Vector, genetically modified cell and genetically modified non-human animal comprising same

Abstract

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

Description

[0001] Related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 521,475, filed June 16, 2023, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application contains a sequence list submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy was created on June 14, 2024, named RPD-00225.xml, and is 43,680 bytes in size. Background Technology

[0005] Genetically modified cells, genetically modified mice containing them, and modified and implanted mice, and their use in mimicking human diseases (e.g., for drug testing), are known in the art. The use of genetically modified mice to mimic 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 human hematopoietic progenitor cells into severely immunodeficient mouse strains (such as Rag2 KO Il2rg KO mice) have been reported. Although multilineage hematopoietic development has been observed in these HIS mice, there is still a need for genetically modified mice capable of supporting human dendritic cell development, and for mice suitable for implantation that can mimic or approximate certain aspects of human dendritic cells. Summary of the Invention

[0006] This disclosure is partly based on the finding that knocking out the Flt3 gene and humanizing Flt3l in immunocompromised mice (e.g., mice with Rag1 and / or Rag2 gene knockout and IL2rg gene knockout) resulted in an increase in human DCs (e.g., myeloid DCs, plasmacytoid DCs, BDCA-3+ DCs) in the spleen, blood, and bone marrow after human hematopoietic cell engraftment.

[0007] Therefore, in one aspect, this 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) a Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operatively linked to the Flt3l promoter.

[0008] 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's Flt3 gene includes insertions, deletions, and / or substitutions in the endogenous Flt3 gene. In some embodiments, the null mutation in the rodent's 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).

[0009] In some embodiments, the rodent portion of the Flt3l gene comprises the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene, and the exon downstream of exon 6. In some embodiments, the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene, and the exon downstream of exon 6, 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. In some embodiments, 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. 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 portions of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B. In some embodiments, the Flt3l gene comprises rodent exon 1, the non-coding portion of rodent exon 2, the signal peptide coding portion of human exon 2, human exons 3-6, and rodent exons 7-9.

[0010] In some embodiments, the Flt3l gene encodes a chimeric membrane-bound Flt3l, which comprises a signal peptide and a cytokine-like core domain of the human FLT3L polypeptide, as well as a C-terminal portion of the rodent Flt3l polypeptide. In some embodiments, the C-terminal portion of the rodent Flt3l polypeptide comprises a rodent stem 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 a soluble form of the Flt3l polypeptide and a membrane-bound form of the Flt3l polypeptide. In some embodiments, both the soluble and membrane-bound forms of the Flt3l polypeptide comprise the signal peptide and the cytokine-like core domain of the human FLT3L polypeptide. In some embodiments, the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1-26 of the human FLT3L polypeptide. In some embodiments, the signal peptide of the human FLT3L polypeptide comprises at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical amino acid sequences to the corresponding amino acid sequences of the signal peptides of the human FLT3L polypeptides shown in Table 2. In some embodiments, the cytokine-like core domain of the human FLT3L polypeptide comprises 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 comprises at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical amino acid sequences to the corresponding amino acid sequences of the cytokine-like core domains of the human FLT3L polypeptides shown in Table 2.

[0011] In some embodiments, the genetically modified rodent is heterozygous for the Flt3 ligand (Flt3l) gene, which includes both rodent and human portions. In some embodiments, the genetically modified rodent is homozygous for the Flt3 ligand (Flt3l) gene, which includes both rodent and human portions.

[0012] In some embodiments, the rodent portion of the Flt3l gene is the endogenous rodent portion of the Flt3l gene; the rodent Flt3l gene is an endogenous rodent gene; and / or the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0013] In some embodiments, the rodent and human portions are operatively 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 an endogenous rodent locus.

[0014] In some embodiments, the genetically modified rodent expresses a human SIRPA polypeptide or a humanized SIRPA polypeptide encoded by a nucleic acid operatively linked to a Sirpa promoter. In some embodiments, the genetically modified rodent further comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a rodent Sirpa polypeptide, wherein the Sirpa gene is operatively linked to a Sirpa promoter. In some embodiments, the Sirpa gene comprises exons 1, 5, 6, 7, and 8 of a rodent Sirpa gene and exons 2-4 of a human SIRPA gene. In some embodiments, the genetically modified rodent further expresses a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an 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, the genetically modified rodent also expresses a human SIRPA polypeptide encoded by a nucleic acid operatively linked to a Sirpa promoter.

[0015] In some embodiments, the genetically modified rodents also 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.

[0016] In some embodiments, the Sirpa promoter, the GM-CSF promoter, and / or the IL3 promoter are rodent promoters. In some embodiments, the Sirpa promoter, the GM-CSF promoter, and / or the IL3 promoter are endogenous rodent promoters. In some embodiments, the Sirpa promoter, the GM-CSF promoter, and / or the IL3 promoter are located at the corresponding endogenous rodent locus.

[0017] 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.

[0018] In some embodiments, the genetically modified rodent is heterozygous for at least one allele, the allele containing a nucleic acid sequence encoding a human protein or a humanized protein. In some embodiments, the genetically modified rodent is homozygous for at least one allele, the allele containing a nucleic acid sequence encoding the human protein or a humanized protein.

[0019] In some embodiments, the genetically modified rodent further comprises the implantation 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 precursor cells, and human dendritic cells. In some embodiments, the genetically modified rodent comprises human dendritic cells.

[0020] In some embodiments, an autoimmune disease is induced or established in the genetically modified rodent. In some embodiments, the autoimmune disease is systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, polymyositis, collagen-induced arthritis, or dermatomyositis.

[0021] In some embodiments, the rodent is a mouse or a rat. In some embodiments, the rodent is a mouse.

[0022] In some aspects, this document provides a method for identifying agents that regulate the function of human dendritic cells, comprising: a. administering a candidate agent to a genetically modified rodent as described herein; and b. determining whether the candidate agent regulates the function of the 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 the human dendritic cells is selected from the group consisting of phagocytosis, cytokine production, cross-presentation of exogenous antigens, and activation of cytotoxic CD8+ T lymphocytes (CTLs).

[0023] In some aspects, this document provides a method for evaluating the therapeutic efficacy of a drug-stimulated T-cell response to a target cell, the method comprising: a. administering a drug candidate to a genetically modified rodent as described herein, wherein the genetically modified rodent contains the target cells; and b. measuring the T-cell response in the rodent to the target cells 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.

[0024] In some respects, this article provides a method for evaluating the therapeutic efficacy of a drug for 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 the autoimmune disease in the rodent.

[0025] In another aspect, this disclosure relates to a genetically modified rodent cell 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) a Flt3 ligand (Flt3l) gene comprising rodent and human portions operatively linked to the Flt3l promoter.

[0026] In some embodiments, the genetically modified rodent cells contain a homozygous null mutation in the Rag1 gene. In some embodiments, the null mutation in the rodent Flt3 gene includes insertions, deletions, and / or substitutions 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 cells are mouse cells, and the mouse cells contain a homozygous deletion of a nucleic acid sequence between coordinates chr5: 147331171-147400265 (GRCm38 assembly).

[0027] In some embodiments, the rodent portion of the Flt3l gene comprises the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene, and the exon downstream of exon 6. In some embodiments, the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene, and the exon downstream of exon 6, 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. In some embodiments, 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. 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 portions of exon 2 and exons 3-6 of the human FLT3L gene shown in Table 1B. In some embodiments, the Flt3l gene comprises rodent exon 1, the non-coding portion of rodent exon 2, the signal peptide coding portion of human exon 2, human exons 3-6, and rodent exons 7-9.

[0028] In some embodiments, the Flt3l gene encodes a chimeric membrane-bound Flt3l, which comprises a signal peptide and a cytokine-like core domain of the human FLT3L polypeptide, and a C-terminal portion of the rodent Flt3l polypeptide. In some embodiments, the C-terminal portion of the rodent Flt3l polypeptide comprises a rodent stem 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 a soluble form of the Flt3l polypeptide and a membrane-bound form of the Flt3l polypeptide. In some embodiments, both the soluble and membrane-bound forms of the Flt3l polypeptide comprise the signal peptide and the cytokine-like core domain of the human FLT3L polypeptide. In some embodiments, the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1-26 of the human FLT3L polypeptide. In some embodiments, the signal peptide of the human FLT3L polypeptide comprises at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical amino acid sequences to the corresponding amino acid sequences of the signal peptides of the human FLT3L polypeptides shown in Table 2. In some embodiments, the cytokine-like core domain of the human FLT3L polypeptide comprises 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 comprises at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical amino acid sequences to the corresponding amino acid sequences of the cytokine-like core domains of the human FLT3L polypeptides shown in Table 2.

[0029] In some embodiments, the genetically modified rodent cells are heterozygous for the Flt3 ligand (Flt3l) gene, which includes both rodent and human portions. In some embodiments, the genetically modified rodent cells are homozygous for the Flt3 ligand (Flt3l) gene, which includes both rodent and human portions.

[0030] In some embodiments, the rodent portion of the Flt3l gene is the endogenous rodent portion of the Flt3l gene; the rodent Flt3l gene is an endogenous rodent gene; and / or the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0031] In some embodiments, the rodent and human portions are operatively 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 an endogenous rodent locus.

[0032] In some embodiments, the genetically modified rodent cells further comprise nucleic acids encoding a human SIRPA polypeptide or a humanized SIRPA polypeptide, wherein the nucleic acid is operatively linked to a Sirpa promoter. In some embodiments, the genetically modified rodent cells express a human SIRPA polypeptide or a humanized SIRPA polypeptide. In some embodiments, the genetically modified rodent cells further comprise a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a rodent Sirpa polypeptide, wherein the Sirpa gene is operatively linked to a Sirpa promoter. In some embodiments, the Sirpa gene comprises exons 1, 5, 6, 7, and 8 of a rodent Sirpa gene and exons 2-4 of a human SIRPA gene. In some embodiments, the genetically modified rodent cells express a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an 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, the genetically modified rodent cells express the human SIRPA polypeptide.

[0033] In some embodiments, the genetically modified rodent cells further comprise: (1) a nucleic acid encoding human GM-CSF protein and operatively linked to a GM-CSF promoter; and / or (2) a nucleic acid encoding human IL3 protein and operatively linked to an IL3 promoter. In some embodiments, the genetically modified rodent cells express human GM-CSF protein and / or human IL3 protein.

[0034] In some embodiments, the Sirpa promoter, the GM-CSF promoter, and / or the IL3 promoter are rodent promoters. In some embodiments, the Sirpa promoter, the GM-CSF promoter, and / or the IL3 promoter are endogenous rodent promoters. In some embodiments, the Sirpa promoter, the GM-CSF promoter, and / or the IL3 promoter are located at the corresponding endogenous rodent locus.

[0035] 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.

[0036] In some embodiments, the genetically modified rodent cells are heterozygous for at least one allele, the allele containing a nucleic acid sequence encoding the human protein or humanized protein. In some embodiments, the genetically modified rodent cells are homozygous for at least one allele, the allele containing a nucleic acid sequence encoding the human protein or humanized protein.

[0037] In some embodiments, the genetically modified rodent cells are rodent embryonic stem (ES) cells.

[0038] In some respects, this document provides a method for preparing rodent embryonic stem cells, comprising genetically modifying the rodent embryonic stem cells such 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) a Flt3 ligand (Flt3l) gene comprising rodent and human portions operatively linked to the Flt3l promoter.

[0039] In some respects, this document provides a rodent embryo comprising rodent embryonic stem cells as described herein or rodent embryonic stem cells prepared according to the methods described herein.

[0040] In some respects, this document provides a method for preparing rodents 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) a Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operatively linked to the Flt3l promoter, the method comprising the steps of: (a) obtaining rodent embryonic stem cells as described herein or rodent embryonic stem cells prepared according to the method described herein; and (b) generating rodents using the rodent embryonic cells described in (a).

[0041] In some respects, this document provides a method for preparing 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) a Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operatively linked to the Flt3l promoter, the method comprising modifying the genome of the rodent such that it 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) a Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operatively linked to the Flt3l promoter. Attached Figure Description

[0042] Figures 1A-1B The use of Flt3l in mice was shown. Figure 1A and 1B The production of the final humanized targeting vector (modified BAC#3), referred to as "mFlt3l" or "Flt3l" in the figure. Unless otherwise indicated by the labels in the figure (e.g., for select cassettes, loxP sites, etc.), solid shapes and single lines represent mouse sequences, and hollow shapes and double lines represent human sequences. "SPEC" indicates spectinomycin resistance gene; "EM7-Hyg" indicates hygromycin resistance gene with EM7 promoter; "loxP" indicates lox P site; SDCNEO is neomycin self-deletion cassette. Human FLT3L in Figure 1B It is referred to as "FLT3LG" in China.

[0043] Figures 1C-1D An exemplary modified mouse Flt3l (in accordance with certain exemplary embodiments provided herein) is shown. Figure 1C and 1D A schematic overview of the locus referred to as “mFLT3l” or “Flt3l” (not to scale). Figure 1C The modified mouse Flt3l locus is shown before the removal of the loxp-Neo-loxp self-deletion box. Figure 1D The modified mouse Flt3l locus is shown after removal of the loxp-Neo-loxp self-deletion cassette. Unless otherwise indicated by the figure (e.g., for selection cassettes, loxP sites, etc.), solid shapes and single lines represent mouse sequences, and hollow shapes and double lines represent human sequences. “loxP” indicates the lox P site; SDC NEO is the neomycin self-deletion cassette. Human FLT3L in Figure 1C and 1DIt is referred to as "FLT3LG" or "hFLT3LG" in Chinese.

[0044] Figure 1E Sequence alignments are shown 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 indicate the exon boundaries of the corresponding nucleic acid sequences of the mouse or human FLT3LG gene encoding the shown mouse or human protein (the upper or solid triangle is the mouse exon boundary, and the lower or hollow triangle is the human exon boundary).

[0045] Figure 2 A schematic overview (not to scale) of an exemplary modified mouse Flt3 locus (i.e., Flt3 deletion) is shown, yielding certain exemplary embodiments provided herein. “loxP” indicates the lox P site; SDC HYG is a hygromycin self-deletion cassette.

[0046] Figure 3 High levels of humanized FLT3L (in the serum and bone marrow aspirates of SRG / hFLT3L / mFLT3 KO mice were observed.) Figure 3 The term "hFLT3L" is used to refer to mice. The "SRG / hFLT3L / mFLT3 KO" mouse, also known as the "SRG hFLT3L mFLT3KO" mouse, "SRG-hFLT3L / mFLT3 KO" mouse, or "hFLT3L / mFLT3 KO" mouse, refers to a mouse containing humanized Sirpa, Rag2 gene knockout, Il2rg gene knockout, humanized Flt3l, and Flt3 KO.

[0047] Figure 4A The gating strategies of pDCs and other dendritic cells are shown.

[0048] Figure 4B This demonstrates that after hHSC implantation, SRG / hFLT3L / mFLT3 KO (in) Figure 4B The blood and spleen of mice known as “SRG hFLT3LmFLT3 KO” showed an increase in myeloid and plasma cell-like DCs.

[0049] Figure 4C This demonstrates that after hHSC implantation, SRG / hFLT3L / mFLT3 KO (in) Figure 4C The blood and spleen of mice known as “SRG hFLT3LmFLT3 KO” showed an increase in BDCA-3+ myeloid DCs.

[0050] Figure 4D SRG / hFLT3L / mFLT3 KO (in) was shown Figure 4D The mice referred to as “SRG hFLT3L mFLT3 KO” had a comparable implantation volume with HSC donor-matched StRG mice, but human myeloid and plasma cell-like DCs were increased in the BM.

[0051] Figure 4E This demonstrates that after hHSC implantation, SRG / hFLT3L / mFLT3 KO (in) Figure 4E The human myeloid and plasma cell-like dendritic cells (DCs) in the thymus of mice known as “SRG hFLT3LmFLT3 KO” were increased.

[0052] Figure 4F The implanted SRG / hFLT3L / mFLT3 KO was shown. Figure 4F The number of mouse DCs (mouse CD45+ / CD11c+ / MHC II+) was significantly reduced in mice known as “SRG-hFLT3L / mFLT3KO”.

[0053] Figure 5A This study demonstrates an increase in human mDC and pDC populations in the blood and spleen of hHSC-implanted mice with both humanized Flt3l and mouse Flt3 KO (i.e., hFLT3L / mFLT3 KO) compared to HSC donor-matched mice with only mFLT3 KO. "FLT3L" m / m mFLT3- / - mice refer to SRG mice containing both homozygous Flt3l mice and Flt3 knockout mice. h / h mFLT3- / -” mice refer to SRG mice containing homozygous humanized Flt3l and mouse Flt3 knockout.

[0054] Figure 5B This study demonstrates an increase in the human mDC and pDC population in the thymus of hHSC-implanted mice with both humanized Flt3l and mouse Flt3 KO (i.e., hFLT3L / mFLT3 KO) compared to HSC donor-matched mice with only mFLT3 KO. "FLT3L" m / m mFLT3- / - mice refer to SRG mice containing both homozygous Flt3l mice and Flt3 knockout mice. h / h mFLT3- / -” mice refer to SRG mice containing homozygous humanized Flt3l and mouse Flt3 knockout.

[0055] Figure 6AThis demonstrates the effect of SRG / hFLT3L / mFLT3 KO implanted via hHSC (in Figure 6A In SRG / hFLT3L / mFLT3 KO mice (referred to as "SRG hFLT3LmFLT3 KO"), the expression of PD-1 in CD3+ T cells in the blood was significantly lower than that in StRG mice; and the percentage of CD4+ T cells in SRG / hFLT3L / mFLT3 KO mice implanted with hHSC was higher than that of CD8+ T cells.

[0056] Figure 6B The results showed that, compared to StRG matched to an HSC donor, SRG / hFLT3L / mFLT3 KO implanted via hHSC (in) Figure 6B In mice known as “hFLT3L / mFLT3 KO”, more CD4+ and CD8+ T cells are central memory T cells (Tcm).

[0057] Figure 7A Key transcriptional differences were revealed between the DC population in the spleen / blood of hHSC-implanted SRG / hFLT3L / mFLT3 KO mice and in the blood of healthy individuals.

[0058] Figure 7B The percentages of mDC, BDCA-3+ mDC, and pDC in hHSC-implanted SRG / hFLT3L / mFLT3 KO (referred to as "SRG-hFLT3L / mFLT3 KO") mice were higher than those in human PBMCs.

[0059] Figure 8 Human pDCs were shown to be present in various organs of SRG / hFLT3L / mFLT3 KO human immune system mice.

[0060] Figure 9A The levels of human IFNα or IFNβ produced by stimulation of human pDC isolated from SRG / hFLT3L / mFLT3 KO human immune system mice with ODN2216 (CpG-DNA) are shown.

[0061] Figure 9B This demonstrates that, as measured by induction of the type I IFN characteristic gene, human pDCs in SRG / hFLT3L / mFLT3 KO human immune system mice respond to ODN2216 CpG stimulation and produce human type I IFN in vivo. Detailed Implementation

[0062] Overview

[0063] This disclosure relates to a genetically modified nonhuman animal (e.g., a rodent, such as a rat or mouse) comprising: (i) a homozygous null mutation in the Rag1 and / or Rag2 genes (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 nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene at the nonhuman animal Flt3 locus; and (iv) a Flt3 ligand (Flt3l) gene comprising a nonhuman animal portion and a human portion operatively linked to the Flt3l promoter. In some embodiments, this document provides a genetically modified nonhuman animal (e.g., a mouse) comprising: (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 nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) a Flt3 ligand (Flt3l) gene comprising a nonhuman animal portion and a human portion operatively linked to the Flt3l promoter.

[0064] In some embodiments, the genetically modified nonhuman animal expresses a human SIRPA protein or a humanized SIRPA protein encoded by a nucleic acid operably linked to a promoter (e.g., the Sirpa promoter). In some embodiments, the genetically modified nonhuman animal also expresses a human GM-CSF protein encoded by a nucleic acid operably linked to a promoter (e.g., the GM-CSF promoter), and / or a human IL3 protein encoded by a nucleic acid operably linked to a promoter (e.g., the IL3 promoter). In some embodiments, at least one promoter operably linked to a nucleic acid encoding a human or humanized protein is an endogenous nonhuman animal promoter. In other embodiments, the genetically modified animal expresses human or humanized nucleic acids via a natural human promoter and a natural regulatory element. Those skilled in the art will understand that the genetically modified animal includes a genetically modified animal that expresses at least one human or humanized nucleic acid via any promoter. Examples of promoters that can be used in this invention include, but are not limited to, the DNA pol II promoter, PGK promoter, ubiquitin promoter, albumin promoter, globin promoter, ovalbumin promoter, SV40 early promoter, Raoult sarcoma virus (RSV) promoter, retroviral LTR, and lentiviral LTR. Promoter and enhancer expression systems that can be used in this disclosure also include inducible and / or tissue-specific expression systems.

[0065] Rodents possessing components of the human immune system, such as mice and rats (e.g., HIS mice and rats), hold great promise for studying the human immune system in vivo, testing human vaccines, and testing and developing drugs to treat human diseases and conditions. HIS mice are generated by transplanting human hematopoietic stem cells and human hematopoietic progenitor cells (e.g., CD34+ HSCs) into severely immunodeficient mouse strains (e.g., recombinant activating gene 2 (Rag2) knockout (KO) and interleukin 2 receptor γ (IL2rg) KO mice). Compared to non-human primates, HIS mice offer the advantages of small animal models, allowing for more diverse experiments, easier access to the research community, and being ethically more acceptable than experiments using human subjects. Most importantly, experimental results obtained from HIS mice are likely to be more relevant and applicable to humans. Similar rat models that offer these advantages are also documented and considered in this paper.

[0066] Although HIS mice develop human immune cells, such as B cells and T cells, a HIS mouse model supporting human dendritic cell development is still required. Human dendritic cells are the main antigen-presenting cells in the immune system that regulate T cell activation and antigen-specific responses. Fms tyrosine-like 3-ligand (Flt3l) is a key cytokine for dendritic cell development. However, humanization of Flt3l in HIS mouse models (e.g., huSIRP Rag2- / - IL-2Rγ- / - (SRG) VelociHum model) has not significantly increased human dendritic cell development. Flt3-deficient mice have been reported to have increased human dendritic cell (DC) development after human hematopoietic stem cell (hHSC) engraftment. However, increased DC development requires repeated injections of high doses of human FLT3L (Li et al., (2016) Eur J Immunol 46: 1291-1299).

[0067] This disclosure provides a novel genetically modified nonhuman animal (e.g., mouse) model that specifically lacks the nonhuman animal (e.g., mouse) Flt3l receptor (Flt3 or FLK2 (fetal liver kinase 2)) in a humanized Flt3l HIS model (e.g., the huSIRP Rag2- / - IL-2Rγ- / - (SRG) VelociHum model). This novel model, with receptor deletion and humanization of Flt3l, allows for sustained expression of the Flt3l isotype at physiologically relevant levels. Furthermore, the humanized Flt3l gene provided herein encodes a chimeric membrane-bound FLT3L containing a human signal peptide and a cytokine-like core domain, as well as a nonhuman animal (e.g., rodent, such as rat or mouse) stem, transmembrane domain, and cytoplasmic tail, which preserves the endogenous functions associated with the stem, transmembrane domain, and cytoplasmic tail. Flt3-ineffective, Flt3l-humanized nonhuman animal (e.g., mouse) models showed increased human dendritic cells (DCs) in the spleen, blood, and bone marrow. This increase has been reported in both myeloid DCs (a subset important for T cell activation / regulation) and plasmacytoid DCs (a subset that is the primary producer of type I IFN in response to viral infection). Furthermore, a significant increase in BDCA-3+ DCs was observed; BDCA-3+ DCs are an important subset for cross-priming of CD8+ T cells against exogenous antigens, such as viral elements derived from phagocytosed infected cells. Without being bound by theory, the absence of the Flt3l receptor (Flt3 or FLK2 (fetal liver kinase 2)) in nonhuman animals (e.g., mice) eliminated competition for human FLT3L from nonhuman animal (e.g., mouse) DCs, thereby enhancing human DC development in HIS models with humanized Flt3l. This disclosure demonstrates that the absence of cytokine receptors in non-human animals (e.g., mice) leads to an increase in human dendritic cells (e.g., human myeloid and plasmacytoid dendritic cells) in the bone marrow and thymus, at levels significantly higher than those observed in StRG. This novel model provides a useful tool for studying human dendritic cell biology and for developing therapeutics for human dendritic cell diseases.

[0068] The genetically modified non-human animals described herein have numerous uses in the art, including, for example, mimicking human autoimmune diseases and dendritic cell function; screening in vivo for agents that regulate dendritic cell function (e.g., in healthy or diseased states); evaluating the therapeutic efficacy of drugs in stimulating T cell responses to target cells; and / or evaluating the therapeutic efficacy of drugs in treating autoimmune diseases. These and other objects, advantages, and features of the invention will become apparent to those skilled in the art upon reading the details of the compositions and methods described more fully below.

[0069] definition

[0070] In this article, the articles “a” and “a kind” are used to refer to the grammatical object of one or more (i.e., at least one). For example, “a component” means one or more components.

[0071] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, including both amino and acid functional molecules, and that can be contained in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives, and congeners; amino acid analogs with variant side chains; and all stereoisomers of any of the above.

[0072] A gene's "coding region" comprises nucleotide residues of both the coding and non-coding strands of the gene, which are homologous or complementary to the coding region of the mRNA molecule produced by gene transcription. The "coding region" of an mRNA molecule also includes nucleotide residues that match the anticodon region of a transfer RNA molecule during mRNA translation, or that encode a stop codon. Therefore, a coding region can include nucleotide residues containing codons for amino acid residues not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in the protein output signal sequence).

[0073] As used herein, the term "chimera" refers to a nucleic acid or protein whose structure (i.e., nucleotide or amino acid sequence) includes portions from a different species. In some embodiments, the "chimera" nucleic acid or protein described herein includes nucleotide or amino acid sequences from non-human sources (e.g., rodents, such as mice) and humans. In such embodiments, the "chimera" nucleic acid or protein may also be referred to as a "humanized" nucleic acid or protein.

[0074] As used herein, the phrase "endogenous gene" or "endogenous gene segment" refers to a gene or gene segment found in a parent or reference organism prior to the introduction of disruption, deletion, substitution, alteration, or modification as described herein. In some embodiments, the reference organism is a wild-type organism. In some embodiments, the reference organism is a modified organism. In some embodiments, the reference organism is a laboratory-grown organism (whether wild-type or modified).

[0075] As used herein, the term "complete coding sequence" refers to the coding nucleic acid sequence from the start codon to the stop codon. The term "full-length polypeptide" refers to a polypeptide that contains the amino acid sequence encoded by such a complete coding sequence.

[0076] The term "humanized" is used herein as it is understood in the art to mean a nucleic acid or protein whose structure (i.e., nucleotide or amino acid sequence) comprises a portion derived from a non-human source, and which has been modified to make its structure and function more similar to a real human nucleic acid or protein than the original source nucleic acid or protein. For example, humanization may involve selecting amino acid substitutions to make a non-human sequence more similar to a human sequence. Humanization may also involve transplanting at least a portion of a non-human protein into a human protein. To cite just one example, in the case of a membrane receptor, a "humanized" gene may encode a polypeptide whose extracellular portion has an amino acid sequence that is a human extracellular portion and the remaining sequence that is a sequence of a non-human (e.g., mouse) polypeptide. In some embodiments, a humanized gene comprises at least a portion of the DNA sequence of a human gene. In some embodiments, a humanized protein comprises a sequence partially present in a human protein. The term "human" is generally accepted in the art to refer to a nucleic acid or protein whose structure (i.e., nucleotide or amino acid sequence) is entirely derived from a human source.

[0077] As used herein, the term "locus" refers to a location on a chromosome containing a group of related genetic elements (e.g., genes, gene segments, regulatory elements). A locus can be endogenous or non-endogenous. The term "endogenous locus" refers to a location on a chromosome where a specific genetic element is naturally present. In some embodiments, an endogenous locus has a sequence that is naturally occurring. In some embodiments, an endogenous locus is a wild-type locus. In some embodiments, an endogenous locus is a modified locus.

[0078] As used herein, the phrase "non-human animal" refers to any non-human vertebrate organism. 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.

[0079] As used herein, the phrase “operably linked” refers to the juxtaposition of the components in a relationship that allows them to function in the intended manner. Control sequences “operably linked” to a coding sequence are linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. “Operably linked” sequences include expression control sequences adjacent to the target gene and expression control sequences that act trans- or at a distance to control the target gene. As used herein, the term “expression control sequence” refers to a polynucleotide sequence essential to the expression and processing of the coding sequence to which it is linked. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences enhancing translation efficiency (i.e., Kozak concordant sequences); sequences enhancing protein stability; and sequences enhancing protein secretion if necessary. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term “control sequence” is intended to include components whose presence is essential for expression and processing, and may also include other components whose presence is advantageous, such as leader sequences and fusion mate sequences.

[0080] The terms “polynucleotide” and “nucleic acid” are used interchangeably. They refer to polymeric forms of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci defined by 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 contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure may be modified before or after polymer assembly. Polynucleotides may be further modified, for example, by conjugation with labeled components. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

[0081] As used herein, the term "peptide" refers to any polymer chain of amino acids. In some embodiments, the peptide has an amino acid sequence that is naturally occurring. In some embodiments, the peptide has an amino acid sequence that is not naturally occurring. In some embodiments, the peptide has an amino acid sequence that has been modified through artificial design and / or production.

[0082] As used herein, the term "promoter" includes a DNA sequence operatively linked to a nucleic acid sequence to be transcribed (e.g., a nucleic acid sequence encoding a desired molecule). Promoters are typically located upstream of the nucleic acid sequence to be transcribed and provide specific binding sites for RNA polymerases and other transcription factors. In a particular embodiment, a promoter is typically located upstream of the nucleic acid sequence transcribed to produce the desired molecule and provides specific binding sites for RNA polymerases and other transcription factors. The phrase "endogenous promoter" refers to a promoter associated with an endogenous gene in its natural state (e.g., in a wild-type organism).

[0083] As used herein, the term “recombinant” is intended to refer to polypeptides (e.g., signal regulatory proteins described herein) designed, modified, prepared, expressed, created, or isolated through recombinant means, such as polypeptides expressed using recombinant expression vectors transfected into host cells, polypeptides isolated from recombinant 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 antibodies isolated from animals transgenic with human immunoglobulin genes (e.g., mice) (see, for example, Taylor, LD, et al. (1992) Nucl. Acids 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) or polypeptides prepared, expressed, created, or isolated by any other means involving splicing selected sequence elements into another. In some embodiments, one or more of these selected sequence elements are present in nature. In some embodiments, one or more of these selected sequence elements are designed by computer simulation. In some embodiments, one or more of these selected sequence elements are generated by mutagenesis (e.g., in vivo or in vitro) of known sequence elements (e.g., from natural or synthetic sources). For example, in some embodiments, the recombinant polypeptide contains a sequence present in the genome of a target source organism (e.g., human, mouse, etc.). In some embodiments, the recombinant polypeptide has an amino acid sequence produced by mutagenesis (e.g., in vitro or in vivo, such as in non-human animals) such that the amino acid sequence of the recombinant polypeptide is such that, although derived from and associated with the polypeptide sequence, it may not be naturally present in the genome of a non-human animal in vivo.

[0084] In this document, the term "replacement" refers to the process of removing a "replaced" nucleic acid sequence (e.g., a gene) present at a host locus (e.g., in the genome) from that locus and placing a different "replaced" nucleic acid in its place. In some embodiments, the replaced nucleic acid sequence and the replacement nucleic acid sequence are comparable to each other, for example, they are homologous to each other and / or contain corresponding elements (e.g., protein-coding elements, regulatory elements, etc.). In some embodiments, the replaced nucleic acid sequence includes one or more of a promoter, enhancer, splice donor site, splice acceptor site, intron, exon, and untranslated region (UTR); in some embodiments, the replacement nucleic acid sequence includes one or more coding sequences. In some embodiments, the replacement nucleic acid sequence is a homolog of the replaced nucleic acid sequence. In some embodiments, the replacement nucleic acid sequence is an ortholog of the replaced sequence. In some embodiments, the replacement nucleic acid sequence is or contains a human nucleic acid sequence. In some embodiments, when the replacement nucleic acid sequence is or contains a human nucleic acid sequence, the replaced nucleic acid sequence is or contains a rodent sequence (e.g., a mouse sequence). The nucleic acid sequence thus positioned may include one or more regulatory sequences, which are portions of the source nucleic acid sequence (e.g., promoters, enhancers, 5′-untranslated regions, or 3′-untranslated regions, etc.) used to obtain the sequence thus positioned. For example, in various embodiments, substitution is the replacement of an endogenous sequence with a heterologous sequence, resulting in the production of a gene product from the thus positioned nucleic acid sequence (containing the heterologous sequence) but without expression of the endogenous sequence; substitution is the replacement of an endogenous genomic sequence with a nucleic acid sequence encoding a protein having a similar function to the protein encoded by the endogenous sequence. In various embodiments, an endogenous gene or a fragment thereof is replaced by a corresponding human gene or a fragment thereof. The corresponding human gene or fragment thereof is an ortholog of the replaced endogenous gene or fragment thereof or a human gene or fragment thereof that is substantially similar or identical in structure and / or function.

[0085] As used herein, the term "variant" includes nucleic acid or peptide sequences that differ sequentially 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 may result in amino acid substitutions, additions, deletions, fusions, and truncations. Sequence changes in peptide variants are typically limited or conserved, and therefore the sequences of the reference peptide and the variant are highly similar overall and identical in many regions. Variants and reference peptides may differ in amino acid sequence from any combination of one or more substitutions, additions, or deletions. Variants of nucleic acids or peptides may be naturally occurring, such as allelic variants, or may be variants known not to exist naturally. Non-naturally occurring nucleic acid and peptide variants may be prepared by mutagenesis or direct synthesis.

[0086] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid associated with it. In some embodiments, a vector is capable of extrachromosomal replication and / or expression of the nucleic acid to which it is linked in a host cell (e.g., eukaryotic and / or prokaryotic cells). In this document, a vector capable of directing the expression of an operatively linked gene is referred to as an "expression vector".

[0087] As used herein, the term "wildtype" has its meaning as understood in the art, referring to an entity that possesses the structure and / or activity found in nature in a "normal" state or environment (as opposed to mutation, disease, alteration, etc.). Those skilled in the art will understand that wild-type genes and polypeptides typically exist in many different forms (e.g., alleles).

[0088] Genetically modified loci

[0089] In some respects, this article provides genetically modified nonhuman animals (e.g., mice or rats) 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 nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) the Flt3 ligand (Flt3l) gene comprising a nonhuman animal portion and a human portion operatively linked to the Flt3l promoter. In some embodiments, this document provides genetically modified nonhuman animals (e.g., mice) comprising: (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 nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) a Flt3 ligand (Flt3l) gene comprising a nonhuman animal portion and a human portion operatively linked to the Flt3l promoter. In some embodiments, the genetically modified nonhuman animal expresses a human SIRPA protein or a humanized SIRPA protein encoded by a nucleic acid operatively linked to the Sirpa promoter. In some embodiments, the genetically modified nonhuman animal also expresses a human GM-CSF protein encoded by a nucleic acid operatively linked to the GM-CSF promoter and / or a human IL3 protein encoded by a nucleic acid operatively linked to the IL3 promoter. In some embodiments, at least one promoter operatively linked to a nucleic acid encoding a human or humanized protein is an endogenous nonhuman animal promoter. In some embodiments, the genetically modified nonhuman animal comprises the implantation of human hematopoietic stem cells (HSCs).

[0090] Flt3 knockout

[0091] In some respects, the genetically modified nonhuman animals (e.g., mice or rats) provided herein contain homozygous null mutations in the nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene.

[0092] The Flt3 gene encodes a class III receptor tyrosine kinase that regulates hematopoiesis. Activation of this receptor occurs through the binding of the fms-associated tyrosine kinase 3 ligand to its extracellular domain, inducing homodimer formation in the plasma membrane, leading to receptor autophosphorylation. The activated receptor kinase then phosphorylates and activates various cytoplasmic effector molecules involved in pathways of apoptosis, proliferation, and differentiation of bone marrow hematopoietic cells. Mutations leading to constitutive activation of this receptor can cause acute myeloid leukemia and acute lymphoblastic leukemia.

[0093] In some respects, the genetically modified nonhuman animals contain homozygous null mutations in the nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene. Null mutations include deletions, insertions, and / or substitutions in a gene that result in the absence of a functional gene product (e.g., the gene product (protein, RNA) is completely absent at the molecular level, or the expression of a nonfunctional gene product). In this disclosure, null mutations and inactivating mutations have the same meaning and are used interchangeably. A homozygous null mutation means that all alleles contain null mutations. For example, a homozygous null mutation in the mouse or rat Flt3 gene means that both alleles (i.e., both null alleles) of the mouse or rat Flt3 gene contain null mutations. Genes with homozygous null mutations are also called gene knockouts or gene defects. For example, a homozygous null mutation in Flt3 is also called Flt3 knockout or Flt3 defect. Therefore, null mutations in the nonhuman animal Flt3 gene include deletions, insertions, and / or substitutions in the nonhuman animal Flt3 gene. In some cases, the Flt3 locus in endogenous nonhuman animals contains null mutations and therefore null alleles. A null allele is a mutated copy of a gene that completely lacks its normal function. This may result from the complete absence of the gene product (protein, RNA) at the molecular level, or from the expression of a nonfunctional gene product. At the phenotypic level, null alleles include the deletion of an entire locus.

[0094] In some embodiments, the invalid mutation is a deletion of the complete endogenous coding sequence of Flt3. In some embodiments, the non-human animals provided herein do not express the Flt3 protein.

[0095] In some implementations, the homozygous null mutation in the nonhuman animal Flt3 gene contains the same null mutation across all alleles. In other implementations, the homozygous null mutation in the nonhuman animal Flt3 gene contains different null mutations across different alleles.

[0096] The mouse Flt3 locus is located on chromosome 5, GRCm38, NC_000071.6 (147330741-147400489), and the coding sequence for mouse Flt3 is available in Genbank accession number NM_010229.2. The mouse Flt3 locus contains 24 exons, of which exons 1-24 are coding exons. Therefore, in some embodiments, the genetically modified animal provided herein is a mouse, and one or more of exons 1-24 of the mouse Flt3 gene are deleted or mutated in the genetically modified mouse. In some cases, other aspects of the mouse Flt3 genomic locus, such as introns, 3' and / or 5' untranslated sequences (UTRs), are also deleted or mutated. In some cases, 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 69.1 kb deletion of the mouse Flt3 gene sequence, wherein the sequence from the start codon ATG to the stop codon is deleted on mouse chromosome 5 G3 between coordinates chr5: 147331171-147400265 (GRCm38 assembly), as shown in Example 1.

[0097] Deletion, modification, or alteration of the Flt3 gene at the endogenous Flt3 locus can be detected using various methods, including, for example, PCR, Western blotting, Southern blotting, restriction fragment length polymorphism (RFLP), or allele gain / loss assays. In some embodiments, the non-human animal is homozygous for the deletion or null mutation of the endogenous Flt3 gene.

[0098] In some embodiments, non-human animals (e.g., mice or rats) containing homozygous null mutations in the Flt3 gene, i.e., Flt3-deficient non-human animals, are immunocompromised animals. For example, Flt3-deficient non-human animals (e.g., mice or rats) may contain at least one null allele of the Rag2 gene (“recombinant activation gene 2”, the coding sequence of which is available in Genbank accession number NM_009020.3). In some embodiments, Flt3-deficient non-human animals (e.g., mice or rats) contain two null alleles of Rag2. In other words, Flt3-deficient non-human animals (e.g., mice) are homozygous null for Rag2. In other embodiments, Flt3-deficient non-human animals (e.g., mice or rats) contain one or two null alleles of the Rag1 gene. In some embodiments, Flt3-deficient non-human animals (e.g., mice or rats) are homozygous null for Rag1. In some embodiments, the Flt3-deficient nonhuman animal (e.g., mouse or rat) contains (i) one or two null alleles of the Rag1 gene; and (ii) one or two null alleles of the Rag2 gene. In some embodiments, the Flt3-deficient nonhuman animal (e.g., mouse or rat) is homozygous null for both Rag1 and Rag2. In some embodiments, the Flt3-deficient nonhuman animal is an immunocompromised mouse containing two null alleles of Rag2 (i.e., homozygous null). In some embodiments, the Flt3-deficient nonhuman animal is an immunocompromised rat containing two null alleles of Rag1 (i.e., homozygous null) and two null alleles of Rag2 (i.e., homozygous null). As another example, the Flt3-deficient nonhuman animal (e.g., mouse or rat) contains at least one null allele of the IL2rg gene (“interleukin-2 receptor, γ”, also known as the shared γ chain or γC, wherein the coding sequence of the mouse gene is available in Genbank accession number NM_013563.3). In some implementations, Flt3-deficient nonhuman animals (e.g., mice or rats) contain two null alleles for IL2rg. In other words, Flt3-deficient nonhuman animals (e.g., mice or rats) are homozygous null for IL2rg, i.e., they are IL2rg null. - / - (Or, when the IL2rg gene is located on the X chromosome (as in mice or rats), it is IL2rg.) Y / - In some implementations, Flt3-deficient nonhuman animals (e.g., mice or rats) contain null alleles for Rag2 and IL2rg, i.e., Rag2. - / - IL2rg - / - (Or, when the IL2rg gene is located on the X chromosome (as in mice or rats), it is Rag2) - / - IL2rg Y / -In some embodiments, Flt3-deficient nonhuman animals (e.g., mice or rats) contain null alleles of Rag1 and IL2rg. In some embodiments, Flt3-deficient nonhuman animals (e.g., mice or rats) contain null alleles of Rag1, Rag2, and IL2rg. In some embodiments, the Flt3-deficient nonhuman animal is an immunocompromised mouse containing two null alleles (i.e., homozygous null) of Rag2 and two null alleles (i.e., homozygous null) of IL2rg (or one null allele for male mice). In some embodiments, the Flt3-deficient nonhuman animal is an immunocompromised rat containing two null alleles (i.e., homozygous null) of Rag1, two null alleles (i.e., homozygous null) of Rag2, and two null alleles (i.e., homozygous null) of IL2rg (or one null allele for male rats). Other genetic modifications are also considered. For example, Flt3-deficient nonhuman animals (e.g., mice or rats) may contain modifications to other genes related to dendritic cell development and / or function, as well as those related to the immune system, such as replacing one or more other nonhuman animal genes with nucleic acid sequences encoding one or more humanized peptides. Such genes include, but are not limited to, modifications to, for example, FLT3L, SIRPA, GM-CSF, and / or IL-3. Alternatively or additionally, Flt3-deficient nonhuman animals (e.g., mice or rats) may contain modifications to genes related to the development and / or function of other cells and tissues, such as genes associated with human symptoms or diseases, or genes that, when modified in a nonhuman animal (e.g., mice), provide models of human symptoms and diseases. The introduction of other gene modifications can be achieved through ES cell modification and / or propagation. For example, Flt3-deficient (optionally, Rag2 and IL2rg-deficient) nonhuman animals (e.g., mice or rats) may be propagated together with nonhuman animals containing one or more other gene modifications, including, but not limited to, modifications in, for example, the SIRPA, GM-CSF, and / or IL-3 genes. In some implementation schemes, all genetic modifications are propagated to homozygosity in the genetically modified animals described herein.

[0099] Humanized Flt3l locus

[0100] In some respects, the genetically modified nonhuman animals provided herein also express a chimeric Flt3l protein encoded by the Flt3 ligand (Flt3l) gene, which comprises rodent and human portions operatively linked to the Flt3l promoter.

[0101] Dendritic cells (DCs) provide a crucial link between innate and adaptive immunity by recognizing pathogens and initiating 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 classic DCs and their CD103-positive tissue counterparts.

[0102] In some embodiments, the genetically modified non-human animal (e.g., rodent, such as rat or mouse) provided herein contains a Flt3 ligand (Flt3l) gene, which comprises a non-human animal (e.g., rodent, such as rat or mouse) portion and a human portion operatively linked to a Flt3l promoter.

[0103] In some embodiments, the non-human animal (e.g., rodent, such as rat or mouse) portion of the Flt3l gene comprises the non-coding portions of exon 1 and 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%) of the sequence identical to the non-human animal (e.g., rodent, such as rat or mouse) non-coding portion of exon 2 and exons 7-9 of the Flt3l gene.

[0104] The genomic locus encoding the wild-type mouse Flt3l protein is located on chromosome 7 of the mouse genome (GRCm39, NC_000073.7 (44780607-44785914)). The polypeptide sequence and nucleic acid sequence encoding wild-type mouse Flt3l are available in GenBank accessions: NP_001389760.1 and NM_001402831.1; NP_001389761.1 and NM_001402832.1; NP_001389762.1 and NM_001402833.1; NP_ The sequences are located at 001389763.1 and NM_001402834.1; NP_001389764.1 and NM_001402835.1; NP_001389765.1 and NM_001402836.1; 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 are encoded by NM_013520.4.

[0105] In some implementations, the non-coding portions of exons 1 and 2 of the Flt3L gene in non-human animals (e.g., rodents, such as rats or mice) and the exons downstream of exon 6 (e.g., exons 7-9) 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 exons 1 and 2 and exons 7-9 of the mouse Flt3l gene shown in Table 1A.

[0106] Table 1A:

[0107] In some implementations, the human portion of the humanized Flt3l gene includes a signal peptide coding portion 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%) of the sequence identical to the signal peptide coding portion of exon 2 and exons 3-6 present in the human FLT3L gene.

[0108] The genomic locus encoding the wild-type human FLT3L protein is located on chromosome 19 of the human genome; 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 in Genbank accessions: NP_001191431.1 and NM_001204502.2 (isotype 1 and transcriptomorph 1); NP_001191432.1 and NM_001204503.2 (isotype 1 and transcriptomorph 2); NP_001265566.1 and NM_001278637.2 (isotype 2 and transcriptomorph 4); NP_001265567.1 and NM_001278638.2 (isotype 2 and transcriptomorph 5); and NP_001450.2 and NM_001459.4 (isotype 1 and transcriptomorph 3). In some embodiments, the membrane-bound human FLT3 ligand polypeptide sequence used in this disclosure is derived from NCBI reference sequence: NP_001191431.1, Uniprot: P49771-1; and encoded by NM_001204502.2.

[0109] In some implementations, the signal peptide coding portion of exon 2 of the human FLT3L gene and exons 3-6 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 of the human FLT3L gene shown in Table 1B.

[0110] Table 1B:

[0111] In some implementations, the Flt3l gene includes non-human animal (e.g., rodent, such as rat or mouse) exon 1, the non-coding portion of rodent exon 2, the signal peptide coding portion of human exon 2, human exons 3-6, and non-human animal (e.g., rodent, such as rat or mouse) exons 7-9. In some cases, the Flt3l gene also includes aspects of the human FLT3L genomic loci, such as introns, 3' and / or 5' untranslated sequences (UTRs).

[0112] In some embodiments, the Flt3l gene encodes a chimeric membrane-bound Flt3l comprising a signal peptide and a cytokine-like core domain of the human FLT3L polypeptide, as well as 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.

[0113] 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 bolded, and the C-terminal portion of the mouse Flt3L polypeptide is shown in italics.

[0114] Table 2

[0115] Humanized Flt3l protein (SEQ ID NO: 11)

[0116]

[0117] In some implementation schemes, such as Figure 1EAs shown, the C-terminal portion of the rodent Flt3l polypeptide includes the rodent stem region, the rodent transmembrane domain, and the rodent cytoplasmic tail of the rodent Flt3l polypeptide. 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%) of the amino acid sequence identical to that of the rodent Flt3l polypeptides shown in Table 2.

[0118] In some embodiments, the signal peptide of the human FLT3L polypeptide comprises 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 comprises 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%) of the corresponding amino acid sequence of the signal peptide of the human FLT3L polypeptide shown in Table 2.

[0119] In some embodiments, the cytokine-like core domain of the human FLT3L peptide comprises amino acids corresponding to residues 27-159 of the human FLT3L peptide (e.g., NP_001191431.1). In some embodiments, the cytokine-like core domain of the human FLT3L peptide comprises 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%) of the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L peptide shown in Table 2.

[0120] In some implementations, the non-human animals described herein express humanized Flt3l protein by genetically modifying the endogenous locus of the non-human animal encoding the Flt3l protein. Suitable examples described herein include rodents, particularly mice.

[0121] The mouse and human Flt3 ligand exists in multiple isoforms, including membrane-bound and soluble forms, which are thought to be generated by 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 which are incorporated herein by reference in their entirety). The soluble isoform can also be generated by proteolytic cleavage (by cleavage via TNFα convertase or TACE) (Kazi et al. (2019) Physiol. Rev. 99:1433-1466, the contents of which are incorporated herein by reference in their entirety). In some embodiments, rodents express one or more soluble forms of the Flt3l polypeptide 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, both the soluble and membrane-bound forms of the Flt3l polypeptide contain a signal peptide and a cytokine-like core domain of the human FLT3L polypeptide. All known soluble isoforms of the human and mouse Flt3 ligand contain a receptor-binding domain (cytokine-like core domain), which is typically encoded by exons 3-6 of the gene.

[0122] In some embodiments, the genetically modified rodent is heterozygous for the Flt3 ligand (Flt3l) gene, which comprises a non-human animal (e.g., rodent, such as a rat or mouse) portion and a human portion. In some embodiments, the genetically modified rodent is homozygous for the Flt3 ligand (Flt3l) gene, which comprises a non-human animal (e.g., rodent, such as a rat or mouse) portion and a human portion.

[0123] In some embodiments, the non-human animal (e.g., rodent, such as rat or mouse) portion of the Flt3l gene is the endogenous non-human animal (e.g., rodent, such as rat or mouse) portion of the Flt3l gene; the rodent Flt3l gene is an endogenous non-human animal (e.g., rodent, such as rat or mouse) gene; and / or the rodent Flt3l polypeptide is an endogenous non-human animal (e.g., rodent, such as rat or mouse) Flt3l polypeptide.

[0124] In some embodiments, the non-human animal (e.g., rodent, such as rat or mouse) portion and the human portion are operatively linked to the Flt3l promoter. In some embodiments, the Flt3l promoter is a non-human animal (e.g., rodent, such as rat or mouse) promoter. In some embodiments, the Flt3l promoter is an endogenous non-human animal (e.g., rodent, such as rat or mouse) promoter. In some embodiments, the Flt3l promoter is located at an endogenous non-human animal (e.g., rodent, such as rat or mouse) locus.

[0125] In some embodiments, the genetically modified nonhuman animal comprises genetic material from a heterologous species (e.g., human), wherein the nonhuman Flt3l gene encodes the Flt3l protein, which comprises a coding portion of the genetic material from the heterologous species. In some embodiments, the humanized Flt3l gene of this disclosure comprises genomic DNA of a heterologous species corresponding to the signal peptide and cytokine-like core domain of the Flt3l protein. Methods for preparing nonhuman animals, nonhuman embryos, and cells containing the humanized Flt3l gene are also provided.

[0126] 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 altered, wherein a portion of the endogenous non-human (e.g., rodent) Flt3l gene is replaced by a heterologous sequence (e.g., all or part of a human FLT3L sequence). In some embodiments, all or substantially all of the endogenous non-human (e.g., rodent) Flt3l gene is replaced by a heterologous gene (e.g., a 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 at the endogenous non-human (e.g., rodent) Flt3l gene locus. In some embodiments, the heterologous gene is a human gene. In some embodiments, one of the two copies of the endogenous non-human (e.g., rodent) Flt3l gene is modified or humanized, thereby producing a non-human animal that is heterozygous for the humanized Flt3l gene. In other embodiments, a non-human animal is provided that is homozygous for the humanized Flt3l gene. In some embodiments, portions of the signal peptide and cytokine-like core domain encoding the endogenous non-human (e.g., rodent) Flt3l gene are replaced with corresponding heterologous sequences (e.g., human FLT3L sequences), while portions encoding the stem, transmembrane domain, and cytoplasmic tail retain the endogenous non-human (e.g., rodent) Flt3l sequence. In such embodiments, non-human (e.g., rodent) TACE cleavage is preserved.

[0127] The non-human animals disclosed herein 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. Flt3l genes that have been substituted, inserted, or modified at the endogenous non-human (e.g., rodent) Flt3l locus can be detected using various methods, including, for example, PCR, Western blotting, Southern blotting, restriction fragment length polymorphism (RFLP), or allele gain / loss assays. In some embodiments, the non-human animals are heterozygous with respect to the humanized Flt3l gene.

[0128] Compositions and methods are provided for preparing non-human animals expressing humanized Flt3l protein, said humanized Flt3l protein comprising specific polymorphic forms or allelic variants (e.g., single amino acid differences). In some embodiments, compositions and methods are provided for preparing non-human animals expressing such proteins by endogenous promoters and endogenous regulatory sequences. The methods include inserting genetic material encoding all or part of the human FLT3L protein into the genome of a non-human animal at a precise location corresponding to an endogenous non-human (e.g., rodent) Flt3l gene, thereby creating a humanized Flt3l gene expressing all or part of the human Flt3l protein. In some embodiments, the method includes inserting genomic DNA corresponding to the signal peptide encoding portion of exon 2, starting from the start codon "ATG", and exons 3-6 of the human FLT3L gene into an endogenous non-human (e.g., rodent) Flt3l gene of a non-human animal, thereby creating a humanized gene encoding the Flt3l protein, which contains a human portion containing the amino acids encoded by the inserted exons.

[0129] In various implementations, the humanized Flt3l gene approach employs relatively minimal modification to the endogenous gene and achieves natural Flt3l-mediated signal transduction in non-human animals. Therefore, in such implementations, Flt3l gene modification does not affect other surrounding genes. Furthermore, in various implementations, the modification does not affect Flt3l's alternative splicing patterns or post-translational cleavage.

[0130] In addition to mice possessing the humanized Flt3l gene as described herein, other genetically modified non-human animals containing the humanized Flt3l gene are also provided herein. In some embodiments, such non-human animals contain a humanized Flt3l gene operatively 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, wherein the humanized Flt3l protein comprises at least amino acid residues 27-160 of the human FLT3L protein, such as 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.

[0131] Immunodeficient nonhuman animals

[0132] As described above, the genetically modified non-human animals with Flt3 deficiency and humanized Flt3l described herein are also immunodeficient because they contain defects in the Rag1 and / or Rag2 and Il2rg genes. Rag1, Rag2, and Il2rg are important 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 attacked by xenograft cells, they are unable to mount an immune response to the foreign cells.

[0133] V(D)J recombination activator protein 1 (also known as RAG1, recombination activation 1, recombination activation gene 1, and recombination activation protein 1) is encoded by the Rag1 gene (also known as recombination activation 1). RAG1 is the catalytic component of the RAG complex, a multi-protein complex that mediates the DNA cleavage phase during V(D)J recombination. V(D)J recombination assembles a diverse library of immunoglobulins and T cell receptor genes in developing B and T lymphocytes through the rearrangement of different V (variable), and in some cases D (diversity) and J (connection) gene segments. In the RAG complex, RAG1 mediates the binding of DNA to a conserved recombination signal sequence (RSS) and catalyzes DNA cleavage activity by introducing double-strand breaks between the RSS and adjacent coding segments. RAG2 is not a catalytic component but is essential for all known catalytic activities. RAG1 and RAG2 are crucial for the generation of mature B cells and T cells, both key components of the adaptive immune system.

[0134] Mouse Rag1 is located on chromosome 2, 2E2; 2 53.88 cM (NCBI RefSeq gene ID 19373; assembly GRCm39 (GCF_000001635.27); location NC_000068.8 (101468597..101479877, complementary)). The mouse Rag1 gene is mentioned in terms of the classic wild-type form, all alleles, and isotypes. The classic wild-type mouse RAG1 protein has been assigned UniProt accession number P15919 and NCBI accession number NP_033045.2. The mouse RAG1 protein is mentioned in terms of the wild-type form, all alleles, and isotypes. The mRNA (cDNA) encoding the classic isotype has been assigned NCBI accession number NM_009019.2. The mouse Rag1 mRNA (cDNA) and coding sequence are mentioned, including the classic wild-type form as well as all alleles and isotypes.

[0135] Rat Rag1 is located on chromosome 3, 3q31 (NCBI RefSeq gene ID 84600; assembly mRatBN7.2 (GCF_015227675.2); location NC_051338.1 (87917061..87928158, complementary)). The rat Rag1 gene is mentioned in terms of the classic wild-type form, all alleles, and isotypes. The classic wild-type rat RAG1 protein has been assigned UniProt accession number G3V6K9 and NCBI accession number NP_445920.1. The rat RAG1 protein is mentioned in terms of the classic wild-type form, all alleles, and isotypes. The mRNA (cDNA) encoding the classic isotype has been assigned NCBI accession number NM_053468.1. The rat Rag1 mRNA (cDNA) and coding sequence are mentioned in terms of the classic wild-type form, all alleles, and isotypes.

[0136] An inactivated endogenous Rag1 gene is a Rag1 gene that does not produce RAG1 protein or does not produce functional RAG1 protein. This inactivated Rag1 gene may be present in the lineage of a non-human animal (or cell or genome). The inactivation mutation in the Rag1 gene in a non-human animal (or cell or genome) may be homozygous. As an example, an inactivated endogenous Rag1 gene may contain insertions, deletions, or one or more point mutations in the endogenous Rag1 gene that result in the loss of functional RAG1 protein expression. Some inactivated endogenous Rag1 genes may contain the deletion or disruption of the entire endogenous Rag1 gene, or may contain the deletion or disruption of a segment (i.e., a portion or part) of the endogenous Rag1 gene. For example, some, most, or all of the coding sequences in the endogenous Rag1 gene may be deleted or disrupted. In one example, the 5' segment of the Rag1 gene may be deleted or disrupted (e.g., including the start codon). As an example, an inactivated endogenous Rag1 gene can be a gene in which the start codon of the endogenous Rag1 gene is missing, destroyed, or mutated to the point that the start codon is no longer functional. For example, the start codon can be destroyed by deletion or insertion within the start codon. Alternatively, the start codon can be mutated by, for example, substitution of one or more nucleotides. In another example, the 3' segment of the Rag1 gene can be missing or destroyed (e.g., including the stop codon). In another example, an internal segment of the Rag1 gene (i.e., a segment in the middle of the Rag1 gene) can be missing or destroyed. In yet another example, all coding sequences in the endogenous Rag1 gene are missing or destroyed.

[0137] V(D)J recombination activator protein 2 (also known as RAG2, recombination activation 2, recombination activation gene 2, and recombination activation protein 2) is encoded by the Rag2 gene (also known as recombination activation 2). As mentioned above, RAG1 is the catalytic component of the RAG complex, a multi-protein complex that mediates the DNA cleavage phase during V(D)J recombination. RAG2 is not a catalytic component, but is essential for all known catalytic activities. RAG1 and RAG2 are crucial for the generation of mature B cells and T cells (both of which are key components of the adaptive immune system).

[0138] Mouse Rag2 is located on chromosome 2, 2E2; 2 53.87 cM (NCBI RefSeq gene ID 19374; assembly GRCm39 (GCF_000001635.27); location NC_000068.8 (101455057..101462873)). The mouse Rag2 gene is mentioned in terms of the classic wild-type form, all alleles, and isotypes. The classic wild-type mouse RAG2 protein has been assigned UniProt accession number P21784 and NCBI accession number NP_033046.1. The mouse RAG2 protein is mentioned in terms of the classic wild-type form, all alleles, and isotypes. The mRNA (cDNA) encoding the classic isotype has been assigned NCBI accession number NM_009020.3. The mouse Rag2 mRNA (cDNA) and coding sequence are mentioned, including the classic wild-type form as well as all alleles and isotypes.

[0139] Rat Rag2 is located on chromosome 3, 3q31 (NCBI RefSeq gene ID 295953; assembly mRatBN7.2 (GCF_015227675.2); location NC_051338.1 (87902373..87910227)). The rat Rag2 gene is mentioned in terms of the classic wild-type form, all alleles, and isotypes. The classic wild-type rat RAG2 protein has been assigned UniProt accession number G3V6K7 and NCBI accession number NP_001093998.1. The rat RAG2 protein is mentioned in terms of the classic wild-type form, all alleles, and isotypes. The mRNA (cDNA) encoding the classic isotype is assigned NCBI accession number NM_001100528.1. The rat Rag2 mRNA (cDNA) and coding sequence are mentioned in terms of the classic wild-type form, all alleles, and isotypes.

[0140] An inactivated endogenous Rag2 gene is a Rag2 gene that does not produce RAG2 protein or does not produce functional RAG2 protein. This inactivated Rag2 gene may be present in the lineage of a non-human animal (or cell or genome). The inactivation mutation in the Rag2 gene in a non-human animal (or cell or genome) may be homozygous. As an example, an inactivated endogenous Rag2 gene may contain insertions, deletions, or one or more point mutations in the endogenous Rag2 gene that result in the loss of functional RAG2 protein expression. Some inactivated endogenous Rag2 genes may contain the deletion or disruption of the entire endogenous Rag2 gene, or may contain the deletion or disruption of a segment (i.e., a portion or part) of the endogenous Rag2 gene. For example, some, most, or all of the coding sequences in the endogenous Rag2 gene may be deleted or disrupted. In one example, the 5' segment of the Rag2 gene may be deleted or disrupted (e.g., including the start codon). As an example, an inactivated endogenous Rag2 gene can be a gene in which the start codon of the endogenous Rag2 gene is missing, destroyed, or mutated to the point that the start codon is no longer functional. For example, the start codon can be destroyed by deletion or insertion within the start codon. Alternatively, the start codon can be mutated by, for example, substitution of one or more nucleotides. In another example, the 3' segment of the Rag2 gene can be missing or destroyed (e.g., including the stop codon). In another example, an internal segment of the Rag2 gene (i.e., a segment in the middle of the Rag2 gene) can be missing or destroyed. In yet another example, all coding sequences in the endogenous Rag2 gene are missing or destroyed.

[0141] The interleukin-2 receptor γ subunit (also known as interleukin-2 receptor, γ; interleukin-2 receptor, γ (severe combined immunodeficiency), isotype CRA_a; cytokine receptor shared γ subunit precursor) is encoded by the Il2rg gene (also known as interleukin-2 receptor γ subunit or IL2RG). IL2RG is a cytokine receptor subunit shared by receptor complexes of several different interleukin receptors. IL2RG is located on the surface of immature hematopoietic cells in the bone marrow. IL2RG works with other proteins to guide 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 lead to X-linked severe combined immunodeficiency, in which lymphocytes fail to develop normally. The lack of functionally mature lymphocytes impairs the immune system's ability to protect the body from infection.

[0142] The mouse Il2rg gene is located on chromosome XD; X 43.9 cM (NCBI RefSeq gene ID 16186; assembly GRCm39 (GCF_000001635.27); location NC_000086.8 (100307991..100311861, complementary)). The mouse Il2rg gene is mentioned in terms of the classic wild-type form, all alleles, and isotypes. The classic wild-type mouse IL2rG protein has been assigned UniProt accession number P34902 and NCBI accession number NP_038591.1. The mouse IL2RG protein is mentioned in terms of the classic wild-type form, all alleles, and isotypes. The mRNA (cDNA) encoding the classic isotype has been assigned NCBI accession number NM_013563.4. The mouse Il2rg mRNA (cDNA) and coding sequence are mentioned, including the classic wild-type form as well as all alleles and isotypes.

[0143] Rat Il2rg is located on chromosome Xq22 (NCBI RefSeq gene ID 140924; assembly mRatBN7.2 (GCF_015227675.2); location NC_051356.1 (66395330…66399026, complementary). The rat Il2rg gene is mentioned in terms of the classic wild-type form, all alleles, and isotypes. The classic wild-type rat IL2rG protein has been assigned UniProt accession number Q68FU6 and NCBI accession number NP_543165.1. The rat IL2RG protein is mentioned in terms of the classic wild-type form, all alleles, and isotypes. The mRNA (cDNA) encoding the classic isotype is assigned NCBI accession number NM_080889.1. The rat Il2rg mRNA (cDNA) and coding sequence are mentioned in terms of the classic wild-type form, all alleles, and isotypes.

[0144] An inactivated endogenous Il2rg gene is an Il2rg gene that does not produce IL2RG protein or does not produce functional IL2RG protein. This inactivated Il2rg gene may be present in the lineage of a non-human animal (or cell or genome). The inactivation mutation in the Il2rg gene in a non-human animal (or cell or genome) may be homozygous. As an example, an inactivated endogenous Il2rg gene may contain insertions, deletions, or one or more point mutations in the endogenous Il2rg gene that result in the loss of functional IL2RG protein expression. Some inactivated endogenous Il2rg genes may contain the deletion or disruption of the entire endogenous Il2rg gene, or may contain the deletion or disruption of a segment (i.e., a portion or part) of the endogenous Il2rg gene. For example, some, most, or all of the coding sequences in the endogenous Il2rg gene may be deleted or disrupted. In one example, the 5' segment of the Il2rg gene may be deleted or disrupted (e.g., including the start codon). As an example, an inactivated endogenous Il2rg gene can be one in which the start codon is missing, destroyed, or mutated, rendering the start codon nonfunctional. For example, the start codon can be destroyed by deletion or insertion within the start codon. Alternatively, the start codon can be mutated, for example, by substitution of one or more nucleotides. In another example, the 3' segment of the Il2rg gene can be missing or destroyed (e.g., including the stop codon). In another example, an internal segment of the Il2rg gene (i.e., a segment in the middle of the Il2rg gene) can be missing or destroyed. In yet another example, the entire coding sequence of the endogenous Il2rg gene is missing or destroyed.

[0145] Humanized Sirpa locus

[0146] In some respects, the genetically modified nonhuman animals described herein also express human SIRPA proteins or humanized SIRPA proteins encoded by nucleic acids operatively linked to the Sirpa promoter.

[0147] Signal regulatory 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 (e.g., see 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 their respective ligands and the types of signal transduction 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 inhibitory motif (ITIM). SIRPA expression has also been observed on neurons. Reported SIRPA ligands include the most prominent CD47, but also surfactant proteins A and D. The effects of SIRPA, particularly its inhibitory role in macrophage phagocytosis of host cells, have been investigated. For example, CD47 binding to SIRPA on macrophages triggers an inhibitory signal that negatively regulates phagocytosis. Alternatively, positive signaling effects mediated by 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).

[0148] The polypeptide sequence of wild-type human SIRPA and the nucleic acid sequence encoding wild-type human SIRPA can be found in Genbank accessions NP_001035111.1 and NM_001040022.1 (isotype 1 and transcriptotype 1); NP_001035112.1 and NM_001040023.2 (isotype 1 and transcriptotype 2); NP_001317657.1 and NM_001330728.1 (isotype 2 and transcriptotype 4); and NP_542970.1 and NM_080792.3 (isotype 1 and transcriptotype 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 may be located on chromosome 20 of the human genome (NC_000020.11 (1894167-1940592)). In some embodiments, the human SIRPA protein is encoded by exons 2 to 9 at this locus. Therefore, in some embodiments, the nucleic acid sequence containing the coding sequence of human SIRPA includes one or more exons 2-9 of the human SIRPA gene. In some cases, the nucleic acid sequence also includes aspects of the human SIRPA genomic locus, such as introns, 3' and / or 5' untranslated sequences (UTRs). In some cases, the nucleic acid sequence includes the entire region of the human SIRPA genomic locus. In some cases, the nucleic acid sequence includes exons 2-4 of the human SIRPA genomic locus.

[0149] Exemplary humanized Sirpa sequences are listed in Table 3. For protein sequences, signal peptides are underlined, and transmembrane and cytoplasmic sequences are shown in italics. 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.

[0150] Table 3

[0151] Humanized Sirpa protein (SEQ ID NO: 12)

[0152]

[0153] In some embodiments, the non-human animals described herein express humanized Sirpa proteins on the surface of non-human animal immune cells (e.g., myeloid cells) by genetically modifying an endogenous locus encoding the Sirpa protein. Suitable examples described herein include rodents, such as mice.

[0154] In some embodiments, the humanized Sirpa gene comprises genetic material from a heterologous species (e.g., human), wherein the humanized Sirpa gene encodes a Sirpa protein, the Sirpa protein comprising a coding portion of the genetic material from the heterologous species. In some embodiments, the humanized Sirpa gene of this disclosure comprises genomic DNA of a heterologous species corresponding to the extracellular portion of the SIRPA protein expressed on the cell membrane. Methods for preparing nonhuman animals, nonhuman embryos, cells, and targeted constructs comprising the humanized Sirpa gene are also provided.

[0155] In some embodiments, the endogenous nonhuman animal (e.g., rodent) Sirpa gene is deleted. In some embodiments, the endogenous nonhuman animal (e.g., rodent) Sirpa gene is altered, wherein a portion of the endogenous nonhuman animal (e.g., rodent) Sirpa gene is replaced by a heterologous sequence (e.g., all or part of a human SIRPA sequence). In some embodiments, all or substantially all of the endogenous nonhuman animal (e.g., rodent) Sirpa gene is replaced by a heterologous gene (e.g., human SIRPA gene). In some embodiments, a portion of a heterologous SIRPα gene is inserted into the endogenous nonhuman Sirpa gene at the endogenous nonhuman animal (e.g., rodent) Sirpa gene locus. In some embodiments, the heterologous gene is a human gene. In some embodiments, one of the two copies of the endogenous nonhuman animal (e.g., rodent) Sirpa gene is modified or humanized, thereby producing a nonhuman animal that is heterozygous in relation to the humanized Sirpa gene. In other embodiments, a non-human animal is provided that is homozygous for the humanized Sirpa gene. In some embodiments, the entire endogenous non-human animal (e.g., rodent) Sirpa gene is replaced with a portion of a heterologous gene (e.g., a portion of the human SIRPA gene), such that the genetically modified non-human animal (e.g., rodent) expresses a functional fragment of the full-length human SIRPA polypeptide (e.g., the extracellular domain of the human SIRPA polypeptide).

[0156] The non-human animals disclosed herein 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 possessing a heterologous SIRP gene. SIRPα genes that have been substituted, inserted, or modified at the Sirpa locus in endogenous non-human animals (e.g., rodents) can be detected using various methods, including, for example, PCR, Western blotting, Southern blotting, restriction fragment length polymorphism (RFLP), or allele gain or loss assays. In some embodiments, the non-human animals are heterozygous with respect to the humanized Sirpa gene.

[0157] In various embodiments, the humanized Sirpa gene according to this disclosure includes the SIRPα gene having second, third, and fourth exons, each exon having a 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 higher) identical to the second, third, and fourth exons present in the human SIRPA gene.

[0158] In various embodiments, the humanized Sirpa gene according to this disclosure includes the SIRPα gene, which 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 higher) of the nucleotide coding sequence (e.g., cDNA sequence) that is identical to nucleotides 352-1114 present in the human SIRPA cDNA sequence.

[0159] In various embodiments, the humanized Sirpa protein produced by the non-human animal of this disclosure has an extracellular portion having a 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 higher) identical to the extracellular portion of the human SIRPA protein.

[0160] In various embodiments, the humanized Sirpa protein produced by the non-human animal of this disclosure has an extracellular portion having a 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 higher) identical to amino acid residues 28-362 present in the human SIRPA protein.

[0161] 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 higher) identical to the amino acid sequence of the humanized Sirpa protein shown in Table 3.

[0162] Compositions and methods are provided for preparing non-human animals expressing humanized Sirpa proteins (including specific polymorphic forms or allelic variants (e.g., single amino acid differences)), including compositions and methods for preparing non-human animals expressing such proteins from human promoters and human regulatory sequences. In some embodiments, compositions and methods are also provided for preparing non-human animals expressing such proteins from endogenous promoters and endogenous regulatory sequences. These methods include inserting genetic material encoding all or part of the human SIRPA protein into the genome of a non-human animal at a precise location corresponding to an endogenous non-human animal (e.g., rodent) Sirpa gene, thereby creating a humanized Sirpa gene expressing all or part of the human SIRPA protein. In some embodiments, the method includes inserting genomic DNA corresponding to exons 2-4 of the human SIRPA gene into an endogenous non-human animal (e.g., rodent) Sirpa gene of a non-human animal, thereby creating a humanized gene encoding a Sirpa protein containing a human portion of the amino acids encoded by the inserted exons.

[0163] In various embodiments, the humanized Sirpa gene approach involves relatively minimal modification of the endogenous gene and generates natural Sirpa-mediated signal transduction in nonhuman animals. Therefore, in such embodiments, Sirpa gene modification does not affect other surrounding genes or other endogenous nonhuman animal (e.g., rodent) Sirp genes. Furthermore, in various embodiments, the modification does not affect the assembly of the functional receptor on the plasma membrane and maintains normal effector function by binding and subsequent signal transduction through the cytoplasmic portion of the receptor unaffected by the modification.

[0164] In addition to mice with a humanized Sirpa gene as described herein, other genetically modified non-human animals containing a humanized Sirpa gene are also provided herein. In some embodiments, such non-human animals contain a humanized Sirpa gene operatively linked to an endogenous Sirpa promoter. In some embodiments, such non-human animals express a humanized Sirpa protein from an endogenous locus, wherein the humanized Sirpa protein contains amino acid residues 28-362 of the human SIRPA protein.

[0165] Humanized Sirpa peptides, loci encoding humanized Sirpa peptides, and non-human animals expressing humanized Sirpa peptides are described in U.S. Patent Nos. 11,019,810, WO 2014 / 039782, WO 2014 / 071397, WO 2016 / 168212, and U.S. Patent No. 10,918,095, each of which is incorporated herein by reference in its entirety.

[0166] Humanized GM-CSF locus

[0167] In some respects, the genetically modified non-human animals described herein also express human GM-CSF protein encoded by nucleic acids operatively linked to the GM-CSF promoter. By human GM-CSF protein, we mean that the protein is human GM-CSF or substantially identical to human GM-CSF, for example, having 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with human GM-CSF, for example, 97%, 98%, or 99% identity with human GM-CSF. Therefore, the nucleic acid sequence encoding human GM-CSF protein is a polynucleotide containing the coding sequence of human GM-CSF protein, i.e., human GM-CSF or a protein substantially identical to human GM-CSF.

[0168] GM-CSF is a cytokine crucial for the development and function of myeloid cells. GM-CSF does not exhibit cross-reactivity between humans and mice. GM-CSF is highly expressed in the lungs and is essential for pulmonary homeostasis, as demonstrated by pulmonary alveolar proteinosis (PAP) in GM-CSF-KO mice, a condition characterized by protein accumulation in the lungs due to a defect in surfactant clearance. Alveolar macrophages from GM-CSF-KO mice exhibit terminal differentiation defects, leading to impaired innate immunity against pathogens in the lungs. GM-CSF also stimulates the proliferation of human alveolar macrophages (AM) in vitro. GM-CSF is essentially non-essential for homeostatic hematopoiesis. Conversely, GM-CSF is essential for inflammatory responses, such as macrophage production of pro-inflammatory cytokines and the mobilization and recruitment of monocytes. GM-CSF is also crucial for protective immunity against a range of pathogens, including Mycobacterium tuberculosis. In particular, GM-CSF KO mice infected with Mycobacterium tuberculosis do not develop granulomas, a hallmark of tuberculosis.

[0169] The polypeptide sequence of human GM-CSF and the nucleic acid sequence encoding human GM-CSF can be found in GenBank accessions NP_000749.2 and NM_000758.4, respectively. The genomic locus encoding the human GM-CSF protein is located on human chromosome 5: NG_033024.1 (4998-7379). The protein sequence is encoded by exons 1 to 4 at this locus. Therefore, the nucleic acid sequence containing the coding sequence of human GM-CSF contains one or more exons 1-4 of the human GM-CSF gene. In some cases, the nucleic acid sequence also contains aspects of the human GM-CSF genomic locus, such as introns, 3' and / or 5' untranslated sequences (UTRs). In some cases, the nucleic acid sequence contains the entire region of the human GM-CSF genomic locus.

[0170] In some embodiments, in the genetically modified nonhuman animals provided herein, the nucleic acid sequence encoding the human GM-CSF protein is operatively linked to one or more regulatory sequences of the nonhuman animal (e.g., mouse) GM-CSF gene. The nonhuman animal (e.g., mouse) GM-CSF regulatory sequences are those sequences at nonhuman animal (e.g., mouse) GM-CSF genomic loci that regulate nonhuman animal (e.g., mouse) GM-CSF expression, such as 5' regulatory sequences, such as the GM-CSF promoter, the GM-CSF 5' untranslated region (UTR), etc.; 3' regulatory sequences, such as the 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 is available in 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 computational methods (e.g., by consulting the aforementioned GenBank accession number via the UCSC Genome Browser (genome.ucsc.edu)) or by the experimental methods described in the art. In some cases, such as 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 operatively linked to the human GM-CSF coding sequence are endogenous or natural to the non-human animal (e.g., mouse) genome, i.e., they were present in the non-human animal (e.g., mouse) genome prior to integration of the human nucleic acid sequence.

[0171] In some cases, genetically modified non-human animals expressing human GM-CSF protein are generated by randomly integrating or inserting a human nucleic acid sequence encoding the human GM-CSF protein or a fragment thereof (i.e., the "human GM-CSF nucleic acid sequence" or "human GM-CSF sequence") into the genome of the 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 cases, genetically modified non-human animals expressing human GM-CSF protein are generated by targeted integration or insertion of the human GM-CSF nucleic acid sequence into the genome of the non-human animal, for example, through homologous recombination. In homologous recombination, a polynucleotide is inserted into a target locus in the host genome while simultaneously removing host genomic material from the target locus, such as 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more of genomic material. Therefore, for example, in a genetically modified non-human animal (e.g., a mouse) containing a nucleic acid sequence encoding a human GM-CSF protein created by targeting a human GM-CSF nucleic acid sequence to a non-human animal GM-CSF (e.g., a mouse) locus, the human GM-CSF nucleic acid sequence may replace some or all of the non-human animal (e.g., mouse) sequence at the GM-CSF locus, such as exons and / or introns. In some such cases, the human GM-CSF nucleic acid sequence is integrated into the non-human animal (e.g., mouse) GM-CSF locus such that the expression of the human GM-CSF sequence is regulated by a natural or endogenous regulatory sequence at the non-human animal (e.g., mouse) GM-CSF locus. In other words, the regulatory sequence operatively linked to the nucleic acid sequence encoding the human GM-CSF protein is a natural GM-CSF regulatory sequence at the non-human animal (e.g., mouse) GM-CSF locus.

[0172] In some cases, integration of a human GM-CSF sequence does not affect the transcription of the gene into which the human GM-CSF sequence has been integrated. For example, if the human GM-CSF sequence is integrated as an inpeptide into a coding sequence, or if the human GM-CSF sequence contains a 2A peptide, the human GM-CSF sequence will be transcribed and translated simultaneously with the gene into which the human GM-CSF sequence has been integrated. In other cases, integration of a human GM-CSF sequence can interrupt the transcription of the gene into which the human GM-CSF sequence has been integrated. For example, after integration of a human GM-CSF sequence via homologous recombination, some or all of the coding sequence at the integration locus can be removed, thereby transcribing the human GM-CSF sequence instead. In some such cases, integration of a human GM-CSF sequence produces null mutations and thus null alleles. Null alleles are mutated copies of a gene that completely lack the gene's normal function. This can result from the 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, null alleles include the deletion of an entire locus.

[0173] In some cases, genetically modified non-human animals (e.g., mice) expressing the human GM-CSF protein contain 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 will contain the nucleic acid sequence, while the other is an endogenous allele. For example, as described above, in some cases, the human GM-CSF nucleic acid sequence is integrated into the non-human animal (e.g., mouse) GM-CSF locus, resulting in a null allele of the non-human animal (e.g., mouse) GM-CSF. In some such embodiments, humanized GM-CSF mice may be heterozygous for the nucleic acid sequence, i.e., humanized GM-CSF mice contain one null allele (containing the nucleic acid sequence) and one endogenous GM-CSF allele (wild-type or other) from the non-human animal (e.g., mouse) GM-CSF. In other cases, genetically modified non-human animals (e.g., mice) expressing the human GM-CSF protein contain two copies of the nucleic acid sequence encoding the human GM-CSF protein. For example, non-human animals (e.g., mice) may be homozygous for the nucleic acid sequence, meaning that both alleles at the locus in the diploid genome will contain the nucleic acid sequence. In other words, genetically modified non-human animals (e.g., mice) expressing human GM-CSF protein contain two invalid alleles of mouse GM-CSF (alleles containing the nucleic acid sequence).

[0174] Although this article discusses extensively implementation schemes using the human GM-CSF gene in mice, other non-human animals containing the human GM-CSF gene (e.g., rodents, such as rats) are also provided.

[0175] Human GM-CSF peptide, the locus encoding human GM-CSF peptide, and non-human animals expressing human GM-CSF peptide are described in WO2011 / 044050, WO 2014 / 039782 and WO 2014 / 071397, each of which is incorporated herein by reference.

[0176] Humanized IL-3 locus

[0177] In some respects, the genetically modified non-human animals described herein also express human IL-3 protein encoded by nucleic acids operatively linked to the IL-3 promoter. By human IL-3 protein, we mean a protein that is human IL-3 or substantially identical to human IL-3, for example, having 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with human IL-3, for example, 97%, 98%, or 99% identity with human IL-3. Therefore, the nucleic acid sequence encoding human IL-3 protein is a polynucleotide containing the coding sequence of human IL-3 protein, i.e., human IL-3 or a protein substantially identical to human IL-3.

[0178] Similar to GM-CSF, IL-3 is a cytokine crucial for myeloid cell development and function. IL-3 exhibits no cross-reactivity between humans and mice. IL-3 stimulates early hematopoietic progenitor cells in vitro, but is not essential for homeostasis in vivo. However, it is necessary for an effective DTH response in vivo, along with GM-CSF. IL-3 also specifically stimulates the proliferation of alveolar macrophages (AM) in vitro.

[0179] The polypeptide sequence of human IL-3 and the nucleic acid sequence encoding human IL-3 are available in Genbank accessions NP_000579.2 and NM_000588.4. The genomic locus encoding the human IL-3 protein is located on human chromosome 5, GRCh38.p14;NC_000005.10 (132060655-132063204). The protein sequence is encoded by exons 1 to 5 at this locus. Therefore, the nucleic acid sequence containing the coding sequence of human IL-3 contains one or more exons 1-5 of the human IL-3 gene. In some cases, the nucleic acid sequence also contains aspects of the human IL-3 genomic locus, such as introns, 3' and / or 5' untranslated sequences (UTRs). In some cases, the nucleic acid sequence contains the entire region of the human IL-3 genomic locus.

[0180] In some embodiments, in the genetically modified nonhuman animals provided herein, the nucleic acid sequence encoding the human IL-3 protein is operatively linked to one or more regulatory sequences of the nonhuman animal (e.g., mouse) IL-3 gene. The nonhuman animal (e.g., mouse) IL-3 regulatory sequences are those sequences at nonhuman animal (e.g., mouse) IL-3 genomic loci that regulate nonhuman animal (e.g., mouse) IL-3 expression, such as 5' regulatory sequences, such as the IL-3 promoter, the IL-3 5' untranslated region (UTR), etc.; 3' regulatory sequences, such as the 3' UTR; and enhancers, etc. For example, mouse IL-3 is located on chromosome 11, GRCm39, NC_000077.7, approximately at position c54158105-54155911, and the mouse IL-3 coding sequence is available in 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 computational methods (e.g., by consulting the aforementioned GenBank accession number in the UCSC Genome Browser (genome.ucsc.edu)) or by the experimental methods described in the art. In some cases, such as 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 operatively linked to the human IL-3 coding sequence are endogenous or natural to the non-human animal (e.g., mouse) genome, i.e., they were present in the non-human animal (e.g., mouse) genome prior to integration of the human nucleic acid sequence.

[0181] In some cases, genetically modified non-human animals expressing human IL-3 protein are generated by randomly integrating or inserting a human nucleic acid sequence encoding human IL-3 protein or a fragment thereof (i.e., the “human IL-3 nucleic acid sequence” or “human IL-3 sequence”) into the genome of the non-human animal. Typically, in such embodiments, the location of the nucleic acid sequence encoding human IL-3 protein in the genome is unknown. In other cases, genetically modified non-human animals expressing human IL-3 protein are generated by targeted integration or insertion of the human IL-3 nucleic acid sequence into the genome of the non-human animal, for example, through homologous recombination. In homologous recombination, a polynucleotide is inserted into a target locus in the host genome while simultaneously removing host genomic material from the target locus, such as 50 base pairs (bp) or more, 100 bp or more, 200 bp or more, 500 bp or more, 1 kB or more, 2 kB or more, 5 kB or more, 10 kB or more, 15 kB or more, 20 kB or more, or 50 kB or more genomic material. Therefore, for example, in a genetically modified non-human animal (e.g., a mouse) containing a nucleic acid sequence encoding the human IL-3 protein created by targeting a human IL-3 nucleic acid sequence to a non-human animal IL-3 (e.g., a mouse) locus, the human IL-3 nucleic acid sequence may replace some or all of the non-human animal (e.g., mouse) sequence at the IL-3 locus, such as exons and / or introns. In some such cases, the human IL-3 nucleic acid sequence is integrated into the non-human animal (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 animal (e.g., mouse) IL-3 locus. In other words, the regulatory sequence operatively linked to the nucleic acid sequence encoding the human IL-3 protein is a native IL-3 regulatory sequence at the non-human animal (e.g., mouse) IL-3 locus.

[0182] In some cases, the integration of a human IL-3 sequence does not affect the transcription of the gene into which the human IL-3 sequence has been integrated. For example, if the human IL-3 sequence is integrated as an inpeptide into a coding sequence, or if the human IL-3 sequence contains a 2A peptide, the human IL-3 sequence will be transcribed and translated simultaneously with the gene into which the human IL-3 sequence has been integrated. In other cases, the integration of a human IL-3 sequence disrupts the transcription of the gene into which the human IL-3 sequence has been integrated. For example, after the integration of a human IL-3 sequence via homologous recombination, some or all of the coding sequence at the integration locus can be removed, thereby allowing the transcription of the human IL-3 sequence instead. In some such cases, the integration of a human IL-3 sequence produces null mutations, and thus null alleles. Null alleles are mutated copies of a gene that completely lack the gene's normal function. This can result from the 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, null alleles include the deletion of an entire locus.

[0183] In some cases, genetically modified non-human animals (e.g., mice) expressing human IL-3 protein contain 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 will contain the nucleic acid sequence, while the other will be an endogenous allele. For example, as described above, in some cases, the human IL-3 nucleic acid sequence is integrated into the non-human animal (e.g., mouse) IL-3 locus, resulting in a null allele for non-human animal (e.g., mouse) IL-3. In some such embodiments, humanized IL-3 mice may be heterozygous for the nucleic acid sequence, i.e., humanized IL-3 mice contain one null 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 other cases, genetically modified non-human animals (e.g., mice) expressing human IL-3 protein contain two copies of the nucleic acid sequence encoding the human IL-3 protein. For example, non-human animals (e.g., mice) may be homozygous for the nucleic acid sequence, meaning that both alleles at the locus in the diploid genome will contain the nucleic acid sequence. In other words, genetically modified non-human animals (e.g., mice) expressing human IL-3 protein contain two invalid alleles of mouse IL-3 (alleles containing the nucleic acid sequence).

[0184] Although this article discusses implementation schemes using the human IL-3 gene in mice extensively, other non-human animals containing the human IL-3 gene (e.g., rodents, such as rats) are also provided.

[0185] Human IL-3 peptide, the locus encoding human IL-3 peptide, and non-human animals expressing human IL-3 peptide are described in WO2011 / 044050, WO 2014 / 039782 and WO 2014 / 071397, each of which is incorporated herein by reference.

[0186] Genetically modified non-human animals and ES cells

[0187] In some respects, this document provides genetically modified nonhuman 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 nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) a Flt3 ligand (Flt3l) gene comprising a nonhuman animal portion and a human portion operatively linked to the Flt3l promoter, and optionally one or more of the humanized loci disclosed herein, and genetically modified nonhuman animal ES cells for the preparation of such nonhuman animals.

[0188] In some respects, this document provides genetically modified nonhuman animal (e.g., rodent, such as rat or mouse) and nonhuman animal (e.g., rodent, such as rat or mouse) ES cells containing 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 nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) a Flt3 ligand (Flt3l) gene comprising a nonhuman animal portion and a human portion operatively linked to the Flt3l promoter, and optionally one or more of the modified loci described herein. In some embodiments, this document provides genetically modified nonhuman animal (e.g., mouse) and nonhuman animal (e.g., mouse) ES cells that contain in their germline and / or genome: (i) Rag2 gene knockout; (ii) IL2rg gene knockout; (iii) a homozygous null mutation in the nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) a Flt3 ligand (Flt3l) gene comprising a nonhuman animal portion and a human portion operatively linked to the Flt3l promoter; and optionally one or more of the modified loci described herein. For example, in some embodiments, the nonhuman animal or ES cell contains the humanized Sirpa locus provided herein in its germline and / or genome. In some embodiments, the nonhuman animal or ES cell contains the GM-CSF locus provided herein in its germline and / or genome. In some embodiments, the nonhuman animal or ES cell contains the IL-3 locus provided herein in its germline and / or genome. In some embodiments, the non-human animal or ES cell is heterozygous for one or more loci (e.g., genetically modified loci) provided herein. In some embodiments, the non-human animal or ES cell is homozygous for one or more loci (e.g., genetically modified loci) provided herein.

[0189] 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 animal described herein may be selected from mice, rats, rabbits, pigs, cattle (e.g., dairy cows, bulls, buffalo), deer, sheep, goats, llamas, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). For non-human animals from which suitable genetically modified ES cells are not readily available, other methods may be used to prepare non-human animals containing the genetic modifications described herein. Such methods include, for example, modifying the genome of a non-ES cell (e.g., fibroblasts or induced pluripotent cells) and transferring the modified genome into a suitable cell (e.g., an oocyte) using nuclear transfer, and gestating the modified cell (e.g., a modified oocyte) in a non-human animal under suitable conditions to form an embryo.

[0190] In some embodiments, the non-human animal is a mammal. In some embodiments, the non-human animal is a small mammal, such as animals from the superfamily Dipodoidea or Muroidea. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent is a mouse, rat, or hamster. In some embodiments, the rodent is selected from the superfamily Muroidea. In some embodiments, the non-human animal is selected from the following families: Calomyscidae (e.g., mouse-like hamster), Cricetidae (e.g., hamster, New World rat and mouse, vole), Muridae (e.g., true mouse and rat, gerbil, tufted rat, crested rat), Nesomyidae (e.g., climbing rat, rock rat, white-tailed rat, Falkland's rat and mouse), Platacanthomyidae (e.g., tufted dormice), and Spalacidae (e.g., mole, bamboo rat, and vole). In some embodiments, the rodent is selected from true mice or rats (Muspidae), gerbils, spiny rats, and crested rats. In some embodiments, the mouse is selected from members 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.

[0191] In some embodiments, the non-human animal is a C57BL strain of mouse. 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 129 strain of mouse. In some embodiments, the 129 strain 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 hybrid of the 129 strain and the C57BL strain. In some embodiments, the mouse is a hybrid of the 129 strain and / or the C57BL / 6 strain. In some embodiments, the 129 strain in the hybrid is the 129S6 (129 / SvEvTac) strain. In some embodiments, the mouse is a BALB strain (e.g., BALB / c). In some embodiments, the mouse is a hybrid of a 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 above strains.

[0192] In some embodiments, the non-human animal provided herein is a rat. In some embodiments, the rat is selected from Wistar rats, the LEA strain, the Sprague Dawley strain, the 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.

[0193] In some embodiments, the genetically modified nonhuman animal or ES cells contain multiple loci provided herein, such as multiple genetically modified loci provided herein, in their genome and / or germline. For example, in some embodiments, the nonhuman animal or ES cells provided herein contain 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 nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) a Flt3 ligand (Flt3l) gene containing a nonhuman animal portion and a human portion operatively linked to the Flt3l promoter. In some embodiments, the nonhuman animal or ES cell contains in its germline and / or genome (i) a homozygous null mutation in the Rag1 and / or Rag2 genes (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 nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene; (iv) a Flt3 ligand (Flt3l) gene comprising a nonhuman animal portion and a human portion operatively linked to the Flt3l promoter; and (v) the humanized Sirpa locus provided herein. In some embodiments, the non-human animal or ES cell contains, in its germline and / or genome, (i) a homozygous null mutation in the Rag1 and / or Rag2 genes (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; (iv) a Flt3 ligand (Flt3l) gene comprising a non-human animal portion and a human portion operatively linked to the Flt3l promoter; (v) the humanized Sirpa locus provided herein; and (vi) the humanized GM-CSF locus provided herein and / or the humanized IL-3 locus provided herein.

[0194] In some aspects, the genetically modified non-human animals do not express the Flt3 polypeptide. In some aspects, the genetically modified non-human animals express both a soluble form of the Flt3L polypeptide and a membrane-bound form of the Flt3L polypeptide, optionally the soluble form and the membrane-bound form of the Flt3L polypeptide comprising a signal peptide and a cytokine-like core domain of the human FLT3L polypeptide.

[0195] In some aspects, the genetically modified non-human animals express one or more human polypeptides or humanized polypeptides encoded by the humanized loci provided herein. For example, in some embodiments, the non-human animals express human SIRPA polypeptides or humanized SIRPA polypeptides. In some embodiments, the non-human animals express human GM-CSF polypeptides or humanized GM-CSF polypeptides. In some embodiments, the non-human animals express human IL-3 polypeptides or humanized IL-3 polypeptides.

[0196] The genetically modified nonhuman animal and ES cells can be generated using any suitable method known in the art. For example, such genetically modified nonhuman animal ES cells can be generated using VELOCIGENE® technology, described in U.S. Patent Nos. 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. Modification can also be performed using genome-targeting nuclease systems, such as the CRISPR / Cas system, transcription activator-like effector nuclease (TALEN) system, or zinc finger nuclease (ZFN) system. In some embodiments, modification is performed using a CRISPR / Cas system, as described, for example, in U.S. Patent Applications Nos. 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 according to US2014 / 0235933 A1 (Regeneron Pharmaceuticals, Inc.), US 2014 / 0310828 A1 (Regeneron Pharmaceuticals, Inc.), Tong et al. (2010) Nature 467:211-215, and Tong et al. (2011) NatProtoc. 6(6): doi:10.1038 / nprot.2011.338 (all of which are incorporated herein by reference in their entirety). An exemplary method for preparing such genetically modified nonhuman animals and ES cells is also provided in Example 1.

[0197] Then, non-human animals can be generated using the ES cells described herein, using methods known in the art. For example, genetically modified mice can be generated using the VELOCIMOUSE® method with the mouse non-human ES cells described herein, as described in U.S. Patent No. 7,294,754 and Poueymirou et al., Nature Biotech 25:91-99 (2007), each of which is incorporated herein by reference. The resulting mice can be bred to homozygosity.

[0198] Methods for preparing genetically modified non-human animals and ES cells

[0199] In some aspects, this document provides methods for preparing nonhuman animal (e.g., mouse or rat) and ES cells comprising one or more genetically modified loci provided herein. For example, in some embodiments, this document provides methods for preparing nonhuman animal (e.g., mouse or rat) and ES cells 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 nonhuman animal FMS-like tyrosine kinase 3 (Flt3) gene; and (iv) a Flt3 ligand (Flt3l) gene comprising a nonhuman animal portion and a human portion operatively linked to the Flt3l promoter. In some embodiments, methods are provided herein for preparing non-human animals (e.g., mice or rats) and ES cells 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) a Flt3 ligand (Flt3l) gene comprising a non-human animal portion and a human portion operatively linked to the Flt3l promoter, and further comprising the humanized Sirpa locus provided herein. In some embodiments, methods are provided herein for preparing non-human animals (e.g., mice or rats) and ES cells that also comprise the humanized GM-CSF locus and / or the humanized IL-3 locus provided herein. Exemplary methods for preparing genetically modified nonhuman animal and ES cells provided herein are described in the specification, examples and / or figures herein.

[0200] Generating nonhuman animals containing null mutations in the nonhuman animal Flt3 gene, and / or nonhuman animals containing the Flt3 ligand (Flt3l) gene (containing a nonhuman animal portion and a human portion operatively linked to the Flt3l promoter) can be accomplished using any convenient method for preparing genetically modified animals, such as those known in the art or as described herein in Example 1.

[0201] Generating nonhuman animals containing nucleic acid sequences encoding human or humanized proteins (e.g., hSIRPA, hGM-CSF, or hIL-3) can be accomplished using any convenient method for preparing genetically modified animals, such as those known in the art or as described herein.

[0202] For example, nucleic acids encoding the human protein or humanized proteins (e.g., hSIRPA, hGM-CSF, and / or hIL-3) can be incorporated into a recombinant vector in a form suitable for insertion into the host cell genome and expression of the human protein in non-human host cells. In various embodiments, the recombinant vector may contain one or more regulatory sequences operatively linked to the nucleic acid encoding the human protein, in a manner that allows transcription of the nucleic acid into mRNA and translation of the mRNA into the human protein, as described above. It should be understood that the design of the vector may depend on factors such as the choice of host cells to be transfected and / or the amount of human protein to be expressed.

[0203] The human nucleic acid sequence can then be introduced into animal cells using any of a variety of methods to produce genetically modified animals expressing the human gene. 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 that can be used to generate genetically modified animals include, but are not limited to, Sundberg and Ichiki (2006, Genetically Engineered Mice Handbook, CRC Press), Hofker and van Deursen (2002, Genetically modified Mouse Methods and Protocols, Humana Press), Joyner (2000, Gene Targeting: A Practical Approach, Oxford University Press), Turksen (2002, Embryonic stem cells: Methods and Protocols in Methods Mol Biol, Humana Press), Meyer et al. (2010, Proc. Nat. Acad. Sci. USA 107: 15022-15026) and Gibson (2004, A Primer Of Genome Science 2nd Edition, Sunderland, Massachusetts: Sinauer), US Patent No. 6,586,251, and Rathinam et al. (2011, Blood The methods described in 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).

[0204] For example, genetically modified animals of this subject matter can be created by introducing nucleic acids encoding human proteins into oocytes (e.g., via microinjection) and allowing the oocytes to develop in a female foster animal. In a preferred embodiment, the expression is injected into fertilized oocytes. Fertilized oocytes can be collected from superovulated females the day after mating and injected with the expression construct. The injected oocytes are cultured overnight or directly transferred to the oviducts of pseudopregnant females at 0.5 days post-mating. Methods for superovulation, oocyte collection, expression construct injection, and embryo transfer are known in the art and described in *Manipulating the Mouse Embryo* (2002, *A Laboratory Manual*, 3rd edition, Cold SpringHarbor Laboratory Press). The presence of the introduced nucleic acids in offspring can be assessed by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing, etc.) or protein analysis (e.g., ELISA, Western blotting, etc.).

[0205] 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 known methods (e.g., electroporation, calcium phosphate precipitation, liposome transfection, etc.). The presence of the introduced nucleic acid in the cells can be assessed by DNA analysis (e.g., PCR, Southern blotting, DNA sequencing, etc.) or protein analysis (e.g., ELISA, Western blotting, etc.). Cells incorporating the expression construct can then be introduced into preimplantation embryos. For a detailed description of the compositions and methods known in the art suitable for use in this 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, 7,294,754, and Kraus et al. (2010, Genesis 48:394-399).

[0206] Furthermore, as described in some of the examples below, nucleic acid constructs can be constructed using VELOCIGENE® genetic engineering technology (see, for example, 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 (such as ES cells), and allele loss and allele gain assays can be used to identify the correctly targeted clones (Valenzuela et al., ibid.); the correctly targeted ES cells can be used as donor ES cells to be introduced into 8-cell stage mouse embryos using the VELOCIMOUSE® method (see, for example, 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). In addition, genetically modified rat ES cells and rats can be prepared according to US 2014 / 0235933A1 (Regeneron Pharmaceuticals, Inc.), US 2014 / 0310828 A1 (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 references are incorporated herein by reference in their entirety).

[0207] In some implementations, the genetically modified founder animal can be bred with other animals carrying one or more gene modifications. For example, the genetically modified nonhuman animals (e.g., rodents, such as rats or mice) provided herein can be produced by breeding the Flt3-deficient nonhuman animals provided herein with nonhuman animals containing the Flt3 ligand (Flt3l) gene (comprising a nonhuman portion and a human portion operatively linked to the Flt3l promoter). These genetically modified nonhuman animals (e.g., rodents, such as rats or mice) can be further bred with other genetically modified nonhuman animals carrying other gene modifications, including the introduction of fully human or humanized genes, such as hSirpa knock-in mice, hIL-3 knock-in mice, hGM-CSF knock-in mice, etc., or bred with gene knockout animals, such as nonhuman animals lacking one or more proteins, or those not expressing one or more of their genes, such as Rag1-deficient animals, Rag2-deficient animals, or IL-2rg-deficient animals.

[0208] In another embodiment, stem cells, such as ES cells, can be generated such that they contain several genetic modifications (e.g., humanization or gene deletion as described herein), and such stem cells can be introduced into an embryo to produce a genetically modified animal with several genetic modifications.

[0209] As described above, in some embodiments, the genetically modified nonhuman animal is an immunodeficient animal. Immunodeficient nonhuman animals containing one or more human or humanized proteins (e.g., hSIRPA, hIL-3, and / or hGM-CSF) can be generated using any convenient method for generating genetically modified animals (e.g., those known in the art or those described herein). For example, genetically modified immunodeficient animals can be generated by introducing nucleic acids encoding human proteins into oocytes or stem cells containing mutant SCID gene alleles or Rag1 and / or Rag2 and Il2rg null alleles, which, when homozygous, will result in immunodeficiency, as described above and in more detail in the working embodiments herein. Mice are then generated from the modified oocytes or ES cells using methods such as those described herein and those known in the art, and mated to produce immunodeficient mice containing the desired genetic modifications. As another example, genetically modified non-human animals can be generated in an immune-active background and then hybridized with animals containing mutant gene alleles, which, when the alleles are hemizygous or homozygous, will result in immunodeficiency. The offspring can then be mated to generate immunodeficient animals expressing at least one target human protein.

[0210] In some embodiments, genetically modified mice are treated to eliminate any endogenous hematopoietic cells that may be present in the mice. In one embodiment, the treatment includes irradiating the genetically modified mice. In a specific embodiment, newborn genetically modified mouse pups are irradiated with a sublethal dose. In a specific embodiment, newborn pups are irradiated with 2 × 200 cGy at four-hour intervals.

[0211] Various embodiments of the present invention provide genetically modified animals containing human nucleic acids in substantially all of their cells, as well as genetically modified animals containing human nucleic acids in some but not all of their cells. In some cases (e.g., targeted recombination), one copy of human nucleic acid will be integrated into the genome of the genetically modified animal. In other cases (e.g., random integration), multiple copies of human nucleic acid (adjacent or distant from each other) may be integrated into the genome of the genetically modified animal.

[0212] Therefore, in some embodiments, the genetically modified nonhuman animal of this subject matter may be an immunodeficient animal whose genome contains nucleic acid encoding a human polypeptide operatively linked to a corresponding nonhuman animal promoter, wherein the animal expresses the encoded human polypeptide. In other words, the genetically modified immunodeficient nonhuman animal of this subject matter contains a genome containing nucleic acid encoding at least one human polypeptide, wherein the nucleic acid is operatively linked to a corresponding nonhuman promoter and a polyadenylation signal, and wherein the animal expresses the encoded human polypeptide.

[0213] U.S. Patent Nos. 11,019,810 and WO 2011 / 044050 (each incorporated herein by reference) describe other methods for generating genetically modified nonhuman animals containing a genome that includes nucleic acids encoding one or more human proteins, such as hSIRPA, hIL-3, and / or hGM-CSF.

[0214] Implantation

[0215] In some embodiments, the genetically modified nonhuman animals of this subject matter are also immunodeficient. “Immunodeficient” includes a deficiency in one or more aspects of an animal’s natural or endogenous immune system, such as the animal lacking one or more types of functional host immune cells, such as a lack of nonhuman B cell quantity and / or function, nonhuman T cell quantity and / or function, nonhuman NK cell quantity and / or function, etc.

[0216] One method for achieving immunodeficiency in the subject animals is sublethal irradiation. Alternatively or additionally, immunodeficiency can be achieved through any of a variety of gene mutations known in the art, any of which can be cultured alone or in combination into genetically modified nonhuman animals of the subject of this disclosure, or said mutation can serve as a source of stem cells into which the gene modifications of this disclosure can be introduced. Non-limiting examples include X-linked SCIDs associated with mutations in the IL2RG gene, characterized by the lymphocyte phenotype T(-), B(+), NK(-); autosomal recessive SCIDs associated with mutations in the Jak3 gene, characterized by the lymphocyte phenotype T(-), B(+), NK(-); ADA gene mutations, characterized by the lymphocyte phenotype T(-), B(-), NK(-); IL-7R α-chain mutations, characterized by the lymphocyte phenotype T(-), B(+), NK(+); CD3 δ or ε mutations, characterized by the lymphocyte phenotype T(-), B(+), NK(+); RAG1 and RAG2 mutations, characterized by the lymphocyte phenotype T(-), B(-), NK(+); Artemis gene mutations, characterized by the lymphocyte phenotype T(-), B(-), NK(+); and CD45 gene mutations, characterized by the lymphocyte phenotype T(-), B(+). NK(+) characterization; and Prkdcscld mutation, characterized by lymphocyte phenotypes T(-) and B(-). Therefore, in some embodiments, the genetically modified immunodeficient nonhuman animal has one or more defects selected from the following: IL2 receptor γ 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 through gene mutations or deletions in the Rag1 and / or Rag2 and IL2rg genes. Those skilled in the art will understand that these and other immunodeficient animal models can be used to generate the immunodeficient animals of this disclosure.

[0217] In some embodiments, the genetically modified non-human animals according to the invention can be used as recipients of human hematopoietic cells, which are capable of developing human immune cells from the implanted human hematopoietic cells. Therefore, in some aspects of the invention, the genetically modified animals of this subject matter are genetically modified, immunodeficient non-human animals implanted with human hematopoietic cells.

[0218] Any source of human hematopoietic cells, human hematopoietic stem cells (HSCs), and / or hematopoietic progenitor cells (HSPCs) known in the art or described herein can be transplanted into the genetically modified immunodeficient nonhuman animals of this disclosure. A suitable source of human hematopoietic cells known in the art is human umbilical cord blood cells, particularly CD34-positive (CD34+) cells. Another source of human hematopoietic cells is human fetal liver. Another source is human bone marrow. This also includes, for example, the generation of induced pluripotent stem cells (iPSCs) and induced hematopoietic stem cells (iHSCs) through somatic cell dedifferentiation using methods known in the art. Methods for transplanting human cells into nonhuman animals are described in detail in the art and elsewhere herein, and any such method can be used by those skilled in the art to obtain the genetically modified, implanted nonhuman animals of this subject matter.

[0219] Cell populations of particular interest include cells containing hematopoietic stem cells or progenitor cells that will contribute to or rebuild the hematopoietic system of genetically modified nonhuman animals, such as peripheral blood leukocytes, fetal hepatocytes, fetal skeleton, fetal thymus, fetal lymph nodes, vascularized skin, arterial segments, and purified hematopoietic stem cells, such as mobilized HSCs or umbilical cord blood HSCs.

[0220] The cells can be derived from any mammalian species, such as murines, rodents, canines, felines, equines, bovines, sheep, primates, humans, etc. In one embodiment, the cells are human cells. The cells can be derived from established cell lines, or they can be primary cells, wherein “primary cells,” “primary cell lines,” and “primary cultures” are used interchangeably herein to refer to cells and cell cultures derived from a subject and permitted to grow through limited passage (i.e., division) in vitro. For example, a primary culture is a culture that may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not to the point of reaching a critical stage. Typically, the primary cell lines of this disclosure have been passaged less than 10 times in vitro.

[0221] If the cells are primary cells, they can be collected from the individual using any convenient method. For example, cells (e.g., blood cells, such as leukocytes) can be collected using plasma exchange, leukocyte separation, density gradient separation, etc. As another example, cells, such as those from skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach tissue, etc., can be collected via biopsy. The collected cells can be dispersed or suspended using suitable solutions. Such solutions are typically balanced salt solutions (e.g., standard saline, PBS, Hank's balanced salt solution, etc.), routinely supplemented with fetal bovine serum or other naturally occurring factors, in conjunction with an acceptable low concentration (usually 5-25 mM) of buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc.

[0222] In some cases, xenogeneic cell populations are transplanted into genetically modified non-human animals. In other cases, cell populations rich in specific cell types (e.g., progenitor cells, such as hematopoietic progenitor cells) are implanted into genetically modified non-human animals. Enrichment of the target cell population can be achieved using any convenient isolation technique. For example, target cells can be enriched through culture methods. In such culture methods, specific growth factors and nutrients are typically added to the culture to promote the survival and / or proliferation of one cell population relative to other cell populations. Other culture conditions affecting survival and / or proliferation include growth using adherent or non-adherent substrates, specific culture durations, etc. These types of culture conditions are well known in the art. As another example, target cells can be enriched by isolating target cells from an initial population using affinity separation techniques. Affinity separation techniques can include magnetic separation using magnetic beads coated with affinity reagents, affinity chromatography, “panning” using affinity reagents attached to a solid matrix (e.g., plates), cytotoxic agents (such as complement and cytotoxins) linked to or used in conjunction with affinity reagents, or other convenient techniques. Technologies providing precise separation include fluorescence-activated cell sorters, which can be configured with varying degrees of complexity, such as multiple color channels, small-angle and blunt light scattering detection channels, and impedance channels. Cells can be selected for dead cells using dyes associated with dead cells (such as propidium iodide). Any technique that does not unduly impair the viability of the target cells can be employed.

[0223] For example, using affinity separation techniques, cells that are not the target cells for transplantation can be removed from a cell population by contacting it with a marker that specifically recognizes and selectively binds to, but is not expressed on, the target cells. For instance, to enrich a population of hematopoietic progenitor cells, cells expressing markers of mature hematopoietic cells can be removed. Alternatively or additionally, positive selection and separation can be performed by contacting the cell population with an affinity reagent that specifically recognizes and selectively binds to markers associated with hematopoietic progenitor cells (such as CD34, CD133, etc.). "Selective binding" means that a molecule preferentially binds to the target site or has a higher binding affinity to the target site than other molecules. For example, an antibody will bind to a molecule containing its specific epitope, but not to an irrelevant 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., such as a peptide ligand to a receptor, an effector molecule to a receptor molecule, a T-cell receptor specific to CD34, CD133, etc. In some implementations, a variety of affinity reagents specific to the target biomarker may be used.

[0224] Antibodies and T-cell receptors used as affinity reagents can be monoclonal or polyclonal and can be produced from 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 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 to tags for separation. Tags include magnetic beads, which can be directly separated; biotin, which can be removed by avidin or streptavidin bound to a support; fluorescent dyes, which can be used with a fluorescence-activated cell sorter; or similar methods to facilitate the separation of specific cell types. Suitable fluorescent dyes include phycobiliproteins (e.g., phycoerythrin and allophycocyanin), fluorescein, and Texas red. Typically, each antibody is labeled with a different fluorescent dye to achieve independent sorting for each marker.

[0225] The initial cell population is contacted with the affinity reagent and incubated for a sufficient time to bind available cell surface antigens. Incubation is typically at least about 5 minutes and usually less than about 60 minutes. The reaction mixture must contain a sufficient concentration of antibody so that separation efficiency is not limited by antibody deficiency. Suitable concentrations are determined by titration, but antibody is typically diluted to the cell suspension volume at approximately 1:50 (i.e., 1 part antibody to 50 parts reaction volume), approximately 1:100, approximately 1:150, approximately 1:200, approximately 1:250, approximately 1:500, approximately 1:1000, approximately 1:2000, or approximately 1:5000. The culture medium in which the cells are suspended can be any medium that maintains cell viability. A preferred medium is phosphate-buffered saline containing 0.1 to 0.5% BSA or 1-4% goat serum. Various commercially available culture media can be used according to cell characteristics, including Dulbecco's Modified Eagle Medium (dMEM), Hank's Basal Salt Solution (HBSS), Dulbecco's Phosphate Buffered Saline (dPBS), RPMI, Iscove medium, PBS containing 5 mM EDTA, etc., which are usually supplemented with fetal bovine serum, BSA, HSA, goat serum, etc.

[0226] Cells labeled with the affinity reagent in the contact population are selected using any convenient affinity separation technique (e.g., as described above or techniques known in the art). After separation, the isolated cells can be collected in any suitable culture medium that maintains cell viability, and the collection tube usually has a serum pad at the bottom. Various commercially available culture media can be used depending on the cell characteristics, including dMEM, HBSS, dPBS, RPMI, Iscove medium, etc., usually supplemented with fetal bovine serum.

[0227] In this manner, a composition highly enriched with a target cell type (e.g., hematopoietic cells) is obtained. The cells constitute approximately 70%, 75%, 80%, 85%, 90%, or higher of the cell composition, and preferably approximately 95% or higher of the enriched cell composition. In other words, the composition will be a substantially pure target cell composition.

[0228] Cells intended for transplantation into genetically modified nonhuman animals (whether allogeneic or enriched cell populations) can be transplanted immediately. Alternatively, cells can be frozen and stored long-term at liquid nitrogen temperatures, thawed, and reused. In such cases, cells are typically frozen in 10% DMSO, 50% serum, 40% buffered medium, or other solutions commonly used in the art for preserving cells at such freezing temperatures, and thawed in accordance with art's known methods for thawing frozen cell cultures. Furthermore, or alternatively, cells can be cultured in vitro under various culture conditions. The culture medium can be liquid or semi-solid (e.g., containing agar, methylcellulose, etc.). Cell populations can be readily suspended in suitable nutrient media (e.g., Iscove modified DMEM or RPMI-1640), typically supplemented with fetal bovine serum (approximately 5-10%), L-glutamine, thiols (especially 2-mercaptoethanol), and antibiotics (e.g., penicillin and streptomycin). The culture may contain growth factors to which the cells respond. Growth factors, as defined in this article, are molecules that promote cell survival, growth, and / or differentiation in cultures or intact tissues through specific action on transmembrane receptors. Growth factors include peptide and non-peptide factors.

[0229] Cells can be genetically modified before transplantation into genetically modified non-human animals, for example, to provide optional or traceable markers, induce genetic defects in cells (e.g., for disease modeling), repair genetic defects, or ectopically express genes in cells (e.g., to determine whether such modifications affect disease progression). Cells can be genetically modified to express target genes through transfection or transduction with appropriate vectors, homologous recombination, or other suitable techniques, or to block the expression of unwanted genes using antisense mRNA, siRNA, or ribozymes. Various techniques for introducing nucleic acids into target cells are known in the art. Various techniques can be used to demonstrate that cells have been genetically modified. The cell genome can be restricted and used with or without amplification. Polymerase chain reaction; gel electrophoresis; restriction analysis; Southern blotting, Northern blotting, and Western blotting; sequencing, etc., are all possible methods. General methods of molecular and cellular biochemistry used for these and other purposes disclosed in this application can be found in standard textbooks such as *Molecular Cloning: A Laboratory Manual*, 3rd edition (Sambrook et al., Cold Spring Harbor Laboratory Press 2001); *Short Protocols in Molecular Biology*, 4th edition (Ausubel et al., John Wiley & Sons 1999); *Protein Methods* (Bollag et al., John Wiley & Sons 1996); *Nonviral Vectors for Gene Therapy* (Wagner et al., Academic Press 1999); *Viral Vectors* (Kaplift and Loewy, Academic Press 1995); *Immunology Methods Manual* (I. Lefkovits, Academic Press 1997); and *Cell and Tissue Culture: Laboratory Procedures in Biotechnology* (Doyle and Griffiths, John Wiley & Sons). (1998); The disclosure of the standard textbook mentioned herein is incorporated herein by reference. The reagents, cloning vectors and kits for genetic manipulation mentioned in this disclosure are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich and ClonTech.

[0230] Cells can be transplanted into genetically modified non-human animals using any convenient method, including intrahepatic injection, tail vein injection, and retro-orbital injection. Typically, approximately 0.5 × 10⁻⁶ cells are transplanted. 5 -2×10 6 One pluripotent cell or progenitor cell, for example, about 1 × 10⁶ 5 -1×10 6 One cell, or approximately 2 × 10⁻⁶ 5 -5×10 5 Individual cells. In some cases, mice are irradiated with a sublethal dose of radiation prior to transplantation of human cells. In other words, the mice are exposed to a sublethal dose of radiation (as described in the Examples section below and as is known in the art). The implanted, genetically modified non-human animals are then kept in laboratory animal housing conditions for at least one week (e.g., one week or longer, or two weeks or longer, sometimes four weeks or longer, and in some cases six weeks or longer) to allow the implanted cells to fully rebuild the immune system.

[0231] In some embodiments, the transplanted human hematopoietic cells generate one or more implanted 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, erythroid 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 post-implantation. In a specific embodiment, the human cells comprise human dendritic cells.

[0232] In some embodiments, the transplanted human hematopoietic cells generate an implanted human blood-lymphatic system in a genetically modified non-human animal, comprising human hematopoietic stem cells and human hematopoietic 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 blood-lymphatic system is present at 4, 5, 6, 7, 8, 9, 10, 11, or 12 months post-implantation. In a specific embodiment, the human blood-lymphatic system comprises human dendritic cells.

[0233] In some respects, the genetically modified non-human animals described in this article were implanted with human hematopoietic cells containing disease-specific mutations.

[0234] Non-restricted use of genetically modified mice with implantation

[0235] The genetically modified non-human animals disclosed herein have numerous uses in the art. For example, the implanted genetically modified animals of this disclosure can be used to study the function of human dendritic cells, such as human myeloid DCs or human plasmacytoid DCs. As another example, the implanted genetically modified mice of this disclosure provide a useful system for screening candidate drugs with desired activity in vivo, for example, identifying drugs that can regulate (i.e., promote or inhibit) the function of human dendritic cells (e.g., human myeloid DCs or human plasmacytoid DCs) (e.g., in healthy or disease states). For example, the implanted genetically modified mice of this disclosure can be used to identify novel therapeutics. As another example, the implanted genetically modified animals of this disclosure provide a useful system for predicting an individual's response to disease therapies, for example, by providing an in vivo platform to screen an individual's immune system for the response to a drug (e.g., a therapeutic agent) to predict the individual's response to that drug. In some embodiments, since myeloid dendritic cells (mDCs) are important for cross-initiation and activation of T cells, the implanted 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 implanted genetically modified animals of this disclosure provide a useful system for evaluating the therapeutic efficacy of drugs stimulating T cell responses to tumor cells or pathogens, or for identifying drugs that stimulate T cell responses to tumor cells or pathogens. In some embodiments, since plasmacytoid dendritic cells (pDCs) are key type I IFN producers in viral infections and are also associated with autoimmune diseases such as lupus, the implanted genetically modified animals of this disclosure provide a useful model for testing therapies for viral infections or autoimmune diseases by targeting pDCs. For example, the implanted 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.

[0236] As a non-limiting example, the genetically modified mice implanted with the present disclosure can be used to generate 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 in research (e.g., to better understand the progression of human autoimmune diseases) and drug discovery (e.g., to identify candidate agents for treating autoimmune diseases).

[0237] The genetically modified animals implanted with the present disclosure can be used to screen candidate drugs to identify those that will treat autoimmune diseases. The terms “treatment,” “treatment,” etc., are used herein to generally refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse reactions attributable to 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 who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., preventing its development; or (c) alleviating the disease, even if the disease regresses. The terms “individual,” “subject,” “host,” and “patient” are used interchangeably herein and include any mammalian subject requiring diagnosis, treatment, or therapy, particularly humans.

[0238] As another non-limiting example, the genetically modified mice implanted with the present disclosure can be used to generate mouse models for evaluating the therapeutic efficacy of drug-stimulated T cell responses to target cells (e.g., tumor cells, virus-infected cells, bacterial-infected cells, bacterial cells, fungal cells, and parasitic cells). Such mouse models could be used for drug discovery, for example, to identify candidate agents that stimulate T cell responses to target cells.

[0239] In the screening assay of bioactive agents, a genetically modified non-human animal implanted with human hematopoietic cells, in accordance with this disclosure, is exposed to a target candidate drug, and the efficacy of the candidate drug is evaluated by monitoring one or more output parameters. These output parameters can reflect the function of human dendritic cells, such as phagocytosis, cytokine production, cross-presentation of exogenous antigens, and activation of cytotoxic CD8+ T lymphocytes (CTLs), using methods known in the art; or the response of T cells to target cells in rodents. Alternatively or additionally, the output parameters can reflect the effect of the drug on autoimmune diseases in genetically modified non-human animals implanted with human hematopoietic cells, in accordance with this disclosure.

[0240] Candidate agents used in the screening or methods of this disclosure may include, for example, organic molecules (e.g., small molecule inhibitors), nucleic acids (e.g., RNA interference agents, oligonucleotides, or nucleic acids encoding polypeptides), peptides, peptide inhibitors, aptamers, antibodies, intracellular antibodies, 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).

[0241] The bioactivity of candidate agents is screened by applying the agent to at least one, and often multiple, samples (sometimes along with samples lacking the agent). Changes in parameters responding to the agent are measured, and the results are evaluated by comparison with reference samples (e.g., results obtained with other agents in the presence and absence of the agent, etc.). When screening to identify candidate agents that will prevent, mitigate, or reverse the effects of pathogenic or non-pathogenic immune stimulation, screening is typically performed in the presence of pathogenic or non-pathogenic immune stimulation, with the agent added at the most suitable time to determine the outcome. For example, in testing the protective / preventive capacity of a candidate agent, it can be added before, simultaneously with, or after pathogenic or non-pathogenic immune stimulation. As another example, in testing the ability of a candidate agent to reverse the effects of pathogenic or non-pathogenic immune stimulation, it can be added after pathogenic infection or after non-pathogenic immune stimulation. As mentioned above, in some cases, the "sample" is a genetically modified non-human animal with implanted cells, for example, providing a candidate drug to a genetically modified non-human animal that has been implanted with human hematopoietic cells. In other cases, the "sample" is human hematopoietic cells to be implanted, i.e., providing the candidate drug to cells, such as human dendritic cells, before implantation into an immunodeficient genetically modified animal.

[0242] If the candidate drug is to be administered directly to implanted, genetically modified animals, it can be administered using many methods known in the art for administering peptides, small molecules, and nucleic acids to mice. For example, it can be administered orally, via mucosal, topical, intradermal, or by injection (e.g., intraperitoneal, subcutaneous, intramuscular, or intravenous injection). The drug can be administered in a buffer solution or incorporated into any of a variety of formulations, for example, by combination with a suitable pharmaceutically acceptable medium. A “pharmaceuticalally acceptable medium” can be a medium approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in mammals (such as humans). The term “medium” refers to a diluent, adjuvant, excipient, or carrier formulated with the compounds of the present invention for administration to mammals. Such drug delivery systems can be lipids, such as liposomes, or liposomal dendrimers; liquids, such as water and oils, including petroleum, animal, vegetable, or synthetically derived oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.; saline solutions; gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Additionally, adjuvants, stabilizers, thickeners, lubricants, and colorants can be used. The drug composition can be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, gels, microspheres, and aerosols. The drug can be systemic after administration, or localized by using regional administration, intramural administration, or by using an implant that retains an active dose at the implantation site. The active agent can be formulated for immediate action or for sustained release. If the drug is provided to the cells prior to implantation, it can be conveniently added to the culture medium of the cultured cells in solution or readily soluble form. The reagent can be added to the flow-through system as an intermittent or continuous stream, or alternatively, a certain amount of the compound can be added once or incrementally to another static solution. In the flow-through system, two fluids are used: one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid flows through the cells, followed by the second. In the single-solution method, a certain amount of the test compound is added to a certain volume of culture medium surrounding the cells. The total concentration of the culture medium components should not change significantly with the amount added, or should not change significantly between the two solutions in the flow-through method.

[0243] Multiple assays can be run in parallel, using different reagent concentrations to obtain differential responses to various concentrations. As is known in the art, determining the effective concentration of a reagent typically involves using a series of concentrations produced by dilutions at a ratio of 1:10 or other logarithmic proportions. If necessary, these concentrations can be further refined with a second series of dilutions. Typically, one of these concentrations is used as a negative control, i.e., at zero concentration or below the detectable level of the reagent, or at a concentration of the reagent that does not cause a detectable change in phenotype or below that concentration.

[0244] The response of cells in implanted, genetically modified animals to the candidate drug can be analyzed at any time after treatment with the candidate drug. For example, cells can be analyzed 1 day, 2 or 3 days after exposure to the candidate drug; sometimes 4, 5 or 6 days; sometimes 8, 9 or 10 days; sometimes 14 days; sometimes 21 days; sometimes 28 days; and sometimes 1 month or longer (e.g., 2 months, 4 months, 6 months or longer). In some embodiments, the analysis includes analysis at multiple time points. The selection of the analysis time points will be based on the type of analysis to be performed, as will be readily understood by those skilled in the art.

[0245] Analysis may include measuring any 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 can be used to determine the total number of hematopoietic cells or the number of cells of a specific hematopoietic cell type. Histochemistry or immunohistochemistry may be performed to determine the apoptotic state of cells, such as measuring DNA fragmentation using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), or detecting the binding of annexin V to phosphatidylserine residues on the cell surface using immunohistochemistry. Flow cytometry may also be used to assess the proportion and type of differentiated cells, for example, to determine the ability of hematopoietic cells to survive and / or differentiate in the presence of agents. ELISA, Western blotting, and Northern blotting may be performed to determine the levels of cytokines, chemokines, immunoglobulins, etc., expressed in implanted genetically modified mice, for example, to assess the function of implanted cells, assess the function of human dendritic cells, etc. In vivo assays can also be performed to test the function of immune cells, as well as assays related to specific target diseases or conditions, such as autoimmune diseases like systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, polymyositis, collagen-induced arthritis, or dermatomyositis. See, for example, Current Protocols in Immunology (edited by Richard Coico, John Wiley & Sons, Inc. 2012) and Immunology Methods Manual (edited by I. Lefkovits, Academic Press 1997), the contents of which are incorporated herein by reference.

[0246] This document provides examples of other uses of the subject mice elsewhere. Other applications of the genetically modified and implanted mice described herein will be apparent to those skilled in the art upon reading this disclosure.

[0247] Other exemplary implementations

[0248] In exemplary embodiment 1, this document provides 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) a Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operatively linked to the Flt3l promoter.

[0249] In exemplary embodiment 2, this document provides a genetically modified rodent according to embodiment 1, which contains a homozygous null mutation in the Rag1 gene.

[0250] In exemplary embodiment 3, this document provides a genetically modified rodent according to embodiment 1 or 2, wherein invalid mutations in the rodent's Flt3 gene include insertions, deletions, and / or substitutions in the endogenous Flt3 gene.

[0251] In exemplary embodiment 4, this document provides a genetically modified rodent according to embodiment 3, wherein the null mutation in the rodent's Flt3 gene is a deletion of the complete endogenous coding sequence of Flt3.

[0252] In exemplary embodiment 5, this document provides a genetically modified rodent according to embodiment 4, wherein the genetically modified rodent is a mouse, and the mouse contains a homozygous deletion of a nucleic acid sequence between coordinates chr5: 147331171-147400265 (GRCm38 assembly).

[0253] In exemplary embodiment 6, this document provides a genetically modified rodent according to any one of embodiments 1-5, wherein the rodent portion of the Flt3l gene comprises the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene and the exon downstream of exon 6.

[0254] In exemplary embodiment 7, this document provides a genetically modified rodent according to embodiment 6, wherein the non-coding portions of exons 1 and 2 of the rodent Flt3l gene and the exons downstream of exon 6 are at least 90%, at least 95%, or 100% identical to the corresponding non-coding portions of exons 1 and 2 and the exons downstream of exon 6 of the rodent Flt3l gene shown in Table 1A.

[0255] In exemplary embodiment 8, this document provides a genetically modified rodent according to any one of embodiments 1-7, wherein the human portion of the Flt3l gene comprises the signal peptide encoding portion of exon 2 of the human FLT3L gene and exons 3-6.

[0256] In exemplary embodiment 9, this document provides a genetically modified rodent according to embodiment 8, 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.

[0257] In exemplary embodiment 10, this document provides a genetically modified rodent according to any one of embodiments 1-9, wherein the Flt3l gene comprises rodent exon 1, a non-coding portion of rodent exon 2, a signal peptide coding portion of human exon 2, human exons 3-6, and rodent exons 7-9.

[0258] In exemplary embodiment 11, this document provides a genetically modified rodent according to any one of embodiments 1-10, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L, the chimeric membrane-bound FLT3L comprising a signal peptide and a cytokine-like core domain of a human FLT3L polypeptide and a C-terminal portion of a rodent Flt3l polypeptide.

[0259] In exemplary embodiment 12, this document provides a genetically modified rodent according to embodiment 11, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises as follows Figure 1E The rodent stem region, rodent transmembrane domain, and rodent cytoplasmic tail are shown.

[0260] In exemplary embodiment 13, this document provides a genetically modified rodent according to embodiment 11 or 12, wherein the C-terminal portion of the rodent Flt3l polypeptide has at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the same amino acid sequence as the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

[0261] In exemplary embodiment 14, this document provides a genetically modified rodent according to any one of embodiments 1-13, wherein the rodent expresses both a soluble form of the Flt3l polypeptide and a membrane-bound form of the Flt3l polypeptide.

[0262] In exemplary embodiment 15, this document provides a genetically modified rodent according to embodiment 14, wherein the soluble form of the Flt3l polypeptide and the membrane-bound form of the Flt3l polypeptide comprise the signal peptide and cytokine-like core domain of the human FLT3L polypeptide.

[0263] In exemplary embodiment 16, this document provides a genetically modified rodent according to any one of embodiments 11-15, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1-26 of the human FLT3L polypeptide.

[0264] In exemplary embodiment 17, this document provides a genetically modified rodent according to any one of embodiments 11-16, 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.

[0265] In exemplary embodiment 18, this document provides a genetically modified rodent according to any one of embodiments 11-17, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises amino acids corresponding to residues 27-159 of the human FLT3L polypeptide.

[0266] In exemplary embodiment 19, this document provides a genetically modified rodent according to any one of embodiments 11-18, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical amino acid sequences to the corresponding amino acid sequences of the cytokine-like core domains of the human FLT3L polypeptide shown in Table 2.

[0267] In exemplary embodiment 20, this document provides a genetically modified rodent according to any one of embodiments 1-19, wherein the genetically modified rodent is heterozygous for the Flt3 ligand (Flt3l) gene comprising both rodent and human portions.

[0268] In exemplary embodiment 21, this document provides a genetically modified rodent according to any one of embodiments 1-19, wherein the genetically modified rodent is homozygous for the Flt3 ligand (Flt3l) gene comprising both rodent and human portions.

[0269] In exemplary embodiment 22, this document provides a genetically modified rodent according to any one of embodiments 1-21, wherein the rodent portion of the Flt3l gene is an endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is an endogenous rodent gene.

[0270] In exemplary embodiment 23, this document provides a genetically modified rodent according to any one of embodiments 1-22, wherein the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0271] In exemplary embodiment 24, this document provides a genetically modified rodent according to any one of embodiments 1-23, wherein the Flt3l promoter is a rodent promoter.

[0272] In exemplary embodiment 25, this document provides a genetically modified rodent according to embodiment 23 or 24, wherein the Flt3l promoter is an endogenous rodent promoter.

[0273] In exemplary embodiment 26, this document provides a genetically modified rodent according to any one of embodiments 23-25, wherein the Flt3l promoter is located at an endogenous rodent locus.

[0274] In exemplary embodiment 27, this document provides a genetically modified rodent according to any one of embodiments 1-26, wherein the genetically modified rodent expresses a human SIRPA polypeptide or a humanized SIRPA polypeptide encoded by a nucleic acid operatively linked to a Sirpa promoter.

[0275] In exemplary embodiment 28, this document provides a genetically modified rodent according to embodiment 27, wherein the genetically modified rodent further comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a rodent Sirpa polypeptide, wherein the Sirpa gene is operatively linked to a Sirpa promoter.

[0276] In exemplary embodiment 29, this document provides a genetically modified rodent according to embodiment 28, 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.

[0277] In exemplary embodiment 30, this document provides a genetically modified rodent according to embodiment 28 or 29, wherein the genetically modified rodent expresses a Sirpa polypeptide comprising the extracellular portion of a human SIRPA polypeptide and the intracellular portion of a rodent Sirpa polypeptide.

[0278] In exemplary embodiment 31, this document provides 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.

[0279] In exemplary embodiment 32, this document provides a genetically modified rodent according to embodiment 27, wherein the genetically modified rodent expresses a human SIRPA polypeptide encoded by a nucleic acid operatively linked to a Sirpa promoter.

[0280] In exemplary embodiment 33, this document provides a genetically modified rodent according to any one of embodiments 27-32, wherein the genetically modified rodent also 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.

[0281] In exemplary embodiment 34, this document provides a genetically modified rodent according to any one of embodiments 27-33, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are rodent promoters.

[0282] In exemplary embodiment 35, this document provides a genetically modified rodent according to embodiment 34, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are endogenous rodent promoters.

[0283] In exemplary embodiment 36, this document provides a genetically modified rodent according to embodiment 34 or 35, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are located at the respective endogenous rodent locus.

[0284] In exemplary embodiment 37, this document provides a genetically modified rodent according to any one of embodiments 27-36, which contains a null mutation in at least one corresponding rodent gene at the corresponding rodent locus.

[0285] In exemplary embodiment 38, this document provides a genetically modified rodent according to any one of embodiments 27-37, wherein the genetically modified rodent is heterozygous for at least one allele containing a nucleic acid sequence encoding a human protein or a humanized protein.

[0286] In exemplary embodiment 39, this document provides a genetically modified rodent according to any one of embodiments 27-38, wherein the genetically modified rodent is homozygous for at least one allele containing a nucleic acid sequence encoding the human protein or humanized protein.

[0287] In exemplary embodiment 40, this document provides a genetically modified rodent according to any one of embodiments 1-39, which further comprises the implantation of human hematopoietic cells.

[0288] In exemplary embodiment 41, this document provides a genetically modified rodent according to embodiment 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 precursor cells, and human dendritic cells.

[0289] In exemplary embodiment 42, this document provides a genetically modified rodent according to embodiment 40 or 41, wherein the genetically modified rodent comprises human dendritic cells.

[0290] In exemplary embodiment 43, this document provides a genetically modified rodent according to any one of embodiments 1-42, wherein an autoimmune disease is induced or established in the genetically modified rodent.

[0291] In exemplary embodiment 44, this document provides a genetically modified rodent according to embodiment 43, wherein the autoimmune disease is systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, polymyositis, collagen-induced arthritis, or dermatomyositis.

[0292] In exemplary embodiment 45, this document provides a genetically modified rodent according to any one of embodiments 1-44, wherein the rodent is a mouse or a rat.

[0293] In exemplary embodiment 46, this document provides a genetically modified rodent according to embodiment 45, wherein the rodent is a mouse.

[0294] In an exemplary embodiment 47, this document provides a method for identifying an agent that modulates the function of human dendritic cells, comprising: a. administering a candidate agent to a genetically modified rodent according to any one of embodiments 40-46; and b. determining whether the candidate agent modulates the function of the human dendritic cells in the rodent.

[0295] In exemplary embodiment 48, this document provides a method according to embodiment 47, wherein the human dendritic cells are myeloid dendritic cells (mDC) or plasmacytoid dendritic cells (pDC).

[0296] In exemplary embodiment 49, this document provides a method according to embodiment 47 or 48, wherein the function of the human dendritic cells is selected from the group consisting of: phagocytosis, cytokine production, cross-presentation of exogenous antigens, and activation of cytotoxic CD8+ T lymphocytes (CTLs).

[0297] In an exemplary embodiment 50, this document provides a method for evaluating the therapeutic efficacy of a drug-stimulated T cell response to a target cell, the method comprising: a. administering a drug candidate to a genetically modified rodent according to any one of embodiments 40-42, wherein the genetically modified rodent contains the target cells; and b. measuring the T cell response in the rodent to the target cells to evaluate the therapeutic efficacy of the drug candidate.

[0298] In exemplary embodiment 51, this document provides a method according to embodiment 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.

[0299] In an exemplary embodiment 52, this document provides a method for evaluating the therapeutic efficacy of a drug for treating an autoimmune disease, the method comprising: a. administering a candidate drug to a genetically modified rodent according to embodiment 43 or 44; and b. determining whether the drug treats the autoimmune disease in the rodent.

[0300] In exemplary embodiment 53, this document provides a genetically modified rodent cell 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) a Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operatively linked to the Flt3l promoter.

[0301] In exemplary embodiment 54, this document provides a genetically modified rodent cell according to embodiment 53, which contains a homozygous null mutation in the Rag1 gene.

[0302] In exemplary embodiment 55, this document provides genetically modified rodent cells according to embodiment 53 or 54, wherein invalid mutations in the rodent Flt3 gene include insertions, deletions, and / or substitutions in the endogenous Flt3 gene.

[0303] In an exemplary embodiment 56, this document provides a genetically modified rodent cell according to embodiment 55, wherein the null mutation in the rodent Flt3 gene is a deletion of the complete endogenous coding sequence of Flt3.

[0304] In exemplary embodiment 57, this document provides a genetically modified rodent cell according to embodiment 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).

[0305] In exemplary embodiment 58, this document provides a genetically modified rodent cell according to any one of embodiments 53-57, wherein the rodent portion of the Flt3l gene comprises the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene and the exon downstream of exon 6.

[0306] In exemplary embodiment 59, this document provides a genetically modified rodent cell according to embodiment 58, wherein the non-coding portions of exons 1 and 2 of the rodent Flt3l gene and the exons downstream of exon 6 are at least 90%, at least 95%, or 100% identical to the corresponding non-coding portions of exons 1 and 2 of the rodent Flt3l gene and the exons downstream of exon 6 shown in Table 1A.

[0307] In an exemplary embodiment 60, this document provides a genetically modified rodent cell according to any one of embodiments 1-59, wherein the human portion of the Flt3l gene comprises the signal peptide encoding portion of exon 2 of the human FLT3L gene and exons 3-6.

[0308] In an exemplary embodiment 61, this document provides a genetically modified rodent cell according to embodiment 60, 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.

[0309] In exemplary embodiment 62, this document provides a genetically modified rodent cell according to any one of embodiments 53-61, wherein the Flt3l gene comprises rodent exon 1, the non-coding portion of rodent exon 2, the signal peptide coding portion of human exon 2, human exons 3-6, and rodent exons 7-9.

[0310] In exemplary embodiment 63, this document provides a genetically modified rodent cell according to any one of embodiments 53-62, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L comprising a signal peptide and a cytokine-like core domain of a human FLT3L polypeptide and a C-terminal portion of a rodent Flt3l polypeptide.

[0311] In exemplary embodiment 64, this document provides a genetically modified rodent cell according to embodiment 63, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises, Figure 1E The rodent stem region, rodent transmembrane domain, and rodent cytoplasmic tail are shown.

[0312] In an exemplary embodiment 65, this document provides a genetically modified rodent cell according to embodiment 63 or 64, wherein the C-terminal portion of the rodent Flt3l polypeptide has at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the same amino acid sequence as the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

[0313] In exemplary embodiment 66, this document provides a genetically modified rodent cell according to any one of embodiments 53-65, wherein the rodent cell expresses both a soluble form of the Flt3l polypeptide and a membrane-bound form of the Flt3l polypeptide.

[0314] In an exemplary embodiment 67, this document provides a genetically modified rodent cell according to embodiment 66, wherein the soluble form of the Flt3l polypeptide and the membrane-bound form of the Flt3l polypeptide comprise a signal peptide and a cytokine-like core domain of the human FLT3L polypeptide.

[0315] In an exemplary embodiment 68, this document provides a genetically modified rodent cell according to any one of embodiments 63-67, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1-26 of the human FLT3L polypeptide.

[0316] In exemplary embodiment 69, this document provides a genetically modified rodent cell according to any one of embodiments 63-68, 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.

[0317] In exemplary embodiment 70, this document provides a genetically modified rodent cell according to any one of embodiments 63-69, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises amino acids corresponding to residues 27-159 of the human FLT3L polypeptide.

[0318] In exemplary embodiment 71, this document provides a genetically modified rodent cell according to any one of embodiments 63-70, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical amino acid sequences to the corresponding amino acid sequences of the cytokine-like core domains of the human FLT3L polypeptide shown in Table 2.

[0319] In exemplary embodiment 72, this document provides a genetically modified rodent cell according to any one of embodiments 53-71, wherein the genetically modified rodent cell is heterozygous for the Flt3 ligand (Flt3l) gene comprising rodent and human portions.

[0320] In exemplary embodiment 73, this document provides a genetically modified rodent cell according to any one of embodiments 53-71, wherein the genetically modified rodent cell is homozygous for the Flt3 ligand (Flt3l) gene comprising rodent and human portions.

[0321] In exemplary embodiment 74, this document provides a genetically modified rodent cell according to any one of embodiments 53-73, wherein the rodent portion of the Flt3l gene is an endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is an endogenous rodent gene.

[0322] In exemplary embodiment 75, this document provides a genetically modified rodent cell according to any one of embodiments 53-74, wherein the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0323] In exemplary embodiment 76, this document provides a genetically modified rodent cell according to any one of embodiments 53-75, wherein the Flt3l promoter is a rodent promoter.

[0324] In exemplary embodiment 77, this document provides genetically modified rodent cells according to embodiment 75 or 76, wherein the Flt3l promoter is an endogenous rodent promoter.

[0325] In exemplary embodiment 78, this document provides a genetically modified rodent cell according to any one of embodiments 75-77, wherein the Flt3l promoter is located at an endogenous rodent locus.

[0326] In exemplary embodiment 79, this document provides a genetically modified rodent cell according to any one of embodiments 53-78, wherein the genetically modified rodent cell further comprises a nucleic acid encoding a human SIRPA polypeptide or a humanized SIRPA polypeptide, and wherein the nucleic acid is operatively linked to a Sirpa promoter.

[0327] In an exemplary embodiment 80, this document provides a genetically modified rodent cell according to embodiment 79, wherein the genetically modified rodent cell further comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a rodent Sirpa polypeptide, wherein the Sirpa gene is operatively linked to a Sirpa promoter.

[0328] In an exemplary embodiment 81, this document provides a genetically modified rodent cell according to embodiment 80, 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.

[0329] In an exemplary embodiment 82, this document provides a genetically modified rodent cell according to embodiment 80 or 81, wherein the genetically modified rodent cell expresses a Sirpa polypeptide comprising the extracellular portion of a human SIRPA polypeptide and the intracellular portion of a rodent Sirpa polypeptide.

[0330] In exemplary embodiment 83, this document provides a genetically modified rodent cell according to any one of embodiments 80-82, wherein the rodent Sirpa polypeptide is an endogenous rodent Sirpa polypeptide, and / or the rodent Sirpa gene is an endogenous rodent gene.

[0331] In exemplary embodiment 84, this document provides a genetically modified rodent cell according to embodiment 79, wherein the genetically modified rodent cell expresses a human SIRPA peptide.

[0332] In exemplary embodiment 85, this document provides a genetically modified rodent cell according to any one of embodiments 79-84, wherein the genetically modified rodent cell further comprises: (1) a nucleic acid operatively linked to a GM-CSF promoter encoding a human GM-CSF protein; and / or (2) a nucleic acid operatively linked to an IL3 promoter encoding a human IL3 protein.

[0333] In exemplary embodiment 86, this document provides a genetically modified rodent cell according to any one of embodiments 79-85, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are rodent promoters.

[0334] In exemplary embodiment 87, this document provides genetically modified rodent cells according to embodiment 86, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are endogenous rodent promoters.

[0335] In exemplary embodiment 88, this document provides genetically modified rodent cells according to embodiment 86 or 87, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are located at the respective endogenous rodent loci.

[0336] In exemplary embodiment 89, this document provides a genetically modified rodent cell according to any one of embodiments 79-88, which contains a null mutation in at least one corresponding rodent gene at a corresponding rodent locus.

[0337] In an exemplary embodiment 90, this document provides a genetically modified rodent cell according to any one of embodiments 79-89, wherein the genetically modified rodent cell is heterozygous for at least one allele containing a nucleic acid sequence encoding the human protein or humanized protein.

[0338] In exemplary embodiment 91, this document provides a genetically modified rodent cell according to any one of embodiments 79-90, wherein the genetically modified rodent cell is homozygous for at least one allele containing a nucleic acid sequence encoding the human protein or humanized protein.

[0339] In exemplary embodiment 92, this document provides a genetically modified rodent cell according to any one of embodiments 53-91, wherein the rodent cell is a rat cell or a mouse cell.

[0340] In exemplary embodiment 93, this document provides a genetically modified rodent cell according to embodiment 92, wherein the rodent cell is a mouse cell.

[0341] In exemplary embodiment 94, this document provides a genetically modified rodent cell according to any one of embodiments 53-65, 68-81, 83 and 85-93, wherein the genetically modified rodent cell is a rodent embryonic stem (ES) cell.

[0342] In exemplary embodiment 95, this document provides a genetically modified rodent embryonic stem cell 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 rodent and human portions operatively linked to the Flt3l promoter.

[0343] In exemplary embodiment 96, this document provides genetically modified rodent embryonic stem cells according to embodiment 95, which contain a homozygous null mutation in the Rag1 gene.

[0344] In exemplary embodiment 97, this document provides genetically modified rodent embryonic stem cells according to embodiment 95 or 96, wherein invalid mutations in the rodent Flt3 gene include insertions, deletions, and / or substitutions in the endogenous Flt3 gene.

[0345] In exemplary embodiment 98, this document provides genetically modified rodent embryonic stem cells according to embodiment 97, wherein the null mutation in the rodent Flt3 gene is a deletion of the complete endogenous coding sequence of Flt3.

[0346] In exemplary embodiment 99, this document provides genetically modified rodent embryonic stem cells according to embodiment 98, wherein the genetically modified rodent embryonic stem cells are mouse embryonic stem cells, and the mouse embryonic stem cells contain homozygous deletions of nucleic acid sequences between coordinates chr5: 147331171-147400265 (GRCm38 assembly).

[0347] In an exemplary embodiment 100, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 95-99, wherein the rodent portion of the Flt3l gene comprises the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene and the exon downstream of exon 6.

[0348] In an exemplary embodiment 101, this document provides genetically modified rodent embryonic stem cells according to embodiment 100, wherein the non-coding portions of exons 1 and 2 of the rodent Flt3l gene and the exons downstream of exon 6 are at least 90%, at least 95%, or 100% identical to the corresponding non-coding portions of exons 1 and 2 of the rodent Flt3l gene and the exons downstream of exon 6 shown in Table 1A.

[0349] In exemplary embodiment 102, this document provides genetically modified rodent embryonic stem cells according to any one of embodiments 95-101, wherein the human portion of the Flt3l gene comprises the signal peptide encoding portion of exon 2 of the human FLT3L gene and exons 3-6.

[0350] In exemplary embodiment 103, this document provides genetically modified rodent embryonic stem cells according to embodiment 102, 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.

[0351] In exemplary embodiment 104, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 95-103, wherein the Flt3l gene comprises rodent exon 1, the non-coding portion of rodent exon 2, the signal peptide coding portion of human exon 2, human exons 3-6, and rodent exons 7-9.

[0352] In exemplary embodiment 105, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 95-104, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L, the chimeric membrane-bound FLT3L comprising a signal peptide and a cytokine-like core domain of a human FLT3L polypeptide, and a C-terminal portion of a rodent Flt3l polypeptide.

[0353] In exemplary embodiment 106, this document provides genetically modified rodent embryonic stem cells according to embodiment 105, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises, Figure 1E The rodent stem region, rodent transmembrane domain, and rodent cytoplasmic tail are shown.

[0354] In exemplary embodiment 107, this document provides genetically modified rodent embryonic stem cells according to embodiment 105 or 106, wherein the C-terminal portion of the rodent Flt3l polypeptide has at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the same amino acid sequence as the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

[0355] In exemplary embodiment 108, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 105-107, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1-26 of the human FLT3L polypeptide.

[0356] In exemplary embodiment 109, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 105-108, 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.

[0357] In exemplary embodiment 110, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 105-109, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises amino acids corresponding to residues 27-159 of the human FLT3L polypeptide.

[0358] In exemplary embodiment 111, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 105-110, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical amino acid sequences to the corresponding amino acid sequences of the cytokine-like core domains of the human FLT3L polypeptide shown in Table 2.

[0359] In exemplary embodiment 112, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 95-111, wherein the genetically modified rodent embryonic stem cell is heterozygous for the Flt3 ligand (Flt3l) gene comprising rodent and human portions.

[0360] In exemplary embodiment 113, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 95-111, wherein the genetically modified rodent embryonic stem cell is homozygous for the Flt3 ligand (Flt3l) gene comprising rodent and human portions.

[0361] In exemplary embodiment 114, this document provides genetically modified rodent embryonic stem cells according to any one of embodiments 95-113, wherein the rodent portion of the Flt3l gene is an endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is an endogenous rodent gene.

[0362] In exemplary embodiment 115, this document provides genetically modified rodent embryonic stem cells according to any one of embodiments 95-114, wherein the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0363] In exemplary embodiment 116, this document provides genetically modified rodent embryonic stem cells according to any one of embodiments 95-115, wherein the Flt3l promoter is a rodent promoter.

[0364] In exemplary embodiment 117, this document provides genetically modified rodent embryonic stem cells according to embodiment 115 or 116, wherein the Flt3l promoter is an endogenous rodent promoter.

[0365] In exemplary embodiment 118, this document provides genetically modified rodent embryonic stem cells according to any one of embodiments 115-117, wherein the Flt3l promoter is located at an endogenous rodent locus.

[0366] In exemplary embodiment 119a, this document provides a genetically modified rodent embryonic stem cell according to any one of claims 95-118, wherein the genetically modified rodent embryonic stem cell further comprises a nucleic acid encoding a human SIRPA polypeptide or a humanized SIRPA polypeptide, and wherein the nucleic acid is operatively linked to a Sirpa promoter.

[0367] In exemplary embodiment 119b, this document provides a genetically modified rodent embryonic stem cell according to embodiment 119a, wherein the genetically modified rodent embryonic stem cell further comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a rodent Sirpa polypeptide, wherein the Sirpa gene is operatively linked to a Sirpa promoter.

[0368] In exemplary embodiment 120, this document provides genetically modified rodent embryonic stem cells according to embodiment 119b, 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.

[0369] In exemplary embodiment 121a, this document provides genetically modified rodent embryonic stem cells according to embodiment 119b or 120, wherein the rodent Sirpa polypeptide is an endogenous rodent Sirpa polypeptide, and / or the rodent Sirpa gene is an endogenous rodent gene.

[0370] In exemplary embodiment 121b, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 119a-121a, wherein the genetically modified rodent embryonic stem cell further comprises (1) a nucleic acid encoding a human GM-CSF protein operatively linked to a GM-CSF promoter; and / or (2) a nucleic acid encoding a human IL3 protein operatively linked to an IL3 promoter.

[0371] In exemplary embodiment 122, this document provides genetically modified rodent embryonic stem cells according to any one of embodiments 119a-121b, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are rodent promoters.

[0372] In exemplary embodiment 123, this document provides genetically modified rodent embryonic stem cells according to embodiment 122, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are endogenous rodent promoters.

[0373] In exemplary embodiment 124, this document provides genetically modified rodent embryonic stem cells according to embodiment 122 or 123, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are located at the respective endogenous rodent loci.

[0374] In exemplary embodiment 125, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 119a-124, which contains a null mutation in at least one corresponding rodent gene at a corresponding rodent locus.

[0375] In exemplary embodiment 126, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 119a-125, wherein the genetically modified rodent embryonic stem cell is heterozygous for at least one allele containing a nucleic acid sequence encoding the human protein or humanized protein.

[0376] In exemplary embodiment 127, this document provides a genetically modified rodent embryonic stem cell according to any one of embodiments 119a-126, wherein the genetically modified rodent embryonic stem cell is homozygous for at least one allele containing a nucleic acid sequence encoding the human protein or humanized protein.

[0377] In exemplary embodiment 128, this document provides genetically modified rodent embryonic stem cells according to any one of embodiments 95-127, wherein the rodent embryonic stem cells are rat embryonic stem cells or mouse embryonic stem cells.

[0378] In exemplary embodiment 129, this document provides genetically modified rodent embryonic stem cells according to embodiment 128, wherein the rodent embryonic stem cells are mouse embryonic stem cells.

[0379] In an exemplary embodiment 130, this document provides a method for preparing rodent embryonic stem cells, comprising genetically modifying the rodent embryonic stem cells such 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) a Flt3 ligand (Flt3l) gene comprising a rodent portion and a human portion operatively linked to the Flt3l promoter.

[0380] In an exemplary embodiment 131, this document provides a method according to embodiment 130, wherein the rodent embryonic stem cells are further modified to have a genome containing a homozygous null mutation in the Rag1 gene.

[0381] In exemplary embodiment 132, this document provides a method according to embodiment 130 or 131, wherein the invalid mutation in the rodent Flt3 gene includes insertions, deletions, and / or substitutions in the endogenous Flt3 gene.

[0382] In exemplary embodiment 133, this document provides a method according to embodiment 132, wherein the invalid mutation in the rodent Flt3 gene is a deletion of the complete endogenous coding sequence of Flt3.

[0383] In exemplary embodiment 134, this document provides a method according to embodiment 133, wherein the genetically modified rodent embryonic stem cells are mouse embryonic stem cells, and the mouse embryonic stem cells comprise homozygous deletions of nucleic acid sequences between coordinates chr5: 147331171-147400265 (GRCm38 assembly).

[0384] In exemplary embodiment 135, this document provides a method according to any one of embodiments 130-134, wherein the rodent portion of the Flt3l gene comprises the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene and the exon downstream of exon 6.

[0385] In an exemplary embodiment 136, this document provides a method according to embodiment 135, wherein the non-coding portions of exons 1 and 2 of the rodent Flt3l gene and the exons downstream of exon 6 are at least 90%, at least 95%, or 100% identical to the corresponding non-coding portions of exons 1 and 2 of the rodent Flt3l gene and the exons downstream of exon 6 shown in Table 1A.

[0386] In exemplary embodiment 137, this document provides a method according to any one of embodiments 130-136, 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.

[0387] In an exemplary embodiment 138, this document provides a method according to embodiment 137, 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 of the human FLT3L gene and exons 3-6 shown in Table 1B.

[0388] In exemplary embodiment 139, this document provides a method according to any one of embodiments 130-138, wherein the Flt3l gene comprises rodent exon 1, a non-coding portion of rodent exon 2, a signal peptide coding portion of human exon 2, human exons 3-6, and rodent exons 7-9.

[0389] In exemplary embodiment 140, this document provides a method according to any one of embodiments 130-139, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L, the chimeric membrane-bound FLT3L comprising a signal peptide and a cytokine-like core domain of a human FLT3L polypeptide, and a C-terminal portion of a rodent Flt3l polypeptide.

[0390] In exemplary embodiment 141, this document provides a method according to embodiment 140, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises, for example, Figure 1E The rodent stem region, rodent transmembrane domain, and rodent cytoplasmic tail are shown.

[0391] In exemplary embodiment 142, this document provides a method according to embodiment 140 or 141, wherein the C-terminal portion of the rodent Flt3l polypeptide has at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the same amino acid sequence as the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

[0392] In exemplary embodiment 143, this document provides a method according to any one of embodiments 140-142, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1-26 of the human FLT3L polypeptide.

[0393] In exemplary embodiment 144, this document provides a method according to any one of embodiments 140-143, wherein the signal peptide of the human FLT3L polypeptide comprises at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical amino acid sequences to the corresponding amino acid sequences of the signal peptides of the human FLT3L polypeptides shown in Table 2.

[0394] In exemplary embodiment 145, this document provides a method according to any one of embodiments 140-144, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises amino acids corresponding to residues 27-159 of the human FLT3L polypeptide.

[0395] In exemplary embodiment 146, this document provides a method according to any one of embodiments 140-145, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

[0396] In exemplary embodiment 147, this document provides a method according to any one of embodiments 130-146, wherein the genetically modified rodent embryonic stem cells are heterozygous for the Flt3 ligand (Flt3l) gene comprising rodent and human portions.

[0397] In exemplary embodiment 148, this document provides a method according to any one of embodiments 130-146, wherein the genetically modified rodent embryonic stem cells are homozygous for the Flt3 ligand (Flt3l) gene comprising rodent and human portions.

[0398] In exemplary embodiment 149, this document provides a method according to any one of embodiments 130-148, wherein the rodent portion of the Flt3l gene is an endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is an endogenous rodent gene.

[0399] In exemplary embodiment 150, this document provides a method according to any one of embodiments 130-149, wherein the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0400] In exemplary embodiment 151, this document provides a method according to any one of embodiments 130-150, wherein the Flt3l promoter is a rodent promoter.

[0401] In exemplary embodiment 152, this document provides a method according to embodiment 150 or 151, wherein the Flt3l promoter is an endogenous rodent promoter.

[0402] In exemplary embodiment 153, this document provides a method according to any one of embodiments 150-152, wherein the Flt3l promoter is located at an endogenous rodent locus.

[0403] In exemplary embodiment 154, this document provides a method according to embodiment 153, wherein the genetically modified rodent embryonic stem cells are further engineered to include a Sirpa gene in their genome, the Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a rodent Sirpa polypeptide, wherein the Sirpa gene is operatively linked to a Sirpa promoter.

[0404] In exemplary embodiment 155, this document provides a method according to embodiment 154, 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.

[0405] In exemplary embodiment 156a, this document provides a method according to 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.

[0406] In exemplary embodiment 156b, this document provides a method according to any one of embodiments 154-156a, wherein the genetically modified rodent embryonic stem cells are further engineered to include in their genome nucleic acids encoding human GM-CSF protein and operably linked to the GM-CSF promoter, and / or nucleic acids encoding human IL3 protein and operably linked to the IL3 promoter.

[0407] In exemplary embodiment 157, this document provides a method according to any one of embodiments 154-156b, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are rodent promoters.

[0408] In exemplary embodiment 158, this document provides a method according to embodiment 157, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are endogenous rodent promoters.

[0409] In exemplary embodiment 159, this document provides a method according to embodiment 156 or 157, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are located at the respective endogenous rodent locus.

[0410] In exemplary embodiment 160, this document provides a method according to any one of embodiments 154-159, which includes a null mutation in at least one corresponding rodent gene at a corresponding rodent locus.

[0411] In exemplary embodiment 161, this document provides a method according to any one of embodiments 154-160, wherein the genetically modified rodent embryonic stem cells are heterozygous for at least one allele, the allele comprising a nucleic acid sequence encoding the human protein or humanized protein.

[0412] In exemplary embodiment 162, this document provides a method according to any one of embodiments 154-161, wherein the genetically modified rodent embryonic stem cells are homozygous for at least one allele, the allele comprising a nucleic acid sequence encoding the human protein or humanized protein.

[0413] In exemplary embodiment 163, this document provides a method according to any one of embodiments 130-162, wherein the rodent embryonic stem cells are rat embryonic stem cells or mouse embryonic stem cells.

[0414] In exemplary embodiment 164, this document provides a method according to embodiment 163, wherein the rodent embryonic stem cells are mouse embryonic stem cells.

[0415] In an exemplary embodiment 165, this document provides a rodent embryo comprising rodent embryonic stem cells according to any one of embodiments 94-129, or rodent embryonic stem cells prepared according to any one of embodiments 130-164.

[0416] In an exemplary embodiment 166, this document provides a method for preparing rodents 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 operatively linked to the Flt3l promoter, the method comprising the steps of: (a) obtaining rodent embryonic stem cells according to any one of embodiments 94-129, or rodent embryonic stem cells prepared according to any one of embodiments 130-164; and (b) generating rodents using the rodent embryonic cells described in (a).

[0417] In an exemplary embodiment 167, this document provides a method for preparing 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 operatively linked to the Flt3l promoter, the method comprising modifying the genome of the rodent such 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 operatively linked to the Flt3l promoter.

[0418] In exemplary embodiment 168, this document provides a method according to embodiment 167, wherein the rodent is modified to include a homozygous null mutation in the Rag1 gene.

[0419] In exemplary embodiment 169, this document provides a method according to embodiment 167 or 168, wherein the invalid mutation in the rodent Flt3 gene includes insertions, deletions, and / or substitutions in the endogenous Flt3 gene.

[0420] In exemplary embodiment 170, this document provides a method according to embodiment 169, wherein the null mutation in the rodent Flt3 gene is a deletion of the complete endogenous coding sequence of Flt3.

[0421] In an exemplary embodiment 171, this document provides a method according to embodiment 170, 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).

[0422] In exemplary embodiment 172, this document provides a method according to any one of embodiments 167-171, wherein the rodent portion of the Flt3l gene comprises the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene and the exon downstream of exon 6.

[0423] In exemplary embodiment 173, this document provides a method according to embodiment 172, wherein the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene and the exon downstream of exon 6 are at least 90%, at least 95%, or 100% identical to the corresponding non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene and the exon downstream of exon 6 shown in Table 1A.

[0424] In exemplary embodiment 174, this document provides a method according to any one of embodiments 167-173, 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.

[0425] In exemplary embodiment 175, this document provides a method according to embodiment 174, 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 of the human FLT3L gene and exons 3-6 shown in Table 1B.

[0426] In exemplary embodiment 176, this document provides a method according to any one of embodiments 167-175, wherein the Flt3l gene comprises rodent exon 1, a non-coding portion of rodent exon 2, a signal peptide coding portion of human exon 2, human exons 3-6, and rodent exons 7-9.

[0427] In exemplary embodiment 177, this document provides a method according to any one of embodiments 167-176, wherein the Flt3l gene encodes a chimeric membrane-bound FLT3L, the chimeric membrane-bound FLT3L comprising a signal peptide and a cytokine-like core domain of a human FLT3L polypeptide, and a C-terminal portion of a rodent Flt3l polypeptide.

[0428] In exemplary embodiment 178, this document provides a method according to embodiment 177, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises, for example, Figure 1E The rodent stem region, rodent transmembrane domain, and rodent cytoplasmic tail are shown.

[0429] In exemplary embodiment 179, this document provides a method according to embodiment 177 or 178, wherein the C-terminal portion of the rodent Flt3l polypeptide has at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the same amino acid sequence as the C-terminal portion of the rodent Flt3l polypeptide shown in Table 2.

[0430] In exemplary embodiment 180, this document provides a method according to any one of embodiments 167-179, wherein the rodent expresses both a soluble form of the Flt3l polypeptide and a membrane-bound form of the Flt3l polypeptide.

[0431] In an exemplary embodiment 181, this document provides a method according to embodiment 180, wherein the soluble form of the Flt3l polypeptide and the membrane-bound form of the Flt3l polypeptide comprise a signal peptide and a cytokine-like core domain of the human FLT3L polypeptide.

[0432] In exemplary embodiment 182, this document provides a method according to any one of embodiments 177-181, wherein the signal peptide of the human FLT3L polypeptide comprises amino acids corresponding to residues 1-26 of the human FLT3L polypeptide.

[0433] In exemplary embodiment 183, this document provides a method according to any one of embodiments 177-182, wherein the signal peptide of the human FLT3L polypeptide comprises at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical amino acid sequences to the corresponding amino acid sequences of the signal peptides of the human FLT3L polypeptides shown in Table 2.

[0434] In exemplary embodiment 184, this document provides a method according to any one of embodiments 177-183, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises amino acids corresponding to residues 27-159 of the human FLT3L polypeptide.

[0435] In exemplary embodiment 185, this document provides a method according to any one of embodiments 177-184, wherein the cytokine-like core domain of the human FLT3L polypeptide comprises at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the corresponding amino acid sequence of the cytokine-like core domain of the human FLT3L polypeptide shown in Table 2.

[0436] In exemplary embodiment 186, this document provides a method according to any one of embodiments 167-185, wherein the genetically modified rodent is heterozygous for the Flt3 ligand (Flt3l) gene comprising both rodent and human portions.

[0437] In exemplary embodiment 187, this document provides a method according to any one of embodiments 167-185, wherein the genetically modified rodent is homozygous for the Flt3 ligand (Flt3l) gene comprising both rodent and human portions.

[0438] In exemplary embodiment 188, this document provides a method according to any one of embodiments 167-187, wherein the rodent portion of the Flt3l gene is an endogenous rodent portion of the Flt3l gene, and the rodent Flt3l gene is an endogenous rodent gene.

[0439] In exemplary embodiment 189, this document provides a method according to any one of embodiments 167-188, wherein the rodent Flt3l polypeptide is an endogenous rodent Flt3l polypeptide.

[0440] In exemplary embodiment 190, this document provides a method according to any one of embodiments 167-189, wherein the Flt3l promoter is a rodent promoter.

[0441] In exemplary embodiment 191, this document provides a method according to embodiment 189 or 190, wherein the Flt3l promoter is an endogenous rodent promoter.

[0442] In exemplary embodiment 192, this document provides a method according to any one of embodiments 189-191, wherein the Flt3l promoter is located at an endogenous rodent locus.

[0443] In exemplary embodiment 193, this document provides a method according to any one of embodiments 167-192, wherein the genetically modified rodent expresses a human SIRPA polypeptide or a humanized SIRPA polypeptide, the human SIRPA polypeptide or humanized SIRPA polypeptide being encoded by a nucleic acid operatively linked to a Sirpa promoter.

[0444] In exemplary embodiment 194, this document provides a method according to embodiment 193, wherein the genetically modified rodent further comprises a Sirpa gene encoding a Sirpa polypeptide comprising an extracellular portion of a human SIRPA polypeptide and an intracellular portion of a rodent Sirpa polypeptide, wherein the Sirpa gene is operatively linked to a Sirpa promoter.

[0445] In an exemplary embodiment 195, this document provides a method according to embodiment 194, 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.

[0446] In exemplary embodiment 196, this document provides a method according to embodiment 194 or 195, wherein the genetically modified rodent expresses a Sirpa polypeptide comprising the extracellular portion of a human SIRPA polypeptide and the intracellular portion of a rodent Sirpa polypeptide.

[0447] In exemplary embodiment 197, this document provides a method according to any one of embodiments 194-196, wherein the rodent Sirpa polypeptide is an endogenous rodent Sirpa polypeptide, and / or the rodent Sirpa gene is an endogenous rodent gene.

[0448] In exemplary embodiment 198, this document provides a method according to embodiment 193, wherein the genetically modified rodent expresses a human SIRPA polypeptide encoded by a nucleic acid operatively linked to a Sirpa promoter.

[0449] In exemplary embodiment 199, this document provides a method according to any one of embodiments 193-198, 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.

[0450] In exemplary embodiment 200, this document provides a method according to any one of embodiments 193-199, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are rodent promoters.

[0451] In exemplary embodiment 201, this document provides a method according to embodiment 200, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are endogenous rodent promoters.

[0452] In exemplary embodiment 202, this document provides a method according to embodiment 200 or 201, wherein the SIRPα promoter, the GM-CSF promoter and / or the IL3 promoter are located at the respective endogenous rodent locus.

[0453] In exemplary embodiment 203, this document provides a method according to any one of embodiments 193-202, wherein the genetically modified rodent contains an invalid mutation in at least one corresponding rodent gene at the corresponding rodent locus.

[0454] In exemplary embodiment 204, this document provides a method according to any one of embodiments 193-203, wherein the genetically modified rodent is heterozygous for at least one allele, the allele comprising a nucleic acid sequence encoding a human protein or a humanized protein.

[0455] In exemplary embodiment 205, this document provides a method according to any one of embodiments 193-204, wherein the genetically modified rodent is homozygous for at least one allele, the allele comprising a nucleic acid sequence encoding the human protein or humanized protein.

[0456] In exemplary embodiment 206, this document provides a method according to any one of embodiments 167-205, wherein the rodent is a mouse or a rat.

[0457] In exemplary embodiment 207, this document provides a method according to embodiment 206, wherein the rodent is a mouse.

[0458] Example

[0459] The following examples and figures are provided to describe to those skilled in the art how to prepare and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Every effort has been made to ensure the accuracy of the figures used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. The examples do not include detailed descriptions of conventional methods (molecular cloning techniques, etc.) well known to those skilled in the art. Unless otherwise stated, parts are parts by weight, molecular weights are average molecular weights, temperatures are expressed in degrees Celsius, and pressures are at or near atmospheric pressure.

[0460] Example 1: Generation of genetically modified mice

[0461] Example 1.1: Generation of humanized Flt3l mice

[0462] The mouse and human Flt3 ligand exists in multiple isoforms, including membrane-bound and soluble forms, which are thought to be generated by 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 soluble isoform can also be generated by proteolytic cleavage (via TNFα-converting enzyme or TACE) (Kazi et al. (2019) Physiol. Rev. 99:1433-1466). All known soluble isoforms of the human and mouse Flt3 ligand contain a receptor-binding domain (cytokine-like core domain), typically encoded by exons 3–6 of the gene. The mouse Flt3 ligand (Flt3l) locus was humanized 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 couple with high-resolution expression analysis. Nat. Biotech. 21(6): 652–659, both cited and incorporated herein by reference). The resulting humanized Flt3l locus contains the endogenous mouse Flt3l promoter; chimeric exon 2, which contains the non-coding portion of mouse exon 2 and the signal peptide-coding portion of human exon 2 (encoding the human signal peptide); and human FLT3L exons 3 to 6 (encoding the human FLT3L cytokine-like core domain), which contains a 111 bp human intron 6 sequence; followed by the remaining endogenous mouse Flt3l, which contains the remainder of intron 6 and exons 7-9, as well as the 3'UTR.

[0463] To generate the final targeting vector for humanizing FLT3L: (i) first, the portion of mouse intron 2 to mouse exon 6 extending to intron 6 (2293 bp; GRCm38 / mm10-chr7:45,133,715-45,136,007, which encodes the mouse Flt3L signaling sequence and the cytokine-like core domain sequence) was deleted in the bacterial artificial chromosome (BAC) clone bMQ432L09 (Thermo-Fisher / Invitrogen); (ii) Subsequently, human exon 2 (at ATG) is introduced into the portion of human exon 6 extending to intron 6 (4520 bp; GRCh38 / hg38-chr19:49,474,640-49,479,159, which encodes the human FLT3L signaling sequence and the cytokine-like core domain sequence), as well as the neomycin resistance self-deletion cassette (containing the CRE recombinase controlled by the protamine promoter).

[0464] Specifically, firstly, a 2293bp deletion was performed in the bacterial artificial chromosome (BAC) clone bMQ432L09, extending mouse intron 2 to mouse exon 6, to produce modified BAC#1 (see [link to BAC#1]). Figure 1A The Flt3l sequence was replaced by spectinomycin C-frames linked to upstream and downstream homology frames (260 bp and 230 bp) via bacterial homology recombination (BHR). The homology frames were prepared by PCR using BAC as a template and are shown in Table 4 below.

[0465] Table 4

[0466] To humanize the aforementioned BAC#1, an initial plasmid was generated to carry nucleic acids encoding the human FLT3L signal peptide and cytokine-like core domain, as well as the 5' mouse Flt3l sequence. More specifically, the initial plasmid contains from 5' to 3': (i) the NotI site; (ii) a 382 bp mouse Flt3l sequence (the 3' portion of mouse intron 1 (348 bp) and the 5' portion (non-coding portion) of mouse exon 2 (34 bp)); (iii) a human FLT3L nucleic acid sequence (from modified human BACCTD2523c15 (ThermoFisher / Invitrogen)), which contains a portion of exon 2 (33 bp) starting from ATG, 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); and (v) the SpeI site. The donor plasmid was digested with NotI and SpeI to release the fragment, which was then ligated, from 5' to 3', to the following: (i) a selection cassette containing a hygromycin resistance gene operatively linked to the EM7 promoter; and (ii) a 230 bp mouse Flt3l sequence (the 3' portion of mouse intron 6). This BHR donor fragment containing the human FLT3L nucleic acid sequence and the selection cassette (side-joined with the mouse Flt3l sequence (upstream and downstream frames)) was used to replace the spectinomycin cassette with BHR, producing BAC#2 (see [link to BHR]). Figure 1B (Step A). ​​Homologous frames were prepared by PCR using BAC as a template and are shown in Table 5 below:

[0467] Table 5

[0468] To prepare the final targeting vector from the aforementioned BAC#2, the EM7-Hyg cassette (see [link to BHR]) was replaced with a neomycin (Neo) resistance self-deletion cassette (with a CRE recombinase controlled by the protamine promoter) with a loxp-side loxp-linked site (loxp-neo-loxp). Figure 1B (Step B).

[0469] The final targeting vector (BAC#3) contains, from 5' to 3': a 5' mouse homologous arm, a human FLT3L signal peptide and a cytokine-like core domain, a loxp-Neo-loxp self-deletion cassette, a 3' mouse homologous arm, and a chloramphenicol resistance cassette (CM); Figure 1B (Not shown in the text); final clones were selected based on Neo / CM resistance.

[0470] The final targeting vector was electroporated into mouse embryonic stem (ES) cells containing hSIRP-α (or human SIRPA), RAG2- / - IL2Rg- / - modified cells (e.g., described in WO 2014 / 071397, US Patent Nos. 11,019,810, and WO 2016 / 168212, each of which is incorporated herein by reference). Targeted homologous recombination resulted in the replacement of a 2336 bp mouse sequence (GRCm38 / mm10 coordinates 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 self-deletion box. Figure 1C Successful integration was confirmed by allele modification (MOA) assays, as described above, for example by Valenzuela et al. The probes used for MOA assays to detect the loss of the mouse FLT3L sequence and the presence of the human FLT3L sequence are shown in Table 6 below, with their approximate locations schematically indicated. Figure 1C and 1D The box was subsequently removed in mice by the expression of CRE recombinase (controlled by the protamine promoter). Figure 1D ).

[0471] Table 6

[0472] Positively targeted ES cells were used as donor ES cells and microinjected into premorula (8-cell) stage mouse embryos using the VELOCIMOUSE® method (see, for example, US 7,576,259, US 7,659,442, US 7,294,754, and US 2008-0078000 A1, all of which are incorporated herein by reference). Mouse embryos containing donor ES cells were incubated in vitro and then implanted into surrogate mothers to produce F0 mice entirely derived from donor ES cells. Mice with the Flt3l gene deletion and those with humanized Flt3l insertion were identified by genotyping using the MOA assay described above. Mice heterozygous for Flt3l gene deletion and humanized Flt3l were bred to be homozygous. These humanized Flt3l mice, which also included Rag2 gene knockout, Il2rg gene knockout, and humanized Sirpa, were bred to be homozygous for all genes.

[0473] Based on a specific design, mice encoding chimeric membrane-bound FLT3L were generated, such as... Figure 1EThe figure shows the membrane-bound mouse FLT3 ligand labeled "mFlt3l_MB", obtained from the NCBI reference sequence: NP_038548.3, Uniprot: A9QW46; the figure shows the membrane-bound human FLT3 ligand labeled "hFLT3LG_MB", obtained from the NCBI reference sequence: NP_001191431.1, Uniprot: P49771-1; the protein domains are shown in the figure.

[0474] Example 1.2: Generating Flt3 knockout mice

[0475] The mouse Flt3 gene was deleted in the mouse genome using VELOCIGENE® technology (see, for example, US 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 cited and incorporated herein by reference). A 69.1 kb mouse genome sequence of the mouse Flt3 gene was deleted from the start codon ATG to the stop codon between coordinates chr5: 147331171-147400265 (GRCm38 assembly) on mouse chromosome 5 G3. See [link to relevant documentation]. Figure 2 .

[0476] Specifically, mouse homologous arms were prepared by PCR amplification using BAC clone RP23-76I6 (ThermoFisher / Invitrogen) as a template, as shown in Table 7 below:

[0477] Table 7

[0478] To prepare a targeting vector (named MAID20455) from the mouse BAC clone RP23-76I6, a 69.1 kb mouse sequence containing the mouse Flt3 gene was replaced by a hygromycin (Hyg) resistance self-deletion cassette (with a CRE recombinase controlled by the protamine promoter) (loxp-hyg-loxp) with a loxp-side loxp site.

[0479] The final targeting vector, from 5' to 3', contains: a 5' mouse homologous arm, a loxp-Hyg-loxp self-deletion cassette, a 3' mouse homologous arm, and a chloramphenicol resistance cassette (CM). Figure 2(Not shown in the image); the final clone was selected based on CM / Hyg resistance.

[0480] The MAID20455 targeting vector was electroporated into mouse embryonic stem (ES) cells containing hSIRP-α (or human SIRPA), RAG2- / - IL2Rg- / - modifications. Targeted homologous recombination resulted in the deletion of approximately 69.1 kb of mouse sequence (GRCm38 coordinates chr5: 147331171-147400265). Successful integration was confirmed by allele modification (MOA) assays, as described above, for example by Valenzuela et al. Primers and probes used for MOA assays to detect the loss of the mouse Flt3 sequence are shown in Table 8 below. The cassette was subsequently removed by expression of the CRE recombinase (controlled by the protamine promoter) in the mouse.

[0481] Table 8

[0482] Positively targeted ES cells were used as donor ES cells and microinjected into premorula (8-cell) stage mouse embryos using the VELOCIMOUSE® method (see, for example, US 7,576,259, US 7,659,442, US 7,294,754, and US 2008-0078000 A1, all of which are incorporated herein by reference). Mouse embryos containing donor ES cells were incubated in vitro and then implanted into surrogate mothers to produce F0 mice entirely derived from donor ES cells. Mice with Flt3 gene deletion were identified by genotyping using the MOA assay described above. Mice heterozygous for Flt3 gene deletion were bred to be homozygous. Flt3 KO mice, which also contained Rag2 gene knockout, Il2rg gene knockout, and humanized Sirpa, were bred to be homozygous for all genes. These mice are referred to as Flt3 in the remaining examples and relevant sections of the specification. - / -Mice containing humanized Flt3l, Rag2 gene knockout, Il2rg gene knockout, and humanized Sirpa were then bred together 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 known as "SRG hFLT3L mFLT3 KO" mice, "SRG-hFLT3L / mFLT3 KO" mice, or "hFLT3L / mFLT3 KO" mice); while mice containing Rag2 gene knockout, Il2rg gene knockout, and humanized Sirpa are referred to as SRG mice, and SRG mice with humanized thrombopoietin are referred to as StRG mice (these mice serve as controls and are expected to have the same level of human dendritic cell implantation as SRG mice, as described, for example, in US 10,463,028, which is incorporated herein by reference).

[0483] Example 1.3: Expression of humanized Flt3l in genetically modified mice

[0484] To validate the expression of humanized Flt3l in genetically modified mice, blood was collected from unimplanted StRG and SRG / hFLT3L / mFLT3 KO mice and added to serum separation tubes to collect serum. Bone marrow aspirates were also collected from unimplanted StRG and SRG / hFLT3L / mFLT3 KO mice. Humanized Flt3l in the samples was analyzed using a Human FLT3L Quantikine ELISA (R&D systems) according to the manufacturer's instructions. Normal human serum and bone marrow aspirates from healthy donors were used as comparative controls.

[0485] Humanized Flt3l levels were approximately 10,000 pg / mL in both serum and bone marrow aspirates from unimplanted SRG / hFLT3L / mFLT3 KO mice, compared to approximately 100 pg / mL in normal healthy human serum and slightly lower in human bone marrow aspirates. Figure 3 The supraphysiological levels of humanized Flt3l in SRG / hFLT3L / mFLT3 KO mice are likely due to the absence of mouse FLT3, which leads to higher levels of free humanized Flt3l because there is no receptor to consume it. As expected, the StRG mouse samples expressing only mouse Flt3l were negative for human FLT3L. The bar chart shows the mean for each sample (n=4) and gives the standard error.

[0486] Example 2: Development of human dendritic cells in genetically modified mice

[0487] Example 2.1: Increased number of human myeloid and plasmacytoid dendritic cells (DCs) in implanted genetically modified mice.

[0488] SRG / hFLT3L / mFLT3KO and StRG mouse strains were implanted by intrahepatic injection of 100,000 hHSCs into 1–5 day old pups. After 10–12 weeks, blood was collected from the mice via cardiac puncture, and spleens were harvested. The spleens were mechanically disrupted, filtered through a 70 mM sieve, and red blood cells (RBCs) were lysed in ACK lysis buffer (Gibco) to prepare single-cell suspensions. Blood single-cell suspensions were prepared by lysing RBCs in ACK lysis buffer (Gibco). All cells were resuspended in FACS buffer containing a mouse / human Fc inhibitor (BD Biosciences) and counted. Cells were stained for FACS analysis using the following monoclonal antibodies diluted 1:50 in PBS + 1 mM EDTA and 2% fetal bovine serum (FACS buffer): 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), anti-human BDCA-3-BV605 (clone M80; Biolegend), anti-human CD123 FITC (clone 6H6; Biolegend), and anti-human BDCA-2-PE-Dazzle (clone 201A; Biolegend). Cells were obtained from BD FACSymphony A3 and analyzed using FlowJo software. Human dendritic cells (DCs) were analyzed from the human CD45+ / mouse CD45- / human CD3- / human CD19- population. Myeloid DCs were then classified into human CD11c+ / human HLA-DR+ (…). Figure 4A Further analysis was conducted within these myeloid DCs to differentiate BDCA-3+ DCs (BDCA-3+ / CD11c+), which are considered the most efficient subset in cross-presenting exogenous antigens via MHC class I. Figure 4APlasma cell-like dendritic cells (the most potent producers of type I IFN) are distinguished from human CD45+ / mouse CD45- / human CD3- / human CD19- / human HLA-DR+ populations by co-expression of BDCA-2 and CD123. Figure 4A ).

[0489] The hCD45+ implantation levels were comparable between the two mouse strains, but SRG / hFLT3L / mFLT3 KO mice had increased myeloid and plasmacytic DCs in the blood and spleen. Figure 4B Furthermore, following hHSC implantation, SRG / hFLT3L / mFLT3 KO mice exhibited increased BDCA-3+ myeloid DCs in their blood and spleen. Figure 4C Data represent three HSC donors matched between StRG and SRG / hFLT3L / mFLT3 KO mice. Individual data points and means are given for each mouse. Unpaired Student's t-tests were used for statistical analysis.

[0490] Bone marrow (BM) was prepared from 10-12 week old hHSC-implanted mice as follows: femurs / tibias were collected and ground, and the ground bone was filtered through a 70 mM mesh filter. RBCs were then lysed with ACK lysis buffer (Gibco). Cells were then FACS stained and DC analysis was performed as previously described. SRG / hFLT3L / mFLT3 KO mice had comparable implantation volumes to HSC donor-matched StRG mice, but BM showed an increase in human myeloid and plasmacytoid DCs. Figure 4D Individual data points and means for each mouse are given. Statistical analysis was performed using an unpaired Student's t-test.

[0491] Thymus cells were also collected from implanted mice and mechanically disrupted. Single-cell suspensions were prepared by passing the cells through a 70 mM filter and lysing RBCs with ACK lysis buffer (Gibco). The cells were then FACS stained and DC analysis was performed as previously described. SRG / hFLT3L / mFLT3 KO mice had comparable implantation volumes to HSC donor-matched StRG mice, but showed an increase in human medullary and plasmacytic DCs in the thymus. Figure 4E Individual data points and means for each mouse are given. Statistical analysis was performed using an unpaired Student's t-test.

[0492] We hypothesize that human DC development is enhanced by the loss of mouse DCs after human hHSC implantation, thereby removing a potentially competitive cellular niche. To this end, spleens from implanted mice were subjected to FACS staining using 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). Significant loss of mouse DCs (mouse CD45+ / CD11c+ / MHC class II+) was observed in implanted SRG / hFLT3L / mFLT3 KO mice, indicating that mouse DCs can be significantly reduced by inducing Flt3 deletion in mice. Figure 4F Individual data points and means for each mouse are given. Statistical analysis was performed using an unpaired Student's t-test.

[0493] Example 2.2: Enhanced human DC development requires humanization of Flt3l and deletion of mouse FLT3 / FLK2.

[0494] It has been reported that FLT3-deficient mice enhance human dendritic cell (DC) development after human hematopoietic stem cell (hHSC) implantation. However, increased human DCs require repeated injections of high doses of human FLT3L (Li et al. (2016) Eur J Immunol 46:1291-1299). Our model, with receptor deficiency and Flt3L humanization, resulted in persistent expression of the Flt3L isotype at physiologically relevant levels. To investigate whether this additional humanization was necessary, we analyzed hHSC-implanted SRG mice (FLT3L) with mFlt3 KO. m / m mFLT3- / -) and SRG mice with mFlt3 KO and humanized Flt3l (FLT3L) h / h mFLT3- / -). Blood, spleen, and thymus were prepared from 10-12 week old mice that underwent implantation, and the human DC population was analyzed as previously described. Mice matched with HSC donors possessing only mFlt3 KO (FLT3L) were compared. m / m mFLT3- / -), hHSC-implanted mice with humanized FLT3L / mFLT3 KO (FLT3L h / h The levels of mFLT3- / - in the blood and spleen of both human mDC and pDC populations were increased. Figure 5A Furthermore, the same pattern can also be observed in the thymus. Figure 5BTherefore, we conclude that optimal human DC development in this novel human immune system strain requires both mouse Flt3 / FLK2 deletion and humanized Flt3l. Individual data points and means for each mouse are presented.

[0495] Example 3: Development of human T cells in genetically modified mice

[0496] Since dendritic cells (DCs) are key regulators of T cell activation, the phenotype of human T cells in hHSC-implanted SRG / hFLT3L / mFLT3 KO and StRG mice was evaluated. Dysmorphic human T cell phenotypes in human immune system mouse models were evidenced by higher expression of checkpoint inhibitors such as PD-1. In normal human T cells, approximately 5% or fewer T cells are PD-1+, while this proportion is consistently higher in human T cells generated from hHSC implantation in various previously characterized human immune system mice.

[0497] As previously described, StRG and SRG / hFLT3L / mFLT3 KO mice were implanted, and blood was collected via the retro-orbital route 10–12 weeks post-implantation. RBCs were lysed with ACK lysis buffer (Gibco), and the cells were stained with the following monoclonal antibodies as previously described: 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-PacificBlue (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).

[0498] Compared to StRG mice, SRG / hFLT3L / mFLT3 KO mice implanted with hHSCs showed significantly lower levels of PD-1 expression on CD3+ T cells in the blood. Figure 6A Furthermore, spleen T cell assessment showed that the proportion of CD4+ T cells was higher than that of CD8+ T cells in hHSC-implanted SRG / hFLT3L / mFLT3 KO mice. Figure 6A This indicates that the T cell subsets are normal, as the normal CD4 / CD8 T cell distribution is 60 / 40, as observed in SRG / hFLT3L / mFLT3 KO. Figure 6AIn StRG mice, the ratio is closer to 50 / 50. Individual data points and means for each mouse are presented. 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.

[0499] To assess the activation status of human T cells in different human immune system mouse strains, spleen cells from hHSC-implanted StRG and SRG / hFLT3L / mFLT3 KO mice were subjected to FACS staining using 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 represent different T cell subsets: CD44- / CD62L+ (naive T cells), CD44+ / CD62L- (effector cells), and CD44+ / CD62L+ (central memory T cells; Tcm). T cells in mouse models of the human immune system are generally considered to have low responses to antigen stimulation and very poor memory responses. However, compared to HSC donor-matched StRG mice, more CD4+ and CD8+ T cells are found in hHSC-implanted SRG / hFLT3L / mFLT3 KO mice, specifically Tcm (…). Figure 6B Individual data points and means for each mouse are given.

[0500] Example 4: Characterization of dendritic cell populations in genetically modified mice

[0501] To compare human DCs from hHSC-implanted SRG / hFLT3L / mFLT3 KO mice with those from healthy human donors, bulk human DCs were isolated from the spleen / blood of implanted mice and from the blood of healthy human donors using the EasySep Human Pan-DC Pre-Enrichment Kit, following the manufacturer's instructions (StemCell Technologies). Additionally, bulk human DCs were enriched from hHSC-implanted SRG / hFLT3L / mFLT3 KO BM and healthy human BM. Although the small number of isolated cells affected the reliability of the analysis, DCs isolated from hHSC-implanted StRG mice were still used as a control. Three different donors were used in the evaluation: human blood, BM, and HSC-implanted donors.

[0502] The isolated dendritic cells (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 the RNA-seq library, sample demultiplexing, alignment, filtering, and UMI counting were performed using the Cell Ranger Single-Cell Software Suite (10X Genomics, v2.2.0). Alignment was performed using mouse mm10 genome assembly and a mouse RefSeq gene model. Subsequently, cluster analysis of the DC population was performed based on transcriptomic profiles (data not shown).

[0503] Key transcriptional differences were found between the dendritic (DC) populations in the spleen / blood of hHSC-implanted SRG / hFLT3L / mFLT3 KO mice and in the blood of healthy individuals. The proportion of Clec10a myeloid DCs was lower in hHSC-implanted SRG / hFLT3L / mFLT3 KO mice compared to healthy human donors. Figure 7A Furthermore, the transcriptional profile of plasma cell-like DCs in healthy human donors was represented by IRF4 expression, while this transcriptional signature was decreased in plasma cell-like DCs from hHSC-implanted SRG / hFLT3L / mFLT3 KO mice. Figure 7A Since IRF4 is a key regulator of type I IFN production by plasma cell-like dendritic cells (DCs), further research was conducted to evaluate the ability of hHSC-implanted mice to produce type I IFN. Figure 9A and 9BFinally, compared with healthy human DCs, SRG / hFLT3L / mFLT3 KO mice implanted with hHSCs had a higher proportion of XCR1+ / Clec9a+ DCs in their spleen / blood. Figure 7A It is noteworthy that this population is a BDCA-3+ DC subset, and FACS analysis of the blood of hHSC-implanted SRG / hFLT3L / mFLT3 KO mice showed that this population indeed had a higher proportion of DCs compared to those derived from human peripheral blood mononuclear cells (PBMCs). Figure 7B Furthermore, the proportions of human myeloid DCs and plasmacytoid DCs in SRG / hFLT3L / mFLT3 KO mice implanted with hHSCs were higher than those in human PBMCs. Figure 7BBDCA-3+ / Clec9a+ / XCR1+ myeloid DCs have been characterized as a key DC subset for cross-initiation and activation of cytotoxic CD8+ T lymphocytes (CTLs) against tumor cells, viral pathogens, and for establishing peripheral immune tolerance (Crozat and Dald (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. and Wolftman, AM (2015) Semin Cell Dev Biol 41:39-48). This DC subset performs well in cross-presentation because it redirects antigens from early endocytic vesicles to late endosomes / lysosomes, and then to cytosols, for presentation via the MHC-I pathway (Cohn et al. (2013) J. Exp. Med. 210(5):1049-63, and Savina et al. (2009) Immunity 30(4):544-55). This DC subset is indeed an attractive target for immunotherapies aimed at evoking T-cell responses to tumors and pathogens (Crozat and Dald (2011) Med Sci (Paris) 27:25-28; Schreibelt et al. (2012) Blood 119: 2284-2292; van der Aa, E., van Montfoort, N. and Wolftman, AM (2015) Semin Cell Dev Biol 41:39-48; Masterman et al. (2020) JImmunother Cancer 8; Pearson et al. (2020) Clin Transl Immunology 9:e1141; Tullett et al. (2016) JCI Insight 1:e87102). Therefore, due to the enhanced peptide presentation capabilities of this DC subset, our SRG / hFLT3L / mFLT3 KO human immune system strain is an attractive model for testing potential immunotherapies targeting cross-initiation.

[0504] Plasma-like dendritic cells (pDCs) are key type I IFN producers in viral infections and are also associated with 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) ProcNatl 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 the presence of human pDCs in various organs of SRG / hFLT3L / mFLT3 KO human immune system mice, spleen, kidney, lung, blood, and bone marrow were extracted from SRG / hFLT3L / mFLT3 KO human immune system mice and processed to prepare single-cell suspensions. Human pDCs were assessed by flow cytometry based on the following gating criteria: lymphocytes, single cells, live cells, mouse CD45-, human CD45+, human CD3-, human CD16-, human CD19-, human CD123+, human HLA-DR+, human ILT7+, and human BDCA2+. Figure 8 The data shown are plotted as the percentage of pDCs in total viable human CD45+ cells, and plotted on a single mouse basis. These data indicate that human pDCs can be identified in the blood, lymphoid organs, and other tissue sites of SRG / hFLT3L / mFLT3 KO human immune system mice.

[0505] To test whether human pDCs retained function in these mice, human pDCs were isolated from SRG / hFLT3L / mFLT3 KO human immune system mice and stimulated in vitro to determine their ability to respond to stimulation and produce type I interferon (IFN). Single-cell suspensions of spleen and bone marrow were obtained by tissue dissociation. Mouse cells were removed by staining APC-conjugated mouse CD45 and mouse Ter119 followed by magnetic separation. Human pDCs were then isolated using the EasySep Human pDC Enrichment Kit (Stem Cell). pDCs were plated at specified cell numbers and a specified concentration of ODN2216 (Invivogen) (a type A stimulating CpGODN) was added. Cells were incubated with the stimulant for 18 hours. The supernatant was collected, and human IFNα or IFNβ were assessed separately by ELISA. Figure 9A As shown, human pDCs isolated from SRG / hFLT3L / mFLT3 KO human immune system mice respond to TLR9 stimulation and produce type I IFN. Furthermore, as measured by induction of type I IFN characteristic genes, human pDCs from these mice were determined to respond in vivo to ODN2216 CpG stimulation and produce human type I IFN (…). Figure 9B Specifically, mice were treated intravenously with 5 μg of ODN2216 delivered via the InVivoJet delivery system (PolyPlus). Blood was collected 6 hours later and processed to isolate RNA. qPCR was run to assess gene expression of the specified genes and based on standard... The analysis was performed to determine the relative quantification.

[0506] Our data demonstrate that human plasma cell-like dendritic cells (DCs) in hHSC-implanted SRG / hFLT3L / mFLT3 KO mice elicit a type I IFN response. Therefore, our SRG / hFLT3L / mFLT3 KO human immune system mouse is an excellent model for testing modulators of plasma cell-like DC function.

[0507] By citation and inclusion in this article

[0508] All publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application were expressly and separately indicated to be incorporated herein by reference. In case of any conflict, this application (including any definitions herein) shall prevail.

[0509] Also incorporated in their entirety by reference are any polynucleotide and polypeptide sequences whose accession numbers are associated with entries in public databases, such as those maintained by the Institute for Genomic Research (TIGR) on tiger.org and / or the National Center for Biotechnology Information (NCBI) on ncbi.nlm.nih.gov. Those skilled in the art will understand that the aforementioned publicly available sequence databases, or sequence databases known in the art, are periodically updated to publish revised sequences. Such revised sequences are incorporated herein by reference in their entirety.

[0510] Equivalent scheme

[0511] Those skilled in the art will recognize or be able to determine many equivalent embodiments of the invention described herein using only conventional experiments. Such equivalent embodiments are intended to be covered by the following claims.

Claims

1. A genetically modified rodent comprising: (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) The Flt3 ligand (Flt3l) gene, which contains rodent and human portions operatively linked to the Flt3l promoter.

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 in the rodent's Flt3 gene includes insertions, deletions, and / or substitutions in the endogenous Flt3 gene.

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

5. The genetically modified rodent of 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 1-5, wherein the rodent portion of the Flt3l gene comprises the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene and the exon downstream of exon 6.

7. The genetically modified rodent according to claim 6, wherein the non-coding portions of exon 1 and exon 2 of the rodent Flt3l gene and the exon downstream of exon 6 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 1-7, wherein the human portion of the Flt3l gene comprises the signal peptide encoding portion of exon 2 of the human FLT3L gene and exons 3-6.

9. The genetically modified rodent of claim 8, 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.

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

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

12. The genetically modified rodent of claim 11, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises the rodent stem region, the rodent transmembrane domain, and the rodent cytoplasmic tail as shown in FIG1E.

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

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

15. The genetically modified rodent of claim 14, wherein the soluble form of the Flt3l polypeptide and the membrane-bound form of the Flt3l polypeptide comprise the signal peptide and cytokine-like core domain of the human FLT3L polypeptide.

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

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

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

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

20. The genetically modified rodent according to any one of claims 1-19, wherein the genetically modified rodent is heterozygous for the Flt3 ligand (Flt3l) gene comprising both rodent and human portions.

21. The genetically modified rodent according to any one of claims 1-19, wherein the genetically modified rodent is homozygous for the Flt3 ligand (Flt3l) gene comprising both rodent and human portions.

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

24. The genetically modified rodent according to any one of claims 1-23, 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 23-25, wherein the Flt3l promoter is located at an endogenous rodent locus.

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

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

29. The genetically modified rodent of claim 28, 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.

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

31. The genetically modified rodent according to any one of claims 28-30, 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 of claim 27, wherein the genetically modified rodent expresses a human SIRPA polypeptide encoded by a nucleic acid operatively linked to a Sirpa promoter.

33. The genetically modified rodent according to any one of claims 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.

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

35. The genetically modified rodent of 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 respective endogenous rodent locus.

37. The genetically modified rodent according to any one of claims 27-36, wherein the rodent contains an invalid mutation in at least one corresponding rodent gene at the corresponding rodent locus.

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

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

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

41. The genetically modified rodent of 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 precursor cells, and human dendritic cells.

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

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

44. The genetically modified rodent of 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 1-44, wherein the rodent is a mouse or a rat.

46. ​​The genetically modified rodent of claim 45, wherein the rodent is a mouse.

47. A method for identifying a drug that regulates the function of human dendritic cells, comprising: a. Administering the candidate agent to genetically modified rodents according to any one of claims 40-46; as well as b. Determine whether the candidate drug modulates the function of the human dendritic cells in the rodent.

48. The method of claim 47, wherein the human dendritic cells are myeloid dendritic cells (mDC) or plasmacytoid dendritic cells (pDC).

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

50. A method for evaluating the therapeutic efficacy of drug-stimulated T cell response to target cells, the method comprising: a. Administering the drug candidate to a genetically modified rodent according to any one of claims 40-42, wherein the genetically modified rodent comprises the target cells; as well as b. Measure the response of T cells in the rodent to the target cells to evaluate the therapeutic efficacy of the drug candidate.

51. The method of 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 efficacy of a drug for treating an autoimmune disease, the method comprising: a. Administering the candidate agent to genetically modified rodents according to claim 43 or 44; as well as b. Determine whether the agent treats the autoimmune disease in the rodent.

53. A genetically modified rodent cell comprising: (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) The Flt3 ligand (Flt3l) gene, which contains rodent and human portions operatively linked to the Flt3l promoter.

54. The genetically modified rodent cell of 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 in the rodent Flt3 gene includes insertions, deletions, and / or substitutions in the endogenous Flt3 gene.

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

57. The genetically modified rodent cell of claim 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).

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

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

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

61. The genetically modified rodent cell of claim 60, 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.

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

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

64. The genetically modified rodent cell of claim 63, wherein the C-terminal portion of the rodent Flt3l polypeptide comprises the rodent stem region, the rodent transmembrane domain, and the rodent cytoplasmic tail as shown in FIG1E.

65. The genetically modified rodent cell according to claim 63 or 64, wherein the C-terminal portion of the rodent Flt3l polypeptide has at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the same amino acid sequence as 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 53-65, wherein the rodent cell expresses both a soluble form of the Flt3l polypeptide and a membrane-bound form of the Flt3l polypeptide.

67. The genetically modified rodent cell of claim 66, wherein the soluble form of the Flt3l polypeptide and the membrane-bound form of the Flt3l polypeptide comprise the signal peptide and cytokine-like core domain of the human FLT3L polypeptide.

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

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

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

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

72. The genetically modified rodent cell according to any one of claims 53-71, wherein the genetically modified rodent cell is heterozygous for the Flt3 ligand (Flt3l) gene comprising both rodent and human portions.

73. The genetically modified rodent cell according to any one of claims 53-71, wherein the genetically modified rodent cell is homozygous for the Flt3 ligand (Flt3l) gene comprising both rodent and human portions.

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

76. The genetically modified rodent cell according to any one of claims 53-75, 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 75-77, wherein the Flt3l promoter is located at an endogenous rodent locus.

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

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

81. The genetically modified rodent cell of claim 80, 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.

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 a human SIRPA polypeptide and the intracellular portion of a rodent Sirpa polypeptide.

83. The genetically modified rodent cell according to any one of claims 80-82, 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 of claim 79, wherein the genetically modified rodent cell expresses human SIRPA polypeptide.

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

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

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

88. The genetically modified rodent cells 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 locus.

89. The genetically modified rodent cell according to any one of claims 79-88, comprising an invalid 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 79-89, wherein the genetically modified rodent cell is heterozygous for at least one allele, the allele comprising a nucleic acid sequence encoding the human protein or humanized protein.

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

92. The genetically modified rodent cell according to any one of claims 53-91, 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 53-65, 68-81, 83 and 85-93, wherein the genetically modified rodent cell is a rodent embryonic stem (ES) cell.

95. A method for preparing rodent embryonic stem cells, comprising genetically modifying the rodent embryonic stem cells such that the rodent embryonic stem cells have a genome comprising: (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) The Flt3 ligand (Flt3l) gene, which contains rodent and human portions operatively linked to the Flt3l promoter.

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

97. A method for preparing a rodent, said 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 comprising a rodent portion and a human portion operatively linked to the Flt3l promoter, said method comprising the steps of: (a) Obtaining rodent embryonic stem cells according to claim 94, or rodent embryonic stem cells prepared by the method according to claim 95; and (b) Producing rodents using rodent embryonic cells as described in (a).

98. A method for preparing a rodent, said 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 comprising a rodent portion and a human portion operatively linked to the Flt3l promoter, said method comprising modifying the genome of said rodent such that it comprises: (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 operatively linked to the Flt3l promoter.

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