Genetically Modified Non-Human Animals and Methods of Use Thereof

Abstract

Generally speaking, the present invention relates to genetically modified non-human animals that express human polypeptides and methods of using the same.

Description

[0001] This application is a divisional application of the invention application with the filing date of September 6, 2013, Chinese Application No. 201380046265.9, and the invention title of "Genetically Modified Non-Human Animals and Methods of Use Thereof".

[0002] Cross-reference to related applications

[0003] This application claims the priority of U.S. Provisional Application Serial No. 61 / 698,002 filed on September 7, 2012, and U.S. Provisional Application Serial No. 61 / 775,171 filed on March 8, 2013, the content of each of which is incorporated herein by reference in its entirety. Background of the invention

[0004] The goal of biomedical research is to better understand human physiology and use this knowledge to prevent, treat, or cure human diseases. Due to the practical and ethical barriers to experimenting on human subjects, much research is conducted on small animal models such as mice. However, mice are not humans, and the knowledge obtained from animal experiments is not always applicable to humans. In this context, mice repopulated with the human hematopoietic-lymphoid system (HHLS) represent a useful small animal model for studying human hematopoiesis and immune function in vivo.

[0005] HHLS mice are generated by transplanting human hematopoietic stem and progenitor cells (HSPCs) and / or human fetal tissues into recipient mice that are defective in both the innate and adaptive branches of the immune response. The first model of HHLS mice was developed in the late 1980s (Mosier et al., 1988, Nature 335:256-259; McCune et al., 1988, Science 241:1632-1639; Kamel-Reid and Dick, 1988, Science 242:1706-1709) and has since undergone a series of improvements (Legrand et al., 2006, Journal of Immunology 176:2053-2058; Shultz et al., 2007, Nature Reviews Immunology 7:118-130). Currently, the mouse strains used as recipients for human hematopoietic engraftment share three characteristics. First, they lack B and T cells due to a Scid mutation in the gene encoding the PRKDC protein (Mosier et al., 1988, Nature 335:256-259; McCune et al., 1988, Science 241:1632-1639), or due to the deletion of one of the two Rag genes (Shultz et al., 2000, Journal of immunology 164:2496-2507; Traggiai et al., 2004, Science 304:104-107). Second, they encode the common γ chain of the cytokine receptor (γ c) Deletion or mutation of the Il2rg gene abrogates IL-15 signaling and results in the absence of NK cells (Traggiai et al., 2004, Science 304:104-107; Ito et al., 2002, Blood 100:3175-3182). Third, the interaction between the SIRPA receptor expressed on mouse macrophages and the CD47 ligand on human cells provides an inhibitory signal to mouse macrophages and confers phagocyte tolerance to human xenografts (Takenaka et al., 2007, Nature Immunology 8:1313-1323; Takizawa and Manz, 2007, Nature Immunology 8:1287-1289). Species-specific interaction between SIRPA expressed on mouse cells and human CD47 is achieved when using the NOD genetic background containing natural polymorphisms in the Sirpa gene (Takenaka et al., 2007, Nature Immunology 8:1313-1323; Takizawa and Manz, 2007, Nature Immunology 8:1287-1289; Legrand et al., 2011, Proc Natl Acad Sci USA 108:13224-13229) or by BAC transgenic expression of the human SIRPA gene (Strowig et al., 2011, Proc Natl Acad Sci USA 108:13218-13223). High levels of human hematopoietic cell engraftment are achieved after human HSPC transplantation when using NOD Scidγ c - / - (NOG (Ito et al., 2002, Blood 100:3175-3182) or NSG (Ishikawa et al., 2005, Blood 106:1565-1573)) or hSIRPA tg RAG2 - / - γ c - / - (SRG (Strowig et al., 2011, Proc Natl Acad Sci USA 108:13218-13223)) mice as recipients.

[0006] Although human multi-lineage hematopoiesis has been observed in these recipient strains, the terminal differentiation, homeostasis, and / or effector functions of most human cell types are suboptimal. This has been hypothesized to be due to reduced or absent cross-reactivity between cytokines secreted by murine tissues and human receptors expressed on hematopoietic cells (Manz, 2007, Immunity 26:537-541; Willinger et al., 2011, Trends in Immunology 32:321-327). To circumvent this limitation, several strategies have been developed to deliver human cytokines in murine hosts. These methods include injection of recombinant cytokines (Lapidot et al., 1992, Science 255:1137-1141; van Lent et al., 2009, J. Immunol 183:7645-7655), lentiviral delivery of cytokine-encoding cDNA (O’Connell et al., 2010, PloS One 5(8):e12009), hydrodynamic injection of plasmid DNA (Chen et al., 2009, Proc Natl Acad Sci USA 106:21783-21788), transgenic expression of cDNA (Nicolini et al., 2004, Leukemia 18(2):341-347; Brehm et al., 2012, Blood 119:2778-2788; Takagi et al., 2012, Blood 119:2768-2777) or knock-in replacement of cytokine-encoding genes (Rongvaux et al., 2011, Proc Natl Acad Sci USA 108:2378-2383; Willinger et al., 2011, Proc Natl Acad Sci USA 108:2390-2395; Rathinam et al., 2011, Blood 118:3119-3128). The latter method has the advantage of a more physiological expression of the human gene. In addition, if the human cytokine is not fully cross-reactive on murine receptors, it can induce defects in the murine cell population and confer an additional competitive advantage to human cells.Using a knock-in gene replacement strategy, humanization of the gene encoding thrombopoietin (Tpo) led to better maintenance of functional human hematopoietic stem cells and increased engraftment in the bone marrow (Rongvaux et al., 2011, Proc Natl Acad Sci USA 108:2378-2383); replacement of the genes encoding interleukin-3 and GM-CSF (Il3 and Csf2) induced the loss of murine alveolar macrophages (AM) and the emergence of functional human AM (Willinger et al., 2011, Proc Natl Acad Sci USA 108:2390-2395); and replacement of the Csf1 gene, which encodes M-CSF, led to an increase in the number of human monocytes in multiple tissues (Rathinam et al., 2011, Blood 118:3119-3128).

[0007] The human and murine hematopoietic-lymphoid systems differ in many respects (Haley, 2003, Toxicology 188:49-71; Mestas and Hughes, 2004, J Immunol 172:2731-2738). One of the major differences between the two species lies in their white blood cell (WBC) differentials. Human blood is rich in myeloid cells, which account for 50-75% of total WBC. In contrast, murine blood is dominated by lymphocytes, and only 20-30% of WBC are of the myeloid lineage. This species difference, the functional and evolutionary significance of which is not yet understood, is not recapitulated in conventional HHLS mice such as NOG / NSG or SRG. Indeed, human myelopoiesis is particularly defective in these hosts, where myeloid cells account for only 5-10% of human WBC.

[0008] One application of mice with a functional human immune system is the development and testing of human vaccines. Historically, the in vivo induction of immune responses has been relatively inefficient (2004, Traggiai et al., Science 304:104 - 107; 2002, Ito et al., Blood 100:3175 - 3182; 2005, Ishikawa et al., Blood 106:1565 - 1573; 2005, Shultz et al., J Immunol 174:6477 - 6489; 2006, Baenziger et al., Proc Natl Acad Sci USA 103:15951 - 15956). Several studies have reported successful pathogen - specific immune responses after infection. Although about 50% of the mice were reported to generate virus - specific IgM and IgG after dengue virus infection (2007, Kuruvilla et al., Virology 369:143 - 152), other studies have reported frequencies of antigen - specific IgM and IgG - producing mice after HIV and EBV infection of less than 20% (2006, Baenziger et al., Proc Natl Acad Sci USA 103:15951 - 15956; 2008, Yajima et al., J Infect Dis 198:673 - 682). After immunization with adjuvants and antigens, class - switching of antigen - specific immunoglobulins has also been historically inefficient, with only a fraction of immunized animals showing antigen - specific IgG responses (2004, Traggiai et al., Science 304:104 - 107; 2002, Ito et al., Blood 100:3175 - 3182; 2005, Ishikawa et al., Blood 106:1565 - 1573; 2005, Shultz et al., J Immunol 174:6477 - 6489; 2009, Watanabe et al., Int Immunol 21:843 - 858; 2010, Becker et al., PLoS ONE 5). These studies included NSG and BALB / c RAG2 - / - gamma c - / - mice and different adjuvant / antigen combinations.

[0009] There is a need in the art for humanized non - human animals that can support and maintain the engraftment of human hematopoietic cells. The present invention addresses this unmet need in the art. SUMMARY OF THE INVENTION

[0010] In general, the present invention relates to genetically modified non-human animals that express at least one of human M-CSF, human IL-3, human GM-CSF, human SIRPA, or human TPO, and methods of using the same. Thus, in one embodiment, the present invention is a genetically modified non-human animal that comprises a genome that comprises at least one nucleic acid encoding at least one of the group consisting of: human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO, wherein the at least one nucleic acid is operably linked to a promoter, and wherein the animal expresses at least one polypeptide selected from the group consisting of: human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO. In another embodiment, the present invention is a genetically modified non-human animal that comprises a genome that comprises a nucleic acid encoding human M-CSF, a nucleic acid encoding human IL-3, a nucleic acid encoding human GM-CSF, a nucleic acid encoding human SIRPA, and a nucleic acid encoding human TPO, wherein each of the nucleic acids encoding human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO is operably linked to a promoter, and wherein the animal expresses a human M-CSF polypeptide, a human IL-3 polypeptide, a human GM-CSF polypeptide, a human SIRPA polypeptide, and a human TPO polypeptide. In some embodiments, the genetically modified non-human animal is immunodeficient. In some embodiments, the genetically modified non-human animal does not express recombination activating gene 2 (Rag-2 - / - ). In some embodiments, the genetically modified non-human animal does not express the IL2 receptor gamma chain (gamma chain - / - ). In some embodiments, the genetically modified non-human animal does not express Rag-2, and the genetically modified non-human animal does not express the IL2 receptor gamma chain (Rag-2 - / - gamma chain - / - ). In some embodiments, the genetically modified non-human animal is a rodent. In some embodiments, the genetically modified non-human animal is a mouse. In one embodiment, the genetically modified non-human animal further comprises at least one human hematopoietic cell. In one embodiment, the genetically modified non-human animal further comprises at least one human cancer cell. In some embodiments, the human cancer cell is a leukemia cell or a melanoma cell.

[0011] In another embodiment, the invention is a method of engrafting hematopoietic stem and progenitor cells (HSPCs) in a genetically modified non-human animal that expresses at least one of the following: human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO, the method comprising the step of administering at least one HSPC to the genetically modified animal that expresses at least one of the following: human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO. In some embodiments, the HSPCs are human HSPCs. In one embodiment, the genetically modified non-human animal is a rodent. In one embodiment, the genetically modified non-human animal is a mouse. In one embodiment, the genetically modified non-human animal is immunodeficient. In one embodiment, the genetically modified immunodeficient non-human animal does not express recombination activating gene 2 (Rag-2 - / - ). In one embodiment, the genetically modified immunodeficient non-human animal does not express the endogenous IL2 receptor (γ chain - / - ). In one embodiment, the genetically modified immunodeficient non-human animal does not express endogenous Rag-2 and does not express the endogenous γ chain (Rag-2 - / - γ chain - / - ). In one embodiment, the genetically modified animal comprises human cancer cells. In one embodiment, the human cancer cells are leukemia cells or melanoma cells.

[0012] In another embodiment, the invention is a genetically modified Rag-2 - / - , γ chain - / -A mouse having a genome that includes at least one nucleic acid encoding at least one of the group consisting of human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO, wherein the at least one nucleic acid is operably linked to at least one promoter, and wherein the mouse expresses at least one polypeptide selected from the group consisting of human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO. In one embodiment, the genetically modified non-human animal comprises a genome having a nucleic acid encoding human M-CSF, a nucleic acid encoding human IL-3, a nucleic acid encoding human GM-CSF, a nucleic acid encoding human SIRPA, and a nucleic acid encoding human TPO, wherein each of the nucleic acids encoding human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO is operably linked to a promoter, and wherein the animal expresses a human M-CSF polypeptide, a human IL-3 polypeptide, a human GM-CSF polypeptide, a human SIRPA polypeptide, and a human TPO polypeptide. In one embodiment, the genetically modified non-human animal is a rodent. In one embodiment, the genetically modified non-human animal is a mouse. In one embodiment, the genetically modified non-human animal comprises human hematopoietic cells. In one embodiment, the genetically modified non-human animal comprises human cancer cells. In some embodiments, the human cancer cells are leukemia cells or melanoma cells.

[0013] This application relates to the following embodiments.

[0014] 1. A genetically modified non-human animal comprising a genome that includes at least one nucleic acid encoding at least one of the group consisting of human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO, wherein the at least one nucleic acid is operably linked to a promoter, and wherein the animal expresses at least one polypeptide selected from the group consisting of human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO.

[0015] 2. The genetically modified non-human animal of embodiment 1, comprising a genome that includes a nucleic acid encoding human M-CSF, a nucleic acid encoding human IL-3, a nucleic acid encoding human GM-CSF, a nucleic acid encoding human SIRPA, and a nucleic acid encoding human TPO, wherein each of the nucleic acids encoding human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO is operably linked to a promoter, and wherein the animal expresses a human M-CSF polypeptide, a human IL-3 polypeptide, a human GM-CSF polypeptide, a human SIRPA polypeptide, and a human TPO polypeptide.

[0016] 3. The genetically modified non-human animal of embodiment 1, wherein the animal is immunodeficient.

[0017] 4. Genetically modified immunodeficient non-human animals of embodiment 3, wherein the animal does not express recombination activating gene 2 (Rag-2 - / - ).

[0018] 5. Genetically modified immunodeficient non-human animals of embodiment 3, wherein the animal does not express the IL2 receptor gamma chain (gamma chain - / - ).

[0019] 6. Genetically modified immunodeficient non-human animals of embodiment 3, wherein the animal does not express Rag-2, and wherein the animal does not express the IL2 receptor gamma chain (Rag-2 - / - gamma chain - / - ).

[0020] 7. Genetically modified non-human animals of embodiment 1, wherein the animal is a rodent.

[0021] 8. Genetically modified non-human animals of embodiment 1, wherein the animal is a mouse.

[0022] 9. Genetically modified animals of embodiment 1, which further comprise human hematopoietic cells.

[0023] 10. Genetically modified animals of embodiment 1, which further comprise human cancer cells.

[0024] 11. Genetically modified animals of embodiment 10, wherein the human cancer cells are leukemia cells or melanoma cells.

[0025] 12. A method of transplanting hematopoietic stem and progenitor cells (HSPCs) into a genetically modified non-human animal, wherein the animal expresses at least one of the following: human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO, the method comprising the step of administering at least one HSPC to a genetically modified animal that expresses at least one of the following: human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO.

[0026] 13. The method of embodiment 12, wherein the genetically modified non-human animal is a rodent.

[0027] 14. The method of embodiment 12, wherein the genetically modified non-human animal is a mouse.

[0028] 15. The method of embodiment 12, wherein the genetically modified non-human animal is immunodeficient.

[0029] 16. The method of embodiment 15, wherein the genetically modified immunodeficient non-human animal does not express recombination activating gene 2 (Rag-2 - / - ).

[0030] 17. The method of embodiment 15, wherein the genetically modified immunodeficient non-human animal does not express endogenous IL2 receptor (γ chain - / - ).

[0031] 18. The method of embodiment 15, wherein the genetically modified immunodeficient non-human animal does not express endogenous Rag-2 and does not express endogenous γ chain (Rag-2 - / - γ chain - / - ).

[0032] 19. The method of embodiment 12, wherein the genetically modified animal comprises human cancer cells.

[0033] 20. The method of embodiment 19, wherein the human cancer cells are leukemia cells or melanoma cells.

[0034] 21. A genetically modified Rag-2 - / - , γ chain - / - mouse having a genome that comprises at least one nucleic acid encoding at least one of the group consisting of human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO, wherein the at least one nucleic acid is operably linked to at least one promoter, and wherein the mouse expresses at least one polypeptide selected from the group consisting of human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO.

[0035] 22. The genetically modified non-human animal of embodiment 21, having a genome that comprises a nucleic acid encoding human M-CSF, a nucleic acid encoding human IL-3, a nucleic acid encoding human GM-CSF, a nucleic acid encoding human SIRPA, and a nucleic acid encoding human TPO, wherein each of the nucleic acids encoding human M-CSF, human IL-3, human GM-CSF, human SIRPA, and human TPO is operably linked to a promoter, and wherein the animal expresses human M-CSF polypeptide, human IL-3 polypeptide, human GM-CSF polypeptide, human SIRPA polypeptide, and human TPO polypeptide.

[0036] 23. The genetically modified non-human animal of embodiment 21, wherein the animal is a rodent.

[0037] 24. The genetically modified non-human animal of embodiment 21, wherein the animal is a mouse.

[0038] 25. The genetically modified non-human animal of embodiment 21, which further comprises human hematopoietic cells.

[0039] 26. The genetically modified non-human animal of embodiment 21, which further comprises human cancer cells.

[0040] 27. The genetically modified non-human animal of embodiment 26, wherein the human cancer cells are leukemia cells or melanoma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustration of the invention, the presently preferred embodiments are shown in the figures. However, it is to be understood that the invention is not limited to the exact arrangements and instrumentalities shown in the figures.

[0042] Figure 1 (including Figure 1 Panel A - Figure 1 Panel E) depict experimental results that show that MISTRG mice support high levels of human hematopoietic engraftment. Human fetal liver (FL)-CD34 + cells were engrafted into newborn mice of the designated strains pre-conditioned with X-ray by intrahepatic injection of 100,000 cells. Human engraftment levels (hCD45 + cells) were measured in blood at 7 - 9 weeks and in BM at 10 - 12 weeks. ([[]] Figure 1 Panel A) Representative flow cytometry analysis of the frequencies of mouse and human CD45 + cells in blood and BM of the designated recipient mice. The numbers next to the gated regions indicate the percentages among total CD45 + cells. ([[]] Figure 1 Panel B) Pooled data of blood engraftment levels (% hCD45 + cells) from 19 independent experiments. In each experiment, a single FL-CD34 + cell sample was split and injected into mice of the corresponding strain. Each symbol represents an individual mouse, and the red bar indicates the mean value (n = 56 - 155; ns, not significant; *p < 0.05 Tukey's test (for full statistical analysis, see Figure 6 ). The horizontal grey line indicates 10% hCD45 + cells. ([[]] Figure 1 Panel C) Engraftment levels in BM of a representative subset of mice from group ([[]] Figure 6 Panel B) ([[]] Figure 6 Panel C) (n = 12 - 16; *p < 0.05 Tukey's test; see also Figure 6 Panel D - Figure 6 Panel E). ([[]] Figure 1 Panel D) Representative flow cytometry analysis of hCD45 + cell engraftment in blood and BM 3 months after intrahepatic injection of 200,000 FL-CD34 + cells into non-irradiated newborn MISTRG mice.Figure 1 Panel E) As Figure 1 Panel D) Human CD45 in the blood and BM of transplanted MISTRG mice + cell engraftment levels (n = 16). In this case, BM of all mice (including those with blood hCD45 + < 10% of the mice) is shown.

[0043] Figure 2 (including Figure 2 Panel A- Figure 2 Panel K) depicts the experimental results, which show that MISTRG mice support efficient myeloid development and maintenance in lymphoid and non-lymphoid tissues. Figure 2 Panel A) Human hematopoietic cells (hCD45 + cells) in the blood of designated recipient mice transplanted as neonates by intrahepatic injection of FL-CD34 + ) and human myeloid cells (hCD33 + ) percentage. Each symbol represents an individual mouse, and the red bar indicates the mean value (n = 20 - 113; Figure 7 statistical analysis shown in Panel A). Figure 2 Panel B) Human WBC composition in the same mice (n = 20 - 113 mice / group; n = 8 human donors; error bars indicate SEM). Figure 2 Panel C) Immunohistochemical staining of human myeloid cells (hCD68 + ) in non-myeloid tissues of designated recipient mice. The black bar represents 20 μm, and the images shown represent at least three mice analyzed per group. Figure 2 Panel D and Figure 2 Panel E) Representative flow cytometry analysis of human monocyte subsets Figure 2 Panel D) and frequency Figure 2 Panel E), which were identified by CD14 and CD16 expression among hCD45 + CD33 + cells in the blood of recipient mice (n = 8 - 12 mice / group; error bars indicate SEM). Figure 2 Panel F and Figure 2 Panel G) Cytokine production by human monocytes isolated from the BM of MITRG recipients and stimulated in vitro with LPS Figure 2 Panel F) or R848 Figure 2 Panel G) (error bars indicate SD in triplicate; representative of 3 independent experiments). Figure 2 Panel H) In vitro phagocytosis of GFP-expressing Escherichia coli by human cells present in the blood of MITRG mice (n = 7). Figure 2 Panel I,Figure 2 Panel J, Figure 2 Panel K) LPS( Figure 2 Panel I; 90 minutes, n = 15 - 18), or with Listeria monocytogenes( Figure 2 Panel J; day 2, n = 6 - 15) or influenza A / PR8 H1N1( Figure 2 Panel K; day 3, n = 3 - 5) - infected mice, as measured by ELISA in serum or by RT - PCR in the lung.( Figure 2 Panel A, Figure 2 Panel J, Figure 2 Panel K) p - values (*p < 0.05) calculated by one - way ANOVA followed by Tukey's post - hoc test;( Figure 2 Panel I) p - values calculated by unpaired Student's t - test on log10 - transformed values.

[0044] Figure 3 (including Figure 3 Panel A - Figure 3 Panel I) depict experimental results that show that MISTRG mice effectively support the development and function of human NK cells.( Figure 3 Panel A) Quantitative RT - PCR analysis of human IL - 15 and IL - 15Rα mRNA expression in the liver transplanted into NSG, MITRG, and MISTRG mice (n = 7 - 8; p - values calculated by one - way ANOVA; *, p < 0.05 Tukey's post - hoc test). Expression was normalized relative to mouse Hprt.( Figure 3 Panel B) Quantitative RT - PCR analysis of human IL - 15 and IL - 15Rα mRNA expression in human cell populations purified from the bone marrow transplanted into MITRG (n = 4 - 5, error bars denote SEM). Expression was normalized relative to human HPRT and relative to hCD14 + hCD16 - cells are shown.( Figure 3 Panels C and Figure 3 Panel D) Representative flow cytometry analysis of human NK cells (hNKp46 + hCD3 - ) transplanted into NSG, MITRG, and MISTRG (gated on hCD45 + mCD45 - cells, lymphocyte gate; numbers near the outlined areas denote the percentage of cells)( Figure 3 Panel C) and absolute numbers or frequencies( Figure 3Panel D) (n = 8 - 16; p-value calculated by one-way ANOVA; *, p < 0.05 Tukey's post hoc test).( Figure 3 Panel E) Absolute numbers of human liver NK (hNKp46 + hCD3 - ) and T cells (hCD3 + , shown as control) in MISTRG mice that were left untreated or treated with liposome-encapsulated clodronate for 3 consecutive days to deplete macrophage infiltration (n = 8; p-value calculated by unpaired Student t-test; ns, not significant).( Figure 3 Panel F) Specific NK cell cytotoxicity was calculated as the proportion of HLA class I-positive or -negative labeled cells recovered in the spleen 12 h after i.v. injection of labeled LCL721.221 (HLA class I-negative) and LCL721.45 (class I-positive) cells at a 1:1 ratio (n = 8, p-value calculated by unpaired Student t-test).( Figure 3 Panel G) Quantitative RT-PCR analysis of human IFNγ mRNA expression in the livers of NSG and MISTRG mice 2 days after Listeria infection (n = 8 - 9, p-value calculated by unpaired Student t-test). Expression was normalized to murine Hprt.( Figure 3 Panel H and Figure 3 Panel I) Representative flow cytometry analysis of IFNγ expression and degranulation (CD107a + ) of human liver NK cells from uninfected or Listeria-infected NSG and MISTRG mice( Figure 3 Panel H) and frequencies( Figure 3 Panel I) (n = 4 - 11; p-value calculated by one-way ANOVA). Results are from a combination of 2( Figure 3 Panel A, Figure 3 Panel E - Figure 3 Panel I), 3( Figure 3 Panel B), or 4( Figure 3 Panel C, Figure 3 Panel D) experiments.

[0045] Figure 4 (Including Figure 4 Panel A - Figure 4 Panel F) depict the experimental results, which show human myeloid cell infiltration of tumors in MISTRG and support their growth. Human melanoma cell line Me290 was implanted subcutaneously in the flanks of transplanted or non-transplanted NSG and MISTRG mice. Some mice were treated with the VEGF inhibitor Avastin TM . Tumors were measured and dissected for analysis after 11 days.( Figure 4Panel A) Infiltration of human hematopoietic cells in tumors, determined by expression of mRNAs encoding human hematopoietic (PTPRC, encoding CD45) and myeloid (ITGAM, encoding CD11b) markers (n = 6 - 7; p-values calculated by unpaired Student t-test).( Figure 4 Panel B and Figure 4 Panel D) Representative immunohistochemistry images of human myeloid cell markers in tumors from NSG, MISTRG, and patients.( Figure 4 Panel C) CD163 + quantification of cell density (n = 3 samples / group, 3 counted slides / sample).( Figure 4 Panel E and Figure 4 Panel F) Representative images of tumors in indicated mouse groups( Figure 4 Panel E) and volume( Figure 4 Panel F) (n = 7 - 24 mice / group). By Student t-test( Figure 4 Panel A) or by one-way ANOVA( Figure 4 Panel C, Figure 4 Panel E), followed by calculation of p-values by Tukey's post hoc test (*p < 0.05).

[0046] Figure 5 Cytokines involved in myelopoiesis and HSC function are depicted. Schematic of hematopoietic stem cell development into myeloid cells and a non-exhaustive list of cytokines known to regulate this process. Shading indicates the percentage of amino acid identity between human and mouse cytokines. The percentage of amino acid identity is the most objective measure of protein conservation between species, but it is not always associated with functional in vivo cross-reactivity between species. Black rectangles indicate genetically humanized cytokines in MISTRG. HSC, hematopoietic stem cell; MPP, multipotent progenitor; CMP, common myeloid progenitor; GMP, granulocyte / macrophage progenitor; MEP, megakaryocyte / erythroid progenitor.

[0047] Figure 6 (including Figure 6 Panel A - Figure 6 Panel E) depict the results of statistical analysis of engraftment levels in recipient mice.( Figure 6 Panel A) Figure 1 Statistical analysis of the data presented in Panel A (percentage of hCD45 + cells in the blood of recipient mice) (one-way ANOVA, followed by Tukey's post hoc test; ns, not significant).( Figure 6 Panel B) Number of recipient mice that reached an engraftment level of at least 10% hCD45 + cells in the blood 7 - 9 weeks after transplantation.( Figure 6Panel C) Figure 1 The level of blood engraftment of the mice used for BM analysis in Panel C.( Figure 6 Panel D) Figure 1 Statistical analysis of the data presented in Panel C (percentage of hCD45 + cells in the BM of recipient mice), similar to( Figure 6 Panel A).( Figure 6 Panel E) Figure 1 The absolute number of hCD45 + cells in the BM (2 femurs and 2 tibias) of recipient mice shown in Panel C. The reduced number of cells in the BM of MISTRG is due to the smaller size of the mice at this age (10 - 12 weeks post - transplantation) and is caused by Figure 10 the first clinical signs of anemia described in

[0048] Figure 7 (including Figure 7 Panel A - Figure 7 Panel H) depict the experimental results evaluating enhanced human myelopoiesis in MISTRG mice.( Figure 7 Panel A) Figure 2 Statistical analysis of the data presented in Panel A (percentage of hCD33 + cells in the blood of recipient mice) (one - way ANOVA, followed by Tukey post - hoc test; ns, not significant).( Figure 7 Panel B and Figure 7 Panel C) The frequency of human myeloid cells (hCD33 + ) in the BM of recipient mice( Figure 7 Panel B) and statistical analysis( Figure 7 Panel C).( Figure 7 Panel D) Representative flow cytometry analysis of human lymphoid and myeloid lineages in the blood of MISTRG.( Figure 7 Panel E and Figure 7 Panel F) Representative flow cytometry analysis of human monocytes (CD33 Figure 7 Panel E) and blood( Figure 7 Panel F) of MISTRG and human donors, for 高 SSC 低 CD66 - ) and granulocytes (CD33 + SSC 高 CD66 + ).( Figure 7 Panel G and Figure 7 Panel H) Human myeloid cells (hCD33 Figure 7 Panel G) and liver( Figure 7 Panel H) of recipient mice, for+ ) absolute numbers (n = 8 - 12; p-values calculated by one-way ANOVA followed by Tukey's post hoc test, *p < 0.05).

[0049] Figure 8 (including Figure 8 Panel A and Figure 8 Panel B) depict the experimental results, which show enhanced human monocyte subset occurrence in MISTRG mice. ( Figure 8 Panel A) Representative flow cytometry analysis of human monocyte subsets, which was identified by the expression of hCD45 + CD33 + intercellular CD14 and CD16 in the BM, spleen, lung, and liver of the designated recipient mice. ( Figure 8 Panel B) Frequencies of hCD33 + intercellular in the lung and liver of recipient mice (error bars represent SEM) and absolute numbers of monocyte subsets (n = 12 mice / group; p-values calculated by one-way ANOVA; *, p < 0.05 Tukey's post hoc test).

[0050] Figure 9 (including Figure 9 Panel A and Figure 9 Panel B) depict the experimental results, which show that human monocyte subsets are similar in MISTRG and human donors. Blood of MISTRG recipients and human donors ( Figure 9 Panel A) and BM ( Figure 9 Panel B) extended immunophenotypes of the designated human monocyte subsets. Staining with isotype control antibodies and specific antibodies is shown.

[0051] Figure 10 (including Figure 10 Panel A - Figure 10 Panel I) depict the experimental results, which show that human myeloid cells disrupt human tolerance to murine phagocytosis. ( Figure 10 Panel A) CFSE-labeled murine RBCs were transferred into the designated mice, and the frequency of labeled cells was measured at the designated time points. ( Figure 10 Panel B) MISTRG was pre-treated with clodronate to deplete phagocytes or otherwise, and CFSE-labeled murine RBCs were transferred and monitored as in ( Figure 10 Panel A) (p-values, clodronate effect measured by repeated measures ANOVA on days 1 - 3). These results show that transferred murine RBCs were rapidly cleared in vivo by phagocytes present in MISTRG but not in NSG. ( Figure 10 Panel C) Non-transferred mice (n = 9 - 15) or human FL-CD34 +RBC counts in blood 8 - 10 weeks after cell transfer (n = 11 - 37). The p - value indicates comparison between non - transferred and transferred mice for each genotype (Student's unpaired t - test).( Figure 10 Panel D) Association between human engraftment level (percentage of hCD45 + cells in blood) and RBC counts (n = 13 - 22).( Figure 10 Panel E) Flow cytometry analysis of murine (mTer119 + ) and human (hCD235a + ) erythroid cells in blood of non - transferred or transferred MISTRG, which shows that almost all erythroid cells in blood of transferred MISTRG are of murine origin and human erythroid cells are barely detectable.( Figure 10 Panel F) Representative photographs and spleen weights of transferred mice of designated strains (n = 3 - 22), which shows splenomegaly in transferred MISTRG mice. Spleen from Balb / c mice was used as control (p - value, one - way ANOVA; *, p < 0.05, compared to all other groups, Tukey's post - hoc test).( Figure 10 Panel G) Histological sections of spleens of transferred NSG and MISTRG stained with H&E, which shows expansion of red pulp in MISTRG mice with splenomegaly.( Figure 10 Panel H) Flow cytometry analysis of murine erythroid progenitors (mTer119 + mCD71 + ), which account for up to 80% of cells in spleen of transferred MISTRG.( Figure 10 Panel I) Blood smears of non - transferred and transferred MISTRG show enrichment of reticulocytes. Collectively, these results strongly suggest that anemia in MISTRG results from lack of human tolerance to murine phagocytosis and that extensive extramedullary murine erythropoiesis cannot compensate for destruction of mRBC. Results represent each group( Figure 10 Panels C, 10E - 10I) and 2 independent experiments( Figure 10 Panels A, Figure 10 Panels B) with at least 5 mice examined.

[0052] Figure 11 (including Figure 11 Panels A and Figure 11 Panels B) depict experimental results, which show that MISTRG mice provide human IL - 15 / IL - 15Rα.( Figure 11Quantitative RT-PCR analysis of human IL-15 and IL-15Rα mRNA expression in the lungs of engrafted NSG, MITRG, and MISTRG mice (n = 7 - 8; p-value calculated by one-way ANOVA; *, p < 0.05 Tukey post hoc test). Expression was normalized to murine Hprt.( Figure 11 Panel B) Flow cytometry analysis of IL-15Rα expression on human cell populations (hCD45 + mCD45 - ) in blood from engrafted MISTRG mice (representative of n = 4). Histograms represent staining with isotype control or with an IL-15Rα antibody. Results are representative of two experiments or are combined from two experiments.

[0053] Figure 12 (including Figure 12 Panel A and Figure 12 Panel B) depict the experimental results, which show enhanced human NK cell development in MISTRG mice.( Figure 12 Panel A and Figure 12 Panel B) Frequency ( + hNKp46 - ) and absolute numbers ( Figure 12 Panel A) and ( Figure 12 Panel B) of human NK cells (hNKp46

[0054] Figure 13 (including Figure 13 Panel A - Figure 13 Panel F) depict the experimental results, which show the presence of truly mature human NK cells with enhanced development in MISTRG mice.( Figure 13 Panel A) Flow cytometry analysis of CD94 and CD161 expression on human blood NK cells from human donors and from engrafted MISTRG (n = 3). Histograms represent staining with isotype control antibody or with CD94 / CD161 antibody.( Figure 13 Panel B) Flow cytometry analysis of KIR expression on human blood NK cells from human donors or from engrafted MISTRG mice (n = 3). Numbers indicate the frequency of KIR + cells.( Figure 13 Panel C and Figure 13Panel D) Surface expression of CD16 on human NK cells from engrafted NSG, MITRG, and MISTRG mice (n = 4 - 8; p - value calculated by one - way ANOVA; *, p < 0.05 Tukey's post - hoc test).( Figure 13 Panel E and Figure 13 Panel F) Intracellular perforin expression of human hepatic NK (hNKp46 + hCD3 - ) and T cells (hCD3 + ) (n = 3; p - value calculated by unpaired Student's t - test). MFI, mean fluorescence intensity. Results represent 1 experiment( Figure 13 Panel A and Figure 13 Panel B), 2 experiments( Figure 13 Panel E and Figure 13 Panel F), or 4 experiments( Figure 13 Panel C and Figure 13 Panel D) or are from a combination of 1 experiment( Figure 13 Panel A and Figure 13 Panel B), 2 experiments( Figure 13 Panel E and Figure 13 Panel F), or 4 experiments( Figure 13 Panel C and Figure 13 Panel D).

[0055] Figure 14 Depicts experimental results that show the effect of human monocyte / macrophage depletion on human NK cell homeostasis in MISTRG mice. Engrafted MISTRG mice were kept untreated or treated with liposome - encapsulated clodronate for 3 consecutive days to deplete phagocytes. Flow cytometry analysis of human monocyte / macrophages (upper panel, gated on hCD33 + cells) and NK cells (hNKp46 + hCD3 - ) in the liver (n = 8) is shown. Results represent two experiments. In 1 of 8 mice, clodronate depletion of monocyte / macrophages was not effective, and no decrease in NK cell numbers was observed in that mouse.

[0056] Figure 15 Depicts experimental results that show the immunohistochemistry of human myeloid cells infiltrating melanoma. Representative immunohistochemical staining of human myeloid cells in tumors from NSG, MISTRG, or human patients is shown. Three subjects per group and 3 photographs per subject are shown.

[0057] Figure 16Shows a comparison of engraftment levels and immune cell development and function in recipient mice with single gene replacements, NSG, MISTRG, and humans.

[0058] Figure 17 (Including Figure 17 Panel A- Figure 17 Panel D) depicts the experimental results, which show that samples isolated from patients with AML, CMML, and MDS can be engrafted in MISTRG. ( Figure 17 Panel A) Characteristics of the samples used (including the type of disease and genetic abnormalities found in the patient samples), experimental protocol (cell purification method, number of cells injected per mouse, and time post-transplantation for analyzing the mice), and engraftment results (including the number of mice with detectable human engraftment, percentages of human hematopoietic CD45 + cells and myeloid CD33 + cells, and genomic abnormalities observed in human cells isolated from the mice). ( Figure 17 Panel B) Representative flow cytometry analysis of the granularity (SSC) of myeloid CD33 + cells isolated from mice transplanted with RAEB I patient or normal donor cells, which shows defective granularity in the RAEB I samples. ( Figure 17 Panel C) Representative Fish analysis of human cells isolated from mice transplanted with RAEBII samples, and shows deletion of chromosome 5q. ( Figure 17 Panel D) Karyotype of human cells isolated from mice transplanted with CMML samples, and shows deletion in chromosome 6. DETAILED DESCRIPTION OF THE INVENTION

[0059] Generally, the present invention relates to genetically modified non-human animals that express at least one of human M-CSF, human IL-3, human GM-CSF, human SIRPA, or human TPO. The present invention also relates to methods of generating the genetically modified non-human animals described herein and methods of using the genetically modified non-human animals described herein. In some embodiments, the genetically modified non-human animal is a mouse. In some embodiments, human hematopoietic cells are engrafted into the genetically modified non-human animals described herein. In various embodiments, the genetically modified non-human animals of the present invention engrafted with human hematopoietic cells can be used for in vivo evaluation of the growth and differentiation of hematopoietic and immune cells, for in vivo evaluation of human hematopoiesis, for in vivo evaluation of cancer cells, for in vivo evaluation of immune responses, for in vivo evaluation of vaccines and vaccination regimens, for testing the effects of agents that regulate cancer cell growth or survival, for in vivo evaluation of cancer treatment, for in vivo generation and collection of immune mediators (including human antibodies), and for testing the effects of agents that regulate the function of hematopoietic and immune cells.

[0060] Definition

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary definitions and uses of such terms are found in various standard references, including J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd Edition, 2001; F. M. Ausubel (ed.), Short Protocols in Molecular Biology, Current Protocols, 5th Edition, 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Edition, Garland, 2002; D. L. Nelson and M. M. Cox, Lehninger Principles of Biochemistry, 4th Edition, W. H. Freeman & Company, 2004; and Herdewijn, P. (ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0062] As used herein, each of the following terms has the meaning associated with it in this section.

[0063] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an element" means one element or more than one element.

[0064] As used herein, "about" when referring to a measurable value such as an amount, a duration of time, etc. is intended to cover variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% of the specified value, because such variations are appropriate for practicing the disclosed methods.

[0065] As used in the context of organisms, tissues, cells or their components, the term "abnormal" refers to those organisms, tissues, cells or their components that differ from those organisms, tissues, cells or their components that exhibit "normal" (expected) corresponding characteristics in at least one observable or detectable characteristic (such as age, treatment, time of day, etc.). Characteristics that are normal or expected for one cell or tissue type may be abnormal for a different cell or tissue type.

[0066] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a particular epitope on an antigen. An antibody can be a complete immunoglobulin derived from a natural source or a recombinant source, and can be an immunoreactive portion of a complete immunoglobulin. The antibodies in the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab and F(ab)2, as well as single-chain antibodies (scFv), heavy-chain antibodies (such as camel 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).

[0067] As used herein, the term "cancer" is defined as a disease characterized by the uncontrolled proliferation and / or growth of abnormal cells. Cancer cells can spread locally or via the bloodstream and lymphatic system to other parts of the body. Cancers herein include both solid tumors and hematopoietic malignancies. Examples of various cancers suitable for the present invention include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, bone cancer, brain cancer, lymphoma, leukemia, lung cancer, myeloid dysplastic syndrome, myeloproliferative disorders, and the like.

[0068] "Constitutive" expression is the state in which a gene product is produced in a living cell under most or all physiological conditions of the cell.

[0069] The "coding region" of a gene consists of nucleotide residues of the coding strand of the gene and nucleotides of the non-coding strand of the gene which are homologous or complementary to the coding regions of an mRNA molecule generated by transcription of the gene, respectively.

[0070] The "coding region" of an mRNA molecule also consists of nucleotide residues in the mRNA molecule that match the anticodon regions of transfer RNA molecules during translation of the mRNA molecule or code for a stop codon. Thus, the coding region can contain nucleotide residues of codons that constitute amino acid residues (such as amino acid residues in a protein export signal sequence) that are not present in the mature protein encoded by the mRNA molecule.

[0071] "Disease" refers to a state of health of an animal in which the animal is unable to maintain homeostasis and in which, if the disease is not ameliorated, the health of the animal deteriorates continuously.

[0072] In contrast, a "disorder" in an animal refers to a state of health in which the animal is able to maintain homeostasis, but the state of health of the animal is not as favorable as its state of health without the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the state of health of the animal.

[0073] A disease or disorder is "attenuated" if the severity of the symptoms of the disease or disorder, the frequency with which a patient experiences such symptoms, or both, are reduced.

[0074] An "effective amount" or "therapeutically effective amount" of a compound refers to the amount of the compound sufficient to provide a beneficial effect to a subject to which the compound is administered. An "effective amount" of a delivery vehicle refers to the amount sufficient to effectively bind or deliver a compound.

[0075] "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template in a biological process for the synthesis of other polymers and macromolecules having a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and biological properties derived therefrom. Thus, if transcription and translation of an mRNA corresponding to a gene results in the production of a protein in a cell or other biological system, then the gene encodes the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and which is typically provided in a sequence listing) and the non-coding strand (which serves as a template for transcribing the gene or cDNA) can be said to encode the protein or other product of the gene or cDNA.

[0076] As used herein, "endogenous" refers to any material that is from within or generated within an organism, cell, tissue, or system.

[0077] As used herein, the term "exogenous" refers to any material that is introduced from outside of an organism, cell, tissue or system or generated outside of an organism, cell, tissue or system.

[0078] The terms "expression construct" and "expression cassette" are used herein to refer to a double-stranded recombinant DNA molecule that contains a desired nucleic acid encoding sequence and contains one or more regulatory elements that are necessary or desirable for the expression of the coding sequence operably linked thereto.

[0079] As used herein, the term "fragment" when applied to a nucleic acid or polypeptide refers to a subsequence of a larger nucleic acid or polypeptide. A "fragment" of a nucleic acid can be at least about 15 nucleotides in length; for example, at least about 50 nucleotides to about 100 nucleotides; at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides, at least about 1000 nucleotides to about 1500 nucleotides; or about 1500 nucleotides to about 2500 nucleotides; or about 2500 nucleotides (and any integer value in between). The length of a "fragment" of a polypeptide can be at least about 15 nucleotides; for example at least about 50 amino acids to about 100 amino acids; at least about 100 to about 500 amino acids, at least about 500 to about 1000 amino acids, at least about 1000 amino acids to about 1500 amino acids; or about 1500 amino acids to about 2500 amino acids; or about 2500 amino acids (and any integer value in between).

[0080] As used herein, the terms "gene" and "recombinant gene" refer to a nucleic acid molecule that contains an open reading frame encoding a polypeptide. Such natural allelic variations typically can result in a 1-5% variation in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily accomplished by using hybridization probes to identify the same genetic locus in a number of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and do not alter functional activity are intended to be within the scope of the present invention.

[0081] As used herein, "homologous" refers to subunit sequence similarity between two polymeric molecules, such as between two nucleic acid molecules (e.g., two DNA molecules or two RNA molecules) or between two polypeptide molecules. When the subunit positions in both of two molecules are occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a positive function of the number of matching or homologous positions. For example, if half of the positions (e.g., 5 positions in a polymer of 10 subunits in length) in two compound sequences are homologous, then the two sequences are 50% homologous, and if 90% of the positions (e.g., 9 out of 10) are matching or homologous, then the two sequences share 90% homology. By way of example, the DNA sequences 5'-ATTGCC-3' and 5'-TATGGC-3' share 50% homology.

[0082] As used herein, the terms "human hematopoietic stem and progenitor cells" and "human HSPC" refer to human self-renewing multipotent hematopoietic stem cells and hematopoietic progenitor cells.

[0083] "Inducible" expression is a state in which a gene product is generated in a living cell in response to the presence of a signal in the cell.

[0084] As used herein, "instructional material" includes publications, records, graphs, or any other medium of expression that can be used to convey the utility of the compounds, compositions, vectors, or delivery systems of the invention in a kit for achieving alleviation of the various diseases or disorders described herein. Optionally or alternatively, the instructional material can describe one or more methods for alleviating a disease or disorder in mammalian cells or tissues. For example, the instructional material for a kit of the invention can be attached to or shipped with a container containing the identified compound, composition, vector, or delivery system of the invention. Alternatively, the instructional material can be shipped separately from the container so that the recipient can use the instructional material and the compound in concert.

[0085] As used herein, the term "operably linked" refers to a polynucleotide in a functional relationship with a second polynucleotide. Describing two polynucleotides as "operably linked" means that a single-stranded or double-stranded nucleic acid module contains the two polynucleotides arranged within the nucleic acid module in such a way that at least one of the two polynucleotides can exert its characterized physiological effect on the other. For example, a promoter operably linked to the coding region of a gene can facilitate transcription of the coding region. Preferably, when a nucleic acid encoding a desired protein further contains a promoter / regulatory sequence, the promoter / regulatory sequence is located at the 5' end of the desired protein coding sequence so that it drives expression of the desired protein in a cell. The nucleic acid encoding the desired protein and its promoter / regulatory sequence together constitute a "transgene".

[0086] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Additionally, a nucleic acid is a polymer of nucleotides. Thus, as used herein, the terms nucleic acid and polynucleotide may be used interchangeably. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, 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 nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., and by synthetic means.

[0087] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limit is placed on the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (e.g., which are commonly referred to as peptides, oligopeptides, and oligomers in the art) and longer chains (which are generally referred to as proteins in the art, of which there are many types). For example, "polypeptide" includes bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof. The term "peptide" generally refers to short polypeptides. The term "protein" generally refers to large polypeptides.

[0088] As used herein, the term "progeny" refers to descendants and includes differentiated or undifferentiated progeny cells derived from a parental cell. In one usage, the term progeny refers to progeny cells that are genetically identical to the parent. In another usage, the term progeny refers to progeny cells that are genetically and phenotypically identical to the parent. In yet another usage, the term progeny refers to progeny cells that have differentiated from the parental cell.

[0089] As used herein, the term "promoter" refers to a DNA sequence that is operably linked to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding a desired molecule. A promoter is generally located upstream of the nucleic acid sequence to be transcribed and provides a site for specific binding of RNA polymerase and other transcription factors. In certain embodiments, the promoter is generally located upstream of the transcribed nucleic acid sequence to generate the desired molecule and provides a site for specific binding of RNA polymerase and other transcription factors. Included promoters can be constitutive promoters or can provide inducible expression; and can provide ubiquitous, tissue-specific, or cell type-specific expression.

[0090] Ranges: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible sub-ranges as well as individual numerical values within the range. For example, the description of a range such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0091] A "recombinant polypeptide" is a polypeptide produced after expression of a recombinant polynucleotide.

[0092] As used herein, the term "regulatory element" refers to a nucleotide sequence that controls some aspect of the expression of a nucleic acid sequence. Exemplary regulatory elements illustratively include enhancers, internal ribosome entry sites (IRESs), introns; replication origins, polyadenylation signals (pA), promoters, enhancers, transcription termination sequences, and upstream regulatory domains, which contribute to the replication, transcription, and post-transcriptional processing of the nucleic acid sequence. Those of ordinary skill in the art can select and use these and other regulatory elements in an expression construct using only routine experimentation. Expression constructs can be generated recombinantly or synthetically using known methods.

[0093] As used herein, the term "specifically binds" with respect to an antibody means an antibody that recognizes a specific antigen but substantially does not recognize or bind other molecules in a sample. For example, an antibody that specifically binds an antigen from one species may also bind the antigen from one or more species. However, such cross-species reactivity by itself does not change the classification of the antibody as specific. In another example, an antibody that specifically binds an antigen may also bind different allelic forms of the antigen. However, such cross-reactivity by itself does not change the classification of the antibody as specific.

[0094] In some instances, the term "specifically binds" may be used with reference to the interaction of an antibody, protein, or peptide with a second chemical species and refers to an interaction that is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than a general protein. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody.

[0095] As used herein, the term "synthetic antibody" means an antibody generated using recombinant DNA technology, such as, for example, an antibody expressed by phage, as described herein. The term should also be construed to mean an antibody that has been generated by synthesizing a DNA molecule encoding the antibody, which DNA molecule expresses the antibody protein, or specifying the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well-known in the art.

[0096] "Variant," as the term is used herein, refers to a nucleic acid sequence or a peptide sequence that differs from a reference nucleic acid sequence or peptide sequence, respectively, in sequence, but retains the necessary biological properties of the reference molecule. The sequence changes of a nucleic acid variant may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. The sequence changes of a peptide variant are generally limited or conservative, such that the sequences of the reference peptide and the variant are generally extremely similar and identical in many regions. The variant and the reference peptide may differ at any combination of one or more substitutions, additions, and deletions in the amino acid sequence. Nucleic acid or peptide variants may be naturally occurring, such as allelic variants, or may be variants that are not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides can be generated by mutagenesis techniques or by direct synthesis.

[0097] As used herein, the term "genetically modified" means an animal whose germ cells contain exogenous human nucleic acid or a human nucleic acid sequence. As a non-limiting example, a genetically modified animal can be a transgenic animal or a knock-in animal, provided that the animal contains a human nucleic acid sequence.

[0098] As used herein, "knock-in" means a genetic modification in which the genetic information encoded at a chromosomal locus in a non-human animal is replaced with a different DNA sequence.

[0099] Description

[0100] The present invention relates to genetically modified non-human animals that express human M -CSF, human I L-3 / GM-CSF, human S IRPA and human T PO (referred to herein as MIST). The present invention also relates to methods of generating the genetically modified non-human animals described herein and methods of using the genetically modified non-human animals described herein. In some embodiments, the genetically modified non-human animal is a mouse. In some embodiments, the genetically modified non-human animal is an immunodeficient mouse. In a specific embodiment, the immunodeficient mouse is a RAG2 - / - γ c - / -Mouse. In another specific embodiment, the genetically modified non-human animal of the present invention expresses human M-CSF, human IL-3 / GM-CSF, and human TPO, and does not express RAG2 or γ c (referred to herein as MITRG). In another specific embodiment, the genetically modified non-human animal of the present invention expresses human M-CSF, human IL-3 / GM-CSF, human SIRPA, and human TPO, and does not express RAG2 or γ c (referred to herein as MISTRG). In some embodiments, human hematopoietic cells are transplanted into the genetically modified non-human animals described herein.

[0101] In various embodiments, the genetically modified non-human animals of the present invention transplanted with human hematopoietic cells can be used for in vivo evaluation of the growth and differentiation of hematopoietic and immune cells, for in vivo evaluation of human hematopoiesis, for in vivo evaluation of cancer cells, for in vivo evaluation of immune responses, for in vivo evaluation of vaccines and vaccination regimens, for testing the effects of agents that regulate cancer cell growth or survival, for in vivo evaluation of cancer treatment, for in vivo generation and collection of immune mediators (including human antibodies), and for testing the effects of agents that regulate hematopoietic and immune cell functions.

[0102] Genetically modified non-human animals

[0103] The present invention includes genetically modified non-human animals that express at least one of the following: human M-CSF, human IL-3 / GM-CSF, human SIRPA, human TPO, and any combination thereof. In some embodiments, the genetically modified non-human animals that express human nucleic acids also express the corresponding non-human animal nucleic acids. In other embodiments, the genetically modified non-human animals that express human nucleic acids do not express the corresponding non-human animal nucleic acids. In some embodiments, the genetically modified animals are animals in which one or more genes have been knocked out (to render the animals immunodeficient), as described elsewhere herein. To create the genetically modified non-human animals, nucleic acids encoding human proteins can be incorporated into recombinant expression vectors in a form suitable for expressing the human proteins in non-human host cells. In various embodiments, the recombinant expression vector contains one or more regulatory sequences operably linked to the nucleic acid encoding the human protein in such a manner that permits transcription of the nucleic acid into mRNA and translation of the mRNA into the human protein. The term "regulatory sequence" is well recognized in the art and is intended to include promoters, enhancers, and other expression control elements (such as polyadenylation signals). Such regulatory sequences are known to those of skill in the art and are described in 1990, Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. It should be understood that the design of the expression vector can depend on factors such as the choice of host cell to be transfected and / or the amount of human protein to be expressed.

[0104] For example, a genetically modified animal can be created by introducing a nucleic acid encoding a human protein (usually linked to a suitable regulatory element, such as a constitutive or tissue-specific enhancer) into an oocyte (e.g., by microinjection), and allowing the oocyte to develop in a female gestating animal. Intron sequences and polyadenylation signals can also be included in the transgene to enhance the expression efficiency of the transgene. Methods for generating genetically modified animals (especially animals such as mice) have become routine in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009 and 1986, Hogan et al., A Laboratory Manual, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory. Genetically modified founder animals can be used to breed other animals carrying the transgene. Genetically modified animals carrying a transgene encoding a human protein of the present invention can be further bred with other genetically modified animals carrying other transgenes, or with knockout animals (e.g., knockout animals that do not express one or more of their genes). In various embodiments, the genetically modified animals of the present invention are mice, rats, or rabbits.

[0105] In some embodiments, the genetically modified animals of the present invention express one or more human nucleic acids from a native promoter and native regulatory elements of a non-human animal. In other embodiments, the genetically modified animals of the present invention express human nucleic acids from a native human promoter and native regulatory elements. Those skilled in the art will understand that the genetically modified animals of the present invention include genetically modified animals that express at least one human nucleic acid from any promoter. Examples of promoters useful in the present invention include, but are not limited to, DNA pol II promoter, PGK promoter, ubiquitin promoter, albumin promoter, globulin promoter, ovalbumin promoter, SV40 early promoter, Rous sarcoma virus (RSV) promoter, retroviral LTR, and lentiviral LTR. Promoter and enhancer expression systems useful in the present invention also include inducible and / or tissue-specific expression systems.

[0106] In some embodiments, the present invention includes a genetically modified immunodeficient animal having a genome comprising a nucleic acid encoding a human polypeptide operably linked to a promoter, wherein the animal expresses the encoded human polypeptide. In various embodiments, the present invention includes a genetically modified immunodeficient non-human animal having a genome comprising an expression cassette, the expression cassette comprising a nucleic acid encoding at least one human polypeptide, wherein the nucleic acid is operably linked to a promoter and a polyadenylation signal, and further contains an intron, and wherein the animal expresses the encoded human polypeptide.

[0107] In multiple embodiments, various methods are used to introduce human nucleic acid sequences into immunodeficient animals to generate genetically modified immunodeficient animals that express human genes. Such techniques are well known in the art and include, but are not limited to, pronuclear microinjection, transformation of embryonic stem cells, homologous recombination, and knock-in techniques. Methods for generating genetically modified animals that can be used include, but are not limited to, 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 nd ed. Sunderland, Massachusetts: Sinauer), 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).

[0108] In some embodiments, the compositions and methods of the invention comprise genetically modified immunodeficient animals that alone or in combination with NK cell number and / or function deficiencies (e.g., due to IL2 receptor gamma chain deficiency (i.e., gamma c - / -) caused) are defective in B cell and / or T cell number and / or function and have a genome comprising a human nucleic acid operably linked to a promoter, wherein the animal expresses the encoded human polypeptide. Generation of the genetically modified animals of the invention can be achieved by methods such as DNA injection of an expression construct into a preimplantation embryo or by using stem cells such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells.

[0109] In one embodiment, the human nucleic acid is expressed by the native regulatory elements of a human gene. In other embodiments, the human nucleic acid is expressed by the native regulatory elements of a non-human animal. In other embodiments, the human nucleic acid is expressed from a ubiquitous promoter. Non-limiting examples of ubiquitous promoters for expression constructs useful in the compositions and methods of the invention include the 3-phosphoglycerate kinase (PGK-1) promoter, the β-actin promoter, the ROSA26 promoter, the heat shock protein 70 (Hsp70) promoter, the EF-1α gene (EF1) promoter encoding elongation factor 1α, the eukaryotic initiation factor 4A (eIF-4A1) promoter, the chloramphenicol acetyltransferase (CAT) promoter, and the CMV (cytomegalovirus) promoter.

[0110] In other embodiments, a human nucleic acid is expressed from a tissue-specific promoter. Non-limiting examples of tissue-specific promoters for use in the expression constructs of the present invention include promoters of genes expressed in the hematopoietic system, such as the M-CSF promoter, IL-3 promoter, GM-CSF promoter, SIRPA promoter, TPO promoter, IFN-β promoter, Wiskott-Aldrich syndrome protein (WASP) promoter, CD45 (also known as leukocyte 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 in 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). Subsequent to the inclusion of the promoter, one or more other regulatory elements, such as enhancer elements or intron sequences, are included in many embodiments of the present invention. Examples of enhancers useful in the compositions and methods of the present invention include, but are not limited to, the cytomegalovirus (CMV) early enhancer element and the SV40 enhancer element. Examples of intron sequences useful in the compositions and methods of the present invention include, but are not limited to, the β-globin intron or a ubiquitous intron. Other regulatory elements useful in some embodiments of the present invention include, but are not limited to, transcription termination sequences and mRNA polyadenylation (pA) sequences.

[0111] In some embodiments, the method of introducing a human nucleic acid expression construct into a preimplantation embryo includes linearizing the expression construct and then injecting it into the preimplantation embryo. In a preferred embodiment, the expression construct is injected into a fertilized oocyte. Fertilized oocytes can be collected from superovulated females on the day after mating and the expression construct injected. The injected oocytes are cultured overnight or directly transferred into the oviduct of 0.5 day p.c. pseudopregnant females. Methods for superovulation, oocyte harvest, expression construct injection, and embryo transfer are known in the art and are described in Manipulating the Mouse Embryo (2002, A Laboratory Manual, 3 rd(Cold Spring Harbor Laboratory Press). The presence of the introduced nucleic acid can be evaluated in the offspring by DNA analysis (such as PCR, Southern blotting, DNA sequencing, etc.) or by protein analysis (such as ELISA, Western blotting, etc.).

[0112] In other embodiments, expression constructs 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 acid can be evaluated in the cells by DNA analysis (such as PCR, Southern blotting, DNA sequencing, etc.) or by protein analysis (such as ELISA, Western blotting, etc.). Then, the cells determined to have incorporated the expression construct can be microinjected into preimplantation embryos. For a detailed description of the methods known in the art that can be used in the compositions and methods of the present invention, see Nagy et al. (2002, Manipulating the Mouse Embryo: A Laboratory Manual, 3 rd 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).

[0113] The genetically modified non-human animals of the present invention can be crossed with immunodeficient animals to create immunodeficient animals that express at least one human nucleic acid. Multiple embodiments of the present invention provide genetically modified animals that contain human nucleic acids in substantially all of their cells, as well as genetically modified animals that contain human nucleic acids in some but not all of their cells. One or more copies (adjacent to or distant from each other) of the human nucleic acid can be integrated into the genome of the cells of the genetically modified animal.

[0114] In some embodiments, the invention is a genetically modified non-human mouse engrafted with at least one type of human blood cell. In other embodiments, the invention is a method of engrafting human blood cells in a genetically modified non-human animal. The engrafted human blood cells useful in the compositions and methods of the invention include any human blood cells. Non-limiting examples of human blood cells useful in the invention include, but are not limited to, HSC, HSPC, leukemia-initiating cells (LIC), and hematopoietic cells of any lineage at any stage of differentiation, including terminally differentiated hematopoietic cells of any lineage. Such hematopoietic cells can be derived from any tissue or location of a human donor, including, but not limited to, bone marrow, peripheral blood, liver, fetal liver, or umbilical cord blood. Such hematopoietic cells can be isolated from any human donor, including healthy donors, as well as donors having a disease (such as cancer, including leukemia).

[0115] In other embodiments, the invention is a method of engrafting human blood cells in a genetically modified non-human animal. In some embodiments, the genetically modified non-human animal engrafted with human blood cells is an immunodeficient animal. Engrafting hematopoietic cells in the genetically modified animal of the invention is characterized by the presence of engrafted human blood cells in the animal. In a specific embodiment, engrafting hematopoietic cells in an immunodeficient animal is characterized by the presence of differentiated engrafted human blood cells in the animal as compared to a suitable control animal.

[0116] In some embodiments, the animals of the invention are also transplanted with human cancer cells (such as human solid tumors, etc.) in addition to the human blood cells. In various embodiments, the human cancer cells can be a cancer cell line or primary human cancer cells isolated from a patient, which are from any one of many different types of cancer (as non-limiting examples, including melanoma, breast cancer, lung cancer, etc.). In some embodiments, human cancer cells and HSPC are isolated from the same patient and transplanted into the same non-human animal.

[0117] The genetically modified non-human animals provided in various embodiments of the invention have various utilities, such as, but not limited to, serving as a model for the growth and differentiation of hematopoietic cells, for in vivo evaluation of human hematopoiesis, for in vivo evaluation of cancer cells, for in vivo studies of immune responses, for in vivo evaluation of vaccines and vaccination regimens, for testing the effects of agents that regulate cancer cell growth or survival, for in vivo evaluation of cancer treatments, for in vivo generation and collection of immune mediators (such as antibodies), and for testing the effects of agents that affect the functions of hematopoietic and immune cells.

[0118] Transferring human artificial blood cells into genetically modified and / or immunodeficient non-human animals has conventionally required conditioning prior to administration of hematopoietic cells, i.e., sublethal irradiation of the recipient animal with high-frequency electromagnetic radiation (generally using γ or X-ray radiation), or treatment with radiomimetic drugs such as busulfan or nitrogen mustard. Conditioning is thought to reduce the number of host hematopoietic cells, create microenvironmental factors suitable for the transfer of human artificial blood cells, and / or create a microenvironmental niche for the transfer of human hematopoietic cells. Standard methods for conditioning are known in the art, such as those described herein and in J. Hayakawa et al., 2009, Stem Cells, 27(1):175-182. Embodiments in accordance with the present invention provide methods for transferring human artificial blood cells into immunodeficient animals, which include providing human artificial blood cells to the immunodeficient animal in the case of irradiating or not irradiating the animal prior to administration of the hematopoietic cells. Embodiments in accordance with the present invention provide methods for transferring human artificial blood cells into immunodeficient animals, which include providing human artificial blood cells to the genetically modified non-human animals of the present invention in the case of administering or not administering a radiomimetic drug (such as busulfan or nitrogen mustard) to the animal prior to administration of the hematopoietic cells.

[0119] In some embodiments, the method for transferring hematopoietic cells into a genetically modified non-human animal in accordance with the embodiments of the present invention includes providing human artificial blood cells to the genetically modified animal of the present invention, as described elsewhere herein. In some embodiments, the genetically modified non-human animals of the present invention are immunodeficient animals that are defective in non-human B cell number and / or function, non-human T cell number and / or function, and / or non-human NK cell number and / or function. In other embodiments, the immunodeficient animal has severe combined immunodeficiency (SCID). SCID refers to a condition characterized by the absence of T cells and the lack of B cell function. Examples of SCID include: X-linked SCID, which is characterized by a mutation in the γ chain gene of the IL2RG gene and a lymphocyte phenotype of T(-)B(+)NK(-); and autosomal recessive SCID, which is characterized by a mutation in the Jak3 gene and a lymphocyte phenotype of T(-)B(+)NK(-), a mutation in the ADA gene and a lymphocyte phenotype of T(-)B(-)NK(-), a mutation in the IL-7Rα chain and a lymphocyte phenotype of T(-)B(+)NK(+), a mutation in CD3δ or ε and a lymphocyte phenotype of T(-)B(+)NK(+), a mutation in RAG1 / RAG2 and a lymphocyte phenotype of T(-)B(-)NK(+), a mutation in the Artemis gene and a lymphocyte phenotype of T(-)B(-)NK(+), and a mutation in the CD45 gene and a lymphocyte phenotype of T(-)B(+)NK(+). In some embodiments, the genetically modified non-human animals of the present invention are RAG1 - / - 。

[0120] In some embodiments, a method of engrafting hematopoietic cells in a genetically modified animal according to an embodiment of the present invention includes providing human hematopoietic cells to a genetically modified non-human animal having a severe combined immunodeficiency mutation (Prkdc scid ), commonly referred to as the scid mutation). The scid mutation is well-known and is located on mouse chromosome 16, as described in Bosma et al. (1989, Immunogenetics 29:54-56). Mice that are homozygous for the scid mutation are characterized by the absence of functional T and B cells, lymphopenia, hypogammaglobulinemia, and a normal hematopoietic microenvironment. For example, the scid mutation can be detected by using well-known methods to detect scid mutation markers.

[0121] In other embodiments, a method of engrafting hematopoietic cells in a genetically modified animal according to an embodiment of the present invention includes providing human hematopoietic cells to a genetically modified immunodeficient non-human animal having an IL2 receptor gamma chain deficiency, either alone or in combination with a severe combined immunodeficiency (scid) mutation. The term "IL2 receptor gamma chain deficiency" refers to a reduced IL2 receptor gamma chain. The reduced IL2 receptor gamma chain can be due to gene deletion or mutation. For example, the reduced IL2 receptor gamma chain can be detected by using well-known methods to detect IL2 receptor gamma chain gene deletion or mutation and / or to detect reduced IL2 receptor gamma chain expression.

[0122] In addition to naturally occurring human nucleic acid and amino acid sequences, the term also encompasses variants of human nucleic acid and amino acid sequences. As used herein, the term "variant" defines an isolated naturally occurring genetic mutant of a human or a recombinantly prepared variant of a human, each of which contains one or more mutations compared to the corresponding wild-type human. For example, such mutations can be one or more amino acid substitutions, additions, and / or deletions. The term "variant" also includes non-human orthologs. In some embodiments, variant polypeptides of the present invention have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the wild-type human polypeptide.

[0123] The percent identity between two sequences is determined using the techniques described elsewhere herein. 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.

[0124] Conservative amino acid substitutions can be made in human proteins to generate human protein variants. A conservative amino acid substitution is recognized as the substitution of one amino acid for another with similar characteristics. For example, each amino acid can be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic, and hydrophilic. A conservative substitution is the substitution of one amino acid having a specified structural or functional characteristic for 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; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, and tryptophan; and conservative substitutions include substitutions between amino acids within each group. Amino acids can also be described in terms of relative size, and alanine, cysteine, aspartic acid, glycine, asparagine, proline, threonine, serine, and valine are generally considered to be smaller.

[0125] Human variants can include synthetic amino acid analogs, amino acid derivatives, and / or non-standard amino acids, illustrative of which include, but are not limited to, α-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxyphenylalanine, lathyrine, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, methylhistidine, and ornithine.

[0126] Human variants are encoded by nucleic acids having a high degree of identity with the nucleic acids encoding wild-type humans. The complement of the nucleic acid encoding the human variant specifically hybridizes with the nucleic acid encoding the wild-type human under high-stringency conditions.

[0127] The term "nucleic acid" refers to any form of RNA or DNA molecule having more than one nucleotide, including single-stranded, double-stranded, oligonucleotide, or polynucleotide. The term "nucleotide sequence" refers to the nucleotide ordering of an oligonucleotide or polynucleotide in a single-stranded form of nucleic acid.

[0128] Well-known methods can be used to isolate, recombine, or synthesize nucleic acids encoding human variants.

[0129] The isolation of artificial blood cells, the administration of artificial blood cells to a host animal, and methods for assessing their engraftment are well known in the art. Hematopoietic cells for administration to a host animal can be obtained from any tissue containing hematopoietic cells, such as but not limited to umbilical cord blood, bone marrow, peripheral blood, cytokine- or chemotherapy-mobilized peripheral blood, and fetal liver. Hematopoietic cells can be administered into neonatal or adult animals by administration via various routes, such as but not limited to intravenous, intrahepatic, intraperitoneal, intramedullary, and / or intratibial.

[0130] Engraftment of artificial blood cells in the genetically modified animals of the present invention can be assessed by any of a variety of methods, such as but not limited to flow cytometry analysis of cells in an animal that has received administration of artificial blood cells at one or more time points after administration of the hematopoietic cells.

[0131] Exemplary methods for isolating artificial blood cells, administering artificial blood cells to a host animal, and assessing engraftment of artificial blood cells in a host animal are described herein and in Pearson et al. (2008, Curr. Protoc. Immunol. 81:1-15), Ito et al. (2002, Blood 100:3175-3182), Traggiai et al. (2004, Science 304:104-107), Ishikawa et al. (2005, Blood 106:1565-1573), Shultz et al. (2005, J. Immunol. 174:6477-6489), and Holyoake et al. (1999, Exp Hematol. 27:1418-27).

[0132] In some embodiments of the present invention, artificial blood cells are isolated from an initial source material to obtain a cell population enriched in a particular hematopoietic cell population (e.g., HSC, HSPC, LIC, CD34 + 、CD34 - 、lineage-specific markers, etc.). The isolated hematopoietic cells may or may not be a pure population. In one embodiment, cells having a particular marker (such as CD34) are depleted from the hematopoietic cells that can be used in the compositions and methods of the present invention. In another embodiment, hematopoietic cells that can be used in the compositions and methods of the present invention are enriched by selection for a marker (such as CD34). In some embodiments, the hematopoietic cells that can be used in the compositions and methods of the present invention are a cell population in which CD34 + cells constitute about 1-100% of the cells, although 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 that can be used in the compositions and methods of the present invention are CD34 +T cell-depleted cell populations, CD34 cells that constitute about 1-3% of the total cells + Lineage-depleted cell populations, or CD34 cells that constitute about 50% of the total cells + CD34 cells that constitute about 90% of the total cells + Positive selection cell populations.

[0133] With regard to generating a human hematopoietic and / or immune system in a genetically modified non-human animal that expresses at least one human gene, it is considered that the number of hematopoietic cells administered is not limiting. Thus, as a non-limiting example, the range of the number of hematopoietic cells administered can be from about 1x10 3 to about 1x10 7 , although in various embodiments, more or fewer can also be used. As another non-limiting example, the range of the number of HSPCs administered can be from about 3x10 3 to about 1x10 6 CD34 + cells when the recipient is a mouse, although in various embodiments, more or fewer can also be used. For recipients of other species, the number of cells to be administered can be determined by routine experimentation only.

[0134] Generally, at a time point beyond the lifespan of the administered human hematopoietic cells, engraftment can be considered successful when the number (or percentage) of human hematopoietic cells present in the genetically modified non-human animal is greater than the number (or percentage) of human cells administered to the non-human animal. For example, detection of the progeny of the administered hematopoietic cells can be achieved by detecting human DNA in the recipient animal, or by detecting intact human hematopoietic cells, such as by detecting human cell surface markers (such as, for example, CD45). Serial transfer of human hematopoietic cells from a first recipient into a second recipient and engraftment of the human hematopoietic cells in the second recipient is another optional test for engraftment in the primary recipient. Engraftment can be detected by flow cytometry at 1-4 months after administration of human hematopoietic cells as 0.05% or more human CD45 + cells in blood, spleen or bone marrow. Stem cells can be mobilized using cytokines (such as GM-CSF), for example as described in Watanabe (1997, Bone Marrow Transplantation 19:1175-1181). Examples

[0135] The present invention is further described in detail by reference to the following examples. These examples are provided for illustrative purposes only and are not intended to be limiting, unless otherwise specified. Thus, the present invention should in no way be construed as limited to the following examples, but should be construed to cover any and all variations that become apparent from the teachings provided herein.

[0136] In cases where no further description is provided, it is considered that a person of ordinary skill in the art can generate and utilize the compounds of the present invention and practice the claimed methods using the foregoing description and the following illustrative embodiments. Accordingly, the following working examples clearly indicate the preferred embodiments of the present invention and should not be construed as limiting the remainder of the disclosure in any way.

[0137] Example 1: Functional Innate Immune Response and Solid Tumor Support in Human Hematolymphoid System Mice

[0138] As described herein, mice reconstituted with the human hematolymphoid system (HHLS) represent a powerful tool for predictive human preclinical in vivo studies. A major limitation of current HHLS mice is the occurrence of defects in human cells that are crucial for innate immunity. Here, a novel mouse strain is reported in which multiple cytokine-encoding genes are genetically humanized. These humanized cytokines act in concert to effectively support human hematopoiesis and the development and function of human monocytes / macrophages and NK cells. In the tumor microenvironment, human macrophages acquire an immunosuppressive phenotype and support human cancer growth. With a more complete and functional human innate immune system, this novel HHLS mouse model has particular potential to facilitate in vivo studies of the physiology and pathology of human innate immunity.

[0139] Monocytes and macrophages are major cellular components of the innate immune response (Auffray et al., 2009, Annual review of immunology 27, 669). In one aspect, these cells are able to sense infection and mediate direct antimicrobial functions through a variety of mechanisms such as phagocytosis or the secretion of pro-inflammatory factors. In another aspect, monocytes / macrophages can acquire immunosuppressive functions that are important for the resolution of inflammation and tissue repair. Additionally, these anti-inflammatory properties can be co-opted by tumor-infiltrating macrophages and confer survival advantages to developing tumors through a variety of mechanisms (Allavena and Mantovani, 2012, Clinical and experimental immunology 167, 195; Qian and Pollard, 2010, Cell 141, 39).

[0140] Small animal models, such as mice, are often used to study mammalian immune responses in vivo. However, there are fundamental differences in immune function between species (Mestas and Hughes, 2004, Journal of Immunology 172, 2731; Rongvaux et al., 2013, Annual review of immunology 31, 635). In particular, there are major phenotypic and functional species-specific differences between monocyte / macrophage populations, and in general, knowledge obtained from mouse studies is only partially applicable to humans (Auffray et al., 2009, Annual review of immunology 27, 669; Rongvaux et al., 2013, Annual review of immunology 31, 635; Chow et al., 2011, Nature reviews Immunology11, 788). A promising approach to studying the specificity of human hematopoiesis and immune function in vivo is to use mice carrying the human hematolymphoid system (HHLS) (Rongvaux et al., 2013, Annual review of immunology 31, 635; Shultz et al., 2012, Nature reviews Immunology 12, 786). However, the development and function of several human immune cell types, such as monocytes / macrophages and NK cells, are largely defective in current HHLS mice (Rongvaux et al., 2013, Annual review of immunology 31, 635). These defects are most likely due to the reduced cross-reactivity of murine cytokines with their corresponding human receptors (Manz, 2007, Immunity 26, 537). To circumvent this limitation, a strategy was developed to replace murine genes encoding cytokines with their human counterparts (Willinger et al., 2011, Trends in immunology 32, 321), and this approach led to a significant improvement in the development and function of individual human cell types( Figure 16 )(Rathinam et al., 2011, Blood 118, 3119; Willinger et al., 2011, Proceedings ofthe National Academy of Sciences 108, 2390; Rongvaux et al., 2011, Proceedings of theNational Academy of Sciences 108, 2378).

[0141] Hematopoiesis is a tightly regulated developmental process in which multipotent hematopoietic stem cells differentiate into more committed progenitors and then into mature blood cells (Kondo et al., 2003, Annual review of immunology 21, 759; Doulatov et al., 2012, Cell stem cell 10, 120). This process requires specific cytokines that support successive developmental steps ( Figure 5 ). Synergy among multiple human cytokines may be required to fully recapitulate human myelopoiesis in mice. Thus, a novel mouse strain, called MISTRG, was generated in which the genes encoding M-CSF (Rathinam et al., 2011, Blood118, 3119), IL-3 / GM-CSF (Willinger et al., 2011, Proceedings of the National Academy ofSciences108, 2390), and TPO (Rongvaux et al., 2011, Proceedings of the National Academy ofSciences 108, 2378) were replaced by their human counterparts (Willinger et al., 2011, Trends in immunology32, 321) in the hSIRPAtg RAG2 - / - IL-2Rγ - / - background (Traggiai et al., 2004, Science304, 104; Strowig et al., 2011, Proceedings of the National Academy of Sciences 108, 13218).

[0142] Newborn MISTRG mice and their littermate MITRG (lacking the hSIRPA transgene) were irradiated sublethally and transplanted with human fetal liver-derived CD34 + cells following a standard protocol (Traggiai et al., 2004, Science 304, 104). RAG2 - / - IL2-Rγ - / - (RG) mice, which share the same genetic background but lack all humanized alleles, and commercially available NOD-Scid IL2-Rγ - / - (NSG) mice served as controls. Blood engraftment levels (percentage of hCD45 + cells; ( Figure 1 Panel A and Figure 1 Panel B; and Figure 6Panel A) was lower in RG and higher in NSG recipients, as previously reported (Strowig et al., 2011, Proceedings of the National Academy of Sciences 108, 13218; Brehm et al., 2010, Clinical immunology 135, 84). The percentage of blood hCD45 + cells was similar in MISTRG and in NSG. Hematopoietic engraftment was also significantly increased in MITRG compared to RG, suggesting that combinatorial gene humanization overcomes the need to induce phagocyte tolerance via SIRPα / CD47 crossreactivity (Strowig et al., 2011, Proceedings of the National Academy of Sciences 108, 13218; Takenaka et al., 2007, Nature immunology 8, 1313; Legrand et al., 2011, Proceedings of the National Academy of Sciences 108, 13224), which may be achieved by attenuating the murine innate response. Mice with at least 10% human CD45 + cells in the blood were selected for further experiments ( Figure 6 Panel B). In the bone marrow (BM), the percentage of hCD45 + cells exceeded 90% and reached up to 99% in most of these two MISTRG recipients ( Figure 1 Panel A and Figure 1 Panel C; and Figure 6 Panel C to Figure 6 Panel E), and the high engraftment efficiency in BM was independent of SIRPα / CD47 interaction. To test the ability of humanized cytokines to support human hematopoiesis in more competitive conditions, human CD34 + cells were transplanted into non-irradiated MISTRG. This protocol generated human CD45 + cells in the blood and BM of all recipients ( Figure 1 Panel D and Figure 1 Panel E), and significantly, half of the mice showed chimerism as high as the highest levels measured in recipients engrafted after X-ray preconditioning (compare Figure 1 Panel E with Figure 1 Panel B and Figure 1 Panel C). The data described herein show that genetic replacement of multiple cytokines in MISTRG creates a human hematopoietic microenvironment that can almost completely replace murine hematopoiesis in the bone marrow and eliminates the need for pathologically induced irradiation.

[0143] Next, the ability of MISTRG mice to support human myelopoiesis was evaluated. Compared with RG and NSG, human myeloid cells (hCD33 + ) were present at significantly higher proportions in the blood and bone marrow of MISTRG ( Figure 2 Panel A; and Figure 7 Panels A to Figure 7 Panel C). The increased proportion of myeloid cells in MISTRG led to a blood composition similar to the physiological composition of human blood, which is rich in myeloid cells and fundamentally different from the composition of murine blood rich in lymphoid cells (Mestas and Hughes, 2004, Journal of Immunology 172, 2731; Rongvaux et al., 2013, Annual review of immunology 31, 6354) ( Figure 2 Panel B; and Figure 7 Panel D). Although both monocytes (CD33 高 SSC 低 CD66 - ) and granulocytes (CD33 + SSC 高 CD66 + ) were present in the BM ( Figure 7 Panel E), the human myeloid cell population in peripheral blood was mainly composed of monocytes ( Figure 7 Panel F), suggesting that the terminal differentiation and egress from the BM or peripheral survival of human granulocytes remained suboptimal in this murine environment. Importantly, however, human myeloid cells were abundant in non-lymphoid tissues of MISTRG, such as the lung, liver, and colon, as shown by immunohistochemistry (hCD68 + cells; ( Figure 2 Panel C)) or by flow cytometry (hCD33 + ; ( Figure 7 Panels G and Figure 7 Panel H)), and significantly exceeded the number of human myeloid cells found in NSG mice by approximately 10-fold.

[0144] In humans, three monocyte subsets have been phenotypically and functionally described based on the expression of CD14 and CD16 markers (Auffray et al., 2009, Annual review of immunology 27, 669; Cros et al., 2010, Immunity 33, 375). All three subsets of human monocytes (CD14 + CD16 - , CD14 + CD16 + , and CD14dim CD16 + ) is present in lymphoid and non-lymphoid tissues of MISTRG (such as the lung and liver) ( Figure 2 Panel D and Figure 2 Panel E; and Figure 8 Panel A and Figure 8 Panel B). In contrast, in NSG, only CD14 can be consistently detected outside of myeloid cells at lower frequencies + CD16 - and to some extent CD14 + CD16 + monocytes, while CD14 dim CD16 + cells are only marginally present. The extended immunophenotype (CD33, CD11b, CD115, CD62L, and CX3CR1) of the monocyte subsets found in MISTRG closely resembles the equivalent subsets in human peripheral blood ( Figure 9 ). Human CD14 + CD16 - and CD14 + CD16 + monocytes isolated from the BM of MITRG produce high levels of inflammatory cytokines in response to TLR4 and TLR7 / 8 ligands (LPS and R848, respectively) ( Figure 2 Panel F and Figure 2 Panel G). In in vitro assays performed on the WBC of MITRG, CD14 + CD16 - and CD14 + CD16 + cells both have a high ability to phagocytose GFP-expressing Escherichia coli, while CD14 dim CD16 + monocytes have limited phagocytic capacity ( Figure 2 Panel H), again reflecting the physiological properties of the corresponding subsets in human blood (Cros et al., 2010, Immunity 33, 375). When challenged in vivo with LPS or infected with the bacterial and viral human pathogens Listeria monocytogenes and influenza A, respectively, MISTRG mice respond with a robust production of human inflammatory cytokines (TNFα, IL-6, and IFNγ, respectively), while NSG mice show a significantly lower (by approximately 1 log) response ( Figure 2 Panel I to Figure 2 Panel K). These results demonstrate that the human monocyte subsets present in MISTRG are functional both in vitro and in vivo. However, one drawback of the presence of functional human phagocytes in mice is the disruption of human tolerance to murine phagocytes, to which murine RBCs are particularly susceptible (Figure 10 Small figure A and Figure 10 Small figure B). This destruction of murine RBCs leads to anemia ( Figure 10 Small figure C to Figure 10 Small figure I), and limits the lifespan of engrafted mice to 10 - 12 weeks (MISTRG) or 12 - 16 weeks (MITRG).

[0145] Myeloid cells can support the development and differentiation of other immune cells by generating cytokines. Assess whether the myeloid compartment of MISTRG mice is a source of human cytokines such as IL - 15. Consistent with this idea, it was found that the mRNA expression of human IL - 15 and IL - 15Rα was increased more than 10 - fold in MISTRG compared to NSG ( Figure 3 Small figure A; and Figure 11 Small figure A). To more precisely define the cellular source of human IL - 15 / IL - 15Rα in MISTRG, the abundance of human IL - 15 and IL - 15Rα transcripts was measured in purified human cell populations. The expression of human IL - 15Rα mRNA was higher in human myeloid cells (hCD33 + ) than in non - myeloid cells (hCD33 - ) ( Figure 3 Small figure B). Specifically, CD14 + CD16 + monocytes showed enrichment of both IL - 15 and IL - 15Rα transcripts ( Figure 3 Small figure B). The surface expression of human IL - 15Rα protein on human myeloid cells from MISTRG was confirmed by flow cytometry ( Figure 11 Small figure B).

[0146] Based on these findings, we evaluated whether MISTRG mice support the development of human immune cells (such as NK cells) that rely on IL-15 trans-presentation (Ma et al., 2006, Annual review of immunology 24, 657; Soderquest et al., 2011, Blood 117, 4511). Efficient development of human NK cells in the current HHLS mouse model requires exogenous pharmacological delivery of human IL-15 / IL-15Rα (Huntington et al., 2009, Journal of experimental medicine 206, 25; Chen et al., 2009, Proceedings of the National Academy of Sciences 106, 21783; Pek et al., 2011, Immunobiology 216, 218)23-25), as murine IL-15 is insufficient in vivo to support human NK cells. As previously reported (Huntington et al., 2009, Journal of experimental medicine 206, 25; Chen et al., 2009, Proceedings of the National Academy of Sciences 106, 21783; Pek et al., 2011, Immunobiology 216, 218), few human NK cells (hNKp46 + hCD3 - )( Figure 3 panel C and Figure 3 panel D; and Figure 12 panel A and Figure 12 panel B) were observed upon engraftment in NSG. In contrast, human NK cells were readily detectable in multiple tissues engrafted with MISTRG and were increased approximately 10-fold compared to NSG ( Figure 3 panel C and Figure 3 panel D; and Figure 12 panel A and Figure 12 panel B). Except for the bone marrow, MITRG had fewer human NK cells than MISTRG, most likely due to the previously reported requirement for human SIRPα for the survival of human NK cells in the periphery (Legrand et al., 2011, Proceedings of the National Academy of Sciences 108, 13224). hNKp46 in MISTRG mice + hCD3 -The cells represent true NK cells as they closely mimic human controls expressing the typical NK cell surface markers CD94, CD161, and killer inhibitory receptors (KIR). Figure 12 Panel A and Figure 12 Panel B). In addition to its effect on ontogeny, IL-15 also promotes NK cell maturation. Consistently, higher surface expression of the maturation marker CD16 and amounts of the soluble granule protein perforin were found on NK cells from MISTRG compared to NSG( Figure 13 Panel C to Figure 13 Panel F).

[0147] The cellular source of IL-15 trans-presentation in humans is currently unknown, but human myeloid cells can support human NK cell proliferation in vitro (Huntington et al., 2009, Journal of experimental medicine 206, 25). To test whether human IL-15 trans-presentation by human monocytes / macrophages supports improved human NK cell ontogeny in MISTRG, mice were treated with liposome-encapsulated clodronate to deplete phagocytes( Figure 14 ). Depletion of phagocytes also induced a significant reduction in human NK cells( Figure 3 Panel E), suggesting that human monocytes / macrophages are indeed a crucial cell type whose trans-presentation of IL-15 supports human NK cell homeostasis in vivo.

[0148] NK cells participate in innate defense against pathogens by killing cells lacking MHC class I expression (missing self) (Raulet, 2006, Seminars in immunology 18, 145), and by generating the key cytokine IFNγ (Vivier et al., 2008, Nature immunology 9, 503). Consistent with higher perforin expression( Figure 13 Panel E and Figure 13 Panel F), significantly enhanced NK cell cytotoxic activity against human cells lacking MHC class I was observed in vivo in MISTRG compared to NSG( Figure 3 Panel F). NK cells are an early source of IFNγ after Listeria infection. Consequently, human IFNγ mRNA expression in the liver was found to be more than 10-fold higher in MISTRG than in NSG 2 days after infection( Figure 3 Panel G). At single-cell resolution, NK cells from Listeria-infected MISTRG showed production of human IFNγ at a significantly higher frequency in the absence of ex vivo restimulation compared to NSG( Figure 3 Panel I) Figure 3Panel H). NK cells in MISTRG also have lytic activity (degranulation) after Listeria infection, as shown by the plasma membrane exposure of CD107a ( Figure 3 Panel H). Overall, MISTRG generated by efficient generation of human myeloid cells supports the development, differentiation, and function of human NK cells, thus overcoming a major limitation of current HHLS mouse models.

[0149] Next, the role of human myeloid cells in the context of the tumor microenvironment was evaluated. Therefore, the human melanoma cell line Me290 was used as a tumor model (Valmori et al., 1998, Journal of immunology 160, 1750). Clinical observations have shown that myeloid cells infiltrate tumors in several solid tumors, and a high density of infiltrating macrophages is associated with poor patient prognosis in most types of cancer (Qian and Pollard, 2010, Cell 141, 39; Coussens et al., Science 339, 286; Egeblad et al., 2010, Developmental cell 18, 884; Nelson and Bissell, 2006, Annual review of cell and developmental biology 22, 287; Bingle et al., 2002, T Journal of pathology 196, 254). Thus, higher human myeloid cell infiltration was detected in tumors in MISTRG than in NSG, as shown by the expression of human PTPRC and ITGAM mRNAs (encoding CD45 and CD11b, respectively) ( Figure 4 Panel A). Cells expressing the macrophage markers CD163 and CD14 are abundant in tumors in MISTRG, very similar to human tumors in patients, but are hardly detectable in the same tumors in NSG ( Figure 4 Panel B and Figure 4 Panel C; and Figure 15 ). Most CD163 + cells also express low levels of HLA-DR and high levels of CD206 ( Figure 4 Panel B and Figure 4 Panel D), an immunophenotype generally associated with "M2-like" macrophages (Hao et al., 2012, Clinical & developmental immunology 2012, 948098; Tang, 2013, Cancer Lett 332, 3).

[0150] Macrophages of the M2 subtype promote tumor progression via multiple effector mechanisms, including proliferative signals for cancer cells, anti-apoptotic signals, pro-angiogenic activities, and the formation of cancer cell extravasation and metastasis from primary tumors (Qian and Pollard, 2010, Cell 141, 39; Coussens et al., Science 339, 286; Egeblad et al., 2010, Developmental cell 18, 884). Evaluating macrophage infiltration in tumors can promote tumor growth in MISTRG. Significantly, tumors were observed to be transplanted into MISTRG with CD34 + and were largely human CD163 + HLA - DR 低 CD206 + macrophage infiltration) were significantly larger than tumors in NSG, which were not infiltrated by human macrophages and were of the same smaller size as those seen in non-transplanted NSG or MISTRG mice ( Figure 4 panel E and Figure 4 panel F). One of the mechanisms by which macrophages support tumor growth is via the production of cytokines or enzymes that promote angiogenesis and immunosuppression. VEGF is an important multifunctional tumor-supportive molecule (Kandalaft et al., Current topics in microbiology and immunology 344, 129; Motz and Coukos, Immunity 39, 61), and to test whether this factor is involved in tumor growth in MISTRG, mice were treated with the human VEGF inhibitor Avastin TM . This treatment completely reversed the tumor growth phenotype ( Figure 4 panel F), demonstrating that myeloid cells in MISTRG support melanoma growth via a VEGF-dependent mechanism. Overall, these results show that MISTRG mice recapitulate the role of human macrophages in tumorigenesis and meet an important need for a model that allows in vivo studies of the interaction between human tumors and human macrophages, especially at the onset of tumorigenesis.

[0151] The data described herein have demonstrated the synergistic effects of providing multiple human cytokines in MISTRG mice on direct or indirect support for human hematopoiesis and human immune cell function ( Figure 16 ). The MISTRG model of HHLS mice provides a unique opportunity to study the human innate immune response in vivo.

[0152] Materials and methods are now described.

[0153] Mouse strain

[0154] In RAG2 - / - γc - / - The generation of mice with BAC transgenic expression of human SIRPα or knock-in replacement of genes encoding TPO, IL-3 / GM-CSF, and M-CSF in a Balb / c x 129 genetic background has been reported (Rathinam et al., 2011, Blood 118, 3119; Willinger et al., 2011, Proceedings of the National Academy of Sciences 108, 2390; Rongvaux et al., 2011, Proceedings of the National Academy of Sciences 108, 2378; Strowig et al., 2011, Proceedings of the National Academy of Sciences 108, 13218). These strains were crossed to obtain MITRG (M-CSFh / hIL-3 / GM-CSFh / hTPOh / hRAG2 - / - γc - / - ) and MISTRG (M-CSFh / hIL-3 / GM-CSFh / hhSIRPAtgTPOh / hRAG2 - / - γc - / - ) mice. Those mice were viable, healthy, and fertile. Mice were maintained under specific pathogen-free conditions in the case of continuous treatment with enrofloxacin (Baytril, 0.27 mg / ml) in drinking water. NOD Scidγc - / - (NSG) mice were obtained from the Jackson Laboratory.

[0155] Human HSPC preparation and transplantation into recipient mice

[0156] Human hematopoietic stem and progenitor cells were transplanted into recipient mice as described (Rathinam et al., 2011, Blood 118, 3119; Willinger et al., 2011, Proceedings of the National Academy of Sciences 108, 2390; Rongvaux et al., 2011, Proceedings of the National Academy of Sciences 108, 2378; Traggiai et al., 2004, Science 304, 104; Strowig et al., 2011, Proceedings of the National Academy of Sciences 108, 13218). Fetal liver samples were minced, treated with collagenase D (Roche, 100 ng / mL) at 37 °C for 45 minutes, and cell suspensions were prepared. Human CD34 + cells were purified by density gradient centrifugation (Lymphocyte Separation Medium, MP Biomedicals), followed by positive immunomagnetic selection with anti-human CD34 microbeads (Miltenyi Biotec). Cells were frozen in FBS containing 10% DMSO and maintained in liquid nitrogen.

[0157] For transplantation, neonatal pups (within the first 2 days of life) were irradiated sub-lethally (X-ray irradiation; RG, 2 times 180 cGy 4 hours apart; NSG, 1 time 100 cGy; MISTRG, 1 time 150 cGy), and 100,000 FL-CD34 + cells in 20 μL PBS were injected into the liver with a 22-gauge needle (Hamilton Company). In specific experiments ( Figure 1 panel D and Figure 1 panel E), 200,000 - 300,000 cells were injected into non-irradiated MISTRG neonatal recipients. Mice were bled at 7 - 9 weeks, and the percentage of human CD45 + cells was measured by flow cytometry. Mice with at least 5% (RG) or 10% (NSG, MITRG, and MISTRG) human CD45 + cells in the total (mouse and human combined) CD45 + population were selected for further experiments. Mice were sacrificed or used for experiments at 9 - 12 weeks post-transplantation.

[0158] All experiments were conducted in accordance with Yale University Human Investigation Committee and Yale Institutional Animal Care and Use Committee protocols.

[0159] Immunophenotypic analysis of human cell populations

[0160] To prepare WBCs, heparinized blood was treated twice with ACK lysis buffer to eliminate RBCs. The single cell suspensions of spleen and bone marrow (flushed out from femurs and tibias) were treated with ACK lysis buffer. Liver and lung leukocytes were isolated by mechanically dissociating the tissues and digesting the tissues with 100 U / ml collagenase IV and 0.02 mg / ml DNase I (Sigma) at 37 °C for 1 hour followed by density gradient centrifugation.

[0161] For FACS analysis, antibodies against the following antigens were used:

[0162] Mouse antigens: CD45 (clone 30-F11), CD71 (RI7217), Ter119

[0163] Human antigens: CD1c (BDCA1, clone L161), CD3 (UCHT1), CD11b (ICRF44), CD11c (3.9), CD14 (M5E2), CD16 (3G8), CD19 (HIB19), CD33 (WM53), CD45 (HI30), CD62L (DREG-56), CD66 (ASL-32), CD94 (DX22), CD107a (H4A3), CD115 (9-4D2-1E4), CD123 (6H6), CD141 (BDCA3, M80), CD161 (HP-3G10), CD235a (HI264), CD303 (BDCA2, 201A), NKp46 (9E2), IL-15Rα (JM7A4), CX3CR1 (2A9-1), HLA-A,B,C (W6 / 32), HLA-DR (L243), IFNγ (B27) KIR2DL1 / S1 (HP-MA4), KIR2DL2 / L3 (DX27), KIR3DL1 (DX9), perforin (dG9).

[0164] Human lineage cocktail: CD3, CD15, CD19, CD56, NKp46

[0165] All antibodies were obtained from Biolegend, BD Biosciences or Miltenyi Biotec. Data were acquired on an LSRII flow cytometer (BD Biosciences) using FACSDiva and analyzed with FlowJo software.

[0166] For histological analysis, spleen, lung, liver, and colon tissues were fixed overnight in IHC zinc fixative (BD Biosciences) or 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin or with anti-human CD68 antibody (clone PGM1), followed by staining with a secondary antibody conjugated to HRP, and developed with the peroxidase substrate 3,3'-diaminobenzidine.

[0167] In vitro phagocytosis assay

[0168] Escherichia coli expressing GFP was cultured overnight in LB medium at 37°C to an OD600 of 1.5 - 1.8, at which point the bacteria were diluted and cultured for 1 - 2 hours to an OD600 of approximately 1.0. The Escherichia coli was washed three times with PBS and incubated with WBCs from MITRG mice at 37°C in a volume of 200 μl at a ratio of approximately 2x10 7 Escherichia coli per 1x10 8 WBC for 4 hours. After incubation, the cells were washed with PBS and analyzed by flow cytometry.

[0169] In vitro TLR stimulation and in vivo infection

[0170] Human monocyte subsets were isolated from the BM of mice. Briefly, BM cells were recovered and pooled from the hind legs and spines of 6 mice. Human CD33 + cells were enriched by magnetic separation (EasySep CD33 Selection Kit, StemCell Technologies). CD14 + CD16 - and CD14 + CD16 + subsets were purified on a FACSAria cell sorter (BD Biosciences). 100,000 cells in 200 μl of medium were cultured overnight in the presence of the TLR4 ligand LPS (Escherichia coli 0111:B4, Sigma-Aldrich, 100 ng / ml) or the TLR7 / 8 ligand R848 (Invivogen, 10 μg / ml).

[0171] For in vivo stimulation, 35 μg of LPS (Escherichia coli 0111:B4, Sigma-Aldrich) in 100 μl of PBS was injected intraperitoneally, and serum was collected 90 minutes later.

[0172] By intravenous injection with 3x10 3Mice were infected with 2x10

[0173] CFU of Listeria monocytogenes (strain 10403S). At 48 hours post-infection, sera and tissues were harvested for ELISA and qPCR, respectively. Hepatocytes from uninfected or infected mice were incubated for 4 hours at 37°C / 5% CO2 in medium containing monensin (GolgiStop, BD Biosciences) and anti-human CD107a antibody. Cells were then stained for surface antigens, permeabilized using the Cytofix / Cytoperm kit (BD Biosciences), and stained intracellularly for human IFNγ. 4 PFU of influenza A / PR8 (H1N1) virus by intranasal infection and lungs were harvested on day 3 post-infection for qPCR analysis.

[0174] Cytokine concentrations (human TNFα, IL-6, and IL-1β) in mouse sera and culture supernatants were measured using the ELISA MAX standard kit (Biolegend) following the manufacturer's instructions.

[0175] RBC analysis

[0176] RBC counts were measured on a Hemavet 950 (Drew Scientific). Blood smears were stained with Wright-Giemsa. For mouse RBC transfer experiments, blood was obtained from RG mice, labeled with CFSE (20 μM, 15 minutes at 37°C), washed three times with PBS, and 200 μl of labeled RBCs were injected by retro-orbital intravenous injection. Mice were bled at 5 minutes to determine the initial frequency of CFSE-positive cells in Ter119 + cells (day 0, 100%). They were then bled at the indicated time points and the maintenance of CFSE-labeled Ter119 + cells was calculated as a percentage of the day 0 value.

[0177] In vivo depletion of phagocytes

[0178] Phagocytes were depleted by intravenous retro-orbital injection of 100 μl of clodronate-loaded liposomes (Van Rooijen and Sanders, 1994, Journal of immunological methods 174, 83). Clodronate-liposomes were injected 3 times a day and human NK cells in the livers of mice were analyzed 24 hours after the last injection. For the RBC phagocytosis assay, clodronate-liposomes were injected 3 days and 1 day before transfer of CFSE-labeled RBCs.

[0179] Quantitative RT-PCR

[0180] Total RNA was extracted from cells organized or purified with TRIzol reagent (Invitrogen) according to the manufacturer's instructions and used for cDNA synthesis performed with the SuperScript First-Strand Synthesis System (Invitrogen). Quantitative RT-PCR was performed on a 7500 Fast Real-Time PCR System using primer-probe sets purchased from ABI. Expression values were calculated using the comparative threshold cycle method and normalized to mouse Hprt or human HPRT as indicated.

[0181] In vivo NK cell cytotoxicity assay

[0182] Human NK cell cytotoxicity in vivo was determined following a previously reported protocol (Strowig et al., 2010, Blood 116, 4158). LCL721.221 (HLA class I negative) and LCL721.45 (class I positive) cells were mixed at a 1:1 ratio, labeled with CellTrace Violet (Invitrogen), and injected intravenously (1x10 7 cells / mouse) into NSG or MISTRG mice. Mice were sacrificed after 12 hours, single-cell suspensions of the spleen were prepared, and analyzed by flow cytometry. The proportion of HLA class I positive and negative cells in the purple cells was measured, and specific lysis was calculated as (MHC class I positive – MHC class I negative) x 100 / MHC class I positive.

[0183] Tumorigenesis

[0184] The human melanoma cell line Me290 (Valmori et al., 1998, Journal of immunology 160, 1750) was cultured to approximately 90% confluence and cells (approximately 7 million cells per mouse) were injected subcutaneously under anesthesia in the flanks of mice. For some experiments, starting on the day of tumor implantation, mice were treated every other day with the anti-human VEGF antibody Avastin TM (Roche; 100 μg intravenously). Tumor size was measured after 11 days and the volume was calculated using the following formula: volume = 0.5 * length 2 * width.

[0185] Patient and mouse tissues were frozen in OCT (Optimum Cutting Temperature, Sakura Finetek). Frozen sections (7 μm) were sequentially treated with 0.1% Triton-100X for 15 minutes, 0.03% hyaluronidase for 15 minutes, background eliminator (Innovex bioscience) for 15 minutes, Fc receptor blocker (Innovex bioscience) for 15 minutes, and background eliminator again for 15 minutes. Then, the sections were stained with primary antibodies diluted in PBS supplemented with 5% BSA and 0.01% saponin for 1 hour at room temperature, washed, and stained with secondary antibodies for 40 minutes at room temperature. Nuclei were stained with 4’,6-diamidino-2-phenylindole (1 μg / mL) for 2 minutes.

[0186] Primary antibodies: human CD14 (1:200, UCHM1, AbD Serotec); human CD163 (1:200, EDHu-1, AbD Serotec); human CD206 (1:100, 15-2, AbD Serotec); human HLA-DR (1:100, LN3, Biolegend). For CD163 / CD206 combined staining, the two antibodies were labeled with Alexa Fluor 488 or 568 antibody labeling kit (Molecular Probes) before tissue staining.

[0187] Secondary antibodies: goat anti-rat Alexa Fluor 568; goat anti-mouse Alexa Fluor 488; goat anti-mouse Alexa Fluor 588 or goat anti-mouse Alexa Fluor 647 (1:700, Molecular Probes).

[0188] Immunofluorescence imaging was performed on an Eclipse Ti inverted microscope system (Nikon Instruments Inc.) operated via NIS-Element Ar software (Nikon Instruments Inc.).

[0189] For CD163 + Quantification of cell infiltration density, tumors from 3 different melanoma patients, 3 NSGs, and 3 MISTRGs were selected. From each tumor, 3 frozen sections were stained for human CD163. Three representative photographs were obtained from each stained section, totaling 27 representative photographs from each group (patients, MISTRG, and NSG). For each photograph, CD163 was analyzed using NIS-Element Ar software (Niko Instruments Inc.)+ Cell counting. Use "separate channels + overlay" display and analyze each photo by simultaneously scaling on each separate channel and on the overlay.

[0190] Statistical analysis

[0191] Statistical analysis was performed using one-way ANOVA, followed by Tukey's post hoc test, two-tailed unpaired Student's t-test, or repeated measures ANOVA with GraphPad Prism 5 software.

[0192] Example 2: Human myeloid neoplasms can be engrafted in MISTRG

[0193] Myeloid leukemia is a type of cancer that affects cells of the myeloid lineage. Myeloid leukemia is classified into different types, including acute myeloid leukemia (AML), myeloproliferative disorder (MPD), chronic myelo-monocytic leukemia (CMML), and myelodysplastic syndrome (MDS). The risk of developing myeloid leukemia increases with age, and the incidence of these diseases may increase as the population ages. Although there are therapeutic and supportive care approaches clinically, there is a need for a better understanding of this group of diseases and novel therapies.

[0194] One method for studying human leukemia relies on xenotransplantation of patient samples into immunodeficient mice. However, currently available recipient mice are not optimal for this purpose: only a subset of AML samples can be successfully engrafted; and robust engraftment of MPD, CMML, or MDS (including RCUD, RAEB I, and RAEB II) has not been reported to date. Thus, an optimized strain of recipient mice is needed to achieve better engraftment of human myeloid leukemia.

[0195] It is demonstrated herein that MISTRG supports better engraftment of human hematopoietic cells, which results in near-complete replacement of murine hematopoiesis by human hematopoiesis in the bone marrow. It is also shown herein that samples isolated from AML, CMML, and MDS patients can be engrafted in MISTRG ( Figure 17 ).

[0196] Accordingly, the genetically modified non-human animals described herein represent a novel in vivo animal model of human myeloid leukemia and would be useful for: (i) studying the cellular and molecular pathogenesis of the disease; (ii) identifying biomarkers with predictive or prognostic value; (iii) identifying novel targets for therapy; and (iv) testing therapies in preclinical and patient-specific settings.

[0197] The disclosure of each patent, patent application, and publication cited herein is hereby incorporated by reference in its entirety. While the invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention may be devised without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A method for detecting the effect of an agent that regulates the function of hematopoietic or immune cells, the method comprising treating a genetically modified immunodeficient mouse with the agent to detect the effect of the agent, wherein the genetically modified immunodeficient mouse has a genome comprising: replacing the mouse M-CSF gene with a nucleic acid encoding a human M-CSF polypeptide at the mouse M-CSF locus, replacing the mouse IL-3 gene with a nucleic acid encoding a human IL-3 polypeptide at the mouse IL-3 locus, replacing the mouse GM-CSF gene with a nucleic acid encoding a human GM-CSF polypeptide at the mouse GM-CSF locus, inserting a nucleic acid encoding a human SIRPA polypeptide, replacing the mouse TPO gene with a nucleic acid encoding a human TPO polypeptide at the mouse TPO locus, recombinase activating gene 2 (Rag-2) gene knockout and, IL2 receptor gamma chain (IL2rg) gene knockout; wherein each of the nucleic acids encoding a human M-CSF polypeptide, a human IL-3 polypeptide, a human GM-CSF polypeptide, a human SIRPA polypeptide, and a human TPO polypeptide is operably linked to a promoter; wherein the mouse expresses a human M-CSF polypeptide, a human IL-3 polypeptide, a human GM-CSF polypeptide, a human SIRPA polypeptide, and a human TPO polypeptide; and wherein the genetically modified immunodeficient mouse is engrafted with human hematopoietic cells.

2. The method of claim 1, wherein the human hematopoietic cells comprise CD34+ cells.

3. The method of claim 1 or 2, wherein the nucleic acids encoding a human M-CSF polypeptide, a human IL-3 polypeptide, a human GM-CSF polypeptide, and a human TPO polypeptide are operably linked to a native mouse M-CSF promoter, an IL-3 promoter, a GM-CSF promoter, and a TPO promoter, respectively.

4. The method of any one of claims 1-3, wherein the human SIRPA polypeptide is a biologically active fragment of the full-length human SIRPA polypeptide.

5. The method of any one of claims 1-3, wherein the human SIRPA polypeptide is a fusion protein.

6. The method of claim 5, wherein the human SIRPA polypeptide is a fusion protein comprising a biologically active fragment of the full-length human SIRPA polypeptide.

7. A method for detecting the effect of an agent that regulates cancer cell growth or survival, or for in vivo evaluation of cancer treatment comprising the agent, the method comprising: transplanting human cancer cells into a genetically modified immunodeficient mouse; and, treating the genetically modified immunodeficient mouse with the agent to detect the effect of the agent; wherein the genetically modified immunodeficient mouse has a genome comprising: replacing the mouse M-CSF gene with a nucleic acid encoding a human M-CSF polypeptide at the mouse M-CSF locus, replacing the mouse IL-3 gene with a nucleic acid encoding a human IL-3 polypeptide at the mouse IL-3 locus, replacing the mouse GM-CSF gene with a nucleic acid encoding a human GM-CSF polypeptide at the mouse GM-CSF locus, inserting a nucleic acid encoding a human SIRPA polypeptide, replacing the mouse TPO gene with a nucleic acid encoding a human TPO polypeptide at the mouse TPO locus, Recombinant activation gene 2 (Rag-2) gene knockout and, IL2 receptor gamma chain (IL2rg) gene knockout; wherein each of the nucleic acids encoding human M-CSF polypeptide, human IL-3 polypeptide, human GM-CSF polypeptide, human SIRPA polypeptide, and human TPO polypeptide is operably linked to a promoter; wherein the mouse expresses human M-CSF polypeptide, human IL-3 polypeptide, human GM-CSF polypeptide, human SIRPA polypeptide, and human TPO polypeptide; and wherein the genetically modified immunodeficient mouse is transplanted with human hematopoietic cells.

8. The method of claim 7, wherein the human hematopoietic cells comprise CD34+ cells.

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