Vectors, genetically modified cells, and genetically modified non-human animals containing them.

Abstract

Genetically modified cells and genetically modified non-human animals (e.g., rodents such as rats and mice) comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a non-human animal portion and a human portion operably linked to the Flt3l promoter and expressing one or more human or humanized polypeptides as appropriate, are provided herein. Methods and compositions for producing and using such genetically modified cells and non-human animals are also provided.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 521,475, filed on 16 June 2023, which is incorporated herein by reference in its entirety. Sequence List

[0002] This application includes a sequence listing submitted electronically in XML format, the entirety of which is incorporated herein by reference. The XML copy, created on 14 June 2024, is named RPD-00225.xml and has a size of 43,680 bytes. [Background technology]

[0003] background Genetically modified cells, genetically modified mice containing them, and modified and engraftable mice, as well as their use in modeling human diseases for purposes such as drug testing, are well known in the art. The use of genetically modified mice to model the human immune system (HIS) has been reported (Manz (2007) Immunity, 26:537-541). For example, HIS mice generated by transplanting human hematopoietic stem cells and progenitor cells into severely immunodeficient mouse lines (e.g., Rag2 KO Il2rg KO mice) have been reported. While multi-lineage hematopoietic development has been observed in these HIS mice, there is a need for genetically modified mice that can support the development of human dendritic cells, and for engraftable mice that can model or approximate specific aspects of human dendritic cells. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Manz (2007) Immunity, 26:537-541 [Overview of the Initiative] [Means for solving the problem]

[0005] Abstract This disclosure is partly based on the finding that knocking out the Flt3 gene in immunodeficient mice (e.g., mice with Rag1 and / or Rag2 gene knockout and IL2rg gene knockout) using humanized Flt3l resulted in increased human dendritic cells (e.g., myeloid dendritic cells, plasmacytoid dendritic cells, BDCA-3+ dendritic cells) in the spleen, blood, and bone marrow after engraftment of human hematopoietic cells. Accordingly, in one embodiment, the present disclosure relates to a genetically modified rodent comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operably linked to the Flt3l promoter.

[0006] In some embodiments, the genetically modified rodent contains a homozygous null mutation in the Rag1 gene. In some embodiments, the null mutation in the rodent Flt3 gene contains an insertion, deletion, and / or substitution in the endogenous Flt3 gene. In some embodiments, the null mutation in the rodent Flt3 gene is a deletion of the complete endogenous Flt3 coding sequence. In some embodiments, the genetically modified rodent is a mouse, and the mouse contains a homozygous deletion of the nucleic acid sequence between coordinates chr5:147331171-147400265 (GRCm38 assembly).

[0007] In some embodiments, the rodent portion of the Flt3l gene includes the non-coding portion of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene. In some embodiments, the non-coding portion of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene are at least 90%, at least 95%, or 100% identical to the corresponding non-coding portion of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene shown in Table 1A. In some embodiments, the human portion of the Flt3l gene includes the signal peptide coding portion of exon 2, and exons 3-6 of the human FLT3L gene. In some embodiments, the signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B. In some embodiments, the Flt3l gene includes rodent exon 1, the rodent non-coding portion of exon 2, the human signal peptide coding portion of exon 2, human exons 3-6, and rodent exons 7-9.

[0008] In some embodiments, the Flt3l gene encodes a chimeric membrane-bound Flt3l comprising the signal peptide and cytokine-like core domain of the human FLT3L polypeptide and the C-terminal portion of the rodent Flt3l polypeptide. In some embodiments, the C-terminal portion of the rodent Flt3l polypeptide includes a rodent stalk region, a rodent transmembrane domain, and a rodent cytoplasmic tail. In some embodiments, the C-terminal portion of the rodent Flt3l polypeptide has an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2. In some embodiments, the rodent expresses both the soluble and membrane-bound forms of the Flt3l polypeptide. In some embodiments, the soluble and membrane-bound forms of the Flt3l polypeptide include the signal peptide and cytokine-like core domain of the human FLT3L polypeptide. In some embodiments, the signal peptide of the human FLT3L polypeptide contains amino acids corresponding to residues 1-26 of the human FLT3L polypeptide. In some embodiments, the signal peptide of the human FLT3L polypeptide contains an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the signal peptide of the human FLT3L polypeptide shown in Table 2. In some embodiments, the cytokine-like core domain of the human FLT3L polypeptide contains amino acids corresponding to residues 27-159 of the human FLT3L polypeptide. In some embodiments, the cytokine-like core domain of the human FLT3L polypeptide contains an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

[0009] In some embodiments, genetically modified rodents are heterozygous for the Flt3 ligand (Flt3l) gene, which contains both a rodent and a human portion. In some embodiments, genetically modified rodents are homozygous for the Flt3 ligand (Flt3l) gene, which contains both a rodent and a human portion.

[0010] In some embodiments, the rodent portion of the Flt3l gene is the endogenous rodent portion of the Flt3l gene, the rodent Flt3l gene is the endogenous rodent gene, and / or the rodent Flt3l polypeptide is the endogenous rodent Flt3l polypeptide.

[0011] In some embodiments, the rodent and human portions are operably linked to the Flt3l promoter. In some embodiments, the Flt3l promoter is a rodent promoter. In some embodiments, the Flt3l promoter is an endogenous rodent promoter. In some embodiments, the Flt3l promoter is located at the endogenous rodent locus.

[0012] In some embodiments, the genetically modified rodent expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to the Sirpa promoter. In some embodiments, the genetically modified rodent further comprises a Sirpa gene encoding a Sirpa polypeptide comprising the extracellular portion of the human SIRPA polypeptide and the intracellular portion of the rodent Sirpa polypeptide, the Sirpa gene being operably linked to the Sirpa promoter. In some embodiments, the Sirpa gene comprises exons 1, 5, 6, 7, and 8 of the rodent Sirpa gene and exons 2-4 of the human SIRPA gene. In some embodiments, the genetically modified rodent further expresses a Sirpa polypeptide comprising the extracellular portion of the human SIRPA polypeptide and the intracellular portion of the rodent Sirpa polypeptide. In some embodiments, the rodent Sirpa polypeptide is the endogenous rodent Sirpa polypeptide, and / or the rodent Sirpa gene is the endogenous rodent gene. In some embodiments, genetically modified rodents further express human SIRPA polypeptide encoded by nucleic acids operably linked to the Sirpa promoter.

[0013] In some embodiments, genetically modified rodents further express human GM-CSF protein encoded by nucleic acids operably linked to the GM-CSF promoter and / or human IL3 protein encoded by nucleic acids operably linked to the IL3 promoter.

[0014] In some embodiments, the Sirpa promoter, GM-CSF promoter, and / or IL3 promoter are rodent promoters. In some embodiments, the Sirpa promoter, GM-CSF promoter, and / or IL3 promoter are endogenous rodent promoters. In some embodiments, the Sirpa promoter, GM-CSF promoter, and / or IL3 promoter are located at the corresponding endogenous rodent locus.

[0015] In some embodiments, the genetically modified rodents described herein contain a null mutation in at least one corresponding rodent gene at the corresponding rodent locus.

[0016] In some embodiments, the genetically modified rodents are heterozygous for at least one allele containing a nucleic acid sequence encoding a human or humanized protein. In some embodiments, the genetically modified rodents are homozygous for at least one allele containing a nucleic acid sequence encoding a human or humanized protein.

[0017] In some embodiments, the genetically modified rodents further comprise engraftment of human hematopoietic cells. In some embodiments, the human hematopoietic cells comprise one or more cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human hematopoietic progenitor cells, human dendritic cell progenitor cells, and human dendritic cells. In some embodiments, the genetically modified rodents comprise human dendritic cells.

[0018] In some embodiments, an autoimmune disease is induced or established in the genetically modified rodents. In some embodiments, the autoimmune disease is systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, polymyositis, collagen-induced arthritis, or dermatomyositis.

[0019] In some embodiments, the rodent is a mouse or a rat. In some embodiments, the rodent is a mouse.

[0020] In certain embodiments, a method is provided herein for identifying a drug that modulates the function of human dendritic cells, comprising: a. administering the candidate drug to a genetically modified rodent described herein; and b. determining whether the candidate drug modulates the function of human dendritic cells in the rodent. In some embodiments, the human dendritic cells are myeloid dendritic cells (mDCs) or plasmacytoid dendritic cells (pDCs). In some embodiments, the function of human dendritic cells is selected from the group consisting of phagocytosis, cytokine production, cross-presentation of exogenous antigens, and activation of cytotoxic CD8+ T-cell lymphocytes (CTLs).

[0021] In certain embodiments, a method is provided herein for evaluating the therapeutic efficacy of a drug for stimulating a T cell response to target cells, the method comprising: a. administering a drug candidate to a genetically modified rodent described herein, wherein the genetically modified rodent includes target cells; and b. measuring the T cell response to the target cells in the rodent to evaluate the therapeutic efficacy of the drug candidate. In some embodiments, the target cells are selected from the group consisting of tumor cells, virus-infected cells, bacterial-infected cells, bacterial cells, fungal cells, and parasitic cells.

[0022] In a particular embodiment, the present invention provides a method for evaluating the therapeutic efficacy of a drug in the treatment of an autoimmune disease, the method comprising: a. administering a candidate drug to a genetically modified rodent as described herein; and b. determining whether the drug treats an autoimmune disease in the rodent.

[0023] In another aspect, the disclosure relates to genetically modified rodent cells comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent and human portion operably linked to the Flt3l promoter.

[0024] In some embodiments, genetically modified rodent cells contain a homozygous null mutation in the Rag1 gene. In some embodiments, a null mutation in the rodent Flt3 gene includes insertions, deletions, and / or substitutions in the endogenous Flt3 gene. In some embodiments, a null mutation in the rodent Flt3 gene is a deletion of the complete endogenous Flt3 coding sequence. In some embodiments, the genetically modified rodent cells are mouse cells, and the mouse cells contain a homozygous deletion of the nucleic acid sequence between coordinates chr5:147331171-147400265 (GRCm38 assembly).

[0025] In some embodiments, the rodent portion of the Flt3l gene includes the non-coding portion of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene. In some embodiments, the non-coding portion of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene are at least 90%, at least 95%, or 100% identical to the corresponding non-coding portion of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene shown in Table 1A. In some embodiments, the human portion of the Flt3l gene includes the signal peptide coding portion of exon 2, and exons 3-6 of the human FLT3L gene. In some embodiments, the signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B. In some embodiments, the Flt3l gene includes rodent exon 1, the rodent non-coding portion of exon 2, the human signal peptide coding portion of exon 2, human exons 3-6, and rodent exons 7-9.

[0026] In some embodiments, the Flt3l gene encodes a chimeric membrane-bound Flt3l comprising the signal peptide and cytokine-like core domain of the human FLT3L polypeptide and the C-terminal portion of the rodent Flt3l polypeptide. In some embodiments, the C-terminal portion of the rodent Flt3l polypeptide includes a rodent stalk region, a rodent transmembrane domain, and a rodent cytoplasmic tail. In some embodiments, the C-terminal portion of the rodent Flt3l polypeptide has an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2. In some embodiments, the rodent expresses both the soluble and membrane-bound forms of the Flt3l polypeptide. In some embodiments, the soluble and membrane-bound forms of the Flt3l polypeptide include the signal peptide and cytokine-like core domain of the human FLT3L polypeptide. In some embodiments, the signal peptide of the human FLT3L polypeptide contains amino acids corresponding to residues 1-26 of the human FLT3L polypeptide. In some embodiments, the signal peptide of the human FLT3L polypeptide contains an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the signal peptide of the human FLT3L polypeptide shown in Table 2. In some embodiments, the cytokine-like core domain of the human FLT3L polypeptide contains amino acids corresponding to residues 27-159 of the human FLT3L polypeptide. In some embodiments, the cytokine-like core domain of the human FLT3L polypeptide contains an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

[0027] In some embodiments, genetically modified rodent cells are heterozygous for the Flt3 ligand (Flt3l) gene, which contains both a rodent and a human portion. In some embodiments, genetically modified rodent cells are homozygous for the Flt3 ligand (Flt3l) gene, which contains both a rodent and a human portion.

[0028] In some embodiments, the rodent portion of the Flt3l gene is the endogenous rodent portion of the Flt3l gene, the rodent Flt3l gene is the endogenous rodent gene, and / or the rodent Flt3l polypeptide is the endogenous rodent Flt3l polypeptide.

[0029] In some embodiments, the rodent and human portions are operably linked to the Flt3l promoter. In some embodiments, the Flt3l promoter is a rodent promoter. In some embodiments, the Flt3l promoter is an endogenous rodent promoter. In some embodiments, the Flt3l promoter is located at the endogenous rodent locus.

[0030] In some embodiments, the genetically modified rodent cells further comprise a nucleic acid encoding a human or humanized SIRPA polypeptide, the nucleic acid being operably linked to a Sirpa promoter. In some embodiments, the genetically modified rodent cells express a human or humanized SIRPA polypeptide. In some embodiments, the genetically modified rodent cells further comprise a Sirpa gene encoding a Sirpa polypeptide comprising the extracellular portion of a human SIRPA polypeptide and the intracellular portion of a rodent Sirpa polypeptide, the Sirpa gene being operably linked to a Sirpa promoter. In some embodiments, the Sirpa gene comprises exons 1, 5, 6, 7, and 8 of the rodent Sirpa gene and exons 2-4 of the human SIRPA gene. In some embodiments, the genetically modified rodent cells express a Sirpa polypeptide comprising the extracellular portion of a human SIRPA polypeptide and the intracellular portion of a rodent Sirpa polypeptide. In some embodiments, the rodent Sirpa polypeptide is an endogenous rodent Sirpa polypeptide, and / or the rodent Sirpa gene is an endogenous rodent gene. In some embodiments, genetically modified rodent cells express human SIRPA polypeptide.

[0031] In some embodiments, genetically modified rodent cells further comprise (1) a nucleic acid encoding human GM-CSF protein and operably linked to a GM-CSF promoter; and / or (2) a nucleic acid encoding human IL3 protein and operably linked to an IL3 promoter. In some embodiments, genetically modified rodent cells express human GM-CSF protein and / or human IL3 protein.

[0032] In some embodiments, the Sirpa promoter, GM-CSF promoter, and / or IL3 promoter are rodent promoters. In some embodiments, the Sirpa promoter, GM-CSF promoter, and / or IL3 promoter are endogenous rodent promoters. In some embodiments, the Sirpa promoter, GM-CSF promoter, and / or IL3 promoter are located at the corresponding endogenous rodent locus.

[0033] In some embodiments, the genetically modified rodent cells described herein contain a null mutation in at least one corresponding rodent gene at the corresponding rodent locus.

[0034] In some embodiments, genetically modified rodent cells are heterozygous for at least one allele containing a nucleic acid sequence encoding a human or humanized protein. In some embodiments, genetically modified rodent cells are homozygous for at least one allele containing a nucleic acid sequence encoding a human or humanized protein.

[0035] In some embodiments, the genetically modified rodent cells are rodent embryonic stem (ES) cells.

[0036] In a particular embodiment, the present invention provides a method for producing rodent embryonic stem cells, comprising genetically engineering the rodent embryonic stem cells to have a genome comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent and human portion operably linked to the Flt3l promoter.

[0037] In certain embodiments, rodent embryos comprising rodent embryonic stem cells as described herein, or rodent embryonic stem cells prepared according to the methods described herein, are provided herein.

[0038] In a particular embodiment, a method is provided herein for producing a rodent comprising in its genome (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operably linked to the Flt3l promoter, the method comprising (a) obtaining rodent embryonic stem cells as described herein or rodent embryonic stem cells produced according to the method described herein; and (b) producing a rodent using the rodent embryonic cells of (a).

[0039] In a particular embodiment, a method is provided herein for producing a rodent whose genome contains (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operably linked to the Flt3l promoter, the method comprising modifying the genome of the rodent to contain (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operably linked to the Flt3l promoter. [Brief explanation of the drawing]

[0040] [Figure 1A]Figures 1A and 1B show the generation of the final targeting vector (modified BAC#3) for humanization of mouse FLT3L (referred to as "mFlt3L" or "Flt3L" in Figures 1A and 1B). Unless otherwise indicated by the labels in the figures (e.g., with respect to selection cassettes, loxP sites, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. "SPEC" indicates a spectinomycin resistance gene. "EM7-Hyg" indicates a hygromycin resistance gene with an EM7 promoter. "loxP" indicates a loxP site. SDC NEO is a neomycin autodeletion cassette. Human FLT3L is referred to as "FLT3LG" in Figure 1B. [Figure 1B] Same as above.

[0041] [Figure 1CD] Figures 1C–1D show schematic diagrams, not to scale, of the exemplary modified mouse Flt3l (referred to as “mFLT3l” or “Flt3l” in Figures 1C and 1D) locus according to certain exemplary embodiments provided herein. Figure 1C shows the modified mouse Flt3l locus before removal of the loxp-Neo-loxp autodeletion cassette. Figure 1D shows the modified mouse Flt3l locus after removal of the loxp-Neo-loxp autodeletion cassette. Unless otherwise indicated by the labels in the figures (e.g., with respect to a select cassette, loxP site, etc.), filled shapes and single lines represent mouse sequences, and empty shapes and double lines represent human sequences. “loxP” indicates a loxP site. SDC NEO is the neomycin autodeletion cassette. Human FLT3L is referred to as “FLT3LG” or “hFLT3LG” in Figures 1C and 1D.

[0042] [Figure 1E]Figure 1E shows the sequence alignment between mouse membrane-bound FLT3L (mFlt3l_MB) (SEQ ID NO: 31), chimeric membrane-bound FLT3L (Chimeric_FLT3LG_MB) (SEQ ID NO: 11), and human membrane-bound FLT3L (hFLT3LG_MB) (SEQ ID NO: 32). Specific protein domains are boxed and labeled. Triangles represent the location of exon boundaries in the corresponding nucleic acid sequences of the mouse FLT3LG gene or human FLT3LG gene encoding the indicated mouse or human protein (top or filled triangles are mouse exon boundaries, and bottom or empty triangles are human exon boundaries).

[0043] [Figure 2] Figure 2 shows a non-scale schematic diagram of the generation of an exemplary modified mouse Flt3 locus (i.e., Flt3 deletion) by a particular exemplary embodiment provided herein. "loxP" indicates the loxP site. SDC HYG is a hygromycin autodeletion cassette.

[0044] [Figure 3] Figure 3 shows high levels of humanized FLT3L (referred to as "hFLT3L" in Figure 3) in both serum and bone marrow aspirate of SRG / hFLT3L / mFLT3 KO mice. SRG / hFLT3L / mFLT3 KO mice, also called "SRG hFLT3L mFLT3 KO" mice, "SRG-hFLT3L / mFLT3 KO" mice, or "hFLT3L / mFLT3 KO" mice, refer to mice that include humanized Sirpa, Rag2 gene knockout, Il2rg gene knockout, humanized Flt3L, and Flt3 KO.

[0045] [Figure 4A-1] Figure 4A shows the gating strategies of pDCs and other dendritic cells. [Figure 4A-2] Figure 4A shows the gating strategies of pDCs and other dendritic cells.

[0046] [Figure 4B] Figure 4B shows that SRG / hFLT3L / mFLT3 KO (referred to as "SRG hFLT3L mFLT3 KO" in Figure 4B) mice showed increased myeloid and plasma cell-like DCs in the blood and spleen upon hHSC engraftment.

[0047] [Figure 4C] Figure 4C shows that SRG / hFLT3L / mFLT3 KO (referred to as "SRG hFLT3L mFLT3 KO" in Figure 4C) mice showed increased levels of BDCA-3+ myeloid DCs in the blood and spleen upon engraftment of hHSCs.

[0048] [Figure 4D] Figure 4D shows that SRG / hFLT3L / mFLT3 KO (referred to as "SRG hFLT3L mFLT3 KO" in Figure 4D) mice had engraftment comparable to HSC donor-matched StRG mice, but with an increased number of human myeloids and plasmacytoid DCs in the brain mass (BM).

[0049] [Figure 4E] Figure 4E shows that SRG / hFLT3L / mFLT3 KO (referred to as "SRG hFLT3L mFLT3 KO" in Figure 4E) increased the number of human myeloid and plasmacytoid dendritic cells in the thymus during hHSC engraftment.

[0050] [Figure 4F] Figure 4F shows the significant loss of mouse DCs (mouse CD45+ / CD11c+ / MHC class II+) in engrafted SRG / hFLT3L / mFLT3 KO (referred to as "SRG-hFLT3L / mFLT3 KO" in Figure 4F).

[0051] [Figure 5A]Figure 5A shows that the human mDC and pDC populations were increased in the blood and spleen of hHSC engrafted mice with humanized Flt3l and mouse Flt3 KO (i.e., hFLT3L / mFLT3 KO) compared to HSC donor-compatible mice with only mFLT3 KO. "FLT3Lm / mmFLT3- / -" mice refer to SRG mice containing homozygous mouse Flt3l and mouse Flt3 knockout. "FLT3Lh / hmFLT3- / -" mice refer to SRG mice containing homozygous humanized Flt3l and mouse Flt3 knockout.

[0052] [Figure 5B] Figure 5B shows that the human mDC and pDC populations were increased in the thymus of hHSC engrafted mice with humanized Flt3l and mouse Flt3 KO (i.e., hFLT3L / mFLT3 KO) compared to HSC donor-compatible mice with only mFLT3 KO. "FLT3Lm / mmFLT3- / -" mice refer to SRG mice containing homozygous mouse Flt3l and mouse Flt3 knockout. "FLT3Lh / hmFLT3- / -" mice refer to SRG mice containing homozygous humanized Flt3l and mouse Flt3 knockout.

[0053] [Figure 6A] Figure 6A shows that CD3+ T cells in the blood expressed significantly less PD-1 in hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice (referred to as "SRG hFLT3L mFLT3 KO" in Figure 6A) than in StRG mice; and that the proportion of CD4+ T cells was higher than that of CD8+ T cells in hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice.

[0054] [Figure 6B-1]Figure 6B shows that more CD4+ and CD8+ T cells in hHSC engrafted SRG / hFLT3L / mFLT3 KO (referred to as "hFLT3L / mFLT3 KO" in Figure 6B) were central memory T cells (Tcm) than in HSC donor-matched StRG. [Figure 6B-2] Same as above. [Figure 6B-3] Same as above.

[0055] [Figure 7A] Figure 7A shows key transcriptional differences between the DC population in the spleen / blood of hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice and healthy human blood.

[0056] [Figure 7B-1] Figure 7B shows that the proportion of mDCs, BDCA-3+ mDCs, and pDCs is higher in hHSC-engrafted SRG / hFLT3L / mFLT3 KO (referred to as "SRG-hFLT3L / mFLT3 KO") mice than in human PBMCs. [Figure 7B-2] Same as above. [Figure 7B-3] Same as above.

[0057] [Figure 8] Figure 8 shows the presence of human pDCs in various organs of SRG / hFLT3L / mFLT3 KO human immune system mice.

[0058] [Figure 9A] Figure 9A shows the levels of human IFNα or IFNβ produced by human pDCs isolated from SRG / hFLT3L / mFLT3 KO human immune system mice stimulated with ODN2216 (CpG-DNA).

[0059] [Figure 9B-1]Figure 9B shows that human pDCs in SRG / hFLT3L / mFLT3 KO human immune system mice respond to ODN2216 CpG stimulation in vivo and produce human type I IFN, as measured by induction of the type I IFN signature gene. [Figure 9B-2] Same as above. [Modes for carrying out the invention]

[0060] Detailed explanation General This disclosure relates to genetically modified non-human animals (e.g., rodents such as rats or mice) comprising (i) homozygous null mutations in the Rag1 and / or Rag2 genes (e.g., Rag1 and / or Rag2 gene knockout); (ii) homozygous null mutations in the IL2rg gene (e.g., IL2rg gene knockout); (iii) homozygous null mutations in the non-human FMS-like tyrosine kinase 3 (Flt3) gene at the non-human Flt3 locus; and (iv) a genetically modified non-human animal (e.g., rodents such as rats or mice) comprising an Flt3 ligand (Flt3l) gene containing a non-human and human portion operably linked to the Flt3l promoter. In some embodiments, genetically modified non-human animals (e.g., mice) are provided herein that include (i) a homozygous null mutation in the Rag2 gene (e.g., Rag2 gene knockout); (ii) a homozygous null mutation in the IL2rg gene (e.g., IL2rg gene knockout); (iii) a homozygous null mutation in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a non-human animal portion and a human portion operably linked to the Flt3l promoter.

[0061] In some embodiments, genetically modified non-human animals express human or humanized SIRPA protein encoded by nucleic acids operably ligated to a promoter, e.g., the Sirpa promoter. In some embodiments, genetically modified non-human animals further express human GM-CSF protein encoded by nucleic acids operably ligated to a promoter, e.g., the GM-CSF promoter, and / or human IL3 protein encoded by nucleic acids operably ligated to a promoter, e.g., the IL3 promoter. In certain embodiments, at least one promoter operably ligated to the nucleic acid encoding the human or humanized protein is an endogenous non-human animal promoter. In other embodiments, genetically modified animals express human or humanized nucleic acids from a natural human promoter and natural regulatory elements. Those skilled in the art will understand that genetically modified animals include genetically modified animals that express at least one human nucleic acid or humanized nucleic acid from any promoter. Examples of promoters useful in the present invention include, but are not limited to, the DNA pol II promoter, PGK promoter, ubiquitin promoter, albumin promoter, globin promoter, ovalbumin promoter, SV40 initial promoter, Roussarcoma virus (RSV) promoter, retroviral LTR, and lentiviral LTR. Promoter and enhancer expression systems useful in this disclosure also include inducible and / or tissue-specific expression systems.

[0062] Rodents such as mice and rats possessing components of the human immune system (e.g., HIS mice and rats) are highly promising for studying the human immune system in vivo, testing human vaccines, and testing and developing drugs to treat human diseases and disorders. HIS mice are generated by transplanting severely immunodeficient mouse strains (e.g., recombinant activating gene 2 (Rag2) knockout (KO) mice and interleukin 2 receptor gamma (IL2rg) KO mice) together with human hematopoietic stem cells and progenitor cells (e.g., CD34+HSCs). Compared to non-human primates, HIS mice have the advantages of a small animal model, namely, they allow for a wider variety of experiments, are more accessible to research groups, and are more ethically acceptable than conducting experiments using human subjects. Most importantly, experimental findings derived from HIS mice may be more relevant and applicable to humans. Similar rat models offering such advantages have also been described and are intended herein.

[0063] While HIS mice generate human immune cells such as B cells and T cells, there is a need for an HIS mouse model that supports the development of human dendritic cells, which are the key antigen-presenting cells of the immune system that modulate T cell activation and antigen-specific responses. Fms tyrosine-like 3 ligand (Flt3l) is a crucial cytokine for dendritic cell development. However, humanization of Flt3l in HIS mouse models (e.g., huSIRP Rag2- / -IL-2Rγ- / -(SRG)VelociHum model) has not been found to significantly increase human dendritic cell development. Mice with a deletion of Flt3l have been reported to have increased human dendritic cell (DC) development upon engraftment of human hematopoietic stem cells (hHSCs). However, this increase in human DCs required repeated injections of large amounts of human FLT3l (Li et al. (2016), Eur J Immunol, 46:1291~1299).

[0064] This disclosure provides a novel genetically modified non-human animal (e.g., mouse) model that specifically deletes the non-human animal (e.g., mouse) receptor for Flt3l (Flt3 or FLK2 (fetal liver kinase 2)) in a HIS model having humanized Flt3l (e.g., the huSIRP Rag2- / -IL-2Rγ- / -(SRG)VelociHum model). The novel model has receptor deletion concurrent with humanization of Flt3l so that Flt3l isoforms are expressed sequentially at physiologically relevant levels. Furthermore, the humanized Flt3l gene provided herein encodes a chimeric membrane-bound FLT3L that includes a human signaling peptide and cytokine-like core domain, as well as a non-human animal (e.g., rodent such as rat or mouse) stalk, transmembrane domain, and cytoplasmic tail that retains endogenous functions related to the stalk, transmembrane domain, and cytoplasmic tail. The Flt3 null, Flt3l humanized non-human animal (e.g., mouse) model showed increased human DCs in the spleen, blood, and bone marrow. This increase was reported in myeloid DCs, a subset crucial for T cell activation / regulation, as well as plasmacytoid DCs, a subset that are the primary type I IFN-producing cells in response to viral infection. Furthermore, a significant increase in BDCA-3+ DCs, a crucial subset for crosspriming CD8+ T cells to exogenous antigens such as viral elements derived from phagocytosed infected cells, was also observed. While we do not wish to be constrained by theory, deletion of the non-human animal (e.g., mouse) receptor for Flt3l (Flt3 or FLK2 (fetal liver kinase 2)) eliminates competition for human FLT3l by non-human animal (e.g., mouse) DCs, and therefore enhances the development of human DCs in HIS models using humanized Flt3l. This disclosure demonstrates that this non-human animal (e.g., mouse) cytokine receptor deletion allows for an increase in human dendritic cells (e.g., human myeloid and plasmacytoid dendritic cells) in the bone marrow and thymus at levels considerably higher than those observed in StRG. This new model provides a useful tool for studying human dendritic cell biology, as well as a useful model for developing treatments for human dendritic cell diseases.

[0065] The genetically modified non-human animals provided herein will find many applications in the art, including, for example, modeling human autoimmune diseases and dendritic cell function; in vivo screening for agents that modulate dendritic cell function, e.g., in healthy or diseased states; evaluating the therapeutic efficacy of drugs for stimulating T cell responses to target cells; and / or evaluating the therapeutic efficacy of drugs in treating autoimmune diseases. These and other purposes, advantages and features of the present invention will become apparent to those skilled in the art upon reading the details of the compositions and methods, as fully described below.

[0066] definition The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "element" means one element or two or more elements.

[0067] The term "amino acid" is intended to encompass all molecules that can be included in polymers of naturally occurring amino acids, whether natural or synthetic, and that include both amino and acid functionalities. Exemplary amino acids include naturally occurring amino acids; their analogues, derivatives, and homologues; amino acid analogues with mutant side chains; and all stereoisomers of any of the aforementioned.

[0068] The "coding region" of a gene contains nucleotide residues from the coding and non-coding strands of the gene that are homologous or complementary to the coding region of the mRNA molecule produced by gene transcription. The "coding region" of an mRNA molecule also contains nucleotide residues of the mRNA molecule that correspond to the anti-codon region of the transfer RNA molecule during translation of the mRNA molecule, or that encode a stop codon. Thus, the coding region may contain nucleotide residues that constitute a codon for an amino acid residue that is not present in the mature protein encoded by the mRNA molecule (e.g., an amino acid residue in a protein transport signal sequence).

[0069] As used herein, the term “chimeric” refers to a nucleic acid or protein that contains a portion of its structure (i.e., nucleotide or amino acid sequence) derived from a different species. In some embodiments, the “chimeric” nucleic acid or protein described herein contains a nucleotide or amino acid sequence derived from both a non-human source (e.g., rodents, e.g., mice) and humans. In such embodiments, the “chimeric” nucleic acid or protein may also be called a “humanized” nucleic acid or protein.

[0070] As used herein, the terms “endogenous gene” or “endogenous gene segment” refer to a gene or gene segment found in the parent or reference organism prior to the introduction of any disruption, deletion, substitution, alteration, or modification described herein. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is an engineered organism. In some embodiments, the reference organism is a laboratory-bred organism (wild-type or engineered).

[0071] As used herein, the term “complete coding sequence” refers to a coding nucleic acid sequence ranging from a start codon to a stop codon. The term “full-length polypeptide” refers to a polypeptide containing the amino acid sequence encoded by such a complete coding sequence.

[0072] The term “humanized” is used herein in accordance with its meaning as understood in the art, referring to a nucleic acid or protein that includes a portion derived from a non-human source that has been manipulated to have a structure and function more similar to a true human nucleic acid or protein than to the nucleic acid or protein of the original source. For example, humanization may include selecting amino acid substitutions to make a non-human sequence more similar to a human sequence. Humanization may also include grafting at least a portion of a non-human protein onto a human protein. For example, in the case of a membrane receptor, a “humanized” gene may encode a polypeptide having an extracellular portion having the same amino acid sequence as the extracellular portion of a human polypeptide and the remaining portion having the same amino acid sequence as the non-human (e.g., mouse) polypeptide. In some embodiments, a humanized gene includes at least a portion of the DNA sequence of a human gene. In some embodiments, a humanized protein includes a sequence having a portion that appears in a human protein. The term “human” is recognized in the art and refers to a nucleic acid or protein whose structure (i.e., nucleotide or amino acid sequence) is entirely derived from a human source.

[0073] As used herein, the term “locus” refers to a location on a chromosome that contains a set of related genetic elements (e.g., genes, genetic segments, regulatory elements). A locus can be endogenous or non-endogenous. The term “endogenous locus” refers to a location on a chromosome where a particular genetic element is found naturally. In some embodiments, an endogenous locus has a naturally occurring sequence. In some embodiments, an endogenous locus is a wild-type locus. In some embodiments, an endogenous locus is a genetically engineered locus.

[0074] As used herein, the term “non-human animal” refers to any vertebrate organism that is not human. In some embodiments, non-human animals are cyclostomes, bony fish, cartilaginous fish (e.g., sharks or rays), amphibians, reptiles, mammals, and birds. In some embodiments, non-human mammals are primates, goats, sheep, pigs, dogs, cattle, or rodents. In some embodiments, non-human animals are rodents such as rats or mice.

[0075] As used herein, the term “operatably linked” refers to a juxtaposition in which the described components are related in a way that enables them to function as intended. A “operatably linked” regulatory sequence to a coding sequence is linked such that the expression of the coding sequence is achieved under conditions that are compatible with the regulatory sequence. “Operatally linked” sequences include both expression regulatory sequences adjacent to the gene of interest, and expression regulatory sequences that act trans or at a distance to control the gene of interest. As used herein, the term “expression regulatory sequence” refers to a polynucleotide sequence necessary to influence the expression and processing of the coding sequence to which they are linked. Expression regulatory sequences include appropriate transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translational efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The properties of such regulatory sequences vary depending on the host organism. For example, in prokaryotes, such regulatory sequences generally include promoters, ribosome-binding sites, and transcription termination sequences, while in eukaryotes, such regulatory sequences typically include promoters and transcription termination sequences. The term “regulatory sequence” is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0076] The terms “polynucleotide” and “nucleic acid” are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides may have any three-dimensional structure and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure may be given before or after the assembly of the polymer. Polynucleotides may be further modified, such as by conjugation with labeling components. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0077] As used herein, the term “polypeptide” refers to any polymer chain of amino acids. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has an amino acid sequence that does not exist in nature. In some embodiments, the polypeptide has an amino acid sequence that has been manipulated to be designed and / or produced by human action.

[0078] As used herein, the term “promoter” includes a DNA sequence operably ligated to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding a desired molecule. Promoters are generally positioned upstream of the nucleic acid sequence to be transcribed and provide a site for specific binding by RNA polymerase and other transcription factors. In certain embodiments, promoters are generally positioned upstream of the nucleic acid sequence that is transcribed to produce the desired molecule and provide a site for specific binding by RNA polymerase and other transcription factors. The term “endogenous promoter” refers to a promoter that naturally associates with an endogenous gene in, for example, a wild-type organism.

[0079] As used herein, the term “recombinant” refers to polypeptides designed, manipulated, prepared, expressed, produced, or isolated by recombinant means (e.g., signal-regulating proteins described herein), polypeptides expressed using recombinant expression vectors transfected into host cells, polypeptides isolated from recombinant combinatorial human polypeptide libraries (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), and human immunoglobulin genes (e.g., Taylor, LD et al. (1992) Nucl. Acids This term is intended to refer to a polypeptide prepared, expressed, produced, or isolated by any other means, including splicing selected sequence elements together, or by an antibody isolated from an animal (e.g., mouse) that is transgenic (Res.20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597, Little M. et al. (2000) Immunology Today 21:364-370). In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more such selected sequence elements are designed in silico. In some embodiments, one or more such selected sequence elements result from mutagenesis (e.g., in vivo or in vitro) of known sequence elements from, for example, natural or synthetic sources. For example, in some embodiments, the recombinant polypeptide consists of sequences found in the genome of the source organism of interest (e.g., human, mouse, etc.).In some embodiments, recombinant polypeptides have amino acid sequences resulting from mutagenesis (e.g., in non-human animals, e.g., in vitro or in vivo), so the amino acid sequences of recombinant polypeptides are derived from and related to polypeptide sequences, but are sequences that cannot naturally exist in the genome of non-human animals in vivo.

[0080] The term “substitution” is used herein to refer to the process by which a “substituted” nucleic acid sequence (e.g., a gene) found at a host locus (e.g., in the genome) is removed from that locus and a different “substituted” nucleic acid is placed in its place. In some embodiments, the substituted nucleic acid sequence and the substitute nucleic acid sequence are equivalent to each other in that they are homologous to each other and / or contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.). In some embodiments, the substituted nucleic acid sequence includes one or more of the following: promoter, enhancer, splice donor site, splice receptor site, intron, exon, or untranslated region (UTR). In some embodiments, the substitute nucleic acid sequence includes one or more coding sequences. In some embodiments, the substitute nucleic acid sequence is a homolog of the substituted nucleic acid sequence. In some embodiments, the substitute nucleic acid sequence is an ortholog of the substituted sequence. In some embodiments, the substitute nucleic acid sequence is or includes a human nucleic acid sequence. In some embodiments, including cases where the substituted nucleic acid sequence is or contains a human nucleic acid sequence, the substituted nucleic acid sequence is or contains a rodent sequence (e.g., a mouse sequence). The thus-configured nucleic acid sequence may include one or more regulatory sequences that are part of a source nucleic acid sequence used to obtain the thus-configured sequence (e.g., promoters, enhancers, 5' or 3' untranslated regions, etc.). For example, in various embodiments, substitutions include substitution of an endogenous sequence by a heterologous sequence that results in the production of a gene product from the thus-configured nucleic acid sequence (including heterologous sequences) but does not result in the expression of the endogenous sequence; substitution of an endogenous genomic sequence by a nucleic acid sequence that encodes a protein having a similar function to the protein encoded by the endogenous sequence. In various embodiments, an endogenous gene or fragment thereof is replaced by a corresponding human gene or fragment thereof. The corresponding human gene or fragment thereof is an ortholog of the endogenous gene or fragment thereof being substituted, or a human gene or fragment whose structure and / or function is substantially similar or identical.

[0081] Where used herein, "variant" includes nucleic acid or peptide sequences that differ in sequence from a reference nucleic acid or peptide sequence but retain the essential biological properties of the reference molecule. Sequence changes in nucleic acid variants may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or they may result in amino acid substitutions, additions, deletions, fusions, and shortenings. Sequence changes in peptide variants are typically limited or conserved, so the sequences of the reference peptide and the variant are closely similar overall and identical in many regions. Variants and reference peptides may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. Nucleic acid or peptide variants may be naturally occurring variants, such as allele variants, or variants not known to exist naturally. Variants of nucleic acids and peptides not found in nature may be produced by mutagenesis or direct synthesis.

[0082] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid with which it is associated. In some embodiments, vectors enable extrachromosomal replication and / or expression of the nucleic acid to which they are linked in host cells such as eukaryotic and / or prokaryotic cells. A vector capable of directing the expression of a functionally linked gene is referred herein to as an “expression vector.”

[0083] When used herein, the term “wild-type” has the meaning understood in the art to refer to an entity with a structure and / or activity that is naturally found in a “normal” state or context (as opposed to a variant, disease, change, etc.). Those skilled in the art will understand that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0084] Genetically modified gene loci In certain embodiments, genetically modified non-human animals (e.g., mice or rats) are provided herein that include (i) homozygous null mutations in the Rag1 and / or Rag2 genes (e.g., Rag1 and / or Rag2 gene knockout); (ii) homozygous null mutations in the IL2rg gene (e.g., IL2rg gene knockout); (iii) homozygous null mutations in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a non-human animal portion and a human portion operably linked to the Flt3l promoter. In some embodiments, genetically modified non-human animals (e.g., mice) are provided herein that include (i) a homozygous null mutation in the Rag2 gene (e.g., Rag2 gene knockout); (ii) a homozygous null mutation in the IL2rg gene (e.g., IL2rg gene knockout); (iii) a homozygous null mutation in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a non-human animal portion and a human portion operably ligated to the Flt3l promoter. In some embodiments, the genetically modified non-human animals express a human or humanized SIRPA protein encoded by a nucleic acid operably ligated to the Sirpa promoter. In certain embodiments, the genetically modified non-human animals further express a human GM-CSF protein encoded by a nucleic acid operably ligated to the GM-CSF promoter and / or a human IL3 protein encoded by a nucleic acid operably ligated to the IL3 promoter. In certain embodiments, at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous non-human animal promoter. In some embodiments, the genetically modified non-human animal includes the engraftment of human hematopoietic stem cells (HSCs).

[0085] FLT3 Knockout In certain embodiments, the genetically modified non-human animals (e.g., mice or rats) provided herein contain a homozygous null mutation in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene.

[0086] The Flt3 gene encodes a class III receptor tyrosine kinase that regulates hematopoiesis. This receptor is activated by the binding of fms-associated tyrosine kinase 3 ligand to its extracellular domain, inducing homodimerization in the plasma membrane and leading to receptor autophosphorylation. The activated receptor kinase subsequently phosphorylates and activates multiple cytoplasmic effector molecules in pathways involved in apoptosis, proliferation, and differentiation of hematopoietic cells in the bone marrow. Mutations resulting in constitutive activation of this receptor lead to acute myeloid leukemia and acute lymphoblastic leukemia.

[0087] In certain embodiments, genetically modified non-human animals include a homozygous null mutation in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene. A null mutation includes deletions, insertions, and / or substitutions of a gene that do not result in a functional gene product (e.g., the complete absence of a gene product (protein, RNA) at the molecular level, or the expression of a non-functional gene product). In this disclosure, null mutation is used interchangeably with inactivating mutation. A homozygous null mutation means having a null mutation in all alleles. For example, a homozygous null mutation in the mouse or rat Flt3 gene means having a null mutation in two alleles (i.e., two null alleles) for the mouse or rat Flt3 gene. A gene having a homozygous null mutation is also called a gene knockout or gene deletion / deficit. For example, a homozygous null mutation in Flt3 is also called an Flt3 knockout or Flt3 deletion. Therefore, null mutations in the non-human animal Flt3 gene include deletions, insertions, and / or substitutions of the non-human animal Flt3 gene. In some cases, the endogenous non-human animal Flt3 locus contains null mutations, and thus null alleles. A null allele is a variant copy of the gene that completely lacks the normal function of that gene. This can result from a complete absence of the gene product (protein, RNA) at the molecular level, or from the expression of a non-functional gene product. At the phenotypic level, a null allele includes a deletion of the entire locus.

[0088] In some embodiments, the null mutation is a complete deletion of the endogenous Flt3 coding sequence. In some embodiments, the non-human animals provided herein do not express the Flt3 protein.

[0089] In some embodiments, a homozygous null mutation in the non-human Flt3 gene includes the same null mutation for all alleles. In some embodiments, a homozygous null mutation in the non-human Flt3 gene includes different null mutations for different alleles.

[0090] The mouse Flt3 locus is located on chromosome 5, GRCm38, NC_000071.6 (147330741-147400489), and the mouse Flt3 coding sequence can be found at Genbank accession number NM_010229.2. The mouse Flt3 locus contains 24 exons, with exons 1-24 being coding exons. Therefore, in some embodiments, the genetically modified animals provided herein are mice, and one or more of exons 1-24 of the mouse Flt3 gene are deleted or mutated in the genetically modified mouse. In some examples, other aspects of the genomic locus of the mouse Flt3 gene, such as introns, 3' and / or 5' untranslated sequences (UTRs), are also deleted or mutated. In some examples, the entire region of the mouse Flt3 genomic locus is deleted. In some embodiments, the entire genomic region of the mouse Flt3 gene, from the start codon to the stop codon, is deleted. For example, a genetically modified mouse may contain a 69.1kb deletion of the mouse genome sequence in the mouse Flt3 gene, as shown in Example 1, where the sequence from the start codon ATG to the stop codon is deleted between coordinates chr5:147331171-147400265 (GRCm38 assembly) on mouse chromosome 5 G3.

[0091] Deletions, alterations, or changes in the endogenous Flt3 gene locus can be detected using a variety of methods, including, for example, PCR, Western blotting, Southern blotting, restriction fragment length polymorphism (RFLP), or allele increase / decrease assays. In some embodiments, non-human animals are homozygous for deletions or null mutations in the endogenous Flt3 gene.

[0092] In some embodiments, non-human animals (e.g., mice or rats) containing a homozygous null mutation in the Flt3 gene, i.e., Flt3-deficient non-human animals, are immunosuppressed animals. For example, an Flt3-deficient non-human animal (e.g., a mouse or rat) may contain at least one null allele for the Rag2 gene (the coding sequence of the mouse gene may be found at Genbank accession number NM_009020.3, "recombinant activating gene 2"). In some embodiments, an Flt3-deficient non-human animal (e.g., a mouse or rat) contains two null alleles for Rag2. In other embodiments, an Flt3-deficient non-human animal (e.g., a mouse or rat) contains one or two null alleles for the Rag1 gene. In some embodiments, an Flt3-deficient non-human animal (e.g., a mouse or rat) is homozygous null for Rag1. In some embodiments, an Flt3-deficient non-human animal (e.g., mouse or rat) comprises (i) one or two null alleles for the Rag1 gene; and (ii) one or two null alleles for the Rag2 gene. In some embodiments, an Flt3-deficient non-human animal (e.g., mouse or rat) is homozygous null for both Rag1 and Rag2. In some embodiments, an Flt3-deficient non-human animal is an immunosuppressed mouse comprising two null alleles for Rag2 (i.e., homozygous null). In some embodiments, an Flt3-deficient non-human animal is an immunosuppressed rat comprising two null alleles for Rag1 (i.e., homozygous null) and two null alleles for Rag2 (i.e., homozygous null). As another example, Flt3-deficient non-human animals (e.g., mice or rats) contain at least one null allele for the IL2rg gene (the "interleukin-2 receptor, gamma," also known as the common gamma strand or γC, whose coding sequence for the mouse gene can be found at Genbank accession number NM013563.3).In some embodiments, Flt3-deficient non-human animals (e.g., mice or rats) contain two null alleles for IL2rg. In other words, Flt3-deficient non-human animals (e.g., mice or rats) are homozygous nulls for IL2rg, i.e., IL2rg. - / - (or the IL2rg gene is located on the X chromosome, as in mice or rats) Y / - In some embodiments, Flt3-deficient non-human animals (e.g., mice or rats) have null alleles for both Rag2 and IL2rg, i.e., it is Rag2 - / - IL2rg - / - (or the IL2rg gene is located on the X chromosome, like in mice or rats, Rag2 - / - IL2rg Y / -In some embodiments, an Flt3-deficient non-human animal (e.g., mouse or rat) contains null alleles for both Rag1 and IL2rg. In some embodiments, an Flt3-deficient non-human animal (e.g., mouse or rat) contains null alleles for Rag1, Rag2, and IL2rg. In some embodiments, an Flt3-deficient non-human animal is an immunosuppressed mouse (or one null allele for male mice) containing two null alleles for Rag2 (i.e., homozygous null) and two null alleles for IL2rg (i.e., homozygous null). In some embodiments, an Flt3-deficient non-human animal is an immunosuppressed rat (or one null allele for male rats) containing two null alleles for Rag1 (i.e., homozygous null), two null alleles for Rag2 (i.e., homozygous null), and two null alleles for IL2rg (i.e., homozygous null). Other genetic modifications are also being considered. For example, Flt3-deficient non-human animals (e.g., mice or rats) may include modifications to other genes related to the development and / or function of dendritic cells and the immune system, such as substitutions of one or more other non-human animal genes with nucleic acid sequences encoding human or humanized polypeptides. Such genes include, but are not limited to, FLT3L, SIRPA, GM-CSF, and / or IL-3. Additionally or alternatively, Flt3-deficient non-human animals (e.g., mice or rats) may include modifications to genes related to the development and / or function of other cells and tissues, such as genes related to human disorders or diseases, or genes that, when modified in non-human animals, such as mice, provide models of human disorders and diseases. The introduction of other genetic modifications may be achieved either by modifying and / or breeding ES cells. For example, a non-human animal (e.g., mouse or rat) lacking Flt3 (and, if necessary, lacking Rag2 and IL2rg) can be crossbred with a non-human animal having one or more other genetic modifications, including but not limited to, modifications of the SIRPA, GM-CSF, and / or IL-3 gene.In some embodiments, all genetic modifications are homozygous in the genetically modified animals described herein.

[0093] Humanized Flt3l locus In certain embodiments, the genetically modified non-human animals provided herein further express a chimeric Flt3l protein encoded by a Flt3 ligand (Flt3l) gene comprising a rodent and a human portion operably linked to the Flt3l promoter.

[0094] Dendritic cells (DCs) provide a crucial link between innate and adaptive immunity by recognizing pathogens and priming pathogen-specific immune responses. FLT3LG (also known as FL, FLG3L, or FLT3L) controls DC development and is particularly important for plasmacytoid DCs and CD8-positive classical DCs, as well as their CD103-positive tissue counterparts.

[0095] In some embodiments, the genetically modified non-human animals (e.g., rodents such as rats or mice) provided herein include an Flt3 ligand (Flt3l) gene comprising a non-human animal (e.g., rodent such as a rat or mouse) portion and a human portion operably linked to the Flt3l promoter.

[0096] In some embodiments, the non-human animal (e.g., rodent such as a rat or mouse) portion of the Flt3l gene includes exon 1, the non-coding portion of exon 2, and exons 7-9, each having at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical sequences to the non-human animal (e.g., rodent such as a rat or mouse) Flt3l gene.

[0097] The genomic locus encoding the wild-type mouse Flt3l protein can be found in the mouse genome on chromosome 7; GRCm39, NC_000073.7 (44780607-44785914). The polypeptide sequence of wild-type mouse Flt3l and the nucleic acid sequence encoding wild-type mouse Flt3l are Genbank accession numbers NP_001389760.1 and NM_001402831.1; NP_001389761.1 and NM_001402832.1; NP_001389762.1 and NM_001402833.1; NP_001389763.1 and NM_001402834.1; NP_001389764.1 and NM_001402835.1; NP_001389765.1 and NM_001402836.1; These can be found in NP_001389766.1 and NM_001402837.1; NP_038548.3 and NM_013520.4. In some embodiments, the membrane-bound mouse Flt3 ligand polypeptide sequences used in this disclosure are obtained from the NCBI reference sequence: NP_038548.3, Uniprot: A9QW46, and encoded by NM_013520.4.

[0098] In some embodiments, the non-coding portions of exon 1 and exon 2, and the downstream exons of exon 6 (e.g., exons 7-9) of the Flt3L gene in non-human animals (e.g., rodents such as rats or mice) are at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the corresponding non-coding portions of exon 1 and exon 2, and exons 7-9 of the mouse Flt3l gene shown in Table 1A. [Table 1A]

[0099] In some embodiments, the human portion of the humanized FLT3L gene includes the signal peptide coding portion of exon 2 that appears in the human FLT3L gene, and the signal peptide coding portions of exon 2 and exons 3-6, each having at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical sequences to exons 3-6.

[0100] The genomic locus encoding the wild-type human FLT3L protein can be found in the human genome on chromosome 19;GRCh38.p14 NC_000019.10 (49474215-49486231). The polypeptide sequence of wild-type human FLT3L and the nucleic acid sequence encoding wild-type human FLT3L can be found at Genbank accession numbers NP_001191431.1 and NM_001204502.2 (isoform 1 and transcription variant 1); NP_001191432.1 and NM_001204503.2 (isoform 1 and transcription variant 2); NP_001265566.1 and NM_001278637.2 (isoform 2 and transcription variant 4); NP_001265567.1 and NM_001278638.2 (isoform 2 and transcription variant 5); NP_001450.2 and NM_001459.4 (isoform 1 and transcription variant 3). In some embodiments, the membrane-bound human FLT3 ligand polypeptide sequences used in this disclosure are obtained from the NCBI reference sequence: NP_001191431.1, Uniprot: P49771-1, and encoded by NM_001204502.2.

[0101] In some embodiments, the signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene are at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the corresponding signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B. [Table 1B]

[0102] In some embodiments, the Flt3l gene includes non-human animal (e.g., rodents such as rats or mice) exon 1, the rodent non-coding portion of exon 2, the human signal peptide coding portion of exon 2, human exons 3-6, and non-human animal (e.g., rodents such as rats or mice) exons 7-9. In some examples, the Flt3l gene also includes aspects of the human FLT3L genomic locus, e.g., introns, 3' and / or 5' untranslated sequences (UTRs). In some examples, the Flt3l gene also includes aspects of the non-human animal (e.g., rodents such as rats or mice) Flt3l genomic locus, e.g., introns, 3' and / or 5' untranslated sequences (UTRs).

[0103] In some embodiments, the Flt3l gene encodes a chimeric membrane-bound Flt3l comprising the signal peptide and cytokine-like core domain of the human FLT3L polypeptide and the C-terminal portion of the rodent Flt3l polypeptide. The terms “cytokine-like core domain” and “cytokine domain” refer to the same portion of the human FLT3L polypeptide and are used interchangeably in this disclosure.

[0104] Exemplary chimeric membrane-bound FLT3L sequences are shown in Table 2. The signal peptide of the human FLT3L polypeptide is underlined, the cytokine-like core domain of the human FLT3L polypeptide is in bold, and the C-terminal portion of the mouse FLT3L polypeptide is italicized.

[0105] [Table 2]

[0106] In some embodiments, the C-terminal portion of the rodent Flt3l polypeptide includes a rodent stalk region, a rodent transmembrane domain, and a rodent cytoplasmic tail, as shown in Figure 1E. In some embodiments, the C-terminal portion of the rodent Flt3l polypeptide contains amino acids corresponding to residues 165-232 of the mouse Flt3l polypeptide (e.g., NP_038548.3). In some embodiments, the C-terminal portion of the rodent Flt3l polypeptide has at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) the same amino acid sequence as the C-terminal portions of the rodent Flt3l polypeptide shown in Table 2.

[0107] In some embodiments, the signal peptide of the human FLT3L polypeptide contains amino acids corresponding to residues 1-26 of the human FLT3L polypeptide (e.g., NP_001191431.1). In some embodiments, the signal peptide of the human FLT3L polypeptide contains at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) the same amino acid sequence as the corresponding amino acid sequence of the human FLT3L polypeptide signal peptide shown in Table 2.

[0108] In some embodiments, the cytokine-like core domain of the human FLT3L polypeptide contains amino acids corresponding to residues 27-159 of the human FLT3L polypeptide (e.g., NP_001191431.1). In some embodiments, the cytokine-like core domain of the human FLT3L polypeptide contains at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) the same amino acid sequence as the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

[0109] In some embodiments, the non-human animals provided herein express a humanized Flt3l protein resulting from a genetic modification of the endogenous locus of the non-human animal encoding the Flt3l protein. Suitable examples described herein include rodents, particularly mice.

[0110] Mouse and human Flt3 ligands exist as multiple isoforms in both membrane-bound and soluble forms, which are thought to arise from alternative splicing (Lyman et al. (1995) Oncogene 10:149-157; McClanahan et al. (1996) Blood 88(9):3371-3382; Lyman et al. (1995) Oncogene 11:1165-1172; Lyman and Jackbsen (1998) Blood 91(4)1101-1134; the contents of each of these are incorporated herein by reference in their entirety). Soluble isoforms can also arise from proteolytic cleavage (resulting from cleavage by TNFα-converting enzyme or TACE) (Kazi et al. (2019) Physiol. Rev. 99:1433-1466; the contents of this are incorporated herein by reference in their entirety). In some embodiments, rodents express one or more soluble and / or membrane-bound forms of the Flt3l polypeptide. In some embodiments, rodents express both soluble and membrane-bound forms of the Flt3l polypeptide. In some embodiments, the soluble and membrane-bound forms of the Flt3l polypeptide include the signal peptide and cytokine-like core domain of the human FLT3L polypeptide. All known soluble isoforms of human and mouse Flt3 ligands generally include a receptor-binding domain (cytokine-like core domain) encoded by exons 3-6 of the gene.

[0111] In some embodiments, genetically modified rodents are heterozygous for the Flt3 ligand (Flt3l) gene, which includes both a non-human (e.g., rodent such as a rat or mouse) portion and a human portion. In some embodiments, genetically modified rodents are homozygous for the Flt3 ligand (Flt3l) gene, which includes both a non-human (e.g., rodent such as a rat or mouse) portion and a human portion.

[0112] In some embodiments, the non-human (e.g., rodent such as a rat or mouse) portion of the Flt3l gene is the endogenous non-human (e.g., rodent such as a rat or mouse) portion of the Flt3l gene. The rodent Flt3l gene is the endogenous non-human (e.g., rodent such as a rat or mouse) gene; and / or, the rodent Flt3l polypeptide is the endogenous non-human (e.g., rodent such as a rat or mouse) Flt3l polypeptide.

[0113] In some embodiments, non-human animal (e.g., rodents such as rats or mice) and human portions are operably linked to the Flt3l promoter. In some embodiments, the Flt3l promoter is a non-human animal (e.g., rodent such as rats or mice) promoter. In some embodiments, the Flt3l promoter is an endogenous non-human animal (e.g., rodent such as rats or mice) promoter. In some embodiments, the Flt3l promoter is located at the endogenous non-human animal (e.g., rodent such as rats or mice) locus.

[0114] In some embodiments, genetically modified non-human animals include genetic material derived from a different species (e.g., humans), and the non-human Flt3l gene encodes an Flt3l protein containing an encoded portion of the genetic material derived from the different species. In some embodiments, the humanized Flt3l gene of this disclosure includes genomic DNA from a different species corresponding to the signal peptide and cytokine-like core domain of the Flt3l protein. Targeted constructs for producing non-human animals, embryos, cells, and cells containing the humanized Flt3l gene are also provided.

[0115] In some embodiments, the endogenous non-human (e.g., rodent) FLT3l gene is deleted. In some embodiments, the endogenous non-human (e.g., rodent) FLT3l gene is modified, and a portion of the endogenous non-human (e.g., rodent) FLT3l gene is replaced with a heterologous sequence (e.g., all or part of the human FLT3L sequence). In some embodiments, all or substantially all of the endogenous non-human (e.g., rodent) FLT3l gene is replaced with a heterologous gene (e.g., the human FLT3L gene). In some embodiments, a portion of the heterologous FLT3L gene is inserted into the endogenous non-human (e.g., rodent) FLT3l gene locus. In some embodiments, the heterologous gene is a human gene. In some embodiments, modification or humanization is performed on one of two copies of the endogenous non-human (e.g., rodent) FLT3l gene, resulting in a non-human animal that is heterozygous for the humanized FLT3l gene. In other embodiments, a non-human animal that is homozygous for the humanized FLT3l gene is provided. In some embodiments, the portions encoding the signal peptide and cytokine-like core domain of the endogenous non-human (e.g., rodent) FLT3l gene are replaced with the corresponding heterologous sequence (e.g., human FLT3L sequence), while the portions encoding the stalk, transmembrane domain, and cytoplasmic tail remain the endogenous non-human (e.g., rodent) FLT3l sequence. In such embodiments, the non-human (e.g., rodent) TACE cleavage is preserved.

[0116] The non-human animals of this disclosure contain all or part of the human FLT3L gene at the endogenous non-human FLT3L locus. Therefore, such non-human animals can be described as having a heterologous FLT3L gene. Substitutions, insertions, or modifications of the FLT3L gene at the endogenous non-human (e.g., rodent) FLT3L locus can be detected using a variety of methods, including, for example, PCR, Western blotting, Southern blotting, restriction fragment length polymorphism (RFLP), or allele increase / decrease assays. In some embodiments, the non-human animals are heterozygous with respect to the humanized FLT3L gene.

[0117] Compositions and methods are provided for creating non-human animals that express a humanized FLT3l protein containing a specific polymorphic form or allele variant (e.g., a single amino acid difference). In some embodiments, compositions and methods are provided for creating non-human animals that express such a protein from an endogenous promoter and an endogenous regulatory sequence. The method involves inserting genetic material encoding a human FLT3L protein, either whole or partially, at a precise location in the genome of a non-human animal corresponding to an endogenous non-human (e.g., rodent) FLT3l gene, thereby creating a humanized FLT3l gene that expresses an FLT3l protein that is whole or partially human. In some embodiments, the method involves inserting genomic DNA corresponding to the signal peptide coding portion of exon 2, starting with the start codon "ATG", and exons 3-6 of the human FLT3L gene, into the endogenous non-human (e.g., rodent) FLT3l gene of a non-human animal, thereby creating a humanized gene that encodes an FLT3l protein containing a human portion containing the amino acid encoded by the inserted exon.

[0118] In various embodiments, the humanized Flt3l gene approach uses relatively minimal modification of the endogenous gene to produce native Flt3l-mediated signaling in non-human animals. Therefore, in such embodiments, the Flt3l gene modification does not affect other surrounding genes. Furthermore, in various embodiments, the modification does not affect the alternative splicing pattern or post-translational cleavage of Flt3l.

[0119] In addition to mice having the humanized FLT3l gene described herein, other genetically modified non-human animals containing the humanized FLT3l gene are also provided herein. In some embodiments, such non-human animals include a humanized FLT3l gene operably linked to an endogenous non-human (e.g., rodent) FLT3l promoter. In some embodiments, such non-human animals express a humanized FLT3l protein from an endogenous locus, the humanized FLT3l protein including at least amino acid residues 27-160, for example, amino acid residues 27-161, 27-162, 27-163, 27-164, 27-165, 1-160, 1-161, 1-162, 1-163, 1-164, or 1-165 of the human FLT3L protein.

[0120] Immunodeficient non-human animals As described above, genetically modified non-human animals, including those with Flt3 deficiency and Flt3l humanization described herein, are also immunodeficient because they contain deficiencies in the Rag1 and / or Rag2 and Il2rg genes. Rag1, Rag2, and Il2rg are essential components of the adaptive immune system. When one or more of these genes are mutated in an animal, T cells and B cells fail to mature, and the animal is severely impaired. When these animals are loaded with foreign body transplant cells, they are unable to initiate an immune response to the foreign cells.

[0121] V(D)J recombination-activated protein 1 (also known as RAG1, recombination-activated 1, recombination-activated gene 1, and recombination-activated protein 1) is encoded by the Rag1 gene (also known as recombination-activated 1). RAG1 is a catalytic component of the RAG complex, a multiprotein complex that mediates the DNA cleavage phase during V(D)J recombination. V(D)J recombination assembles a diverse repertoire of immunoglobulin and T cell receptor genes in the development of B and T lymphocytes through the rearrangement of different V (variability), and sometimes D (diversity), and J (linking) gene segments. In the RAG complex, RAG1 catalyzes DNA cleavage activity by mediating DNA binding to a conserved recombination signal sequence (RSS) and introducing double-strand breaks between the RSS and adjacent coding segments. RAG2 is not a catalytic component but is required for all known catalytic activity. RAG1 and RAG2 are essential for the generation of mature B cells and T cells, two types of lymphocytes that are important components of the adaptive immune system.

[0122] Mouse Rag1 maps to 2 E2; 2 53.88 cM on chromosome 2 (NCBI RefSeq gene ID 19373; assembly GRCm39 (GCF_000001635.27); position NC_000068.8 (101468597..101479877, complementary strand)). References to the mouse Rag1 gene include the canonical wild-type morph as well as all allele morphs and isoforms. The canonical wild-type mouse RAG1 protein is assigned UniProt accession number P15919 and NCBI accession number NP_033045.2. References to the mouse RAG1 protein include the wild-type morph as well as all allele morphs and isoforms. The mRNA (cDNA) encoding the canonical isoform is assigned NCBI accession number NM_009019.2. Mouse Rag1 References to mRNA (cDNA) and coding sequences include the canonical wild-type form as well as all allele forms and isoforms.

[0123] Rat Rag1 maps to 3q31 on chromosome 3 (NCBI RefSeq gene ID 84600; assembly mRatBN7.2(GCF_015227675.2); position NC_051338.1(87917061..87928158, complementary strand). References to the rat Rag1 gene include the canonical wild-type morph as well as all allele forms and isoforms. The canonical wild-type rat RAG1 protein is assigned UniProt accession number G3V6K9 and NCBI accession number NP_445920.1. References to the rat RAG1 protein include the canonical wild-type morph as well as all allele forms and isoforms. The mRNA (cDNA) encoding the canonical isoform is assigned NCBI accession number NM_053468.1. References to mRNA (cDNA) and coding sequences include the canonical wild-type form as well as all allele forms and isoforms.

[0124] An inactivated endogenous Rag1 gene is a Rag1 gene that does not produce the RAG1 protein or does not produce the functional RAG1 protein. Non-human animals (or cells or genomes) may have an inactivated Rag1 gene in their germline. Non-human animals (or cells or genomes) may be homozygous for inactivating mutations in the Rag1 gene. For example, an inactivated endogenous Rag1 gene may contain an insertion, deletion, or one or more point mutations in the endogenous Rag1 gene that result in loss of expression of the functional RAG1 protein. Some inactivated endogenous Rag1 genes may contain all deletions or disruptions of the endogenous Rag1 gene, or may contain deletions or disruptions of a fragment (i.e., part or portion) of the endogenous Rag1 gene. For example, some, most, or all of the coding sequence in the endogenous Rag1 gene may be deleted or disrupted. For example, the 5' fragment of the Rag1 gene may be deleted or disrupted (e.g., including the start codon). For example, an inactivated endogenous Rag1 gene may be one in which the start codon of the endogenous Rag1 gene is deleted, or the start codon is disrupted or mutated so that it is no longer functional. For example, the start codon may be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon may be mutated, for example, by the substitution of one or more nucleotides. In another example, the 3' fragment of the Rag1 gene may be deleted or disrupted (e.g., including the stop codon). In yet another example, an internal fragment of the Rag1 gene (i.e., a fragment from the center of the Rag1 gene) may be deleted or disrupted. In yet another example, the entire coding sequence within the endogenous Rag1 gene may be deleted or disrupted.

[0125] V(D)J recombinant activating protein 2 (also known as RAG2, recombinant activating gene 2, recombinant activating gene 2, and recombinant activating protein 2) is encoded by the Rag2 gene (also known as recombinant activating 2). As mentioned above, RAG1 is a catalytic component of the RAG complex, a multiprotein complex that mediates the DNA cleavage phase during V(D)J recombination. RAG2 is not a catalytic component but is required for all known catalytic activity. RAG1 and RAG2 are essential for the generation of mature B cells and T cells, two types of lymphocytes that are important components of the adaptive immune system.

[0126] Mouse Rag2 maps to 2 E2; 2 53.87 cM on chromosome 2 (NCBI RefSeq gene ID 19374; assembly GRCm39 (GCF_000001635.27); position NC_000068.8 (101455057..101462873)). References to the mouse Rag2 gene include the canonical wild-type morph as well as all allele forms and isoforms. The canonical wild-type mouse RAG2 protein is assigned UniProt accession number P21784 and NCBI accession number NP_033046.1. References to the mouse RAG2 protein include the canonical wild-type morph as well as all allele forms and isoforms. The mRNA (cDNA) encoding the canonical isoform is assigned NCBI accession number NM_009020.3. Mouse Rag2 References to mRNA (cDNA) and coding sequences include the canonical wild-type form as well as all allele forms and isoforms.

[0127] Rat Rag2 maps to 3q31 on chromosome 3 (NCBI RefSeq gene ID 295953; assembly mRatBN7.2(GCF_015227675.2); position NC_051338.1(87902373..87910227). References to the rat Rag2 gene include the canonical wild-type morph as well as all allele forms and isoforms. The canonical wild-type rat RAG2 protein is assigned UniProt accession number G3V6K7 and NCBI accession number NP_001093998.1. References to the rat RAG2 protein include the canonical wild-type morph as well as all allele forms and isoforms. The mRNA (cDNA) encoding the canonical isoform is assigned NCBI accession number NM_001100528.1. Rat Rag2 References to mRNA (cDNA) and coding sequences include the canonical wild-type form as well as all allele forms and isoforms.

[0128] An inactivated endogenous Rag2 gene is a Rag2 gene that does not produce the RAG2 protein or does not produce the functional RAG2 protein. Non-human animals (or cells or genomes) may have inactivated Rag2 genes in their germline. Non-human animals (or cells or genomes) may be homozygous for inactivating mutations in the Rag2 gene. For example, an inactivated endogenous Rag2 gene may contain an insertion, deletion, or one or more point mutations in the endogenous Rag2 gene that result in loss of expression of the functional RAG2 protein. Some inactivated endogenous Rag2 genes may contain all deletions or disruptions of the endogenous Rag2 gene, or may contain deletions or disruptions of a fragment (i.e., part or portion) of the endogenous Rag2 gene. For example, some, most, or all of the coding sequence in the endogenous Rag2 gene may be deleted or disrupted. For example, the 5' fragment of the Rag2 gene may be deleted or disrupted (e.g., including the start codon). For example, an inactivated endogenous Rag2 gene may be one in which the start codon of the endogenous Rag2 gene is deleted, or the start codon is disrupted or mutated so that it is no longer functional. For example, the start codon may be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon may be mutated, for example, by the substitution of one or more nucleotides. In another example, the 3' fragment of the Rag2 gene may be deleted or disrupted (e.g., including the stop codon). In yet another example, the internal fragment of the Rag2 gene (i.e., the fragment from the center of the Rag2 gene) may be deleted or disrupted. In yet another example, the entire coding sequence within the endogenous Rag2 gene may be deleted or disrupted.

[0129] The interleukin-2 receptor subunit gamma (also known as interleukin-2 receptor gamma; interleukin-2 receptor gamma (severe combined immunodeficiency), isoform CRA_a; cytokine receptor common subunit gamma precursor) is encoded by the Il2rg gene (also known as interleukin-2 receptor subunit gamma or IL2RG). IL2RG is a cytokine receptor subunit common to several different interleukin receptor receptor complexes. IL2RG is located on the surface of immature hematopoietic cells in the bone marrow. IL2RG, in cooperation with other proteins, guides hematopoietic cells to form lymphocytes. IL2RG also guides the growth and maturation of T cells, B cells, and natural killer cells. Mutations in Il2rg can cause X-linked severe combined immunodeficiency, in which lymphocytes cannot develop normally. The lack of functionally mature lymphocytes disrupts the immune system's ability to protect the body from infection.

[0130] Mouse Il2rg maps to X 43.9 cM on the XD;X chromosome (NCBI RefSeq gene ID 16186; assembly GRCm39 (GCF_000001635.27); position NC_000086.8 (100307991..100311861, complementary strand)). References to the mouse Il2rg gene include the canonical wild-type morph as well as all allele morphs and isoforms. The canonical wild-type mouse IL2rG protein is assigned UniProt accession number P34902 and NCBI accession number NP_038591.1. References to the mouse IL2RG protein include the canonical wild-type morph as well as all allele morphs and isoforms. The mRNA (cDNA) encoding the canonical isoforms is assigned NCBI accession number NM_013563.4. Mouse Il2rg References to mRNA (cDNA) and coding sequences include the canonical wild-type form as well as all allele forms and isoforms.

[0131] Rat Il2rg maps to Xq22 on the X chromosome (NCBI RefSeq Gene ID 140924; assembly mRatBN7.2 (GCF_015227675.2); position NC_051356.1 (66395330..66399026, complementary strand)). References to the rat Il2rg gene include the canonical wild-type morph as well as all allele forms and isoforms. The canonical wild-type rat IL2rG protein is assigned UniProt accession number Q68FU6 and NCBI accession number NP_543165.1. References to the rat IL2RG protein include the canonical wild-type morph as well as all allele forms and isoforms. The mRNA (cDNA) encoding the canonical isoform is assigned NCBI accession number NM_080889.1. References to mRNA (cDNA) and coding sequences include the canonical wild-type form as well as all allele forms and isoforms.

[0132] An inactivated endogenous Il2rg gene is an Il2rg gene that does not produce IL2RG protein or does not produce functional IL2RG protein. Non-human animals (or cells or genomes) may have inactivated Il2rg genes in their germline. Non-human animals (or cells or genomes) may be homozygous for inactivating mutations in the Il2rg gene. For example, an inactivated endogenous Il2rg gene may contain an insertion, deletion, or one or more point mutations in the endogenous Il2rg gene that result in loss of expression of functional IL2RG protein. Some inactivated endogenous Il2rg genes may contain all deletions or disruptions of the endogenous Il2rg gene, or may contain deletions or disruptions of a fragment (i.e., part or portion thereof) of the endogenous Il2rg gene. For example, some, most, or all of the coding sequence in the endogenous Il2rg gene may be deleted or disrupted. In one example, the 5' fragment of the Il2rg gene may be deleted or disrupted (e.g., including the start codon). In another example, an inactivated endogenous Il2rg gene may have the start codon of the endogenous Il2rg gene deleted, or the start codon disrupted or mutated so that it is no longer functional. For example, the start codon may be disrupted by a deletion or insertion within the start codon. Alternatively, the start codon may be mutated, for example, by the substitution of one or more nucleotides. In yet another example, the 3' fragment of the Il2rg gene may be deleted or disrupted (e.g., including the stop codon). In yet another example, the internal fragment of the Il2rg gene (i.e., the fragment from the center of the Il2rg gene) may be deleted or disrupted. In yet another example, the entire coding sequence within the endogenous Il2rg gene may be deleted or disrupted.

[0133] Humanized Sirpa locus In certain embodiments, the genetically modified non-human animals provided herein further express a human or humanized SIRPA protein encoded by a nucleic acid operably linked to a Sirpa promoter.

[0134] Signal-regulating proteins (SIRPs) constitute a family of cell surface glycoproteins expressed on lymphocytes, myeloid cells (including macrophages, neutrophils, granulocytes, myeloid dendritic cells, and mast cells), and neurons (see, e.g., Barclay and Brown, 2006, Nat Rev Immunol 6, 457-464). Reported SIRP genes include at least SIRPA, SIRP3, SIRPβ, SIRPγ, and SIRP8, and can be classified according to the respective ligands and signaling types they are involved in. SIRPA (also known as CD172A, SHPS1, P84, MYD-1, BIT, and PTPNS1) is expressed on myeloid immune cells and functions as an inhibitory receptor via an immunoreceptor tyrosine-based inhibitory motif (ITIM). SIRPA expression has also been observed in neurons. Reported ligands for SIRPA include, most notably, CD47, but also surfactant proteins A and D. In particular, the role of SIRPA has been studied in relation to its inhibitory role in macrophage-mediated phagocytosis of host cells. For example, the binding of CD47 to SIRPA on macrophages induces an inhibitory signal that negatively modulates phagocytosis. Alternatively, positive signaling effects mediated via SIRPA binding have been reported (Shultz et al., 1995, J Immunol, 154, 180-91). SIRPA has been shown to improve cell engraftment in immunodeficient mice (Strowig et al., Proc Natl Acad Sci USA, 2011;108:13218-13223).

[0135] The polypeptide sequence of wild-type human SIRPA and the nucleic acid sequence encoding wild-type human SIRPA can be found in Genbank accession numbers NP_001035111.1 and NM_001040022.1 (isoform 1 and transcription variant 1); NP_001035112.1 and NM_001040023.2 (isoform 1 and transcription variant 2); NP_001317657.1 and NM_001330728.1 (isoform 2 and transcription variant 4); and NP_542970.1 and NM_080792.3 (isoform 1 and transcription variant 3). The SIRPA gene is conserved in at least chimpanzees, rhesus monkeys, dogs, cattle, mice, rats, and chickens. The genomic locus encoding the wild-type human SIRPA protein can be found in the human genome on chromosome 20. NC_000020.11(1894167-1940592). In some embodiments, the human SIRPA protein is encoded by exons 2–9 of this locus. Therefore, in some embodiments, the nucleic acid sequence containing the coding sequence for human SIRPA includes one or more of exons 2–9 of the human SIRPA gene. In some examples, the nucleic acid sequence also includes aspects of the human SIRPA genomic locus, e.g., introns, 3' and / or 5' untranslated sequences (UTRs). In some examples, the nucleic acid sequence includes the entire region of the human SIRPA genomic locus. In some examples, the nucleic acid sequence includes exons 2–4 of the human SIRPA genomic locus.

[0136] Exemplary humanized Sirpa sequences are shown in Table 3. For protein sequences, the signal peptide is underlined, and the transmembrane and cytoplasmic sequences are italicized. Representative mouse Sirpa cDNA, mouse Sirpa protein, human SIRPA cDNA, and human SIRPA protein sequences are described in U.S. Patent No. 11,019,810, which is incorporated herein by reference in its entirety.

[0137] [Table 3]

[0138] In some embodiments, the non-human animals provided herein express the humanized Sirpa protein on the surface of immune cells (e.g., myeloid cells) of the non-human animals resulting from genetic modification of the endogenous locus of the non-human animals encoding the Sirpa protein. Suitable examples described herein include rodents, such as mice.

[0139] In some embodiments, the humanized Sirpa gene comprises genetic material from a different species (e.g., human), and the humanized Sirpa gene encodes a Sirpa protein that includes an encoded portion of the genetic material from the different species. In some embodiments, the humanized Sirpa gene of this disclosure comprises genomic DNA from a different species corresponding to the extracellular portion of the SIRPA protein expressed on the plasma membrane of a cell. Targeted constructs for producing non-human animals, embryos, cells, and non-human animals, non-human embryos, and cells containing the humanized Sirpa gene are also provided.

[0140] In some embodiments, the endogenous non-human (e.g., rodent) Sirpa gene is deleted. In some embodiments, the endogenous non-human (e.g., rodent) Sirpa gene is modified, and a portion of the endogenous non-human (e.g., rodent) Sirpa gene is replaced with a heterologous sequence (e.g., all or part of the human SIRPA sequence). In some embodiments, all or substantially all of the endogenous non-human (e.g., rodent) Sirpa gene is replaced with a heterologous gene (e.g., human SIRPA gene). In some embodiments, a portion of the heterologous SIRPA gene is inserted into the endogenous non-human Sirpa gene at the endogenous non-human (e.g., rodent) Sirpa locus. In some embodiments, the heterologous gene is a human gene. In some embodiments, the modification or humanization is performed on one of two copies of the endogenous non-human (e.g., rodent) Sirpa gene, resulting in a non-human animal that is heterozygous with respect to the humanized Sirpa gene. In other embodiments, non-human animals homozygous for the humanized Sirpa gene are provided. In some embodiments, the entire Sirpa gene of an endogenous non-human animal (e.g., a rodent) is replaced with a portion of a heterologous gene (e.g., a portion of the human Sirpa gene) so that the genetically modified non-human animal (e.g., a rodent) expresses a functional fragment of the full-length human SIRPA polypeptide (e.g., the extracellular domain of the human SIRPA polypeptide).

[0141] The non-human animals of this disclosure contain all or part of the human SIRPA gene at the endogenous non-human Sirpa locus. Therefore, such non-human animals can be described as having a heterologous SIRPA gene. Substitutions, insertions, or modifications of the SIRPA gene at the Sirpa locus in endogenous non-human animals (e.g., rodents) can be detected using a variety of methods, including, for example, PCR, Western blotting, Southern blotting, restriction fragment length polymorphism (RFLP), or allele increase / decrease assays. In some embodiments, the non-human animals are heterozygous with respect to the humanized Sirpa gene.

[0142] In various embodiments, the humanized Sirpa gene according to this disclosure includes a SIRPα gene having second, third, and fourth exons that are at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical in sequence to the second, third, and fourth exons appearing in the human SIRPA gene.

[0143] In various embodiments, the humanized Sirpa gene according to this disclosure includes a SIRPα gene having a nucleotide coding sequence (e.g., cDNA sequence) that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical nucleotide coding sequence (e.g., cDNA sequence) to nucleotides 352-1114 appearing in the human SIRPA cDNA sequence.

[0144] In various embodiments, the humanized Sirpa protein produced by a non-human animal of the Disclosure has an extracellular portion having at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical sequence to the extracellular portion of the human SIRPA protein.

[0145] In various embodiments, the humanized Sirpa protein produced by a non-human animal of the Disclosure has an extracellular portion having at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical sequences to amino acid residues 28–362 that appear in the human SIRPA protein.

[0146] In various embodiments, the humanized Sirpa protein produced by the non-human animal of this disclosure has an amino acid sequence that is at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identical to the amino acid sequence of the humanized Sirpa protein shown in Table 3.

[0147] Compositions and methods are provided for creating non-human animals that express a humanized Sirpa protein containing a specific polymorphic form or allele variant (e.g., a single amino acid difference), and include compositions and methods for creating non-human animals that express such a protein from a human promoter and human regulatory sequence. In some embodiments, compositions and methods are also provided for creating non-human animals that express such a protein from an endogenous promoter and endogenous regulatory sequence. This method includes inserting all or part of the genetic material encoding a human SIRPA protein into a precise location in the genome of a non-human animal corresponding to an endogenous non-human animal (e.g., a rodent) Sirpa gene, thereby creating a humanized Sirpa gene that expresses all or part of a human SIRPA protein. In some embodiments, the method includes inserting genomic DNA corresponding to exons 2-4 of the human SIRPA gene into the endogenous non-human animal (e.g., a rodent) Sirpa gene of a non-human animal, thereby creating a humanized gene that encodes a Sirpa protein containing a human portion containing the amino acid encoded by the inserted exon.

[0148] In various embodiments, the humanized Sirpa gene approach uses relatively minimal modification of the endogenous gene to produce native Sirpa-mediated signaling in non-human animals. Therefore, in such embodiments, Sirpa gene modification does not affect other surrounding genes or other endogenous non-human animal (e.g., rodent) Sirp genes. Furthermore, in various embodiments, the modification does not affect the assembly of the functional receptor in plasma, maintaining normal effector function through binding via the cytoplasmic portion of the receptor unaffected by the modification and subsequent signaling.

[0149] In addition to mice having the humanized Sirpa gene described herein, other genetically modified non-human animals containing the humanized Sirpa gene are also provided herein. In some embodiments, such non-human animals include the humanized Sirpa gene operably linked to an endogenous Sirpa promoter. In some embodiments, such non-human animals express the humanized Sirpa protein from an endogenous locus, the humanized Sirpa protein containing amino acid residues 28–362 of the human SIRPA protein.

[0150] Humanized Sirpa polypeptides, loci encoding humanized Sirpa polypeptides, and non-human animals expressing humanized Sirpa polypeptides are described in U.S. Patent No. 11,019,810, International Publication No. 2014 / 039782, International Publication No. 2014 / 071397, International Publication No. 2016 / 168212, and U.S. Patent No. 10,918,095, the contents of each of these are incorporated herein by reference in their entirety.

[0151] Humanized GM-CSF gene locus In some embodiments, the genetically modified non-human animals provided herein further express a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter. Human GM-CSF protein means a protein that is human GM-CSF or substantially identical to human GM-CSF, for example, 80% or more identical, 85% or more identical, 90% or more identical, or 95% or more identical to human GM-CSF, for example, 97%, 98%, or 99% identical to human GM-CSF. Therefore, a nucleic acid sequence encoding a human GM-CSF protein is a polynucleotide containing the coding sequence of a human GM-CSF protein, i.e., human GM-CSF or a protein substantially identical to human GM-CSF.

[0152] GM-CSF is a cytokine crucial for the development and function of myeloid cells. GM-CSF is not cross-reactive between humans and mice. As demonstrated by the fact that GM-CSF KO mice develop alveolar proteinosis (PAP), GM-CSF is highly expressed in the lungs and is important for lung homeostasis in vivo, which is characterized by protein accumulation in the lungs due to defects in surfactant clearance. Alveolar macrophages from GM-CSF KO mice have defects in terminal differentiation, resulting in congenital immune deficiency against pathogens in the lungs. GM-CSF also stimulates the proliferation of human alveolar macrophages (AMs) in vitro. GM-CSF is largely unnecessary for steady-state hematopoiesis. In contrast, GM-CSF is required for inflammatory responses such as the production of pro-inflammatory cytokines by macrophages and the recruitment of monocytes. GM-CSF is also essential for protective immunity against various pathogens, including Mycobacterium tuberculosis. In particular, GM-CSF knockout mice infected with Mycobacterium tuberculosis do not develop granulomas, which are characteristic of tuberculosis.

[0153] The polypeptide sequence of human GM-CSF and the nucleic acid sequence encoding human GM-CSF can be found at Genbank accession numbers NP_000749.2 and NM_000758.4, respectively. The genomic locus encoding the human GM-CSF protein can be found in the human genome on chromosome 5; NG_033024.1 (4998-7379). The protein sequence is encoded by exons 1-4 at this locus. Therefore, the nucleic acid sequence containing the coding sequence of human GM-CSF contains one or more of exons 1-4 of the human GM-CSF gene. In some examples, the nucleic acid sequence also contains aspects of the human GM-CSF genomic locus, e.g., introns, 3' and / or 5' untranslated sequences (UTRs). In some examples, the nucleic acid sequence contains the entire region of the human GM-CSF genomic locus.

[0154] In some embodiments, in the genetically modified non-human animals provided herein, the nucleic acid sequence encoding the human GM-CSF protein is operably ligated to one or more regulatory sequences of the non-human animal (e.g., mouse) GM-CSF gene. The non-human animal (e.g., mouse) GM-CSF regulatory sequences are sequences of the non-human animal (e.g., mouse) GM-CSF genomic locus that regulate non-human animal (e.g., mouse) GM-CSF expression, e.g., 5' regulatory sequence, e.g., GM-CSF promoter, GM-CSF 5' untranslated region (UTR), etc.; 3' regulatory sequence, e.g., 3'UTR; and enhancers, etc. For example, mouse GM-CSF is located on chromosome 11, GRCm39, NC_000077.7, approximately at position c54140725–54138096, and the mouse GM-CSF coding sequence may be found at Genbank accession number NM_009969.4. The regulatory sequences of mouse GM-CSF are well defined in the art and can be readily identified using in silico methods by referring, for example, to the above Genbank accession numbers in the UCSC Genome Browser, on the World Wide Web at genome.ucsc.edu, or to experimental methods described in the art. In some cases, for example, when the nucleic acid sequence encoding the human GM-CSF protein is located at a non-human animal (e.g., mouse) GM-CSF genome locus, the regulatory sequences operably ligated to the human GM-CSF coding sequence are endogenous or native to the non-human animal (e.g., mouse) genome, i.e., they existed in the non-human animal (e.g., mouse) genome before the incorporation of the human nucleic acid sequence.

[0155] In some examples, genetically modified non-human animals expressing human GM-CSF protein are generated by randomly incorporating or inserting a human nucleic acid sequence encoding the human GM-CSF protein or a fragment thereof, i.e., a "human GM-CSF nucleic acid sequence" or "human GM-CSF sequence," into the genome of a non-human animal. Typically, in such embodiments, the location of the nucleic acid sequence encoding the human GM-CSF protein in the genome is unknown. In other examples, genetically modified non-human animals expressing human GM-CSF protein are generated by targeted incorporation or insertion of the human GM-CSF nucleic acid sequence into the genome of a non-human animal, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome of the target locus while simultaneously removing host genomic material from the target locus, such as genomic material exceeding 50 base pairs (bp), 100 bp, 200 bp, 500 bp, 1 kB, 2 kB, 5 kB, 10 kB, 15 kB, 20 kB, or 50 kB. Therefore, in a genetically modified non-human animal (e.g., mouse) containing a nucleic acid sequence encoding a human GM-CSF protein, produced by targeting a human GM-CSF nucleic acid sequence to a non-human animal (e.g., mouse) GM-CSF locus, the human GM-CSF nucleic acid sequence may replace some or all of the non-human animal (e.g., mouse) sequence, such as exons and / or introns, at the GM-CSF locus. In some such cases, the human GM-CSF nucleic acid sequence is incorporated into the non-human (e.g., mouse) GM-CSF locus such that the expression of the human GM-CSF sequence is regulated by a native or endogenous regulatory sequence at the non-human (e.g., mouse) GM-CSF locus. In other words, the regulatory sequence to which the nucleic acid sequence encoding the human GM-CSF protein is operably ligated is the native GM-CSF regulatory sequence at the non-human (e.g., mouse) GM-CSF locus.

[0156] In some cases, the integration of a human GM-CSF sequence does not affect the transcription of the gene into which it is integrated. For example, if the human GM-CSF sequence is integrated into the coding sequence as an intein, or if the human GM-CSF sequence contains a 2A peptide, the human GM-CSF sequence is transcribed and translated simultaneously with the gene into which it is integrated. In other cases, the integration of a human GM-CSF sequence disrupts the transcription of the gene into which it is integrated. For example, during homologous recombination integration of a human GM-CSF sequence, some or all of the coding sequence at the integration locus may be removed so that the human GM-CSF sequence is transcribed instead. In some such cases, the integration of a human GM-CSF sequence produces a null mutation, and therefore a null allele. A null allele is a variant copy of a gene that completely lacks the normal function of that gene. This may result from a complete absence of the gene product (protein, RNA) at the molecular level, or from the expression of a non-functional gene product. At the phenotypic level, a null allele involves the deletion of an entire gene locus.

[0157] In some examples, a genetically modified non-human animal (e.g., mouse) expressing human GM-CSF protein contains one copy of the nucleic acid sequence encoding the human GM-CSF protein. For example, the non-human animal (e.g., mouse) may be heterozygous for the nucleic acid sequence. In other words, one allele at the locus contains the nucleic acid sequence, and the other is an endogenous allele. For example, as described above, in some examples, the human GM-CSF nucleic acid sequence is incorporated into the non-human animal (e.g., mouse) GM-CSF locus, thereby creating a null allele of non-human animal (e.g., mouse) GM-CSF. In some such embodiments, the humanized GM-CSF mouse may be heterozygous for the encoding nucleic acid sequence, i.e., the humanized GM-CSF mouse contains one null allele (an allele containing the nucleic acid sequence) and one endogenous GM-CSF allele (wild type or other) for non-human animal (e.g., mouse) GM-CSF. In another example, a genetically modified non-human animal (e.g., a mouse) expressing human GM-CSF protein contains two copies of the nucleic acid sequence encoding the human GM-CSF protein. For example, a non-human animal (e.g., a mouse) may be homozygous for the nucleic acid sequence, i.e., both alleles for a locus in the diploid genome contain the nucleic acid sequence, i.e., a genetically modified non-human animal (e.g., a mouse) expressing human GM-CSF protein contains two null alleles (alleles containing the nucleic acid sequence) for mouse GM-CSF.

[0158] Embodiments using the human GM-CSF gene in mice are discussed extensively herein, but other non-human animals containing the human GM-CSF gene (e.g., rodents, e.g., rats) are also provided.

[0159] Human GM-CSF polypeptide, the gene loci encoding human GM-CSF polypeptide, and non-human animals expressing human GM-CSF polypeptide are described in International Publication Nos. 2011 / 044050, 2014 / 039782, and 2014 / 071397, each of which is incorporated herein by reference.

[0160] Humanized IL-3 gene locus In some embodiments, the genetically modified non-human animals provided herein further express a human IL-3 protein encoded by a nucleic acid operably linked to an IL-3 promoter. Human IL-3 protein means a protein that is human IL-3 or substantially identical to human IL-3, for example, 80% or more identical, 85% or more identical, 90% or more identical, or 95% or more identical to human IL-3, for example, 97%, 98%, or 99% identical to human IL-3. Therefore, a nucleic acid sequence encoding a human IL-3 protein is a polynucleotide containing the coding sequence of a human IL-3 protein, i.e., a human IL-3 or a protein substantially identical to human IL-3.

[0161] Similar to GM-CSF, IL-3 is a crucial cytokine for myeloid cell development and function. IL-3 is not cross-reactive between humans and mice. While IL-3 stimulates early hematopoietic progenitor cells in vitro, it is not essential for steady-state hematopoiesis in vivo. However, along with GM-CSF, an effective DTH response is required in vivo. IL-3 also specifically stimulates alveolar macrophage (AM) proliferation in vitro.

[0162] The polypeptide sequence of human IL-3 and the nucleic acid sequence encoding human IL-3 can be found at Genbank accession numbers NP_000579.2 and NM_000588.4. The genomic locus encoding the human IL-3 protein can be found in the human genome on chromosome 5, GRCh38.p14;NC_000005.10 (132060655-132063204). The protein sequence is encoded by exons 1-5 at this locus. Therefore, a nucleic acid sequence containing the coding sequence of human IL-3 includes one or more of exons 1-5 of the human IL-3 gene. In some examples, the nucleic acid sequence also includes aspects of the human IL-3 genomic locus, e.g., introns, 3' and / or 5' untranslated sequences (UTRs). In some examples, the nucleic acid sequence includes the entire region of the human IL-3 genomic locus.

[0163] In some embodiments, in the genetically modified non-human animals provided herein, the nucleic acid sequence encoding the human IL-3 protein is operably ligated to one or more regulatory sequences of the non-human animal (e.g., mouse) IL-3 gene. The non-human animal (e.g., mouse) IL-3 regulatory sequences are sequences of the non-human animal (e.g., mouse) IL-3 genomic locus that regulate non-human animal (e.g., mouse) IL-3 expression, e.g., 5' regulatory sequences, e.g., IL-3 promoter, IL-3 5' untranslated region (UTR), etc.; 3' regulatory sequences, e.g., 3'UTR; and enhancers, etc. For example, mouse IL-3 is located at approximately c54158105~54155911 on chromosome 11, GRCm39, NC_000077.7, and the mouse IL-3 coding sequence may be found at Genbank accession number NM_010556.4. The regulatory sequences of mouse IL-3 are well defined in the art and can be readily identified using in silico methods by referring, for example, to the above Genbank accession number in the UCSC Genome Browser, on the World Wide Web at genome.ucsc.edu, or to experimental methods described in the art. In some cases, for example, when the nucleic acid sequence encoding the human IL-3 protein is located at a non-human animal (e.g., mouse) IL-3 genome locus, the regulatory sequences operably linked to the human IL-3 coding sequence are endogenous or native to the non-human animal (e.g., mouse) genome, i.e., they existed in the non-human animal (e.g., mouse) genome before the incorporation of the human nucleic acid sequence.

[0164] In some examples, genetically modified non-human animals expressing human IL-3 protein are generated by randomly incorporating or inserting a human nucleic acid sequence encoding the human IL-3 protein or a fragment thereof, i.e., a "human IL-3 nucleic acid sequence" or "human IL-3 sequence," into the genome of a non-human animal. Typically, in such embodiments, the location of the nucleic acid sequence encoding the human IL-3 protein in the genome is unknown. In other examples, genetically modified non-human animals expressing human IL-3 protein are generated by targeted incorporation or insertion of the human IL-3 nucleic acid sequence into the genome of a non-human animal, for example, by homologous recombination. In homologous recombination, a polynucleotide is inserted into the host genome of the target locus while simultaneously removing host genomic material from the target locus, such as genomic material exceeding 50 base pairs (bp), 100 bp, 200 bp, 500 bp, 1 kB, 2 kB, 5 kB, 10 kB, 15 kB, 20 kB, or 50 kB. Therefore, in a genetically modified non-human animal (e.g., mouse) containing a nucleic acid sequence encoding the human IL-3 protein, produced by targeting the human IL-3 nucleic acid sequence to the non-human animal (e.g., mouse) IL-3 locus, the human IL-3 nucleic acid sequence may replace some or all of the non-human animal (e.g., mouse) sequence, such as exons and / or introns, at the IL-3 locus. In some such cases, the human IL-3 nucleic acid sequence is incorporated into the non-human (e.g., mouse) IL-3 locus such that the expression of the human IL-3 sequence is regulated by a native or endogenous regulatory sequence at the non-human (e.g., mouse) IL-3 locus. In other words, the regulatory sequence to which the nucleic acid sequence encoding the human IL-3 protein is operably linked is the native IL-3 regulatory sequence at the non-human (e.g., mouse) IL-3 locus.

[0165] In some cases, the integration of a human IL-3 sequence does not affect the transcription of the gene into which it is integrated. For example, if the human IL-3 sequence is integrated into the coding sequence as an intein, or if the human IL-3 sequence contains a 2A peptide, the human IL-3 sequence is transcribed and translated simultaneously with the gene into which it is integrated. In other cases, the integration of a human IL-3 sequence disrupts the transcription of the gene into which it is integrated. For example, during homologous recombination, some or all of the coding sequence at the integration locus may be removed so that the human IL-3 sequence is transcribed instead. In some such cases, the integration of a human IL-3 sequence produces a null mutation, and therefore a null allele. A null allele is a variant copy of a gene that completely lacks the normal function of that gene. This can result from a complete absence of the gene product (protein, RNA) at the molecular level, or from the expression of a non-functional gene product. At the phenotypic level, a null allele involves the deletion of an entire gene locus.

[0166] In some examples, a genetically modified non-human animal (e.g., mouse) expressing human IL-3 protein contains one copy of the nucleic acid sequence encoding the human IL-3 protein. For example, the non-human animal (e.g., mouse) may be heterozygous for the nucleic acid sequence. In other words, one allele at the locus contains the nucleic acid sequence, and the other is the endogenous allele. For example, as described above, in some examples, the human IL-3 nucleic acid sequence is incorporated into the non-human animal (e.g., mouse) IL-3 locus, thereby creating a null allele of non-human animal (e.g., mouse) IL-3. In some such embodiments, the humanized IL-3 mouse may be heterozygous for the encoding nucleic acid sequence, i.e., the humanized IL-3 mouse contains one null allele (an allele containing the nucleic acid sequence) and one endogenous IL-3 allele (wild type or other) for non-human animal (e.g., mouse) IL-3. In another example, a genetically modified non-human animal (e.g., a mouse) expressing human IL-3 protein contains two copies of the nucleic acid sequence encoding the human IL-3 protein. For example, a non-human animal (e.g., a mouse) may be homozygous for the nucleic acid sequence, i.e., both alleles for a locus in the diploid genome contain the nucleic acid sequence, i.e., a genetically modified non-human animal (e.g., a mouse) expressing human IL-3 protein contains two null alleles (alleles containing the nucleic acid sequence) for mouse IL-3.

[0167] Embodiments using the human IL-3 gene in mice are discussed extensively herein, but other non-human animals containing the human IL-3 gene (e.g., rodents, e.g., rats) are also provided.

[0168] Human IL-3 polypeptides, loci encoding human IL-3 polypeptides, and non-human animals expressing human IL-3 polypeptides are described in International Publication Nos. 2011 / 044050, 2014 / 039782, and 2014 / 071397, each of which is incorporated herein by reference.

[0169] Genetically modified non-human animals and ES cells In certain embodiments, the following are provided herein: (i) genetically modified non-human animals (e.g., rodents such as rats or mice) comprising homozygous null mutations in the Rag1 and / or Rag2 genes (e.g., Rag1 and / or Rag2 gene knockout); (ii) homozygous null mutations in the IL2rg gene (e.g., IL2rg gene knockout); (iii) homozygous null mutations in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a non-human animal portion and a human portion operably linked to the Flt3l promoter, and, if applicable, one or more of the humanization loci disclosed herein, as well as genetically modified non-human animal ES cells useful for producing such non-human animals.

[0170] In certain embodiments, genetically modified non-human animal (e.g., rodents such as rats or mice) and non-human animal (e.g., rodents such as rats or mice) ES cells are provided herein, comprising, in their germline and / or genome, (i) homozygous null mutations in the Rag1 gene and / or Rag2 gene (e.g., Rag1 and / or Rag2 gene knockout); (ii) homozygous null mutations in the IL2rg gene (e.g., IL2rg gene knockout); (iii) homozygous null mutations in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a non-human animal portion and a human portion operably linked to the Flt3l promoter, and optionally one or more of the manipulated loci described herein. In some embodiments, genetically modified non-human animals (e.g., mice) and non-human animal ES cells (e.g., mice) are provided herein, wherein their germline and / or genomes include (i) a Rag2 gene knockout; (ii) an IL2rg gene knockout; (iii) a homozygous null mutation in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) a non-human animal portion and a human portion operably linked to the Flt3l promoter, and optionally one or more of the manipulated loci described herein. For example, in some embodiments, the non-human animal or ES cell includes the humanized Sirpa locus provided herein in its germline and / or genome. In certain embodiments, the non-human animal or ES cell includes the GM-CSF locus provided herein in its germline and / or genome. In certain embodiments, the non-human animal or ES cell includes the IL-3 locus provided herein in its germline and / or genome. In some embodiments, non-human animals or ES cells are heterozygous for one or more of the loci provided herein, such as genetically engineered loci.In some embodiments, non-human animals or ES cells are homozygous for one or more of the loci provided herein, such as genetically engineered loci.

[0171] In some embodiments, the non-human animal can be any non-human animal. In some embodiments, the non-human animal is a vertebrate. In some embodiments, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animals described herein may be selected from the group consisting of mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, llamas, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). In the case of non-human animals for which suitable genetically modifiable ES cells are not readily available, non-human animals containing the genetic modifications described herein can be produced by other methods. Such methods include, for example, modifying a non-ES cell genome (e.g., fibroblasts or induced pluripotent cells), using nuclear transfer to transfer the modified genome into suitable cells such as oocytes, and impregnating the modified cells (e.g., modified oocytes) in a non-human animal under conditions suitable for embryo formation.

[0172] In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human animal is, for example, a small mammal of the Diprotodontoidea or Muroidea superfamily. In some embodiments, the non-human animal is a rodent. In certain embodiments, the rodent is a mouse, rat, or hamster. In some embodiments, the rodent is selected from the Muroidea superfamily. In some embodiments, the non-human animal is derived from a family selected from Calomyscidae (e.g., mouse-like hamster), Cricetidae (e.g., hamster, New World rat and mouse, voles), Muridae (e.g., true mouse and rat, gerbil, spiny mouse, cristed rat), Nesomyidae (e.g., climbing mouse, rock mouse, white-tailed rat, Malagasy rat and mouse), Platacanthomyidae (e.g., spiny dormouse), and Spalacidae (e.g., mole rat, bamboo rat, and zocol). In some embodiments, the rodent is selected from true mice or rats (Muridae), gerbils, spiny mice, and cristed rats. In some embodiments, the mouse is derived from a member of the Muridae family. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent is selected from mice and rats. In some embodiments, the non-human animal is a mouse.

[0173] In some embodiments, the non-human animal is a mouse of the C57BL strain. In some embodiments, the C57BL strain is selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the non-human animal is a mouse of the 129 strain. In some embodiments, strain 129 is selected from the group consisting of strains 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. In some embodiments, the genetically modified mouse is a mixture of strain 129 and strain C57BL. In some embodiments, the mouse is a mixture of strain 129 and / or strain C57BL / 6. In some embodiments, the strain 129 in the mixture is strain 129S6 (129 / SvEvTac). In some embodiments, the mouse is strain BALB (e.g., BALB / c). In some embodiments, the mouse is a mixture of the BALB strain and another strain (e.g., the C57BL strain and / or the 129 strain). In some embodiments, the non-human animal provided herein may be a mouse derived from any combination of the aforementioned strains.

[0174] In some embodiments, the non-human animal provided herein is a rat. In some embodiments, the rat is selected from Wistar rat, LEA strain, Sprague Dawley strain, Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mixture of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0175] In certain embodiments, a genetically modified non-human animal or ES cell includes in its genome and / or germline a plurality of loci provided herein, e.g., a plurality of genetically engineered loci provided herein. For example, in some embodiments, a non-human animal or ES cell includes in its germline and / or genome (i) a homozygous null mutation in the Rag1 and / or Rag2 gene (e.g., Rag1 and / or Rag2 gene knockout); (ii) a homozygous null mutation in the IL2rg gene (e.g., IL2rg gene knockout); (iii) a homozygous null mutation in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene provided herein, comprising a non-human animal portion and a human portion operably linked to the Flt3l promoter. In some embodiments, non-human animal or ES cells include in their germline and / or genome (i) homozygous null mutations in the Rag1 and / or Rag2 genes (e.g., Rag1 and / or Rag2 gene knockout); (ii) homozygous null mutations in the IL2rg gene (e.g., IL2rg gene knockout); (iii) homozygous null mutations in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; (iv) an Flt3 ligand (Flt3l) gene comprising a non-human animal portion and a human portion operably linked to the Flt3l promoter; and (v) a humanized Sirpa locus provided herein.In some embodiments, non-human animals or ES cells include in their germline and / or genome (i) homozygous null mutations in the Rag1 and / or Rag2 genes (e.g., Rag1 and / or Rag2 gene knockout); (ii) homozygous null mutations in the IL2rg gene (e.g., IL2rg gene knockout); (iii) homozygous null mutations in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; (iv) an Flt3 ligand (Flt3l) gene comprising a non-human animal portion and a human portion operably linked to the Flt3l promoter; (v) a humanized Sirpa locus provided herein; and (vi) a humanized GM-CSF locus and / or a humanized IL-3 locus provided herein.

[0176] In certain embodiments, genetically modified non-human animals do not express the Flt3 polypeptide. In certain embodiments, genetically modified non-human animals express both the soluble and membrane-bound forms of the Flt3l polypeptide, and, as necessary, the soluble and membrane-bound forms of the Flt3l polypeptide include the signal peptide and cytokine-like core domain of the human FLT3L polypeptide.

[0177] In certain embodiments, the genetically modified non-human animal expresses one or more human or humanized polypeptides encoded by the humanization loci provided herein. For example, in some embodiments, the non-human animal expresses human or humanized Sirpa polypeptide. In certain embodiments, the non-human animal expresses human or humanized GM-CSF polypeptide. In certain embodiments, the non-human animal expresses human or humanized IL-3 polypeptide.

[0178] Genetically modified non-human animals and ES cells can be generated using any suitable method known in the art. For example, such genetically modified non-human animal ES cells can be generated using VELOCIGENE® technology, which is described in U.S. Patents 6,586,251, 6,596,541, 7,105,348, and Valenzuela et al. (2003) "High-throughput engineering of the mouse genome coupled with high-resolution expression analysis" Nat. Biotech. 21(6):652-659, each of which is incorporated herein by reference. Modifications can be made using genome-targeted nuclease systems such as CRISPR / Cas systems, transcription activator-like effector nuclease (TALEN) systems, or zinc finger nuclease (ZFN) systems. In some embodiments, modifications are made using the CRISPR / Cas system, as described, for example, in U.S. Patent Applications No. 14 / 314,866, 14 / 515,503, 14 / 747,461 and 14 / 731,914, each of which is incorporated herein by reference. Genetically modified rat ES cells and rats can be prepared in accordance with U.S. Patent Publication No. 2014 / 0235933 (Regeneron Pharmaceuticals, Inc.), U.S. Patent Publication No. 2014 / 0310828 (Regeneron Pharmaceuticals, Inc.), Tong et al. (2010) Nature 467:211-215 and Tong et al. (2011) Nat Protoc.6(6):doi:10.1038 / nprot.2011.338 (all of which are incorporated herein by reference in their entirety). Exemplary methods for producing such genetically modified non-human animals and ES cells are also provided herein in Example 1.

[0179] Subsequently, the ES cells described herein may be used to produce non-human animals using methods known in the art. For example, the mouse non-human animal ES cells described herein may be used to produce genetically modified mice using the VELOCIMOUSE® method, as described in U.S. Patent No. 7,294,754 and Poueymirou et al., Nature Biotech 25:91-99 (2007), which are incorporated herein by reference, respectively. The resulting mice may be mated until homozygous.

[0180] Methods for creating genetically modified non-human animals and ES cells In certain embodiments, methods for producing non-human animals (e.g., mice or rats) and ES cells comprising one or more of the gene modification loci provided herein are provided herein. For example, in some embodiments, methods for producing non-human animals (e.g., mice or rats) and ES cells comprising (i) a homozygous null mutation in the Rag1 and / or Rag2 gene (e.g., Rag1 and / or Rag2 gene knockout); (ii) a homozygous null mutation in the IL2rg gene (e.g., IL2rg gene knockout); (iii) a homozygous null mutation in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a non-human animal portion and a human portion operably linked to the Flt3l promoter are provided herein. In some embodiments, methods for producing non-human animals (e.g., mice or rats) and ES cells are provided herein, further comprising (i) homozygous null mutations in the Rag1 and / or Rag2 genes (e.g., Rag1 and / or Rag2 gene knockout); (ii) homozygous null mutations in the IL2rg gene (e.g., IL2rg gene knockout); (iii) homozygous null mutations in the non-human animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene containing non-human animal and human portions operably linked to the Flt3l promoter, and the humanized Sirpa locus provided herein. In some embodiments, methods for producing non-human animals (e.g., mice or rats) and ES cells are provided herein, further comprising the humanized GM-CSF locus and / or the humanized IL-3 locus provided herein. Exemplary methods for producing genetically modified non-human animals and ES cells provided herein are described herein, in examples and / or in drawings.

[0181] The generation of non-human animals containing a null mutation in the non-human Flt3 gene, and / or non-human animals containing an Flt3 ligand (Flt3l) gene containing a non-human portion and a human portion operably linked to the Flt3l promoter, can be achieved using any convenient method for producing genetically modified animals, such as those known in the art or described herein in Example 1.

[0182] The generation of non-human animals containing nucleic acid sequences encoding human or humanized proteins (e.g., hSIRPA, hGM-CSF, or hIL-3) can be achieved using any convenient method for producing genetically modified animals, such as those known in the art or described herein.

[0183] For example, nucleic acids encoding human or humanized proteins (e.g., hSIRPA, hGM-CSF, and / or hIL-3) can be incorporated into recombinant vectors in a form suitable for insertion into the genome of host cells and expression of human proteins in non-human host cells. In various embodiments, the recombinant vector may contain one or more regulatory sequences operably ligated to the nucleic acid encoding a human protein in a manner that enables transcription of the nucleic acid into mRNA and translation of the mRNA into a human protein, as described above. It will be understood that the design of the vector may depend on factors such as the selection of host cells to be transfected and / or the amount of human protein to be expressed.

[0184] Subsequently, human nucleic acid sequences can be introduced into animal cells using one of various methods to produce genetically modified animals that express human genes. Such techniques are well known in the art and include, but are not limited to, pronuclear microinjection, embryonic stem cell transformation, homologous recombination, and knock-in techniques. Methods for creating genetically modified animals that can be used 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 Mol Biol, Methods and Protocols, Humana Press), Meyer et al. (2010, Proc.Nat.Acad.Sci.USA 107:15022-15026), and Gibson (2004, A Primer Of Genome Science 2nd). This may include those described in (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).

[0185] For example, the genetically modified animal of interest may be produced by introducing nucleic acids encoding human proteins into oocytes, for example, by microinjection, and then developing the oocytes in female rearing animals. In a preferred embodiment, the expression is injected into fertilized oocytes. Fertilized oocytes can be collected from females that have superovulated the day after mating and injected with the expression construct. The injected oocytes are either cultured overnight or directly transferred to the fallopian tubes of pseudopregnant females 0.5 days post-mating. Methods for superovulation, oocyte collection, expression construct injection, and embryo transfer are known in the art and are described in *Manipulating the Mouse Embryo* (2002, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press). The offspring can be evaluated for the presence of the introduced nucleic acids by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing, etc.) or protein analysis (e.g., ELISA, Western blotting, etc.).

[0186] As another example, constructs containing nucleic acid sequences encoding human proteins can be transfected into stem cells (e.g., ES cells or iPS cells) using well-known methods such as electroporation, calcium phosphate precipitation, or lipofection. The cells can then be evaluated for the presence of the transfected nucleic acid by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing) or protein analysis (e.g., ELISA, Western blotting). Cells determined to have incorporated the expression construct can then be introduced into preimplantation embryos. For a detailed description of known methods in the art that are useful for the compositions and methods of the present invention, see Nagy et al. (2002, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press), Nagy et al. (1990, Development 110:815-821), U.S. Patent Nos. 7,576,259, 7,659,442, and 7,294,754, and Kraus et al. (2010, Genesis 48:394-399).

[0187] In addition, as described in some of the examples below, nucleic acid constructs may be constructed using VELOCIGENE® genetic engineering technology (see, e.g., Valenzuela et al. (2003), High throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotech. 21(6):652-59 and U.S. Patent No. 6,586,251), introduced into stem cells (e.g., ES cells), and precisely targeted clones are determined using allele loss assays and allele gain assays (Valenzuela et al., previously described). Precisely targeted ES cells can be used as donor ES cells for introduction into 8-cell stage mouse embryos using the VELOCIMOUSE® method (see, e.g., U.S. Patent No. 7,294,754 and Poueymirou et al., 2007, F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses, Nature Biotech. 25(l):91-99). Furthermore, genetically modified rat ES cells and rats can be prepared in accordance with U.S. Patent Application Publication No. 2014 / 0235933 (Regeneron Pharmaceuticals, Inc.), U.S. Patent Application Publication No. 2014 / 0310828 (Regeneron Pharmaceuticals, Inc.), Tong et al. (2010) Nature 467:211-215, and Tong et al. (2011) Nat Protoc.6(6):doi:10.1038 / nprot.2011.338 (all of which are incorporated herein by reference in their entirety).

[0188] In some embodiments, genetically modified emergent agents can be crossed with additional animals having one or more genetic modifications. For example, genetically modified non-human animals (e.g., rodents such as rats or mice) provided herein can be produced by breeding Flt3-deficient non-human animals provided herein with non-human animals containing an Flt3 ligand (Flt3l) gene, which includes a non-human animal portion and a human portion operably linked to the Flt3l promoter. These genetically modified non-human animals (e.g., rodents such as rats or mice) can be further crossed with other genetically modified non-human animals having other genetic modifications, such as the introduction of either fully human genes or humanized genes, e.g., hSirpa knock-in mice, hIL-3 knock-in mice, hGM-CSF knock-in mice, etc., or can be crossed with knockout animals, e.g., non-human animals lacking one or more proteins, e.g., one or more animals not expressing their genes, e.g., Rag1-deficient animals, Rag2-deficient animals, or IL-2rg-deficient animals.

[0189] In another embodiment, stem cells, such as ES cells, may be generated so that they include several genetic modifications, such as humanization or gene deletion as described herein, and such stem cells may be introduced into an embryo to produce a genetically modified animal having several genetic modifications.

[0190] As described above, in some embodiments, the genetically modified non-human animals are immunodeficient animals. Immunodeficient genetically modified non-human animals containing one or more human or humanized proteins, such as hSIRPA, hIL-3, and / or hGM-CSF, can be produced using any convenient method for producing genetically modified animals, such as those known in the art or described herein. For example, the production of a genetically modified immunodeficient animal can be achieved by introducing nucleic acids encoding human proteins into oocytes or stem cells containing, in the case of homozygote, a mutant SCID gene allele or Rag1 and / or Rag2 and Il2rg null alleles that result in immunodeficiency, as described in more detail above and in the examples herein. Then, mice are produced using the modified oocytes or ES cells and mated, for example, using methods described herein and known in the art, to produce immunodeficient mice containing the desired genetic modification. As another example, genetically modified non-human animals can be generated in an immunoqualified background and crossed with animals containing mutant gene alleles that result in immunodeficiency in the case of hemizygous or homozygous individuals. The offspring can then cross to produce immunodeficient animals that express at least one human protein of interest.

[0191] In some embodiments, genetically modified mice are treated to eliminate endogenous hematopoietic cells that may be present in the mouse. In one embodiment, the treatment includes irradiating the genetically modified mouse. In a particular embodiment, neonatal genetically modified offspring are irradiated sublethally. In a particular embodiment, neonates are irradiated with 2 × 200 cGy at 4-hour intervals.

[0192] Various embodiments of the present invention provide genetically modified animals in which substantially all of their cells contain human nucleic acids, as well as genetically modified animals in which some, but not all, of their cells contain human nucleic acids. In some examples, for example, targeted recombination, one copy of human nucleic acid is incorporated into the genome of the genetically modified animal. In other examples, for example, random integration, multiple copies of human nucleic acid may be incorporated into the genome of the genetically modified animal, adjacent to or apart from one another.

[0193] Therefore, in some embodiments, the genetically modified non-human animal may be an immunodeficient animal comprising a genome containing a nucleic acid encoding a human polypeptide operably linked to a corresponding non-human animal promoter, and the animal expresses the encoded human polypeptide. In other words, the genetically modified immunodeficient non-human animal of interest comprises a genome containing a nucleic acid encoding at least one human polypeptide, the nucleic acid operably linked to a corresponding non-human promoter and polyadenylation signal, and the animal expresses the encoded human polypeptide.

[0194] Further methods for producing genetically modified non-human animals containing genomes comprising nucleic acids encoding one or more human proteins, such as hSIRPA, hIL-3, and / or hGM-CSF, are described in U.S. Patent No. 11,019,810 and International Publication No. 2011 / 044050, each of which is incorporated herein by reference.

[0195] engraftment In some embodiments, the subjects of the genetically modified non-human animals are also immunodeficient. "Immunodeficiency" includes defects in one or more aspects of the animal's innate or endogenous immune system, for example, the animal is deficient in one or more types of functioning host immune cells, such as the number and / or function of non-human B cells, the number and / or function of non-human T cells, the number and / or function of non-human NK cells, etc.

[0196] One method for achieving immunodeficiency in target animals is sublethal irradiation. Alternatively, or in addition to this, immunodeficiency may be achieved by any one of a number of gene mutations known in the art, any one of which may be crossed with the genetically modified non-human animals of the Disclosure, or used as a source of stem cells into which the genetically modified animals of the Disclosure may be introduced, either alone or in combination. Non-limiting examples include X-linked SCID associated with the IL2RG gene mutation and characterized by the lymphocyte phenotype T(-)B(+)NK(-); autosomal recessive SCID associated with the Jak3 gene mutation and characterized by the lymphocyte phenotype T(-)B(+)NK(-); ADA gene mutation characterized by the lymphocyte phenotype T(-)B(-)NK(-); IL-7R alpha chain mutation characterized by the lymphocyte phenotype T(-)B(+)NK(+); lymphocyte phenotype T( This includes CD3 delta or epsilon mutations characterized by -)B(+)NK(+); RAG1 and RAG2 mutations characterized by lymphocyte phenotype T(-)B(-)NK(+); Artemis gene mutations characterized by lymphocyte phenotype T(-)B(-)NK(+); CD45 gene mutations characterized by lymphocyte phenotype T(-)B(+)NK(+); and Prkdcscld mutations characterized by lymphocyte phenotype T(-),B(-). Thus, in some embodiments, genetically modified immunodeficient non-human animals have one or more deletions selected from IL2 receptor gamma chain deficiency, Jak3 deficiency, ADA deficiency, IL7R deficiency, CD3 deficiency, RAG1 and / or RAG2 deficiency, Artemis deficiency, CD45 deficiency, and Prkdc deficiency. In one embodiment, immunodeficiency is achieved by gene mutations or gene deletions in the Rag1 and / or Rag2 and Il2rg genes. These and other animal models of immunodeficiency are known to those skilled in the art, and any of them may be used to produce the immunodeficient animals of this disclosure.

[0197] In some embodiments, the genetically modified non-human animals according to the present invention find use as recipients of human hematopoietic cells capable of generating human immune cells from engrafted human hematopoietic cells. Thus, in some aspects of the present invention, the genetically modified animal is a genetically modified immunodeficient non-human animal on which human hematopoietic cells have been engrafted.

[0198] Any source of human hematopoietic cells, human hematopoietic stem cells (HSCs), and / or hematopoietic stem progenitor cells (HSPCs) known in the art or described herein may be transplanted into genetically modified, immunodeficient non-human animals as described herein. One suitable source of human hematopoietic cells known in the art is human umbilical cord blood cells, in particular CD34-positive (CD34+) cells. Another source of human hematopoietic cells is human fetal liver. Another source is human bone marrow. Induced pluripotent stem cells (iPSCs) and induced hematopoietic stem cells (iHSCs), produced by dedifferentiation of somatic cells, for example by methods known in the art, are also included. Methods for transplanting human cells into non-human animals are well described in the art and elsewhere herein, and any of them may be used by those skilled in the art to reach the target genetically modified and engrafted non-human animals.

[0199] Cell populations of particular interest include hematopoietic stem cells or progenitor cells that contribute to or reconstitute the hematopoietic system of genetically modified non-human animals, such as peripheral blood leukocytes, fetal hepatocytes, fetal bone, fetal thymus, fetal lymph nodes, vascularized skin, arterial segments, and spermatogenic hematopoietic stem cells, such as recruited HSCs or umbilical cord blood HSCs.

[0200] The cells may originate from any mammalian species, such as mice, rodents, dogs, cats, horses, cattle, sheep, primates, or humans. In one embodiment, the cells are human cells. The cells may be from an established cell line or may be primary cells, where “primary cells,” “primary cell line,” and “primary culture” are used interchangeably herein to refer to cells and cell cultures derived from the subject and grown in vitro for a limited number of passages, i.e., for the division of the culture. For example, a primary culture may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, but is a culture that has not passed through a sufficient number of critical stages. Typically, the primary cell lines of this disclosure are maintained in vitro for fewer than 10 passages.

[0201] If the cells are primary cells, they can be collected from the individual by any convenient method. For example, cells such as blood cells, or leukocytes, can be collected by apheresis, leukocyte apheresis, density gradient separation, etc. Another example is that cells such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, and stomach tissue can be collected by biopsy. A suitable solution can be used for the dispersion or suspension of the collected cells. Such solutions are generally equilibrium salt solutions, such as ordinary saline, PBS, or Hanks equilibrium salt solutions, and are conveniently supplemented with fetal bovine serum or other naturally occurring factors, generally in low concentrations of 5-25 mM, along with an acceptable buffer. Convenient buffers include HEPES, phosphate buffer, and lactate buffer.

[0202] In some cases, heterologous populations of cells are transplanted into genetically modified non-human animals. In other cases, a population of cells enriched with a specific type of cell, such as progenitor cells, such as hematopoietic progenitor cells, is engrafted into a genetically modified non-human animal. The enrichment of the target cell population may be by any simple separation technique. For example, the target cells may be enriched by a culture method, in which specific growth factors and nutrients are added to the culture, typically promoting the survival and / or proliferation of one cell population more than others. Other culture conditions that affect survival and / or proliferation include growth on adhesive or non-adhesive substrates and culture over a specific period of time. Such culture conditions are well known in the art. As another example, the target cells may be enriched by separating them from an initial population by affinity separation techniques. Techniques for affinity separation may include magnetic separation using magnetic beads coated with affinity reagents, affinity chromatography "panning" using affinity reagents bound to a solid matrix, such as a plate, cytotoxic agents bound to affinity reagents, or used in combination with affinity reagents, such as complementary chains and cytotoxins, or other convenient techniques. Techniques that provide precise separation include fluorescence-activated cell sorters, which can have various degrees of refinement, such as multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels. Cells may be selected for dead cells by using dyes associated with dead cells (e.g., propidium iodide). Any technique that is not excessively detrimental to the viability of the cells of interest may be used.

[0203] For example, using affinity separation techniques, cells that are not the target cells for transplantation can be depleted from a population by contacting the population with an affinity reagent that specifically recognizes and selectively binds to markers not expressed on the target cells. For example, to enrich a population of hematopoietic progenitor cells, cells expressing mature hematopoietic cell markers can be depleted. In addition, or alternatively, positive selection and separation can be performed by contacting a population with an affinity reagent that specifically recognizes and selectively binds to markers associated with hematopoietic progenitor cells (e.g., CD34, CD133, etc.). "Selectively binding" means that a molecule preferentially binds to the target of interest or binds to the target with greater affinity than other molecules. For example, an antibody binds to molecules containing a specific epitope and not to an unrelated epitope. In some embodiments, the affinity reagent may be an antibody, i.e., an antibody specific to CD34, CD133, etc. In some embodiments, the affinity reagent may be a specific receptor or ligand for CD34, CD133, etc., e.g., peptide ligands and receptors. Effector and receptor molecules, such as T cell receptors specific to CD34, CD133, etc. In some embodiments, multiple affinity reagents specific to the target marker may be used.

[0204] Antibodies and T cell receptors used as affinity reagents can be monoclonal or polyclonal and can be produced by transgenic animals, immunized animals, immortalized human or animal B cells, cells transfected with DNA vectors encoding antibodies or T cell receptors, etc. Details of antibody preparation and their suitability for use as specific binding members are well known to those skilled in the art. Of particular interest is the use of labeled antibodies as affinity reagents. Conveniently, these antibodies are conjugated with labels for use in separation. Labels include magnetic beads that allow direct separation; biotin that can be removed with avidin or streptavidin bound to a support; fluorescent dyes that can be used with fluorescence-activated cell sorters; and others that can facilitate the separation of specific cell types. Fluorescent dyes for which applications have been found include phycobiliproteins, e.g., phycoerythrin and allophycocyanin, fluorescein, and Texas Red. Each antibody is often labeled with a different fluorescent dye to allow independent sorting for each marker.

[0205] The initial population of cells is brought into contact with the affinity reagent and incubated for a period sufficient to bind to the available cell surface antigen. Incubation is typically at least about 5 minutes, and usually less than about 60 minutes. It is desirable to have a sufficient concentration of antibody in the reaction mixture so that the efficiency of separation is not limited by a lack of antibody. The appropriate concentration is determined by titration, but is typically a dilution of antibody to the volume of the cell suspension, which is about 1:50 (i.e., 1 part antibody per 50 parts reaction volume), about 1:100, about 1:150, about 1:200, about 1:250, about 1:500, about 1:1000, about 1:2000, or about 1:5000. The medium in which the cells are suspended is any medium that maintains cell viability. Preferred media are phosphate-buffered saline containing 0.1–0.5% BSA or 1–4% goat serum. Various culture media are commercially available and can be used according to the characteristics of the cells, including Dulbecco's modified Eagle medium (dMEM), Hanks' basic salt solution (HBSS), Dulbecco's phosphate-buffered saline (dPBS), RPMI, Iscove medium, and PBS containing 5 mM EDTA, often supplemented with fetal bovine serum, BSA, HSA, or goat serum.

[0206] Cells in a contacted population that are to be labeled with affinity reagents are selected by any convenient affinity separation technique, for example, as described above, or as known in the art. After separation, the separated cells can be collected in any suitable medium that maintains cell viability, usually with a serum cushion at the bottom of the collection tube. Various media are commercially available and can be used depending on the properties of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove medium, etc., and are often supplemented with fetal bovine serum.

[0207] A composition highly concentrated with respect to the target cell type, for example, hematopoietic cells, is achieved in this manner. The cells constitute about 70%, about 75%, about 80%, about 85%, about 90%, or more of the cell composition, and about 95%, or more of the concentrated cell composition, preferably about 95%, or more of the concentrated cell composition. In other words, the composition is a substantially pure composition of the target cells.

[0208] Cells transplanted into genetically modified non-human animals can be transplanted immediately, whether they are heterogeneous or enriched populations of cells. Alternatively, cells can be frozen at liquid nitrogen temperature, stored for extended periods, thawed, and reused. In such cases, cells are typically frozen in 10% DMSO, 50% serum, 40% buffer medium, or some other such solution commonly used in the art to store cells at such freezing temperatures, and thawed in methods commonly known in the art for thawing frozen cultured cells. Additionally or alternatively, cells can be cultured in vitro under a variety of culture conditions. Culture media can be liquid or semi-solid, for example, containing agar, methylcellulose, etc. Cell populations can typically be conveniently suspended in a suitable nutrient medium, such as Iskoff Modified DMEM or RPMI-1640, supplemented with fetal bovine serum (about 5-10%), L-glutamine, thiols, especially 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. Cultures may contain growth factors to which cells are responsive. Growth factors as defined herein are molecules that can promote the survival, proliferation, and / or differentiation of cells in culture or in intact tissue through specific effects on transmembrane receptors. Growth factors include polypeptides and non-polypeptide factors.

[0209] Cells may be genetically modified before transplantation into genetically modified non-human animals for purposes such as inducing genetic defects in cells (e.g., for disease modeling), repairing genetic defects, or ectopically expressing genes in cells (e.g., to determine whether such modifications affect the course of a disease), for example, to provide selectable or traceable markers. Cells may be genetically modified by transfection or transduction with a suitable vector, homologous recombination, or other suitable technique to express a gene of interest, or with antisense mRNA, siRNA, or ribozymes to block the expression of an undesirable gene. Various techniques for introducing nucleic acids into target cells are known in the art. Various techniques may be used to prove that cells have been genetically modified. The cell genome may be restricted and used with or without amplification. Polymerase chain reaction; gel electrophoresis; restriction analysis; Southern blotting, Northern blotting, and Western blotting; sequencing; etc. may all be used.General methods in molecular and cell biochemistry for these and other purposes disclosed in this application can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, and kits for genetic manipulation mentioned in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0210] Cells can be transplanted into gene-modified non-human animals by any convenient method, including, for example, intrahepatic injection, tail vein injection, retro-orbital injection, etc. Typically, about 0.5×10 5 ~2×10 6 pluripotent cells or progenitor cells, e.g., about 1×10 5 cells to 1×10 6 cells, or about 2×10 5 ~5×10 5Individual cells are transplanted. In some cases, mice are irradiated to a sublethal dose before transplantation of human cells. In other words, mice are exposed to a sublethal dose of radiation, as described, for example, in the Examples section below, and as is well known in the Art. The engrafted genetically modified non-human animals are then maintained under experimental animal housing conditions for at least one week, e.g., one week or more, or two weeks or more, sometimes four weeks or more, and in some cases six weeks or more, to allow for sufficient reconstruction of the immune system by the engrafted cells.

[0211] In some embodiments, transplanted human hematopoietic cells produce one or more engrafted human cells in a genetically modified non-human animal, selected from human CD34-positive cells, human hematopoietic stem cells, human hematopoietic cells, myeloid progenitor cells, erythrocyte progenitor cells, myeloid cells, dendritic cells, monocytes, neutrophils, mast cells, erythrocytes, and combinations thereof. In one embodiment, the human cells are present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after engraftment. In certain embodiments, the human cells include human dendritic cells.

[0212] In some embodiments, transplanted human hematopoietic cells in genetically modified non-human animals give rise to an engrafted human hematopoietic system containing human hematopoietic stem cells and progenitor cells, human myeloid progenitor cells, human myeloid cells, human dendritic cells, human monocytes, human granulocytes, human neutrophils, human mast cells, human erythrocytes, human thymocytes, human T cells, human B cells, and human platelets. In one embodiment, the human hematopoietic system is present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after engraftment. In certain embodiments, the human hematopoietic system includes human dendritic cells.

[0213] In some embodiments, the genetically modified non-human animals provided herein are engrafted with human hematopoietic cells containing disease-specific mutations.

[0214] Non-limited application of genetically modified engrafted mice The genetically modified non-human animals of this disclosure have many potential applications in the art. For example, the engrafted genetically modified animals of this disclosure are useful for studying the function of human dendritic cells, e.g., human myeloid DCs or human plasmacytoid DCs. As another example, the engrafted genetically modified mice of this disclosure provide a useful system for screening candidate drugs for desired activity in vivo, for example, to identify drugs that can modulate (i.e., promote or inhibit) the function of human dendritic cells (e.g., human myeloid DCs or human plasmacytoid DCs) in a healthy or diseased state. For example, the engrafted genetically modified mice of this disclosure can be used to identify novel therapeutic agents. As yet another example, the engrafted genetically modified animals of this disclosure provide a useful system for predicting an individual's response to disease therapy by providing an in vivo platform for screening the response of an individual's immune system to drugs, e.g., therapeutic agents, and predicting an individual's response to drugs. In some embodiments, myeloid DCs (mDCs) are important for T cell cross-priming and activation; therefore, the engrafted genetically modified animals of this disclosure provide a useful model for testing potential immunotherapies that stimulate T cell responses by targeting mDCs. For example, the engrafted genetically modified animals of this disclosure provide a useful system for evaluating the therapeutic efficacy of drugs for stimulating T cell responses against tumor cells or pathogens, or for identifying drugs that stimulate T cell responses against tumor cells or pathogens. In some embodiments, plasmacytoid DCs (pDCs) are important type I IFN-producing cells in viral infections and are also involved in autoimmune diseases such as lupus; therefore, the engrafted genetically modified animals of this disclosure provide a useful model for testing treatments for viral infections or autoimmune diseases by targeting pDCs. For example, the engrafted genetically modified animals of this disclosure provide a useful system for evaluating the therapeutic efficacy of drugs in treating viral infections or autoimmune diseases, or for identifying drugs that treat viral infections or autoimmune diseases.

[0215] As one non-limiting example, the engrafted genetically modified mice of this disclosure can be used in generating mouse models of autoimmune diseases (e.g., systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, polymyositis, collagen-induced arthritis, or dermatomyositis). Such mouse models of autoimmune diseases would be useful both, for example, in studies to better understand the progression of autoimmune diseases in humans, and in drug discovery to identify candidate drugs to treat autoimmune diseases.

[0216] The engrafted genetically modified animals of this disclosure are used for screening candidate drugs to identify candidate drugs for treating autoimmune diseases. Terms such as “treatment” and “to treat” are used herein to generally include obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. As used herein, “treatment” includes any treatment of a disease in a mammal and includes (a) preventing the development of the disease in a subject that may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., stopping its development; or (c) mitigating the disease, i.e., causing regression of the disease. The terms “individual,” “subject,” “host,” and “patient” are used herein interchangeably and include any mammalian subject, in particular humans, for which diagnosis, treatment, or therapy is desired.

[0217] As another non-limiting example, the engrafted genetically modified mice of this disclosure can be used in generating mouse models to evaluate the therapeutic efficacy of drugs that stimulate T cell responses against target cells (e.g., tumor cells, virus-infected cells, bacterial-infected cells, bacterial cells, fungal cells, and parasitic cells). Such mouse models may be useful, for example, in drug discovery to identify candidate drugs that stimulate T cell responses against target cells.

[0218] In biological activator screening assays, the effects of a candidate drug are evaluated by exposing the human hematopoietic cell engraftment gene-modified non-human animals of the present disclosure to the candidate drug of interest and monitoring one or more output parameters. These output parameters may reflect the function of human dendritic cells, e.g., phagocytosis, cytokine production, cross-presentation of exogenous antigens, and activation of cytotoxic CD8+ T-cell lymphocytes (CTLs); or, by methods well known in the art, the T-cell response to target cells in rodents. Alternatively, or in addition, the output parameters may reflect the effect of the drug on autoimmune diseases in the human hematopoietic cell engraftment gene-modified non-human animals of the present disclosure.

[0219] Candidate agents for the screening or methods of this disclosure may include, for example, organic molecules (e.g., small molecule inhibitors), nucleic acids (e.g., nucleic acids encoding RNA interference agents, oligonucleotides, or polypeptides), peptides, peptide mimetic inhibitors, aptamers, antibodies, intrabodies, etc. As used herein, “RNA interference agent” may be small interfering RNA (siRNA), CRISPR RNA (crRNA), CRISPR guide RNA (gRNA), small hairpin RNA (shRNA), microRNA (miRNA), or piwi-interacting RNA (piRNA).

[0220] Candidate drugs are screened for biological activity by administering the drug to at least one, usually multiple, samples, sometimes in combination with samples lacking the drug. Changes in parameters in response to the drug are measured, and the results are evaluated, for example, by comparison with reference samples obtained with other drugs in the presence and absence of the drug. When screening is performed to identify candidate drugs that prevent, mitigate, or reverse the effects of pathogens or non-pathogenic immune stimulation, the screening is typically performed in the presence of pathogens or non-pathogenic immune stimulation, and the pathogens or non-pathogenic immune stimulation are added at the time most appropriate for the desired outcome. For example, when testing the protective / preventive capacity of a candidate drug, the candidate drug may be added before, simultaneously with, or after infection by the pathogen or non-pathogenic immune stimulation. As another example, when testing the ability of a candidate drug to reverse the effects of pathogens or non-pathogenic immune stimulation, the candidate drug may be added after infection by the pathogen or non-pathogenic immune stimulation. As described above, in some cases, the "sample" is a genetically modified non-human animal on which cells have been engrafted, for example, the candidate drug is provided to a genetically modified non-human animal on which human hematopoietic cells have been engrafted. In some cases, the "sample" is the human hematopoietic cells to be engrafted, that is, the candidate drug is provided to cells, such as human dendritic cells, before engraftment into an immunodeficient genetically modified animal.

[0221] If the candidate drug is to be administered directly to engrafted genetically modified animals, the drug may be administered by any of the many well-known methods in the art for administering peptides, small molecules, and nucleic acids to mice. For example, the drug may be administered orally, mucosally, topically, intradermally, or by injection, such as intraperitoneal, subcutaneous, intramuscular, or intravenous injection. The drug may be administered in a buffer solution or incorporated into any of the various formulations in combination with a suitable pharmaceutically acceptable vehicle, for example. A “pharmaceutically acceptable vehicle” may be a vehicle approved by a federal or state regulatory agency, or a vehicle listed in the United States Pharmacopeia or other commonly recognized pharmacopoeia for use in mammals such as humans. The term “vehicle” refers to a diluent, adjuvant, excipient, or carrier on which the compound of the present invention is formulated for administration to a mammal. Such pharmaceutical vehicles may include lipids, such as liposomes, such as liposomal dendrimers; liquids, such as water and oils, such as petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.; saline solution; acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Furthermore, auxiliaries, stabilizers, thickeners, lubricants, and colorants may be used. Pharmaceutical compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. The drug may be systemic after administration, or it may be localized by topical administration, intramural administration, or by the use of implants that act to retain the active dose at the implantation site. The activator may be formulated for immediate activity or for sustained release. If the drug is to be supplied to cells before engraftment, the drug is conveniently added to the cell culture medium in solution or in an easily soluble form during culture. The drug may be added intermittently or continuously as a flow in a flow-through system, or a bolus of the compound may be added alone or gradually to otherwise static solutions. In a flow-through system, two fluids are used: one is a physiologically neutral solution, and the other is the same solution to which the test compound has been added.The first fluid passes over the cells, followed by the second fluid. In the single-solution method, a bolus of the test compound is added to the volume of the culture medium surrounding the cells. The overall concentration of the components of the culture medium should not change significantly due to the addition of the bolus or between the two solutions in the flow-through method.

[0222] Multiple assays can be performed in parallel with different drug concentrations to obtain differential responses to various concentrations. As is known in the art, determining the effective concentration of a drug typically involves using a range of concentrations obtained from 1:10 or other logarithmic dilutions. The concentrations may be further purified with a second series of dilutions, if necessary. Typically, one of these concentrations is a negative control, i.e., zero concentration, below the detection level of the drug, or a concentration of the drug that does not produce a detectable change in phenotype.

[0223] Analysis of the response of cells in engrafted genetically modified animals to a candidate drug can be performed at any point in time after treatment with the drug. For example, cells may be analyzed 1, 2, or 3 days, sometimes 4, 5, or 6 days, sometimes 8, 9, or 10 days, sometimes 14 days, sometimes 21 days, sometimes 28 days, sometimes 1 month or more, for example 2 months, 4 months, 6 months or more, after contact with the candidate drug. In some embodiments, the analysis includes analysis at multiple time points. The selection of time points for analysis is based on the type of analysis to be performed, as will be readily understood by those skilled in the art.

[0224] The analysis may include measuring any of the parameters described herein or known in the art for measuring cell viability, cell proliferation, cell identity, cell morphology, and cell function, particularly those relevant to immune cells. For example, flow cytometry may be used to determine the total number of hematopoietic cells or the number of cells of a particular hematopoietic cell type. Histochemistry or immunohistochemistry may be performed to determine the apoptotic state of cells, for example, terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) to measure DNA fragmentation, or immunohistochemistry to detect annexin V binding to phosphatidylserine on the cell surface. Flow cytometry may also be used to assess the proportion of differentiated cells and differentiated cell types, for example, to determine the ability of hematopoietic cells to survive and / or differentiate in the presence of a drug. ELISA, Western, and Northern blotting may be performed to determine the levels of cytokines, chemokines, immunoglobulins, etc., expressed in engrafted genetically modified mice, for example, to assess the function of engrafted cells, to assess the function of human dendritic cells, etc. In vivo assays for testing the function of immune cells, as well as assays related to specific diseases or disorders such as autoimmune diseases, including systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, polymyositis, collagen-induced arthritis, or dermatomyositis, may also be performed. See, for example, Current Protocols in Immunology (Richard Coico, ed., John Wiley & Sons, Inc., 2012) and Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997), whose disclosures are incorporated herein by reference.

[0225] Other examples of use on target mice are provided elsewhere in this specification. Further uses of the genetically modified mice and engrafted mice described herein will be apparent to those skilled in the art upon reading this disclosure. Further exemplary embodiments

[0226] In an exemplary embodiment 1, a genetically modified rodent is provided herein, comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent and human portion operably linked to the Flt3l promoter.

[0227] In an exemplary embodiment 2, the genetically modified rodent described in Embodiment 1, comprising a homozygous null mutation in the Rag1 gene, is provided herein.

[0228] In an exemplary embodiment 3, a genetically modified rodent according to embodiment 1 or 2 is provided herein, wherein a null mutation in the rodent Flt3 gene includes an insertion, deletion, and / or substitution in the endogenous Flt3 gene.

[0229] In an exemplary embodiment 4, the genetically modified rodent described herein is provided, wherein the null mutation in the rodent Flt3 gene is a complete deletion of the endogenous Flt3 coding sequence.

[0230] In an exemplary embodiment 5, the genetically modified rodent described herein is a mouse, and the mouse contains a homozygous deletion of the nucleic acid sequence between coordinates chr5:147331171-147400265 (GRCm38 assembly).

[0231] In an exemplary embodiment 6, a genetically modified rodent according to any one of embodiments 1 to 5 is provided herein, wherein the rodent portion of the Flt3l gene includes the non-coding portions of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene.

[0232] In an exemplary embodiment 7, the genetically modified rodent described herein is provided, wherein the exons 1, the non-coding portions of exon 2, and the downstream exon of exon 6 of the rodent Flt3l gene are at least 90%, at least 95%, or 100% identical to the corresponding exons 1, the non-coding portions of exon 2, and the downstream exon of exon 6 of the rodent Flt3l gene shown in Table 1A.

[0233] In an exemplary embodiment 8, a genetically modified rodent according to any one of embodiments 1 to 7 is provided herein, wherein the human portion of the Flt3l gene comprises the signal peptide coding portion of exon 2 and exons 3 to 6 of the human FLT3L gene.

[0234] In an exemplary embodiment 9, a genetically modified rodent according to Embodiment 8 is provided herein, wherein the signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B.

[0235] In exemplary embodiment 10, a genetically modified rodent according to any one of embodiments 1 to 9 is provided herein, wherein the Flt3l gene comprises rodent exon 1, the rodent non-coding portion of exon 2, the human signal peptide coding portion of exon 2, human exons 3 to 6, and rodent exons 7 to 9.

[0236] In exemplary embodiment 11, a genetically modified rodent according to any one of embodiments 1 to 10 is provided herein, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L comprising the signal peptide and cytokine-like core domain of a human FLT3L polypeptide and the C-terminal portion of a rodent Flt3l polypeptide.

[0237] In an exemplary embodiment 12, the genetically modified rodent described herein is provided, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises a rodent stalk region, a rodent transmembrane domain, and a rodent cytoplasmic tail, as shown in Figure 1E.

[0238] In exemplary embodiment 13, a genetically modified rodent according to embodiment 11 or 12 is provided herein, having an amino acid sequence in which the C-terminal portion of the rodent Flt3l polypeptide is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

[0239] In exemplary embodiment 14, a genetically modified rodent according to any one of embodiments 1 to 13 is provided herein, wherein the rodent expresses both the soluble and membrane-bound forms of the Flt3l polypeptide.

[0240] In exemplary embodiment 15, the genetically modified rodent described herein, wherein the soluble and membrane-bound forms of the FLT3L polypeptide comprise the signal peptide and cytokine-like core domain of the human FLT3L polypeptide, is provided herein.

[0241] In exemplary embodiment 16, a genetically modified rodent according to any one of embodiments 11 to 15 is provided herein, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1 to 26 of the human FLT3L polypeptide.

[0242] In an exemplary embodiment 17, a genetically modified rodent according to any one of embodiments 11 to 16 is provided herein, wherein the signal peptide of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the signal peptide of the human FLT3L polypeptide shown in Table 2.

[0243] In exemplary embodiment 18, a genetically modified rodent according to any one of embodiments 11 to 17 is provided herein, wherein the cytokine-like core domain of the human FLT3L polypeptide contains amino acids corresponding to residues 27 to 159 of the human FLT3L polypeptide.

[0244] In exemplary embodiment 19, a genetically modified rodent according to any one of embodiments 11 to 18 is provided herein, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

[0245] In exemplary embodiment 20, a genetically modified rodent according to any one of embodiments 1 to 19 is provided herein, wherein the genetically modified rodent is heterozygous for the Flt3 ligand (Flt3l) gene, which comprises a rodent portion and a human portion.

[0246] In exemplary embodiment 21, a genetically modified rodent according to any one of embodiments 1 to 19 is provided herein, wherein the genetically modified rodent is homozygous for the Flt3 ligand (Flt3l) gene, which comprises a rodent portion and a human portion.

[0247] In exemplary embodiment 22, a genetically modified rodent according to any one of embodiments 1 to 21 is provided herein, wherein the rodent portion of the Flt3l gene is the endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is the endogenous rodent gene.

[0248] In exemplary embodiment 23, a genetically modified rodent according to any one of embodiments 1 to 22 is provided herein, wherein the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0249] In exemplary embodiment 24, a genetically modified rodent according to any one of embodiments 1 to 23 is provided herein, wherein the Flt3l promoter is a rodent promoter.

[0250] In exemplary embodiment 25, a genetically modified rodent according to embodiment 23 or 24 is provided herein, wherein the Flt3l promoter is an endogenous rodent promoter.

[0251] In exemplary embodiment 26, a genetically modified rodent according to any one of embodiments 23 to 25 is provided herein, wherein the Flt3l promoter is located at an endogenous rodent locus.

[0252] In exemplary embodiment 27, a genetically modified rodent according to any one of embodiments 1 to 26 is provided herein, wherein the genetically modified rodent expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0253] In an exemplary embodiment 28, the genetically modified rodent described herein is provided, further comprising a Sirpa gene encoding a Sirpa polypeptide that includes the extracellular portion of human SIRPA polypeptide and the intracellular portion of rodent Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

[0254] In exemplary embodiment 29, a genetically modified rodent according to embodiment 28 is provided herein, wherein the Sirpa gene comprises exons 1, 5, 6, 7, and 8 of the rodent Sirpa gene and exons 2-4 of the human SIRPA gene.

[0255] In exemplary embodiment 30, the genetically modified rodent described herein is provided as described in embodiment 28 or 29, wherein the genetically modified rodent expresses a Sirpa polypeptide comprising the extracellular portion of human SIRPA polypeptide and the intracellular portion of rodent Sirpa polypeptide.

[0256] In exemplary embodiment 31, there is provided herein a genetically modified rodent according to any one of embodiments 28-30, wherein the rodent Sirpa polypeptide is an endogenous rodent Sirpa polypeptide and / or the rodent Sirpa gene is an endogenous rodent gene.

[0257] In exemplary embodiment 32, there is provided herein a genetically modified rodent according to embodiment 27, wherein the genetically modified rodent expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0258] In exemplary embodiment 33, there is provided herein a genetically modified rodent according to any one of embodiments 27-32, wherein the genetically modified rodent further expresses a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter and / or a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter.

[0259] In exemplary embodiment 34, there is provided herein a genetically modified rodent according to any one of embodiments 27-33, wherein the SIRPα promoter, GM-CSF promoter and / or IL3 promoter is a rodent promoter.

[0260] In exemplary embodiment 35, there is provided herein a genetically modified rodent according to embodiment 34, wherein the SIRPα promoter, GM-CSF promoter and / or IL3 promoter is an endogenous rodent promoter.

[0261] In exemplary embodiment 36, there is provided herein a genetically modified rodent according to embodiment 34 or 35, wherein the SIRPα promoter, GM-CSF promoter and / or IL3 promoter is at the corresponding endogenous rodent locus.

[0262] In exemplary embodiment 37, a genetically modified rodent according to any one of embodiments 27 to 36 is provided herein, comprising a null mutation in at least one corresponding rodent gene at the corresponding rodent locus.

[0263] In exemplary embodiment 38, a genetically modified rodent according to any one of embodiments 27 to 37 is provided herein, wherein the genetically modified rodent is heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0264] In exemplary embodiment 39, a genetically modified rodent according to any one of embodiments 27 to 38 is provided herein, wherein the genetically modified rodent is homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0265] In exemplary embodiment 40, a genetically modified rodent according to any one of embodiments 1 to 39 is provided herein, further comprising the engraftment of human hematopoietic cells.

[0266] In an exemplary embodiment 41, the genetically modified rodent described herein, as in embodiment 40, is provided, wherein the human hematopoietic cells comprise one or more cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human hematopoietic progenitor cells, human dendritic cell progenitor cells, and human dendritic cells.

[0267] In exemplary embodiment 42, the genetically modified rodent is provided herein, which comprises human dendritic cells, as described in embodiment 40 or 41.

[0268] In exemplary embodiment 43, a genetically modified rodent according to any one of embodiments 1 to 42 is provided herein, wherein an autoimmune disease is induced or established in the genetically modified rodent.

[0269] In an exemplary embodiment 44, a genetically modified rodent according to embodiment 43 is provided herein, wherein the autoimmune disease is systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, polymyositis, collagen-induced arthritis, or dermatomyositis.

[0270] In exemplary embodiment 45, a genetically modified rodent according to any one of embodiments 1 to 44 is provided herein, wherein the rodent is a mouse or a rat.

[0271] In an exemplary embodiment 46, the rodent is a mouse, and a genetically modified rodent as described in embodiment 45 is provided herein.

[0272] In an exemplary embodiment 47, a method for identifying a drug that modulates the function of human dendritic cells is provided herein, comprising: a. administering the candidate drug to a genetically modified rodent described in any one of embodiments 40 to 46; and b. determining whether the candidate drug modulates the function of human dendritic cells in the rodent.

[0273] In an exemplary embodiment 48, the method of embodiment 47 is provided herein, wherein the human dendritic cells are myeloid dendritic cells (mDCs) or plasmacytoid dendritic cells (pDCs).

[0274] In an exemplary embodiment 49, the method described herein is provided as described in Embodiment 47 or 48, wherein the function of the human dendritic cell is selected from the group consisting of phagocytosis, cytokine production, cross-presentation of exogenous antigens, and activation of cytotoxic CD8+ T-cell lymphocytes (CTLs).

[0275] An exemplary embodiment 50 provides a method for evaluating the therapeutic efficacy of a drug for stimulating a T cell response to target cells, the method comprising: a. administering a drug candidate to a genetically modified rodent according to any one of embodiments 40 to 42, wherein the genetically modified rodent contains target cells; and b. measuring the T cell response to the target cells in the rodent to evaluate the therapeutic efficacy of the drug candidate.

[0276] In an exemplary embodiment 51, the method of embodiment 50 is provided herein, wherein the target cells are selected from the group consisting of tumor cells, virus-infected cells, bacterial-infected cells, bacterial cells, fungal cells, and parasitic cells.

[0277] In an exemplary embodiment 52, a method for evaluating the therapeutic efficacy of a drug in the treatment of an autoimmune disease is provided herein, the method comprising: a. administering a candidate drug to a genetically modified rodent as described in Embodiment 43 or 44; and b. determining whether the drug treats an autoimmune disease in the rodent.

[0278] In an exemplary embodiment 53, genetically modified rodent cells are provided herein, comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent and human portion operably linked to the Flt3l promoter.

[0279] In an exemplary embodiment 54, the genetically modified rodent cells described in embodiment 53, which include a homozygous null mutation in the Rag1 gene, are provided herein.

[0280] In exemplary embodiment 55, there is provided herein a genetically modified rodent cell as described in embodiment 53 or 54, wherein the null mutation of the rodent Flt3 gene comprises an insertion, deletion, and / or substitution in the endogenous Flt3 gene.

[0281] In exemplary embodiment 56, there is provided herein a genetically modified rodent cell as described in embodiment 55, wherein the null mutation of the rodent Flt3 gene is a deletion of the entire Flt3 endogenous coding sequence.

[0282] In exemplary embodiment 57, there is provided herein a genetically modified rodent cell as described in embodiment 56, wherein the genetically modified rodent cell is a mouse cell, and the mouse cell comprises a homozygous deletion of a nucleic acid sequence between coordinates chr5:147331171-147400265 (GRCm38 assembly).

[0283] In exemplary embodiment 58, there is provided herein a genetically modified rodent cell as described in any one of embodiments 53-57, wherein the rodent portion of the Flt3l gene comprises exon 1 of the rodent Flt3l gene, the non-coding portion of exon 2, and the exons downstream of exon 6.

[0284] In exemplary embodiment 59, there is provided herein a genetically modified rodent cell as described in embodiment 58, wherein exon 1 of the rodent Flt3l gene, the non-coding portion of exon 2, and the exons downstream of exon 6 are at least 90%, at least 95%, or 100% identical to the corresponding exon 1 of the rodent Flt3l gene, the non-coding portion of exon 2, and the exons downstream of exon 6 shown in Table 1A.

[0285] In exemplary embodiment 60, there is provided herein a genetically modified rodent cell as described in any one of embodiments 1-59, wherein the human portion of the Flt3l gene comprises the signal peptide coding portion of exon 2 of the human FLT3L gene, and exons 3-6.

[0286] In an exemplary embodiment 61, genetically modified rodent cells according to Embodiment 60 are provided herein, wherein the signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding signal peptide coding portions of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B.

[0287] In exemplary embodiment 62, a genetically modified rodent cell according to any one of embodiments 53 to 61 is provided herein, wherein the Flt3l gene comprises rodent exon 1, the rodent non-coding portion of exon 2, the human signal peptide coding portion of exon 2, human exons 3 to 6, and rodent exons 7 to 9.

[0288] In an exemplary embodiment 63, a genetically modified rodent cell according to any one of embodiments 53 to 62 is provided herein, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L comprising the signal peptide and cytokine-like core domain of a human FLT3L polypeptide and the C-terminal portion of a rodent Flt3l polypeptide.

[0289] In an exemplary embodiment 64, a genetically modified rodent cell as described in embodiment 63 is provided herein, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises a rodent stalk region, a rodent transmembrane domain, and a rodent cytoplasmic tail as shown in Figure 1E.

[0290] In exemplary embodiment 65, genetically modified rodent cells according to embodiment 63 or 64 are provided herein, having an amino acid sequence in which the C-terminal portion of the rodent Flt3l polypeptide is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

[0291] In an exemplary embodiment 66, genetically modified rodent cells according to any one of embodiments 53 to 65 are provided herein, wherein the rodent cells express both the soluble and membrane-bound forms of the Flt3l polypeptide.

[0292] In an exemplary embodiment 67, genetically modified rodent cells as described in embodiment 66 are provided herein, wherein the soluble and membrane-bound forms of the FLT3L polypeptide comprise the signal peptide and cytokine-like core domain of the human FLT3L polypeptide.

[0293] In exemplary embodiment 68, genetically modified rodent cells according to any one of embodiments 63 to 67 are provided herein, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1 to 26 of the human FLT3L polypeptide.

[0294] In exemplary embodiment 69, genetically modified rodent cells according to any one of embodiments 63 to 68 are provided herein, wherein the signal peptide of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the signal peptide of the human FLT3L polypeptide shown in Table 2.

[0295] In exemplary embodiment 70, a genetically modified rodent cell according to any one of embodiments 63 to 69 is provided herein, wherein the cytokine-like core domain of the human FLT3L polypeptide contains amino acids corresponding to residues 27 to 159 of the human FLT3L polypeptide.

[0296] In an exemplary embodiment 71, a genetically modified rodent cell according to any one of embodiments 63 to 70 is provided herein, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

[0297] In an exemplary embodiment 72, the genetically modified rodent cells described herein are provided, wherein the genetically modified rodent cells are heterozygous for the Flt3 ligand (Flt3l) gene, which comprises a rodent portion and a human portion, as described in any one of embodiments 53 to 71.

[0298] In exemplary embodiment 73, genetically modified rodent cells according to any one of embodiments 53 to 71 are provided herein, wherein the genetically modified rodent cells are homozygous for the Flt3 ligand (Flt3l) gene, which comprises a rodent portion and a human portion.

[0299] In exemplary embodiment 74, a genetically modified rodent cell according to any one of embodiments 53 to 73 is provided herein, wherein the rodent portion of the Flt3l gene is the endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is the endogenous rodent gene.

[0300] In exemplary embodiment 75, a genetically modified rodent cell according to any one of embodiments 53 to 74 is provided herein, wherein the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0301] In exemplary embodiment 76, a genetically modified rodent cell according to any one of embodiments 53 to 75 is provided herein, wherein the Flt3l promoter is a rodent promoter.

[0302] In exemplary embodiment 77, genetically modified rodent cells according to embodiment 75 or 76 are provided herein, wherein the Flt3l promoter is an endogenous rodent promoter.

[0303] In exemplary embodiment 78, a genetically modified rodent cell according to any one of embodiments 75 to 77 is provided herein, wherein the Flt3l promoter is located at an endogenous rodent locus.

[0304] In an exemplary embodiment 79, the genetically modified rodent cell described herein is provided, further comprising a nucleic acid encoding a human or humanized SIRPA polypeptide, wherein the nucleic acid is operably linked to a Sirpa promoter, as described in any one of embodiments 53 to 78.

[0305] In an exemplary embodiment 80, the genetically modified rodent cell described herein is provided, further comprising a Sirpa gene encoding a Sirpa polypeptide which includes the extracellular portion of human SIRPA polypeptide and the intracellular portion of rodent Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

[0306] In an exemplary embodiment 81, the genetically modified rodent cell described herein is provided, wherein the Sirpa gene comprises exons 1, 5, 6, 7, and 8 of the rodent Sirpa gene and exons 2-4 of the human SIRPA gene.

[0307] In an exemplary embodiment 82, the genetically modified rodent cells described herein are provided, wherein the genetically modified rodent cells express a Sirpa polypeptide comprising the extracellular portion of human SIRPA polypeptide and the intracellular portion of rodent Sirpa polypeptide.

[0308] In exemplary embodiment 83, a genetically modified rodent cell according to any one of embodiments 80 to 82 is provided herein, wherein the rodent Sirpa polypeptide is an endogenous rodent Sirpa polypeptide and / or the rodent Sirpa gene is an endogenous rodent gene.

[0309] In exemplary embodiment 84, genetically modified rodent cells as described in embodiment 79 are provided herein, wherein the genetically modified rodent cells express human SIRPA polypeptide.

[0310] In exemplary embodiment 85, genetically modified rodent cells according to any one of embodiments 79 to 84 are provided herein, further comprising (1) a nucleic acid encoding a human GM-CSF protein operably linked to a GM-CSF promoter; and / or (2) a nucleic acid encoding a human IL3 protein operably linked to an IL3 promoter.

[0311] In exemplary embodiment 86, genetically modified rodent cells according to any one of embodiments 79 to 85 are provided herein, wherein the SIRPα promoter, GM-CSF promoter, and / or IL3 promoter are rodent promoters.

[0312] In exemplary embodiment 87, genetically modified rodent cells as described in embodiment 86 are provided herein, wherein the SIRPα promoter, GM-CSF promoter, and / or IL3 promoter are endogenous rodent promoters.

[0313] In exemplary embodiment 88, genetically modified rodent cells as described in embodiment 86 or 87 are provided herein, wherein the SIRPα promoter, GM-CSF promoter, and / or IL3 promoter are located at the corresponding endogenous rodent loci.

[0314] In exemplary embodiment 89, a genetically modified rodent cell according to any one of embodiments 79 to 88 is provided herein, comprising a null mutation in at least one corresponding rodent gene at the corresponding rodent locus.

[0315] In an exemplary embodiment 90, the genetically modified rodent cells described herein are provided as described in any one of embodiments 79 to 89, wherein the genetically modified rodent cells are heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0316] In an exemplary embodiment 91, the genetically modified rodent cells described herein are provided as described in any one of embodiments 79 to 90, wherein the genetically modified rodent cells are homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0317] In exemplary embodiment 92, genetically modified rodent cells according to any one of embodiments 53 to 91 are provided herein, wherein the rodent cells are rat cells or mouse cells.

[0318] In exemplary embodiment 93, genetically modified rodent cells as described in embodiment 92 are provided herein, wherein the rodent cells are mouse cells.

[0319] In exemplary embodiment 94, genetically modified rodent cells are provided herein according to any one of embodiments 53-65, 68-81, 83, and 85-93, wherein the genetically modified rodent cells are rodent embryonic stem (ES) cells.

[0320] In an exemplary embodiment 95, genetically modified rodent embryonic stem cells are provided herein, the genome comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene containing a rodent and human portion operably linked to the Flt3l promoter.

[0321] In an exemplary embodiment 96, a genetically modified rodent embryonic stem cell as described in embodiment 95, comprising a homozygous null mutation in the Rag1 gene, is provided herein.

[0322] In an exemplary embodiment 97, a genetically modified rodent embryonic stem cell as described in Embodiment 95 or 96 is provided herein, wherein the null mutation of the rodent Flt3 gene includes an insertion, deletion, and / or substitution in the endogenous Flt3 gene.

[0323] In an exemplary embodiment 98, the gene-modified rodent embryonic stem cells described herein are provided, wherein the null mutation in the rodent Flt3 gene is a deletion of the complete Flt3 endogenous coding sequence, as described in Embodiment 97.

[0324] In exemplary embodiment 99, the genetically modified rodent embryonic stem cells described herein are provided, wherein the genetically modified rodent embryonic stem cells are mouse embryonic stem cells, and the mouse comprises a homozygous deletion of the nucleic acid sequence between coordinates chr5:147331171-147400265 (GRCm38 assembly).

[0325] In exemplary embodiment 100, a genetically modified rodent embryonic stem cell according to any one of embodiments 95 to 99 is provided herein, wherein the rodent portion of the Flt3l gene includes the non-coding portions of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene.

[0326] In exemplary embodiment 101, the gene-modified rodent embryonic stem cells described herein are provided, wherein the exons 1, the non-coding portions of exon 2, and the downstream exon of exon 6 of the rodent Flt3l gene are at least 90%, at least 95%, or 100% identical to the corresponding exons 1, the non-coding portions of exon 2, and the downstream exon of exon 6 of the rodent Flt3l gene shown in Table 1A.

[0327] In exemplary embodiment 102, a genetically modified rodent embryonic stem cell according to any one of embodiments 95 to 101 is provided herein, wherein the human portion of the Flt3l gene comprises the signal peptide coding portion of exon 2 and exons 3 to 6 of the human FLT3L gene.

[0328] In exemplary embodiment 103, genetically modified rodent embryonic stem cells as described herein are provided, wherein the signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B.

[0329] In exemplary embodiment 104, a genetically modified rodent embryonic stem cell according to any one of embodiments 95 to 103 is provided herein, wherein the Flt3l gene comprises rodent exon 1, the rodent non-coding portion of exon 2, the human signal peptide coding portion of exon 2, human exons 3 to 6, and rodent exons 7 to 9.

[0330] In exemplary embodiment 105, a genetically modified rodent embryonic stem cell according to any one of embodiments 95 to 104 is provided herein, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L comprising the signal peptide and cytokine-like core domain of a human FLT3L polypeptide and the C-terminal portion of a rodent Flt3l polypeptide.

[0331] In exemplary embodiment 106, a genetically modified rodent embryonic stem cell as described in embodiment 105 is provided herein, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises a rodent stalk region, a rodent transmembrane domain, and a rodent cytoplasmic tail, as shown in Figure 1E.

[0332] In exemplary embodiment 107, genetically modified rodent embryonic stem cells according to embodiment 105 or 106 are provided herein, having an amino acid sequence in which the C-terminal portion of the rodent Flt3l polypeptide is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

[0333] In exemplary embodiment 108, a genetically modified rodent embryonic stem cell according to any one of embodiments 105 to 107 is provided herein, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1 to 26 of the human FLT3L polypeptide.

[0334] In exemplary embodiment 109, a genetically modified rodent embryonic stem cell according to any one of embodiments 105 to 108 is provided herein, wherein the signal peptide of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the signal peptide of the human FLT3L polypeptide shown in Table 2.

[0335] In exemplary embodiment 110, a genetically modified rodent embryonic stem cell according to any one of embodiments 105 to 109 is provided herein, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises amino acids corresponding to residues 27 to 159 of the human FLT3L polypeptide.

[0336] In an exemplary embodiment 111, a genetically modified rodent embryonic stem cell according to any one of embodiments 105 to 110 is provided herein, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

[0337] In exemplary embodiment 112, genetically modified rodent embryonic stem cells according to any one of embodiments 95 to 111 are provided herein, wherein the genetically modified rodent embryonic stem cells are heterozygous for the Flt3 ligand (Flt3l) gene, which comprises a rodent portion and a human portion.

[0338] In exemplary embodiment 113, genetically modified rodent embryonic stem cells are provided herein according to any one of embodiments 95 to 111, wherein the genetically modified rodent embryonic stem cells are homozygous for the Flt3 ligand (Flt3l) gene, which comprises a rodent portion and a human portion.

[0339] In exemplary embodiment 114, a genetically modified rodent embryonic stem cell according to any one of embodiments 95 to 113 is provided herein, wherein the rodent portion of the Flt3l gene is the endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is the endogenous rodent gene.

[0340] In exemplary embodiment 115, a genetically modified rodent embryonic stem cell according to any one of embodiments 95 to 114 is provided herein, wherein the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0341] In exemplary embodiment 116, a genetically modified rodent embryonic stem cell according to any one of embodiments 95 to 115 is provided herein, wherein the Flt3l promoter is a rodent promoter.

[0342] In an exemplary embodiment 117, the genetically modified rodent embryonic stem cells described herein are provided, wherein the Flt3l promoter is an endogenous rodent promoter.

[0343] In exemplary embodiment 118, a genetically modified rodent embryonic stem cell according to any one of embodiments 115 to 117 is provided herein, wherein the Flt3l promoter is located at an endogenous rodent locus.

[0344] In exemplary embodiment 119a, the genetically modified rodent embryonic stem cells described herein are provided as described in any one of embodiments 95 to 118, further comprising a nucleic acid encoding a human or humanized SIRPA polypeptide, the nucleic acid being operably linked to a Sirpa promoter.

[0345] In exemplary embodiment 119b, the genetically modified rodent embryonic stem cell described herein is provided, further comprising a Sirpa gene encoding a Sirpa polypeptide which includes the extracellular portion of human SIRPA polypeptide and the intracellular portion of rodent Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

[0346] In an exemplary embodiment 120, the genetically modified rodent embryonic stem cell described herein is provided, wherein the Sirpa gene comprises exons 1, 5, 6, 7, and 8 of the rodent Sirpa gene and exons 2-4 of the human SIRPA gene.

[0347] In exemplary embodiment 121a, a genetically modified rodent embryonic stem cell as described in embodiment 119b or 120 is provided herein, wherein the rodent Sirpa polypeptide is an endogenous rodent Sirpa polypeptide and / or the rodent Sirpa gene is an endogenous rodent gene.

[0348] In exemplary embodiment 121b, genetically modified rodent embryonic stem cells according to any one of embodiments 119a to 121a are provided herein, further comprising (1) a nucleic acid encoding a human GM-CSF protein operably linked to a GM-CSF promoter; and / or (2) a nucleic acid encoding a human IL3 protein operably linked to an IL3 promoter.

[0349] In exemplary embodiment 122, genetically modified rodent embryonic stem cells according to any one of embodiments 119a to 121b are provided herein, wherein the SIRPα promoter, GM-CSF promoter, and / or IL3 promoter are rodent promoters.

[0350] In exemplary embodiment 123, genetically modified rodent embryonic stem cells as described in embodiment 122 are provided herein, wherein the SIRPα promoter, GM-CSF promoter, and / or IL3 promoter are endogenous rodent promoters.

[0351] In exemplary embodiment 124, genetically modified rodent embryonic stem cells as described in embodiment 122 or 123 are provided herein, wherein the SIRPα promoter, GM-CSF promoter, and / or IL3 promoter are located at the corresponding endogenous rodent loci.

[0352] In exemplary embodiment 125, a genetically modified rodent embryonic stem cell according to any one of embodiments 119a to 124 is provided herein, comprising a null mutation in at least one corresponding rodent gene at the corresponding rodent locus.

[0353] In exemplary embodiment 126, genetically modified rodent embryonic stem cells according to any one of embodiments 119a to 125 are provided herein, wherein the genetically modified rodent embryonic stem cells are heterozygous for at least one allele containing a nucleic acid sequence encoding a human or humanized protein.

[0354] In exemplary embodiment 127, genetically modified rodent embryonic stem cells according to any one of embodiments 119a to 126 are provided herein, wherein the genetically modified rodent embryonic stem cells are homozygous for at least one allele containing a nucleic acid sequence encoding a human or humanized protein.

[0355] In exemplary embodiment 128, the rodent embryonic stem cells are rat embryonic stem cells or mouse embryonic stem cells, as described herein, and a genetically modified rodent embryonic stem cell according to any one of embodiments 95 to 127 is provided.

[0356] Genetically modified rodent embryonic stem cells as described in Embodiment 128 are provided herein, in exemplary embodiment 129, where the rodent embryonic stem cells are mouse embryonic stem cells.

[0357] In an exemplary embodiment 130, a method for producing rodent embryonic stem cells is provided herein, comprising genetically modifying rodent embryonic stem cells so that the rodent embryonic stem cells have a genome comprising (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent and human portion operably linked to the Flt3l promoter.

[0358] In exemplary embodiment 131, rodent embryonic stem cells are further genetically modified to have a genome containing a homozygous null mutation in the Rag1 gene, as described herein in embodiment 130.

[0359] In an exemplary embodiment 132, the method described herein is provided as described in Embodiment 130 or 131, wherein a null mutation in the rodent Flt3 gene includes an insertion, deletion, and / or substitution in the endogenous Flt3 gene.

[0360] In exemplary embodiment 133, the method described herein is provided as described in embodiment 132, in which the null mutation of the rodent Flt3 gene is a deletion of the complete Flt3 endogenous coding sequence.

[0361] In exemplary embodiment 134, the genetically modified rodent cells are mouse cells, and the method described herein is provided, wherein the mouse cells include a homozygous deletion of a nucleic acid sequence between coordinates chr5:147331171-147400265 (GRCm38 assembly).

[0362] In exemplary embodiment 135, the rodent portion of the Flt3l gene includes the rodent Flt3l gene, the non-coding portion of exon 1 and exon 2, and the downstream exon of exon 6, as described herein, and the method according to any one of embodiments 130 to 134.

[0363] The method described herein is provided as described in exemplary embodiment 136, in which the exons 1, the non-coding portions of exon 2, and the downstream exon of exon 6 of the rodent Flt3l gene are at least 90%, at least 95%, or 100% identical to the corresponding exons 1, the non-coding portions of exon 2, and the downstream exon of exon 6 of the rodent Flt3l gene shown in Table 1A.

[0364] In exemplary embodiment 137, the method described herein is provided as described in any one of embodiments 130 to 136, wherein the human portion of the Flt3l gene comprises the signal peptide coding portion of exon 2 and exons 3 to 6 of the human FLT3L gene.

[0365] The method described herein is provided as described in exemplary embodiment 138, in which the signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B.

[0366] In exemplary embodiment 139, the method described herein is provided as described in any one of embodiments 130 to 138, wherein the Flt3l gene comprises rodent exon 1, the rodent non-coding portion of exon 2, the human signal peptide coding portion of exon 2, human exons 3 to 6, and rodent exons 7 to 9.

[0367] In an exemplary embodiment 140, the method described herein is provided as described in any one of embodiments 130 to 139, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L comprising the signal peptide and cytokine-like core domain of a human FLT3L polypeptide and the C-terminal portion of a rodent Flt3l polypeptide.

[0368] In exemplary embodiment 141, the method described herein is provided, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises a rodent stalk region, a rodent transmembrane domain, and a rodent cytoplasmic tail, as shown in Figure 1E.

[0369] In exemplary embodiment 142, the method described herein is provided as described in embodiment 140 or 141, wherein the C-terminal portion of the rodent Flt3l polypeptide has an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

[0370] In exemplary embodiment 143, the method described herein is provided as described in any one of embodiments 140 to 142, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1 to 26 of the human FLT3L polypeptide.

[0371] In exemplary embodiment 144, the method according to any one of embodiments 140 to 143 is provided herein, wherein the signal peptide of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the signal peptide of the human FLT3L polypeptide shown in Table 2.

[0372] In exemplary embodiment 145, the method described herein is provided as described in any one of embodiments 140 to 144, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises amino acids corresponding to residues 27 to 159 of the human FLT3L polypeptide.

[0373] In exemplary embodiment 146, the method described herein is provided as described in any one of embodiments 140 to 145, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

[0374] In an exemplary embodiment 147, the method described herein is provided as described in any one of embodiments 130 to 146, wherein the genetically modified rodent embryonic stem cells are heterozygous for the Flt3 ligand (Flt3l) gene, which comprises a rodent portion and a human portion.

[0375] In an exemplary embodiment 148, the method described herein is provided as described in any one of embodiments 130 to 146, wherein the genetically modified rodent embryonic stem cells are homozygous for the Flt3 ligand (Flt3l) gene, which comprises a rodent portion and a human portion.

[0376] In exemplary embodiment 149, the rodent portion of the Flt3l gene is the endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is the endogenous rodent gene, as described herein.

[0377] In exemplary embodiment 150, the method according to any one of embodiments 130 to 149 is provided herein, wherein the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0378] In exemplary embodiment 151, the method described herein is provided as described in any one of embodiments 130 to 150, wherein the Flt3l promoter is a rodent promoter.

[0379] In exemplary embodiment 152, the method described herein is provided as described in embodiment 150 or 151, wherein the Flt3l promoter is an endogenous rodent promoter.

[0380] In exemplary embodiment 153, the method described herein is provided as described in any one of embodiments 150 to 152, wherein the Flt3l promoter is located at an endogenous rodent locus.

[0381] In an exemplary embodiment 154, the method of embodiment 153 is provided herein, wherein the genetically modified rodent embryonic stem cell is further genetically modified to include a Sirpa gene encoding a Sirpa polypeptide comprising the extracellular portion of human SIRPA polypeptide and the intracellular portion of rodent Sirpa polypeptide in its genome, and the Sirpa gene is operably linked to a Sirpa promoter.

[0382] In an exemplary embodiment 155, the method of embodiment 154 is provided herein, wherein the Sirpa gene comprises exons 1, 5, 6, 7, and 8 of the rodent Sirpa gene and exons 2-4 of the human SIRPA gene.

[0383] In exemplary embodiment 156a, the method described herein is provided as described in embodiment 154 or 155, wherein the rodent Sirpa polypeptide is an endogenous rodent Sirpa polypeptide and / or the rodent Sirpa gene is an endogenous rodent gene.

[0384] In an exemplary embodiment 156b, the method described herein is provided as described in any one of embodiments 154 to 156a, wherein genetically modified rodent embryonic stem cells are further manipulated to include in their genome a nucleic acid encoding the human GM-CSF protein and operably linked to the GM-CSF promoter, and / or a nucleic acid encoding the human IL3 protein and operably linked to the IL3 promoter.

[0385] In exemplary embodiment 157, the method described herein is provided as described in any one of embodiments 154 to 156b, wherein the SIRPα promoter, GM-CSF promoter, and / or IL3 promoter are rodent promoters.

[0386] In exemplary embodiment 158, the method of embodiment 157 is provided herein, wherein the SIRPα promoter, GM-CSF promoter and / or IL3 promoter are endogenous rodent promoters.

[0387] In exemplary embodiment 159, the method described herein is provided as described in embodiment 156 or 157, wherein the SIRPα promoter, GM-CSF promoter, and / or IL3 promoter are located at the corresponding endogenous rodent loci.

[0388] Exemplary embodiment 160 provides a method according to any one of embodiments 154 to 159, which includes a null mutation in at least one corresponding rodent gene at the corresponding rodent locus.

[0389] In an exemplary embodiment 161, the method described herein is provided as described in any one of embodiments 154 to 160, wherein the genetically modified rodent embryonic stem cells are heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0390] In an exemplary embodiment 162, the method described herein is provided as described in any one of embodiments 154 to 161, wherein the genetically modified rodent embryonic stem cells are homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0391] In exemplary embodiment 163, the method according to any one of embodiments 130 to 162 is provided herein, wherein the rodent embryonic stem cells are rat embryonic stem cells or mouse embryonic stem cells.

[0392] In an exemplary embodiment 164, the method described herein is provided, wherein the rodent embryonic stem cells are mouse embryonic stem cells.

[0393] In exemplary embodiment 165, a rodent embryo is provided herein, comprising rodent embryonic stem cells described in any one of embodiments 94 to 129, or rodent embryonic stem cells prepared according to the method described in any one of embodiments 130 to 164.

[0394] An exemplary embodiment 166 provides a method for producing a rodent whose genome includes (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent and human portion operably linked to the Flt3l promoter, the method comprising (a) obtaining rodent embryonic stem cells described in any one of embodiments 94 to 129 or rodent embryonic stem cells produced according to the method described in any one of embodiments 130 to 164; and (b) producing a rodent using the rodent embryonic cells of (a).

[0395] An exemplary embodiment 167 provides a method for producing a rodent whose genome includes (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operably linked to the Flt3l promoter, the method comprising modifying the genome of the rodent to include (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) an Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operably linked to the Flt3l promoter.

[0396] In an exemplary embodiment 168, the method of embodiment 167 is provided herein, wherein the rodent is manipulated to contain a homozygous null mutation in the Rag1 gene.

[0397] In exemplary embodiment 169, a method according to embodiment 167 or 168 is provided herein, wherein a null mutation in the rodent Flt3 gene includes insertions, deletions, and / or substitutions in the endogenous Flt3 gene.

[0398] In an exemplary embodiment 170, the method described herein as described in Embodiment 169 is provided, in which a null mutation in the rodent Flt3 gene is a deletion of the complete Flt3 endogenous coding sequence.

[0399] In exemplary embodiment 171, the genetically modified rodent is a mouse, and the mouse comprises a homozygous deletion of a nucleic acid sequence between coordinates chr5:147331171-147400265 (GRCm38 assembly), as described herein.

[0400] In exemplary embodiment 172, the rodent portion of the Flt3l gene includes the rodent Flt3l gene, the non-coding portion of exon 1 and exon 2, and the downstream exon of exon 6, as described herein.

[0401] This specification provides a method according to Embodiment 172, in exemplary Embodiment 173, in which the exons 1, the non-coding portion of exon 2, and the downstream exon of exon 6 of the rodent Flt3l gene are at least 90%, at least 95%, or 100% identical to the corresponding exons 1, the non-coding portion of exon 2, and the downstream exon of exon 6 of the rodent Flt3l gene shown in Table 1A.

[0402] In exemplary embodiment 174, the method described herein is provided as described in any one of embodiments 167 to 173, wherein the human portion of the Flt3l gene comprises the signal peptide coding portion of exon 2 and exons 3 to 6 of the human FLT3L gene.

[0403] In exemplary embodiment 175, the method according to embodiment 174 is provided herein, wherein the signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding signal peptide coding portions of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B.

[0404] In exemplary embodiment 176, the method described herein is provided as described in any one of embodiments 167 to 175, wherein the Flt3l gene comprises rodent exon 1, the rodent non-coding portion of exon 2, the human signal peptide coding portion of exon 2, human exons 3 to 6, and rodent exons 7 to 9.

[0405] In exemplary embodiment 177, the method described herein is provided as described in any one of embodiments 167 to 176, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L comprising the signal peptide and cytokine-like core domain of a human FLT3L polypeptide and the C-terminal portion of a rodent Flt3l polypeptide.

[0406] In exemplary embodiment 178, the method described herein is provided, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises a rodent stalk region, a rodent transmembrane domain, and a rodent cytoplasmic tail, as shown in Figure 1E.

[0407] In exemplary embodiment 179, the method described herein is provided as described in embodiment 177 or 178, wherein the C-terminal portion of the rodent Flt3l polypeptide has an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

[0408] In exemplary embodiment 180, a method according to any one of embodiments 167-179 is provided herein, wherein a rodent expresses both the soluble and membrane-bound forms of the Flt3l polypeptide.

[0409] In exemplary embodiment 181, the method described herein is provided, wherein the soluble and membrane-bound forms of the FLT3L polypeptide include the signal peptide and cytokine-like core domain of the human FLT3L polypeptide.

[0410] In exemplary embodiment 182, the method described herein is provided as described in any one of embodiments 177 to 181, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1 to 26 of the human FLT3L polypeptide.

[0411] In an exemplary embodiment 183, the method according to any one of embodiments 177 to 182 is provided herein, wherein the signal peptide of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the signal peptide of the human FLT3L polypeptide shown in Table 2.

[0412] In exemplary embodiment 184, the method described herein is provided as described in any one of embodiments 177 to 183, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises amino acids corresponding to residues 27 to 159 of the human FLT3L polypeptide.

[0413] In exemplary embodiment 185, the method described herein is provided as described in any one of embodiments 177 to 184, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

[0414] In an exemplary embodiment 186, the method described herein is provided as described in any one of embodiments 167 to 185, wherein the genetically modified rodent is heterozygous for the Flt3 ligand (Flt3l) gene, which comprises a rodent portion and a human portion.

[0415] In exemplary embodiment 187, the method described herein is provided as described in any one of embodiments 167 to 185, wherein the genetically modified rodent is homozygous for the Flt3 ligand (Flt3l) gene, which comprises a rodent portion and a human portion.

[0416] In exemplary embodiment 188, the rodent portion of the Flt3l gene is the endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is the endogenous rodent gene, as described herein.

[0417] In exemplary embodiment 189, the method described herein is provided as described in any one of embodiments 167 to 188, wherein the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0418] In exemplary embodiment 190, the method described herein is provided as described in any one of embodiments 167 to 189, wherein the Flt3l promoter is a rodent promoter.

[0419] In exemplary embodiment 191, the method described herein is provided as described in embodiment 189 or 190, wherein the Flt3l promoter is an endogenous rodent promoter.

[0420] In exemplary embodiment 192, the method described herein is provided as described in any one of embodiments 189 to 191, wherein the Flt3l promoter is located at an endogenous rodent locus.

[0421] In an exemplary embodiment 193, a method according to any one of embodiments 167 to 192 is provided herein, wherein a genetically modified rodent expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0422] In an exemplary embodiment 194, the genetically modified rodent further comprises a Sirpa gene encoding a Sirpa polypeptide that includes the extracellular portion of the human SIRPA polypeptide and the intracellular portion of the rodent Sirpa polypeptide, and the Sirpa gene is operably linked to a Sirpa promoter, the method of embodiment 193 is provided herein.

[0423] In an exemplary embodiment 195, the method of embodiment 194 is provided herein, wherein the Sirpa gene comprises exons 1, 5, 6, 7, and 8 of the rodent Sirpa gene and exons 2-4 of the human SIRPA gene.

[0424] In an exemplary embodiment 196, the method described herein is provided as described in Embodiment 194 or 195, wherein a genetically modified rodent expresses a Sirpa polypeptide comprising the extracellular portion of human SIRPA polypeptide and the intracellular portion of rodent Sirpa polypeptide.

[0425] In exemplary embodiment 197, the method according to any one of embodiments 194 to 196 is provided herein, wherein the rodent Sirpa polypeptide is an endogenous rodent Sirpa polypeptide and / or the rodent Sirpa gene is an endogenous rodent gene.

[0426] In an exemplary embodiment 198, the method described herein is provided, in which a genetically modified rodent expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a Sirpa promoter.

[0427] Exemplary embodiment 199 provides a method according to any one of embodiments 193 to 198, wherein a genetically modified rodent further expresses a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter and / or a human IL3 protein encoded by a nucleic acid operably linked to an IL3 promoter.

[0428] In an exemplary embodiment 200, the method described herein is provided as described in any one of embodiments 193 to 199, wherein the SIRPα promoter, GM-CSF promoter, and / or IL3 promoter are rodent promoters.

[0429] In exemplary embodiment 201, the method described herein is provided, in which the SIRPα promoter, GM-CSF promoter, and / or IL3 promoter are endogenous rodent promoters.

[0430] In exemplary embodiment 202, the method described herein is provided as described in embodiment 200 or 201, wherein the SIRPα promoter, GM-CSF promoter, and / or IL3 promoter are located at the corresponding endogenous rodent loci.

[0431] In exemplary embodiment 203, the genetically modified rodent comprises a null mutation in at least one corresponding rodent gene at the corresponding rodent locus, as described herein by any one of embodiments 193 to 202.

[0432] In an exemplary embodiment 204, the method described herein is provided as described in any one of embodiments 193 to 203, wherein the genetically modified rodent is heterozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0433] In exemplary embodiment 205, the method described herein is provided as described in any one of embodiments 193 to 204, wherein the genetically modified rodent is homozygous for at least one allele comprising a nucleic acid sequence encoding a human or humanized protein.

[0434] In exemplary embodiment 206, the method described herein is provided as described in any one of embodiments 167 to 205, wherein the rodent is a mouse or a rat.

[0435] In exemplary embodiment 207, the method of embodiment 206 is provided herein, wherein the rodent is a mouse. [Examples]

[0436] The following examples and accompanying drawings are provided to those skilled in the art to illustrate the methods and compositions of the present invention and how to prepare and use them, and are not intended to limit the scope of what the inventors consider to be the present invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantities, temperatures, etc.), but some experimental errors and deviations should be taken into consideration. The examples do not include detailed descriptions of conventional methods (e.g., molecular cloning techniques) that would be well known to those skilled in the art. Unless otherwise specified, parts are by weight, molecular weights are average molecular weights, temperatures are given in Celsius, and pressures are atmospheric pressure or near atmospheric pressure.

[0437] Example 1: Generation of genetically modified mice Example 1.1: Generation of humanized Flt3l mice Mouse and human Flt3 ligands exist as multiple isoforms, both membrane-bound and soluble, which are thought to arise from alternative splicing (Lyman et al. (1995) Oncogene 10:149-157; McClanahan et al. (1996) Blood 88(9):3371-3382; Lyman et al. (1995) Oncogene 11:1165-1172; Lyman and Jackbsen (1998) Blood 91(4)1101-1134). Soluble isoforms can also arise from proteolytic cleavage (resulting from cleavage by TNFα-converting enzyme or TACE) (Kazi et al. (2019) Physiol. Rev. 99:1433-1466). All known soluble isoforms of human and mouse Flt3 ligands generally contain a receptor-binding domain (cytokine-like core domain) encoded by exons 3–6 of the gene. The mouse Flt3 ligand (Flt3l) locus was humanized using VELOCIGENE® technology (see, e.g., U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome couple with high-resolution expression analysis. Nat. Biotech. 21(6):652–659, both incorporated herein by reference). The resulting humanized FLT3l locus contained the endogenous mouse FLT3l promoter, a chimeric exon 2 (encoding the human signal peptide) containing the mouse non-coding portion of exon 2 and the human signal peptide coding portion of exon 2, human FLT3L exons 3-6 (encoding the human FLT3L cytokine-like core domain) containing a 111 bp human intron 6 sequence, followed by the remaining endogenous mouse FLT3l including intron 6 and the remainder of exons 7-9, and the 3'UTR.

[0438] To create the final targeting vector for humanizing FLT3L: (i) From a portion of mouse intron 2 through mouse exon 6 to intron 6 (2293bp; GRCm38 / mm110-chr7:45,133,715-45,136,007, which encodes a portion of the mouse FLT3L signal sequence and cytokine-like core domain sequence, first create a bacterial artificial chromosome (BAC) clone bMQ432L09 (Thermo-Fisher / Invitrogen (ii) it was deleted in (ii) then introduced in ATG from a portion of human exon 2 through human exon 6 to intron 6 (4520 bp; GRCh38 / hg38-chr19:49,474,640-49,479,159, which encodes the human FLT3L signal sequence and cytokine-like core domain sequence), and into a neomycin (Neo) resistant autodeletion cassette (which has a CRE recombinase controlled by a protamine promoter).

[0439] In detail, a portion of mouse intron 2 to mouse exon 6, specifically intron 6 (2293 bp), was initially deleted in the bacterial artificial chromosome (BAC) clone bMQ432L09 to obtain modified BAC#1 (see Figure 1A). By bacterial homologous recombination (BHR), the Flt3l sequence was substituted using spectinomycin cassettes ligated to Up and Down homologous boxes (260 bp and 230 bp). The homologous boxes were prepared by PCR using BAC as a template and are shown in Table 4 below. [Table 4]

[0440] To humanize the above BAC#1, an initial plasmid was generated containing nucleic acids encoding the human FLT3L signal peptide and cytokine-like core domain, along with the 5' mouse FLT3L sequence. More specifically, this initial plasmid contained, from 5' to 3': (i) a NotI site, (ii) a 382 bp mouse FLT3L sequence (3' portion of mouse intron 1 (348 bp) and 5' portion of mouse exon 2 (i.e., non-coding portion) (34 bp)), (iii) a human FLT3L nucleic acid sequence (modified human BAC CTD2523c15 (ThermoFisher / Invitrogen)) containing a portion of ATG-derived exon 2 (33 bp), intron 2 (1018 bp), exon 3 (111 bp), intron 3 (343 bp), exon 4 (54 bp), intron 4 (224 bp), exon 5 (144 bp), intron 5 (2343 bp), exon 6 (139 bp), and a portion of intron 6 (111 bp), as well as (v) a SpeI site. This donor plasmid was digested with NotI and SpeI to release fragments, which were then ligated from 5' to 3': (i) a selection cassette containing a hygromycin resistance gene operably ligated to the EM7 promoter and (ii) a 230 bp mouse FLT3l sequence (3' portion of mouse intron 6). Using this BHR donor fragment containing the human FLT3L nucleic acid sequence and selection cassette, flanked by mouse FLT3l sequences (up-box and down-box), the spectinomycin cassette was replaced by BHR to obtain BAC#2 (see step A in Figure 1B). Homologous boxes were prepared by PCR using BAC as a template and are shown in Table 5 below. [Table 5]

[0441] To construct the final targeting vector from the aforementioned BAC#2, a neomycin (Neo)-resistant autodeletion cassette (containing a CRE recombinase controlled by a protamine promoter) flanked by loxp sites (loxp-neo-loxp) was used, replacing the EM7-Hyg cassette with BHR (see step B in Figure 1B).

[0442] The final targeting vector (BAC#3) contained, from 5' to 3': a 5' mouse homologous arm, a human FLT3L signaling peptide and cytokine-like core domain, a loxp-Neo-loxp autodeletion cassette, a 3' mouse homologous arm, and a chloramphenicol resistance cassette (CM; not shown in Figure 1B). The final clone was selected based on Neo / CM resistance.

[0443] The final targeting vector was electroporated into mouse embryonic stem (ES) cells containing hSIRP-alpha (or human SIRPA), RAG2- / -IL2Rg- / - modifications (e.g., described in International Publication No. 2014 / 071397, U.S. Patent No. 11,019,810, and International Publication No. 2016 / 168212, each incorporated herein by reference). Targeted homologous recombination resulted in the substitution of a 2336 bp kb mouse sequence (GRCm38 / mm10 coordinate chr7: 45,133,715-45,136,050) with a 4520 bp human sequence (GRCh38 / hg38-chr19: 49,474,640-49,479,159) and a 4927 bp loxp-Neo-loxp autodeletion cassette (Figure 1C). The success of the integration was confirmed, for example, by an allele modification (MOA) assay, as described by Valenzuela et al. (see above). The probes used in the MOA assay for loss of the mouse FLT3L sequence and presence of the human FLT3L sequence are shown in Table 6 below, and their approximate locations are schematically shown in Figures 1C and 1D. Subsequently, the cassette was removed by expression of CRE recombinase (regulated by the protamine promoter) in mice (Figure 1D). [Table 6]

[0444] Positive target ES cells were used as donor ES cells and microinjected into premorlar (8-cell) stage mouse embryos by the VELOCIMOUSE® method (see, for example, U.S. Patent Nos. 7,576,259, 7,659,442, 7,294,754, and U.S. Patent Application Publication No. 2008-0078000, all of which are incorporated herein by reference). Mouse embryos containing donor ES cells were incubated in vitro and then transplanted into surrogate mothers to produce F0 mice completely derived from the donor ES cells. Mice with a deletion of the Flt3l gene and insertion of humanized Flt3l were identified by genotyping using the MOA assay described above. Heterozygous mice for Flt3l gene deletion and acquisition of humanized Flt3l were mated homozygous. These humanized Flt3l mice, including those with Rag2 gene knockout, Il2rg gene knockout, and humanized Sirpa, were crossed to achieve homozygosity for all genes.

[0445] Based on a specific design, a mouse encoding a chimeric membrane-bound FLT3L, shown in Figure 1E and labeled "Chimeric_FLT3LG_MB," was generated. The membrane-bound mouse FLT3 ligand, shown in this figure and labeled "mFlt3l_MB," is obtained from the NCBI reference sequence NP_038548.3, Uniprot:A9QW46. The membrane-bound human FLT3 ligand, shown in this figure and labeled "hFLT3LG_MB," is obtained from the NCBI reference sequence NP_001191431.1, Uniprot:P49771-1. The protein domains are as shown in the figure.

[0446] Example 1.2: Generation of Flt3 knockout mice The mouse Flt3 gene was deleted in the mouse genome using VELOCIGENE® technology (see, for example, U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotech. 21(6):652-659, both incorporated herein by reference). The 69.1kb mouse genome sequence of the mouse Flt3 gene, from the start codon ATG to the stop codon, was deleted on mouse chromosome 5 G3 between coordinates chr5:147331171-147400265 (GRCm38 assembly). See Figure 2.

[0447] In detail, mouse homologous arms were prepared by PCR amplification using BAC clone RP23-76I6 (ThermoFisher / Invitrogen) as a template, and are shown in Table 7 below. [Table 7]

[0448] To construct a targeted vector (named MAID20455) from the mouse BAC clone RP23-76I6, a hygromycin (Hyg)-resistant autodeletion cassette (containing a CRE recombinase regulated by a protamine promoter) flanked by loxp sites (loxp-hyg-loxp) replaced approximately 69.1 kb of mouse sequence containing the mouse Flt3 gene via bacterial homologous recombination (BHR).

[0449] The final targeting vector contained a 5' to 3':5' mouse homologous arm, a loxp-Hyg-loxp autodeletion cassette, a 3' mouse homologous arm; a chloramphenicol-resistant cassette (CM; not shown in Figure 2); and the final clone was selected based on CM / Hyg resistance.

[0450] The MAID20455 targeted vector was electroporated into mouse embryonic stems (ES) containing hSIRP-alpha (or human SIRPA), RAG2- / -IL2Rg- / - modification. Targeted homologous recombination resulted in a deletion of approximately 69.1 kb of mouse sequence (GRCm38 coordinates chr5:147331171-147400265). Successful integration was confirmed by allelic modification (MOA) assays, e.g., Valenzuela et al. (see above). The primers and probes used for the MOA assay for mouse Flt3 sequence loss are shown in Table 8 below. Subsequently, the cassette was removed by expression of CRE recombinase (regulated by the protamine promoter) in mice. [Table 8]

[0451] Positive target ES cells were used as donor ES cells and microinjected into premorlar (8-cell) stage mouse embryos by the VELOCIMOUSE® method (see, for example, U.S. Patent Nos. 7,576,259, 7,659,442, 7,294,754, and U.S. Patent Application Publication No. 2008-0078000, all of which are incorporated herein by reference). Mouse embryos containing donor ES cells were incubated in vitro and then transplanted into surrogate mothers to produce F0 mice completely derived from the donor ES cells. Mice lacking the Flt3 gene were identified by genotyping using the MOA assay described above. Heterozygous mice for Flt3 gene deletion were mated to homozygous mice. Flt3 KO mice, including Rag2 gene knockout, Il2rg gene knockout, and humanized Sirpa, were mated to homozygous mice for all genes. These mice are used in the remaining examples and relevant portions of this specification, Flt3 - / -These are called mFLT3- / -, Flt3 KO, mFlt3 KO, or mFLT3 KO mice. Subsequently, mice containing humanized Flt3l, Rag2 gene knockout, Il2rg gene knockout, and humanized Sirpa were crossed with mice containing Flt3 KO, Rag2 gene knockout, Il2rg gene knockout, and humanized Sirpa. Mice containing Flt3 KO and also containing Rag2 gene knockout, Il2rg gene knockout, humanized Sirpa, and humanized Flt3l are referred to herein as SRG / hFLT3L / mFLT3 KO (also called "SRG hFLT3L mFLT3 KO" mice, "SRG-hFLT3L / mFLT3 KO" mice, or "hFLT3L / mFLT3 KO" mice). On the other hand, mice containing Rag2 gene knockout, Il2rg gene knockout, or humanized Sirpa are called SRG mice, and SRG mice possessing humanized thrombopoietin are called StRG mice (these mice are used as controls and are expected to have the same level of human dendritic cell engraftment as SRG mice, as described, for example, in U.S. Patent No. 10,463,028, which is incorporated herein by reference).

[0452] Example 1.3: Humanized Flt3l is expressed in genetically modified mice. To verify the expression of humanized Flt3l in genetically modified mice, blood was collected from non-grafted StRG and SRG / hFLT3L / mFLT3 KO mice and added to serum separation tubes to collect serum. Bone marrow aspirates were also collected from non-grafted StRG and SRG / hFLT3L / mFLT3 KO mice. Samples were analyzed for humanized Flt3l using human FLT3L Quantikine ELISA (R&D systems) according to the manufacturer's instructions. Healthy human serum and bone marrow aspirates from normal donors were used as control samples.

[0453] In non-engrafted SRG / hFLT3L / mFLT3 KO mice, the level of humanized Flt3l was approximately 10,000 pg / mL in both serum and bone marrow aspirate, compared to approximately 100 pg / mL in normal healthy human serum and slightly lower in human bone marrow aspirate (Figure 3). The hyperphysiological levels of humanized Flt3l in SRG / hFLT3L / mFLT3 KO mice are likely due to the deletion of mouse FLT3, which results in higher free levels of humanized Flt3l due to the lack of receptors to consume it. As expected, sample StRG mice expressing only mouse Flt3l were negative for human FLT3L. The column shows the mean values ​​for n=4 per sample with standard error.

[0454] Example 2: Development of human dendritic cells in genetically modified mice Example 2.1: Increased human myeloid and plasmacytoid dendritic cells (DCs) in genetically modified engrafted mice SRG / hFLT3L / mFLT3KO and StRG mouse strains were engrafted by intraperitoneal injection of 100,000 hHSCs into 1-5 day old mouse pups. After 10-12 weeks, blood was collected from the mice by cardiac puncture, and the spleen was removed. Single-cell suspensions were prepared by mechanically rupturing the spleen, passing it through a 70 mM mesh filter, and then lysing red blood cells (RBCs) in ACK lysis buffer (Gibco). Blood single-cell suspensions were prepared by RBC lysis in ACK lysis buffer (Gibco). All cells were resuspended in FACS buffer containing mouse / human Fc block (BD Biosciences) and counted. Cells were stained for FACS analysis using the following monoclonal antibodies in PBS + 1 mM EDTA and 2% fetal bovine serum (FACS buffer) at a 1:50 dilution: anti-mouse CD45-APC-Cy7 (clone 30-F11; BD Biosciences), anti-human CD45-PE-Cy5.5 (clone HI30; ThermoFisher), anti-human CD19-PE-Cy7 (clone HIB19; BD Biosciences), anti-human CD3-Pacific Blue (clone S4.1; ThermoFisher), anti-human CD11c-APC (clone Bu15; Biolegend), anti-human HLA-DR-PE (clone Tu36; Biolegend), and anti-human BDCA-3-BV605 (clone M80; Biolegend), and anti-human CD123 FITC (clone 6H6; Biolegend) and anti-human BDCA-2-PE-Dazzle (clone 201A; Biolegend). Cells were acquired using BD FACSymphony A3 and analyzed with FlowJo software. Human DC populations were analyzed from human CD45+ / mouse CD45- / human CD3- / human CD19- populations. Myeloid DCs were then distinguished as human CD11c+ / human HLA-DR+ (Figure 4A). Further analysis was performed to distinguish BDCA-3+ DCs (BDCA-3+ / CD11c+), a subset of these myeloid DCs considered to be the most potent in cross-presentation of exogenous antigens on MHC class I (Figure 4A).Plasmacytoid DCs, the largest producers of type I IFN, were distinguished from the human CD45+ / mouse CD45- / human CD3- / human CD19- / human HLA-DR+ population by co-expression of BDCA-2 and CD123 (Figure 4A).

[0455] Engraftment of hCD45+ was comparable between the two mouse strains, but SRG / hFLT3L / mFLT3 KO mice showed increased myeloid DCs and plasmacytoid DCs in the blood and spleen (Figure 4B). Furthermore, SRG / hFLT3L / mFLT3 KO mice showed increased BDCA-3+ myeloid DCs in the blood and spleen upon hHSC engraftment (Figure 4C). The data represent three HSC donors matched between StRG mice and SRG / hFLT3L / mFLT3 KO mice, with individual data points per mouse and given mean values. Statistical analysis was performed using unpaired Student's t-tests.

[0456] Bone marrow (BM) was prepared by harvesting femoral / tibia from 10-12 week old hHSC-grafted mice. This BM was then crushed, the crushed bone was passed through a 70 mM mesh filter, and RBCs were subsequently lysed with ACK lysis buffer (Gibco). Cells were then FACS stained, and DC analysis was performed as described above. SRG / hFLT3L / mFLT3 KO mice exhibited comparable graft survival to HSC-donor-matched StRG mice, but with increased human myeloid and plasmacytoid DCs in the BM (Figure 4D). Individual data points and means for each mouse are shown. Statistical analysis was performed using unpaired Student's t-tests.

[0457] Thymuses were also harvested from engrafted mice and subsequently mechanically destroyed. Single-cell suspensions were prepared by passing the thymus through a 70 mM mesh filter and lysing the RBCs with ACK lysis buffer (Gibco). Cells were then FACS stained, and DC analysis was performed as described above. SRG / hFLT3L / mFLT3 KO mice exhibited similar engraftment to HSC donor-matched StRG mice, but with increased human myeloid and plasmacytoid DCs in the thymus (Figure 4E). Individual data points and means for each mouse are shown. Statistical analysis was performed using unpaired Student's t-tests.

[0458] The inventors theorized that the development of human DCs in hHSC engraftment is enhanced by the loss of mouse DCs, thus eliminating a potentially competitive cellular niche. For this purpose, spleens derived from engrafted mice were also FACS stained with the following monoclonal antibodies: anti-mouse CD45-APC-Cy7 (clone 30-F11; BD Biosciences), anti-mouse CD11c APC (clone N418; Biolegend), and anti-mouse MHC class II IA. b / d -PE (Clone M5 / 114.15.2; BD Biosciences). Engrafted SRG / hFLT3L / mFLT3 KOs showed a significant loss of mouse DCs (mouse CD45+ / CD11c+ / MHC class II+), indicating that deletion of mouse Flt3 significantly reduces mouse DCs (Figure 4F). Individual data points and means for each mouse are shown. Statistical analysis was performed using unpaired Student's t-tests.

[0459] Example 2.2: Increased human DC generation requires humanization of Flt3l and deletion of mouse FLT3 / FLK2. Mice with a deleted FLT3 have been reported to exhibit increased human dendritic cell (DC) development upon engraftment of human hematopoietic stem cells (hHSCs). However, this increase in human DCs required repeated injections of large amounts of human FLT3L (Li et al. (2016), Eur J Immunol, 46:1291~1299). Our model has receptor deletion concurrent with humanization of Flt3l so that Flt3l isoforms are continuously expressed at physiologically relevant levels. To address whether further humanization is necessary, we have developed hHSC-engrafted SRG mice with mFlt3 KO (FLT3L m / m mFLT3- / -) is used to create SRG mice (FLT3L) containing mFlt3 KO and humanized Flt3l. h / h Analysis was performed against mFLT3- / -). Blood, spleen, and thymus were prepared from engrafted mice for 10-12 weeks, and the human DC population was analyzed as described above. The human mDC and pDC populations were humanized FLT3L / mFLT3 KO(FLT3L h / h In the blood and spleen of hHSC-engrafted mice with mFLT3- / -, mFlt3 KO(FLT3L m / m The number of DCs was increased compared to HSC donor-matched mice possessing only mFLT3- / -) (Figure 5A). Furthermore, the same pattern could be observed in the thymus (Figure 5B). Therefore, we conclude that both mouse Flt3 / FLK2 deletion and humanized Flt3l are necessary for optimal human DC development in this novel human immune system strain. Individual data points and mean values ​​per mouse are shown.

[0460] Example 3: Development of human T cells in genetically modified mice Since DCs are a key regulator of T cell activation, we evaluated the human T cell phenotype in hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice compared to StRG mice. Human T cells in human immune system mouse models exhibit a dysregulated phenotype, as evidenced by higher expression of checkpoint inhibitors such as PD-1. In normal human T cells, approximately 5% or less of T cells are PD-1+, but this is consistently higher in human T cells arising from hHSC engraftment in various previously characterized human immune system mice.

[0461] StRG and SRG / hFLT3L / mFLT3 KO mice were engrafted as described above, and post-orbital hemorrhage was induced 10-12 weeks after engraftment. RBCs were lysed with ACK lysis buffer (Gibco), and the cells were stained with the following monoclonal antibodies as described above: anti-mouse CD45-APC-Cy7 (clone 30-F11; BD Biosciences), anti-human CD45-PE-Cy5.5 (clone HI30; ThermoFisher), anti-human CD19-FITC (clone HIB19; BD Biosciences), anti-human CD3-Pacific Blue (clone S4.1; ThermoFisher), anti-human CD4 BUV496 (clone SK3; BD Biosciences), anti-human CD8-APC (clone SK1; Biolegend), and anti-human PD1-BV605 (clone EH12; BD Biosciences).

[0462] CD3+ T cells in the blood expressed significantly less PD-1 in hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice than in StRG mice (Figure 6A). Furthermore, evaluation of spleen T cells showed a higher proportion of CD4+ T cells than CD8+ T cells in hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice (Figure 6A). This would indicate a normalization of the T cell subset, as the normal human distribution of CD4 / CD8 T cells is 60 / 40, as observed in SRG / hFLT3L / mFLT3 KO mice (Figure 6A), while the ratio is more 50 / 50 in StRG mice. Individual data points and means for each mouse are shown. All analyses were performed using hHSC donor-matched StRG and SRG / hFLT3L / mFLT3 KO mice, and statistical analysis was performed using unpaired Student's t-tests.

[0463] To evaluate the activation status of human T cells in different human immune system mouse strains, spleen cells from hHSC-engrafted StRG mice and SRG / hFLT3L / mFLT3 KO mice were FACS stained with the following monoclonal antibodies: anti-mouse CD45-APC-Cy7 (clone 30-F11; BD Biosciences), anti-human CD45-PE-Cy5.5 (clone HI30; ThermoFisher), anti-human CD3-Pacific Blue (clone S4.1; ThermoFisher), anti-human CD4-BUV496 (clone SK3; BD Biosciences), anti-human CD8-APC (clone SK1; Biolegend), anti-human CD44-PE (clone C44Mab-5; Biolegend), and anti-human CD62L-FITC (clone SK11; BD Biosciences). The following combinations of CD44 and CD62L expression result in different T cell subsets: CD44- / CD62L+ (naive T cells), CD44+ / CD62L- (effector cells), and CD44+ / CD62L+ (central memory T cells; Tcm). Generally, T cells in human immune system mouse models are considered to be underresponsive to antigen stimulation, and the memory response is thought to be very suboptimal. However, more CD4+ and CD8+ T cells in hHSC engrafted SRG / hFLT3L / mFLT3 KO were Tcm than in HSC donor-matched StRG (Figure 6B). Individual data points and mean values ​​per mouse are shown.

[0464] Example 4: Characterization of dendritic cell populations in genetically modified mice To compare human dendritic cells (DCs) from hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice with DCs from healthy human donors, bulk human DCs were isolated from the spleen / blood of engrafted mice and blood from healthy human donors using the EasySep Human Pan-DC Pre-Enrichment Kit according to the manufacturer's instructions (StemCell Technologies). Additionally, bulk human DCs were enriched from hHSC-engrafted SRG / hFLT3L / mFLT3 KO BM and healthy human BM. Although the number of isolated cells was too small for analytical reliability, DCs isolated from hHSC-engrafted StRG were also used as a control. Three different donors—HSC donors for human blood, BM, and engraftment—were used for evaluation.

[0465] Isolated DCs were resuspended in PBS containing 0.04% BSA (approximately 6000 cells) and loaded into a Chromium Single Cell Analyzer (10X Genomics). RNA-seq was prepared using the Chromium Single Cell v1.0 5' Library, Gel Beads & Multiplex Kit (10X Genomics). For RNA-seq libraries, the Cell Ranger Single-Cell Software Suite (10X Genomics, v2.2.0) was used for sample demultiplexing, alignment, filtering, and UMI counting. Mouse mm10 genome assembly and mouse RefSeq gene models were used for alignment. Analysis was then performed to cluster the DC population based on transcriptional profiles (data not shown).

[0466] Significant transcriptional differences were observed between the dendritic cell (DC) population in the spleen / blood of hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice and healthy human blood. hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice had a lower proportion of Clec10a myeloid DCs compared to healthy human donors (Figure 7A). Furthermore, while the transcriptional profile of plasmacytoid DCs in healthy human donors is indicated by IRF4 expression, this transcriptional signature was reduced in plasmacytoid DCs derived from hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice (Figure 7A). Since IRF4 is a key regulator of type I IFN production by plasmacytoid DCs, further work was performed to evaluate type I IFN production by plasmacytoid DCs derived from hHSC-engrafted mice (Figures 9A and 9B). Finally, hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice had a higher proportion of XCR1+ / Clec9a+ DCs in the spleen / blood compared to healthy human DCs (Figure 7A). Notably, this population was a BDCA-3+ DC subset, and FACS analysis of blood from hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice showed that this population was indeed overrepresented compared to DCs derived from human peripheral blood mononuclear cells (PBMCs) (Figure 7B). Furthermore, hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice had a higher proportion of human myeloid and plasmacytoid DCs than human PBMCs (Figure 7B). BDCA-3+ / Clec9a+ / XCR1+ myeloid DCs have been characterized as an important DC subset for cross-priming and activation of cytotoxic CD8+ T-cell lymphocytes (CTLs) against tumor cells and viral pathogens, as well as for establishing peripheral immune tolerance (Crozat & Dalod (2011) Med Sci (Paris) 27:25-28; Cui et al. (2021) Am J Physiol Lung Cell Mol Physiol 320:L193-L204; Schreibelt et al. (2012) Blood 119:2284-2292; van der Aa, E., van Montfoort, N. & Woltman, AM (2015) Semin Cell Dev Biol 41:39-48).This subset of DCs excels in cross-presentation for antigen retargeting from early endocytotic vesicles presented on the MHC-I pathway to late endosomes / lysosomes and subsequent cytosol (Cohn et al. (2013) J.Exp.Med.210(5):1049-63 and Savina et al. (2009) Immunity 30(4):544-55). Indeed, this DC subset is an attractive target for immunotherapy that appears to stimulate T cell responses against tumors and pathogens (Crozat & Dalod (2011) Med Sci (Paris) 27:25-28; Schreibelt et al. (2012) Blood 119:2284-2292; van der Aa, E., van Montfoort, N. & Woltman, AM (2015) Semin Cell Dev Biol 41:39-48; Masterman et al. (2020) J Immunother Cancer 8; Pearson et al. (2020) Clin Transl Immunology 9:e1141; Tullett et al. (2016) JCI Insight 1:e87102). Therefore, our SRG / hFLT3L / mFLT3 KO human immune system strain is an attractive model for testing potential immunotherapies aimed at cross-priming due to the enhancement of peptide presentation by this DC subset.

[0467] Plasma cell-like DCs (pDCs) are important type I IFN-producing cells in viral infections and are also involved in autoimmune diseases such as lupus (Furie et al. (2022), N Engl J Med, 387:894~904; Laurent et al. (2022), Sci Immunol, 7:eadd4906; Li et al. (2020), Front Pharmacol, 11:8; Palucka (2005) Proc Natl Acad Sci USA 102:3372-3377; Sakata et al. (2018) Front Immunol 9:1957; Sprow et al. (2022) Front Med (Lausanne) 9:968323; Takagi et al. (2016) Sci Rep 6:24477; Werth et al. (2022), N Engl J Med, 387:321~331; Zhang et al. (2017) Proc Natl Acad Sci USA 114:1988-1993). To test whether human pDCs are present in various organs of SRG / hFLT3L / mFLT3 KO human immune system mice, spleen, kidney, lung, blood, and bone marrow were isolated from SRG / hFLT3L / mFLT3 KO human immune system mice and processed for single-cell suspension. Human pDCs were evaluated by flow cytometry based on the following gating: lymphocytes, singlet, live, mouse CD45-, human CD45+, human CD3-, human CD16-, human CD19-, human CD123+, human HLA-DR+, human ILT7+, human BDCA2+ (Figure 8). The data shown are plotted as pDCs as the percentage of overall viable human CD45+ cells, and individual mice are graphed. This data demonstrates that human pDCs can be identified in the blood, lymphoid organs, and other tissue sites of SRG / hFLT3L / mFLT3 KO human immune system mice.

[0468] To test whether human pDCs retain functionality in these mice, human pDCs were isolated from SRG / hFLT3L / mFLT3 KO human immune system mice and stimulated in vitro to determine if they could produce type I interferon (IFN) in response to stimulation. Single-cell suspensions were obtained from the spleen and bone marrow by tissue dissociation. Mouse cells were removed by staining for APC-bound mouse CD45 and mouse Ter119, followed by magnetic separation. Human pDCs were then isolated using the EasySep Human pDC Concentration Kit (Stem Cell). pDCs were seeded at the indicated cell counts and ODN2216 (Invivogen), a class A stimulated CpG ODN, was added at the indicated concentrations. Cells were incubated with stimulation for 18 hours. The supernatant was collected and evaluated by ELISA for human IFNα or IFNβ, respectively. As shown in Figure 9A, human pDCs isolated from SRG / hFLT3L / mFLT3 KO human immune system mice produce type I IFN in response to TLR9 stimulation. Furthermore, it was determined that human pDCs from these mice produce human type I IFN in response to ODN2216 CpG stimulation in vivo, as measured by induction of type I IFN signature genes (Figure 9B). Specifically, mice were intravenously treated with 5 ug of ODN2216 delivered via the InVivoJet delivery system (PolyPlus). Blood was collected 6 hours later, processed, and RNA was isolated. qPCR was performed to evaluate and analyze the gene expression of the indicated genes using Standard 2. ΔΔct Based on the analysis, relative quantitative values ​​were determined.

[0469] Our data demonstrate that human plasmacytoid dendritic cells (DCs) in hHSC-engrafted SRG / hFLT3L / mFLT3 KO mice can induce a type I IFN response. Therefore, our SRG / hFLT3L / mFLT3 KO human immune system mice are an excellent model for testing modulators of plasmacytoid DC function.

[0470] Embedding by reference All publications, patents, and patent applications referenced herein are incorporated herein by whole, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. In case of any conflict, the present application, including any definitions herein, shall prevail.

[0471] Furthermore, the entire sequence is incorporated by reference to any polynucleotide and polypeptide sequence that references accession numbers corresponding to entries in public databases, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web at tigr.org and / or those maintained by the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov. Those skilled in the art will understand that the sequence databases disclosed above or sequence databases known in the art are periodically updated to publish corrected sequences. Such corrected sequences are incorporated in their entirety by reference.

[0472] Equal parts Those skilled in the art will recognize, or can confirm by routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

Claims

1. (i) Homozygous null mutations in the Rag2 gene; (ii) Homozygous null mutations in the IL2rg gene; (iii) Homozygous null mutations in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) Flt3 ligand (Flt3l) gene containing rodent and human portions operably linked to the Flt3l promoter Genetically modified rodents, including [specific species / species].

2. The genetically modified rodent according to claim 1, comprising a homozygous null mutation in the Rag1 gene.

3. The genetically modified rodent according to claim 1 or 2, wherein the null mutation of the rodent Flt3 gene comprises an insertion, deletion, and / or substitution in the endogenous Flt3 gene.

4. The genetically modified rodent according to claim 3, wherein the null mutation in the rodent Flt3 gene is a complete deletion of the endogenous Flt3 coding sequence.

5. The genetically modified rodent according to claim 4, wherein the genetically modified rodent is a mouse, and the mouse comprises a homozygous deletion of a nucleic acid sequence between coordinates chr5:147331171–147400265 (GRCm38 assembly).

6. The genetically modified rodent according to any one of claims 1 to 5, wherein the rodent portion of the Flt3l gene includes the non-coding portions of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene.

7. The genetically modified rodent according to claim 6, wherein the non-coding portions of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene are at least 90%, at least 95%, or 100% identical to the corresponding non-coding portions of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene shown in Table 1A.

8. The genetically modified rodent according to any one of claims 1 to 7, wherein the human portion of the Flt3l gene comprises the signal peptide coding portion of exon 2 and exons 3 to 6 of the human FLT3L gene.

9. The genetically modified rodent according to claim 8, wherein the signal peptide coding portion of exon 2 of the human FLT3L gene and exons 3-6 are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B.

10. The genetically modified rodent according to any one of claims 1 to 9, wherein the Flt3l gene comprises rodent exon 1, the rodent non-coding portion of exon 2, the human signal peptide coding portion of exon 2, human exons 3 to 6, and rodent exons 7 to 9.

11. The genetically modified rodent according to any one of claims 1 to 10, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L comprising the signal peptide and cytokine-like core domain of a human FLT3L polypeptide and the C-terminal portion of a rodent Flt3l polypeptide.

12. The genetically modified rodent according to claim 11, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises a rodent stalk region, a rodent transmembrane domain, and a rodent cytoplasmic tail as shown in Figure 1E.

13. The genetically modified rodent according to claim 11 or 12, wherein the C-terminal portion of the rodent Flt3l polypeptide has an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

14. The genetically modified rodent according to any one of claims 1 to 13, wherein the rodent expresses both the soluble form and the membrane-bound form of the Flt3l polypeptide.

15. The genetically modified rodent according to claim 14, wherein the soluble form and membrane-bound form of the Flt3L polypeptide include a signal peptide and a cytokine-like core domain of human FLT3L polypeptide.

16. The genetically modified rodent according to any one of claims 11 to 15, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1 to 26 of the human FLT3L polypeptide.

17. The genetically modified rodent according to any one of claims 11 to 16, wherein the signal peptide of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the signal peptide of the human FLT3L polypeptide shown in Table 2.

18. The genetically modified rodent according to any one of claims 11 to 17, wherein the cytokine-like core domain of the human FLT3L polypeptide contains amino acids corresponding to residues 27 to 159 of the human FLT3L polypeptide.

19. The genetically modified rodent according to any one of claims 11 to 18, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

20. The genetically modified rodent according to any one of claims 1 to 19, wherein the genetically modified rodent is heterozygous for the Flt3 ligand (Flt3l) gene, which includes a rodent portion and a human portion.

21. The genetically modified rodent according to any one of claims 1 to 19, wherein the genetically modified rodent is homozygous for the Flt3 ligand (Flt3l) gene, which includes a rodent portion and a human portion.

22. The genetically modified rodent according to any one of claims 1 to 21, wherein the rodent portion of the Flt3l gene is the endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is an endogenous rodent gene.

23. The genetically modified rodent according to any one of claims 1 to 22, wherein the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

24. The genetically modified rodent according to any one of claims 1 to 23, wherein the Flt3l promoter is a rodent promoter.

25. The genetically modified rodent according to claim 23 or 24, wherein the Flt3l promoter is an endogenous rodent promoter.

26. The genetically modified rodent according to any one of claims 23 to 25, wherein the Flt3l promoter is located at an endogenous rodent locus.

27. The genetically modified rodent according to any one of claims 1 to 26, wherein the genetically modified rodent expresses a human or humanized SIRPA polypeptide encoded by a nucleic acid operably linked to a SIRPA promoter.

28. The genetically modified rodent according to claim 27, further comprising a Sirpa gene encoding a Sirpa polypeptide comprising the extracellular portion of human Sirpa polypeptide and the intracellular portion of rodent Sirpa polypeptide, wherein the Sirpa gene is operably linked to a Sirpa promoter.

29. The genetically modified rodent according to claim 28, wherein the Sirpa gene comprises exons 1, 5, 6, 7, and 8 of the rodent Sirpa gene and exons 2 to 4 of the human SIRPA gene.

30. The genetically modified rodent according to claim 28 or 29, wherein the genetically modified rodent expresses a Sirpa polypeptide comprising the extracellular portion of human Sirpa polypeptide and the intracellular portion of rodent Sirpa polypeptide.

31. The genetically modified rodent according to any one of claims 28 to 30, wherein the rodent Sirpa polypeptide is an endogenous rodent Sirpa polypeptide, and / or the rodent Sirpa gene is an endogenous rodent gene.

32. The genetically modified rodent according to claim 27, wherein the genetically modified rodent expresses a human SIRPA polypeptide encoded by a nucleic acid operably linked to a SIRPA promoter.

33. The genetically modified rodent according to any one of claims 27 to 32, wherein the genetically modified rodent further expresses a human GM-CSF protein encoded by a nucleic acid operably linked to a GM-CSF promoter and / or a human IL-3 protein encoded by a nucleic acid operably linked to an IL-3 promoter.

34. The genetically modified rodent according to any one of claims 27 to 33, wherein the SIRPα promoter, the GM-CSF promoter, and / or the IL3 promoter are rodent promoters.

35. The genetically modified rodent according to claim 34, wherein the SIRPα promoter, the GM-CSF promoter, and / or the IL3 promoter are endogenous rodent promoters.

36. The genetically modified rodent according to claim 34 or 35, wherein the SIRPα promoter, the GM-CSF promoter, and / or the IL3 promoter are located at the corresponding endogenous rodent loci.

37. A genetically modified rodent according to any one of claims 27 to 36, comprising a null mutation in at least one corresponding rodent gene at the corresponding rodent locus.

38. The genetically modified rodent according to any one of claims 27 to 37, wherein the genetically modified rodent is heterozygous for at least one allele containing the nucleic acid sequence encoding the human or humanized protein.

39. The genetically modified rodent according to any one of claims 27 to 38, wherein the genetically modified rodent is homozygous for at least one allele containing the nucleic acid sequence encoding the human or humanized protein.

40. A genetically modified rodent according to any one of claims 1 to 39, further comprising the engraftment of human hematopoietic cells.

41. The genetically modified rodent according to claim 40, wherein the human hematopoietic cells comprise one or more cells selected from the group consisting of human CD34-positive cells, human hematopoietic stem cells, human hematopoietic progenitor cells, human dendritic cell progenitor cells, and human dendritic cells.

42. The genetically modified rodent according to claim 40 or 41, wherein the genetically modified rodent includes human dendritic cells.

43. A genetically modified rodent according to any one of claims 1 to 42, wherein an autoimmune disease is induced or established in the genetically modified rodent.

44. The genetically modified rodent according to claim 43, wherein the autoimmune disease is systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, polymyositis, collagen-induced arthritis, or dermatomyositis.

45. The genetically modified rodent according to any one of claims 1 to 44, wherein the rodent is a mouse or a rat.

46. The genetically modified rodent according to claim 45, wherein the rodent is a mouse.

47. A method for identifying drugs that modulate the function of human dendritic cells, a. Administering a candidate drug to a genetically modified rodent according to any one of claims 40 to 46; and b. To determine whether the candidate drug modulates the function of the human dendritic cells in the rodents. Methods that include...

48. The method according to claim 47, wherein the human dendritic cells are myeloid dendritic cells (mDCs) or plasmacytoid dendritic cells (pDCs).

49. The method according to claim 47 or 48, wherein the function of the human dendritic cell is selected from the group consisting of phagocytosis, cytokine production, cross-presentation of exogenous antigens, and activation of cytotoxic CD8+ T cell lymphocytes (CTLs).

50. A method for evaluating the therapeutic efficacy of a drug for stimulating a T cell response against target cells, wherein the method is: a. Administering a candidate drug to a genetically modified rodent according to any one of claims 40 to 42, wherein the genetically modified rodent includes the target cells; and b. Evaluating the therapeutic efficacy of the drug candidate by measuring the T cell response to the target cells in the rodents. Methods that include...

51. The method according to claim 50, wherein the target cells are selected from the group consisting of tumor cells, virus-infected cells, bacterial-infected cells, bacterial cells, fungal cells, and parasitic cells.

52. A method for evaluating the therapeutic effectiveness of drugs in the treatment of autoimmune diseases, wherein the method is a. Administering the candidate drug to the genetically modified rodent described in claim 43 or 44; and b. To determine whether the drug treats the autoimmune disease in the rodents. Methods that include...

53. (i) Homozygous null mutations in the Rag2 gene; (ii) Homozygous null mutations in the IL2rg gene; (iii) Homozygous null mutations in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) Flt3 ligand (Flt3l) gene containing rodent and human portions operably linked to the Flt3l promoter Genetically modified rodent cells, including [specific cells / organizations].

54. Genetically modified rodent cells according to claim 53, comprising a homozygous null mutation in the Rag1 gene.

55. The genetically modified rodent cell according to claim 53 or 54, wherein the null mutation of the rodent Flt3 gene comprises an insertion, deletion, and / or substitution in the endogenous Flt3 gene.

56. The genetically modified rodent cell according to claim 55, wherein the null mutation in the rodent Flt3 gene is a complete deletion of the endogenous Flt3 coding sequence.

57. The genetically modified rodent cell according to claim 56, wherein the genetically modified rodent cell is a mouse cell, and the mouse cell contains a homozygous deletion of a nucleic acid sequence between coordinates chr5:147331171–147400265 (GRCm38 assembly).

58. The genetically modified rodent cell according to any one of claims 53 to 57, wherein the rodent portion of the Flt3l gene includes the non-coding portions of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene.

59. The genetically modified rodent cell according to claim 58, wherein the non-coding portions of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene are at least 90%, at least 95%, or 100% identical to the corresponding non-coding portions of exon 1 and exon 2, and the exon downstream of exon 6 of the rodent Flt3l gene shown in Table 1A.

60. The genetically modified rodent cell according to any one of claims 53 to 59, wherein the human portion of the Flt3l gene comprises the signal peptide coding portion of exon 2 and exons 3 to 6 of the human FLT3L gene.

61. The genetically modified rodent cell according to claim 60, wherein the signal peptide coding portion of exon 2 of the human FLT3L gene and exons 3-6 are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding signal peptide coding portion of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B.

62. A genetically modified rodent cell according to any one of claims 53 to 61, wherein the Flt3l gene comprises rodent exon 1, the rodent non-coding portion of exon 2, the human signal peptide coding portion of exon 2, human exons 3 to 6, and rodent exons 7 to 9.

63. The genetically modified rodent cell according to any one of claims 53 to 62, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L comprising the signal peptide and cytokine-like core domain of a human FLT3L polypeptide and the C-terminal portion of a rodent Flt3l polypeptide.

64. The genetically modified rodent cell according to claim 63, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises a rodent stalk region, a rodent transmembrane domain, and a rodent cytoplasmic tail as shown in Figure 1E.

65. The genetically modified rodent cell according to claim 63 or 64, wherein the C-terminal portion of the rodent Flt3l polypeptide has an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

66. The genetically modified rodent cell according to any one of claims 53 to 65, wherein the rodent cell expresses both the soluble and membrane-bound forms of the Flt3l polypeptide.

67. The genetically modified rodent cell according to claim 66, wherein the soluble and membrane-bound forms of the Flt3L polypeptide comprise a signal peptide and a cytokine-like core domain of human FLT3L polypeptide.

68. The genetically modified rodent cell according to any one of claims 63 to 67, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1 to 26 of the human FLT3L polypeptide.

69. The genetically modified rodent cell according to any one of claims 63 to 68, wherein the signal peptide of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the signal peptide of the human FLT3L polypeptide shown in Table 2.

70. The genetically modified rodent cell according to any one of claims 63 to 69, wherein the cytokine-like core domain of the human FLT3L polypeptide contains amino acids corresponding to residues 27 to 159 of the human FLT3L polypeptide.

71. The genetically modified rodent cell according to any one of claims 63 to 70, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

72. The genetically modified rodent cell according to any one of claims 53 to 71, wherein the genetically modified rodent cell is heterozygous for the Flt3 ligand (Flt3l) gene, which includes a rodent portion and a human portion.

73. The genetically modified rodent cell according to any one of claims 53 to 71, wherein the genetically modified rodent cell is homozygous for the Flt3 ligand (Flt3l) gene, which includes a rodent portion and a human portion.

74. A genetically modified rodent cell according to any one of claims 53 to 73, wherein the rodent portion of the Flt3l gene is the endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is an endogenous rodent gene.

75. The genetically modified rodent cell according to any one of claims 53 to 74, wherein the rodent Flt3l polypeptide is endogenous rodent Flt3l polypeptide.

76. The genetically modified rodent cell according to any one of claims 53 to 75, wherein the Flt3l promoter is a rodent promoter.

77. The genetically modified rodent cell according to claim 75 or 76, wherein the Flt3l promoter is an endogenous rodent promoter.

78. The genetically modified rodent cell according to any one of claims 75 to 77, wherein the Flt3l promoter is located at an endogenous rodent gene locus.

79. The genetically modified rodent cell according to any one of claims 53 to 78, further comprising a nucleic acid encoding a human or humanized SIRPA polypeptide, wherein the nucleic acid is operably linked to a Sirpa promoter.

80. The genetically modified rodent cell according to claim 79, wherein the genetically modified rodent cell further comprises a Sirpa gene encoding a Sirpa polypeptide that includes an extracellular portion of human Sirpa polypeptide and an intracellular portion of rodent Sirpa polypeptide, and the Sirpa gene is operably linked to a Sirpa promoter.

81. The genetically modified rodent cell according to claim 80, wherein the Sirpa gene comprises exons 1, 5, 6, 7, and 8 of the rodent Sirpa gene and exons 2 to 4 of the human SIRPA gene.

82. The genetically modified rodent cell according to claim 80 or 81, wherein the genetically modified rodent cell expresses a Sirpa polypeptide comprising the extracellular portion of human Sirpa polypeptide and the intracellular portion of rodent Sirpa polypeptide.

83. A genetically modified rodent cell according to any one of claims 80 to 82, wherein the rodent Sirpa polypeptide is an endogenous rodent Sirpa polypeptide, and / or the rodent Sirpa gene is an endogenous rodent gene.

84. The genetically modified rodent cell according to claim 79, wherein the genetically modified rodent cell expresses human SIRPA polypeptide.

85. The genetically modified rodent cell according to any one of claims 79 to 84, further comprising (1) a nucleic acid encoding a human GM-CSF protein operably linked to a GM-CSF promoter; and / or (2) a nucleic acid encoding a human IL3 protein operably linked to an IL3 promoter.

86. A genetically modified rodent cell according to any one of claims 79 to 85, wherein the SIRPα promoter, the GM-CSF promoter, and / or the IL3 promoter are rodent promoters.

87. The genetically modified rodent cell according to claim 86, wherein the SIRPα promoter, the GM-CSF promoter, and / or the IL3 promoter are endogenous rodent promoters.

88. The genetically modified rodent cell according to claim 86 or 87, wherein the SIRPα promoter, the GM-CSF promoter, and / or the IL3 promoter are located at the corresponding endogenous rodent loci.

89. A genetically modified rodent cell according to any one of claims 79 to 88, comprising a null mutation in at least one corresponding rodent gene at the corresponding rodent locus.

90. The genetically modified rodent cell according to any one of claims 79 to 89, wherein the genetically modified rodent cell is heterozygous for at least one allele containing the nucleic acid sequence encoding the human or humanized protein.

91. The genetically modified rodent cell according to any one of claims 79 to 90, wherein the genetically modified rodent cell is homozygous for at least one allele containing the nucleic acid sequence encoding the human or humanized protein.

92. The genetically modified rodent cell according to any one of claims 53 to 91, wherein the rodent cell is a rat cell or a mouse cell.

93. The genetically modified rodent cell according to claim 92, wherein the rodent cell is a mouse cell.

94. The genetically modified rodent cell according to any one of claims 53 to 65, 68 to 81, 83, and 85 to 93, wherein the genetically modified rodent cell is a rodent embryonic stem (ES) cell.

95. A method for producing rodent embryonic stem cells, wherein the rodent embryonic stem cells are (i) Homozygous null mutations in the Rag2 gene; (ii) Homozygous null mutations in the IL2rg gene; (iii) Homozygous null mutations in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) Flt3 ligand (Flt3l) gene containing rodent and human portions operably linked to the Flt3l promoter A method comprising genetically manipulating the rodent embryonic stem cells to have a genome containing the following.

96. A rodent embryo comprising rodent embryonic stem cells according to claim 94, or rodent embryonic stem cells prepared according to the method described in claim 95.

97. A method for producing a rodent comprising in its genome (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) a Flt3 ligand (Flt3l) gene containing a rodent portion and a human portion operably linked to the Flt3l promoter, wherein the method is (a) the step of obtaining rodent embryonic stem cells according to claim 94, or rodent embryonic stem cells prepared according to the method of claim 95; and (b) A step to create rodents using the rodent embryonic cells from (a). Methods that include...

98. A method for producing a rodent whose genome includes (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) a Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operably linked to the Flt3l promoter, the method comprising modifying the genome of the rodent so that it includes (i) a homozygous null mutation in the Rag2 gene; (ii) a homozygous null mutation in the IL2rg gene; (iii) a homozygous null mutation in the rodent FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) a Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operably linked to the Flt3l promoter.

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