includes one or more of a promoter, an enhancer, a splice donor site, a splice acceptor site, an intron, an exon, an untranslated region (UTR); in some embodiments, the replacement nucleic acid sequence includes one or more coding sequences. In some embodiments, the replacement nucleic acid sequence is a homolog of the replaced nucleic acid sequence. In some embodiments, the replacement nucleic acid sequence is an ortholog of the replaced sequence. In some embodiments, the replacement nucleic acid sequence is or includes a human nucleic acid sequence. In some embodiments, including cases where the replacement nucleic acid sequence is or includes a human nucleic acid sequence, the replaced nucleic acid sequence is or includes a rodent sequence (e.g., a mouse sequence). The nucleic acid sequences so arranged may include one or more regulatory sequences (e.g., a promoter, an enhancer, a 5'- or 3'-untranslated region, etc.) that are part of the source nucleic acid sequence used to obtain the sequences so arranged. For example, in various embodiments, the replacement is a replacement of an endogenous sequence with a heterologous sequence that results in the production of a gene product from the nucleic acid sequence so arranged (including heterologous sequences) but does not result in the expression of the endogenous sequence; the replacement is a replacement of an endogenous genomic sequence with a nucleic acid sequence that encodes a protein having a function similar to the protein encoded by the endogenous sequence. In various embodiments, an endogenous gene or a fragment thereof is replaced with a corresponding human gene or a fragment thereof.The corresponding human gene or fragment thereof is a human gene or fragment that is an ortholog of the endogenous gene or fragment thereof to be replaced, or that is substantially similar or identical in structure and / or function thereto.

[0075] As used herein, the term "variant" includes a nucleic acid sequence or peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence, respectively, but retains the essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may or may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Changes in the sequence of a peptide variant are typically limited or conservative, such that the sequences of the reference peptide and the variant are overall closely similar and identical in many regions. A variant and a reference peptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. Nucleic acid or peptide variants can be naturally occurring, such as allelic variants, or variants not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides can be made by mutagenesis techniques or by direct synthesis.

[0076] As used herein, the term "vector" refers to a nucleic acid molecule that can carry another nucleic acid to which it is related. In some embodiments, a vector is capable of extrachromosomal replication and / or expression of the linked nucleic acid in a host cell, such as a eukaryotic cell and / or a prokaryotic cell. A vector that can direct the expression of an operably linked gene is referred to herein as an "expression vector".

[0077] As used herein, the term "wild-type" has the meaning understood in the art to refer to an entity having a structure and / or activity found in a natural, "normal" (as contrasted with mutant, diseased, altered, etc.) state or situation. One of ordinary skill in the art will recognize that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles). Genetically modified locus

[0078] In certain embodiments, there are provided genetically modified non-human animals (e.g., mice or rats) that include (i) a Rag1 and / or Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, there are provided genetically modified animals (e.g., mice) that include (i) a Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the mouse Hmox-1 gene. In some embodiments, there are provided genetically modified animals (e.g., rats) that include (i) a Rag1 gene knockout; (ii) a Rag2 gene knockout; (iii) an IL2rg gene knockout; and (iv) a homozygous null mutation in the rat Hmox-1 gene. In some embodiments, the genetically modified non-human animal expresses a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter. In certain embodiments, the genetically modified non-human animal further expresses one or more human or humanized proteins selected from the group consisting of a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter. In some embodiments, the genetically modified non-human animal further includes inactivation (e.g., deletion) of the fumarylacetoacetase (FAH) gene.In certain embodiments, at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter. In some embodiments, the genetically modified non-human animal comprises engraftment of human hematopoietic stem cells (HSCs). In some embodiments, the genetically modified non-human animal comprises engraftment of human liver cells. Hmox-1 knockout

[0079] In certain aspects, the genetically modified non-human animals (e.g., mice or rats) provided herein comprise a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

[0080] Heme oxygenase metabolizes heme and releases free iron, carbon monoxide, and biliverdin, which is rapidly converted to bilirubin. Humans and mice contain two well-characterized heme oxygenase enzymes: inducible HMOX-1 and constitutively expressed HMOX-2 in most tissues (Kovtunovych et al. (2010) Blood 116:6054-6062). Previous studies in mouse models have shown that the absence of both Hmox-1 and Hmox-2 is embryonically lethal. HMOX-1-deficient human patients died at 6 years of age, presenting with growth retardation, severe anemia, endothelial cell damage, fibrosis of the spleen, liver, and kidneys, and iron overload in the liver and kidneys (Kawashima et al. (2002) Hum. Path. 33:125-130; Yachie et al. (1999) J. Clin. Invest. 103:129-135). Hmox-1 - / - mice are anemic, but RBCs under oxidative stress have a longer lifespan. Splenomegaly from increased red pulp has been observed in Hmox-1 - / - mice, and older mice have splenic fibrosis. Hmox-1 - / - Increased iron in the kidneys of Hmox-1 mice caused kidney damage in these mice. Macrophage deficiency in the spleen and liver has also been observed in Hmox-1 - / -Observed in mice (Poss et al. (1997) PNAS 94:10919-10924; Kovtunovych et al. (2010) Blood 116:6054-6062; Kovtunovych et al. (2014) Blood 124:1522-1530). For example, Hmox-1 - / - mice had reduced macrophages (F4 / 80 + ) in the spleen, liver, blood, and bone marrow (BM), but still had myeloid cells (CD11b + ) (Fraser et al. (2015) Haematologica 100:601-610). Absence of Hmox-1 prevented erythrophagocytic macrophages from processing heme, leading to intracellular toxicity due to increased heme (Kovtunovych et al. (2010) Blood 116:6054-6062). Wild-type bone marrow transplantation reversed the disease in Hmox-1 - / - mice as a result of restored heme utilization by repopulation of tissues with wild-type macrophages (Kovtunovych et al. (2014) Blood 124:1522-1530).

[0081] Representative human HMOX-1 cDNA and human HMOX-1 protein sequences are well-known in the art and publicly available from the National Center for Biotechnology Information (NCBI). For example, human (NP_002124.1) can be encoded by the transcript (NM_002133.3). Nucleic acid and polypeptide sequences of Hmox-1 orthologs in organisms other than human are well-known, for example, chimpanzee Hmox-1 (XM_525579.6 and XP_525579.2), rhesus macaque Hmox-1 (XM_028827760.1 and XP_028683593.1), bovine Hmox-1 (NM_001014912.1 and NP_001014912.1), canine Hmox-1 (NM_001194969.1 and NP_001181898.1), rat Hmox-1 (NM_012580.2 and NP_036712.1), mouse Hmox-1 (NM_010442.2 and NP_034572.1), Chinese hamster Hmox-1 (XM_003511957.5 and XP_003512005.1), chicken Hmox-1 (XM_046921508.1 and XP_046777464.1), Xenopus tropicalis Hmox-1 (XM_002934720.5 and XP_002934766.2), and zebrafish Hmox-1 (NM_001127516.1 and NP_001120988.1).

[0082] In certain embodiments, the genetically modified non-human animal comprises a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene. A null mutation includes a deletion, insertion, and / or substitution of a gene that does not result in a functional gene product (e.g., complete absence of a gene product (protein, RNA) at the molecular level, or expression of a non-functional gene product). In the present disclosure, a null mutation has the same meaning and is used interchangeably with an inactivating mutation. A homozygous null mutation refers to having a null mutation in all alleles. For example, a homozygous null mutation in the mouse or rat Hmox-1 gene refers to having a null mutation in two alleles (i.e., two null alleles) for the mouse or rat Hmox-1 gene. A gene having a homozygous null mutation is also referred to as a gene knockout or gene deficient / deficiency. For example, a homozygous null mutation in Hmox-1 is also referred to as Hmox-1 knockout or Hmox-1 deficiency. Thus, a null mutation in the Hmox-1 gene of a non-human animal includes a deletion, insertion, and / or substitution in the Hmox-1 gene of the non-human animal. In some examples, the endogenous non-human animal Hmox-1 locus comprises a null mutation and thus a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of that gene. This can be the result of a complete absence of a gene product (protein, RNA) at the molecular level, or expression of a non-functional gene product. At the phenotypic level, a null allele includes a deletion of the entire locus.

[0083] In some embodiments, the null mutation is at least a deletion of the exon corresponding to mouse Hmox-1 exons 3-5. In some embodiments, the null mutation is a deletion of the entire endogenous Hmox-1 coding sequence. In some embodiments, the non-human animals provided herein do not express the Hmox-1 protein.

[0084] In some embodiments, the homozygous null mutation in the Hmox-1 gene of the non-human animal comprises the same null mutation for all alleles. In some embodiments, the homozygous null mutation in the Hmox-1 gene of the non-human animal comprises different null mutations for different alleles.

[0085] Mouse Hmox-1 is located on chromosome 8, GRCm39, NC_000074.7 (75820246-75827221), and the mouse Hmox-1 coding sequence can be found under Genbank accession number NM_010442.2. The mouse Hmox-1 locus contains five exons, and exons 1-5 are coding exons. Thus, in some embodiments, the genetically modified animals provided herein are mice, and one or more of exons 1-5 of the mouse Hmox-1 gene are deleted or mutated in the genetically modified mouse. In some examples, other aspects of the genomic locus of the mouse Hmox-1 gene, such as introns, 3' and / or 5' untranslated regions (UTRs), are also deleted or mutated. In some examples, the entire region of the mouse Hmox-1 genomic locus is deleted. In some embodiments, the entire genomic region from the start codon to the stop codon of the mouse Hmox-1 gene is deleted. For example, the genetically modified mouse can contain a deletion of approximately 7 kb of the mouse sequence (GRCm38 coordinates chr8:75093750-75100019) as shown in Example 1.

[0086] Deletions, modifications, or altered Hmox-1 genes at the endogenous Hmox-1 locus can be detected using a variety of methods, including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or allele gain or loss assays. In some embodiments, the non-human animal is homozygous for a deletion or null mutation of the endogenous Hmox-1 gene.

[0087] In some embodiments, a non-human animal (e.g., a mouse or a rat) comprising a homozygous null mutation in the Hmox-1 gene, i.e., a Hmox-1-deficient non-human animal, is an immunocompromised animal. For example, a Hmox-1-deficient non-human animal (e.g., a mouse or a rat) may comprise at least one null allele for the Rag2 gene (“recombination activating gene 2”, where the coding sequence for the mouse gene can be found at Genbank accession number NM_009020.3). In some embodiments, a Hmox-1-deficient non-human animal (e.g., a mouse or a rat) comprises two null alleles for Rag2. In other words, a Hmox-1-deficient non-human animal (e.g., a mouse or a rat) is homozygous null for Rag2. In other embodiments, a Hmox-1-deficient non-human animal (e.g., a mouse or a rat) comprises one or two null alleles for the Rag1 gene. In some embodiments, a Hmox-1-deficient non-human animal (e.g., a mouse or a rat) is homozygous null for Rag1. In some embodiments, a Hmox-1-deficient non-human animal (e.g., a mouse or a rat) comprises (i) one or two null alleles for the Rag1 gene; and (ii) one or two null alleles for the Rag2 gene. In some embodiments, a Hmox-1-deficient non-human animal (e.g., a mouse or a rat) is homozygous null for both Rag1 and Rag2. In some embodiments, a Hmox-1-deficient non-human animal is an immunocompromised mouse comprising two null alleles for Rag2 (i.e., homozygous null). In some embodiments, a Hmox-1-deficient non-human animal is an immunocompromised rat comprising two null alleles for Rag1 (i.e., homozygous null) and two null alleles for Rag2 (i.e., homozygous null).As another example, an Hmox-1 deficient non-human animal (e.g., a mouse or a rat) comprises at least one null allele for the IL2rg gene (also known as "interleukin 2 receptor gamma", common gamma chain, or γC, where the coding sequence for the mouse gene can be found at Genbank accession number NM_013563.4). In some embodiments, an Hmox-1 deficient non-human animal (e.g., a mouse or a rat) comprises two null alleles for IL2rg. In other words, an Hmox-1 deficient non-human animal (e.g., a mouse or a rat) is homozygous null for IL2rg, i.e., it is IL2rg. - / - (or IL2rg Y / - , where the IL2rg gene is located on the X chromosome as in the mouse). In some embodiments, an Hmox-1 deficient non-human animal (e.g., a mouse or a rat) comprises null alleles for both Rag2 and IL2rg, i.e., it is Rag2 - / - IL2rg - / - (or Rag2 - / - IL2rg Y / -, here, the IL2rg gene is located on the X chromosome, as in mice or rats. In some embodiments, the Hmox-1 deficient non-human animal (e.g., mouse or rat) contains null alleles for both Rag1 and IL2rg. In some embodiments, the Hmox-1 deficient non-human animal (e.g., mouse or rat) contains null alleles for Rag1, Rag2, and IL2rg. In some embodiments, the Hmox-1 deficient non-human animal is a mouse in an immunocompromised state that contains two null alleles for Rag2 (i.e., homozygous null) and two null alleles for IL2rg (i.e., homozygous null) (or one null allele for male mice). In some embodiments, the Hmox-1 deficient non-human animal is a rat in an immunocompromised state that contains two null alleles for Rag1 (i.e., homozygous null), two null alleles for Rag2 (i.e., homozygous null), and two null alleles for IL2rg (i.e., homozygous null) (or one null allele for male rats). Other genetic modifications are contemplated. For example, the Hmox-1 deficient non-human animal (e.g., mouse or rat) may include modifications to other genes associated with the development and / or function of hematopoietic cells and the immune system, e.g., replacement of one or more other non-human animal genes with nucleic acid sequences encoding human or humanized polypeptides. Such genes include, but are not limited to, for example, SIRPA, CD47, M-CSF, GM-CSF, TPO, EPO, IL-3, and IL-15. Additionally or alternatively, the Hmox-1 deficient non-human animal (e.g., mouse or rat) may include modifications to genes associated with the development and / or function of other cells and tissues, e.g., genes associated with human disorders or diseases, or genes that provide models of human disorders and diseases when modified in non-human animals, e.g., mice. In one embodiment described below herein, the Hmox-1 deficient non-human animal (e.g., mouse or rat) may contain a mutation, insertion, or deletion in the Fah gene.The introduction of other genetic modifications can be achieved by either ES cell modification and / or breeding. For example, Hmox-1 deficient (and optionally, Rag2 and IL2rg deficient) non-human animals (e.g., mice or rats) can be bred with non-human animals comprising one or more other genetic modifications, including, but not limited to, modifications in the SIRPA, CD47, M-CSF, GM-CSF, TPO, EPO, IL-3, IL-15, and / or Fah genes. In some embodiments, all gene variants are bred to be homozygous in the gene-modified animals described herein. Immunodeficient non-human animals

[0088] As described above, the gene-modified non-human animals comprising Hmox-1 deficiency described herein are also immunodeficient since they contain deficiencies in the Rag1 and / or Rag2, and Il2rg genes. Rag1, Rag2, and Il2rg are essential components of the adaptive immune system. When one or more of these genes are mutated in an animal, T cells and B cells do not mature and the animal is severely impaired. When these animals are loaded with xenografted cells, they are unable to mount an immune response against the foreign cells.

[0089] V(D)J recombination activating protein 1 (RAG1, recombination activating 1, recombination activating gene 1, and also known as recombination activating protein 1) is encoded by the Rag1 gene (also known as recombination activating 1). RAG1 is a catalytic component of the RAG complex, a multi-protein complex that mediates the DNA cleavage phase during V(D)J recombination. V(D)J recombination assembles diverse repertoires of immunoglobulin and T cell receptor genes during the development of B and T lymphocytes by the rearrangement of different V (variable), in some cases D (diversity), and J (joining) gene segments. In the RAG complex, RAG1 mediates DNA binding to conserved recombination signal sequences (RSSs) and catalyzes DNA cleavage activity by introducing double-strand breaks between the RSS and the adjacent coding segment. RAG2 is not a catalytic component but is required for all known catalytic activities. RAG1 and RAG2 are essential for the development of two types of lymphocytes, mature B cells and T cells, which are important components of the adaptive immune system.

[0090] Mouse Rag1 maps to chromosome 2 at 2 E2;2 53.88 cM (NCBI RefSeq gene ID 19373; assembly GRCm39 (GCF_000001635.27); position NC_000068.8 (101468597..101479877, complement). References to the mouse Rag1 gene include the canonical wild-type form, as well as all allelic forms and isoforms. The canonical wild-type mouse RAG1 protein is assigned UniProt accession number P15919 and NCBI accession number NP_033045.2. References to the mouse RAG1 protein include the wild-type form, as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the canonical isoform is assigned NCBI accession number NM_009019.2. References to mouse Rag1 mRNA (cDNA) and coding sequences include the canonical wild-type form, as well as all allelic forms and isoforms.

[0091] Rat Rag1 maps to 3q31 on chromosome 3 (NCBI RefSeq gene ID 84600; assembly mRatBN7.2 (GCF_015227675.2); position NC_051338.1 (87917061..87928158, complement). References to the rat Rag1 gene include the standard wild-type form, as well as all allelic forms and isoforms. The standard wild-type rat RAG1 protein has been assigned UniProt accession number G3V6K9 and NCBI accession number NP_445920.1. References to the rat RAG1 protein include the standard wild-type form, as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the standard isoform has been assigned NCBI accession number NM_053468.1. References to the rat Rag1 mRNA (cDNA) and coding sequence include the standard wild-type form, as well as all allelic forms and isoforms.

[0092] An inactivated endogenous Rag1 gene is a Rag1 gene that does not produce the RAG1 protein or does not produce a functional RAG1 protein. A non-human animal (or cell or genome) can contain the inactivated Rag1 gene in its germline. A non-human animal (or cell or genome) can be homozygous for an inactivating mutation in the Rag1 gene. As an example, an inactivated endogenous Rag1 gene can include an insertion, deletion, or one or more point mutations in the endogenous Rag1 gene that result in the loss of expression of the functional RAG1 protein. Some inactivated endogenous Rag1 genes can include all deletions or disruptions of the endogenous Rag1 gene or can include deletions or disruptions of a fragment (i.e., a part or portion) of the endogenous Rag1 gene. For example, some, most, or all of the coding sequence in the endogenous Rag1 gene can be deleted or disrupted. In one example, the 5’ fragment of the Rag1 gene can be deleted or disrupted (e.g., including the start codon). As an example, an inactivated endogenous Rag1 gene can be one in which the start codon of the endogenous Rag1 gene is deleted such that the start codon no longer functions or is disrupted or mutated. For example, the start codon can be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated, for example, by substitution of one or more nucleotides. In another example, the 3’ fragment of the Rag1 gene can be deleted or disrupted (e.g., including the stop codon). In another example, an internal fragment of the Rag1 gene (i.e., a fragment from the middle of the Rag1 gene) can be deleted or disrupted. In another example, all of the coding sequence in the endogenous Rag1 gene is deleted or disrupted.

[0093] V(D)J recombination activating protein 2 (RAG2, recombination activating 2, recombination activating gene 2, and also known as recombination activating protein 2) is encoded by the Rag2 gene (also known as recombination activating 2). As described above, RAG1 is the catalytic component of the RAG complex, a multi-protein complex that mediates the DNA cleavage step during V(D)J recombination. RAG2 is not a catalytic component but is required for all known catalytic activities. RAG1 and RAG2 are essential for the development of mature B cells and T cells, two types of lymphocytes that are important components of the adaptive immune system.

[0094] Mouse Rag2 maps to chromosome 2 at 2 E2;2 53.87 cM (NCBI RefSeq gene ID 19374; assembly GRCm39 (GCF_000001635.27); position NC_000068.8 (101455057..101462873)). References to the mouse Rag2 gene include the standard wild-type form, as well as all allelic forms and isoforms. The standard wild-type mouse RAG2 protein has been assigned UniProt accession number P21784 and NCBI accession number NP_033046.1. References to the mouse RAG2 protein include the standard wild-type form, as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the standard isoform is assigned NCBI accession number NM_009020.3. References to mouse Rag2 mRNA (cDNA) and coding sequences include the standard wild-type form, as well as all allelic forms and isoforms.

[0095] Rat Rag2 maps to 3q31 on chromosome 3 (NCBI RefSeq gene ID 295953; assembly mRatBN7.2 (GCF_015227675.2); position NC_051338.1 (87902373..87910227). References to the rat Rag2 gene include the standard wild-type form, as well as all allelic forms and isoforms. The standard wild-type rat RAG2 protein has been assigned UniProt accession number G3V6K7 and NCBI accession number NP_001093998.1. References to the rat RAG2 protein include the standard wild-type form, as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the standard isoform is assigned NCBI accession number NM_001100528.1. References to rat Rag2 mRNA (cDNA) and coding sequences include the standard wild-type form, as well as all allelic forms and isoforms.

[0096] An inactivated endogenous Rag2 gene is a Rag2 gene that does not produce the RAG2 protein or does not produce a functional RAG2 protein. A non-human animal (or cell or genome) can contain the inactivated Rag2 gene in its germline. A non-human animal (or cell or genome) can be homozygous for an inactivating mutation in the Rag2 gene. As an example, an inactivated endogenous Rag2 gene can include an insertion, deletion, or one or more point mutations in the endogenous Rag2 gene that result in the loss of expression of the functional RAG2 protein. Some inactivated endogenous Rag2 genes can include all deletions or disruptions of the endogenous Rag2 gene or can include deletions or disruptions of a fragment (i.e., a part or portion) of the endogenous Rag2 gene. For example, some, most, or all of the coding sequence in the endogenous Rag2 gene can be deleted or disrupted. In one example, the 5’ fragment of the Rag2 gene can be deleted or disrupted (e.g., including the start codon). As an example, an inactivated endogenous Rag2 gene can be one in which the start codon of the endogenous Rag2 gene has been deleted such that the start codon no longer functions or has been disrupted or mutated. For example, the start codon can be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated, for example, by substitution of one or more nucleotides. In another example, the 3’ fragment of the Rag2 gene can be deleted or disrupted (e.g., including the stop codon). In another example, an internal fragment of the Rag2 gene (i.e., a fragment from the middle of the Rag2 gene) can be deleted or disrupted. In another example, all of the coding sequence in the endogenous Rag2 gene is deleted or disrupted.

[0097] Interleukin-2 receptor subunit gamma (Interleukin-2 receptor, gamma; Interleukin-2 receptor, gamma (severe combined immunodeficiency); Isoform CRA_a; also known as cytokine receptor common subunit gamma precursor) is encoded by the Il2rg gene (also known as Interleukin-2 receptor subunit gamma or IL2RG). IL2RG is a cytokine receptor subunit common to the receptor complexes for several different interleukin receptors. IL2RG is located on the surface of immature hematopoietic cells in the bone marrow. IL2RG partners with other proteins to direct hematopoietic cells to form lymphocytes. IL2RG also directs the growth and maturation of T cells, B cells, and natural killer cells. Mutations in Il2rg can cause X-linked severe combined immunodeficiency, in which lymphocytes cannot develop normally. The absence of functional mature lymphocytes destroys the body's ability to protect itself from infection.

[0098] Mouse Il2rg maps to X D;X 43.9 cM on the X chromosome (NCBI RefSeq gene ID 16186; assembly GRCm39 (GCF_000001635.27); position NC_000086.8 (100307991..100311861, complement). References to the mouse Il2rg gene include the standard wild-type form, as well as all allelic forms and isoforms. The standard wild-type mouse IL2rG protein has been assigned UniProt accession number P34902 and NCBI accession number NP_038591.1. References to the mouse IL2RG protein include the standard wild-type form, as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the standard isoform is assigned NCBI accession number NM_013563.4. References to the mouse Il2rg mRNA (cDNA) and coding sequence include the standard wild-type form, as well as all allelic forms and isoforms.

[0099] Rat Il2rg maps to Xq22 on the X chromosome (NCBI RefSeq gene ID 140924; assembly mRatBN7.2 (GCF_015227675.2); position NC_051356.1 (66395330..66399026, complement). References to the rat Il2rg gene include the standard wild-type form, as well as all allelic forms and isoforms. The standard wild-type rat IL2rG protein has been assigned UniProt accession number Q68FU6 and NCBI accession number NP_543165.1. References to the rat IL2RG protein include the standard wild-type form, as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the standard isoform is assigned NCBI accession number NM_080889.1. References to the rat Il2rg mRNA (cDNA) and coding sequence include the standard wild-type form, as well as all allelic forms and isoforms.

[0100] An inactivated endogenous Il2rg gene is an Il2rg gene that does not produce the IL2RG protein or does not produce a functional IL2RG protein. A non-human animal (or cell or genome) can contain the inactivated Il2rg gene in its germline. A non-human animal (or cell or genome) can be homozygous for an inactivating mutation in the Il2rg gene. As an example, an inactivated endogenous Il2rg gene can include an insertion, deletion, or one or more point mutations in the endogenous Il2rg gene that result in the loss of expression of the functional IL2RG protein. Some inactivated endogenous Il2rg genes can include all deletions or disruptions of the endogenous Il2rg gene or can include deletions or disruptions of a fragment (i.e., a part or portion) of the endogenous Il2rg gene. For example, some, most, or all of the coding sequence in the endogenous Il2rg gene can be deleted or disrupted. In one example, the 5’ fragment of the Il2rg gene can be deleted or disrupted (e.g., including the start codon). As an example, an inactivated endogenous Il2rg gene can be one in which the start codon of the endogenous Il2rg gene is deleted such that the start codon no longer functions or is disrupted or mutated. For example, the start codon can be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated, for example, by substitution of one or more nucleotides. In another example, the 3’ fragment of the Il2rg gene can be deleted or disrupted (e.g., including the stop codon). In another example, an internal fragment of the Il2rg gene (i.e., a fragment from the middle of the Il2rg gene) can be deleted or disrupted. In another example, all of the coding sequence in the endogenous Il2rg gene is deleted or disrupted. Humanized Sirpa locus

[0101] In certain embodiments, the genetically modified non-human animals provided herein further express a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter.

[0102] Signal regulatory protein (SIRP) constitutes a family of cell surface glycoproteins expressed on lymphocytes, myeloid cells (including macrophages, neutrophils, granulocytes, myeloid dendritic cells, and mast cells), and neurons (see, for example, Barclay and Brown, 2006, Nat Rev Immunol 6, 457-464). The reported SIRP genes include at least SIRPA, SIRP3, SIRPβ, SIRPγ, and SIRP8, and can be classified by their respective ligands and the types of signal transduction in which they are involved. SIRPA (also known as CD172A, SHPS1, P84, MYD-1, BIT, and PTPNS1) is expressed on immune cells of the myeloid lineage and functions as an inhibitory receptor via an immunoreceptor tyrosine-based inhibitory motif (ITIM). SIRPA expression has also been observed on neurons. The most notable of the reported ligands for SIRPA is CD47, but protein A and D of surfactants are also included. The role of SIRPA has been investigated particularly with regard to its inhibitory role in phagocytosis of host cells by macrophages. For example, CD47 binding to SIRPA on macrophages induces an inhibitory signal that negatively regulates phagocytosis. Alternatively, a positive signal transduction effect mediated by SIRPA binding has been reported (Shultz et al., 1995, J Immunol 154, 180-91). SIRPA has been shown to improve cell engraftment in immunodeficient mice (Strowig et al. Proc Natl Acad Sci USA 2011; 108: 13218-13223).

[0103] The polypeptide sequence for wild-type human SIRPA and the nucleic acid sequence encoding wild-type human SIRPA can be found in Genbank accession numbers NP_001035111.1 and NM_001040022.1 (isoform 1 and transcript variant 1); NP_001035112.1 and NM_001040023.2 (isoform 1 and transcript variant 2); NP_001317657.1 and NM_001330728.1 (isoform 2 and transcript variant 4); and NP_542970.1 and NM_080792.3 (isoform 1 and transcript variant 3). The SIRPA gene is conserved in at least chimpanzee, rhesus monkey, dog, cow, mouse, rat, and chicken. The genomic locus encoding wild-type human SIRPA protein can be found in the human genome on chromosome 20; NC_000020.11 (1894167 - 1940592). In some embodiments, the human SIRPA protein is encoded by exons 2 - 9 at this locus. Thus, in some embodiments, a nucleic acid sequence comprising the coding sequence for human SIRPA comprises one or more of exons 2 - 9 of the human SIRPA gene. In some examples, the nucleic acid sequence also comprises aspects of the genomic locus of human SIRPA, such as introns, 3' and / or 5' untranslated regions (UTRs). In some examples, the nucleic acid sequence comprises the entire region of the human SIRPA genomic locus. In some examples, the nucleic acid sequence comprises exons 2 - 4 of the human SIRPA genomic locus.

[0104] Exemplary humanized Sirpa sequences are shown in Table 3. For the protein sequences, the signal peptide is underlined and the transmembrane and cytoplasmic sequences are in italics. Representative mouse Sirpa cDNA, mouse Sirpa protein, human SIRPA cDNA, and human SIRPA protein sequences are described in U.S. Patent No. 11,019,810, which is hereby incorporated by reference in its entirety.

[0105] [Table 3]

[0106] In some embodiments, the non-human animals provided herein express a humanized Sirpa protein on the surface of immune cells (e.g., myeloid cells) of a non-human animal obtained from a genetic modification of an endogenous locus of a non-human animal that encodes the Sirpa protein. Suitable examples described herein include rodents, such as mice.

[0107] The humanized Sirpa gene, in some embodiments, contains genetic material from a heterologous species (e.g., human), where the humanized Sirpa gene encodes a Sirpa protein that contains a portion encoded by the genetic material from the heterologous species. In some embodiments, the humanized Sirpa gene of the present disclosure contains genomic DNA of a heterologous species corresponding to the extracellular portion of the SIRPA protein expressed on the cell membrane of a cell. Also provided are non-human animals, non-human embryos, and cells containing the humanized Sirpa gene, as well as non-human animals, embryos, cells, and targeting constructs for making such non-human animals, embryos, and cells.

[0108] In some embodiments, the endogenous non-human animal (e.g., rodent) Sirpa gene is deleted. In some embodiments, the endogenous non-human animal (e.g., rodent) Sirpa gene is modified, where a portion of the endogenous non-human animal (e.g., rodent) Sirpa gene is replaced with a heterologous sequence (e.g., the whole or a part of the human SIRPA sequence). In some embodiments, all or substantially all of the endogenous non-human animal (e.g., rodent) Sirpa gene is replaced with a heterologous gene (e.g., the human SIRPA gene). In some embodiments, a portion of the heterologous SIRPα gene is inserted into the endogenous non-human Sirpa gene at the endogenous non-human animal (e.g., rodent) Sirpa locus. In some embodiments, the heterologous gene is a human gene. In some embodiments, the modification or humanization is performed on one of the two copies of the endogenous non-human animal (e.g., rodent) Sirpa gene, resulting in a non-human animal that is heterozygous with respect to the humanized Sirpa gene. In other embodiments, non-human animals that are homozygous with respect to the humanized Sirpa gene are provided. In some embodiments, all of the endogenous non-human animal (e.g., rodent) Sirpa gene is replaced with a portion of a heterologous gene (e.g., a portion of the human SIRPA gene) such that the genetically modified non-human animal (e.g., rodent) expresses a functional fragment of the full-length human SIRPA polypeptide (e.g., the extracellular domain of the human SIRPA polypeptide).

[0109] The non-human animals of the present disclosure contain the whole or a part of the human SIRPA gene at the endogenous non-human Sirpa locus. Thus, such non-human animals can be described as having a heterologous SIRP gene. The SIRPα gene replaced, inserted or modified at the endogenous non-human animal (e.g., rodent) Sirpa locus can be detected using various methods, including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or allele gain or loss assays. In some embodiments, the non-human animal is heterozygous with respect to the humanized Sirpa gene.

[0110] In various embodiments, the humanized Sirpa gene according to the present disclosure comprises a SIRPα gene having second, third, and fourth exons each having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to the second, third, and fourth exons found in the human SIRPA gene.

[0111] In various embodiments, the humanized Sirpa gene according to the present disclosure comprises a SIRPα gene having a nucleotide coding sequence (e.g., cDNA sequence) that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to nucleotides 352-1114 found in the human SIRPA cDNA sequence.

[0112] In various embodiments, the humanized Sirpa protein produced by the non-human animals of the present disclosure has an extracellular portion having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to the extracellular portion of the human SIRPA protein.

[0113] In various embodiments, the humanized Sirpa protein produced by the non-human animals of the present disclosure has an extracellular portion having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to amino acid residues 28-362 found in the human SIRPA protein.

[0114] In various embodiments, the humanized Sirpa protein produced by the non-human animals of the present disclosure has an amino acid sequence that is at least 50% identical (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) to the amino acid sequence of the humanized SIRPA protein shown in Table 3.

[0115] Compositions and methods for generating non-human animals that express a humanized Sirpa protein, including specific polymorphic forms or allelic variants (e.g., differences in a single amino acid), are provided, including compositions and methods for generating non-human animals that express such proteins from human promoters and human regulatory sequences. In some embodiments, compositions and methods for generating non-human animals that express such proteins from endogenous promoters and endogenous regulatory sequences are also provided. The methods include inserting gene material encoding all or a portion of the human SIRPA protein at the precise location in the genome of the non-human animal corresponding to the endogenous non-human animal (e.g., rodent) Sirpa gene, thereby creating a humanized Sirpa gene that expresses a SIRPA protein that is wholly or partially human. In some embodiments, the method includes inserting genomic DNA corresponding to exons 2-4 of the human SIRPA gene into the endogenous non-human animal (e.g., rodent) Sirpa gene of the non-human animal, thereby creating a humanized gene encoding a Sirpa protein that contains a human portion containing the amino acids encoded by the inserted exons.

[0116] In various embodiments, the humanized Sirpa gene approach results in native Sirpa-mediated signaling in non-human animals using relatively minimal modification of the endogenous gene. Thus, in such embodiments, the Sirpa gene modification does not affect other surrounding genes or other endogenous non-human animal (e.g., rodent) Sirp genes. Further, in various embodiments, the modification does not affect the assembly of the functional receptor in plasma and maintains normal effector function through binding through the cytoplasmic portion of the receptor not affected by the modification and subsequent signal transduction.

[0117] In addition to the mice having the humanized Sirpa gene described herein, other genetically modified non-human animals (e.g., rodents, e.g., rats) containing the humanized Sirpa gene are also provided herein. In some embodiments, such non-human animals contain a humanized Sirpa gene operably linked to an endogenous Sirpa promoter. In some embodiments, such non-human animals express the humanized Sirpa protein from the endogenous locus, where the humanized Sirpa protein contains amino acid residues 28-362 of the human SIRPA protein.

[0118] Humanized Sirpa polypeptides, loci encoding humanized Sirpa polypeptides, and non-human animals expressing humanized Sirpa polypeptides are described in U.S. Patent No. 11,019,810, WO2014 / 039782, WO2014 / 071397, and WO2016 / 168212, each of which is incorporated herein by reference in its entirety. Humanized CD47 locus

[0119] In one aspect, non-human animals genetically modified to express one or more human proteins from their genomes are provided. In certain aspects, the genetically modified non-human animals provided herein further express a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0120] Originally named integrin-associated protein (IAP) for its role in signal transduction from integrins in immune cells, CD47 is a transmembrane protein that contains an N-terminal immunoglobulin V (IgV) domain, five transmembrane domains, and a short C-terminal cytoplasmic tail. The cytoplasmic tail varies in length according to four alternatively spliced isoforms that have been identified. CD47 (or IAP) was first described as being expressed in all tissues (isoform 2), neurons (isoform 4), as well as keratinocytes and macrophages (isoform 1; see Reinhold et al. (1995) J. Cell Sci. 108:3419-3425). In addition to integrins, CD47 is known to interact with several other cell surface proteins such as thrombospondin and members of the SIRP family. Most notably, CD47 interacts with SIRPA, resulting in bidirectional signaling that regulates various intercellular responses such as inhibition of phagocytosis and T cell activation. Indeed, the CD47-SIRPA interaction has recently been in the spotlight for its role in giving rise to tumor cells that have the ability to evade immune surveillance. Binding of CD47 to SIRPA normally provides protection through an anti-phagocytic signal (“don't eat me”) against normal cells. However, tumors have also been found to express anti-phagocytic signals that include CD47 in order to avoid destruction by phagocytosis. Interestingly, CD47 is known to be upregulated in several blood cancers and contribute to both tumor growth and dissemination (Chao et al. (2012) Curr Opin Immunol. 24(2): 225-232).

[0121] The polypeptide sequence for wild-type human CD47 and the nucleic acid sequence encoding wild-type human CD47 can be found in Genbank accession numbers NP_001369235.1 and NM_001382306.1 (isoform 3 and transcript variant 3); NP_001768.1 and NM_001777.4 (isoform 1 and transcript variant 1); NP_942088.1 and NM_198793.3 (isoform 2 and transcript variant 2); and XP_005247966.1 and XM_005247909.3 (isoform X1 and transcript variant X1). The CD47 gene is conserved in at least chimpanzee, rhesus monkey, dog, cow, mouse, rat, and chicken. The genomic locus encoding wild-type human CD47 protein can be found in the human genome on chromosome 3; NC_000003.12 (c108091031~108043091). In some embodiments, the human CD47 protein is encoded by exons 1-11 at this locus. Thus, in some embodiments, the nucleic acid sequence comprising the coding sequence for human CD47 comprises one or more of exons 1-11 of the human CD47 gene. In some examples, the nucleic acid sequence also comprises aspects of the genomic locus of human CD47, such as introns, 3' and / or 5' untranslated regions (UTRs). In some examples, the nucleic acid sequence comprises the entire region of the human CD47 genomic locus. In some examples, the nucleic acid sequence comprises exons 2-7 of the human CD47 genomic locus.

[0122] Exemplary humanized CD47 sequences are shown in Table 4. For the humanized protein sequences, non-human (e.g., mouse) sequences are shown in normal font, human sequences are shown in bold font, and the signal peptide is underlined. Representative mouse CD47 cDNA, mouse CD47 protein, human CD47 cDNA, and human CD47 protein sequences are described in U.S. Patent Publication No. 2021 / 0161112A1, which is hereby incorporated by reference in its entirety.

[0123] [Table 4]

[0124] In some embodiments, the non-human animals provided herein express a humanized CD47 protein on the surface of cells of the non-human animal obtained from genetic modification of the endogenous locus of the non-human animal that encodes the CD47 protein. Suitable examples described herein include rodents, such as mice.

[0125] The humanized CD47 gene, in some embodiments, contains genetic material from a heterologous species (e.g., human), where the humanized CD47 gene encodes a CD47 protein that contains the portion encoded by the genetic material from the heterologous species. In some embodiments, the humanized CD47 gene of the present disclosure contains genomic DNA of a heterologous species that encodes the extracellular portion of the CD47 protein expressed on the cell membrane of the cell. In some embodiments, the humanized CD47 gene of the present disclosure contains genomic DNA of a heterologous species that encodes the extracellular portion and transmembrane portion of the CD47 protein expressed on the cell membrane of the cell. Also provided are non-human animals, non-human embryos, and cells containing the humanized CD47 gene, as well as non-human animals, embryos, cells, and targeting constructs for producing the same.

[0126] In some embodiments, the endogenous CD47 gene is deleted. In some embodiments, the endogenous CD47 gene is altered, where a portion of the endogenous CD47 gene is replaced with a heterologous sequence (e.g., all or part of the human CD47 sequence). In some embodiments, all or substantially all of the endogenous CD47 gene is replaced with a heterologous gene (e.g., the human CD47 gene). In some embodiments, a portion of the heterologous CD47 gene is inserted into the endogenous non-human CD47 gene at the endogenous CD47 locus. In some embodiments, the heterologous gene is a human gene. In some embodiments, the modification or humanization is performed on one of the two copies of the endogenous CD47 gene, resulting in a non-human animal that is heterozygous with respect to the humanized CD47 gene. In other embodiments, non-human animals that are homozygous with respect to the humanized CD47 gene are provided.

[0127] In some embodiments, the non-human animals of the present disclosure contain all or a portion of the human CD47 gene at the endogenous non-human CD47 locus. Thus, such non-human animals can be described as having a heterologous CD47 gene. The CD47 gene replaced, inserted, modified or altered at the endogenous CD47 locus can be detected using a variety of methods, including, for example, PCR, Western blot, Southern blot, restriction fragment length polymorphism (RFLP), or allele gain or loss assays. In some embodiments, the non-human animal is heterozygous with respect to the humanized CD47 gene. In some embodiments, the non-human animal is homozygous with respect to the humanized CD47 gene.

[0128] In various embodiments, the humanized CD47 gene according to the present disclosure comprises a CD47 gene having exons 2, 3, 4, 5, 6 and 7 that each have a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to the exons 2, 3, 4, 5, 6 and 7 found in the human CD47 gene.

[0129] In various embodiments, the humanized CD47 gene according to the present disclosure comprises a CD47 gene having an exon 1 and downstream exon(s) (e.g., exons 8 and 9 of isoform 2) that each have a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to the respective exons found in the mouse CD47 gene.

[0130] In various embodiments, the humanized CD47 gene according to the present disclosure includes a CD47 gene having a 5' untranslated region and a 3' untranslated region each having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to the 5' untranslated region and the 3' untranslated region found in the mouse CD47 gene.

[0131] In various embodiments, the humanized CD47 gene according to the present disclosure includes a CD47 gene having a nucleotide coding sequence (e.g., cDNA sequence) that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to the nucleotide coding sequence found in the human CD47 nucleotide coding sequence.

[0132] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an extracellular portion having an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to the extracellular portion of the human CD47 protein.

[0133] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an extracellular portion having an amino acid sequence that is identical to amino acid residues 19 - 141 found in the human CD47 protein.

[0134] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an N-terminal immunoglobulin V domain having an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to the N-terminal immunoglobulin V domain of the human CD47 protein.

[0135] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an N-terminal immunoglobulin V domain having an amino acid sequence that is identical to amino acid residues 19-127 found in the human CD47 protein.

[0136] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an N-terminal immunoglobulin V domain and five transmembrane domains each having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to the N-terminal immunoglobulin V domain and five transmembrane domains of the human CD47 protein.

[0137] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has a cytoplasmic tail having a sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to the cytoplasmic tail of the mouse CD47 protein.

[0138] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to amino acid residues 16-292 found in the human CD47 protein.

[0139] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to amino acid residues 19-292 found in the human CD47 protein.

[0140] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an amino acid sequence that is identical to amino acid residues 19-292 (or 16-292) found in the human CD47 protein.

[0141] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identical to the amino acid sequence of the humanized CD47 protein found in Table 4.

[0142] In various embodiments, the humanized CD47 protein produced by the non-human animals of the present disclosure has an amino acid sequence that is identical to the amino acid sequence of the humanized CD47 protein found in Table 4.

[0143] Compositions and methods for generating non-human animals that express a humanized CD47 protein, including specific polymorphic forms, allelic variants (e.g., differences in a single amino acid), or alternatively spliced isoforms, are provided, including compositions and methods for generating non-human animals that express such proteins from human promoters and human regulatory sequences. In some embodiments, compositions and methods for generating non-human animals that express such proteins from endogenous promoters and endogenous regulatory sequences are also provided. The methods include inserting genetic material encoding all or part of a human CD47 protein at the precise location in the genome of a non-human animal corresponding to the endogenous CD47 gene, thereby creating a humanized CD47 gene that expresses a CD47 protein that is wholly or partially human. In some embodiments, the method includes inserting genomic DNA corresponding to exons 2-7 of the human CD47 gene into the endogenous CD47 gene of a non-human animal, thereby creating a humanized gene encoding a CD47 protein that contains a human portion containing the amino acids encoded by the inserted exons.

[0144] Where appropriate, the coding region of a genetic material or polynucleotide sequence encoding all or part of a human CD47 protein can be modified to include codons optimized for expression in a non-human animal (see, for example, U.S. Patent Nos. 5,670,356 and 5,874,304). The codon-optimized sequence is a synthetic sequence and preferably encodes the same polypeptide (or a biologically active fragment of a full-length polypeptide having substantially the same activity as the full-length polypeptide) encoded by the non-codon-optimized parental polynucleotide. In some embodiments, the coding region of a genetic material encoding all or part of a human CD47 protein can include a modified sequence that optimizes the codon usage frequency for a particular cell type (e.g., rodent cells). For example, the codons of genomic DNA corresponding to exons 2-7 of the human CD47 gene inserted into the endogenous CD47 gene of a non-human animal (e.g., a rodent) can be optimized for expression in the cells of the non-human animal. Such sequences can be described as codon-optimized sequences.

[0145] In various embodiments, the humanized CD47 gene approach results in native CD47-mediated signaling in a non-human animal using a relatively minimal modification of the endogenous gene, since the genomic sequence of the CD47 sequence is modified in a single fragment and thus retains normal function by including the necessary regulatory sequences. Thus, in such embodiments, the CD47 gene modification does not affect other surrounding genes or other endogenous CD47 interacting genes (e.g., thrombospondin, SIRP, integrin, etc.). Further, in various embodiments, the modification does not affect the assembly of the functional CD47 transmembrane protein in the cell membrane and maintains normal effector function through binding through the cytoplasmic portion of the protein not affected by the modification and subsequent signal transduction.

[0146] Embodiments using a humanized CD47 gene in a mouse (i.e., a mouse having a CD47 gene encoding a CD47 protein comprising a human portion and a mouse portion) are widely discussed herein, but other non-human animals (e.g., rodents, e.g., rats) comprising a humanized CD47 gene are also provided. In some embodiments, such non-human animals comprise a humanized CD47 gene operably linked to an endogenous CD47 promoter. In some embodiments, such non-human animals express a humanized CD47 protein from an endogenous locus, where the humanized CD47 protein comprises amino acid residues 16-292 (or 19-141 or 19-127) of the human CD47 protein.

[0147] Humanized CD47 polypeptides, loci encoding humanized CD47 polypeptides, and non-human animals expressing humanized CD47 polypeptides are described in U.S. Patent Publication No. 2021 / 0161112, which is incorporated herein by reference. Humanized M-CSF locus

[0148] In some aspects, the genetically modified non-human animals provided herein further express a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter. A human M-CSF protein is a protein that is human M-CSF or is substantially identical to human M-CSF, e.g., 80% or higher identical to human M-CSF, 85% or higher identical to human M-CSF, 90% or higher identical to human M-CSF, or 95% or higher identical to human M-CSF, e.g., 97%, 98%, or 99% identical to human M-CSF. A nucleic acid sequence encoding a human M-CSF protein is thus a polynucleotide comprising a coding sequence for a human M-CSF protein, i.e., a human M-CSF or a protein substantially identical to human M-CSF.

[0149] M-CSF (also known as CSF-1 with respect to "colony-stimulating factor 1") is a cytokine that controls the production, differentiation, and function of macrophages. The polypeptide sequence for human M-CSF and the nucleic acid sequence encoding human M-CSF can be found in Genbank accession numbers NP_000748.4 and NM_000757.6 (isoform a and transcript variant 1); NP_757349.2 and NM_172210.3 (isoform b and transcript variant 2); NP_757350.2 and NM_172211.4 (isoform c and transcript variant 3) and NP_757351.2; and NM 172212.3 (isoform a and transcript variant 4). The genomic locus encoding the human M-CSF protein can be found in the human genome on chromosome 1; NC_000001.11 (109910506-109930992). The protein sequence is encoded by exons 1-8 at this locus, although exon 9 contains untranslated sequence. Thus, a nucleic acid sequence containing the coding sequence for human M-CSF contains one or more of exons 1-8 of the human M-CSF gene. In some examples, the nucleic acid sequence also includes aspects of the genomic locus of human M-CSF, such as introns, 3' and / or 5' untranslated regions (UTRs). In some examples, the nucleic acid sequence includes the entire region of the human M-CSF genomic locus. In some examples, the nucleic acid sequence includes from exon 2 of the human M-CSF genomic locus to 633 nucleotides downstream of non-coding exon 9.

[0150] In some embodiments, in the genetically modified non-human animals provided herein, the nucleic acid sequence encoding the human M-CSF protein is operably linked to one or more regulatory sequences of the non-human animal (e.g., mouse) M-CSF gene. The non-human animal (e.g., mouse) M-CSF regulatory sequences are sequences of the non-human animal (e.g., mouse) M-CSF genomic locus that regulate non-human animal (e.g., mouse) M-CSF expression, such as 5' regulatory sequences, such as the M-CSF promoter, M-CSF 5' untranslated region (UTR), etc.; 3' regulatory sequences, such as 3' UTR; and enhancers, etc. For example, mouse M-CSF is located on chromosome 3, NC_000069.7, positions approximately c107668048 to 107648364, and the mouse M-CSF coding sequences can be found in Genbank accession numbers NM_007778.4 (transcript variant 1 coding isoform 1), NM_001113529.1 (transcript variant 2 coding isoform 2), and NM_001113530.1 (transcript variant 3 coding isoform 1). The regulatory sequences of mouse M-CSF are well defined in the art and can be readily identified using in silico methods, such as by referring to the above Genbank accession numbers in the UCSC Genome Browser at genome.ucsc.edu, or by experimental methods described in the art, such as Abboud et al. (2003) Analysis of the Mouse CSF-1 Gene Promoter in a Transgenic Mouse Model. J. Histochemistry and Cytochemistry 51 (7):941 - 949, the disclosures of which are incorporated herein by reference.In one example, for instance, when a nucleic acid sequence encoding a human M-CSF protein is located at the non-human animal (e.g., mouse) M-CSF genomic locus, the regulatory sequences operably linked to the human CSF coding sequence are endogenous or native to the non-human animal (e.g., mouse) genome, i.e., they were present in the non-human animal (e.g., mouse) genome prior to the integration of the human nucleic acid sequence.

[0151] In some examples, a genetically modified non-human animal that expresses human M-CSF protein is produced by random integration or insertion of a human nucleic acid sequence encoding human M-CSF protein or a fragment thereof into the genome of the non-human animal, i.e., a "human M-CSF nucleic acid sequence" or a "human M-CSF sequence". Typically, in such embodiments, the location of the nucleic acid sequence encoding human M-CSF protein in the genome is unknown. In other examples, a genetically modified non-human animal that expresses human M-CSF protein is produced by targeted integration or insertion of a human M-CSF nucleic acid sequence into the genome of the non-human animal, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at the target locus while simultaneously removing host genomic material from the target locus, for example, 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more. Thus, for example, in a genetically modified non-human animal (e.g., a mouse) containing a nucleic acid sequence encoding a human M-CSF protein created by targeting a human M-CSF nucleic acid sequence to the non-human animal M-CSF (e.g., mouse) locus, the human M-CSF nucleic acid sequence can replace some or all of the non-human animal (e.g., mouse) sequence at the M-CSF locus, e.g., exons and / or introns. In some such examples, the human M-CSF nucleic acid sequence is integrated into the non-human animal (e.g., mouse) M-CSF locus such that expression of the human M-CSF sequence is regulated by native or endogenous regulatory sequences at the non-human animal (e.g., mouse) M-CSF locus. In other words, the regulatory sequences to which the nucleic acid sequence encoding human M-CSF protein is operably linked are the native M-CSF regulatory sequences of the non-human animal (e.g., mouse) M-CSF locus.

[0152] In some examples, the integration of the human M-CSF sequence does not affect the transcription of the gene into which the human M-CSF sequence is integrated. For example, the human M-CSF sequence is integrated into the coding sequence as an intein, or if the human M-CSF sequence contains a 2A peptide, the human M-CSF sequence is transcribed and translated simultaneously with the gene into which the human M-CSF sequence is integrated. In other examples, the integration of the human M-CSF sequence interferes with the transcription of the gene into which the human M-CSF sequence is integrated. For example, during the integration of the human M-CSF sequence by homologous recombination, some or all of the coding sequence of the integration locus may be removed, such that the human M-CSF sequence is transcribed instead. In some such examples, the integration of the human M-CSF sequence creates a null mutation, and thus, a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of that gene. This can be the result of the complete absence of the gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product. At the phenotypic level, a null allele is indistinguishable from a deletion of the entire locus.

[0153] In some examples, a genetically modified non-human animal (e.g., a mouse) that expresses human M-CSF protein contains one copy of the nucleic acid sequence encoding the human M-CSF protein. For example, the non-human animal (e.g., a mouse) can be heterozygous for the nucleic acid sequence. In other words, one allele of the locus contains the nucleic acid sequence, while the other is the endogenous allele. For example, as discussed above, in some examples, the human M-CSF nucleic acid sequence is integrated into the non-human animal (e.g., a mouse) M-CSF locus such that it creates a null allele for non-human animal (e.g., a mouse) M-CSF. In some such embodiments, the humanized M-CSF mouse can be heterozygous for the nucleic acid sequence encoding, i.e., the humanized M-CSF mouse contains one null allele (the allele containing the nucleic acid sequence) for non-human animal (e.g., a mouse) M-CSF, and one endogenous M-CSF allele (wild-type or otherwise). In other examples, a genetically modified non-human animal (e.g., a mouse) that expresses human M-CSF protein contains two copies of the nucleic acid sequence encoding the human M-CSF protein. For example, the non-human animal (e.g., a mouse) can be homozygous for the nucleic acid sequence, i.e., both alleles for the locus in the diploid genome contain the nucleic acid sequence, i.e., the genetically modified non-human animal (e.g., a mouse) that expresses human M-CSF protein contains two null alleles (the alleles containing the nucleic acid sequence) for mouse M-CSF.

[0154] Embodiments using the human M-CSF gene in mice are discussed extensively herein, but other non-human animals (e.g., rodents, e.g., rats) containing the human M-CSF gene are also provided.

[0155] The human M-CSF polypeptide, the locus encoding the human M-CSF polypeptide, and the non-human animal expressing the human M-CSF polypeptide are described in WO2012 / 112544, WO2014 / 039782, and WO2014 / 071397, each of which is incorporated herein by reference. Humanized GM-CSF locus

[0156] In some embodiments, the genetically modified non-human animals provided herein further express a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter. The human GM-CSF protein is either human GM-CSF or substantially identical to human GM-CSF, e.g., 80% or more identical to human GM-CSF, 85% or more identical to human GM-CSF, 90% or more identical to human GM-CSF, or 95% or more identical to human GM-CSF, e.g., 97%, 98%, or 99% identical to human GM-CSF. The nucleic acid sequence encoding the human GM-CSF protein is thus a polynucleotide comprising the coding sequence for the human GM-CSF protein, i.e., human GM-CSF or a protein substantially identical to human GM-CSF.

[0157] GM-CSF is a cytokine that is important for the development and function of myeloid cells. GM-CSF is not cross-reactive between humans and mice. GM-CSF is highly expressed in the lung and is important for in vivo lung homeostasis, as demonstrated by the fact that GM-CSF KO mice develop pulmonary alveolar proteinosis (PAP), which is characterized by protein accumulation in the lung due to defects in surfactant clearance. Alveolar macrophages derived from GM-CSF KO mice have a defect in terminal differentiation, which results in impaired innate immunity against pathogens in the lung. GM-CSF also stimulates the proliferation of human alveolar macrophages (AMs) in vitro. GM-CSF is mostly unnecessary for steady-state hematopoiesis. In contrast, GM-CSF is required for immune responses such as the production of inflammatory cytokines by macrophages and the mobilization and recruitment of monocytes. GM-CSF is also essential for protective immunity against a range of pathogens, including M. tuberculosis. In particular, GM-CSF KO mice infected with M. tuberculosis do not develop granulomas, a prominent feature of tuberculosis.

[0158] The polypeptide sequence for human GM-CSF and the nucleic acid sequence encoding human GM-CSF can be found at Genbank accession numbers NP_000749.2 and NM_000758.4, respectively. The genomic locus encoding the human GM-CSF protein can be found in the human genome on chromosome 5; NG_033024.1 (4998 - 7379). The protein sequence is encoded by exons 1 - 4 at this locus. Thus, a nucleic acid sequence containing the coding sequence for human GM-CSF contains one or more of exons 1 - 4 of the human GM-CSF gene. In some examples, the nucleic acid sequence also includes aspects of the genomic locus of human GM-CSF, such as introns, 3' and / or 5' untranslated regions (UTRs). In some examples, the nucleic acid sequence includes the entire region of the human GM-CSF genomic locus.

[0159] In some embodiments, in the genetically modified non-human animals provided herein, the nucleic acid sequence encoding the human GM-CSF protein is operably linked to one or more regulatory sequences of the non-human animal (e.g., mouse) GM-CSF gene. The non-human animal (e.g., mouse) GM-CSF regulatory sequences are sequences of the non-human animal (e.g., mouse) GM-CSF genomic locus that regulate non-human animal (e.g., mouse) GM-CSF expression, such as 5' regulatory sequences, such as the GM-CSF promoter, GM-CSF 5' untranslated region (UTR), etc.; 3' regulatory sequences, such as 3' UTR; and enhancers, etc. For example, mouse GM-CSF is located on chromosome 11, GRCm39, NC_000077.7, at positions approximately c54140725-54138096, and the mouse GM-CSF coding sequence can be found under Genbank accession number NM_009969.4. The regulatory sequences of mouse GM-CSF are well defined in the art and can be readily identified using in silico methods, such as by referring to the above Genbank accession number in the UCSC Genome Browser at genome.ucsc.edu, or by experimental methods described in the art. In some examples, for instance, when the nucleic acid sequence encoding the human GM-CSF protein is located at the non-human animal (e.g., mouse) GM-CSF genomic locus, the regulatory sequences operably linked to the human GM-CSF coding sequence are endogenous or native to the non-human animal (e.g., mouse) genome, i.e., they were present in the non-human animal (e.g., mouse) genome prior to the integration of the human nucleic acid sequence.

[0160] In some examples, a genetically modified non-human animal that expresses human GM-CSF protein is produced by random integration or insertion of a human nucleic acid sequence encoding human GM-CSF protein or a fragment thereof, i.e., a "human GM-CSF nucleic acid sequence" or "human GM-CSF sequence", into the genome of the non-human animal. Typically, in such embodiments, the location of the nucleic acid sequence encoding human GM-CSF protein in the genome is unknown. In other examples, a genetically modified non-human animal that expresses human GM-CSF protein is produced by targeted integration or insertion of a human GM-CSF nucleic acid sequence into the genome of the non-human animal, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at the target locus while simultaneously removing host genomic material from the target locus, such as 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more. Thus, for example, in a genetically modified non-human animal (e.g., a mouse) containing a nucleic acid sequence encoding human GM-CSF protein created by targeting the human GM-CSF nucleic acid sequence to the non-human animal GM-CSF (e.g., mouse) locus, the human GM-CSF nucleic acid sequence can replace some or all of the non-human animal (e.g., mouse) sequence at the GM-CSF locus, such as exons and / or introns. In some such examples, the human GM-CSF nucleic acid sequence is integrated into the non-human animal (e.g., mouse) GM-CSF locus such that expression of the human GM-CSF sequence is regulated by native or endogenous regulatory sequences at the non-human animal (e.g., mouse) GM-CSF locus. In other words, the regulatory sequences to which the nucleic acid sequence encoding human GM-CSF protein is operably linked are the native GM-CSF regulatory sequences of the non-human animal (e.g., mouse) GM-CSF locus.

[0161] In some examples, the integration of the human GM-CSF sequence does not affect the transcription of the gene into which the human GM-CSF sequence is integrated. For example, the human GM-CSF sequence is integrated into the coding sequence as an intein, or if the human GM-CSF sequence contains a 2A peptide, the human GM-CSF sequence is transcribed and translated simultaneously with the gene into which the human GM-CSF sequence is integrated. In other examples, the integration of the human GM-CSF sequence interferes with the transcription of the gene into which the human GM-CSF sequence is integrated. For example, during the integration of the human GM-CSF sequence by homologous recombination, some or all of the coding sequence of the integration locus may be removed, such that the human GM-CSF sequence is transcribed instead. In some such examples, the integration of the human GM-CSF sequence creates a null mutation, and thus, a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of that gene. This can be the result of the complete absence of the gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product. At the phenotypic level, a null allele is indistinguishable from a deletion of the entire locus.

[0162] In some examples, a genetically modified non-human animal (e.g., a mouse) that expresses human GM-CSF protein contains one copy of the nucleic acid sequence encoding the human GM-CSF protein. For example, the non-human animal (e.g., a mouse) can be heterozygous for the nucleic acid sequence. In other words, one allele of the locus contains the nucleic acid sequence, while the other is the endogenous allele. For example, as discussed above, in some examples, the human GM-CSF nucleic acid sequence is integrated into the non-human animal (e.g., a mouse) GM-CSF locus to create a null allele for non-human animal (e.g., a mouse) GM-CSF. In some such embodiments, the humanized GM-CSF mouse can be heterozygous for the nucleic acid sequence encoding, i.e., the humanized GM-CSF mouse contains one null allele (the allele containing the nucleic acid sequence) for non-human animal (e.g., a mouse) GM-CSF, and one endogenous GM-CSF allele (wild-type or otherwise). In other examples, a genetically modified non-human animal (e.g., a mouse) that expresses human GM-CSF protein contains two copies of the nucleic acid sequence encoding the human GM-CSF protein. For example, the non-human animal (e.g., a mouse) can be homozygous for the nucleic acid sequence, i.e., both alleles for the locus in the diploid genome contain the nucleic acid sequence, i.e., a genetically modified non-human animal (e.g., a mouse) that expresses human GM-CSF protein contains two null alleles (the alleles containing the nucleic acid sequence) for mouse GM-CSF.

[0163] Embodiments using the human GM-CSF gene in mice are discussed extensively herein, but other non-human animals (e.g., rodents, e.g., rats) containing the human GM-CSF gene are also provided.

[0164] The human GM-CSF polypeptide, the locus encoding the human GM-CSF polypeptide, and the non-human animal expressing the human GM-CSF polypeptide are described in WO2011 / 044050, WO2014 / 039782, and WO2014 / 071397, each of which is incorporated herein by reference. Humanized TPO locus

[0165] In some embodiments, the genetically modified non-human animals provided herein further express a human TPO protein encoded by a nucleic acid operably linked to the TPO promoter. A human TPO protein is a protein that is human TPO or is substantially identical to human TPO, e.g., 80% or higher identical to human TPO, 85% or higher identical to human TPO, 90% or higher identical to human TPO, or 95% or higher identical to human TPO, e.g., 97%, 98%, or 99% identical to human TPO. The nucleic acid sequence encoding the human TPO protein is thus a polynucleotide comprising the coding sequence for the human TPO protein, i.e., human TPO or a protein that is substantially identical to human TPO.

[0166] Thrombopoietin (TPO) was first identified as a growth factor that promotes the development of megakaryocytes and platelets. TPO is constitutively produced by the liver and kidneys and released into the bloodstream. The receptor for TPO, c-Mpl, is expressed by hematopoietic stem cells and progenitor cells in the bone marrow. C-Mpl is also expressed on circulating platelets. However, the binding of TPO to platelets does not activate any signaling pathway. Therefore, platelets act as a sink or scavenger for TPO and contribute to the negative regulation of thrombopoiesis through this mechanism. Subsequently, TPO has been recognized for its important function in supporting the expansion and self-renewal of HSCs. TPO deficiency results in a reduction in the number of HSCs in adult mice, and the presence of TPO is necessary to maintain quiescent adult HSCs. Furthermore, TPO is required to support the post-transplant expansion of HSCs necessary to repopulate the hematopoietic compartment of irradiated hosts. Interestingly, osteoblasts, which are involved in the formation of the HSC niche in the bone marrow, have been shown to produce TPO, which is important for HSC function and maintenance.

[0167] The polypeptide sequence for human TPO and the nucleic acid sequence encoding human TPO can be found in Genbank accession numbers NM_000547.6 and NP_000538.3 (transcript variant 1 and isoform a); NM_001206744.2 and NP_001193673.1 (transcript variant 6 and isoform a); NM_001206745.2 and NP_001193674.1 (transcript variant 7 and isoform b); NM_175719.4 and NP_783650.1 (transcript variant 2 and isoform b); NM_175721.3 and NP_783652.1 (transcript variant 4 and isoform d); and NM_175722.3 and NP_783653.1 (transcript variant 5 and isoform e). The genomic locus encoding the human TPO protein can be found in the human genome on chromosome 2; NG_011581.1 (4999 - 134265). The protein sequence is encoded by exons 2 - 17 at this locus. Thus, a nucleic acid sequence containing the coding sequence for human TPO contains one or more of exons 2 - 17 of the human TPO gene. In some examples, the nucleic acid sequence also includes aspects of the genomic locus of human TPO, such as introns, 3' and / or 5' untranslated regions (UTRs). In some examples, the nucleic acid sequence includes the entire region of the human TPO genomic locus.

[0168] In some embodiments, in the genetically modified non-human animals provided herein, the nucleic acid sequence encoding the human TPO protein is operably linked to one or more regulatory sequences of the non-human animal (e.g., mouse) TPO gene. The non-human animal (e.g., mouse) TPO regulatory sequences are sequences of the non-human animal (e.g., mouse) TPO genomic locus that regulate non-human animal (e.g., mouse) TPO expression, such as 5' regulatory sequences, such as the TPO promoter, the TPO 5' untranslated region (UTR), etc.; 3' regulatory sequences, such as the 3' UTR; and enhancers, etc. For example, mouse TPO is located on chromosome 12, GRCm39, NC_000078.7, positions approximately c30182983 to 30104658, and the mouse TPO coding sequence can be found under Genbank accession number NM_009417.3. The regulatory sequences of mouse TPO are well defined in the art and can be readily identified using in silico methods, such as by referring to the above Genbank accession number in the UCSC Genome Browser at genome.ucsc.edu, or by experimental methods described in the art. In some examples, for instance, when the nucleic acid sequence encoding the human TPO protein is located at the non-human animal (e.g., mouse) TPO genomic locus, the regulatory sequences operably linked to the human TPO coding sequence are endogenous or native to the non-human animal (e.g., mouse) genome, i.e., they were present in the non-human animal (e.g., mouse) genome prior to the integration of the human nucleic acid sequence.

[0169] In some examples, a genetically modified non-human animal that expresses human TPO protein is produced by random integration or insertion of a human nucleic acid sequence encoding human TPO protein or a fragment thereof into the genome of the non-human animal, i.e., a "human TPO nucleic acid sequence" or a "human TPO sequence". Typically, in such embodiments, the location of the nucleic acid sequence encoding human TPO protein in the genome is unknown. In other examples, a genetically modified non-human animal that expresses human TPO protein is produced by targeted integration or insertion of a human TPO nucleic acid sequence into the genome of the non-human animal, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at the target locus while simultaneously removing host genomic material from the target locus, for example, 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more. Thus, for example, in a genetically modified non-human animal (e.g., a mouse) containing a nucleic acid sequence encoding human TPO protein generated by targeting the human TPO nucleic acid sequence to the non-human animal TPO (e.g., mouse) locus, the human TPO nucleic acid sequence can replace some or all of the non-human animal (e.g., mouse) sequence at the TPO locus, such as exons and / or introns. In some such examples, the human TPO nucleic acid sequence is integrated into the non-human animal (e.g., mouse) TPO locus such that expression of the human TPO sequence is regulated by native or endogenous regulatory sequences at the non-human animal (e.g., mouse) TPO locus. In other words, the regulatory sequences to which the nucleic acid sequence encoding human TPO protein is operably linked are the native TPO regulatory sequences of the non-human animal (e.g., mouse) TPO locus.

[0170] In some examples, the integration of the human TPO sequence does not affect the transcription of the gene into which the human TPO sequence is integrated. For example, the human TPO sequence is integrated into the coding sequence as an intein, or, if the human TPO sequence contains a 2A peptide, the human TPO sequence is transcribed and translated simultaneously with the gene into which the human TPO sequence is integrated. In other examples, the integration of the human TPO sequence interferes with the transcription of the gene into which the human TPO sequence is integrated. For example, upon integration of the human TPO sequence by homologous recombination, some or all of the coding sequence of the integration locus may be removed, such that the human TPO sequence is transcribed instead. In some such examples, the integration of the human TPO sequence creates a null mutation, and thus, a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of that gene. This can be the result of the complete absence of the gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product. At the phenotypic level, a null allele is indistinguishable from a deletion of the entire locus.

[0171] In some examples, a genetically modified non-human animal (e.g., a mouse) that expresses human TPO protein contains one copy of the nucleic acid sequence encoding the human TPO protein. For example, the non-human animal (e.g., a mouse) can be heterozygous for the nucleic acid sequence. In other words, one allele of the locus contains the nucleic acid sequence, while the other is the endogenous allele. For example, as discussed above, in some examples, the human TPO nucleic acid sequence is integrated into the non-human animal (e.g., a mouse) TPO locus to create a null allele for non-human animal (e.g., a mouse) TPO. In some such embodiments, the humanized TPO mouse can be heterozygous for the nucleic acid sequence encoding, i.e., the humanized TPO mouse contains one null allele (the allele containing the nucleic acid sequence) for non-human animal (e.g., a mouse) TPO, and one endogenous TPO allele (wild-type or otherwise). In other examples, a genetically modified non-human animal (e.g., a mouse) that expresses human TPO protein contains two copies of the nucleic acid sequence encoding the human TPO protein. For example, the non-human animal (e.g., a mouse) can be homozygous for the nucleic acid sequence, i.e., both alleles for the locus in the diploid genome contain the nucleic acid sequence, i.e., the genetically modified non-human animal (e.g., a mouse) that expresses human TPO protein contains two null alleles (the alleles containing the nucleic acid sequence) for mouse TPO.

[0172] Embodiments using the human TPO gene in mice are discussed extensively herein, but other non-human animals (e.g., rodents, e.g., rats) containing the human TPO gene are also provided.

[0173] Human TPO polypeptides, the loci encoding human TPO polypeptides, and non-human animals expressing human TPO polypeptides are described in WO2011 / 044050, WO2014 / 039782, and WO2014 / 071397, each of which is incorporated herein by reference. Humanized EPO locus

[0174] In some embodiments, the genetically modified non-human animals provided herein further express a human erythropoietin (EPO) protein encoded by a nucleic acid operably linked to an EPO promoter. The human EPO protein is either human EPO or is substantially identical to human EPO, e.g., 80% or higher identical to human EPO, 85% or higher identical to human EPO, 90% or higher identical to human EPO, or 95% or higher identical to human EPO, e.g., 97%, 98%, or 99% identical to human EPO. The nucleic acid sequence encoding the human EPO protein is thus a polynucleotide comprising the coding sequence for the human EPO protein, i.e., human EPO or a protein that is substantially identical to human EPO.

[0175] Erythropoietin (EPO) encodes a secreted, glycosylated cytokine composed of four alpha helix bundles. The encoded EPO protein is mainly synthesized in the kidneys, secreted into the plasma, binds to the erythropoietin receptor, and promotes erythropoiesis or red blood cell production in the bone marrow. Expression of the EPO gene is upregulated under hypoxic conditions, which then leads to an increase in erythropoiesis and enhanced oxygen-carrying capacity of the blood. Expression of the EPO gene has also been observed in the brain and eyes, and elevated expression levels have been observed in diabetic retinopathy and glaucoma. Recombinant forms of the encoded EPO protein exhibit neuroprotective activity against various potential brain injuries, as well as anti-apoptotic functions in some tissue types, and are used in the treatment of anemia and to enhance the efficacy of cancer therapy.

[0176] The polypeptide sequence for human EPO and the nucleic acid sequence encoding human EPO can be found, respectively, at Genbank accession numbers NP_000790.2 and NM_000799.4. The genomic locus encoding the human EPO protein can be found in the human genome on chromosome 7; NG_021471.2 (4669 - 7901) or NC_000007.14 (100720468 - 100723700). The protein sequence is encoded by exons 1 - 5 at this locus. Thus, a nucleic acid sequence containing the coding sequence for human EPO contains one or more of exons 1 - 5 of the human EPO gene. In some examples, the nucleic acid sequence also includes aspects of the genomic locus of human EPO, such as introns, 3' and / or 5' untranslated regions (UTRs). In some examples, the nucleic acid sequence includes the entire region of the human EPO genomic locus.

[0177] In some embodiments, in the genetically modified non-human animals provided herein, the nucleic acid sequence encoding the human EPO protein is operably linked to one or more regulatory sequences of the non-human animal (e.g., mouse) EPO gene. The non-human animal (e.g., mouse) EPO regulatory sequences are sequences of the non-human animal (e.g., mouse) EPO genomic locus that regulate non-human animal (e.g., mouse) EPO expression, such as 5' regulatory sequences, such as the EPO promoter, the EPO 5' untranslated region (UTR), etc.; 3' regulatory sequences, such as the 3' UTR; and enhancers, etc. For example, mouse EPO is located on chromosome 5, GRCm39, NC_000071.7, at positions approximately c137484078-137481282, and the mouse EPO coding sequences can be found in Genbank accession numbers NM_007942.2 (transcript variant 1 encoding isoform 1) and NM_001312875.1 (transcript variant 2 encoding isoform 2). The regulatory sequences of mouse EPO are well defined in the art and can be readily identified using in silico methods, such as by referring to the above Genbank accession numbers in the UCSC Genome Browser at genome.ucsc.edu on the World Wide Web, or by experimental methods described in the art. In some examples, for instance, when the nucleic acid sequence encoding the human EPO protein is located at the non-human animal (e.g., mouse) EPO genomic locus, the regulatory sequences operably linked to the human EPO coding sequence are endogenous or native to the non-human animal (e.g., mouse) genome, i.e., they were present in the non-human animal (e.g., mouse) genome prior to the integration of the human nucleic acid sequence.

[0178] In some examples, a genetically modified non-human animal that expresses human EPO protein is created by random integration or insertion of a human nucleic acid sequence encoding human EPO protein or a fragment thereof into the genome of the non-human animal, i.e., a "human EPO nucleic acid sequence" or a "human EPO sequence". Typically, in such embodiments, the location of the nucleic acid sequence encoding human EPO protein in the genome is unknown. In other examples, a genetically modified non-human animal that expresses human EPO protein is created by targeted integration or insertion of a human EPO nucleic acid sequence into the genome of the non-human animal, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at the target locus while simultaneously removing host genomic material from the target locus, for example, 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kb or more, 2 kb or more, 5 kb or more, 10 kb or more, 15 kb or more, 20 kb or more, or 50 kb or more. Thus, for example, in a genetically modified non-human animal (e.g., a mouse) that contains a nucleic acid sequence encoding human EPO protein created by targeting a human EPO nucleic acid sequence to a non-human animal EPO (e.g., mouse) locus, the human EPO nucleic acid sequence can replace some or all of the non-human animal (e.g., mouse) sequence at the EPO locus, such as exons and / or introns. In some such examples, the human EPO nucleic acid sequence is integrated into the non-human animal (e.g., mouse) EPO locus such that expression of the human EPO sequence is regulated by native or endogenous regulatory sequences at the non-human animal (e.g., mouse) EPO locus. In other words, the regulatory sequences to which the nucleic acid sequence encoding human EPO protein is operably linked are the native EPO regulatory sequences of the non-human animal (e.g., mouse) EPO locus.

[0179] In some examples, the integration of the human EPO sequence does not affect the transcription of the gene into which the human EPO sequence is integrated. For example, the human EPO sequence is integrated into the coding sequence as an intein, or if the human EPO sequence contains a 2A peptide, the human EPO sequence is transcribed and translated simultaneously with the gene into which the human EPO sequence is integrated. In other examples, the integration of the human EPO sequence interferes with the transcription of the gene into which the human EPO sequence is integrated. For example, during the integration of the human EPO sequence by homologous recombination, some or all of the coding sequence of the integration locus may be removed, such that the human EPO sequence is transcribed instead. In some such examples, the integration of the human EPO sequence creates a null mutation and thus a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of that gene. This can be the result of the complete absence of the gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product. At the phenotypic level, a null allele is indistinguishable from a deletion of the entire locus.

[0180] In some examples, a genetically modified non-human animal (e.g., a mouse) that expresses a human EPO protein includes one copy of a nucleic acid sequence encoding the human EPO protein. For example, the non-human animal (e.g., a mouse) can be heterozygous for the nucleic acid sequence. In other words, one allele at the locus includes the nucleic acid sequence, while the other is the endogenous allele. For example, as discussed above, in some examples, the human EPO nucleic acid sequence is integrated into the non-human animal (e.g., a mouse) EPO locus to create a null allele for non-human animal (e.g., a mouse) EPO. In some such embodiments, the humanized EPO mouse can be heterozygous for the nucleic acid sequence encoding, i.e., the humanized EPO mouse includes one null allele (the allele including the nucleic acid sequence) for non-human animal (e.g., a mouse) EPO, and one endogenous EPO allele (wild-type or otherwise). In other examples, a genetically modified non-human animal (e.g., a mouse) that expresses a human EPO protein includes two copies of a nucleic acid sequence encoding the human EPO protein. For example, the non-human animal (e.g., a mouse) can be homozygous for the nucleic acid sequence, i.e., both alleles at the locus in the diploid genome include the nucleic acid sequence, i.e., the genetically modified non-human animal (e.g., a mouse) that expresses a human EPO protein includes two null alleles (the alleles including the nucleic acid sequence) for mouse EPO.

[0181] Embodiments using the human EPO gene in mice are discussed extensively herein, but other non-human animals (e.g., rodents, e.g., rats) that include the human EPO gene are also provided.

[0182] Human EPO polypeptides, loci encoding human EPO polypeptides, and non-human animals expressing human EPO polypeptides are described in WO2015 / 179317, which is incorporated herein by reference. Humanized IL-3 locus

[0183] In one aspect, the genetically modified non-human animals provided herein further express a human IL-3 protein encoded by a nucleic acid operably linked to an IL-3 promoter. The human IL-3 protein is either human IL-3 or substantially identical to human IL-3, e.g., 80% or higher identical to human IL-3, 85% or higher identical to human IL-3, 90% or higher identical to human IL-3, or 95% or higher identical to human IL-3, e.g., 97%, 98%, or 99% identical to human IL-3. The nucleic acid sequence encoding the human IL-3 protein is thus a polynucleotide comprising the coding sequence for the human IL-3 protein, i.e., human IL-3 or a protein substantially identical to human IL-3.

[0184] Like GM-CSF, IL-3 is a cytokine important for the development and function of myeloid cells. IL-3 is not cross-reactive between humans and mice. IL-3 stimulates early hematopoietic precursors in vitro but is not required for steady-state hematopoiesis in vivo. However, together with GM-CSF, it is required for an effective DTH response in vivo. IL-3 also specifically stimulates the proliferation of alveolar macrophages (AMs) in vitro.

[0185] The polypeptide sequence for human IL-3 and the nucleic acid sequence encoding human IL-3 can be found, respectively, under Genbank accession numbers NP_000579.2 and NM_000588.4. The genomic locus encoding the human IL-3 protein can be found in the human genome on chromosome 5; GRCh38.p14; NC_000005.10(132060655~132063204). The protein sequence is encoded by exons 1-5 at this locus. Thus, a nucleic acid sequence containing the coding sequence for human IL-3 contains one or more of exons 1-5 of the human IL-3 gene. In some examples, the nucleic acid sequence also includes aspects of the genomic locus of human IL-3, such as introns, 3' and / or 5' untranslated regions (UTRs). In some examples, the nucleic acid sequence includes the entire region of the human IL-3 genomic locus.

[0186] In some embodiments, in the genetically modified non-human animals provided herein, the nucleic acid sequence encoding a human IL-3 protein is operably linked to one or more regulatory sequences of a non-human animal (e.g., mouse) IL-3 gene. The non-human animal (e.g., mouse) IL-3 regulatory sequence is a sequence of the non-human animal (e.g., mouse) IL-3 genomic locus that regulates non-human animal (e.g., mouse) IL-3 expression, such as a 5' regulatory sequence, e.g., an IL-3 promoter, an IL-3 5' untranslated region (UTR), etc.; a 3' regulatory sequence, e.g., a 3' UTR; and an enhancer. For example, mouse IL-3 is located on chromosome 11, GRCm39, NC_000077.7, at approximately c54158105-54155911, and the mouse IL-3 coding sequence can be found under GenBank accession number NM_010556.4. Regulatory sequences for mouse IL-3 are well defined in the art and can be readily identified using in silico methods, for example, by referencing the above Genbank accession numbers in the UCSC Genome Browser on the worldwide web at genome.ucsc.edu, or by experimental methods described in the art. In some examples, for example, when a nucleic acid sequence encoding a human IL-3 protein is located at a non-human animal (e.g., mouse) IL-3 genomic locus, the regulatory sequences operably linked to the human IL-3 coding sequence are endogenous or native to the non-human animal (e.g., mouse) genome, i.e., they were present in the non-human animal (e.g., mouse) genome prior to integration of the human nucleic acid sequence.

[0187] In some examples, a genetically modified non-human animal that expresses human IL-3 protein is created by random integration or insertion of a human nucleic acid sequence encoding human IL-3 protein or a fragment thereof into the genome of the non-human animal, i.e., a "human IL-3 nucleic acid sequence" or a "human IL-3 sequence". Typically, in such embodiments, the location of the nucleic acid sequence encoding human IL-3 protein in the genome is unknown. In other examples, a genetically modified non-human animal that expresses human IL-3 protein is created by targeted integration or insertion of a human IL-3 nucleic acid sequence into the genome of the non-human animal, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at the target locus while simultaneously removing host genomic material from the target locus, such as 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more. Thus, for example, in a genetically modified non-human animal (e.g., a mouse) containing a nucleic acid sequence encoding human IL-3 protein created by targeting the human IL-3 nucleic acid sequence to the non-human animal IL-3 (e.g., mouse) locus, the human IL-3 nucleic acid sequence can replace some or all of the non-human animal (e.g., mouse) sequence at the IL-3 locus, such as exons and / or introns. In some such examples, the human IL-3 nucleic acid sequence is integrated into the non-human animal (e.g., mouse) IL-3 locus such that expression of the human IL-3 sequence is regulated by native or endogenous regulatory sequences at the non-human animal (e.g., mouse) IL-3 locus. In other words, the regulatory sequences to which the nucleic acid sequence encoding human IL-3 protein is operably linked are the native IL-3 regulatory sequences of the non-human animal (e.g., mouse) IL-3 locus.

[0188] In some examples, the integration of the human IL-3 sequence does not affect the transcription of the gene into which the human IL-3 sequence is integrated. For example, the human IL-3 sequence is integrated into the coding sequence as an intein, or if the human IL-3 sequence contains a 2A peptide, the human IL-3 sequence is transcribed and translated simultaneously with the gene into which the human IL-3 sequence is integrated. In other examples, the integration of the human IL-3 sequence interferes with the transcription of the gene into which the human IL-3 sequence is integrated. For example, during the integration of the human IL-3 sequence by homologous recombination, some or all of the coding sequence of the integration locus may be removed, such that the human IL-3 sequence is transcribed instead. In some such examples, the integration of the human IL-3 sequence creates a null mutation, and thus, a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of that gene. This can be the result of the complete absence of the gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product. At the phenotypic level, a null allele is indistinguishable from a deletion of the entire locus.

[0189] In some examples, a genetically modified non-human animal (e.g., a mouse) that expresses a human IL-3 protein contains one copy of a nucleic acid sequence encoding the human IL-3 protein. For example, the non-human animal (e.g., a mouse) can be heterozygous for the nucleic acid sequence. In other words, one allele of the locus contains the nucleic acid sequence, while the other is the endogenous allele. For example, as discussed above, in some examples, the human IL-3 nucleic acid sequence is integrated into the non-human animal (e.g., a mouse) IL-3 locus so as to create a null allele for non-human animal (e.g., a mouse) IL-3. In some such embodiments, the humanized IL-3 mouse can be heterozygous for the nucleic acid sequence encoding, i.e., the humanized IL-3 mouse contains one null allele (the allele containing the nucleic acid sequence) for non-human animal (e.g., a mouse) IL-3, and one endogenous IL-3 allele (wild-type or otherwise). In other examples, a genetically modified non-human animal (e.g., a mouse) that expresses a human IL-3 protein contains two copies of a nucleic acid sequence encoding the human IL-3 protein. For example, the non-human animal (e.g., a mouse) can be homozygous for the nucleic acid sequence, i.e., both alleles for the locus in the diploid genome contain the nucleic acid sequence, i.e., the genetically modified non-human animal (e.g., a mouse) that expresses a human IL-3 protein contains two null alleles (the alleles containing the nucleic acid sequence) for mouse IL-3.

[0190] Embodiments using the human IL-3 gene in mice are discussed extensively herein, but other non-human animals (e.g., rodents, e.g., rats) containing the human IL-3 gene are also provided.

[0191] Human IL-3 polypeptides, loci encoding human IL-3 polypeptides, and non-human animals expressing human IL-3 polypeptides are described in WO2011 / 044050, WO2014 / 039782, and WO2014 / 071397, each of which is incorporated herein by reference. Humanized IL-15 locus

[0192] In some embodiments, the genetically modified non-human animals provided herein further express a human IL-15 protein encoded by a nucleic acid operably linked to an IL-15 promoter. As used herein, "human IL-15 protein" retains one or more signaling functions of the wild-type (or native) human IL-15 protein, e.g., enables stimulation (or signaling through) of the human IL-15 receptor, and / or can bind to the human IL-15 receptor alpha subunit of the human IL-15 receptor, and / or can bind to IL-2R beta / IL-15R beta and the common gamma chain (γc), and means a protein that is a wild-type (or native) human IL-15 protein or a variant of the wild-type (or native) human IL-15 protein. The term "human IL-15 protein" also encompasses fragments of wild-type human IL-15 protein (or variants thereof) that retain one or more signaling functions of the wild-type human IL-15 protein, e.g., enable stimulation (or signaling through) of the human IL-15 receptor, and / or can bind to the human IL-15 receptor alpha subunit of the human IL-15 receptor, and / or can bind to IL-2R beta / IL-15R beta and the common gamma chain (γc).

[0193] The term "human IL-15 protein" also encompasses fusion proteins, i.e., chimeric proteins, which contain one or more fragments of wild-type human IL-15 protein (or variants thereof) and retain one or more signaling functions of the wild-type human IL-15 protein described above. Fusion proteins containing one or more fragments of wild-type human IL-15 protein (or variants thereof) may also be referred to herein as humanized IL-15 proteins.

[0194] The nucleic acid sequences encoding human IL-15 protein thus include polynucleotides encoding human IL-15 proteins that retain one or more signaling functions of the wild-type human IL-15 protein, i.e., the wild-type human IL-15 protein, variants of the wild-type human IL-15 protein, fragments of the wild-type human IL-15 protein (or variants thereof), or fusion proteins, i.e., chimeric proteins, that include one or more fragments of the wild-type human IL-15 protein (or variants thereof) and retain one or more signaling functions of the wild-type human IL-15 protein as described above.

[0195] IL-15 (also known as "interleukin 15") is a cytokine that stimulates the proliferation of T lymphocytes. The polypeptide sequence for wild-type human IL-15 and the nucleic acid sequence encoding wild-type human IL-15 can be found in Genbank accession numbers NP_000576.1 and NM_000585.5 (isoform 1 and transcript variant 3), NP_751915.1 and NM_172175.3 (isoform 2 and transcript variant 2). The genomic locus encoding wild-type human IL-15 protein can be found in the human genome at chromosome 4; NC_000004.12 (141636583-141733987) or NG_029605.2 (4988-102392). In some embodiments, the human IL-15 locus (e.g., NM_000585.5) includes eight exons, and exons 3-8 are coding exons. Thus, in some embodiments, a nucleic acid sequence comprising a coding sequence for human IL-15 includes one or more of exons 3-8 of the human IL-15 gene. In some examples, the nucleic acid sequence also includes aspects of the genomic locus of human IL-15, e.g., introns, 3' and / or 5' untranslated regions (UTRs). In some examples, the nucleic acid sequence includes the entire region of the human IL-15 genomic locus. In some examples, the nucleic acid sequence includes exons 5-8 of the human IL-15 genomic locus (i.e., coding exons 3-6).

[0196] In the humanized IL-15 non-human animals described herein, the nucleic acid sequence encoding the human IL-15 protein is operably linked to one or more regulatory sequences of the IL-15 gene, such as the regulatory sequences of the IL-15 gene of the non-human animal. Non-human animal IL-15 regulatory sequences, such as those of a mouse, are sequences of the non-human animal IL-15 genomic locus that regulate non-human animal IL-15 expression, such as 5' regulatory sequences, such as the IL-15 promoter, the IL-15 5' untranslated region (UTR), etc.; 3' regulatory sequences, such as the 3' UTR; and enhancers, etc. Mouse IL-15 is located on chromosome 8, NC_000074.7 (c83129883~83058253), and the mouse IL-15 coding sequence can be found in Genbank accession numbers NM_008357.3 (transcript variant 1); NM_001254747.2 (transcript variant 2). The regulatory sequences of mouse IL-15 are well defined in the art and can be readily identified using in silico methods, such as by referring to the above Genbank accession numbers in the UCSC Genome Browser at genome.ucsc.edu on the World Wide Web, or by experimental methods described in the art. In some examples, for instance, when the nucleic acid sequence encoding the human IL-15 protein is located at the mouse IL-15 genomic locus, the regulatory sequences operably linked to the human IL-15 coding sequence are endogenous or native to the mouse genome, i.e., they were present in the mouse genome prior to the integration of the human nucleic acid sequence.

[0197] In some examples, a humanized IL-15 non-human animal, such as a mouse, is created by random integration or insertion of a human nucleic acid sequence encoding a human IL-15 protein (including the above fragments) into the genome of the non-human animal, i.e., a "human IL-15 nucleic acid sequence" or a "human IL-15 sequence". Typically, in such embodiments, the location of the nucleic acid sequence encoding the human IL-15 protein in the genome is unknown. In other examples, a humanized IL-15 non-human animal is created by targeted integration or insertion of a human IL-15 nucleic acid sequence into the genome of the non-human animal, such as by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at a target locus while simultaneously removing host genomic material from the target locus, such as 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more. Thus, for example, in a humanized IL-15 mouse containing a nucleic acid sequence encoding a human IL-15 protein created by targeting the human IL-15 nucleic acid sequence to the non-human animal IL-15 mouse locus, the human IL-15 nucleic acid sequence can replace some or all of the mouse sequence at the IL-15 locus, such as exons and / or introns. In some such examples, the human IL-15 nucleic acid sequence is integrated into the mouse IL-15 locus such that expression of the human IL-15 sequence is regulated by native or endogenous regulatory sequences at the mouse IL-15 locus. In other words, the regulatory sequences to which the nucleic acid sequence encoding the human IL-15 protein is operably linked are the native IL-15 regulatory sequences of the mouse IL-15 locus.

[0198] In some examples, the integration of the human IL-15 sequence does not affect the transcription of the gene into which the human IL-15 sequence is integrated. For example, the human IL-15 sequence is integrated into the coding sequence as an intein, or if the human IL-15 sequence contains a 2A peptide, the human IL-15 sequence is transcribed and translated simultaneously with the gene into which the human IL-15 sequence is integrated. In other examples, the integration of the human IL-15 sequence interferes with the transcription of the gene into which the human IL-15 sequence is integrated. For example, during the integration of the human IL-15 sequence by homologous recombination, some or all of the coding sequence of the integration locus may be removed, such that the human IL-15 sequence is transcribed instead. In some such examples, the integration of the human IL-15 sequence creates a null mutation, and thus, a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of that gene. This can be the result of the complete absence of the gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product. At the phenotypic level, a null allele is indistinguishable from a deletion of the entire locus.

[0199] In some examples, a humanized IL-15 non-human animal, such as a mouse, contains one copy of a nucleic acid sequence encoding a human IL-15 protein. For example, the non-human animal can be heterozygous for the nucleic acid sequence. In other words, one allele of the locus contains the nucleic acid sequence, while the other is the endogenous allele. For example, as discussed above, in some examples, the human IL-15 nucleic acid sequence is integrated into the non-human animal, such as a mouse IL-15 locus, to create a null allele for non-human animal IL-15. In some such embodiments, the humanized IL-15 non-human animal can be heterozygous for the nucleic acid sequence encoding, i.e., the humanized IL-15 non-human animal contains one null allele (the allele containing the nucleic acid sequence) for non-human animal IL-15, and one endogenous IL-15 allele (wild-type or otherwise). In other examples, the humanized IL-15 contains two copies of the nucleic acid sequence encoding a human IL-15 protein. For example, the non-human animal, such as a mouse, can be homozygous for the nucleic acid sequence, i.e., both alleles for the locus in the diploid genome contain the nucleic acid sequence, i.e., the humanized IL-15 non-human animal contains two null alleles (the alleles containing the nucleic acid sequence) for non-human animal IL-15.

[0200] Embodiments using the human IL-15 gene in mice are discussed extensively herein, but other non-human animals containing the human IL-15 gene (e.g., rodents, such as rats) are also provided.

[0201] Human IL-15 polypeptides, loci encoding human IL-15 polypeptides, and non-human animals expressing human IL-15 polypeptides are described in WO2016 / 168212, which is incorporated herein by reference. Humanized liver

[0202] In some embodiments, the genetically modified non-human animals provided herein further comprise a modification to the animal's immune system such that, as a result, it is unable to enhance or has a reduced ability to mount an immune response against xenogeneic transplanted cells (e.g., human hepatocytes) (e.g., the non-human animal is immunodeficient). In some cases, the animal is suitable for xenotransplantation of hepatocytes. In some cases, the animal is a genetically modified non-human animal in which (1) non-human (i.e., endogenous) hepatocytes in the liver can be selectively and conditionally removed; and (2) the immune system of the non-human animal is modified such that it is unable to enhance an immune response against xenogeneic transplanted cells (e.g., human hepatocytes) (e.g., the non-human animal is immunodeficient). Cells and genomes comprising the genetic modifications disclosed herein are also provided. The genetically modified non-human animals disclosed herein can be used for in vivo engraftment and expansion of xenogeneically transplanted hepatocytes (e.g., human hepatocytes). Xenotransplantation refers to the transplantation of living cells, tissues, or organs from one species to another. Such cells, tissues, or organs are referred to as xenografts or xenotransplants (e.g., xenogeneically transplanted cells).

[0203] Any suitable immunodeficient non-human animal can be used. For example, see Weber et al. (2009) Liver Transplantation 15:7-14, which is hereby incorporated by reference in its entirety for all purposes. Such non-human animals can be made immunodeficient such that native non-human T and B cells do not develop. For example, such immunodeficient non-human animals can lack functional native non-human T cells, B cells, and / or natural killer (NK) cells. An immunodeficient non-human animal refers to a non-human animal that lacks at least one essential function of the immune system. For example, an immunodeficient non-human animal lacks a particular component of the immune system or lacks the function of a particular component of the immune system (e.g., native non-human B cells, T cells, or NK cells, etc.). In some cases, the immunodeficient animal lacks native non-human macrophages. In some cases, the immunodeficient animal contains one or more genetic modifications that prevent or inhibit the development of functional immune cells (such as native non-human B cells, T cells, or NK cells). Rag genes (e.g., the immunodeficient non-human animal is Rag1 - / - and / or Rag2 - / - ), and animals with genetic modifications of the Il2rg gene (Il2rg - / - ) (see Traggiai et al. (2004) Science, 304:104, which is hereby incorporated by reference); animals with severe combined immunodeficiency (SCID) mutations in the Prkdc gene (Prkdc scidor SCID (e.g., see Mercer et al. (2001) Nat. Med. 7(8):927-933, which is incorporated herein by reference in its entirety for all purposes); animals having SCID mutations and inactivated endogenous Il2rg genes, such as the NOG background (e.g., see Ito et al. (2002) Blood 100(9):3175-3182, which is incorporated herein by reference in its entirety for all purposes); or animals having other mutations, such as X-linked SCID characterized by autosomal recessive SCID characterized by JAK3 mutations, ADA mutations, IL7R mutations, CD3 mutations, ARTEMIS (DCLRE1C) mutations, and CD45 (PTPRC) mutations. Various immunodeficient animal models are known in the art, such as the above-provided specific non-limiting examples of genetic alterations that result in immunodeficiency, but other known immunodeficient non-human animals can also be used.

[0204] Any suitable genetic modification can be used that enables the selective and conditional removal of non-human (i.e., endogenous) hepatocytes in the liver. In some cases, the non-human animal contains an inactivated endogenous Fah gene. This inactivation results in the toxic accumulation of tyrosine catabolites within hepatocytes. The compound 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) can be used to block the enzyme hydroxyphenylpyruvate dioxygenase upstream of FAH, thus preventing the accumulation of liver-toxic metabolites. In some cases, the non-human animal contains the urokinase-type plasminogen activator gene (Plau; NCBI Gene ID 18792 in mice) operably linked to a liver-specific promoter such as the albumin promoter. See, for example, Mercer et al. (2001) Nat. Med. 7(8):927-933, which is incorporated herein by reference in its entirety for all purposes. Such non-human animals have liver toxicity leading to liver failure. In some cases, the non-human animal contains the herpes simplex virus type 1 thymidine kinase (HSVtk) gene operably linked to a liver-specific promoter such as the albumin promoter. See, for example, Hasegawa et al. (2011) Biochem. Biophys. Res. Commun. 405(3):405-410, which is incorporated herein by reference in its entirety for all purposes. Administration of ganciclovir (GCV), a drug that is not toxic to human or mouse tissue, induces the tissue-specific removal of transgenic hepatocytes. Since HSVtk catalyzes GCV phosphorylation, which is the rate-limiting step that cannot be carried out in mammalian cells lacking this transgene, hepatocytes expressing the transgene are selectively destroyed. Certain non-limiting examples of genetic modifications that enable the selective and conditional removal of non-human (i.e., endogenous) hepatocytes in the liver are provided above, but any other suitable genetic modification can also be used.

[0205] In one example, a genetically modified non-human animal for xenotransplantation of hepatocytes (e.g., transplantation of human hepatocytes) has the following genes inactivated (i.e., knocked out): Fah (encoding fumarylacetoacetase); Rag1 (encoding recombination activating protein 1 of V(D)J); and / or Rag2 (encoding recombination activating protein 2 of V(D)J); and Il2rg (encoding interleukin 2 receptor subunit gamma). Also provided are genetically modified non-human animal cells or genomes having the following genes inactivated (i.e., knocked out): Fah; Rag1 and / or Rag2; and Il2rg. In one example, the genetically modified non-human animal is a rat. See, for example, Carbonaro et al. (2022) Sci. Rep. 12(1):14079 and US2016 / 0249591, each of which is hereby incorporated by reference in its entirety for all purposes. In another example, the genetically modified non-human animal is a mouse. See, for example, Strom et al. (2010) Methods Mol. Biol. 640:491-509, Azuma et al. (2007) Nat. Biotechnol. 25(8):903-910, and US8,569,573, each of which is hereby incorporated by reference in its entirety for all purposes. In another example, the genetically modified non-human animal is a pig. See, for example, US9,000,257, which is hereby incorporated by reference in its entirety for all purposes.

[0206] Fah is an essential gene in the tyrosine catabolism pathway. When Fah is mutated in an animal, toxic intermediate metabolites accumulate in the liver, causing loss of hepatocytes and ultimately liver failure and death. This toxicity can be ameliorated by blocking the activity of another tyrosine catabolic enzyme, 4-hydroxyphenylpyruvate dioxygenase, which can be achieved by administration of the small molecule nitisinone (NTBC). Fah mutant mice are healthy and viable when NTBC is administered. However, they rapidly become moribund and die when NTBC is withdrawn. Therefore, administration and withdrawal of NTBC enable precise temporal control of hepatotoxicity in Fah mutant non-human animals.

[0207] Fumarylacetoacetase (FAH, also known as FAA, beta-ketothiolase, or fumarylacetoacetate hydrolase) is encoded by the Fah gene (also known as the fumarylacetoacetate hydrolase gene). FAH is an enzyme required for the final step in the tyrosine catabolic pathway, which hydrolyzes fumarylacetoacetate to fumarate and acetoacetate. Deficiency of Fah results in the accumulation of toxic metabolites including fumarylacetoacetate and maleylacetoacetate. 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (nitisinone or NTBC), an inhibitor of 4-hydroxyphenylpyruvate dioxygenase, acts by blocking the accumulation of toxic metabolites such as fumarylacetoacetate and maleylacetoacetate and may be effective in ameliorating liver and kidney damage in human patients with Fah deficiency. Several Fah mutations that cause tyrosinemia type I (type 1 hereditary tyrosinemia or HT) in humans have been identified. This condition is characterized by severe liver and kidney disease, neurological problems, and other signs and symptoms that begin in infancy. The altered Fah gene that causes this condition produces an unstable or inactive enzyme, which results in a reduction or absence of fumarylacetoacetate hydrolase activity. Without sufficient fumarylacetoacetate hydrolase activity, tyrosine and its by-products are not properly degraded. As a result, fumarylacetoacetate accumulates in the liver and kidneys. Elevated levels of fumarylacetoacetate are thought to be toxic to cells, and the accumulation of this substance can cause liver and kidney problems, as well as other features characteristic of tyrosinemia type I.

[0208] Mouse Fah maps to chromosome 7 at 7 D3;7 48.36 cM (NCBI RefSeq gene ID 14085; assembly GRCm39 (GCF_000001635.27); position NC_000073.7 (84234367..84255150, complement). References to the mouse Fah gene include the standard wild-type form, as well as all allelic forms and isoforms. The standard wild-type mouse FAH protein is assigned UniProt accession number P35505 and NCBI accession number NP_034306.2. References to the mouse FAH protein include the standard wild-type form, as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the standard isoform is assigned NCBI accession number NM_010176.4. References to the mouse Fah mRNA (cDNA) and coding sequence include the standard wild-type form, as well as all allelic forms and isoforms.

[0209] Rat Fah maps to chromosome 1 at 1q31 (NCBI RefSeq gene ID 29383; assembly mRatBN7.2 (GCF_015227675.2); position NC_051336.1 (138548830..138571599, complement). References to the rat Fah gene include the standard wild-type form, as well as all allelic forms and isoforms. The standard wild-type rat FAH protein is assigned UniProt accession number P25093 and NCBI accession number NP_058877.1. References to the rat FAH protein include the standard wild-type form, as well as all allelic forms and isoforms. The mRNA (cDNA) encoding the standard isoform is assigned NCBI accession number NM_017181.2. References to the rat Fah mRNA (cDNA) and coding sequence include the standard wild-type form, as well as all allelic forms and isoforms.

[0210] An inactivated endogenous Fah gene is a Fah gene that does not produce the FAH protein or does not produce a functional FAH protein. A non-human animal (or cell or genome) can contain the inactivated Fah gene in its germline. A non-human animal (or cell or genome) can be homozygous for an inactivating mutation in the Fah gene. As an example, an inactivated endogenous Fah gene can contain an insertion, deletion, or one or more point mutations in the endogenous Fah gene that result in the loss of expression of the functional FAH protein. Some inactivated endogenous Fah genes can contain all deletions or disruptions of the endogenous Fah gene or can contain deletions or disruptions of a fragment (i.e., a part or portion) of the endogenous Fah gene. For example, some, most, or all of the coding sequence in the endogenous Fah gene can be deleted or disrupted. In one example, the 5’ fragment of the Fah gene can be deleted or disrupted (e.g., including the start codon). As an example, an inactivated endogenous Fah gene can be one in which the start codon of the endogenous Fah gene has been deleted such that the start codon no longer functions or has been disrupted or mutated. For example, the start codon can be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon can be mutated, for example, by substitution of one or more nucleotides. In another example, the 3’ fragment of the Fah gene can be deleted or disrupted (e.g., including the stop codon). In another example, an internal fragment of the Fah gene (i.e., a fragment from the middle of the Fah gene) can be deleted or disrupted. In another example, all of the coding sequence in the endogenous Fah gene is deleted or disrupted.

[0211] Inactivated endogenous Fah gene non-human animals and non-human animals lacking Fah expression are described in US2016 / 024959, US Patent No. 8,569,573, Azuma et al. Nature Biotechnology 25(8):903-10 (2007), Carbonaro et al. Scientific Reports 12:14079-89 (2022), Carbonaro et al. Sci. Adv. 9, eadf4490 (2023), and Wilson et al. Stem Cell Research 13:404-412 (2014), which are hereby incorporated by reference.

[0212] Genetically modified non-human animals including Rag2 (and optionally Rag1) knockout, Il2rg KO, Hmox-1 KO, Fah KO, and humanization of genes such as Sirpa (e.g., such as M-CSF, CD47, etc. described herein) can be mated together to engraft human HSCs and human hepatocytes and used as a model for studying human parasitic infections, such as malaria, at both the liver and blood stages. Genetically Modified Non-Human Animals and ES Cells

[0213] In certain embodiments, (i) Rag1 and / or Rag2 gene knockout; (ii) IL2rg gene knockout; and (iii) homozygous null mutations in the non-human animal heme oxygenase-1 (Hmox-1) gene, and optionally, Fah gene knockout and / or one or more of the humanized loci disclosed herein, genetically modified non-human animals (e.g., rodents, e.g., rats or mice), and genetically modified non-human animal ES cells useful for producing such non-human animals are provided herein.

[0214] In certain embodiments, genetically modified non-human animals (e.g., rodents, e.g., rats or mice) and non-human animal (e.g., rodent, e.g., rat or mouse) ES cells are provided herein that comprise in their germline and / or genome: (i) a Rag1 and / or Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, and optionally one or more of the engineered loci described herein. In some embodiments, genetically modified non-human animals (e.g., mice) and non-human animal (e.g., mouse) ES cells are provided herein that comprise in their germline and / or genome: (i) a Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, and optionally one or more of the engineered loci described herein. In some embodiments, genetically modified non-human animals (e.g., rats) and non-human animal (e.g., rat) ES cells are provided herein that comprise in their germline and / or genome: (i) a Rag1 gene knockout; (ii) a Rag2 gene knockout; (iii) an IL2rg gene knockout; and (iv) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, and optionally one or more of the engineered loci described herein. For example, in some embodiments, the non-human animal or ES cell comprises in its germline and / or genome the humanized Sirpa locus provided herein. In some embodiments, the non-human animal or ES cell comprises in their germline and / or genome the CD47 locus provided herein. In certain embodiments, the non-human animal or ES cell comprises in their germline and / or genome the M-CSF locus provided herein. In certain embodiments, the non-human animal or ES cell comprises in their germline and / or genome the GM-CSF locus provided herein.In certain embodiments, the non-human animals or ES cells contain in their germline and / or genome the TPO locus provided herein. In certain embodiments, the non-human animals or ES cells contain in their germline and / or genome the EPO locus provided herein. In certain embodiments, the non-human animals or ES cells contain in their germline and / or genome the IL-3 locus provided herein. In certain embodiments, the non-human animals or ES cells contain in their germline and / or genome the IL-15 locus provided herein. In certain embodiments, the non-human animals or ES cells contain in their germline and / or genome inactivation (e.g., deletion) of the Fah gene provided herein. In some embodiments, the non-human animals or ES cells are heterozygous for one or more of the loci provided herein, e.g., the engineered loci. In some embodiments, the non-human animals or ES cells are homozygous for one or more of the loci provided herein, e.g., the engineered loci.

[0215] In some embodiments, the non-human animal can be any non-human animal. In some embodiments, the non-human animal is a vertebrate. In some embodiments, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animals described herein are selected from the group consisting of mice, rats, rabbits, pigs, cows (e.g., female cows, male cows, buffalos), deer, sheep, goats, llamas, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For non-human animals for which suitable genetically modified ES cells are not readily available, other methods can be used to generate non-human animals comprising the genetic modifications described herein. Such methods include, for example, modifying a non-ES cell genome (e.g., fibroblast or induced pluripotent cell), and using nuclear transfer to transfer the modified genome into a suitable cell such as an oocyte, and gestating the modified cell (e.g., modified oocyte) in a non-human animal under conditions suitable for forming an embryo.

[0216] In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human animal is a small mammal, such as a small mammal of the superfamily Dipodoidea or Muroidea. In some embodiments, the non-human animal is a rodent. In certain embodiments, the rodent is a mouse, rat or hamster. In some embodiments, the rodent is selected from the superfamily Muroidea. In some embodiments, the non-human animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, gerbils), Muridae (e.g., true mice and rats, spiny mice, brush mice, tufted mice), Nesomyidae (e.g., climbing mice, rock mice, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rats, bamboo rats, and zokors). In some embodiments, the rodent is selected from true mice or rats (family Muridae), spiny mice, brush mice, and tufted mice. In some embodiments, the mouse is from a member of the family Muridae. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent is selected from mice and rats. In some embodiments, the non-human animal is a mouse.

[0217] In some embodiments, the non-human animal is a mouse of the C57BL strain. In some embodiments, the C57BL strain is selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the non-human animal is a mouse of the 129 strain. In some embodiments, the 129 strain is selected from the group consisting of 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2. In some embodiments, the genetically modified mouse is a mix of the 129 strain and the C57BL strain. In some embodiments, the mouse is a mix of the 129 strain and / or the C57BL / 6 strain. In some embodiments, the mixed 129 strain is the 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse is of the BALB strain (e.g., BALB / c). In some embodiments, the mouse is a mix of the BALB strain and another strain (e.g., the C57BL strain and / or the 129 strain). In some embodiments, the non-human animals provided herein can be mice derived from any combination of the aforementioned strains.

[0218] In some embodiments, the non-human animals provided herein are rats. In some embodiments, the rats are selected from Wistar rats, LEA strain, Sprague Dawley strain, Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0219] In certain embodiments, the genetically modified non-human animals or ES cells comprise in their genomes and / or germlines a plurality of loci provided herein, such as a plurality of genetically engineered loci provided herein. For example, in some embodiments, the non-human animals or ES cells comprise in their germlines and / or genomes: (i) a Rag1 and / or Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene. In some embodiments, the non-human animals or ES cells comprise in their germlines and / or genomes: (i) a Rag1 and / or Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, as well as a humanized Sirpa locus provided herein. In some embodiments, the non-human animals or ES cells comprise in their germlines and / or genomes: (i) a Rag1 and / or Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, a humanized Sirpa locus provided herein, and a humanized M-CSF locus provided herein. In some embodiments, the non-human animals or ES cells comprise in their germlines and / or genomes: (i) a Rag1 and / or Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, a humanized Sirpa locus provided herein, a humanized M-CSF locus provided herein, and a humanized CD47 locus provided herein.In some embodiments, the non-human animal or ES cell has in its germline and / or genome: (i) a Rag1 and / or Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, the humanized Sirpa locus provided herein, and optionally, the humanized CD47 locus provided herein, the humanized M-CSF locus provided herein, the humanized GM-CSF locus provided herein, the humanized TPO locus provided herein, the humanized EPO locus provided herein, the humanized IL-3 locus provided herein, the humanized IL-15 locus provided herein, and one or more humanized loci selected from the group consisting of any combination thereof. In some embodiments, the non-human animal or ES cell described herein further has inactivation (e.g., deletion) of the Fah gene in its germline and / or genome.

[0220] In certain embodiments, the genetically modified non-human animal does not express the Hmox-1 polypeptide. In certain embodiments, the genetically modified non-human animal expresses one or more human or humanized polypeptides encoded by the humanized locus provided herein. For example, in some embodiments, the non-human animal expresses a human or humanized Sirpa polypeptide. In certain embodiments, the non-human animal expresses a human or humanized CD47 polypeptide. In certain embodiments, the non-human animal expresses a human or humanized M-CSF polypeptide. In certain embodiments, the non-human animal expresses a human or humanized GM-CSF polypeptide. In certain embodiments, the non-human animal expresses a human or humanized TPO polypeptide. In certain embodiments, the non-human animal expresses a human or humanized EPO polypeptide. In certain embodiments, the non-human animal or ES cell non-human animal expresses a human or humanized IL-3 polypeptide. In certain embodiments, the non-human animal expresses a human or humanized IL-15 polypeptide. In certain embodiments, the non-human animal or ES cell does not express the FAH protein. In certain embodiments, the genetically modified non-human animal does not express the Hmox-1 protein and the FAH protein.

[0221] Genetically modified non-human animals and ES cells can be produced using any suitable method known in the art. For example, such genetically modified non-human animal ES cells can be produced using the VELOCIGENE® technology described in U.S. Patent Nos. 6,586,251, 6,596,541, 7,105,348, and Valenzuela et al. (2003) “High-throughput engineering of the mouse genome coupled with high-resolution expression analysis” Nat. Biotech. 21(6): 652-659, each of which is hereby incorporated by reference herein. The modification can also be performed using a genome targeting nuclease system, such as the CRISPR / Cas system, the transcription activator-like effector nuclease (TALEN) system, or the zinc finger nuclease (ZFN) system. In some embodiments, the modification is performed using the CRISPR / Cas system described, for example, in U.S. Patent Application Nos. 14 / 314,866, 14 / 515,503, 14 / 747,461, and 14 / 731,914, each of which is incorporated by reference. Genetically modified rat ES cells and rats can be produced according to US2014 / 0235933A1 (Regeneron Pharmaceuticals, Inc.), US2014 / 0310828A1 (Regeneron Pharmaceuticals, Inc.), Tong et al. (2010) Nature 467:211-215, and Tong et al. (2011) Nat Protoc. 6(6): doi:10.1038 / nprot.2011.338 (all of which are hereby incorporated by reference in their entirety). Exemplary methods for producing such genetically modified non-human animals and ES cells are also provided in Example 1 herein.

[0222] Subsequently, using the ES cells described herein, non-human animals can be produced using methods known in the art. For example, using the mouse non-human animal ES cells described herein, genetically modified mice can be produced using the VELOCIMOUSE® method described in U.S. Patent No. 7,294,754 and Poueymirou et al., Nature Biotech 25:91-99 (2007), each of which is hereby incorporated by reference herein. The resulting mice can be bred to be homozygous. Methods for producing genetically modified non-human animals and ES cells

[0223] In certain embodiments, provided herein are methods of generating non-human animals (e.g., mice or rats) and ES cells comprising one or more of the genetically modified loci provided herein. For example, in some embodiments, methods of generating non-human animals (e.g., mice or rats) and ES cells comprising (i) a Rag1 and / or Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene are provided herein. In some embodiments, methods of generating non-human animals (e.g., mice or rats) and ES cells comprising (i) a Rag1 and / or Rag2 gene knockout; (ii) an IL2rg gene knockout; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene and further comprising the humanized Sirpa locus provided herein are provided herein. In some embodiments, methods of generating non-human animals (e.g., mice or rats) and ES cells further comprising the humanized CD47 locus provided herein, the humanized M-CSF locus provided herein, the humanized GM-CSF locus provided herein, the humanized TPO locus provided herein, the humanized EPO locus provided herein, the humanized IL-3 locus provided herein, and / or the humanized IL-15 locus provided herein are provided herein. In some embodiments, methods of generating non-human animals (e.g., mice or rats) and ES cells comprising a homozygous null mutation in the Fah gene are provided herein. Exemplary methods of generating the genetically modified non-human animals and ES cells provided herein are described in the description, examples, and / or figures of this specification. Generation of non-human animals comprising a null mutation in the Hmox-1 gene of the non-human animal can be accomplished, for example, using any convenient method for generating genetically modified animals known in the art or described in Example 1 of this specification.

[0224] The production of a non-human animal comprising a nucleic acid sequence encoding a human or humanized protein (e.g., hSIRPA, hCD47, hM-CSF, hGM-CSF, hTPO, hEPO, hIL-3, or hIL-15) can be achieved, for example, using any convenient method for producing genetically modified animals known in the art or described herein.

[0225] For example, a nucleic acid encoding a human or humanized protein (e.g., hSIRPA, hCD47, hM-CSF, hGM-CSF, hTPO, hEPO, hIL-3, and / or hIL-15) can be incorporated into a recombinant vector in a form suitable 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 can comprise one or more regulatory sequences operably linked to the nucleic acid encoding the human protein in a manner that allows transcription of the nucleic acid into mRNA and translation of the mRNA into a human protein, as described above. It is understood that the design of the vector can depend on factors such as the choice of host cell to be transfected and / or the amount of human protein to be expressed.

[0226] Next, a human nucleic acid sequence can be introduced into an animal cell using any of a variety of methods to produce a genetically modified animal that expresses a human gene. 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 producing genetically modified animals that can be used include, but are not limited to, 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 2nd ed. Sunderland, Massachusetts: Sinauer), U.S. Patent No. 6,586,251, Rathinam et al. (2011, Blood 118:3119-28), Willinger et al, (2011, Proc Natl Acad Sci USA, 108:2390-2395), Rongvaux et al, (2011, Proc Natl Acad Sci USA, 108:2378-83) and Valenzuela et al. (2003, Nat Biot 21 :652-659).

[0227] For example, a genetically modified animal of interest can be produced by introducing, for example, a nucleic acid encoding a human protein into an oocyte by microinjection and developing the oocyte in a female foster animal. In a preferred embodiment, the expression construct is injected into a fertilized oocyte. The fertilized oocytes can be collected from females superovulated on the day after mating and the expression construct can be injected. The injected oocytes are cultured overnight or directly transferred into the oviducts of pseudopregnant females at 0.5 days post coitum. Methods for superovulation, oocyte collection, expression construct injection and embryo transfer are known in the art and are described in Manipulating the Mouse Embryo (2002, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press). Offspring can be evaluated for the presence of the introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or by protein analysis (e.g., ELISA, Western blot, etc.).

[0228] As another example, a construct containing a nucleic acid sequence encoding a human protein may be transfected into stem cells (e.g., ES cells or iPS cells) using well-known methods such as electroporation, calcium phosphate precipitation, lipofection, etc. 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 by protein analysis (e.g., ELISA, Western blot, etc.). Cells in which it has been determined that the expression construct has been integrated can then be introduced into pre-implantation embryos. For a detailed description of methods known in the art useful in the compositions and methods of the present invention, see Nagy et al, (2002, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press), Nagy et al. (1990, Development 110:815-821), U.S. Patent No. 7,576,259, U.S. Patent No. 7,659,442, U.S. Patent No. 7,294,754, and Kraus et al. (2010, Genesis 48:394-399).

[0229] In addition, as described in part in the examples below, nucleic acid constructs are constructed using VELOCIGENE® genetic engineering techniques (see, for example, Valenzuela et al. (2003) High throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6): 652-59 and U.S. Patent No. 6,586,251), introduced into stem cells (e.g., ES cells), and precisely targeted clones can be determined using allele loss and allele gain assays (Valenzuela et al., supra); precisely targeted ES cells can be used as donor ES cells for introduction into 8-cell stage mouse embryos using the VELOCIMOUSE® method (see, for example, U.S. Patent 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 analyses Nature Biotech. 25(l):91-99). In addition, genetically modified rat ES cells and rats can be generated according to US2014 / 0235933A1 (Regeneron Pharmaceuticals, Inc.), US2014 / 0310828A1 (Regeneron Pharmaceuticals, Inc.), Tong et al. (2010) Nature 467:211-215, and Tong et al. (2011) Nat Protoc. 6(6): doi:10.1038 / nprot.2011.338 (all of which are hereby incorporated by reference in their entirety).

[0230] In some embodiments, the genetically modified founder animals can be mated with additional animals having one or more genetic modifications. For example, the HMOX-1 deficient non-human animals provided herein can be further mated with other genetically modified non-human animals having other genetic modifications including the introduction of either a fully human or humanized gene, such as hSirpa knock-in mice, hM-CSF knock-in mice, hCD47 knock-in mice, hIL-3 knock-in mice, hGM-CSF knock-in mice, hTPO knock-in mice, hEPO knock-in mice, hIL-15 knock-in mice, etc., or with knockout animals, such as non-human animals, in which one or more proteins are deficient, e.g., in which one or more of those genes are not expressed, such as Rag1 deficient animals, Rag2 deficient animals, I12rg deficient animals, or FAH deficient animals.

[0231] In another embodiment, stem cells, such as ES cells, can be generated such that they contain several genetic modifications, such as those described herein including humanization or gene deletion, and such stem cells can be introduced into an embryo to generate a genetically modified animal having several genetic modifications.

[0232] As discussed above, in some embodiments, the genetically modified non-human animal is an immunodeficient animal. An immunodeficient genetically modified non-human animal that contains one or more human or humanized proteins, such as hSIRPA, hIL-3, hGM-CSF, hM-CSF, hEPO, hCD47, hIL-15, and / or hTPO, can be produced using any convenient method for producing genetically modified animals, such as methods known in the art or described herein. For example, the production of a genetically modified immunodeficient animal can be achieved by introducing a nucleic acid encoding a human protein into an oocyte or stem cell that contains a mutant SCID gene allele, or Rag and / or Rag2 and Il2rg null alleles, which result in immunodeficiency in the homozygous case, as described in more detail in the working examples above and herein. The mice are then produced using the modified oocytes or ES cells, for example, using methods described herein and known in the art, and mated to produce immunodeficient mice that contain the desired genetic modification. As another example, a genetically modified non-human animal can be produced in an immunocompetent background, mated with an animal that contains a mutant gene allele that results in immunodeficiency in the heterozygous or homozygous case, and the offspring mated to produce an immunodeficient animal that expresses at least one desired human protein.

[0233] 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 includes irradiating the genetically modified mouse. In a specific embodiment, the pups of the genetically modified mouse are irradiated with a sub-lethal dose. In a specific embodiment, the newborn pups are irradiated with 2 x 200 cGy at 4-hour intervals.

[0234] Various embodiments of the present invention provide genetically modified animals that contain human nucleic acids in substantially all of those cells, and genetically modified animals that contain human nucleic acids in some, but not all, of those cells. In some examples, such as targeted recombination, one copy of the human nucleic acid is integrated into the genome of the genetically modified animal. In other examples, such as random integration, multiple copies of the human nucleic acids, either adjacent to each other or separated, can be integrated into the genome of the genetically modified animal.

[0235] Thus, in some embodiments, a subject genetically modified non-human animal can be an immunodeficient animal that contains a genome comprising a nucleic acid encoding a human polypeptide operably linked to a corresponding non-human animal promoter, wherein the animal expresses the encoded human polypeptide. In other words, a subject genetically modified immunodeficient non-human animal contains a genome comprising a nucleic acid encoding at least one human polypeptide, wherein the nucleic acid is operably linked to a corresponding non-human promoter and polyadenylation signal, and the animal expresses the encoded human polypeptide.

[0236] Additional methods of generating a genetically modified non-human animal that contains a genome comprising a nucleic acid encoding one or more human proteins, such as hSIRPA, hIL-3, hGM-CSF, hM-CSF, hEPO, hCD47, hIL-15, and / or hTPO, are described in U.S. Patent No. US11019810, U.S. Patent Publication No. US2021 / 0161112, WO2011 / 044050, WO2012 / 112544, WO2014 / 039782, WO2014 / 071397, WO2015 / 179317, and WO2016 / 168212, each of which is incorporated herein by reference. Engraftment

[0237] In some embodiments, the subject genetically modified non-human animal is also immunodeficient. "Immunodeficient" includes a defect in one or more aspects of the animal's native or endogenous immune system. For example, the animal is deficient in one or more types of functional host immune cells. For example, 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. are deficient.

[0238] One way to achieve immunodeficiency in a subject animal is sublethal irradiation. Alternatively or in addition, immunodeficiency can be achieved by any one of several gene mutations known in the art, any of which may be mated into the genetically modified non-human animals of the present disclosure, either alone or in combination, or may be used as a source of stem cells into which the disclosed genetic modification 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(+); CD3 delta or epsilon mutations characterized by lymphocyte phenotype T(-)B(+)NK(+); RAG1 and RAG2 mutations characterized by lymphocyte phenotype T(-)B(-)NK(+); Artemis gene mutations characterized by lymphocyte phenotype T(-)B(-)NK(+), CD45 gene mutations characterized by lymphocyte phenotype T(-)B(+)NK(+); and Prkdcscld mutations characterized by lymphocyte phenotypes T(-), B(-). Thus, in some embodiments, the genetically modified immunodeficient non-human animals have one or more deficiencies selected from IL2 receptor gamma chain deficiency, Jak3 deficiency, ADA deficiency, IL7R deficiency, CD3 deficiency, RAG1 and / or RAG2 deficiency, Artemis deficiency, CD45 deficiency, and Prkdc deficiency. In one embodiment, immunodeficiency is achieved by gene mutations or deletions in the Rag1 and / or Rag2 and Il2rg genes. These and other animal models of immunodeficiency are known to those of skill in the art, and any of these may be used to generate the immunodeficient animals of the present disclosure.

[0239] In some embodiments, the genetically modified non-human animals according to the invention are found to be useful as recipients of human hematopoietic cells capable of developing human immune cells from engrafted human hematopoietic cells. Thus, in some aspects of the invention, the subject genetically modified animals are genetically modified immunodeficient non-human animals in which human hematopoietic cells have engrafted.

[0240] Any source of human hematopoietic cells, human hematopoietic stem cells (HSCs) and / or hematopoietic stem progenitor cells (HSPCs) known in the art or described herein can 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, particularly CD34-positive (CD34+) cells. Another source of human hematopoietic cells is the 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, and any of these can be used by one of ordinary skill in the art to reach the subject genetically modified engrafted non-human animals.

[0241] Particular cell populations of interest include those containing hematopoietic stem or progenitor cells and contribute to or reconstitute the hematopoietic system of the genetically modified non-human animals, such as peripheral blood leukocytes, fetal liver cells, fetal bone, fetal thymus, fetal lymph nodes, skin with vessels, arterial segments, and purified hematopoietic stem cells, such as mobilized HSCs or umbilical cord blood HSCs.

[0242] The cells can be derived from any mammalian species, such as mouse, rodent, dog, cat, horse, cow, sheep, primate, human, etc. In one embodiment, the cells are human cells. The cells can be from an established cell line or the cells can be primary cells, where "primary cells", "primary cell line", and "primary culture" are used interchangeably herein to refer to cells and cell cultures derived from a subject and capable of growing in vitro for a limited number of passages, i.e., during division. For example, a primary culture is a culture that has not progressed to the crisis stage but may have been passaged 0, 1, 2, 4, 5, 10, or 15 times. Typically, the primary cell lines of the present disclosure are maintained in vitro for less than 10 passages.

[0243] When the cells are primary cells, the cells can be obtained from an individual by any convenient method. For example, cells, such as blood cells, such as white blood cells, can be obtained by apheresis, leukapheresis, density gradient separation, etc. As another example, cells, such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach tissue, etc., can be obtained by biopsy. A suitable solution can be used for dispersion or suspension of the obtained cells. Such solutions are generally balanced salt solutions, such as physiological saline, PBS, Hank's balanced salt solution, etc., supplemented with fetal bovine serum or other naturally occurring factors, generally in combination with a low concentration, generally 5 - 25 mM, of an acceptable buffer. Suitable buffers include HEPES, phosphate buffer, lactate buffer, etc.

[0244] In some examples, a heterogeneous population of cells is transplanted into a genetically modified non-human animal. In other examples, a population of cells that is enriched for a particular type of cell, such as a progenitor cell, such as a hematopoietic progenitor cell, is engrafted into a genetically modified non-human animal. Enrichment of the desired cell population may be by any convenient separation technique. For example, the desired cells can be enriched by culture methods. In such culture methods, typically, specific growth factors and nutrients that promote the survival and / or proliferation of one cell population over others are added to the culture. Other culture conditions that affect survival and / or proliferation include growth on adherent or non-adherent substrates, culture for a specific period, and the like. Such culture conditions are well known in the art. As another example, the desired cells can be enriched by separation of the desired cells from the initial population by affinity separation techniques. Techniques for affinity separation include magnetic separation using magnetic beads coated with an affinity reagent, affinity chromatography, solid matrices such as affinity reagents attached to plates, cell damaging agents conjugated to or used in combination with an affinity reagent, such as "panning" with complement and cytotoxins, or other convenient techniques. Techniques that provide accurate separation include fluorescence activated cell sorters that can have various degrees of sophistication, such as multiple color channels, low angle and obtuse angle light scatter detection channels, impedance channels, and the like. Cells can be selected against dead cells by using a dye that associates with dead cells (e.g., propidium iodide). Any technique that is not overly detrimental to the viability of the desired cells may be used.

[0245] For example, using an affinity separation technique, cells that are not the cells of interest for transplantation can be depleted from a population by contacting the population with an affinity reagent that specifically recognizes and selectively binds to a marker not expressed on the cells of interest. For example, to enrich a population of hematopoietic progenitor cells, cells expressing mature hematopoietic cell markers can be depleted. Additionally or alternatively, positive selection and separation can be performed by contacting the population with an affinity reagent that specifically recognizes and selectively binds to a marker associated with hematopoietic progenitor cells, such as CD34, CD133, etc. "Selectively binds" means that a molecule binds preferentially to the target of interest over other molecules or binds to the target with a higher affinity. For example, an antibody binds to a molecule containing the epitope to which it is specific and does not bind to irrelevant epitopes. In some embodiments, the affinity reagent may be an antibody, i.e., an antibody specific for CD34, CD133, etc. In some embodiments, the affinity reagent may be a specific receptor or ligand for CD34, CD133, etc., such as a peptide ligand and receptor; effector and receptor molecules, a T cell receptor specific for CD34, CD133, etc. In some embodiments, multiple affinity reagents specific for the marker of interest can be used.

[0246] Antibodies and T cell receptors used as affinity reagents can be monoclonal or polyclonal and may be produced by, for example, transgenic animals, immunized animals, immortalized human or animal B cells, cells transfected with a DNA vector encoding an antibody or T cell receptor. The details of antibody preparation and their suitability for use as specific binding members are well known to those skilled in the art. Of particular interest is the use of labeled antibodies as affinity reagents. Conveniently, these antibodies are conjugated to a label for use in separation. Labels include magnetic beads that allow direct separation, biotin that can be removed using avidin or streptavidin bound to a support, fluorescent dyes that can be used with a fluorescence-activated cell sorter, etc., which facilitate the separation of specific cell types. Fluorescent dyes used include phycobiliproteins such as phycoerythrin and allophycocyanin, fluorescein, and Texas red. Frequently, each antibody is labeled with a different fluorescent dye to allow independent sorting for each marker.

[0247] An initial population of cells is contacted with an affinity reagent and incubated for a period of time sufficient to bind to available cell surface antigens. The incubation is typically at least about 5 minutes and typically less than about 60 minutes. It is desirable to have a sufficient concentration of antibody in the reaction mixture such that the efficiency of separation is not limited by antibody deficiency. The appropriate concentration is determined by titration, but typically the dilution of antibody relative to the volume of the cell suspension is about 1:50 (i.e., 1 part antibody to 50 parts reaction volume), about 1:100, about 1:150, about 1:200, about 1:250, about 1:500, about 1:1000, about 1:2000, or about 1:5000. The medium in which the cells are suspended is any medium that maintains the viability of the cells. Preferred media are phosphate buffered saline containing 0.1 - 0.5% BSA or 1 - 4% goat serum. A variety of media are commercially available that are frequently supplemented with fetal bovine serum, BSA, HSA, goat serum, etc., such as Dulbecco's Modified Eagle Medium (dMEM), Hank's Balanced Salt Solution (HBSS), Dulbecco's Phosphate Buffered Saline (dPBS), RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., and can be used according to the nature of the cells.

[0248] Cells in the contacted population labeled with the affinity reagent are selected by any convenient affinity separation technique described above or known in the art. After separation, the separated cells can be collected in any suitable medium that maintains the viability of the cells, usually having a serum cushion at the bottom of the collection tube. A variety of media are commercially available that are frequently supplemented with fetal bovine serum, such as dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., and can be used according to the nature of the cells.

[0249] Compositions highly enriched for a cell type of interest, e.g., hematopoietic cells, are achieved in this manner. The cells are about 70%, about 75%, about 80%, about 85%, about 90%, or more of the cell composition, about 95% or more of the enriched cell composition, preferably about 95% or more of the enriched cell composition. In other words, the composition is a substantially pure composition of the cells of interest.

[0250] Cells to be transplanted into a genetically modified non - human animal may be transplanted immediately, whether they are a heterogeneous cell population or an enriched cell population. Alternatively, the cells may be frozen at liquid nitrogen temperature, stored for a long period of time, thawed, and reused. In such cases, the cells are usually frozen in 10% DMSO, 50% serum, 40% buffer medium, or some other such solution commonly used in the art to store cells at such freezing temperatures, and thawed in a manner generally known in the art for thawing frozen cultured cells. Additionally or alternatively, the cells may be cultured in vitro under various culture conditions. The culture medium may be liquid or semi - solid containing, for example, agar, methylcellulose, etc. The cell population may typically be conveniently suspended in a suitable nutrient medium such as Iscove's modified DMEM or RPMI - 1640 supplemented with fetal bovine serum (about 5 - 10%), L - glutamine, thiols, particularly 2 - mercaptoethanol, and antibiotics such as penicillin and streptomycin. The culture may contain growth factors to which the cells respond. Growth factors, as defined herein, are molecules that can promote cell survival, growth, and / or differentiation in either the culture or intact tissue by their specific effects on transmembrane receptors. Growth factors include polypeptide and non - polypeptide factors.

[0251] Cells can be genetically modified before transplantation into a genetically modified non-human animal, for example, to provide a selectable or tracking marker, to induce a genetic defect in the cell (e.g., for modeling a disease), to repair a genetic defect, or to ectopically express a gene in the cell (e.g., to determine whether such a modification affects the course of a disease). Cells can be genetically modified by transfection or transduction with a suitable vector, homologous recombination, or other appropriate techniques, such that the cells express the gene of interest or block the expression of an unwanted gene using antisense mRNA, siRNA, or ribozymes. A variety of techniques are known in the art for introducing nucleic acids into target cells. Various techniques can be used to verify that the cells have been genetically modified. The genome of the cells may be used with or without restriction and amplification. All of polymerase chain reaction; gel electrophoresis; restriction analysis; Southern, Northern, and Western blotting; sequencing, etc. can be used.The general methods in molecular and cell biochemistry for these and other purposes disclosed in this application can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. The reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0252] Cells can be transplanted into genetically modified non-human animals by any convenient method including, for example, intrahepatic injection, tail vein injection, retro-orbital injection, etc. Typically, about 0.5×10 5 ~2×10 6 pluripotent or progenitor cells, e.g., about 1×10 5 ~1×10 6 cells, or about 2×105 ~5×10 5 ~5×10 cells are transplanted. In some examples, the mouse is irradiated with a sub-lethal dose before transplantation of human cells. In other words, the mouse is exposed to a sub-lethal dose of radiation as described in the Examples section below and as is well known in the art. The engrafted genetically modified non-human animal is then maintained under experimental animal housing conditions for at least 1 week, e.g., 1 week or longer, or 2 weeks or longer, and sometimes 4 weeks or longer, and in some examples 6 weeks or longer, to allow sufficient reconstitution of the immune system by the engrafted cells.

[0253] In some embodiments, the transplanted human hematopoietic cells give rise to 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, 5, 6, 7, 8, 9, 10, 11, or 12 months after engraftment. In specific embodiments, the human cells include cells of the erythroid lineage.

[0254] In some embodiments, the transplanted human hematopoietic cells give rise to an engrafted human blood and lymphoid system that includes 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 the genetically modified non-human animal. In one embodiment, the human blood and lymphoid system is present 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after engraftment. In specific embodiments, the human blood and lymphoid system includes cells of the erythroid lineage.

[0255] Examples of erythroid lineage cells include erythrocytes and cells that give rise to erythrocytes. "Erythrocytes" include mature red blood cells, also known as red cells or red corpuscles. Cells that give rise to erythrocytes include erythroid progenitor cells, i.e., proliferative multipotent cells, and erythroid precursors, i.e., proliferative or non-proliferative cells committed to becoming erythrocytes.

[0256] Erythrocytes are the major cellular element of circulating blood and their major function is to carry oxygen. The number of erythrocytes per cubic millimeter of blood is normally maintained between 4.5 and 5.5 million in males and between 4.2 and 4.8 million in females. This varies with age, activity, and environmental conditions. For example, above an altitude of 10,000 feet, an increase to levels of usually 8 million / mm may occur. Erythrocytes normally survive for 110 - 120 days and are broken down by the reticuloendothelial system when removed from the bloodstream. New erythrocytes are produced at a rate slightly higher than 1% per day; thus, a constant level is normally maintained. Acute blood loss, hemolytic anemia, or chronic oxygen deprivation can cause a substantial increase in erythrocyte production.

[0257] Erythrocytes originate from hematopoietic stem cells in the bone marrow of long bones and develop through a continuous series of cell stages including common myeloid progenitor cells (CD123+, CD34+, c-kit+, Flt3+); megakaryocyte-erythroid progenitor cells (CD34+, CD38+, CD45RA-); proerythroblasts (normally also called pronormoblasts or, when abnormal, promegaloblasts; large CD71+, EpoR+, c-kit+, Terl19+ precursors); basophilic erythroblasts (cytoplasm is basophilic, nucleus is large with condensed chromatin and nucleoli are absent); polychromatophilic erythroblasts (also called intermediate normoblasts, showing increased condensed nuclear chromatin, cytoplasm begins to acquire hemoglobin and takes on an eosinophilic color); orthochromatic normoblasts (nucleus is small and finally becomes a homogeneous, unstructured mass of blue to black color, the final stage before nuclear loss); and reticulocytes (circulating CD235+, CD71+ cells; cells are characterized by a reticular pattern of threads and particles at the site of the former nucleus).

[0258] Mature erythrocytes appear on peripheral smears as biconcave circular or oval discs approximately 6 - 8 μm in diameter. They contain hemoglobin, have a central pale zone due to the biconcavity of the cell, and can be easily identified by flow cytometry or immunohistochemistry-based methods by the expression of the elevated cell surface markers CD235 and CD59 compared to non-erythroid cells.

[0259] In some embodiments, the genetically modified non-human animals provided herein have engrafted human hematopoietic cells and 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, Theileria. As described in more detail below, the genetically modified non-human animals with engrafted human hematopoietic cells can be infected with a human pathogen using any suitable method known in the art or described herein for infecting the animal with the pathogen of interest. Such infected animals typically exhibit signs of parasitemia, including altered morphology by Giemsa-stained blood smears, as well as severe decreases in total erythrocyte concentration (e.g., 50%) and anemia.

[0260] In some embodiments, the genetically modified non-human animals provided herein have engrafted human hematopoietic cells that contain a disease-specific mutation, such as a mutation in the β-globin gene that results in sickle cell disease. Non-limiting applications of genetically modified engrafted mice

[0261] The genetically modified non-human animals of the present disclosure find many uses 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 can be used to identify, for example, agents that can modulate (i.e., promote or inhibit) human erythropoiesis and / or the function of human erythrocytes in, for example, healthy or diseased states (e.g., as cancerous cells, during pathogen infection, etc.), and thus provide a useful system for screening candidate agents for a desired activity in vivo. For example, the engrafted genetically modified mice of the present disclosure can be used to identify novel therapeutic agents, or as another example, to identify agents that are toxic to erythroid lineage human cells, to identify agents that can prevent, mitigate, or reverse the toxic effects of toxic agents on erythroid lineage human cells, and the like. As another example, the engrafted genetically modified animals of the present disclosure provide an in vivo platform for screening, for example, the responsiveness of an individual's immune system to an agent, such as a therapeutic agent, and thus provide a useful system for predicting an individual's responsiveness to disease treatment by predicting the individual's responsiveness to the agent. As yet another example, hematopoietic stem and progenitor cells (HSPCs) derived from patients with genetic abnormalities in erythrocytes (e.g., sickle cell anemia, beta-thalassemia, etc.), or iPS-derived HSPCs having such genetic modifications, can be engrafted into HMOX-1-deficient HIS mice to model certain erythroid diseases and test potential therapeutic agents.

[0262] As a non-limiting example, the engrafted genetically modified mice of the present disclosure find use in the generation of mouse models of pathogen infection by parasites that target human erythroid cells, such as Plasmodium, Babesia, Theileria, and the like. Such mouse models of infection are useful in both research, for example, to better understand the progression of infection in humans, and in drug discovery, for example, to identify candidate agents that protect against or treat infection by such parasites.

[0263] 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 protozoa into the circulatory system via its saliva and ultimately into the liver, where the protozoa mature and reproduce. The protozoa then enter the bloodstream and infect cells of the erythroid lineage at various stages of maturity.

[0264] Five species of Plasmodium can infect humans and be transmitted by humans. Most deaths are caused by P. falciparum, while P. vivax, P. ovale, and P. malariae generally cause a milder form of malaria that is 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 maturities, while P. vivax, for example, is restricted to growth in reticulocytes, which correspond to only approximately 1% to 2% of total peripheral RBCs. In addition, P. falciparum causes severe malaria through a characteristic property not shared by any other human malaria, namely sequestration. Within the 48-hour asexual blood-stage cycle, the mature form changes the surface properties of the infected red blood cells, causing their adhesion to blood vessels (a process called cytoadhesion). This leads to the occlusion of the microcirculation and results in the dysfunction of multiple organs.

[0265] Symptoms of malaria include fever, chills, headache, sweating, fatigue, anemia, nausea, dry (non-productive) cough, muscle pain and / or back pain, and splenomegaly. Other symptoms and complications associated with malaria include cerebral infection (encephalitis), hemolytic anemia, kidney failure, liver failure, meningitis, pulmonary edema, and bleeding from the spleen. In general, individuals at risk of developing malaria begin to show symptoms 7 days or more after infection, for example, 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. malariae, or 11 - 12 days after initial infection with P. knowlesi. Anti-malarial agents used in the art to treat or prevent malaria include chloroquine, quinidine, doxycycline, tetracycline, clindamycin, atovaquone + proguanil (Malarone), mefloquine, artesunate, and pyrimethamine + sulfadoxine (Fansidar).

[0266] Methods for determining whether a subject is infected with Plasmodium are well known in the art and include, for example, microscopic examination of blood using a blood film, rapid diagnostic tests (RDTs) based on antigens, such as RDTs based on immunochromatography, detection of parasite DNA by polymerase chain reaction (PCR), etc. Any convenient method can be used to determine whether the subject's human red blood cells are infected with the pathogen.

[0267] HMOX-1 engrafted with HSC - / -The presence of human red blood cells in the peripheral blood of human immune system (HIS) mouse models provides a unique opportunity for modeling malaria using in vivo mouse models. Malaria, after infection, initiates a life cycle stage where sporozoites injected from a mosquito bite first infect the liver and then the emerging merozoites infect human RBCs (Vaughan et al. (2012) J Clin Invest 122(10):3618-28; Minkah et al (2018) Front Immunol. 9:807; Kaushansky et al. (2014) Cell Microbiol. 16(5):602-11; Good et al. (2015) Trends Parasitol. 31(11):583-94; Foquet et al. (2017) Methods Mol Biol. 1506:117-30). Human liver infection has been modeled in vivo using immunodeficient mice in which human hepatocytes can engraft, for example, mice with a deletion of fumarylacetoacetate hydrolase (FAH) that allows removal of the mouse liver and replacement with a human liver, but in vivo studies of malaria in mice are hampered by the lack of survival of human RBCs in mice (Vaughan et al. (2012) J Clin Invest 122(10):3618-28). By combining the HMOX-1-deficient HIS model with modifications that allow human liver engraftment, an excellent model is created for studying the complete malaria life cycle and testing potential treatments at all stages of malaria.

[0268] Therefore, in one embodiment, the HMOX-1-deficient MSRG47 mice or rats described herein are mated with a Fah− / −SRG model (FSRG) (e.g., see Carbonaro et al. (2023) Sci. Adv. 9, eadf4490; Carbonaro et al. (2022) Sci. Rep. 12:14079, both of which are incorporated herein by reference) to generate a human immune system model (e.g., after human HSC and human hepatocyte engraftment) that allows for the development of human RBCs, human white blood cells, and human hepatocytes. Such animals can be used to test potential malaria therapies, particularly immune-based therapies, which are envisioned in the methods for screening the potential therapies described herein.

[0269] Another example of a pathogen of interest is the protozoan of the genus Babesia. Babesia infection results in a malaria-like disease called babesiosis. Babesiosis is a vector-borne disease typically transmitted by the tick Ixodes scapularis. The disease is typically caused by B. microti in humans, B. canis rossi and B. canis canis in dogs, B. bovis in female cattle, and B. bigemina in cattle. Babesia microti, which infects humans, uses the same tick vector as Lyme disease and ehrlichiosis and may occur in conjunction with these other diseases. The protozoan can also be transmitted by blood transfusion.

[0270] In humans, babesiosis can be asymptomatic or can be characterized by symptoms ranging from mild fever and diarrhea to high fever, chills, and severe anemia. In severe cases, organ failure, including respiratory distress syndrome, may occur. Severe cases occur mainly in people who have had a splenectomy or who have immunodeficiency, such as HIV / AIDS patients. Treatment typically involves a two-drug regimen of quinine and clindamycin, or atovaquone and azithromycin. In cases where babesiosis is thought to be life-threatening, exchange transfusion is performed to remove infected red blood cells and replace them with uninfected red blood cells.

[0271] A definitive diagnosis of Babesia infection is made by identification of the parasite in a Giemsa-stained thin blood smear. The parasite appears in red blood cells as merozoite pairs that form a "Maltese cross" in humans or "two pears hanging together" in animals. Other diagnostic methods include PCR of peripheral blood and serological tests for antibodies (IgG, IgM) against Babesia.

[0272] Another malaria-like disease, Theileriosis, is caused by protozoa of the genus Theileria. In humans, Theileriosis is caused by T. microti; in horses, by T. equi ("equine piroplasmosis"); in sheep and goats, by T. lestoquardi; and in cattle, African buffalo, swine, and waterbuck, by T. annulata (also known as "tropical theileriosis", "Mediterranean theileriosis") or T. parva (also known as "East Coast fever", "corridor disease"). Theileriosis is transmitted to the host by various tick species including Ixodes scapularis, Rhipicephalus, Dermacentor, Haemaphysalis, and Hyalomma. The organism multiplies in the tick as its life stage progresses and matures and invades the saliva after the tick attaches to the host. Usually, the tick must attach for several days before becoming infectious. However, at high environmental temperatures, infectious sporozoites can develop in ticks on the ground and invade the host within hours of attachment.

[0273] Theileriosis in humans typically presents with fever and hemolysis. A definitive diagnosis of Theileria infection is made by identification of the parasite in a Giemsa-stained thin blood smear.

[0274] The engrafted genetically modified animals of the present disclosure are found to be useful in screening for candidate agents for preventing (e.g., as a vaccine) or treating infection by Plasmodium, Babesia, Theileria, and other parasites that target human red blood cells. The terms "treatment," "treating," etc. are used herein generally to include obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms and / or therapeutic in terms of partially or completely curing the disease and / or the adverse effects caused by the disease. "Treatment" as used herein includes any treatment of a disease in a mammal and includes (a) preventing the disease from occurring 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) alleviating the disease, i.e., causing regression of the disease. Candidate agents intended as anti-parasitic therapeutic agents are administered before, during, or after infection by the parasite and, when administered in an effective amount, can inhibit the effect of the parasite on the individual (i.e., the host), for example, by killing the parasite or the cells infected by the parasite, by preventing the transmission of the parasite, by preventing the production or action of agents (i.e., toxins) produced by the parasite that are toxic to the individual, etc. The terms "individual," "subject," "host," and "patient" are used interchangeably herein and include any mammalian subject for which diagnosis, treatment, or therapy is desired, particularly a human.

[0275] In a screening assay for a biologically active agent, the genetically modified non-human animals engrafted with the human hematopoietic cells of the present disclosure are contacted with a candidate agent of interest, and the effect of the candidate agent is evaluated by monitoring one or more output parameters. These output parameters can reflect, by methods well known in the art, the survival rate of human red blood cells, e.g., the total number of human red blood cells, or the apoptotic state of human red blood cells, e.g., the amount of cell blebbing, the amount of phosphatidylserine on the surface of human red blood cells, etc. Alternatively or additionally, the output parameter can reflect the effect of the agent on infection in human red blood cells of the genetically modified non-human animals engrafted with the human hematopoietic cells of the present disclosure.

[0276] Candidate agents for the screening or methods of the present disclosure can include, for example, organic molecules (e.g., small molecule inhibitors), nucleic acids (e.g., RNA interference agents, oligonucleotides, or nucleic acids encoding polypeptides), peptides, peptidomimetic inhibitors, aptamers, antibodies, intrabodies, etc. As used herein, "RNA interference agent" may be small interfering RNA (siRNA), CRISPR RNA (crRNA), CRISPR guide RNA (gRNA), small hairpin RNA (shRNA), microRNA (miRNA), or piwi-interacting RNA (piRNA).

[0277] Candidate agents are screened for biological activity, often in combination with a sample lacking the agent, by administering the agent to at least one, usually a plurality of samples. Changes in parameters responsive to the agent are measured and the results are evaluated by comparison, for example, with reference samples obtained with other agents in the presence and absence of the agent. In examples where the screening is being done to identify candidate agents that prevent, mitigate or reverse the effects of a pathogen, the screening is typically done in the presence of the pathogenic agent, where the pathogenic agent is added at the time most appropriate for the result to be determined. For example, where the defensive / preventive ability of a candidate agent is being tested, the candidate agent can be added before, simultaneously with, or after infection with the pathogen. As another example, where the ability of a candidate agent to reverse the effects of a pathogen is being tested, the candidate agent can be added after infection with the pathogen. As noted above, in some examples, the "sample" is a genetically modified non-human animal in which cells have engrafted, e.g., the candidate agent is provided to a genetically modified non-human animal in which human hematopoietic cells have engrafted. In some examples, the "sample" is the engrafted human hematopoietic cells, i.e., the candidate agent is provided to cells, e.g., reticulocytes, erythrocytes, etc., prior to engraftment into an immunodeficient genetically modified animal.

[0278] When the candidate agent is administered directly to the genetically modified animal in which it has engrafted, the agent can be administered by any of several methods well known in the art for the 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 intraperitoneal, subcutaneous, intramuscular, or intravenous injection. The agent may be administered in a buffer or incorporated into any of a variety of formulations, for example, in combination with a suitable pharmaceutically acceptable vehicle. A "pharmaceutically acceptable vehicle" can be a vehicle approved by a federal or state government regulatory authority for use in mammals such as humans or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which the compounds of the invention are formulated for administration to mammals. Such pharmaceutical vehicles can include lipids, such as liposomes, for example, liposome dendrimers; liquids, such as water, and oils, saline; including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil; acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, adjuvants, stabilizers, thickeners, lubricants, and coloring agents may be used. The pharmaceutical composition may be formulated in a preparation in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. The agent may be systemic after administration or may be local by use of local administration, administration within a wall, or use of an implant that acts to retain an active dose at the site of implantation. The active agent may be formulated for immediate activity or for sustained release. When the agent(s) is provided to the cell prior to engraftment, the agent is preferably added to the cell culture medium in solution or in a readily soluble form.The agent may be added in a flow-through system as a discontinuous or continuous stream, or alternatively, added separately as a single bolus or incremental bolus of the compound to a static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution and the other is the same solution with the added test compound. The first fluid passes over the cells and is followed by the second fluid. In the single-solution method, the bolus of the test compound is added to the volume of the medium surrounding the cells. The total concentration of the components of the culture medium should not change significantly due to the addition of the bolus or between the two solutions in the flow-through method.

[0279] Multiple assays may be run in parallel at different drug concentrations to obtain different responses to different concentrations. As is known in the art, determining the effective concentration of a drug typically involves using a range of concentrations obtained from a 1:10 or other logarithmic scale dilution. The concentration can be further refined, if necessary, in a second dilution series. Typically, one of these concentrations serves as a negative control, i.e., a zero concentration, or a concentration of the drug below the level of detection of the drug, or a concentration of the drug that gives no detectable change in the phenotype.

[0280] Analysis of the response of cells to a candidate agent in an engrafted genetically modified animal can be performed at any time point after treatment with the agent. For example, the cells can be analyzed 1, 2, or 3 days after contact with the candidate agent, sometimes 4, 5, or 6 days, sometimes 8, 9, or 10 days, sometimes 14 days, sometimes 21 days, sometimes 28 days, sometimes 1 month or longer, e.g., 2 months, 4 months, 6 months or longer. In some embodiments, the analysis includes analysis at multiple time points. The selection of the time point(s) for analysis is based on the type of analysis being performed, as will be readily understood by those skilled in the art.

[0281] The analysis can include measuring any of the parameters described herein or known in the art for measuring cell viability, cell proliferation, cell identity, cell morphology, and cell function, which may be particularly relevant to cells of the immune system. 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. Histochemical or immunohistochemical assays, such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) for measuring DNA fragmentation or immunohistochemical assays for detecting the binding of annexin V to phosphatidylserine on the cell surface, may be performed to determine the apoptotic state of the cells. Also, flow cytometry can be used to evaluate the proportion of differentiated cells and differentiated cell types, for example, to determine the ability of hematopoietic cells to survive and / or differentiate in the presence of a drug. ELISA, Western blot, and Northern blot can be performed to determine the levels of cytokines, chemokines, immunoglobulins, etc. expressed in engrafted genetically modified mice, for example, to evaluate the function of engrafted cells or to evaluate the survival of red blood cells. In vivo assays for testing the function of immune cells and assays related to a particular disease or disorder of interest, such as anemia, for example, sickle cell anemia, can also be performed. For example, reference is made to 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.

[0282] Thus, for example, a method is provided for determining the effect of an agent on pathogen-infected or infectable erythroid cells, the method including: administering the agent to genetically modified non-human animals of the disclosure in which human reticulocytes and / or erythrocytes have engrafted; measuring a parameter of the survival rate of the engrafted cells over time in the presence of the agent; and comparing the measured value with a measured value from genetically modified non-human animals that have engrafted and have not been exposed to the agent. The agent is determined to be anti-pathogenic if it reduces the infection and / or death of human erythrocytes in the peripheral blood of mice by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% (i.e., to an undetectable amount) after one or more administrations of the agent over a selected period. In a specific embodiment, the administration of the drug or drug combination is for at least 3 days, at least 1 week, at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 10 weeks after engraftment by human hematopoietic cells.

[0283] Other examples of uses for the subject mice are provided elsewhere in this specification. Additional applications of the genetically modified, engrafted mice described in this disclosure will be apparent to those skilled in the art upon reading this disclosure. Additional exemplary embodiments

[0284] In Exemplary Embodiment 1, there is provided herein a genetically modified non-human animal comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

[0285] In Exemplary Embodiment 2, there is provided herein a genetically modified non-human animal of Embodiment 1, comprising a homozygous null mutation in the Rag1 gene.

[0286] In exemplary embodiment 3, there is provided herein a genetically modified non-human animal of embodiment 1 or 2, wherein the null mutation is a deletion of at least the exons corresponding to mouse Hmox-1 exons 3-5.

[0287] In exemplary embodiment 4, there is provided herein a genetically modified non-human animal of any one of embodiments 1-3, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0288] In exemplary embodiment 5, there is provided herein a genetically modified non-human animal of any one of embodiments 1-4, comprising a homozygous null mutation in the Fah gene.

[0289] In exemplary embodiment 6, there is provided herein a genetically modified non-human animal of embodiment 5, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion, and / or substitution in the endogenous Fah gene.

[0290] In exemplary embodiment 7, there is provided herein a genetically modified non-human animal of any one of embodiments 1-6, expressing a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to the Sirpa promoter.

[0291] In exemplary embodiment 8, there is provided herein a genetically modified non-human animal of embodiment 7, wherein the genetically modified non-human animal comprises a Sirpa gene encoding a Sirpa polypeptide comprising the extracellular portion of the human SIRPA polypeptide and the intracellular portion of the non-human animal Sirpa polypeptide, and the Sirpa gene is operably linked to the Sirpa promoter.

[0292] In exemplary embodiment 9, there is provided herein a genetically modified non-human animal of embodiment 8, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

[0293] In exemplary embodiment 10, there is provided herein a genetically modified non-human animal expressing a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, of embodiment 8 or 9.

[0294] In exemplary embodiment 11, there is provided herein a genetically modified non-human animal of any one of embodiments 8-10, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

[0295] In exemplary embodiment 12, there is provided herein a genetically modified non-human animal of embodiment 7 expressing a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0296] In exemplary embodiment 13, there is provided herein a genetically modified non-human animal of any one of embodiments 7-12 further expressing one or more human or humanized proteins selected from the group consisting of a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0297] In exemplary embodiment 14, there is provided herein a genetically modified non-human animal of any one of embodiments 7-13, wherein at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter.

[0298] In exemplary embodiment 15, there is provided herein a genetically modified non-human animal of embodiment 14, wherein all promoters operably linked to a nucleic acid encoding a human or humanized protein are endogenous non-human animal promoters.

[0299] In exemplary embodiment 16, there is provided herein a genetically modified non-human animal of embodiment 14 or 15, wherein the endogenous non-human animal promoter is at the locus of the corresponding non-human animal gene.

[0300] In exemplary embodiment 17, there is provided herein a genetically modified non-human animal of any one of embodiments 7-16, which comprises a null mutation in at least one corresponding non-human animal gene at the locus of the corresponding non-human animal gene.

[0301] In exemplary embodiment 18, there is provided herein a genetically modified non-human animal of any one of embodiments 7-17, which is heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0302] In exemplary embodiment 19, there is provided herein a genetically modified non-human animal of any one of embodiments 7-17, which is homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0303] In exemplary embodiment 20, there is provided herein a genetically modified non-human animal of any one of embodiments 7-19, which expresses human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

[0304] In exemplary embodiment 21, provided herein is a genetically modified non-human animal of any one of embodiments 7-20 that expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0305] In exemplary embodiment 22, provided herein is a genetically modified non-human animal of embodiment 21 that expresses a humanized CD47 protein, wherein the humanized CD47 protein comprises an extracellular portion of the human CD47 protein and an intracellular portion of the endogenous non-human animal CD47 protein.

[0306] In exemplary embodiment 23, provided herein is a genetically modified non-human animal of any one of embodiments 7-22 that expresses (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0307] In exemplary embodiment 24, provided herein is a genetically modified non-human animal of any one of embodiments 7-23 that expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0308] In exemplary embodiment 25, provided herein is a genetically modified non-human animal of any one of embodiments 1-24 that further comprises engraftment of human hematopoietic cells.

[0309] In exemplary embodiment 26, provided herein is a genetically modified non-human animal of embodiment 25, wherein the human hematopoietic cells comprise one or more cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human hematopoietic progenitor cells, human erythroid progenitor cells, and human erythrocytes.

[0310] In exemplary embodiment 27, provided herein is a genetically modified non-human animal of embodiment 36, wherein the animal comprises human cells of the erythroid lineage.

[0311] In exemplary embodiment 28, provided herein is a genetically modified non-human animal of embodiment 27, wherein the non-human animal further comprises an infection by a pathogen that targets human cells of the erythroid lineage.

[0312] In exemplary embodiment 29, provided herein is a genetically modified non-human animal of any one of embodiments 25 - 28, wherein the animal comprises an inactivated endogenous FAH gene and further comprises transplanted human hepatocytes.

[0313] In exemplary embodiment 30, provided herein is a genetically modified non-human animal of embodiment 28 or embodiment 29, wherein the pathogen can cause malaria in humans.

[0314] In exemplary embodiment 31, provided herein is a genetically modified non-human animal of embodiment 28, wherein the pathogen is selected from Plasmodium sp., Babesia sp., and Theileri sp.

[0315] In exemplary embodiment 32, provided herein is a genetically modified non-human animal of any one of embodiments 25 - 27, wherein the engrafted human hematopoietic cells give rise to abnormal human cells of the erythroid lineage.

[0316] In exemplary embodiment 33, provided herein is a genetically modified non-human animal of embodiment 32, wherein the engrafted human hematopoietic cells comprise a mutation in the β-globin gene that results in sickle cell disease.

[0317] In exemplary embodiment 34, provided herein is a genetically modified non-human animal of any one of embodiments 1 - 33, which is a mammal.

[0318] In exemplary embodiment 35, there is provided a genetically modified non-human animal of embodiment 34, wherein the mammal is a rodent, such as a rat or a mouse.

[0319] In exemplary embodiment 36, there is provided a genetically modified non-human animal of embodiment 35, wherein the rodent is a mouse.

[0320] In exemplary embodiment 37, there is provided a method for identifying an agent that inhibits infection by a pathogen targeting human cells of the erythroid lineage, the method comprising: a. administering the agent to a genetically modified non-human animal, wherein the genetically modified non-human animal has: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. homozygous null mutations in the Rag2 gene and the IL2rg gene; iii. engraftment of human hematopoietic cells; and iv. infection by a pathogen targeting human cells of the erythroid lineage; and b. determining whether the agent reduces the amount of the pathogen and / or inhibits the activity of the pathogen in the genetically modified non-human animal infected with the pathogen.

[0321] In exemplary embodiment 38, there is provided a method for identifying an agent that prevents infection by a pathogen targeting human cells of the erythroid lineage, the method comprising: a. administering the agent to a genetically modified non-human animal, wherein the genetically modified non-human animal has: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. homozygous null mutations in the Rag2 gene and the IL2rg gene; and iii. engraftment of human hematopoietic cells; b. injecting parasitized reticulocytes or erythrocytes into the genetically modified non-human animal; and c. determining whether the agent prevents infection of human reticulocytes and / or erythrocytes of the genetically modified non-human animal.

[0322] In exemplary embodiment 39, methods of embodiments 37 or 38 are provided herein, wherein the pathogen can cause malaria in humans.

[0323] In exemplary embodiment 40, methods of embodiments 37 or 38 are provided herein, wherein the pathogen is selected from Plasmodium sp., Babesia sp., and Theileri sp.

[0324] In exemplary embodiment 41, a method for identifying an agent for treating sickle cell disease, comprising: a. administering the agent to a genetically modified non-human animal, wherein the genetically modified non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. engraftment of human hematopoietic cells comprising a mutation in the β-globin gene that results in sickle cell disease; and b. determining whether the agent prevents or reduces sickling of red blood cells in the non-human animal.

[0325] In exemplary embodiment 42, a method for evaluating the therapeutic efficacy of a drug candidate that targets human red blood cells, comprising: a. administering the drug candidate to a genetically modified non-human animal, wherein the genetically modified non-human animal comprises: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. engraftment of human hematopoietic progenitor cells; and b. monitoring human red blood cells in the non-human animal to evaluate the therapeutic efficacy of the drug candidate. Methods are provided herein.

[0326] In exemplary embodiment 43, the method of embodiment 42 is provided herein, wherein human red blood cells are monitored to determine whether the generation and / or survival of human red blood cells in a non-human animal is increased by a drug candidate.

[0327] In exemplary embodiment 44, a method for evaluating the toxicity of a drug candidate to human red blood cells, comprising: a. administering the drug candidate to a genetically modified non-human animal, wherein the genetically modified non-human animal has: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. engraftment of human hematopoietic progenitor cells; and b. monitoring human red blood cells in the non-human animal to evaluate the toxicity of the drug candidate. A method is provided herein.

[0328] In exemplary embodiment 45, an embodiment 44 method is provided herein, wherein the toxicity is on-target toxicity or off-target toxicity.

[0329] In exemplary embodiment 46, an embodiment 44 or 45 method is provided herein, wherein human red blood cells are monitored to determine whether the number of human red blood cells in the non-human animal is reduced by the drug candidate.

[0330] In exemplary embodiment 47, an embodiment 46 method is provided herein, wherein the drug candidate is a chemotherapeutic agent or an antimalarial agent.

[0331] In exemplary embodiment 48, an embodiment 44 or 45 method is provided herein, wherein human red blood cells are monitored to evaluate whether the drug candidate induces aggregation of red blood cells.

[0332] In exemplary embodiment 49, an embodiment 48 method is provided herein, wherein the drug candidate is a modulator of the human CD47 protein.

[0333] In exemplary embodiment 50, an embodiment 49 method is provided herein, wherein the modulator is an antibody.

[0334] In exemplary embodiment 51, a method for identifying an agent that reduces the toxicity of a toxic drug to human red blood cells, comprising: a. administering an agent and a toxic drug to a genetically modified non-human animal, wherein the genetically modified non-human animal has: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the IL2rg gene; and iii. engraftment of human hematopoietic progenitor cells, and b. determining whether the agent reduces the toxicity of the toxic drug to human red blood cells in the non-human animal, is provided herein.

[0335] In exemplary embodiment 52, a method according to embodiment 51 is provided herein, wherein the agent and the toxic drug are administered to the non-human animal either concomitantly or sequentially.

[0336] In exemplary embodiment 53, a method according to any one of embodiments 37 to 52 is provided herein, wherein the genetically modified non-human animal comprises a homozygous null mutation in the Rag1 gene.

[0337] In exemplary embodiment 54, a method according to any one of embodiments 37 to 53 is provided herein, wherein the null mutation is a deletion of at least the exon corresponding to mouse Hmox-1 exons 3-5.

[0338] In exemplary embodiment 55, a method according to any one of embodiments 37 to 54 is provided herein, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0339] In exemplary embodiment 56a, a method according to any one of embodiments 37 to 55 is provided herein, wherein the genetically modified non-human animal comprises engraftment of human hepatocytes.

[0340] In exemplary embodiment 56b, a method according to any one of embodiments 37 to 56a is provided herein, wherein the genetically modified non-human animal comprises a homozygous null mutation in the Fah gene.

[0341] In exemplary embodiment 57, a method of embodiment 56b is provided herein, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion, and / or substitution in the endogenous Fah gene.

[0342] In exemplary embodiment 58, a method of any one of embodiments 37-57 is provided herein, wherein the genetically modified non-human animal expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0343] In exemplary embodiment 59, a method of embodiment 58 is provided herein, wherein the genetically modified non-human animal comprises a Sirpa gene encoding a Sirpa polypeptide comprising the extracellular portion of the human SIRPA polypeptide and the intracellular portion of the non-human animal Sirpa polypeptide, and the Sirpa gene is operably linked to a Sirpa promoter.

[0344] In exemplary embodiment 60, a method of embodiment 59 is provided herein, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

[0345] In exemplary embodiment 61, a method of embodiment 59 or 60 is provided herein, wherein the genetically modified non-human animal expresses a Sirpa polypeptide comprising the extracellular portion of the human SIRPA polypeptide and the intracellular portion of the non-human animal Sirpa polypeptide.

[0346] In exemplary embodiment 62, a method of any one of embodiments 59-61 is provided herein, wherein the non-human animal Sirpa polypeptide is the endogenous non-human animal Sirpa polypeptide and / or the non-human animal Sirpa gene is the endogenous non-human animal gene.

[0347] In exemplary embodiment 63, provided herein is the method of embodiment 58, wherein the genetically modified non-human animal expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0348] In exemplary embodiment 64, provided herein is any one of the methods of embodiments 58 - 63, wherein the genetically modified non-human animal further expresses one or more human or humanized proteins selected from the group consisting of: a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0349] In exemplary embodiment 65, provided herein is any one of the methods of embodiments 58 - 64, wherein at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter.

[0350] In exemplary embodiment 66, provided herein is the method of embodiment 65, wherein all promoters operably linked to a nucleic acid encoding a human or humanized protein are endogenous non-human animal promoters.

[0351] In exemplary embodiment 67, provided herein is the method of embodiment 65 or 66, wherein the endogenous non-human animal promoter is at the locus of the corresponding non-human animal gene.

[0352] In exemplary embodiment 68, a method according to any one of embodiments 58-67 is provided herein, wherein the genetically modified non-human animal comprises a null mutation in at least one corresponding non-human animal gene at the locus of the corresponding non-human animal gene.

[0353] In exemplary embodiment 69, a method according to any one of embodiments 58-68 is provided herein, wherein the genetically modified non-human animal is heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0354] In exemplary embodiment 70, a method according to any one of embodiments 58-69 is provided herein, wherein the genetically modified non-human animal is homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0355] In exemplary embodiment 71, a method according to any one of embodiments 58-70 is provided herein, wherein the genetically modified non-human animal expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

[0356] In exemplary embodiment 72, a method according to any one of embodiments 58-71 is provided herein, wherein the genetically modified non-human animal expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0357] In exemplary embodiment 73, a method according to embodiment 72 is provided herein, wherein the genetically modified non-human animal expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of the human CD47 protein and an intracellular portion of the endogenous non-human animal CD47 protein.

[0358] In exemplary embodiment 74, there is provided herein a method according to any one of embodiments 58 - 73, wherein the genetically modified non - human animal expresses: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M - CSF protein encoded by a nucleic acid operably linked to an M - CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0359] In exemplary embodiment 75, there is provided herein a method according to any one of embodiments 58 - 74, wherein the genetically modified non - human animal expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0360] In exemplary embodiment 76, there is provided herein a method according to any one of embodiments 37 - 75, wherein the genetically modified non - human animal is a mammal.

[0361] In exemplary embodiment 77, there is provided herein a method according to embodiment 76, wherein the mammal is a rodent, such as a rat or a mouse.

[0362] In exemplary embodiment 78, there is provided herein a method according to embodiment 77, wherein the rodent is a mouse.

[0363] In exemplary embodiment 79, there are provided herein genetically modified non - human animal cells comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non - human animal heme oxygenase - 1 (Hmox - 1) gene.

[0364] In exemplary embodiment 80, there are provided herein genetically modified non - human animal cells according to embodiment 79, comprising a homozygous null mutation in the Rag1 gene.

[0365] In exemplary embodiment 81, there is provided a genetically modified non-human animal cell of embodiment 79 or 80, wherein the null mutation is a deletion of at least the exon corresponding to mouse Hmox-1 exons 3-5.

[0366] In exemplary embodiment 82, there is provided a genetically modified non-human animal cell of any one of embodiments 79-81, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0367] In exemplary embodiment 83, there is provided a genetically modified non-human animal cell of any one of embodiments 79-82, which comprises a homozygous null mutation in the Fah gene.

[0368] In exemplary embodiment 84, there is provided a genetically modified non-human animal cell of embodiment 83, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion, and / or substitution in the endogenous Fah gene.

[0369] In exemplary embodiment 85, there is provided a genetically modified non-human animal cell of any one of embodiments 79-84, which expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to the Sirpa promoter.

[0370] In exemplary embodiment 86, there is provided a genetically modified non-human animal cell of embodiment 85, wherein the genetically modified non-human animal cell comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, and the Sirpa gene is operably linked to the Sirpa promoter.

[0371] In exemplary embodiment 87, there is provided a genetically modified non-human animal cell of embodiment 86, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

[0372] In exemplary embodiment 88, provided herein are the genetically modified non-human animal cells of embodiment 86 or 87 that express a Sirpa polypeptide comprising the extracellular portion of a human SIRPA polypeptide and the intracellular portion of a non-human animal Sirpa polypeptide.

[0373] In exemplary embodiment 89, provided herein are the genetically modified non-human animal cells of any one of embodiments 86-88, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

[0374] In exemplary embodiment 90, provided herein are the genetically modified non-human animal cells of embodiment 85 that express a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0375] In exemplary embodiment 91, provided herein are the genetically modified non-human animal cells of any one of embodiments 85-90 that further express one or more human or humanized proteins selected from the group consisting of a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0376] In exemplary embodiment 92, there is provided a genetically modified non-human animal cell of any one of embodiments 85-91, wherein at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter.

[0377] In exemplary embodiment 93, there is provided a genetically modified non-human animal cell of embodiment 92, wherein all promoters operably linked to a nucleic acid encoding a human or humanized protein are endogenous non-human animal promoters.

[0378] In exemplary embodiment 94, there is provided a genetically modified non-human animal cell of embodiment 92 or 93, wherein the endogenous non-human animal promoter is at the locus of the corresponding non-human animal gene.

[0379] In exemplary embodiment 95, there is provided a genetically modified non-human animal cell of any one of embodiments 85-94, which comprises a null mutation in at least one corresponding non-human animal gene at the locus of the corresponding non-human animal gene.

[0380] In exemplary embodiment 96, there is provided a genetically modified non-human animal cell of any one of embodiments 85-95, which is heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0381] In exemplary embodiment 97, there is provided a genetically modified non-human animal cell of any one of embodiments 85-95, which is homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0382] In exemplary embodiment 98, there is provided a genetically modified non-human animal cell of any one of embodiments 85-97, which expresses human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

[0383] In exemplary embodiment 99, provided herein are genetically modified non-human animal cells of any one of embodiments 85-98 that express a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0384] In exemplary embodiment 100, provided herein are genetically modified non-human animal cells of embodiment 99 that express a humanized CD47 protein, wherein the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

[0385] In exemplary embodiment 101, provided herein are genetically modified non-human animal cells of any one of embodiments 85-100 that express: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0386] In exemplary embodiment 102, provided herein are genetically modified non-human animal cells of any one of embodiments 85-101 that express a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0387] In exemplary embodiment 103, provided herein are genetically modified non-human animal cells of any one of embodiments 79-102 that are mammalian cells.

[0388] In exemplary embodiment 104, provided herein are genetically modified non-human animal cells of embodiment 103, wherein the mammalian cells are rodent cells, e.g., rat cells or mouse cells.

[0389] In exemplary embodiment 105, there is provided a genetically modified non-human animal cell of embodiment 104, wherein the rodent cell is a mouse cell.

[0390] In exemplary embodiment 106, there is provided a genetically modified non-human animal cell of any one of embodiments 79-84, 86-87, 89, 92-97, and 103-105, wherein the non-human animal cell is a non-human animal embryonic stem (ES) cell.

[0391] In exemplary embodiment 107, there is provided a genetically modified non-human animal embryonic stem cell having, in its genome, (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

[0392] In exemplary embodiment 108, there is provided a non-human animal embryonic stem cell of embodiment 107, wherein the non-human animal embryonic stem cell has, in its genome, a homozygous null mutation in the Rag1 gene.

[0393] In exemplary embodiment 109, there is provided a genetically modified non-human animal embryonic stem cell of embodiment 107 or 108, wherein the null mutation is a deletion of at least the exon corresponding to mouse Hmox-1 exons 3-5.

[0394] In exemplary embodiment 110, there is provided a genetically modified non-human animal embryonic stem cell of any one of embodiments 107-109, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0395] In exemplary embodiment 111, there is provided a genetically modified non-human animal embryonic stem cell of any one of embodiments 107-110, wherein the non-human animal embryonic stem cell has, in its genome, a homozygous null mutation in the Fah gene.

[0396] In exemplary embodiment 112, there are provided gene-modified non-human animal embryonic stem cells of embodiment 111, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion, and / or substitution in the endogenous Fah gene.

[0397] In exemplary embodiment 113, there are provided non-human animal embryonic stem cells of any one of embodiments 107-112, wherein the non-human animal embryonic stem cells comprise, in their genome, a nucleic acid encoding a human or humanized SIRPA polypeptide, and the nucleic acid is operably linked to a Sirpa promoter.

[0398] In exemplary embodiment 114, there are provided gene-modified non-human animal embryonic stem cells of embodiment 113, wherein the gene-modified non-human animal embryonic stem cells comprise a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, and the Sirpa gene is operably linked to a Sirpa promoter.

[0399] In exemplary embodiment 115, there are provided gene-modified non-human animal embryonic stem cells of embodiment 114, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

[0400] In exemplary embodiment 116, there are provided non-human animal embryonic stem cells of any one of embodiments 114-115, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide.

[0401] In exemplary embodiment 117, there are provided non-human animal embryonic stem cells of any one of embodiments 114-115, wherein the non-human animal Sirpa gene is an endogenous non-human animal gene.

[0402] In exemplary embodiment 118, there is provided a non-human animal embryonic stem cell of embodiment 113, wherein the genetically modified non-human animal embryonic stem cell comprises a nucleic acid encoding a human SIRPA polypeptide, and the nucleic acid is operably linked to a Sirpa promoter.

[0403] In exemplary embodiment 119, there is provided a non-human animal embryonic stem cell of any one of embodiments 113-118, wherein the genetically modified non-human animal embryonic stem cell further comprises one or more nucleic acids selected from the group consisting of a nucleic acid encoding a human TPO protein and operably linked to a TPO promoter; a nucleic acid encoding a human GM-CSF protein and operably linked to a GM-CSF promoter; a nucleic acid encoding a human IL3 protein and operably linked to an IL3 promoter; a nucleic acid encoding a human IL15 protein and operably linked to an IL15 promoter; a nucleic acid encoding a human M-CSF protein and operably linked to an M-CSF promoter; a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter; and a nucleic acid encoding a human EPO protein and operably linked to an EPO promoter.

[0404] In exemplary embodiment 120, there is provided a non-human animal embryonic stem cell of any one of embodiments 113-119, wherein at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter.

[0405] In exemplary embodiment 121, there is provided a non-human animal embryonic stem cell of embodiment 120, wherein all promoters operably linked to a nucleic acid encoding a human or humanized protein are endogenous non-human animal promoters.

[0406] In exemplary embodiment 122, there is provided a non-human animal embryonic stem cell of embodiment 120 or 121, wherein the endogenous non-human animal promoter is at the locus of the corresponding non-human animal gene.

[0407] In exemplary embodiment 123, provided herein are non-human animal embryonic stem cells of any one of embodiments 113-122, comprising a null mutation in at least one corresponding non-human animal gene at the locus of the corresponding non-human animal gene.

[0408] In exemplary embodiment 124, provided herein are non-human animal embryonic stem cells of any one of embodiments 113-123, which are heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0409] In exemplary embodiment 125, provided herein are non-human animal embryonic stem cells of any one of embodiments 113-123, which are homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0410] In exemplary embodiment 126, provided herein are non-human animal embryonic stem cells of any one of embodiments 113-125, wherein the genetically modified non-human animal embryonic stem cells comprise, in their genome, a nucleic acid encoding human M-CSF protein and operably linked to an M-CSF promoter.

[0411] In exemplary embodiment 127, provided herein are non-human animal embryonic stem cells of any one of embodiments 113-126, wherein the genetically modified non-human animal embryonic stem cells comprise, in their genome, a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter.

[0412] In exemplary embodiment 128, provided herein are non-human animal embryonic stem cells of embodiment 127, wherein the genetically modified non-human animal cell embryonic stem cells comprise, in their genome, a nucleic acid encoding a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of the human CD47 protein and an intracellular portion of the endogenous non-human animal CD47 protein.

[0413] In exemplary embodiment 129, there is provided herein a non-human animal embryonic stem cell of any one of embodiments 113-128, wherein the genetically modified non-human animal embryonic stem cell comprises, in its genome, (i) a nucleic acid encoding a human or humanized SIRPA protein and operably linked to a Sirpa promoter; (ii) a nucleic acid encoding a human M-CSF protein and operably linked to an M-CSF promoter; and (iii) a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter.

[0414] In exemplary embodiment 130, there is provided herein a non-human animal embryonic stem cell of any one of embodiments 113-129, wherein the genetically modified non-human animal embryonic stem cell comprises, in its genome, a nucleic acid encoding a human EPO protein and operably linked to an EPO promoter.

[0415] In exemplary embodiment 131, there is provided herein a non-human animal embryonic stem cell of any one of embodiments 107-130, wherein the genetically modified non-human animal embryonic stem cell is a mammalian embryonic stem cell.

[0416] In exemplary embodiment 132, there is provided herein the non-human animal embryonic stem cell of embodiment 131, wherein the mammalian embryonic stem cell is a rodent embryonic stem cell, such as a rat embryonic stem cell or a mouse embryonic stem cell.

[0417] In exemplary embodiment 133, there is provided herein the non-human animal embryonic stem cell of embodiment 132, wherein the rodent embryonic stem cell is a mouse embryonic stem cell.

[0418] In exemplary embodiment 134, provided herein is a method of generating non-human animal embryonic stem cells, the method comprising the step of genetically engineering non-human animal embryonic stem cells such that the non-human animal embryonic stem cells have a genome comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

[0419] In exemplary embodiment 135, provided herein is the method of embodiment 134, wherein the non-human animal embryonic stem cells are further engineered such that their genome comprises a homozygous null mutation in the Rag1 gene.

[0420] In exemplary embodiment 136, provided herein is the method of embodiment 134 or 135, wherein the null mutation is a deletion of at least the exons corresponding to mouse Hmox-1 exons 3-5.

[0421] In exemplary embodiment 137, provided herein is any one of the methods of embodiments 134-136, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0422] In exemplary embodiment 138, provided herein is any one of the methods of embodiments 134-137, wherein the non-human animal embryonic stem cells are further engineered such that their genome comprises a homozygous null mutation in the Fah gene.

[0423] In exemplary embodiment 139, provided herein is the method of embodiment 138, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion, and / or substitution in the endogenous Fah gene.

[0424] In exemplary embodiment 140, the non-human animal embryonic stem cells are further engineered to contain in their genome a nucleic acid encoding a human or humanized SIRPA polypeptide, and the nucleic acid is operably linked to a Sirpa promoter, and a method according to any one of embodiments 134-139 is provided herein.

[0425] In exemplary embodiment 141, the genetically modified non-human animal embryonic stem cells contain a Sirpa gene encoding a Sirpa polypeptide that includes the extracellular portion of the human SIRPA polypeptide and the intracellular portion of the non-human animal Sirpa polypeptide, and the Sirpa gene is operably linked to a Sirpa promoter, and a method according to embodiment 140 is provided herein.

[0426] In exemplary embodiment 142, the Sirpa gene includes exons 2-4 of the human SIRPA gene, and a method according to embodiment 141 is provided herein.

[0427] In exemplary embodiment 143, the non-human animal Sirpa polypeptide is the endogenous non-human animal Sirpa polypeptide, and a method according to any one of embodiments 141 or 142 is provided herein.

[0428] In exemplary embodiment 144, the non-human animal Sirpa gene is the endogenous non-human animal gene, and a method according to any one of embodiments 141 or 142 is provided herein.

[0429] In exemplary embodiment 145, the genetically modified non-human animal embryonic stem cells contain a nucleic acid encoding a human SIRPA polypeptide, and the nucleic acid is operably linked to a Sirpa promoter, and a method according to embodiment 140 is provided herein.

[0430] In exemplary embodiment 146, the genetically modified non-human animal embryonic stem cell has, in its genome, a nucleic acid encoding a human TPO protein and operably linked to a TPO promoter; a nucleic acid encoding a human GM-CSF protein and operably linked to a GM-CSF promoter; a nucleic acid encoding a human IL3 protein and operably linked to an IL3 promoter; a nucleic acid encoding a human IL15 protein and operably linked to an IL15 promoter; a nucleic acid encoding a human M-CSF protein and operably linked to an M-CSF promoter; a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter; and a nucleic acid encoding a human EPO protein and operably linked to an EPO promoter, and is further engineered to contain one or more nucleic acids selected from the group consisting of: Provided herein is any one of methods of embodiments 140-145.

[0431] In exemplary embodiment 147, at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter. Provided herein is any one of methods of embodiments 140-146.

[0432] In exemplary embodiment 148, all promoters operably linked to a nucleic acid encoding a human or humanized protein are endogenous non-human animal promoters. Provided herein is the method of embodiment 147.

[0433] In exemplary embodiment 149, the endogenous non-human animal promoter is at the locus of the corresponding non-human animal gene. Provided herein is the method of embodiment 147 or 148.

[0434] In exemplary embodiment 150, any one of methods of embodiments 140-149 includes a null mutation in at least one corresponding non-human animal gene at the locus of the corresponding non-human animal gene. Provided herein is any one of methods of embodiments 140-149.

[0435] In exemplary embodiment 151, a method according to any one of embodiments 140 - 150 is provided herein, wherein the genetically modified non - human animal cell is heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0436] In exemplary embodiment 152, a method according to any one of embodiments 140 - 150 is provided herein, wherein the genetically modified non - human animal cell is homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0437] In exemplary embodiment 153, a method according to any one of embodiments 140 - 152 is provided herein, wherein the genetically modified non - human animal embryonic stem cell is engineered to comprise in its genome a nucleic acid encoding human M - CSF protein and operably linked to an M - CSF promoter.

[0438] In exemplary embodiment 154, a method according to any one of embodiments 140 - 153 is provided herein, wherein the genetically modified non - human animal embryonic stem cell is engineered to comprise in its genome a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter.

[0439] In exemplary embodiment 155, a method according to embodiment 154 is provided herein, wherein the genetically modified non - human animal cell embryonic stem cell comprises in its genome a nucleic acid encoding a humanized CD47 protein, and the humanized CD47 protein comprises the extracellular portion of the human CD47 protein and the intracellular portion of the endogenous non - human animal CD47 protein.

[0440] In exemplary embodiment 156, there is provided herein a method according to any one of embodiments 140 - 155, wherein a genetically modified non - human animal embryonic stem cell is engineered to contain in its genome: (i) a nucleic acid encoding a human or humanized SIRPA protein and operably linked to a Sirpa promoter; (ii) a nucleic acid encoding a human M - CSF protein and operably linked to an M - CSF promoter; and (iii) a nucleic acid encoding a human or humanized CD47 protein and operably linked to a CD47 promoter.

[0441] In exemplary embodiment 157, there is provided herein a method according to any one of embodiments 140 - 156, wherein a genetically modified non - human animal embryonic stem cell is engineered to contain in its genome a nucleic acid encoding a human EPO protein and operably linked to an EPO promoter.

[0442] In exemplary embodiment 158, there is provided herein a method according to any one of embodiments 134 - 157, wherein the genetically modified non - human animal embryonic stem cell is a mammalian embryonic stem cell.

[0443] In exemplary embodiment 159, there is provided herein the method of embodiment 158, wherein the mammalian embryonic stem cell is a rodent embryonic stem cell, such as a rat embryonic stem cell or a mouse embryonic stem cell.

[0444] In exemplary embodiment 160, there is provided herein the method of embodiment 159, wherein the rodent embryonic stem cell is a mouse embryonic stem cell.

[0445] In exemplary embodiment 161, there is provided herein a non - human animal embryo comprising a non - human animal embryonic stem cell according to any one of embodiments 106 - 133, or a non - human animal embryonic stem cell produced according to the method of any one of embodiments 134 - 160.

[0446] In exemplary embodiment 162, a method of producing a non-human animal comprising in its genome: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, the method comprising: (a) obtaining a non-human animal embryonic stem cell of any one of embodiments 106-133, or a non-human animal embryonic stem cell produced according to the method of any one of embodiments 134-160; and (b) using the non-human animal embryonic cell of (a) to create a non-human animal is provided herein.

[0447] In exemplary embodiment 163, a method of producing a non-human animal comprising in its genome: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, the method comprising modifying the genome of the non-human animal such that it comprises: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene is provided herein.

[0448] In exemplary embodiment 164, the method of embodiment 163 is provided herein, wherein the genetically modified non-human animal is further engineered to comprise a homozygous null mutation in the Rag1 gene.

[0449] In exemplary embodiment 165, the method of embodiment 163 or 164 is provided herein, wherein the null mutation is at least a deletion of the exon corresponding to mouse Hmox-1 exons 3-5.

[0450] In exemplary embodiment 166, the method of any one of embodiments 163-165 is provided herein, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

[0451] In exemplary embodiment 167, provided herein is a method according to any one of embodiments 163 - 166, wherein the genetically modified non - human animal is further engineered to contain a homozygous null mutation in the Fah gene.

[0452] In exemplary embodiment 168, provided herein is a method according to embodiment 167, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion, and / or substitution in the endogenous Fah gene.

[0453] In exemplary embodiment 169, provided herein is a method according to any one of embodiments 163 - 168, wherein the genetically modified non - human animal is further engineered to express a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to the Sirpa promoter.

[0454] In exemplary embodiment 170, provided herein is a method according to embodiment 169, wherein the genetically modified non - human animal further comprises a Sirpa gene encoding a Sirpa polypeptide comprising the extracellular portion of the human SIRPA polypeptide and the intracellular portion of the non - human animal Sirpa polypeptide, and the Sirpa gene is operably linked to the Sirpa promoter.

[0455] In exemplary embodiment 171, provided herein is a method according to embodiment 170, wherein the Sirpa gene comprises exons 2 - 4 of the human SIRPA gene.

[0456] In exemplary embodiment 172, provided herein is a method according to embodiment 170 or 171, wherein the genetically modified non - human animal expresses a Sirpa polypeptide comprising the extracellular portion of the human SIRPA polypeptide and the intracellular portion of the non - human animal Sirpa polypeptide.

[0457] In exemplary embodiment 173, a method according to any one of embodiments 170-172 is provided herein, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

[0458] In exemplary embodiment 174, a method according to embodiment 169 is provided herein, wherein the genetically modified non-human animal expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0459] In exemplary embodiment 175, a method according to any one of embodiments 169-174 is provided herein, wherein the genetically modified non-human animal is further engineered to express one or more human or humanized proteins selected from the group consisting of a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; a human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0460] In exemplary embodiment 176, a method according to any one of embodiments 169-175 is provided herein, wherein at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter.

[0461] In exemplary embodiment 177, there is provided a method of embodiment 176, wherein all promoters operably linked to a nucleic acid encoding a human or humanized protein are endogenous non-human animal promoters.

[0462] In exemplary embodiment 178, there is provided a method of embodiment 176 or 177, wherein the endogenous non-human animal promoter is at the locus of the corresponding non-human animal gene.

[0463] In exemplary embodiment 179, there is provided a method of any one of embodiments 169-178, which comprises a null mutation in at least one corresponding non-human animal gene at the locus of the corresponding non-human animal gene.

[0464] In exemplary embodiment 180, there is provided a method of any one of embodiments 169-179, wherein the genetically modified non-human animal is heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0465] In exemplary embodiment 181, there is provided a method of any one of embodiments 169-180, wherein the genetically modified non-human animal is homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0466] In exemplary embodiment 182, there is provided a method of any one of embodiments 169-181, wherein the genetically modified non-human animal expresses human M-CSF protein encoded by a nucleic acid operably linked to the M-CSF promoter.

[0467] In exemplary embodiment 183, there is provided a method of any one of embodiments 169-182, wherein the genetically modified non-human animal expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to the CD47 promoter.

[0468] In illustrative embodiment 184, there is provided a method of embodiment 183, wherein the genetically modified non-human animal expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

[0469] In illustrative embodiment 185, there is provided a method of any one of embodiments 169-184, wherein the genetically modified non-human animal expresses: (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

[0470] In illustrative embodiment 186, there is provided a method of any one of embodiments 169-185, wherein the genetically modified non-human animal expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

[0471] In illustrative embodiment 187, there is provided a method of any one of embodiments 163-186, wherein the genetically modified non-human animal is a mammal.

[0472] In illustrative embodiment 188, there is provided a method of embodiment 187, wherein the mammal is a rodent, such as a rat or a mouse.

[0473] In illustrative embodiment 189, there is provided a method of embodiment 188, wherein the rodent is a mouse.

Examples

[0474] The following examples and the accompanying drawings are provided to illustrate to those skilled in the art how to make and use the methods and compositions of the present invention and are not intended to limit the scope of what the inventors regard as their invention. Although efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be taken into account. The examples do not include detailed descriptions of conventional methods (such as molecular cloning techniques) that will be well known to those skilled in the art. Unless otherwise indicated, parts are by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. (Example 1) Generation of Hmox-1 knockout mice

[0475] The mouse Hmox-1 gene was deleted in the mouse genome using the VELOCIGENE® technology (see, for example, U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotech. 21(6): 652-659, both of which are incorporated herein by reference). A 7 kb mouse genomic sequence of the mouse Hmox-1 gene from the start codon ATG to the stop codon was deleted in mouse chromosome 8 C1 between coordinates chr8:75093750 - 75100019 (GRCm38 assembly). Figure 1.

[0476] Specifically, mouse homologous arms were generated by PCR amplification using the BAC clone RP23-102I24 as a template. This is shown in Table 1 below.

[0477] [Table 1]

[0478] To generate the targeting vector (designated MAID20433) from the mouse BAC clone RP23-102I24, a hygromycin (Hyg) resistance self-deleting cassette (with CRE recombinase controlled by the protamine promoter) flanked by mutant lox sites (lox2372-hyg-lox2372) was replaced by bacterial homologous recombination (BHR) with an approximately 7 kb mouse sequence containing the mouse Hmox-1 gene.

[0479] The final targeting vector contains, from 5' to 3', a chloramphenicol resistance cassette (CM; not shown in Figure 1), a 5' mouse homologous arm, a lox2372-Hyg-lox2372 self-deleting cassette, and a 3' mouse homologous arm, and the final clones were selected based on CM / Hyg resistance.

[0480] The MAID20433 targeting vector was electroporated into mouse embryonic stem (ES) cells. Targeted homologous recombination resulted in the deletion of an approximately 7 kb mouse sequence (GRCm38 coordinates chr8:75093750-75100019). The success of the integration was confirmed by an allelic modification (MOA) assay as described, for example, by Valenzuela et al, supra. The primers and probes used for the MOA assay for the loss of the mouse Hmox-1 sequence are shown in Table 2 below. The cassette was then removed in mice by the expression of CRE recombinase (controlled by the protamine promoter).

[0481]

Table 2

[0482] Targeted ES cells were used as donor ES cells and microinjected into mouse embryos at the pre-morula (8-cell) stage by the VELOCIMOUSE® method (see, for example, US7,576,259, US7,659,442, US7,294,754, and US2008-0078000A1, all of which are incorporated herein by reference). Mouse embryos containing the donor ES cells were incubated in vitro and then transferred to surrogate mothers to produce F0 mice that were completely derived from the donor ES cells. Mice lacking the Hmox-1 gene were identified by genotyping using the MOA assay described above. Heterozygous mice for the deletion of the Hmox-1 gene were mated to make them homozygous. HMOX-1 KO mice (described in WO2011 / 044050, WO2012 / 112544, WO2014 / 039782, WO2014 / 071397, US Patent No. 11,019,810, WO2014 / 039782, WO2014 / 071397, WO2016 / 168212, US Pa...

Claims

1. A genetically modified non-human animal comprising: (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the Il2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

2. The genetically modified non-human animal according to claim 1, comprising a homozygous null mutation in the Rag1 gene.

3. The genetically modified non-human animal according to claim 1 or 2, wherein the null mutation is a deletion of at least the exon corresponding to mouse Hmox-1 exons 3-5.

4. The genetically modified non-human animal according to any one of claims 1-3, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

5. The genetically modified non-human animal according to any one of claims 1-4, comprising a homozygous null mutation in the Fah gene.

6. The genetically modified non-human animal according to claim 5, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion, and / or substitution in the endogenous Fah gene.

7. The genetically modified non-human animal according to any one of claims 1-6, which expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

8. The genetically modified non-human animal according to claim 7, wherein the genetically modified non-human animal comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, and the Sirpa gene is operably linked to a Sirpa promoter.

9. The genetically modified non-human animal according to claim 8, wherein the Sirpa gene comprises exons 2-4 of the human SIRPA gene.

10. The genetically modified non-human animal according to claim 8 or 9, which expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide.

11. The genetically modified non-human animal according to any one of claims 8-10, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide, and / or the non-human animal Sirpa gene is an endogenous non-human animal gene. **Claim 12** The genetically modified non-human animal according to claim 7, which expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter. **Claim 13** A human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; A human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; A human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; A human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; A human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; A human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and A human EPO protein encoded by a nucleic acid operably linked to an EPO promoter The genetically modified non-human animal according to any one of claims 7 to 12, which further expresses one or more human or humanized proteins selected from the group consisting of. **Claim 14** The genetically modified non-human animal according to any one of claims 7 to 13, wherein at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter. **Claim 15** The genetically modified non-human animal according to claim 14, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters. **Claim 16** The genetically modified non-human animal according to claim 14 or 15, wherein the endogenous non-human animal promoter is at the locus of the corresponding non-human animal gene. **Claim 17** The genetically modified non-human animal according to any one of claims 7 to 16, which comprises a null mutation in at least one corresponding non-human animal gene at the locus of the corresponding non-human animal gene. **Claim 18** The genetically modified non-human animal according to any one of claims 7 to 17, which is heterozygous for at least one allele containing the nucleic acid sequence encoding the human or humanized protein. **Claim 19** The genetically modified non-human animal according to any one of claims 7 to 17, which is homozygous for at least one allele containing a nucleic acid sequence encoding the human or humanized protein.

20. The genetically modified non-human animal according to any one of claims 7 to 19, which expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

21. The genetically modified non-human animal according to any one of claims 7 to 20, which expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

22. The genetically modified non-human animal according to claim 21, wherein the genetically modified non-human animal expresses a humanized CD47 protein, and the humanized CD47 protein contains an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

23. The genetically modified non-human animal according to any one of claims 7 to 22, which expresses (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

24. The genetically modified non-human animal according to any one of claims 7 to 23, which expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

25. The genetically modified non-human animal according to any one of claims 1 to 24, which further comprises engraftment of human hematopoietic cells.

26. The genetically modified non-human animal according to claim 25, wherein the human hematopoietic cells comprise one or more cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human hematopoietic progenitor cells, human erythroid progenitor cells, and human erythrocytes.

27. The genetically modified non-human animal according to claim 36, wherein the animal comprises human cells of the erythroid lineage.

28. The genetically modified non-human animal according to claim 27, wherein the non-human animal further comprises infection by a pathogen that targets the human cells of the erythroid lineage.

29. The genetically modified non-human animal according to any one of claims 25 to 28, wherein the animal contains an inactivated endogenous Fah gene and further contains transplanted human hepatocytes.

30. The genetically modified non-human animal according to claim 28 or claim 29, wherein the pathogen can cause malaria in humans.

31. The genetically modified non-human animal according to claim 28, wherein the pathogen is selected from Plasmodium sp., Babesia sp., and Theileria sp.

32. The genetically modified non-human animal according to any one of claims 25 to 27, wherein the engrafted human hematopoietic cells give rise to abnormal human cells of the erythroid lineage.

33. The genetically modified non-human animal according to claim 32, wherein the engrafted human hematopoietic cells contain a mutation in the β-globin gene that causes sickle cell disease.

34. The genetically modified non-human animal according to any one of claims 1 to 33, which is a mammal.

35. The genetically modified non-human animal according to claim 34, wherein the mammal is a rodent, such as a rat or a mouse.

36. The genetically modified non-human animal according to claim 35, wherein the rodent is a mouse.

37. A method for identifying an agent that inhibits infection by a pathogen targeting human cells of the erythroid lineage, comprising: a. administering the agent to a genetically modified non-human animal, wherein the genetically modified non-human animal has: i. a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the Il2rg gene; iii. engraftment of human hematopoietic cells; and iv. infection by a pathogen targeting human cells of the erythroid lineage and b. determining whether the agent reduces the amount of the pathogen and / or inhibits the activity of the pathogen in the non-human animal infected with the pathogen. A method comprising the steps.

38. A method for identifying an agent that prevents infection by a pathogen targeting human cells of the erythroid lineage, comprising: a. administering the agent to a genetically modified non-human animal, wherein the genetically modified non-human animal has: i. A homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. A homozygous null mutation in the Rag2 gene and a homozygous null mutation in the Il2rg gene; and iii. Engraftment of human hematopoietic cells comprising steps b. Injecting parasitized reticulocytes or red blood cells into the genetically modified non-human animal, and c. Determining whether the agent prevents the infection of the human reticulocytes and / or red blood cells of the non-human animal comprising a method. **Claim 39** The method according to claim 37 or 38, wherein the pathogen can cause malaria in humans. **Claim 40** The method according to claim 37 or 38, wherein the pathogen is selected from Plasmodium sp., Babesia sp., and Theileria sp. **Claim 41** A method for identifying an agent for treating sickle cell disease, comprising a. Administering the agent to a genetically modified non-human animal, wherein the genetically modified non-human animal i. has a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. has a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the Il2rg gene; and iii. engraftment of human hematopoietic cells containing a mutation in the β-globin gene that causes sickle cell disease comprising steps, and b. Determining whether the agent prevents or reduces sickling of red blood cells in the non-human animal comprising a method. **Claim 42** A method for evaluating the therapeutic efficacy of a drug candidate targeting human red blood cells, comprising a. Administering the drug candidate to a genetically modified non-human animal, wherein the genetically modified non-human animal i. has a homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. has a homozygous null mutation in the Rag2 gene and a homozygous null mutation in the Il2rg gene; and iii. engraftment of human hematopoietic progenitor cells comprising steps, and b. Monitoring the human red blood cells in the non-human animal to evaluate the therapeutic efficacy of the drug candidate comprising a method. **Claim 43** The method according to claim 42, wherein the human red blood cells are monitored to determine whether the generation and / or survival of the human red blood cells in the non-human animal are increased by the drug candidate. **Claim 44** A method for evaluating the toxicity of a drug candidate to human red blood cells, comprising: a. Administering the drug candidate to a genetically modified non-human animal, wherein the genetically modified non-human animal has: i. A homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. A homozygous null mutation in the Rag2 gene and a homozygous null mutation in the Il2rg gene; and iii. Engraftment of human hematopoietic progenitor cells ; and b. Monitoring the human red blood cells in the non-human animal to evaluate the toxicity of the drug candidate .

45. The method according to claim 44, wherein the toxicity is on-target toxicity or off-target toxicity.

46. The method according to claim 44 or 45, wherein the human red blood cells are monitored to determine whether the number of human red blood cells in the non-human animal is reduced by the drug candidate.

47. The method according to claim 46, wherein the drug candidate is a chemotherapeutic agent or an antimalarial agent.

48. The method according to claim 44 or 45, wherein the human red blood cells are monitored to evaluate whether the drug candidate induces aggregation of the red blood cells.

49. The method according to claim 48, wherein the drug candidate is a modulator of human CD47 protein.

50. The method according to claim 49, wherein the modulator is an antibody.

51. A method for identifying an agent that reduces the toxicity of a toxic drug to human red blood cells, comprising: a. Administering the agent and the toxic drug to a genetically modified non-human animal, wherein the genetically modified non-human animal has: i. A homozygous null mutation in the Hmox-1 gene of the non-human animal; ii. A homozygous null mutation in the Rag2 gene and a homozygous null mutation in the Il2rg gene; and iii. Engraftment of human hematopoietic progenitor cells ; and b. Determining whether the agent reduces the toxicity of the toxic drug to human red blood cells in the non-human animal .

52. The method according to claim 51, wherein the agent and the toxic drug are administered to the non-human animal together or sequentially.

53. The method according to any one of claims 37 to 52, wherein the genetically modified non-human animal comprises a homozygous null mutation in the Rag1 gene.

54. The method according to any one of claims 37 to 53, wherein the null mutation is a deletion of at least an exon corresponding to mouse Hmox-1 exons 3 to 5.

55. The method according to any one of claims 37 to 54, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

56. The method according to any one of claims 37 to 55, comprising a homozygous null mutation in the Fah gene.

57. The method according to claim 56, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion, and / or substitution in the endogenous Fah gene.

58. The method according to any one of claims 37 to 57, wherein the genetically modified non-human animal expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

59. The method according to claim 58, wherein the genetically modified non-human animal comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, and the Sirpa gene is operably linked to a Sirpa promoter.

60. The method according to claim 59, wherein the Sirpa gene comprises exons 2 to 4 of the human SIRPA gene.

61. The method according to claim 59 or 60, wherein the genetically modified non-human animal expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide.

62. The method according to any one of claims 59 to 61, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

63. The method according to claim 58, wherein the genetically modified non-human animal expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

64. The genetically modified non-human animal is a human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; A human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; A human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; A human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; A human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and A human EPO protein encoded by a nucleic acid operably linked to an EPO promoter The method according to any one of claims 58 to 63, further expressing one or more human or humanized proteins selected from the group consisting of.

65. The method according to any one of claims 58 to 64, wherein at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter.

66. The method according to claim 65, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters.

67. The method according to claim 65 or 66, wherein the endogenous non-human animal promoter is at the locus of the corresponding non-human animal gene.

68. The method according to any one of claims 58 to 67, comprising a null mutation in at least one corresponding non-human animal gene at the locus of the corresponding non-human animal gene.

69. The method according to any one of claims 58 to 68, wherein the genetically modified non-human animal is heterozygous for at least one allele containing a nucleic acid sequence encoding the human or humanized protein.

70. The method according to any one of claims 58 to 69, wherein the genetically modified non-human animal is homozygous for at least one allele containing a nucleic acid sequence encoding the human or humanized protein.

71. The method according to any one of claims 58 to 70, wherein the genetically modified non-human animal expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

72. The method according to any one of claims 58 to 71, wherein the genetically modified non-human animal expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

73. The method according to claim 72, wherein the genetically modified non-human animal expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

74. The method according to any one of claims 58 to 73, wherein the genetically modified non-human animal expresses (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

75. The method according to any one of claims 58 to 74, wherein the genetically modified non-human animal expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

76. The method according to any one of claims 37 to 75, wherein the genetically modified non-human animal is a mammal.

77. The method according to claim 76, wherein the mammal is a rodent, such as a rat or a mouse.

78. The method according to claim 77, wherein the rodent is a mouse.

79. Genetically modified non-human animal cells comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the Il2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

80. The genetically modified non-human animal cells according to claim 79, comprising a homozygous null mutation in the Rag1 gene.

81. The genetically modified non-human animal cells according to claim 79 or 80, wherein the null mutation is a deletion of at least the exons corresponding to mouse Hmox-1 exons 3 to 5.

82. The genetically modified non-human animal cells according to any one of claims 79 to 81, wherein the null mutation is a deletion of the entire Hmox-1 endogenous coding sequence.

83. The genetically modified non-human animal cell according to any one of claims 79 to 82, comprising a homozygous null mutation in the Fah gene.

84. The genetically modified non-human animal cell according to claim 83, wherein the homozygous null mutation in the Fah gene comprises an insertion, deletion, and / or substitution in the endogenous Fah gene.

85. The genetically modified non-human animal cell according to any one of claims 79 to 84, which expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

86. The genetically modified non-human animal cell according to claim 85, wherein the genetically modified non-human animal cell comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide, and the Sirpa gene is operably linked to a Sirpa promoter.

87. The genetically modified non-human animal cell according to claim 86, wherein the Sirpa gene comprises exons 2 to 4 of the human SIRPA gene.

88. The genetically modified non-human animal cell according to claim 86 or 87, which expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a non-human animal Sirpa polypeptide.

89. The genetically modified non-human animal cell according to any one of claims 86 to 88, wherein the non-human animal Sirpa polypeptide is an endogenous non-human animal Sirpa polypeptide, and / or the non-human animal Sirpa gene is an endogenous non-human animal gene.

90. The genetically modified non-human animal cell according to claim 85, which expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

91. A human TPO protein encoded by a nucleic acid operably linked to a TPO promoter; A human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter; A human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter; A human IL15 protein encoded by a nucleic acid operably linked to an IL15 promoter; A human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; A human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter; and A human EPO protein encoded by a nucleic acid operably linked to an EPO promoter The genetically modified non-human animal cell according to any one of claims 85 to 90, further expressing one or more human or humanized proteins selected from the group consisting of.

92. The genetically modified non-human animal cell according to any one of claims 85 to 91, wherein at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter.

93. The genetically modified non-human animal cell according to claim 92, wherein all promoters operably linked to the nucleic acid encoding the human or humanized protein are endogenous non-human animal promoters.

94. The genetically modified non-human animal cell according to claim 92 or 93, wherein the endogenous non-human animal promoter is at the locus of the corresponding non-human animal gene.

95. The genetically modified non-human animal cell according to any one of claims 85 to 94, comprising a null mutation in at least one corresponding non-human animal gene at the locus of the corresponding non-human animal gene.

96. The genetically modified non-human animal cell according to any one of claims 85 to 95, which is heterozygous for at least one allele containing the nucleic acid sequence encoding the human or humanized protein.

97. The genetically modified non-human animal cell according to any one of claims 85 to 95, which is homozygous for at least one allele containing the nucleic acid sequence encoding the human or humanized protein.

98. The genetically modified non-human animal cell according to any one of claims 85 to 97, which expresses a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter.

99. The genetically modified non-human animal cell according to any one of claims 85 to 98, which expresses a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

100. The genetically modified non-human animal cell according to claim 99, wherein the genetically modified non-human animal cell expresses a humanized CD47 protein, and the humanized CD47 protein comprises an extracellular portion of a human CD47 protein and an intracellular portion of an endogenous non-human animal CD47 protein.

101. The genetically modified non-human animal cell according to any one of claims 85 to 100, which expresses (i) a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter; (ii) a human M-CSF protein encoded by a nucleic acid operably linked to an M-CSF promoter; and (iii) a human or humanized CD47 protein encoded by a nucleic acid operably linked to a CD47 promoter.

102. The genetically modified non-human animal cell according to any one of claims 85 to 101, which expresses a human EPO protein encoded by a nucleic acid operably linked to an EPO promoter.

103. The genetically modified non-human animal cell according to any one of claims 79 to 102, which is a mammalian cell.

104. The genetically modified non-human animal cell according to claim 103, wherein the mammalian cell is a rodent cell, such as a rat cell or a mouse cell.

105. The genetically modified non-human animal cell according to claim 104, wherein the rodent cell is a mouse cell.

106. The genetically modified non-human animal cell according to any one of claims 79 to 84, 86 to 87, 89, 92 to 97, and 103 to 105, which is a non-human animal embryonic stem (ES) cell.

107. A method for producing a non-human animal embryonic stem cell, the method comprising the step of genetically engineering the non-human animal embryonic stem cell such that the non-human animal embryonic stem cell has a genome comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the Il2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

108. A non-human animal embryo comprising the non-human animal embryonic stem cell according to claim 106, or the non-human animal embryonic stem cell produced according to the method according to claim 107.

109. A method of producing a non-human animal whose genome contains (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the Il2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, comprising: (a) obtaining the non-human animal embryonic stem cells according to claim 106, or the non-human animal embryonic stem cells produced according to the method of claim 107; and (b) using the non-human animal embryonic cells of (a) to create a non-human animal A method comprising.

110. A method of producing a non-human animal whose genome contains (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the Il2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene, the method comprising modifying the genome of the non-human animal such that it contains (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the Il2rg gene; and (iii) a homozygous null mutation in the non-human animal heme oxygenase-1 (Hmox-1) gene.

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