Genetically modified non-human animals and their uses

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] Description of research or development sponsored by the federal government. This invention was made with government support under grant number 5R01CA156689-04, awarded by the National Institutes of Health (NIH). The government reserves certain rights in this invention.

[0002] Cross-reference of related applications This application claims priority based on the filing date of U.S. Provisional Patent Application No. 61 / 722,437, filed 5 November 2012, pursuant to 35 U.S.SC § 119(e), the full disclosure of which is incorporated herein by reference. [Background technology]

[0003] 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. Due to practical and ethical barriers to experimental methods in human subjects, much research is conducted in small animal models such as mice. Therefore, animal models of these human diseases are needed.

[0004] For example, in the United States, approximately 20,000 patients are newly diagnosed each year with multiple myeloma (MM), a nearly incurable malignant disease of highly differentiated antibody-secreting B cells (Hideshima et al., 2007, Nat Rev Cancer. 7:585-98 (Non-Patent Literature 1); Kuehl and Bergsagel, 2002, Nat Rev Cancer. 2:175-87 (Non-Patent Literature 2)). MM is characterized by the infiltration of malignant plasma cells into the bone marrow (BM), and clinical signs include bone disease, hypercalcemia, cytopenia, renal dysfunction, and peripheral neuropathy (Hideshima et al., 2007, Nat Rev Cancer. 7:585-98 (Non-Patent Literature 1); Kuehl and Bergsagel, 2002, Nat Rev Cancer. 2:175-87 (Non-Patent Literature 2)). A precancerous condition called benign monoclonal gamma globulinemia (MGUS), affecting approximately 3% of individuals over 50 years of age, precedes MM in most cases (Landgren et al., 2009, Blood 113:5412-7 (Non-Patent Literature 3)). Complex, heterogeneous genetic abnormalities, including karyotype changes and IgH translocations, are characteristic of MM cells (Kuehl and Bergsagel, 2002, Nat Rev Cancer. 2:175-87 (Non-Patent Literature 2); Zhan et al., 2006, Blood 108:2020-8 (Non-Patent Literature 4)). Plasma cell clones amplified in MGUS are thought to have a genetic and phenotypic profile similar to myeloma plasma cells (Chng et al., 2005, Blood 106:2156-61 (Non-Patent Literature 5); Fonseca et al., 2002, Blood 100:1417-24 (Non-Patent Literature 6); Kaufmann et al., 2004, Leukemia.18:1879-82 (Non-Patent Literature 7)). Mutations in the cyclin D gene have been suggested to cause MM, but the possible contributions of other factors have not been definitively proven (Bergsagel et al., 2005, Blood 106:296-303 (Non-Patent Literature 8)). Nevertheless, hereditary genetic modification is not the sole determinant of MM cell behavior.Rather, drug resistance and abnormal biological responses to cytokines are strongly influenced by interactions with the microenvironment, which presents opportunities for the development of novel therapeutics.

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

[0006] Major 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 microenvironment niche. Mouse models play a crucial 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 diseases. While xenotransplantation of human tumors into immunocompromised mice is widely used, reliable engraftment has typically only been achievable with high-grade tumors or cell lines.

[0007] The best model currently available for growing human tumor cells is a severely immunodeficient mouse lacking B cells, T cells, 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 engraft into human fetal bone fragments through co-transplantation into immunocompromised mice (Yaccoby et al., 1998, Blood 92:2908-13 (Non-Patent Literature 12)). In this model, MM cells are found in human bone but not in mouse bone or peripheral tissue, demonstrating a high degree of residual xenorejection and the necessity of the human BM microenvironment (Yaccoby et al., 1998, Blood 92:2908-13 (Non-Patent Literature 12); Yaccoby and Epstein, 1999, Blood 94:3576-82 (Non-Patent Literature 13)). NOD / Scid / γc - / -Recent studies have demonstrated that mice can engraft several MM cell lines, revealing their potential as in vivo models of MM (Dewan et al., 2004, Cancer Sci. 95:564-8 (Non-Patent Literature 14); Miyakawa et al., 2004, Biochem Biophys Res Commun. 313:258-62 (Non-Patent Literature 15)). However, even these mouse models with low xenophobic rejection have a limited growth environment due to numerous factors essential for supporting the proliferation and survival of transformed cells, although they do not cross the species barrier (Manz, 2007). In vivo models that allow us to explore the complex pathogenic interplay between tumors and their environments will be essential for designing new drugs and therapies.

[0008] Therefore, the need to develop humanized non-human animals and methods for reliably proliferating and studying human hematopoietic cells, including primary human hematopoietic tumor cells, in mice remains unmet. This 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

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Summary of the Invention

[0010] Genetically modified non-human animals are provided that can be used to model the development, function, or disease of human hematopoietic cells. The genetically modified non-human animals contain nucleic acids encoding human IL-6 functionally linked to an IL-6 promoter. In some cases, 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. In some cases, the genetically modified non-human animals are immunodeficient. In some such cases, the genetically modified non-human animals can engraft healthy or diseased human hematopoietic cells. Methods for using the genetically modified non-human animals of the present invention in modeling the development, function, and / or disease of human hematopoietic cells are also provided, as well as reagents and kits useful in the preparation of the genetically modified non-human animals of the present invention and / or in carrying out the methods of the present invention.

[0011] In various aspects of the present invention, a genetically modified non-human animal is provided, comprising a genome containing a nucleic acid encoding human IL-6 functionally linked to an IL-6 promoter, and expressing 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 functionally ligated is the mouse IL-6 promoter, and the human IL-6 gene is functionally ligated to the mouse IL-6 promoter at the mouse IL-6 locus.

[0013] In some embodiments, a genetically modified non-human animal further comprises one or more additional nucleic acids selected from: nucleic acids encoding human SIRPa under the regulation of a SIRPa promoter; nucleic acids encoding human M-CSF functionally linked to an M-CSF promoter (in which case the animal expresses human M-CSF); nucleic acids encoding human IL-3 functionally linked to an IL-3 promoter (in which case the animal expresses human IL-3); nucleic acids encoding human GM-CSF functionally linked to a GM-CSF promoter (in which case the animal expresses human GM-CSF); and nucleic acids encoding human TPO functionally linked to a TPO promoter (in which case the animal expresses human TPO). In some embodiments, the promoter is a human promoter for its gene. In other embodiments, the promoter is a non-human animal promoter for its 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, genetically modified non-human animals are immunodeficient with respect to the non-human animal immune system. In some such embodiments, immunodeficient, genetically modified non-human animals do not express recombinant activating genes (RAGs). In some such embodiments, immunodeficient, genetically modified non-human animals do not express the IL2 receptor γ chain (IL2rg or "γc"). In some such embodiments, immunodeficient, genetically modified non-human animals do not express either RAGs (e.g., RAG1, RAG2) or IL2rg.

[0015] In some embodiments, immunodeficient, genetically modified non-human animals can be engrafted with human hematopoietic cells to form genetically modified and engrafted non-human animals. In one embodiment, the human hematopoietic cells are selected from human umbilical cord blood cells, human fetal hepatocytes, and cells from 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, genetically modified and 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, human cells are present at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after engraftment.

[0017] In several embodiments, genetically modified and engrafted animals produce a human hematopoietic lymphoid system comprising human hematopoietic stem cell / 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 hematopoietic lymphoid system is present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after engraftment.

[0018] In some aspects of the present invention, a method is provided for generating non-human animals in which human hematopoietic cells have been engrafted. In some embodiments, a method is provided for generating animal models of the development and function of human immune cells. In certain embodiments, a method is provided for generating animal models of the development and function of human B cells.

[0019] In some embodiments, the method involves 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 functionally ligated 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 functionally ligated 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. Therefore, in some embodiments, the IL-6 promoter to which the nucleic acid encoding human IL-6 is functionally ligated is a mouse IL-6 promoter, and the human IL-6 gene is functionally ligated to the mouse IL-6 promoter at the mouse IL-6 locus.

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

[0021] In some embodiments, immunodeficient, genetically modified animals express at least one additional human nucleic acid selected from the group consisting of: nucleic acid encoding human SIRPa functionally ligated to the SIRPa promoter; nucleic acid encoding human M-CSF functionally ligated to the M-CSF promoter; nucleic acid encoding human IL-3 functionally ligated to the IL-3 promoter; nucleic acid encoding human GM-CSF functionally ligated to the GM-CSF promoter; and nucleic acid encoding human TPO functionally ligated to the TPO promoter.

[0022] In some aspects of the present invention, engrafted genetically modified non-human animals are provided that express nucleic acids encoding human IL-6 functionally linked to an IL-6 promoter, prepared according to methods described herein or known in the art. In some embodiments, the engrafted genetically modified non-human animals serve as animal models of human B cell development and differentiation.

[0023] In various embodiments, methods are provided that encompass 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 hematopoietic and immune cell proliferation and differentiation; methods for in vivo evaluation of the human hematopoietic system; methods for in vivo evaluation of cancer cells; methods for in vivo assessment of immune responses; methods for in vivo evaluation of vaccines and immunization programs; methods for use in testing the efficacy 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 efficacy of agents that modulate the function of hematopoietic and immune cells. For example, in some embodiments, methods are provided for screening candidate agents for their ability to treat hematopoietic cancers. In some embodiments, the method includes the steps of contacting a genetically modified non-human animal of the present disclosure, in which human hematopoietic cancer cells have been engrafted, with a candidate drug, and comparing the viability and / or growth rate of human hematopoietic cancer cells in the contacted, engrafted, genetically modified non-human animal with that of human hematopoietic cancer cells in a similarly engrafted, genetically modified non-human animal that has not been contacted with the candidate drug. In this method, a decrease in the viability and / or growth rate of human hematopoietic cancer cells in the contacted, engrafted non-human animal indicates that the candidate drug will treat the hematopoietic cancer. These and other methods will be apparent to those skilled in the art from the disclosures herein. [Invention 1001] Nucleic acids encoding human IL-6 functionally linked to the IL-6 promoter, and (i) Nucleic acids encoding human SIRPa, functionally linked to the SIRPa promoter, randomly incorporated into the genome of a non-human animal; (ii) Nucleic acids encoding human M-CSF functionally linked to the M-CSF promoter; (iii) Nucleic acids encoding human IL-3 functionally linked to the IL-3 promoter; (iv) nucleic acids encoding human GM-CSF functionally linked to a GM-CSF promoter; and (v) Nucleic acids encoding human TPO functionally linked to the TPO promoter: At least one additional nucleic acid selected from the group consisting of Genetically modified non-human animals, including [specific animal species]. [Invention 1002] A genetically modified non-human animal according to the present invention 1001, wherein the IL-6 promoter is a non-human animal IL-6 promoter, and the nucleic acid encoding human IL-6 is functionally linked to the non-human animal IL-6 promoter at the non-human animal IL-6 locus. [Invention 1003] A genetically modified non-human animal according to Invention 1001, wherein the nucleic acid encoding human M-CSF is located at the non-human animal M-CSF locus, the nucleic acid encoding human IL-3 is located at the non-human animal IL-3 locus, the nucleic acid encoding human GM-CSF is located at the non-human animal GM-CSF locus, and the nucleic acid encoding human TPO is located at the non-human animal TPO locus. [Invention 1004] A genetically modified non-human animal according to the present invention 1001, which is immunodeficient. [Invention 1005] A genetically modified non-human animal according to the present invention 1004 that does not express recombinant activating genes (RAGs). [Invention 1006] A genetically modified non-human animal according to Invention 1004 that does not express the IL2 receptor γ chain (γ chain-- / -). [Invention 1007] A genetically modified non-human animal according to the present invention 1004, which does not express RAG2 and does not express IL2rg. [Invention 1008] A genetically modified non-human animal according to the present invention 1001, further comprising the engraftment of human hematopoietic cells. [Invention 1009] A genetically modified non-human animal according to Invention 1008, wherein human hematopoietic cells are CD34+ cells. [Invention 1010] A genetically modified non-human animal according to the present invention 1008, wherein human hematopoietic cells are multiple myeloma cells. [Invention 1011] A mouse, a genetically modified non-human animal according to the present invention 1001. [Invention 1012] A process of transplanting a population of human hematopoietic cells into a genetically modified non-human animal that is immunodeficient, contains nucleic acid encoding human IL-6 functionally linked to the IL-6 promoter, expresses human IL-6, and does not express non-human animal IL-6. A method for generating an animal model of human B cell development and function, including the development of human B cells. [Invention 1013] The method of the present invention 1012, wherein the IL-6 promoter is a non-human animal IL-6 promoter, and the nucleic acid encoding human IL-6 is functionally linked to the non-human animal IL-6 promoter at the non-human animal IL-6 locus. [Invention 1014] The method of the present invention 1012, wherein the population of hematopoietic cells to be transplanted includes CD34+ cells. [Invention 1015] The method of the present invention 1012, wherein the population of hematopoietic cells to be transplanted includes multiple myeloma cells. [Invention 1016] The method of the present invention 1012, wherein the transplantation step includes intrafemoral injection and / or intratibial injection. [Invention 1017] The method of the present invention 1012, further comprising an animal nucleic acid encoding human SIRPa, which is randomly incorporated into the genome of a non-human animal and functionally linked to a SIRPa promoter. [Invention 1018] Immunodeficient, genetically modified animals, (i) Nucleic acids encoding human M-CSF functionally linked to an M-CSF promoter; (ii) Nucleic acids encoding human IL-3 functionally linked to the IL-3 promoter; (iii) nucleic acids encoding human GM-CSF functionally linked to a GM-CSF promoter; and (iv) Nucleic acids encoding human TPO functionally linked to the TPO promoter: The method of the present invention 1017, wherein at least one additional human nucleic acid selected from the group consisting of the following is expressed. [Invention 1019] Any method of the present invention 1012 to 1018, wherein the animal is a mouse. [Invention 1020] An engrafted non-human animal prepared according to any of the methods described in Invention 1012 to 1019. [Invention 1021] A step of contacting a genetically modified non-human animal of the present invention 1020, on which human hematopoietic cancer cells have engrafted, with a candidate drug, and A step of comparing the survival rate and / or proliferation rate of human hematopoietic cancer cells in a non-human animal that has been exposed to the candidate drug with the survival rate and / or proliferation rate of human hematopoietic cancer cells in a genetically modified non-human animal of the present invention 1020 that has been engrafted with human hematopoietic cancer cells but has not been exposed to the candidate drug. A method for screening candidate drugs for their ability to treat hematopoietic cancer, wherein a reduction in the viability and / or proliferation rate of human hematopoietic cancer cells in a non-human animal exposed to such drug indicates that the candidate drug would treat hematopoietic cancer. [Invention 1022] The method of the present invention 1021, wherein the hematopoietic cancer is multiple myeloma. [Brief explanation of the drawing]

[0024] The following detailed description of preferred embodiments of the present invention will be better understood when viewed in conjunction with the accompanying drawings. In accordance with general practice, it should be emphasized that the various features in the drawings are not to a constant scale. Conversely, the dimensions of the various features have been arbitrarily enlarged or reduced for clarity. For the purpose of illustrating the present invention, currently preferred embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings. The drawings include the following figures:

[0025] [Figure 1] This is a set of graphs showing the results of experiments 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 received intravenous transplantation of 5 × 10⁶ INA-6 cells. N indicates the number of transplanted mice in each group. [Figure 2] Figure 2, including Figures 2A–2F, is a set of images showing the histological analysis of femurs after intravenous engraftment of INA-6 cells. Rag2- / -Il2rgnullIl6h / hhSIRPa+ mice were sacrificed 8 weeks after intravenous engraftment of 5 × 10⁶ INA-6 cells. 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. [Figure 3] This is a set of images and graphs showing the analysis of lung tissue after intravenous engraftment of INA-6 cells. Rag2- / -Il2rgnullIl6h / hhSIRPa+ mice were sacrificed 8 weeks after engraftment of 5 × 10⁶ intravenously injected INA-6 cells. Lung tissue was fixed in 10% formalin, and 10 μM sections were directly analyzed for GFP expression using a Leica confocal microscope. The image (left) shows 10x (top) and 63x (bottom) magnified images of the section. The graph (right) shows human Il6 gene expression measured in the indicated tissue and normalized against mouse hprt expression. [Figure 4]This is a set of graphs showing the results of experiments 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 genotype, in which 5 × 10⁵ INA-6 cells were transplanted into the femur. Each line represents an individual mouse. [Figure 5] This is a set of images showing the histological analysis of femurs after INA-6 cell engraftment. Rag2- / -Il2rgnullIl6h / hhSIRPa+ mice were sacrificed 4-6 weeks after engraftment of 5 × 10⁵ INA-6 cells injected intrafemur. Femurs were fixed with 10% formalin and decalcified. 10 μM sections were stained with toluidine blue. [Figure 6] This is a set of images and graphs showing the results of μCT analysis of mouse femurs after INA-6 transplantation. Four weeks after engraftment of 5 × 10⁵ INA-6 cells injected intrafemur, Rag2- / -Il2rgnullIl6h / hhSIRPa+ mice and control mice were sacrificed. Femurs were fixed with 70% ethanol and analyzed using mouse μCT. Cancellous 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] This graph shows the results of μCT analysis of mouse femurs after treatment with antimyeloma drugs. INA-6 cells, injected intrafemur with 5 × 10⁵ cells, were engrafted into Rag2- / -Il2rgnullIl6h / hhSIRPa+ mice and treated every other week with either Velcade® or Zometa®. After 4 weeks, the mice were sacrificed and the femurs were fixed with 70% ethanol for μCT analysis. Cancellous bone volume and tissue volume were quantified to calculate the ratio between bone volume and tissue volume. [Figure 8]This 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 1.5 × 10⁶ CD3-depleted bone marrow cells injected intrafemur and sacrificed after 12 weeks. Single-cell suspensions were generated from the injected femur, its collateral limb, and spleen. Cells were stained for mCD45, hCD19, hCD38, and hCD138. FACS plots show post-gating events against mCD45-negative cells. The numbers indicate the frequency of CD38+CD138+ cells. [Figure 9] This 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. The horizontal bars represent the average frequency of each cell type. [Figure 10] This document presents the results of experiments assessing the percentage of hCD45 in B cells (CD19+), T cells (CD3+), and myeloid cells (CD33+) in the blood of engrafted mice, as determined by flow cytometry. Only mice with an hCD45 percentage 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-old engrafted mice. The bars represent the mean ± SEM values ​​for 4 / 5 mice in each group. [Figure 12] Figure 12, including Figures 12A and 12B, shows the results of FACS experiments assessing human cells. (A) Figure showing the gating strategy used to separate different B cell populations by flow cytometry. (B) Percentage of different B cell subsets within human CD45+CD19+ cells in 20-week-old mice. Bars represent the mean ± SEM of 4 / 5 mice in each group. [Figure 13]Figure 13, including Figures 13A and 13B, shows the results of FACS experiments assessing human cells. (A) Representative flow cytometry analysis of CD5+ B cells in human fetal liver (FL) and 20-week-old mice. The numbers within the quarters represent the percentage of cells. All plots are gated to human CD45+ cells. (B) Percentage of CD5 on human B cells in the BM and spleen of 20-week-old engrafted mice. Bars represent the mean ± SEM of 4 / 5 mice in each group. [Figure 14] The 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. The numbers within the quarters represent the percentage of cells. All plots are gated to human CD45+ cells. (B) Percentage of CD27 on human B cells in the BM and spleen of 20-week-old engrafted mice. The bars represent the mean ± SEM of 4 / 5 mice in each group. [Figure 15] The results of experiments assessing whole-human IgM and whole-human IgG levels in plasma samples from 12-week-old (A) and 20-week-old (B) mice are shown. Horizontal bars indicate the geometric mean. Mice with less than 2% PB human engraftment were excluded from the analysis. [Modes for carrying out the invention]

[0026] Detailed explanation Provided are genetically modified non-human animals that can be used to model the development, function, or disease of human hematopoietic cells. The genetically modified non-human animals contain nucleic acids encoding human IL-6 functionally 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 invention also relates to methods for 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 can engraft human hematopoietic cells, including normal cells, neoplastic cells, or combinations thereof. In some embodiments, the genetically modified non-human animals described herein can engraft human multiple myeloma (MM) cells. In various embodiments, the genetically modified non-human animals engrafted with human hematopoietic cells of the present 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 immunization programs, use in testing the efficacy of agents that modulate the proliferation or survival of cancer cells, in vivo evaluation of cancer treatments, and in vivo production and collection of immune mediators, including human antibodies, and use in testing the efficacy of agents that modulate the function of hematopoietic and immune cells.

[0027] These and other objectives, advantages, and features of the present invention will become apparent to those skilled in the art by referring to the details of the compositions and methods described below in their entirety.

[0028] definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention pertains. Such terminology is found, defined, and used in context in various standard reference books, including, as exemplified, J. Sambrook and DWRussell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; FMAusubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; DLNelson 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. Any methods and materials similar to or equivalent to those described herein may be used in the practice or trial of the present invention, but preferred methods and materials are described.

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

[0030] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “element” means one or more elements.

[0031] "Approximately" as used herein means, when referring to a measurable value such as a quantity or duration of time, to include a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from a specified value, provided that such variation is appropriate for carrying out the disclosed method.

[0032] When used in reference to organisms, tissues, cells, or components thereof, the term “abnormal” refers to an organism, tissue, cell, or component thereof that exhibits at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) that differs from an organism, tissue, cell, or component thereof that exhibits each “normal” (expected) characteristic. A characteristic that is normal or expected for one cell type or tissue type may be abnormal for a different cell type 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 full immunoglobulins of natural or recombinant origin, or the immunoreactive portion of full immunoglobulins. The antibodies in this 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 viable cells under most or all physiological conditions.

[0035] The "coding region" of a gene contains nucleotide residues from the coding and non-coding strands of the gene that are homologous or complementary to the coding region of the mRNA molecule produced by gene transcription. The "coding region" of the mRNA molecule also includes nucleotide residues of the mRNA molecule that pair with the anticodon region of the transfer RNA molecule or encode a stop codon during translation of the mRNA molecule. Therefore, the coding region may contain 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 the protein export signal sequence).

[0036] "Disease" includes a state of animal health in which the animal is unable to maintain homeostasis and, if the disease does not go into remission, the animal's health continues to deteriorate.

[0037] In contrast, "disorder" in animals includes a health condition in which the animal can maintain homeostasis, but the animal's health is worse than it would be without the disorder. If left untreated, the disorder does not necessarily lead to a further decline in the animal's health.

[0038] A disease or disorder is “relieved” if the severity of its symptoms, the frequency with which the patient experiences such symptoms, or both are reduced.

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

[0040] "Coded" includes the intrinsic properties of a specific nucleotide sequence of a polynucleotide, such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules in biological processes, as well as either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and which has the biological properties derived therefrom. Thus, for example, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, that gene codes for that protein. 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 the transcription of a gene or cDNA, may be said to code for a protein or other product of that gene or cDNA.

[0041] As used herein, “intrinsic” includes any material that originates from or is produced within an organism, cell, tissue, or system.

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

[0043] The terms “expression construct” and “expression cassette” include, as used herein, double-stranded recombinant DNA molecules containing a desired nucleic acid human coding sequence and containing one or more regulatory elements necessary or desirable for the expression of a functionally linked coding sequence.

[0044] As used herein, the term “fragment” includes a larger subsequence of a nucleic acid or polypeptide, as applied to nucleic acids or polypeptides. A “fragment” of a nucleic acid may be at least about 15 nucleotides long; for example, at least about 50 to about 100 nucleotides; at least about 100 to about 500 nucleotides; at least about 500 to about 1000 nucleotides; at least about 1000 to about 1500 nucleotides; or about 1500 to about 2500 nucleotides; or about 2500 nucleotides (and any intermediate integer value). A “fragment” of a polypeptide may be at least about 15 nucleotides long; for example, at least about 50 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 to about 1500 amino acids; or at least about 1500 to about 2500 amino acids; or about 2500 amino acids (and any intermediate integer value).

[0045] As used herein, the terms “gene” and “recombinant gene” include nucleic acid molecules containing an open reading frame that encodes a polypeptide. Variations in such native alleles can typically result in variations of 1–5% of 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 carried out by using hybridization probes to identify the same locus in diverse individuals. All such nucleotide variations and resulting amino acid polymorphisms or variations that result from variations in native alleles and do not alter functional activity are included within the scope of the present invention.

[0046] "Homologie," as used herein, includes subunit sequence similarity between two polymer molecules, for example, two nucleic acid molecules, for example, two DNA molecules or two RNA molecules, or two polypeptide molecules. In both molecules, a certain subunit position is occupied by the same monomeric subunit, for example, in each of two DNA molecules, a certain position is occupied by adenine, in which case they are homologous at that position. The homology between two sequences is a linear function of the number of matching or homologous positions, for example, if half of the positions in the sequences of two compounds are homologous (e.g., five positions in a polymer with a length of 10 subunits), then the two sequences are 50% homologous, and if 90% of the positions, for example, nine out of ten, are matching or homologous, then 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 cells and progenitor cells” and “human HSPCs” include, as used herein, human self-renewing pluripotent hematopoietic stem cells and hematopoietic progenitor cells.

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

[0049] As used herein, “informative materials” include publications, records, diagrams, or other expressive media that may be used to inform the recipient of the usefulness of the compounds, compositions, vectors, or delivery systems of the present invention contained in the kit for achieving the alleviation of various diseases or disorders described herein. Optionally or or otherwise, the informative materials may describe one or more methods for alleviating diseases or disorders in mammalian cells or tissues. The informative materials for the kit of the present invention may, for example, be attached to the container containing the identified compounds, compositions, vectors, or delivery systems of the present invention, or shipped together with the container containing the identified compounds, compositions, vectors, or delivery systems. Alternatively, the informative materials may be shipped separately from the container, with the intention that the informative materials and compounds be used collaboratively by the recipient.

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

[0051] The term “functionally linked” includes, as used herein, a single-stranded or double-stranded nucleic acid moiety containing two polynucleotides that are functionally related to a second polynucleotide, for example, 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 functionally linked to the coding region of a gene can promote transcription of the coding region. Preferably, when the nucleic acid encoding the desired protein further includes a promoter / control sequence, the promoter / control sequence is positioned at the 5' end of the sequence encoding the desired protein to express the desired protein in a 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, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides are interchangeable, as used herein. Those skilled in the art have general knowledge that nucleic acids are polynucleotides that can be hydrolyzed to monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning technologies and PCR, etc., and synthesis 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 constitute a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the terms include both short chains, also commonly called peptides, oligopeptides, and oligomers in the art, and longer chains, of which many types exist. “Polypeptides” include, among other things, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof. The term “peptide” typically refers to a short polypeptide. The term “protein” typically refers to a long polypeptide.

[0054] The term “offspring” includes progeny or derivatives as used herein, and includes differentiated or undifferentiated progeny cells derived from parent cells. In one use, the term “offspring” includes progeny cells that are genetically identical to the parent. In another use, the term “offspring” includes progeny cells that are genetically and phenotypic identical to the parent. In yet another use, the term “offspring” includes progeny cells that have differentiated from parent cells.

[0055] The term “promoter” includes, as used herein, DNA sequences that are functionally linked to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding a desired molecule. Generally, a promoter is located upstream of the nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors. In specific embodiments, a promoter is generally located upstream of the 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] Scope: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that the range format is for convenience and conciseness only and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, range descriptions should be considered to specifically disclose all possible subranges and the individual numbers within those ranges. For example, a range description 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 the individual numbers within those ranges, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0057] "Recombinant polypeptides" include those produced by the expression of recombinant polynucleotides.

[0058] The term “regulatory element” includes nucleotide sequences that modulate several aspects of nucleic acid sequence expression, as used herein. Exemplary regulatory elements include, exemplarially, enhancers, intra-sequence ribosome entry sites (IRESs), introns; replication start sites, polyadenylation signals (pA), promoters, enhancers, transcription termination sequences, and upstream regulatory domains that contribute to nucleic acid sequence replication, transcription, and post-transcriptional processing. Those skilled in the art can select these and other regulatory elements and use them in expression constructs using only routine experimental methods. Expression constructs can be produced by recombination or synthesis using well-known methodologies.

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

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

[0061] The term “synthetic antibody” includes antibodies produced using recombinant DNA technology, such as antibodies expressed by bacteriophages as described herein, as used herein. The term should also be interpreted to mean antibodies produced by the synthesis of DNA molecules that encode antibodies and express antibody proteins, or amino acid sequences that specify antibodies, obtained using DNA or amino acid sequence synthesis technologies 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 reference peptide sequence, but retain the essential biological properties of the reference molecule. Sequence changes in nucleic acid variants may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or they may result in amino acid substitutions, additions, deletions, fusions, and shortenings. Sequence changes in peptide variants are typically limited or conserved, so that the sequences of the reference peptide and the variant are generally very 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, or deletions. Nucleic acid or peptide variants may be naturally occurring, such as allele variants, or they may be variants whose natural occurrence is not known. Variants of nucleic acids and peptides that do not exist naturally may be produced by mutagenesis or direct synthesis.

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

[0064] As used herein, the term “transgenic animal” includes animals that have exogenous human nucleic acid sequences incorporated into their genome.

[0065] As used herein, “knock-in” includes genetic modifications that target a specific chromosomal locus in a non-human animal genome and insert a nucleic acid of interest into that targeted locus. In some cases, the genetic modification replaces the genetic information encoded at 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 the sequence described, for example, 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 comprising a binding subunit (IL-6R) and a signaling subunit (gp130). While gp130 is a common component of other receptors such as the receptors 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 crucial factor for the final maturation of B cells into antibody-secreting cells (ASCs), particularly for plasmablast growth in germinal center responses in T-dependent (TD) antibody responses. IL-6 is required for in vitro T cell proliferation and in vivo cytotoxic T cell (CTL) generation, making them more responsive to IL-2.

[0067] In some aspects of the present invention, a 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, a non-human animal expressing human IL-6 may express one, two, three, four, or all five 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, which can be used to design or generate the non-human animals of the present invention, are well known in the art, for example, U.S. Patent Application US 2013 / 0042330 and Rathinam et al. 2011, Blood 118:3119-28 disclosing a knock-in mouse expressing human M-CSF; U.S. Patent No. 8,541,646 and Willinger et al. 2011, Proc Natl Acad Sci USA, 108:2390-2395 disclosing a knock-in mouse expressing human IL-3 and human GM-CSF; and U.S. Patent No. 8,541,646 and Rongvaux et al. 2011, Proc Natl Acad Sci Further details are provided in USA, 108:2378-83; and in PCT application WO 2012 / 040207 and Strowig et al. 2011, Proc Natl Acad Sci USA 108(32):13218-13223, which disclose transgenic mice expressing human Sirpa (the full disclosures of which are incorporated herein by reference).

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

[0069] Therefore, in some embodiments, nucleic acids encoding human IL-6 are functionally ligated to the IL-6 promoter in non-human animals. In other embodiments, nucleic acids encoding human IL-6 are functionally ligated to the human IL-6 promoter. As another example, in some embodiments, nucleic acids encoding human M-CSF are functionally ligated to the M-CSF promoter in animals. In other embodiments, nucleic acids encoding human M-CSF are functionally ligated to the human M-CSF promoter. As a third example, in some embodiments, nucleic acids encoding human IL-3 are functionally ligated to the IL-3 promoter in animals. In other embodiments, nucleic acids encoding human IL-3 are functionally ligated to the human IL-3 promoter. As a fourth example, in some embodiments, nucleic acids encoding human GM-CSF are functionally ligated to the GM-CSF promoter in animals. In other embodiments, nucleic acids encoding human GM-CSF are functionally ligated to the human GM-CSF promoter. As a fifth example, in some embodiments, nucleic acids encoding human TPO are functionally ligated to the TPO promoter in animals. In another embodiment, the nucleic acid encoding human TPO is functionally linked to the human TPO promoter.

[0070] Those skilled in the art will understand that the genetically modified animals of the present invention include genetically modified animals expressing at least one human nucleic acid from a promoter. Non-limiting examples of promoters that are ubiquitously expressed and useful in the present invention include, but are not limited to, the DNA pol II promoter, PGK promoter, ubiquitin promoter, albumin promoter, globin promoter, ovalbumin promoter, SV40 initial promoter, Roussarcoma virus (RSV) promoter, retroviral LTR and lentiviral LTR, β-actin promoter, ROSA26 promoter, heat shock protein 70 (Hsp70) promoter, the EF-1α gene encoding the elongation factor 1α (EF1) promoter, eukaryotic initiation factor 4A (eIF-4A1) promoter, chloramphenicol acetyltransferase (CAT) promoter, and CMV (cytomegalovirus) promoter. Expression systems of promoters and enhancers useful in the present invention also include inducible expression systems and / or tissue-specific expression systems. Non-limiting examples of tissue-specific promoters useful in 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, M-CSF promoter, IL-3 promoter, GM-CSF promoter, SIRPA promoter, TPO promoter, IFN-β promoter, Wiscott-Aldrich syndrome protein (WASP) promoter, CD45 (also known as lymphocyte common antigen) promoter, Flt-1 promoter, endoglin (CD105) promoter, and ICAM-2 (intracellular adhesion molecule 2) promoter. These and other promoters useful in the compositions and methods of the present 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 the inclusion of promoters, one or more additional regulatory 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) initial 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 regulatory elements useful in some embodiments of the present invention include, but are not limited to, transcription termination sequences and mRNA polyadenylation (pA) sequences.

[0071] Those skilled in the art will recognize that, in addition to naturally occurring human nucleic acid and amino acid sequences, the terms human nucleic acids and human amino acids also encompass variants of human nucleic acid and amino acid sequences. As used herein, the term “variant” defines either an isolated, naturally occurring human gene variant or a human recombinant variant containing one or more mutations compared to the corresponding wild-type human. For example, such mutations may be one or more amino acid substitutions, additions, and / or deletions. 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 the wild-type human polypeptide.

[0072] The percentage of identity between the two sequences is determined using techniques as described elsewhere in this specification. Mutations can be introduced using standard molecular biology techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Those skilled in the art will recognize that one or more amino acid mutations can be introduced without altering the functional properties of the human protein.

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

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

[0075] Human variants are encoded by nucleic acids that have a high degree of identity with the nucleic acid encoding wild-type humans. The complementary strand of the nucleic acid encoding the human variant specifically hybridizes with the nucleic acid encoding wild-type humans under high stringency conditions. The nucleic acid encoding the human variant may be isolated using well-known methodologies, or it may be generated by recombination or synthesis.

[0076] In some embodiments, genetically modified non-human animals expressing 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, human nucleic acid sequences are randomly incorporated into the genome of a non-human animal so that, for example, the animal contains exogenous human nucleic acid sequences at loci other than the non-human animal loci that encode the corresponding non-human animal protein. In other embodiments, genetically modified non-human animals expressing 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, nucleic acids encoding human proteins are introduced into an animal to replace genomic material encoding the corresponding non-human animal protein, thereby deactivating the corresponding non-human animal gene and deleting the corresponding non-human animal protein in that animal. In other words, the non-human animal undergoes a "knock-in" of the human gene.

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

[0078] In some embodiments, the genetically modified animals of the present invention are immunodeficient. "Immunodeficiency" means that in a non-human animal, one or more aspects of its native immune system are deficient, for example, one or more types of functional host immune cells in the animal, such as a deficiency in 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] For example, immunodeficient animals may have severe combined immunodeficiency (SCID). SCID is a condition characterized by a lack of T cells and impaired B cell function. Examples of SCID include X-linked SCID characterized by γ chain mutation or loss of the IL2RG gene and lymphocyte phenotype T(-)B(+)NK(-); as well as Jak3 gene mutation and lymphocyte phenotype T(-)B(+)NK(-), ADA gene mutation and lymphocyte phenotype T(-)B(-)NK(-), IL-7Rα chain mutation and lymphocyte phenotype T(-)B(+)NK(+), CD3δ or ε mutation and lymphocyte phenotype T(-)B(+)NK(+), RAG1 / RAG2 mutation and lymphocyte phenotype T(-)B(-)NK(+), Artemis gene mutation and lymphocyte phenotype T(-)B(-)NK(+), CD45 gene mutation 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 are included. Therefore, in some embodiments, genetically modified immunodeficient non-human animals have deletions of one or more selected from IL2 receptor γ chain deficiency, ADA gene mutations, IL7R mutations, CD3 mutations, RAG1 and / or RAG2 mutations, Artemis mutations, CD45 mutations, and Prkdc mutations.

[0080] The genetically modified non-human animals of the present invention are any non-human mammals genetically modified to include a human IL-6 coding sequence functionally linked to the IL-6 promoter, such as experimental animals, domesticated animals, livestock, etc., species such as mice, rodents, dogs, cats, pigs, horses, cattle, sheep, non-human primates, etc.; for example, mice, rats, rabbits, hamsters, guinea pigs, cattle, pigs, sheep, goats, and other transgenic animal species known in the art, specifically mammalian species. In certain embodiments, the genetically modified animal of the present invention is a mouse, rat, or rabbit.

[0081] In one embodiment, the non-human animal is a mammal. In some such embodiments, the non-human animal is, for example, a small mammal of the superfamily Dipodoidea or Muroidea. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the superfamily Muroidea. In one embodiment, genetically modified animals include the families Calomyscidae (e.g., kangaroo 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). It is derived from families selected from (rates), bamboo rats, and zokors. In a specific embodiment, genetically modified rodents are selected from typical mice and rats (Muridae), gerbils, spiny mice, and maned mice. In one embodiment, genetically modified mice are derived from members 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 rat, LEA strain, Sprague Dawley strain, Fischer strain, 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, for example, a mouse of the C57BL lineage (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 strains of 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, for example, Festing et al. (1999) Mammalian Genome 10:836 and 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 hybrid of the aforementioned 129 strain and the aforementioned C57BL / 6 strain. In another specific embodiment, the mouse is a hybrid of the aforementioned 129 strain or a hybrid of the aforementioned BL / 6 strain. In a specific embodiment, the hybrid 129 strain is the 129S6(129 / SvEvTac) strain. In yet another embodiment, the mouse is a hybrid of the BALB strain and another aforementioned strain.

[0084] Therefore, for example, in some embodiments, the genetically modified non-human animals of the present invention are (for example, IL2 receptor γ chain deficiency (i.e., γ) c - / -due to, and / or because of RAG deficiency), there is a 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, and has a genome containing nucleic acids encoding human IL-6, hM-CSF, hIL-3, hGM-CSF, hTPO, and / or hSIRPa, each operably linked to a human nucleic acid, for example, its corresponding promoter, such as the M-CSF promoter, IL-3 promoter, GM-CSF promoter, TPO promoter, or SIRPa promoter, and is an immunodeficient mouse that expresses the encoded human protein.

[0085] In certain specific embodiments, the genetically modified animal of the present invention is an immunodeficient mouse 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 (i.e., the mouse expresses mouse SIRPa), i.e., an immunodeficient hIL-6, hSirpa mouse, for example, Rag2 - / - IL2rg - / - IL-6 h / + hSIRPa + mouse or Rag2 - / - IL2rg - / - IL-6 h / h hSIRPa + is a mouse. 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, for example, Rag2 - / - IL2rg - / - IL-6 h / + M-CSF h / + IL-3 h / +GM-CSF h / + T h / + hSIRPa + Mouse, Rag2 - / - IL2rg - / - IL-6 h / + M-CSF h / h IL-3 h / h GM-CSF h / h T h / h hSIRPa + mouse.

[0086] In certain specific embodiments, the genetically modified animal of the present invention comprises a nucleic acid encoding human IL-6 functionally linked to the IL-6 promoter, lacking mouse IL-6; a nucleic acid encoding human SIRPa functionally linked to a human SIRPa promoter randomly incorporated into the genome of a non-human animal (i.e., the mouse still expresses mouse SIRPa); a nucleic acid encoding human M-CSF functionally linked to the M-CSF promoter, lacking mouse M-CSF; a nucleic acid encoding human IL-3 functionally linked to the IL-3 promoter, lacking mouse IL-3; a nucleic acid encoding human GM-CSF functionally linked to the GM-CSF promoter, lacking mouse GM-CSF; and a nucleic acid encoding human TPO functionally linked to the TPO promoter, lacking mouse TPO, i.e., Rag2 - / - IL2rg - / - IL-6 h / h M-CSF h / h IL-3 h / h GM-CSF h / h T h / h This is an hSIRPa+ mouse.

[0087] Method for creating genetically modified non-human animals The genetically modified non-human animals of the present invention can be produced, for example, using any convenient method for producing genetically modified animals, such as those known in the art or described herein.

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

[0089] Next, in order to create genetically modified animals that express human genes, one of various methods can be used to introduce human nucleic acid sequences into animal cells. Such techniques are well known in the art and include, but are not limited to, microinjection of oocyte pronuclei, transformation of embryonic stem cells, homologous recombination, and knock-in techniques. Methods for generating genetically modified animals that can be used include those described in 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 This includes, but is not limited to, the works described in Sunderland, Massachusetts: Sinauer (ed.), U.S. Patent No. 6,586,251, Rathinam et al. (2011, Blood 118:3119-28), Willinger et al. (2011, Proc Natl Acad Sci USA, 108:2390-2395), Rongvaux et al. (2011, Proc Natl Acad Sci USA, 108:2378-83), and Valenzuela et al. (2003, Nat Biot 21:652-659).

[0090] For example, the genetically modified animals of the present invention can be produced, for example, by introducing nucleic acids encoding human proteins into oocytes by microinjection and developing the oocytes in female rearing animals. In a preferred embodiment, a construct containing a human nucleic acid sequence is injected into fertilized oocytes. Fertilized oocytes can be collected from superovulating females the day after mating and injected with the expression construct. The injected oocytes are cultured overnight or directly transferred into the fallopian tubes 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 are described in *Manipulating the Mouse Embryo* (2002, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press). The presence of the introduced nucleic acids can be evaluated in the derived animals by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing, etc.) or protein analysis (e.g., ELISA, Western blotting, etc.). Such methods typically result in the random integration of the injected nucleic acid sequence (in this case, a construct containing nucleic acids encoding the human protein of interest) into the genome of an oocyte, and therefore into a non-human animal, i.e., at a locus other than the host animal locus that expresses the corresponding protein.

[0091] As another example, constructs containing nucleic acids encoding human proteins can be transfected into stem cells (ES cells or iPS cells) using well-known methods such as electroporation, calcium phosphate precipitation, and lipofection. The presence of the introduced nucleic acids can then be evaluated in the cells by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing) or protein analysis (e.g., ELISA, Western blotting). Cells determined to have incorporated the expression construct can then be microinjected into preimplantation embryos. For a detailed description of known methods in the art 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 target-specific integration of a transfected nucleic acid sequence (in this case, a construct containing a nucleic acid encoding a human protein of interest) into the genome of a stem cell, and thus into a human animal. Often, such methods result in the substitution of host genomic material, e.g., genomic material encoding a corresponding host protein, with a nucleic acid encoding a human protein of interest.

[0092] Genetically modified established animals can be used to breed additional animals that retain the genetic modifications. Genetically modified animals that retain the nucleic acids encoding the human proteins of this disclosure may further be crossed with other genetically modified animals that retain other genetic modifications, or with knockout animals, for example, knockout animals that do not express one or more of their genes.

[0093] In some embodiments, genetically modified immunodeficient animals comprise a genome containing a nucleic acid encoding a human polypeptide functionally linked to a promoter, and express the encoded human polypeptide. In various embodiments, genetically modified immunodeficient non-human animals comprise a genome comprising an expression cassette containing a nucleic acid encoding at least one human polypeptide functionally linked to a promoter and a polyadenylation signal, and further containing introns, and express the encoded human polypeptide.

[0094] As described above, in some embodiments, the genetically modified animals of the present invention are immunodeficient animals. A genetically modified non-human animal that is immunodeficient and contains one or more human cytokines, e.g., 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 those known in the art or described herein, e.g., by DNA injection of an expression construct into a preimplantation embryo, or by the use of stem cells such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells containing a mutated SCID gene allele that would result in immunodeficiency when homozygous, e.g., as described in more detail above and in the examples herein. Then, mice having modified oocytes or ES cells are generated, e.g., using methods described herein and known in the art, and are crossed to produce immunodeficient mice containing the desired genetic modifications. As another example, genetically modified non-human animals can be generated in a non-immunodeficient background, crossed with animals containing a mutated SCID gene allele that would cause immunodeficiency when homozygous, and then the offspring can be crossed to create immunodeficient animals that express at least one human protein of interest.

[0095] Various aspects of the present invention provide genetically modified animals in which substantially all cells contain human nucleic acids, and genetically modified animals in which some but not all cells contain human nucleic acids. In some cases, for example, in target-specific recombination, one copy of human nucleic acid will be incorporated into the genome of the genetically modified animal. In other cases, for example, in random integration, multiple copies of human nucleic acid that are adjacent or distant from each other may be incorporated into the genome of the genetically modified animal.

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

[0097] Availability The genetically modified non-human animals provided in various embodiments of the present invention have a variety of applications, such as use as models for hematopoietic cell proliferation and differentiation, in vivo evaluation of the human hematopoietic system, in vivo evaluation of cancer cells, in vivo studies of immune responses, in vivo evaluation of vaccine and immunization programs, use in testing the efficacy of agents that modulate the proliferation or survival of cancer cells, in vivo evaluation of cancer treatments, in vivo production and collection of immune mediators such as antibodies, and use in testing the efficacy of agents that affect the function of hematopoietic and immune cells.

[0098] To this end, in some cases, the genetically modified non-human animal ("host") of the present invention can be engrafted with at least one species of human hematopoietic cells. In some embodiments, a method is provided for creating an animal model for studying the human hematopoietic system, comprising the step of engrafting human hematopoietic cells into the genetically modified non-human animal ("host") of the present invention. In certain embodiments, a method is provided for engrafting human hematopoietic cells into the genetically modified non-human animal disclosed herein.

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

[0100] The human hematopoietic cells useful in the engraftment of 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 any lineage of highly differentiated hematopoietic cells. In some cases, the human hematopoietic cells are primary cells. Herein, “primary cells,” “primary cell line,” and “primary culture” are used interchangeably herein to include acutely isolated cells or cell cultures obtained from a subject and grown in vitro for a limited number of passages, i.e., between divisions. For example, a primary culture may be a culture that has been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not exceeding the crisis period. In other embodiments, human hematopoietic cells are derived from cell lines, i.e., the cells are derived from immortalized cultures that have been passaged, for example, about 15 times or more. In some cases, the engrafted hematopoietic cells include healthy cells. In other cases, the engrafted hematopoietic cells include diseased hematopoietic cells, e.g., cancerous hematopoietic cells, e.g., cancerous effector B cells, i.e., multiple myeloma cells. In some cases, the engrafted hematopoietic cells include both healthy cells 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, and cell lines derived therefrom, may originate 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 can be isolated from any human donor, including healthy donors and donors with diseases such as cancer, including leukemia. Engraftment of hematopoietic cells in genetically modified animals according to the present invention is characterized by the presence of human hematopoietic cells in the engrafted animals. In a specific embodiment, engraftment of hematopoietic cells in genetically modified animals according to the present invention is characterized by the presence of differentiated human hematopoietic cells in the engrafted animals from which hematopoietic cells were supplied, compared to a suitable control animal.

[0102] Methods for isolating human hematopoietic cells, administering them to host animals, and assessing their engraftment are well known in the art. Hematopoietic cells for administration to host animals, including either normal cells or neoplastic cells or combinations thereof, can be obtained from any tissue containing hematopoietic cells, such as umbilical cord blood, bone marrow, peripheral blood, peripheral blood mobilized by cytokines or chemotherapy, and fetal liver, but are not limited to the following. Exemplary methods for isolating human hematopoietic cells, administering them to a host animal, and assessing the engraftment of human hematopoietic cells in the host animal are described herein, as well as by 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 initial source material to obtain a population of cells enriched with a specific hematopoietic cell population (e.g., HSCs, HSPCs, LICs, 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 having a specific marker. In another embodiment, the hematopoietic cells useful in the compositions and methods of the present invention are enriched by selection for markers. In some embodiments, the hematopoietic cells useful in the compositions and methods of the present invention are a cell population in which selected cells constitute about 1-100% of the total cells, although in certain embodiments, a cell population in which selected cells constitute less than 1% of the total cells may also be used. In one embodiment, the hematopoietic cells useful in the compositions and methods of the present invention are depleted of cells having a specific 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 markers such as CD34. In some embodiments, the hematopoietic cells useful in the compositions and methods of the present invention are a cell population in which CD34+ cells constitute about 1-100% of the total cells, but in certain embodiments, a cell population in which CD34+ cells constitute less than 1% of the total cells can also be used. In certain embodiments, the hematopoietic cells useful in the compositions and methods of the present invention are a T-cell depleted cell population in which CD34+ cells constitute about 1-3% of the total cells, a lineage depleted cell population in which CD34+ cells constitute about 50% of the total cells, or a selected CD34+-positive cell population in which CD34+ cells constitute about 90% of the total cells.

[0104] With regard to the generation of the human hematopoietic system and / or immune system in genetically modified non-human animals expressing at least one human gene, the number of hematopoietic cells administered is not limited to a specific consideration. Therefore, as a non-limited example, the number of hematopoietic cells administered is approximately 1 × 10⁻⁶. 3 ~Approx. 1×10 7While it can be in the range of a few, in various embodiments, a larger or smaller number may be used. As another non-limiting example, when the recipient is a mouse, the number of HSPCs administered may be approximately 3 × 10⁶. 3 ~Approx. 1×10 6 The range may be CD34+ cells, but in various embodiments, more or fewer cells may be used. For other species of recipients, the number of cells that need to be administered can be determined using routine experimental methods only.

[0105] For example, in one embodiment, genetically modified and treated mice are engrafted with human hematopoietic cells or human hematopoietic stem cells (HPSCs) to form genetically modified and engrafted mice. In one embodiment, the hematopoietic cells are selected from human umbilical cord blood cells and human fetal hepatocytes. In one embodiment, engraftment occurs at a rate of approximately 1-2 × 10⁻⁶. 5 Based on individual human CD34+ cells.

[0106] In some cases, pretreatment may be performed before administration of hematopoietic cells (e.g., normal or neoplastic), such as sublethal irradiation of the recipient animal with radiofrequency electromagnetic radiation, generally using gamma rays or X-rays, or treatment with radioactive drugs such as busulfan or nitrogen mustard. Pretreatment is thought to reduce the number of host hematopoietic cells, create suitable microenvironmental factors for human hematopoietic cell engraftment, and / or create a microenvironmental niche for human hematopoietic cell engraftment. 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, genetically modified mice are treated to eliminate any endogenous hematopoietic cells that may be present in the mice. In one embodiment, the treatment includes the step of irradiating the genetically modified mice. In a specific embodiment, genetically modified neonatal mice are irradiated with a sublethal dose. In a specific embodiment, the newborn is irradiated with 200 cGy twice, at 4-hour intervals.

[0107] Hematopoietic cells (e.g., normal or neoplastic) can be administered to neonatal or adult animals by various routes, including, but not limited to, intravenous, intrahepatic, intraperitoneal, intrafemoral, and / or intratibia. A method for engrafting human hematopoietic cells, including either normal cells and neoplastic cells or a combination thereof, into immunodeficient animals is provided, comprising the step of providing human hematopoietic cells to immunodeficient animals, and including or not comprising the step of irradiating the animals before administration of the hematopoietic cells. A method for engrafting human hematopoietic cells into immunodeficient animals is provided, comprising the step of providing human hematopoietic cells, including either normal cells and neoplastic cells or a combination thereof, to genetically modified non-human animals, and including or not comprising the step of administering a radioactive drug such as busulfan or nitrogen mustard to the animals before administration of the hematopoietic cells.

[0108] Engraftment of human hematopoietic cells, including normal cells, 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 flow cytometry analysis of cells in the animals administered with human hematopoietic cells at one or more time points after administration of the hematopoietic cells, but is not limited to the following.

[0109] Generally, successful engraftment can be considered achieved when the number (or percentage) of human hematopoietic cells present in genetically modified non-human animals, including either normal cells, neoplastic cells, or a combination thereof, exceeds the lifespan of the administered human hematopoietic cells, and is greater than the number (or percentage) of human cells administered to the non-human animals. Detection of offspring of administered hematopoietic cells can be achieved, for example, by detecting human DNA in the recipient animal, or by detecting complete human hematopoietic cells, such as by detecting human cell markers like human CD45, human CD34, or sIL-6R. Sequential transfer of human hematopoietic cells from a first recipient to a second recipient, and engraftment of human hematopoietic cells in the second recipient, is a further option for testing 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 to 4 months after administration of human hematopoietic cells. For example, as described by Watanabe (1997, Bone Marrow Transplantation 19:1175-1181), cytokines (e.g., GM-CSF) can be used to mobilize stem cells.

[0110] In one embodiment, immunodeficient, genetically modified, and 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, platelets, and combinations thereof. In one embodiment, human cells are present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after engraftment.

[0111] In one embodiment, an immunodeficient, genetically modified, engrafted animal produces a human hematopoietic lymphoid system comprising human hematopoietic stem cell / 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 hematopoietic lymphoid system is present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months post-engraftment.

[0112] In one embodiment, immunodeficient, genetically modified, and engrafted animals produce a human hematopoietic system containing cancerous human hematopoietic cells, such as neoplastic (effector B) cells. In one embodiment, cancerous human hematopoietic cells are present at weeks 4, 6, 8, 12, or beyond after engraftment. In certain embodiments, cancerous human hematopoietic cells are present at weeks 2, 4, 6, 8, 12, or beyond after engraftment.

[0113] After engraftment of human hematopoietic cells, the genetically modified non-human animals of the present invention have many applications in the art. For example, the engrafted genetically modified animals of this disclosure are useful for studying the function of human hematopoietic cells in peripheral blood. As demonstrated in Example 2, a genetically modified mouse (e.g., Rag2) is immunodeficient and contains nucleic acid encoding human IL-6 functionally linked to the IL-6 promoter at the IL-6 mouse locus. - / - 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 T h / h ,hSIRPa+) is an immunodeficient mouse that does not express human IL-6, i.e., Rag2- / - IL2rg null Human hematopoietic cells, e.g., CD34, are produced more effectively than in mice. + These genetically modified mice support the engraftment of progenitor cells into peripheral blood and the 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 better promote the differentiation of CD5+ B cells and CD27+ B cells. CD5 is a protein found in a subset of IgM-secreting B cells called B-1 cells, and functions to mitigate activation signals from the B cell receptor, so B-1 cells can only be activated by extremely potent stimuli (such as bacterial proteins) and not by normal tissue proteins. CD27 is a marker for memory B cells. In addition, these genetically modified mice support the development of more functional human hematopoietic cells 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. Therefore, the genetically modified animals engrafted in this disclosure are useful in studying hematopoietic cell development and function, more specifically, B lymphocyte differentiation and function.

[0114] As another example, the engrafted genetically modified animals of this disclosure are useful for studying hematopoietic cancers. As demonstrated in Example 1 below, an immunodeficient and genetically modified mouse containing nucleic acid encoding human IL-6 functionally linked to the IL-6 promoter at 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 T h / h ,hSIRPa + Primary human multiple myeloma cells and human multiple myeloma cell lines engraft in these cells, but the immunodeficient mice that do not express human IL-6, i.e., Rag2 mice, engraft in these cells. - / - IL2rg null The cells do not engraft in mice. Expression of human SIRPa by a genetically modified host further improves the observed rate and extent of engraftment. Furthermore, the direct engraftment of multiple myeloma cells into the bone of these immunodeficient, genetically modified mice disclosed herein replicates osteopathologies typically associated with human multiple myeloma, such as bone destruction and bone resorption, as quantified, for example, by μCT scans.

[0115] Therefore, the engrafted, genetically modified animals of this disclosure are useful in screening candidate drugs to identify those that may treat hematopoietic cancers. The terms “treatment,” “to treat,” etc., are used herein to generally include obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it may completely or partially prevent the disease or its symptoms, and / or therapeutic in that it may partially or completely cure the disease and / or adverse effects resulting from the disease. “Treatment,” as used herein, includes any treatment of a disease in a mammal and includes: (a) preventing the onset of the disease in a subject that may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., preventing its development; or (c) reducing the disease, i.e., causing disease regression. Key candidate drugs for the treatment of hematopoietic cancers include those that may be administered before, during, or after the onset of cancer. Treatment of an ongoing disease is of particular importance if the treatment stabilizes or reduces the patient’s undesirable clinical symptoms. The terms “individual,” “subject,” “host,” and “patient” are used interchangeably herein and refer to any mammalian subject, specifically humans, for whom diagnosis, treatment, or therapy is desired.

[0116] As another example, the engrafted, genetically modified animals of this 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 efficacy of agents in the defense and / or treatment of infection by human pathogens. Pathogens may include viruses, fungi, bacteria, etc. Non-limiting examples of viral pathogens include human, swine, or avian influenza viruses. Non-limiting examples of bacterial pathogens include Mycobacterium, e.g., Mycobacterium tuberculosis (M. tuberculosis), and Enterobacteria, e.g., Salmonella typhi (S. typhi). An example of a method for infecting mice with Salmonella typhi and assessing the infection can be found, for example, in published U.S. Patent Application No. 2011 / 0200982, the disclosure of which is incorporated herein by reference. An example of a method for infecting mice with Mycobacterium tuberculosis and assessing the infection can be found, for example, in published U.S. Patent Application No. 2011 / 0200982, the disclosure of which is incorporated herein by reference. Other examples of human pathogens, which do not infect wild-type mice, or infect wild-type mice but do not model the immune response initiated by humans in response to the pathogen, will be well known to those skilled in the art. Such mouse models of pathogen infections are useful in research, for example, to better understand the progression of human infection. Such mouse models of infections are also useful in drug discovery, for example, to identify candidate drugs that defend against or treat infection.

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

[0118] In a screening assay for a biologically active drug, genetically modified mice engrafted with human hematopoietic cells of the present disclosure, for example, engrafted Rag2 - / - IL2rg - / - IL-6 h / h hSIRPa + Mouse, Rag2 implanted - / - IL2rg - / - IL-6 h / h M-CSF h / h IL-3 h / h GM-CSF h / h T h / hThe effects of a candidate drug of interest are assessed by exposing hSIRPa+ mice, etc., to the candidate drug and monitoring one or more output parameters. These output parameters may reflect cell viability, e.g., the total number of hematopoietic cells or the number of cells of a particular hematopoietic cell type, or the apoptotic state of cells, e.g., the amount of DNA fragmentation, the amount of cell vesicle formation, the amount of phosphatidylserine on the cell surface, etc., by methods well known in the art. Alternatively, the output parameters may reflect the differentiation potential of cells, e.g., the proportion of differentiated cells and differentiated cell types. Alternatively, the output parameters may reflect cell function, e.g., cytokines and chemokines produced by cells, antibodies produced by cells (e.g., quantity or type), the ability of cells to home to the challenge site and extravasate, or the ability of cells to modulate, i.e., promote or suppress the activity of other cells in vitro or in vivo. Other output parameters may reflect injury induced by diseased hematopoietic cells, e.g., the degree of bone destruction and bone resorption induced by multiple myeloma cells. Furthermore, other parameters may reflect infection in animals, such as the effect of drugs on pathogen infection, such as the titer of pathogens in mice or the presence of granulomas in mice, which are relevant to the research being conducted.

[0119] Parameters are quantifiable components of cells, particularly those that can be accurately measured, as is desirable in high-throughput systems. Parameters may include cell surface determinants, receptors, proteins or their structural or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids such as mRNA and DNA, or cellular components or cell products, or parts derived from such cellular components, or combinations thereof. While most parameters are expected to provide quantitative readouts, in some cases semi-quantitative or qualitative results may be acceptable. Readouts may include a single determination value, or they may include a mean, median, or variance, etc. Characteristically, a series of parameter readout values ​​are expected to be obtained for each parameter from a large number of identical assays. Variables are expected, and a series of values ​​for each of the set of test parameters are expected to be obtained using standard statistical methods, along with common statistical methods used to provide single values.

[0120] Candidate drugs of interest for screening include a large number of chemical classes, known and unknown compounds primarily comprising organic molecules, and may include organometallic molecules, inorganic molecules, gene sequences, vaccines, other drugs presumed to have antibiotic or antimicrobial properties, peptides, polypeptides, antibodies, and drugs that are pharmaceuticals approved for use in humans. A key aspect of the present invention is the evaluation of candidate drugs, including, for example, toxicity tests.

[0121] Candidate drugs include organic molecules containing structural interactions, specifically functional groups necessary for hydrogen bonding, typically at least one amine, carbonyl, hydroxyl, or carboxyl group, and often at least two functional chemical groups. Candidate drugs often include cyclic carbon structures, heterocyclic structures, and / or aromatic or polycyclic aromatic structures substituted with one or more of the above functional groups. Candidate drugs are also found in biomolecules, including peptides, polynucleotides, sugars, fatty acids, steroids, purines, pyrimidines, their derivatives, structural analogs, or combinations. These include drugs with pharmacological activity, molecules with genetic activity, etc. Compounds of interest include chemotherapeutic agents, hormones, or hormone antagonists, etc. Examples of pharmaceutical agents suitable for the present invention are described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, NY, (1996), Ninth edition. Toxins, as well as biological and chemical weapons agents, are also included. For example, see Somani, SM (Ed.), "Chemical Warfare Agents," Academic Press, New York, 1992.

[0122] Candidate drugs of interest for screening include nucleic acids, such as nucleic acids encoding siRNA, shRNA, antisense molecules, or miRNA, or nucleic acids encoding polypeptides. Many vectors useful for transferring nucleic acids into target cells are available. The vector may be maintained as an episome, for example, as a viral vector such as a plasmid, minicircle DNA, cytomegalovirus, or adenovirus, or it may be incorporated into the target cell genome through homologous recombination or random incorporation (e.g., retroviral vectors such as MMLV, HIV-1, ALV). The vector may be directly delivered to the cells of the present invention. In other words, pluripotent cells are brought into contact with a vector containing the nucleic acid of interest so that the vector is taken up by the cells.

[0123] Methods for contacting cells, such as cultured cells or cells in a mouse body, with nucleic acid vectors, such as electroporation, calcium chloride transfection, and lipofection, are well known in the art. Alternatively, the nucleic acid of interest may be delivered to cells via a virus. In other words, cells are brought into contact with viral particles containing the nucleic acid of interest. Retroviruses, such as lentiviruses, are particularly suitable for the methods of the present invention. Commonly used retroviral vectors are "defective," meaning they cannot produce the viral proteins necessary for productive infection. Instead, vector replication requires proliferation in a packaging cell line. To generate viral particles containing the nucleic acid of interest, the retroviral nucleic acid containing that nucleic acid is packaged into a viral capsid by a packaging cell line. Different packaging cell lines provide different envelope proteins to be incorporated into the capsid, and these envelope proteins determine the specificity of the viral particles for the cells. There are at least three types of envelope proteins: homotropic, bitropic, and heterotropic. Retroviruses packaged by allotropic envelope proteins, such as MMLV, can infect most mouse and rat cell types and are produced using allotropic packaging cell lines such as BOSC23 (Pear et al. (1993) PNAS 90:8392-8396). Retroviruses possessing bitropic envelope proteins, such as 4070A (Danos et al. (above)), can infect most mammalian cell types, including human, dog, and mouse, and are produced using bitropic 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); and GRIP (Danos et al. (1988) PNAS 85:6460-6464).Retroviruses packaged by heteromorphic envelope proteins, such as AKR env, can infect most mammalian cell types other than mouse cells. Appropriate packaging cell lines may be used to ensure that the cells of interest—in some cases, engrafted cells, and in others, the host, i.e., genetically modified animal cells—are targeted by the packaged viral particles.

[0124] Vectors used to deliver nucleic acids of interest to the cells of the present invention typically contain a suitable promoter to activate the expression, i.e., transcription, of the nucleic acid of interest. This may include ubiquitous promoters, such as the CMV-β-actin promoter, or inducible promoters that are active in a specific cell population or respond to the presence of a drug such as tetracycline. Transcriptional activation means that transcription will increase at least about 10-fold, at least about 100-fold, and more commonly, at least about 1000-fold, above the basal level in the target cells. Furthermore, vectors used to deliver reprogramming factors to the cells of the present invention may contain genes that must be subsequently removed using a recombinase system such as Cre / Lox, or genes that enable selective toxicity, such as herpesvirus TK, bcl-xs, etc., thereby disrupting the cells expressing them.

[0125] Polypeptides are also included among the candidate drugs of interest for screening. Such polypeptides may optionally be fused with polypeptide domains that increase the solubility of the product. The domains may be linked to the polypeptide through distinct protease cleavage sites, e.g., TEV sequences cleaved by TEV proteases. The linker may contain one or more flexible sequences, e.g., 1 to 10 glycine residues. In some embodiments, cleavage of the fusion protein is carried out in a buffer that maintains the solubility of the product, e.g., in the presence of 0.5–2 M urea, or in the presence of polypeptides and / or polynucleotides that increase solubility. Domains of interest include endosomal lysis domains, e.g., influenza HA domains; and other polypeptides that assist in production, e.g., IF2 domains, GST domains, GRPE domains, etc. Furthermore, or alternatively, such polypeptides may be formulated for improved stability. For example, the peptide may be PEGylated, in which case the polyethyleneoxy group provides an enhanced lifespan in the bloodstream. Polypeptides may be fused with other polypeptides to provide additional functionality, for example, to increase in vivo stability. Generally, such fusion partners are stable plasma proteins that can extend the in vivo plasma half-life of the polypeptide when present as a fusion, and specifically, such stable plasma proteins are immunoglobulin constant domains. In most cases where stable plasma proteins are commonly found as immunoglobulins or lipoproteins, identical or different polypeptide chains are typically disulfide-bonded and / or non-covalently bonded to form an assembled polychain polypeptide, the fusions herein containing polypeptides will also be prepared and utilized as polymers having substantially the same structure as the stable plasma protein precursors. These polymers will be homogeneous with respect to the polypeptide agents they contain, or they may contain multiple polypeptide agents.

[0126] Candidate polypeptide agents may be produced from eukaryotic cells or prokaryotic cells. They may be further processed by unfolding, e.g., thermal denaturation, DTT reduction, etc., and further refolded using methods known in the art. Modifications of interest that do not alter the primary sequence include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Glycosylation modifications are also included, such as those produced by modifying the glycosylation pattern of polypeptides, e.g., during synthesis and processing, or in a further processing step, by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylationases or deglycosylases. Sequences having phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine are also included. Polypeptides may be modified using conventional molecular biological techniques and synthetic chemistry to improve resistance to proteolysis, optimize solubility, or make them more suitable as therapeutic agents. Analogues of such polypeptides include those containing residues other than naturally occurring L-amino acids, such as D-amino acids or synthetic amino acids that do not exist in nature. D-amino acids can be used in place of some or all of the amino acid residues.

[0127] Candidate polypeptide agents can be prepared by in vitro synthesis using conventional methods known in the art. Various commercially available synthesis equipment, such as automated synthesis equipment from Applied Biosystems, Inc., Beckman, etc., are available. By using synthesis equipment, naturally occurring amino acids can be substituted with non-natural amino acids. The specific sequence and mode of preparation are considered to be determined by convenience, cost-effectiveness, required purity, etc. Alternatively, candidate polypeptide agents may be isolated and purified according to conventional recombinant synthesis methods. Lysates of the expression host can be prepared and purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In most cases, the composition used is considered to contain at least 20% by weight, more generally at least about 75% by weight, preferably at least about 95% by weight, and generally at least about 99.5% by weight for therapeutic purposes, in relation to contaminants associated with the preparation and purification methods of the product. Generally, the percentages are considered to be based on total protein.

[0128] In some cases, the candidate polypeptide drugs being screened are antibodies. The terms “antibody” or “antibody moiety” include any polypeptide chain that contains a molecular structure having a specific form that fits to and recognizes an epitope, and in which one or more non-covalent bonding interactions stabilize the complex between the molecular structure and the epitope. The specific or selective fit of a given structure to its specific epitope is sometimes referred to as a “lock and key” fit. Prototype antibody molecules are immunoglobulins, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all origins, e.g., humans, rodents, rabbits, cattle, sheep, pigs, dogs, other mammals, chickens, and other birds, are considered “antibodies.” The antibodies used in this invention may be either polyclonal or monoclonal antibodies. Antibodies are typically provided in the culture medium in which cells are cultured. Antibody production and screening are described in more detail below.

[0129] Candidate drugs can be obtained from diverse sources, including libraries of synthetic or natural compounds. For example, numerous methods, including the expression of randomized oligonucleotides and oligopeptides, are available for the random and specific synthesis of diverse organic compounds, including biomolecules. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily prepared. Furthermore, naturally occurring or synthetically prepared libraries and compounds can be readily modified through conventional chemical, physical, and biochemical means and used to prepare combinatorial libraries. Known pharmacological agents can be subjected to specific or random chemical modifications, such as acylation, alkylation, esterification, and amidation, to produce structural analogues.

[0130] Candidate drugs are screened for biological activity by administering the drug to at least one, and generally more, samples, sometimes along with samples lacking the drug. Changes in parameters in response to the drug are measured, and the results are evaluated by comparison with reference cultures, e.g., cultures in and without the drug, or cultures obtained with other drugs. When screening is performed to identify candidate drugs that may prevent, mitigate, or reverse the effects of a toxic drug, the screening is typically performed in the presence of the toxic drug, in which case the toxic drug is added at the most appropriate time for the result to be determined. For example, in cases where the protective / preventive ability of a candidate drug is being tested, the candidate drug may be added before the toxic drug, simultaneously with the candidate drug, or after treatment with the candidate drug. In another example, when the ability of a candidate drug to reverse the effects of a toxic drug is being tested, the candidate drug may be added after treatment with the toxic drug. As described above, in some cases, the "sample" is a genetically modified non-human animal on which cells have engrafted. For example, the candidate drug is provided to an immunodeficient animal, such as a mouse, on which human hematopoietic cells have engrafted and which contain nucleic acids encoding human IL-6 functionally linked to the IL-6 promoter. In some cases, the sample is the human hematopoietic cells to be engrafted, i.e., the candidate drug is provided to the cells before engraftment into an immunodeficient, genetically modified animal.

[0131] When candidate drugs are administered directly to engrafted, genetically modified animals, the drugs may be administered by any of the many methods known in the art for the administration of peptides, small molecules, and nucleic acids to mice. For example, the drugs may be administered orally, mucousally, topically, intradermally, or by injection, such as intraperitoneal, subcutaneous, intramuscular, intravenous, or intracranial injection. The drugs may be administered in a buffer solution or incorporated into any of a variety of formulations, for example, in combination with a suitable pharmaceutically acceptable medium. A “pharmaceutically acceptable medium” may be a medium approved by a federal or state regulatory authority for use in mammals such as humans, or listed in the US Pharmacopeia or other generally recognized pharmacopoeias. The term “medium” refers to a diluent, adjuvant, excipient, or carrier for formulating the compounds of the present invention for administration to mammals. Such pharmaceutical media may include lipids, such as liposomes, such as liposomal dendrimers; water and liquids such as oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.; and saline solutions; as well as gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Furthermore, adjuvants, stabilizers, thickeners, lubricants, and colorants may be used. Pharmaceutical compositions can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. The drug may be systemic after administration, or topical by local administration, intramural administration, or the use of implants that act to retain the active dose at the implantation site. The active drug may be formulated for immediate action or for sustained release. For certain conditions, specifically those affecting the central nervous system, it may be necessary to formulate drugs that can cross the blood-brain barrier (BBB).One strategy for drug delivery across the blood-brain barrier (BBB) ​​requires disrupting the BBB biochemically, either by osmotic means such as mannitol or leukotrienes, or by the use of vasoactive substances such as bradykinin. When the composition is administered by intravascular injection, the BBB disruptor may be administered co-administered with the drug. Other strategies for crossing the BBB may require the use of endogenous transport systems, including transcellular transport mediated by caveolin 1, transporters mediated by carriers such as glucose and amino acid carriers, transcellular transport mediated by receptors for insulin or transferrin, and active efflux transporters such as p-glycoprotein. To facilitate transport across the endothelial wall of blood vessels, the active transport portion may be conjugated to the therapeutic compound for use in the present invention. Alternatively, drug delivery of the drug after the blood-brain barrier may be by local delivery, e.g., subarachnoid delivery, e.g., via an Ommaya reservoir (see, e.g., U.S. Patents 5,222,982 and 5385582 incorporated herein by reference); e.g., intravitreous or intracranial delivery, e.g., by bolus injection, e.g., with a syringe; e.g., continuous injection, e.g., by cannula insertion, e.g., by convection (see, e.g., U.S. Application No. 20070254842 incorporated herein by reference); or by implantation of a device to which the drug is reversibly attached (see, e.g., U.S. Applications No. 20080081064 and 20090196903 incorporated herein by reference).

[0132] When providing drugs to cells before engraftment, it is convenient to add the drug to the culture medium of the cells in solution or in an easily soluble form. The drug may be added through a flow-through system as an intermittent or continuous flow, or a bolus of the compound may be added to the static solution in a single or gradually increasing amount. In the flow-through system, two solutions are used: one is a physiologically neutral solution, and the other is the same solution to which the test compound has been added. The first solution is passed over the cells, followed by the second solution. In the 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 due to 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 performed in parallel. As is well known in the art, determining the effective concentration of a drug typically involves using a series of concentrations resulting from dilutions of 1:10 or other logarithmic scales. If necessary, a second dilution series can be used to further refine the concentrations. Typically, one of these concentrations serves as a negative control, i.e., zero concentration, or a concentration of the drug below the detection level, or a concentration of the drug that does not produce a detectable change in phenotype.

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

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

[0136] Therefore, for example, humanized IL-6 mice in which human multiple myeloma cells have engrafted, such as Rag2 - / - IL2rg- / - IL-6 h / h A method is provided for determining the effect of a drug on multiple myeloma, comprising the steps of: administering the drug to a mouse; measuring parameters of the viability and / or proliferative capacity of multiple myeloma cells over time in the presence of the drug; and comparing the measurement with that of engrafted humanized IL-6 mice that have not been exposed to the drug. The drug is determined to be anticancer if, after a single dose or two or more doses of the drug over a selected period, it reduces the proliferation of multiple myeloma cells in the blood or tissue of the mouse and / or reduces the number of multiple myeloma cells by at least 20%, 30%, 40%, or more, and in some cases 50%, 60%, 70%, or more, for example 80%, 90%, or 100%, i.e., to an undetectable level. In a specific embodiment, the administration of the drug or combination of drugs is at least one week, ten days, two weeks, three weeks, or four weeks after engraftment of multiple myeloma cells.

[0137] Other examples of the use of the present invention in mice are provided elsewhere in this specification. Additional applications of the genetically modified and engrafted mice described herein will become apparent to those skilled in the art by reference to this disclosure.

[0138] Human antibody production As described elsewhere in this specification, compositions and methods useful for producing human monoclonal antibodies from engrafted immunodeficient animals are also provided. In various embodiments, the method includes the steps of contacting an immunodeficient animal with human hematopoietic cells to produce an immune system-transplanted non-human animal (engrafted animal), then contacting the engrafted animal with an antigen, collecting human cells from the engrafted animal that produce human antibodies against the antigen, and isolating antibodies from the antibody-producing cells.

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

[0140] In various aspects, the engrafted animals are non-human mammals. In some aspects, the engrafted animals are mice, rats, or rabbits.

[0141] In various embodiments of the present invention, 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 the following: a peptide, polypeptide, MHC / peptide complex, DNA, live virus, dead virus or part thereof, live bacterium, dead bacterium or part thereof, or cancer cell or part thereof.

[0143] In some embodiments, engrafted animals are exposed to the antigen 1 to 5 months after they have been exposed to human hematopoietic cells. In some embodiments, engrafted animals are exposed to the antigen only once, while in other embodiments, engrafted animals are exposed to the antigen two, three, four, five, six, seven, eight, 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 from the animal by removing appropriate cellular components of the immune system. 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 a portion thereof.

[0145] In various embodiments, the method of the present invention utilizes conventional hybridoma technology using a suitable fusion partner. In various embodiments, the fusion partner 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, as well as cell lines derived from these cells.

[0146] The method for isolating antibodies from engrafted animals according to the present invention is well known in the art. Isolation of antibodies from antibody-producing cells, culture media in which cell-produced antibodies are cultured, and / or from ascites fluid 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 the following methods: 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 may be administered to the engrafted animal by appropriate means known in the art. In various embodiments, the antigen may be administered to the engrafted animal by at least one of the following methods: 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 thereof of the present invention can vary considerably. In some embodiments, the reagents or kits are thought to comprise one or more reagents for use in the generation and / or maintenance of genetically modified non-human animals of the present invention. For example, a kit may comprise an immunodeficient mouse comprising a nucleic acid encoding human IL-6 functionally linked to an IL-6 promoter and a nucleic acid encoding human SIRPa functionally linked to a SIRPa promoter; or a mouse comprising a nucleic acid encoding human IL-6 functionally linked to an IL-6 promoter and a nucleic acid encoding human M-CSF functionally linked to an M-CSF promoter; a nucleic acid encoding human IL-3 functionally linked to an IL-3 promoter; a nucleic acid encoding human GM-CSF functionally linked to a GM-CSF promoter; a nucleic acid encoding human TPO functionally linked to a TPO promoter; and / or a nucleic acid encoding human SIRPa functionally linked to a SIRPa promoter. The kit may include reagents for breeding such mice, such as genotyping primers for the human IL-6 gene, human M-CSF gene, human IL-3 gene, human GM-CSF gene, human SIRPa gene, and / or human TPO gene, PCR buffer, MgCl2 solution, etc.

[0149] In some embodiments, the reagents or kits are thought to include one or more reagents for use in engraftment into genetically modified non-human animals according to the present invention, for example, human hematopoietic cells, enriched populations of human hematopoietic progenitor cells, hematopoietic cell lines, neoplastic hematopoietic cell lines, etc., for transplantation into genetically modified non-human animals according to the present invention, or reagents for preparing populations of hematopoietic cells, enriched populations of human-derived hematopoietic cells, hematopoietic cell lines, neoplastic hematopoietic cell lines, etc., for transplantation into genetically modified non-human animals according to the present invention.

[0150] In some embodiments, the reagents or kits may include, for example, reagents for determining the viability and / or function of hematopoietic cells in the presence or absence of a candidate drug, such as one or more antibodies specific to markers expressed by different types of hematopoietic cells, or reagents for detecting specific cytokines, chemokines, etc. Other reagents may include culture media, culture auxiliaries, substrate compositions, etc.

[0151] In addition to the components described above, the kit of the present invention is considered to further include instructions for carrying out the method of the present invention. These instructions may be present in a variety of forms within the kit of the present invention, and one or more of these forms may be present within the kit. One form in which these instructions may be present is information printed on a suitable medium or substrate, for example, as printed paper, within the kit packaging, within a package insert, etc. Another means may be a computer-readable medium on which the information is recorded, for example, a disk, CD, etc. Yet another possible means may be a website address that can be used via the internet to access information from a distant site. Any convenient means may be present within the kit. [Examples]

[0152] Experimental example The present invention will be described in more detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not limiting unless otherwise noted. Accordingly, the present invention should not be construed as being limited to the following examples, but rather as encompassing all any variations that may become apparent as a result of the teachings provided herein.

[0153] Without further explanation, those skilled in the art will likely be able to prepare, utilize, and carry out the methods described in the claims for the compounds of the present invention using the above description and the following exemplary examples. Accordingly, the following examples specifically point to preferred embodiments of the present invention and should not be construed as limiting the remainder of the disclosure.

[0154] Example 1: Genetic humanization of cytokine genes enables engraftment of human multiple myeloma cells into mice. The data described 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 substituting 6.8kb of the mouse IL-6 gene locus with a 4.8kb human IL-6 gene sequence containing exons 1-5, 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® gene 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 targeting construct linearized by NotI, generated by gap repair cloning, containing upstream and downstream homology arms of mouse IL-6 adjacent to a 4.8 kb human IL-6 sequence (genomic coordinates: NCBI h37.1:ch7:22,766,882~22,771,637) spanning from the ATG in exon 1 to exon 5, along with a neo selection cassette into which a 16 nucleotide 3' downstream sequence and loxP were introduced, was used for Rag2 + / - IL2rg Y / - electroporated into ES cells. The parental ES cell line with knockouts of the RAG2 gene and the IL2rg gene was a commercially available V17 ES cell (BALB / cx129 heterozygote). To remove the drug selection cassette, correctly targeted ES cells can be electroporated using a transient Cre expression vector.

[0157] Correctly targeted ES cell clones were identified by a loss-of-native-allele / LONA assay (Valenzuela et al. 2003) that determines the copy number of the native unmodified Il6 gene by two TaqMan® quantitative polymerase chain reaction (qPCR) specific to sequences within the targeted mouse Il6 gene due to deletion. The qPCR assay contained the following primer-probe sets (written 5' to 3'): Upstream forward primer: TIFF0007877413000001.tif4128 Upstream reverse primer: TIFF0007877413000002.tif4128 Upstream probe: TIFF0007877413000003.tif4129 Downstream forward primer: TIFF0007877413000004.tif4128 Downstream reverse primer: TIFF0007877413000005.tif4128 Downstream probe: TIFF0007877413000006.tif4130. Here, FAM refers to a 5-carboxyfluorescein fluorescent probe, and BHQ refers to a black hole quencher type fluorescent quencher (Biosearch Technologies). DNA purified from ES cell clones incorporating a targeted vector and integrated into the genome was cycled in an Applied Biosystems Prism 7900HT in a 384-well PCR plate (MicroAmp® Optical 384-Well Reaction Plate, Life Technologies) in combination with TaqMan® Gene Expression Master Mix (Life Technologies) as suggested by the manufacturer. Fluorescence data was collected during PCR, and threshold cycles (Ct) (the number of PCR cycles at which accumulated fluorescence reaches a preset threshold) were determined. Upstream and downstream Il6-specific qPCRs, as well as 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. 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 using the following formula: copy number = 2·2 -ΔΔCtA precisely targeted clone that has lost one of its native copies is thought to 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 using the following primer-probe set (written from 5' to 3'): Human forward-facing primer: TIFF0007877413000007.tif4128 Human reverse primer: TIFF0007877413000008.tif4128 and human probe: TIFF0007877413000009.tif4128.

[0158] The upstream junction between the mouse gene locus and the sequence containing the hIL-6 gene is It is designed to be located within TIFF0007877413000010.tif35152. Here, the last mouse nucleotide before the first nucleotide of the human gene is "T" in CCGCT, and the first nucleotide of the human sequence is the first "A" in ATGAA. The downstream junction between the sequence containing the hIL-6 gene and the mouse locus is, It is designed to be located within TIFF0007877413000011.tif35150. Here, 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 (its beginning is shown) for the removal of the neocassette, which is activated by a ubiquitin promoter into which loxP has been introduced. The junction between the neocassette and the mouse IL-6 locus is, It is designed to be located within TIFF0007877413000012.tif28151. Here, the last "C" in AGCTC is the last nucleotide of the neocassette; 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 pre-implantation embryos using techniques known in the art.

[0160] Next, the humanized IL-6 KI mice are backcrossed to generate mice that lack Rag2 and Il2rg and express hIL-6, and are mated with mice that express the human SIRPa transgene (Strowig et al., 2011, Proc Natl Acad Sci USA, 108(32):13218-13223) to generate mice that lack Rag2 and Il2rg and express both hIL-6 and hSIRPa (Rag2 - / - Il2rg null Il6 h / h hSIRPa + ). Furthermore, Rag2 - / - , IL-2rg Y / - , hIL-6 KI mice are mated with mice that express 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), and human M-CSF (Rathinam et al, 2011, Blood, 118(11):3119-3128), and also express hSIRPa (Strowig et al., 2011, Proc Natl Acad Sci USA, 108(32):13218-13223) to generate mice that express combinations of these human proteins (Rag2 - / - Il2rg null hSIRPa + Tpo h / h Mcsf h / h Il3 / Gmcsf h / h Il6 h / h ).

[0161] Cell lines and primary cellsMultiple myeloma cell line INA-6 (Burger et al., 2001, Hematol J, 2(1):42-53) was maintained in a standard incubator at 37°C and 5% CO2 in RPMI1640 medium supplemented with 20% FCS, penicillin / streptomycin, L-glutamine, and 2.5 ng / ml hIL-6.

[0162] After obtaining informed consent from patients, primary cells derived 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®). CD3+ cells were depleted by negative selection using an AutoMACS system with anti-CD3 microbeads (Miltenyi Biotec) to obtain a T-cell depleted population. CD138+ cells were isolated by positive selection using an AutoMACS® system with anti-CD138 microbeads (Miltenyi Biotec) to obtain CD138+ cells. The purity of the cells after MACS® selection was analyzed by flow cytometry.

[0163] Cell transplantation For intrafemoral transplantation of INA6 cells, Rag2 - / - Il2rg null Il6 h / h hSIRPa + Mice were irradiated twice with 200 rads from an X-ray source. The indicated amount of cells was then transplanted into the femur of the recipient mouse. Briefly, the mice were anesthetized with ketamine / xylazine, and the patellar surface of the femur was perforated using a 26-gauge needle. The cells were then slowly injected in 20 μl volumes 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 twice with 150 rad 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 morphometry was quantified using cone-beam microfocus computed tomography (μCT40; ScancoMedicalAG). Serial tomographic images were acquired, 3D images were reconstructed, and parameters were determined. Trabecular bone morphometry was characterized by measuring bone volume fraction, trabecular width, trabecular number, and trabecular spacing. Cortical measurements included mean cortical width, cortical bone cross-sectional area, subperiosteal cross-sectional area, and bone marrow area.

[0166] histology Following standard procedures, the femur was removed from the soft tissue, fixed in 10% buffered formalin, dehydrated, embedded in methyl methacrylate, then sectioned and stained with toluidine blue.

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

[0168] Therefore, in order to directly test the possibility of humanizing IL-6 to enable the proliferation of myeloma cells, INA-6 cells were (i) Rag2 - / - Il2rg null Mouse, (ii) Rag2 - / - Il2rg null hSIRPa+ mouse, (iii) Rag2 - / - Il2rg null Il6 h / h Mouse, and (iv)Rag2 - / - Il2rg null Il6 h / h INA-6 cells were intravenously transplanted into hSIRPa+ mice. Engraftment was analyzed by measuring the 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 using a fluorescence microscope. Few GFP+ cells were detected in the bone marrow (Figure 2), but an increased number of GFP+ cells were 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 lungs (Figure 3), and therefore correlated with the presence of INA-6 cells in the lungs. In summary, the data disclosed herein demonstrate successful engraftment of INA-6 cells after intravenous injection (iv) of mice genetically modified to express human IL6, or human IL6 and human SIRPa, suggesting that genetic humanization can overcome limitations in the proliferation of human MM cells in mice.

[0170] The data in Figure 3 suggest that INA-6 cells do not efficiently home to the bone marrow, but rather can proliferate in non-physiological sites. Therefore, we next investigated whether transplantation of tumor cell lines in the natural microenvironment could reproduce the pathology typically associated with human MM. To this end, we tested intraosseous injection of INA-6 cells. This resulted in significant bone destruction and bone resorption, which are pathological aspects seen in MM patients (Figures 4-6). Specifically, loss of cancellous bone volume was observed by histology quantified by μCT. Furthermore, only limited metastases to peripheral sites such as the lungs were observed, leading to the conclusion that this model can be further explored to investigate novel drugs that interfere with this pathology. To test this conclusion, mice engrafted with INA-6 were treated with two drugs commonly used to treat multiple myeloma patients: Zometa or Velcade. As quantified by μCT, treatment with Zometa in mice injected with INA-6 cells significantly reduced bone resorption compared to untreated mice (Figure 7).

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

[0172] Genetic humanization of cytokine genes enables the engraftment of patient-derived primary multiple myeloma cells. Next, we conducted experiments to investigate the transplantation of primary MM cells into genetically humanized mice. It has been previously 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, GM-CSF, and M-CSF improved the engraftment of myeloid cells, which have been shown to be important for certain aspects of MM pathology. Transgenic expression of hSIRPa improves human cell engraftment, and the SIRPa-CD47 axis has recently been shown to be involved in tumorigenesis. Rag2 - / - Il2rg null hSIRPa + TPO h / h Mcsf h / h Il3 / Gmcsf h / h Il6 h / h To generate mice, 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 myeloma cells derived from MM patients were injected into the bone marrow of the mice. Myeloma cells, identified as CD138+CD38+CD19- cells, were detected at a high frequency in the injected bone, but very few cells were detected in the accompanying 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 data demonstrate that humanization of cytokine genes encoding IL-6, TPO, IL-3, GM-CSF, and / or M-CSF enables the engraftment of patient-derived primary 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 As described above, humanized IL-6 KI mice were generated. First, the chimeric mice were crossed with BALB / c mice, and then backcrossed to obtain derivative animals homozygous for hIL-6. Mice with the same mixed BALB / c × 129 background were used as controls.

[0175] Newborn mice (within 1 day of birth) were irradiated twice with a sublethal dose of 150 cGy of X-rays at 4-hour intervals. Four hours after irradiation, 1-2 × 10⁻¹⁶ X-rays were obtained using a 30-gauge needle (Hamilton Company, NV, USA). 5 individual CD34 + 20 μl of PBS containing fetal liver (FL) cells was injected into the livers of mice. Mice were weaned at 3 weeks of age. Mice were maintained under specific pathogen-free conditions, and all experiments were conducted in accordance with the Yale Institutional Animal Care and Use Committee protocol.

[0176] Analysis of hematological cell populations in humans and mice Blood was collected from the posterior orbital plexus of mice under isoflurane anesthesia at various post-transplantation points. Plasma samples were collected and stored at -20°C for further Ig measurements. Red blood cells were lysed twice 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 were obtained from the bone marrow (BM), thymus, and spleen. The samples were then stained with mAbs labeled with fluorescent dyes 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 from 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 using an LSRII (BD Biosciences) cytometer and analyzed with FlowJo (Treestar, OR, USA) software.

[0178] Measurement of human immunoglobulinsTotal immunoglobulin (Ig) levels were measured in mouse plasma collected 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 the samples were added at room temperature (RT) for 2 hours. The plates were washed and incubated with isotype-specific secondary biotinylated antibodies (Southern Biotechnology) at RT for 1 hour, followed by incubation with streptavidin-HRP (Pierce Protein Research Products, IL, USA). After the final wash, enzyme activity was determined using TMB substrate solution, followed by a stop solution (both Pierce Protein Research Products). Absorbance was measured at 450 nm. Human serum samples from Bethyl (TX, USA) were used as a reference.

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

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

[0181] Human CD34 + RAG2 reconstituted by cells - / - γ c - / - Organocyte engraftment and composition in mice Human - origin cells were found in blood - lymphoid organs such as BM, spleen, and thymus (Figure 11A). Interestingly, the spleen of IL6h / h mice showed higher human engraftment than that of IL6m / m mice (61.75% ± 10.87 vs 23.56% ± 5.2). These data were confirmed by the doubling of 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 that was implanted - / - γ c - / - B cell maturation in miceUsing the gating strategy illustrated in Figure 12A, based on the use of combinations of CD24, CD38, and surface IgM antibodies, the maturation stages of human B cells were studied in the BM and spleen of engrafted mice. This strategy is particularly useful for examining the presence of transitional B cell subsets. In the BM, the main compartment consisted of pro / pre-B cells (Figure 12B), and there was no difference between IL6m / m mice and IL6h / h mice (76.04% ± 9.09 and 79.07% ± 3.43, respectively). The spleens of both groups contained some mature B cells (approximately 20%), but also had 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 + (Figure 13B). This marker is not generally expressed in human BM and peripheral B cells, but is found at a low percentage in FL B cells (Figure 13A).

[0184] IL6h / h mice showed a sharp increase in the percentage of CD27 + B cells in the spleen when compared to IL6m / m mice (24.73% ±8.94 vs 9.46% ±2.32), but CD27 + cells were rarely found in the BM (Figures 14A and 14B).

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

[0186] In general, a higher percentage of antibody-secreting IL6h / h mice existed compared to IL6m / m mice. The former mice showed lower IgM levels (14.69 μg / ml vs. 33.66 μg / ml at week 12, and 18.25 μg / ml vs. 190.2 μg / ml at week 20) ​​and increased IgG levels (243 μg / ml vs. 49.6 μg / ml at week 12, and 553.6 μg / ml vs. 297.2 μg / ml at week 20) ​​(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] All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. While the present invention has been disclosed in specific embodiments, it will be apparent that other embodiments and variations of the invention can be devised by those skilled in the art without departing from the spirit and scope of the invention. The appended claims shall 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. nucleic acids encoding human IL-6, functionally linked to the rodent IL-6 promoter at the rodent IL-6 locus; and (i) A nucleic acid encoding human SIRPa, functionally linked to the SIRPa promoter, (ii) A nucleic acid encoding human M-CSF, functionally linked to the M-CSF promoter. (iii) A nucleic acid encoding human IL-3, functionally linked to the IL-3 promoter, (iv) A nucleic acid encoding human GM-CSF, functionally linked to a GM-CSF promoter, and (v) Nucleic acids encoding human TPO, functionally linked to the TPO promoter. At least one additional nucleic acid selected from the group consisting of Genetically modified rodents, including [specific species / species].

2. A genetically modified rodent according to claim 1, wherein the nucleic acid encoding human M-CSF is located at the rodent M-CSF locus, the nucleic acid encoding human IL-3 is located at the rodent IL-3 locus, the nucleic acid encoding human GM-CSF is located at the rodent GM-CSF locus, and the nucleic acid encoding human TPO is located at the rodent TPO locus.

3. A genetically modified rodent according to claim 1 or 2, which is immunodeficient.

4. Rag2 - / - IL2rg null A genetically modified rodent according to claim 3, which is a rodent.

5. A genetically modified rodent according to claim 3, further comprising the engraftment of human hematopoietic cells.

6. The genetically modified rodent according to claim 5, wherein the human hematopoietic cells are CD34+ cells.

7. The genetically modified rodent according to claim 5, wherein the human hematopoietic cells are multiple myeloma cells.

8. The process of transplanting a population of human hematopoietic cells into genetically modified, immunodeficient rodents containing nucleic acids encoding human IL-6. A method for generating a rodent model of human B cell development and function, including, The nucleic acid encoding human IL-6 is functionally linked to the rodent IL-6 promoter at the rodent IL-6 locus. The rodent expresses human IL-6 and does not express rodent IL-6. The aforementioned method.

9. The method according to claim 8, wherein the population of hematopoietic cells to be transplanted includes CD34+ cells.

10. The method according to claim 8, wherein the population of hematopoietic cells to be transplanted includes multiple myeloma cells.

11. The method according to claim 8, wherein the rodent further comprises a nucleic acid encoding human SIRPa, the nucleic acid encoding human SIRPa being functionally linked to a SIRPa promoter.

12. Genetically modified rodents, (i) A nucleic acid encoding human M-CSF, functionally linked to the M-CSF promoter. (ii) A nucleic acid encoding human IL-3, functionally linked to the IL-3 promoter, (iii) A nucleic acid encoding human GM-CSF, functionally linked to a GM-CSF promoter, and (iv) Nucleic acids encoding human TPO, functionally linked to the TPO promoter. The method according to claim 11, wherein at least one additional human nucleic acid selected from the group consisting of the following is expressed.

13. An engrafted rodent prepared according to the method described in any one of claims 8 to 12.

14. A step of contacting a genetically modified rodent according to claim 13, on which human hematopoietic cancer cells have been engrafted, with a candidate drug, and A step of comparing the survival rate and / or proliferation rate of human hematopoietic cancer cells in the contacted rodents with the survival rate and / or proliferation rate of human hematopoietic cancer cells in genetically modified rodents according to claim 13, in which human hematopoietic cancer cells have been engrafted but have not been in contact with the candidate drug. A method for screening candidate drugs for their ability to treat hematopoietic cancers, including, The reduction in the survival rate and / or proliferation rate of human hematopoietic cancer cells in the contacted rodents indicates that the candidate drug will treat hematopoietic cancer. The aforementioned method.

15. The method according to claim 14, wherein the hematopoietic cancer is multiple myeloma.

16. The process of immunizing genetically modified immunodeficient rodents with antigens. A method for producing an antigen-binding protein, including The rodent, A genome comprising a nucleic acid encoding human IL-6, wherein the nucleic acid is functionally linked to the rodent IL-6 promoter at the rodent IL-6 locus, and Engraftment of human hematopoietic cells Includes, After immunization, the rodent produces human cells containing antigen-binding proteins that specifically bind to the antigen. The aforementioned method.

17. The method according to claim 16, further comprising the step of collecting at least one of the rodent's spleen, lymph nodes, peripheral blood, bone marrow, or a part thereof from the rodent.

18. The method according to claim 17, further comprising the step of isolating human cells from at least one of the spleen, lymph nodes, peripheral blood, bone marrow, or a portion thereof of a rodent.

19. The method according to claim 18, further comprising the step of isolating an antigen-binding protein from human cells.

20. The method according to claim 19, wherein the human cell is a B cell.

21. The method according to claim 18, further comprising the step of producing a hybridoma cell line from human cells.

22. The method according to claim 21, further comprising the step of isolating an antigen-binding protein that specifically binds to an antigen from a hybridoma cell line.

23. The method according to claim 16, wherein the antigen-binding protein is human IgG.

24. The method according to claim 16, wherein the human hematopoietic cells include CD34+ cells.

25. The method according to claim 16, wherein the antigen is at least one of a peptide, an MHC / peptide complex, DNA, a live virus, a dead virus or part thereof, a live bacterium, a dead bacterium or part thereof, and a cancer cell or part thereof.

26. The method according to claim 16, wherein the engrafted and genetically modified immunodeficient rodents do not express rodent IL-6.

27. The method according to claim 16, wherein an engrafted and genetically modified immunodeficient rodent comprises a genome further comprising a nucleic acid encoding human SIRPa, the nucleic acid being functionally linked to a promoter.

28. The method according to claim 16, wherein the engrafted and genetically modified immunodeficient rodent comprises a recombinant activating gene (RAG) knockout.

29. The method according to claim 16, wherein the engrafted and genetically modified immunodeficient rodent comprises a knockout of the IL2 receptor γ chain (IL2rg) gene.

30. The method according to claim 16, wherein the engrafted and genetically modified immunodeficient rodents include a knockout of recombinant activating gene (RAG) and a knockout of the IL2 receptor γ chain (IL2rg) gene.

31. The process of isolating human cells that produce antigen-binding proteins that specifically bind to antigens from engrafted and genetically modified immunodeficient rodents. A method for isolating an antigen-binding protein, including The engrafted and genetically modified rodents, A genome comprising a nucleic acid encoding human IL-6, wherein the nucleic acid is functionally linked to the rodent IL-6 promoter at the rodent IL-6 locus, and Engraftment of human hematopoietic cells Includes, The engrafted, genetically modified immunodeficient rodents are immunized with the antigen. The aforementioned method.

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