Transgenic mouse model expressing human HLA-A201-restricted genes

Abstract

The present disclosure provides an immunodeficient NOD.Cg-Prkdc mouse comprising an inactivated mouse Flt3 allele, a nucleic acid encoding human interleukin 3 (IL3), a nucleic acid encoding human granulocyte / macrophage stimulating factor (GM-CSF), a nucleic acid encoding human stem cell factor (SCF), and an HLA-A2 / H2-D / B2M transgene encoding (i) human B2-microglobulin (B2M) covalently linked to the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene and (ii) the α3 cytoplasmic and transmembrane domains of mouse H2-Db. scid Il2rg tm1Wjl The / SzJ (NSG™) mouse model is provided.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 049,187, filed July 8, 2020, which is incorporated by reference herein in its entirety. [Background technology]

[0002] background Mouse models are widely used to study human diseases in vivo, avoiding the complexities of working with human patients. Nevertheless, mouse models often poorly recapitulate human diseases, in part due to significant differences between the mouse and human immune systems (Hagai et al., 2018; Kanazawa, 2007; Mestas & Hughes, 2004; Williams, Flavell, & Eisenbarth, 2010). Therefore, humanized mice, defined as mice with a human immune system, may be an attractive alternative (Shultz, Brehm, Garcia-Martinez, & Greiner, 2012; Theocharides, Rongvaux, Fritsch, Flavell, & Manz, 2016; Victor Garcia, 2016; Zhang & Su, 2012). To this end, we developed a NOD-SCID-Il2γc mouse model. - / - (NSG) or BALB / c-Rag2 - / - -γc - / - Immunodeficient mice lacking the common gamma chain (γc) (BRG) ( Matsumura et al., 2003 ; Traggiai et al., 2004 ) were cultured with human CD34 + Humanization can be achieved by transplantation of hematopoietic progenitor cells (HPCs). Based on the source of T cells, models can be further classified into two types: (1) models in which mature T cells are isolated from HPC donors and adoptively transferred (Aspord et al., 2007; Pedroza-Gonzalez et al., 2011; Wu et al., 2014; Wu et al., 2018; Yu et al., 2008); in this case, T cells are selected in the human thymus; and (2) models in which human CD34 T cells are selected in the human thymus.+ A model in which endogenous T cells are generated de novo from HPCs (Matsumura et al., 2003; Traggiai et al., 2004); in this case, human T cells are selected in the mouse thymus. Summary of the Invention [Means for solving the problem]

[0003] overview The present disclosure provides a humanized mouse model expressing the HLA-A201-restricted gene. One important aspect of humanized mouse research is the maturation of human adaptive immunity in the context of human MHC (Billerbeck et al., 2013; Danner et al., 2011; Najima et al., 2016). This mouse model was created, in part, to support antigen presentation on human HLA and to match hematopoietic progenitor cell (HPC) donors to the mice. This mouse model, among other things, addresses the limitations of the previous models. The biggest limitation of the first model, in which mature T cells are isolated from HPC donors and adoptively transferred, is graft-versus-host disease, where endogenous T cells express human CD34. + The biggest limitation of the second model, generated de novo from HPCs, is the limited number of T cells that can recognize human major histocompatibility complexes (MHC).

[0004] Accordingly, some aspects of the disclosure include a non-obese diabetic (NOD) mouse, comprising: an inactivated mouse Prkdc allele; an inactivated mouse IL2rg allele; an inactivated mouse Flt3 allele; a nucleic acid encoding human interleukin 3 (IL3); and a nucleic acid encoding human granulocyte / macrophage stimulating factor (GM-CSF). a nucleic acid encoding human stem cell factor (SCF); and an MHC class 1, α1 and α2 binding domain of the human HLA-A2.1 gene. , and the α3, cytoplasmic and transmembrane domains of mouse H2-Db Human B2-microglobulin (B2M) covalently bound to )( HLA-A2 / H2-D / B2M )ofand a nucleic acid encoding a human Flt3 allele. A further aspect of the present disclosure provides an NSG™ mouse comprising an inactivated mouse Flt3 allele; a nucleic acid encoding human IL3; a nucleic acid encoding human GM-CSF; a nucleic acid encoding human SCF; and a nucleic acid encoding HLA-A2 / H2-D / B2M. These mouse models support antigen presentation on human HLA and allow for matching of hematopoietic progenitor cell (HPC) donor mice.

[0005] Also provided herein are methods for generating NOD mice comprising an inactivated mouse Prkdc allele, an inactivated mouse IL2rg allele, an inactivated mouse Flt3 allele, a nucleic acid encoding human IL3, a nucleic acid encoding human GM-CSF, a nucleic acid encoding human SCF, and a nucleic acid encoding human HLA-A2 / H2-D / B2M, methods for using the mice as a model system, and methods for breeding the mice.

[0006] Further provided herein is an NSG™ cell comprising a nucleic acid encoding a human ILS, a nucleic acid encoding human GM-CSF, a nucleic acid encoding human SCF, and a transgene encoding human HLA-A2 / H2-D / B2M. [Brief explanation of the drawings]

[0007] [Figure 1-1] Figures 1A-1C show human engraftment of human CD34+ HPCs from different sources in NSG-SGM3F-A2 mice. Figure 1A is a schematic diagram showing the mating scheme of NSG-SGM3F-A2 mice. Figure 1B shows HLA-A2 expression on mCD45+ cells in the blood of 4-week-old mice. Figure 1C shows graphs measuring the absolute number of hCD45+ cells and the percentage of human CD33+, CD19+, and CD3+ cells in hNSG-SGM3F-A2 mice 12 weeks after transplantation of human fetal liver, umbilical cord blood, and bone marrow HPCs. [Figure 1-2] Same as above. [Figure 1-3]Same as above.

[0008] [Figure 2-1] Figures 2A-2D show a comparison of human engraftment in humanized SGM3F-A2 mice transplanted with human umbilical cord blood or fetal liver HPCs. Figure 2A shows human engraftment measured in the blood by the percentage and absolute number of hCD45+ cells in hSGM3F-A2 mice 12 weeks after transplantation of 1 x 105 umbilical cord blood (CB) or fetal liver (FL) HPCs. n = 91 mice from five CB donors, n = 95 mice from four FL donors. Nested t-test. Figure 2B shows the absolute numbers of hCD33+, hCD19+, and hCD3+ cells in hSGM3F-A2 mice. Figure 2C shows the absolute numbers of human CD4+ and CD8+ T cells in the blood of hSGM3F-A2 mice. Figure 2D shows total human IgM, IgG, and IgA measured in the plasma of hSGM3F-A2 mice 12 weeks after transplantation by ELISA. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description The present disclosure provides mouse models that support antigen presentation on human HLA and match hematopoietic progenitor cell (HPC) donors to mice. The mouse models provided herein are In one aspect, the present invention provides a method for the production of a mouse Flt3 allele having a NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG™) background, comprising: a nucleic acid encoding human interleukin 3 (IL3); a nucleic acid encoding human granulocyte-macrophage colony-stimulating factor (GM-CSF); a nucleic acid encoding human stem cell factor (SCF); and an MHC class 1, α1, and α2 binding domain of the human HLA-A2.1 gene. , and the α3, cytoplasmic and transmembrane domains of mouse H2-Db Human B2-microglobulin (B2M) covalently bound to )of(Herein referred to as an NSG-SGM3F-A2 mouse.) In some embodiments, the genotype of the NSG-SGM3F-A2 mouse model is NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(HLA-A / H2-D / B2M) 1Dvs / SzJ Flt3 em1Akp Tg(CMV-IL3, CSF2, KITLG) 1Eav / MloySzJ (NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(HLA-A / H2-D / B2M) 1Dvs / SzJ Flt3 em1Akp Tg(CMV-IL3, CSF2, KITLG) 1Eav / MloySzJ (See Example 1 for an exemplary method of generating mice.) In some embodiments, the NOD.Cg-Prkdc scid Il2rg tm1Wjl -Flt3 em1Akp Tg(CMV-IL3, CSF2, KITLG) 1Eav / MloySzJ (SGM3F) mice are NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(HLA-A / H2-D / B2M) 1Dvs / SzJ Flt3 em1Akp Tg(CMV-IL3, CSF2, KITLG) 1Eav / MloySzJ To obtain mice, they are crossed with HLA-A0201 transgenic mice (NSG-A2(HHD)) and inbred until all offspring are homozygous.

[0010] NSG™ mice are immunodeficient mice that lack mature T cells, B cells, and natural killer (NK) cells, are defective in multiple cytokine signaling pathways, and have numerous defects in innate immunity (see, e.g., (Shultz, Ishikawa, & Greiner, 2007; Shultz et al., 2005; Shultz et al., 1995), each of which is incorporated herein by reference). NSG™ mice, derived from the non-obese diabetic (NOD) mouse strain NOD / ShiLtJ (see, e.g., (Makino et al., 1980), incorporated herein by reference), express Prkdc scid mutation (also called "severe combined immunodeficiency" mutation or "scid" mutation) and Il2rg tm1Wjl Contains targeted mutations. Prkdc scid The mutation is a loss-of-function mutation in the mouse homolog of the human PRKDC gene, which mutation essentially eliminates adaptive immunity (see, e.g., Blunt et al., 1995; Greiner, Hesslton, & Shultz, 1998, each of which is incorporated herein by reference). tm1Wjl The mutation is a null mutation in the gene encoding the interleukin-2 receptor gamma chain (IL2Rγ, homologous to IL2RG in humans), which blocks NK cell differentiation, thereby removing the obstacle that prevents efficient engraftment of primary human cells (Cao et al., 1995; Greiner et al., 1998; Shultz et al., 2005), each of which is incorporated herein by reference. As is known in the art, a loss-of-function mutation results in a gene product with little or no function. In comparison, a null mutation results in a gene product with no function. The inactivated allele can be a loss-of-function allele or a null allele.

[0011] An inactivated allele is an allele that does not produce a detectable level of a functional gene product (e.g., a functional protein). In some embodiments, an inactivated allele is not transcribed. In some embodiments, an inactivated allele does not encode a functional protein. Thus, a mouse comprising an inactivated mouse Flt3 allele does not produce a detectable level of functional FLT3. In some embodiments, a mouse comprising an inactivated mouse Flt3 allele does not produce functional FLT3.

[0012] Flt3 is a receptor important for the development of dendritic cells and monocyte lineages. Flt3L-Flt3 signaling is important for the development of various DC and monocyte lineages (Ding et al., 2014; Ginhoux et al., 2009; McKenna et al., 2000; Waskow et al., 2008), and its role is further supported by the increase in circulating conventional (c)DCs and plasmacytoid (p)DCs after in vivo administration of Flt3L in mice and humans (Karsunky, Merad, Cozzio, Weissman, & Manz, 2003; Maraskovsky et al., 1996; Pulendran et al., 2000). Knocking out mouse Flt3 can result in (1) a decrease in mouse DCs and other myeloid cells; and (2) an increased availability of mouse Flt3L (which can act via the human receptor) to human cells, thereby enhancing the expression of human CD34. + Improve long-term development of human myeloid cells upon transplantation of HPCs.

[0013] The NSG-SGM3F-A2 mouse model provided herein comprises a genomic modification that inactivates mouse Flt3 alleles.A modification relating to nucleic acid is any manipulation of nucleic acid compared to corresponding wild-type nucleic acid (e.g., naturally occurring nucleic acid).Thus, a genomic modification is any manipulation of nucleic acid in genome compared to corresponding wild-type nucleic acid (e.g., naturally occurring nucleic acid) in genome.Non-limiting examples of nucleic acid (e.g., genomic) modification include deletion, insertion, "indel" (deletion and insertion), and substitution (e.g., point mutation).In some embodiments, deletion, insertion, indel, or other modification in gene results in a frameshift mutation such that the gene no longer encodes a functional product (e.g., protein).Modification also includes chemical modification, for example, chemical modification of at least one nucleic acid base. The method of nucleic acid modification, for example, the method of causing gene inactivation, is known, and includes but is not limited to RNA interference, chemical modification and gene editing (for example, using recombinase or other programmable nuclease system, for example, CRISPR / Cas, TALEN and / or ZFN).In some embodiments, as described elsewhere herein, CRISPR / Cas gene editing is used to inactivate mouse Flt3 allele.

[0014] In some embodiments, the genomic modification (e.g., a deletion or indel) is in (at least one) region of the mouse Flt3 allele selected from a coding region, a non-coding region, and a regulatory region. In some embodiments, the genomic modification (e.g., a deletion or indel) is in the coding region of the mouse Flt3 allele. For example, the genomic modification (e.g., a deletion or indel) can be in exon 3 or can span exon 3 of the mouse Flt3 allele. In some embodiments, the genomic modification is a genomic deletion. For example, the mouse Flt3 allele can contain a genomic deletion of a nucleotide sequence in exon 3. In some embodiments, the nucleotide sequence of SEQ ID NO: 1 is deleted from the inactivated mouse Flt3 allele. In some embodiments, the inactivated mouse Flt3 allele contains the nucleotide sequence of SEQ ID NO: 1.

[0015] In some embodiments, the NSG-SGM3F-A2 mouse model provided herein does not express detectable levels of mouse FLT3. A detectable level of mouse FLT3 is any level of FLT3 protein detected using standard protein detection assays such as flow cytometry and / or ELISA. In some embodiments, the NSG-SGM3F-A2 mouse model expresses undetectable or low levels of mouse FLT3. For example, the mouse model may express less than 1,000 pg / ml of mouse FLT3. In some embodiments, the mouse model expresses less than 500 pg / ml of mouse FLT3 or less than 100 pg / ml of mouse FLT3. The mouse FLT3 receptor is also referred to as the cluster of differentiation antigen CD135. Thus, in some embodiments, the NSG-SGM3F-A2 mouse model expresses CD135. + Does not contain (does not exist in) multipotent progenitor cells.

[0016] In some embodiments, Flt3 knockout mice are generated by CRISPR using Cas9 mRNA and guide RNA (gRNA). In some embodiments, the gRNA (e.g., 5'-AAGTGCAGCTCGCCACCCCA-3', SEQ ID NO: 2) is generated from mouse Flt3NSG™ mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl -Flt3 em1Akp ;RRID:IMSR JAX:005557) target exon 3. Blastocysts derived from the injected embryos are, in some embodiments, implanted into foster mothers to obtain newborns. In some embodiments, mice with the null deletion are backcrossed to NSG™. F0 and F1 littermates can be tested for successful gene knockout by, for example, PCR and Sanger sequencing. For example, to detect the mouse Flt3 wild-type allele from the mutant allele, primers (5'-GGTACCAGCAGAGTTGGATAGC-3', SEQ ID NO:3) and (5'-ATCCCTTACACAGAAGCTGGAG-3', SEQ ID NO:4) can be used in a PCR reaction (Table 1). The WT allele generates a 799-bp DNA fragment, while the mutated allele generates a 363-bp DNA fragment. Transgenic mouse models

[0017] Transgenic mouse models (Tg mice) can be created to modify gene sequences, for example, by replacing gene sequences with transgenes or by adding gene sequences not found within the locus. The NSG-SGM3F-A2 mouse model provided herein contains a transgenic allele. These mouse models contain exogenous nucleic acids introduced into the mouse genome.

[0018] Nucleic acids used as provided herein can be DNA, RNA, or a chimera of DNA and RNA. In some embodiments, the nucleic acid (e.g., DNA) comprises a gene encoding a specific protein of interest. A gene is a distinct sequence of nucleotides, the order of which determines the order of monomers in a polynucleotide or polypeptide. Genes typically encode proteins. Genes can be endogenous (naturally occurring in a host organism) or exogenous (introduced into a host organism naturally or through genetic engineering). An allele arises through mutation and is one of two or more alternative forms of a gene found at the same locus on a chromosome. In some embodiments, a gene comprises a promoter sequence, a coding region (e.g., exons), a non-coding region (e.g., introns), and a regulatory region (also called a regulatory sequence). As known in the art, a promoter sequence is a DNA sequence at which transcription of a gene begins. A promoter sequence is typically located immediately upstream (5' end) of the transcription start site. An exon is a region of a gene that encodes amino acids. Introns (and other non-coding DNA) are regions of genes that do not code for amino acids.

[0019] A mouse containing a human gene is considered to contain a human transgene. A transgene is a gene that is exogenous to the host organism. That is, a transgene is a gene that has been introduced into the host organism, either naturally or through genetic engineering. A transgene does not naturally occur in the host organism (the organism containing the transgene, e.g., a mouse).

[0020] Methods for generating transgenic mouse models are described elsewhere herein.

[0021] The NSG-SGM3F-A2 mice described herein are derived from inactivated mouse Fl the t3 allele, a nucleic acid encoding IL3, a nucleic acid encoding GM-CSF, a nucleic acid encoding SCF, and the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene , and the α3, cytoplasmic and transmembrane domains of mouse H2-DbHuman B2-microglobulin (B2M) covalently bound to )of In some embodiments, the NSG-SGM3F-A2 mice described herein comprise an inactivated mouse Flt3 allele, a nucleic acid encoding human IL3, a nucleic acid encoding human GM-CSF, a nucleic acid encoding human SCF, and the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene. , and the α3, cytoplasmic and transmembrane domains of mouse H2-Db Human B2-microglobulin (B2M) covalently bound to )of In some embodiments, the NSG-SGM3F-A2 mouse comprises a human IL3 transgene, a human GM-CSF transgene, a human SCF transgene, and a human HLA-A2 / H2-D / B2M transgene (which encodes the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene). , and the α3, cytoplasmic and transmembrane domains of mouse H2-Db Human B2-microglobulin (B2M) covalently bound to )of In some embodiments, a transgene such as a human IL3 transgene, a human GM-CSF transgene, a human SCF transgene, and / or a human HLA-A2 / H2-D / B2M transgene is integrated into the mouse genome. Human IL3, CSF2, and KITLG transgenes have been described in (Nicolini, Cashman, Hogge, Humphries, & Eaves, 2004), which is incorporated herein by reference. Human HLA-A2 / H2-D / B2M transgenes have been described (Pascolo et al., 1997; Takaki et al., 2006), which is incorporated herein by reference.

[0022] In some embodiments, NSG-SGM3F-A2 mice are derived from NSG-HLA-A2 / HHD mice (RRID:IMSR JAX:014570) bred into SGM3F mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl -Flt3 em1Akp Tg(CMV-IL3, CSF2, KITLG)1Eav / MloySzJ NSG-SGM3 mice are generated by crossing NSG-SGM3 mice to BALB / c-scid / scid mice. NSG-SGM3 mice carry three separate transgenes, each designed to carry one of the following genes: human interleukin-3 (IL3), human granulocyte / macrophage-stimulating factor (GM-CSF), or human stem cell factor (SCF). Expression of each gene is driven by a human cytomegalovirus promoter / enhancer sequence, followed by a human growth hormone cassette and polyadenylation (polyA) sequence. The transgenes were microinjected into fertilized C57BL / 6xC3H / HeN oocytes. In some embodiments, the resulting founders carrying all three transgenes (3GS) are backcrossed to BALB / c-scid / scid mice for several generations and then backcrossed to NOD.CB17-Prkdcscid mice for multiple (e.g., at least 11) generations (Nicolini et al., 2004; Wunderlich et al., 2010). These mice are then cloned, for example, into NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl ;RRID:IMSR JAX:005557), and then can be inbred until all offspring are homozygous for the 3GS and IL2rg-targeted mutations. The transgenic mice can be bred to NSG mice for at least one generation to establish NSG-SGM3 mice. NSGF mice can be generated, for example, using the CRISPR / cas system. In some embodiments, Cas9 mRNA and sgRNA targeting mouse Flt3 are co-injected into fertilized NSG oocytes. The resulting founders with Flt3 deletion can be bred to NSG mice, and then can be inbred until all offspring are homozygous for the Flt3-targeted mutations. NSG-SGM3 mice can be bred to NSGF mice for multiple (e.g., 2) generations to establish NSG-SGM3F mice. H-2Db - / - B2m - / -HLA-A2 / HHD transgenic expression in mice restores CD8+ T cells and enables HLA-A2.1-restricted cytotoxic T cell responses (Pascolo et al., 1997). NSG-HLA-A2 / HHD mice can then be bred to NSG-SGM3F mice for multiple generations (e.g., at least four) to establish NSG-SGM3F-A2 mice. Human Immune System Model

[0023] The NSG-SGM3F-A2 mouse model of the present disclosure, in some embodiments, expresses human CD34 + It is used to support the development of hematopoietic progenitor cells (HPCs) and the human innate immune system. The human immune system includes the innate and adaptive immune systems. The innate immune system is responsible for recruiting immune cells to sites of infection, activating the complement cascade, enabling white blood cells to identify and remove foreign substances from the body, activating the adaptive immune system, and acting as a physical and chemical barrier against infectious agents.

[0024] In some embodiments, the NSG-SGM3F-A2 mouse model provided herein is sublethally irradiated (e.g., 100-300 cGy) to kill resident mouse HPCs, and the irradiated mice are then transfected with human CD34 to initiate the development of a human innate immune system. + In some embodiments, the mice are transplanted with human CD34 HPCs (e.g., 50,000 to 200,000 HPCs). + Further contains HPC. Human CD34 + HPCs can be derived from any source, including, but not limited to, human fetal liver, umbilical cord blood, mobilized peripheral blood, and bone marrow. In some embodiments, human CD34 + HPCs are derived from human umbilical cord blood.

[0025] Human CD34 +The differentiation of HPCs into various immune cells (e.g., T cells, B cells, dendritic cells) is a complex process in which successive developmental stages are regulated by multiple cytokines. This process can be monitored through cell surface antigens, such as cluster of differentiation (CD) antigens. For example, CD45 is expressed on the surface of HPCs, macrophages, monocytes, T cells, B cells, natural killer cells, and dendritic cells, and can therefore be used as a marker for engraftment. On T cells, CD45 regulates T cell receptor signaling, cell proliferation, and cell differentiation. In some embodiments, the NSG-SGM3F-A2 mouse model expresses human CD45 + In some embodiments, the NSG-SGM3F-A2 mouse model also translocates human CD45 cells to tissues in, but not limited to, the lung, thymus, spleen, lymph nodes, and / or small intestine. + Shows cell engraftment.

[0026] As CD45+ cells mature, they begin to express additional biomarkers indicative of various developmental stages and differentiated cell types. For example, developing T cells also express CD3, CD4, and CD8. As another example, developing myeloid cells express CD33 + The mouse models herein, in some embodiments, express human CD45 + cells as well as double-positive human CD45 + / CD3 + Also included are T cells and double-positive human CD45+ / CD33+ myeloid cells.

[0027] Thus, in some embodiments, human CD45 in the NSG-SGM3F-A2 mouse model + The cell population is human CD45 + / CD3 + In some embodiments, the T cells are human CD45 + The population of cells contained an increased percentage of human CD45 cells compared to NSG™ control mice. + / CD3 +In some embodiments, the NSG-SGM3F-A2 mouse model comprises human CD45 T cells. + / CD3 + The percentage of T cells is increased by at least 25% compared to NSG™ control mice. For example, the percentage of human CD45 T cells in the mouse model is increased by at least 25% compared to NSG™ control mice. + / CD3 + The percentage of T cells may be increased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to NSG™ control mice. + / CD3 + The percentage of T cells is increased by at least 50% compared to NSG™ control mice. In some embodiments, the percentage of human CD45 T cells in the mouse model is increased by at least 50% compared to NSG™ control mice. + / CD3 + The percentage of T cells is increased by at least 100% compared to NSG™ control mice. In some embodiments, the percentage of human CD45 T cells in the mouse model is increased by at least 100% compared to NSG™ control mice. + / CD3 + The percentage of T cells is increased by 25%-100%, 25%-75%, 25%-50%, 50%-100%, 50%-75%, or 75%-100% compared to NSG™ control mice.

[0028] In some embodiments, human CD45 in the NSG-SGM3F-A2 mouse model + The cell population is human CD45 + / CD33 + In some embodiments, the human CD45 + The population of cells contained an increased percentage of human CD45 cells compared to NSG™ control mice. + / CD33 + In some embodiments, the mouse model includes human CD45 cells. + / CD33 +The percentage of T cells is increased by at least 25% compared to NSG™ control mice. For example, the percentage of human CD45 T cells in the mouse model is increased by at least 25% compared to NSG™ control mice. + / CD33 + The percentage of myeloid cells may be increased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to NSG™ control mice. + / CD33 + The percentage of myeloid cells is increased by at least 50% compared to NSG™ control mice. In some embodiments, the human CD45 + / CD33 + The percentage of myeloid cells is increased by at least 100% compared to NSG™ control mice. In some embodiments, human CD45 + / CD33 + The percentage of myeloid cells is increased by 25%-100%, 25%-75%, 25%-50%, 50%-100%, 50%-75%, or 75%-100% compared to NSG™ control mice.

[0029] In some embodiments, human CD45 in the NSG-SGM3F-A2 mouse model + The cell population is human CD45 + / CD19 + In some embodiments, the human CD45 + The population of cells contained an increased percentage of human CD45 cells compared to NSG™ control mice. + / CD19 + In some embodiments, the mouse model includes human CD45 B cells. + / CD19 + The percentage of B cells is increased by at least 25% compared to NSG™ control mice. For example, the percentage of B cells in the mouse model is increased by at least 25% compared to NSG™ control mice. + / CD19+ The percentage of B cells may be increased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to NSG™ control mice. + / CD19 + The percentage of B cells is increased by at least 50% compared to NSG™ control mice. In some embodiments, human CD45 + / CD19 + The percentage of B cells is increased by at least 100% compared to NSG™ control mice. In some embodiments, the percentage of human CD45 + / CD19 + The percentage of B cells is increased by 25%-100%, 25%-75%, 25%-50%, 50%-100%, 50%-75%, or 75%-100% compared to NSG™ control mice.

[0030] The NSG-SGM3F-A2 mouse model provided herein surprisingly also supports the engraftment of dendritic cells (e.g., plasmacytoid dendritic cells and myeloid dendritic cells), natural killer cells, and monocyte-derived macrophages (monocyto-macrophages). Plasmacytoid dendritic cells (pDCs) secrete high levels of interferon-α; myeloid dendritic cells (mDCs) secrete interleukin-12, interleukin-6, tumor necrosis factor, and chemokines; natural killer cells destroy damaged host cells, such as tumor cells and virus-infected cells; and macrophages phagocytose significant numbers of bacteria or other cells or microorganisms.

[0031] In some embodiments, the NSG-SGM3F-A2 mouse model expresses an increased percentage of human CD11c compared to NSG™ control mice and / or NSGF control mice. +In some embodiments, the expression of human CD11c in NSG-SGM3F-A2 mice includes myeloid dendritic cells. + HLA-DR + The percentage of myeloid dendritic cells is increased by at least 25% compared to NSG™ control mice and / or NSGF control mice. For example, human CD11C + HLA-DR + The percentage of myeloid dendritic cells may be increased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to NSG™ control mice and / or NSGF control mice. + HLA-DR + The percentage of myeloid dendritic cells is increased by at least 50% compared to NSG™ control mice and / or NSGF control mice. In some embodiments, human CD11c in NSG-SGM3F-A2 mice + HLA-DR + The percentage of myeloid dendritic cells is increased by at least 100% compared to NSG™ control mice and / or NSGF control mice. In some embodiments, human CD11c in NSG-SGM3F-A2 mice + HLA-DR + The percentage of myeloid dendritic cells is increased by 25% to 100%, 25% to 75%, 25% to 50%, 50% to 100%, 50% to 75%, or 75% to 100% compared to NSG™ control mice and / or NSGF control mice.

[0032] In some embodiments, the NSG-SGM3F-A2 mouse model of the present disclosure is used to support engraftment of HLA-A2-matched hematopoietic lineages. Methods for producing transgenic animals

[0033] In some aspects, provided herein is a method for producing a transgenic animal that expresses a human transgene. Transgenic animal, as used herein, refers to an animal that has a foreign (exogenous) nucleic acid (e.g., a transgene) inserted (integrated) into its genome. In some embodiments, the transgenic animal is a transgenic rodent such as a mouse or a rat. In some embodiments, the transgenic animal is a mouse. Three conventional methods used to produce transgenic animals include DNA microinjection (Gordon & Ruddle, 1981), incorporated herein by reference), embryonic stem cell-mediated gene transfer (Gossler, Doetschman, Korn, Serfling, & Kemler, 1986), incorporated herein by reference), and retrovirus-mediated gene transfer (Jaenisch, 1976), incorporated herein by reference), any of which can be used as provided herein. Electroporation can also be used to generate transgenic mice (see WO 2016 / 054032 and WO 2017 / 124086, each of which is incorporated herein by reference).

[0034] In some embodiments, the nucleic acid comprises a transgene, for example, a transgene comprising a promoter (e.g., a constitutively active promoter) operably linked to a nucleotide sequence encoding a polypeptide of interest. In some embodiments, the nucleic acid used to generate a transgenic animal (e.g., a mouse) is present on a vector, such as a plasmid, bacterial artificial chromosome (BAC), or yeast artificial chromosome (YAC), which is delivered to the pronucleus / nucleus of a fertilized embryo, where the nucleic acid is randomly integrated into the animal genome. In some embodiments, the fertilized embryo is a single-cell embryo (e.g., a zygote). In some embodiments, the fertilized embryo is a multicellular embryo (e.g., a developmental stage subsequent to the zygote, such as a blastocyst). In some embodiments, to generate a mouse model of the present disclosure, a nucleic acid (e.g., carried on a BAC) is delivered to a fertilized mouse embryo. After injection of the fertilized embryo, the fertilized embryo can be introduced into a pseudopregnant female, which then gives birth to offspring comprising a nucleic acid encoding a polypeptide of interest. The presence or absence of a nucleic acid can be confirmed, for example, using any number of genotyping methods (eg, sequencing and / or genomic PCR).

[0035] Also provided herein is a method for inactivating endogenous Flt3 allele.In some embodiments, endogenous Flt3 allele is inactivated in transgenic animals.In some embodiments, gene / genome editing method is used for gene (allele) inactivation.The engineered nuclease-based gene editing system that can be used as described herein includes, for example, clustered regularly interspaced short palindromic repeats (CRISPR) system, zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN).For example, see (Carroll, 2011; Gaj, Gersbach, & Barbas, 2013; Joung & Sander, 2013), each of which is incorporated herein by reference.

[0036] In some embodiments, CRISPR system is used to inactivate the endogenous Flt3 allele of the NSG-SGM3F-A2 mouse model provided herein.For example, see (Harms et al., 2014; Inui et al., 2014), each of which is incorporated herein by reference.For example, to generate precise genome editing in the Flt3 gene, Cas9 mRNA or protein and one or more guide RNAs (gRNAs) can be directly injected into mouse embryos.The mice that develop from these embryos can be genotyped or sequenced to determine whether they carry the desired mutation, and the mice that carry the desired mutation can be bred to confirm germline transmission.

[0037] The CRISPR / Cas system is a naturally occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA targeting platform for gene editing. Engineered CRISPR systems contain two main components: a guide RNA (gRNA) and a CRISPR-associated endonuclease (e.g., a Cas protein). The gRNA is a short synthetic RNA composed of a scaffold sequence for nuclease binding and a user-defined nucleotide spacer (e.g., about 15-25 nucleotides, or about 20 nucleotides) that defines the genomic target to be modified. Therefore, the genomic target of the Cas protein can be changed simply by changing the target sequence present in the gRNA. In some embodiments, the CRISPR-associated endonuclease is selected from Cas9, Cpf1, C2c1, and C2c3. In some embodiments, the Cas nuclease is Cas9.

[0038] Guide RNA comprises at least a spacer sequence that hybridizes (binds) to the target nucleic acid sequence and a CRISPR repeat sequence that binds to an endonuclease and guides the endonuclease to the target nucleic acid sequence. As will be understood by those skilled in the art, each gRNA is designed to comprise a spacer sequence that is complementary to its genomic target sequence (e.g., a region of an Flt3 allele). For example, see (Deltcheva et al., 2011; Jinek et al., 2012), each of which is incorporated herein by reference. In some embodiments, the gRNA used in the methods provided herein binds to a region of a mouse Flt3 allele (e.g., exon 3). In some embodiments, the gRNA that binds to a region of a mouse Flt3 allele comprises the nucleotide sequence 5'-AAGTGCAGCTCGCCACCCCA-3' (SEQ ID NO: 2). How to use

[0039] The NSG-SGM3F-A2 mouse model provided herein can be used for a number of purposes. For example, the mouse model can be used to test how a particular agent (e.g., a therapeutic agent) or medical treatment (e.g., a tissue transplant) affects the human innate immune system (e.g., human innate immune cell response) and the human adaptive immune system (e.g., antibody response).

[0040] In some embodiments, a mouse model is used to evaluate the effect of an agent on the development of the human innate immune system. Accordingly, provided herein is a method comprising administering an agent to a mouse model and evaluating the effect of the agent on the development of the human innate immune system in mice. The effect of an agent can be evaluated, for example, by measuring human innate immune cell (e.g., T cell and / or dendritic cell) responses (e.g., cell death, cell signaling, cell proliferation, etc.) and human adaptive immune responses (e.g., antibody production). Non-limiting examples of agents include therapeutic agents such as anti-cancer agents and anti-inflammatory agents, and prophylactic agents such as immunogenic compositions (e.g., vaccines).

[0041] In other embodiments, mouse models are used to evaluate immunotherapy responses to human tumors. Thus, provided herein are methods that include administering an agent to a mouse model bearing a human tumor and evaluating the effect of the agent on the human innate immune system and / or on the tumor in the mouse. The effect of the agent can be evaluated by measuring human innate immune cell (e.g., T cell and / or dendritic cell) response, human adaptive immune response (e.g., antibody production) and / or tumor cell response (e.g., cell death, cell signaling, cell proliferation, etc.). In some embodiments, the agent is an anti-cancer agent.

[0042] In yet another embodiment, the mouse model is used to evaluate human innate immune responses to infectious microorganisms. Thus, provided herein is a method comprising exposing the mouse model to infectious microorganisms (e.g., bacteria and / or viruses) and evaluating the impact of the infectious microorganisms on human innate immune responses. The impact of infectious microorganisms can be evaluated by measuring human innate immune cell (e.g., T cell and / or dendritic cell) responses (e.g., cell death, cell signaling, cell proliferation, etc.). These methods can further comprise administering a drug or antimicrobial agent (e.g., an antibacterial agent or antiviral agent) to the mouse and evaluating the effect of the drug or antimicrobial agent on the infectious microorganism.

[0043] In yet another embodiment, mouse model is used to evaluate the human immune response to tissue transplantation.Therefore, provided herein is a method, which includes transplanting tissue (for example, allogeneic tissue) into mouse model, and evaluating the effect of transplanted tissue on human innate immune response.The effect of transplanted tissue can be evaluated by measuring the response (for example, cell death, cell signaling, cell proliferation, etc.) of human innate immune cell (for example, T cell and / or dendritic cell) to transplanted tissue and human adaptive immune response (for example, antibody production). [Example]

[0044] Example Example 1. NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(HLA-A / H2-D / B2M) 1Dvs / SzJ Flt3 em1Akp Tg(CMV-IL3, CSF2, KITLG) 1Eav / MloySzJ (NSG-SGM3F-A2) mouse model One important aspect of humanized mouse research is the maturation of human adaptive immunity in the context of human MHC (Billerbeck et al., 2013; Danner et al., 2011; Najima et al., 2016). To support antigen presentation on human HLA and to match HPC donors with mice, we used SGM3F (NOD.Cg-Prkdc scid Il2rg tm1Wjl -Flt3 em1Akp Tg(CMV-IL3, CSF2, KITLG) 1Eav / MloySzJ ) mice were crossed with HLA-A0201 transgenic mice (NSG-A2(HHD)) and inbred until all offspring were homozygous to generate NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(HLA-A / H2-D / B2M) 1Dvs / SzJ Flt3 em1Akp Tg(CMV-IL3, CSF2, KITLG) 1Eav / MloySzJ We obtained NSG-SGM3F-A2 mice (Figure 1A). SGM3F mice combine the characteristics of NSG mice (NSG-SGM3, SGM3) with transgenic expression of human stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-3 (Nicolini et al., 2004; Wunderlich et al., 2010) and NSG mice with Flt3 mutant mice (NSGF). To confirm the expression of human HLA-A0201, we measured and confirmed the surface expression of HLA-A2 on mouse bone marrow cells (Figure 1B). To test their ability to support engraftment of the human immune system, NSG-SGM3F-A2 mice were sublethally irradiated and transfected with 1 × 10 human fetal liver, umbilical cord blood, or adult bone marrow cells. 5 HLA-A2 + CD34+ Mice receiving both fetal liver and cord blood HPCs had comparable immune cell composition in their blood, whereas mice receiving bone marrow HPCs had fewer CD3 + Furthermore, 6 months after transplantation of FL, CB, or BM HPCs, we observed CD11c T cells. + DC and CD3 + High abundance of hCD45 in the lungs harbors different human immune cells that are important for both innate and adaptive immune responses, including T cells. + Immune cells were observed (data not shown). Importantly, HLA-A2 + Six months after CB HPC transplantation, HLA-A2 was detected using the BB7.2 antibody specific for HLA-A2. + Thymic HLA-A2 expression was detected on mouse thymic epithelial cells of hNSG-SGM3F-A2 mice reconstituted with HPCs (data not shown), allowing T cell maturation in the context of human HLA-A2. Example 2. Comparison of human engraftment in humanized SGM3F-A2 mice transplanted with human umbilical cord blood or fetal liver HPCs.

[0045] Due to the limited availability of human fetal tissue, we validated the use of umbilical cord blood-derived HPCs to construct humanized mice. Controlled comparisons were performed on different cell types from cohorts of mice transplanted with 4–5 different umbilical cord blood or fetal liver donors. Data showed that humanized mice transplanted with fetal liver HPCs had slightly higher hCD45 HPCs at 12 weeks post-transplant due to the expansion of hCD19+ B cells and hCD3+ T cells. + In mice transplanted with fetal liver HPCs, only hCD4 + A slight increase in T cells was observed, but hCD8 +No differences were observed in the total number of T cells (Figure 2C). To compare the functional capacity of fetal liver- or umbilical cord blood-derived human HPCs in mounting an adaptive humoral response, we assessed the ability of humanized NSG-SGM3F-A2 mice to produce human antibodies. To this end, we measured total human Ig in plasma at 12 weeks posttransplantation by ELISA. As shown in Figure 2D, both groups of mice secreted similar amounts of human IgM into plasma, as well as similar levels of total human IgG and IgA subclasses. Thus, the ability of antibody secretion and Ig class switching was comparable between different HPC sources in humanized NSG-SGM3F-A2 mice. Overall, we observed a higher level of variability in mice between different donors from the same HPC source than between HPC sources. This analysis demonstrated that cord blood HPCs provided comparable human engraftment to fetal liver HPCs in NSG-SGM3F-A2 mice.

[0046] Generation of the mouse model SGM3F: NSG-SGM3 mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(CMV-IL3, CSF2, KITLG) 1Eav / MloySzJ ;RRID:IMSR JAX:013062) to NSGF(NOD.Cg-Prkdc scid Il2rg tm1Wjl -Flt3 em1Akp ) mice by crossing them to NSG-SGM3-Flt3ko or SGM3F mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl -Flt3 em1Akp Tg(CMV-IL3, CSF2, KITLG) 1Eav / MloySzJNSG-SGM3 mice were generated and inbred until all offspring were homozygous. NSG-SGM3 mice carried three separate transgenes, each designed to carry either the human interleukin-3 (IL3) gene, the human granulocyte / macrophage-stimulating factor (GM-CSF) gene, or the human stem cell factor (SCF) gene. Expression of each gene was driven by a human cytomegalovirus promoter / enhancer sequence, followed by a human growth hormone cassette and polyadenylation (polyA) sequence. The transgenes were microinjected into fertilized C57BL / 6xC3H / HeN oocytes. The resulting founders carrying all three transgenes (3GS) were backcrossed to BALB / c-scid / scid mice for several generations and then backcrossed to NOD.CB17-Prkdcscid mice for at least 11 generations (Nicolini et al., 2004). These mice were then used as NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl;RRID:IMSR JAX:005557), and then inbred until all offspring were homozygous for the 3GS and IL2rg-targeted mutations. Upon arrival at The Jackson Laboratory, the transgenic mice were mated to NSG mice for one generation to establish NSG-SGM3 mice. NSGF mice were generated using the CRISPR / cas system by CRISPR using Cas9 mRNA and an sgRNA (5'-AAGTGCAGCTCGCCACCCCA-3', SEQ ID NO: 2) targeting exon 3 of mouse Flt3 in fertilized eggs of NSG mice. Blastocysts derived from the injected embryos were implanted into surrogate mothers to generate newborn pups. Mice with the null deletion were backcrossed to NSG. The tail tips of F0 and F1 littermates were removed and tested for successful gene knockout by PCR and Sanger sequencing. To detect the mouse Flt3 wild-type allele from the mutant allele, primers (5'-GGTACCAGCAGAGTTGGATAGC-3', SEQ ID NO: 3) and (5'-ATCCCTTACACAGAAGCTGGAG-3', SEQ ID NO: 4) were used in PCR reactions (Table 1). The WT allele generates a 799-bp-long DNA fragment, whereas the mutated allele generates a 363-bp-long DNA fragment.

[0047] Generation of the mouse model NSG-SGM3F-A2: NSG-HLA-A2 / HHD mice (RRID: IMSR JAX: 014570) were transfected with NSG-SGM3-Flt3ko or SGM3F mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl -Flt3 em1Akp Tg(CMV-IL3, CSF2, KITLG) 1Eav / MloySzJ ) to NSG-SGM3-Flt3ko-A2 or NSG-SGM3F-A2 mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl Tg(HLA-A / H2-D / B2M) 1Dvs / SzJ Flt3 em1Akp Tg(CMV-IL3, CSF2, KITLG) 1Eav / MloySzJNSG-SGM3-Flt3ko mice were generated. NSG-SGM3-Flt3ko mice carried three separate transgenes, each carrying either the human interleukin-3 (IL3) gene, the human granulocyte / macrophage-stimulating factor (GM-CSF) gene, or the human stem cell factor (SCF) gene. Expression of each gene was driven by a human cytomegalovirus promoter / enhancer sequence, followed by a human growth hormone cassette and polyadenylation (polyA) sequence. The transgenes were microinjected into fertilized C57BL / 6xC3H / HeN oocytes. The resulting founders carrying all three transgenes (3GS) were backcrossed to BALB / c-scid / scid mice for several generations and then backcrossed to NOD.CB17-Prkdcscid mice for at least 11 generations (Nicolini et al., 2004). These mice were then used as NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl ;RRID:IMSR JAX:005557) and then inbred until all offspring were homozygous for the 3GS and IL2rg-targeted mutations. Upon arrival at The Jackson Laboratory, the transgenic mice were mated to NSG mice for one generation to establish NSG-SGM3 mice. NSGF mice were generated using the CRISPR / cas system. Cas9 mRNA and sgRNA targeting mouse Flt3 were co-injected into fertilized NSG oocytes. The resulting founders with Flt3 deletion were mated to NSG mice and then inbred until all offspring were homozygous for the Flt3-targeted mutations. NSG-SGM3 mice were mated to NSGF mice for two generations to establish NSG-SGM3-Flt3ko mice. NSG-HLA-A2 / HHD mice express the MHC class 1, α1, and α2-binding domains of the human HLA-A2.1 gene. , and the α3, cytoplasmic and transmembrane domains of mouse H2-Db Human B2-microglobulin (B2M) covalently bound to )ofNSG-HLA-A2 / HHD mice carried the encoding HLA-A2 / H2-D / B2M transgene ( Pascolo et al., 1997 ; Shultz et al., 2010 ). NSG-HLA-A2 / HHD mice were crossed with NSG-SGM3F mice for four generations to establish NSG-SGM3F-A2 mice. Additional Materials and Methods humanized mice

[0048] Humanized mice were generated using different strains of NSG background mice obtained from The Jackson Laboratory (Bar Harbor, ME). All protocols were reviewed and approved by the Institutional Animal Care and Use Committees of The Jackson Laboratory (14005) and the University of Connecticut Health Center (101163-0220&101831-0321; Farmington, CT). Mice were sublethally irradiated (10 cGy per gram of body weight) using gamma irradiation at 4 weeks of age. One hundred thousand CD34 cells from fetal liver or full-term umbilical cord blood were used. + HPCs (Advanced Bioscience Resources or Lonza) were given by tail vein intravenous (IV) injection in 200 μL of PBS. Alternatively, mice were given adult CD34 IgG from bone marrow as indicated. + Mice were given HPCs (Lonza). To assess engraftment, blood was collected from mice 4–12 weeks after HPC transplantation and then euthanized according to individual experimental protocols. Flow cytometry analysis

[0049] Mice were euthanized, and blood was collected with heparin. Bones (femurs and tibias), spleens, and lungs were collected to prepare single-cell suspensions. Spleens were digested with 50 μg / ml Liberase (Roche Diagnostics, Indianapolis, IN) and 24 U / ml DNase I (Sigma) for 10 minutes at 37°C. Lungs were digested with 50 μg / ml Liberase and 24 U / ml DNase I (Sigma) for 30 minutes at 37°C, followed by mechanical dissociation using a GentleMACS (Miltenyi Biotec). Cells were first treated with mouse Fc blocker (BD) and then stained with an antibody cocktail for 30 minutes on ice. After two washes with PBS, samples were acquired on an LSRII or FACSARIA II (BD) and analyzed using FlowJo software (Tree Star, Ashland, OR). For human HLA-0201 expression, cells were stained with antibodies against mouse CD45-BV421 (30-F11, BD) and human HLA-A2-PE (BB7.2, BD). For human engraftment in blood, cells were stained with antibodies against mouse CD45-BV650 (30-F11, BD) and human CD45-BV510 (HI30, BD), CD33-PE (P67.6, Biolegend), CD14-PE-Cy7 (MqP9, BD), CD19-APC (HIB19, Biolegend), and CD3-APC-H7 (SK7, BD). Immunofluorescence staining

[0050] Tissues were embedded in OCT (Sakura Finetek USA) and flash-frozen in liquid nitrogen. Frozen sections were cut at 6 μm, air-dried on slides coated with Superfrost, and fixed in cold acetone for 5 minutes. Tissue sections were first treated with 0.03% hyaluronidase (Sigma) for 15 minutes, followed by background buster and Fc receptor block (Innovex Bioscience). Sections were then stained with monoclonal antibodies against human CD3 (UCHT1, Biolegend), CD11c (S-HCL-3, BD), HLA-A2 (BB7.2, BD), HLA-DR (L243, Biolegend), or pan-cytokeratin (AE1 / AE3, Miltenyi Biotech) for 1 hour at room temperature, followed by isotype-specific secondary antibodies for 30 minutes at room temperature. Each isotype antibody was used as a control. Finally, sections were counterstained with 1 μg / ml 4′,6-diamidino-2-phenylindole (DAPI), mounted with Fluoromount (Thermo Fisher Scientific), and visualized using a Leica SP8 confocal microscope with Leica LAS AF2.0 software or a Zeiss Axio fluorescence microscope with ZEN software. statistical analysis

[0051] Statistical analysis was performed using Prism (GraphPad). Comparisons between any two groups were analyzed using the Mann-Whitney test or two-tailed t-test. Comparisons between any three or more groups were analyzed by analysis of variance (ANOVA). Table 1. List of primers for mouse genotyping. [Table 1]

[0052] array SEQ ID NO: 1, Flt3 em1Akp [ka] [ka] [ka] SEQ ID NO: 2, gRNA against mouse Flt3, 5'-AAGTGCAGCTCGCCACCCCA-3' PCR primers for mouse Flt3, including SEQ ID NOs: 3 to 4, 5'-GGTACCAGCAGAGTTGGATAGC-3' (SEQ ID NO: 3) and 5'-ATCCCTTACACAGAAGCTGGAG-3' (SEQ ID NO: 4)

[0053] References [ka] [ka] [ka]

[0054] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may include the entire document.

[0055] The indefinite articles "a" and "an," as used in this specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0056] It is also to be understood that, unless expressly stated otherwise, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0057] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of," respectively, shall be closed or semi-closed transitional phrases, as set forth in the United States Patent Office Manual of Patent Examining Procedures Section 2111.03.

[0058] The terms "about" and "substantially" before a numerical value mean ±10% of the stated numerical value.

[0059] Where a range of values ​​is provided, each value between and including the upper and lower endpoints of that range is specifically contemplated and described herein. The present invention provides, for example, the following items. (Item 1) A non-obese diabetic (NOD) mouse, with an inactivated mouse Prkdc allele; with an inactivated mouse IL2rg allele; with an inactivated mouse Flt3 allele; a nucleic acid encoding human interleukin 3 (IL3); a nucleic acid encoding human granulocyte / macrophage stimulating factor (GM-CSF); a nucleic acid encoding human stem cell factor (SCF); a nucleic acid encoding human B2-microglobulin (B2M) covalently linked to the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene and the α3, cytoplasmic and transmembrane domains of mouse H2-Db (HLA-A2 / H2-D / B2M); and non-obese diabetic (NOD) mice. (Item 2) the mouse comprises a NOD.Cg-Prkdc mouse, the mouse comprising an inactivated mouse Flt3 allele; a nucleic acid encoding human IL3; a nucleic acid encoding human GM-CSF; a nucleic acid encoding human SCF; and a nucleic acid encoding HLA-A2 / H2-D / B2M. scid Il2rg tm1Wjl 2. The mouse according to item 1, which is a / SzJ (NOD scid gamma) mouse. (Item 3) 3. The mouse of item 1 or 2, wherein the nucleic acid encoding HLA-A2 / H2-D / B2M comprises a transgene encoding HLA-A2 / H2-D / B2M. (Item 4) 4. The mouse according to any one of items 1 to 3, wherein the mouse expresses a transgene encoding the HLA-A2 / H2-D / B2M. (Item 5) Bone marrow mouse CD45 + 5. The mouse of any one of items 1 to 4, wherein the cells express detectable levels of HLA-A2 at 4 weeks of age. (Item 6) 6. The mouse of any one of items 1 to 5, wherein the mouse is irradiated and transplanted with human hematopoietic progenitor cells (HPCs), and the human HPCs are transplanted as human CD45+ cells. (Item 7) the human HPCs are derived from fetal liver, umbilical cord blood, or bone marrow, and the transplanted human CD45 + The cells are CD19 + B cells, CD33 + Myeloid cells and CD3 + 7. The mouse of item 6, comprising a mixed population of T cells. (Item 8) The human HPCs are from fetal liver, umbilical cord blood, or bone marrow, and the mouse lung tissue is CD3 + T cells and HLA-DR + CD11c + 7. The mouse of item 6, comprising dendritic cells. (Item 9) The human HPC is HLA-A2 + and the mouse is HLA-A2 + 7. The mouse according to item 6, comprising mouse thymic epithelial cells. (Item 10) 6. A method for producing the mouse of any one of items 1 to 5, comprising introducing a transgene encoding HLA-A2 / H2-D / B2M into a NOD scid gamma mouse comprising an inactivated mouse Flt3 allele, a nucleic acid encoding human interleukin 3 (IL3), a nucleic acid encoding human granulocyte / macrophage stimulating factor (GM-CSF), and a nucleic acid encoding human stem cell factor (SCF). (Item 11) 6. A method for producing the mouse of any one of Items 1 to 5, comprising crossbreeding an NSG-SGM3F mouse comprising a nucleic acid encoding human interleukin 3 (IL3), a nucleic acid encoding human granulocyte / macrophage stimulating factor (GM-CSF), a nucleic acid encoding human stem cell factor (SCF), and an inactivated mouse Flt3 allele with an NSG-HLA-A2 / HHD mouse comprising a transgene encoding human B2-microglobulin (B2M) covalently linked to the MHC class 1, α1, and α2 binding domains of the human HLA-A2.1 gene and the α3, cytoplasmic, and transmembrane domains of mouse H2-Db. (Item 12) (a) generating founder mice having a NOD scid gamma genetic background, an inactivated mouse Flt3 allele, a nucleic acid encoding human IL3, a nucleic acid encoding human GM-CSF, and a nucleic acid encoding human SCF; (b) mating the founder mice to NOD scid gamma mice containing a transgene encoding human B2-microglobulin (B2M) covalently linked to the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene and the α3, cytoplasmic and transmembrane domains of mouse H2-Db to generate F1 progeny mice; (c) inbreeding the F1 progeny mice to generate F2 progeny mice homozygous for the inactivated Flt3 allele, a nucleic acid encoding human interleukin 3 (IL3), a nucleic acid encoding human granulocyte / macrophage stimulating factor (GM-CSF), a nucleic acid encoding human stem cell factor (SCF), and a transgene encoding human B2-microglobulin (B2M) covalently linked to the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene and the α3, cytoplasmic and transmembrane domains of mouse H2-Db; 6. A method for producing the mouse according to any one of items 1 to 5, comprising: (Item 13) To generate offspring mice, Prkdc scid homozygous for Il2rg tm1Wjl homozygous for Flt3 em1Akp Female mice homozygous for IL-3, GM-CSF, SCF, and HLA-A2 / H2-D / B2M-encoding transgenes were cultured in a Prkdc scid homozygous for X-linked Il2rg tm1Wjl Hemizygous for Flt3 em1Akp The method comprises mating a mouse homozygous for IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-1A, IL-1B, IL-2C, IL-3C, IL-4C, IL-5C, IL-6C, IL-7C, IL-8C, IL-9C, IL-10C, IL-11C, IL-12C, IL-13C, IL-14C, IL-15C, IL-16C, IL-17C, IL-18C, IL-19C, IL-20C, IL-21C, IL-22C, IL-23C, IL-24C, IL-25C, IL-26C, IL-27C, IL-28C, IL-29C, IL-30C, IL-31C (Item 14) A cell obtained from the mouse of any one of the preceding items. (Item 15) A mouse comprising cells having the same genotype as cells obtained from the mouse of any one of the preceding items. (Item 16) A mouse that is a descendant of the mouse of any one of the preceding items. (Item 17) A method of producing a mouse according to any one of the preceding items. (Item 18) A method of breeding a mouse according to any one of the preceding items. (Item 19) 20. The method of claim 18, comprising mating the mouse of any one of the preceding items with a second mouse to produce an offspring mouse. (Item 20) 20. The method of claim 19, wherein the second mouse is the mouse of any one of the preceding items. (Item 21) A method comprising sublethally irradiating the mouse of any one of the preceding items to produce an irradiated mouse. (Item 22) 22. The method of claim 21, further comprising administering human hematopoietic progenitor cells (HPCs) to the mouse. (Item 23) 23. The method of item 21 or 22, further comprising administering to the mouse an agent of interest. (Item 24) 24. The method of claim 23, further comprising evaluating the effect of the agent on human immune cells in the mouse. (Item 25) 25. The method of claim 24, wherein the human immune cells are selected from T cells, dendritic cells, natural killer cells, and macrophages.

Claims

1. Non-obese diabetic (NOD) mice, an inactivated mouse Prkdc allele; an inactivated mouse IL2rg allele; an inactivated mouse Flt3 allele; a nucleic acid encoding human interleukin 3 (IL3); a nucleic acid encoding human granulocyte / macrophage stimulating factor (GM-CSF); a nucleic acid encoding human stem cell factor (SCF); a nucleic acid encoding human B2-microglobulin (B2M) covalently linked to the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene and the α3, cytoplasmic and transmembrane domains of mouse H2-Db (HLA-A2 / H2-D / B2M); Non-obese diabetic (NOD) mice, including:

2. The mouse comprises an NOD.Cg-Prkdc mouse comprising an inactivated mouse Flt3 allele; a nucleic acid encoding human IL3; a nucleic acid encoding human GM-CSF; a nucleic acid encoding human SCF; and a nucleic acid encoding HLA-A2 / H2-D / B2M. ｓｃｉｄ Il2rg ｔｍ１Ｗｊｌ The mouse of claim 1, which is a / SzJ (NOD scid gamma) mouse.

3. The mouse of claim 1 or 2, wherein the nucleic acid encoding HLA-A2 / H2-D / B2M comprises a transgene encoding HLA-A2 / H2-D / B2M.

4. The mouse of any one of claims 1 to 3, wherein the mouse expresses a transgene encoding the HLA-A2 / H2-D / B2M.

5. Bone marrow mouse CD45 ＋ The mouse of any one of claims 1 to 4, wherein the cells express a detectable level of HLA-A2 at 4 weeks of age.

6. 6. The mouse of any one of claims 1 to 5, wherein the mouse is irradiated and transplanted with human hematopoietic progenitor cells (HPCs), and the human HPCs engraft as human CD45+ cells.

7. the human HPCs are derived from fetal liver, umbilical cord blood, or bone marrow; and the transplanted human CD45 ＋ The cells are CD19 ＋ B cells, CD33 ＋ Myeloid cells and CD3 ＋ The mouse of claim 6 , comprising a mixed population of T cells.

8. The human HPCs are from fetal liver, umbilical cord blood, or bone marrow, and the mouse lung tissue is CD3 ＋ T cells and HLA-DR ＋ CD11c ＋ The mouse of claim 6 , comprising dendritic cells.

9. The human HPC is HLA-A2 ＋ and the mouse is HLA-A2 ＋ The mouse of claim 6 , comprising mouse thymic epithelial cells.

10. 6. A method for producing a mouse according to any one of claims 1 to 5, comprising introducing a transgene encoding said HLA-A2 / H2-D / B2M into a NOD scid gamma mouse comprising an inactivated mouse Flt3 allele, a nucleic acid encoding human interleukin 3 (IL3), a nucleic acid encoding human granulocyte / macrophage stimulating factor (GM-CSF), and a nucleic acid encoding human stem cell factor (SCF).

11. 6. A method for producing the mouse of any one of claims 1 to 5, comprising crossbreeding an NSG-SGM3F mouse comprising a nucleic acid encoding human interleukin 3 (IL3), a nucleic acid encoding human granulocyte / macrophage stimulating factor (GM-CSF), a nucleic acid encoding human stem cell factor (SCF), and an inactivated mouse Flt3 allele, with an NSG-HLA-A2 / HHD mouse comprising a transgene encoding human B2-microglobulin (B2M) covalently linked to the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene and the α3, cytoplasmic and transmembrane domains of the mouse H2-Db.

12. (a) generating founder mice having a NOD scid gamma genetic background, an inactivated mouse Flt3 allele, a nucleic acid encoding human IL3, a nucleic acid encoding human GM-CSF, and a nucleic acid encoding human SCF; (b) mating the founder mice to NOD scid gamma mice containing a transgene encoding human B2-microglobulin (B2M) covalently linked to the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene and the α3, cytoplasmic and transmembrane domains of the mouse H2-Db to generate F1 progeny mice; (c) inbreeding the F1 progeny mice to generate F2 progeny mice homozygous for the inactivated Flt3 allele, a nucleic acid encoding human interleukin 3 (IL3), a nucleic acid encoding human granulocyte / macrophage stimulating factor (GM-CSF), a nucleic acid encoding human stem cell factor (SCF), and a transgene encoding human B2-microglobulin (B2M) covalently linked to the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene and the α3, cytoplasmic and transmembrane domains of the mouse H2-Db; A method for producing the mouse of any one of claims 1 to 5, comprising:

13. To generate offspring mice, Prkdc ｓｃｉｄ homozygous for Il2rg ｔｍ１Ｗｊｌ homozygous for Flt3 ｅｍ１Ａｋｐ Female mice homozygous for Prkdc, homozygous for Il-3, homozygous for GM-CSF, homozygous for SCF, and homozygous for a transgene encoding HLA-A2 / H2-D / B2M were cultured in a 200-well plate. ｓｃｉｄ homozygous for X-linked Il2rg ｔｍ１Ｗｊｌ Hemizygous for Flt3 ｅｍ１Ａｋｐ and mating the mouse with a male mouse homozygous for Il-3, homozygous for GM-CSF, homozygous for SCF, and homozygous for a transgene encoding HLA-A2 / H2-D / B2M.

14. Cells obtained from a mouse described in any one of claims 1 to 9.

15. A descendant mouse of the mouse according to any one of claims 1 to 9, wherein the descendant mouse is an inactivated mouse Prkdc allele; an inactivated mouse IL2rg allele; an inactivated mouse Flt3 allele; a nucleic acid encoding human interleukin 3 (IL3); a nucleic acid encoding human granulocyte / macrophage stimulating factor (GM-CSF); a nucleic acid encoding human stem cell factor (SCF); a nucleic acid encoding human B2-microglobulin (B2M) covalently linked to the MHC class 1, α1 and α2 binding domains of the human HLA-A2.1 gene and the α3, cytoplasmic and transmembrane domains of mouse H2-Db (HLA-A2 / H2-D / B2M); and progeny mice.

16. A method for producing a mouse described in any one of claims 1 to 9.

17. A method for breeding a mouse described in any one of claims 1 to 9.

18. 18. The method of claim 17, comprising mating a mouse of any one of claims 1 to 9 with a second mouse to produce an offspring mouse.

19. The method of claim 18, wherein the second mouse is a mouse according to any one of claims 1 to 9.

20. A method comprising sublethally irradiating a mouse according to any one of claims 1 to 9 to produce an irradiated mouse.

21. 21. The method of claim 20, further comprising administering to the mouse human hematopoietic progenitor cells (HPCs).

22. 22. The method of claim 20 or 21, further comprising administering to said mouse an agent of interest.

23. 23. The method of claim 22, further comprising evaluating the effect of the agent on human immune cells in the mouse.

24. 24. The method of claim 23, wherein the human immune cells are selected from T cells, dendritic cells, natural killer cells, and macrophages.

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