RU2819731C2 - Humanized m-csf mice - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method for screening a candidate substance for human haemopoietic cell toxicity. Method comprises contacting a first humanised M-CSF mouse with a candidate substance, wherein the first humanised M-CSF mouse is grafted with human hematopoietic cells, and comparing the viability and/or function of the human hematopoietic cells in the first humanized M-CSF mouse in contact with the candidate substance, with viability and/or function of human hematopoietic cells in a second humanized M-CSF mouse, which is grafted with human hematopoietic cells, but which is not in contact with a candidate substance.

EFFECT: invention is effective for screening a candidate substance for toxicity with respect to human hematopoietic cells.

6 cl, 9 dwg, 4 ex

Description

Technical field

The invention relates to genetically modified mice containing the gene encoding the human M-CSF protein, and mice that contain additional modifications that promote the engraftment of human hematopoietic cells.

State of the art

The development of animal models for studying human diseases has greatly contributed to the understanding of the mechanisms underlying several diseases, including cancer. It is now recognized that animal models, particularly mice, are excellent candidates for assessing the quality and efficacy of drugs and therapeutic options. While the use of these surrogate models to study human biology and disease is largely justified (due to the ethical and technical limitations of conducting experimental treatments in humans), research has raised the issue of possible limitations in extrapolating data from mice to humans ( Mestas J, Hughes CC (2004) Of mice and not men: differences between mouse and human immunology. J Immunol 172:2731-2738).

There has been a need for many years to develop humanized mouse models to overcome these limitations. Hard work by several groups of researchers has successfully demonstrated that it is possible to study human biology and disease in mice. Since the presence of a functional and effective immune system in recipients results in the destruction of transplanted tissues/cells of human origin, the use of genetic mutants lacking adaptive immune system cells such as T, B and NK cells greatly contributes to the success of the humanized mouse model. Based on this, the most effective options for humanized mouse models include NOD-SCID and Balb/c strains that lack the recombination activation genes (RAG), common gamma chain (γC, also known as “interleukin 2 receptor gamma”, or IL2rg ), beta2 microglobin (B2M) and perforin 1 (Prf1) (Shultz LD, et al. (2007) Humanized mice in translational biomedical research, Nat. Rev. Immunol. 7:118-130). Research over the past several decades has shown the possibility of transplanting several types of human tissue, including peripheral blood leukocytes, fetal liver cells, fetal bone, fetal thymus, fetal lymph nodes, vascularized skin, arterial segments, and mobilized hematopoietic stem cells or hematopoietic stem cells. umbilical cord blood (HSC) to some 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 will provide improved model systems, since the data obtained from the human cells present in these mice can reflect the physiology of the human system. The main focus of research in this area is the creation of 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 cells, B cells, NK cells, and dendritic cells (DCs), several challenges remain in the field, one of which is insufficient myeloid differentiation in humanized mice.

Interestingly, great strides have been made in generating human T cells, B cells, NK cells, and dendritic cells (DCs) from hematopoietic stem cells (HSCs) in humanized mice. Administration of human HSCs to these mice, in addition to the individual hematopoietic compartment, resulted in the restoration of lymphoid organs such as the thymus and spleen. However, the numbers of myeloid cells, particularly granulocytes, macrophages, erythrocytes and megakaryocytes, are very low, a result likely due to ineffective myelopoiesis from human HSCs in these mice (Shultz et al. (2007); Macchiarini et al. ( 2005)). Given that myeloid-derived cells (such as red blood cells and megakaryocytes) are vital for the normal functioning of the circulatory system, and granulocytes and macrophages are critical for the development of the adaptive immune system, the creation of humanized mice with efficient human myelopoiesis is of great importance.

Thus, there is a need in the art for genetically modified mice capable of enhanced human myelopoiesis as a result of engraftment of human HSCs (Manz MG. Human-hemato-lymphoid-system mice: opportunities and challenges. Immunity. 2007 May;26(5) :537-41).

Disclosure of the Invention

Provided are genetically modified mice containing a nucleic acid sequence encoding the human M-CSF protein. Also provided are genetically modified mice containing a nucleic acid sequence encoding the human M-CSF protein that have been transplanted with human cells, such as human hematopoietic cells, and methods for creating such “grafted” mice (ie, mice transplanted with human cells). These mice have a range of applications, such as modeling human immune disease and pathogen infection; in in vivo search for substances that modulate the development and/or activity of hematopoietic cells, for example, in a normal state or in a disease state; during in vivo search for substances toxic to hematopoietic cells; in in vivo screening for substances that prevent, reduce or reverse the toxic effects of toxic substances on hematopoietic cells; in in vivo screening of human hematopoietic cells obtained from an individual to predict the individual's sensitivity to disease treatment, etc.

In some aspects of the invention, a humanized mouse M-CSF is provided, wherein the humanized M-CSF comprises a nucleic acid sequence that encodes a human M-CSF protein and is operably linked to a 5' regulatory sequence of the mouse M-CSF structural gene locus, e.g., the mouse M-CSF promoter , 5'UTR, etc. In some embodiments, the mouse contains 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 mouse. In some embodiments, the mouse contains two alleles in which the nucleic acid sequence is located in the mouse genome within the mouse M-CSF locus. In some embodiments, the nucleic acid sequence of both alleles 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/h mouse. In some embodiments, the humanized M-CSF mouse contains a null mutation (null mutation) in at least one mouse M-CSF allele. In some embodiments, the humanized M-CSF mouse contains a null mutation in both mouse M-CSF alleles. In some such embodiments, the null mutation is a deletion of exons 2-9 of mouse M-CSF.

In some embodiments, the mouse expresses human M-CSF in the bone marrow, spleen, blood, liver, brain, lungs, testes, and kidneys. In some embodiments, the amount of human M-CSF expressed is substantially equal to the amount of murine M-CSF expressed in a wild-type mouse. In some embodiments, the humanized M-CSF mouse bone marrow mesenchymal stromal cells express an amount of human M-CSF that is substantially equal to the amount of murine M-CSF expressed by wild-type mouse bone marrow mesenchymal stromal cells. In some embodiments, the humanized M-CSF mouse has a physiological concentration of M-CSF in the blood and/or tissue. In some embodiments, the mouse expresses both mouse M-CSF and human M-CSF. In other embodiments, the only M-CSF that the mouse expresses is human M-CSF.

In some embodiments, the mouse secretes sufficient amounts of human M-CSF to differentiate the engrafted human hematopoietic stem cells into human monocytes, human macrophages, and human osteoclasts. In some embodiments, the mouse secretes an effective amount of M-CSF to promote the development of human macrophages from human monocytes that results from the engraftment of human hematopoietic stem cells in the mouse. In some embodiments, the mouse secretes an effective amount of M-CSF to promote the development of a human hematopoietic stem cell into a monoblast, a monoblast into a human promonocyte, a human promonocyte into a human monocyte, and a human monocyte into a human macrophage in a mouse inoculated with human hematopoietic stem cells. In some embodiments, the effective amount of human M-CSF secreted in a mouse is substantially the same as the amount of mouse M-CSF secreted by a wild-type mouse to achieve the corresponding effect (e.g., an effective amount of mouse M-CSF to stimulate the development of a mouse macrophage from a mouse monocyte).

In some embodiments, the control of transcription and translation of human M-CSF in a genetically modified mouse is similar or substantially similar to the control of transcription and translation of mouse M-CSF in a mouse that lacks modification of its endogenous mouse M-CSF gene.

In some embodiments, the physiological concentration of human M-CSF in a humanized mouse M-CSF results from the secretion of human M-CSF by the same cell types that secrete mouse M-CSF in a wild-type mouse that has the mouse M-CSF gene and does not have the mouse M-CSF gene. nucleic acid encoding human M-CSF protein. In other words, one or more M-CSF isoforms are expressed within a normal tissue-specific and developmental profile.

In some embodiments, the mouse expresses an isoform of human M-CSF selected from M-CSF proteoglycan, M-CSF glycoprotein, and cell surface M-CSF, and a combination thereof. In one embodiment, the mouse expresses at least two of the isoforms within the normal tissue-specific and developmental profile. In a separate embodiment, the mouse expresses human CSF-1 proteoglycan and human M-CSF glycoprotein and human M-CSF cell surface.

In some embodiments, the mouse contains human macrophages that are not thymic T cell-derived macrophages. In some embodiments, the mouse contains human macrophages that exhibit M-CSF-dependent podosome formation caused by human M-CSF expressed in the mouse.

In some embodiments, the mouse is homozygous for the Rag2 null mutation. In some embodiments, the mouse is homozygous for the IL2rg null mutation. In some embodiments, the mouse is homozygous for the Rag2 and IL2rg null mutation. In some embodiments, the mouse contains human cells. In some embodiments, the human cells are hematopoietic cells.

In some aspects of the invention, a mouse model of the human immune system is provided, a mouse model comprising 2 Rag2 null alleles, 2 IL2rg null alleles, a nucleic acid sequence that encodes the human M-CSF protein operably linked to the mouse M-CSF gene promoter, and human hematopoietic cells . In other words, the mouse is a Rag2 ^−/− IL2rg ^−/− hM-CSF mouse, where hM-CSF indicates that the mouse contains at least one nucleic acid encoding a human M-CSF gene. In some embodiments, the grafted Rag2 ^-/- IL2rg ^-/- hM-CSF mouse is a BALB/c strain mouse containing these genetic modifications. In some embodiments, the mouse also contains other genetic modifications.

In some embodiments, an engrafted Rag2 ^−/− IL2rg ^−/− hM-CSF mouse at about 12 weeks of age has increased numbers of human CD14 ⁺ CD33 ⁺ (hCD14 ⁺ CD33 ⁺ ) cells in the bone marrow, spleen, and peripheral blood compared to with a mouse containing human hematopoietic cells that express murine M-CSF rather than human M-CSF. In a certain embodiment, the increase in hCD14 ⁺ CD33 ⁺ bone marrow cells compared to a mouse expressing murine M-CSF alone is about 5-fold to 15-fold, in one embodiment about 12- to 14-fold. In a certain embodiment, the increase in hCD14 ⁺ CD33 ⁺ spleen cells compared to a mouse containing human hematopoietic cells that express only murine M-CSF is about 2 to 6 times, in one embodiment about 5 to 6 times. In a certain embodiment, the increase in hCD14 ⁺ CD33 ⁺ peripheral blood cells compared to a mouse containing human hematopoietic cells that express only murine M-CSF is about 2- to 8-fold, in one embodiment about 5- to 7-fold.

In some embodiments, an engrafted Rag2 ^-/- IL2rg ^-/- hM-CSF mouse at about 12 weeks of age has levels of hCD14 ⁺ CD33 ⁺ monocyte/macrophage lineage cells in the blood of about 15 to 40%, in one in an embodiment, approximately 30%. In one embodiment, a genetically modified grafted mouse at about 16 weeks of age exhibits a level of hCD14 ⁺ CD33 ⁺ monocyte/macrophage lineage cells in the blood of about 15 to 30%, in one embodiment about 22%. In one embodiment, a genetically modified grafted mouse at about 20 weeks of age exhibits a level of hCD14 ⁺ CD33 ⁺ monocyte/macrophage lineage cells in the blood of about 5 to 15%, in one embodiment about 10%. In one embodiment, a genetically modified grafted mouse at about 20 weeks of age exhibits a level of hCD14 ⁺ CD33 ⁺ monocyte/macrophage lineage cells in the blood that is approximately 4 to 8 times higher than the level of a grafted mouse expressing murine M -CSF, rather than human M-CSF, is approximately 6 times higher in one embodiment.

In some embodiments, an engrafted Rag2 ^-/- IL2rg ^-/- hM-CSF mouse at about 12 weeks of age exhibits a level of hCD14 ⁺ CD33 ⁺ CD45 ⁺ cells in the liver that is approximately 1.5 to 6 times higher than that of "grafted" mouse expressing mouse M-CSF rather than human M-CSF. In one embodiment, a genetically modified grafted mouse at about 12 weeks of age exhibits a level of hCD14 ⁺ CD33 ⁺ CD45 ⁺ cells in the lungs that is approximately 1.5-10 times higher than that of a grafted mouse expressing murine M-CSF , not human M-CSF. In one embodiment, a genetically modified grafted mouse at about 12 weeks of age exhibits a level of human hCD14 ⁺ CD33 ⁺ CD45 ⁺ cells in the peritoneal cavity or skin that is approximately 2-3 times higher than that of a grafted mouse expressing murine M -CSF, not human M-CSF.

In some embodiments, an engrafted Rag2 ^−/− IL2rg ^−/− hM-CSF mouse exhibits a response to LPS that is approximately 1.5 to 6 times higher relative to the percentage of hCD14 ⁺ CD33 ⁺ cells in the liver than a mouse which lacks human M-CSF, in one embodiment by about 2-4 times; in the lungs, the LPS response relative to hCD14 ⁺ CD33 ⁺ cells is about 1.5-10-fold, in one embodiment about 2-3-fold; in skin, the LPS response relative to hCD14 ⁺ CD33 ⁺ is 2-5-fold, in one embodiment about 3-4-fold; in the peritoneal cavity, the LPS response relative to hCD14 ⁺ CD33 ⁺ is 2-5-fold, in one embodiment about 3-4-fold.

In some embodiments, a Rag2 ^-/- IL2rg ^-/- hM-CSF grafted mouse exhibits an enhanced pro-inflammatory cytokine response in response to LPS stimulation, which is enhanced compared to a genetically modified and grafted mouse lacking the hM-CSF gene , is about 2 to at least 5 times the level of activation and/or differentiation of a cell type that is sensitive to the proinflammatory cytokine.

In some embodiments, an engrafted Rag2 ^-/- IL2rg ^-/- hM-CSF mouse exhibits increased production of hCD14 ⁺ CD33 ⁺ hCD45 ⁺ cells in the spleen approximately 48 hours after LPS administration, with an increase of approximately 2- to 5-fold, in in one embodiment, about 4 to 5 times that of a grafted mouse expressing murine M-CSF rather than human M-CSF.

In some embodiments, an engrafted Rag2 ^-/- IL2rg ^-/- hM-CSF mouse exhibits increased production of human serum IL-6 in response to LPS, with hIL-6 levels increasing by approximately 2 to 5 hours approximately 6 hours after LPS administration. times compared to a grafted mouse expressing mouse M-CSF rather than human M-CSF, in one embodiment about 3-4 times.

In some embodiments, an engrafted Rag2 ^-/- IL2rg ^-/- hM-CSF mouse exhibits increased production of human serum TNFα in response to LPS, with hTNFα levels approximately 2-4 times higher at approximately 6 hours post-LPS administration. a "grafted" mouse that expresses murine M-CSF rather than human M-CSF, in one embodiment, by about 2-3 fold.

In some embodiments, a monocyte and/or macrophage obtained from a Rag2 ^−/− IL2rg ^−/− hM-CSF mouse, upon LPS stimulation, exhibits in vitro secretion of hTNFα that is approximately 2-3 times higher than that of grafted" mouse expressing mouse M-CSF rather than human M-CSF.

In some embodiments, a monocyte and/or macrophage obtained from a Rag2 ^−/− IL2rg ^−/− hM-CSF mouse, upon LPS stimulation, exhibits in vitro hIL-6 secretion that is approximately 2-4 times higher than in a “grafted” mouse expressing murine M-CSF rather than human M-CSF.

In some embodiments, a monocyte and/or macrophage derived from a Rag2 ^−/− IL2rg ^−/− hM-CSF mouse, upon stimulation with poly I:C in vitro, exhibits hIFNα secretion that is approximately 3-6 times higher than in a “grafted” mouse expressing murine M-CSF rather than human M-CSF.

In some embodiments, a monocyte and/or macrophage derived from a Rag2 ^−/− IL2rg ^−/− hM-CSF mouse, upon stimulation with poly I:C in vitro, exhibits hIFNβ secretion that is approximately 2-3 times higher than in a “grafted” mouse expressing murine M-CSF rather than human M-CSF.

In some embodiments, a human monocyte and/or macrophage derived from a grafted Rag2 ^-/- IL2rg ^-/- hM-CSF mouse exhibits increased phagocytosis compared to a grafted mouse expressing mouse M-CSF rather than human M -CSF. In one embodiment, the rate of phagocytosis is increased approximately two-fold, as determined by incorporation of labeled bacteria at 37ºC for a 60-minute period of time, compared to human cells from an engrafted mouse expressing murine M-CSF rather than human M-CSF. CSF. In one embodiment, the rate of phagocytosis as defined above is two times or more that of human cells from a grafted mouse that expresses mouse M-CSF rather than human M-CSF, e.g., 2-fold, 3-fold or 4 times or more.

In some embodiments, a human monocyte and/or macrophage derived from a grafted Rag2 ^-/- IL2rg ^-/- hM-CSF mouse exhibits increased in vitro chemotaxis in response to Mip3β compared to a grafted mouse that expresses murine M -CSF, not human M-CSF. In one embodiment, the increase is about 1.5 to 3 times or more, such as about 1.5 times, 2 times, 3 times, 4 times or more, as measured by the number of cells migrated 30 or 60 minutes after exposure to Mip3β , compared to a human monocyte and/or macrophage from a grafted mouse expressing murine M-CSF rather than human M-CSF.

In some embodiments, a human monocyte and/or macrophage derived from a Rag2 ^−/− IL2rg ^−/− M-CSF ^h mouse exhibits in vitro secretion of hIFNα in response to poly I:C stimulation that is approximately 3-6 times higher than that of a grafted mouse expressing murine M-CSF but not human M-CSF F.

In some embodiments, a human monocyte and/or macrophage derived from an engrafted Rag2 ^−/− IL2rg ^−/− hM-CSF mouse exhibits in vitro upregulation of a costimulatory molecule 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 a combination thereof.

In some aspects of the invention, a genetically modified "grafted" mouse is provided, wherein the mouse contains a human hematopoietic cell graft, is Rag2 ^-/- Il2rg ^-/- , contains a null allele of mouse M-CSF, and contains a nucleic acid sequence encoding human M-CSF in endogenous at the M-CSF locus, with the mouse exhibiting an increase or increased number of human myeloid cells compared to a mouse expressing murine M-CSF rather than human M-CSF.

In some embodiments, the increase is at least a doubling of the number of hCD14 ⁺ CD33 ⁺ cells in any part of the mouse body selected from bone marrow, spleen, and peripheral blood. In a particular embodiment, the increase is a threefold increase in hCD14 ⁺ CD33 ⁺ cells. In another embodiment, the increase is an increase in the number of hCD14 ⁺ CD33 ⁺ cells by 4-5 times or more.

In some embodiments, the increase is a 2- to 3-fold increase in the number of hCD14 ⁺ CD33 ⁺ hCD45 ⁺ cells in a part of the mouse's body selected from the skin and abdomen.

In some embodiments, the increase is a 1.5- to 10-fold increase in the number of hCD14 ⁺ CD33 ⁺ hCD45 ⁺ cells in a portion of the mouse body selected from the liver and lungs.

In some embodiments, the increase is a 4- to 5-fold increase in the number of hCD14 ⁺ CD33 ⁺ hCD45 ⁺ spleen cells approximately 48 hours after LPS stimulation.

In some embodiments, the increase is a 2- to 4-fold increase in LPS-stimulated serum hIL-6 or LPS-stimulated serum hTNFα.

In some embodiments, the increase is a 2- to 3-fold increase in the migration of hCD14 ⁺ CD33 ⁺ cells stimulated with human MIP3β in vitro.

In some aspects of the invention, a mouse model of a human pathogen is provided, a mouse model comprising 2 Rag2 null alleles, 2 IL2rg null alleles, a nucleic acid sequence encoding a human M-CSF protein operably linked to a mouse M-CSF gene promoter, human hematopoietic cells, and pathogen infection person. In other words, the mouse is a Rag2 ^−/− IL2rg ^−/− hM-CSF mouse that has been infected with a human pathogen. In some embodiments, the pathogen is a virus, fungus, or bacterium. 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 (M. tuberculosis). In some embodiments, the bacterium is an enterobacterium, such as Salmonella typhi (S. typhi).

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

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

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

In some aspects of the invention, a human immune cell from a mouse as described herein is provided. In one embodiment, the human immune cell is selected from a human monocyte and a human macrophage. In one embodiment, the human immune cell is selected from a human NK cell, a human B cell, and a human T cell.

In some aspects of the invention, an antibody encoded with a human nucleotide sequence from a mouse described herein is provided. In one embodiment, the antibody is selected from an antibody of the IgA, IgD, IgE, IgM, or IgG isotype.

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 in accordance with the invention. In one embodiment, the nucleotide sequence encodes a human variable region of a human immunoglobulin gene or a fragment thereof. In one embodiment, the nucleotide sequence encodes a human TCR variable region or fragment thereof.

In some aspects of the invention, a method for creating a humanized mouse M-CSF expressing biologically active human M-CSF is provided. In some embodiments, the method includes contacting a mouse pluripotent stem cell, such as an ES cell or an iPS cell, with a nucleic acid sequence comprising the coding sequence of a human M-CSF protein or a fragment thereof, and culturing the pluripotent stem cell under conditions that promote integration of the nucleic acid sequence into mouse genome; creating a mouse from a mouse ES cell that contains a nucleic acid sequence encoding a human M-CSF protein; and maintaining the mouse under conditions suitable to cause the mouse to express human M-CSF through 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 the target locus. In some such embodiments, the target locus is an endogenous mouse M-CSF locus, e.g., a nucleic acid sequence comprising a human M-CSF protein coding sequence is flanked by sequences that are homologous to the endogenous mouse M-CSF locus, and the nucleic acid sequence is integrated into the endogenous M-CSF locus -mouse CSF by homologous recombination. In some embodiments, the mouse is homozygous for the Rag2 null mutation. In some embodiments, the mouse is homozygous for the IL2rg null mutation. In some embodiments, the mouse is homozygous for the Rag2 and IL2rg null mutation, i.e. is Rag2 ^-/- IL2rg ^-/- .

In some aspects of the invention, a method for creating a humanized mouse M-CSF containing the human hematopoietic system is provided. In some embodiments, the method comprises transplanting a humanized M-CSF mouse, eg, a Rag2 ^-/- IL2rg ^-/- hM-CSF mouse or a sublethal irradiated hM-CSF mouse, into a cell population comprising human hematopoietic progenitor cells. In some embodiments, the human hematopoietic progenitor cells are CD34+ cells. In some embodiments, the human hematopoietic progenitor cells are CD133+ cells. In some embodiments, human hematopoietic progenitor cells are pluripotent stem cells, such as ES cells or iPS cells. In some embodiments, the source of the cell population containing human hematopoietic progenitor cells is fetal liver. In some embodiments, the source of the cells is bone marrow. In some embodiments, the source of the cells is peripheral blood. In some embodiments, the cell source is an in vitro population of cells.

In some aspects of the invention, a method is provided for creating a mouse infected with a human pathogen. In some embodiments, the method includes exposing a humanized M-CSF mouse containing human hematopoietic cells to a human pathogen, such as a Rag2 ^−/− IL2rg ^−/− hM-CSF inoculated mouse, or a sublethal irradiated mouse inoculated, and maintaining the mouse under conditions sufficient to for a human pathogen to infect a mouse. In some embodiments, the human pathogen is a human pathogen that does not infect a mouse that lacks one or more of the genetic modifications described herein. In some embodiments, the human pathogen is a human pathogen that is not pathogenic to a mouse that lacks one or more genetic modifications described herein.

In some aspects of the invention, there is provided a method for creating biologically active human M-CSF in a mouse, the method including creating a humanized mouse M-CSF expressing biologically active human M-CSF as described above and elsewhere herein. In some embodiments, the method includes purifying biologically active human M-CSF from blood, such as serum, or mouse tissue. In some embodiments, the method includes obtaining a cell that expresses biologically active human M-CSF from a mouse, culturing the cell under conditions sufficient for the cell to express and secrete biologically active human M-CSF, and isolating the secreted biologically active human M-CSF. It should be noted that this aspect of the invention does not require the mouse to have any other genetic modifications and that the mouse is used to create preparations of certain human immune cells. Accordingly, in some aspects of the invention, isolated, biologically active human M-CSF obtained from a transgenic mouse is provided.

In some aspects of the invention, there is provided a method of creating an activated human monocyte or an activated human macrophage in a mouse, comprising exposing a humanized M-CSF mouse engrafted with human hematopoietic cells to an immunostimulant that activates human monocytes or macrophages in the mouse, and isolating human monocytes or human macrophages from the mouse. , and the fraction of activated monocytes or activated macrophages is approximately two to five times greater than that obtained from a “grafted” mouse that is not a humanized M-CSF mouse, i.e. which lacks the human M-CSF gene. In some embodiments, the immunostimulant is an endotoxin. In a particular embodiment, the endotoxin is LPS.

In some aspects of the invention, a method is provided for screening a candidate substance with activity that modulates the function of human hematopoietic cells. In some embodiments, the method includes contacting a humanized M-CSF mouse grafted with human hematopoietic cells, such as a Rag2 ^-/- IL2rg ^-/- hM-CSF grafted mouse or an hM-CSF grafted sublethal irradiated mouse, with a substance -candidate; and comparing the function of hematopoietic cells in a mouse model that was exposed to the candidate substance with the function of hematopoietic cells in a mouse model that was not exposed to the candidate substance; wherein the change in hematopoietic cell function in a mouse exposed to the candidate substance indicates that the candidate substance alters hematopoietic cell function.

In some aspects of the invention, a method is provided for determining the effect of a substance on a human pathogen, comprising exposing a humanized M-CSF inoculated mouse, e.g., a Rag2 ^−/− IL2rg ^−/− hM-CSF inoculated mouse or an hM-CSF inoculated sublethal irradiated mouse, to an effective amount of the pathogen. a human, wherein the effective amount of the pathogen is the amount of pathogen required to infect a mouse; allowing the mouse to become infected with a pathogen; measuring the infection rate over a period of time in the presence of the substance; and comparing these measurements with measurements obtained from a non-exposed humanized M-CSF mouse. In some embodiments, the substance is used before the mouse is exposed to the pathogen, for example, to determine a protective effect. In some embodiments, the substance is used concurrently with exposure of the mouse to the pathogen, for example, to determine a protective or therapeutic effect. In some embodiments, the substance is used after exposure of the mouse to a pathogen, for example, to determine a therapeutic effect. In some embodiments, upon exposure to a pathogen, the mouse exhibits an increased cellular and/or humoral immune response that replicates infection in a human exposed to the pathogen. In some embodiments, the human pathogen is a pathogen that does not infect a mouse that lacks one or more of the described genetic modifications. In some embodiments, the human pathogen is a pathogen that infects a wild-type mouse, wherein the wild-type mouse, upon infection, does not mount an immune response that is elevated in a human in response to the pathogen. In some embodiments, the virus is a human, swine, or avian influenza virus. In some embodiments, the bacterium is a mycobacterium, such as Mycobacterium tuberculosis (M. tuberculosis). In some embodiments, the bacterium is an enterobacterium, such as Salmonella typhi (S. typhi). In some embodiments, the mouse is exposed to a known number of human pathogen units, and the indicator of infection is the number of infectious human pathogen units in the body fluid or tissue of the mouse. In some embodiments, the indicator of infection is a titer in a mouse body fluid. In some embodiments, the indicator of infection is the formation of a granuloma. In some such embodiments, the granuloma is a pulmonary granuloma. In some such embodiments, the granuloma is a well-defined granuloma.

In some aspects of the invention, a method is provided for determining the effect of a substance on a human pathogen, comprising exposing a humanized M-CSF grafted mouse, e.g., a Rag2 ^-/- IL2rg ^-/- hM-CSF grafted mouse or an hM-CSF grafted sublethal irradiated mouse, to an effective amount of an antigen. a human pathogen, wherein the effective amount of antigen is the amount of antigen necessary to promote a cellular and/or humoral immune response in a mouse; allowing the development of a cellular and/or humoral response; measuring the cellular and/or humoral response over time in the presence of the substance; and comparing this measurement with a measurement obtained from a "grafted" humanized M-CSF mouse that was not exposed to the substance. In some embodiments, the substance is used before exposing the mouse to an antigen from a human pathogen, for example, to determine the protective effect of the substance. In some embodiments, the substance is used concurrently with exposure of the mouse to an antigen from a human pathogen, for example, to determine the protective or therapeutic effect of the substance. In some embodiments, the substance is used after exposure of the mouse to an antigen from a human pathogen, for example, to determine the therapeutic effect of the substance. In some embodiments, upon exposure to a human pathogen, the mouse exhibits an increased cellular and/or humoral immune response that mimics infection in a human exposed to the pathogen.

In some embodiments, the antigen is an antigen from a human pathogen that does not infect a mouse that lacks one or more of the genetic modifications described herein. In other embodiments, the antigen is an antigen from a human pathogen that infects a wild-type mouse, wherein the wild-type mouse, after infection, does not produce an immune response that is elevated in a human in response to the pathogen. In some embodiments, the pathogen is a virus, fungus, or bacterium. 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 (M. tuberculosis). In some embodiments, the bacterium is an enterobacterium, such as Salmonella typhi (S. typhi).

In some aspects of the invention, a method is provided for screening candidate substances for toxicity to human hematopoietic cells. In some embodiments, the method comprises contacting a humanized M-CSF mouse grafted with human hematopoietic cells, such as a Rag2 ^-/- IL2rg ^-/- hM-CSF mouse, with a candidate substance; and comparing the viability and/or function of hematopoietic cells in a mouse that has been exposed to the candidate substance with the viability and/or function of hematopoietic cells in a humanized M-CSF mouse inoculated with human hematopoietic cells that has not been exposed to the candidate substance; wherein a decrease in the viability and/or deterioration in the function of hematopoietic cells in a mouse that has been in contact with the candidate substance indicates that the candidate substance is toxic to hematopoietic cells.

In some aspects of the invention, a method is provided for screening candidate substances for their ability to protect human hematopoietic cells from a toxic agent, reduce the effects of a toxic substance on human hematopoietic cells, or reverse the effects of a toxic substance on human hematopoietic cells. In some embodiments, the method includes contacting a humanized M-CSF mouse inoculated with human hematopoietic cells, such as a Rag2 ^-/- IL2rg ^-/- hM-CSF inoculated mouse or a sublethally irradiated hM-CSF inoculated mouse, with a toxic substance; contacting the mouse with the candidate substance; and comparing the viability and/or function of hematopoietic cells in a mouse that has been exposed to the candidate substance with the viability and/or function of hematopoietic cells in a humanized M-CSF mouse inoculated with human hematopoietic cells that has not been exposed to the candidate substance; wherein the increase in the viability and/or function of hematopoietic cells in a mouse model that was exposed to the candidate substance indicates that the candidate substance protects hematopoietic cells from the toxic substance.

In some aspects of the invention, a method is provided for predicting the sensitivity of an individual to treatment with a therapeutic agent. In some embodiments, the method includes contacting a humanized M-CSF mouse grafted with human hematopoietic cells obtained from an individual, for example, a Rag2 ^-/- IL2rg ^-/- hM-CSF mouse grafted or a sublethally irradiated hM-CSF mouse grafted. mice, with a therapeutic agent; and comparing the viability and/or function of hematopoietic cells in a mouse model that was exposed to the candidate substance with the viability and/or function of hematopoietic cells in a humanized M-CSF mouse inoculated with human hematopoietic cells that was not exposed to the candidate substance; wherein a change in the viability and/or function of hematopoietic cells in a mouse that has been exposed to the candidate substance indicates that the individual will respond to treatment with the therapeutic agent.

Brief description of the figures

Fig. 1 illustrates (for bone marrow mesenchymal stromal cells) (A) M-CSF expression; These organs were isolated from M-CSF ^m/m and M-CSF ^h/h , RNA was isolated and reverse transcription-PCR analysis was performed using specific primers for both mouse M-CSF (top) and human M-CSF ( middle); HPRT level (bottom) was used as a control for cDNA input; data represent results from 2 independent experiments. (B) The indicated organs were isolated from M-CSF ^h/m , RNA was isolated and analyzed using primers specific for either mouse M-CSF (top) or human M-CSF (middle). RNA isolated from mouse liver or human fetal liver served as a positive control for mouse and human primer pairs; respectively, the absence of RT and the PCR reaction template served as negative controls. Data represent the results of 2 independent experiments. (C) Bone-associated stromal cells from M-CSF ^m/m , M-CSF ^m/h , and M-CSF ^h/h mice were isolated and cultured in vitro for 10 days, cells were lysed, RNA was isolated, and PCR analysis was performed. real time using either mouse M-CSF (white bars) or human M-CSF (black bars) specific primers; average values from samples are shown; whiskers indicate ± SEM; the amount of introduced cDNA was normalized in accordance with the expression level of HPRT (hypoxanthine guanine phosphoribosyl transferase); data represent the results of 2 independent experiments; and, (D) bone-associated stromal cells from M-CSF ^m/m , M-CSF ^m/h , and M-CSF ^h/h mice were isolated and cultured in vitro for 10 days, cell culture supernatants were collected, and secreted levels of mouse (white) and human (black bars) M-CSF were quantified using species-specific M-CSF ELISA kits; average values from three samples are shown; whiskers indicate ± SEM; Data represent the results of 2 independent experiments. (E) 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 measured by ELISA. Shown are the averages of three samples; whiskers indicate ± SEM.

Fig. 2A shows the absolute bone marrow (BM) cell counts of M-CSF ^m/m , M-CSF ^m/h , and M-CSF ^h/h mice as an average per animal (two tibias and fibulas); each group contains n=5 mice aged 4 weeks; whiskers indicate ± SEM; Data represent the results of 3 independent experiments.

Fig. 2B illustrates flow cytometry analysis of a suspension of stained single cells from bone marrow (top), spleen (middle), and peripheral blood (PB) obtained from M-CSF ^m/m , M-CSF ^m/h , and M-CSF ^h/h mice ; staining with Gr1 and CD11b antibodies.

Fig. 2C illustrates flow cytometry analysis of a suspension of stained single cells from bone marrow (top) and spleen (middle) obtained from M-CSF ^m/m , M-CSF ^m/h , and M-CSF ^h/h mice; staining with F4/80 and CD11b antibodies.

Fig. 2D illustrates flow cytometry analysis of isolated bone marrow cells that were cultured in the presence of recombinant mouse M-CSF (left) or human M-CSF (right) for 7 days; cells were stained with F4/80 and CD11b antibodies.

Fig. 2E illustrates flow cytometry analysis of isolated bone marrow cells that were cultured in the presence of recombinant mouse M-CSF (solid) or human M-CSF (unshaded) for 7 days; cells were stained with the indicated surface markers.

Fig. 3A illustrates flow cytometry results of single cell suspensions of bone marrow (top), spleen (middle), and peripheral blood (PB) from human CD34 ⁺ cells transplanted with M-CSF ^m/m , M-CSF ^m/h , and M-CSF ^{h/ h} mice; CD45, CD14 and CD33 staining with human antibodies; human CD45 ⁺ cells were pre-gated and separated based on CD14 and CD33 expression.

Fig. 3B illustrates the relative frequencies of human CD45+ CD14+CD33+ cells from bone marrow (top), spleen (middle), and peripheral blood (PB); the absolute number of bone marrow cells was determined as the average per animal (two tibias and fibulas), and peripheral blood cells were determined per ml of blood volume; each group contains n=20 mice; each symbol represents an individual mouse, horizontal bars indicate average values; Data represent the results of 5 independent experiments.

Fig. 3C illustrates the absolute frequencies of human CD45+ CD14+CD33+ cells from bone marrow (top), spleen (middle), and peripheral blood (PB); the absolute number of bone marrow cells was determined as the average per animal (two tibias and fibulas), and peripheral blood cells were determined per ml of blood volume; each group contains n=20 mice; each symbol represents an individual mouse, horizontal bars indicate average values; Data represent the results of 5 independent experiments.

Fig. 4A illustrates flow cytometry analysis of stained cells from human CD34 ⁺ cells transplanted into M-CSF ^m/m , M-CSF ^m/h and M-CSF ^h/h mice, blood collected at 12, 16 and 20 weeks post-transplantation; cells were stained with human CD45, CD14, and CD33 antibodies; human CD45 ⁺ cells were pre-gated and separated based on CD14 and CD33 expression.

Fig. 4B illustrates the relative frequencies of human CD45 ⁺ CD14 ⁺ CD33 ⁺ cells; each group contains n=10 mice; each symbol represents an individual mouse, horizontal bars indicate average values; Data represent the results of 3 independent experiments.

Fig. 5 presents flow cytometry analysis of M-CSF ^m/m , M-CSF ^m/h , and M-CSF ^h/h mice engrafted with human CD34 ⁺ cells 12 weeks after transplantation, when mice were sacrificed and perfused with PBS; liver (A), lungs (B) and skin (C) were removed and single cell suspensions were prepared; Abdominal cells (D) were collected using PBS aspiration; cells were stained with human CD45, CD14 and CD33 antibodies and examined by flow cytometry; each symbol represents an individual mouse, horizontal bars indicate average values; Data represent the results of 3 independent experiments.

Fig. 6 illustrates the results of LPS stimulation. (A) M-CSF ^m/m and M-CSF ^m/h mice were transplanted with human CD34 ⁺ cells and LPS was administered intraperitoneally (ip) 12 weeks after transplantation. After 48 hours, mice were sacrificed and the frequencies of human CD45 ⁺ CD14 ⁺ CD33 ⁺ cells in the spleen were determined; Mice injected with PBS served as controls; each symbol represents an individual mouse, horizontal bars indicate average values. (B), (C) M-CSF ^m/m and M-CSF ^m/h mice were transplanted with human CD34 ⁺ cells and LPS was administered 12 weeks after transplantation (ip). Six hours later, mice were sacrificed and serum levels of human (right) and murine (left) IL-6 and TNFα were quantified by ELISA; Mice injected with PBS served as controls; the average values of three parallel samples are shown; whiskers indicate ± SEM.

Fig. 7A (hTNFα) and 7B (hIL-6) illustrate the ability of monocytes/macrophages to secrete proinflammatory cytokines in vitro after stimulation with LPS. Human CD45 ⁺ CD14 ⁺ CD33 ⁺ cells were isolated from the spleens of M-CSF ^m/m and M-CSF ^h/h mice transplanted with human CD34 ⁺ cells 12 weeks after transplantation; human CD45 ⁺ CD14 ⁺ CD33 ⁺ cells derived from fetal liver served as controls; cells were stimulated in vitro with LPS for 24 or 48 hours, cell culture supernatants were collected, and levels of human TNFα (A) and IL-6 (B) were quantified by ELISA; the average values of three parallel samples are shown; whiskers indicate ± SEM.

Fig. 7C illustrates interferon-α and interferon-β mRNA levels in response to poly I:C trimulation. Human CD45 ⁺ CD14 ⁺ CD33 ⁺ cells were stimulated with poly I:C for 6 or 12 hours and IFNα (left) and IFNβ (right) mRNA levels were quantified by real-time PCR; the average values of two parallel samples are shown; whiskers indicate ± SEM.

Fig. 7D illustrates the phagocytosis, migration and activation capacity of cells obtained from engrafted mice. Human CD45 ⁺ CD14 ⁺ CD33 ⁺ cells were isolated from humanized mice and incubated with FITC-labeled bacteria at 37°C for 30 or 60 minutes and measured by flow cytometry; cells incubated with FITC-labeled bacteria on ice served as controls. Open histograms represent cells from M-CSF ^m/m mice, dotted histograms represent cells from M-CSF ^h/h mice, and filled histograms represent cells from human fetal liver.

Fig. 7E illustrates cell chemotaxis in response to MIP3β. Human CD45 ⁺ CD14 ⁺ CD33 ⁺ cells isolated from M-CSF ^m/m mice, M-CSF ^h/h mice, and human fetal liver were contained in the upper wells, and medium containing MIP3β was added to the lower wells; cells were incubated for 30 or 60 minutes, then the number of cells migrating from the upper cells to the lower cells was counted and plotted; the average values of two parallel samples are shown; whiskers ± SEM.

Fig. 7F illustrates increased activation of human monocytes/macrophages from engrafted mice as evidenced by upregulation of hCD40, hCD80, hCD86, and hHLA-DR following in vitro LPS stimulation. Human CD45 ⁺ CD14 ⁺ CD33 ⁺ cells isolated from M-CSF ^m/m mice, M-CSF ^h/h mice, and human fetal liver were cultured in the presence or absence of LPS; after 24 hours of stimulation, cells were stained with the indicated surface markers and measured by flow cytometry. Open histograms represent cells from M-CSF ^m/m mice, dotted histograms represent cells from M-CSF ^h/h mice, and filled histograms represent human fetal liver cells.

Fig. 8 provides a schematic representation of the mouse M-CSF locus, indicating the relative location of exons 1-9, and the final target allele with the human M-CSF gene.

Fig. 9A,B illustrates the frequencies of the HSC compartment and the myeloid progenitor compartment in M-CSF ^m/m , M-CSF ^h/m and M-CSF ^h/h mice. Bone marrow cells from M-CSF ^m/m , M-CSF ^m/h and M-CSF ^h/h mice were stained with antibodies to lineage markers, c-Kit, Sca1, CD150, CD48, CD16/32 and CD34 and analyzed by flow cytometry. (A) Lineage ^— cells (top) were selected and isolated for their expression of Sca1 and c-Kit (middle). Lineage ^- Sca1 ⁺ c-Kit ⁺ (LSK) cells were selected and isolated for their expression of CD150 and CD48 (bottom). (B) Lineage ^— cells were preselected and isolated for their expression of Sca1 and c-Kit (top). Lineage ^- c-Kit ⁺ Sca1 ^- cells were selected and isolated based on the expression of CD16/32 and CD34 (bottom).

Detailed Description of the Invention

Before describing the present methods and compositions, it should be understood that this invention is not limited to the specific method or composition described, and as such is, of course, subject to variation. It should also be understood that the terminology used in this description is for the purpose of describing individual embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Unless otherwise specified, all technical and scientific terms used in this description have the same meaning as commonly understood by one skilled in the art to which this invention relates. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, specific methods and materials are described below. All publications mentioned in this description are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It should be understood that this disclosure supersedes any disclosure in the included publication to the extent there is a conflict.

Upon reading this disclosure to one skilled in the art, it will be apparent that each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated from or combined with features of any of several other embodiments within the scope and essence of the present invention. Any method described can be implemented in the listed order of steps or in any other order that is logically possible.

It should be noted that as used herein and in the accompanying claims, the singular forms of the invention include the plural forms of the subject unless the context clearly dictates otherwise. Thus, for example, a reference to a “cell” includes a plurality of such cells, and a reference to a “peptide” includes a reference to one or more peptides and their equivalents, eg, polypeptides known to those skilled in the art, and so on.

The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing in this specification should be construed to imply that the present invention is not entitled to precede such publication by reason of the earlier creation of the present invention.

Genetically modified mice containing a nucleic acid sequence encoding human M-CSF protein are provided. Also provided are genetically modified mice containing a nucleic acid sequence encoding human M-CSF protein into which human cells, such as human hematopoietic cells, have been transplanted, and methods for creating such “grafted” mice. These mice have a range of applications, such as modeling human immune disease and pathogen infection; in in vivo screening for substances that modulate the development and/or activity of hematopoietic cells, for example, in a normal state or in a diseased state; when screening in vivo for substances toxic to hematopoietic cells; in in vivo screening for substances that prevent, reduce or reverse the toxic effects of toxic substances on hematopoietic cells; in in vivo screening of human hematopoietic cells obtained from an individual to determine the individual's sensitivity to disease therapy, etc.

Humanized m-csf mice

In some aspects of the invention, a humanized M-CSF mouse is provided. By humanized M-CSF mouse or “hM-CSF mouse” is meant a mouse containing a nucleic acid sequence that encodes a human M-CSF protein. By human M-CSF protein is meant a protein that is human M-CSF or is a protein substantially identical to human M-CSF, e.g., 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 protein. The nucleic acid sequence encoding human M-CSF protein is thus a polynucleotide that contains the coding sequence for human M-CSF protein, i.e. human M-CSF or a protein that is substantially identical to human M-CSF. M-CSF (also known as CSF-1, “colony stimulating factor 1”) is a cytokine that controls the formation, differentiation and function of macrophages. The polypeptide sequence of human M-CSF and the nucleic acid sequence encoding human M-CSF can be found under Genbank accession numbers NM_000757.5 (option 1), NM_172210.2 (option 2), and NM_172212.2 (option 4). The genomic locus encoding the human M-CSF protein can be found in the human genome on Chromosome 1; NC_000001.10 (110453233-110472355). Protein sequence is encoded at this locus by exons 1 to 8, while exon 9 contains untranslated sequence. In this regard, the nucleic acid sequence containing the coding sequence of human M-CSF contains one or more exons 1-8 of the human M-CSF gene. In some cases, the nucleic acid sequence also contains components of the human M-CSF genomic locus, for example, introns, 3' and/or 5' untranslated sequences (UTRs). In some cases, the nucleic acid sequence contains completely all regions of the human M-CSF genomic locus. In some cases, the nucleic acid sequence contains exon 2 of the human M-CSF genomic locus up to 633 nucleotides (nt), located 3' (downstream) of non-coding exon 9.

In the humanized M-CSF mice of the present application, the nucleic acid sequence encoding the human M-CSF protein is operably linked to one or more mouse M-CSF gene regulatory sequences. Mouse M-CSF regulatory sequences are those sequences of the mouse M-CSF genomic locus that regulate the expression of mouse M-CSF, e.g., 5' regulatory sequences, e.g., M-CSF promoter, 5' M-CSF untranslated region (UTR), etc.; 3' regulatory sequences, for example, 3'UTR; and enhancers, etc. Mouse M-CSF is located on chromosome 3 at approximately positions 107,543,966-107,563,387, and the coding sequence of mouse M-CSF can be found under Genbank accession numbers NM_007778.4 (isoform 1), NM_001113529.1 (isoform 2), and NM_001113530.1 (isoform 3). Mouse M-CSF regulatory sequences are well characterized in the field and can be readily determined using in silico methods, such as the aforementioned Genbank accessions on the UCSC Genome Browser, the World Wide Web at genome.ucsc.edu, or 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 cases, 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 encoding sequence are endogenous, or native, to the mouse genome, i.e. e. they were present in the mouse genome before the integration of human nucleic acid sequences.

In some cases, a humanized mouse M-CSF is created by random integration, or insertion, of a human nucleic acid sequence encoding the human M-CSF protein, or a fragment thereof, i.e. the “human M-CSF nucleic acid sequence”, or “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 cases, a humanized mouse M-CSF is created 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 bp or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 tbp or more , 2 tpo or more, 5 tpo or more, 10 tpo or more, 15 tpo or more, 20 tpo or more, or 50 tpo or more genomic material from the target locus. Thus, for example, in a humanized mouse M-CSF containing a nucleic acid sequence that encodes a human M-CSF protein, created by targeting the human M-CSF nucleic acid sequence to the mouse M-CSF locus, the human M-CSF nucleic acid sequence can be completely or partially replaced mouse sequence, eg exons and/or introns, in the M-CSF locus. In some such cases, 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 native, or endogenous, regulatory sequences at the mouse M-CSF locus. In other words, the regulatory sequence(s) to which the nucleic acid sequence encoding the human M-CSF protein is operably linked are the native M-CSF regulatory sequences at the mouse M-CSF locus.

In some cases, integration of the human M-CSF sequence does not affect the transcription of the gene into which the human M-CSF sequence has been integrated. For example, in the event that 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 into which the human M-CSF sequence has been integrated . In other cases, integration of the human M-CSF sequence interferes with the transcription of the gene into which the human M-CSF sequence has been integrated. For example, when integrating a human M-CSF sequence by homologous recombination, the coding sequence at the integration locus may be removed in whole or in part so that the human M-CSF sequence is transcribed instead. In some such cases, integration of the human M-CSF sequence causes a null mutation, and hence a null allele. A null allele is a mutant copy of a gene that completely lacks the normal function of that gene. This may result from the complete absence of a gene product (protein, RNA) at the molecular level or from the expression of a nonfunctional gene product. At the phenotypic level, a null allele is no different from a deletion of the entire locus.

In some cases, the humanized mouse M-CSF contains one copy of the nucleic acid sequence encoding the human M-CSF protein. For example, a mouse may be heterozygous for a nucleic acid sequence. In other words, one allele at a locus will contain the nucleic acid sequence, while the others will be endogenous alleles. For example, as discussed above, in some cases, the human M-CSF nucleic acid sequence is integrated into the mouse M-CSF locus such that a null allele of mouse M-CSF is generated. In some such embodiments, the humanized M-CSF mouse may be heterozygous for the coding nucleic acid sequence, i.e. a humanized M-CSF mouse contains one murine M-CSF null allele (an allele containing the nucleic acid sequence) and one endogenous M-CSF allele (wild type or otherwise). In other words, the mouse is an M-CSF ^h/m mouse, where “h” indicates the allele containing the human sequence and “m” indicates the endogenous allele. In other cases, the humanized mouse M-CSF contains two copies of the nucleic acid sequence encoding the human M-CSF protein. For example, a mouse may be homozygous for a nucleic acid sequence, i.e. both alleles at a locus in a diploid genome will contain a nucleic acid sequence, i.e. The humanized M-CSF mouse contains two null alleles of mouse M-CSF (the allele containing the nucleic acid sequence). In other words, the mouse is an M-CSF ^h/h mouse.

It should be noted that humanized M-CSF mice, such as those described above, M-CSF ^h/h and M-CSF ^h/m mice, exhibit normal or wild-type development and function of macrophages and monocytes and tissues that are formed from cells of the macrophage lineage, for example, bone. For example, humanized mice have normal teeth and bone properties, as well as normal bone marrow content, the frequency of myeloid cells in the bone marrow, spleen and peripheral blood, and the frequency of macrophages in the bone marrow and spleen.

In some cases, the humanized M-CSF mouse contains other genetic modifications. For example, a humanized M-CSF mouse may contain at least one null allele of the Rag2 gene (“recombination-activating gene 2”, the coding sequence of which can be found in Genbank Accession No. 1.NM_009020.3). In some embodiments, the humanized M-CSF mouse contains two Rag2 null alleles. In other words, the humanized M-CSF mouse is homozygous for the Rag2 null allele. In another example, a humanized M-CSF mouse contains at least one null allele of the IL2rg gene (“interleukin 2 receptor gamma,” also known as common gamma chain or γC, the coding sequence of which can be found under Genbank accession number 1 .NM_013563.3). In some embodiments, the humanized M-CSF mouse contains two IL2rg null alleles. In other words, it is homozygous for the IL2rg null allele. In some embodiments, the mouse contains a null allele of both Rag2 and IL2rg, i.e. is Rag2 ^-/- IL2RG ^-/- . Other genetic modifications are also being considered. For example, a humanized M-CSF mouse may contain modifications of other genes associated with the development and/or functioning of hematopoietic cells and the immune system, for example, replacing one or another mouse gene with a nucleic acid sequence encoding a human ortholog. Additionally or alternatively, the humanized mouse M-CSF may contain modifications of genes associated with the development and/or function of other cells and tissues, for example, genes associated with diseases or disorders in humans, or genes that, when modified in a mouse, provide mouse models of diseases and disorders in humans.

In some aspects of the invention, a humanized M-CSF mouse, for example, a Rag2 ^-/- IL2rg ^-/- hM-CSF mouse or a sublethal irradiated hM-CSF mouse, is engrafted, or transplanted, with cells. The cells may be mitotic cells or post-mitotic cells and include cells of interest such as pluripotent stem cells, for example, ES cells, iPS cells and embryonic germ cells; and somatic cells, for example, fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, vascular endothelial cells, intestinal cells and the like, and their lineage-specific progenitors and progenitors. Cell populations of particular interest include those containing hematopoietic stem or progenitor cells that will reconstitute or contribute to the hematopoietic system of the humanized M-CSF mouse, e.g., peripheral blood leukocytes, fetal liver cells, fetal bone cells, fetal thymus cells, fetal lymphatic vessels, vascularized skin, arterial segments and purified hematopoietic stem cells, such as mobilized HSCs or umbilical cord blood HSCs. The cells can come from any mammalian species, such as mice, rodents, dogs, cats, cows, sheep, primates, humans, etc. Cells may be derived from established cell lines or may be primary cells, with the terms “primary cells,” “primary cell line,” and “primary cultures” being used interchangeably herein to refer to cells and cell cultures obtained from a subject and grown in vitro for a limited number of passages, i.e. doubling culture. For example, primary crops are crops that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times to pass the critical stage. Typically, the primary cell lines of the present invention are maintained in vitro for less than 10 passages.

If the cells are primary cells, they can be collected from the individual by any suitable method. Blood cells, such as white blood cells, can be collected by apheresis, leukocytapheresis, density gradient separation, etc. As another example, cells from skin, muscle, spinal cord, spleen, liver, pancreas, lungs, intestines, stomach tissue, etc. can be collected by biopsy. A suitable solution may be used to disperse or suspend the collected cells. This solution will typically be a balanced salt solution, such as saline, PBS, Hank's balanced salt solution, etc., supplemented for convenience with fetal bovine serum or other natural factors, combined with an appropriate low concentration buffer solution, usually from 5-25 mM. Suitable buffer solutions include HEPES, phosphate buffer solutions, lactate buffer solutions, etc.

In some cases, a heterogeneous cell population will be transplanted into a humanized mouse. In other cases, a population of cells enriched in a particular cell type, such as progenitor cells, such as hematopoietic progenitor cells, will engraft into a humanized mouse. Enrichment of the target cell population can be accomplished using any suitable separation method. For example, target cells can be enriched by culture methods. In such culture methods, certain growth factors and nutrients are typically added to the culture that promote the survival and/or proliferation of one cell population more than another. Other culture conditions that affect survival and/or proliferation include growth on an attached or unattached substrate, culture for specified periods of time, etc. Such culture conditions are well known in the art. As another example, target cells can be enriched by separating target cells from the source population using affinity separation methods. Affinity separation methods may include magnetic separation using magnetic beads coated with an affinity reagent, affinity chromatography, panning with the affinity reagent attached to a solid matrix such as a plate, cytotoxic agents attached to the affinity reagent or used simultaneously with the affinity reagent, e.g. complement and cytotoxins, or other appropriate methods. Methods that provide thorough separation include fluorescence-activated cell sorters, which can have varying degrees of complexity such as multiple color channels, low-angle and low-angle light scatter detection channels, resistance channels, etc. Cells can be selected against dead cells by using dyes that bind to dead cells (eg, propidium iodide). Any method that does not unduly compromise the viability of the target cells may be used.

For example, using affinity separation methods, cells that are not targeted for transplantation can be removed from the population by interacting the population with affinity reagents that specifically recognize and selectively bind markers that are not expressed on the target cells. For example, in order to enrich the population of hematopoietic progenitor cells, cells expressing markers of mature hematopoietic cells can be recovered. Additionally or alternatively, positive selection and separation can be accomplished by interacting the population with affinity reagents that specifically recognize and selectively bind markers associated with hematopoietic progenitor cells, for example, CD34, CD133, etc. By "selective binding" is meant that the molecule preferentially binds to the target of interest or binds to the target molecule with greater affinity than others. For example, an antibody will bind to a molecule containing the epitope for which it is specific and will not bind to extraneous epitopes. In some embodiments, the affinity reagent may be an antibody, i.e. antibody specific for CD34, CD133, etc. In some embodiments, the affinity reagent may be a specific receptor or ligand for CD34, CD133, etc., for example, a peptide ligand and receptor; effector and receptor molecules, T-cell receptor specific for CD34, CD133, etc. and the like. In some embodiments, multiple affinity reagents specific to target markers may be used.

Antibodies and T-cell receptors that find use as affinity reagents can be monoclonal or polyclonal and can be produced by transgenic animals, immunized animals, immortalized human and animal B cells, cells transfected with DNA vectors encoding the antibody or T-cell receptor etc. Details of the preparation of antibodies and the possibility of their use as specific binding elements are well known to those skilled in the art. Particularly interesting is the use of labeled antibodies as affinity reagents. Conveniently, these antibodies are conjugated to a tag for use in separation. The tags include magnetic beads that enable direct separation; biotin, which can be removed by avidin or streptavidin bound to a support; fluorochromes, which can be used when using a fluorescently activated cell sorter; or the like, to allow easy separation of a particular cell type. Suitable fluorochromes include phycobiliproteins, such as phycoerythrin and allophycocyanins, fluorescein and Texas red. Often, each antibody is labeled with a separate fluorochrome to allow independent sorting for each marker.

The initial population of cells is interacted with the affinity reagent(s) and incubated for a period of time sufficient to bind the available surface antigens. Incubation is typically at least about 5 minutes and typically less than about 60 minutes. It is desirable to have a sufficient concentration of antibodies in the reaction mixture such that separation efficiency is not limited by a lack of antibodies. The appropriate concentration will be determined by titration, but will typically be a dilution of antibody per volume of cell suspension of approximately 1:50 (i.e. 1 part antibody to 50 parts reaction volume), approximately 1:100, approximately 1:150, approximately 1:200, approximately 1:250, approximately 1:500, approximately 1:1000, approximately 1:2000 or approximately 1:5000. The medium in which the cells are suspended will be any medium that supports cell viability. The preferred medium is phosphate buffered saline containing 0.1 to 0.5% BSA or 1 to 4% goat serum. Various media are available commercially and can be used according to cell characteristics, including Dulbecco's modified Eagle's medium (dMEM), Hanks' balanced salt solution (HBSS), saline, Dulbecco's modified phosphate-buffered saline (dPBS), RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., often supplemented with fetal bovine serum, BSA, HSA, goat serum, etc.

Cells in the contact population, labeled with an affinity reagent, are selected by any convenient affinity separation method, for example, as described above or as known in the art. Once separated, the separated cells can be collected in any suitable medium that supports cell viability, usually having a cushion of serum at the bottom of the tube. Various media are available commercially and can be used according to cell characteristics, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., often supplemented with fetal bovine serum.

In this way, compositions highly enriched in the target cell type, for example hematopoietic cells, are obtained. Cells will comprise 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 will preferably comprise about 95% or more of the enriched cell composition. In other words, the composition will be a substantially pure composition of the target cells.

Cells for transplantation of humanized M-CSF mice, whether they are a heterogeneous cell population or an enriched heterogeneous population, can be transplanted immediately. Alternatively, cells can be frozen at liquid nitrogen temperatures and stored for long periods of time, once thawed they can be reused. In such cases, the cells are typically frozen in a composition of 10% DMSO, 50% serum, 40% buffered medium, or some other similar liquid as is commonly used in the art for storing cells at such freezing temperatures, and thawed in a manner well known in the art. in the art for thawing frozen cultured cells. Additionally or alternatively, cells can be cultured in vitro under various culture conditions. The culture medium may be liquid or semi-liquid, for example containing agar, methylcellulose, etc. The cell population can be conveniently resuspended in an appropriate growth medium such as Iscove's modified DMEM or RPMI-1640, typically supplemented with fetal bovine serum (approximately 5-10%), L-glutamine, thiol, particularly 2-mercaptoethanol, and antibiotics eg penicillin and streptomycin. The culture may contain growth factors to which the cells are sensitive. Growth factors as defined herein are molecules that promote the survival, growth and/or differentiation of cells, both in culture and in intact tissue, through specific action on a transmembrane receptor. Growth factors include polypeptides and non-polypeptide factors.

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

The cells may be transplanted into a humanized M-CSF mouse by any suitable method, including, for example, intrahepatic administration, tail vein administration, retroorbital administration, and the like. Typically, approximately 0.5 x 10 ⁵ - 2 x 10 ⁶ pluripotent or progenitor cells are transplanted, eg approximately 1 x 10 ⁵ - 1 x 10 ⁶ cells or approximately 2 x 10 ⁵ - 5 x 10 ⁵ cells. In some cases, the mouse is sublethally irradiated before human cell transplantation. In other words, the mouse is exposed to a sublethal dose of radiation, for example, as described in the Examples section below and as is well known in the art. The "grafted" humanized M-CSF mouse is then maintained in laboratory animal husbandry for at least 1 week, such as 1 week or more, or two weeks or more, sometimes 4 weeks or more, and in some cases 6 weeks or more to ensure sufficient restoration of the immune system by the engrafted cells.

As shown in the Examples section below, humanized M-CSF mice exhibit a significantly increased ability to engraft and maintain human hematopoietic stem cells compared to other strains of mice that have been generated for this purpose and other M-CSF transgenic mice. For example, intrahepatic transplantation of human fetal liver-derived hematopoietic stem and progenitor cells (CD34+) into neonatal mice results in more efficient differentiation and increased frequencies of human monocytes/macrophages in the bone marrow, spleen, peripheral blood, lung, liver, and peritoneal cavity. A significant proportion of human CD14+CD33+ cells are observed around 16-20 weeks. Specifically, humanized M-CSF mice engrafted with hematopoietic cells exhibit one or more, in some cases two or more, in some cases three or more, in some cases four or more, in some cases all of the following features : They express human M-CSF in bone marrow, spleen, blood, liver, brain, lungs, testes and kidneys at levels comparable to the expression of murine M-CSF in wild-type mice; observed incidence of hCD14⁺CD33⁺spleen cells, which is 2-6 times higher than hCD14⁺CD33⁺in a “grafted” mouse that does not express hM-CSF; observed incidence of hCD14⁺CD33⁺peripheral blood cells, which is 2-8 times higher than hCD14⁺CD33⁺at a “grafted” mouse that does not express hM-CSF; hCD14 level observed⁺CD33⁺ cells of the monocyte/macrophage lineage in the blood from approximately 15 to approximately 40%; hCD14 level observed⁺CD33⁺ monocyte/macrophage lineage cells in the blood approximately 5 to 15% at approximately 20 weeks of age; there is a response to LPS administration that is approximately 1.5-6 times higher relative to the percentage of hCD14⁺CD33⁺ cells in the liver than in mice lacking human M-CSF; increased production of hCD14 is observed⁺CD33⁺hCD45⁺ cells in the spleen approximately 48 hours after LPS administration, with an increase of approximately 2 to 5 times compared to a grafted mouse lacking hM-CSF; there is increased production of serum human IL-6 in response to LPS, with the level of hIL-6 approximately 6 hours after LPS administration increasing approximately 2-5 times compared to a “grafted” mouse lacking hM-CSF; secretion of hTNFα by monocytes and/or macrophages after LPS stimulation is observed in vitro, which is approximately 2-3 times higher than in a “grafted” mouse that lacks the hM-CSF gene; in vitro secretion of hIL-6 by monocytes and/or macrophages after LPS stimulation is observed, which is approximately 2-4 times higher than in a “grafted” mouse that lacks the hM-CSF gene; secretion of hIFNα by monocytes and/or macrophages after I:C stimulation is observed in vitro, which is approximately 3-6 times higher than in a “grafted” mouse that lacks the hM-CSF gene; secretion of hIFNβ by monocytes and/or macrophages during I:C stimulation is observed in vitro, which is approximately 3-6 times higher than in a “grafted” mouse that lacks the hM-CSF gene; increased phagocytosis is observed compared to a genetically modified and “grafted” mouse that lacks the hM-CSF gene; there is increased in vitro chemotaxis in response to Mip3β compared to a genetically modified grafted mouse lacking the hM-CSF gene; and there is an in vitro upregulation of a costimulatory molecule in response to LPS stimulation, where the costimulatory molecule is selected from human CD40, human CD80, human CD86, human HLA-DR, and combinations thereof.

Practical value

Humanized M-CSF mice and humanized M-CSF mice transplanted with human hematopoietic cells, for example, grafted Rag2 ^-/- IL2rg ^-/- hM-CSF mice, and optionally other genetic modifications find various uses. For example, these mice provide a suitable system for modeling human immune diseases and human pathogens. For example, mice are used to model human hematopoietic malignancies arising from an early human hematopoietic cell, such as a human hematopoietic stem or progenitor cell. As another example, mice are used to study human pathogens, such as viruses, fungi and bacteria, that do not normally infect mice.

One such example of a human pathogen that does not normally infect mice is the typhoid pathogen, S. typhi. Typhoid fever affects more than 21 million people worldwide, mostly in developing countries, including approximately 400 cases per year in the United States. Typhoid fever has been treated with many drugs - amoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazimide, chloamphenicol, ciprofloxacin, co-trimoxazole, ertapenem, imipenem, fluoroquinolones (for example, ciprofloxacin, gatifloxacin, ofloxacin), tomycin, sulfadiazine, sulfamethoxazole, tetracycline and their combinations . Recurrent infections are common, limiting disease management with antibiotic therapy. In addition, multidrug resistance is also widespread in S. typhi infections.

New therapeutics, new vaccines, and new ways to test the effectiveness of therapeutics and vaccines are needed. For example, a mouse that can be infected with S. typhi could be used to discover new therapeutics and new vaccines. New therapeutics and new vaccines could be tested in such mice, for example by determining the amount of S. typhi in the mouse (in the blood or a specific tissue) in response to treatment with a putative anti-S. typhi agent, or by inoculating a mouse with a candidate vaccine, followed by inoculation with infective S. typhi, and observing the change in infectivity resulting from inoculation of the candidate vaccine compared to a control mouse that did not receive the vaccine but was infected with S. typhi.

A humanized M-CSF mouse with transplanted human hematopoietic cells, e.g. Rag2 ^-/- IL2rg ^-/- hM-CSF mouse, is suitable for studying human pathogens, i.e. pathogens that infect humans; response of the human immune system to infection by human pathogens; and the effectiveness of the agents in protecting against and/or treating infection by human pathogens. The pathogen may be a virus, fungus or bacteria, etc. Non-limiting examples of viral pathogens include human, swine or avian influenza virus. Non-limiting examples of bacterial pathogens include mycobacteria, such as Mycobacterium tuberculosis (M. tuberculosis) and enterobacteria, such as Salmonella typhi (S. typhi).

For example, “grafted” humanized M-CSF mice are useful as a non-human animal model of S. typhi infection. In comparison, wild-type mice and other known immunocompromised mice (eg, RAG1/RAG2 knockout mice) cannot be infected with S. typhi. As discussed above, the human M-CSF engrafted mice described herein demonstrate improved engraftment of human cells compared to engrafted mice that do not contain human M-CSF protein. This improvement is sufficient to maintain productive S. typhi infection, i.e. the ability of S. typhi to reproduce in the mouse, i.e. S. typhi can "invade" and reproduce in one or more cells of an infected mouse. In a particular embodiment, S. typhi can multiply in a mouse for at least a week, 10 days, two weeks, three weeks, or four weeks after initial introduction or infectious exposure to S. Typhi. In other words, the S. typhi titer or level in the mouse's blood or at least one tissue may persist for at least a week, 10 days, two weeks, three weeks, or four weeks after infectious exposure to S. typhi. Examples of methods for infecting mice with S. typhi and assessing infection can be found, for example, in US Published Application No. 2011/0200982, the disclosure of which is incorporated by reference.

As another example, grafted humanized M-CSF mice, such as grafted Rag2 ^-/- IL2rg ^-/- hM-CSF mice, are useful as a non-human animal model of M. tuberculosis infection. Enhanced engraftment of human hematopoietic cells in mice containing a nucleic acid that encodes the human M-CSF protein is sufficient to support productive infection with M. tuberculosis, that is, the ability of M. tuberculosis to reproduce in the mouse, i.e. M. tuberculosis can “invade” and replicate in one or more cells of an infected mouse. In some such embodiments, the mouse exhibits an antimycobacterial immune response to a human pathogenic mycobacterium, wherein the response involves human immune cell-mediated formation of a granuloma that contains the human immune cell. 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 M. tuberculosis and assessing infection can be found, for example, in US Published Application No. 2011/0200982, the disclosure of which is incorporated by reference.

Other examples of human pathogens that do not infect mice expressing human M-CSF and, in some cases, one or more other genetic modifications, such as those described herein, are well known to those skilled in the art, or that infect wild-type mice but do not. This wild-type mouse after infection is not a model of the immune response that occurs in humans in response to a pathogen.

Such mouse models of pathogen infection are useful in research, for example to better understand the progression of infection in humans. Such mouse models of infection are also useful for drug discovery, for example, to identify candidate substances that protect against or cure infection.

Humanized M-CSF mice engrafted with human hematopoietic cells provide a convenient system for screening candidate substances with other desired in vivo activities, as well as, for example, substances that have the ability to modulate (i.e., promote or inhibit) development and/or activity of hematopoietic cells, for example, the activity of B cells, T cells, NK cells, macrophages, neutrophils, eosinophils, basophils, etc., for example, in normal or disease states, for example, to establish new therapeutic agents and/or a better understanding of the molecular basis of immune system development and function; substances that are toxic to hematopoietic cells, for example, B cells, T cells, NK cells, macrophages, neutrophils, eosinophils, basophils, etc. and their predecessors; and substances that prevent, reduce or reverse the toxic effects of toxic substances on hematopoietic cells, for example, B cells, T cells, NK cells, macrophages, neutrophils, eosinophils, basophils, etc. and their predecessors; etc. As another example, the genetically modified mice described herein provide a system useful for determining the sensitivity of an individual to disease therapy, for example, providing an in vivo platform for screening the sensitivity of an individual's immune system to a substance, for example, a therapeutic agent, in order to predict the sensitivity of the individual to that substance .

In screening studies for biologically active substances, humanized M-CSF mice, e.g. Rag2 ^-/- IL2rg ^-/- hM-CSF mice, have been engrafted with human hematopoietic cells and, in some cases, infected with human pathogens, or cells intended for transplantation of a humanized M-CSF mouse are contacted with a candidate target substance, and the effect of the candidate substance is assessed by monitoring one or more parameters. These parameters may reflect cell viability, for example, the total number of hematopoietic cells or the number of individual hematopoietic cells, or the apoptotic state of cells, for example, the amount of DNA fragmentation, the number of blebbing cells, the amount of phosphatidylserine on the cell surface, and similar methods well known in the art. this field of technology. Alternatively or additionally, parameters may reflect the ability of cells to differentiate, for example, quantitative ratios of differentiated cells and differentiated cell types. Alternatively or additionally, parameters may reflect cell function, for example, cytokines and chemokines produced by cells, the ability of cells to invade the site of infection, the ability of cells to modulate, i.e. promote or inhibit the activity of other cells in vitro or in vivo, etc. Other parameters may reflect the degree of pathogen infection in the animal, such as the titer of the pathogen in the mouse, the presence of granuloma in the mouse, etc.

Parameters are quantifiable components of a cell, especially components that can be accurately measured, preferably in a high-throughput system. The criterion can be any cell component or product of a cell, including a cell surface determinant, a receptor, a protein or conformational or post-translational modifications thereof, a lipid, a hydrocarbon, an organic or inorganic molecule, a nucleic acid such as mRNA, DNA, etc. or a portion derived from such a cell component, or combinations thereof. While most parameters will provide a quantitative reading, in some cases a semi-quantitative or qualitative result is acceptable. Readings may include individual specific values, or may include mean, median, or change, etc. Typically, a number of reading values for each parameter are obtained from many identical studies. Variability (variability) is to be expected, with a range of values for each set of parameters under study being obtained using standard statistical methods in combination with the normal statistical method used to obtain the individual values.

Candidate substances of interest for screening include known and unknown compounds that span numerous classes of chemical compounds, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, vaccines, antibiotics, or other agents suspected of having properties antibiotics, peptides, polypeptides, antibodies, pharmaceuticals approved for human use, etc. An important aspect of the invention is the evaluation of candidate substances, including toxicity testing; etc.

Candidate substances include organic molecules containing functional groups required for structural interactions, in particular hydrogen bonding groups, and typically include at least an amino, carbonyl, hydroxyl or carboxyl group, often at least two of the functional chemical groups. Candidate substances often include cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate substances are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives thereof, structural analogues, or combinations thereof. Included are pharmacologically active drugs, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, hormones or hormone antagonists, etc. Examples of pharmaceutical agents suitable for the invention are described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition. Also included are toxins and biological and chemical warfare agents, for example, see Somani, S. M. (Ed.), "Chemical Warfare Agents," Academic Press, New York, 1992).

Candidate substances of interest for screening also include nucleic acids, for example, nucleic acids that encode miRNAs, shRNAs, antisense molecules or microRNAs, or nucleic acids that encode polypeptides. A variety of vectors are available that are suitable for transferring nucleic acids into target cells. Vectors may be maintained episomally, e.g., as plasmids, minicircular DNA, virus-derived vectors such as cytomegalovirus, adenovirus, etc., or they may be integrated into the genome of the target cell through homologous recombination or random integration, e.g. retrovirus-based vectors such as MMLV, HIV-1, ALV, etc. Vectors can be delivered directly into the subject cells. In other words, pluripotent cells are contacted with vectors containing the target nucleic acid, so that the vectors are taken up by these cells.

Methods for contacting cells, for example cells in culture or mouse cells, with nucleic acid vectors, such as electroporation, calcium phosphate transfection and lipofection, are well known in the art. Alternatively, the target nucleic acid may be provided to the cells via a virus. In other words, cells come into contact with viral particles containing the target nucleic acid. In particular, retroviruses, for example lentiviruses, are suitable for the method of the invention. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins necessary for productive infection. That is, development (culturing) in a packaging cell line is required for vector replication. To generate viral particles containing the target nucleic acid, retroviral nucleic acid containing nucleic acids are packaged into a viral capsid using a cell packaging line. Different packaging lines of cells ensure the incorporation of different envelope proteins into the capsid, this envelope protein determines the cell specificity of the virus particle. Envelope proteins can be of at least three types: ecotropic, amphotropic and xenotropic. Retroviruses packaged with ecotropic envelope proteins, such as MMLV, are capable of infecting most mouse and rat cell types, and are generated by using ecotropic packaging cell lines such as BOSC23 (Pear et al. (1993) P.N.A.S. 90:8392-8396). Retroviruses bearing amphotropic envelope proteins such as 4070A (Danos et al, supra.) are capable of infecting most mammalian cell types, including humans, dogs and mice, and are generated using amphotropic packaging cell lines such as PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with xenotropic envelope proteins, such as AKR env, are capable of infecting most mammalian cell types, with the exception of mouse cells. An appropriate cell packaging line can be used to ensure that the packaged viral particles are targeted to target cells - in some cases, transplanted cells, and in some cases, host cells, i.e. humanized M-CSF.

Vectors used to provide the target nucleic acid to the target cells will typically contain suitable promoters to drive the expression, ie, transcriptional activation, of the target nucleic acid. This may include ubiquitous promoters, such as the CMV-b-actin promoter, or inducible promoters, such as promoters that are active in specific cell populations or that respond to the presence of drugs such as tetracycline. By transcriptional activation is meant that transcription will increase in the target cell above the baseline level by at least about 10-fold, at least about 100-fold, and more commonly, at least about 1000-fold. In addition, the vectors used to deliver reprogramming factors into cells may include genes that must subsequently be deleted, for example, using a recombinase system such as Cre/Lox, or the cells expressing them may be disrupted, for example, by switching on genes that provide the possibility of selective toxicity, such as herpes virus TK, bcl-xs, etc.

Candidate substances of interest for screening also include polypeptides. Such polypeptides may optionally be coupled to a polypeptide domain that increases the solubility of the product. The domain may be linked to the polypeptide through a specific protease cleavage site, for example, a TEV sequence that is cleaved by TEV protease. The linker may also include one or more flexible sequences, for example, from 1 to 10 glycine residues. In some embodiments, cleavage of the fusion protein is carried out in a buffer solution that maintains the solubility of the product, for example, in the presence of 0.5 to 2 M urea, in the presence of polypeptides and/or polynucleotides that increase solubility, and the like. Domains of interest include endosomolytic domains, for example, the influenza HA domain; and other polypeptides that promote production, for example, IF2 domain, GST domain, GRPE domain, etc. Additionally or alternatively, such polypeptides may be included in the formulation to improve stability. For example, peptides can be PEGylated because the polyethyleneoxy group provides an increased lifetime in the bloodstream. Polypeptides can be linked to another polypeptide to provide additional functionality, for example, to increase in vivo stability. In most cases, such fusion partners are stable plasma proteins which can, for example, extend the in vivo plasma half-life of the polypeptide if it is present as a fusion, particularly when the stable plasma protein is an immunoglobulin constant domain. In most cases where a stable plasma protein normally occurs in a multimeric form, such as immunoglobulins or lipoproteins, in which the same or different polypeptide chains are usually linked by disulfide and/or non-covalent bonds to form a multi-chain composite polypeptide, fusion forms containing polypeptide will also be produced and used as a multimer having essentially the same structure as the stable plasma protein precursor. These multimers will be homogeneous with respect to the polypeptide agent they contain, or they may contain more than one polypeptide agent.

The candidate polypeptides may be produced by eukaryotic cells or may be produced by prokaryotic cells. Additionally, they can be unfolded, for example, by heat denaturation, DTT reduction, etc., and subsequently refolded using methods known in the art. Modifications of interest that do not alter the primary sequence include chemical derivatization of polypeptides, such as acylation, acetylation, carboxylation, amidation, etc. Also included are modifications in the form of glycosylation, such as those created by modification of the glycosylation profiles of the polypeptide during synthesis and processing or in additional processing steps; for example, by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes. Also included are sequences having phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine or phosphothreonine. Polypeptides can be modified using conventional techniques of molecular biology and synthetic chemistry in order to improve their resistance to proteolytic degradation or optimize solubility properties or render them more suitable for therapeutic use. Analogs of such polypeptides include polypeptides containing residues other than naturally occurring L-amino acids, such as D-amino acids or unnatural synthetic amino acids. D-amino acids may be substituted at some or all amino acid residues.

The candidate polypeptide can be prepared by in vitro synthesis using conventional methods known in the art. Various commercial synthesis facilities are available, such as automated synthesis units from Applied Biosystems, Inc., Beckman, etc. When using synthesizers, natural amino acids can be replaced by unnatural amino acids. The specific sequence and method of preparation will be determined by convenience, economics, required purity, etc. Alternatively, the candidate polypeptide can be isolated and purified according to conventional recombinant synthesis methods. A lysate may be prepared from cells of the expressing host, and the lysate may be purified using HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification method. In general, the compositions used will comprise at least 20% by weight of the desired product, more often 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, relative to contaminants (impurities) associated with the process of obtaining the product and its purification. Generally, the percentage is based on total protein.

In some cases, the candidate polypeptides that are screened are antibodies. The term “antibody” or “antibody fragment” includes any molecular structure containing a polypeptide chain with a specific shape that closely matches and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The specific or selective matching of a given structure and its specific epitope is sometimes called “lock and key” compatibility. The primary antibody molecule is an immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, such as human, rodent, rabbit, cow, sheep, pig, dog, other mammal, chicken, other birds, etc. are considered “antibodies”. The antibodies used in the present invention can be either polyclonal antibodies or monoclonal antibodies. Antibodies are usually found in the medium in which cells are cultured.

Candidate compounds can be obtained from a variety of sources, including libraries of synthetic or natural compounds. For example, numerous methods are available for the random and targeted synthesis of a wide range of organic compounds, including biomolecules, including the expression of random oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or easy to obtain. In addition, natural or synthetically produced libraries and compounds can be easily modified using conventional chemical, physical and biochemical methods and used to generate combinatorial libraries. Moreover, known pharmacological agents can be subjected to targeted or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to obtain structural analogues.

Screening of candidate substances for biological activity is carried out by introducing the substance into at least one or usually a plurality of samples, sometimes together with samples that do not have the drug. The change in parameters in response to the substance is measured, and the result is assessed by comparison with control cultures, for example, in the presence and absence of the drug, with results obtained with other agents, etc. In cases where screening is performed to identify candidate substances that will prevent, reduce, or reverse the effects of a toxic agent, the screening is typically conducted in the presence of the toxic agent, with the toxic agent added at the time most appropriate to obtain the results. For example, in cases where the protective/preventive ability of a candidate substance is determined, the candidate substance may be added before the toxic agent, simultaneously with the toxic agent, or after treatment with the toxic agent. As another example, in cases where the ability of a candidate substance to abolish (reverse) the effects of a toxic agent is determined, the candidate substance may be added after treatment with the toxic agent. As stated above, in some cases, the sample is a humanized M-CSF mouse that has been engrafted with cells, i.e. the candidate substance is “delivered” into a humanized M-CSF mouse in which the cells have been engrafted. In some cases, the sample represents cells to be engrafted, i.e. the cells are supplied with the candidate substance before transplantation.

If the candidate substance is to be administered directly to a mouse, the substance can be administered using any of a number of methods well known in the art for administering peptides, small molecules and nucleic acids to mice. For example, the substance may be administered orally, transmucosally, topically, intradermally, or by injection, such as intraperitoneal, subcutaneous, intramuscular, intravenous, or intracranial injection, and the like. The substance may be administered in a buffer solution, or it may be included in any of a number of compositions, for example by combination with a suitable pharmaceutically acceptable base. "Pharmaceutically acceptable bases" may be bases approved by a State or Federal Government regulatory agency or listed in the US Pharmacopoeia or other generally accepted pharmacopoeia for use in mammals such as humans. The term "vehicle" refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is formulated for administration to a mammal. Such pharmaceutical bases may be lipids, eg liposomes, eg liposomal dendrimers; liquids such as water and oils, including liquids of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline; gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon oxide, urea and the like. In addition, auxiliary agents, stabilizing agents, thickeners, lubricants and coloring agents can be used. Pharmaceutical compositions may be formulated in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections and inhalations, gels, microspheres and aerosols. The substance, once administered, may be distributed systemically or may be localized using local administration, intramural administration, or an implant may be used to maintain an active dose at the site of implantation. The active substance may be contained in an immediate release formulation or in a sustained release formulation. Under some conditions, particularly when used in the central nervous system, it may be necessary to formulate substances designed to cross the blood-brain barrier (BBB). One strategy for drug delivery across the blood-brain barrier (BBB) involves passage of the BBB, either osmotic agents such as mannitol or leukotrienes, or biochemically through the use of vasoactive substances such as bradykinin. The BBB disrupting agent may be co-administered with the active agent when the composition is administered by intravascular injection. Other strategies for traversing the BBB may involve the use of endogenous transport systems, including Caveolin-1-mediated transcytosis, the use of carrier carriers such as glucose and amino acid transporters, receptor-mediated transcytosis of insulin or transferrin, and active efflux transporters such as p-glycoprotein. For use in the invention, active transport molecules can also be conjugated to therapeutic compounds to facilitate transport across the endothelial wall of blood vessels. Alternatively, delivery of the agents may occur by local delivery, for example by intrathecal delivery, for example, into the Ommaya reservoir (see, for example, US Pat. Nos. 5,222,982 and 5,385,582, incorporated by reference); by bolus injection, for example, with a syringe, for example, intravitreal or intracranial; by continuous infusion, for example, by catheterization, for example, with convection (see, for example, US application No. 20070254842, incorporated herein by reference); or by implanting a device, after which a substance is reversibly attached to it (see, for example, US patent applications 20080081064 and 20090196903, incorporated herein by reference).

If the substance(s) are provided to the cells prior to transplantation, the substances are conveniently added in solution or readily soluble form to the environment of the cells in culture. Substances may be added to a flow-through system (as a stream) intermittently or continuously, or alternatively by adding a single dose (bolus) of the compound once or in increments to another continuously flowing solution. The flow system uses two fluids, where one is a physiologically neutral solution and the other is the same solution with the test compound added. The first liquid washes the cells, followed by the second. In the single solution method, a bolus of the test compound is added to the volume of media surrounding the cells. The total concentrations of culture medium components should not change significantly between bolus injections, or between two solutions in a flow-through method.

Multiple assays with different concentrations of a substance can be performed in parallel to obtain different responses to different concentrations. As is known in the art, to determine the effective concentration of a substance, a concentration range resulting from 1:10 dilutions or other log scale is typically used. If necessary, concentrations can be further optimized using a second series of dilutions. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the detection level of the substance, or at a concentration of the substance or below that does not produce a detectable change in phenotype.

Analysis of the response of humanized mouse M-CSF cells to a candidate substance can be performed at any time after treatment with the substance. For example, cells can be analyzed after 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 more after exposure to candidate substance, for example, after 2 months, 4 months, 6 months or more. In some embodiments, the analysis includes analysis at multiple time points. The selection of time point(s) for analysis will be based on the type of analysis being performed, as will be apparent to one skilled in the art.

The assay may include measuring any of the parameters described herein or known in the art to determine cell viability, cell proliferation, cell characteristics, cell morphology, and cell function, particularly as they may relate to immune cells. For example, flow cytometry can be used to determine the total number of hematopoietic cells or the number of hematopoietic cells of a particular type. Histochemical or immunohistochemical assays may be performed to determine the apoptotic state of cells, such as terminal deoxyuridine dUTP nick end labeling (TUNEL) to measure DNA fragmentation, or an immunohistochemical assay to detect Annexin V binding to phosphatidylserine on the cell surface. Flow cytometry can also be used to estimate the percentage of differentiated cells and the types of differentiated cells, for example to determine the ability of hematopoietic cells to differentiate in the presence of a substance. ELISA, Western blotting and Northern blotting can be performed to determine the levels of cytokines, chemokines, immunoglobilins, etc. expressed in engrafted humanized M-CSF mice, for example, to assess the functioning of engrafted cells. In vivo assays may also be performed to test the functioning of immune cells, as well as assays relevant to specific diseases or disorders of interest, such as diabetes, autoimmune disease, graft-versus-host disease, AMD, etc. See, for example, Current Protocols in Immunology (Richard Coico, ed. John Wiley & Sons, Inc. 2012) and Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), the disclosure of which is incorporated herein by reference.

For example, a method of determining the effect of a substance on a human pathogen is provided, comprising exposing a grafted humanized M-CSF mouse, e.g., a grafted Rag2 ^-/- IL2rg ^-/- hM-CSF mouse, to an effective amount of a human pathogen, an effective amount pathogen is the amount of pathogen required to cause infection in a mouse; allowing the mouse to become infected with a pathogen; measuring the contamination rate over a period of time in the presence of the substance; and comparing this measurement with that of a naïve humanized M-CSF-engrafted mouse. A substance is defined as an antipathogen, eg, an anti-S. typhi substance, if it reduces the amount of pathogen in the blood or tissue of a mouse by at least half after a single administration or two or more administrations of the substance over a selected period of time.

As another example, a method is provided for determining whether a pathogen isolate or strain of interest is drug resistant, such as multidrug resistant. In such methods, a grafted humanized M-CSF mouse, for example a grafted Rag2 ^-/- IL2rg ^-/- hM-CSF mouse, is exposed to an effective amount of a human pathogen isolate or strain of interest, the effective amount of pathogen being the amount of pathogen required to mouse infection; provides the possibility of infecting a mouse with a pathogen; an indicator of infection, such as the titer of an isolate or strain of interest in the blood or tissue of a mouse, the ability of an isolate or strain of interest to maintain an infection in a mouse, or the ability of an isolate or strain of interest to reproduce after administration of a drug, is measured in the presence of the drug; and this measurement is compared with a measurement obtained from a “grafted” M-CSF humanized mouse infected with the pathogen but not exposed to the test substance. Examples of drugs of interest include amoxicillin, ampicillin, cefotaxime, ceftriaxone, ceftazidime, chloramphenicol, ciprofloxacin, co-trimoxazole, ertapenem, imipenem, fluoroquinolones (eg, ciprofloxacin, gatifloxacin, ofloxacin), streptomycin, sulfadiazine, azole, tetracycline and their combination. In a particular embodiment, administration of the drug or combination of drugs occurs at least one week, 10 days, two weeks, three weeks, or four weeks after infectious exposure to the isolate or strain of interest.

Other examples of the use of mice of the invention are provided elsewhere in this specification. Additional uses of genetically modified and grafted mice described in this disclosure will be apparent to one skilled in the art upon reading this disclosure.

Reagents, devices and kits

Also provided are reagents, devices and kits thereof for practicing one or more of the methods described above. Subject reagents, devices and their kits may vary greatly.

In some embodiments, the reagents or kits will contain one or more substances for use in the described methods. For example, the kit may contain a humanized M-CSF mouse. The kit may contain reagents for breeding humanized M-CSF mice, such as primers, and, in some cases, reagents for genotyping humanized M-CSF mice. The kit may contain human hematopoietic cells or an enriched population of human hematopoietic progenitor cells for transplantation of a humanized M-CSF mouse, or reagents for preparing a population of hematopoietic cells or an enriched population of human hematopoietic cells for transplantation of a humanized M-CSF mouse. Other reagents may include reagents for determining the viability and/or function of hematopoietic cells, for example, in the presence/absence of a candidate substance, for example, one or more antibodies specific for markers that are expressed by different types of hematopoietic cells, or reagents for detecting a particular cytokine, chemokine, etc. Other reagents may include culture medium, culture supplements, matrix compositions, and the like.

In addition to the above components, the kits will further include instructions regarding the use of the methods. These instructions may be present in the kits in various forms, one or more of which may be present in the kit. One form in which these instructions may be present is the information printed on a suitable medium or substrate, such as a sheet or sheets of paper on which the information is printed, in a kit package, on a package insert, etc. Another method is a medium that can be read by a computer, such as a floppy disk, CD, etc., on which the information is recorded. Another method that may occur is a website address, which can be used to obtain information from a remote site via the Internet. The sets may contain any convenient devices.

EXAMPLES

The following examples are used to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to The experiments presented below represent all possible experiments or the only experiments performed. Efforts have been made to ensure accuracy in terms of numbers used (eg quantities, temperatures, etc.), however, some experimental errors and deviations must be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or approximately atmospheric.

Colony-stimulating factor-1 (CSF-1) or macrophage colony-stimulating factor (M-CSF) is one of the first cytokines discovered that promotes hematopoiesis. In the hematopoietic system, M-CSF is believed to specifically act on myeloid progenitors, starting at the common myeloid progenitor (CMP) stage, and promote the differentiation of CMPs into the monocyte/macrophage lineage (Sherr, C.J. et al. (1988) Macrophage colony-stimulating factor , CSF-1, and its proto-oncogene-encoded receptor, Cold Spring Harb Biol. 53 Pt 1:521-530). In addition, M-CSF is required for the survival, adhesion and motility of macrophages (Pixley, F.J., and Stanley, E.R. (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; 1, Mol. Reprod. 1997;46:4-10). In addition to its key role in myeloid differentiation, M-CSF is vital for osteoclast differentiation, for the differentiation, survival and proliferation of female reproductive tract cells and for placental formation (Pixley et al. (2004); Socolovsky et al. (1998); Stanley et al. . (1997)). M-CSF is produced by a range of cell types, 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), binding of M-CSF to its receptor results in tyrosine phosphorylation of Fms and subsequent phosphorylation of several host cell proteins such as Grb2, Shc, Sos1 and p85 (Pixley et al (2004); Rohrschneider, L.R. et al. (1997) Growth and differentiation signals regulated by M-CSF. Dev. Stanley, E.R. (2003) Proteomic approaches to the analysis of early events in colony-stimulating factor-1 signal transduction, Mol. Cell Proteomics 2:1143-1155).

The inventors hypothesized that defective differentiation of human myeloid cells in humanized mice may be due to a lack of specific signals that promote myeloid differentiation. To confirm this, the inventors created a new generation of humanized mice capable of secreting human M-CSF at a physiological level from the corresponding tissues. Analysis of these humanized M-CSF mice both qualitatively and quantitatively showed normal expression of human M-CSF. Analysis of humanized M-CSF mice engrafted with human CD34 ⁺ cells revealed increased frequencies of human monocytes/macrophages in various tissues. Moreover, human monocytes/macrophages obtained from these mice showed improved functional properties.

The humanized M-CSF mice described here demonstrate increased frequency and function of human myeloid cells. Insertion of human M-CSF into the mouse M-CSF locus Balb/c of mice lacking recombination activating 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 number 1.NM_013563.3) (Balb/c Rag2 ^-/- γc ^-/- mice) resulted in significant expression of human M-CSF in these mice, both qualitatively and quantitatively. Intrahepatic administration (transfer) of hematopoietic stem and progenitor cells derived from human fetal liver (CD34 ⁺ ) to humanized M-CSF (M-CSF ^h/h ) to newborn mice led to more efficient differentiation and an increased frequency of occurrence of human monocytes/macrophages in the bone brain, spleen and peripheral blood. In addition, M-CSF ^h/h mice exhibited a long-lasting ability to maintain human monocyte/macrophage differentiation even 20 weeks after transplantation. Moreover, M-CSF ^h/h mice contain resident human monocytes/macrophages in various tissues, including the liver and lungs, in contrast to control unmodified mice. Human monocytes/macrophages derived from humanized M-CSF mice also exhibit enhanced functional properties such as migration, phagocytosis, activation, and response to LPS.

Example 1: Cell Preparations, Analytical Methods and Assays

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 using human tissue were performed in agreement with the Yale University Research Committee.

To isolate human CD34 ⁺ cells, fetal liver samples were washed once with PBS and cut into small pieces and treated with collagenase D (100 ng/ml) at 37°C for 45 minutes. Single cell suspensions were prepared and mononuclear cells were isolated by density gradient centrifugation (Lymphocyte Fractionation Medium, MP biomedicals). CD34 ⁺ cells were isolated after treating the cells with anti-human CD34 microbeads followed by MACS™ technology (Miltenyi Biotech).

For transplantation, newborn mice (1 day after birth) were sublethally irradiated with two separate doses (2 x 150 cGy) 4 hours apart, and then 1 x 10 ⁵ - 2 x 10 ⁵ purified human CD34 ⁺ cells in 20 μl PBS were injected into the liver using a 22 gauge needle (Hamilton Company, Reno, NV).

Isolation and culture of mesenchymal stromal cells (MSC). The long bones of the mouse were isolated and the bone marrow cells were washed away with a strong stream of liquid. The bones were cut into pieces and digested using a cocktail of collagenases D and P (25 ng/ml) for 45 minutes at 37°C. Cell suspensions 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.

Antibodies and flow cytometry. Single cell suspensions were analyzed by flow cytometry using FACS Calibur or LSRII and CELLQUEST™ software, FACS DIVA™ software (BD Biosciences, San Jose, CA), or FLOWJO™ software (Tree Star, Inc., Ashland, OR). respectively. Sorting of cells into specific subpopulations was performed using a FACS ARIA™ cell sorter (BD Biosciences, San Jose, CA).

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

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

Cell culture. To differentiate murine bone marrow macrophages, cells were seeded in 6-well plates in the presence of DMEM with 10% FCS and necessary supplements (2 mM L-glutamine, 1% penicillin-streptomycin, and 1 mM essential amino acids). Cells were treated with recombinant mouse M-CSF (10 ng/ml) or recombinant human M-CSF (10 ng/ml) for 7 days. The cell culture supernatant was removed once every three days and replaced with fresh medium and cytokines.

To study human macrophages, such as activation, phagocytosis and migration, 2 x 10 ⁵ CD45 ⁺ CD14 ⁺ CD33 ⁺ spleen cells were sorted and cultured in vitro in DMEM with 15% human AB serum and necessary supplements (2 mM L-glutamine, 1% penicillin-streptomycin and 1mM essential amino acids).

Analysis of activation, phagocytosis and migration. To stimulate LPS in vivo, mice were administered intraperitoneal (i.p.) LPS (100 ng/g body weight). For LPS stimulation in vitro, LPS (10 ng/ml) was added to the cells and cultured for 1 or 2 days. For poly I:C stimulation in vitro, cells were cultured in the presence of poly I:C (10 μg/ml) for 6 or 12 hours.

Phagocytosis assays were performed using the commercially available VYBRANT™ Phagocytosis Assay Kit (Invitrogen) according to the manufacturer's instructions.

Migration assays were performed using a commercially available QCM™ Cell Migration Chemotactic Assay Kit (Millipore) according to the manufacturer's instructions.

RNA extraction and real-time PCR. Total RNA was isolated using a commercially available system (RNEASY™ Mini kit, Qiagen). cDNA was synthesized using oligo dT primer and reverse transcriptase (Roche). The PCR reaction was performed in duplicate using the 7500 real time PCR and power SYBR™ Green PCR master mix systems (Applied Biosystems) according to the manufacturer's instructions using the following gene-specific primer pairs: Human CSF1 (forward: 5′-TACTGTAGCCACATGATTGGGA-3′ (SEQ ID NO:1) and reverse: 5′-CCTGTGTCAGTCAAAGGAAC-3′ (SEQ ID NO:2)), Mouse csf1 (forward: 5′-CGACATGGCTGGGCTCCC-3′ (SEQ ID NO:3) and reverse: 5′ -CGCATGGTCTCATCTATTTAT- 3′ (SEQ ID NO:4), Human IFNa (forward: 5′-GTACTGCAGAATCTCTCCTTTCTCCTG-3′ (SEQ ID NO:5) and reverse: 5′-GTGTCTAGATCTGACAACCTCCCAGGCACA-3′ (SEQ ID NO:6)), Human IFNb (forward: 5′-TTGTGCTTCTCCACTACAGC-3′ (SEQ ID NO:7) and reverse: 5′-CTGTAAGTCTGTTAATGAAG-3′ (SEQ ID NO:8)), Mouse hprt primers (forward: 5′-AAGGACCTCTCGAAGTGTTGGATA (SEQ ID NO :9) and reverse: 5′-CATTTAAAAGGAACTGTTGACAACG-3′ (SEQ ID NO:10)) and Human HPRT primers (forward: 5′-CTTCCTCCTCCTGAGGAGTC-3′ (SEQ ID NO:11) and reverse: 5′-CCTGACCAAGGAAAGCAAAG- 3′ (SEQ ID NO:12)). For conventional PCR, DNA from target cells was isolated using a commercially available kit (DNEASY™ blood and tissue kit, Qiagen) and PCR analysis was performed using gene-specific primer pairs.

ELISA. For quantitative cytokine studies, serum or cell culture supernatants were collected and ELISA assays were performed using commercially available human IL6 and human TNF ELISA kits (Ray Biotech, Inc., GA) according to the manufacturer's instructions.

Histology. Solid organs were fixed in 4% PFA. Fixed organs were embedded in paraffin (Blue RiBbon; Surgipath Medical Industries). Blocks were dissected, and 5-μm sections were stained with H&E and then coverslipped using standard methods. The sections were stored without any medium. At room temperature, digital images were acquired using a Zeiss Axio Imager.A1 optical microscope (with 2× and 10× objective lenses), an AxioCam MRc5 camera, and AxioVision 4.7.1 image processing software (Carl Zeiss Microimaging LLC).

Statistical analysis. Data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed Student's t test. P values >0.05 were considered not to be statistically significant, and P values <0.05 were presented as *.

Example 2: Genetically Modified Mice for Engraftment

Human M-CSF knock-in strategy. A targeting construct to replace the mouse M-CSF nucleic acid sequence with the human M-CSF nucleic acid sequence (VELOCIGENE® Allele registration number 5093) in a single targeting step was constructed using VELOCIGENE® technology as described previously (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 DNAs were obtained from bacterial artificial chromosome (BAC) RPCI-23, clone 373B18, and from BAC RPCI-11, clone 101M23, respectively. Briefly, RAG2 ^+/- γc ^-/- mouse embryonic stem (ES) cells derived from the commercially available V17 ES cell line (BALB/cx 129 F1) were electroporated with a linearized targeting construct generated by gap repair cloning and containing arms located in the 3' and 5' directions, homologous to mouse M-SCF, flanking 17.5 kb of human M-CSF sequence extending from exon 2 to 633 nuclei in the 5' direction from non-coding exon 9, and flanked by a loxP selection cassette. Murine ES cells carrying a heterozygous deletion of the M-CSF gene were identified by screening for loss of the allele using 2 TaqMan qPCR probes that recognized sequences in intron 2 (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)′; TDR primer, 5′-AATAGGAAGAACGAACAGGTCTAATACC-3′ (SEQ ID NO:18)) mouse Csf1 gene . Simultaneous replacement of the mouse gene with the human CSF1 gene was confirmed using a TaqMan Gain-of-Allele probe, which detected one copy of the 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 the neomycin resistance cassette (neor) (forward primer, 5′-GGTGGAGAGGCTATTCGGC-3′ (SEQ ID NO:22); probe, 5′-TGGGCACAACAGACAATCGGCTG-3′ (SEQ ID NO:23); reverse primer, 5′-GAACACGGGCGGCATCAG-3′ (SEQ ID NO:24); ) see Fig. 8. qPCR probes recognizing the CSF1 sequence do not amplify mouse genome. Similar probes were used to confirm the genotypes of mice derived from the target ES cells were confirmed by neo ^r TaqMan probe. probe were supplied by Biosearch Technologies. The probes were labeled with 6-carboxyfluorescein (FAM) at their 5′ ends and BHQ-1 at their 3′ ends.

Correctly targeted ES cells were then electroporated with a vector transiently expressing Cre to remove the selection cassette. Target ES cell clones without a selection cassette were introduced into the 8 cell stage mouse embryo using the VELOCIMOUSE® method (Poueymirou et al. (2007)). VELOCIMICE® (F0 mice derived entirely from donor ES cells) carrying the humanized M-CSF gene (VG 5093) were identified by genotyping for loss of the mouse allele and gain of the human allele using a modification of the allele analysis method (Valenzuela et al. ( 2003)).

Keeping mice. 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 special sterile conditions in the Yale University animal facility. All experiments on mice were approved by the Institutional Animal Care and Use Committee of Yale University.

Generation of humanized M-CSF mice. To confirm that physiological expression of human M-CSF in the mouse results in improved differentiation of human macrophages in humanized mice, Balb/c Rag2 ^−/− γc ^−/− mice were generated to express human M-CSF. The Rag2 ^-/- γc ^-/- lacking Balb/c strain serves as a successful model system for studies of the human immune system in mice (Traggiai E et al. (2004) Development of a human immune adaptive system in cord blood cell-transplanted mice, Science 304:104-107). To avoid supraphysiological expression of human M-CSF in these mice, a strategy was adopted to replace the mouse M-CSF coding sequence with a human counterpart. To replace most of the M-CSF open reading frame with the human M-CSF coding sequence (Velocigene® Allele accession number 5093) in a single targeting step, a construct was generated (Fig. 8) using the VELOCIGENE® technology as previously described (Valenzuela et al. (2003)). It should be noted that the mouse promoter and other regulatory elements (such as the 5'UTR) were retained in this vector. The linearized targeting vector was introduced into Balb/cx 129 Rag 2 ^+/- γc ^-/- embryonic stem cells by electroporation. Correctly targeted target ES cells were then electroporated with a vector transiently expressing Cre to remove the selection cassette. Target ES cell clones without a selection cassette were introduced into the 8-cell stage mouse embryo using the VELOCIMOUSE® method (Poueymirou et al. (2007)). VELOCIMICE® (F0 mice entirely derived from donor ES cells) carrying the humanized M-CSF gene (VG 5093) were identified by genotyping for loss of the mouse allele and gain of the human allele using a modification of the allele analysis method (Valenzuela et al. (2003) )). Chimeric Balb/c Rag2 ^-/- γc ^-/- mice and germline transmitted mice with mouse and human M-CSF (M-CSF ^m/h ; heterozygous knock-in) were created by sequentially crossing offspring. and only human M-CSF (M-CSF ^h/h ; homozygous knock-in).

Characterization of humanized M-CSF mice. The expression of human M-CSF in humanized M-CSF mice was assessed. M-CSF organs were taken for the study^m/m or M-CSF^h/hmice and analyzed the mRNA expression of mouse and human M-CSF using species-specific primers. As shown in FIG. 1A and 1B, M-CSF is expressed in most organs analyzed, including bone marrow, spleen, blood, liver, brain, lung, testes, and kidney. However, M-CSF expression was not detected in the thymus and skin. It should be noted that the expression profile of mouse and human M-CSF was comparable between M-CSF^m/mAnd M-CSF^h/hmice, respectively. Next, the expression levels of mouse and human M-CSF were quantified in M-CSF^m/m, M-CSF^m/h And M-CSF^h/hmice. Bone marrow mesenchymal stromal cells (MSCs) were isolated and M-CSF mRNA expression levels were quantified by real-time PCR (Fig. 1C) and (secreted) M-CSF protein by ELISA (Fig. 1D). M-CSF^m/mmice expressed only mouse M-CSF, M-CSF^m/hmice expressed both murine and human M-CSF and M-CSF^h/hmice expressed only human M-CSF. The expression level of human M-CSF was comparable to mouse M-CSF. Consistent with these data, serum CSF-1 analysis showed comparable levels of CSF-1 protein expression in m/m, h/m, and h/h mice ( Fig. 1E ). Hemizygosity does not result in a decrease in gene and protein expression levels, indicating that gene dosage levels do not appear to be limiting for this cytokine.

To determine whether replacement of murine M-CSF with human M-CSF results in deleterious effects, particularly on bone and hematopoiesis, M-CSF was analyzed^h/hmice of different ages. Previous studies have confirmed that mice with a defective M-CSF signaling pathway (Csf1^{op/op and}Csf1r^-/-) there is a lack of teething and bone defects (Dai, X.M. et al. (2002) Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects, Blood 99 :111-120; Felix, R. et al. (1990) Macrophage colony stimulating factor restores in vivo bone resorption in the op/op osteopetrotic mouse, Endocrinology 127:2592-2594; ) Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse, Proc. Natl Acad. USA 87:4828-4832; is in the coding region of the macrophage colony stimulating factor gene, Nature 345:442-444). In contrast, M-CSF^h/hthe mice showed normal bone and tooth properties. Moreover, unlike Csf1^op/op And Csf1r^-/-mice, total bone marrow cell content (Figure 2A), myeloid cell frequencies in bone marrow, spleen (SP), and peripheral blood (PB) (Figure 2B), and macrophage frequencies in bone marrow and spleen (Figure 2C) were comparable to M-CSF^m/m, M-CSF^h/mand M-CSF^h/h mice. Consistent with these observations, the frequencies of compartment HSCs (including long-repopulating HSCs, short-term repopulating HSCs, and multipotent progenitors) and myeloid progenitors (including common myeloid progenitors, granulocyte-monocyte progenitors, and megakaryocyte-erythrocyte progenitors) were comparable in M-CSF^m/m, M-CSF^h/mand M-CSF^h/h mice (Fig. 9).

A possible explanation for the normal hematopoiesis and bone development in M-CSF ^h/h mice may be that human M-CSF cross-reacts with mouse cells. To test this, bone marrow cells isolated from M-CSF ^m/m were cultured in the presence of recombinant mouse M-CSF or recombinant human M-CSF. While bone marrow cells cultured in the absence of the cytokine did not survive, cells cultured in the presence of human or murine M-CSF showed comparable levels of differentiation in vitro (Fig. 2d). Analysis of these in vitro differentiated macrophages for the expression of costimulatory molecules and MHC showed comparable levels of these molecules in the presence of human or murine M-CSF (Fig. 2e). Consistent with our results, previous studies have confirmed that human M-CSF is active in murine target cells, whereas murine M-CSF does not cross-react with human cells (Sieff, CA (1987) Hematopoietic growth factors, J. Clin Invest 79:1549–1557).

Example 3: Human Monocyte/Macrophage Differentiation in Humanized M-CSF Mice

To evaluate the effect of humanizing M-CSF in sublethally irradiated neonates Rag2^-/- γc^-/-M-CSF^m/m, Rag2^-/- γc^-/-M-CSF^{h/m and}Rag2^-/- γc^-/-M-CSF^h/hmice were transplanted intrahepatically (i.h) with ~2 x 10⁵ purified CD34⁺ human embryonic liver cells. Blood was then drawn from recipients 8 weeks after transplantation to confirm cell origin from the donor (based on human CD45 expression). Twelve weeks after transplantation, recipients were sacrificed and their bone marrow, spleen, and peripheral blood were collected for testing. The analysis showed an increase in the relative and absolute frequencies of CD14⁺CD33⁺ cells of the monocyte/macrophage lineage in the bone marrow, spleen and peripheral blood and M-CSF^h/mAnd M-CSF^h/hmice compared to M-CSF^m/m mice (Fig. 3A-C). Although M-CSF^h/mmice showed increased frequencies of CD14⁺CD33⁺ cells, maximum frequencies of occurrence of CD14⁺CD33⁺ cells were found in M-CSF^h/hmice. Interestingly, in addition to this increase, the frequency of CD14^-CD33⁺ cells were also increased in bone marrow, spleen and peripheral blood M-CSF^h/mAnd M-CSF^h/hmice (Fig. 3A).

To analyze whether human M-CSF knock-in mice support long-term human myelopoiesis, recipients were analyzed at 12, 16, and 20 weeks posttransplantation. Whereas M-CSF^m/m mice human CD14 count⁺CD33⁺ cells of the monocyte/macrophage lineage were slightly reduced at 16 weeks and significantly reduced at 20 weeks after transplantation, in M-CSF^h/mAnd M-CSF^h/h mice have a significant percentage of human CD14⁺CD33⁺cells were observed even after 16 and 20 weeks. However, the maximum frequencies of human CD14⁺CD33⁺cells were observed in M-CSF^h/hmice (Figures 4A and 4B).

We next determined whether humanized M-CSF mice supported efficient differentiation of human tissue macrophages. For this purpose, mice were perfused with M-CSF^m/m, M-CSF^m/hAnd M-CSF^h/husing PBS, and their organs (including liver, lungs and skin) were collected for analysis. Peritoneal cells were obtained by washing the peritoneal cavity with PBS. Single cell suspensions were prepared and frequencies of human CD14 were calculated.⁺CD33⁺cells. As expected, the frequencies of human CD14⁺CD33⁺cells were significantly increased in the liver, lungs and abdominal cavity M-CSF^m/h and M-CSF^h/hmice. However, analysis of skin explants showed comparable frequencies of human CD14⁺CD33⁺cells in M-CSF^m/m and M-CSF^m/hmice, despite the fact that in skin explants M-CSF^h/hmice there was a significant increase in these cells (Fig. 5). Taken together, these data suggest that expression of human M-CSF in mice improves the differentiation of the myeloid/macrophage lineage of human HSCs.

Example 4: Function of Human Monocytes/Macrophages in Humanized M-CSF Mice

To find out whether human CD14 functions normally⁺CD33⁺monocytes/macrophages in humanized M-CSF mice, functional studies were carried out both in vivo and in vitro. Sublethal irradiated M-CSF^m/m and M-CSF^m/hMice were injected with CD34+ fetal liver cells, and 12 weeks after transplantation, the resulting donor hematopoiesis was assessed by administering LPS or PBS to recipient mice. Human CD14 frequencies were analyzed 2 days after LPS administration.⁺CD33⁺cells in the spleen of recipients. Compared with the PBS group, LPS administration caused only a moderate increase in monocyte/macrophage lineage cells in M-CSF^m/mmice, while administration of LPS M-CSF^m/hmice showed an increase in human CD14⁺CD33⁺cells in the spleen several times (Fig. 6A). We further tested the ability of these cells to produce proinflammatory cytokines in response to LPS stimulation in vivo.

LPS was injected with M-CSF^m/m and M-CSF^m/hmice “inoculated” with human CD34+ cells. Six hours after administration, mice were bled and serum levels of human and murine IL6 and TNFα were determined using ELISA. Consistent with the increased frequency of monocytes/macrophages in humanized M-CSF mice, M-CSF^m/hmice, increased levels of human IL6 and TNFα were found. Although baseline levels of these cytokines were higher in M-CSF^m/hmice, LPS stimulation resulted in increased serum levels of human IL6 and TNFα (Figures 6B and 6C). The ability of monocytes/macrophages (derived from humanized M-CSF mice) to secrete proinflammatory cytokines in vitro was further analyzed. Human CD14⁺CD33⁺cells M-CSF was isolated from the spleen^m/m or M-CSF^h/hmice 12 weeks after restoration with human CD34⁺ cells and stimulated with LPS in vitro for 24 or 48 hours. Levels of IL-6 and TNFα cytokines in cell culture supernatants were assessed using ELISA. Consistent with in vivo data, CD14⁺CD33⁺M-CSF cells^h/hmice secreted increased levels of these cytokines in response to LPS (Figures 7A and 7B). Likewise, human CD14⁺CD33⁺cells from humanized M-CSF mice expressed interferon-α and interferon-β mRNA at elevated levels in response to poly I:C stimulation (Fig. 7C). Finally, the phagocytosis, migration, and activation characteristics of human monocytes/macrophages of humanized M-CSF mice were analyzed. Human CD14⁺CD33⁺M-CSF-derived cells^h/hmice reconstituted with human CD34⁺, demonstrated increased phagocytosis capacity (Figure 7D) and increased chemotaxis in response to the chemokine Mip3β (Figure 7E). As expected, human monocytes/macrophages M-CSF^h/hmice exhibited increased activation, as assessed by 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 exhibit enhanced functional characteristics.

Generating mice with a fully restored and functional hematopoietic/immune system of human origin has been a challenge in this field. Currently, 3 mouse strains have been created (NOD-scid γc ^−/− , [NSG], NOD/Shi-scid γc ^−/− [NOG], and Balb/c-Rag2 ^−/− γc ^−/− ). Despite the advantages provided by each of these strains, human hematopoiesis in these mice was defective.

To overcome this major technical difficulty, the mouse CSF-1 gene was replaced with its human counterpart. This resulted in efficient differentiation of human macrophages in mice transformed with human hematopoietic and progenitor cells. Analysis of humanized CSF-1 mice showed efficient differentiation of human monocytes/macrophages in bone marrow, spleen and peripheral blood. Moreover, human macrophages were detected in several other tissues, including lung and liver, in these mice, indicating that the presence of CSF-1 in humanized mice is sufficient to promote differentiation of human tissue macrophages. Additionally, the functional studies described here on human monocytes/macrophages derived from CSF1 ^m/m and CSF1 ^h/h mice indicate that CSF1 ^h/h mouse cells were better able to perform functions such as phagocytosis, migration, activation, and cytokine secretion. Based on the results obtained, it can be concluded that monocytes/macrophages that differentiate in the presence of human CSF-1 have better functioning.

VELOCIGENE® genetic engineering technology was used to create a new line of Balb/c-Rag2 ^−/− γc ^−/− mice expressing human CSF-1. Accordingly, the mouse CSF-1 coding region was replaced with a human counterpart without disrupting regulatory elements, such as the promoter, of the mouse csf1 gene. This led to the formation of a chimeric gene containing mouse regulatory elements and encoding a region of human CSF-1. Expression studies of this chimeric gene in these mice showed that it is expressed in a qualitative and quantitative manner.

The role of CSF-1 in murine macrophage differentiation has been well established. Mice lacking either CSF-1 (Csf1 ^op/op ) or its receptor (Csf1r ^−/− ) exhibit a pronounced decrease in the frequencies of macrophages and osteoclasts, osteopetrosis, lack of teething, and developmental defects in various tissues, including the nervous system, male and female fertility, dermis and synovium. Although these studies have provided significant insight into the role of CSF-1 in mice, the significance of CSF-1 in human hematopoiesis remains largely unknown. In this regard, the mice described here will serve as an important tool as it will improve our understanding of the physiology and function of cytokines in human hematopoiesis and hematopoietic cell function. Additionally, this mouse can be used to model disease and test the effects of substances on the human immune system. This mouse model provides a useful tool for understanding the pathophysiology and treatment of several human disorders and diseases.

The above only illustrates the principles of the invention. It should be understood that those skilled in the art will be able to create various embodiments which, although not described in detail or shown in the specification, embody the principles of the invention and are included within its scope and spirit. Moreover, all examples and conventional language mentioned in the description are primarily intended to provide an understanding of the principles of the invention and the ideas introduced by the inventors to advance the art, and should be considered not to be limited by such specifically stated examples and conditions. Moreover, all statements, principles, aspects and embodiments of the invention set forth in the specification, as well as specific examples thereof, are intended to include their structural and functional equivalents. Moreover, such equivalents are intended to include both currently known equivalents and equivalents that will be created in the future, that is, any created elements that perform the same function, regardless of structure. Therefore, the scope of the present invention is not limited to the illustrative embodiments shown and described herein. More precisely, the essence and scope of the present invention is set forth in the appended claims.

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LIST OF SEQUENCES

<110> Murphy, Andrew J.

Stevens, Sean

Rathinam, Chozhavendan

Eynon, Elizabeth

Manz, Markus

Flavell, Richard

<120> Humanized M-CSF Mice

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<150> 61/442,946

<151> 2011-02-15

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Claims (12)

1. A method for screening a candidate substance for toxicity towards human hematopoietic cells, comprising: (a) contacting the first humanized M-CSF mouse with the candidate substance, wherein the first humanized M-CSF mouse is engrafted with human hematopoietic cells; And (b) comparing the viability and/or function of human hematopoietic cells in a first humanized M-CSF mouse exposed to the candidate substance with the viability and/or function of human hematopoietic cells in a second humanized M-CSF mouse inoculated with human hematopoietic cells, but which has not been exposed to the candidate substance, where both the first and second humanized M-CSF mice contain: a nucleic acid sequence included in the humanized mouse M-CSF genome that encodes the human M-CSF protein and is operably linked to the endogenous mouse M-CSF gene promoter at the mouse M-CSF locus; And Rag2 gene knockout and IL2rg gene knockout or selected from the group consisting of a NOD-scid γc ^-/- mouse, a NOD/Shi-scid γc ^-/- mouse, and a Balb/c Rag2 ^-/- γc ^-/- mouse _, wherein both the first and second humanized M-CSF mice express the nucleotide sequence-encoded M-CSF RNA in bone marrow, spleen, blood, liver, brain, lung, testis and kidney, and wherein a decrease in the viability and/or function of hematopoietic cells in the first mouse exposed to the candidate substance indicates that the candidate substance is toxic to hematopoietic cells. 2. The method of claim 1, wherein both the first and second humanized M-CSF mice contain two copies of said coding sequence. 3. The method of claim 2, wherein both the first and second humanized M-CSF mice have the same null mutation of at least one mouse M-CSF allele. 4. The method according to claim 3, characterized in that the null mutation is a deletion of exons 2-9 of mouse M-CSF. 5. The method of claim 1, wherein both the first and second humanized M-CSF mice have the same null mutation of at least one mouse M-CSF allele. 6. The method according to claim 5, characterized in that the null mutation is a deletion of exons 2-9 of mouse M-CSF.