Humanized M-CSF mice

Abstract

The present application relates to humanized M-CSF mice. Genetically modified mice comprising a nucleic acid sequence encoding a human M-CSF protein are provided. Genetically modified mice comprising a nucleic acid sequence encoding a human M-CSF protein that have been grafted with human cells, such as human hematopoietic cells, and methods for generating such grafted mice are also provided. These mice are used in many applications, such as in modeling human immune diseases and pathogen infections; in in vivo screening of agents that regulate (e.g., in a healthy or diseased state) hematopoietic cell development and / or activity; in in vivo screening of agents that are toxic to hematopoietic cells; in in vivo screening of agents that provide protection, mitigate, or reverse the toxic effects of toxic agents on hematopoietic cells; in in vivo screening of human hematopoietic cells from an individual to predict the individual's response to disease therapy, and the like.

Description

[0001] This application is a re-divisional application of the invention divisional application with the application date of February 14, 2012, the Chinese application number of 201610297251.X, and the invention name of “Humanized M-CSF Mouse” (the original application number is 201280018139.8). Field of the Invention

[0002] The present invention relates to genetically modified mice comprising a gene encoding human M-CSF protein, and mice comprising additional modifications that support engraftment of human hematopoietic cells. Background of the Invention

[0003] The development of animal models for studying human diseases has significantly advanced the understanding of the fundamental mechanisms of several diseases (including cancer). So far, it has been demonstrated that animal models, particularly mice, are excellent candidates for the efficiency and effectiveness of evaluating drugs and therapy options. Although the use of these alternative models to study human biology and disease can be justified to a large extent (due to the ethics and technical constraints of conducting human experimental therapy), research has highlighted the potential limitations of inferring data from mice to people (Mestas J, Hughes CC. (2004) Of mice and not men: differences between mouse and human immunology. J Immunol. 172: 2731-2738).

[0004] In order to overcome these problems, there is a long-standing interest in developing humanized mouse models. The intensive work of several groups has successfully proved the feasibility of studying human biology and disease in mice. Due to the functional and effective immune system in the recipient, the transplanted tissue / cell of human origin can be eliminated, and the genetic mutants of cells such as T, B and NK cells lacking adaptive immune system have significantly contributed to the success of humanized mouse models. Thus, the most effective candidates of humanized mouse models include NOD-SCID and Balb / c strains lacking genes, and the genes include recombinant activation genes (RAG), common gamma chains (γC, also known as "interleukin 2 receptor, γ", or IL2rg), β2 microglobulin (B2M) and perforin (Perforin) 1 (Prf1) (Shultz LD etc. (2007) Humanized mice in translational biomedicalresearch, Nat.Rev.Immunol.7:118-130). Several studies in the past few decades have demonstrated the feasibility of transplanting several types of human tissues, including peripheral blood leukocytes, fetal liver cells, fetal bones, fetal thymus, fetal lymph nodes, vascularized skin, arterial segments, and mobile or umbilical cord blood hematopoietic stem cells (HSC) into certain humanized mice (Macchiarini F. et al. (2005) Humanized mice: are we there yet? J. Exp. Med. 202: 1307-1311). It is believed that this approach provides a better model system because the data obtained from human cells in these mice can reflect the physiology of the human system. The main research approach in this field is to generate mice with a complete hematopoietic system and a functional immune system of human origin. Although significant progress has been made in generating immunocompromised mice with human T lymphocytes, B lymphocytes, NK cells, and dendritic cells (DC), there are still several challenges in this field, one of which is the poor myeloid differentiation in humanized mice. Interestingly, there have been many advances in the generation of human T cells, B cells, NK cells and dendritic cells (DC) from hematopoietic stem cells (HSC) in humanized mice. In addition to individual hematopoietic compartments, injection of human HSC in these mice leads to the reconstruction of lymphoid organs such as thymus and spleen. However, the frequency of myeloid cells, particularly granulocytes, macrophages, erythrocytes and megakaryocytes is very low, which may be due to the result caused by the inefficient bone marrow tissue generation of human HSC in these mice (Shultz et al. (2007); Macchiarini et al. (2005)).Considering the fact that cells of myeloid origin, such as erythrocytes and megakaryocytes, are crucial for the normal functioning of the blood system, and that granulocytes and macrophages are important for the development of the adaptive immune system, the generation of humanized mice with efficient human bone marrow tissue production is of great significance.

[0005] Thus, there is a need in the art for genetically modified mice that can improve human myelopoiesis after engraftment with human HSCs (Manz MG. Human-hemato-lymphoid-system mice: opportunities and challenges. Immunity. 2007 May; 26(5):537-41). SUMMARY OF THE INVENTION

[0006] The present application provides genetically modified mice comprising a nucleic acid sequence encoding a human M-CSF protein. Also provided are genetically modified mice comprising a nucleic acid sequence encoding a human M-CSF protein that have been grafted with human cells, such as human hematopoietic cells, and methods for generating such grafted mice. These mice can be used in many applications, such as in modeling human immune diseases and pathogen infections; in in vivo screening for agents that modulate (e.g., in a healthy or diseased state) hematopoietic cell development and / or activity; in in vivo screening for agents that are toxic to hematopoietic cells; in in vivo screening for agents that provide protection against, mitigate, or reverse the toxic effects of toxic agents on hematopoietic cells; in in vivo screening for human hematopoietic cells from an individual that predict the responsiveness of the individual to disease therapy, and the like.

[0007] In some aspects of the present invention, a humanized M-CSF mouse is provided, wherein the humanized M-CSF mouse comprises a nucleic acid sequence encoding a human M-CSF protein and is operably linked to a regulatory sequence 5' of the mouse M-CSF structural gene locus, such as a mouse M-CSF promoter, 5'UTR, etc. In some embodiments, the mouse comprises two copies of the nucleic acid sequence. In some embodiments, the nucleic acid sequence is located in the mouse genome within the mouse M-CSF locus. In some embodiments, the nucleic acid sequence is operably linked to the endogenous mouse M-CSF promoter at the mouse M-CSF locus, i.e., the mouse is an M-CSF h / m In some embodiments, the mouse comprises two alleles, wherein the nucleic acid sequence is located in the mouse genome within the mouse M-CSF locus. In some embodiments, the nucleic acid sequences of the two alleles are operably linked to the endogenous mouse M-CSF promoter at the mouse M-CSF locus, i.e., the mouse is an M-CSF h / hIn some embodiments, the humanized M-CSF mouse comprises a null mutation in at least one mouse M-CSF allele. In some embodiments, the humanized M-CSF mouse comprises a null mutation in both mouse M-CSF alleles. In some such embodiments, the null mutation is a deletion of mouse M-CSF exons 2-9.

[0008] In some embodiments, mice express human M-CSF in bone marrow, spleen, blood, liver, brain, lung, testis, and kidney. In some embodiments, the amount of human M-CSF expressed is substantially the same as the amount of mouse M-CSF expressed in wild-type mice. In some embodiments, the amount of human M-CSF expressed by the bone marrow mesenchymal stromal cells of humanized M-CSF mice is substantially the same as the amount of mouse M-CSF expressed by wild-type mouse bone marrow mesenchymal stromal cells. In some embodiments, humanized M-CSF mice exhibit physiological concentrations of M-CSF in blood and / or tissues. In some embodiments, mice express both mouse M-CSF and human M-CSF. In other embodiments, the only M-CSF expressed by mice is human M-CSF.

[0009] In some embodiments, mice secrete enough human M-CSF to differentiate grafted human hematopoietic stem cells into human monocytes, human macrophages, and human osteoclasts. In some embodiments, mice secrete an effective amount of M-CSF to stimulate the formation of human macrophages from human monocytes derived from grafting human hematopoietic stem cells into mice. In some embodiments, mice secrete an effective amount of M-CSF to stimulate the formation of promonocytes, promonocytes, human monocytes, and human monocytes in mice grafted with human hematopoietic stem cells. Human monocytes form human macrophages. In some embodiments, the effective amount of human M-CSF secreted in mice is to achieve the same amount of mouse M-CSF secreted by wild-type mice to achieve the corresponding results (e.g., an effective amount of mouse M-CSF to stimulate the formation of mouse macrophages from mouse monocytes).

[0010] In some embodiments, the transcriptional and translational control of human M-CSF in the genetically modified mouse is the same or substantially the same as the transcriptional and translational control of mouse M-CSF in a modified mouse lacking its endogenous mouse M-CSF gene.

[0011] In some embodiments, the physiological concentration of human M-CSF in the humanized M-CSF mouse results from secretion of human M-CSF from the same cell types that secrete mouse M-CSF in wild-type mice that possess the mouse M-CSF gene and lack nucleic acid encoding the human M-CSF protein. In other words, one or more M-CSF isoforms are expressed in a normal tissue-specific and developmental manner.

[0012] In some embodiments, the mouse expresses a human M-CSF isoform selected from the group consisting of proteoglycan M-CSF, glycoprotein M-CSF, and cell surface M-CSF, and combinations thereof. In one embodiment, the mouse expresses at least two isoforms in a normal tissue-specific and developmental manner. In a specific embodiment, the mouse expresses human proteoglycan CSF-1 and human glycoprotein M-CSF and human cell surface M-CSF.

[0013] In some embodiments, the mouse comprises human macrophages that are not thymic T cell-derived macrophages. In some embodiments, the mouse comprises human macrophages that exhibit M-CSF-dependent cytopodial formation stimulated by human M-CSF expressed in the mouse.

[0014] In some embodiments, the mouse is homozygous null for Rag2. In some embodiments, the mouse is homozygous null for IL2rg. In some embodiments, the mouse is homozygous null for Rag2 and for IL2rg. In some embodiments, the mouse comprises a human cell. In some embodiments, the human cell is a hematopoietic cell.

[0015] In some aspects of the present invention, a mouse model of the human immune system is provided, the mouse model comprising two null Rag2 alleles, two null IL2rg alleles, a nucleic acid sequence encoding a human M-CSF protein operably linked to a promoter of a mouse M-CSF gene, and human hematopoietic cells. In other words, the mouse is a grafted Rag2 - / - IL - / - hM-CSF mouse, wherein hM-CSF means that the mouse comprises at least one nucleic acid encoding a human M-CSF gene. In some embodiments, the grafted Rag2 - / - IL - / - The hM-CSF mouse is a BALB / c strain mouse comprising these genetic modifications. In some embodiments, the mouse also comprises other genetic modifications.

[0016] In some embodiments, the engrafted Rag2 at about 12 weeks of age is expressed in comparison to mice comprising human hematopoietic cells expressing mouse M-CSF instead of human M-CSF. - / - IL- / - hM-CSF mice exhibit increased human CD14 in bone marrow, spleen, and peripheral blood + CD33 + (hCD14 + CD33 + ) cell frequency. In a specific embodiment, in bone marrow hCD14 + CD33 + In one embodiment, the expression of hCD14 in the spleen is increased by about 5 to about 15 times, and in one embodiment, by about 12 to about 14 times, compared to mice expressing only mouse M-CSF. + CD33 + In one embodiment, the expression of hCD14 in peripheral blood is increased by about 2 to about 6 times, and in one embodiment, about 5 to about 6 times, compared to mice containing human hematopoietic cells expressing only mouse M-CSF. In a specific embodiment, the expression of hCD14 in peripheral blood is increased by about 2 to about 6 times, and in one embodiment, about 5 to about 6 times. + CD33 + The cells are elevated by about 2 to about 8 fold, and in one embodiment, about 5 to about 7 fold, compared to mice comprising human hematopoietic cells expressing only mouse M-CSF.

[0017] In some embodiments, the grafted Rag2 - / - IL - / - hM-CSF mice exhibit about 15% to about 40%, and in one embodiment about 30%, of hCD14 in the blood. + CD33 + In one embodiment, genetically modified engrafted mice at about 16 weeks of age exhibit about 15% to about 30%, in one embodiment about 22%, of hCD14 in the blood. + CD33 + In one embodiment, genetically modified engrafted mice of about 20 weeks of age exhibit about 5% to about 15%, in one embodiment about 10%, of hCD14 in the blood. + CD33 + In one embodiment, genetically modified engrafted mice at about 20 weeks of age exhibit about 4 to 8 times, in one embodiment about 6 times, higher levels of hCD14 in the blood than in engrafted mice expressing mouse M-CSF instead of human M-CSF. + CD33 + Monocyte / macrophage lineage cell levels.

[0018] In some embodiments, the grafted Rag2 - / - IL - / -hM-CSF mice exhibited approximately 1.5- to 6-fold higher levels of hCD14 in the liver than engrafted mice expressing mouse M-CSF but not human M-CSF. + CD33 + CD45 + In one embodiment, genetically modified engrafted mice at about 12 weeks of age exhibit about 1.5 to about 10 times higher hCD14 in the lungs than engrafted mice expressing mouse M-CSF instead of human M-CSF. + CD33 + CD45 + In one embodiment, genetically modified engrafted mice at about 12 weeks of age exhibit about 2 to about 3 times higher levels of human hCD14 in the peritoneum or skin than engrafted mice expressing mouse M-CSF instead of human M-CSF. + CD33 + CD45 + Cellular level.

[0019] In some embodiments, the grafted Rag2 - / - IL - / - hM-CSF mice exhibited a higher level of hCD14 in the liver than mice lacking human M-CSF. + CD33 + In the lung, the percentage of hCD14 + CD33 + The LPS response for hCD14 + CD33 + The LPS response in the peritoneum was about 2 to about 5 times, in one embodiment about 3 to about 4 times; in the peritoneum, the LPS response in the hCD14 + CD33 + The LPS response is about 2 to about 5 times, in one embodiment about 3 to about 4 times.

[0020] In some embodiments, the grafted Rag2 - / - IL - / - The hM-CSF mice exhibit an enhanced pro-inflammatory cytokine response in response to LPS stimulation, with the level of activation and / or differentiation of cell types responsive to the pro-inflammatory cytokines being enhanced by about 2 to at least about 5 fold compared to genetically modified and engrafted mice lacking the hM-CSF gene.

[0021] In some embodiments, the grafted Rag2 - / - IL - / -hM-CSF mice exhibited enhanced hCD14 in the spleen approximately 48 hours after LPS injection + CD33 + hCD45 + Cell production, wherein the enhancement is about 2 to about 5 fold, and in one embodiment 4 to about 5 fold, compared to engrafted mice expressing mouse M-CSF but not human M-CSF.

[0022] In some embodiments, the grafted Rag2 - / - IL - / - hM-CSF mice exhibit enhanced blood human IL-6 production in response to LPS, with hIL-6 levels 6 hours after LPS injection being about 2 to about 5 fold, and in one embodiment about 3 to about 4 fold, enhanced compared to engrafted mice expressing mouse M-CSF rather than human M-CSF.

[0023] In some embodiments, the grafted Rag2 - / - IL - / - hM-CSF mice exhibit enhanced serum human TNFα production in response to LPS, with hTNFα levels at about 6 hours after LPS injection being about 2 to about 4 fold, and in one embodiment about 2 to about 3 fold, enhanced compared to engrafted mice expressing mouse M-CSF rather than human M-CSF.

[0024] In some embodiments, self-grafted Rag2 - / - IL - / - Monocytes and / or macrophages isolated from hM-CSF mice exhibit approximately 2- to 3-fold higher secretion of hTNFα upon LPS stimulation in vitro than engrafted mice expressing mouse M-CSF but not human M-CSF.

[0025] In some embodiments, self-grafted Rag2 - / - IL - / - Monocytes and / or macrophages isolated from hM-CSF mice exhibit approximately 2- to 4-fold higher secretion of hIL-6 upon LPS stimulation in vitro than engrafted mice expressing mouse M-CSF but not human M-CSF.

[0026] In some embodiments, self-grafted Rag2 - / - IL - / - Monocytes and / or macrophages isolated from hM-CSF mice exhibit approximately 3- to 6-fold higher secretion of hIFNα upon poly I:C stimulation in vitro than engrafted mice expressing mouse M-CSF but not human M-CSF.

[0027] In some embodiments, self-grafted Rag2 - / - IL - / -Monocytes and / or macrophages isolated from hM-CSF mice exhibit approximately 2- to 3-fold higher secretion of hIFNβ upon poly I:C stimulation in vitro than engrafted mice expressing mouse M-CSF but not human M-CSF.

[0028] In some embodiments, compared to engrafted mice expressing mouse M-CSF instead of human M-CSF, the autografted Rag2 - / - IL - / - Human monocytes and / or macrophages isolated from hM-CSF mice exhibit enhanced phagocytosis. In one embodiment, the rate of phagocytosis is enhanced by about two-fold compared to human cells from engrafted mice expressing mouse M-CSF but not human M-CSF, as measured by incorporation of labeled bacteria over a period of 60 minutes at 37°C. In one embodiment, the rate of phagocytosis as measured above is two or more times, e.g., 2-fold, 3-fold, or 4-fold or more, the rate of human cells from engrafted mice expressing mouse M-CSF but not human M-CSF.

[0029] In some embodiments, compared to engrafted mice expressing mouse M-CSF instead of human M-CSF, the autografted Rag2 - / - IL - / - Human monocytes and / or macrophages isolated from hM-CSF mice exhibit enhanced in vitro chemotaxis in response to Mip3β. In one embodiment, the enhancement is about 1.5-fold to 3-fold or more, such as about 1.5-fold, 2-fold, 3-fold, 4-fold or more, as measured by the number of migrated cells at 30 or 60 minutes after Mip3β exposure, compared to human monocytes / macrophages from engrafted mice expressing mouse M-CSF but not human M-CSF.

[0030] In some embodiments, the self-grafted Rag2 - / - IL - / - M-CSF h Mouse-isolated human monocytes and / or macrophages exhibited approximately 3- to 6-fold higher secretion of hIFNα upon poly I:C stimulation in vitro compared to engrafted mice expressing mouse M-CSF but not human M-CSF.

[0031] In some embodiments, the self-grafted Rag2 - / - IL - / - Human monocytes and / or macrophages isolated from hM-CSF mice exhibit in vitro upregulation of co-stimulatory molecules in response to LPS stimulation. In one embodiment, the co-stimulatory molecule is selected from human CD40, human CD80, human CD86, human HLA-DR, and combinations thereof.

[0032] In some aspects of the invention, a genetically modified engrafted mouse is provided, wherein the mouse comprises an engraftment of human hematopoietic cells, is Rag2 - / - Iq - / - , comprising a null allele of mouse M-CSF and comprising a nucleic acid sequence encoding human M-CSF at an endogenous M-CSF locus, wherein the mouse exhibits an enhancement or increase in the number of human myeloid cells compared to a mouse expressing mouse M-CSF but not human M-CSF.

[0033] In some embodiments, the enhancement comprises hCD14 in a mouse portion selected from the group consisting of + CD33 + At least double the number of cells: bone marrow, spleen and peripheral blood. In a specific embodiment, the enhancement includes hCD14 + CD33 + In another embodiment, the enhancement includes hCD14 + CD33 + The cell number increased 4 to 5 times or more.

[0034] In some embodiments, the enhancement comprises hCD14 in a mouse compartment selected from the group consisting of + CD33 + hCD45 + Cell number increased 2-3 fold: skin and peritoneum.

[0035] In some embodiments, the enhancement comprises hCD14 in a mouse compartment selected from the group consisting of + CD33 + hCD45 + Cell number increased 1.5 to 10-fold: liver and lung.

[0036] In some embodiments, the enhancement comprises hCD14 at about 48 hours after LPS stimulation. + CD33 + hCD45 + The number of spleen cells increased 4 to 5 times.

[0037] In some embodiments, the enhancement comprises a 2 to 4 fold increase in LPS-stimulated serum hIL-6 or LPS-stimulated serum hTNFα.

[0038] In some embodiments, the enhancement includes stimulation of hCD14 by human MIP3β + CD33 + Cell migration in vitro increased 2 to 3 times.

[0039] In some aspects of the present invention, a mouse model of a human pathogen is provided, the mouse model comprising two Rag2 null alleles, two IL2rg null alleles, a nucleic acid sequence encoding a human M-CSF protein operably linked to a promoter of a mouse M-CSF gene, human hematopoietic cells, and infection with a human pathogen. In other words, the mouse is a grafted Rag2 that has been infected with a human pathogen. - / - IL - / - hM-CSF mice. In some embodiments, the pathogen is a virus, fungus or bacteria. In some embodiments, the virus is a human or swine or avian influenza virus. In some embodiments, the cell is a mycobacterium, such as Mycobacterium tuberculosis (M. tuberculosis). In some embodiments, the bacterium is an enterobacterium, such as Salmonella typhi (S. typhi).

[0040] In some aspects of the present invention, a pluripotent, induced pluripotent, or totipotent mouse cell is provided, comprising a nucleic acid sequence encoding a human M-CSF protein operably linked to a promoter of a mouse M-CSF gene. In one embodiment, the mouse cell is a mouse ES cell.

[0041] In some aspects of the present invention, a mouse embryo is provided, comprising a nucleic acid sequence encoding a human M-CSF protein operably linked to a promoter of a mouse M-CSF gene.

[0042] In some aspects of the present invention, a targeting construct for targeting a mouse M-CSF gene is provided, comprising (a) upstream and downstream targeting arms that are complementary or substantially complementary to (i) a nucleotide sequence encoding a mouse M-CSF protein, or (ii) an upstream and downstream nucleotide sequence that is complementary to a nucleotide sequence encoding a mouse M-CSF protein; (b) a human nucleic acid sequence encoding a human M-CSF protein or a fragment thereof, or a nucleotide sequence encoding a complement of a human M-CSF protein or a fragment thereof; and (c) a marker and / or a selection cassette.

[0043] In some aspects of the invention, human immune cells from mice as described herein are provided. In one embodiment, the human immune cells are selected from human monocytes and human macrophages. In one embodiment, the human immune cells are selected from human NK cells, human B cells, and human T cells.

[0044] In some aspects of the invention, antibodies encoded by human nucleotide sequences from mice as described herein are provided. In one embodiment, the antibody is selected from an IgA, IgD, IgE, IgM, or IgG isotype antibody.

[0045] In some aspects of the invention, a nucleotide sequence encoding a human immunoglobulin sequence is provided, wherein the nucleotide sequence is obtained from an engrafted humanized M-CSF mouse according to the invention. In one embodiment, the nucleotide sequence encodes a human variable region or a fragment thereof of a human immunoglobulin gene. In one embodiment, the nucleotide sequence encodes a human TCR variable region or a fragment thereof.

[0046] In some aspects of the invention, methods for generating humanized M-CSF mice expressing biologically active human M-CSF are provided. In some embodiments, the method comprises contacting mouse pluripotent stem cells, such as ES cells or iPS cells, with a nucleic acid sequence comprising a coding sequence for a human M-CSF protein or a fragment thereof, and culturing the pluripotent stem cells under conditions that promote integration of the nucleic acid sequence into the mouse genome; generating mice from mouse ES cells comprising a nucleic acid sequence encoding a human M-CSF protein; and maintaining the mice under conditions sufficient for the mice to express human M-CSF from the human M-CSF gene. In some embodiments, the nucleic acid sequence is randomly integrated into the genome. In other embodiments, the nucleic acid sequence is integrated into a target locus. In some such embodiments, the target locus is an endogenous mouse M-CSF locus, for example, a nucleic acid sequence comprising a human M-CSF protein coding sequence is flanked by sequences homologous to the endogenous mouse M-CSF locus, and the nucleic acid sequence is integrated into the endogenous mouse M-CSF locus by homologous recombination. In some embodiments, the mouse is homozygous null for Rag2. In some embodiments, the mouse is homozygous null for IL2rg. In some embodiments, the mouse is homozygous for Rag2 and IL2rg, i.e., it is Rag2 - / - IL - / - .

[0047] In some aspects of the invention, methods are provided for generating humanized M-CSF mice comprising a human hematopoietic system. In some embodiments, the method comprises transplanting a cell population comprising hematopoietic progenitor cells into a humanized M-CSF mouse, such as Rag2 - / - IL - / - hM-CSF mice or sublethally irradiated hM-CSF mice. In some embodiments, the human hematopoietic progenitor cells are CD34+ cells. In some embodiments, the human hematopoietic progenitor cells are CD133+. In some embodiments, the human hematopoietic progenitor cells are pluripotent stem cells, such as ES cells or iPS cells. In some embodiments, the cell population source comprising human hematopoietic progenitor cells is fetal liver. In some embodiments, the cell source is bone marrow. In some embodiments, the cell source is peripheral blood. In some embodiments, the cell source is an in vitro cell population.

[0048] In some aspects of the invention, methods are provided for generating mice infected with human pathogens. In some embodiments, the methods include introducing humanized M-CSF containing human hematopoietic cells, such as engrafted Rag2 - / - IL - / - The hM-CSF mice or the engrafted sublethally irradiated mice are exposed to a human pathogen and the mice are maintained under conditions sufficient for the human pathogen to infect the mice. In some embodiments, the human pathogen is a human pathogen that does not infect mice lacking one or more genetic modifications described herein. In some embodiments, the human pathogen is a human pathogen that is not pathogenic in mice lacking one or more genetic modifications described herein.

[0049] In some aspects of the invention, methods for producing biologically active human M-CSF in mice are provided, the methods comprising generating a humanized M-CSF mouse expressing biologically active human M-CSF, as described above and elsewhere herein. In some embodiments, the methods comprise purifying biologically active human M-CSF from the blood, e.g., serum, or tissue of the mouse. In some embodiments, the methods comprise obtaining cells expressing biologically active human M-CSF from the mouse, culturing the cells under conditions sufficient for the cells to express and secrete biologically active human M-CSF, and isolating secreted biologically active human M-CSF. Note that in this aspect of the invention, the mouse is not required to have any other genetic modifications, and the mouse can be used to generate certain human immune cell preparations. Thus, in some aspects of the invention, isolated biologically active human M-CSF obtained from transgenic mice is provided.

[0050] In some aspects of the invention, a method for generating activated human monocytes or activated human macrophages in a mouse is provided, comprising exposing a humanized M-CSF mouse grafted with human hematopoietic cells to an immunostimulant, allowing the human monocytes or macrophages in the mouse to be activated, and isolating the human monocytes or human macrophages from the mouse, wherein the fraction of activated monocytes or activated macrophages is about 2-fold to 5-fold higher than that obtained from a non-humanized M-CSF mouse, i.e., a grafted mouse lacking the human M-CSF gene. In some embodiments, the immunostimulant is an endotoxin. In a specific embodiment, the endotoxin is LPS.

[0051] In some aspects of the invention, methods are provided for screening candidate agents for activity in regulating human hematopoietic cell function. In some embodiments, the methods include using humanized M-CSF mice engrafted with human hematopoietic cells, such as engrafted Rag2 - / - IL - / -hM-CSF mice or engrafted sublethally irradiated hM-CSF mice are contacted with a candidate agent; and the function of hematopoietic cells in the mouse model contacted with the candidate agent is compared with the function of hematopoietic cells in the mouse model not contacted with the candidate agent; wherein modulation of hematopoietic cell function in the mouse contacted with the candidate agent indicates that the candidate agent modulates hematopoietic cell function.

[0052] In some aspects of the invention, a method for determining the effect of an agent on a human pathogen is provided, comprising: implanting a humanized M-CSF mouse, such as a Rag2 - / - IL - / - The hM-CSF mouse or the engrafted sublethally irradiated hM-CSF mouse is exposed to an effective amount of a human pathogen, the effective amount of the pathogen being the amount of the pathogen required to produce an infection in the mouse; the pathogen is allowed to infect the mouse; the infection parameter is measured over time in the presence of the agent; and the measured values ​​are compared to the measured values ​​from the engrafted humanized M-CSF mouse that was not exposed to the agent. In some embodiments, the agent is provided before the mouse is exposed to the human pathogen, for example, to determine the protective effect. In some embodiments, the agent is provided at the same time as the mouse is exposed to the human pathogen, for example, to determine the protective or therapeutic effect. In some embodiments, the agent is provided after the mouse is exposed to the human pathogen, for example, to determine the therapeutic effect. In some embodiments, the mouse after exposure to the human pathogen initiates a cellular and / or humoral immune response that mimics infection in a human exposed to the pathogen. In some embodiments, the human pathogen is a pathogen that does not infect mice lacking one or more genetic modifications described herein. In some embodiments, the human pathogen is a pathogen that infects wild-type mice, wherein the wild-type mouse after infection does not mimic the immune response initiated by humans in response to the pathogen. In some embodiments, the virus is a human or swine or avian influenza virus. In some embodiments, the bacterium is a mycobacterium, such as Mycobacterium tuberculosis. In some embodiments, the bacterium is an enterobacterium, such as Salmonella typhi. In some embodiments, mice are exposed to a known number of human pathogen infection units, and the infection parameter is the number of infection units of the human pathogen in the body fluid or tissue of the mouse. In some embodiments, the infection parameter is the titer in the body fluid of the mouse. In some embodiments, the parameter of infection is the formation of granulomas. In some such embodiments, the granuloma is a pulmonary granuloma. In some such embodiments, the granuloma is a well-defined granuloma.

[0053] In some aspects of the invention, a method for determining the effect of an agent on a human pathogen is provided, comprising: implanting a humanized M-CSF mouse, such as a Rag2 - / - IL - / -The hM-CSF mouse or the engrafted sublethally irradiated hM-CSF mouse is exposed to an effective amount of a human pathogen antigen, the effective amount of the antigen being the amount of antigen required to promote a cellular and / or humoral response in the mouse; allowing the cellular and / or humoral response to develop; measuring the cellular and / or humoral response parameters over time in the presence of the agent; and comparing the measurements to those from the engrafted humanized M-CSF mouse that was not exposed to the agent. In some embodiments, the agent is provided prior to exposing the mouse to the antigen from the human pathogen, for example, to determine the protective effect of the agent. In some embodiments, the agent is provided simultaneously with exposing the mouse to the antigen from the human pathogen, for example, to determine the protective or therapeutic effect of the agent. In some embodiments, the agent is provided after the mouse is exposed to the antigen from the human pathogen, for example, to determine the therapeutic effect of the agent. In some embodiments, a cellular and / or humoral immune response is initiated in the mouse after exposure to the human pathogen, which mimics infection in a human exposed to the pathogen.

[0054] In some embodiments, the antigen is from a human pathogen that does not infect mice lacking one or more genetic modifications described herein. In other embodiments, the antigen is from a human pathogen that infects wild-type mice, wherein the infected wild-type mice do not mimic the immune response launched by humans in response to the pathogen. In some embodiments, the pathogen is a virus, fungus, or bacteria. In some embodiments, the virus is a human or porcine or avian influenza virus. In some embodiments, the bacterium is a mycobacterium, such as Mycobacterium tuberculosis. In some embodiments, the bacterium is an enterobacterium, such as Salmonella typhi.

[0055] In some aspects of the invention, methods are provided for screening candidate agents for toxicity to human hematopoietic cells. In some embodiments, the methods include using humanized M-CSF mice engrafted with human hematopoietic cells, such as engrafted Rag2 - / - IL - / - contacting hM-CSF mice with a candidate agent; and comparing the viability and / or function of hematopoietic cells in the mice contacted with the candidate agent with the viability and / or function of hematopoietic cells in humanized M-CSF mice engrafted with human hematopoietic cells that were not contacted with the candidate agent; wherein a decrease in the viability and / or function of the hematopoietic cells in the mice contacted with the candidate agent indicates that the candidate agent is toxic to the hematopoietic cells.

[0056] In some aspects of the invention, methods are provided for screening candidate agents for their ability to protect human hematopoietic cells from toxic agents, mitigate the effects of toxic agents on human hematopoietic cells, or reverse the effects of toxic agents on human hematopoietic cells. In some embodiments, the methods include using human hematopoietic cell-engrafted humanized M-CSF mice, e.g., engrafted Rag2 - / - IL - / -The hM-CSF mice or the engrafted sublethally irradiated hM-CSF mice are contacted with a toxic agent; the mice are contacted with a candidate agent; and the viability and / or function of hematopoietic cells in the mice contacted with the candidate agent are compared with the viability and / or function of hematopoietic cells in humanized M-CSF mice engrafted with human hematopoietic cells that were not contacted with the candidate agent; wherein an increase in the viability and / or function of hematopoietic cells in the mouse model contacted with the candidate agent indicates that the candidate agent protects the hematopoietic cells from the toxic agent.

[0057] In some aspects of the invention, methods are provided for predicting the responsiveness of an individual to treatment with a therapeutic agent. In some embodiments, the method comprises engrafting a humanized M-CSF mouse with human hematopoietic cells from the individual, such as a Rag2 - / - IL - / - contacting hM-CSF mice or engrafted sublethally irradiated hM-CSF mice with a therapeutic agent; and comparing the viability and / or function of hematopoietic cells in the mouse model contacted with the candidate agent with the viability and / or function of hematopoietic cells in humanized M-CSF mice engrafted with human hematopoietic cells that were not contacted with the candidate agent; wherein modulation of the viability and / or function of hematopoietic cells in the mouse contacted with the candidate agent indicates that the individual will respond to treatment with the therapeutic agent.

[0058] The present application relates to the following embodiments.

[0059] 1. A humanized M-CSF mouse, comprising:

[0060] The nucleic acid sequence encodes human M-CSF protein and is operably linked to the promoter of mouse M-CSF gene.

[0061] 2. The humanized M-CSF mouse according to embodiment 1, wherein the mouse comprises two copies of the nucleic acid sequence.

[0062] 3. The humanized M-CSF mouse according to embodiment 1, wherein the nucleic acid sequence is operably linked to an endogenous mouse M-CSF promoter at the mouse M-CSF locus.

[0063] 4. The humanized M-CSF mouse of embodiment 1, wherein the mouse comprises a null mutation in at least one mouse M-CSF allele.

[0064] 5. The humanized M-CSF mouse according to embodiment 4, wherein the null mutation is a deletion of exons 2-9 of mouse M-CSF.

[0065] 6. The humanized M-CSF mouse according to embodiment 1, wherein the mouse is homozygous null for Rag2.

[0066] 7. The humanized M-CSF mouse according to embodiment 6, wherein the mouse is homozygous null for IL2rg.

[0067] 8. The humanized M-CSF mouse of embodiment 1, wherein the mouse comprises human cells.

[0068] 9. The humanized M-CSF mouse of embodiment 8, wherein the human cells are hematopoietic cells.

[0069] 10. The humanized M-CSF mouse of embodiment 9, wherein the mouse comprises an infection with a human pathogen.

[0070] 11. A mouse model of an artificial hematopoietic system, comprising:

[0071] 2 null mutations in Rag2;

[0072] 2 null mutations in IL2rg;

[0073] a nucleic acid sequence operably linked to the promoter of the mouse M-CSF gene, the nucleic acid sequence encoding the human M-CSF protein; and

[0074] Human hematopoietic cells.

[0075] 12. The mouse model according to embodiment 11, wherein the nucleic acid sequence is operably linked to an endogenous mouse M-CSF promoter at the mouse M-CSF locus.

[0076] 13. The mouse model of embodiment 11, wherein the mouse comprises a null deletion of mouse M-CSF.

[0077] 14. The mouse model according to embodiment 11, wherein the mouse exhibits one or more characteristics selected from the group consisting of:

[0078] a. express human M-CSF in bone marrow, spleen, blood, liver, brain, lung, testis, and kidney at levels comparable to mouse M-CSF expression in wild-type mice;

[0079] b. showed that hCD14 in engrafted mice expressed less than hM-CSF + CD33 + 2- to 6-fold higher spleen hCD14 + CD33 + Cell frequency;

[0080] c. showed that hCD14 in engrafted mice expressed less than hM-CSF + CD33 + 2 to 8 times higher peripheral blood hCD14 + CD33+ Cell frequency;

[0081] d. Exhibits about 15% to about 40% of hCD14 in the blood + CD33 + monocyte / macrophage lineage cell levels;

[0082] e. At about 20 weeks of age, about 5% to about 15% of hCD14 in the blood is expressed + CD33 + monocyte / macrophage lineage cell levels;

[0083] f. exhibited a higher expression of hCD14 in the liver in response to LPS injection than mice lacking human M-CSF. + CD33 + About 1.5 to about 6 times higher in terms of cell percentage;

[0084] g. Demonstrated enhanced hCD14 in spleen approximately 48 hours after LPS injection + CD33 + hCD45 + cell production, wherein the enhancement is greater than about 2 to about 5 fold compared to engrafted mice lacking hM-CSF;

[0085] h. exhibited enhanced serum human IL-6 production in response to LPS, wherein hIL-6 levels at about 6 hours after LPS injection were enhanced by about 2 to about 5 fold compared to engrafted mice lacking hM-CSF;

[0086] i. exhibit in vitro secretion of monocytes and / or macrophages after LPS stimulation that is approximately 2 to 3 times higher than that of engrafted mice lacking the hM-CSF gene in terms of hTNFα;

[0087] j. exhibited in vitro secretion of hIL-6 by monocytes and / or macrophages after LPS stimulation, which was approximately 2 to 4 times higher than that of engrafted mice lacking the hM-CSF gene;

[0088] k. exhibit in vitro secretion of monocytes and / or macrophages after poly I:C stimulation that is approximately 3 to 6 times higher in terms of hIFNα than engrafted mice lacking the hM-CSF gene;

[0089] l. exhibit in vitro secretion of monocytes and / or macrophages after poly I:C stimulation, which is about 2 to 3 times higher in hIFNβ than that of engrafted mice lacking the hM-CSF gene;

[0090] m. exhibited enhanced phagocytosis compared to genetically modified and engrafted mice lacking the hM-CSF gene;

[0091] n. exhibiting enhanced in vitro chemotaxis in response to Mip3β compared to genetically modified grafted mice lacking the hM-CSF gene; and

[0092] o. exhibits in vitro upregulation of a co-stimulatory molecule in response to LPS stimulation, wherein the co-stimulatory molecule is selected from the group consisting of human CD40, human CD80, human CD86, human HLA-DR, and combinations thereof.

[0093] 15. A mouse model according to embodiment 14, wherein the mouse exhibits two or more of the characteristics.

[0094] 16. A mouse model according to embodiment 14, wherein the mouse exhibits three or more of the characteristics.

[0095] 17. A mouse model for a human pathogen, comprising:

[0096] 2 null mutations in Rag2;

[0097] 2 null mutations in IL2RG;

[0098] a nucleic acid sequence operably linked to the promoter of the mouse M-CSF gene, encoding the human M-CSF protein;

[0099] Human hematopoietic cells; and

[0100] Infection with human pathogens.

[0101] 18. The mouse model according to embodiment 17, wherein the pathogen is selected from the group consisting of viruses, fungi, and bacteria.

[0102] 19. The mouse model according to embodiment 18, wherein the bacteria is a mycobacterium or an enterobacterium. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figures 1A to 1E For bone marrow mesenchymal stromal cells, ( Figure 1A ) Expression of M-CSF; Isolation of M-CSF m / m and M-CSF h / h RNA was extracted from the indicated organs and analyzed by reverse transcription (RT)-PCR using primers specific for mouse M-CSF (top) or human M-CSF (middle); HPRT levels (bottom) were used as a control for input cDNA; data are representative of 2 independent experiments. ( Figure 1B ) isolated from M-CSF h / mRNA was extracted from the indicated organs and reverse transcription RT-PCR analysis was performed using mouse M-CSF (top) or human M-CSF (middle) specific primers. RNA extracted from mouse liver or human fetal liver served as positive controls for mouse and human primer pairs, respectively, and no RT and no template PCR reactions served as negative controls. Data represent 2 independent experiments. ( Figure 1C ) will come from M-CSF m / m 、M-CSF m / h and M-CSF h / h Mouse bone-associated stromal cells were isolated and cultured in vitro for 10 days, the cells were lysed, and RNA was extracted and analyzed by real-time PCR using primers specific for mouse M-CSF (white) or human M-CSF (black); mean values ​​of duplicate samples are shown; error bars indicate ± SEM; input cDNA amounts were normalized according to HPRT (hypoxanthine guanine phosphoribosyltransferase) expression levels; data represent 2 independent experiments; and ( Figure 1D ) will come from M-CSF m / m 、M-CSF m / h and M-CSF h / h Mouse bone-associated stromal cells were isolated and cultured in vitro for 10 days; cell culture supernatants were collected and the secretion levels of mouse (white) and human (black) M-CSF were quantified using species-specific M-CSF ELISA kits; mean values ​​of triplicate samples are shown; error bars indicate ±SEM; data are representative of 2 independent experiments. ( Figure 1E ) M-CSF m / m 、M-CSF h / m , and M-CSF h / h Mice were bled and serum levels of human and mouse M-CSF were quantified via ELISA. Mean values ​​of triplicate samples are shown. Error bars indicate ± SEM.

[0104] Figure 2A M-CSF is shown as the average of each animal (two tibiae and fibula). m / m 、M-CSF m / h and M-CSF h / h Absolute number of bone marrow (BM) cells in mice; each group contained n=5 mice, 4 weeks old; error bars indicate ± SEM; data are representative of 3 independent experiments.

[0105] Figure 2B showed that the effect of M-CSF m / m ,M-CSF m / h and M-CSF h / hFlow cytometric analysis of stained single cell suspensions of BM (top), spleen (middle) and peripheral blood (PB) of mice; stained with Gr1 and CD11b antibodies.

[0106] Figure 2C showed that the effect of M-CSF m / m 、M-CSF m / h and M-CSF h / h Flow cytometric analysis of stained single cell suspensions of BM (top) and spleen (middle) of mice; stained with F4 / 80 and CD11b antibodies.

[0107] Figure 2D Shown are flow cytometric analyses of BM cells isolated and cultured for 7 days in the presence of recombinant mouse M-CSF (left) or human M-CSF (right); cells were stained with F4 / 80 and CD11b antibodies.

[0108] Figure 2E Shown are flow cytometric analyses of BM cells isolated and cultured for 7 days in the presence of recombinant mouse M-CSF (solid) or human M-CSF (open); cells were stained with the indicated surface markers.

[0109] Figure 3A Shown is the human CD34 + Cell engraftment with M-CSF m / m 、M-CSF m / h and M-CSF h / h Flow cytometry of single cell suspensions of mouse BM (top), spleen (middle), and peripheral blood (PB); stained with CD45, CD14, and CD33 human antibodies; human CD45 + The cells were pre-gated and differentiated based on CD14 and CD33 expression.

[0110] Figure 3B The relative frequencies of human CD45+CD14+CD33+ cells of BM (top), spleen (middle) and peripheral blood (PB) are shown; the absolute number of BM cells was determined as the mean of each animal (two tibiae and fibula) and the absolute number of peripheral blood was determined per ml volume of blood; each group contained n=20 mice; each symbol represents an individual mouse and the horizontal bar represents the mean value; data represent 5 independent experiments.

[0111] Figure 3CThe absolute frequencies of human CD45+CD14+CD33+ cells of BM (top), spleen (middle) and peripheral blood (PB) are shown; the absolute number of BM cells was determined as the mean of each animal (two tibiae and fibula) and the absolute number of peripheral blood was determined per ml volume of blood; each group contained n=20 mice; each symbol represents an individual mouse and the horizontal bar represents the mean value; data represent 5 independent experiments.

[0112] Figure 4A Human CD34 cells were collected from blood samples 12, 16, and 20 weeks after transplantation. + Cell engraftment with M-CSF m / m 、M-CSF m / h and M-CSF h / h Flow cytometric analysis of stained cells from mice; cells were stained with human antibodies against CD45, CD14, and CD33; human CD45 + The cells were pre-gated and differentiated based on CD14 and CD33 expression.

[0113] Figure 4B Human CD45 + CD14 + CD33 + Relative frequency of cells; each group contained n = 10 mice; each symbol represents an individual mouse and horizontal bars indicate mean values; data are representative of 3 independent experiments.

[0114] Figure 5 showed the use of human CD34 + Cells were engrafted and M-CSF was administered 12 weeks after transplantation (at which time the animals were sacrificed and perfused with PBS). m / m 、M-CSF m / h and M-CSF h / h Analysis of flow cytometry results of mice; liver (A), lung (B) and skin (C) were harvested and single-cell suspensions were prepared; peritoneal cells were collected by aspiration with PBS (D); cells were stained with human CD45, CD14 and CD33 antibodies and analyzed by flow cytometry; each symbol represents an individual mouse and the horizontal bar represents the mean value; data represent 3 independent experiments.

[0115] Figures 6A to 6C Results of LPS stimulation are shown. ( Fig. 6A ) Using human CD34 + Cell engraftment with M-CSF m / m and M-CSF m / h Mice were injected with LPS ip at 12 weeks after transplantation. Mice were sacrificed 48 hours later and the expression of human CD45 in spleen was measured. + CD14 + CD33 +Frequency of cells; PBS-injected mice served as controls; each symbol represents an individual mouse and the horizontal bar represents the mean value. ( Figure 6B ),( Figure 6C ) Using human CD34 + Cell engraftment with M-CSF m / m and M-CSF m / h Mice were injected with LPS ip 12 weeks after transplantation. Six hours later, mice were bled and serum levels of human (right) and mouse (left) IL-6 and TNFα were quantified by ELISA; mice injected with PBS served as controls; mean values ​​of triplicate samples are shown; error bars indicate ± SEM.

[0116] Fig. 7A (for hTNFα) and 7B (for hIL-6) show the ability of monocytes / macrophages to secrete proinflammatory cytokines in vitro after LPS stimulation. + Cell engraftment with M-CSF m / m and M-CSF h / h Mouse spleen human CD45 + CD14 + CD33 + Cells: Human CD45 derived from fetal liver + CD14 + CD33 + Cells served as controls; cells were stimulated with LPS in vitro for 24 or 48 hours, cell culture supernatants were collected, and human TNFα ( Fig. 7A ) and IL-6 ( Figure 7B ) levels; mean values ​​of triplicate samples are shown; error bars indicate ± SEM.

[0117] Figure 7C Shown are interferon-α and -β mRNA levels in response to poly I:C stimulation. + CD14 + CD33 + Cells were stimulated for 6 or 12 h, and IFNα (left) and IFNβ (right) mRNA levels were quantified by performing PCR; mean values ​​of duplicate samples are shown; error bars indicate ± SEM.

[0118] Fig.7D The phagocytic, migratory, and activation properties of cells from engrafted mice were demonstrated. + CD14 + CD33 +Cells were isolated from humanized mice and incubated with FITC-labeled bacteria at 37°C for 30 or 60 min and measured by flow cytometry; cells incubated with FITC-labeled bacteria on ice served as controls. m / m Mouse cells, dotted histograms represent cells from M-CSF h / h mouse, while the solid histograms represent cells from human fetal liver.

[0119] Fig. 7E The chemotaxis of cells in response to MIP3β was shown. m / m Mouse, M-CSF h / h Human CD45 isolated from mouse and human fetal liver + CD14 + CD33 + Cells were maintained in the upper wells and medium containing MIP3β was added to the lower wells; cells were incubated for 30 or 60 min, and the number of cells from the upper to the lower wells was counted and plotted; mean values ​​of duplicate samples are shown; error bars indicate ± SEM.

[0120] Figure 7F Enhanced activation of human monocytes / macrophages from engrafted mice was demonstrated based on upregulation of hCD40, hCD80, hCD86, and hHLA-DR after in vitro LPS stimulation. m / m Mouse, M-CSF h / h Human CD45 isolated from mouse and human fetal liver + CD14 + CD33 + Cells were cultured in the presence or absence of LPS; after 24 h of stimulation, cells were stained with the indicated surface markers and measured by flow cytometry. m / m Mouse cells, dotted histograms represent cells from M-CSF h / h mouse, while the solid histograms represent cells from human fetal liver.

[0121] Figure 8 A schematic diagram of the mouse M-CSF locus is provided, indicating the relative positions of exons 1-9 and the ultimate targeted alleles of the human M-CSF gene.

[0122] Fig. 9 Panels A and B show M-CSF m / m 、M-CSF h / m , and M-CSF h / hFrequency of HSC compartment and myeloid progenitor compartment in mice. M-CSF derived cells were stained with antibodies to lineage, c-Kit, Sca1, CD150, CD48, CD16 / 32, and CD34. m / m ,M-CSF m / h and M-CSF h / h BM cells from mice were analyzed by flow cytometry. - Cells were gated (top) and differentiated based on Sca1 and c-Kit expression (middle). - Sca1 + c-Kit + (LSK) Cells were gated and further differentiated based on CD150 and CD48 expression (bottom). (B) Lineage - Cells were pre-gated and differentiated based on Sca1 and c-Kit expression (top). - c-Kit + Sca1 - Cells were pre-gated and further differentiated based on CD16 / 32 and CD34 expression (bottom). DETAILED DESCRIPTION OF THE INVENTION

[0123] Before describing the present method and composition, it should be understood that the present invention is not limited to the specific method and composition described, as it can certainly vary. It should also be understood that the terminology used herein is only for describing specific embodiments, and is not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

[0124] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein may be used in the implementation or testing of the present invention, specific methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials related to the cited publications. It should be understood that in the event of a conflict, the present disclosure replaces any conflicting disclosures of the publications collected.

[0125] As will be apparent to those skilled in the art after reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order that is logically possible.

[0126] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to a "cell" includes a plurality of such cells, and reference to "the peptide" includes reference to one or more peptides and equivalents thereof known to those skilled in the art, such as polypeptides, and so forth.

[0127] The publications discussed herein are provided solely for their disclosure as of the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0128] The present application provides genetically modified mice comprising a nucleic acid sequence encoding a human M-CSF protein. Also provided are genetically modified mice comprising a nucleic acid sequence encoding a human M-CSF protein and having been grafted with human cells, such as human hematopoietic cells, and methods for generating such grafted mice. These mice can be used in many applications, such as in modeling human immune diseases and pathogen infections; in in vivo screening for agents that modulate (e.g., in a healthy or diseased state) hematopoietic cell development and / or activity; in in vivo screening for agents that are toxic to hematopoietic cells; in in vivo screening for agents that provide protection against, mitigate, or reverse the toxic effects of toxic agents on hematopoietic cells; in in vivo screening for human hematopoietic cells from an individual that predict the responsiveness of the individual to disease therapy, and the like.

[0129] Humanized M-CSF mice

[0130] In some aspects of the invention, a humanized M-CSF mouse is provided. A humanized M-CSF mouse, or "hM-CSF mouse" means a mouse comprising a nucleic acid sequence encoding a human M-CSF protein. A human M-CSF protein means a protein that is human M-CSF or substantially identical to human M-CSF, for example, it is 80% or more identical, 85% or more identical, 90% or more identical, or 95% or more identical to human M-CSF, for example, 97%, 98%, or 99% identical to human M-CSF. Thus, a nucleic acid sequence encoding a human M-CSF protein is a polynucleotide comprising a coding sequence for a human M-CSF protein, i.e., human M-CSF or a protein substantially identical to human M-CSF. M-CSF (also known as CSF-1, which stands for "colony stimulating factor 1") is a cytokine that controls the generation, differentiation, and function of macrophages. The polypeptide sequence of human M-CSF and the nucleic acid sequence encoding human M-CSF can be found in Genbank accession numbers NM_000757.5 (variant 1), NM_172210.2 (variant 2), and NM_172212.2 (variant 4). The genomic locus encoding the human M-CSF protein can be present in the human genome at chromosome 1; NC_000001.10 (110453233-110472355). The protein sequence is encoded by exons 1 to 8 at this locus, while exon 9 contains untranslated sequences. Thus, the nucleic acid sequence comprising the human M-CSF coding sequence comprises one or more of exons 1-8 of the human M-CSF gene. In some examples, the nucleic acid sequence also comprises aspects of the genomic locus of human M-CSF, such as introns, 3' and / or 5' untranslated sequences (UTRs). In some examples, the nucleic acid sequence comprises the entire region of the human M-CSF genomic locus. In some examples, the nucleic acid sequence comprises exon 2 to the downstream 633 nt of non-coding exon 9 of the human M-CSF genomic locus.

[0131] In the humanized M-CSF mouse of the present application, the nucleic acid sequence encoding the human M-CSF protein is operably linked to one or more regulatory sequences of the mouse M-CSF gene. The mouse M-CSF regulatory sequences are those sequences of the mouse M-CSF genomic locus that regulate the expression of mouse M-CSF, such as 5' regulatory sequences, such as the M-CSF promoter, the M-CSF 5' untranslated region (UTR), etc.; 3' regulatory sequences, such as the 3'UTR; and enhancers, etc. Mouse M-CSF is located at approximately positions 107,543,966-107,563,387 on chromosome 3, and the mouse M-CSF coding sequence can be found in Genbank accession numbers NM_007778.4 (isoform 1), NM_001113529.1 (isoform 2), and NM_001113530.1 (isoform 3). Regulatory sequences for mouse M-CSF are well defined in the art and can be readily identified using in silico methods, for example by reference to the above-mentioned Genbank accession number on the UCSC Genome Fluorescence Facility on the world wide web at genome.ucsc.edu, or by experimental methods, as described below and in the art, for example, Abboud et al. (2003) Analysis of the Mouse CSF-1 Gene Promoter in a Transgenic Mouse Model. J. Histochemistry and Cytochemistry 51(7):941-949, the disclosure of which is incorporated herein by reference. In some instances, for example, when the nucleic acid sequence encoding the human M-CSF protein is located in the mouse M-CSF genomic locus, the regulatory sequences operably linked to the human CSF coding sequence are endogenous or native to the mouse genome, i.e., they are present in the mouse genome prior to integration of the human nucleic acid sequence.

[0132] In some examples, humanized M-CSF mice are generated by randomly integrating or inserting a human nucleic acid sequence encoding a human M-CSF protein or a fragment thereof, i.e., a "human M-CSF nucleic acid sequence" or a "human M-CSF sequence" into the genome. Typically, in such embodiments, the location of the nucleic acid sequence encoding the human M-CSF protein in the genome is unknown. In other examples, humanized M-CSF mice are generated by targeted integration or insertion of a human M-CSF nucleic acid sequence into the genome, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome at a target locus, while simultaneously removing host genomic material, e.g., 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more of genomic material, from the target locus. Thus, for example, in a humanized M-CSF mouse encoding a gene created by targeting a human M-CSF nucleic acid sequence to a mouse M-CSF locus, the human M-CSF nucleic acid sequence can replace a certain or entire mouse sequence at the M-CSF locus, such as an exon and / or intron, wherein the humanized M-CSF mouse comprises a nucleic acid sequence encoding a human M-CSF protein. In some such examples, the human M-CSF nucleic acid sequence is integrated into the mouse M-CSF locus such that expression of the human M-CSF sequence is regulated by a native, or endogenous, regulatory sequence at the mouse M-CSF locus. In other words, the regulatory sequence operably linked to the nucleic acid sequence encoding the human M-CSF protein is a native M-CSF regulatory sequence at the mouse M-CSF locus.

[0133] In some examples, the integration of the human M-CSF sequence does not affect the transcription of genes that have already received the human M-CSF sequence integration. For example, if the human M-CSF sequence is integrated into the coding sequence as an intein, or the human M-CSF sequence contains a 2A peptide, the human M-CSF sequence will be transcribed and translated simultaneously with the gene that has already received the human M-CSF sequence integration. In other cases, the integration of the human M-CSF sequence interrupts the transcription of genes that have already received the human M-CSF sequence integration. For example, after the integration of the human M-CSF sequence by homologous recombination, some or the entire coding sequence at the integration locus can be removed, thereby transcribing the human M-CSF sequence instead. In some such examples, the integration of the human M-CSF sequence creates a null mutation and, therefore, a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of the gene. This can be due to the complete lack of a gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product. At the phenotypic level, a null allele can be distinguished from the deletion of the entire locus.

[0134] In some examples, the humanized M-CSF mouse comprises a nucleic acid sequence encoding one copy of the human M-CSF protein. For example, the mouse can be heterozygous for the nucleic acid sequence. In other words, one allele at the locus will comprise the nucleic acid sequence, and the other will be an endogenous allele. For example, as discussed above, in some examples, a human M-CSF nucleic acid sequence is integrated into the mouse M-CSF locus so that it creates an allele of mouse M-CSF. In some such embodiments, the humanized M-CSF mouse can be heterozygous for the encoding nucleic acid sequence, i.e., the humanized M-CSF mouse comprises one mouse M-CSF null allele (an allele comprising the nucleic acid sequence) and one endogenous M-CSF allele (wild type or otherwise). In other words, the mouse is M-CSF h / m Mouse, wherein "h" represents an allele comprising a human sequence and "m" represents an endogenous allele. In other cases, humanized M-CSF comprises two copies of a nucleic acid sequence encoding a human M-CSF protein. For example, the mouse can be homozygous for the nucleic acid sequence, i.e., both alleles of the locus in the diploid genome will comprise the nucleic acid sequence, i.e., the humanized M-CSF mouse comprises two mouse M-CSF null alleles (alleles comprising the nucleic acid sequence). In other words, the mouse is M-CSF h / h Mouse.

[0135] Notably, humanized M-CSF mice, e.g., such as those described above, e.g., M-CSF h / h and M-CSF h / m The mice exhibit normal, or wild-type development and function of macrophages and monocytes and tissues that develop from macrophage lineage cells, such as bone. For example, humanized mice have normal dental and bone characteristics and normal bone marrow content, myeloid cell frequency in the bone marrow, spleen and peripheral blood, and macrophage frequency in the bone marrow and spleen.

[0136] In some examples, the humanized M-CSF mouse comprises other genetic modifications. For example, the humanized M-CSF mouse may comprise at least one null allele of the Rag2 gene ("recombination activated gene 2", which can be found in the coding sequence of Genbank Accession No. 1.NM_009020.3). In some embodiments, the humanized M-CSF mouse comprises two null alleles of Rag2. In other words, the humanized M-CSF mouse is homozygous for Rag2. As another example, the humanized M-CSF mouse comprises at least one null allele of the IL2rg gene ("interleukin 2 receptor, γ", also known as common γ chain, or γC, which can be found in the coding sequence of Genbank Accession No. 1.NM_013563.3). In some embodiments, the humanized M-CSF mouse comprises two null alleles of IL2rg. In other words, the humanized M-CSF mouse is homozygous null for IL2rg. In some embodiments, the mouse comprises a null allele of both Rag2 and IL2rg, i.e., it is a null allele of Rag2 - / - IL2RG - / - Other genetic modifications are also contemplated. For example, a humanized M-CSF mouse can include modifications in other genes associated with the development and / or function of hematopoietic cells and the immune system, e.g., replacement of one or other mouse gene with a nucleic acid sequence encoding a human ortholog. Additionally or alternatively, a humanized M-CSF mouse can include modifications in genes associated with the development and / or function of other cells and tissues, e.g., genes associated with human conditions or diseases, or genes that, when modified in a mouse, provide a mouse model for human conditions and diseases.

[0137] In some aspects of the invention, humanized M-CSF mice, such as Rag2 - / - IL - / -hM-CSF mice or sublethally irradiated hM-CSF mice. The cells can be mitotic cells or post-mitotic cells, and include cells of interest such as pluripotent stem cells, such as ES cells, iPS cells, and embryonic germ cells; and somatic cells, such as fibroblasts, hematopoietic cells, neurons, myocytes, osteocytes, vascular endothelial cells, intestinal cells, etc., and their lineage-restricted ancestors and precursors. Cell populations of particular interest include those containing hematopoietic stem cells or progenitor cells that contribute to or reconstruct the hematopoietic system of humanized M-CSF mice, for example, peripheral blood leukocytes, fetal liver cells, fetal bones, fetal thymus, fetal lymph nodes, vascularized skin, arterial segments, and purified hematopoietic stem cells, such as mobile HSCs or umbilical cord blood HSCs. The cells can be from any mammalian species, such as mice, rodents, dogs, cats, horses, cattle, sheep, primates, humans, etc. The cells can be from an established cell line or they can be primary cells, where "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein to refer to cells and cell cultures that have been derived from a subject and allowed to grow in vitro for a limited number of culture passages (i.e., splits). For example, a primary culture is a culture that may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, but has not had sufficient time to undergo a critical stage. Typically, the primary cell lines of the invention are maintained in vitro for less than 10 passages.

[0138] If the cells are primary cells, they can be harvested from individuals by any convenient method. For example, cells such as blood cells, such as leukocytes, can be harvested by shedding, leukocyte shedding, density gradient separation, etc. As another example, cells such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, gastric tissue, etc. can be harvested by biopsy. Suitable solutions can be used to disperse or suspend harvested cells. Such solutions are generally balanced salt solutions, such as normal saline, PBS, Hank's balanced salt solution, etc., which are conveniently supplemented with fetal bovine serum or other naturally occurring factors and low concentrations (generally 5-25mM) of acceptable buffers. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.

[0139] In some examples, heterogeneous cell colonies can be transplanted into humanized cells. In other examples, a cell colony rich in specific cell types, such as progenitor cells, such as hematopoietic progenitor cells, can be grafted into humanized mice. Enrichment of a cell colony of interest can be performed by any convenient separation technique. For example, cells of interest can be enriched by a culture method. In such a culture method, specific growth factors and nutrients are usually added to the culture, which promotes the survival and / or proliferation of a cell colony on other cells. Other culture conditions that affect survival and / or proliferation include growth on an adherent or non-adhesive matrix, culture specific time lengths, etc. Such culture conditions are well known in the art. As another example, cells of interest are enriched from the initial colony separation of cells of interest by affinity separation technology. The technology used for affinity separation can include magnetic separation using magnetic beads coated with affinity reagents, affinity chromatography, "elutriation" with affinity reagents attached to a solid matrix (such as a plate), cytotoxic preparations such as complement and cytotoxins or other convenient technologies connected with affinity reagents or used in conjunction with affinity reagents. Techniques that provide precise separation include fluorescence activated cell sorters, which may have varying degrees of sophistication, such as multicolor channels, low angle and obtuse angle light scatter detection channels, impedance channels, etc. Cells may be selected for dead cells by employing dyes associated with dead cells (e.g., propidium iodide). Any technique that is not overly detrimental to the viability of the cells of interest may be employed.

[0140] For example, using affinity separation technology, cells that are not used as cells of interest for transplantation can be eliminated from the population by contacting the population with an affinity reagent, which specifically recognizes and selectively binds to markers that are not expressed on the cells of interest. For example, in order to enrich the hematopoietic progenitor cell population, cells expressing mature hematopoietic cell markers can be eliminated. In addition / or, positive selection and separation can be implemented by contacting the population with an affinity reagent, which specifically recognizes and selectively binds to markers related to hematopoietic progenitor cells, such as CD34, CD133, etc. "Selective binding" means that a molecule preferentially binds to a target of interest or binds with an affinity for a target greater than for other molecules. For example, an antibody will bind to a molecule containing an epitope specific to it rather than an irrelevant epitope. In some embodiments, the affinity reagent can be an antibody, i.e., an antibody specific to CD34, CD133, etc. In some embodiments, the affinity reagent can be a receptor or ligand specific to CD34, CD133, etc., such as a peptide ligand and receptor specific to CD34, CD133, etc.; effector molecules and receptor molecules, T cell receptors, etc. In some embodiments, multiple affinity reagents specific for a marker of interest may be used.

[0141] Antibodies and T cell receptors that can be used as affinity reagents can be monoclonal or polyclonal and can be generated by transgenic animals, immune animals, immortalized human or animal B cells, cells transfected with DNA vectors encoding antibodies or T cell receptors, etc. The details of preparing antibodies and their suitability for use as specific binding members are well known to those skilled in the art. Of particular interest is the use of labeled antibodies as affinity reagents. Conveniently, these antibodies are conjugated to markers for separation. Markers include magnetic beads, which allow direct separation; biotin, which can be removed with avidin or streptavidin bound to a support; fluorescent dyes, which can be used with a fluorescence-activated cell sorter; etc., to allow easy separation of specific cell types. Fluorescent dyes that can be used include phycobiliproteins, such as phycoerythrin and allophycocyanin, fluorescein, and Texas red. Each antibody is often labeled with a different fluorescent dye to allow independent sorting of each marker.

[0142] The initial cell colony is contacted with the affinity reagent and incubated for a period of time, so as to be sufficient to bind available cell surface antigens. The incubation is usually at least about 5 minutes, and is usually less than about 60 minutes. It is desirable to have sufficient concentrations of antibody in the reaction mixture so that the separation efficiency is not limited to the lack of antibody. Suitable concentration is determined by titer, but the antibody is usually diluted into the cell suspension volume, the dilution is about 1:50 (that is, 1 part of antibody and 50 parts of reaction volume), about 1:100, about 1:150, about 1:200, about 1:250, about 1:500, about 1:1000, about 1:2000 or about 1:5000. The culture medium of the suspended cells can be any culture medium that maintains cell viability. Preferably, the culture medium is a phosphate buffered saline containing 0.1 to 0.5% BSA or 1-4% goat serum. A variety of culture media are commercially available and can be used depending on the properties of the cells, including Dulbecco's modified Eagle's medium (dMEM), Hank's basic saline solution (HBSS), Dulbecco's phosphate-buffered saline (dPBS), RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., which are often supplemented with fetal bovine serum, BSA, HSA, goat serum, etc.

[0143] The cells that obtain the affinity reagent labeling in the contact population are selected by any convenient affinity separation technique, such as described above or known in the art. After separation, the separated cells can be collected in any suitable culture medium that maintains cell viability, usually with a serum pad at the bottom of the collection tube. A variety of culture media are commercially available and can be used according to the properties of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove's culture medium, etc., which are often supplemented with fetal bovine serum.

[0144] In this way, a composition highly enriched in a cell type of interest, such as hematopoietic cells, is achieved. The cells will account for about 70%, about 75%, about 80%, about 85%, about 90% or more of the cell composition, about 95% or more of the enriched cell composition, and preferably, about 95% or more of the enriched cell composition. In other words, the composition will be a substantially pure composition of the cells of interest.

[0145] The cells to be transplanted into the humanized M-CSF mice (which are either heterogeneous cell populations or enriched cell populations) can be transplanted immediately. Alternatively, the cells can be frozen at liquid nitrogen temperatures and stored for a longer period of time, which thaws and can be used again. In such cases, the cells are usually frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution commonly used in the art to protect the cells at such freezing temperatures, and thawed in a manner commonly known in the art for thawing frozen cultured cells. Additionally or alternatively, the cells can be cultured in vitro under various culture conditions. The culture medium can be liquid or semisolid, for example containing agar, methylcellulose, etc. The cell population can be conveniently suspended in a suitable nutrient medium, such as Iscove's modified DMEM or RPMI-1640 (which is usually supplemented with fetal bovine serum (about 5-10%), L-glutamine, thiols, especially 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin). The culture can contain growth factors to which the cells respond. As defined herein, a growth factor is a molecule capable of promoting cell survival, growth and / or differentiation in culture or in intact tissues via specific effects on transmembrane receptors.Growth factors include polypeptide and non-polypeptide factors.

[0146] Cells can be genetically modified prior to transplantation into humanized M-CSF mice, for example, to provide a selectable or traceable marker, to induce a genetic defect in the cell (e.g., for disease modeling), to repair a genetic defect or to ectopically express a gene in the cell (e.g., to determine whether such a modification affects the course of a disease), etc. Cells can be genetically modified by transfection or transduction with an appropriate vector, homologous recombination, or other suitable techniques so that they express a gene of interest, or genetically modified with antisense mRNA, siRNA, or ribozymes to block unwanted gene expression. A variety of techniques for introducing nucleic acids into target cells are known in the art. To demonstrate that cells have been genetically modified, a variety of techniques can be used. The genome of the cell is restricted and used with or without amplification. Polymerase chain reaction; gel electrophoresis; restriction analysis; Southern, Northern, and Western blotting; sequencing, etc. can all be used. General methods in molecular and cellular biochemistry for these and other purposes disclosed in this application can be found in standard textbooks, such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosure of which is incorporated herein by reference. The reagents, cloning vectors, and kits for genetic manipulation mentioned in this disclosure can be purchased from commercial manufacturers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0147] Cells can be transplanted in humanized M-CSF mice by any convenient method, including, for example, intrahepatic injection, tail vein injection, retro-orbital injection, etc. Typically, about 0.5 x 10 5 -2x 10 6 pluripotent cells or progenitor cells, for example about 1 x 10 5 – 1x 10 6 cells, or approximately 2 x 10 5 – 5x 10 5 In some instances, the mice are sublethally irradiated prior to transplantation of the human cells. In other words, the mice are exposed to a sublethal dose of irradiation, such as described in the Examples section below and known in the art. The engrafted humanized M-CSF mice are then maintained under laboratory animal resource management conditions for at least 1 week, such as 1 week or more, or 2 weeks or more, sometimes 4 weeks or more, and in some instances 6 weeks or more, to allow for adequate reconstitution of the immune system with the engrafted cells.

[0148] As shown in the Examples section below, humanized M-CSF mice demonstrate a significantly increased ability to engraft and maintain human hematopoietic cells compared to other mouse strains and other M-CSF transgenic mice that have been developed for this purpose. For example, intrahepatic transfer of human fetal liver-derived hematopoietic stem and progenitor cells (CD34+) into newborn mice resulted in more efficient differentiation and enhanced frequencies of human monocytes / macrophages in the bone marrow, spleen, peripheral blood, lungs, liver, and peritoneal cavity. A substantial proportion of human CD14+CD33+ cells were observed at 16-20 weeks.Specifically, humanized M-CSF mice engrafted with hematopoietic cells demonstrate one or more of the following, in some examples two or more, in some examples three or more, in some examples four or more, and in some examples all of the following characteristics: they express human M-CSF in the bone marrow, spleen, blood, liver, brain, lung, testis, and kidney at levels comparable to mouse M-CSF expression in wild-type mice; exhibit a 2- to 6-fold higher frequency of splenic hCD14+CD33+ cells than hCD14+CD33+ in engrafted mice that do not express hM-CSF; exhibit a 2- to 8-fold higher frequency of hCD14+CD33+ cells than hCD14+CD33+ in engrafted mice that do not express hM-CSF; peripheral blood hCD14+CD33+ cell frequency; exhibiting a level of hCD14+CD33+ monocyte / macrophage lineage cells in the blood of about 15% to about 40%; exhibiting a level of hCD14+CD33+ monocyte / macrophage lineage cells in the blood of about 5% to about 15% at about 20 weeks of age; exhibiting a response to LPS injection that is about 1.5 to about 6 times greater than the percentage of hCD14+CD33+ cells in the liver of mice lacking human M-CSF; exhibiting enhanced generation of hCD14+CD33+hCD45+ cells in the spleen about 48 hours after LPS injection, wherein the enhancement exceeds that of mice lacking hM-CSF; The invention discloses a novel method for treating hM-CSF grafted mice with hIL-6. The invention discloses a method for treating hM-CSF grafted mice with hIL-6. The invention discloses a method for treating hM-CSF grafted mice with hIL-6. The invention discloses a method for treating hM-CSF grafted mice with hIL-6. The invention discloses a method for treating hM-CSF grafted mice with hIL-6. The invention discloses a method for treating hM-CSF grafted mice with hIL-6. The invention discloses a method for treating hM-CSF grafted mice with hIL-6. The invention discloses a method for treating hM-CSF grafted mice with hIL-6. The invention discloses a method for treating hM-CSF grafted mice with hIL-6. The invention discloses a method for treating hM-CSF grafted mice with hIL-6. The invention discloses a method for treating hM-CSF grafted mice with hIL-6. -CSF gene-engrafted mice have about 3 to 6 times higher levels of hIFNα; exhibit in vitro secretion of monocytes and / or macrophages after poly I:C stimulation that is about 2 to 3 times higher than that of engrafted mice lacking the hM-CSF gene; exhibit enhanced phagocytosis compared to genetically modified and engrafted mice lacking the hM-CSF gene; exhibit enhanced in vitro chemotaxis in response to Mip3β compared to genetically modified and engrafted mice lacking the hM-CSF gene; and exhibit in vitro upregulation of costimulatory molecules in response to LPS stimulation, wherein the costimulatory molecules are selected from human CD40, human CD80, human CD86, human HLA-DR, and combinations thereof.

[0149] utility

[0150] Humanized M-CSF mice and humanized M-CSF mice engrafted with human hematopoietic cells, such as engrafted Rag2 - / - IL - / - hM-CSF mice, and optionally other genetic modifications, can be used for many applications. For example, these mice provide a system that can be used to model human immune diseases and human pathogens. For example, the mice of the present invention can be used to model human hematopoietic malignancies derived from early human hematopoietic cells, such as human hematopoietic stem cells or progenitor cells. As another example, the mice of the present invention can be used to study human pathogens that do not normally infect mice, such as viruses, fungi, and bacteria.

[0151] One such example of a human pathogen that does not normally infect mice is Salmonella typhi, the cause of typhoid fever. Typhoid fever afflicts more than 21 million people worldwide, primarily in developing countries, including about 400 cases per year in the United States. Typhoid fever has been treated with the drugs amoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime, chloramphenicol, ciprofloxacin, co-trimoxazole, ertapenem, imipenem, fluoroquinolones (e.g., ciprofloxacin, gatifloxacin, ofloxacin), streptomycin, sulfadiazine, sulfamethoxazole, tetracycline, and combinations thereof. Recurrent infections are common, which limits disease management by antibiotic therapy. Furthermore, multidrug resistance is also prevalent in cases of S. Typhi infection.

[0152] New therapeutic agents, new vaccines, and new ways to test the efficacy of therapeutic agents and vaccines are needed. For example, mice that can be infected with Salmonella Typhi can be used to identify new therapeutic agents and new vaccines. For example, new therapeutic agents and new vaccines can be tested in such mice, for example, by measuring the amount of Salmonella Typhi in mice (in the blood or a given tissue) in response to treatment with a putative anti-Salmonella Typhi agent, or by inoculating mice with a putative vaccine, followed by infectious administration of Salmonella Typhi, and observing any changes in infection status due to the putative vaccination compared to controls that were not vaccinated but infected with Salmonella Typhi.

[0153] Humanized M-CSF mice engrafted with human hematopoietic cells, such as Rag2 - / - IL - / -hM-CSF mice can be used to study human pathogens (i.e., pathogens that infect humans); the response of the human immune system to infection with human pathogens; and the efficiency of agents in providing protection from and / or treating infection with human pathogens. The pathogens can be viruses, fungi, bacteria, etc. Non-limiting examples of viral pathogens include human or porcine or avian influenza viruses. Non-limiting examples of bacterial pathogens include mycobacteria, e.g., Mycobacterium tuberculosis and enterobacteriaceae, e.g., Salmonella typhi.

[0154] For example, engrafted humanized M-CSF mice can be used as a non-human animal model of Salmonella typhi infection. In comparison, wild-type mice and other known immunocompromised mice (e.g., RAG1 / RAG2 knockout mice) cannot be infected by Salmonella typhi. As discussed above, the engrafted human M-CSF mice described herein exhibit enhanced human cell engraftment compared to engrafted mice that do not contain human M-CSF protein. This enhancement is sufficient to maintain the resulting Salmonella typhi infection, that is, the Salmonella typhi is able to propagate in the mouse, that is, the infected mouse is able to contain and propagate Salmonella typhi in one or more of its cells. In a specific embodiment, the mouse is able to propagate Salmonella typhi for at least one week, 10 days, 2 weeks, 3 weeks, or 4 weeks after the initial introduction or infectious exposure of Salmonella typhi. In other words, the mouse is able to maintain a Salmonella typhi titer or level in its blood or in at least one tissue for at least one week, 10 days, 2 weeks, 3 weeks, or 4 weeks. Examples of methods for infecting mice with Salmonella typhi and for assessing infection can be found, for example, in U.S. Published Application No. 2011 / 0200982, the disclosure of which is incorporated herein by reference.

[0155] As another example, humanized M-CSF-engrafted mice, such as Rag2 - / - IL - / - The hM-CSF mouse can be used as a non-human animal model of Mycobacterium tuberculosis infection. The enhanced engraftment of human hematopoietic cells in the mouse comprising a nucleic acid encoding a human M-CSF protein is sufficient to maintain the resulting Mycobacterium tuberculosis infection, that is, Mycobacterium tuberculosis is able to propagate in the mouse, that is, the infected mouse is able to contain and propagate Mycobacterium tuberculosis in one or more of its cells. In some such embodiments, the mouse mounts an anti-mycobacterial immune response to human pathogenic mycobacteria, wherein the response comprises granuloma formation mediated by human immune cells, and which comprises human immune cells. In some such embodiments, the granuloma is a pulmonary granuloma. In some such embodiments, the granuloma is a well-defined granuloma. Examples of methods for infecting mice with Mycobacterium tuberculosis and for assessing infection can be found in U.S. Published Application No. 2011 / 0200982, the disclosure of which is incorporated herein by reference.

[0156] Other examples of human pathogens that do not infect mice that express human M-CSF and, in some cases, contain one or more other genetic modifications (e.g., as described herein) or that infect wild-type mice, where the infected wild-type mice do not mimic the immune response that a human would mount in response to the pathogen, will be known to those of ordinary skill in the art.

[0157] Such mouse models of pathogen infection can be used for research, for example, to better understand the progression of human infection. Such mouse models of infection can also be used for drug research, for example, to identify candidate agents that provide protection from infection or candidate agents that treat infection.

[0158] Humanized M-CSF mice engrafted with human hematopoietic cells provide a useful system for screening candidate agents for other desired in vivo activities as well, such as agents that can modulate (i.e., promote or inhibit) (e.g., in healthy or diseased states) hematopoietic cell development and / or activity, such as the activity of B cells, T cells, NK cells, macrophages, neutrophils, eosinophils, basophils, etc.), e.g., to identify new therapeutic agents and / or to gain a better understanding of the molecular basis of the development and function of the immune system; agents that are toxic to hematopoietic cells, such as B cells, T cells, NK cells, macrophages, neutrophils, eosinophils, basophils, etc., and their progenitors; and agents that provide protection from, mitigate, or reverse the toxic effects of toxic agents on hematopoietic cells, such as B cells, T cells, NK cells, macrophages, neutrophils, eosinophils, basophils, etc., and their progenitors, etc. As yet another example, the genetically modified mice described herein provide a system that can be used to predict an individual's responsiveness to a disease therapy, e.g., by providing an in vivo platform for screening an individual's immune system's responsiveness to an agent, e.g., a therapeutic agent, to predict an individual's responsiveness to the agent.

[0159] In screening assays for biologically active agents, humanized M-CSF mice that have been engrafted with human hematopoietic cells and in some cases infected with human pathogens, such as Rag2 - / - IL - / -hM-CSF mice, or cells to be grafted into humanized M-CSF mice, are contacted with a candidate agent of interest, and the effect of the candidate agent is evaluated by monitoring one or more output parameters. These output parameters can reflect the viability of the cells, such as the total number of hematopoietic cells or the number of cells of a specific hematopoietic cell type, or reflect the apoptotic state of the cells, such as the amount of DNA fragmentation, the amount of cell blebbing, the amount of phosphatidylserine on the cell surface, etc., by methods known in the art. Alternatively or additionally, the output parameters can reflect the differentiation ability of the cells, such as the ratio of differentiated cells and differentiated cell types. Alternatively or additionally, the output parameters can reflect the function of the cells, such as the cytokines and chemokines produced by the cells, the ability of the cells to home and extravasate to the site of attack, the ability of the cells to regulate in vitro or in vivo, i.e., to promote or inhibit the activity of other cells, etc. Other output parameters can reflect the degree of pathogen infection in the animal, such as the titer of the pathogen in the mouse, the presence of granulomas in the mouse, etc.

[0160] Parameter is quantifiable cell component, particularly can accurately measure (expect in high throughput system) measured component.Parameter can be any cell component or cell product, including cell surface determinant, receptor, protein or its conformation or post-translational modification, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, such as mRNA, DNA etc., or from such cell component derived part or its combination.Although most parameters can provide quantitative reading, in some examples, semi-quantitative or qualitative result will be acceptable.Reading can comprise single determination value, or can comprise mean, median value or variance, etc.In characteristic, can obtain parameter readout value range for each parameter from many identical determination methods.Variability is expected, and can use standard statistical method and the common statistical method for providing single value to obtain the numerical range of each group of test parameter.

[0161] Candidate agents of interest for screening include known and unknown compounds covering many chemical classes, primarily organic molecules (which may include organometallic molecules), inorganic molecules, genetic sequences, vaccines, antibiotics or other agents suspected of having antibiotic properties, peptides, polypeptides, antibodies, agents already approved as drugs for human use, etc. An important aspect of the present invention is the evaluation of candidate drugs, including testing their toxicity, etc.

[0162] Candidate agents include organic molecules containing functional groups necessary for structural interactions, particularly hydrogen bonding, and typically include at least amine, carbonyl, hydroxyl or carboxyl groups, often at least two functional chemical groups. Candidate agents often include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also sought from biomolecules, including peptides, polynucleotides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Including pharmacologically active drugs, genetically active molecules, etc. Interested compounds include chemotherapeutic agents, hormones or hormone antagonists, etc. Exemplary agents suitable for the present invention are those recorded in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY, (1996), 9th edition. Also included are toxins, and biological and chemical agents; see, e.g., Somani, SM (ed.), "Chemical Warfare Agents," Academic Press, New York, 1992).

[0163] Candidate agents of interest for screening also include nucleic acids, for example, nucleic acids encoding siRNA, shRNA, antisense molecules or miRNA, or nucleic acids encoding polypeptides. Many vectors that can be used to transfer nucleic acids into target cells are available. The vector can be maintained as an episome, for example, with a plasmid, minicircle DNA, a virus-derived vector such as cytomegalovirus, adenovirus, etc., or they can be integrated into the target cell genome via homologous recombination or random integration, for example, retrovirus-derived vectors such as MMLV, HIV-1, ALV, etc. The vector can be directly provided to the cells of the present invention. In other words, pluripotent cells are contacted with a vector comprising a nucleic acid of interest so that the cells absorb the vector.

[0164] Methods for contacting cells (e.g., cells in culture or cells in mice) with nucleic acid vectors, such as electroporation, calcium chloride transfection, and lipofection are well known in the art. Alternatively, the nucleic acid of interest can be provided to the cell via a virus. In other words, the cell is contacted with a viral particle containing the nucleic acid of interest. Retroviruses, such as lentiviruses, are particularly suitable for the method of the present invention. Commonly used retroviral vectors are "defective", i.e., they cannot produce the viral proteins required for infection. Instead, the replication of the vector needs to be generated in a packaging cell line. In order to generate viral particles containing the nucleic acid of interest, the reverse transcribed nucleic acid containing the nucleic acid is packaged into a viral capsid by a packaging cell line. Different packaging cell lines provide different envelope proteins incorporated into the capsid, which determine the specificity of the viral particles to the cell. The envelope protein has at least three types, isotropic, amphotropic, and anisotropic. Retroviruses packaged with isotropic envelope proteins (e.g., MMLV) are capable of infecting most mouse and rat cell types and are generated by using isotropic packaging cell lines such as BOSC23 (Pear et al. (1993) PNAS 90:8392-8396). Retroviruses carrying amphotropic envelope proteins (e.g., 4070A) (Danos et al., supra) are capable of infecting most mammalian cell types, including humans, dogs, and mice and are generated by using amphotropic packaging cell lines such as PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with heterotropic envelope proteins (e.g., AKR env) are capable of infecting most mammalian cell types, in addition to murine cells. Appropriate packaging cell lines can be used to ensure that the packaged viral particles are targeted to the cells of interest, which in some cases are engrafted cells, and in some cases, are host cells, i.e., humanized M-CSF.

[0165] The vector for providing the nucleic acid of interest to the cell of the present invention generally comprises a promoter suitable for driving the expression (i.e., transcriptionally active) of the nucleic acid of interest. This can include ubiquitous promoters, such as CMV-b-actin promoters, or inducible promoters, such as promoters that are active in a particular cell population or that respond to drugs such as tetracycline. By transcriptional activation, it is intended to increase transcription in the target cell by at least about 10 times, at least about 100 times, and more generally at least about 1000 times at the basal level. In addition, the vector for providing the reprogramming factor to the cell of the present invention can comprise genes that must be removed later, for example, using a recombinase system such as Cre / Lox, or express them and, for example, by comprising genes that allow selective toxicity, such as herpes virus TK, bcl-xs, etc., cells that are destroyed.

[0166] Candidate agents of interest for screening also include polypeptides. Optionally, such polypeptides can be fused to polypeptide domains that improve product solubility. The domain can be connected to the polypeptide via a defined protease cleavage site, such as a TEV sequence (which is cleaved by a TEV protease). The joint can also include one or more flexible sequences, such as 1 to 10 glycine residues. In some embodiments, the cutting of the fusion protein is implemented in a buffer that maintains product solubility, such as in the presence of 0.5 to 2M urea, in the presence of polypeptides and / or polynucleotides that improve solubility, etc. The domain of interest includes an endosomolytic domain, such as an influenza HA domain; and other polypeptides that contribute to generation, such as an IF2 domain, a GST domain, a GRPE domain, etc. In addition / or, such polypeptides can be formulated to achieve improved stability. For example, a peptide can be PEGylated, wherein a polyethyleneoxy group provides an enhanced life span in the bloodstream. A polypeptide can be fused to another polypeptide to provide increased functionality, such as to improve in vivo stability. Generally, such fusion partners are stable plasma proteins, which can, for example, extend the in vivo plasma half-life of a polypeptide when present as a fusion, particularly where such stable plasma proteins are immunoglobulin constant domains. In most cases where stable plasma proteins are typically found in polymeric form (e.g., immunoglobulins or lipoproteins), where identical or different polypeptide chains are typically disulfides and / or non-covalently bound to form assembled multi-chain polypeptides, fusions containing polypeptides herein will also be generated and utilized in a polymeric form with substantially the same structure as a stable plasma protein precursor. These polymers are homogeneous with respect to the polypeptide agents they contain, or they may contain more than one polypeptide agent.

[0167] Candidate polypeptide agents can be generated from eukaryotic cells, or can be generated by prokaryotic cells. It can be further processed by unfolding, such as heat denaturation, DTT reduction, etc., and can be further refolded using methods known in the art. Interested modifications that do not change the primary sequence include chemical derivatization of the polypeptide, such as acylation, acetylation, carboxylation, amidation, and the like. Also included are modifications of glycosylation, such as those made by modifying the glycosylation mode of the polypeptide during its synthesis and processing or in further processing steps; for example, by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes. Also covered are sequences with phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine, or phosphothreonine. It has been possible to use common molecular biology techniques and synthetic chemistry to modify polypeptides, thereby improving their resistance to proteolytic degradation or to optimize solubility properties or to make them more suitable as therapeutic agents. Analogs of such polypeptides include those containing residues different from naturally occurring L-amino acids, such as D-amino acids or those analogs of non-naturally occurring synthetic amino acids. Some or all of the amino acid residues can be replaced with D amino acids.

[0168] Candidate polypeptide agents can be prepared by in vitro synthesis using conventional methods as known in the art. A variety of commercial synthesis devices are available, for example, automated synthesizers from Applied Biosystems, Inc., Beckman, etc. By using a synthesizer, naturally occurring amino acids can be replaced with non-natural amino acids. The specific sequence and preparation method will be determined based on convenience, economy, required purity, etc. Alternatively, candidate polypeptide agents can be separated and purified according to conventional methods of recombinant synthesis. Lysates of expression hosts can be prepared, and lysates can be purified using HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. Typically, the composition used will contain at least 20% (by weight) of the desired product, more typically at least about 75% (by weight), preferably at least about 95% (by weight), and for therapeutic purposes, typically at least about 99.5% (by weight). Typically, the percentage is based on the percentage of total protein.

[0169] In some cases, the candidate polypeptide agent to be screened is an antibody. The term "antibody" or "antibody module" is intended to include any polypeptide chain containing a molecular structure of a specific shape suitable for and recognizing an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The specificity or selective coordination of a given structure and its specific epitope is sometimes referred to as a "key" coordination. The prototype antibody molecule is an immunoglobulin, and it is believed that all types of immunoglobulins from all sources, such as humans, rodents, rabbits, cattle, goats, pigs, dogs, other mammals, chickens, other birds, etc., i.e., IgG, IgM, IgA, IgE, IgD, etc. are "antibodies". The antibodies utilized in the present invention can be polyclonal antibodies or monoclonal antibodies. Antibodies are usually provided in the culture medium of cultured cells.

[0170] Candidate agents can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, many means can be used for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including oligonucleotides and oligopeptides that express randomization. Alternatively, libraries of natural compounds in the form of bacteria, fungi, plants, and animal extracts are available or easily prepared. In addition, natural or synthetically generated libraries and compounds are easily modified via conventional chemical, physical, and biochemical means, and can be used to generate combinatorial libraries. Known pharmacological agents are subjected to directed or random chemical modifications, such as acylation, acetylation, esterification, amidation, etc., to generate structural analogs.

[0171] Candidate agents are screened for biological activity by administering the agent to at least one and usually multiple samples, sometimes to samples lacking the agent. Changes in parameters of the response agent are measured, and the results are evaluated by comparing with reference cultures obtained with other agents, for example, in the presence and absence of the agent, and the like. In the case of implementing screening to identify candidate agents that prevent, mitigate or reverse the effects of toxic agents, screening is usually implemented in the presence of toxic agents, wherein the toxic agent is added at the time most suitable for the determination of the results. For example, in the case of testing the protection / prevention ability of candidate agents, the candidate agent can be added before the toxic agent, at the same time as the candidate agent, or after treatment with the candidate agent. As another example, in the case of testing the ability of candidate agents to reverse the effects of toxic agents, the candidate agent can be added after treatment with the candidate agent. As mentioned above, in some examples, the sample is a humanized M-CSF mouse that has been grafted with cells, that is, the candidate agent is provided to the humanized M-CSF mouse that has been grafted with cells. In some examples, the sample is a cell to be grafted, that is, the candidate agent is provided to the cell before transplantation.

[0172] If the candidate agent is to be directly applied to mice, the agent can be applied by various known methods used in the art for applying peptides, small molecules and nucleic acids to mice. For example, the agent can be applied orally, mucosally, topically, intradermally or by injection, such as intraperitoneally, subcutaneously, intramuscularly, intravenously or intracranial injections, etc. The agent can be applied in a buffer, or it can be incorporated into a variety of formulations, for example, by combining it with a suitable pharmaceutically acceptable vehicle. "Pharmaceutically acceptable vehicles" can be vehicles that are approved by the federal or state government administration or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for mammals such as humans. The term "vehicle" refers to a diluent, adjuvant, excipient or carrier for preparing the compound of the present invention to be applied to a mammal. Such pharmaceutical vehicles can be lipids, such as liposomes, such as liposome dendrimers; liquids, such as water and oils (including those derived from petroleum, animals, plants or synthetic origins, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.), saline; gum arabic, gelatin, starch paste, talc, keratin, silica gel, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. Pharmaceutical compositions can be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. The medicament can be distributed throughout the body after administration or can be localized by topical administration, intramural administration, or by using an implant that maintains activity at the implantation site. The active agent can be formulated into a preparation that immediately realizes activity or can be formulated into a preparation that realizes sustained release. In some cases, particularly for the central nervous system, it is necessary to formulate a medicament to pass through the blood-brain barrier (BBB). A strategy for delivering drugs across the blood-brain barrier (BBB) ​​requires disruption of the BBB, which is achieved by osmotic means such as mannitol or leukotrienes, or by biochemical methods using vasoactive substances such as bradykinin. BBB disruptors can be co-administered with agents when the composition is administered by intravascular injection. Other strategies for experiencing the BBB may require the use of endogenous transport systems, including caveolin-1-mediated transcytosis, carrier-mediated transporters such as glucose and amino acid carriers, receptor-mediated transcytosis (for insulin or transferrin), and active efflux transporters such as p-glycoprotein. Active transport modules can also be conjugated to the therapeutic compounds used in the present invention to promote transport through the endothelial wall of the blood vessel.Alternatively, drug delivery behind the BBB can be by local delivery, e.g., by intrathecal delivery, e.g., via an Ommaya reservoir (see, e.g., U.S. Pat. Nos. 5,222,982 and 5385582, which are incorporated herein by reference); by, e.g., intravitreal or intracranial bolus, e.g., by syringe; by continuous infusion, e.g., by cannulation, e.g., with convection (see, e.g., U.S. Application No. 20070254842, which is incorporated herein by reference); or by implantation of a device that reversibly attaches a pharmaceutical agent (see, e.g., U.S. Application Nos. 20080081064 and 20090196903, which are incorporated herein by reference).

[0173] If medicament is provided to cell before transplantation, then medicament can be easily added to the cell culture medium of cultivation in solution or easily soluble form.Medicament can be added with discontinuous flow or continuous flow in flow-through system, or alternatively, compound single or increment is added to static solution.In flow-through system, two kinds of fluids are used, wherein one is physiological neutral solution, and another is the solution identical with the test compound added.The first fluid is passed through on the pond, followed by the second fluid.In the single solution method, test compound is added to the culture medium around the cell.The overall concentration of culture medium components should not be added with the compound or significantly changed between the two solutions of the flow-through method.

[0174] Multiple assays can be run in parallel with different drug concentrations to obtain differential responses to various concentrations. As known in the art, the effective concentration of a drug is determined to be generally derived from a concentration range of 1:10 or other logarithmic scale dilutions. Where necessary, the concentration can be further refined with subsequent serial dilutions. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the detection level of the drug or at or below a drug concentration that does not give a detectable change in phenotype.

[0175] Analysis of the response of humanized M-CSF mouse cells to a candidate agent can be performed at any time after treatment with the agent. For example, the cells are analyzed 1, 2, or 3 days, sometimes 4, 5, or 6 days, sometimes 8, 9, or 10 days, sometimes 14 days, sometimes 21 days, sometimes 28 days, sometimes 1 month or longer, such as 2 months, 4 months, 6 months or longer, after contact with the candidate agent. In some embodiments, the analysis includes analysis at multiple time points. The selection of the time point for analysis is based on the type of analysis to be performed, as will be readily understood by those of ordinary skill.

[0176] The analysis may include measuring any parameter described herein or known in the art for measuring cell viability, cell proliferation, cell identity, cell morphology, and cell function, particularly when they relate to immune cells. For example, flow cytometry can be used to determine the total number of hematopoietic cells or the number of cells of a specific hematopoietic cell type. Histochemistry or immunohistochemistry can be performed to determine the apoptotic state of the cells, such as measuring the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of DNA fragmentation, or immunohistochemistry to detect the binding of annexin V to cell surface phosphatidylserine. Flow cytometry can also be used to assess the ratio of differentiated cells and differentiated cell types, such as to determine the ability of hematopoietic cells to differentiate in the presence of an agent. ELISA, Western, and Northern blots can be performed to determine the levels of cytokines, chemokines, immunoglobulins, etc. expressed in grafted humanized M-CSF mice, such as to assess the function of grafted cells. In vivo assays for testing immune cell function and assays associated with specific diseases or conditions of interest such as diabetes, autoimmune diseases, graft-versus-host disease, AMD, etc. can also be performed. See, e.g., Current Protocols in Immunology (Richard Coico, ed. John Wiley & Sons, Inc. 2012) and Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), the disclosures of which are incorporated herein by reference.

[0177] Thus, for example, a method for determining the effect of an agent on a human pathogen is provided, comprising: introducing an engrafted humanized M-CSF mouse, such as an engrafted Rag2 - / - IL - / - The hM-CSF mice are exposed to an effective amount of a human pathogen, where the effective amount of the pathogen is the amount of the pathogen required to produce an infection in the mouse; the pathogen is allowed to infect the mouse; infection parameters are measured over time in the presence of the agent; and the measurements are compared to measurements from engrafted humanized M-CSF mice that were not exposed to the agent. If the agent reduces the amount of the pathogen in the blood or tissue of the mouse by at least half after a single administration or two or more administrations over a selected time period, the agent is determined to be an anti-pathogenic agent, such as an anti-Salmonella typhi agent.

[0178] As another example, methods are provided for determining whether an isolate or strain of a pathogen of interest is drug resistant (e.g., multi-drug resistant). In these methods, a humanized M-CSF-engrafted mouse, such as a Rag2-engrafted mouse, is injected with a humanized M-CSF-engrafted mouse. - / - IL - / -hM-CSF mice are exposed to an effective amount of an isolate or strain of a human pathogen of interest, where the effective amount of the pathogen is the amount of the pathogen required to produce an infection in the mouse; the pathogen is permitted to infect the mouse; an infection parameter is measured in the presence of the agent, such as the titer of the isolate or strain of interest in the blood or tissue of the mouse, the ability of the isolate or strain of interest to maintain infection in the mouse, or the ability of the isolate or strain of interest to propagate in the mouse at a certain time point after administration of the agent; and the measurements are compared to measurements from engrafted humanized M-CSF mice infected with the pathogen that have not been exposed to the agent. Examples of drugs of interest include amoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime, chloramphenicol, ciprofloxacin, cotrimoxazole, ertapenem, imipenem, fluoroquinolones (e.g., ciprofloxacin, gatifloxacin, ofloxacin, streptomycin, sulfadiazine, sulfamethoxazole, tetracycline, and combinations thereof. In a specific embodiment, administration of the drug or drug combination continues for at least 1 week, 10 days, 2 weeks, 3 weeks, or 4 weeks after exposure to the isolate or strain of interest that produces the infection.

[0179] Other examples of uses of the mice of the invention are also provided herein.Other applications of the genetically modified grafted mice described in this disclosure will be apparent to those skilled in the art upon reading this disclosure.

[0180] Reagents, Devices and Kits

[0181] The present invention also provides reagents, devices and kits thereof for carrying out one or more of the methods described above. The reagents, devices and kits thereof of the present invention can vary greatly.

[0182] In some embodiments, the reagent or kit comprises one or more agents used in the methods described herein. For example, the kit may comprise a humanized M-CSF mouse. The kit may comprise reagents for raising humanized M-CSF mice, such as primers and, in some cases, reagents for determining the genotype of humanized M-CSF mice. The kit may comprise an enriched population of human hematopoietic cells or human hematopoietic progenitor cells for transplantation into humanized M-CSF mice, or a reagent for preparing a hematopoietic cell population or an enriched population of hematopoietic cells from a human for transplantation into a humanized M-CSF mouse. Other reagents may include reagents for determining hematopoietic cell viability and / or function, for example, in the presence / absence of a candidate agent, such as one or more antibodies specific to markers expressed by different types of hematopoietic cells, or reagents for detecting specific cytokines, chemokines, and the like. Other reagents may include culture medium, culture supernatant, matrix composition, and the like.

[0183] In addition to the above components, the kit of the present invention further comprises instructions for implementing the method. These instructions can be present in one or more forms in one or more copies in this kit. One form in which these instructions can exist is as a suitable medium or substrate, such as printed information on one or more sheets of paper, in the packaging of the kit, in the printed information in the package insert, etc. Another means is a computer-readable medium that has recorded information, such as a disk, CD, etc. Another means that can exist is a website address, which can be used via the Internet to obtain information at the take-out position. Any convenient means can be present in the kit. Example

[0184] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to implement and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments in implementing the present invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is or is close to atmospheric pressure.

[0185] Colony stimulating factor-1 (CSF-1) or macrophage colony stimulating factor (M-CSF) is one of the early cytokines that promote hematopoiesis. In the hematopoietic system, it is believed that M-CSF acts specifically on myeloid progenitors, starting from the common myeloid progenitor (CMP) stage, and promotes CMP differentiation into monocyte / macrophage lineages (Sherr, CJ et al. (1988) Macrophage colony-stimulating factor, CSF-1, and its proto-oncogene-encoded receptor, Cold Spring Harb. Symp. Quant. Biol. 53 Pt1: 521-530). In addition, M-CSF is essential for the survival, adhesion, and motility of macrophages (Pixley, FJ, and Stanley, ER (2004) CSF-1 regulation of the wandering macrophage: complexity in action, Trends Cell Biol. 14: 628-638; Socolovsky, M. et al. (1998) Cytokines in hematopoiesis: specificity and redundancy in receptor function, Adv. Protein Chem. 52: 141-198; Stanley, ER et al. (1997) Biology and action of colony-stimulating factor-1, Mol. Reprod. Dev. 1997; 46: 4-10). In contrast to its key role in myeloid differentiation, M-CSF is essential for the differentiation of osteoclasts, for the differentiation, survival and proliferation of female reproductive tract cells, and for the formation of the placenta (Pixley et al. (2004); Socolovsky et al. (1998); Stanley et al. (1997)). M-CSF is produced by a variety of cells, including fibroblasts, bone marrow (BM) stromal cells, activated T cells and macrophages, and secretory epithelial cells.M-CSF signals through the M-CSF receptor (Fms; CD115), and ligation of its receptor by M-CSF leads to tyrosine phosphorylation of Fms and subsequent phosphorylation of several host cell proteins such as Grb2, Shc, Sos1, and p85 (Pixley et al. (2004); Stanley et al. (1997); Rohrschneider, LR et al. (1997) Growth and differentiation signals regulated by the M-CSF receptor, Mol. Reprod. Dev. 46:96-103; Yeung, YG and Stanley, ER (2003) Proteomic approaches to the analysis of early events in colony-stimulating factor-1 signal transduction, Mol. Cell. Proteomics 2:1143-1155).

[0186] The inventors hypothesized that defective human myeloid differentiation in humanized mice may be due to the lack of specific signals that promote myeloid differentiation. To confirm this, the inventors engineered a new generation of humanized mice to secrete human M-CSF at physiological levels from appropriate tissues. Qualitative and quantitative analysis of these humanized M-CSF mice revealed normal expression of human M-CSF. + Analysis of cell-engrafted humanized M-CSF mice indicated elevated human monocyte / macrophage frequencies in various tissues. In addition, human monocyte / macrophages obtained from these mice exhibited enhanced functional properties.

[0187] The humanized M-CSF mice described herein show increased frequency and function of human myeloid cells. Human M-CSF was inserted into Balb / c mice deficient in recombinant activated gene 2 (Rag2; Genbank Accession No. 1.NM_009020.3) and gamma chain (γc, also known as "interleukin 2 receptor, gamma chain" or IL2RG; Genbank Accession No. 1.NM_013563.3) - / - γc - / - Qualitative and quantitative manipulation of the mouse M-CSF locus in humanized mice resulted in faithful expression of human M-CSF in these mice. h / h Human fetal liver-derived hematopoietic stem and progenitor cells (CD34 +) resulted in more efficient differentiation and enhanced frequency of human monocytes / macrophages in the bone marrow, spleen, and peripheral blood. In addition, M-CSF h / h The mice demonstrated a sustained ability to support monocyte / macrophage differentiation even 20 weeks after transplantation. Furthermore, unlike unmodified control mice, M-CSF h / h The mice contained residual human monocytes / macrophages in multiple tissues, including the liver and lungs. Human monocytes / macrophages obtained from humanized M-CSF mice also displayed enhanced functional properties, such as migration, phagocytosis, activation, and response to LPS.

[0188] Example 1: Cell preparation, analytical methods, and assays

[0189] CD34 + Cell Isolation and Transplantation. Human fetal liver samples were obtained from the Human Fetal Liver Tissue Repository at Albert Einstein College of Medicine, Bronx, NY, and from Advance Biosciences Resources, Inc., Alameda, CA. All experiments involving human tissues were performed with approval from the Yale Human Investigations Committee.

[0190] To isolate human CD34 + Cells were prepared by rinsing fetal liver tissue once with PBS, cutting into small pieces, and treating with collagenase D (100 ng / mL) at 37°C for 45 min. Single cell suspension was prepared, and mononuclear cells were isolated using density gradient centrifugation (lymphocyte separation medium, MP biomedicals). After treating cells with anti-human CD34 microbeads, MACS TM Isolation of CD34 by Miltenyi Biotech + cell.

[0191] For transplantation, newborn pups (day 1 of life) were sublethally irradiated with two separate doses (2 x 150 cGy) 4 hours apart and 1 x 10 5 Up to 2x 10 5 Purified human CD34 + Cells were injected into the liver.

[0192] Mesenchymal stromal cell (MSC) isolation and culture. Long bones of mice were isolated and BM cells were flushed out. Bones were cut into blocks and digested with a mixture of collagenase D and P (25 ng / mL) at 37°C for 45 minutes. Suspended cells were isolated and plated in the presence of MSC culture medium (Stem Cell Technologies). After 2 weeks of culture, CD45 - Sca1 + CD90 + Cells were isolated and cultured.

[0193] Antibodies and flow cytometry. Flow cytometry was performed using FACS Calibur or LSRII and CELLQUEST, respectively. TM Software, FACS DIVA TM Software (BD Biosciences, San Jose, CA) or FLOWJO TM Single cell suspensions were analyzed using FACS ARIA software (TreeStar, Inc., Ashland, OR). TM Sorting of cells into defined subpopulations was performed using a cell sorter (BD Biosciences, San Jose, CA).

[0194] The following human antibodies were used in the study: CD11b, CD14, CD33, CD34, CD38, CD40, CD45, CD80, CD86, CD90 and HLA-DR.

[0195] The following mouse antibodies were used in this study: CD11b, CD40, CD45, CD80, CD86, F4 / 80, Gr1, H2K d and IA d .

[0196] Cell culture. For murine macrophage differentiation, BM cells were plated in 6-well plates in the presence of DMEM with 10% FCS and essential supplements (2 mM L-glutamine, 1% penicillin-streptomycin and 1 mM non-essential amino acids). Cells were treated with recombinant murine M-CSF (10 ng / mL) or recombinant human M-CSF (10 ng / mL) for 7 days. Cell culture supernatants were removed every three days and the cultures were replaced with fresh medium and cytokines.

[0197] For human macrophage studies, such as activation, phagocytosis, and migration, 2 x 10 5 CD45 + CD14 + CD33 +Cells were sorted and cultured in vitro in DMEM with 15% human AB serum and essential supplements (2 mM L-glutamine, 1% penicillin-streptomycin, and 1 mM non-essential amino acids).

[0198] Activation, phagocytosis and migration assays. For LPS stimulation in vivo, mice were injected with LPS (100 ng / g body weight) ip. For LPS stimulation in vitro, LPS (10 ng / mL) was added to cells and cultured for 1 or 2 days. For poly I:C stimulation in vitro, cells were cultured for 6 or 12 hours in the presence of poly I:C (10 ug / mL).

[0199] Use of commercial VYBRANT TM Phagocytosis assay was performed using the Phagocytosis Assay Kit (Invitrogen) according to the manufacturer's instructions.

[0200] Use of commercial QCM TM Migration assays were performed using the Chemotaxis Cell Migration Assay Kit (Millipore) according to the manufacturer's instructions.

[0201] RNA extraction and real-time PCR were performed using a commercial kit system (RNEASY TM Total RNA was isolated using the PCR amplification kit (Qiagen). cDNA was synthesized using oligo dT primers and Amplify reverse transcriptase (Roche). PCR was performed using the 7500 Real-Time PCR System and PowerSYBR TMPCR reactions were performed in duplicate using the following gene-specific primer pairs using Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions: human CSF1 (sense: 5′-TACTGTAGCCACATGATTGGGA-3′ (SEQ ID NO: 1) and antisense: 5′-CCTGTGTCAGTCAAAGGAAC-3′ (SEQ ID NO: 2)), mouse csf1 (sense: 5′-CGACATGGCTGGGCTCCC-3′ (SEQ ID NO: 3) and antisense: 5′-CGCATGGTCTCATCTATTAT-3′ (SEQ ID NO: 4), human IFNα (sense: 5′-GTACTGCAGAATCTCTCCTTTCTCCTG-3′ (SEQ ID NO: 5) and antisense: 5′-GTGTCTAGATCTGACAACCTCCCAGGCACA-3′ (SEQ ID NO: 6)), human IFNb (sense: 5′-TTGTGCTTCTCCACTACAGC-3′ (SEQ ID NO: 7)). NO:7) and antisense: 5′-CTGTAAGTCTGTTAATGAAG-3′ (SEQ ID NO:8)), mouse hprt primer (sense: 5′-AAGGACCTCTCGAAGTGTTGGATA (SEQ ID NO:9) and antisense: 5′-CATTTAAAAGGAACTGTTGACAACG-3′ (SEQ ID NO:10)) and human HPRT primer (sense: 5′-CTTCCTCCTCCTGAGGAGTC-3′ (SEQ ID NO:11) and antisense: 5′-CCTGACCAAGGAAAGCAAAG-3′ (SEQ ID NO:12)). For normal PCR, a commercial kit (DNEASY TM DNA from target cells was extracted using the Qiagen Blood and Tissue Kit (Qiagen) and PCR analysis was performed using gene-specific primer pairs.

[0202] ELISA. For cytokine quantification studies, blood serum or cell culture supernatant was collected and ELISA was performed using commercial human IL6 and human TNF ELISA kits (Ray Biotech, Inc., GA) according to the manufacturer's instructions.

[0203] Histology. Solid tissues were fixed in 4% PFA. Fixed tissues were embedded in paraffin (Blue RiBbon; Surgipath Medical Industries). Blocks were cut into sections, and 5-μm sections were stained with H&E dye, followed by placement of coverslips by conventional methods. Sections were maintained without any medium. Digital light microscopy images were recorded at room temperature using a Zeiss Axio Imager.A1 microscope (with 2× and 10× objectives), an AxioCam MRc5 camera, and AxioVision 4.7.1 imaging software (Carl Zeiss Microimaging LLC).

[0204] Statistical analysis. Data are presented as mean ± SEM. Statistical significance was assessed using a 2-sided Student t test. P values ​​> 0.05 were considered non-significant, while P values ​​< 0.05 are indicated by *.

[0205] Example 2: Genetically modified mice for grafting

[0206] Human M-CSF knock-in strategy. The technology was constructed to use the human M-CSF nucleic acid sequence ( A targeting construct was constructed by replacing the mouse M-CSF nucleic acid sequence with the 5093 allele (allele identifier 5093) as previously described (Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nat. Biotechnol. 21: 652-659). Mouse and human M-CSF DNA were obtained from bacterial artificial chromosomes (BACs) RPCI-23, clone 373B18 and from BACRPCI-11, clone 101M23, respectively. Briefly, the linearized targeting construct generated by gap repair cloning was electroporated into the RAG2 + / - γc - / -Mouse embryonic stem (ES) cells, generated from the commercial V17 ES cell line (BALB / cx129F1), the linearized targeting construct contains mouse M-CSF upstream and downstream homology arms flanking a 17.5 kb human M-CSF sequence extending from exon 2 to 633 nt downstream of noncoding exon 9 and a floxed drug selection cassette. Mouse ES cells carrying a heterozygous deletion of the M-CSF gene were identified using an allelic deletion screen using two TaqMan qPCR assays recognizing intron 2 of the mouse Csf1 gene (TUF primer, 5′-CCAGGAATGTCCACTATGGATTC-3′ (SEQ ID NO: 13); TUP probe, 5′ACTGCTCCTTGACCCTGCTCTGACTCA-3′ (SEQ ID NO: 14); TUR primer, 5′-TGGGCTGACTTCCCAAAGG-3′ (SEQ ID NO: 15)) and in the 3′ flanking sequence (TDF primer, 5′-TTAGGTGCTAGTAGGCTGGAAAGTG-3′ (SEQ ID NO: 16); TDP probe, 5′-TGCAATCGCAGCTTCTCTCCTTACTAGGCT-3′ (SEQ ID NO: 17). NO:17)′; TDR primer, 5′-AATAGGAAGAACGAACAGGTCTAATACC-3′ (SEQ ID NO:18)). Simultaneous replacement of the mouse gene with the human CSF1 gene was confirmed by an allele gain TaqMan assay that detects one copy of a sequence in intron 2 of CSF1 (forward primer, 5′-GCTGCTTGCCTGGGTTAGTG-3′ (SEQ ID NO: 19); probe, 5′-TGCCCAGGAACATCAACCACTGATTCTG-3′ (SEQ ID NO: 20); reverse primer, 5′-GAGGGACAGCAGACCTCAGAAG-3′ (SEQ ID NO: 21)) and one copy of a neomycin resistance (neor) cassette (forward primer, 5′-GGTGGAGAGGCTATTCGGC-3′ (SEQ ID NO: 22); probe, 5′-TGGGCACAACAGACAATCGGCTG-3′ (SEQ ID NO: 23); reverse primer, 5′-GAACACGGCGGCATCAG-3′ (SEQ ID NO: 24); see Figure 8 The qPCR assay that recognizes the CSF1 sequence did not amplify DNA from the mouse genome. The same assay was used to confirm the genotype of mice derived from targeted ES cells.r TaqMan assay confirmed Cre-mediated excision of the drug selection cassette. All primer-probe sets were provided by Biosearch Technologies. The probes were labeled with 6-carboxy-fluorescein (FAM) at their 5' end and BHQ-1 at their 3' end.

[0207] Correctly targeted ES cells were further electroporated with a transient Cre expression vector to remove the drug selection cassette. Targeted ES cell clones without the drug kit were introduced into 8-cell stage mouse embryos using the ES cell clone method (Poueymirou et al. (2007)). Genotyping by loss of mouse alleles and gain of human alleles was performed using a modified allele determination method (Valenzuela et al. (2003)) to identify the humanized M-CSF gene (VG 5093). (F0 mice derived entirely from donor ES cells).

[0208] Mouse maintenance. Balb / c-Rag2 - / - γc - / - M-CSF m / m , Balb / c-Rag2 - / - γc - / - M-CSF h / m and Balb / c-Rag2 - / - γc - / - M-CSF h / h Mice were maintained under specific pathogen-free conditions in the Yale University Animal Care Facility. All mouse experiments were approved by the Yale University Institutional Animal Care and Use Committee.

[0209] Generation of humanized M-CSF mice. To confirm whether physiological expression of human M-CSF in mice results in improved human macrophage differentiation in humanized mice, Balb / c Rag2 - / - γc - / - Mice engineered to express human M-CSF. - / - γc - / - The deficient Balb / c strain serves as a successful model system for studying the human immune system in mice (Traggiai E et al. (2004) Development of a human adaptive immune system in cord blood cell-transplanted mice, Science 304: 104-107). To circumvent the supraphysiological expression of human M-CSF in these mice, a strategy was adopted to replace the mouse M-CSF coding sequence with the human counterpart. The technology was constructed to use the human M-CSF coding sequence ( Allele ID 5093) replaces most of the M-CSF open reading frame ( Figure 8 ), as previously described (Valenzuela et al. (2003)). Of note, the mouse promoter and other regulatory elements (such as the 5'UTR) were retained in this vector. The linearized targeting vector was electroporated into Balb / cx 129Rag 2 + / - γc - / - Correctly targeted ES cells were further electroporated with a transient Cre expression vector to remove the drug selection cassette. Targeted ES cell clones without the drug cassette were introduced into 8-cell stage mouse embryos using the genotyping method (Poueymirou et al. (2007)). Genotyping for loss of mouse alleles and gain of human alleles was performed using a modified allele determination method (Valenzuela et al. (2003)) to identify the humanized M-CSF gene (VG 5093). (F0 mice derived entirely from donor ES cells). Through sequential intercrossing of the progeny, mice with both mouse and human M-CSF (M-CSF m / h ; heterozygous knock-in) and human M-CSF only (M-CSF h / h ; homozygous knock-in) Balb / c Rag2 - / - γc - / - Mouse chimeric mice and germline transmitted mice.

[0210] Characterization of humanized M-CSF mice. Expression of human M-CSF in humanized M-CSF mice was evaluated. m / m or M-CSF h / h Organs from mice were harvested and analyzed for murine and human M-CSF mRNA expression using species-specific primers. Figure 1A and 1B As shown in Figure 2, M-CSF was expressed in most organs analyzed, including BM, spleen, blood, liver, brain, lung, testis, and kidney. However, thymus and skin showed no detectable M-CSF expression. Of note, the expression pattern of mouse and human M-CSF is similar to that of M-CSF. m / m and M-CSF h / h Next, we quantified the expression of mouse and human M-CSF in M-CSF. m / m 、M-CSF m / h , and M-CSF h / hExpression levels in mice. Bone marrow mesenchymal stromal cells (MSCs) were isolated and the expression level of M-CSF mRNA was quantified using real-time PCR ( Figure 1C ), and ELISA was used to quantify M-CSF protein (secreted) ( Figure 1D ). M-CSF m / m Mice express only mouse M-CSF, M-CSF m / h Mice express both mouse and human M-CSF, and M-CSF h / h Mice express only human M-CSF. The expression level of human M-CSF is comparable to that of mouse M-CSF. Consistent with these data, analysis of CSF-1 in serum revealed comparable CSF-1 protein expression levels in m / m, h / m, and h / h mice ( Figure 1E ). Hemizygosity did not result in reduced gene and protein expression levels, indicating that gene dosage levels do not appear to be limiting for this cytokine.

[0211] To investigate whether replacement of mouse M-CSF with human M-CSF leads to adverse effects, particularly on bone and hematopoiesis, we analyzed M-CSF at multiple ages. h / h Mice. Earlier studies have shown that mice with defective M-CSF signaling (Csf1 op / op and Csf1r - / -) shows dental failure and bone defects (Dai, theop / op osteopetrotic mouse, Endocrinology 127:2592-2594; Wiktor-Jedrzejczak, W. et al. (1990) Total absence of colony-stimulating factor 1 in the macrophage-deficientosteopetrotic (op / op) mouse, Proc. Natl Acad.Sci.USA87:4828-4832; Yoshida, H. et al. (1990) The murine mutation Osteopetrosis is in the coding region of themacrophage colony stimulating factor gene,Nature345:442-444). In comparison, M-CSF h / h The mice revealed normal tooth and bone properties. In addition, with Csf1 op / op and Csf1r - / - Different mice have different total cell content of BM ( Figure 2A ), myeloid cell frequencies in BM, spleen (SP) and peripheral blood (PB) Figure 2B ) and the macrophage frequencies in BM and SP ( Figure 2C ) in M-CSF m / m 、M-CSF h / m and M-CSF h / hConsistent with this observation, the frequencies of the HSC compartment (including long-term HSCs, short-term HSCs, and multipotent progenitors) and the myeloid progenitor compartment (including a common myeloid progenitor, a granulocytic monocytic progenitor, and a megakaryocyte erythroid progenitor) increased significantly in the presence of M-CSF. m / m ,M-CSF h / m and M-CSF h / h The mice were comparable ( Fig. 9 ).

[0212] About M-CSF h / h A possible explanation for the normal hematopoiesis and bone development in mice could be that human M-CSF is cross-reactive with mouse cells. To confirm this, m / m Total BM cells were isolated from 100% BM and cultured in the presence of recombinant murine M-CSF or recombinant human M-CSF. BM cells cultured in the absence of cytokines did not survive, whereas cells cultured in the presence of human or mouse M-CSF showed comparable levels of in vitro differentiation ( Figure 2D Analysis of these in vitro differentiated macrophages for the expression of co-stimulatory molecules and MHC indicated comparable levels of these molecules in the presence of human or mouse M-CSF ( Figure 2E Consistent with our findings, previous studies have demonstrated that human M-CSF is active in mouse target cells, whereas mouse M-CSF is not cross-reactive with human cells (Sieff, CA (1987) Hematopoietic growth factors, J. Clin. Invest. 79: 1549-1557).

[0213] Example 3: Differentiation of human monocytes / macrophages in humanized M-CSF mice

[0214] To evaluate the effects of humanization with M-CSF, sublethally irradiated neonatal Rag2 - / - γc - / - M-CSF m / m ,Rag2 - / - γc - / - M-CSF h / m and Rag2 - / - γc - / - M-CSF h / h Puppies received about 2x 10 intrahepatic (IH) transplants 5 Purified human fetal liver CD34 + Cells. Recipients were then bled at 8 weeks post-transplantation to confirm donor-derived cells (based on human CD45 expression). Twelve weeks post-transplantation, recipients were sacrificed and their BM, SP, and PB were harvested. Analysis revealed that M-CSF m / m Compared with mice, M-CSFh / m and M-CSF h / h CD14 in BM, SP and PB of both mice + CD33 + Increased relative and absolute frequencies of monocyte / macrophage lineage cells ( Figures 3A-3C ). Although M-CSF h / m Mice exhibited elevated CD14 + CD33 + Cell frequency, but CD14 + CD33 + The greatest frequency of cells was present in M-CSF h / h Surprisingly, in addition to this increase, CD14 - CD33 + Cell frequency in M-CSF h / m and M-CSF h / h The BM, SP, and PB of mice were also increased ( Figure 3A ).

[0215] To analyze whether human M-CSF knock-in mice support sustained human myelopoiesis, recipients were analyzed at 12, 16, and 20 weeks after transplantation. + CD33 + Monocyte / macrophage lineage cells in M-CSF m / m In mice, the expression of cytokines was slightly decreased at 16 weeks and severely decreased at 20 weeks after transplantation, but even at 16 and 20 weeks in the presence of M-CSF h / m and M-CSF h / h A significant proportion of human CD14 was observed in both mice + CD33 + However, in M-CSF h / h Human CD14 seen in mice + CD33 + The maximum frequency of the cell ( Figure 4A and 4B ).

[0216] Next, we evaluated whether humanized M-CSF mice support efficient differentiation of human tissue macrophages. To this end, we perfused M-CSF with PBS. m / m 、M-CSF m / h and M-CSF h / h Mice were sterilized and their organs (including liver, lungs and skin) were harvested. Peritoneal cells were obtained by flushing the peritoneal cavity with PBS. Single cell suspensions were prepared and human CD14 + CD33 + As expected, human CD14 + CD33 +The frequency of cells in M-CSF m / h and M-CSF h / h However, analysis of skin explants revealed that M-CSF m / m and M-CSF m / h Mouse to human CD14 + CD33 + The frequency of cells, despite the presence of M-CSF h / h A significant increase in these cells was observed in mouse skin explants ( Figure 5 Together, these data suggest that expression of human M-CSF in mice improves myeloid / macrophage lineage differentiation of human HSCs.

[0217] Example 4: Human monocyte / macrophage function in humanized M-CSF mice

[0218] To study human CD14 in humanized M-CSF mice + CD33 + Whether monocytes / macrophages function normally, both in vivo and in vitro functional studies are performed. m / m and M-CSF m / h Pups were injected with fetal liver CD34+ cells and 12 weeks after transplantation, donor-derived hematopoiesis was assessed and recipient mice were injected with LPS or PBS. 2 days after LPS injection, human CD14 in the spleen was analyzed by docking receptor + CD33 + When compared with the PBS-injected group, although LPS injection was only in the M-CSF m / m In mice, M-CSF induced a modest increase in monocyte / macrophage lineage cells, but LPS injection of M-CSF m / h Mice display human CD14 in the spleen + CD33 + The number of cell-fold increases ( Fig. 6A ). Next, the ability of these cells to produce pro-inflammatory cytokines in response to LPS stimulation in vivo was examined.

[0219] M-CSF was administered to human CD34+ cells m / m and M-CSF m / h Mice were injected with LPS. Six hours after injection, mice were bled, and serum levels of human and mouse IL6 and TNFα were determined by ELISA. Consistent with the elevated monocyte / macrophage frequencies in humanized M-CSF mice, m / h Elevated levels of human IL-6 and TNFα were detected in mice. Although the basal levels of these cytokines were m / hmice, but LPS stimulation resulted in elevated levels of human IL6 and TNFα in serum ( Figure 6B and 6C ). Next, the ability of monocytes / macrophages (obtained from humanized M-CSF mice) to secrete pro-inflammatory cytokines in vitro was analyzed. + After 12 weeks of cell reconstitution, cells were reconstituted with M-CSF. m / m or M-CSF h / h Isolation of human CD14 from mouse spleen + CD33 + Cells were stimulated with LPS in vitro for 24 or 48 h. IL-6 and TNFα cytokine levels were assessed in cell culture supernatants by ELISA. h / h Mouse purified CD14 + CD33 + Cells respond to LPS by increasing the secretion of these cytokines ( Fig. 7A and 7B ). Similarly, human CD14 isolated from humanized M-CSF mice + CD33 + Cells respond to poly I:C stimulation by increasing the expression of interferon-α and interferon-β mRNA ( Figure 7C Finally, the phagocytic, migration and activation properties of human monocytes / macrophages obtained from humanized M-CSF mice were analyzed. + Reconstituted M-CSF h / h Mouse purified human CD14 + CD33 + Cells exhibited increased phagocytic properties ( Fig.7D ), and exhibited enhanced chemotaxis in response to the cytokine Mip3β ( Fig. 7E As expected, from M-CSF h / h Mouse-derived human monocytes / macrophages exhibited enhanced activation properties as assessed based on upregulation of co-stimulatory molecules, including CD40, CD80 and CD86, and HLA-DR in response to LPS stimulation in vitro ( Figure 7F ). Overall, human monocytes / macrophages differentiated in humanized mice in the presence of human M-CSF exhibited improved functional properties.

[0220] The generation of mice with a fully reconstituted and functional human-derived hematopoietic / immune system has been a major challenge in the field. To date, three mouse strains have been developed (NOD-scidγc - / - ,[NSG],NOD / Shi-scidγc - / - [NOG], and Balb / c-Rag2- / - γc - / - ). Although each of these strains has its own advantages, artificial hematopoietic function is incomplete in these mice.

[0221] To overcome this major technical challenge, the mouse CSF-1 gene was replaced with its human counterpart. This resulted in efficient human macrophage differentiation in mice reconstituted with human hematopoietic stem and progenitor cells. Analysis of humanized CSF-1 mice showed efficient differentiation of human monocytes / macrophages in BM, spleen, and peripheral blood. In addition, human macrophages were detected in several different tissues in these mice, including lungs and liver, indicating that the presence of CSF-1 in humanized mice is sufficient to promote human tissue macrophage differentiation. In addition, studies involving self-CSF1 m / m and CSF1 h / h The functional studies described in this article of human monocytes / macrophages isolated from mice indicate that CSF1 h / h The cells of the mouse are better at performing functions such as phagocytosis, migration, activation and cytokine secretion. Based on these findings, it can be inferred that differentiated monocytes / macrophages function better in the presence of human CSF-1.

[0222] use Genetic engineering technology to generate a novel Balb / c-Rag2 expressing human CSF-1 - / - γc - / - mouse line. Thus, the mouse CSF-1 coding region was replaced with the human counterpart without disrupting the regulatory elements of the mouse csf1 gene, such as the promoter. This generated a chimeric gene containing the mouse regulatory elements and the human CSF-1 coding region. Expression studies of these mice indicated that this chimeric gene was faithfully expressed in both a qualitative and quantitative manner.

[0223] The role of CSF-1 in mouse macrophage differentiation is well established. op / op ) or its receptor (Csf1r - / - ) deficient mice exhibit severe reductions in macrophage and osteoclast frequencies, osteopetrosis, failed teething, developmental defects in multiple tissues including the nervous system, male and female reproduction, dermis and synovium. Although these studies have provided a very important understanding of the effects of CSF-1 in mice, the significance of CSF-1 in artificial hematopoiesis remains largely unknown. In this regard, the mice described herein will serve as a valuable tool because it will enable understanding of the physiological and functional improvements of cytokines in artificial hematopoietic and hematopoietic cell function. In addition, this mouse can be used to model diseases and test the effects of agents on the human immune system. This mouse model is a valuable tool in understanding the pathophysiology of multiple human disorders and diseases and in treatment.

[0224] The above only illustrates the principle of the present invention. It should be appreciated that those skilled in the art will be able to come up with various arrangements, although not explicitly described or shown herein, which embody the principle of the present invention and are included in its spirit and scope. In addition, all examples and conditional language described herein are intended in principle to help readers understand the principles of the present invention and the concept of the inventor's promotion of technological progress, and should be interpreted as not being limited to such specific examples and conditions. In addition, all statements describing the principles of the present invention, aspects and embodiments and their specific examples herein are intended to cover both their structural and functional equivalents. Thus, such equivalents include both currently known equivalents and future development equivalents, that is, including any elements developed for the implementation of the same function and unrelated to the structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. And the attached claims embody the scope and spirit of the present invention. Sequence Listing <110> A.J. Murphy S. Stevens C. Rescinan E. Einon M. Manz R. Flavel G.D. Yancopoulos <120> Humanized M-CSF mice <130> REGN-002WO <150> 61 / 442,946 <151> 2011-02-15 <160> twenty four <170> FastSEQ for Windows Version 4.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 1 tactgtagcc acatgattgg ga 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 2 cctgtgtcag tcaaaggaac 20 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 3 cgacatggct gggctccc 18 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 4 cgcatggtct catctattat 20 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 5 gtactgcaga atctctcctt tctcctg 27 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 6 gtgtctagat ctgacaacct cccaggcaca 30 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 7 ttgtgcttct ccactacagc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 8 ctgtaagtct gttaatgaag 20 <210> 9 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 9 aaggacctct cgaagtgttg gata 24 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 10 catttaaaag gaactgttga caacg 25 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 11 cttcctcctc ctgaggagtc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 12 cctgaccaag gaaagcaaag 20 <210> 13 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 13 ccaggaatgt ccactatgga ttc 23 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 14 actgctcctt gaccctgctc tgactca 27 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 15 tgggctgact tcccaaagg 19 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 16 ttaggtgcta gtaggctgga aagtg 25 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 17 tgcaatcgca gcttctctcc tactaggct 30 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 18 aataggaaga acgaacaggt ctaatacc 28 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 19 gctgcttgcc tgggttagtg 20 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 20 tgcccaggaa catcaaccac tgattctg 28 <210> twenty one <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> twenty one gagggacagc agacctcaga ag 22 <210> twenty two <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> twenty two ggtggagagg ctattcggc 19 <210> twenty three <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> twenty three tgggcacaac agacaatcgg ctg 23 <210> twenty four <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> twenty four gaacacggcg gcatcag 17

Claims

1. A method for screening candidate agents having activity in regulating human hematopoietic cell function in humanized macrophage colony stimulating factor (M-CSF) mice, the method comprising: (a) contacting a first humanized M-CSF mouse comprising engrafted human hematopoietic cells with a candidate agent; and (b) comparing the function of human hematopoietic cells of the first humanized M-CSF mouse with the function of engrafted human hematopoietic cells of a second humanized M-CSF mouse in the absence of the candidate agent, wherein the first and second humanized M-CSF mice are both immunocompromised with respect to the mouse immune system and both comprise an allele comprising a nucleic acid sequence incorporated into the genome of the humanized M-CSF mouse, the sequence encoding a human M-CSF protein and operably linked to an endogenous promoter of a mouse M-CSF gene at the mouse M-CSF locus, and replacing part or all of the mouse M-CSF coding sequence at the mouse M-CSF locus, wherein the only M-CSF expressed by the allele is human M-CSF, The first and second humanized M-CSF mice both contain Rag2 gene knockout and IL2rg gene knockout, or are selected from NOD- scid γ c − / − Mouse, NOD / Shi- scid γ c − / − Mouse and Balb / c- Rag2 − / − γ c − / − The mice group, wherein the first and second humanized M-CSF mice both express the M-CSF RNA encoded by the nucleic acid sequence in the bone marrow, spleen, blood, liver, brain, lung, testis and kidney, and Wherein modulation of human hematopoietic cell function in the first humanized M-CSF mouse indicates that the candidate agent modulates hematopoietic cell function.

2. The method of claim 1, wherein the first humanized M-CSF mouse and the second humanized M-CSF mouse are the same mouse, wherein the absence of the candidate agent occurs prior to the contacting, and wherein modulation of human hematopoietic cell function after the contacting relative to function of the human hematopoietic cells prior to the contacting indicates that the candidate agent modulates human hematopoietic cell function.

3. The method according to claim 1, wherein the first humanized M-CSF mouse and the second humanized M-CSF mouse are different mice.

4. The method of any one of claims 1 to 3, comprising exposing the first and second humanized M-CSF mice to an amount of a human pathogen sufficient to produce an infection in the first and second humanized M-CSF mice, and measuring infection parameters over time in the first humanized M-CSF mouse and infection parameters over time in the second humanized M-CSF mouse, wherein the comparing comprises comparing the measured parameters to determine the in vivo effect of the candidate agent on the human pathogen.

5. The method according to claim 4, wherein said first humanized M-CSF mouse is contacted with said candidate agent prior to exposing said first and second humanized M-CSF mice to said human pathogen.

6. The method according to claim 4, wherein said first humanized M-CSF mouse is contacted with said candidate agent concurrently with exposure to said human pathogen.

7. The method according to claim 4, wherein said first humanized M-CSF mouse is contacted with said candidate agent after said first and second humanized M-CSF mice have been exposed to said human pathogen.

8. The method according to any one of claims 1 to 7, wherein the first and second humanized M-CSF mice each comprise two copies of the allele.

9. The method according to any one of claims 1-8, wherein the first and second humanized M-CSF mice both comprise the same null mutation in at least one mouse M-CSF allele.

10. The method according to claim 9, wherein the null mutation is a deletion of exons 2-9 of mouse M-CSF.

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