Genetically modified non-human animal, and its usage

Abstract

To provide a genetically modified non-human animal, usable for modeling growth, function or disease of human hematopoietic cells.SOLUTION: A genetically modified non-human animal contains nucleic acid for coding human IL-6 connected functionally to IL-6 promotor. In several cases, a genetically modified non-human animal expressing human IL-6 also expresses at least one kind of human M-CSF, human IL-3, human GM-CSF, human SIRPa and human TPO.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. 5R01CA156689-04 awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority pursuant to 35 U.S.C. §119(e) to the filing date of U.S. Provisional Patent Application No. 61 / 722,437, filed November 5, 2012, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0003] 2. Background of the Invention The goal of biomedical research is to gain a better understanding of human physiology and to use this knowledge to prevent, treat, or cure human diseases. Because of practical and ethical barriers to experimental procedures in human subjects, many studies are carried out in small animal models, such as mice. Thus, there is a need for animal models of these human diseases.

[0004] For example, approximately 20,000 patients in the United States are newly diagnosed each year with multiple myeloma (MM), a nearly incurable malignant disease of antibody-secreting, highly differentiated B cells (Hideshima et al., 2007, Nat Rev Cancer. 7:585-98 (Non-Patent Document 1); Kuehl and Bergsagel, 2002, Nat Rev Cancer. 2:175-87 (Non-Patent Document 2)). MM is characterized by infiltration of malignant plasma cells into the bone marrow (BM), and clinical signs include bone disease, hypercalcemia, cytopenias, renal dysfunction, and peripheral neuropathy (Hideshima et al., 2007, Nat Rev Cancer. 7:585-98 (Non-Patent Document 1); Kuehl and Bergsagel, 2002, Nat Rev Cancer. 2:175-87 (Non-Patent Document 2)). A precancerous condition called monoclonal gammopathy of unknown etiology (MGUS), which affects approximately 3% of people over the age of 50, precedes MM in the majority of cases (Landgren et al., 2009, Blood 113:5412-7). Complex heterogeneous genetic abnormalities, including karyotypic changes and IgH translocations, characterize MM cells (Kuehl and Bergsagel, 2002, Nat Rev Cancer. 2:175-87; Zhan et al., 2006, Blood 108:2020-8). The plasma cell clones amplified in MGUS appear to have genetic and phenotypic profiles similar to myeloma plasma cells (Chng et al., 2005, Blood 106:2156-61 (Non-Patent Document 5); Fonseca et al., 2002, Blood 100:1417-24 (Non-Patent Document 6); Kaufmann et al., 2004, Leukemia. 18:1879-82 (Non-Patent Document 7)). Mutations in the cyclin D gene have been suggested to cause MM, but the possible contribution of other factors has not been conclusively proven (Bergsagel et al., 2005, Blood 106:296-303 (Non-Patent Document 8)). Nevertheless, inherited genetic alterations are not the sole determinant of MM cell behavior.Rather, resistance to drugs and aberrant biological responses to cytokines are strongly influenced by interactions with the microenvironment, which provides an opportunity to develop novel therapeutic agents.

[0005] Like many other tumors, MM is characterized by a heterogeneous cell population that strongly interacts with non-malignant stromal cells creating a supportive environment (De Raeve and Vanderkerken, 2005, Histol Histopathol. 20:1227-50 (Non-Patent Document 9); Dhodapkar, 2009, Am J Hematol. 84:395-6 (Non-Patent Document 10)). The BM microenvironment for MM cells consists of a diverse extracellular matrix (ECM) and cellular components of both hematopoietic and non-hematopoietic origin. Although BM provides a protected environment for normal hematopoiesis, MM cell interactions with ECM proteins and accessory cells play a critical role in MM pathogenesis (De Raeve and Vanderkerken, 2005, Histol Histopathol. 20:1227-50 (Non-Patent Document 9); Dhodapkar, 2009, Am J Hematol. 84:395-6 (Non-Patent Document 10); Hideshima et al., 2007, Nat Rev Cancer. 7:585-98 (Non-Patent Document 1)). Stromal cells, myeloid cells, osteoclasts, and osteoblasts produce growth factors such as interleukin 6 (IL-6), B-cell activating factor (BAFF), fibroblast growth factor, and stromal cell-derived factor 1a, which activate signaling pathways that mediate MM cell migration, survival, and proliferation. In particular, IL-6 produced by stromal cells, osteoclasts, and myeloid cells appears to be a critical factor for the pathogenesis of MM in the early stages (De Raeve and Vanderkerken, 2005, Histol Histopathol. 20:1227-50 (Non-Patent Document 9)). Similarly, upon interaction with MM cells, osteoclasts and dendritic cells produce BAFF and / or proliferation-inducing ligand (APRIL), which provide anti-apoptotic signals, and also increase drug resistance (De Raeve and Vanderkerken, 2005, Histol Histopathol. 20:1227-50 (Non-Patent Document 9); Kukreja et al., 2006, J Exp Med. 203:1859-65 (Non-Patent Document 11)).

[0006] Key events in cancer pathogenesis (unregulated proliferation, survival, and spread of malignant cells) depend on specific combinations of supportive cell types and soluble factors present in the microenvironmental niche. Mouse models play an important role in characterizing critical aspects of malignant transformation and disease drivers in humans. However, they rarely represent the genetic complexity and clinicopathological features of human disease. Although xenografts of human tumors into immune-compromised mice have been widely utilized, reliable engraftment has typically only been feasible with high-grade tumors or cell lines.

[0007] The best model currently available for growing human tumor cells is the severely immunodeficient mouse, lacking B, T, and NK cells. In the case of MM, engraftment of primary myeloma cells into these mice has not been successful, but primary myeloma cells can be engrafted into human fetal bone fragments by co-transplantation into immunocompromised mice (Yaccoby et al., 1998, Blood 92:2908-13 (Non-Patent Document 12)). In this model, MM cells are found in human bone but not in mouse bone or the periphery, demonstrating a high degree of residual xenorejection and the requirement for a human BM microenvironment (Yaccoby et al., 1998, Blood 92:2908-13 (Non-Patent Document 12); Yaccoby and Epstein, 1999, Blood 94:3576-82 (Non-Patent Document 13)). NOD / Scid / γc - / -Mice have recently been shown to allow the engraftment of several MM cell lines, proving their potential as in vivo models of MM (Dewan et al., 2004, Cancer Sci. 95:564-8 (Non-Patent Document 14); Miyakawa et al., 2004, Biochem Biophys Res Commun. 313:258-62 (Non-Patent Document 15)). However, even those mouse models with low xeno-rejection do not cross the species barrier but have a restricted growth environment due to the numerous factors that are essential to support the growth and survival of transformed cells (Manz, 2007). In vivo models that allow exploring the complex pathogenic interplay between the tumor and its environment will be essential for designing new drugs and treatments.

[0008] Thus, there remains an unmet need to develop humanized non-human animals and methods for reliably growing and studying human hematopoietic cells, including primary human hematopoietic tumor cells, in mice. The present invention addresses these unmet needs in the art. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Hideshima et al.,2007,Nat Rev Cancer.7:585-98 [Non-Patent Document 2] Kuehl and Bergsagel,2002, Nat Rev Cancer.2:175-87 [Non-Patent Document 3] Landgren et al.,2009,Blood 113:5412-7 [Non-Patent Document 4] Zhan et al.,2006,Blood 108:2020-8 [Non-Patent Document 5] Chng et al.,2005,Blood 106:2156-61 [Non-Patent Document 6] Fonseca et al., 2002, Blood 100:1417 - 24

Non - Patent Document 7

Non - Patent Document 8

Non - Patent Document 9

Non - Patent Document 10

Non - Patent Document 11

Non - Patent Document 12

Non - Patent Document 13

Non - Patent Document 14

Non - Patent Document 15

Summary of the Invention

[0010] Provided are genetically modified non-human animals that can be used to model human hematopoietic cell development, function, or disease. The genetically modified non-human animals include a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter. In some cases, the genetically modified non-human animals that express human IL-6 also express at least one of human M-CSF, human IL-3, human GM-CSF, human SIRPa, or human TPO. In some cases, the genetically modified non-human animals are immunodeficient. In some such cases, the genetically modified non-human animals are engrafted with healthy or diseased human hematopoietic cells. Also provided are methods of using the genetically modified non-human animals of the invention in modeling human hematopoietic cell development, function, and / or disease, as well as reagents and kits thereof useful in making the genetically modified non-human animals of the invention and / or in carrying out the methods of the invention.

[0011] In various aspects of the present invention, a genetically modified non-human animal is provided that comprises a genome comprising a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter and expresses a human IL-6 polypeptide under the regulatory control of the IL-6 promoter. In some embodiments, the genetically modified non-human animal does not express the animal's native IL-6.

[0012] In some embodiments, the genetically modified non-human animal is a rodent. In some embodiments, the non-human animal is a mouse. In some such embodiments, the IL-6 promoter to which the nucleic acid encoding human IL-6 is operably linked is a mouse IL-6 promoter, and the human IL-6 gene is operably linked to the mouse IL-6 promoter at the mouse IL-6 locus.

[0013] In some embodiments, the genetically modified non-human animal further comprises one or more additional nucleic acids selected from a nucleic acid encoding human SIRPa under the regulation of a SIRPa promoter; a nucleic acid encoding human M-CSF operably linked to an M-CSF promoter (in which case the animal expresses human M-CSF); a nucleic acid encoding human IL-3 operably linked to an IL-3 promoter (in which case the animal expresses human IL-3); a nucleic acid encoding human GM-CSF operably linked to a GM-CSF promoter (in which case the animal expresses human GM-CSF); and a nucleic acid encoding human TPO operably linked to a TPO promoter (in which case the animal expresses human TPO). In some embodiments, the promoter is a human promoter for the gene. In other embodiments, the promoter is a non-human animal promoter for the gene. In some embodiments, the genetically modified non-human animal expresses the corresponding native protein of the animal. In other embodiments, the genetically modified non-human animal does not express the corresponding native protein of the animal.

[0014] In some embodiments, the genetically modified non-human animal is immunodeficient with respect to the non-human animal immune system. In some such embodiments, the immunodeficient genetically modified non-human animal does not express a recombination activating gene (RAG). In some such embodiments, the immunodeficient genetically modified non-human animal does not express the IL2 receptor gamma chain (IL2rg or "γc"). In some such embodiments, the immunodeficient genetically modified non-human animal does not express either a RAG (e.g., RAG1, RAG2) or IL2rg.

[0015] In some embodiments, the immunodeficient genetically modified non-human animal is engrafted with human hematopoietic cells to form a genetically modified engrafted non-human animal. In one embodiment, the human hematopoietic cells are selected from human umbilical cord blood cells, human fetal liver cells, and cells of human hematopoietic cell lines. In one embodiment, the human hematopoietic cells are CD34+ progenitor cells. In one embodiment, the human hematopoietic cells are cancer cells. In certain embodiments, the cancer cells are human multiple myeloma cells.

[0016] In some embodiments, the genetically modified engrafted animals produce human cells selected from CD34+ cells, hematopoietic stem cells, hematopoietic cells, myeloid progenitor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, thymocytes, T cells, B cells, plasma cells, platelets, and combinations thereof. In one embodiment, the human cells are present 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after engraftment.

[0017] In some embodiments, the genetically modified engrafted animals develop a human hematolymphoid system including human hematopoietic stem and progenitor cells, human myeloid progenitor cells, human myeloid cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, human plasma cells, and human platelets. In one embodiment, the human hematolymphoid system is present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months post-engraftment.

[0018] In some aspects of the invention, methods are provided for generating non-human animals engrafted with human hematopoietic cells. In some embodiments, methods are provided for generating animal models of human immune cell development and function. In certain embodiments, methods are provided for generating animal models of human B cell development and function.

[0019] In some embodiments, the method includes transplanting a population of human hematopoietic cells into a genetically modified non-human animal that is immunodeficient and expresses a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter. In some embodiments, the animal does not express native IL-6. In some embodiments, the IL-6 promoter is a non-human animal IL-6 promoter, and the human IL-6 gene is operably linked to the non-human animal IL-6 promoter at the non-human animal IL-6 locus. In some embodiments, the non-human animal is a rodent. In some such embodiments, the non-human animal is a mouse. Thus, in some embodiments, the IL-6 promoter to which the nucleic acid encoding human IL-6 is operably linked is a mouse IL-6 promoter, and the human IL-6 gene is operably linked to the mouse IL-6 promoter at the mouse IL-6 locus.

[0020] In some embodiments, the population of transplanted hematopoietic cells comprises CD34+ cells. In some embodiments, the population of transplanted hematopoietic cells comprises cancer cells. In some embodiments, the population of transplanted cancer cells comprises multiple myeloma cells. In some embodiments, the transplantation comprises intrafemoral and / or intratibial injection.

[0021] In some embodiments, the immunodeficient, genetically modified animal expresses at least one additional human nucleic acid selected from the group consisting of a nucleic acid encoding human SIRPa operably linked to a SIRPa promoter; a nucleic acid encoding human M-CSF operably linked to an M-CSF promoter; a nucleic acid encoding human IL-3 operably linked to an IL-3 promoter; a nucleic acid encoding human GM-CSF operably linked to a GM-CSF promoter; and a nucleic acid encoding human TPO operably linked to a TPO promoter.

[0022] In some aspects of the present invention, there is provided an engrafted genetically modified non-human animal that expresses a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter, prepared according to the methods described herein or known in the art. In some embodiments, the engrafted genetically modified non-human animal is an animal model of human B cell development and differentiation.

[0023] In various embodiments, methods are provided that include the use of genetically modified non-human animals engrafted with human hematopoietic cells of the present disclosure.These methods include, for example, methods for in vivo evaluation of the proliferation and differentiation of hematopoietic cells and immune cells, methods for in vivo evaluation of human hematopoietic system, methods for in vivo evaluation of cancer cells, methods for in vivo assessment of immune response, methods for in vivo evaluation of vaccines and vaccination programs, methods for use in testing the effect of agents that modulate the proliferation or survival of cancer cells, methods for in vivo evaluation of cancer treatment, and methods for in vivo production and collection of immune mediators, including human antibodies, and methods for use in testing the effect of agents that modulate the function of hematopoietic cells and immune cells.For example, in some embodiments, methods are provided for screening candidate agents for their ability to treat hematopoietic cancer. In some embodiments, the method includes contacting a genetically modified non-human animal of the present disclosure engrafted with human hematopoietic cancer cells with a candidate agent, and comparing the survival and / or proliferation rate of the human hematopoietic cancer cells in the contacted engrafted genetically modified non-human animal with the human hematopoietic cancer cells in a similarly engrafted genetically modified non-human animal that has not been contacted with the candidate agent. In this method, a decrease in survival and / or proliferation rate of the human hematopoietic cancer cells in the contacted engrafted non-human animal indicates that the candidate agent will treat hematopoietic cancer. These and other methods will be apparent to those of skill in the art from the disclosure herein. [The present invention 1001] A nucleic acid encoding human IL-6 operably linked to an IL-6 promoter, and (i) a nucleic acid encoding human SIRPa operably linked to a SIRPa promoter, randomly integrated into the genome of the non-human animal; (ii) a nucleic acid encoding human M-CSF operably linked to an M-CSF promoter; (iii) a nucleic acid encoding human IL-3 operably linked to an IL-3 promoter; (iv) a nucleic acid encoding human GM-CSF operably linked to a GM-CSF promoter; and (v) a nucleic acid encoding human TPO operably linked to a TPO promoter: At least one additional nucleic acid selected from the group consisting of 2. A genetically modified non-human animal comprising: [The present invention 1002] The genetically modified non-human animal of the present invention 1001, wherein the IL-6 promoter is a non-human animal IL-6 promoter and a nucleic acid encoding human IL-6 is operably linked to the non-human animal IL-6 promoter at the non-human animal IL-6 locus. [The present invention 1003] The genetically modified non-human animal of the present invention 1001, wherein a nucleic acid encoding human M-CSF is located at the non-human animal M-CSF locus, a nucleic acid encoding human IL-3 is located at the non-human animal IL-3 locus, a nucleic acid encoding human GM-CSF is located at the non-human animal GM-CSF locus, and a nucleic acid encoding human TPO is located at the non-human animal TPO locus. [The present invention 1004] 1001. The genetically modified non-human animal of the present invention, which is immunodeficient. [The present invention 1005] 1004. A genetically modified non-human animal of the present invention, which does not express a recombination activating gene (RAG). [The present invention 1006] 1004. A genetically modified non-human animal of the invention which does not express the IL2 receptor gamma chain (gamma chain- / -). [The present invention 1007] The genetically modified non-human animal of the present invention 1004, which does not express RAG2 and does not express IL2rg. [The present invention 1008] The genetically modified non-human animal of the present invention 1001 further comprising engraftment of human hematopoietic cells. [The present invention 1009] 1008. The genetically modified non-human animal of the present invention, wherein the human hematopoietic cells are CD34+ cells. [The present invention 1010] The genetically modified non-human animal of the present invention, wherein the human hematopoietic cells are multiple myeloma cells. [The present invention 1011] The genetically modified non-human animal of the present invention 1001, which is a mouse. [The present invention 1012] transplanting the population of human hematopoietic cells into a genetically modified non-human animal that is immunodeficient, that contains a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter, that expresses human IL-6, and that does not express the non-human animal IL-6. A method for generating an animal model of human B cell development and function, comprising: [The present invention 1013] The method of claim 1012, wherein the IL-6 promoter is a non-human animal IL-6 promoter and the nucleic acid encoding human IL-6 is operably linked to the non-human animal IL-6 promoter at the non-human animal IL-6 locus. [The present invention 1014] The method of claim 1012, wherein the population of transplanted hematopoietic cells comprises CD34+ cells. [The present invention 1015] The method of claim 1012, wherein the population of transplanted hematopoietic cells comprises multiple myeloma cells. [The present invention 1016] The method of claim 1012, wherein the step of implanting comprises intrafemoral injection and / or intratibial injection. [The present invention 1017] The method of claim 1012, wherein the animal further comprises a nucleic acid encoding human SIRPa operably linked to the SIRPa promoter, randomly integrated into the genome of the non-human animal. [The present invention 1018] Immunodeficient, genetically modified animals (i) a nucleic acid encoding human M-CSF operably linked to an M-CSF promoter; (ii) a nucleic acid encoding human IL-3 operably linked to an IL-3 promoter; (iii) a nucleic acid encoding human GM-CSF operably linked to a GM-CSF promoter; and (iv) a nucleic acid encoding human TPO operably linked to a TPO promoter: The method of claim 1017, wherein the human nucleic acid is expressed in a human mouse model. [The present invention 1019] The method according to any one of claims 1012 to 1018, wherein the animal is a mouse. [The present invention 1020] An engrafted non-human animal prepared according to any one of the methods of the present invention 1012 to 1019. [The present invention 1021] contacting the genetically modified non-human animal of the present invention having engrafted human hematopoietic cancer cells with a candidate drug; and comparing the survival rate and / or proliferation rate of the human hematopoietic cancer cells in the contacted non-human animal with the survival rate and / or proliferation rate of the human hematopoietic cancer cells in a genetically modified non-human animal of the invention engrafted with the human hematopoietic cancer cells but not contacted with the candidate drug. wherein a decrease in the viability and / or proliferation rate of human hematopoietic cancer cells in the contacted non-human animal indicates that the candidate agent will treat the hematopoietic cancer. [The present invention 1022] The method of claim 1021, wherein the hematopoietic cancer is multiple myeloma. [Brief description of the drawings]

[0024] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings, in which: It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, dimensions of the various features have been arbitrarily increased or reduced for clarity. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. The drawings include the following figures:

[0025] [Figure 1] 1 is a set of graphs showing the results of an experiment demonstrating the engraftment of INA-6 cells in human IL-6 knock-in mice. Soluble IL-6R levels were measured in mice of the indicated genotypes that were intravenously implanted with 5×106 INA-6 cells. N indicates the number of implanted mice in each group. [Diagram 2] FIG. 2, including FIG. 2A-2F, is a set of images showing histological analysis of femurs after intravenous engraftment of INA-6 cells. Eight weeks after intravenous engraftment of 5×106 INA-6 cells, Rag2- / -Il2rgnullIl6h / hhSIRPa+ mice were sacrificed. Femurs were fixed in 10% formalin and decalcified. 10 μM sections were stained with toluidine blue or directly analyzed for GFP expression using a Leica confocal microscope. [Diagram 3] A set of images and graphs showing analysis of lungs after intravenous engraftment of INA-6 cells. Eight weeks after engraftment of 5x106 intravenously injected INA-6 cells, Rag2- / -Il2rgnullIl6h / hhSIRPa+ mice were sacrificed. Lung tissues were fixed in 10% formalin and 10μM sections were directly analyzed for GFP expression using a Leica confocal microscope. The images (left) show 10x (top) and 63x (bottom) magnifications of the sections. The graphs (right) show human Il6 gene expression measured in the indicated tissues and normalized to mouse hprt expression. [Figure 4]This is a set of graphs showing the results of an experiment demonstrating the engraftment of INA-6 cells in human IL-6 knock-in mice. Soluble IL-6R levels were measured in mice of the indicated genotypes implanted intrafemorally with 5x105 INA-6 cells. Each line represents an individual mouse. [Diagram 5] A set of images showing histological analysis of femurs after intra-femoral engraftment of INA-6 cells. Rag2- / -Il2rgnullIl6h / hhSIRPa+ mice were sacrificed 4-6 weeks after engraftment of 5x105 intra-femorally injected INA-6 cells. Femurs were fixed in 10% formalin and decalcified. 10μM sections were stained with toluidine blue. [Figure 6] A set of images and graphs showing the results of μCT analysis of mouse femurs after INA-6 implantation. Four weeks after engraftment of 5×105 intrafemorally injected INA-6 cells, Rag2− / −Il2rgnullIl6h / hhSIRPa+ mice and control mice were sacrificed. Femurs were fixed in 70% ethanol and analyzed using mouse μCT. Trabecular bone volume and tissue volume were quantified to calculate the ratio between bone volume and tissue volume. *: p<0.01 by Student's t-test. [Figure 7] Figure 1 shows the results of μCT analysis of mouse femurs after treatment with antimyeloma drugs. Rag2- / -Il2rgnullIl6h / hhSIRPa+ were engrafted with 5x105 INA-6 cells injected intrafemorally and treated every other week with Velcade® or Zometa®, respectively. After 4 weeks, mice were sacrificed and femurs were fixed with 70% ethanol for μCT analysis. Trabecular bone volume and tissue volume were quantified to calculate the ratio between bone volume and tissue volume. [Figure 8]1 is a set of graphs showing the results of FACS analysis of primary cell engraftment in Rag2- / -Il2rgnullhSIRPa+Tpoh / hMcsfh / hIl3 / Gmcsfh / hIl6h / h mice. Mice were transplanted with 1.5x106 CD3-depleted bone marrow cells injected intrafemorally and sacrificed 12 weeks later. Single cell suspensions were generated from the injected femur, collateral limb, and spleen. Cells were stained for mCD45, hCD19, hCD38, and hCD138. FACS plots show events after gating on mCD45-negative cells. Numbers show the frequency of CD38+CD138+ cells. [Figure 9] 1 shows the results of an experiment assessing the percentage of human hematopoietic (hCD45+) cells in the blood of engrafted mice as determined by flow cytometry. Horizontal bars indicate the mean frequency of each. [Figure 10] 1 shows the results of an experiment assessing the percentage of hCD45 on blood B cells (CD19+), T cells (CD3+), and myeloid cells (CD33+) in engrafted mice as determined by flow cytometry. Only mice with hCD45 percentages higher than 2% are shown. [Figure 11] Figure 11, including Figures 11A and 11B, shows the results of an experiment assessing the percentage (A) and number (B) of human CD45+ cells in the BM, spleen, and thymus of 20-week engrafted mice. Bars represent the mean ± SEM of 4 / 5 mice per group. [Figure 12] Figure 12, including Figures 12A and 12B, shows the results of a FACS experiment assessing human cells. (A) Diagram showing the gating strategy used to separate different B cell populations by flow cytometry. (B) Percentage of different B cell subsets among human CD45+CD19+ cells in 20 week old mice. Bars represent the mean ± SEM of 4 / 5 mice per group. [Figure 13]Figure 13, including Figures 13A and 13B, shows the results of a FACS experiment assessing human cells. (A) Representative flow cytometry analysis of CD5+ B cells in human fetal liver (FL) and 20 week old mice. Numbers within quadrants indicate percentage of cells. All plots are gated on human CD45+ cells. (B) Percentage of CD5 on human B cells in BM and spleen of 20 week engrafted mice. Bars represent mean ± SEM of 4 / 5 mice per group. [Figure 14] Results of FACS experiments assessing human cells are shown. (A) Representative flow cytometry analysis of CD27+ B cells in human fetal liver (FL) and 20 week old mice. Numbers within quadrants indicate percentage of cells. All plots are gated on human CD45+ cells. (B) Percentage of CD27 on human B cells in BM and spleen of 20 week engrafted mice. Bars represent mean ± SEM of 4 / 5 mice per group. [Figure 15] Results of an experiment assessing total human IgM and IgG levels in plasma samples from mice at 12 weeks of age (A) and 20 weeks of age (B) are shown. Horizontal bars indicate the respective geometric means. Mice with less than 2% PB human engraftment were excluded from the analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description Provided are genetically modified non-human animals that can be used to model human hematopoietic cell development, function, or disease. The genetically modified non-human animals comprise a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter. In some embodiments, the genetically modified non-human animals expressing human IL-6 also express at least one of human M-CSF, human IL-3, human GM-CSF, human SIRPa, or human TPO. The present invention also relates to methods of generating and using the genetically modified non-human animals described herein. In some embodiments, the genetically modified non-human animals are mice. In some embodiments, the genetically modified non-human animals described herein are engrafted with human hematopoietic cells, including normal or neoplastic cells, or a combination thereof. In some embodiments, the genetically modified non-human animals described herein are engrafted with human multiple myeloma (MM) cells. In various embodiments, genetically modified non-human animals engrafted with the human hematopoietic cells of the invention are useful for in vivo evaluation of hematopoietic and immune cell proliferation and differentiation, in vivo evaluation of the human hematopoietic system, in vivo evaluation of cancer cells, in vivo assessment of immune responses, in vivo evaluation of vaccines and vaccination regimens, use in testing the effect of agents that modulate cancer cell proliferation or survival, in vivo evaluation of cancer treatments, and in vivo generation and collection of immune mediators, including human antibodies, and use in testing the effect of agents that modulate hematopoietic and immune cell function.

[0027] These and other objects, advantages and features of the present invention will become apparent to those skilled in the art upon review of the details of the compositions and methods that are more fully described below.

[0028] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Such terms are found, defined, and used in context in a variety of standard reference texts, including, by way of example, J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; FMA Russell, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; DL Nelson and MMCox, Lehninger Principles of Biochemistry, 4th Ed., WH Freeman & Company, 2004; and Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0029] As used herein, each of the following terms has the meaning associated with it in this section.

[0030] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0031] "About," as used herein, when referring to a measurable value such as an amount, duration, etc., is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for performing the disclosed methods.

[0032] The term "abnormal," when used in reference to an organism, tissue, cell, or component thereof, refers to an organism, tissue, cell, or component thereof that differs in at least one observable or detectable characteristic (e.g., age, treatment, time, etc.) from the organism, tissue, cell, or component thereof that exhibits the "normal" (expected) respective characteristic. A characteristic that is normal or expected for one cell or tissue type may be abnormal for a different cell or tissue type.

[0033] The term "antibody," as used herein, includes immunoglobulin molecules that can specifically bind to a particular epitope on an antigen. Antibodies may be complete immunoglobulins derived from natural or recombinant sources or may be immunoreactive portions of complete immunoglobulins. Antibodies in the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab, and F(ab)2, as well as single chain antibodies (scFv), heavy chain antibodies such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0034] "Constitutive" expression includes a state in which a gene product is produced in a living cell under most or all physiological conditions of the cell.

[0035] The "coding region" of a gene includes nucleotide residues of the coding strand of the gene and nucleotides of the non-coding strand of the gene that are homologous or complementary, respectively, to the coding region of an mRNA molecule produced by transcription of the gene. The "coding region" of an mRNA molecule also includes nucleotide residues of the mRNA molecule that pair with the anticodon region of a transfer RNA molecule during translation of the mRNA molecule or that code for a stop codon. Thus, a coding region can include nucleotide residues that constitute codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0036] "Disease" includes a state of an animal's health where the animal is unable to maintain homeostasis and where the animal's health continues to deteriorate if the disease is not remitted.

[0037] In contrast, a "disorder" in an animal includes a state of health in which the animal is able to maintain homeostasis, but in which the state of health of the animal is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause a further decrease in the state of health of the animal.

[0038] A disease or disorder is "alleviated" if the severity of the symptoms of the disease or disorder, the frequency with which such symptoms are experienced by the patient, or both, are reduced.

[0039] An "effective amount" or "therapeutically effective amount" of a compound includes an amount of the compound sufficient to provide a beneficial effect to a subject to which the compound is administered. An "effective amount" of a delivery vehicle includes an amount sufficient to effectively bind or deliver the compound.

[0040] "Encoding" includes the inherent property of a specific sequence of nucleotides of a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, as well as the biological properties ascribed thereto. Thus, for example, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of the gene or cDNA.

[0041] As used herein, "endogenous" includes any material that is derived from or produced within an organism, cell, tissue, or system.

[0042] As used herein, the term "exogenous" includes any material introduced from or produced outside an organism, cell, tissue, or system.

[0043] The terms "expression construct" and "expression cassette," as used herein, include a double-stranded recombinant DNA molecule that contains a desired nucleic acid coding sequence and contains one or more regulatory elements necessary or desirable for expression of the operably linked coding sequence.

[0044] As used herein, the term "fragment" as applied to a nucleic acid or polypeptide includes a subsequence of a larger nucleic acid or polypeptide. A "fragment" of a nucleic acid can be at least about 15 nucleotides long; e.g., at least about 50 nucleotides to about 100 nucleotides; at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides, at least about 1000 nucleotides to about 1500 nucleotides; or about 1500 nucleotides to about 2500 nucleotides; or about 2500 nucleotides (and any integer value in between). A "fragment" of a polypeptide can be at least about 15 nucleotides long; e.g., at least about 50 amino acids to about 100 amino acids; at least about 100 to about 500 amino acids, at least about 500 to about 1000 amino acids, at least about 1000 amino acids to about 1500 amino acids; or at least about 1500 amino acids to about 2500 amino acids; or about 2500 amino acids (and any integer value in between).

[0045] As used herein, the terms "gene" and "recombinant gene" include nucleic acid molecules that contain an open reading frame that encodes a polypeptide. Such natural allelic variations can typically result in 1-5% variation in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily performed by using hybridization probes to identify identical loci in various individuals. All such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variations and do not alter functional activity are intended to be included within the scope of the present invention.

[0046] "Homology", as used herein, includes subunit sequence similarity between two polymer molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position is occupied by the same monomeric subunit in both of the two molecules, e.g., when a position is occupied by adenine in each of the two DNA molecules, they are homologous at that position. The homology between two sequences is a direct function of the number of positions that match or are homologous, e.g., if half of the positions in the sequences of two compounds (e.g., 5 positions in a polymer 10 subunits long) are homologous, the two sequences are 50% homologous, and if 90% of the positions, e.g., 9 out of 10, are matched or homologous, the two sequences share 90% homology. For example, the DNA sequences 5'-ATTGCC-3' and 5'-TATGGC-3' share 50% homology.

[0047] The terms "human hematopoietic stem and progenitor cells" and "human HSPCs," as used herein, include human self-renewing, multipotent hematopoietic stem and progenitor cells.

[0048] "Inducible" expression includes the condition in which a gene product is produced in a living cell in response to the presence of a signal in the cell.

[0049] As used herein, "instructional material" includes publications, records, diagrams, or other media of expression that can be used to inform of the usefulness of the compounds, compositions, vectors, or delivery systems of the present invention in the kits to achieve alleviation of various diseases or disorders as described herein. Optionally or alternatively, the instructional material can describe one or more methods of alleviating a disease or disorder in a mammalian cell or tissue. The instructional material of the kits of the present invention can be, for example, attached to a container containing the identified compounds, compositions, vectors, or delivery systems of the present invention or shipped together with a container containing the identified compounds, compositions, vectors, or delivery systems. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compounds are used cooperatively by the recipient.

[0050] The term "nucleic acid" includes RNA or DNA molecules having multiple nucleotides in any form, including single-stranded, double-stranded, oligonucleotide, or polynucleotide. The term "nucleotide sequence" includes the order of nucleotides in a single-stranded form of a nucleic acid, an oligonucleotide, or a polynucleotide.

[0051] The term "operably linked" as used herein includes a polynucleotide in a functional relationship with a second polynucleotide, e.g., a single-stranded or double-stranded nucleic acid moiety containing two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides can exert a characteristic physiological effect on the other. For example, a promoter operably linked to a coding region of a gene can promote transcription of the coding region. Preferably, when the nucleic acid encoding a desired protein further comprises a promoter / control sequence, the promoter / control sequence is located at the 5' end of the sequence encoding the desired protein to express the desired protein in the cell. The nucleic acid encoding the desired protein and its promoter / control sequence together constitute a "transgene".

[0052] The term "polynucleotide" as used herein includes a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide are interchangeable as used herein. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, any nucleic acid sequence obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning of a nucleic acid sequence from a recombinant library or a cell genome using conventional cloning technology and PCR, etc., and synthetic means.

[0053] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and include compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids joined to each other by peptide bonds. As used herein, the term includes both short chains, also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, as well as longer chains, of which many types exist. "Polypeptides" include, inter alia, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof. The term "peptide" typically refers to short polypeptides. The term "protein" typically refers to large polypeptides.

[0054] The term "progeny" as used herein includes descendants or derivatives, including differentiated or undifferentiated progeny cells derived from a parent cell. In one usage, the term progeny includes progeny cells that are genetically identical to the parent. In another usage, the term progeny includes progeny cells that are genetically and phenotypically identical to the parent. In yet another usage, the term progeny includes progeny cells that are differentiated from a parent cell.

[0055] The term "promoter" as used herein includes a DNA sequence that is operably linked to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence that encodes a desired molecule. A promoter is generally located upstream of a nucleic acid sequence to be transcribed, and provides a site for specific binding by RNA polymerase and other transcription factors. In a specific embodiment, a promoter is generally located upstream of a nucleic acid sequence to be transcribed to produce a desired molecule, and provides a site for specific binding by RNA polymerase and other transcription factors.

[0056] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is true regardless of the breadth of the range.

[0057] A "recombinant polypeptide" includes one produced by expression of a recombinant polynucleotide.

[0058] The term "control element" as used herein includes nucleotide sequences that regulate some aspect of the expression of a nucleic acid sequence. Exemplary control elements include enhancers, internal ribosome entry sites (IRES), introns, replication origins, polyadenylation signals (pA), promoters, enhancers, transcription termination sequences, and upstream control domains that contribute to the replication, transcription, and post-transcriptional processing of nucleic acid sequences. Those skilled in the art can select these and other control elements to be used in expression constructs using only routine experimentation. Expression constructs can be produced recombinantly or synthetically using well-known methodologies.

[0059] The term "specifically binds", as used herein with respect to antibodies, includes antibodies that recognize a specific antigen but do not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species can also bind to that antigen from one or more species. However, such cross-species cross-reactivity does not, in itself, alter the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen can also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, alter the classification of the antibody as specific.

[0060] In some cases, the terms "specific binding" or "specifically binds" can be used with respect to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than the entire protein. If an antibody is specific for epitope "A", then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0061] The term "synthetic antibody," as used herein, includes antibodies produced using recombinant DNA technology, such as, for example, antibodies expressed by bacteriophage as described herein. The term should also be taken to mean antibodies produced by synthesis of a DNA molecule encoding an antibody and expressing an antibody protein, or an amino acid sequence specifying an antibody, obtained using DNA or amino acid sequence synthesis technology that is available and well known in the art.

[0062] The term "variant" as used herein includes nucleic acid or peptide sequences that differ in sequence from the reference nucleic acid or peptide sequence, respectively, but retain the essential biological properties of the reference molecule. The sequence change of the nucleic acid variant may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitution, addition, deletion, fusion, and truncation. The sequence change of the peptide variant is typically limited or conservative, so that the sequences of the reference peptide and the variant are generally similar and, in many regions, identical. The variant peptide and the reference peptide may differ in amino acid sequence by any combination of one or more substitutions, additions, deletions. The variant of the nucleic acid or peptide may be naturally occurring, such as an allelic variant, or may be a variant that is not known to occur naturally. The non-naturally occurring variants of nucleic acids and peptides may be produced by mutagenesis techniques or direct synthesis.

[0063] The term "genetically modified" includes animals whose germ cells contain exogenous human nucleic acid or human nucleic acid sequences. As a non-limiting example, a genetically modified animal can be a transgenic animal or a knock-in animal, so long as it contains a human nucleic acid sequence.

[0064] As used herein, the term "transgenic animal" includes animals that contain an exogenous human nucleic acid sequence integrated into the genome of the animal.

[0065] As used herein, "knock-in" (knock in / knockin) includes genetic modification that targets a specific chromosomal locus in the genome of a non-human animal and inserts a nucleic acid of interest into the targeted locus. In some cases, the genetic modification replaces the genetic information encoded in the chromosomal locus of the non-human animal with a different DNA sequence.

[0066] Genetically Modified Non-Human Animals In some aspects of the present invention, genetically modified non-human animals expressing human IL-6 are provided. Human IL-6 (hIL6) refers to a 184 amino acid protein having a sequence as described, for example, in Genbank accession numbers NM_000600.3 and NP_000591.1. Human IL-6 is a secreted protein produced, for example, by T cells, B cells, monocytes, macrophages, fibroblasts, keratinocytes, endothelial cells, and myeloma cells. IL-6 acts through a cell surface heterodimeric receptor complex that includes a binding subunit (IL-6R) and a signaling subunit (gp130). While gp130 is a common component of other receptors such as those for IL-11, IL-27, and LIF, IL-6R is primarily restricted to hepatocytes, monocytes, activated B cells, resting T cells, and myeloma cell lines. IL-6 plays a central role in hematopoiesis, immune responses, and acute phase reactions, and has been shown to be a key factor for the final maturation of B cells into antibody-secreting cells (ASCs), particularly for the expansion of plasmablasts in germinal center reactions in T-dependent (TD) antibody responses. IL-6 is required for T cell proliferation in vitro and for the generation of cytotoxic T cells (CTLs) in vivo, making them more responsive to IL-2.

[0067] In some aspects of the present invention, the genetically modified non-human animal expressing human IL-6 also expresses at least one additional human protein selected from human M-CSF, human IL-3, human GM-CSF, human TPO, and human SIRPa, or any combination thereof. In other words, the non-human animal expressing human IL-6 may express one, two, three, four, or all five of the human proteins selected from hM-CSF, hIL-3, hGM-CSF, hTPO, and hSIRPa. Genetically modified non-human animals expressing hM-CSF, hIL-3, hGM-CSF, hTPO, and / or hSIRPa from which the non-human animals of the invention can be designed or from which the non-human animals of the invention can be generated are known in the art, see, for example, US application US 2013 / 0042330 and Rathinam et al. 2011, Blood 118:3119-28, which disclose knock-in mice expressing human M-CSF; US Patent No. 8,541,646 and Willinger et al. 2011, Proc Natl Acad Sci USA, 108:2390-2395, which disclose knock-in mice expressing human IL-3 and human GM-CSF; US Patent No. 8,541,646 and Rongvaux et al. 2011, Proc Natl Acad Sci USA, 108:2390-2395, which disclose knock-in mice expressing human TPO. USA, 108:2378-83; and in PCT application WO 2012 / 040207, which discloses a transgenic mouse expressing human Sirpa, and Strowig et al. 2011, Proc Natl Acad Sci USA 108(32):13218-13223, the entire disclosures of which are incorporated herein by reference.

[0068] In various embodiments, the nucleic acid encoding the human protein is operably linked to one or more control sequences in a manner that allows transcription of the nucleic acid into mRNA and translation of the mRNA into a human protein. The term "control sequence" is recognized in the art and is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such control sequences are known to those skilled in the art and are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, 1990, Academic Press, San Diego, Calif. In one embodiment, the human nucleic acid is expressed by the native control elements of the human nucleic acid. In another embodiment, the human nucleic acid is expressed by the native control elements of the corresponding nucleic acid of the non-human host animal.

[0069] Thus, in some embodiments, the nucleic acid encoding human IL-6 is operably linked to the IL-6 promoter of a non-human animal. In other embodiments, the nucleic acid encoding human IL-6 is operably linked to the human IL-6 promoter. As another example, in some embodiments, the nucleic acid encoding human M-CSF is operably linked to the M-CSF promoter of an animal. In other embodiments, the nucleic acid encoding human M-CSF is operably linked to the human M-CSF promoter. As a third example, in some embodiments, the nucleic acid encoding human IL-3 is operably linked to the IL-3 promoter of an animal. In other embodiments, the nucleic acid encoding human IL-3 is operably linked to the human IL-3 promoter. As a fourth example, in some embodiments, the nucleic acid encoding human GM-CSF is operably linked to the GM-CSF promoter of an animal. In other embodiments, the nucleic acid encoding human GM-CSF is operably linked to the human GM-CSF promoter. As a fifth example, in some embodiments, the nucleic acid encoding human TPO is operably linked to the TPO promoter of an animal. In other embodiments, the nucleic acid encoding human TPO is operably linked to a human TPO promoter.

[0070] Those skilled in the art will understand that the genetically modified animals of the present invention include genetically modified animals that express at least one human nucleic acid from a promoter. Non-limiting examples of ubiquitously expressed promoters useful in the present invention include, but are not limited to, DNA pol II promoter, PGK promoter, ubiquitin promoter, albumin promoter, globin promoter, ovalbumin promoter, SV40 early promoter, Rous sarcoma virus (RSV) promoter, retroviral LTR and lentiviral LTR, β-actin promoter, ROSA26 promoter, heat shock protein 70 (Hsp70) promoter, EF-1α gene encoding elongation factor 1α (EF1) promoter, eukaryotic initiation factor 4A (eIF-4A1) promoter, chloramphenicol acetyltransferase (CAT) promoter, and CMV (cytomegalovirus) promoter. Promoter and enhancer expression systems useful in the present invention also include inducible and / or tissue-specific expression systems. Non-limiting examples of tissue-specific promoters useful in the expression constructs of the compositions and methods of the present invention include promoters of genes expressed in the hematopoietic system, such as the IL-6 promoter, the M-CSF promoter, the IL-3 promoter, the GM-CSF promoter, the SIRPA promoter, the TPO promoter, the IFN-β promoter, the Wiskott-Aldrich syndrome protein (WASP) promoter, the CD45 (also called lymphocyte common antigen) promoter, the Flt-1 promoter, the endoglin (CD105) promoter, and the ICAM-2 (intercellular adhesion molecule 2) promoter. These and other promoters useful in the compositions and methods of the invention are known in the art, as exemplified by Abboud et al. (2003, J. Histochem & Cytochem. 51:941-949), Schorpp et al. (1996, NAR 24:1787-1788), McBurney et al. (1994, Devel. Dynamics, 200:278-293), and Majumder et al. (1996, Blood 87:3203-3211).In addition to including a promoter, one or more additional control elements, such as enhancer elements or intron sequences, are included in various embodiments of the present invention.Examples of enhancers useful in the compositions and methods of the present invention include, but are not limited to, cytomegalovirus (CMV) early enhancer elements and SV40 enhancer elements.Examples of intron sequences useful in the compositions and methods of the present invention include, but are not limited to, β-globin introns or generic introns.Other additional control elements useful in some embodiments of the present invention include, but are not limited to, transcription termination sequences and mRNA polyadenylation (pA) sequences.

[0071] In addition to naturally occurring human nucleic acid and amino acid sequences, those skilled in the art will also recognize that the terms human nucleic acid and human amino acid also encompass variants of human nucleic acid and amino acid sequences. As used herein, the term "variant" defines either an isolated naturally occurring genetic variant of a human or a recombinantly prepared variation of a human, each of which contains one or more mutations compared to the corresponding wild-type human. For example, such mutations can be substitutions, additions, and / or deletions of one or more amino acids. The term "variant" also includes non-human orthologs. In some embodiments, the variant polypeptides of the present invention have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with wild-type human polypeptides.

[0072] The percent identity between two sequences is determined using techniques as described elsewhere herein. Standard molecular biology techniques such as site-directed mutagenesis and PCR-mediated mutagenesis can be used to introduce mutations. Those skilled in the art will recognize that one or more amino acid mutations can be introduced without altering the functional properties of human proteins.

[0073] To generate human protein variants, conservative amino acid substitutions can be generated in human proteins.Conservative amino acid substitutions are recognized in the art as the substitution of one amino acid with another amino acid with similar characteristics.For example, each amino acid can be described as having one or more of the following characteristics: positively charged, negatively charged, aliphatic, aromatic, polar, hydrophobic, and hydrophilic.Conservative substitutions are the substitution of one amino acid with a specified structural or functional characteristic with another amino acid with the same characteristics. Acidic amino acids include aspartic acid, glutamic acid; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine, and valine; aromatic amino acids include phenylalanine, glycine, tyrosine, and tryptophan; polar amino acids include aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, and tyrosine; hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, and tryptophan; conservative substitutions include the substitution of amino acids within each group.Amino acids can also be described in terms of relative size, and alanine, cysteine, aspartic acid, glycine, asparagine, proline, threonine, serine, and valine are all typically considered small.

[0074] Human variants may include synthetic amino acid analogs, amino acid derivatives, and / or non-standard amino acids, illustratively including, but not limited to, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, dienkolinic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, methylhistidine, and ornithine.

[0075] The human variant is encoded by a nucleic acid that has a high degree of identity with the nucleic acid encoding the wild-type human. The complementary strand of the nucleic acid encoding the human variant specifically hybridizes with the nucleic acid encoding the wild-type human under high stringency conditions. The nucleic acid encoding the human variant may be isolated or produced recombinantly or synthetically using well-known methodologies.

[0076] In some embodiments, the genetically modified non-human animals that express human nucleic acid sequences also express the corresponding non-human animal nucleic acid sequences. For example, as described in more detail below, in certain embodiments, the human nucleic acid sequences are randomly integrated into the genome of the non-human animal, e.g., such that the animal contains an exogenous human nucleic acid sequence at a locus other than the non-human animal locus that encodes the corresponding non-human animal protein. In other embodiments, the genetically modified non-human animals that express human nucleic acid sequences do not express the corresponding non-human animal nucleic acid sequences. For example, as described in more detail below, in certain embodiments, a nucleic acid encoding a human protein is introduced into the animal to replace the genomic material encoding the corresponding non-human animal protein, disabling the corresponding non-human animal gene in the animal and causing the corresponding non-human animal protein to be missing. In other words, the non-human animal is "knocked in" for the human gene.

[0077] Thus, in some embodiments, the genetically modified non-human animal expressing human IL-6 also expresses non-human animal IL-6. In other embodiments, the genetically modified non-human animal expressing human IL-6 does not express non-human animal IL-6. As a second example, in some embodiments, the genetically modified non-human animal expressing human M-CSF also expresses non-human animal M-CSF. In other embodiments, the genetically modified non-human animal expressing human M-CSF does not express non-human animal M-CSF. As a third example, in some embodiments, the genetically modified non-human animal expressing human IL-3 also expresses non-human animal IL-3. In other embodiments, the genetically modified non-human animal expressing human IL-3 does not express non-human animal IL-3. As a fourth example, in some embodiments, the genetically modified non-human animal expressing human GM-CSF also expresses non-human animal GM-CSF. In other embodiments, the genetically modified non-human animal expressing human GM-CSF does not express non-human animal GM-CSF. As a fifth example, in some embodiments, the genetically modified non-human animal expressing human TPO also expresses non-human animal TPO. In other embodiments, the genetically modified non-human animal expressing human TPO does not express non-human animal TPO.

[0078] In some embodiments, the genetically modified animals of the invention are immunodeficient. By "immunodeficient" is meant that in a non-human animal, one or more aspects of its native immune system are deficient, e.g., the animal is deficient in one or more types of functional host immune cells, e.g., the number and / or function of non-human B cells, the number and / or function of non-human T cells, the number and / or function of non-human NK cells, etc.

[0079] As an example, the immunodeficient animal may have severe combined immunodeficiency (SCID), which refers to a condition characterized by a lack of T cells and deficient B cell function. Examples of SCID include X-linked SCID, characterized by gamma chain gene mutations or loss of the IL2RG gene and lymphocyte phenotype T(-)B(+)NK(-); as well as Jak3 gene mutations and lymphocyte phenotype T(-)B(+)NK(-), ADA gene mutations and lymphocyte phenotype T(-)B(-)NK(-), IL-7R alpha chain mutations and lymphocyte phenotype T(-)B(+)NK(+), CD3 delta or epsilon mutations and lymphocyte phenotype T(-)B(+)NK(+), RAG1 / RAG2 mutations and lymphocyte phenotype T(-)B(-)NK(+), Artemis gene mutations and lymphocyte phenotype T(-)B(-)NK(+), CD45 gene mutations and lymphocyte phenotype T(-)B(+)NK(+), and Prkdc scid Mutations (Bosma et al. (1989, Immunogenetics 29:54-56) and autosomal recessive SCID characterized by lymphocyte phenotype T(-)B(-), lymphopenia, and hypoglobulinemia. Thus, in some embodiments, the genetically modified immunodeficient non-human animal has one or more deficiencies selected from IL2 receptor gamma chain deficiency, ADA gene mutation, IL7R mutation, CD3 mutation, RAG1 and / or RAG2 mutation, Artemis mutation, CD45 mutation, and Prkdc mutation.

[0080] The genetically modified non-human animal of the present invention can be any non-human mammal, such as laboratory animals, farm animals, livestock, etc., such as mice, rodents, dogs, cats, pigs, horses, cows, sheep, non-human primates, etc., that have been genetically modified to include a human IL-6 coding sequence operably linked to an IL-6 promoter; for example, mice, rats, rabbits, hamsters, guinea pigs, cows, pigs, sheep, goats, and other transgenic animal species, particularly mammalian species, as known in the art. In certain embodiments, the genetically modified animal of the present invention is a mouse, a rat, or a rabbit.

[0081] In one embodiment, the non-human animal is a mammal. In some such embodiments, the non-human animal is a small mammal, for example, of the superfamily Dipodoidea or superfamily Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from mouse, rat, and hamster. In one embodiment, the rodent is selected from the superfamily Muridea. In one embodiment, the genetically modified animals are selected from the Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rats). In a specific embodiment, the genetically modified rodent is selected from typical mice and rats (Muridae), Mongolian gerbils, spiny mice, and maned mice. In one embodiment, the genetically modified mouse is derived from a member of the Muridae family.

[0082] In one embodiment, the genetically modified non-human animal of the present invention is a rat. In one such embodiment, the rat is selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In another embodiment, the rat strain is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0083] In another embodiment, the genetically modified animal of the present invention is a mouse, e.g., a mouse of the C57BL strain (e.g., C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, C57BL / Ola etc.); 129 strain mice (e.g., 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2); BALB strain mice; e.g., BALB / c, etc. See, e.g., Festing et al. (1999) Mammalian Genome 10:836, see also Auerbach et al (2000) Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines. In a specific embodiment, the genetically modified mouse is a mix of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In another specific embodiment, the mouse is a mix of the aforementioned 129 strains or a mix of the aforementioned BL / 6 strains. In a specific embodiment, the mixed 129 strain is a 129S6 (129 / SvEvTac) strain. In yet another embodiment, the mouse is a mix of a BALB strain and another of the aforementioned strains.

[0084] Thus, for example, in some embodiments, the genetically modified non-human animals of the present invention are (e.g., IL2 receptor γ chain deficient (i.e., γ c - / -) and / or RAG deficiency) in the number and / or function of B cells and / or the number and / or function of T cells and / or the number and / or function of NK cells, having a genome that includes human nucleic acids, e.g., nucleic acids encoding human IL-6, hM-CSF, hIL-3, hGM-CSF, hTPO, and / or hSIRPa operably linked to their corresponding promoters, e.g., M-CSF promoter, IL-3 promoter, GM-CSF promoter, TPO promoter, or SIRPa promoter, respectively, and which express the encoded human proteins.

[0085] In certain specific embodiments, the genetically modified animals of the invention are immunodeficient mice (i.e., the mice express mouse SIRPa), i.e., immunodeficient hIL-6,hSirpa mice, e.g., Rag2, comprising a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter at the mouse IL-6 locus, and a nucleic acid encoding human SIRPa operably linked to a human SIRPa promoter randomly integrated into the genome of the non-human animal. - / - IL2rg - / - IL-6 h / + hSIRPa + Mouse or Rag2 - / - IL2rg - / - IL-6 h / h hSIRPa + In some such embodiments, the mouse further comprises a nucleic acid encoding human M-CSF operably linked to an M-CSF promoter, a nucleic acid encoding human IL-3 operably linked to an IL-3 promoter, a nucleic acid encoding human GM-CSF operably linked to a GM-CSF promoter, and a nucleic acid encoding human TPO operably linked to a TPO promoter. That is, an immunodeficient hIL-6, hSirpa, hM-CSF, hIL-3, hGM-CSF, hTPO mouse, e.g., Rag2 - / - IL2rg - / - IL-6 h / + M-CSF h / + IL-3 h / +GM-CSF h / + TPO h / + hSIRPa + Mouse, Rag2 - / - IL2rg - / - IL-6 h / + M-CSF h / h IL-3 h / h GM-CSF h / h TPO h / h hSIRPa + mouse.

[0086] In certain specific embodiments, the genetically modified animal of the invention is an immunodeficient mouse that comprises a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter, is deficient in mouse IL-6, comprises a nucleic acid encoding human SIRPa operably linked to a human SIRPa promoter randomly integrated into the genome of the non-human animal (i.e., the mouse still expresses mouse SIRPa), comprises a nucleic acid encoding human M-CSF operably linked to an M-CSF promoter, is deficient in mouse M-CSF, comprises a nucleic acid encoding human IL-3 operably linked to an IL-3 promoter, is deficient in mouse IL-3, comprises a nucleic acid encoding human GM-CSF operably linked to a GM-CSF promoter, is deficient in mouse GM-CSF, comprises a nucleic acid encoding human TPO operably linked to a TPO promoter, and is deficient in mouse TPO, i.e., Rag2 - / - IL2rg - / - IL-6 h / h M-CSF h / h IL-3 h / h GM-CSF h / h TPO h / h , hSIRPa+ mice.

[0087] Methods for Producing Genetically Modified Non-Human Animals - Patent application The genetically modified non-human animals of the invention can be produced using any convenient method for the production of genetically modified animals, for example, as known in the art or described herein.

[0088] For example, the nucleic acid encoding the human protein of interest, such as IL-6, hM-CSF, hIL-3, hGM-CSF, hTPO, or hSIRPa, can be incorporated into a recombinant vector in a suitable form for insertion into the genome of host cells and expression of the human protein in non-human host cells.In various embodiments, the recombinant vector comprises one or more control sequences operably linked to the nucleic acid encoding the human protein, as described above, in a manner that allows the transcription of the nucleic acid into mRNA and the translation of the mRNA into a human protein.It will be understood that the design of the vector can depend on factors such as the selection of the host cell to be transfected, the amount of the human protein to be expressed, and / or how the encoding nucleic acid is integrated into the genome of the non-human host, as known in the art.

[0089] Any of a variety of methods can then be used to introduce the human nucleic acid sequence into animal cells to generate genetically modified animals that express human genes. Such techniques are well known in the art and include, but are not limited to, pronuclear microinjection of oocytes, transformation of embryonic stem cells, homologous recombination, and knock-in techniques. Methods for generating genetically modified animals that may be used include those described by Sundberg and Ichiki (2006, Genetically Engineered Mice Handbook, CRC Press), Hofker and van Deursen (2002, Genetically modified Mouse Methods and Protocols, Humana Press), Joyner (2000, Gene Targeting: A Practical Approach, Oxford University Press), Turksen (2002, Embryonic stem cells: Methods and Protocols in Methods Mol Biol., Humana Press), Meyer et al. (2010, Proc. Nat. Acad. Sci. USA 107:15022-15026), and Gibson (2004, A Primer Of Genome Science 2 nd ed. Sunderland, Massachusetts: Sinauer), U.S. Pat. No. 6,586,251, Rathinam et al. (2011, Blood 118:3119-28), Willinger et al. (2011, Proc Natl Acad Sci USA, 108:2390-2395), Rongvaux et al. (2011, Proc Natl Acad Sci USA, 108:2378-83), and Valenzuela et al. (2003, Nat Biot 21:652-659).

[0090] For example, the genetically modified animals of the present invention can be produced by introducing a nucleic acid encoding a human protein into an oocyte, e.g., by microinjection, and allowing the oocyte to develop in a female foster animal. In a preferred embodiment, a construct containing a human nucleic acid sequence is injected into a fertilized oocyte. Fertilized oocytes can be collected from superovulated females the day after mating and injected with an expression construct. Injected oocytes are cultured overnight or transferred directly into the oviducts of pseudopregnant females 0.5 days after mating. Methods for superovulation, oocyte collection, expression construct injection, and embryo transfer are known in the art and described in Manipulating the Mouse Embryo (2002, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press). Derived animals can be evaluated for the presence of the introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or protein analysis (e.g., ELISA, Western blot, etc.). Such methods typically result in integration of the injected nucleic acid sequence (in this case a construct comprising a nucleic acid encoding a human protein of interest) into the genome of the oocyte, and thus into the non-human animal, randomly, i.e., at a locus other than the locus of the host animal that expresses the corresponding protein.

[0091] As another example, constructs containing nucleic acids encoding human proteins can be transfected into stem cells (ES or iPS cells) using well-known methods such as electroporation, calcium phosphate precipitation, and lipofection. The cells can be evaluated for the presence of the introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or protein analysis (e.g., ELISA, Western blot, etc.). Cells that are determined to have incorporated the expression construct can then be microinjected into preimplantation embryos. For detailed descriptions of methods known in the art that are useful for the compositions and methods of the present invention, see Nagy et al. (2002, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press), Nagy et al. (1990, Development 110:815-821), U.S. Patent No. 7,576,259, U.S. Patent No. 7,659,442, U.S. Patent No. 7,294,754, and Kraus et al. (2010, Genesis 48:394-399). Such methods are typically used in the targeted integration of transfected nucleic acid sequences (in this case constructs containing nucleic acids encoding human proteins of interest) into the genome of stem cells and thus into human animals. Often, such methods result in the replacement of host genomic material, e.g., genomic material encoding the corresponding host protein, with nucleic acids encoding the human protein of interest.

[0092] The genetically modified founder animals can be used to breed additional animals carrying the genetic modifications. Genetically modified animals carrying nucleic acids encoding the human proteins of the present disclosure can be further crossed with other genetically modified animals carrying other genetic modifications, or crossed with knockout animals, e.g., knockout animals that do not express one or more of their genes.

[0093] In some embodiments, the genetically modified immunodeficient animal comprises a genome comprising a nucleic acid encoding a human polypeptide operably linked to a promoter and expresses the encoded human polypeptide. In various embodiments, the genetically modified immunodeficient non-human animal comprises a genome comprising an expression cassette comprising a nucleic acid encoding at least one human polypeptide operably linked to a promoter and a polyadenylation signal, and further comprising an intron, and expresses the encoded human polypeptide.

[0094] As mentioned above, in some embodiments, the genetically modified animal of the present invention is an immunodeficient animal.A genetically modified non-human animal that is immunodeficient and contains one or more human cytokines, such as IL-6, M-CSF, IL-3, GM-CSF, TPO, and / or SIRPa, can also be generated by any convenient method for generating genetically modified animals, such as, for example, using DNA injection of expression constructs into preimplantation embryos, for example, as known in the art or described herein, or by using stem cells, such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, that contain mutant SCID gene alleles that will cause immunodeficiency when homozygous, for example, as described in more detail herein above and in the examples.Then, mice with modified oocytes or ES cells are generated, for example, using the methods described herein and known in the art, and mated to generate immunodeficient mice that contain desired genetic modifications. As another example, genetically modified non-human animals can be generated in a non-immunodeficient background and bred with animals containing mutant SCID gene alleles that would result in immunodeficiency when homozygous, and the progeny bred to produce immunodeficient animals that express at least one human protein of interest.

[0095] Various aspects of the present invention provide genetically modified animals that contain human nucleic acid in substantially all of their cells, and genetically modified animals that contain human nucleic acid in some, but not all, of their cells. In some cases, e.g., in targeted recombination, one copy of human nucleic acid will be integrated into the genome of the genetically modified animal. In other cases, e.g., in random integration, multiple copies of human nucleic acid, adjacent or separated from each other, may be integrated into the genome of the genetically modified animal.

[0096] Thus, in some embodiments, the genetically modified non-human animal of the invention may be an immunodeficient animal that comprises a genome comprising a nucleic acid encoding a human polypeptide operably linked to a corresponding non-human animal promoter and expresses the encoded human polypeptide. In other words, the genetically modified immunodeficient non-human animal of the invention comprises a genome comprising an expression cassette comprising a nucleic acid encoding at least one human polypeptide operably linked to a corresponding non-human promoter and polyadenylation signal, and expresses the encoded human polypeptide.

[0097] Availability The genetically modified non-human animals provided in various embodiments of the present invention have a variety of uses, such as use as models for hematopoietic cell growth and differentiation, in vivo evaluation of the human hematopoietic system, in vivo evaluation of cancer cells, in vivo study of immune responses, in vivo evaluation of vaccines and vaccination regimens, use in testing the effect of agents that modulate cancer cell growth or survival, in vivo evaluation of cancer treatments, in vivo generation and collection of immune mediators such as antibodies, and use in testing the effect of agents that affect hematopoietic and immune cell function.

[0098] Towards this goal, in some cases, the genetically modified non-human animals of the invention ("hosts") are engrafted with at least one human hematopoietic cell. In some embodiments, methods are provided for producing animal models for the study of the human hematopoietic system, comprising the step of engrafting human hematopoietic cells into the genetically modified non-human animals of the invention ("hosts"). In certain embodiments, methods are provided for engrafting human hematopoietic cells into the genetically modified non-human animals disclosed herein.

[0099] In some specific cases, the genetically modified non-human animal of the present invention is engrafted with at least one human multiple myeloma cell.In some such embodiments, a method for making an animal model for cancer research is provided, comprising engrafting human multiple myeloma cells into the genetically modified non-human animal of the present invention.In some such embodiments, the present invention is a method for engrafting human multiple myeloma cells into the genetically modified non-human animal of the present invention.The engrafted human multiple myeloma cells useful in the compositions and methods of the present invention include any human multiple myeloma cells.

[0100] Human hematopoietic cells useful in the engraftment of the genetically modified non-human animals of the present invention include any convenient human hematopoietic cells. Non-limiting examples of human hematopoietic cells useful in the present invention include, but are not limited to, hematopoietic cells of any lineage at any stage of differentiation, including HSCs, HSPCs, LICs (leukemia initiating cells), and highly differentiated hematopoietic cells of any lineage. In some cases, the human hematopoietic cells are primary cells. Here, "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein to include acutely isolated cells or cell cultures obtained from a subject and propagated in vitro for a limited number of passages, i.e., divisions, of the culture. For example, a primary culture is a culture that may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not beyond the crisis stage. In other embodiments, the human hematopoietic cells are derived from a cell line, i.e., the cells are derived from an immortalized culture, e.g., have been passaged more than about 15 times. In some cases, the hematopoietic cells engrafted include healthy cells. In other cases, the hematopoietic cells engrafted include diseased hematopoietic cells, e.g., cancerous hematopoietic cells, e.g., cancerous effector B cells, i.e., multiple myeloma cells. In some cases, the hematopoietic cells engrafted include both healthy and diseased cells, e.g., healthy B cells and cancerous effector B cells, healthy T cells and cancerous effector B cells, etc.

[0101] Hematopoietic cells, i.e., primary cells, cell lines generated therefrom, etc., may be derived from any tissue or location of a human donor, including, but not limited to, bone marrow, peripheral blood, liver, fetal liver, or umbilical cord blood. Such hematopoietic cells may be isolated from any human donor, including healthy donors and donors with diseases such as cancer, including leukemia. Engraftment of hematopoietic cells in the genetically modified animals of the invention is characterized by the presence of human hematopoietic cells in the engrafted animal. In a specific embodiment, engraftment of hematopoietic cells in the genetically modified animals of the invention is characterized by the presence of differentiated human hematopoietic cells in the engrafted animal to which the hematopoietic cells were provided, compared to a suitable control animal.

[0102] Methods for isolating human hematopoietic cells, administering human hematopoietic cells to a host animal, and assessing their engraftment are well known in the art. Hematopoietic cells, including either normal cells or neoplastic cells or a combination thereof, for administration to a host animal can be obtained from any tissue that contains hematopoietic cells, such as, but not limited to, umbilical cord blood, bone marrow, peripheral blood, peripheral blood mobilized by cytokines or chemotherapy, and fetal liver. Exemplary methods for isolating human hematopoietic cells, administering human hematopoietic cells to a host animal, and assessing engraftment of human hematopoietic cells in the host animal are described herein, as well as in Pearson et al. (2008, Curr. Protoc. Immunol. 81:1-15), Ito et al. (2002, Blood 100:3175-3182), Traggiai et al. (2004, Science 304:104-107), Ishikawa et al. (2005, Blood 106:1565-1573), Shultz et al. (2005, J. Immunol. 174:6477-6489), and Holyoake et al. (1999, Exp Hematol. 27:1418-27).

[0103] In some embodiments of the present invention, human hematopoietic cells, including either normal cells and neoplastic cells or a combination thereof, are isolated from the original source material to obtain a population of cells enriched for a particular hematopoietic cell population (e.g., HSC, HSPC, LIC, CD34+, CD34-, lineage-specific markers, cancer cell markers, etc.). The isolated hematopoietic cells may or may not be a pure population. In one embodiment, the hematopoietic cells useful in the compositions and methods of the present invention are depleted of cells with a particular marker. In another embodiment, the hematopoietic cells useful in the compositions and methods of the present invention are enriched by selection for a marker. In some embodiments, the hematopoietic cells useful in the compositions and methods of the present invention are cell populations in which the selected cells constitute about 1-100% of the cells, although in certain embodiments, cell populations in which the selected cells constitute less than 1% of the total cells can also be used. In one embodiment, the hematopoietic cells useful in the compositions and methods of the present invention are depleted of cells with a particular marker, such as CD34. In another embodiment, the hematopoietic cells useful in the compositions and methods of the present invention are enriched by selection for a marker, such as CD34. In some embodiments, hematopoietic cells useful in the compositions and methods of the invention are cell populations in which CD34+ cells comprise about 1-100% of the cells, although in certain embodiments cell populations in which CD34+ cells comprise less than 1% of the total cells can also be used. In certain embodiments, hematopoietic cells useful in the compositions and methods of the invention are T cell depleted cell populations in which CD34+ cells comprise about 1-3% of the total cells, lineage depleted cell populations in which CD34+ cells comprise about 50% of the total cells, or CD34+ positive selected cell populations in which CD34+ cells comprise about 90% of the total cells.

[0104] With respect to the generation of a human hematopoietic and / or immune system in a genetically modified non-human animal expressing at least one human gene, the number of hematopoietic cells administered is not a limiting consideration. Thus, as a non-limiting example, the number of hematopoietic cells administered may be about 1×10 3 ~Approx. 1×10 7The number of HSPCs administered may range from about 3×10 to about 10, although in various embodiments, more or fewer may be used. As another non-limiting example, when the recipient is a mouse, the number of HSPCs administered may be about 3×10 3 ~Approx. 1×10 6 The number of cells that need to be administered can range from 1 to 200 CD34+ cells, although in various embodiments, more or less may be used. For other species of recipient, the number of cells that need to be administered can be determined using only routine experimentation.

[0105] For example, in one embodiment, the genetically modified and treated mouse is engrafted with human hematopoietic cells or human hematopoietic stem cells (HPSCs) to form a genetically modified and engrafted mouse. In one embodiment, the hematopoietic cells are selected from human umbilical cord blood cells and human fetal liver cells. In one embodiment, engraftment is at least about 1-2×10 5 with human CD34+ cells.

[0106] In some cases, administration of hematopoietic cells (e.g., normal or neoplastic) may be preceded by pretreatment, such as sublethal irradiation of the recipient animal with high frequency electromagnetic radiation, typically using gamma or X-rays, or treatment with radio-like drugs such as busulfan or nitrogen mustard. Pretreatment is believed to reduce the number of host hematopoietic cells, create appropriate microenvironmental factors for engraftment of human hematopoietic cells, and / or create a microenvironmental niche for engraftment of human hematopoietic cells. Standard methods for pretreatment are known in the art, as described herein and in J. Hayakawa et al, 2009, Stem Cells, 27(1):175-182. In one embodiment, the genetically modified mouse is treated to eliminate endogenous hematopoietic cells that may be present in the mouse. In one embodiment, the treatment includes irradiating the genetically modified mouse. In a specific embodiment, the genetically modified newborn mouse is sublethally irradiated. In a specific embodiment, neonates are irradiated with two doses of 200 cGy, four hours apart.

[0107] Hematopoietic cells (e.g., normal or neoplastic) may be administered to newborn or adult animals by administration via a variety of routes, such as, but not limited to, intravenous, intrahepatic, intraperitoneal, intrafemoral, and / or intratibial. Methods are provided by embodiments of the present invention for the engraftment of human hematopoietic cells, including either normal cells and neoplastic cells or a combination thereof, into an immunodeficient animal, comprising the step of providing human hematopoietic cells to the immunodeficient animal, with or without the step of irradiating the animal prior to administration of the hematopoietic cells. Methods are provided by embodiments of the present invention for the engraftment of human hematopoietic cells into an immunodeficient animal, comprising the step of providing human hematopoietic cells, including either normal cells and neoplastic cells or a combination thereof, into a genetically modified non-human animal of the present invention, with or without the step of administering a radio-like drug, such as busulfan or nitrogen mustard, to the animal prior to administration of the hematopoietic cells.

[0108] Engraftment of human hematopoietic cells, including either normal cells and neoplastic cells or a combination thereof, into the genetically modified animals of the present invention can be assessed by any of a variety of methods, such as, but not limited to, flow cytometric analysis of cells in the animal to which the human hematopoietic cells have been administered, at one or more time points following administration of the hematopoietic cells.

[0109] In general, engraftment can be considered successful when the number (or percentage) of human hematopoietic cells, including either normal and neoplastic cells or a combination thereof, present in the genetically modified non-human animal is greater than the number (or percentage) of human cells administered to the non-human animal over the life span of the administered human hematopoietic cells. Detection of the progeny of the administered hematopoietic cells can be achieved, for example, by detection of human DNA in the recipient animal or by detection of fully human hematopoietic cells, for example, by detection of human cell markers such as human CD45, human CD34, or sIL-6R. Serial transfer of human hematopoietic cells from the first recipient to a second recipient and engraftment of the human hematopoietic cells in the second recipient is a further optional test of engraftment in the first recipient. Engraftment can be detected by flow cytometry as 0.05% or more human CD45+ cells in the blood, spleen, or bone marrow 1-4 months after administration of the human hematopoietic cells. For example, cytokines (eg, GM-CSF) can be used to mobilize stem cells as described in Watanabe (1997, Bone Marrow Transplantation 19:1175-1181).

[0110] In one embodiment, the immunodeficient genetically modified engrafted animal produces human cells selected from CD34+ cells, hematopoietic stem cells, hematopoietic cells, myeloid progenitor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, thymocytes, T cells, B cells, platelets, and combinations thereof. In one embodiment, the human cells are present at 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after engraftment.

[0111] In one embodiment, the immunodeficient genetically modified engrafted animal develops a human hematolymphoid system including human hematopoietic stem and progenitor cells, human myeloid progenitor cells, human myeloid cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human thymocytes, human T cells, human B cells, and human platelets. In one embodiment, the human hematolymphoid system is present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months post-engraftment.

[0112] In one embodiment, the immunodeficient genetically modified engrafted animal develops a human blood-lymphoid system that includes cancerous human hematopoietic cells, e.g., neoplastic plasma (effector B) cells. In one embodiment, the cancerous human hematopoietic cells are present 4 weeks, 6 weeks, 8 weeks, 12 weeks, or 12 weeks or later after engraftment. In certain embodiments, the cancerous human hematopoietic cells are present 2 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, or 12 weeks or later after engraftment.

[0113] After engraftment of human hematopoietic cells, the genetically modified non-human animals of the present invention have many uses in the art. For example, the engrafted genetically modified animals of the present disclosure are useful for studying the function of human hematopoietic cells in peripheral blood. As demonstrated in Example 2, genetically modified mice that are immunodeficient and contain a nucleic acid encoding human IL-6 operably linked to the IL-6 promoter in the IL-6 mouse locus (e.g., Rag2 - / - IL2rg null IL-6 h / h Mouse, Rag2 - / - IL2rg null IL-6 h / h hSIRPa + Mouse, and Rag2 - / - IL2rg - / - IL-6 h / h M-CSF h / h IL-3 h / h GM-CSF h / h TPO h / h ,hSIRPa+) are immunodeficient mice that do not express human IL-6, i.e., Rag2- / - IL2rg null Human hematopoietic cells, such as CD34, are better than mice. + They support the engraftment of progenitor cells into peripheral blood and spleen. Furthermore, these genetically modified mice promote the differentiation of human hematopoietic cells more efficiently than immunodeficient mice that do not express human IL-6. For example, these genetically modified mice promote the differentiation of CD5+ and CD27+ B cells better. CD5 is a protein found on a subset of IgM-secreting B cells called B-1 cells, and functions to buffer the activation signal from the B cell receptor so that B-1 cells can only be activated by very strong stimuli (such as bacterial proteins) and not by normal tissue proteins. CD27 is a marker for memory B cells. Furthermore, these genetically modified mice support the development of highly functional human hematopoietic cells more rapidly than immunodeficient mice that do not express human IL-6. For example, B cells differentiate into IgG-secreting plasma cells more rapidly in these genetically modified mice than in immunodeficient mice that do not express human IL-6. As such, the engrafted genetically modified animals of the present disclosure are useful in studying hematopoietic cell development and function, and more specifically, B lymphocyte differentiation and function.

[0114] As another example, the engrafted genetically modified animals of the present disclosure are useful for studying hematopoietic cancers. As demonstrated in Example 1 below, genetically modified mice that are immunodeficient and contain a nucleic acid encoding human IL-6 operably linked to the IL-6 promoter in the mouse IL-6 locus, e.g., Rag2 - / - IL2rg null IL-6 h / h Mouse, Rag2 - / - IL2rg null IL-6 h / h hSIRPa + Mouse, and Rag2 - / - IL2rg - / - IL-6 h / h M-CSF h / h IL-3 h / h GM-CSFh / h TPO h / h ,hSIRPa + In the mouse model, primary human multiple myeloma cells and human multiple myeloma cell lines can be engrafted, but the mouse model does not express human IL-6, i.e., Rag2 - / - IL2rg null However, these mice do not engraft. The expression of human SIRPa by genetically modified hosts further improves the observed engraftment rate and extent. Furthermore, the engraftment of multiple myeloma cells directly into the bone of these immunodeficient, genetically modified mice disclosed herein reproduces the bone pathology typically associated with human multiple myeloma, such as bone destruction and bone resorption, as quantified, for example, by μCT scan.

[0115] Thus, the engrafted genetically modified animals of the present disclosure are useful in screening candidate agents to identify those that will treat hematopoietic cancers. The terms "treatment", "treating" and the like are used herein generally to include obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it is a complete or partial prevention of the disease or its symptoms, and / or it may be therapeutic, in that it is a partial or complete cure of the disease and / or the deleterious effects caused by the disease. "Treatment", as used herein, includes any treatment of disease in a mammal, including: (a) preventing the onset of the disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. Important candidate agents as therapeutics for hematopoietic cancers include those that can be administered before, during, or after the onset of the cancer. Treatment of ongoing disease, where treatment stabilizes or reduces undesirable clinical symptoms in the patient, is particularly important. The terms "individual," "subject," "host," and "patient" are used interchangeably herein and include any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.

[0116] As another example, the engrafted genetically modified animals of the present disclosure are useful for studying human pathogens, i.e. pathogens that infect humans; the response of the human immune system to infection by human pathogens; and the effectiveness of drugs in preventing and / or treating infection by human pathogens. Pathogens can be viruses, fungi, bacteria, and the like. Non-limiting examples of viral pathogens include human or swine or avian influenza viruses. Non-limiting examples of bacterial pathogens include mycobacteria, such as Mycobacterium tuberculosis / M.tuberculosis, and enterobacteria, such as Salmonella typhi / S.typhi. Examples of methods for infecting mice with Salmonella typhi and assessing infection can be found, for example, in published U.S. Application No. 2011 / 0200982, the disclosure of which is incorporated herein by reference. Examples of methods for infecting mice with Mycobacterium tuberculosis and assessing infection can be found, for example, in published US application 2011 / 0200982, the disclosure of which is incorporated herein by reference. Other examples of human pathogens that do not infect wild-type mice, or that infect wild-type mice but do not model the immune response that humans mount in response to the pathogen, will be known to those skilled in the art. Such mouse models of pathogen infection are useful in research, for example, to better understand the progression of human infection. Such mouse models of infection are also useful in drug discovery, for example, to identify candidate agents that prevent or treat infection.

[0117] The engrafted genetically modified mice of the present disclosure also provide a useful system for screening candidate agents for a desired activity in vivo, e.g., to identify novel therapeutic agents and / or develop a better understanding of the molecular basis of immune system development and function, e.g., to identify agents that can modulate (i.e., promote or inhibit) the development and / or activity of hematopoietic cells, e.g., B cells, T cells, NK cells, macrophages, neutrophils, eosinophils, basophils, etc., in healthy or diseased states, e.g., as cancer cells, during pathogen infection; to identify agents that are toxic to hematopoietic cells, e.g., B cells, T cells, NK cells, macrophages, neutrophils, eosinophils, basophils, etc., and their precursor cells; to identify agents that prevent, attenuate, or reverse the toxic effects of toxic agents on hematopoietic cells, e.g., B cells, T cells, NK cells, macrophages, neutrophils, eosinophils, basophils, etc., and their precursor cells. As yet another example, the engrafted genetically modified animals of the present disclosure provide a system useful for predicting an individual's responsiveness to disease treatment, by providing an in vivo platform for predicting an individual's responsiveness to a drug, e.g., by screening the responsiveness of an individual's immune system to a drug, e.g., a therapeutic agent.

[0118] In a screening assay for biologically active agents, genetically modified mice engrafted with human hematopoietic cells of the present disclosure, e.g., engrafted Rag2 - / - IL2rg - / - IL-6 h / h hSIRPa + Mouse, engrafted Rag2 - / - IL2rg - / - IL-6 h / h M-CSF h / h IL-3 h / h GM-CSF h / h TPO h / h, hSIRPa+ mice, etc. are contacted with a candidate agent of interest, and the effect of the candidate agent is assessed by monitoring one or more output parameters. These output parameters may reflect the viability of the cells, e.g., the total number of hematopoietic cells or the number of cells of a particular hematopoietic cell type, or the apoptotic state of the cells, e.g., the amount of DNA fragmentation, the amount of cell blebbing, the amount of phosphatidylserine on the cell surface, etc., by methods well known in the art. Alternatively or in addition, the output parameters may reflect the differentiation potential of the cells, e.g., the percentage of differentiated cells and differentiated cell types. Alternatively or in addition, the output parameters may reflect the function of the cells, e.g., cytokines and chemokines produced by the cells, antibodies (e.g., amount or type) produced by the cells, the ability of the cells to home and extravasate to the site of challenge, the ability of the cells to modulate, i.e., promote or suppress, the activity of other cells in vitro or in vivo, etc. Other output parameters may reflect the extent of injury induced by diseased hematopoietic cells, e.g., bone destruction and bone resorption induced by multiple myeloma cells. Still other parameters may reflect infection in animals, e.g., the effect of an agent on pathogen infection, e.g., titer of pathogen in mice, presence of granulomas in mice, etc., relevant to the study being conducted.

[0119] A parameter is a quantifiable component of a cell, particularly one that can be accurately measured, as is desirable in high-throughput systems. A parameter may be a cellular component or product, including cell surface determinants, receptors, proteins or their conformational or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids, such as mRNA, DNA, etc., or a portion derived from such a cellular component, or a combination thereof. Most parameters will provide a quantitative readout, but in some cases semi-quantitative or qualitative results will be acceptable. The readout may include a single determined value, or may include a mean, median, or variance, etc. Characteristically, a series of parameter readout values ​​will be obtained for each parameter from multiple identical assays. A series of values ​​will be obtained for each of a set of test parameters that are expected to be variable and are expected to be obtained using standard statistical methods with common statistical methods used to provide a single value.

[0120] Candidate agents of interest for screening include known and unknown compounds encompassing numerous chemical classes, primarily organic molecules, and may include organometallic molecules, inorganic molecules, genetic sequences, vaccines, antibiotics or other agents suspected of having antimicrobial properties, peptides, polypeptides, antibodies, agents that are pharmaceuticals approved for use in humans, etc. An important aspect of the present invention is the evaluation of candidate drugs, including, for example, toxicity testing, etc.

[0121] Candidate agents include organic molecules that contain functional groups necessary for structural interactions, specifically hydrogen bonding, typically at least one amine, carbonyl, hydroxyl, or carboxyl group, and often at least two functional chemical groups. Candidate agents often contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Included are pharmacologically active drugs, genetically active molecules, and the like. Compounds of interest include chemotherapeutic agents, hormones, or hormone antagonists, and the like. Examples of pharmaceutical agents suitable for the present invention are those described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth edition. Also included are toxins and biological and chemical warfare agents. See, e.g., Somani, SM (Ed.), "Chemical Warfare Agents," Academic Press, New York, 1992.

[0122] Candidate agents of interest for screening also include nucleic acids, such as siRNA, shRNA, antisense molecules, or miRNA-encoding nucleic acids, or nucleic acids that encode polypeptides. Many vectors are available that are useful for transferring nucleic acids into target cells. Vectors can be maintained as episomes, such as plasmids, minicircle DNA, virus-derived vectors, such as cytomegalovirus or adenovirus, or can be integrated into target cell genomes through homologous recombination or random integration (e.g., retrovirus-derived vectors, such as MMLV, HIV-1, ALV, etc.). Vectors can be directly provided to the cells of the present invention. In other words, pluripotent cells are contacted with vectors that contain the nucleic acid of interest, so that the vectors are taken up by the cells.

[0123] Methods for contacting cells, e.g., cultured cells or cells in mice, with nucleic acid vectors, such as electroporation, calcium chloride transfection, and lipofection, are well known in the art. Alternatively, the nucleic acid of interest can be provided to the cells via a virus. In other words, the cells are contacted with viral particles containing the nucleic acid of interest. Retroviruses, e.g., lentiviruses, are particularly suitable for the method of the present invention. Commonly used retroviral vectors are "defective", i.e., unable to produce viral proteins required for productive infection. Instead, vector replication requires growth in a packaging cell line. To generate viral particles containing the nucleic acid of interest, the retroviral nucleic acid containing the nucleic acid is packaged into a viral capsid by a packaging cell line. Different packaging cell lines provide different envelope proteins that are incorporated into the capsid, and this envelope protein determines the specificity of the viral particle for the cell. There are at least three types of envelope proteins: ecotropic, amphotropic, and xenotropic. Retroviruses packaged with ecotropic envelope proteins, such as MMLV, can infect most mouse and rat cell types and are generated by using ecotropic packaging cell lines such as BOSC23 (Pear et al. (1993) PNAS 90:8392-8396). Retroviruses carrying amphotropic envelope proteins, such as 4070A (Danos et al., supra), can infect most mammalian cell types, including human, dog, and mouse, and are generated by using amphotropic packaging cell lines such as PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); GRIP (Danos et al. (1988) PNAS 85:6460-6464).Retroviruses packaged with xenotropic envelope proteins, such as AKR env, can infect most mammalian cell types except mouse cells. An appropriate packaging cell line can be used to ensure that the cells of interest, in some cases the engrafted cells, and in some cases the cells of the host, i.e., the genetically modified animal, are targeted by the packaged viral particles.

[0124] The vectors used to provide the nucleic acid of interest to the cells of the invention will typically contain a suitable promoter to activate expression, i.e., transcription, of the nucleic acid of interest. This may include ubiquitously acting promoters, such as the CMV-β actin promoter, or inducible promoters, such as promoters that are active in a particular cell population or that respond to the presence of drugs, such as tetracycline. Transcriptional activation means that transcription will be increased at least about 10-fold, at least about 100-fold, and more commonly at least about 1000-fold over basal levels in the target cells. Additionally, the vectors used to provide the reprogramming factors to the cells of the invention may contain genes that must be subsequently removed, e.g., using a recombinase system such as Cre / Lox, or cells expressing them must be destroyed, e.g., by including genes that allow for selective toxicity, such as herpes virus TK, bcl-xs, etc.

[0125] Candidate agents of interest for screening also include polypeptides. Such polypeptides may optionally be fused to a polypeptide domain that increases the solubility of the product. The domain may be linked to the polypeptide through a distinct protease cleavage site, e.g., a TEV sequence that is cleaved by the TEV protease. The linker may contain one or more flexible sequences, e.g., 1-10 glycine residues. In some embodiments, cleavage of the fusion protein is performed in a buffer that maintains the solubility of the product, e.g., in the presence of 0.5-2M urea, in the presence of polypeptides and / or polynucleotides that increase solubility, etc. Domains of interest include endosomolytic domains, e.g., influenza HA domains; and other polypeptides that aid in production, e.g., IF2 domains, GST domains, GRPE domains, etc. Additionally or alternatively, such polypeptides may be formulated for improved stability. For example, the peptides may be PEGylated, where the polyethyleneoxy groups provide enhanced longevity in the bloodstream. A polypeptide may be fused to another polypeptide to provide additional functionality, e.g., to increase in vivo stability. Typically, such a fusion partner is a stable plasma protein that can, e.g., extend the in vivo plasma half-life of the polypeptide when present as a fusion; specifically, such a stable plasma protein is an immunoglobulin constant domain. In most cases where stable plasma proteins are usually found as multimeric forms, e.g., immunoglobulins or lipoproteins, in which identical or different polypeptide chains are usually disulfide-bonded and / or non-covalently linked to form an assembled multi-chain polypeptide, the fusions herein containing the polypeptides will also be made and utilized as multimers with substantially the same structure as the stable plasma protein precursor. These multimers will be homogeneous with respect to the polypeptide drug they contain. Alternatively, they may contain multiple types of polypeptide drugs.

[0126] Candidate polypeptide agents may be produced from eukaryotic cells or by prokaryotic cells. They may be further processed by unfolding, e.g., heat denaturation, DTT reduction, etc., and may be further refolded using methods known in the art. Modifications of interest that do not alter the primary sequence include chemical derivatization of the polypeptide, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are glycosylation modifications, e.g., those made by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes, to modify the glycosylation pattern of the polypeptide, e.g., during synthesis and processing, or in further processing steps. Sequences with phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine, are also included. Polypeptides may be modified using conventional molecular biology techniques and synthetic chemistry to improve resistance to proteolysis, optimize solubility properties, or make them more suitable as therapeutic agents. Analogs of such polypeptides include those that contain residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids, which can be substituted for some or all of the amino acid residues.

[0127] Candidate polypeptide agents may be prepared by in vitro synthesis using conventional methods as known in the art. A variety of commercially available synthesizers are available, such as automated synthesizers from Applied Biosystems, Inc., Beckman, and others. By using synthesizers, naturally occurring amino acids can be substituted with unnatural amino acids. The particular sequence and mode of preparation will be determined by convenience, economics, the required purity, and the like. Alternatively, candidate polypeptide agents may be isolated and purified according to conventional methods of recombinant synthesis. A lysate of the expression host may be prepared and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In most cases, the composition used will comprise at least 20% by weight, more typically at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes generally at least about 99.5% by weight of the desired product, relative to contaminants associated with the method of preparation of the product and its purification. Generally, the percentages will be based on total protein.

[0128] In some cases, the candidate polypeptide agent being screened is an antibody. The term "antibody" or "antibody portion" is intended to include any polypeptide chain that contains a molecular structure with a special shape that fits and recognizes an epitope, and one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The specific or selective fit of a given structure with its specific epitope is sometimes referred to as a "lock and key" fit. The prototypical antibody molecule is an immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g., human, rodent, rabbit, bovine, ovine, porcine, canine, other mammals, chicken, other birds, etc., are considered to be "antibodies." The antibodies utilized in the present invention can be either polyclonal or monoclonal. The antibodies are typically provided in the medium in which the cells are cultured. The production and screening of antibodies is described in more detail below.

[0129] Candidate agents can be obtained from a variety of sources, including libraries of synthetic or natural compounds. Numerous means are available for random and specific synthesis of a variety of organic compounds, including biomolecules, including, for example, expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or easily produced. In addition, natural or synthetically produced libraries and compounds can be easily modified through conventional chemical, physical, and biochemical means and used to produce combinatorial libraries. Known pharmacological agents can be subjected to specific or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to produce structural analogs.

[0130] Candidate agents are screened for biological activity by administering the agent to at least one, and generally multiple, samples, sometimes together with a sample lacking the agent. Changes in parameters in response to the agent are measured, and the results are evaluated by comparison with reference cultures, e.g., cultures in the presence and absence of the agent, cultures obtained with other agents, etc. In cases where screening is performed to identify candidate agents that will prevent, mitigate, or reverse the effects of a toxic agent, the screening is typically performed in the presence of the toxic agent, in which case the toxic agent is added at the most appropriate time for the outcome to be determined. For example, in cases where the protective / preventive ability of the candidate agent is tested, the candidate agent can be added before the toxic agent, simultaneously with the candidate agent, or after treatment with the candidate agent. As another example, in cases where the ability of the candidate agent to reverse the effects of a toxic agent is tested, the candidate agent can be added after treatment with the toxic agent. As noted above, in some cases, the "sample" is a genetically modified non-human animal into which the cells have been engrafted, e.g., a candidate agent is provided to an immunodeficient animal, e.g., a mouse, into which human hematopoietic cells have been engrafted, the animal comprising a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter. In some cases, the sample is the human hematopoietic cells to be engrafted, i.e., the candidate agent is provided to the cells prior to engraftment into the immunodeficient, genetically modified animal.

[0131] When a candidate agent is administered directly to the engrafted genetically modified animal, the agent can be administered by any of the numerous methods known in the art for administration of peptides, small molecules, and nucleic acids to mice. For example, the agent can be administered orally, mucosally, topically, intradermally, or by injection, such as intraperitoneal, subcutaneous, intramuscular, intravenous, or intracranial injection. The agent can be administered in a buffer or incorporated into any of a variety of formulations, for example, by combination with a suitable pharmacopoeia. A "pharmaceutical acceptable vehicle" can be a vehicle approved by a federal or state regulatory agency for use in mammals, such as humans, or listed in the US Pharmacopeia or other generally recognized pharmacopeia. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier for formulating the compound of the present invention for administration to a mammal. Such pharmaceutical vehicles may be lipids, e.g., liposomes, e.g., liposomal dendrimers; water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., liquids, such as saline; gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Additionally, auxiliary substances, stabilizers, thickeners, lubricants, and colorants may be used. The pharmaceutical compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. The agents may be systemic after administration or localized by use of topical administration, intramural administration, or the use of implants that act to retain the active dose at the site of implantation. The active agents may be formulated for immediate activity or for sustained release. For some conditions, particularly central nervous system conditions, it may be necessary to formulate an agent that crosses the blood-brain barrier (BBB).One strategy for drug delivery through the blood-brain barrier (BBB) ​​involves disrupting the BBB by osmotic means such as mannitol or leukotrienes, or biochemically by the use of vasoactive substances such as bradykinin. When the composition is administered by intravascular injection, a BBB disrupting agent can be co-administered with the drug. Other strategies to cross the BBB may involve the use of endogenous transport systems, including caveolin-1 mediated transcytosis, carrier-mediated transporters such as carriers for glucose and amino acids, receptor-mediated transcytosis for insulin or transferrin, and active efflux transporters such as p-glycoprotein. To facilitate transport across the endothelial wall of blood vessels, active transport moieties may be conjugated to the therapeutic compounds for use in the present invention. Alternatively, drug delivery of the agent beyond the BBB can be by local delivery, e.g., intrathecal delivery, e.g., through an Ommaya reservoir (see, e.g., U.S. Patent Nos. 5,222,982 and 5,385,582, which are incorporated herein by reference); by bolus injection, e.g., intravitreally or intracranially, e.g., by a syringe; by continuous infusion, e.g., by cannulation, e.g., by convection (see, e.g., U.S. Application No. 20070254842, which is incorporated herein by reference); or by implantation of a device to which the agent is reversibly attached (see, e.g., U.S. Application Nos. 20080081064 and 20090196903, which are incorporated herein by reference).

[0132] When providing drugs to cells prior to engraftment, the drugs can conveniently be added to the culture medium of cultured cells in a solution or in a readily soluble form. Drugs can be added through a flow-through system as an intermittent or continuous stream, or a single or incremental bolus of compound can be added to a stationary solution. In a flow-through system, two liquids are used, one a physiologically neutral solution and the other an identical solution to which the test compound is added. The first liquid is passed over the cells, followed by the second liquid. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentration of the components of the culture medium should not change significantly with the addition of the bolus or between the two solutions in the flow-through method.

[0133] To obtain differential responses to various concentrations, multiple assays using different drug concentrations can be carried out in parallel.As known in the art, the determination of the effective concentration of a drug typically uses a series of concentrations resulting from 1:10 or other logarithmic scale dilutions.If necessary, the concentration can be further refined by a second dilution series.Typically, one of these concentrations serves as a negative control, i.e., zero concentration, or below the level of detection of the drug, or at or below the concentration of the drug that does not cause detectable changes in phenotype.

[0134] The analysis of the response of cells to candidate drugs in the genetically modified animals engrafted can be carried out at any time after treatment with the drug.For example, cells can be analyzed 1, 2, or 3 days after contact with the candidate drug, sometimes 4, 5, or 6 days after, sometimes 8, 9, or 10 days after, sometimes 14 days after, sometimes 21 days after, sometimes 28 days after, sometimes more than one month after, for example, 2 months after, 4 months after, 6 months after, or later.In some embodiments, the analysis includes analysis at multiple time points.The selection of the time point for analysis will be based on the type of analysis to be carried out, as will be easily understood by those skilled in the art.

[0135] The analysis may include any of the parameters described herein or known in the art to measure cell viability, cell proliferation, cell identity, cell morphology, and cell function, specifically those related to immune cells. For example, flow cytometry can be used to determine the total number of hematopoietic cells or the number of cells of a particular hematopoietic cell type. Histochemistry or immunohistochemistry, such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to measure DNA fragmentation or immunohistochemistry to detect Annexin V binding to phosphatidylserine on the cell surface, can be performed to determine the apoptotic state of the cells. Flow cytometry can also be used to assess the percentage of differentiated cells and differentiated cell types, for example, to determine the differentiation potential of hematopoietic cells in the presence of a drug. For example, ELISA, Western, and Northern blots can be performed to assess the function of engrafted cells, to assess the survival of cancerous plasma cells, etc., to determine the levels of cytokines, chemokines, immunoglobulins, etc. expressed in the engrafted genetically modified mice. To determine the extent of injury induced by diseased hematopoietic cells, for example, bone destruction and bone resorption induced by multiple myeloma cells, μCT scans can be performed. In vivo assays to test immune cell function can also be performed, as well as assays related to specific diseases or disorders of interest, such as diabetes, autoimmune diseases, 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 disclosures of which are incorporated herein by reference.

[0136] Thus, for example, humanized IL-6 mice engrafted with human multiple myeloma cells, e.g., Rag2 - / - IL2rg- / - IL-6 h / h Provided is a method for determining the effect of a drug on multiple myeloma, comprising administering a drug to a mouse; measuring the parameters of the viability and / or proliferation capacity of multiple myeloma cells over time in the presence of the drug; and comparing the measurements with those from engrafted humanized IL-6 mice that are not exposed to the drug.If the drug reduces the proliferation and / or reduces the number of multiple myeloma cells in the blood or tissue of the mouse by at least 20%, 30%, 40% or more, in some cases 50%, 60%, 70% or more, for example 80%, 90% or 100%, i.e., to an undetectable amount, after a single dose or administration of two or more doses of the drug in a selected period of time, the drug is determined to be anticancer.In a specific embodiment, the drug or drug combination is administered at least 1 week, 10 days, 2 weeks, 3 weeks or 4 weeks after the engraftment of multiple myeloma cells.

[0137] Other examples of uses for the mice of the invention are provided elsewhere herein. Additional applications of the genetically modified and engrafted mice described in this disclosure will become apparent to those of skill in the art upon reference to this disclosure.

[0138] Human antibody production As described elsewhere herein, compositions and methods useful for the production of human monoclonal antibodies from engrafted immunodeficient animals are also provided. In various embodiments, the methods include contacting the immunodeficient animal with human hematopoietic cells to generate a non-human animal with an engrafted immune system (the engrafted animal), then contacting the engrafted animal with an antigen, harvesting human cells from the engrafted animal that produce human antibodies against the antigen, and isolating the antibodies from the antibody-producing cells.

[0139] In various embodiments, the present invention includes a method comprising a step of establishing an antibody-producing cell (e.g., a human B cell) by a transformation method (e.g., EBV) or a cell fusion method (e.g., a hybridoma). Preferably, the antibody-producing cell can be maintained under appropriate cell culture conditions for at least about 50 passages.

[0140] In various embodiments, the engrafted animal is a non-human mammal, hi some embodiments, the engrafted animal is a mouse, a rat, or a rabbit.

[0141] In various embodiments of the invention, the human hematopoietic cells are CD34+ cells obtained from a sample of human fetal liver, bone marrow, umbilical cord blood, peripheral blood, or spleen.

[0142] In various embodiments, the antigen is at least one of a peptide, a polypeptide, an MHC / peptide complex, DNA, a live virus, a dead virus or a portion thereof, a live bacterium, a dead bacterium or a portion thereof, or a cancer cell or a portion thereof.

[0143] In some embodiments, the engrafted animal is contacted with the antigen 1-5 months after the animal is contacted with the human hematopoietic cells, in some embodiments, the engrafted animal is contacted with the antigen only once, while in other embodiments, the engrafted animal is contacted with the antigen 2, 3, 4, 5, 6, 7, 8 or more times.

[0144] In one embodiment, the human antibody-producing cells collected from the engrafted animal are B cells. In various embodiments, the human antibody-producing cells collected from the animal express at least one of CD19, CD20, CD22, and CD27 on their surface. The human antibody-producing cells of the present invention can be recovered by removing the appropriate cellular components of the immune system from the animal. In various embodiments, the antibody-producing cells are removed from the engrafted animal by removing at least one of the spleen, lymph nodes, peripheral blood, bone marrow, or portions thereof.

[0145] In various embodiments, the methods of the present invention utilize conventional hybridoma technology using an appropriate fusion partner, which in various embodiments is at least one cell selected from the group consisting of MOPC21, P3X63AG8, SP2 / 0, NS-1, P3.X63AG8.653, F0, S194 / 5.XXO.BU-1, FOX-NY, SP2 / 0-Ag14, MEG-01, HEL, UT-7, M07e, MEG-A2, and DAMI, and cell lines derived from these cells.

[0146] Methods for isolating antibodies from engrafted animals of the present invention are well known in the art. Isolation of antibodies from antibody-producing cells, the medium in which the cells produce antibodies are cultured, and / or the ascites of engrafted animals can be carried out according to methods known in the art, for example, by chromatography and dialysis. In various other embodiments, antibodies can be isolated using one or more of immunoaffinity purification, ammonium sulfate precipitation, protein A / G purification, ion exchange chromatography, and gel filtration. Such methods are described in Nau (1989, Optimization of monoclonal antibody purification, In: Techniques in Protein Chemistry, Hugli, T. (ed.), Academic Press, New York) and Coligan et al. (2005, Current Protocols in Immunology, John Wiley & Sons, Inc.).

[0147] The antigen can be administered to the engrafted animal by any suitable means known in the art. In various embodiments, the antigen can be administered to the engrafted animal by at least one of intrasplenic, intravenous, intraperitoneal, intradermal, intramuscular, and subcutaneous. In some embodiments, the antigen is administered alone, and in other embodiments, the antigen is administered in combination with a suitable immunomodulator or adjuvant. Examples of adjuvants useful in the methods of the present invention include, but are not limited to, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), and alum (Al3(OH)4).

[0148] Reagents and Kits Reagents and kits thereof for carrying out one or more of the above methods are also provided. The reagents and kits of the present invention may vary widely. In some embodiments, it is contemplated that the reagents or kits include one or more reagents for use in generating and / or maintaining the genetically modified non-human animals of the present invention. For example, the kits may include an immunodeficient mouse comprising a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter and a nucleic acid encoding human SIRPa operably linked to a SIRPa promoter; or a mouse comprising a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter and further comprising a nucleic acid encoding human M-CSF operably linked to an M-CSF promoter; a nucleic acid encoding human IL-3 operably linked to an IL-3 promoter; a nucleic acid encoding human GM-CSF operably linked to a GM-CSF promoter; a nucleic acid encoding human TPO operably linked to a TPO promoter; and / or a nucleic acid encoding human SIRPa operably linked to a SIRPa promoter. The kit may include reagents for breeding such mice, such as genotyping primers for the human IL-6 gene, the human M-CSF gene, the human IL-3 gene, the human GM-CSF gene, the human SIRPa gene, and / or the human TPO gene, PCR buffer, MgCl2 solution, etc.

[0149] In some embodiments, the reagents or kits will include one or more reagents for use in engraftment into a genetically modified non-human animal of the invention, e.g., human hematopoietic cells, enriched populations of human hematopoietic progenitor cells, hematopoietic cell lines, neoplastic hematopoietic cell lines, etc. for transplantation into a genetically modified non-human animal of the invention, or reagents for preparing a population of hematopoietic cells, enriched populations of human-derived hematopoietic cells, hematopoietic cell lines, neoplastic hematopoietic cell lines, etc. for transplantation into a genetically modified non-human animal of the invention.

[0150] In some embodiments, the reagents or kits will include, for example, reagents for determining the viability and / or function of hematopoietic cells in the presence / absence of a candidate agent, e.g., one or more antibodies specific for markers expressed by different types of hematopoietic cells, or reagents for detecting specific cytokines, chemokines, etc. Other reagents may include culture media, culture supplements, matrix compositions, etc.

[0151] In addition to the above components, the kit of the present invention will further include instructions for carrying out the method of the present invention. These instructions can be present in the kit of the present invention in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is information printed on a suitable medium or substrate, such as a paper on which the information is printed, present in the kit packaging, present in a package insert, etc. Yet another means would be a computer readable medium on which the information is recorded, such as a disk, CD, etc. Yet another possible means is a website address that can be used via the Internet to access information at a remote site. Any convenient means may be present in the kit. EXAMPLES

[0152] Experimental Example The present invention will be described in more detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but should be construed as embracing any variations that may become evident as a result of the teachings provided herein.

[0153] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the methods recited in the claims. Accordingly, the following examples specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0154] Example 1: Genetic humanization of cytokine genes enables engraftment of human multiple myeloma cells in mice The data presented herein demonstrate that the genetically modified non-human animals described herein represent a novel in vivo animal model of multiple myeloma.

[0155] material and method mouse Humanized IL-6 knock-in mice were generated by replacing 6.8 kb of the mouse IL-6 locus with 4.8 kb of human IL-6 gene sequence containing exons 1 to 5, including the 3' untranslated region, of the human IL-6 gene.

[0156] Briefly, a targeting construct for replacing the mouse with the human IL-6 gene in a single targeting step was constructed using VELOCIGENE® genetic engineering technology (see Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech, 21(6):652-659). Mouse and human IL-6 DNA were obtained from bacterial artificial chromosome (BAC) RPCI-23 clone 368C3 and BAC CTD clone 2369M23, respectively. Briefly, a NotI-linearized targeting construct generated by gap repair cloning containing mouse IL-6 upstream and downstream homology arms flanking 4.8 kb of human IL-6 sequence spanning from the ATG in exon 1 to exon 5 (genomic coordinates: NCBIh37.1:ch7:22,766,882-22,771,637) along with 16 nucleotides of 3' downstream sequence and a loxP-introduced neo selection cassette was cloned into Rag2. + / - IL2rg Y / - ES cells were electroporated. The parental ES cell line in which the knockouts of the RAG2 and IL2rg genes were generated was commercially available V17 ES cells (BALB / cxl29 heterozygote). To remove the drug selection cassette, the precisely targeted ES cells can be electroporated with a transient Cre expression vector.

[0157] Correctly targeted ES cell clones were identified by loss-of-native-allele (LONA) assays (Valenzuela et al. 2003), which determine the copy number of the native unmodified Il6 gene by two TaqMan™ quantitative polymerase chain reactions (qPCR) specific for sequences within the mouse Il6 gene targeted for deletion. The qPCR assays contained the following primer-probe sets (written 5' to 3'): Upstream forward primer: TIFF2025010204000002.tif4128 Upstream reverse primer: TIFF2025010204000003.tif4128 Upstream probe: TIFF2025010204000004.tif4129 Downstream forward primer: TIFF2025010204000005.tif4128 Downstream reverse primer: TIFF2025010204000006.tif4128 Downstream probe: TIFF2025010204000007.tif4130. Here, FAM refers to 5-carboxyfluorescein fluorescent probe, and BHQ refers to black hole quencher type fluorescent quencher (Biosearch Technologies). DNA purified from ES cell clones incorporating targeting vector and integrated into genome was combined with TaqMan™ Gene Expression Master Mix (Life Technologies) according to manufacturer's suggestions in 384-well PCR plate (MicroAmp™ Optical 384-Well Reaction Plate, Life Technologies), and cycled in Applied Biosystems Prism 7900HT, which collected fluorescence data during PCR and determined threshold cycle (Ct) (PCR cycle number when accumulated fluorescence reaches a preset threshold). Upstream and downstream Il6-specific qPCR and two qPCRs for non-targeted reference genes were performed for each DNA sample. The difference in Ct values ​​(ΔCt) between each Il6-specific qPCR and each reference gene qPCR was calculated, and then the difference between each assayed ΔCt and the median ΔCt for all samples was calculated to obtain the ΔΔCt value for each sample. The copy number of the IL-6 gene in each sample was calculated from the following formula: copy number = 2 2 -ΔΔCtCorrectly targeted clones that have lost one of the native copies will have an IL-6 gene copy number equal to 1. Confirmation that the human IL-6 gene sequence replaced the deleted mouse IL-6 gene sequence in the humanized allele was confirmed by a TaqMan™ qPCR assay containing the following primer-probe set (written 5' to 3'): Human forward primer: TIFF2025010204000008.tif4128 Human reverse primer: TIFF2025010204000009.tif4128 and human probe: TIFF2025010204000010.tif4128.

[0158] The upstream junction between the mouse locus and the sequence containing the hIL-6 gene is The mouse locus is designed to be located within TIFF2025010204000011.tif35152, where the last mouse nucleotide before the first nucleotide of the human gene is a "T" in CCGCT, and the first nucleotide of the human sequence is the first "A" in ATGAA. The downstream junction of the sequence containing the hIL-6 gene with the mouse locus is The downstream junction region was designed to fit within TIFF2025010204000012.tif35150, where the last nucleotide of the human sequence is the last "G" in TCACG and the first nucleotide of the mouse sequence is the first "C" in CTCCC; the downstream junction region also contained a loxP site at the 3' end (the first of which is shown) for removal of the neo cassette driven by a loxP-introduced ubiquitin promoter. The junction of the neo cassette with the mouse IL-6 locus was TIFF2025010204000013.tif28151, where the last "C" in AGCTC is the last nucleotide of the neo cassette; the first nucleotide of the mouse genome after the cassette is the first "C" in CTAAG.

[0159] To generate mice that contain hIL-6 and lack Rag2 and Il2rg, precisely targeted ES cells are identified and introduced into preimplantation embryos using techniques known in the art.

[0160] The humanized IL-6 KI mice were then backcrossed to generate mice lacking Rag2 and Il2rg and expressing hIL-6, and crossed with mice expressing a human SIRPa transgene (Strowig et al., 2011, Proc Natl Acad Sci USA, 108(32):13218-13223) to generate mice lacking Rag2 and Il2rg and expressing both hIL-6 and hSIRPa (Rag2 - / - Il2rg null Il6 h / h hSIRPa + ) was generated. - / - ,IL-2rg Y / - The hIL-6 KI mice were crossed with mice expressing human TPO (Rongvaux et al., 2011, Proc Natl Acad Sci USA, 108(6):2378-2383), human IL-3 and human GM-CSF (Willinger et al., 2011, Proc Natl Acad Sci USA, 108(6):2390-2395), as well as human M-CSF (Rathinam et al., 2011, Blood, 118(11):3119-3128) and also expressing hSIRPa (Strowig et al., 2011, Proc Natl Acad Sci USA, 108(32):13218-13223) to produce mice expressing a combination of these human proteins (Rag2 - / - Il2rg null hSIRPa + Tpo h / h Mcsf h / h Il3 / Gmcsf h / h Il6 h / h ) was generated.

[0161] Cell lines and primary cellsThe multiple myeloma cell line INA-6 (Burger et al., 2001, Hematol J, 2(1):42-53) was maintained in RPMI 1640 medium supplemented with 20% FCS, penicillin / streptomycin, L-glutamine, and 2.5 ng / ml hIL-6 in a standard incubator at 37°C and 5% CO2.

[0162] After obtaining informed consent from the patients, primary cells from multiple myeloma patients were isolated from bone marrow aspirates. Mononuclear cells were purified by Ficoll density gradient centrifugation, and then different cell subsets were isolated by magnetic-activated cell sorting (MACS®). To obtain T cell-depleted populations, CD3+ cells were depleted by negative selection with an AutoMACS® system using anti-CD3 microbeads (Miltenyi Biotec). To obtain CD138+ cells, CD138+ cells were isolated by positive selection with an AutoMACS® system using anti-CD138 microbeads (Miltenyi Biotec). The purity of the cells after MACS® selection was analyzed by flow cytometry.

[0163] Cell transplantation For intrafemoral implantation of INA6 cells, Rag2 - / - Il2rg null Il6 h / h hSIRPa + Mice were irradiated twice with 200 rad from an X-ray source. The indicated amount of cells was then transplanted into the femur of recipient mice. Briefly, mice were anesthetized using ketamine / xylazine, and the femoral patellar surface was perforated using a 26-gauge needle. Cells were then slowly injected in a volume of 20 μl using a 27-gauge needle. For transplantation of patient-derived primary cells, Rag2 - / - Il2rg null hSIRPa + Tpo h / h Mcsf h / h Il3 / Gmcsf h / h Il6 h / hMice were irradiated with two 150 rad doses from an X-ray source and transplantation was performed as described above.

[0164] ELISA Commercially available ELISA kits were used to measure the concentrations of human soluble IL-6R (R&D Systems), human Igκ, and Igλ (Bethyl Laboratories). Detection of these proteins was performed according to the manufacturer's instructions.

[0165] μCT Femoral bone morphometry was quantified using cone-beam microfocus X-ray computed tomography (μCT40; ScancoMedicalAG). Serial sections were acquired and 3D images were reconstructed and used to determine parameters. Trabecular bone morphometry was characterized by measuring bone volume fraction, trabecular width, trabecular number, and trabecular spacing. Cortical measurements included mean cortical width, cortical cross-sectional area, subperiosteal cross-sectional area, and bone marrow area.

[0166] histology Femurs were cleared of soft tissue, fixed in 10% buffered formalin, dehydrated, embedded in methyl methacrylate, sectioned, and stained with toluidine blue according to standard procedures.

[0167] result Engraftment of multiple myeloma cell lines in mice carrying a humanized IL-6 geneTo evaluate whether mice expressing human SIRPα and IL-6 are suitable hosts for multiple myeloma (MM) cell lines, a human IL-6-dependent MM cell line (INA6-gfp) was utilized. When transplanted in a xenograft system in scid-hu mice, the INA6-gfp cell line shows high dependency on the human microenvironment, i.e., human fetal bone chips (Epstein et al., 2005, Methods Mol Med, 113:183-190). Specifically, INA-6 cells can only engraft human bone grafts in scid-hu mice, suggesting a dependency on the human bone marrow microenvironment similar to primary MM cells (Tassone et al., 2005, Blood, 106(2):713-716).

[0168] Therefore, to directly test the potential of IL-6 humanization to enable myeloma cell proliferation, INA-6 cells were cultured with (i) Rag2 - / - Il2rg null mouse, (ii) Rag2 - / - Il2rg null hSIRPa+ mice, (iii) Rag2 - / - Il2rg null Il6 h / h mouse, and (iv) Rag2 - / - Il2rg null Il6 h / h The INA-6 cells were transplanted intravenously into hSIRPa+ mice. Engraftment was analyzed by measuring sIL-6R protein secreted by INA-6 cells in the blood. Engraftment was detected only in mice expressing human IL-6 (Figure 1), demonstrating that INA-6 cells can indeed engraft in mice expressing human IL-6. Next, the location of INA-6 cells (modified to express GFP) in the engrafted mice was investigated by fluorescence microscopy. Few GFP+ cells were detected in the bone marrow (Figure 2), whereas an increased number of GFP+ cells was detected in the lungs of the engrafted mice (Figure 3).

[0169] Analysis of human Il6 gene expression revealed that the highest levels of human Il6 gene expression were found in the lung (Figure 3), thus correlating with the presence of INA-6 cells in the lung. In summary, the data disclosed herein demonstrate successful engraftment of INA-6 cells following intravenous injection (iv) into mice genetically modified to express human IL6, or human IL6 and human SIRPa, suggesting that genetic humanization can overcome the growth limitations of human MM cells in mice.

[0170] The data in Figure 3 suggest that INA-6 cells do not home efficiently to the bone marrow, but rather may proliferate in non-physiological sites. Therefore, we next investigated whether the implantation of a tumor cell line in a native microenvironment could recapitulate the pathology typically associated with human MM. To that end, we tested intraosseous injection of INA6 cells. This resulted in significant bone destruction and bone resorption, an aspect of the pathology seen in MM patients (Figures 4-6). Specifically, loss of cancellous bone mass was observed by histology, quantified by μCT. Furthermore, only limited metastasis to peripheral sites such as the lungs was observed, which led to the conclusion that this model could be further explored to investigate novel drugs that interfere with this pathology. To test this conclusion, mice engrafted with INA-6 were treated with Zometa or Velcade, two drugs commonly used to treat multiple myeloma patients. Treatment of mice injected with INA-6 cells with Zometa was able to significantly reduce bone resorption compared to untreated mice, as quantified by μCT ( FIG. 7 ).

[0171] The data disclosed herein demonstrate that humanization of the Il6 gene allows the engraftment of multiple myeloma cell lines that typically require human microenvironment. Engraftment reproduces several pathological symptoms observed in patients, including bone loss. Moreover, these symptoms can be treated using approved drugs, highlighting the usefulness of this model for testing new drugs.

[0172] Genetic humanization of cytokine genes enables engraftment of primary patient-derived multiple myeloma cells Next, experiments were performed to investigate the transplantation of primary MM cells into genetically humanized mice. It has previously been demonstrated that humanization of multiple cytokines, including thrombopoietin, IL-3, GM-CSF, and M-CSF, as well as the macrophage inhibitory receptor SIRPa, results in improved engraftment of human hematopoietic cells in immunodeficient mice (Strowig et al., 2011, Proc Natl Acad Sci USA, 108(32):13218-13223; Rathinam et al., 2011, Blood, 118(11):3119-3128; Rongvaux et al., 2011, Proc Natl Acad Sci USA, 108(6):2378-2383; Willinger et al., 2011, Proc Natl Acad Sci USA, 108(6):2390-2395). Specifically, humanization of IL-3 and GM-CSF as well as M-CSF improved the engraftment of myeloid cells, which have proven to be important for certain aspects of MM pathology. Transgenic expression of hSIRPα improves human cell engraftment, but the SIRPa-CD47 axis has also recently been shown to be involved in tumorigenesis. - / - Il2rg null hSIRPa + Tpo h / h Mcsf h / h Il3 / Gmcsf h / h Il6 h / h To generate mice, the previously generated humanized mice were combined with human IL-6 knock-in mice. To evaluate the ability of this line to support human cell engraftment, CD3-depleted bone marrow cells from MM patients were injected into the bone marrow of mice. Myeloma cells, identified as CD138+CD38+CD19- cells, were frequently detected in the injected bone, but few cells were detected in the associated bone (Figure 8).

[0173] These results suggest that the genetically humanized mice described herein support the engraftment of primary human MM cells in vivo. The present data demonstrate that humanization of cytokine genes encoding IL-6, TPO, IL-3, GM-CSF, and / or M-CSF enables the engraftment of primary patient-derived multiple myeloma cells, which typically require a human microenvironment for successful transplantation.

[0174] Example 2: Genetic humanization of the IL-6 gene enables engraftment of human hematopoietic cells into mice material and method mouse Humanized IL-6 KI mice were generated as described above. The chimeric mice were first crossed with BALB / c mice and then backcrossed to obtain derivatives with homozygous hIL-6. Mice with the same mixed BALB / c×129 background were used as controls.

[0175] Neonatal mice (within 1 day of birth) were irradiated with a sublethal dose of 150 cGy of X-rays twice, 4 hours apart. Four hours after irradiation, 1–2 × 10 5 CD34 + Fetal liver (FL) cells in 20 μl of PBS were injected into the liver of mice. Mice were weaned at 3 weeks of age. Mice were maintained under specific pathogen-free conditions and all experiments were performed in compliance with Yale Institutional Animal Care and Use Committee protocols.

[0176] Analysis of human and mouse hematological cell populations At various time points after transplantation, mice were bled from the retro-orbital plexus under isoflurane anesthesia. Plasma samples were collected and stored at -20°C for further Ig measurements. Red blood cells were lysed twice by using ammonium chloride (ACK) lysis buffer, and the remaining PBMCs were resuspended in FACS buffer (PBS supplemented with 5% FBS and 5 mM EDTA).

[0177] Mice were sacrificed and single cell suspensions of cells were obtained from bone marrow (BM), thymus, and spleen. Samples were then stained with fluorochrome-labeled mAbs against mouse and human cell surface antigens according to the manufacturer's instructions. The following anti-human mAbs were used: CD45 (HI30), CD19 (HIB19), CD3 (UCHT1), CD4 (RPA-T4), CD8 (HIT8a), CD20 (2H7), CD5 (UCHT2), CD27 (O323), CD24 (ML5), CD10 (HI10a) (all Biolegend, CA, USA); CD33 (WM53), CD38 (HIT2), IgM (G20-127), CD138 (MI15) from BD Biosciences, and CD34 (AC136) from Miltenyi Biotec. Mouse cells were stained with anti-mouse CD45 Ab (30-F11, Biolegend). Samples were acquired on an LSRII (BD Biosciences) cytometer and analyzed with FlowJo (Treestar, OR, USA) software.

[0178] Measurement of human immunoglobulinsTotal immunoglobulins (Ig) were measured in plasma collected from mice by ELISA. 96-well plates (Nunc, NY, USA) were coated overnight at 4°C with 20 μg / ml purified goat anti-human IgM and IgG (Southern Biotechnology, AL, USA). After washing and blocking with PBS 1% bovine serum albumin (BSA, Sigma-Aldrich), appropriate dilutions of samples were added for 2 h at room temperature (RT). Plates were washed and incubated with isotype-specific secondary biotinylated antibodies (Southern Biotechnology) for 1 h at RT, followed by incubation with streptavidin-HRP (Pierce Protein Research Products, IL, USA). After a final wash, enzyme activity was determined using TMB substrate solution followed by stop solution (both Pierce Protein Research Products). Absorbance was measured at 450 nm. Samples of human serum from Bethyl (TX, USA) were used as references.

[0179] statistical analysis All data were expressed as mean ± standard error of the mean (SEM) except for Ab levels, which were plotted as geometric means. The nonparametric Mann-Whitney U test was used to determine statistical significance between two groups. Differences were considered significant when p-value was less than 0.05.

[0180] result Human CD34 + RAG2 cells were transplanted - / - Gamma c - / - Peripheral blood engraftment in miceIL6h / h mice showed higher peripheral blood (PB) engraftment throughout the analysis when compared to IL6m / m mice, and their engraftment increased over time (Figure 9). There were no major differences in the composition of human cells between the two mouse groups at any of the time points examined (Figure 10). In both groups, the percentages of B and T cells were similar at week 8 and weeks 16-20, but at weeks 11-15 there was a higher percentage of B cells (69.23±3.97 in IL6m / m, 55.91±4.86 in IL6h / h) than T cells (16.86±3.14 in IL6m / m, 30.26±6.23 in IL6h / h). Myeloid CD33 + The cells represent a minor component of human cells and their percentage decreased over time.

[0181] Human CD34 + RAG2 reconstituted by cells - / - Gamma c - / - Mouse organ cell engraftment and composition Cells of human origin were found in hematolymphoid organs such as the BM, spleen, and thymus (Figure 11A). Interestingly, the spleens of IL6h / h mice showed higher human engraftment than IL6m / m mice (61.75% ± 10.87 vs. 23.56% ± 5.2). These data were confirmed by a doubling in the absolute number of human cells (14.21 × 10 6 ±1.38 vs. 7.26×10 6 ±0.66) (Figure 11B).

[0182] Human CD34 + RAG2 engrafted with - / - Gamma c - / - B cell maturation in miceThe maturation stage of human B cells was studied in the BM and spleen of engrafted mice using a gating strategy illustrated in FIG. 12A, based on the use of a combination of CD24, CD38, and surface IgM antibodies. This strategy is particularly useful for probing the presence of transitional B cell subsets. In the BM, the main compartment consisted of pro / pre-B cells (FIG. 12B), with no difference between IL6m / m and IL6h / h mice (76.04%±9.09 and 79.07%±3.43, respectively). Spleens of both groups contained some mature B cells (approximately 20%), but also a higher percentage of immature / transitional cells (27.13%±8.99 and 36.45%±5.12).

[0183] A significant percentage of B cells in the spleen were CD5 + (FIG. 13B). This marker is not generally expressed on human BM and peripheral B cells, but is found in a low percentage of FL B cells (FIG. 13A).

[0184] IL6h / h mice had significantly lower CD27 expression in the spleen compared to IL6m / m mice. + There was a sharp increase in the percentage of B cells (24.73% ± 8.94 vs. 9.46% ± 2.32), but the BM contained no CD27 + Very few cells were found (Figures 14A and 14B).

[0185] Human CD34 + RAG2 engrafted with - / - Gamma c - / - Antibody production in mice Since B cells were found in the engrafted mice, we then measured the concentrations of human IgM and human IgG in plasma collected 12 and 20 weeks after human cell transplantation. Both IL6m / m and IL6h / h mice secreted human IgM and human IgG (Figure 15).

[0186] In general, there was a higher percentage of antibody-secreting IL6h / h mice compared to IL6m / m mice, with the former mice exhibiting lower IgM levels (14.69 μg / ml vs. 33.66 μg / ml at 12 weeks, 18.25 μg / ml vs. 190.2 μg / ml at 20 weeks) and increased IgG levels (243 μg / ml vs. 49.6 μg / ml at 12 weeks, 553.6 μg / ml vs. 297.2 μg / ml at 20 weeks) (Figure 15A). In IL6m / m mice, the mean levels of both IgM and IgG increased over time (Figure 15B). Conversely, serum Ig remained constant in IL6h / h mice.

[0187] The disclosures of all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with respect to specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the spirit and scope of the present invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0188] Sequence information SEQUENCE LISTING <110> REGENERON PHARMACEUTICALS, INC. YALE UNIVERSITY INSTITUTE FOR RESEARCH IN BIOMEDICINE(IRB) <120> GENETICALLY MODIFIED NON-HUMAN ANIMALS AND METHODS OF USE THEREOF <150> US 61 / 722,437 <151> 2012-11-05 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 1 ttgccggttt tcccttttct c 21 <210> 2 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 2 agggaaggcc gtggttgtc 19 <210> 3 <211> 26 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 3 ccagcatcag tcccaagaag gcaact 26 <210> 4 <211> 21 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 4 tcagagtgtg ggcgaacaaa g 21 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 5 gtggcaaaag cagccttagc 20 <210> 6 <211> 28 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 6 tcattccagg cccttcttat tgcatctg 28 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 7 ccccactcca ctggaatttg 20 <210> 8 <211> 21 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 8 gttcaaccac agccaggaaa g 21 <210> 9 <211> 27 <212> DNA <213> Artificial sequence <220> <223> synthetic polynucleotide <400> 9 agctacaact cattggcatc ctggcaa 27 <210> 10 <211> 197 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 10 aattagagag ttgactccta ataaatatga gactggggat gtctgtagct cattctgctc 60 tggagcccac caagaacgat agtcaattcc agaaaccgct atgaactcct tctccacaag 120 taagtgcagg aaatccttag ccctggaact gccagcggcg gtcgagccct gtgtgaggga 180 ggggtgtgtg gcccagg 197 <210> 11 <211> 151 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 11 ttttaaagaa atatttatat tgtatttata taatgtataa atggttttta taccaataaa 60 tggcatttta aaaaattcag caactttgag tgtgtcacgc tcccgggctc gataactata 120 acggtcctaa ggtagcgact cgagataact t 151 <210> 12 <211> 128 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 12 tatacgaagt tatcctaggt tggagctcct aagttacatc caaacatcct cccccaaatc 60 aataattaag cactttttat gacatgtaaa gttaaataag aagtgaaagc tgcagatggt 120 gagtgaga 128

Claims

1. A nucleic acid encoding human IL-6 operably linked to an IL-6 promoter, and (i) a nucleic acid encoding human SIRPa operably linked to a SIRPa promoter, randomly integrated into the genome of the non-human animal; (ii) a nucleic acid encoding human M-CSF operably linked to an M-CSF promoter; (iii) a nucleic acid encoding human IL-3 operably linked to an IL-3 promoter; (iv) a nucleic acid encoding human GM-CSF operably linked to a GM-CSF promoter; and (v) a nucleic acid encoding human TPO operably linked to a TPO promoter: At least one additional nucleic acid selected from the group consisting of 2. A genetically modified non-human animal comprising:

2. The genetically modified non-human animal of claim 1, wherein the IL-6 promoter is a non-human animal IL-6 promoter and the nucleic acid encoding human IL-6 is operably linked to the non-human animal IL-6 promoter at the non-human animal IL-6 locus.

3. 2. The genetically modified non-human animal of claim 1, wherein a nucleic acid encoding human M-CSF is located at the non-human animal M-CSF locus, a nucleic acid encoding human IL-3 is located at the non-human animal IL-3 locus, a nucleic acid encoding human GM-CSF is located at the non-human animal GM-CSF locus, and a nucleic acid encoding human TPO is located at the non-human animal TPO locus.

4. 2. The genetically modified non-human animal of claim 1, which is immunodeficient.

5. 5. The genetically modified non-human animal of claim 4, which does not express a recombination activating gene (RAG).

6. 5. The genetically modified non-human animal of claim 4, which does not express the IL2 receptor gamma chain (gamma chain- / -).

7. The genetically modified non-human animal of claim 4, which does not express RAG2 and does not express IL2rg.

8. 2. The genetically modified non-human animal of claim 1, further comprising engraftment of human hematopoietic cells.

9. The genetically modified non-human animal of claim 8, wherein the human hematopoietic cells are CD34+ cells.

10. 9. The genetically modified non-human animal of claim 8, wherein the human hematopoietic cells are multiple myeloma cells.

11. The genetically modified non-human animal of claim 1, which is a mouse.

12. transplanting the population of human hematopoietic cells into a genetically modified non-human animal that is immunodeficient, that contains a nucleic acid encoding human IL-6 operably linked to an IL-6 promoter, that expresses human IL-6, and that does not express the non-human animal IL-6. A method for generating an animal model of human B cell development and function, comprising:

13. The method of claim 12, wherein the IL-6 promoter is a non-human animal IL-6 promoter and the nucleic acid encoding human IL-6 is operably linked to the non-human animal IL-6 promoter at the non-human animal IL-6 locus.

14. The method of claim 12, wherein the population of transplanted hematopoietic cells comprises CD34+ cells.

15. The method of claim 12, wherein the population of transplanted hematopoietic cells comprises multiple myeloma cells.

16. 13. The method of claim 12, wherein the implanting step comprises intrafemoral and / or intratibial injection.

17. The method of claim 12, wherein the animal further comprises a nucleic acid encoding human SIRPa operably linked to the SIRPa promoter randomly integrated into the genome of the non-human animal.

18. Immunodeficient, genetically modified animals (i) a nucleic acid encoding human M-CSF operably linked to an M-CSF promoter; (ii) a nucleic acid encoding human IL-3 operably linked to an IL-3 promoter; (iii) a nucleic acid encoding human GM-CSF operably linked to a GM-CSF promoter; and (iv) a nucleic acid encoding human TPO operably linked to a TPO promoter:

18. The method of claim 17, wherein the human p53 expresses at least one additional human nucleic acid selected from the group consisting of:

19. The method according to any one of claims 12 to 18, wherein the animal is a mouse.

20. An engrafted non-human animal prepared according to the method of any one of claims 12 to 19.

21. contacting the genetically modified non-human animal of claim 20 engrafted with human hematopoietic cancer cells with a candidate drug; comparing the survival rate and / or proliferation rate of the human hematopoietic cancer cells in said contacted non-human animal with the survival rate and / or proliferation rate of the human hematopoietic cancer cells in a genetically modified non-human animal of claim 20 that has been engrafted with the human hematopoietic cancer cells but has not been contacted with the candidate drug. wherein a decrease in the viability and / or proliferation rate of human hematopoietic cancer cells in the contacted non-human animal indicates that the candidate agent will treat the hematopoietic cancer.

22. 22. The method of claim 21, wherein the hematopoietic cancer is multiple myeloma.

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