Genetically modified mice and engraftment method

Abstract

To provide a mouse that models a human mycobacterial infection and develops one or more granulomas comprising human immune cells.SOLUTION: A genetically modified mouse comprises: (a) a mouse RAG gene knockout; (b) a mouse Il2rg gene knockout; (c) a replacement of a mouse IL-3 gene with a human IL-3 gene, where the human IL-3 gene is at a mouse IL-3 gene locus; and (d) a replacement of a mouse GM-CSF gene with a human GM-CSF gene, where the human GM-CSF gene is at a mouse GM-CSF gene locus.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the field of genetically modified non-human animals, in particular to immunodeficient mice with RAG gene knockout, Il2rgIl2rg gene knockout, and humanization of IL-3 and GM-CSF genes, and optionally humanization of the TPO gene; RAG / Il2rgIl2rg knockout mice with humanization of the TPO gene; genetically modified mice engrafted with human hematopoietic cells; and engrafted mice infected with human pathogens, such as Salmonella typhi or Mycobacterium tuberculosis. [Background technology]

[0002] background Genetically modified mice, modified and transplanted mice, and their use in modeling human disease, for example for drug testing purposes, are known in the art. Attempts have been made to use genetically modified mice to model the human immune system. A review of the field is provided in Manz (2007) Human-Hemato-Lympoid-System Mice: Opportunities and Challenges, Immunity, 26:537-541, which is incorporated herein by reference.

[0003] To date, genetically modified mice have not been produced that are susceptible to certain human pathogens, such as S. typhi. Even for pathogenic infections for which mouse models exist, the models may not be able to adequately model certain human pathologies, for example, the mouse model of M. tuberculosis fails to form well-defined granulomas or granulomas that contain human immune cells. To study the effects of certain pathogens on humans and to test drugs for efficacy in treating humans infected with certain pathogens, it would be useful to have non-human animals, such as mice, that are genetically modified to be susceptible to infection with such pathogens, such as S. typhi, and / or to make the infection a more rigorous model of human pathologies, such as a more rigorous model of human infection with M. tuberculosis.

[0004] Generally, there is a need for genetically modified mice that can support the maintenance and proliferation of human hematopoietic stem cells, as well as a need for transplant-compatible mice that can model or approximate parts of the human blood-lymphatic system, e.g., in response to human pathogens. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Manz (2007) Human-Hemato-Lympoid-System Mice: Opportunities and Challenges, Immunity, 26:537-541 Summary of the Invention

[0006] overview Genetically modified non-human animals are provided, including mice that contain a knockout of one or more endogenous genes and one or more humanized genes (i.e., replacement of an endogenous gene with a human analog or homolog at its endogenous locus).

[0007] Genetically modified mice that have had their immune systems ablated or compromised (e.g., by irradiation) are provided, as are mice engrafted with human hematopoietic cells or human hematopoietic stem and progenitor cells (HSPCs). Genetically modified mice that contain human cells derived from human hematopoietic cells or HSPCs are provided, as are mice that contain a human blood-lymphatic system.

[0008] Genetically modified, irradiated and transplanted mice are provided that are capable of infection with human pathogens that do not infect wild-type mice. Mice are provided that, in response to exposure to a human pathogen (e.g., Mycobacterium tuberculosis), mount an immune response having characteristics not observed in wild-type mice (e.g., formation of well-defined granulomas or granulomas containing human immune cells).

[0009] Provided are genetically modified, irradiated and engrafted mice, as well as compositions and methods for their use, for identifying drug-resistant strains of human pathogens, for testing human vaccines, and for developing and testing anti-pathogen drugs.

[0010] Genetically modified mice capable of receiving and expanding human immune cells are provided, including mice capable of harboring human hematopoietic malignancies.

[0011] In one aspect, a genetically modified mouse is provided that comprises: (a) a mouse RAG gene knockout; (b) a mouse Il2rgIl2rg gene knockout; and (c) humanization of one or more genes selected from: (i) a mouse IL-3 (mIL-3) gene, (ii) a mouse GM-CSF (mGM-CSF) gene, and (iii) a mouse thrombopoietin (mTPO) gene.

[0012] In one embodiment, the RAG gene knockout is a RAG2 gene knockout.

[0013] In one embodiment, the humanization comprises replacement of the mTPO gene with the hTPO gene, hi a particular embodiment, the humanization consists essentially of humanizing the mTPO gene with the hTPO gene.

[0014] In one embodiment, the humanization comprises replacement of the mIL-3 gene with the human IL-3 (hIL-3) gene and replacement of the mGM-CSF gene with the human GM-CSF (hGM-CSF) gene. In another embodiment, the mouse further comprises replacement of the mTPO gene with the human TPO (hTPO) gene. In a particular embodiment, the humanization consists essentially of humanization of the mIL-3 gene with the hIL-3 gene and humanization of the mGM-CSF gene with the hGM-CSF gene.

[0015] In one embodiment, humanization includes replacing the mGM-CSF gene with the human GM-CSF gene, and human GM-CSF is not predominantly expressed in the liver and circulation in mice.In one embodiment, human GM-CSF is predominantly expressed in mouse lungs.In one embodiment, the expression of human GM-CSF is tissue-specific and reflects the tissue-specific expression in humans.

[0016] In one embodiment, the genetically modified mouse is treated to eliminate endogenous hematopoietic cells that may be present in the mouse. In one embodiment, the treatment comprises irradiating the genetically modified mouse. In a particular embodiment, the genetically modified mouse neonate is sublethally irradiated. In a particular embodiment, the neonate is irradiated with 2x200cGy at 4 hour intervals.

[0017] In one embodiment, the genetically modified and treated mouse is transplanted with human hematopoietic cells or human hematopoietic stem cells (HPSCs) to form a genetically modified and transplanted mouse. In one embodiment, the hematopoietic cells are selected from human umbilical cord blood cells and human fetal liver cells. In one embodiment, about 1-2×10 5 Human CD34+ cells are used.

[0018] In one embodiment, the genetically modified and transplanted mouse gives rise to human cells selected from the following: CD34+ cells, hematopoietic stem cells, hematopoietic cells, bone marrow progenitor cells, bone marrow cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, thymocytes, T cells, B cells, platelets, and combinations thereof. In one embodiment, the human cells are present at 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after transplantation.

[0019] In one embodiment, the genetically modified and transplanted mice give rise to a human blood-lymphatic system comprising human hematopoietic stem cell progenitors, human bone marrow progenitors, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, and human platelets. In one embodiment, the human blood-lymphatic system is present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months post-transplant.

[0020] In one embodiment, the genetically modified and transplanted mouse exhibits an inflammatory response mediated by human cells. In a particular embodiment, the human cells are macrophages. In a particular embodiment, the macrophage-mediated inflammatory response is mediated by alveolar macrophages. In a particular embodiment, the alveolar macrophage-mediated inflammatory response comprises the formation of granulomas. In a particular embodiment, the granulomas comprise human immune cells. In a particular embodiment, the granulomas are well-organized granulomas. In a particular embodiment, the granulomas are formed after exposure to mycobacteria, such as Mycobacterium tuberculosis. In one embodiment, the mouse exhibits an immune response comprising two or more granulomas. In one embodiment, the genetically modified and transplanted mouse is a model for Mycobacterium tuberculosis infection.

[0021] In one embodiment, the genetically modified and transplanted mouse comprises a Mycobacterium tuberculosis infection characterized at least in part by the formation of granulomas comprising human immune cells. In a particular embodiment, the granulomas are clearly organized granulomas. In a particular embodiment, the Mycobacterium tuberculosis is a drug-resistant or multi-drug resistant strain of Mycobacterium tuberculosis that infects human populations. In one embodiment, the mouse infected with Mycobacterium tuberculosis comprises granulomas in the lungs. In a particular embodiment, the granulomas are fully developed granulomas. In a particular embodiment, the pulmonary granulomas comprise human immune cells. In a particular embodiment, the human immune cells in the granulomas are selected from activated human macrophages, activated human T cells, and combinations thereof.

[0022] In one embodiment, the genetically modified and engrafted mice exhibit enhanced mucosal immunity compared to engrafted mice lacking humanization of one or more of the IL-3, GM-CSF and TPO genes. In a particular embodiment, the enhanced mucosal immunity comprises high expression of interferon beta (IFNβ) following influenza A infection.

[0023] In one embodiment, the genetically modified and transplanted mouse comprises an infection selected from Mycobacterium tuberculosis infection and Salmonella typhi infection. In one embodiment, the mouse propagates Salmonella typhi or Mycobacterium tuberculosis. In one embodiment, the mouse mounts an anti-mycobacterial immune response against human pathogenic mycobacteria, the immune response comprising the formation of granulomas mediated by and including human immune cells. In a particular embodiment, the granulomas are fully developed granulomas.

[0024] In one embodiment, the genetically modified and transplanted mouse comprises humanization of the mTPO gene, forming a mouse transplanted with hTPO. In one embodiment, the mouse transplanted with hTPO shows an increase in human bone marrow cells in bone marrow compared to a mouse transplanted with mTPO gene but not with hTPO gene. In a particular embodiment, the human bone marrow cells are increased 1.5-fold, 2-fold, 2.5-fold or 3-fold compared to a mouse transplanted with a lack of hTPO gene. In a particular embodiment, the increase in granulocytes is about 1.5-fold, 2-fold, 2.5-fold or 3-fold. In another embodiment, the increase in peripheral blood monocytes is observed compared to a mouse transplanted with a lack of hTPO gene, and the increase in peripheral blood monocytes is about 1.5-fold, 2-fold, 2.5-fold or 3-fold. In one embodiment, the genetically modified and transplanted mouse comprises a humanization consisting essentially of a hTPO gene replacing the mTPO gene, and the mouse expresses human TPO without expressing mouse TPO.

[0025] In one aspect, a genetically modified and engrafted mouse is provided, comprising a knockout of Rag gene, an Il2rgIl2rg knockout, and a humanized TPO, the mouse is engrafted with human hematopoietic stem cells or human immune cells, and comprises a human hematopoietic malignancy arising from an early human hematopoietic cell. In a particular embodiment, the malignancy is selected from a myeloid leukemia and a myeloproliferative neoplasm.

[0026] In one embodiment, the mouse further comprises the human IL-3 gene and the human GM-CSF gene, as well as a knockout of the endogenous mouse IL-3 gene and a knockout of the endogenous mouse GM-CSF gene.

[0027] In one aspect, a mouse is provided that comprises a RAG gene knockout, an Il2rg gene knockout, and a genetic modification that provides human bone marrow cells with a competitive advantage over mouse bone marrow cells. In one embodiment, the genetic modification is a replacement of a mouse gene required for the development and / or maintenance of mouse bone marrow cells with a corresponding human gene. In one embodiment, the genetic modification is selected from a replacement of a mouse IL-3 gene with a human IL-3 gene, a replacement of a mouse GM-CSF gene with a human GM-CSF gene, and combinations thereof. In one embodiment, the mouse lacks or substantially lacks endogenous mouse hematopoietic cells and comprises human hematopoietic cells.

[0028] In one aspect, a method for producing a mouse capable of being infected with a human pathogen is provided, the method includes genetically modifying a mouse as described herein and transplanting the mouse, exposing the genetically modified and transplanted mouse to a human pathogen, and maintaining the mouse under sufficient conditions for the human pathogen to infect the mouse.In one embodiment, the human pathogen is selected from Mycobacterium tuberculosis and Salmonella typhi.In one embodiment, the human pathogen is a human pathogen that is not pathogenic to mice that lack genetic modification.In one embodiment, the human pathogen is a human pathogen that is not infectious to mice that lack genetic modification.

[0029] In one aspect, a method for determining the effect of a drug on a human pathogen is provided, comprising exposing a mouse that has been genetically modified and transplanted as described herein to a human pathogen, and infecting the mouse with the pathogen and measuring the parameter of infection over time in the presence and absence of the drug.In one embodiment, the human pathogen is a pathogen that does not infect mice that lack genetic modification.In one embodiment, the human pathogen is selected from Mycobacterium tuberculosis and Salmonella typhi.In one embodiment, the mouse is exposed to a known number of infectious units of the human pathogen, and the parameter of infection is the number of infectious units of the human pathogen in the body fluids or tissues of the mouse.

[0030] In one embodiment, the parameter of infection is the titer in the body fluids of the mouse. In one embodiment, the infection is selected from Mycobacterium tuberculosis infection and Salmonella typhi infection. In a particular embodiment, the infection is Mycobacterium tuberculosis infection and the parameter is granuloma formation. In a particular embodiment, the granuloma is a pulmonary granuloma. In another particular embodiment, the granuloma is a sharply demarcated granuloma.

[0031] In one aspect, a genetically modified mouse is provided, comprising: (a) mouse RAG gene knockout; (b) mouse Il2rg gene knockout; and (c) (i) mouse IL-3 (mIL-3) gene and (ii) mouse GM-CSF (mGM-CSF) gene humanization; the mouse maintains its human hematopoietic stem cells after irradiation to remove endogenous mouse hematopoietic cells and transplants human hematopoietic stem cells, and generates a human immune cell population from the human hematopoietic stem cells, including differentiated functional human immune cells. The differentiated human immune cells include human bone marrow progenitor cells, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, and human platelets. In another aspect, the mouse further comprises (iii) mouse thrombopoietin (mTPO) gene humanization.

[0032] In one embodiment, the mouse maintains a population of human immune cells that is as diverse in cell types as a human population of immune cells, hi one embodiment, the human immune cells are maintained for at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after transplantation.

[0033] In one embodiment, the mouse is exposed to a human pathogen or an antigen of the human pathogen and mounts a cellular and / or humoral immune response that models the infection of a human exposed to the pathogen. In one embodiment, the human pathogen is a pathogen that does not infect wild type mice. In another embodiment, the human pathogen is a pathogen that infects wild type mice, and the infected wild type mice do not model the immune response that humans mount in response to the pathogen. In one embodiment, the pathogen is a virus, a mycobacteria, a fungus, or a bacterium. In a particular embodiment, the pathogen is a human, swine, or avian influenza virus, Salmonella typhi, or Mycobacterium tuberculosis.

[0034] Further applications and embodiments of the present invention will become apparent to those of skill in the art upon reading this disclosure. [The present invention 1001] (a) Mouse RAG gene knockout; (b) Mouse Il2rg gene knockout; (c) replacement of the mouse IL-3 gene with the human IL-3 gene, wherein the human IL-3 gene is present at the mouse IL-3 locus; and (d) replacement of the mouse GM-CSF gene with the human GM-CSF gene, where the human GM-CSF gene is present at the mouse GM-CSF locus. A genetically modified mouse, comprising: [The present invention 1002] Replacement of the mouse thrombopoietin gene with the human thrombopoietin gene, the human thrombopoietin gene being present at the mouse thrombopoietin locus The mouse of the present invention 1001 further comprising: [The present invention 1003] 1001. The mouse of the present invention, wherein said RAG gene knockout is selected from a RAG1 gene knockout, a RAG2 gene knockout, and a combination thereof. [The present invention 1004] The mouse of the present invention 1001, further comprising transplantation of human hematopoietic cells. [The present invention 1005] The mouse of claim 1004, wherein said human hematopoietic cells are selected from the group consisting of umbilical cord blood cells and fetal liver cells. [The present invention 1006] The mouse of claim 1005, wherein said human hematopoietic cells are human umbilical cord blood cells that are CD34+ cells. [The present invention 1007] 1003. The mouse of the present invention, comprising a human cell that is a CD34 positive cell, a hematopoietic stem cell, a hematopoietic cell, a bone marrow progenitor cell, a bone marrow cell, a dendritic cell, a monocyte, a granulocyte, a neutrophil, a mast cell, or a combination thereof. [The present invention 1008] 1003. A mouse of the present invention comprising human hematopoietic stem and progenitor cells, human bone marrow progenitor cells, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, and human platelets. [The present invention 1009] 1004. A mouse of the present invention comprising a Salmonella typhi infection. [The present invention 1010] The mouse of claim 1009, wherein said Salmonella typhi infection is a systemic Salmonella typhi infection. [The present invention 1011] 1004. A mouse of the present invention comprising a Mycobacterium tuberculosis infection. [The present invention 1012] A mouse of the present invention comprising pulmonary granulomas comprising human immune cells. [The present invention 1013] (a) administering an agent to a genetically modified, transplanted, and human pathogen-infected mouse; The mouse, (i) RAG1 gene knockout or RAG2 gene knockout, or a combination thereof; (ii) Il2rg gene knockout, (iii) replacement of the mouse IL-3 gene with the human IL-3 gene, where the human IL-3 gene is present at the mouse IL-3 locus; and (iv) replacement of the mouse GM-CSF gene with the human GM-CSF gene, where the human GM-CSF gene is present at the mouse GM-CSF locus. Including, the mouse is engrafted with CD34-positive human umbilical cord blood cells or CD34-positive human fetal liver cells; and (b) determining whether the agent reduces the amount of the human pathogen in the mouse infected with the human pathogen. A method for identifying an agent that inhibits human pathogen infection, comprising: [The present invention 1014] The method of claim 1013, wherein the mouse further comprises a replacement of the mouse thrombopoietin gene with a human thrombopoietin gene. [The present invention 1015] The method of claim 1013, wherein said human pathogen is Salmonella typhi. [The present invention 1016] The method of claim 1013, wherein said human pathogen is a mycobacterium, and said mycobacterium causes granulomas in said mouse comprising human immune cells. [The present invention 1017] The method of claim 1016, wherein said mycobacteria is Mycobacterium tuberculosis. [Brief description of the drawings]

[0035] [Figure 1] S. typhi infection in transplanted RAG KO, Il2rg KO mice and RAG KO, Il2rg KO, hIL-3 / hGM-CSF mice 10 days after infection. Experimental groups: (1) transplant: n=9 (4m / m, 5h / m); engraftment rate in blood=6.5-16.7%; (2) control: n=8; engraftment rate in blood=0.04-0.4% (reflects background in flow cytometry; control mice were not transplanted). [Diagram 2] Shows S. typhi infection in the spleens of transplanted RAG KO, Il2rg KO mice 1 week after infection with 1 x 10 3 S. typhi. [Diagram 3]Shown is S. typhi infection in the spleen and liver of transplanted RAG KO, Il2rg KO mice 4 weeks after infection with 1x104 S. typhi, which were transplanted with CD34 positive fetal liver cells. [Figure 4] Shown is S. typhi infection in the gallbladder of RAG KO, Il2rg KO mice 4 weeks after infection with 1x104 S. typhi and the mice were transplanted with CD34 positive fetal liver cells. [Figure 5A] 1 shows the results of a validation study of hGM-CSF expression in non-transplanted hIL-3 / hGM-CSF mice. [Figure 5B] 1 shows the results of a validation study of hGM-CSF expression in non-transplanted hIL-3 / hGM-CSF mice. [Figure 5C] 1 shows the results of a validation study of hGM-CSF expression in non-transplanted hIL-3 / hGM-CSF mice. [Figure 5D] 1 shows the results of a validation study of hGM-CSF expression in non-transplanted hIL-3 / hGM-CSF mice. [Figure 5E] 1 shows a humanization strategy at the mouse IL-3 / GM-CSF locus. [Figure 6A] FIG. 1 shows the results of lung examination of transplanted humanized (hIL-3 / hGM-CSF) mice. [Figure 6B] FIG. 1 shows the results of lung examination of transplanted humanized (hIL-3 / hGM-CSF) mice. [Figure 6C] FIG. 1 shows the results of lung examination of transplanted humanized (hIL-3 / hGM-CSF) mice. [Figure 6D] FIG. 1 shows the results of lung examination of transplanted humanized (hIL-3 / hGM-CSF) mice. [Figure 6E] FIG. 1 shows the results of lung examination of transplanted humanized (hIL-3 / hGM-CSF) mice. [Figure 6F] ELISA results of mouse and human IL-3 production by activated splenocytes are shown. [Figure 6G] ELISA results of mouse and human GM-CSF production by activated splenocytes are shown. [Figure 7A]PAS staining of lung tissue sections from non-transplanted or transplanted m / m or h / h KI mice is shown. [Figure 7B] Quantification of total protein in BAL fluid from non-transplanted or transplanted h / h KI or m / m control mice (n=6 per group) is shown. [Figure 8A] Expression of human Hprt normalized to mouse Hprt is shown. [Figure 8B] Expression of human IFNγ normalized to mouse Hprt is shown. [Figure 8C] Expression of human IFNγ normalized to human Hprt is shown. [Figure 8D] Flow cytometry analysis of human bone marrow cells from engrafted hIL-3 / GM-CSF h / m KI mice at steady state. [Figure 8E] Flow cytometric analysis of human blood cells from CB-engrafted m / m or h / m KI mice 72 hours after two intraperitoneal injections of LPS. [Figure 8F] The frequency of human CD14+ blood cells in transplanted m / m or h / m KI mice 72 hours after LPS injection is shown. [Figure 8G] ELISA of human IL-6 in serum from transplanted m / m or h / m KI mice 2-3 hours after the first (top) and second (bottom) LPS injections is shown. [Figure 9A] The frequency of human T cells (hCD45+hCD3+) in the lungs is shown. [Figure 9B] 1 shows the distribution of human CD4 and CD8 T cells in the lungs. [Figure 9C] 1 shows the ratio of human CD4 to CD8 T cells in the lungs. [Figure 9D] Flow cytometric analysis of splenocytes from BALB / c mice, transplanted m / m mice, and transplanted h / m KI mice 4 weeks after BCG infection is shown. [Figure 9E]Quantitative RT-PCR analysis of human IFNγ (left) and TNFα (right) gene expression in lung tissue from BALB / c mice, non-transplanted m / m mice, transplanted m / m mice, and transplanted h / m KI mice 4 weeks after BCG infection. [Figure 9F] DiffQuick™ staining of BAL cells from non-transplanted m / m or h / h KI mice; magnification 400x is shown. [Figure 9G] PAS staining of lung F tissue sections from non-transplanted m / m or h / h KI mice; magnification 400x is shown. [Figure 10A] Hematoxylin and eosin (H&E) staining of lung tissue sections from engrafted h / m KI mice 4 weeks after BCG infection; magnifications 100x (left) and 200x (right) are shown. [Figure 10B] Lung tissue sections stained for human CD45, CD3 or CD68 from engrafted h / m KI mice 4 weeks post-BCG infection; magnification 200x shown. [Figure 11A] RT-PCR analysis of mouse TPO (mTpo) and human TPO (hTPO) expression in different tissues of Rag2+ / -γc Y / - TPOh / m mice. [Figure 11B] RT-PCR analysis of mTpo and hTPO expression in liver, kidney and mesenchymal multipotent stromal cells (MSCs) from Rag2− / −γc− / − TPOm / m, TPOh / m and TPOh / h mice. [Figure 11C] Concentrations of mouse and human TPO protein in serum of TPOm / m, TPOh / m and TPOh / h mice as measured by ELISA are shown. [Figure 11D] A targeting construct for replacing the mTPO gene with the hTPO gene is shown. [Figure 12A] FACS analysis of human and mouse CD45+ cells in the bone marrow of Rag2- / -γc- / - TPOm / m and TPOh / h mice 3 to 4 months after transplantation of human CD34+ cells is shown. [Figure 12B] The percentage of human CD45+ cells in bone marrow 3-4 months (left) or 6-7 months (right) after transplantation is shown. [Figure 12C] Absolute numbers of human CD45+ cells in bone marrow of the same animals as in FIG. 12(b) are shown. [Figure 12D] The percentage of human CD45+ cells in the bone marrow of Rag2- / -γc- / - TPOm / m and TPOh / h mice engrafted with human CD34+ cells isolated from umbilical cord blood (CB) or fetal liver (FL) is shown. [Figure 13A] Platelet counts in the blood of non-transplanted Rag2- / -γc- / - TPOm / m, TPOh / m and TPOh / h adult mice are shown. [Figure 13B] Representative FACS analysis of mouse (mCD61+) and human (hCD41a+) platelets in the blood of Rag2− / −γc− / − TPOm / m and TPOh / h mice 3–4 months after transplantation is shown. [Figure 13C] FIG. 1 shows human platelet chimerism in TPOm / m and TPOh / h mice as measured by FACS. [Figure 13D] Mouse (mCD61+, 20d) platelet counts in the blood of TPOm / m and TPOh / h recipients are shown. [Figure 13E] Human (hCD41a+, 20e) platelet counts in the blood of TPOm / m and TPOh / h recipients are shown. [Figure 13F] The percentage of human megakaryocytes (CD41a+) among human CD45+ cells in bone marrow is shown. [Figure 13G] The percentage of human CD45+ cells in the blood of Rag2- / -γc- / - TPOm / m and TPOh / h mice is shown. [Figure 13H] The percentage of human CD45+ cells in the spleens of Rag2- / -γc- / - TPOm / m and TPOh / h mice is shown. [Figure 13I] Provide total thymic cell mass for transplant TPOm / m and TPOh / h recipients. [Figure 14A]FACS analysis of human myeloid cell populations in bone marrow and blood of Rag2- / -γc- / - TPOm / m and TPOh / h mice 3 to 4 months after transplantation. [Figure 14B] The total bone marrow population (CD33+ cells) is shown. [Figure 14C] Shows granulocytes (CD33+CD66hi). [Figure 14D] DiffQuick™ staining of hCD45+SSClCD33+CD66hi cells purified from bone marrow of TPOh / h recipients is shown. [Figure 14E] Monocytes (CD33+CD66loCD14+) are shown. [Figure 14F] Analysis of human bone marrow cell populations versus total human CD45+ cell chimerism in blood of Rag2− / −γc− / − TPOm / m and TPOh / h recipients. Granulocytes (CD66+). [Figure 14G] Analysis of human bone marrow cell populations versus total human CD45+ cell chimerism in blood of Rag2− / −γc− / − TPOm / m and TPOh / h recipients. Monocytes (CD14+). [Figure 15A] FACS analysis of mouse Lin-Sca1+c-Kit+ stem cell progenitors in the bone marrow of non-transplanted Rag2- / -γc- / - TPOh / m and TPOh / h mice compared to WT TPO (TPOm / m) Rag2- / -γc- / - mice. [Figure 15B] A quantitative analysis of the results shown in FIG. 15(a) is shown. [Figure 15C] FACS analysis of human CD34+CD38- cells in the bone marrow of Rag2- / -γc- / - TPOm / m and TPOh / h mice 3 to 4 months after transplantation. [Figure 15D] Quantitative analysis of the percentage of CD38− cells within the human CD45+CD34+ population in TPOm / m and TPOh / h recipient mice is shown. [Figure 15E] Human CD34+CD38- cells in the bone marrow of the same mouse as in Figure 15(d) are shown. [Figure 15F]Figure 15(f) and (g) show methylcellulose colony formation assays using human CD45+CD34+ cells purified from Rag2- / -γc- / - TPOm / m and TPOh / h recipients, and Figure 15(f) is CFU-GEMM. [Figure 15G] Figure 15(f) and (g) show methylcellulose colony formation assays using human CD45+CD34+ cells purified from Rag2- / -γc- / - TPOm / m and TPOh / h recipients, Figure 15(g) shows BFU-E (black), CFU-G (white), CFU-M (grey) and CFU-GM (dashed line). [Figure 15H] Human CD45+ chimerism upon secondary transplantation of human CD45+CD34+ cells from Rag2- / -γc- / - TPOm / m and TPOh / h mice into neonatal Rag2- / -γc- / - mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description The invention is not limited to the particular embodiments described, but is instead defined by the appended claims.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any similar or equivalent methods and materials described can be used in carrying out or using this invention, specific embodiments, methods and materials are described herein.All publications mentioned are incorporated herein by reference.In case of discrepancy, this disclosure takes precedence over any disclosure of the incorporated publication.

[0038] The singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a gene" includes a plurality of such genes and reference to "the gene knockout" includes reference to one or more knockouts and equivalents.

[0039] Engineered mice that support human immune cells: hIL-3 / GM-CSF mice Mice containing components of the human immune system (HIS mice) hold considerable promise for studying the human immune system in vivo, for testing human vaccines, and for testing and developing drugs to treat human diseases and disorders. HIS mice are generated by transplanting human hematopoietic stem cell progenitors into severely immunodeficient mouse strains (e.g., recombination activating gene 2 (Rag2) knockout (KO) interleukin 2 receptor gamma (Il2rg) KO mice). Compared to nonhuman primates, HIS mice have the advantage of being small animal models, i.e., they allow more versatile experiments, are more accessible to the research community, and are more ethically acceptable to perform experiments with human subjects. Most importantly, experimental results obtained from HIS mice are highly relevant and potentially applicable to humans, because studies of infections with human-specific pathogens, as well as human-specific immune responses and immunopathology, are now becoming feasible.

[0040] Although much progress has been made in recent years, current HIS mouse models have several major limitations, including poor development, maintenance, and function of human myeloid and T cells. As a result, human inflammatory and immune responses at mucosal surfaces, or robust human T cell-mediated responses such as delayed-type hypersensitivity (DTH), have rarely been observed. Therefore, current HIS mice are not suitable for studying infections and pathologies caused by the serious human pathogen Mycobacterium tuberculosis. Indeed, granulomas, especially those containing human cells, which are a hallmark of the human immune response to mycobacteria, have not been reported in HIS mice so far (see, e.g., Manz et al. (2009) Renaissance for mouse models of human hematopoiesis and immunobiology, Nat. Immunol. 10:1039-1042).

[0041] Current HIS mouse hosts are not suitable for modeling some infectious diseases, at least in part because current HIS mouse hosts do not present a suitable physiological environment for human cells. Several mouse cytokines, e.g., IL-3 and GM-CSF, do not act on their human cognate receptors. Furthermore, Rag2 KO Il2rg KO mice have intact mouse myeloid compartments, and human myeloid cells may be at a competitive disadvantage to host cells. To overcome these limitations, the present disclosure describes the generation of human cytokine knock-in mice in which mouse cytokine-encoding genes are replaced by their human counterparts. The criteria for cytokine replacement are: (1) the mouse cytokine does not or only weakly acts on human cells; (2) the human cytokine does not or only weakly acts on mouse cells to confer a competitive advantage to human cells; and (3) the human cytokine is not exclusively produced by hematopoietic (transplant) cells. (4) Either the lack of the mouse cytokine is not lethal to the mouse host or the human KI cytokine is sufficiently cross-reactive to rescue the mouse KO phenotype. The KI strategy should allow faithful expression at physiological concentrations in the appropriate organs. Importantly, in homozygous KI mice, cells expressing the human cognate receptor should gain a competitive advantage over the respective mouse cells.

[0042] IL-3 and GM-CSF are two cytokines essential for the development and function of bone marrow cells. Neither cytokine has cross-reactivity between humans and mice. IL-3 stimulates early hematopoietic progenitors in vitro but is dispensable 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. GM-CSF is highly expressed in the lung and is important for lung homeostasis in vivo. This is evidenced by the fact that GM-CSF KO mice develop pulmonary alveolar proteinosis (PAP), characterized by accumulation of proteins in the lung due to defective surfactant clearance. Alveolar macrophages from GM-CSF KO mice have defects in terminal differentiation, which leads to reduced innate immunity against pulmonary pathogens. GM-CSF also stimulates the proliferation of human AMs in vitro. Like IL-3, GM-CSF is largely dispensable for steady-state hematopoiesis, and the same is true for mice lacking both cytokines. In contrast, GM-CSF is required for inflammatory responses, including the production of inflammatory cytokines by macrophages and the recruitment and recruitment of monocytes. GM-CSF is also essential for protective immunity against a broad range of pathogens, including Mycobacterium tuberculosis. Notably, GM-CSF KO mice infected with Mycobacterium tuberculosis fail to develop granulomas, a hallmark of tuberculosis.

[0043] This disclosure is based, at least in part, on the recognition that generating hIL-3 / GM-CSF KI mice would be valuable in supporting human bone marrow cell reconstitution and function as well as human innate immune responses to pathogens in mice. The results obtained with such KI mice and described in this disclosure demonstrate that this strategy provides significant improvements over current models of HIS mice in terms of human bone marrow cell development, human lung mucosal immunity, and even granuloma formation following mycobacterial infection. These and other beneficial properties of such mice are described elsewhere in this disclosure.

[0044] Being able to study human tissues in an in vivo setting in mice opens up a wide range of possible research avenues. Major limitations have hindered the application of the approach, one of the most important of which is the inability of mouse factors to support human cells. Indeed, in the immune system, many essential factors required for the development and function of human immune cells are species-specific and cannot be effectively provided by mice. It was therefore decided to follow a strategy of replacing mouse genes with their human counterparts to allow good development and function of human cells and potentially not that of the corresponding mouse cells. By applying this concept to human cytokine KI mice, a proof of concept is provided herein that replacement of immune genes of the mouse host with human genes improves HIS mice. Specifically, the present disclosure supports the view that inappropriate cytokine cross-reactivity between mice and humans, and the necessity to compete with mouse cells, indeed limit the engraftment and function of human bone marrow cells in current HIS mice.

[0045] Human cytokines can be administered to HIS mice by intravenous injection, for example, injection of IL-15 / IL-15Rα complex and IL-7 can enhance the reconstitution of human NK cells and T cells, respectively. Another approach is hydrodynamic injection of plasmid DNA expressing human cytokines, leading to transient expression in the liver. This approach was used very recently to improve the reconstitution of human DCs by hydrodynamic delivery of GM-CSF and IL-4 (see Chen et al. (2009) Expression of human cytokines dramatically improves reconstitution of specific human-blood lineage cells in humanized mice, Proc Natl Acad Sci USA, 106(51):21783-21788). In contrast to the present disclosure, no functional responses of myeloid cells or in vivo responses to pathogens were reported in these mice. Finally, human cytokines can also be overexpressed as transgenes in HIS mice. This approach was taken to generate human IL-3 / GM-CSF / Stem Cell Factor (SCF) transgenic (tg) mice (see Nicolini et al. (2004) NOD / SCID mice engineered to express human IL-3, GM-CSF and Steel factor constitutively mobilize engrafted human progenitors and compromise human stem cell regeneration, Leukemia 18:341-347). In these mice, expression of the human cytokine is driven by a cytomegalovirus promoter resulting in ubiquitous expression. However, hIL-3 / GM-CSF / SCF tg HIS mice are hampered by reduced maintenance of human hematopoietic stem cells in the bone marrow and increased terminal myelopoiesis. Again, unlike the present disclosure, improved bone marrow cell function or in vivo responses were not described.In contrast, in the system described herein, physiological expression of target genes at steady state and in inflammation allows proper development and function of only the appropriate cell types. Importantly, the approach described in this disclosure generates mouse strains that can be maintained and propagated under highly reproducible conditions and are available worldwide for research.

[0046] The hIL-3 / GM-CSF KI mice described in this disclosure represent a considerable improvement over previous HIS mice and the alternative approaches described above. First, delivery of human IL-3 and GM-CSF by the KI strategy described herein leads to long-term cytokine expression, which avoids the need for repeated injections of expensive cytokines. Second, faithful expression is achieved in organs where IL-3 and GM-CSF are normally expressed. Under physiological conditions, GM-CSF is primarily expressed in the lung (Figure 5a). In contrast, hydrodynamic delivery results in predominant expression in the liver and circulation. In neither organ is GM-CSF expressed under steady-state conditions. Third, in contrast to delivery by hydrodynamic injection or ubiquitous overexpression in hIL-3 / GM-CSF / SCF tg mice, physiological amounts of IL-3 and GM-CSF are expressed in KI mice. Physiological levels of GM-CSF have been demonstrated to be important for protective immune responses against M. tuberculosis. Thus, transgenic mice with localized overexpression of GM-CSF in the lungs show impaired granuloma formation and increased susceptibility to M. tuberculosis. Similarly, intravenous administration of GM-CSF also leads to reduced protection against M. tuberculosis infection in mice. Fourth, homozygous hIL-3 / GM-CSF KI mice allow for simultaneous injury of the mouse myeloid compartment, since mouse IL-3 and GM-CSF are not expressed in homozygous mice. This provides a competitive advantage to human myeloid cells, as shown in this disclosure.

[0047] Tuberculosis caused by M. tuberculosis infection causes 1.7 million deaths per year. Novel effective preventive and therapeutic measures are therefore urgently needed. Although mice can be infected with M. tuberculosis, they do not represent an ideal model of human tuberculosis because of species-specific differences in the immune response to M. tuberculosis. For example, infected mice do not develop clearly organized granulomas. Granulomas are a hallmark of the immune response in humans with tuberculosis and contain activated macrophages and activated T cells that fuse to form epithelioid and multinucleated giant cells. Granulomas play an important role in limiting bacterial replication and in controlling the spread of mycobacteria. GM-CSF promotes the differentiation of AMs into multinucleated giant cells in vitro. Studies with transgenic mice have also revealed a role for GM-CSF in the fusion of macrophages to form multinucleated giant cells in vivo. Furthermore, GM-CSF is essential for granuloma formation after mycobacterial infection. The absence of granulomas in GM-CSF KO mice infected with M. tuberculosis is associated with increased bacterial replication and reduced survival.Finally, humans with PAP due to defective GM-CSF signaling show increased susceptibility to mycobacterial infections.

[0048] Human anti-mycobacterial immune responses, especially the formation of granulomas by human cells, have not been reported before in HIS mice. This is likely due to the weak responses of human macrophages and T cells. In this disclosure, antigen-specific T cell responses against mycobacteria were detected in a subset of mice engrafted with human cells. Furthermore, given the prominent role of GM-CSF in granuloma biology, it is hypothesized that engrafted hIL-3 / GM-CSF KI mice are excellent hosts for supporting granuloma formation. This was indeed the case, at least in the subset of mice infected with BCG. Importantly, the pulmonary granulomas in these mice contained human T cells and human macrophages, although they had the loose organization typical of mouse granulomas. Future efforts should aim to further enhance the human T cell and macrophage responses in HIS mice. This should lead to the development of a small animal model that would allow the study of human immune responses to M. tuberculosis in vivo. The hIL-3 / GM-CSF KI mice may also be useful in other settings to study human immune responses in vivo, including infection with pulmonary pathogens, autoimmunity, and human cancer. In summary, the hIL-3 / GM-CSF KI mice provided in this disclosure represent a significantly improved HIS mouse model that may serve as a versatile tool for future research.

[0049] Engineered mice that support human immune cells: hTPO Hematopoietic stem cells (HSCs) are characterized by two main properties: the ability to self-renew throughout life, and the ability to differentiate into all mature hematopoietic cell types. To ensure homeostasis of the HSC pool, during cell division, HSCs generate functional HSCs, whereas their progeny undergo a highly orchestrated program of differentiation and cell proliferation during which multiple lineage-committed progenitor cells and, ultimately, terminally differentiated cells are generated.

[0050] Murine hematopoiesis has been studied extensively over the past several decades, leading to the identification and functional analysis of immunophenotypically defined cell populations that are highly enriched for stem and progenitor cells in vivo. However, promising experimental in vivo studies of human hematopoiesis are limited by obvious practical and ethical constraints.

[0051] To circumvent this limitation, several xenotransplantation models for in vivo studies of human hematopoiesis have been developed. Among these, transplantation of human hematopoietic cells into immunodeficient mice has been widely established in experimental hematopoietic laboratories. The most commonly used model today is the BALB / c Rag2 - / - Gamma c - / - or NOD-SCIDγ c - / - Both strains are highly immunodeficient, lacking B, T and NK cells, and their genetic background is permissive for human hematopoietic engraftment and differentiation. + Upon transplantation of hematopoietic stem cell progenitors, most human hematopoietic populations (including B cells, T cells, monocytes, dendritic cells, erythrocytes, and platelets) can develop and are detectable in these models. However, in these chimeric animals, there is a bias towards lymphoid development with an early high number of B cells, generally representing up to 80% of the human cells, and only a small number of myelomonocytic development. Furthermore, engraftment levels usually begin to decline 4–6 months after transplantation. Furthermore, xenotransplantation of human cells into mice requires the transplantation of a large number of cells compared to the numbers sufficient for optimal engraftment of mouse hematopoietic stem cell progenitors into mice or human cells into humans, respectively. Moreover, in contrast to mouse HSCs transplanted into mouse recipients, human HSCs do not expand or are not maintained in a xenogeneic mouse environment. Thus, the mouse background does not provide an optimal environment to study the physiology of human HSCs, which may be due to the absence or limited cross-reactivity of growth factors required to support HSC function and maintenance.

[0052] Thrombopoietin (TPO) was first identified as a growth factor that promotes megakaryocyte and platelet development. TPO is constitutively produced in the liver and kidney and released into the blood circulation. The receptor for TPO, c-Mpl, is expressed by hematopoietic stem cell progenitors in the bone marrow. c-Mpl is also expressed on circulating platelets. However, binding of TPO to platelets does not activate any signaling pathways. Thus, platelets act as a sink or scavenger for TPO and contribute to the negative regulation of platelet production through this mechanism. TPO has subsequently been recognized for its important function in supporting HSC proliferation and self-renewal. Deficiency of TPO leads to a reduction in HSC numbers in adult mice, and the presence of TPO is necessary for the maintenance of quiescent adult HSCs. Furthermore, TPO is required to support the post-transplant proliferation of HSCs, which are necessary to replenish the hematopoietic compartment of irradiated hosts. Interestingly, it has been demonstrated that osteoblasts, which are involved in the formation of the HSC niche in the bone marrow, produce TPO, which is important for HSC function and maintenance.

[0053] Although mouse and human TPO are both cross-reactive with their cognate receptors when used at supraphysiological doses in vitro, their affinity and biological activity may differ when the cytokines act at physiologically limiting doses in an in vivo environment. Thus, mouse TPO may not adequately stimulate human c-Mpl receptors in vivo and therefore may be a major cause of the impaired properties of human HSCs in the mouse environment. To correct this potential defect, Rag2 - / - Gamma c - / - The gene encoding mouse TPO was replaced in mice by its human counterpart.

[0054] This disclosure is based, at least in part, on RAG2 - / - Gamma c - / -This is based on the recognition that generating hTPO KI mice in a mouse background would be valuable in supporting human bone marrow cell reconstitution and function, as well as human innate immune responses to pathogens in mice, both in the mouse itself and in their progeny when crossed with hIL-3 / GM-CSF mice. - / - Gamma c - / - In the background, results obtained from hTPO KI mice are described in this disclosure. Homozygous hTPO KI mice have significantly increased human engraftment levels in bone marrow and multilineage differentiation of hematopoietic cells is improved compared to mTPO mice, with hTPO KI mice showing increased ratios of myelomonocytic to lymphocytic lineages. The number and self-renewal capacity of human stem cell progenitors are both improved, as demonstrated by serial transplantation. Thus, among other applications, hTPO KI mice are useful for the expansion of human cells by serial transplantation.

[0055] Crossbreeding of hIL-3 / GM-CSF mice with hTPO mice The offspring of the hIL-3 / GM-CSF and hTPO mice described herein are expected to have at least the same relevant characteristics and exhibit at least the same advantages as the parental strains (i.e., hIL-3 / GM-CSF and hTPO mice). For example, human cell populations can be isolated from either parental strain, or both, or their progeny, and serially transplanted into offspring of hTPO mice or hIL-3 / GM-CSF and hTPO mice. Thus, in one aspect, genetically modified mice are provided that are the offspring of hIL-3 / GM-CSF and hTPO mice described herein (including offspring bred homozygously for each relevant gene), and that exhibit the advantages and characteristics of both hIL-3 / GM-CSF and hTPO mice. In one aspect, such offspring mice are provided that are suitable for transplantation and / or serial transplantation from any transplanted mouse (e.g., a transplanted mouse described herein) that includes an ablated immune system (e.g., an irradiated mouse).

[0056] Transplantation of genetically modified mice The genetically modified mouse according to the present invention is used as a recipient of human hematopoietic cells, which can develop human immune cells from the transplanted human hematopoietic cells. In one embodiment, human hematopoietic cells or human hematopoietic stem cell precursor cells (HSPCs) are placed (transplanted) into the genetically modified and irradiated mouse according to the present invention. The human hematopoietic cells or human hematopoietic stem cells give rise in the genetically modified mouse to cells selected from the following: human CD34 positive cells, human hematopoietic stem cells, human hematopoietic cells, bone marrow progenitor cells, bone marrow cells, dendritic cells, monocytes, neutrophils, mast cells, and human blood-lymphatic system (including human hematopoietic stem cell precursor cells, human bone marrow progenitor cells, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, human platelets), and combinations thereof.

[0057] The genetically modified mouse can be irradiated to eliminate any endogenous hematopoietic cells that may be present, and the mouse can be transplanted with human hematopoietic cells from any suitable source.One suitable source of hematopoietic cells known in the art is human umbilical cord blood cells, particularly CD34 positive cells.Another source of hematopoietic cells is human fetal liver.

[0058] In one embodiment, transplantation of mice according to the present invention with human hematopoietic cells results in mice that exhibit increased numbers of human hematopoietic cells than immunodeficient mice lacking humanized TPO gene, lacking humanized IL-3 and GM-CSF genes, or lacking humanized TPO gene and humanized IL-3 and GM-CSF genes.

[0059] In one embodiment, transplantation of mice according to the present invention with human hematopoietic cells results in mice exhibiting increased numbers of human blood cells (e.g., mature hematopoietic cells) compared to immunodeficient mice lacking humanization. In certain embodiments, the human hematopoietic cells are selected from human CD34 positive cells, hematopoietic stem cells, hematopoietic cells, bone marrow progenitor cells, bone marrow cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, and human blood-lymphatic system (including human hematopoietic stem cell progenitor cells, human bone marrow progenitor cells, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, human platelets), and combinations thereof.

[0060] Non-limiting Applications of Genetically Modified and Engrafted Mice Genetically modified mice engrafted with human hematopoietic cells are useful for studying pathogens that do not normally infect mice, one such example being Salmonella typhi, the causative agent of typhoid fever.

[0061] Typhoid fever afflicts over 21 million people worldwide, primarily in developing countries, including approximately 400 cases per year in the United States. Typhoid fever is treated with the following drugs: amoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime, chloramphenicol, ciprofloxacin, cotrimoxazole, ertapenem, imipenem, fluoroquinolones (e.g., ciprofloxacin, gatifloxacin, ofloxacin), streptomycin, sulfadiazine, sulfamethoxazole, tetracycline, and combinations thereof. Recurrent infections are common, limiting disease control with antibiotic therapy. Furthermore, multidrug resistance is also common in Salmonella typhi infections.

[0062] There is a need for new therapeutics, new vaccines, and new methods for testing the efficacy of therapeutics and vaccines. Mice that can be infected by Salmonella typhi will be useful, for example, to identify new therapeutics and vaccines. New therapeutics and vaccines can be tested in such mice, for example, by measuring the amount of Salmonella typhi in the mice (in blood or in selected tissues) in response to treatment with a putative anti-Salmonella typhi agent, or by inoculating the mice with a putative vaccine and then exposing them to an infectious challenge of Salmonella typhi and observing any change in infectivity due to inoculation with the putative vaccine compared to a control that is not vaccinated but infected with Salmonella typhi.

[0063] The genetically modified and transplanted mice according to the present invention are useful for producing mice that can be infected by human pathogens that do not infect mice. For example, the mice are useful as non-human animals that can be infected by Salmonella typhi. In one embodiment, the genetically modified and transplanted mice show enhanced human cell engraftment compared to transplanted mice lacking genetic modification, and the enhancement is sufficient to maintain Salmonella typhi infection. In a particular embodiment, maintaining Salmonella typhi infection comprises the ability of Salmonella typhi to reproduce in the mouse. In a particular embodiment, Salmonella typhi infection comprises the ability of the infected mouse to reproduce Salmonella typhi. In a particular embodiment, the mouse can reproduce Salmonella typhi for at least 1 week, 10 days, 2 weeks, 3 weeks, or 4 weeks after initial introduction or infectious exposure to Salmonella typhi.

[0064] Also provided is a method for identifying anti-S. typhi agents, which uses a mouse as described herein that can be infected by S. typhi. Wild-type mice and other known immunodeficient mice (e.g., RAG1 / RAG2 gene knockout mice) cannot be infected by S. typhi.

[0065] Genetically modified mice are provided that include an Il2rg gene knockout and a RAG gene knockout (e.g., a RAG2 gene knockout) (a first type), and further include a replacement of the endogenous mouse IL-3 gene with a human IL-3 gene and a replacement of the endogenous mouse GM-CSF gene with a human GM-CSF gene (a second type), which genetically modified mice are capable of infection with Salmonella typhi when engrafted with human hematopoietic cells.

[0066] The data shown in Figure 1 is representative of both the first and second type of mice. The genetic modifications of the mouse in Figure 1 include: (a) a mouse RAG gene knockout; and (b) a mouse Il2rg gene knockout. The mouse in Figure 1 also includes transplantation of human hematopoietic cells. This mouse can be further modified with two additional modifications to generate a second type of mouse: (c) replacement of the endogenous mouse IL-3 gene with a human IL-3 gene; and (d) replacement of the mouse GM-CSF gene with a human GM-CSF gene.

[0067] Figures 2, 3 and 4 were obtained using only the first type of modified mice (comprising (a) mouse RAG gene knockout and (b) mouse Il2rg gene knockout modifications and transplantation of human hematopoietic cells).

[0068] In various embodiments, the genetically modified mouse infected with S. typhi comprises a productive infection of S. typhi. In one embodiment, the mouse is capable of carrying and propagating S. typhi in one or more of its cells. In one embodiment, the mouse is capable of maintaining a titer or level of S. typhi in its blood or in at least one tissue for at least 1 week, 10 days, 2 weeks, 3 weeks, or 4 weeks after infectious exposure to S. typhi.

[0069] In one embodiment, the method comprises administering an agent to a genetically modified mouse according to the invention, the genetically modified mouse being infected with Salmonella typhi; detecting the level of Salmonella typhi in the blood or tissues of the mouse after administration of the agent; and optionally determining whether administration of the agent reduces the level of Salmonella typhi in the blood or tissues of the mouse. In one embodiment, the agent is a vaccine. In another embodiment, the agent is an antibiotic or an agent predicted to have antibiotic properties. In one embodiment, the agent is an antigen-binding protein, and in a particular embodiment, an antibody. In one embodiment, the agent is an approved pharmaceutical for human use.

[0070] In one embodiment, the method includes infecting a genetically modified and transplanted mouse according to the present invention with a known amount of S. typhi, administering an agent to the infected mouse, and measuring the amount of S. typhi in the genetically modified and transplanted mouse after administration of the agent. In one embodiment, an agent is determined to be an anti-S. typhi agent if it reduces the amount of S. typhi in the blood or tissues of the mouse by at least half over a predetermined period of time after one administration of the agent or after two or more administrations of the agent.

[0071] In one aspect, a method is provided for determining whether an isolate or strain of S. typhi of interest is drug-resistant or multi-drug-resistant, comprising administering a drug or combination of drugs used to treat S. typhi to a genetically modified and transplanted mouse according to the present invention, the mouse being infected with an isolate or strain of S. typhi of interest. The method comprises measuring the effect, if any, of the drug or combination of drugs on (a) the titer of the isolate or strain of S. typhi of interest in the blood or tissues of the mouse at a time point after administration of the drug or combination of drugs, (b) the ability of the isolate or strain of S. typhi of interest to maintain infection in the mouse or the level of S. typhi in the tissues of the mouse after one or more administrations of the drug or combination of drugs, or (c) the ability of the isolate or strain of S. typhi of interest to reproduce in the mouse at a time point after administration of the drug or combination of drugs. In certain embodiments, the drug is selected from the group consisting of amoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime, chloramphenicol, ciprofloxacin, cotrimoxazole, ertapenem, imipenem, fluoroquinolones (e.g., ciprofloxacin, gatifloxacin, ofloxacin), streptomycin, sulfadiazine, sulfamethoxazole, tetracycline, and combinations thereof. In certain embodiments, administration of the drug or combination of drugs is for at least 1 week, 10 days, 2 weeks, 3 weeks, or 4 weeks after exposure resulting in infection with Salmonella typhi.

[0072] In various aspects and embodiments, the level of S. typhi in blood or tissue is measured by ascertaining the number of colony forming units per unit (eg, weight or volume) of blood or tissue.

[0073] In one embodiment, mice genetically modified according to the present invention and transplanted with human hematopoietic cells have at least 100-fold, 1,000-fold, or 10,000-fold higher levels of Salmonella typhi, measured in colony forming units (cfu), compared to mice not transplanted with human hematopoietic cells.

[0074] Methods and compositions useful for confirming the effectiveness of anti-S. typhi vaccines are provided. In one aspect, a method for confirming the effectiveness of anti-S. typhi vaccines is provided, the method comprising exposing a genetically modified and transplanted mouse according to the present invention to an anti-S. typhi vaccine, then exposing the genetically modified and transplanted mouse to S. typhi, and determining whether or to what extent the genetically modified and transplanted mouse can be infected by S. typhi.

[0075] In one embodiment, the anti-S. typhi vaccine comprises a cell surface protein of S. typhi or an immunogenic fragment thereof. In one embodiment, the vaccine comprises a membrane fraction of a S. typhi strain. In one embodiment, the vaccine comprises a recombinant S. typhi protein or an immunogenic fragment thereof. In one embodiment, the vaccine comprises an expression vector encoding a S. typhi protein or an immunogenic fragment thereof. In one embodiment, the vaccine comprises an inactivated S. typhi strain or a mixture of inactivated S. typhi strains.

[0076] The genetically modified and transplanted mice described in this disclosure are also useful for modeling human pathogen infections more closely than existing mice. For example, infection with Mycobacterium tuberculosis. The genetically modified and transplanted mice described herein are useful for modeling human infection with mycobacteria, for example, by providing a Mycobacterium tuberculosis mouse model that develops granulomas, including granulomas containing human immune cells and well-defined granulomas. The drug and vaccine testing methods described in connection with the described genetically modified and transplanted mice with Salmonella typhi infection can also be applied to Mycobacterium tuberculosis applications, such as identifying drug-resistant strains, testing the efficacy of Mycobacterium tuberculosis vaccines, testing anti-Mycobacterium tuberculosis agents, measuring cfu in response to anti-Mycobacterium tuberculosis agents, etc.

[0077] The genetically modified and engrafted mice described in this disclosure are also useful for modeling human hematopoietic malignancies that arise from early human hematopoietic cells, e.g., from human hematopoietic or progenitor cells. Further applications of the genetically modified and engrafted mice described in this disclosure will be apparent to those of skill in the art upon reading this disclosure.

[0078] Thrombopoietin and Transplantation Thrombopoietin (TPO) was first identified as a growth factor that promotes the development of megakaryocytes and platelets (Wendling, F. et al. (1994) cMpl ligand is a humoral regulator of megakaryocytopoiesis, Nature 369:571-574; Kaushansky, K. et al. (1994) Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin, Nature 369:568-571; Lok, S. et al. (1994) Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo, Nature 369:565-568; de Sauvage, FJ et al. (1994) Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand, Nature 369:533-538; Bartley, TD et al. (1994) Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl, Cell 77:1117-1124; Kaushansky, K. (1998) Thrombopoietin, N Engl J Med 339:746-754; Kaushansky, K. (2005) The molecular mechanisms that control thrombopoiesis, J Clin Invest 115:3339-3347; Kaushansky, K.(2008) Historical review: megakaryopoiesis and thrombopoiesis, Blood 111:981-986). .

[0079] TPO is constitutively produced in the liver and kidney and released into the circulation. The receptor for TPO, c-Mpl, is expressed by hematopoietic stem cell precursors in the bone marrow. c-Mpl is also expressed on circulating platelets. However, binding of TPO to platelets does not activate any signaling pathways. Thus, platelets act as a sink or scavenger for TPO and, through this mechanism, contribute to the negative regulation of platelet production (Kuter, DJ and Rosenberg, RD (1995) The reciprocal relationship of thrombopoietin (c-Mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit, Blood 85:2720-2730). TPO has subsequently been recognized for its important function in supporting HSC proliferation and self-renewal (Fox, N., et al. (2002) Thrombopoietin expands hematopoietic stem cells after transplantation, J Clin Invest 110, 389-3894; Kirito, K. et al. (2003) Thrombopoietin stimulates Hoxb4 expression: an explanation for the favorable effects of TPO on hematopoietic stem cells, Blood 102:3172-3178).

[0080] TPO deficiency leads to a reduction in HSC numbers in adult mice, and the presence of TPO is required for the maintenance of quiescent adult HSCs (Yoshihara, H. et al. (2007) Thrombopoietin / MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche, Cell Stem Cell 1, 685-697; Qian, H. et al. (2007) Critical role of thrombopoietin in maintaining adult quiescent hematopoietic stem cells, Cell Stem Cell 1:671-684). Furthermore, TPO is required to support the post-transplant proliferation of HSCs, which are necessary to replenish the hematopoietic compartment of irradiated hosts. Interestingly, it has been demonstrated that osteoblasts, which are involved in the formation of the HSC "niche" in the bone marrow, produce TPO, which is important for the function and maintenance of HSCs.

[0081] Although mouse and human TPO are both cross-reactive with their cognate receptors when used at supraphysiological doses in vitro, their affinities and biological activities may differ when the cytokines act at physiologically limiting doses in an in vivo environment. We hypothesized that mouse TPO may not adequately stimulate human c-Mpl receptors in vivo and therefore may be the main cause of the impaired properties of human HSCs in the mouse environment. To correct this potential defect, we synthesized Rag2 - / - Gamma c - / - The gene encoding mouse TPO was replaced by its human counterpart in mice, with the hypothesis that such mice would have an improved ability to maintain the differentiation and function of the human hemato-lymphopoietic system.

[0082] Significant progress has been achieved in developing mice that maintain the differentiation and function of the human hemato-lymphoid cell lineage since the publication of the first model over 20 years ago. However, several limitations remain, including: (i) transient human cell engraftment that does not persist throughout the lifespan of the recipient mouse; (ii) unphysiological bias towards lymphoid lineages and poor differentiation of myeloid cells; and (iii) variability in engraftment levels among different animals, even when a group of mice are transplanted with cells from a single human donor. These limitations may be due to the unphysiological location of human cells, residual xenoreactivity of the immunocompromised host, different composition of hemato-lymphoid cells in mouse and human species, and / or lack or paucity of mouse and human cross-reactivity of hematopoietic supportive factors leading to preferential mouse cell support. Thus, providing physiological levels of human growth factors and deleting the respective mouse homologs in the host may further favor the development and survival of human cell populations. Herein, we describe a novel recipient mouse strain in which we humanized the gene encoding thrombopoietin, a cytokine with important functions in the maintenance and self-renewal of hematopoietic stem cells.

[0083] Transplantation of human CD34+ hematopoietic stem cell progenitors into these humanized thrombopoietin mice demonstrated significant improvements in all three limitations listed above compared to previously available models: higher bone marrow chimerism was achieved and maintained for at least 6 months, multilineage, especially myeloid, differentiation was enhanced, and engraftment levels were reduced in variability.

[0084] A major difference between the mouse and human immune systems is the proportion of granulocytes present in the blood. While lymphocytes predominate in mice, human blood is rich in granulocytes, making the species difference of unclear significance. Interestingly, the presence of human TPO improved human granulocyte differentiation (Figure 14). Thus, the presence of human TPO in recipient mice favors a granulocyte-lymphocyte balance that better reflects the human physiological state, possibly due to improved maintenance and / or differentiation of human bone marrow progenitors.

[0085] More importantly, these results indicate that TPO humanization favors the maintenance of secondary recipients that repopulate human hematopoietic stem cell progenitors in the mouse environment (Figure 15). - / - Gamma c - / - TPO h / h Mice represent a novel model to study various aspects of human stem cell progenitor function in vivo.

[0086] Nevertheless, although a good balance between myeloid and lymphoid lineages in the blood was observed, no significant effect of TPO humanization on the overall engraftment level in peripheral lymphoid tissues (including spleen, blood and thymus) was found (Figure 13(g)-(i)). It may be explained by various factors. First, although the recipient mice are sublethally irradiated before transplantation, a large population of mouse bone marrow cells is still present. Among these cells, macrophages can function as phagocytes for human cells, limiting the overall engraftment level in the periphery. Thus, genetic depletion of mouse macrophages, or their functional inactivation, may allow higher peripheral engraftment levels. Second, human cells may require additional human growth factors to favor their terminal differentiation, exit from the bone marrow and / or their survival in the periphery. A diverse panel of cytokines may be considered for each lineage. Finally, although secondary lymphoid organs are formed in humanized mice, their structure is not optimal compared to human tissues. Their partially defective structure may represent a limitation to the number of human cells that can survive in these organs.

[0087] Further gene replacements can be employed to further improve mouse recipients. To achieve that, the approach used in this study, based on knock-in replacement of mouse genes with their human homologs, offers two main advantages over classical transgenic approaches. First, it maintains most of the regulatory sequences of mouse origin, thus ensuring that the humanized genes are faithfully expressed in the mouse host. Second, since the knock-in strategy replaces the mouse cytokine with its human homolog, it can affect the population of mouse-derived cells that depend on the mouse cytokine when the human cytokine does not show sufficient cross-reactivity to the mouse receptor. This can provide an additional competitive advantage to the human cell population after transplantation. Indeed, that seems to be the case for human TPO, since homozygous replacement of TPO leads to a reduction in mouse platelets and HSCs in non-transplanted animals (Figures 13(a), 15(a) and 15(b)).

[0088] In the case of human TPO knock-in mice, an improved model is provided that may be useful for studying the in vivo physiology of human hematopoiesis in general, and human hematopoietic stem cell progenitors in particular. Furthermore, these mice support in vivo human hematopoietic malignancies that arise from early hematopoietic cells, such as myeloid leukemias and myeloproliferative neoplasms. EXAMPLES

[0089] The following examples are not intended to limit the scope of what the inventors regard as their invention. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0090] Example 1 Generation of human IL-3 / GM-CSF and human TPO mice hIL-3 / GM-CSF targeting Targeting constructs for replacing the mouse IL-3 gene with the human IL-3 gene and the mouse GM-CSF gene with the human GM-CSF gene in a single targeting step were constructed using VELOCIGENE® technology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003) "High-throughput engineering of the mouse genome coupled with high-resolution expression analysis," Nat Biot 21(6):652-659, incorporated herein by reference) that utilize gap repair cloning.

[0091] The mouse sequence was obtained from bacterial artificial chromosome (BAC) RPCI-23, clone 5E15, and the human sequence was obtained from the Caltech D library (CTD), BAC clone 2333J5.

[0092] A gap repair donor vector containing a p15 origin of replication was constructed by cloning a 5' mouse homology arm immediately upstream of the mIL-3 ATG, a human 5' IL-3 homology arm spanning the hIL-3 gene from the hIL-3 ATG to about 274 nt, a polylinker, a 3' hGM-CSF (about 233 bases) starting about 2.9 kb downstream of the polyA sequence of the hGM-CSF gene, and a mouse 3' homology arm carrying a drug selection cassette followed by sequences downstream (about 2.9 kb downstream) of the mGM-CSF polyA sequence. This gap repair vector was linearized and inserted into E. coli DH10B strain containing the human CTD BAC clone 2333J5 and recombinase vector as described in Valenzuela et al.

[0093] Cells were grown in drug selection medium.Individual clones were grown to extract gap repair donor vector DNA, and the vector was sequenced for the appropriate mouse-human junction.Pulsed-field gel electrophoresis was used to confirm the size of inserts and expected restriction fragment lengths.

[0094] A captured donor containing mouse upstream and downstream homology boxes flanking the hIL-3 gene, the hGM-CSF gene, and a loxed drug selection cassette was obtained from the repair donor vector, linearized, and the linearized captured donor was introduced into E. coli DH10B containing RPCI23 clone 5E15 and the pABG vector. The cells were grown in drug selection medium. Individual clones containing the captured donor DNA in RPCI23 clone 5E15 DNA (forming the targeting vector) were isolated, the targeting vector DNA was extracted, and a portion of the vector was sequenced for the appropriate mouse-human junction. The size of the insert and the expected restriction fragment length were confirmed using pulsed field gel electrophoresis.

[0095] Electroporation The targeting vector was linearized and used to electroporate mouse ES cells as described in Valenzuela et al. The electroporated mouse ES cells containing the targeting vector were further electroporated with a transient Cre expression vector to remove the roxide drug selection cassette. The targeting vector was electroporated into Rag2 HET Il2rg y / - ES cells. The parent ES cell line with the knockout of RAG2 and Il2rg genes was commercially available V17 ES cells (BALB / c x 129 heterozygote). ES cells targeted with hIL-3 and hGM-CSF genes were used to introduce into mouse embryos.

[0096] hIL-3 / GM-CSF mice Targeted donor ES cells are introduced into 8-cell stage mouse embryos by the VELOCIMOUSE® method (see, e.g., 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," Nat Biot 25(1):91-99, incorporated herein by reference). VELOCIMICE® (fully donor ES cell-derived F0 mice) carrying humanized IL-3 and GM-CSF constructs are identified by genotyping for loss of mouse alleles and gain of human alleles using a modified allele assay (see, e.g., Valenzuela et al.). These mice will first be crossed with BALB / cAnNCR, and then mice heterozygous for Rag2 and Il2rg and containing human IL-3 / GM-CSF KI will be crossed with Rag2 / Il2rg double KO mice for transplantation studies.

[0097] Phenotyping of hIL-3 / GM-CSF mice Humanized mice were tested for production of human GM-CSF by RT-PCR using hGM-CSF-specific primers. The expression pattern of human GM-CSF in the tissues tested was consistent with that of mouse GM-CSF (predominantly expressed in the lung). ELISA was performed on splenocytes from humanized mice stimulated with ConA and IL-2 for 48 hours to detect the presence of hIL-3 and hGM-CSF. Splenocytes were positive for expression of both hIL-3 and hGM-CSF.

[0098] hTPO targeting A targeting construct (Figure 11(d)) for replacing the mouse Tpo (mTpo) gene with the human TPO (hTPO) gene in a single targeting step was constructed using VELOCIGENE® technology utilizing gap repair cloning (Valenzuela et al.). This vector was designed to replace sequences containing the open reading frame of Tpo but maintain the promoter and 5'UTR from mouse. The mouse sequence was obtained from bacterial artificial chromosome (BAC) RPCI-23, clone 98H7. The human sequence was obtained from BAC RPCI-11, clone 63m3. A gap repair donor vector containing a p15 origin of replication was constructed by cloning a 5' mouse homology arm immediately upstream of the mTpo ATG, a human 5' TPO homology arm spanning the hTPO gene from the hTPO ATG to about 275 nt, a polylinker, a 3' hTPO homology arm starting about 1.5 kb downstream of the polyA sequence of the hTPO gene, and a mouse 3' homology arm with a roxide drug selection cassette followed by sequences downstream (about 3.5 kb downstream) of the mTpo polyA sequence. This gap repair vector was linearized and inserted into E. coli DH10B strain containing human BAC clones RPCI-11, 63m3 and the recombinase vector. Cells were grown in drug selection medium. Individual clones were grown to extract gap repair donor vector DNA and sequenced portions of the vector for the appropriate mouse-human junction. Pulsed-field gel electrophoresis was used to confirm the size of the insert and the expected restriction fragment lengths. A capture donor containing mouse upstream and downstream homology boxes flanking the hTPO gene and a roxide drug selection cassette was obtained from the repair donor vector, the capture donor was linearized, and the linearized capture donor was introduced into E. coli DH10B containing RPCI-23 clone 98H7 and the pABG vector. The cells were grown in drug selection medium. Individual clones containing the capture donor DNA in RPCI-23 clone 98H7 DNA (forming the targeting vector) were isolated, the targeting vector DNA was extracted, and the portion of the vector was sequenced for the appropriate mouse-human junction.Pulsed-field gel electrophoresis was used to confirm the size of the insert and the expected restriction fragment lengths. The targeting vector was linearized and used to electroporate mouse embryonic stem (ES) cells. The targeting vector was RAG2+ / -γ. c The parental RAG2 gene was introduced into Y / - ES cells by electroporation. + / - Gamma c Y / - ES cell lines were generated from the commercially available V17 ES cell line (BALB / c x 129 heterozygote). Correctly targeted ES cells were further electroporated with a transient Cre expression vector to remove the roxide drug selection cassette. hTPO-targeted ES cells without the selection cassette were introduced into 8-cell stage mouse embryos by the VELOCIMOUSE® method (Poueymirou et al.). Wild-type Tpo (TPO m / m ), Heterozygous (TPO h / m ) or homozygous (TPO h / h ) Rag2 with TPO gene replacement - / - Gamma c - / - Mice were obtained.

[0099] Example 2 hIL-3 / GM-CSF mouse: transplantation Isolation of human hematopoietic stem cells Human umbilical cord blood and fetal liver samples were obtained from Yale-New Haven Hospital and Albert Einstein Medical College New York, respectively, under approval of the Yale University Human Investigation Committee. CD34+ cells were isolated from human umbilical cord blood or fetal liver by density gradient centrifugation and immunomagnetic selection using CD34 microbeads (Miltenyi Biotec). The purity of isolated CD34+ cells was confirmed by flow cytometry. Purified human CD34+ cells were cryopreserved and stored in liquid nitrogen before use.

[0100] Engraftment of mice with human hematopoietic stem cells Transplantation was performed as previously described (Traggiai et al. (2004) Development of a human adaptive immune system in cord blood cell-transplanted mice, Science 304:104-107). Briefly, on the day of birth, mouse pups from a RAG2 gene knockout / Il2rg gene knockout background (with or without hIL-3 / hGM-CSF) were sublethally irradiated (2 × 200 cGy at 4-h intervals). After irradiation, neonates were injected with 1–2 × 10 5 Mice received 100 human CD34+ cells (resuspended in 25 μL of PBS). Controls were injected with PBS only. Mice were weaned at 3–4 weeks of age and maintained under specific pathogen-free conditions. Mice received prophylactic antibiotics (Sulfatrim) in drinking water to prevent opportunistic infections. All animal experimental work was approved by the Yale University Institutional Animal Care and Use Committee (IACUC) and was performed in accordance with IACUC regulations.

[0101] Analysis of transplanted mice Human hematopoietic cell engraftment was assessed 8–12 weeks after transplantation. Blood samples were collected from the retro-orbital sinus and red blood cell lysis was performed using ACK lysis buffer (Lonza). Samples were then stained with fluorescently labeled monoclonal antibodies against mouse CD45, human CD45, human CD3, and human CD14 (all from BD Biosciences) and analyzed by flow cytometry on a FACScalibur™ (BD Biosciences). Mice used in infection experiments had blood engraftment levels of hCD45+ cells of >4% unless otherwise stated. Matched mice, i.e. mice transplanted with the same batch of CD34+ cells, were used for the experiments. Unless otherwise stated, experiments were performed with mice transplanted with FL-derived CD34+ cells.

[0102] Flow cytometry For hIL-3 / GM-CSF studies, cell suspensions were prepared from lungs, BAL, bone marrow, thymus, spleen, and blood of mice 10–14 weeks after transplantation. Lysis of RBCs was performed with ACK lysis buffer (Lonza). Samples were then stained with fluorochrome-conjugated monoclonal antibodies (mAbs) against mouse and human cell surface antigens. The following mAbs were used: (1) anti-human: CD3 (UCHT1), CD4 (RPA-T4), CD8 (HIT8a), CD11c (B-ly6), CD14 (MoP9), CD19 (HIB19), CD33 (WM53), CD45 (HI30 and 2D1), CD56 (NCAM 16.2), CD66 (B1.1), CD116 (4H1), CD123 (9F5); (2) Anti-mouse: CD45 (30-F11), F4 / 80 (BM8). CD116, CD45 (30-F11) and F4 / 80 mAbs were purchased from eBioscience. All other mAbs were purchased from BD Biosciences. Samples were analyzed on a FACSCalibur™ or LSRII™ flow cytometer (BD Biosciences).

[0103] Methylcellulose CFU Assay Method For hIL-3 / GM-CSF studies, human CD34+ bone marrow cells from transplanted mice were purified by cell sorting. Sorted cells (1–1.5 × 10 5 ) were cultured in Iscove's Modified Dulbecco's Medium (IMDM, GIBCO)-based methylcellulose medium (Methocult™ H4100, StemCell Technologies). The medium was supplemented with 20% FBS, 1% BSA, 2 mM L-glutamine, 55 μM 2-mercaptoethanol, and the following human cytokines: stem cell factor (10 ng / ml), FLT3 ligand (10 ng / ml), thrombopoietin (50 ng / ml), IL-3 (20 ng / ml), IL-6 (10 ng / ml), IL-11 (10 ng / ml), GM-CSF (50 ng / ml), and erythropoietin (4 U / ml) (all from R&D Systems). Cells were incubated in 60 mm Petri dishes at 37°C / 5% C02. After 12-14 days, the number of colonies was counted microscopically.

[0104] Inflammatory response to LPS Mice received two intraperitoneal injections of Ultrapure LPS E. coli 0111:B4 (Invivogen) (35 and 17.5 μg) 48 h apart. Serum was collected 2–3 h after each injection. Serum concentrations of human IL-6 were measured by ELISA (R&D Systems). 72 h after the first LPS injection, mice were sacrificed and blood was collected by cardiac puncture for flow cytometry.

[0105] Intracellular cytokine staining For the hIL-3 / GM-CSF studies, overlapping peptides covering the entire TB10.4 protein (Skjot et al. (2002) Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the esat-6 gene family, Infect. Immun. 70:5446-5453) were synthesized at the W. M. Keck Facility of Yale University. Splenocytes (2 × 10 6 Cells (cells / well) were incubated with mixed peptides (5 μg / ml of each peptide) in a total volume of 200 μl / well in 96-well U-bottom microtiter plates (Becton Dickinson) for 5 h at 37° C. / 5% CO2. RPMI 1640 medium (Invitrogen) supplemented with 10% FCS, 1% penicillin-streptomycin, 1% L-glutamine, and 55 μM 2-mercaptoethanol was used for cell culture. Intracellular cytokine staining was performed using the Cytofix / Cytoperm™ kit (BD Biosciences) according to the manufacturer's instructions. The following mAbs were used for intracellular staining (all BD Biosciences): anti-human IFNγ (B27), anti-mouse IFNγ (XMG1.2). Isotype-matched mAbs were used as controls.

[0106] Histology and immunohistochemistry For hIL-3 / GM-CSF studies, organs were removed and fixed in 10% neutral buffered formalin or Zinc Fixative (BD Biosciences) for histological analysis. Paraffin-embedded tissue sections were prepared and stained with H&E or PAS or processed for immunohistochemistry at Yale Pathology Tissue Services. The following anti-human antibodies were used for immunohistochemistry (all from Dako): CD45 (2B11+PD7 / 26), CD3 (F7.2.38), CD68 (PG-M1). Evaluation of tissue sections for the presence of granulomas was performed in a blinded fashion.

[0107] statistical analysis For the hIL-3 / GM-CSF study, the nonparametric Mann-Whitney U test was used to determine statistical significance between two groups (a = 0.05). For multiple group comparisons, we employed one-way ANOVA with post hoc testing using Tukey's multiple comparison test (a = 0.05). Only statistically significant P values ​​(P < 0.05) are shown.

[0108] Example 3 hIL-3 / GM-CSF-transplanted mice: infection S. typhi ISP2825 (Galan JE and Curtiss, R. (1991) Distribution of the invA, -B, -C, and -D genes of S. thyphimurium among other S. serovars: invA mutants of S. typhi are deficient for entry into mammalian cells, Infect. Immun. 59(9):2901-2908; incorporated herein by reference), a clinical isolate from a patient with typhoid fever, was grown overnight in LB broth. The next day, 40 μL of bacterial cell culture was transferred to 2 mL of fresh LB broth containing 0.3 M NaCl, and the culture was incubated at 37° C. for 2 h until it reached an OD of approximately 0.9. 600The bacterial cultures were then spun down, resuspended in buffered saline, and used for infection. Humanized and control mice aged 9–12 weeks were infected with 1 × 10 3 or 1×10 4 or 1×10 5 S. typhi was inoculated intraperitoneally on day 0. Infected mice were closely monitored and sacrificed 4 weeks post-infection. Spleens, livers and gallbladders were aseptically removed and mechanically homogenized in 3-5 mL of sterile PBS containing 0.05% sodium deoxycholate. Tissue homogenates were serially diluted, plated on LB agar plates and incubated overnight at 37°C for colony counting. Colonies were counted and the total number of colony forming units recovered was calculated. Data for mice are provided in Figures 1-4. In Figure 1, "control" mice are non-transplanted genetically modified mice (RAG KO, Il2rg KO / hIL-3, hGM-CSF). In Figures 2-4, "control" mice are non-transplanted RAG KO / Il2rg KO mice (i.e., they lack humanized IL-3 and GM-CSF, but instead have endogenous mouse IL-3 and endogenous mouse GM-CSF). "Control" mice were injected with PBS instead of human CD34+ cells.

[0109] As shown in Figure 1 , S. typhi infection in the spleen was detected in two genetically modified mice (RAG KO, Il2rg KO / hIL-3, hGM-CSF) 10 days after infection.

[0110] As shown in Figure 2, 1×10 3 One week after infection with S. typhi, genetically modified mice (RAG KO, Il2rg KO) with engraftment rates of 3.8 and 3% showed infection in the spleen that was approximately 1000-fold higher than control mice (p<0.01). The p-value for the difference between control and humanized mice was p<0.01.

[0111] As shown in Figure 3, fetal liver-derived CD34 positive cells were used for transplantation, and 1 × 10 4Genetically modified mice (RAG KO, Il2rg KO) infected with S. typhi showed S. typhi infection in both the spleen (average 1,000-10,000-fold) and liver (average 1,000-fold) at 4 weeks post-infection. Each mouse in the cohort tested for S. typhi in the spleen had human cell engraftment rates of 23.5, 40.1, 16.5, 50, 26, and 51.7 (from top to bottom in the "humanized" cohort, left panel of Figure 3). Each mouse in the cohort tested for S. typhi in the liver had human cell engraftment rates of 16.5, 40.1, 23.5, 26, 50, and 51.7 (from top to bottom in the "humanized" cohort, right panel of Figure 3). The p-value for the difference between control and humanized mice in the spleen was p<0.01; in the liver, p<0.03.

[0112] As shown in Figure 4, fetal liver-derived CD34 positive cells were used for transplantation, and 1 × 10 4 Genetically modified mice (RAG KO, Il2rg KO) infected with S. typhi showed S. typhi infection in the gallbladder 4 weeks post-infection with S. typhi cfu on average 1 million times higher than control mice. Mice in the cohort tested for S. typhi in the gallbladder had human cell engraftment rates of 23.5, 16.5, 40.1, 50, 26, and 51.7 (from top to bottom in the "humanized" cohort in Figure 3). The p-value for the difference between control and humanized mice was p<0.03.

[0113] These results demonstrate that genetically modified mice (RAG KO, Il2rg KO / hIL-3, hGM-CSF) can be colonized by S. typhi following systemic infection.

[0114] Example 4 hIL-3 / GM-CSF-engrafted mice: validation of a mouse model for the human inflammatory response to pulmonary pathogens The genes encoding GM-CSF (Csf2) and IL-3 are closely linked (<10 kb) in humans and mice on chromosomes 5 and 11, respectively. This allowed the replacement of mouse and human loci of both genes to generate hIL-3 / GM-CSF KI mice (Figure 5(e)). The human IL-3 KI allele is under the control of mouse regulatory elements, while the human Csf2 KI allele remains under the control of its human regulatory elements. Expression of mouse and human GM-CSF mRNA was analyzed by RT-PCR in hIL-3 / GM-CSF KI mice expressing one allele of each mouse gene and one allele of each human gene (termed IL-3 / GM-CSF "human / mouse" (h / m) mice). Wild-type mice carrying only mouse alleles of IL-3 and GM-CSF are termed IL-3 / GM-CSF "mouse / mouse" (m / m) mice.

[0115] RT-PCR and ELISA analysis of hIL-3 / GM-CSF KI mice Total RNA was extracted from homogenized tissues using TRIzol™ Reagent (Invitrogen) according to the manufacturer's instructions. Equal amounts of DNase-treated RNA were used for cDNA synthesis using the SuperScript™ First-Strand Synthesis System (Invitrogen). Conventional RT-PCR was performed using the following primers: TIFF2025032190000002.tif36170Quantitative RT-PCR was performed on a 7500 Fast Real-Time PCR System using primer-probe sets purchased from ABI. Expression levels were calculated using the comparative threshold cycle method and normalized to mouse or human HPRT. Mouse and human IL-3 and GM-CSF proteins were detected using species-specific ELISA kits from R&D Systems according to the manufacturer's instructions. Splenocytes were activated with 5 μg / ml concanavalin A (ConA) and 100 U / ml IL-2, and after 48 h of stimulation, supernatants were collected for ELISA.

[0116] Expression in hIL-3 / GM-CSF KI mice Human GM-CSF mRNA was expressed in a similar pattern to its mouse counterpart, with highest expression in the lung (Figure 5(a)). IL-3 is expressed primarily by activated T cells, which also produce GM-CSF. Therefore, ELISA was performed on supernatants from activated splenocytes isolated from h / m mice. Both human IL-3 and GM-CSF proteins could be detected (Figure 6(f), 6(g)). To confer a competitive advantage to human hematopoietic cells, we generated homozygous KI mice, termed IL-3 / GM-CSF "human / human" (h / h) mice, which express two alleles of human IL-3 and GM-CSF. Conventional and quantitative RT-PCR analysis of lung tissues showed that h / h mice express only human GM-CSF mRNA, but not mouse GM-CSF mRNA (Figure 5(b), 5(c)). Human GM-CSF protein could be detected in the bronchoalveolar lavage (BAL) fluid of h / h mice by ELISA (Figure 5(d)). These results indicate that hIL-3 / GM-CSF KI mice faithfully express human GM-CSF (and IL-3).

[0117] Figure 5(a)-(d) show validation of human GM-CSF expression in non-transplanted hIL-3 / GM-CSF KI mice. Figure 5(a) shows representative RT-PCR analysis of GM-CSF mRNA expression in various tissues from KI mice (h / m) carrying one allele of human Csf2 and one allele of mouse Csf2. Li, liver; Br, brain; Lu, lung; Mu, muscle; Sp, spleen; Th, thymus; LN, lymph node; BM, bone marrow. Bottom: Specificity of primers for detecting human GM-CSF was validated by RT-PCR analysis of tissues from control mice (m / m). Ribosomal protein L13 (Rpl13) was used as an endogenous control. Figure 5(b) shows RT-PCR analysis of GM-CSF mRNA expression in lungs from m / m mice or homozygous KI mice expressing two alleles of human Csf2 (h / h) (n=5 each). Rpl13 was used as an endogenous control; NTC, no template control. Figure 5(c) shows quantitative RT-PCR analysis of the same GM-CSF mRNA expression as in (b). GM-CSF expression was normalized to mouse Hprt (n=5 each). Figure 5(d) shows ELISA of human GM-CSF protein in BAL fluid collected from m / m or h / h KI mice (n=6 each). Results are representative of two independent experiments. Each dot represents one mouse. Horizontal bars indicate the mean value.

[0118] Figure 5(e) shows the strategy for generating hIL-3 / GM-CSF KI mice. The genomic organization of the mouse (top) and human (bottom) IL3 and Csf2 loci are shown on chromosomes 11 and 5, respectively. The mouse loci were replaced with the human loci as described in this disclosure.

[0119] Figure 6(f) and (g) show the expression of human IL-3 and GM-CSF in non-transplanted hIL-3 / GM-CSF KI mice. ELISA results of human IL-3 (f) and GM-CSF (g) production by activated splenocytes are presented. Splenocytes from m / m mice (white bars) or h / m KI mice (black bars) were stimulated with ConA and IL-2 for 48 h and supernatants were collected (n=1 each). Human IL-3 and GM-CSF were not detected in m / m mice (ND).

[0120] Example 5 Mice engrafted with hIL-3 / GM-CSF: enhanced human inflammatory responses hIL-3 / GM-CSF KI mice were generated from embryonic stem (ES) cells in which one allele of both Rag2 and Il2rg had already been deleted. Subsequent crossing into a Rag2 KO Il2rg KO background allowed for the engraftment of human CD34+ hematopoietic cells. Overall human CD45+ hematopoietic cell chimerism, as well as the distribution of T cells, B cells, and natural killer (NK) cells in bone marrow, thymus, spleen, and blood, were not significantly increased in hIL-3 / GM-CSF KI mice (data not shown). Furthermore, the frequencies of total human CD33+ myeloid cells, CD66+ granulocytes, CD14+ monocytes / macrophages, CD14loCD16+ non-classical monocytes, CD11c+ dendritic cells (DCs), and CD123+CD11c- plasmacytoid DCs were not significantly increased in hIL-3 / GM-CSF KI mice (data not shown). This was true for both h / m and h / h mice under steady-state conditions. Finally, human bone marrow cells from transplanted hIL-3 / GM-CSF KI mice had a similar ability to form myeloid colonies in methylcellulose in vitro (data not shown). These findings are consistent with results from KO mouse studies showing that both IL-3 and GM-CSF are largely dispensable for steady-state myelopoiesis in the organs analyzed here.

[0121] In contrast, GM-CSF plays a key role in mediating inflammatory responses. Expression of GM-CSF is induced by inflammatory stimuli, which leads to the production of inflammatory cytokines (such as IL-6 and TNFα) by monocytes / macrophages and their recruitment to inflammatory sites. Human CD14+ monocytes from transplanted hIL-3 / GM-CSF KI mice showed the highest expression of GM-CSF receptor α chain (CD116) (Figure 8(d)). Therefore, analysis of transplanted hIL-3 / GM-CSF KI mice focused on human monocytes / macrophages. First, the inflammatory response of human monocytes in transplanted hIL-3 / GM-CSF KI mice was analyzed. Systemic inflammation was induced by intraperitoneal (ip) injection of lipopolysaccharide (LPS). After LPS injection, the frequency of circulating human CD14+ monocytes was significantly increased in h / m mice compared to control m / m mice (Figure 8(e), (f)). Increased human monocyte recruitment in h / m mice was associated with elevated serum concentrations of human IL-6 after one and two LPS injections (Figure 8(g)). LPS-induced production of human TNFα was also increased in h / m mice, although this result did not reach statistical significance. These data indicate that hIL-3 / GM-CSF KI mice engrafted with human hematopoietic cells have an enhanced human inflammatory response mediated by human myelomonocytic cells.

[0122] Figure 8(d)-(g) show enhanced human inflammatory responses in transplanted hIL-3 / GM-CSF KI mice. Figure 8(d) shows flow cytometry analysis of human bone marrow cells from transplanted hIL-3 / GM-CSF h / m KI mice at steady state. The dot plot (left) is gated on hCD45+mCD45- cells. The histogram (right) shows GM-CSF receptor alpha (CD116) expression on CD14- cells (population 1), CD14mid / SSChi granulocytes (population 2), and CD14hi monocytes (population 3). A representative example from a total of 12 mice analyzed is shown. Figure 8(e) contains a representative flow cytometry analysis of human blood cells from CB-transplanted m / m or h / m KI mice 72 hours after two LPS intraperitoneal injections. The plot is gated on hCD45+mCD45- cells. Numbers next to boxed areas indicate the percentage of human CD14+ cells. Figure 8(f) shows the frequency of human CD14+ blood cells in transplanted m / m mice (n=4) or h / m KI mice (n=8) 72 hours after LPS injection. Figure 8(g) shows ELISA results for human IL-6 in serum from transplanted m / m mice (n=4-5) or h / m KI mice (n=8) 2-3 hours after the first (top) and second (bottom) LPS injections. One m / m mouse died after the first LPS injection. Each dot represents one mouse. Horizontal bars indicate the mean. Results are representative of two independent experiments.

[0123] Example 6 Mice transplanted with hIL-3 / GM-CSF: Enhanced human macrophage engraftment in the lungs BAL analysis Bronchoalveolar analysis for hIL-3 / GM-CSF studies was performed as follows: Lungs were inflated with 1 ml of PBS through a catheter inserted in the trachea. This was repeated twice and the collected lavage fluids were pooled. After centrifugation, cell-free supernatants were saved for measurement of GM-CSF protein concentration by ELISA or total protein content using the BCA Protein Assay Kit (Pierce) according to the manufacturer's instructions. After lysis of red blood cells (RBCs) with ACK lysis buffer (Lonza), cell pellets were counted and used for flow cytometry or for cytospin preparation. Cells were spun onto slides and stained with Diff-Quik™ Stain Set (Dade Behring) according to the manufacturer's instructions.

[0124] Enhanced macrophage engraftment The absence of mouse GM-CSF leads to reduced mouse alveolar macrophage (AM) function, which should favor reconstitution with human macrophages in homozygous hIL-3 / GM-CSF KI mice. In support, human GM-CSF is highly expressed in the lungs and BAL of h / h mice, whereas mouse GM-CSF is lacking.

[0125] Mouse AMs from non-transplanted h / h mice were enlarged and had a typical "foamy" appearance (Figure 9(f)). This foamy appearance has been described for AMs from GM-CSF KO mice. GM-CSF KO mice develop PAP due to defective clearance of surfactant by AMs with impaired terminal differentiation. Similar to what was reported for GM-CSF KO mice, non-transplanted h / h mice developed features of PAP, including subpleural accumulation of AMs replete with periodic acid-Schiff (PAS)-positive material (Figure 9(g)). It was therefore concluded that non-transplanted h / h mice exhibit impaired differentiation of mouse AMs and develop PAP, and are therefore functionally equivalent to GM-CSF KO mice.

[0126] Figures 9(f) and (g) show the development of PAP in non-transplanted homozygous hIL-3 / GM-CSF KI mice. Figure 9(f) shows Diff-Quick™ staining of BAL cells from non-transplanted m / m or h / h KI mice (magnification 400x); a representative example of a total of 6 mice analyzed per group is shown. Figure 9(g) shows PAS staining of lung tissue sections from non-transplanted m / m or h / h KI mice (magnification 400x); a representative example of a total of 12 mice analyzed per group is shown.

[0127] We next examined the lung compartments of h / h mice after transplantation with human hematopoietic cells. FACS analysis showed that h / h mice had significantly more human CD45+ cells in the BAL (Figure 6(a), (b)). Quantitative RT-PCR of lung tissue revealed that this increase in human cells was mainly composed of cells expressing mRNA for the human myeloid markers CD33, CD11b, CD11c, and CD14 (Figure 6(c)). Furthermore, mRNA expression of human CD68, a mature macrophage marker expressed primarily intracellularly, was significantly increased in transplanted h / h mice (Figure 6(d)). This increase in h / h mice was associated with higher expression of two transcription factors expressed by AMs, namely PU.1 (Spi1) and peroxisome proliferator-activated receptor gamma (PPARγ) (Figure 6(d)). PU.1 is highly expressed in terminally differentiated AMs in a GM-CSF-dependent manner. Importantly, transduction of GM-CSF KO AMs with PU.1 in vitro ameliorates their impaired function. PPARγ is also highly expressed in AMs, and like GM-CSF KO mice, PPARγ KO mice develop PAP. Immunohistochemical staining of lung sections revealed the presence of numerous hCD68+ cells in a typical intraalveolar location in transplanted h / h mice, consistent with human AMs (Figure 6(e)). In contrast, only very few human AMs could be detected in transplanted m / m control mice. In summary, lungs from h / h mice transplanted with CD34+ hematopoietic cells show markedly improved human macrophage engraftment.

[0128] Figures 6(a)-(e) show that homozygous hIL-3 / GM-CSF KI mice have good human macrophage engraftment in the lungs. Figure 6(a) shows representative flow cytometry analysis of BAL cells from transplanted m / m and h / h KI mice. Numbers next to the circled areas indicate the percentage of hCD45+ and mCD45+ hematopoietic cells. mCD45+hCD45+ cells have high autofluorescence and constitute F4 / 80+ mouse AM. Figure 6(b) provides the number of human hematopoietic (hCD45+) cells in the BAL from transplanted m / m and h / h KI mice (results are combined from three independent experiments (total n=15 per group)). Figure 6(c) shows the results of quantitative RT-PCR analysis of human lymphoid and myeloid gene expression in lung tissue from transplanted m / m and h / h KI mice (n=4 each). Expression was normalized to mouse HPRT ( * , P<0.05). Figure 6(d) shows quantitative RT-PCR analysis of human macrophage gene expression in lung tissue from transplanted m / m and h / h KI mice (n=4 each). Expression was normalized to mouse HPRT ( * , P<0.05). Each dot represents one mouse. Horizontal bars indicate the mean. Figure 6(e) shows immunohistochemistry of lung tissue sections stained for human CD68 from transplanted m / m and h / h KI mice (magnification 100x (top) and 200x (bottom)). A representative example from a total of 10 mice analyzed per group is shown.

[0129] Example 7 Mice engrafted with hIL-3 / GM-CSF: PAP is alleviated by human hematopoietic cells We investigated whether increased human macrophage engraftment in h / h mice would lead to better human immune function in the lungs. We first investigated whether human macrophages could rescue the PAP syndrome seen in non-transplanted h / h mice. Although type II alveolar epithelial cells and AMs can both respond to GM-CSF, PAP can be rescued by bone marrow transplantation. This indicates that hematopoietic cells, especially AMs, are the main cell type that can ameliorate PAP. It was therefore hypothesized that h / h mice engrafted with human hematopoietic cells should have less severe PAP. As expected, non-transplanted h / h mice showed intra-alveolar accumulation of PAS-positive material (Figure 7(a)), a hallmark of PAP. Consistent with this hypothesis, transplanted h / h mice had mild protein accumulation in the lungs, and the lungs of some h / h mice resembled those of (non-transplanted or transplanted) m / m control mice (Figure 7(a)). Furthermore, the total protein content in the BAL fluid of transplanted h / h mice was significantly lower than that of non-transplanted h / h mice (Figure 7(b)). These results indicate that transplanted human hematopoietic cells (presumably AM) can alleviate PAP in homozygous hIL-3 / GM-CSF KI mice.

[0130] Figures 7(a)-(e) show that human hematopoietic cells alleviate PAP in homozygous hIL-3 / GM-CSF KI mice. Figure 7(a) shows PAS staining of lung tissue sections from non- or transplanted m / m or h / h KI mice. Lung sections from two different transplanted h / h KI mice are shown (magnification 400x). A representative example of a total of 10-12 mice analyzed per group is shown. Figure 7(b) shows quantification of total protein in BAL fluid from non- or transplanted h / h KI mice or m / m control mice (n=6 per group). *P<0.0001 (one-way ANOVA test). *P values ​​determined by Tukey's multiple comparison test are indicated by an asterisk ( ** , P < 0.01; *** , P<0.001).

[0131] Example 8 Mice engrafted with hIL-3 / GM-CSF: robust human type I IFN responses to influenza A Influenza A infection Mice (9-10 weeks old) were incubated with 2 × 10 4 Mice were infected intranasally with plaque-forming units of influenza A / PR8 (H1N1) virus. Infection was performed by intranasal administration of 50 μl of virus stock diluted in PBS (or an equal volume of PBS as a control) to mice deeply anesthetized with Anafane™ (Ivesco). Twenty-four hours after infection, lungs were harvested for RNA extraction and quantitative RT-PCR analysis as described above.

[0132] In addition to their role in lung homeostasis, AMs are essential for host defense in the lung. Many studies have shown that GM-CSF KO mice are more susceptible to various pathogens in the lung. To evaluate the functional response of transplanted human AMs to pulmonary pathogens, transplanted h / h mice were infected intranasally with influenza A / PR8 (H1N1) virus. AMs are the main producers of type I interferon (IFN) after pulmonary virus infection, and AMs are required for an effective innate response to influenza A. Human hypoxanthine phosphoribosyltransferase (HPRT) mRNA expression was significantly higher in the lungs of transplanted h / h mice compared to control m / m mice (Figure 8(a)), indicating good human immune cell chimerism. Transplanted m / m mice did not show significant induction of human IFNβ mRNA expression after influenza A infection when compared to transplanted m / m mice administered PBS intranasally (Figure 8(b)). In contrast, transplanted h / h mice expressed significantly more human IFNβ mRNA than both uninfected h / h and infected m / m mice (Figure 8(b)). The increased expression of human IFNβ mRNA in h / h mice compared to m / m mice was still significant when normalized to human HPRT, i.e., the number of human cells in the lungs (Figure 8(c)). Taken together, homozygous hIL-3 / GM-CSF KI mice allow good human macrophage chimerism and function in the lungs leading to enhanced human mucosal immunity against viral infection.

[0133] Figures 8(a)-(g) show that homozygous hIL-3 / GM-CSF KI mice mount a strong human type I IFN response to influenza A infection. Figures 8(a)-(c) show quantitative RT-PCR analysis of gene expression in lung tissue from m / m and h / h KI 24 hours after intranasal infection with influenza A (PR8) (n=8 each). Intranasal administration of PBS was used as a control (PBS) (n=4 each). Figure 8(a) shows expression of human Hprt normalized to mouse Hprt. P<0.0001 (one-way ANOVA test). Figure 8(b) shows expression of human IFNγ normalized to mouse Hprt. P=0.0171 (one-way ANOVA test). Figure 8(c) shows expression of human IFNγ normalized to human Hprt. P=0.0032 (one-way ANOVA test). P values ​​determined by Tukey's multiple comparison test are indicated by asterisks. (* , P < 0.05; ** , P < 0.01; *** , P<0.001). Each dot represents one mouse. Horizontal bars indicate the mean. Results are representative of two independent experiments.

[0134] Example 9 Mice engrafted with hIL-3 / GM-CSF: Granulomas containing human cells after mycobacterial infection The ability of hIL-3 / GM-CSF KI mice to support a human inflammatory response against a second pathogen with pulmonary tropism, where macrophages play a central role in pathogen-specific immune responses, was examined. Granuloma formation after mycobacterial infection was selected. Granulomas represent a specialized local inflammatory response that is characteristic of mycobacterial infection. It is a classic example of a DTH response, the formation of which depends on the interaction of activated T cells with macrophages. Both IL-3 and GM-CSF are required for an optimal DTH response, and importantly, GM-CSF KO mice do not form granulomas when infected with mycobacteria.

[0135] The transplanted hIL-3 / GM-CSF h / m KI mice were infected intravenously with Bacillus Calmette-Guerin (BCG). BCG is an attenuated strain of M. bovis used as a vaccine against human tuberculosis. Mice used in BCG infection experiments had blood engraftment levels of >20% hCD45+ cells, and >8% of the hCD45+ cells were T cells (hCD3+). Mice were 9-10 weeks old at the time of infection. 1×10 5 Mice were infected with colony forming units of BCG (Statens Serum Institute, Copenhagen) via tail vein injection in a volume of 0.1 ml.

[0136] Since T cells are essential for granuloma formation, we first examined the human T cell response to BCG 4 weeks after infection. Flow cytometry clearly demonstrated the presence of human T cells in the lungs of both transplanted m / m and h / m mice (Figure 9(a)). Indeed, T cells were the predominant human hematopoietic cell type in BCG-infected lungs. Compared to m / m control mice, BCG-infected h / m mice had a higher mean ratio of human CD4 to CD8 T cells in the lungs, although the difference did not quite reach statistical significance (Figures 9(b), 9(c)). No differences in the ratios of splenic hCD4 / hCD8 T cells were observed between these two groups of mice.

[0137] We next analyzed the expression of two T cell-derived cytokines, namely IFNγ and TNFα, both of which are crucial for protective immune responses against mycobacteria. BCG-specific IFNγ production was examined by intracellular cytokine staining after restimulation of splenocytes from infected mice with a peptide derived from the immunodominant mycobacterial antigen TB10.4. As expected, a population of IFNγ-producing mouse T cells was detected among splenocytes from BALB / c mice (Fig. 9(d)). Furthermore, human BCG-specific T cell responses were found in a subset of transplanted h / m and m / m mice (Fig. 9(d)). Finally, the majority of transplanted h / m and m / m mice expressed human IFNγ and TNFα mRNA in the lungs after BCG infection (Fig. 9(e)). These results indicate that a subset of transplanted mice is capable of mounting a pathogen-specific human T cell response against BCG, but this response was not enhanced in hIL-3 / GM-CSF KI mice. Consistent with this, bacterial burden did not differ between h / m and m / m mice.

[0138] Granuloma formation was then assessed by histology in the lungs and liver 4 weeks after infection. Non-engrafted m / m mice (lacking T cells) did not develop granulomas (Table 1), consistent with the requirement of T cells for granuloma formation. Similarly, m / m mice engrafted with human cells did not show granulomas in the lungs or liver (Table 1). In contrast, the majority of h / m mice had small lesions or granulomas in the lungs (Figure 10(a)) or liver or both organs (Table 1). In general, the granulomas observed were small and had a loose organization more typical of mouse granulomas than human. However, pulmonary granulomas in hIL-3 / GM-CSF KI mice contained human hematopoietic cells (hCD45+) as evidenced by immunohistochemistry (Figure 10(b)). The majority of these cells were human T cells (hCD3+), with a few human macrophages (hCD68+) present in the center (Figure 10(b)). In summary, engrafted hIL-3 / GM-CSF KI mice are capable of developing granulomas containing human T cells and human macrophages in response to mycobacterial infection, which has not been previously reported in HIS mice. Table 1 shows the lesions / granulomas observed in liver and lung tissue sections from BALB / c, non-engrafted m / m (non), engrafted m / m, and engrafted hIL-3 / GM-CSF h / m KI mice 4 weeks after BCG infection.

[0139] Table 1 Granulomas in transplanted hIL-3 / GM-CSF KI infected with BCG TIFF2025032190000003.tif175153

[0140] Figures 9(a)-(g) show human T cell responses to BCG in transplanted hIL-3 / GM-CSF KI mice. Figures 9(a)-(c) show flow cytometry analysis of lung cells from transplanted m / m and h / m KI mice 4 weeks after BCG infection. Figure 9(a) shows the frequency of human T cells (hCD45+hCD3+) in the lungs. Numbers next to the boxed areas indicate the percentage of cells. Figure 9(b) shows the distribution of human CD4 and CD8 T cells in the lungs. Dot plots are gated on hCD45+hCD3+ cells. Numbers in the quadrants indicate the percentage of cells. Figure 9(c) shows the ratio of human CD4 to CD8 T cells in the lungs (n=6 each). Each dot represents one mouse. Horizontal bars indicate the mean. Figure 9(d) shows flow cytometry analysis of splenocytes from BALB / c mice, engrafted m / m mice, and engrafted h / m KI mice 4 weeks after BCG infection. Splenocytes were restimulated in vitro with a pool of overlapping peptides covering the TB10.4 protein as described herein. Dot plots show the frequency of murine IFNγ+ CD4 T cells (mCD4+) or human IFNγ+ T cells (hCD3+) measured by intracellular cytokine staining. Staining with isotype-matched antibodies was used as control. Figure 9(e) shows the results of quantitative RT-PCR analysis of human IFNγ (left) and TNFα (right) gene expression in lung tissue from BALB / c mice, non-engrafted m / m mice, engrafted m / m mice, and engrafted h / m KI mice 4 weeks after BCG infection (n=4–7 per group). Each dot represents one mouse. Horizontal bars indicate the mean value.

[0141] Figure 10(a) and (b) show that transplanted hIL-3 / GM-CSF KI mice develop granulomas containing human cells after BCG infection. Figure 10(a) shows hematoxylin and eosin (H&E) staining of lung tissue sections from transplanted h / m KI mice 4 weeks after BCG infection (100x magnification (left) and 200x magnification (right)). Figure 10(b) shows immunohistochemistry of lung tissue sections stained for human CD45, CD3, or CD68 from transplanted h / m KI mice 4 weeks after BCG infection (200x magnification). A representative example of two mice with pulmonary granulomas is shown.

[0142] Example 10 hTPO mice: engraftment and analysis Transplantation into TPO mice Recipient mice were transplanted with human hematopoietic progenitor cells as described in Traggiai et al.. Umbilical cord blood samples were collected from healthy, full-term newborns under approval from the Yale Human Rights Inspection Committee (Department of Labor and Birth, Yale New Haven Hospital, New Haven, CT). Fetal liver samples were obtained from the Human Fetal Tissue Repository at the Albert Einstein College of Medicine (Bronx, NY) and from Advance Biosciences Resources (Alameda, CA).

[0143] Fetal liver samples were cut into small pieces, treated with collagenase D (100 ng / ml, Roche) for 45 min at 37°C, and cell suspensions were prepared. Human CD34+ cells were purified from fetal liver samples or from umbilical cord blood by density gradient centrifugation (Lymphocyte Separation Medium, MP Biomedicals) followed by positive immunomagnetic selection with anti-human CD34 microbeads according to the manufacturer's instructions (Miltenyi Biotec). Cells were frozen in 10% DMSO with FBS or directly injected.

[0144] Neonatal pups (<1 day old) were sublethally irradiated (X-irradiation, 2 × 150 cGy at 4-h intervals) and 100,000–200,000 CD34+ cells in 20 μl of PBS were injected into the liver using a 22-gauge needle (Hamilton, Reno, NV).

[0145] All experiments were performed in accordance with Yale University Human Rights Review Board protocols and Yale University Animal Care and Use Committee protocols.

[0146] TPO expression Serum concentrations of mouse and human TPO protein were measured by species-specific ELISA (RayBiotech) according to the manufacturer's protocol. To measure expression of mouse and human mRNA encoding TPO, tissues were isolated from adult animals and total RNA was purified using TRIzol (Invitrogen) according to the manufacturer's instructions. Contaminating genomic DNA was removed by treatment with RNase-Free DNase I (Roche), and the RNA was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen) and an oligo dT primer. The following primers were used for PCR amplification: TIFF2025032190000004.tif30160

[0147] To examine whether human TPO is faithfully expressed in these mice, total RNA was analyzed using TPO h / m The TPO mRNA was extracted from various organs derived from mice. Similar expression patterns were observed for both mouse and human mRNA encoding TPO by RT-PCR (Figure 11(a)). m / m , TPO h / m and TPO h / h Expression was compared in three tissues or cell types from mice that are known to express TPO (liver, kidney, and mesenchymal multipotent stromal cells). m / m and TPO h / m Expression of mouse Tpo was detected in samples from mice, whereas TPO h / m and TPO h / h Human TPO was expressed in the mice (Figure 11(b)). The concentration of TPO protein in the serum of the target mice was also measured. Mouse TPO was expressed as TPO m / m and TPO h / m Detected in animals, human TPO is TPO h / m and TPO h / h (Figure ​(c)). The measured concentrations of human TPO were approximately 10-fold lower than mouse TPO. However, this difference is in line with the physiological concentrations reported in healthy humans and mice (Figure11(c)) and may be due to species-specific differences in cytokine half-life.

[0148] Figure 11 shows (a) Rag2 + / - Gamma c Y / - TPO h / m RT-PCR analysis of mouse TPO (mTpo) and human TPO (hTPO) expression in different mouse tissues (mouse Rpl13a was used as a housekeeping gene); (b) Rag2 - / - Gamma c - / - TPO m / m , TPO h / m and TPO h / h RT-PCR analysis of mTpo and hTPO expression in mouse liver, kidney and mesenchymal multipotent stromal cells (MSCs); (c) TPO m / m , TPO h / mand TPO h / h Concentrations of mouse and human TPO protein in mouse serum (pg / ml, mean ± SD, n=7-9) measured by ELISA are shown. ND: not detected. Normal ranges shown are from the Thrombopoietin Quantikine kit from R&D Systems.

[0149] Isolation of mesenchymal multipotent stromal cells For TPO studies, femurs and tibias of mice were harvested and bone marrow cells were flushed out. Bones were cut into small pieces and digested with collagenase P and D (10 μg / ml) at 37°C for 45 min. Bone-associated cells were collected by repeated pipetting. Cells were cultured for 14 days in the presence of MSC medium supplemented with stimulatory supplements (Stemcell Technologies). Hematopoietic cells were removed from the cultures by immunomagnetic cell sorting (MACS, Miltenyi Biotec) using CD45 and Ter119 antibodies. Non-hematopoietic cells (CD45-Ter119-) were cultured for another 5 days and the MSC phenotype (CD45-Ter119-Sca1+CD90+) was confirmed by FACS (Diminici et al. (2006) Minimal criteria for defining multipotent mesenchymal stromal cells, The International Society for Cellular Therapy position statement, Cytotherapy 8:315-317).

[0150] Analysis of hematopoietic cell populations in TPO mice Mice were bled 8–12 weeks after transplantation. Red blood cells were lysed three times using ACK (Lonza), and cells were stained with anti-mouse CD45 and anti-human CD45 antibodies. Animals in which at least 1% of CD45+ cells were of human origin were used for further analysis. Approximately 80% of transplanted mice reached this engraftment threshold, and no differences were observed between the TPOm / m and TPOh / h groups.

[0151] Mice were sacrificed 3–4 or 6–7 months after transplantation. Single cell suspensions were prepared from bone marrow (bleed from two femurs and two tibias), spleen and thymus. Red blood cells were excluded by ACK lysis and cells were stained with the following antibodies for FACS analysis: for global hematopoietic engraftment: anti-mouse CD45-eFluor450 (30-F11, eBioscience) and anti-human CD45-APC-Cy7 (2D1); for human hematopoietic stem cell progenitor and hematopoietic lineages: anti-human CD14-PerCP (MoP9), CD19-APC (HIB19), CD33-APC (WM53), CD34-PE (AC136, Miltenyi Biotec), CD38-FITC (HIT2), CD41a-APC (HIP8) and CD66-FITC (B1.1).

[0152] For analysis of mouse stem cell progenitors, the anti-lineage cocktail contained biotinylated antibodies against CD3ε (145-2C11), CD11b (M1 / 70), CD11c (HL3), CD19 (1D3), Gr1 (RB6-8C5) and Ly-76 (Ter119). Cells were then stained with streptavidin-APC-Cy7, anti-cKit-APC (2B8) and anti-Sca1-PE-Cy7 (D7).

[0153] All antibodies were obtained from BD Biosciences unless otherwise specified. Data were acquired on a FACSCalibur™ or LSRII™ flow cytometer (BD Biosciences) and analyzed using FlowJo™ software.

[0154] Functional characterization of human hematopoietic stem cell progenitors in TPO mice Bone marrow cells from 3–7 transplanted mice were pooled and human CD34+ cells were purified by MACS depletion of mouse CD45+ cells (Miltenyi Biotec) followed by FACS sorting of human CD45+CD34+ cells on a FACSAria™ flow cytometer (BD Biosciences).

[0155] To evaluate the colony-forming ability of human CD34+ cells, IMDM was supplemented with 20% FCS, 2 mM L-glutamine, and 55 μM 2-mercaptoethanol (all reagents from GIBCO), mixed with Methocult™ H4100, 1% BSA (Stemcell Technologies), and the following human cytokines were added: SCF (10 ng / ml), FLT3I (10 ng / ml), TPO (50 ng / ml), IL-3 (20 ng / ml), IL-6 (10 ng / ml), IL-11 (10 ng / ml), GM-CSF (50 ng / ml), and EPO (4 U / ml) (all from R&D systems). 100,000–150,000 sorted cells were plated in 60 mm Petri dishes and incubated at 37°C, 5% CO2 for 12–14 days. Colonies were counted at 12–14 days and classified microscopically into specific myeloid lineages.

[0156] For secondary transplantation experiments, TPO m / m or TPO h / h 100,000 CD34+ cells purified from primary recipients were transfected with sublethally irradiated (2 × 200 cGy) Rag2 - / - Gamma c - / - TPO m / m The mice were sacrificed 8 weeks later and the percentage of human CD45+ cells in the bone marrow was determined by FACS.

[0157] Statistical analysis of TPO mouse data Data were compared using unpaired two-tailed t-tests. When more than two samples were compared, one-way ANOVA followed by Tukey's post-hoc test was performed. The percentage of mice that showed engraftment in secondary transplantation experiments was compared using Pearson's chi-square test. Differences were considered significant when p-values ​​were below 0.05.

[0158] Example 11 Mice transplanted with hTPO:TPO h / h Improved levels of human engraftment in the bone marrow of recipient mice Phenotyping of humanized and engrafted mouse bone marrow Flow cytometric analysis of cells isolated from bone marrow of humanized mice showed statistically significant improvement in engraftment of total human hematopoietic cells, human hematopoietic stem cells, human bone marrow cells, and human granulocytes compared to engraftment of non-humanized mice (i.e., RAG and Il2rg knockouts lacking humanization of the TPO gene), see Figures 12 and 14.

[0159] Wild-type Tpo (TPO m / m ), Heterozygous (TPO h / m ) or homozygous (TPO h / h ) Rag2 with TPO gene replacement - / - Gamma c - / - Mice were generated as described. Irradiated (2 × 1.5 Gy) neonatal Rag2 - / - Gamma c - / - TPO m / m and TPO h / h Mice were cultured with human CD34 purified from umbilical cord blood or fetal liver. + The cells were transplanted and analyzed for engraftment in the bone marrow after 3-4 or 6-7 months.

[0160] Figure 12 shows the results of the engraftment study. + Rag2 cells 3–4 months after transplantation - / - Gamma c - / - TPO m / m and TPO h / hHuman and mouse CD45 in mouse bone marrow + Representative FACS analysis of cells is shown. Results from two representative mice are shown for each genotype. Total (mouse + human) CD45 + Mouse and human CD45 in cell populations + Figure 12(b) shows the percentage of human CD45 cells in bone marrow 3-4 months (left, n=42-53) or 6-7 months (right, n=20-25) after transplantation. + Figure 12(c) shows the percentage of human CD45 cells in the bone marrow of the same animals as in (b). Each symbol represents an individual mouse, and the horizontal bar indicates the mean. + Absolute cell numbers are shown, p-values ​​indicate statistical significance.

[0161] TPO m / m Compared with recipients, TPO h / h Human hematopoietic cells (hCD45 + A significant increase in the percentage (Figures 12(a) and 12(b)) and absolute number (Figure 12(c)) of TPO was observed at both time points. h / h Recipients showed lower engraftment variability, with 75% of mice showing at least 80% human chimerism at 3–4 months (Figure 12(b)). + The source of cells did not affect this result, but TPO h / h A similar increase in host chimerism was observed in cord blood-derived and fetal liver-derived cells (Figure 12(d)). m / m In the host, the number of human cells decreased between the early and late time points, whereas TPO h / h They remained constant in irradiated animals (Figure 12(c)). These results are consistent with the previously described functions of TPO in mice. First, TPO favors the expansion of HSCs after transplantation into irradiated recipient mice, resulting in increased levels of engraftment. Second, it favors the maintenance of adult HSCs, resulting in sustained hematopoiesis throughout adulthood.

[0162] Example 12 Mice engrafted with hTPO: Effects of TPO humanization on mouse and human platelets Platelet analysis in TPO mice Peripheral blood platelet counts were measured using a Hemavet™ 950FS instrument (Drew Scientific). Blood samples were then stained with anti-mouse CD61-PE (2C9.G2) and anti-human CD41a-APC (HIP8), and the percentages of mouse and human platelets were measured by flow cytometry without any gating on cell size (FSC) or granulosity (SSC). Absolute mouse and human platelet counts were calculated by multiplying these percentages by the absolute platelet counts.

[0163] Since TPO is well known for its important function on platelet production, we investigated whether humanization of TPO would affect platelet development. Humanization of both alleles of the TPO gene resulted in the expression of Rag2 TPO in non-transplanted mice. - / - Gamma c - / - This led to a roughly two-fold decrease in platelet counts in the blood of the mice (Figure 13(a)). After transplantation of human cells, TPO h / h Mouse platelet counts in mice further decreased to less than 25% of normal (Figure 13(d)). The ratio of human to mouse platelets (Figures 13(b), 13(c)), as well as the absolute number of human platelets (Figure 13(e)), were significantly decreased with TPO. m / m TPO h / h Although there was a tendency for the expression of CD41a in mice to be higher, none of these differences reached statistical significance. + The percentage of human TPO-dependent IL-16 (IL-16) ...

[0164] Figure 13(a) shows adult non-transplanted Rag2 - / - Gamma c- / - TPO m / m , TPO h / m and TPO h / h Platelet counts in the blood of mice are shown. p<0.0001 (one-way ANOVA, n=7-17; p values ​​shown were calculated by Tukey's post-hoc test). Each symbol represents an individual mouse, and the horizontal bars indicate the mean. Figure 13(b) Rag2 mice 3-4 months after transplantation - / - Gamma c - / - TPO m / m and TPO h / h Mouse (mCD61 + ) and human (hCD41a + ) Representative FACS analysis of platelets. Numbers indicate percentages of all events. Figure 13(c) TPO m / m and TPO h / h Chimerism of human platelets measured by FACS in mice (n=19-22). + Only mice with a percentage of TPO cells greater than 5% were included in the analysis. m / m and TPO h / h Mice in the recipient blood (mCD61 + ,(d)) and human (hCD41a + (e)) platelet count; (f) human CD45 in bone marrow + Among the cells, human megakaryocytes (CD41a + ) percentage.

[0165] Figure 13(g)-(i) show the level of human engraftment in secondary lymphoid organs. - / - Gamma c - / - TPO m / m and TPO h / h Human CD45 in mouse blood (20g; n = 43–53) and spleen (13h; n = 35–36) + The percentage of cells is provided (each symbol represents an individual mouse, and the horizontal bar indicates the mean). m / m and TPO h / hThe total cellularity of the thymus of recipients (n=24-34) is shown. More than 90% of the cells found in the thymus were of human origin (hCD45 + ) was.

[0166] Example 13 Mice engrafted with hTPO: Multilineage hematopoiesis in TPO-humanized mice Phenotyping of humanized and transplanted mouse blood cells Flow cytometric analysis of cells isolated from the blood of humanized mice showed a statistically significant improvement in engraftment of human monocytes and granulocytes compared to engraftment in non-humanized mice (i.e., RAG and Il2rg knockouts lacking humanization of the TPO gene), see Figure 14.

[0167] We investigated whether human TPO could favor the multilineage differentiation of human hematopoietic stem cell progenitors in vivo. As previously reported (Traggiai et al.; Ishikawa et al. (2005) Development of functional human blood and immune systems in NOD / SCID / IL2 receptor gamma chain(null) mice, Blood 106:1565-1573), transplanted human cells expressed wild-type Rag2 - / - Gamma c - / - In the host, mainly B cells (CD19 + ) (61.51 ± 4.71% of human cells in the spleen, mean ± SEM, n = 32), with only a small amount of bone marrow cells. m / m Recipient and TPO h / h When comparing recipients, TPO h / h CD33 in mouse bone marrow + A significant increase in the frequency of myeloid cells was observed (Figures 14(a) and 14(b)). Interestingly, this increase was mainly due to granulocytes (CD33 + CD66 hi SSC hi cells) and monocytes (CD33 hi CD66lo CD14 + The percentage of myeloid cells (both granulocytes and monocytes) was also significantly higher in both strains than in the control strains (Figure 14(a), (c), (d), and (e)). h / h It was significantly increased in the peripheral blood of the animals (Figures 14(a), (f), and (g)).

[0168] Figure 14(a)-(g) shows the results of transplanted TPO. m / m and TPO h / h Mouse CD33 + , CD66 + , CD14 + Figure 14(a) shows improved multilineage hematopoiesis in hTPO mice as measured in Rag2 cells 3-4 months after transplantation. - / - Gamma c - / - TPO m / m and TPO h / h Representative FACS analyses of human myeloid cell populations in mouse bone marrow and blood are shown. Numbers indicate percentages of the gated cell populations shown. Figures 14(b)-(e) show Rag2 - / - Gamma c - / - TPO m / m and TPO h / h Total human CD45 in the bone marrow of recipients (n=19) + Analysis of human bone marrow cell populations for cell chimerism is shown. Figure 14(b) provides the total bone marrow population (CD33+ cells). Figure 14(d) provides the granulocyte (CD33 + CD66 hi ) and Fig. 14(c) shows the TPO h / h hCD45 purified from recipient bone marrow + SSC hi CD33 + CD66 hi Diff-Quick™ staining of cells is shown. Figure 14(e) shows monocytes (CD33 + CD66 lo CD14 + ) are shown. Each symbol represents an individual mouse, and the horizontal bar indicates the mean value. Figures 14(f) and (g) show Rag2 - / - Gamma c- / - TPO m / m and TPO h / h Total human CD45 in the blood of recipients (n=6–7) + Analysis of human bone marrow cell populations for cell chimerism; Fig. 14(f): Granulocytes (CD66 + ); Fig. 14(g): Monocytes (CD14 + ).

[0169] Example 14 Mice engrafted with hTPO: Humanizing effects on mouse and human hematopoietic stem cell progenitors The effect of human TPO on the number and function of HSCs and progenitor cells themselves was analyzed. Genetic deletion of TPO leads to a reduction in HSCs in adult mice. To investigate whether TPO humanization could affect mouse populations immunophenotypically defined as containing mouse HSCs, we used non-transplanted TPO m / m , TPO h / m and TPO h / h Lineage-negative Sca1 in the bone marrow of adult mice + c-Kit + The percentages of cells were compared.

[0170] Figure 15 shows the lin expression levels in the bone marrow of human TPO knock-in mice. - c-Kit + Sca1 + Figure 15(a) shows the reduction of human stem cell progenitor cells and the increase in the number and self-renewal capacity of human stem cell progenitor cells. m / m ) Rag2 - / - Gamma c - / - Rag2 mice compared to non-transplanted mice - / - Gamma c - / - TPO h / m and TPO h / h Mouse Lin in mouse bone marrow - Sca1+c-Kit + Representative results of FACS analysis of stem cell progenitor cells are shown. Numbers indicate Lin - Sca1 in the population + c-Kit +The percentage of cells that are grafted with Rag2+ / - 3D-T cells is shown. Figure 15(b) shows a quantitative analysis of the results shown in (a). p=0.0006 (one-way ANOVA; p-values ​​shown were calculated by Tukey's post-hoc test; n=5 per genotype; results shown are representative of two independent experiments). Each symbol represents an individual mouse, and the horizontal bars indicate the mean. Figure 15(c) shows the quantitative analysis of the results shown in (a). p=0.0006 (one-way ANOVA; p-values ​​shown were calculated by Tukey's post-hoc test; n=5 per genotype; results shown are representative of two independent experiments). Each symbol represents an individual mouse, and the horizontal bars indicate the mean. - / - Gamma c - / - TPO m / m and TPO h / h Human CD34 in mouse bone marrow + CD38 - Representative FACS analysis of cells is shown. Numbers indicate human CD45 + CD34 + CD38 in cells - Figure 15(d) shows the percentage of cells that were TPO m / m and TPO h / h Human CD45 in recipient mice (n=43–53) + CD34 + CD38 in the population - Figure 15(e) shows a quantitative analysis of the percentage of human CD34 cells in the bone marrow of the same mice as in 15(d)d. + CD38 - Absolute cell numbers are shown. Figures 15(f) and (g) show Rag2 - / - Gamma c - / - TPO m / m and TPO h / h Human CD45 purified from recipients + CD34 + Methylcellulose colony formation assay using cells. Figure 15(f) shows CFU-GEMM, and Figure 15(g) shows BFU-E (black), CFU-G (white), CFU-M (gray) and CFU-GM (dashed line). CD34 + Cells were pooled from groups of 3–4 mice to obtain four independent pools per recipient mouse genotype. - / - Gamma c - / - TPO m / m and TPO h / hHuman CD45 from primary recipient mice + CD34 + Purify neonatal Rag2 cells - / - Gamma c - / - mice (100,000 cells per mouse) and 8 weeks later human CD45 + Chimerism was measured. Results are pooled from two independent experiments (n = 7–12 primary recipients, n = 11–19 secondary recipients).

[0171] TPO m / m Compared to TPO h / m and TPO h / h A significant decrease in the percentage of these cells was observed in both mice (Figure 15(b)), suggesting that human TPO is either not fully cross-reactive to the mouse receptor or is not available in sufficient amounts to mouse cells in this knock-in setting.

[0172] Transplanted TPO m / m and TPO h / h Human CD34 in the host bone marrow + A population of human HSCs with long-term repopulating potential was characterized. + CD38 - Contained in the cell fraction. Human CD45 + CD34 in the population + The percentage of cells is TPO h / h The TPO level was slightly increased in mice (12.39 ± 0.79% vs. 10.00 ± 0.81%, mean ± SEM, n = 43–53, p = 0.037). m / m Compared to the recipient, TPO h / h So CD34 + CD38 in the population - A small (1.5-fold) but statistically significant increase in the percentage of CD34 cells was observed (Figure 15(c) and (d)). Overall, this suggests that TPO-humanized mice are more susceptible to CD34 + CD38 -This resulted in a significant increase in absolute cell numbers (approximately 2.8-fold) (Figure 15(e)). Thus, based on cell surface immunophenotype, human TPO supports a population of cells known to be highly enriched for HSCs.

[0173] To investigate the functional properties of this cell population, human CD34 + Cells are treated with TPO m / m and TPO h / h CFU-GEMMs were purified from mouse bone marrow and evaluated in vitro by methylcellulose colony formation assay. CFU-GEMMs are multilineage myeloid colonies derived from immature cells that contain at least all erythroblast-megakaryocyte and myeloid cell differentiation potentials. The formation of CFU-GEMMs, although in small numbers, was associated with TPO h / h CD34 isolated from recipient mice + was detected in all four cell samples, whereas TPO m / m Only one sample from the TPO strain produced CFU-GEMM (Fig. 15(f)). h / h In addition, consistent with the enhanced myeloid differentiation observed in vivo (FIG. 14), the number of CFU-M also increased with TPO. m / m Compared to mice, TPO h / h Human CD34 isolated from + The results were significantly higher in cell samples (150,000 cultured CD34 + 225.0±12.25 vs. 81.25±10.80 colonies per cell, mean±sem, p=0.0001; Figure 15(g)).

[0174] Maintenance and / or self-renewal of HSCs is functionally best demonstrated by successful secondary transplantation. Continuously engrafting SCID repopulating cells (SRCs) in mice now represent a reliable surrogate experimental standard for human HSC function. Thus, human CD34 + Cells are treated with TPO m / m and TPO h / h Purified from primary recipient bone marrow and Rag2 - / - Gammac - / - Neonatal mice were incubated with similarly low numbers (100,000 CD34 cells per animal). + The bone marrow of the secondary recipient was analyzed 8 weeks later (FIG. 15(h)). TPO m / m Human CD34 isolated from primary recipients + The cells were human CD45 + The ability to serially engraft was very low, as TPO cells were only detected in 2 of 11 secondary recipients. h / h CD34 isolated from primary recipients + The bone marrow of 15 of 19 mice transplanted with cells expressed human CD45 + The genotype of the secondary recipient mice was similar in both groups (TPO m / m ), this result indicates that the presence of human TPO in primary recipients favors the maintenance of human cells with enhanced self-renewal capacity.

[0175] Taken together, these results demonstrate that homozygous TPO-humanized mice provide a favorable environment for maintaining the self-renewal and multilineage differentiation potential of human hematopoietic stem cell progenitors.

[0176] Those skilled in the art can devise various modifications that embody the present invention and are within the spirit and scope of the present invention, although not explicitly described or expressed in this disclosure. All examples are provided to help the reader understand the principles and concepts of the present invention, and are used without being limited to the specific examples and embodiments described. All principles, aspects, embodiments, and examples of the present invention are intended to encompass equivalents, whether currently known or developed in the future. The scope of the present invention is not limited to the embodiments and examples shown or described in this disclosure.

[0177] Sequence information SEQUENCE LISTING <110> REGENERON PHARMACEUTICALS, INC. Yale University INSTITUTE FOR RESEARCH IN BIOMEDICINE (IRB) <120> Genetically Modified Mice and Engraftment <150> US 61 / 320,132 <151> 2010-04-01 <150> US 61 / 256,237 <151> 2009-10-29 <150> US 61 / 249,069 <151> 2009-10-06 <160> 12 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 1 ccaccaccca tggatctc 18 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 2 aaagcagaac atctggagca g 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 3 caggactgaa aagggaatca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 4 cgttggaagg ccttgaattt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 5 gtacgctgtg aaggcatcaa 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 6 atcccatcca acaccttgag 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 7 ccagtccaaa aatgaggaag c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 8 cagcgttttc agagggctat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 9 ggcgtctcct gaacctgagt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 10 ggggatgaca agcagaaagt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 11 gtacgctgtg aaggcatcaa 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 12 atcccatcca acaccttgag 20

Claims

1. (a) Mouse RAG gene knockout; (b) Mouse Il2rg gene knockout; (c) replacement of the mouse IL-3 gene with the human IL-3 gene, wherein the human IL-3 gene is present at the mouse IL-3 locus; and (d) replacement of the mouse GM-CSF gene with the human GM-CSF gene, where the human GM-CSF gene is present at the mouse GM-CSF locus. A genetically modified mouse, comprising:

2. Replacement of the mouse thrombopoietin gene with the human thrombopoietin gene, the human thrombopoietin gene being present at the mouse thrombopoietin locus The mouse of claim 1, further comprising:

3. The mouse of claim 1, wherein the RAG gene knockout is selected from a RAG1 gene knockout, a RAG2 gene knockout, and a combination thereof.

4. The mouse of claim 1, further comprising an engraftment of human hematopoietic cells.

5. The mouse of claim 4, wherein the human hematopoietic cells are selected from the group consisting of umbilical cord blood cells and fetal liver cells.

6. The mouse of claim 5, wherein the human hematopoietic cells are human umbilical cord blood cells that are CD34+ cells.

7. The mouse of claim 3, comprising human cells that are CD34 positive cells, hematopoietic stem cells, hematopoietic cells, myeloid progenitor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, or combinations thereof.

8. 4. The mouse of claim 3, comprising human hematopoietic stem and progenitor cells, human bone marrow progenitor cells, human bone marrow cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, and human platelets.

9. The mouse of claim 4, comprising a Salmonella typhi infection.

10. The mouse of claim 9, wherein the Salmonella typhi infection is a systemic Salmonella typhi infection.

11. The mouse of claim 4, comprising a Mycobacterium tuberculosis infection.

12. The mouse of claim 11, comprising pulmonary granulomas comprising human immune cells.

13. (a) administering an agent to a genetically modified, transplanted, and human pathogen-infected mouse; The mouse, (i) RAG1 gene knockout or RAG2 gene knockout, or a combination thereof; (ii) Il2rg gene knockout, (iii) replacement of the mouse IL-3 gene with the human IL-3 gene, where the human IL-3 gene is present at the mouse IL-3 locus; and (iv) replacement of the mouse GM-CSF gene with the human GM-CSF gene, where the human GM-CSF gene is present at the mouse GM-CSF locus. Including, the mouse is engrafted with CD34-positive human umbilical cord blood cells or CD34-positive human fetal liver cells; and (b) determining whether the agent reduces the amount of the human pathogen in the mouse infected with the human pathogen. A method for identifying an agent that inhibits human pathogen infection, comprising:

14. The method of claim 13, wherein the mouse further comprises a replacement of the mouse thrombopoietin gene with a human thrombopoietin gene.

15. 14. The method of claim 13, wherein the human pathogen is Salmonella typhi.

16. 14. The method of claim 13, wherein the human pathogen is a mycobacterium, and the mycobacterium causes granulomas in the mouse that contain human immune cells.

17. 17. The method of claim 16, wherein the mycobacteria is Mycobacterium tuberculosis.

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