WO2022012544A1 - 对非人动物进行基因改造和构建免疫缺陷动物模型的方法 - Google Patents

对非人动物进行基因改造和构建免疫缺陷动物模型的方法 Download PDF

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WO2022012544A1
WO2022012544A1 PCT/CN2021/106058 CN2021106058W WO2022012544A1 WO 2022012544 A1 WO2022012544 A1 WO 2022012544A1 CN 2021106058 W CN2021106058 W CN 2021106058W WO 2022012544 A1 WO2022012544 A1 WO 2022012544A1
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human
cells
promoter
mice
mouse
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黄菁
卢娜
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黄菁
卢娜
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Definitions

  • the present application relates to a method for genetically modifying non-human animals and constructing an immunodeficiency animal model.
  • Humanization of the mouse immune system usually refers to the reconstruction of one or several human immune cells in the mouse after the implantation of human peripheral blood leukocytes (hPBMC) or human hematopoietic stem cells (CD34+HSC) in immunodeficient mice.
  • hPBMC peripheral blood leukocytes
  • CD34+HSC human hematopoietic stem cells
  • the humanization process of the mouse immune system can be used to detect the differentiation and colonization function of human hematopoietic stem cells and therapeutic cells based on human hematopoietic stem cells.
  • humanized mice can better simulate the human immune system. It plays an irreplaceable role in many fields such as infectious diseases, antibody drug development, autoimmune diseases and oncology research.
  • Severely immunodeficient mice usually refer to NOD scid IL2R ⁇ KO mice (SCID mutant and IL2R ⁇ knockout NOD mice), BALB/c Rag1KO.IL2R ⁇ KO (IL2R ⁇ and Rag1 knockout BALB/c mice) or BALB/c Rag2KO .IL2R ⁇ KO mice (IL2R ⁇ and Rag2 knockout BALB/c mice.
  • mice with only SCID, Rag1 or Rag2 knockout severely immunodeficient mice further knocked out IL2R ⁇ , not only lacking T Cells, B cells, and lack of NK cell function, and the number of macrophages and dendritic cells is also greatly reduced, which further reduces the immune function of mice, which is more conducive to the construction of a humanized immune system.
  • NOD scid IL2R ⁇ KO mice are widely used to rebuild the human immune system in severely immunodeficient mice
  • BALB/c Rag1/2KO IL2R ⁇ KO mice have higher innate immune activity than NOD scid IL2R ⁇ KO mice, and the ability to rebuild the human immune system is also Weaker than NOD scid IL2R ⁇ KO mice, more genetic modifications are required to improve the degree to which BALB/c Rag1or2KO IL2R ⁇ KO mice reconstitute the human immune system. Therefore, BALB/c Rag1or2KO IL2R ⁇ KO mice are not as widely used as NOD scid IL2R ⁇ KO mice.
  • the above-mentioned severe immunodeficiency mice are collectively referred to as the first generation of severe immunodeficiency mice.
  • human hematopoietic stem cells CD34+HSC
  • human hematopoietic stem cells can reconstitute various immune cells in mice, such as human T cells, human B cells, and human myeloid cells.
  • the human immune system usually needs the support of human cytokines to differentiate and mature.
  • the human immune system reconstituted in the first generation of severely immunodeficient mice has functional defects, such as myeloid There are fewer cells, the lymphoid tissue is underdeveloped, and the T cells and B cells are not fully functioning.
  • mice with NOD scid IL2R ⁇ KO as genetic background such as NSG-Tg (hu-IL-15), NSG-SGM3, hIL2NOG, hIL-6NOG, hIL-15NOG , NOG-EXL, etc.
  • NOD scid IL2R ⁇ KO such as NSG-Tg (hu-IL-15), NSG-SGM3, hIL2NOG, hIL-6NOG, hIL-15NOG , NOG-EXL, etc.
  • the hIL-15NOG developed by the Central Research Institute of Laboratory Animals (CLEA) in Japan overexpressed human IL-15 in NOG mice using the CMV promoter, and the plasma expression reached 80-120pg/ml.
  • mice due to the slightly higher expression of cytokines, or the cytotoxicity caused by the non-specific expression of cytokines, mice are prone to a wasting disease after rebuilding the human immune system, reducing the experimental window period.
  • experiments have shown that purified human peripheral NK cells in hIL-15NOG mice can maintain growth for a long time.
  • American Jackson Lab used BAC transgenic technology to transfer the complete human IL5 locus into NSG mice to obtain NSG-Tg (hu-IL-15).
  • the expression level of human IL-15 in plasma was 7.1 pg/ml.
  • NSG-Tg (hu-IL-15) mice or hIL-15NOG mice use transgenic technology to express exogenous human IL-15 in mice, and cannot replace endogenous IL in NOG or NSG mice -15. Therefore, endogenous IL-15 also interferes with the function of the human immune system, especially the function of human NK cells.
  • using the principle of homologous recombination to directly perform gene editing on the fertilized eggs or ES cells of NOD scid IL2R ⁇ KO mice is technically difficult and has not been successfully reported.
  • mice were all transgenic mice with C57BL as the genetic background established by the classical microinjection of fertilized eggs, and then backcrossed with NOD scid IL2R ⁇ KO mice for more than 10 consecutive generations to obtain severe immunodeficiency mice overexpressing human cytokines. mouse. The entire backhand process took about 2 years.
  • the direct genetic modification of NOD scid IL2R ⁇ KO mice in the prior art has a low success rate and is difficult, so it is necessary to use mice with other genetic backgrounds for multi-generation backcrossing, resulting in a lot of time and labor costs.
  • the overexpression of human cytokines in mice, or the mutual interference with the mouse's own cytokines has adverse effects on the development and lifespan of the mice themselves, and also affects the human cells constructed in the mice. function of the immune system.
  • cytokines that are related to the maintenance of the mouse's own health and lack cross-activity with human homologous cytokines humanization in mice to replace the original mouse cytokines or overexpression using existing technologies will have little effect. The health and longevity of mice, and the function of building the human immune system in their bodies.
  • the present application utilizes an optimized genetic engineering method, while in some steps Creative improvements are made to directly transform non-human animals, such as the fertilized egg cells of NOD scid IL2R ⁇ KO severe immunodeficiency mice to avoid backcrossing, and obtain severe immunodeficiency mice that are more suitable for rebuilding a multifunctional human immune system.
  • a method for genetic modification of a non-human animal comprising:
  • Genetic modification includes the use of CRISPR/Cas9 for gene editing of fertilized eggs of non-human animals;
  • the fertilized egg is an in vitro fertilized fertilized egg
  • the fertilized egg is obtained by in vitro fertilization, and the fertilized egg is cultured in vitro for a certain period of time, preferably a certain period of time is more than 6 hours, more preferably more than 8 hours, more than 10 hours, more than 12 hours, 14 hours. Above, most preferably 16 hours or more.
  • the linear DNA template for homologous recombination needs to be injected greater than or equal to 2.5kb, and the gene editing of the fertilized egg of the non-human animal is to microinject the nucleus of the fertilized egg of the non-human animal cell .
  • the non-human animal is a mouse with NOD as a genetic background
  • the mouse is preferably an immunodeficient NOD mouse with knockout of recombination activation gene 1 (Rag1) or knockout of recombination activation gene 2 (Rag2) or SCID mutation and deletion of T cells and B cells,
  • the mouse is a severely immunodeficient NOD mouse depleted of NK cells in which the IL2 receptor ⁇ chain has been further knocked out.
  • a method of genetically modifying a non-human animal comprising:
  • the cytokines include any one or two or more of the following: interleukin-15 (IL-15), interleukin-7 (IL-7), interleukin-6 (IL-6), B cell activation Factor (BAFF), FMS-like tyrosine kinase 3 ligand (Flt3L) recombinant protein;
  • the immune-related genes include the major histocompatibility complex (MHC);
  • the cytokine is interleukin-15 (IL-15).
  • IL-15 interleukin-15
  • the method of genetic modification of non-human animals is to genetically modify the fertilized eggs of non-human animals
  • CRISPR/Cas9 is used to perform gene editing on fertilized eggs of non-human animals
  • the zygote of a non-human animal is gene-edited using CRISPR/Cas9 by microinjection into the nucleus of a fertilized egg to achieve in situ humanization of cytokines.
  • the genetic modification of the fertilized egg of the non-human animal further comprises inserting a bGHpolyA (bovine growth hormone polyadenylation) sequence after the fourth exon of IL-15 of the non-human animal;
  • bGHpolyA bovine growth hormone polyadenylation
  • the bGHpolyA sequence is shown in SEQ ID No.2;
  • the coding sequence of the degenerate human IL15 exons 5 to 8 inserted after the fourth exon of IL-15 of the non-human animal is shown in SEQ ID No. 1.
  • the mouse is preferably an immunodeficient NOD mouse with knockout of recombination activation gene 1 (Rag1) or knockout of recombination activation gene 2 (Rag2) or SCID mutation and deletion of T cells and B cells,
  • the mouse is further preferably a severely immunodeficient NOD mouse with NK cells depleted in which the IL2 receptor ⁇ chain has been further knocked out;
  • sequence of the fourth exon of IL-15 of the mouse is shown in SEQ ID No.4.
  • the fertilized eggs obtained by in vitro fertilization are cultured in vitro for a certain period of time before the genetic modification of the fertilized eggs of the non-human animal, preferably the certain period of time is more than 6 hours, more preferably more than 8 hours, more than 10 hours, 12 hours above, 14 hours or more, most preferably 16 hours or more.
  • the homologous recombination DNA comprises the human IL-15 homologous recombination DNA sequence shown in SEQ ID No.3;
  • the recognition target sequence of the gRNA1 is:
  • the recognition target sequence of the gRNA2 is:
  • a method for constructing an animal model of severe immunodeficiency comprising:
  • Cytokine genes or immune-related genes are humanized in situ by genetically modifying fertilized eggs of non-human animals, and these cytokine genes or immune-related genes will not affect immunodeficient mice after humanization. Humanization of health and reproductive efficiency;
  • the cytokines include any one or two or more of the following: interleukin-15 (IL-15), interleukin-7 (IL-7), interleukin-6 (IL-6), B cell activation Factor (BAFF), FMS-like tyrosine kinase 3 ligand (Flt3L) recombinant protein;
  • the immune-related genes include the major histocompatibility complex (MHC);
  • the cytokine is interleukin-15 (IL-15).
  • IL-15 interleukin-15
  • the method of genetic modification of non-human animals is to genetically modify the fertilized eggs of non-human animals
  • CRISPR/Cas9 is used to perform gene editing on fertilized eggs of non-human animals
  • the zygote of a non-human animal is gene-edited using CRISPR/Cas9 by microinjection into the nucleus of a fertilized egg to achieve in situ humanization of cytokines.
  • the genetic modification of the fertilized egg of the non-human animal further comprises inserting a bGHpolyA (bovine growth hormone polyadenylation) sequence after the fourth exon of IL-15 of the non-human animal;
  • bGHpolyA bovine growth hormone polyadenylation
  • the bGHpolyA sequence is shown in SEQ ID No.2;
  • the coding sequence of the degenerate human IL fifth to eighth exons inserted after the fourth exon of IL-15 of the non-human animal is shown in SEQ ID No.1.
  • the mouse is preferably an immunodeficient NOD mouse with knockout of recombination activation gene 1 (Rag1) or knockout of recombination activation gene 2 (Rag2) or SCID mutation and deletion of T cells and B cells,
  • the mouse is further preferably a severely immunodeficient NOD mouse with NK cells depleted in which the IL2 receptor ⁇ chain has been further knocked out;
  • sequence of the fourth exon of IL-15 of the mouse is shown in SEQ ID No.4.
  • the fertilized eggs obtained by in vitro fertilization are cultured in vitro for a certain period of time before the genetic modification of the fertilized eggs of the non-human animal, preferably the certain period of time is more than 6 hours, more preferably more than 8 hours, more than 10 hours, 12 hours above, 14 hours or more, most preferably 16 hours or more.
  • the homologous recombination DNA comprises the human IL-15 homologous recombination DNA sequence shown in SEQ ID No.3;
  • the recognition target sequence of the gRNA1 is:
  • the recognition target sequence of the gRNA2 is:
  • Linear DNA comprising the homologous recombination sequence of human IL-15 as shown in SEQ ID No.3.
  • a method of genetically modifying a non-human animal comprising:
  • Genetic modification includes gene editing of fertilized eggs of non-human animals using a Piggy transposase-dependent transgenic system
  • the fertilized egg is an in vitro fertilized fertilized egg
  • the fertilized egg is obtained by in vitro fertilization, and the fertilized egg is cultured in vitro for a certain period of time, preferably a certain period of time is more than 6 hours, more preferably more than 8 hours, more than 10 hours, more than 12 hours, 14 hours. Above, most preferably 16 hours or more.
  • the gene fragment to be edited is a circular plasmid
  • the gene editing of the fertilized egg of a non-human animal is to perform cytoplasmic or nuclear injection into the cytoplasm of the fertilized egg of the non-human animal cell.
  • the non-human animal is a mouse with NOD as a genetic background
  • the mouse is preferably an immunodeficient NOD mouse with knockout of recombination activation gene 1 (Rag1) or knockout of recombination activation gene 2 (Rag2) or SCID mutation and deletion of T cells and B cells,
  • the mouse is a severely immunodeficient NOD mouse depleted of NK cells in which the IL2 receptor ⁇ chain has been further knocked out.
  • Target protein 1 and/or target protein 2 Overexpression of target protein 1 and/or target protein 2 in non-human animals
  • the target protein 1 is mainly expressed in T cells, B cells and NK cells; after the gene of the target protein 1 is in situ humanized, the target protein 1 cannot be detected in severely immunodeficient non-human animals.
  • the expression of the gene preferably, the target protein one includes any one or both of the following: human interleukin-3 (human IL3), human interleukin-2 (human IL2); further preferably, the target protein one is human Interleukin-3 (human IL3);
  • the target protein 2 may affect the health of immunodeficient non-human animals; preferably, the target protein 2 includes any one or more of the following: human thrombopoietin (human TPO), human granulocyte-macrophage colony stimulation factor (human GM-CSF), human macrophage colony stimulating factor (human MCSF); further preferably, the target protein 2 is human granulocyte-macrophage colony stimulating factor (human GM-CSF).
  • human TPO human thrombopoietin
  • human GM-CSF human macrophage colony stimulating factor
  • human MCSF human macrophage colony stimulating factor
  • human IL3 cDNA encoding human interleukin-3 (human IL3) is shown in SEQ ID No.14;
  • human GM-CSF cDNA encoding human granulocyte macrophage colony stimulating factor is shown in SEQ ID No. 15.
  • the method of genetically modifying the non-human animal is to genetically modify the fertilized egg of the non-human animal so that the target protein is overexpressed in the non-human animal, which means that the target protein is overexpressed in the myeloid cells of the non-human animal;
  • the target protein is overexpressed in myeloid cells of non-human animals using a promoter specific for myeloid cell expression
  • the promoter expressed by the myeloid cell is selected from human CD68 promoter, CSF1R promoter, CD11c promoter, CX3CR1 promoter, Langerin/CD207 promoter, MMLV LTR promoter, Visna virus LTR promoter, DC-STAMP promoter promoter, Human MSR promoter, MSR-A promoter, CD4 promoter, CD2 promoter, Iba-AIF-1 promoter, CD11b promoter, c-fms promoter, scavenger receptor A (SR-A) promoter, lysozyme promoter and MHC class II promoter (MHC-II);
  • piggyBac transposon system plasmid and piggyBac transposase it is further preferred to use the piggyBac transposon system plasmid and piggyBac transposase to introduce the cytokine expression cassette into the fertilized egg of the non-human animal.
  • RAKR-GSG-P2A (RAKR-GSG-ATNFSLLKQAGDVEENPGP), RAKR- GSG-T2A (RAKR-GSG-EGRGSLLTCGDVEENPGP), RAKR-GSG-E2A (RAKR-GSG-QCTNYALLKLAGDVESNPGP) or RAKR-GSG-F2A (RAKR-GSG-VKQTLNFDLLKLAGDVESNPGP) mediate the overexpression of different proteins, where RAKR is a Furin shear
  • the sequence of the shortened peptide, GSG is the linker;
  • the double self-cleaving short peptide is RAKR-GSG-P2A
  • the sequence of the gene encoding the double self-cleaving short peptide RAKR-GSG-P2A is shown in SEQ ID No. 13.
  • a response repeat sequence is also inserted into the piggyBac transposon system plasmid, preferably the sequence is shown in SEQ ID No. 16 or SEQ ID No. 17.
  • the non-human animal is a mouse with NOD as a genetic background
  • the mouse is preferably an immunodeficient NOD mouse with knockout of recombination activation gene 1 (Rag1) or knockout of recombination activation gene 2 (Rag2) or SCID mutation and deletion of T cells and B cells;
  • the mouse is a severely immunodeficient NOD mouse depleted of NK cells in which the IL2 receptor gamma chain has been knocked out.
  • the fertilized eggs obtained by in vitro fertilization are cultured in vitro for a certain period of time before the genetic modification of the fertilized eggs of the non-human animal, preferably the certain period of time is more than 6 hours, more preferably more than 8 hours, more than 10 hours, 12 hours above, 14 hours or more, most preferably 16 hours or more.
  • the PiggyBac transposon system plasmid includes the sequence shown in SEQ ID No. 11.
  • a method of constructing an animal model of severe immunodeficiency comprising:
  • the target protein is target protein 1 and/or target protein 2;
  • the target protein 1 is mainly expressed in T cells, B cells and NK cells; the target protein 2 may affect the health of immunodeficient mice;
  • the target proteins overexpressed in non-human animals include any one or more of the following: human interleukin-3 (human IL3), human interleukin-2 (human IL2), human thrombopoietin ( Human TPO), human granulocyte-macrophage colony stimulating factor (human GM-CSF), human macrophage colony stimulating factor (human MCSF);
  • human interleukin-3 human IL3
  • human granulocyte-macrophage colony stimulating factor human GM-CSF
  • human IL3 cDNA encoding human interleukin-3 (human IL3) is shown in SEQ ID No. 14; human GM-CSF cDNA encoding human granulocyte-macrophage colony stimulating factor (human GM-CSF) The sequence is shown in SEQ ID No.15.
  • the method of genetically modifying the non-human animal is to genetically modify the fertilized egg of the non-human animal so that the target protein is overexpressed in the non-human animal, which means that the target protein is overexpressed in the myeloid cells of the non-human animal;
  • the target protein is overexpressed in myeloid cells of non-human animals using a promoter specific for myeloid cell expression
  • the promoter expressed by the myeloid cell is selected from human CD68 promoter, CSF1R promoter, CD11c promoter, CX3CR1 promoter, Langerin/CD207 promoter, MMLV LTR promoter, Visna virus LTR promoter, DC-STAMP promoter promoter, Human MSR promoter, MSR-A promoter, CD4 promoter, CD2 promoter, Iba-AIF-1 promoter, CD11b promoter, c-fms promoter, scavenger receptor A (SR-A) promoter, lysozyme promoter and MHC class II promoter (MHC-II), the promoter overexpresses the target protein in the myeloid cells of non-human animals;
  • SR-A scavenger receptor A
  • MHC-II MHC class II promoter
  • piggyBac transposon system plasmid and piggyBac transposase to overexpress the target protein in myeloid cells of non-human animals.
  • RAKR-GSG-P2A (RAKR-GSG-ATNFSLLKQAGDVEENPGP), RAKR-GSG- T2A (RAKR-GSG-EGRGSLLTCGDVEENPGP), RAKR-GSG-E2A (RAKR-GSG-QCTNYALLKLAGDVESNPGP) or RAKR-GSG-F2A (RAKR-GSG-VKQTLNFDLLKLAGDVESNPGP) mediates the overexpression of different target proteins, where RAKR is Furin spliced short The sequence of the peptide, GSG is the linker;
  • the double self-cleaving short peptide is RAKR-GSG-P2A
  • the sequence of the gene encoding the double self-cleaving short peptide PAKR-GSG-P2A is shown in SEQ ID No. 13.
  • a response repeat sequence is also inserted into the piggyBac transposon system plasmid, preferably the sequence is shown in SEQ ID No. 16 or SEQ ID No. 17.
  • the non-human animal is a mouse with NOD as a genetic background
  • the mouse is preferably an immunodeficient NOD mouse with knockout of recombination activation gene 1 (Rag1) or knockout of recombination activation gene 2 (Rag2) or SCID mutation and deletion of T cells and B cells;
  • the mouse is a severely immunodeficient NOD mouse depleted of NK cells in which the IL2 receptor ⁇ chain has been further knocked out.
  • the fertilized eggs obtained by in vitro fertilization are cultured in vitro for a certain period of time before the genetic modification of the fertilized eggs of the non-human animal, preferably the certain period of time is more than 6 hours, more preferably more than 8 hours, more than 10 hours, 12 hours above, 14 hours or more, most preferably 16 hours or more.
  • the PiggyBac transposon system plasmid includes the sequence shown in SEQ ID No. 11.
  • a PiggyBac transposon system plasmid the sequence of which is shown in SEQ ID No.11.
  • the present application solves the problem that NOD scid IL2R ⁇ KO mice have low fertilization activity, immature cell nucleus, and are not suitable for direct gene editing of large fragments. cytokine-derived NOD scid IL2R ⁇ KO mice, thus avoiding multiple generations of backcrosses, saving time and labor costs. In addition, the present application retains some fragments of the homologous gene of human and mouse, and recombines with the truncated human gene to obtain a chimeric humanized gene.
  • the size of the inserted fragment is reduced and the recombination efficiency is improved; the mouse-derived gene is replaced by a humanized gene, which avoids the coexistence of human and mouse genes compared to the overexpression method commonly used in the prior art.
  • the mutual interference reduces the impact on the differentiation, colonization and function of the human immune system when the human immune system is constructed in the mouse, and avoids the impact of overexpressed human cytokines on the development and physiological function of the mouse, prolonging the time. Mice lifespan and experimental window period.
  • the present application relates to a method for engineering a severely immunodeficient mouse, which can be used to more completely and efficiently reconstitute the human immune system in the mouse.
  • using the method described in the present application to obtain engineered mice is more time-saving and efficient, and the obtained mice have a longer experimental window period, and the functions of the human immune system reconstructed by using the mice are also improved. complete.
  • This application solves the problems of low fertilization activity in NOD scid IL2R ⁇ KO mice and immature cell nuclei that are not suitable for direct gene editing of large fragments, and realizes the direct genetic modification of NOD scid IL2R ⁇ KO mouse fertilized eggs to obtain expressing humanized cells factored NOD scid IL2R ⁇ KO mice, avoiding multiple generations of backcrossing, thus saving time and labor costs.
  • the present application achieves timely and appropriate expression, on the one hand, it reduces the immunity to human beings when the human immune system is constructed in the mouse. On the other hand, it avoids the influence of over-expressed human cytokines on the development and physiological functions of mice, thereby maintaining the health of mice and prolonging the lifespan and experimental window period of mice.
  • the present application directly genetically modifies the fertilized eggs of NOD scid IL2R ⁇ KO mice to obtain NOD scid IL2R ⁇ KO mice expressing humanized cytokines, avoiding multi-generation backcrossing, thereby saving time and labor costs.
  • the present application achieves timely and appropriate expression, which not only reduces the impact on the human immune system when constructing the human immune system in the mouse.
  • the influence of differentiation, colonization and function also avoids the influence of over-expressed human cytokines on the development and physiological functions of mice, thereby maintaining the health of mice and prolonging the lifespan and experimental window period of mice.
  • ordinary DNA microinjection requires large fragments to enter the nucleus of the fertilized egg, while the piggybac technology can perform chromatin injection of the fertilized egg, so it is especially suitable for the fertilized egg with hypoplastic or delayed cells.
  • the engineered severe immunodeficiency mouse can be used to more completely and efficiently reconstitute the human immune system in the mouse.
  • using the method described in the present application to obtain engineered mice is more time-saving and efficient, and the obtained mice are in better health and have a longer test window period.
  • the immune system is also more functional.
  • FIG. 1 Schematic diagram of humanization design of mouse IL-15 locus
  • Figure 2 cDNA sequence and protein amino acid sequence of humanized mouse IL-15 gene.
  • Figure 2A is the cDNA sequence (5'-3', 5'utl ⁇ mouse 4+ ⁇ human 5-8 ⁇ ) of IL-15 after humanization
  • Figure 2B is the amino acid sequence of IL-15 after humanization Compared with the amino acid sequence of murine IL-15;
  • Fig. 4 The average expression concentration of human IL-15 in plasma of NVG mice and NVG-hIL15 mice detected by ELISA method
  • Figure 5(A)-(D) are the frequencies of human CD45 cells, human T cells, human NK cells, and cytotoxic human NK cells expressing CD16 in the blood of mice after injection of human PBMC;
  • Figure 7 shows the results of IVIS imaging of K562-Luc after mice were injected with human PBMC and then K562-Luc;
  • Figure 8 is a flow chart of injecting NK cells and K562-Luc isolated from human peripheral blood into mice successively, and carrying out in vivo tumor-bearing test;
  • Figure 9 Changes in the percentage of NK cells in vivo in mice with time after injection of NK cells isolated from human peripheral blood;
  • Figure 11 is a flow chart of injecting MIA PaCa-2 and CAR-NK92 into mice successively, and carrying out in vivo tumor-bearing test;
  • Figure 14 is the piggyBAC CD68Pro-Intron-hGM-CSF-PAKR-GSG-P2A-hIL3-pA recombinant plasmid.
  • Figure 15 is an electrophoresis image of piggyBAC transgenic mice identified by PCR, the numerals are positive mice, and M is a 1kb DNA marker. Among them, 3, 4, 5, 6, and 10 are F0 generation NOD-SCID IL2R ⁇ KO-Tg (hCD68-hIL3/GMCSF) mice, and 12, 13, and 14 are F0 generation NOD-SCID IL2R ⁇ KO-Tg (SV40-hIL3/GMCSF) mice Mice, WT are negative transgenic mice.
  • Figure 16 is a schematic diagram of the working principle of inverse PCR.
  • Figure 17 is a graph showing the electrophoresis results of inverse PCR products.
  • Figure 18 is a graph showing the results of identification of the expression specificity of human IL3 and human GM-CSF in transgenic mouse tissues.
  • Figures 19(A)-(D) show the phenotypic characteristics of the human immune system in mice after the 12th week of injection of human CD34 stem cells.
  • Figure 20 shows the survival rate of mice after transplantation of human CD34 stem cells.
  • Figure 21 shows the expression levels of cytokines in mouse plasma after the 12th week of injection of human CD34 stem cells.
  • Figure 22 shows the hemoglobin count of mice after the 16th week of injection of human CD34 stem cells.
  • progeny as used herein includes progeny and includes differentiated or undifferentiated progeny cells derived from parental cells. In one usage, the term progeny includes progeny cells that are genetically identical to the parent. In another use, the term progeny includes progeny cells that are inherited and phenotypically identical to the parent. In yet another usage, the term progeny includes progeny cells that have differentiated from a parent cell.
  • promoter includes a DNA sequence operably linked to a nucleic acid sequence to be transcribed, such as a nucleic acid sequence encoding a desired molecule.
  • a promoter is generally located upstream of the nucleic acid sequence to be transcribed and provides a site for specific binding of RNA polymerase and other transcription factors.
  • the promoter is generally located upstream of the transcribed nucleic acid sequence to generate the desired molecule, and provides a site for specific binding of RNA polymerase and other transcription factors.
  • immuno-deficient means that a non-human animal is deficient in one or more aspects of its innate immune system, eg, the animal is deficient in one or more types of functional host immune cells is, for example, deficient in the number and/or function of non-human B cells, the number and/or function of non-human T cells, the number and/or function of non-human NK cells, and the like.
  • non-human animal as used herein is eg laboratory animals, livestock, livestock, etc., eg species such as murine, rodent, canine, feline, porcine, equine, bovine, ovine, non-human primates, etc.; eg small Mice, rats, rabbits, hamsters, guinea pigs, cattle, pigs, sheep, goats, and other transgenic animal species, particularly mammalian species, are known in the art.
  • the subject genetically modified animal is a mouse, rat, or rabbit.
  • the non-human animal is a mammal. In some such embodiments, the non-human animal is a small mammal, eg, of the Dipodoidea or Muroidea family.
  • the genetically modified animal is a rodent. In one embodiment, the rodent is selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the superfamily Murine.
  • the genetically modified animal is from a family selected from the group consisting of Calomyscidae (eg, mouse-like hamsters), Cricetidae (eg, hamsters, New World rats, and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice) climbing mice, rock mice, with-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g. spiny sleepers) spiny dormice), and Spalacidae (eg mole rates, bamboo rat, and zokors).
  • Calomyscidae eg, mouse-like hamsters
  • Cricetidae eg, hamsters, New World rats, and mice, voles
  • Muridae true mice and rats, gerbils, spiny mice, crested rats
  • Nesomyidae climb
  • the genetically modified rodent is selected from the group consisting of true mice or rats (muridae), gerbils, spiny mice, and bristle rats.
  • the genetically modified mouse is from a member of the family Murine.
  • NOD mice refer to no obesity diabetes ie non-obese diabetic mice. There are many derivative strains of NOD mice, including NOD/Scid NOD/Ltj mice.
  • immunodeficient mouse refers to a mouse that is congenitally genetically mutated or artificially deficient in one or more components of the immune system, the common mutant genotypes of which are, SCID mutation, Rag1 knockout , Rag2 knockout, the main phenotype is the lack of T cells and B cells.
  • severe immunodeficient mouse generally refers to an immunodeficient mouse in which the function of the IL2R ⁇ gene has been further knocked out.
  • IL2R ⁇ is a shared receptor for many cytokines. Knockout of IL2R ⁇ further reduced the function of the immune system in mice.
  • the main phenotype of severely immunodeficient mice is the lack of T cells, B cells, and NK cell functions, and the number of macrophages and dendritic cells is also greatly reduced.
  • the main use of severely immunodeficient mice is to reconstitute the human immune system in mice.
  • humanized mouse of the immune system generally refers to the presence of human peripheral blood leukocytes (hPBMCs) or human hematopoietic stem cells (CD34+HSCs) in severely immunodeficient mice after implantation of human peripheral blood leukocytes (hPBMCs). Mice reconstituted with one or several human immune cells.
  • hPBMCs human peripheral blood leukocytes
  • CD34+HSCs human hematopoietic stem cells
  • CRISPR/Cas9 is an adaptive immune defense developed by bacteria and archaea over a long period of evolution to combat invading viruses and foreign DNA.
  • CRISPR/Cas9 gene editing technology is a technology that makes specific DNA modifications to targeted genes.
  • CRISPR/Cas9-based gene editing technology has shown great application prospects in a series of gene therapy applications, such as blood diseases, tumors and other genetic diseases. The technological achievements have been applied to the precise modification of the genomes of human cells, zebrafish, mice and bacteria.
  • Piggy transposition system as used herein is also referred to as sleeping beauty transposition system (or PiggyBac system).
  • the PiggyBac (PB) transposon is derived from insects of the order Lepidoptera, which belongs to the second class of eukaryotes in taxonomy. Transposon-like, is an autonomous factor with a length of 2476bp, a short inverted terminal repeat (ITR) and an open coding frame (ORF). Piggy transposon mainly adopts "cut-paste" mechanism to transpose.
  • the Piggy transposition system has high transposition efficiency and a wide host range, and is widely used in gene transfer and mutation screening of insects and other lower organisms.
  • Transgenic Drosophila melanogaster, Drosophila melanogaster and Bombyx mori have been successfully obtained using the vector-helper plasmid system composed of Piggy transposon.
  • the transposase forms a short-term hairpin structure by combining with the end of the transposon. After the transposon is completely excised, it attacks the 5' end of the TTAA site in the target DNA sequence through its 3'OH end.
  • the TTAA overhangs of the 5' sequence are paired with the single-stranded TTAA opened by the target DNA and religated with the gaps flanking the integration site, and the ligation process does not require synthetic DNA.
  • interleukin is a class of cytokines that are produced by and act on a variety of cells. Refers to a class of cytokines whose molecular structure and biological functions have been basically clarified and have important regulatory effects and are uniformly named. It is the same cytokine as blood cell growth factor. The two coordinate and interact with each other to complete the functions of hematopoiesis and immune regulation. Interleukins play an important role in transmitting information, activating and regulating immune cells, mediating T and B cell activation, proliferation and differentiation, and in inflammatory responses.
  • Interleukin interleukin is abbreviated as IL, and its function is related to the expression and regulation of immune response, and this regulation involves many factors derived from lymphocytes or macrophages.
  • lymphokines derived from lymphocytes and those derived from macrophages are collectively called monokine.
  • the biological activities of each factor are different (such as activation of macrophages, promotion of T cell reproduction, etc.), and the physicochemical properties of the factors themselves The nature is unclear.
  • Interleukin-15 can be produced by a variety of cells including activated monocyte-macrophages, epidermal cells and fibroblasts.
  • the molecular structure of IL-15 has many similarities with IL-2, so it can use the ⁇ chain and ⁇ chain of the IL-2 receptor to bind to target cells and exert biological activities similar to IL-2.
  • IL-15 can induce B cell proliferation and differentiation, and is the only cytokine that can partially replace IL-2 to induce initial antibody production; IL-15 can stimulate the proliferation of T cells and NK cells, induce LAK cell activity, and can also interact with IL-12. Synergistic stimulation of NK cells to produce IFN- ⁇ .
  • Interleukin-7 is a glycoprotein secreted by bone marrow stromal cells with a molecular weight of 25KD; its gene is located on chromosome 8.
  • the target cells of IL-7 are mainly lymphocytes, which have growth-promoting activity on B progenitor cells, thymocytes and peripheral mature T cells from human or mouse bone marrow.
  • Interleukin-6 acts on many target cells, including macrophages, hepatocytes, quiescent T cells, activated B cells and plasma cells, etc. Its biological effects are also very complex, once called B cells Cell stimulating factor 2 (bsf-2), 26KD protein, B cell differentiation factor (bCDf), hepatocyte stimulating factor (hsf), etc. IL-6 could not stimulate the corresponding cells to secrete other cytokines, and its autocrine effect on immune cells was weak at physiological concentrations, suggesting that its main immunological function is to enhance the effects of other cytokines.
  • Bsf-2 B cells Cell stimulating factor 2
  • bCDf B cell differentiation factor
  • hsf hepatocyte stimulating factor
  • IL-6 is synthesized by a variety of cells, including activated T and B cells, monocytes-macrophages, endothelial cells, epithelial cells, and fibroblasts.
  • the human IL-6 gene is located on chromosome 7; the molecular weight of IL-6 is between 21 and 30KD, and the difference is due to the different degrees of glycosylation and phosphorylation of the peptide chain.
  • IL-6 is composed of two glycoprotein chains; one is an ⁇ chain with a molecular weight of 80KD; the other is a ⁇ chain with a molecular weight of 130KD.
  • the ⁇ chain lacks the intracellular domain and can only bind to IL-6 with low affinity.
  • the formed complex immediately binds to the high-affinity ⁇ chain and transmits information to the cell through the ⁇ chain.
  • Interleukin-3 is one of the important members of the interleukin family, also known as pleiotropic colony stimulating factor (multi-CSF). It is produced by T lymphocytes and can stimulate the proliferation, differentiation and function of cells involved in the immune response.
  • multi-CSF pleiotropic colony stimulating factor
  • the sequence of the human IL3 cDNA encoding human interleukin-3 (human IL3) involved in this application is shown in SEQ ID No. 5.
  • Interleukin-2 is a cytokine in the chemokine family. It is derived from multicellular sources (mainly produced by activated T cells) and has a pleiotropic effect of cytokines (mainly promoting the growth, proliferation and differentiation of lymphocytes); it plays an important role in the body's immune response and antiviral infection. It can stimulate the proliferation of T cells that have been initiated by specific antigens or mitogenic factors; it can activate T cells and promote the production of cytokines; it can stimulate the proliferation of NK cells, enhance the killing activity of NK and produce cytokines, and induce the production of LAK cells; promote B cells Proliferates and secretes antibodies; activates macrophages. Can be used for clinical research and tumor treatment.
  • BAFF Cell activating factor
  • TNFSF13B tumor necrosis factor superfamily
  • MAFF tumor necrosis factor superfamily
  • FMS-like tyrosine kinase 3 ligand is a key cytokine that can regulate early hematopoiesis, and combined with cytokines such as IL-3, IL-6, G-CSF, and SCF, has a significant effect on primitive hematopoietic stem/progenitors.
  • Cells have a strong proliferative effect
  • Flt3L is a ligand for the type III tyrosine kinase receptor FMS-like tyrosine kinase 3 (Flt3), which is mainly expressed on hematopoietic stem/progenitor cells. growth factor.
  • Flt3L can significantly expand DCs in vitro and in vivo, stimulate the proliferation of T cells and NK cells, and provide a good foundation for tumor immunotherapy.
  • FMS-like tyrosine kinase 3 ligand (Flt3L) recombinant protein refers to a protein in which FMS-like tyrosine kinase 3 ligand is recombinantly expressed in host cells.
  • HLA Leukocyte antigens
  • Serum method can be used to determine A, B and C site antigens
  • mixed lymphocyte culture method can be used to determine D and DR antigens.
  • TPO Thrombopoietin
  • TPO is an important enzyme that catalyzes thyroid hormones.
  • TPO is synthesized by thyroid follicular cells. It is a 10% glycated hemoglobin-like protein composed of 933 amino acid residues with a molecular weight of 103 kD. It is most abundantly distributed in the microvilli on the follicular lumen surface.
  • TPO uses hydrogen peroxide as an oxidant.
  • the activity of TPO disappeared 48 hours after the rat pituitary was removed, and the activity of TPO was restored after injection of thyroid stimulating hormone. It can be seen that the generation and activity of TPO are regulated by TSH.
  • Thiourea drugs can inhibit the activity of TPO, thus inhibiting the synthesis of thyroid hormones, and are commonly used clinically for the treatment of hyperthyroidism (hyperthyroidism).
  • Granulocyte-macrophage colony-stimulating factor is one of the hematopoietic growth factors, which is involved in hematopoietic regulation.
  • GM-CSF maintains the survival, proliferation and differentiation of hematopoietic cells in vitro and in vivo, and also affects the biological functions of mature granulocytes and monocytes-macrophages.
  • the clinical application of GM-CSF has developed rapidly, such as chemotherapy for malignant tumors, bone marrow transplantation, myelodysplastic syndrome, neutropenia and so on.
  • the sequence of human GM-CSF cDNA encoding human granulocyte macrophage colony stimulating factor (human GM-CSF) involved in this application is shown in SEQ ID No. 6
  • Macrophage colony-stimulating factor also known as colony-stimulating factor-1 (CSF-1)
  • CSF-1 colony-stimulating factor-1
  • M-CSF is a dimeric glycoprotein linked by interchain disulfide bonds. It mainly exists in the bone marrow cavity and plays an important role in the proliferation, differentiation and maintenance of monocytes. Its receptor is c-Fms.
  • Histocompatibility complex (major histocompatibility complex, MHC) is a collective term for a group of genes encoding animal major histocompatibility antigens.
  • Human MHC is called HLA (human leukocyte antigen, HLA), that is, human leukocyte antigen; mouse MHC is called H-2.
  • HLA is located on the short arm of chromosome 6 in humans, and H-2 is located on chromosome 17 in mice.
  • HLA human leukocyte antigen
  • H-2 is located on chromosome 17 in mice.
  • the major histocompatibility complexes are divided into three classes, MHC class I, MHC class II, and MHC class III, according to the location and function of the genes.
  • MHC class I Located on the surface of ordinary cells, it can provide some conditions in ordinary cells. For example, if the cell is infected by a virus, the amino acid chain (peptide) of the outer membrane fragments of the disease will be prompted to the outside of the cell through MHC, It can be used for the identification of killer CD8+ T cells for culling.
  • MHC II MHC class II: mostly located on antigen presenting cells (APC), such as macrophages. This type of provision is external to the cell, such as bacterial invasion in the tissue. After the macrophage engulfs it, it uses MHC to prompt the helper T cells to initiate an immune response.
  • APC antigen presenting cells
  • the present application provides a method for genetic modification of non-human animals, especially mice, especially mice with NOD as a genetic background, which includes: genetic modification of fertilized eggs of non-human animal cells, and the genetic modification includes using CRISPR/Cas9 gene editing of fertilized eggs of non-human animals.
  • mice were immunodeficient NOD knocked out of recombination activator gene 1 (Rag1) or knockout of recombination activator gene 2 (Rag2) or SCID mutated, depleted of T and B cells, IL2 receptor gamma knocked out Chain deletion in severely immunodeficient NOD mice with NK cells.
  • the fertilized egg of the non-human animal is the fertilized egg of the above-mentioned severely immunodeficient NOD mouse, especially the fertilized egg of in vitro fertilization.
  • NOD mice have a poorer pregnancy rate from in vivo fertilization compared to B6 mice.
  • In vitro fertilized eggs are more active than in vivo fertilized eggs. Therefore, the preparation of fertilized eggs by in vitro fertilization of mice with NOD genetic background is the first choice.
  • the genetically modified fertilized egg is obtained by in vitro fertilization, and the fertilized egg is cultured in vitro for a certain period of time, preferably the certain period of time is more than 6 hours, more preferably more than 8 hours, 10 hours or more, Fertilized eggs obtained after 12 hours, 14 hours or more, and most preferably 16 hours or more. It has been reported in the literature that Crispr technology can perform gene knockout and gene mutation in NOD mice, but there is no successful case for site-directed integration of large fragments of genes.
  • mice are immature, and small fragments of DNA or gRNA can be injected into the cytoplasm and then counted into the nucleus, while large fragments of linear DNA are inefficient from the cytoplasm into the nucleus, and can only be injected through the nucleus.
  • studies have found that culturing in vitro for a certain period of time can wait for the maturation of the nucleus, which is helpful for genetic modification of mice.
  • the genetic modification of the fertilized egg of non-human animal cells is accomplished by injecting the nucleus or cytoplasm of the fertilized egg. This is speculated that because the nucleus of NOD mice is immature, for the random integration of large fragments of DNA into chromosomes, it is difficult to perform nuclear injection, and the probability is relatively low.
  • the gene fragment that needs to be edited is linear DNA with a length of more than 500 bp.
  • Gene editing of fertilized eggs of non-human animals is to microscopically perform microscopic analysis of the nucleus of fertilized eggs of non-human animal cells. injection.
  • the certain period of time is preferably 6 hours or more, more preferably 8 hours or more, 10 hours or more, 12 hours or more,
  • the fertilized egg obtained after 14 hours, most preferably after 16 hours, is performed by microinjection of the nucleus.
  • the present application also relates to a method for genetically modifying a non-human animal, comprising: genetically modifying the fertilized egg of the non-human animal, so that cytokine genes or immune-related genes are in situ humanized, and the in situ human-derived Humanization of modified cytokine genes or immune-related genes does not affect the health and reproductive efficiency of immunodeficient mice. Humanization of these cytokine genes or immune-related genes does not affect the health and reproductive efficiency of immunodeficient mice. For example, cytokine genes or immune-related genes with high homology in humans and mice, even if these cytokine genes or immune-related genes are homologous, their functions will not be changed, so they will not significantly change immunodeficient mice. health and reproductive efficiency.
  • cytokine genes or immune-related genes with a homology of more than 50% between humans and mice preferably cytokine genes or immune-related genes with a homology of more than 60%, and more preferably cells with a homology of more than 70% Factor genes or immune-related genes, more preferably cytokine genes or immune-related genes with a homology of more than 80%, more preferably cytokine genes or immune-related genes with a homology of more than 90%, and more preferably a homology of 95% % or more of cytokine genes or immune-related genes.
  • in situ humanization refers to the replacement of a mouse gene or a portion of a gene with a homologous human gene or a portion of a human gene at the same genetic position, for example in the context of the non-human animal IL- After the fourth exon of 15, the fifth to eighth exons and their intron positions of non-human IL-15 were originally replaced by the degenerate human IL fifth to eighth exons The coding sequence, such humanization is in situ humanization.
  • cytokines include but are not limited to: interleukin-15 (IL-15), interleukin-7 (IL-7), interleukin-6 (IL-6), B cells Activating factor (BAFF), FMS-like tyrosine kinase 3 ligand (Flt3L) recombinant protein and leukocyte antigen (HLA).
  • IL-15 interleukin-15
  • IL-7 interleukin-7
  • IL-6 interleukin-6
  • BAFF B cells Activating factor
  • Flt3L FMS-like tyrosine kinase 3 ligand
  • HLA leukocyte antigen
  • the cytokine is interleukin-15 (IL-15).
  • the immune-related gene is the histocompatibility complex (MHC).
  • MHC histocompatibility complex
  • the method for genetically modifying the non-human animal is to genetically modify the fertilized egg of the non-human animal.
  • CRISPR/Cas9 is used to perform gene editing on fertilized eggs of non-human animals, and the nucleus of fertilized eggs is microinjected to perform gene editing of non-human animal fertilized eggs using CRISPR/Cas9 to achieve the original cytokines.
  • Humanized Specifically, for fertilized eggs that are fertilized in vitro and cultured in vitro for a certain period of time, the certain period of time is preferably 6 hours or more, more preferably 8 hours or more, 10 hours or more, 12 hours or more, 14 hours or more, and most preferably 16 hours or more.
  • the nucleus of the resulting zygote was microinjected to use CRISPR/Cas9 for gene editing of the zygote of a non-human animal to achieve in situ humanization of cytokines.
  • the genetic modification of the zygote of a non-human animal is to insert the degenerate human IL fifth to eighth exons after the fourth exon of IL-15 of the non-human animal the coding sequence of the sub.
  • after the fourth exon of IL-15 of the non-human animal refers to the position of the next nucleotide of the fourth exon of IL-15 of the non-human animal, that is, next to behind the fourth exon.
  • the non-human animal is a mouse
  • the sequence of the fourth exon of IL-15 of the mouse is shown in SEQ ID No. 4
  • the fourth exon of IL-15 of the mouse is shown in SEQ ID No. 4.
  • the coding sequence of the degenerate human IL exons 5 to 8 is inserted after the last nucleotide position of SEQ ID No. 4.
  • the genetic modification of the fertilized egg of the non-human animal also includes further inserting a bGHpolyA (bovine growth hormone polyadenylation) sequence after the fourth exon of IL-15 of the non-human animal.
  • bGHpolyA sequence is shown in SEQ ID No.2.
  • the coding sequence of the fifth to eighth exons of the degenerate human IL inserted after the fourth exon of IL-15 of the non-human animal is shown in SEQ ID No. 1.
  • the non-human animal is a mouse of NOD genetic background.
  • the mice were immunodeficient NOD with knockout of recombination activator gene 1 (Rag1) or knockout of recombination activator gene 2 (Rag2) or SCID mutation, deletion of T cells and B cells, and knockout of IL2 receptor ⁇ Chain deletion in severely immunodeficient NOD mice with NK cells.
  • the target sequence for inserting the coding sequence of the degenerate human IL exons 5 to 8 following the fourth exon of mouse IL-15 as described above is shown in SEQ ID No. 4.
  • the genetic modification of the fertilized egg of the non-human animal is to microinject the nucleus of the fertilized egg, and the microinjected substances include: homologous recombination DNA; gRNA1, gRNA2, and mRNACas9.
  • the homologous recombination DNA includes the human IL-15 homologous recombination DNA sequence shown in SEQ ID No. 3.
  • the recognition target sequence of the gRNA1 is CATAGCTATTATCAAGTTAGTGG (SEQ ID No. 5)
  • the recognition target sequence of the gRNA2 is GAAACACAAGTAGCACGAGATGG (SEQ ID No. 6).
  • the present application also relates to a method for constructing an animal model of severe immunodeficiency, by genetically modifying the fertilized eggs of non-human animals so that the expression in the non-human animals will not affect the health and reproductive efficiency of immunodeficient mice Gene humanization of cytokines.
  • the content involved can be all the content described above for this application.
  • the human and mouse cytokine sequences are similar in function or the absence of the murine cytokine does not affect the health of severely immunodeficient mice. Therefore, after humanization, these mouse cytokines are beneficial to the reconstruction of the human immune system without harming the existing immune system of mice.
  • IL-15 can promote the development and maturation of T cells and NK cells. But severely immunodeficient mice lack endogenous T cells and NK cells. Humanized IL-15 in these mice can promote the reconstitution of human T cells and NK cells without affecting the health of severely immunodeficient mice.
  • the present application attempts to directly humanize IL-15 in NOD scid IL2R ⁇ KO mice using an optimized CRISPR/Cas9-dependent zygote microinjection technology.
  • the present application directly genetically modifies the fertilized eggs of NOD scid IL2R ⁇ KO mice through an optimized microinjection method to obtain in situ humanized IL-15 severely immunodeficient mice.
  • Such humanization methods can also be applied to engineer passive immune-related cytokines, such as IL-6, IL-7, BAFF, HLA, and the like.
  • the present application relates to a linear DNA comprising the homologous recombination sequence of human IL-15 as shown in SEQ ID No.3.
  • the serum expression level of NOD scid IL2R ⁇ KO mice with humanized IL-15 gene was 90 pg/ml, which was close to the physiological level.
  • the method of the present application is also applicable to the targeted knock-in of other human genes, such as IL-7, IL-6, BAFF and HLA, and other passive immune-related cytokines in NOD scid IL2R ⁇ KO mice.
  • mice In order to improve the reconstituted human immune system in mice, it is necessary to overexpress human cytokines or humanize mouse cytokines in mice.
  • the present application provides a method for genetically modifying non-human animals, especially mice, especially mice with NOD as a genetic background. Piggy transposase-dependent transgenic system for gene editing of fertilized eggs of non-human animals.
  • mice were immunodeficient NOD knocked out of recombination activator gene 1 (Rag1) or knockout of recombination activator gene 2 (Rag2) or SCID mutated, depleted of T and B cells, IL2 receptor gamma knocked out Chain deletion in severely immunodeficient NOD mice with NK cells.
  • the fertilized egg of the non-human animal is the fertilized egg of the above-mentioned severely immunodeficient NOD mouse, especially the fertilized egg of in vitro fertilization.
  • NOD mice have a poorer pregnancy rate from in vivo fertilization compared to B6 mice.
  • In vitro fertilized eggs are more active than in vivo fertilized eggs. Therefore, the preparation of fertilized eggs by in vitro fertilization of mice with NOD genetic background is the first choice.
  • the genetically modified fertilized egg is obtained by in vitro fertilization, and the fertilized egg is cultured in vitro for a certain period of time, preferably the certain period of time is more than 6 hours, more preferably more than 8 hours, 10 hours or more, Fertilized eggs obtained after 12 hours, 14 hours or more, and most preferably 16 hours or more.
  • the certain period of time is more than 6 hours, more preferably more than 8 hours, 10 hours or more, Fertilized eggs obtained after 12 hours, 14 hours or more, and most preferably 16 hours or more.
  • the genetic modification of the fertilized egg of non-human animal cells is accomplished by injecting the nucleus or cytoplasm of the fertilized egg. This is speculated that because the nucleus of NOD mice is immature, for the random integration of large fragments of DNA into chromosomes, it is difficult to perform nuclear injection, and the probability is relatively low.
  • the certain period of time is preferably 6 hours or more, more preferably 8 hours or more, 10 hours or more, 12 hours or more, and 14 hours. In the above, most preferably, the fertilized eggs obtained after 16 hours are subjected to microinjection of the nucleus.
  • the gene fragment that needs to be edited is a circular plasmid
  • the gene editing of the fertilized egg of a non-human animal is to perform cytoplasmic cytoplasmic editing on the fertilized egg of the non-human animal cell. or nuclear injection.
  • transposase technologies such as piggyBac can be injected into the cytoplasm as a plasmid, which increases the success rate.
  • in situ humanization refers to the replacement of a mouse gene or a portion of a gene with a homologous human gene or a portion of a human gene at the same gene position.
  • the non-human animal is a mouse of NOD genetic background.
  • the mice were immunodeficient NOD with knockout of recombination activator gene 1 (Rag1) or knockout of recombination activator gene 2 (Rag2) or SCID mutation, deletion of T cells and B cells, and knockout of IL2 receptor ⁇ Chain deletion in severely immunodeficient NOD mice with NK cells.
  • the method of the present application comprises: overexpressing target protein 1 and/or target protein 2 in a non-human animal;
  • the target protein 1 is mainly expressed in T cells, B cells and NK cells; after the gene of the target protein 1 is in situ humanized, the target protein 1 cannot be detected in severely immunodeficient non-human animals. gene expression;
  • the target protein II may affect the health of immunodeficient non-human animals
  • Target proteins that may affect the health of immunodeficient mice are those with low homology between humans and mice, and when humanized, these target proteins will affect the health of immunodeficient mice.
  • cytokines with a homology of less than 50% between humans and mice are preferred, cytokines with a homology of less than 40% are preferred, cytokines with a homology of less than 30% are more preferred, and homology is more preferably 20%.
  • cytokines with homology of 10% or less are more preferred, and cytokines with homology of 5% or less are more preferred.
  • GM-CSF is essential for maintaining the maturation and development of macrophages.
  • the homology of GM-CSF in humans and mice is very low, and there is no cross-activity.
  • mouse GM-CSF was humanized, human GM-CSF could not replace the function of mouse GM-CSF.
  • mice developed pneumonia due to decreased macrophage activity.
  • TPO thrombopoietin
  • the humanized TPO cannot promote the growth of platelets in mice, thus causing anemia in mice. Overexpression strategies are required for such cytokines.
  • the target protein includes, but is not limited to, any one or both of the following: human interleukin-3 (human IL3), human interleukin-2 (human IL2); IL3 is mainly expressed on T cells, and immunodeficient mice have no T cells, so after humanization, there is no cytokine expression.
  • the first target protein is human interleukin-3 (human IL3);
  • the target protein two includes but is not limited to any one or two or more of the following: human thrombopoietin (human TPO), human granulocyte-macrophage Cell colony stimulating factor (human GM-CSF), human macrophage colony stimulating factor (human MCSF);
  • the second target protein is human granulocyte-macrophage colony stimulating factor (human GM-CSF).
  • target protein one and target protein two are overexpressed in a non-human animal.
  • human interleukin-3 human IL3
  • human granulocyte macrophage colony stimulating factor human GM-CSF
  • the method of genetically modifying the non-human animal is to genetically modify the fertilized egg of the non-human animal so that the target protein is overexpressed in the non-human animal, and the target protein is overexpressed in the myeloid cells of the non-human animal.
  • the protein of interest is preferably overexpressed in myeloid cells of a non-human animal using a promoter specific for myeloid cell expression.
  • promoters expressed in myeloid cells include but are not limited to human CD68 promoter, CSF1R promoter, CD11c promoter, CX3CR1 promoter, Langerin/CD207 promoter, MMLV LTR promoter, Visna virus LTR promoter, DC-STAMP promoter, Human MSR promoter, MSR-A promoter, CD4 promoter, CD2 promoter, Iba-AIF-1 promoter, CD11b promoter, c-fms promoter, scavenger receptor A (SR-A ) promoter, lysozyme promoter and MHC class II promoter (MHC-II) intermediate promoter.
  • mice The use of the above promoters for expression in myeloid cells is because it is generally observed that after the infusion of human hematopoietic stem cells in immunodeficient mice, the humanized immune system is reconstituted in the mice with the help of human cytokines. The myeloid cells of the mouse will gradually disappear, so that the overexpressed exogenous expressed genes will also decrease or disappear, so that the overexpressed human cytokines will not over-activate the fully humanized immune system due to continuous expression. So as to protect the health of mice.
  • the genetically modified non-human animal is a mouse with NOD as a genetic background, and the mouse is a knockout of recombination activator gene 1 (Rag1) or knockout of recombination activator gene 2 (Rag2) or SCID Mutant, T- and B-cell-depleted, immunodeficient NOD mice, and NK cell-deficient severely immunodeficient NOD mice further knocked out the IL2 receptor gamma chain.
  • fertilized eggs of non-human animals are obtained by in vitro fertilization.
  • the fertilized eggs obtained by in vitro fertilization are cultured in vitro for a certain period of time before the genetic modification of the fertilized eggs of the non-human animal, preferably the certain period of time is 6 hours or more, more preferably 8 hours or more, 10 hours or more, 12 hours or more, 14 hours or more. hours or more, most preferably 16 hours or more.
  • the target protein is overexpressed in myeloid cells of a non-human animal using the human CD68 promoter, ie, human interleukin-3 (human IL3) and human granulocyte macrophage colony stimulating factor are overexpressed (Human GM-CSF).
  • human CD68 promoter ie, human interleukin-3 (human IL3) and human granulocyte macrophage colony stimulating factor are overexpressed (Human GM-CSF).
  • the piggyBac transposon system plasmid and piggyBac transposase are used to overexpress the target protein in myeloid cells of non-human animals.
  • double self-cleaving short peptides RAKR-GSG-P2A (RAKR-GSG-ATNFSLLKQAGDVEENPGP), RAKR-GSG-T2A (RAKR-GSG) are utilized when overexpressing two or more proteins of interest in non-human animals -EGRGSLLTCGDVEENPGP), RAKR-GSG-E2A (RAKR-GSG-QCTNYALLKLAGDVESNPGP) or RAKR-GSG-F2A (RAKR-GSG-VKQTLNFDLLKLAGDVESNPGP) mediates the overexpression of different target proteins, wherein RAKR is the sequence of Furin cleavage short peptide, GSG is a linker.
  • the double self-cleaving short peptide is RAKR-GSG-P2A
  • the sequence of the gene encoding the double self-cleaving short peptide RAKR-GSG-P2A is shown in SEQ ID No. 13.
  • the piggyBac transposon system plasmid is used to overexpress the target protein in the myeloid cells of non-human animals, and a bGH polyA sequence is also inserted into the piggyBac transposon system plasmid, preferably the sequence is as shown in SEQ ID No.2.
  • a response repeat sequence is inserted into the piggyBac transposon system plasmid, preferably the sequence is shown in SEQ ID No.16 or SEQ ID No.17.
  • the ITR suitable for insertion into the 5' end of piggyBAC is shown in SEQ ID No. 16, and the ITR suitable for insertion into the 3' end of piggyBAC is shown in SEQ ID No. 17.
  • the genetic modification of the fertilized egg of the non-human animal is to inject cytoplasm or nucleus into the cytoplasm of the fertilized egg, and the substances injected into the cytoplasm or nucleus include: PiggyBac transposon system plasmid and piggyBac Transposase.
  • the PiggyBac transposon system plasmid includes the sequence shown in SEQ ID No.11.
  • the present application also relates to a method for constructing an animal model of severe immunodeficiency, the method comprising: overexpressing a target protein in a non-human animal, the target protein is target protein 1 and/or target protein 2; wherein, The target protein one is mainly expressed in T cells, B cells and NK cells; the target protein two may affect the health of immunodeficient mice.
  • the content involved can be all the content described above for this application.
  • the present application also relates to a PiggyBac transposon system plasmid, the sequence of which is shown in SEQ ID No.11.
  • mice In order to improve the reconstituted human immune system in mice, it is necessary to overexpress human cytokines or humanize mouse cytokines in mice.
  • cytokines that are relevant to the maintenance of the mouse's own health and lack cross-activity with human cognate cytokines, retain their own murine IL-3 and GM-CSF in NOD scid IL2R ⁇ KO mice, while exploring how to moderately express human IL3 And GM-CSF becomes the optimized choice.
  • This application uses PiggyBac technology to overexpress a single copy of human IL3 and human GM-CSF in NOD scid IL2R ⁇ KO mouse macrophages.
  • Overexpression of human IL3 and human GM-CSF can support the reconstruction of human myeloid cells in NOD scid IL2R ⁇ KO mice, which in turn can improve the growth and maturation of human T cells and B cells. Additionally, expression of these cytokines is restricted to mouse myeloid cells due to the use of tissue-specific promoters. When the mouse immune system is fully humanized, the mouse myeloid cells will be replaced by human immune cells, so the exogenous human IL3 and GM-CSF overexpressed in the mouse will gradually become more mature as the human immune system matures. reduce.
  • the human IL3 and human GM-CSF detected in the humanized mice of the immune system mainly came from T cells and macrophages of the human immune system.
  • Such a design not only provides the necessary human cytokines to support the maturation of the human immune system in mice, but does not make the overexpressed exogenous human cytokines over-activate the mature human immune system, thereby affecting the health and safety of the mice. Later functional analysis.
  • a similar design can also be used to overexpress cytokines related to the reconstruction of human innate immunity in mice, such as G-MCSF, TPO, M-CSF, etc.
  • the fertilized eggs of NOD scid IL2R ⁇ KO mice were directly genetically modified by the optimized microinjection method to obtain severely immunodeficient mice overexpressing IL3/GMCSF double transgene.
  • This application utilizes PiggyBac transgenic technology and optimized fertilized egg microinjection technology to directly overexpress human IL3 and human GM-CSF in NOD scid IL2R ⁇ KO mice, saving time for mouse development.
  • the present application utilizes the human CD68 promoter to overexpress human cytokines in mouse myeloid cells. Because in the process of rebuilding the human immune system in severely immunodeficient mice, the mouse endogenous myeloid cells will be gradually replaced by human immune cells, so the exogenous cytokines expressed in the myeloid cells will increase with the reconstitution level of the human immune system. increase and decrease.
  • the advantages of this design are both to speed up the reconstitution of the human immune system and to avoid the subsequent reduction in the lifespan of mice caused by the overactivation of the human immune system.
  • NOD scid IL2R ⁇ KO mice that overexpress human IL3 and human GMCSF, namely NOD scid IL2R ⁇ KO Tg(hIL3/hGMCSF) mice, have the advantage of not only being genetically modified directly in mice with severe immune systems, but also restricting the use of these cytokines in mice.
  • Expression in mouse myeloid cells Because mouse myeloid cells will gradually disappear after humanization of the mouse immune system, and the overexpressed human cytokines will also decrease with the reduction of mouse myeloid cells, so it is possible to avoid overexpressing human cytokines to mice Genotoxicity from a humanized immune system.
  • the endogenous myeloid cells expressing human cytokines will be gradually replaced by exogenous human immune cells, thereby accelerating the reconstruction of the human immune system on the one hand. speed, on the other hand avoids the subsequent reduction in lifespan of mice caused by overactivation of the human immune system.
  • the transgenic method used in the present application has the advantage that the present application can not only retain the activity of the homologous gene of the mouse, but also obtain transgenic mice with various expression levels. Different expression levels of exogenous genes confer different functions in transgenic mice.
  • Furin(RARK)-GSG-P2A double self-cleaving short peptide was also used in this application to mediate the co-expression of human IL3 and GM-CSF.
  • other self-cleaving peptides with similar functions to Furin-GSG-P2A can also achieve similar effects in this application.
  • the present application also utilizes hCD68 to express cytokines in mouse myeloid cells.
  • the advantage of expressing human cytokines in myeloid cells is that it can reduce genotoxicity and reduce the excessive activation of the human immune system by excess exogenous cytokines.
  • the present application also helps to express other cytokines that are related to maintaining the mouse's own health and lack cross-activity with human homologous cytokines, such as human TPO, M-CSF, etc.
  • the inventors of the present application tried to combine the optimized fertilization technology of fertilized eggs and gene editing tool-dependent homologous recombination technology to the NOD scid IL2R ⁇ KO mice.
  • the fertilized egg undergoes gene editing.
  • the NOD scid IL2R ⁇ KO mouse in this example is the M-NSG mouse purchased from the Shanghai Southern Model, which is a severely immunodeficient mouse obtained by knocking out the IL2R ⁇ gene of the NODscid mouse using CRISPR/Cas9 technology.
  • human IL-15 has 8 exons and is 96kb in length.
  • the inventors used CRISPR/Cas9-dependent homologous recombination technology to humanize the mouse IL-15 gene.
  • the inventors site-directed insertion of degenerate human IL-15 at the fourth exon of mouse IL-15.
  • the eighth exon see Figure 1.
  • the newly generated gene not only removes the interference of murine IL-15, but also utilizes the entire promoter, part of the intron regulatory sequence of murine IL-15 and the secretion signal peptide of murine IL-15. To a large extent, the expression of cytokines at the physiological level is achieved, and the length of the knock-in gene is reduced to improve the success rate of gene knock-in.
  • the inventors used the fertilized eggs of C57BL mice as a control. Specific steps are as follows.
  • mouse IL-15-MGP_NODSiLtJ_T0089152.1 transcript the intron and exon information of this transcript are all included in NCBI, and it is the only transcript. There are 8 exons in the transcript structure, and protein translation starts in exon 3 and ends in exon 8.
  • the human IL-15-202 transcript (IL-15-202) also contains 8 exons, and protein translation starts in exon 3 and ends in exon 8.
  • the coding sequences of the fifth to eighth exons of the degenerate human IL-15-202 transcript SEQ ID No. 1
  • pGHpolyA SEQ ID No. 2
  • the comparison results of the humanized IL-15 nucleic acid sequence and protein sequence with the mouse homologous sequence are shown in Figures 2A and 2B.
  • the genomic DNA about 500 bp upstream of the end of the fourth exon of the mouse IL-15 locus is the 5' homology arm
  • the genomic DNA about 500 bp downstream is the 3' homology arm.
  • homologous recombination sequences were recombined into pBR322 vector (sigma, cata: 10481238001) ( After the recombination is completed, it is called human IL-15 homologous recombination plasmid).
  • the human IL-15 homologous recombination plasmid is shown in Figure 3.
  • the homologous recombination sequence is shown in SEQ ID No.3.
  • CRISPR.mit.edu was used to design guide RNAs (gRNAs) targeting the site in the fourth exon of the mouse IL-15 transcript.
  • gRNAs guide RNAs targeting the site in the fourth exon of the mouse IL-15 transcript.
  • the sequence of the targeting site of the gRNA is as follows:
  • Purified Cas9 mRNA was purchased from Origene, product number GE100054.
  • NOD scid IL2R ⁇ KO mice (experimental group) and C57BL mice (control group) were used as sperm and egg donors.
  • CD1 mice were pseudopregnant dams.
  • NOD scid IL2R ⁇ KO mice were derived from NOD scid mice, and the fourth to eighth exons of the IL2R ⁇ gene were knocked out using CRISPR/Cas9 technology. The above mice were purchased from Shanghai Model Biology Company.
  • Fertilized eggs prepared by natural fertilization (nature mating): Select female mice and inject pregnant horse serum gonadotropin (PMSG) (Sigma Chemical Inc., cat. no. G4877-2000IU), and inject human chorionic gonadotropin (HCG) after 46h to 48h. HCG) (Sigma Chemical Inc., cat. no. CG10-1VL), co-cage 1:1 with normal male mice immediately after injection.
  • PMSG pregnant horse serum gonadotropin
  • HCG human chorionic gonadotropin
  • HCG human chorionic gonadotropin
  • Fertilized eggs were prepared by in vitro fertilization (IVF): ovulation was induced according to routine operations, and female mice of appropriate age (6 weeks) were selected to inject pregnant horse serum gonadotropin (PMSG), and HCG was injected 46-48 hours later, and vaginal plugs were tested the next day.
  • PMSG pregnant horse serum gonadotropin
  • HCG human horse serum gonadotropin
  • Sperm collection Male mice meeting the requirements of sexual maturity (more than 8 weeks) were killed by cervical dislocation, and the tail of the upper epididymis was removed. After removing fat and blood, they were placed in HTF solution (Millipore, cata: MR-070-D). Use ophthalmic scissors to transection the cauda epididymis in the droplet.
  • the sperm When the sperm is fully freed from the cauda epididymis, remove the tissue from the droplet and place the petri dish in an incubator. The superovulated female mice were sacrificed, the fallopian tubes on both sides were removed, and the oocyte mass was moved into the fertilization drop with microscopic forceps. Use a 10 ⁇ l pipette to suck sperm from the sperm drop, add it to the egg drop, and add about 8 ⁇ l of sperm to each drop according to the size of the egg mass and the viability of the sperm. After the sperm and eggs were mixed, they were finally placed in M16 solution and cultured in a 37°C incubator with 5% CO 2 .
  • pseudopregnant mice normal female mice and ligated male mice were caged together (3:1). About 20 fertilized eggs were implanted in the fallopian tubes of each pseudopregnant mouse. After 3 weeks, the mice were born. The viability of fertilized eggs was measured by the number of mice born and the proportion of fertilized eggs implanted.
  • Microinjection was started after the zygotes were cultured at M16 for a period of time. Place a drop of M2 medium in the center of the tissue culture slide and cover the drop with mineral oil. Aspirate the fertilized egg with an egg-holding needle, and inject the needle containing the linearized homologous recombination plasmid DNA (100 ng each), the above-mentioned gRNA (50 ng each), and mRNACas9 (100 ng) into the nucleus (Pronucleus) or cytoplasm of the fertilized egg (Pronucleus). Cytosol). The injection causes the cells to swell, the needle is withdrawn, and the next fertilized egg is aspirated for injection.
  • Genotypes were identified by PCR amplification and sequencing.
  • the PCR primer sequences were designed as follows.
  • primer sequence 5'-->3' Primer type I TCTGATGGGAACTGGATGA (SEQ ID No. 7) positive II AGCCTGACTTACTGTTGTG (SEQ ID No. 8) reverse
  • the genomic DNA of the offspring mice was identified by PCR according to classical molecular biology methods. PCR positive clone identification results show that the 5' arm homologous recombination positive genome should amplify a 4.1kb fragment, and the negative genome should amplify a 6.9kb fragment; the 3' arm homologous recombination positive genome should amplify a 3.6kb fragment, and the negative The genome should be amplified with a 6.9kb fragment.
  • the sequencing results were consistent with the PCR results. The specific results are shown in the F1 generation in Table 1.
  • the obtained F0 generation mice are chimeras, which do not necessarily have the ability to stably inherit. From the progeny of 5 F0 mice, 3 positive F1 generation mice were identified as described above. The results are shown in Tables 1 and 2.
  • He in Table 1 means heterozygote
  • Phenotypic determination (expression of human IL-15 in transgenic mice)
  • the gene editing method for gene-directed knock-in of fertilized eggs is more efficient and concise. But insert size is limited. If the gene exceeds 2KB, the site-directed insertion efficiency will be greatly reduced.
  • the human IL-15 gene in this example has a complete locus of 95kb. Therefore, at the time of genetic design, the inventors of the present application retained the first 4 exons and the first 3 introns of mouse IL-15, and then inserted the combined human 5th to 8th exons. son. This design not only reduces the length of gene knock-in, but also preserves mouse expression regulatory elements, including promoters and partial introns, and mouse signal peptides.
  • the serum expression level of NOD scid IL2R ⁇ KO mice with humanized IL-15 gene was 18 pg/ml, which was close to the physiological level.
  • the method of the present application is also applicable to the targeted knock-in of other human genes, such as IL-7, IL-6, BAFF and HLA and other passive immune-related cytokines, in NOD scid IL2R ⁇ KO mice.
  • the advantage of this application is that the fertilized eggs of NOD scid IL2R ⁇ KO mice can be directly genetically modified to obtain NOD scid IL2R ⁇ KO mice expressing humanized cytokines, avoiding multi-generation backcrossing, thereby saving time and labor costs.
  • the present application retains a partial fragment of the murine homologous gene, and recombines with the truncated human gene to obtain a humanized gene.
  • humanized IL-15 utilizes the part including the UTR of the mouse IL-15 gene, which reduces the size of the site-directed insertion fragment and improves the efficiency of homologous recombination.
  • the IL-15 of the mouse itself is replaced by humanized IL-15, which avoids the coexistence and mutual interference of human and mouse IL-15 that often occurs in the prior art, and reduces the Effects on the function of human immune cells such as human NK cells.
  • humanized IL-15 uses the promoter of the mouse itself, it avoids the overexpression of human IL-15 that often occurs in the prior art, thereby avoiding human IL-15 to a greater extent. Effects on the development of the mouse itself.
  • NOD scid IL2R ⁇ KO mice will support the growth and maturation of human NK cells, which can be used to detect the activity of CAR-NK and the ADCC effect of antibodies.
  • Example 2 Using the humanized IL-15 immunodeficient mice (NVG-hIL15) constructed in Example 1 to evaluate NK cell-related immunotherapy
  • NK cells Due to the natural killing cytotoxicity of NK cells, immunotherapy strategies represented by chimeric antigen receptor NK cell therapy (CAR-NK) and antibody-dependent cell-mediated cytotoxicity (ADCC) have gradually attracted people's attention. development interest. Immunodeficient mice harboring human immune systems have been widely used to evaluate novel human immunotherapies. But the lack of functional natural killer cells (NK cells) in these models limits their application in this regard. Therefore, the development of NK cell-mediated immunotherapies urgently requires an improved preclinical animal model for clinical translational research.
  • CAR-NK chimeric antigen receptor NK cell therapy
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • NVG mice are M-NSG mice purchased from Shanghai Nsweeping Model, which are NOD scid IL2R ⁇ KO mice obtained by knocking out the IL2R ⁇ gene of NODscid mice using CRISPR/Cas9 technology, which are severely immunodeficient mice. It was named NVG mice and served as a control group.
  • NVG-IL15 mice and other transgenic mice are genetically modified on this strain of NVG mice. Since human IL-15 is a key cytokine for natural killer cell (NK cell) homeostasis and maturation, as shown in Figure 1, we obtained human IL-15-expressing NVG by humanizing IL-15 from NVG mice -hIL15 mice, as the experimental group.
  • NK cell natural killer cell
  • the average plasma expression concentration of human IL-15 in NVG mice and NVG-hIL15 mice was determined by ELISA. The results are shown in Figure 5.
  • the average plasma expression concentration of human IL-15 was At 90 pg/ml, the mean plasma expression concentration of human IL-15 was 0 in NVG mice.
  • NVG-hIL15 mice Since the highly humanized immune system may cause NVG-hIL15 mice to exhibit graft-versus-host disease (GVHD) after 6 weeks, as shown in Figure 6, NVG-hIL15 mice were injected with 5 million human PBMCs for about 6 weeks The survival rate plummeted after about 8 weeks, and its survival rate was 0 after about 8 weeks, and the survival rate of NVG mice began to decrease gradually after about 10 weeks after injection of human PBMC (because of the difference, no retest after 10 weeks).
  • GVHD graft-versus-host disease
  • NVG-hIL15 mice After injecting human PBMC into NVG mice and NVG-hIL15 mice, and then injecting human leukemia luciferase-labeled cells K562-Luc, the highly humanized immune system also allows NVG-hIL15 mice to inhibit the growth of K562-Luc cells As shown in Figure 7, after 6 weeks of K562-Luc injection, the growth of K562-Luc cells in NVG-hIL15 mice was significantly inhibited compared with NVG mice.
  • NK cells isolated from human peripheral blood from healthy donors were injected into NVG mice and NVG-hIL15 mice respectively , and 5 ⁇ 10 6 K562-Luc was injected after 3 days.
  • IVIS Intravital Imaging System detects tumor cell lysis.
  • NK cells isolated from human peripheral blood from healthy donors After injection of NK cells isolated from human peripheral blood from healthy donors, the percentage of NK cells in vivo was determined. The results are shown in Figure 9. NK cells isolated from human peripheral blood from healthy donors can continue to grow in NVG-IL15 mice. 6 weeks, while in NVG mice it was only maintained for 2 weeks of growth.
  • NVG mice and NVG-hIL15 mice were pretreated with cyclophosphamide (CTX), respectively, followed by orthotopic injection of MIA PaCa-2 (a human pancreatic cancer cell line) at an injection volume of 1 million
  • CTX cyclophosphamide
  • MIA PaCa-2 a human pancreatic cancer cell line
  • 10 7 irradiated CAR-NK92 cells were injected every week until the control group mice died.
  • the tumor cell lysis was detected by the IVIS in vivo imaging system.
  • mice were injected with the luciferase substrate luciferin, and a high-sensitivity CCD camera was used to detect NVG mice and NVG-hIL15 mice in vivo
  • the change of luciferase content the results are shown in Figure 13, the luciferase content in NVG-hIL15 mice dropped sharply, while the luciferase content in NVG mice did not change much.
  • the results showed that in NVG-hIL15 mice, CAR-NK92 cells could kill live tumor MIA PaCa-2 cells.
  • Example 3 Expression of foreign genes in NOD scid IL2R ⁇ KO mice: human IL3 and human GM-CSF
  • PiggyBac recombinant plasmid (Piggy hCD68-GMCSF/IL3)
  • the present application utilizes a Piggy transposase-dependent transgenic system to overexpress human GM-CSF and human IL3 in mice. Since the PiggyBac transposase tends to insert the target fragment into the transcriptionally active region, the probability of obtaining a transgenic mouse with positive expression will be greatly improved.
  • the 3.1 kb human CD68 promoter was used for the vector so that gene overexpression occurred only in myeloid cells such as monocytes in mice.
  • the vector uses double self-cleaving short peptide RAKR-GSG-P2A to link human IL3 and human GM-CSF genes.
  • ITRs Piggy transposase-dependent response-directed repeats
  • the inventors replaced the human CD68 promoter in the aforementioned expression original with the universal SV40 promoter (SEQ ID No. 12) to construct a control
  • the piggyBac vector (Piggy SV40-GMCSF/IL3), other recombination elements remain unchanged.
  • NOD scid IL2R ⁇ KO mice (experimental group) and C57BL mice (control group) were used as sperm and egg donors.
  • CD1 mice were pseudopregnant dams.
  • NOD scid IL2R ⁇ KO mice are derived from NOD scid mice, which are M-NSG mice purchased from Shanghai Southern Model, which are severely immunodeficient mice obtained by knocking out the fourth to eighth exons of the IL2R ⁇ gene using CRISPR/Cas9 technology. mouse, which was named NVG mouse.
  • Preparation of fertilized eggs by natural fertilization (nature mating): select female mice to inject PMSG, and inject human chorionic gonadotropin HCG (Sigma Chemical Inc., cat.no.CG10-1VL) 46h to 48h after injection. According to 1:1 cage.
  • HCG human chorionic gonadotropin HCG
  • the fertilized egg donor mice were sacrificed by cervical dislocation, and the entire fallopian tube was surgically removed and placed in a hyaluronidase ⁇ 0.3 mg/M2 solution (sigma chemical, cata: M 7167). Under the microscope, carefully observe the fertilized eggs placed in the hyaluronidase M2 solution.
  • the fertilized eggs are sucked out, washed in the M2 solution, and finally placed in the M16 solution (Sigma Chemical, cat. .no.M7292) was placed in 5% CO 2 and cultured in a 37°C incubator. Observation under the microscope, select fertilized eggs with full cells, clear zona pellucida, and clearly visible nuclei for use.
  • Fertilized eggs prepared by in vitro fertilization ovulation induction was performed according to routine operations, and female mice of appropriate age (6 weeks) were selected to be injected with pregnant horse serum gonadotropin (PMSG (Sigma Chemical Inc., cat. no. G4877-2000IU)), 46h ⁇ 48 hours later, HCG was injected, and vaginal suppository was checked the next day.
  • PMSG pregnant horse serum gonadotropin
  • HCG human heart rate
  • vaginal suppository was checked the next day.
  • Sperm collection Male mice meeting the requirements of sexual maturity (more than 8 weeks) were killed by cervical dislocation, and the tail of the upper epididymis was removed. After removing fat and blood, they were placed in HTF solution (Millipore, cata: MR-070-D).
  • NOD scid IL2R ⁇ KO mice were used as sperm and egg donors.
  • CD1 mice were pseudopregnant dams.
  • the above mice were purchased from Shanghai Southern Model Biology Company.
  • the piggyBac transposon system plasmid (Piggy hCD68-GMCSF/IL3 or Piggy SV40-GMCSF/IL3) carrying the exogenous gene and piggyBac transposase mRNA (Shanghai Southern model) were injected together by cytoplasmic injection method
  • the injection timing is 16 to 18 hours after the completion of in vitro fertilization.
  • piggyBac transposon system plasmid concentration of piggyBac transposon system plasmid was 50ng/ ⁇ L
  • piggyBac transposase mRNA concentration of piggyBac transposase mRNA (Shanghai Southern model) was 50ng/ ⁇ l
  • the injection volume was 15pL.
  • the control injection needle injected DNA into the cytoplasm.
  • about 20 fertilized eggs were aspirated with an egg transfer needle and implanted into the ampulla of the fallopian tube of a pseudopregnant female mouse. The ovaries and fallopian tubes were then returned to the pseudopregnant mice, and the wounds were sutured.
  • mice born about 20 days after injection were first established mice (F0 generation mice). It was genotyped by PCR method. After designing primers for PCR, positive mice were identified. The primer sequences are shown in Table 3:
  • primer sequence 5'-->3' Primer type V GCGGGGCAGCCTCACCAA (SEQ ID No. 22) positive VI AATTCATTCCAGTCACCGTCCTT (SEQ ID No. 23) reverse
  • the positive PCR band was identified according to the method of classical molecular biology, and the size was 639bp.
  • the results of PCR identification electrophoresis are shown in Figure 15. Among them, 3, 4, 5, 6, and 10 are F0 generation NOD scid IL2R ⁇ KO-Tg (hCD68-hIL3/GMCSF) mice, and 12, 13, and 14 are F0 generation NOD scid IL2R ⁇ KO-Tg (SV40-hIL3/GMCSF) mice .
  • inverse PCR was used to identify the gene sequences flanking the inserted gene.
  • inverse PCR please refer to the instructions of classical molecular biology methods and kits. Genomic DNA of mouse tails was extracted with Zymo Research's DNA extraction kit (Catalog #D3024). Subsequently, the genome was digested with DpnII restriction endonuclease (NEB Biolabs, Catalog #R0543S) for 3 hr, and the endonuclease was inactivated at 80°C for 20 minutes.
  • DpnII restriction endonuclease NEB Biolabs, Catalog #R0543S
  • the digested product was cyclized with T4 ligase (Enzymatics, Catalog #L6030-LC-L) for 1 hour, and then placed in a -20°C refrigerator to terminate the reaction. Nested PCR reactions are set up to amplify the genomic DNA flanking the inserted gene.
  • the DNA polymerase was Phusion DNA Polymerase (NEB Biolabs, Catalog#M0530S). The amplification primers are shown in Table 4:
  • primer sequence 5'-->3' Primer type VII GCTCTATGGCTTCTGTTTGT (SEQ ID No. 24) Forward_1 st VIII GATAAAACACATGCGTCAATTT (SEQ ID No. 25) Forward_2nd IX CCAATCCTCCCCCTTGCTGTCC (SEQ ID No. 26) reverse_1 st X AAACAACAGATGGCTGGCAACTA (SEQ ID No. 27) reverse_2nd XI GTAAAACGACGGCCAG (SEQ ID No. 28) M13 forward
  • the primers VII and IX are used to amplify the ligation products of the first round, and the primers VIII and X are used to amplify the PCR products of the first round.
  • the principle of inverse PCR ( Figure 16) and the results of the second PCR extension electrophoresis ( Figure 17) are shown in the figure.
  • the second round PCR product was cloned into pUC18 plasmid, and the cloned product was sequenced with M13 forward universal primer (XI).
  • the sequencing results were aligned on the NCBI website. After sequence alignment, the insertion positions of each transgenic mouse are shown in Table 5. The results showed that using the piggyBAC transgenic approach, multiple transgene insertion sites existed in multiple mice, and were located within known genes. However, some mice have insertion sites in regions of unknown function.
  • F0 mice Since some F0 mice have multiple insertion sites, PCR primers were used to screen out transgenic mice whose insertion sites were not within known functional genes. The F0 generation mice were laterally crossed, and the following mice were obtained in the F2 generation selection: SV40-12-6, SV40-14-X, CD68-6-13, CD68-10-12. The expression levels of human cytokines in serum were detected. Anticoagulated blood was collected from the orbital vein of each screened mouse, and the serum was prepared by centrifugation. Human IL-3 Quantikine ELISA Kit (R&D systems, Catalog#D3000) and Human GM-CSF Quantikine ELISA Kit (R&D systems, Catalog#DGM00) were used to detect serum cytokine expression in F2 generation mice, respectively.
  • the test results are shown in Table 6. Experiments have shown that different transgenic mouse lines have different insertion sites and different cytokine expression levels.
  • the obtained CD68-10-12 mice were named NVG-hCD68-10-12 mice, and the obtained SV40-14-X mice were named NVG-SV40-14-X mice.
  • Human CD68 promoter and SV40 promoter were used to express human IL3 and human GM-CSF cytokines in transgenic mice, respectively.
  • Real-time RT-PCR was used to detect the mRNA abundance of human IL3 and endogenous GAPDH in different tissues (bone marrow, muscle, lung, kidney and spleen), respectively.
  • the specific protocol is as follows: RNA was extracted from mouse bone marrow, muscle, lung, kidney and spleen with Trizol (Thermofisher, Catalog #15596026). mRNA was reverse transcribed into cDNA with PrimeScript RT Master Mix (Takara, Catalog #RR036A).
  • NOD scid IL2R ⁇ KO tg(SV40-IL3/GM-CSF) (expressing human IL3 and human GMCSF from SV40 promoter) mice expressed high abundance of IL-3 mRNA in various tissues
  • NOD scid IL2R ⁇ KO tg (hCD68-IL3/GM-CSF) (expressing human IL3 and human GMCSF with human CD68 promoter) is only expressed in myeloid cell-enriched tissues, such as lung, spleen, bone marrow, expressing high abundance of IL3 mRNA, while in muscle and kidneys showed very little expression (as shown in Figure 18).
  • the survival rate of NVG-SV40-14-X mice dropped sharply after 120 days of injection of CD34 stem cells, and the survival rate was only 10% after about 160 days; NVG-hCD68-10-12 mice survived about 130 days The rate decreased to 80% and did not continue to decrease; the survival rate of NVG mice did not change.
  • the average expression levels of hGMCSF and hIL3 in the plasma of the mice in the control group and the experimental group were determined by ELISA.
  • the average plasma expression concentration of hIL3 is about 0; in NVG-SV40-14-X mice, the average plasma expression concentration of hGMCSF is about 200 pg/ml, and the average plasma expression concentration of hIL3 is about 150 pg/ml; NVG-hCD68-10-12 In mice, the mean plasma concentration of hGMCSF was about 25 pg/ml, and the mean plasma concentration of hIL3 was about 25 pg/ml.
  • mice use the SV40 promoter to continuously overexpress GM-CSF and IL3, which may Over-activation of human macrophages causes a decrease in the number of blood cells in mice, and the survival rate of mice is affected.
  • GM-CSF and IL3 which may Over-activation of human macrophages causes a decrease in the number of blood cells in mice, and the survival rate of mice is affected.
  • hCD68 promoter expression of human cytokines can be restricted to mouse lymphocytes (mCD45). After humanization of the mouse immune system, the lymphocytes in the mice were reduced, and consequently, the concentration of overexpressed human cytokines was greatly reduced. Thereby avoiding the excessive activation of the human immune system by human cytokines.
  • This application uses piggyBac technology to express human IL3 and human GM-CSF in NOD scid IL2R ⁇ KO. Since the experiments used optimized in vitro fertilization and microinjection conditions, other transgenic techniques with similar properties, such as Tol2, Sleep Beauty and other transposase-dependent transgenic techniques, can also be used in mice with NOD scid IL2R ⁇ KO genetic background , in order to achieve the similar inventive effect as in this embodiment. However, compared with other transgenic technologies, the advantage of piggyBac transgenic technology is that only circular plasmids need to be injected into the cytoplasm, and linear DNA does not need to be injected into the nucleus, thereby improving the reproductive activity of fertilized eggs.
  • the piggyBac transgenic technology can also integrate large fragments of exogenous genes into regions with high transcriptional activity in the form of a single copy, which ensures the expression rate of exogenous genes.
  • the present application therefore preferably uses piggyBac transgenic technology.
  • the transgenic method used in the present application has the advantage that the present application can not only retain the activity of the homologous gene of the mouse, but also obtain transgenic mice with various expression levels. Different expression levels of exogenous genes confer different functions in transgenic mice.
  • Furin(RARK)-GSG-P2A double self-cleaving short peptide was also used in this example to mediate the co-expression of human IL3 and human GM-CSF.
  • other self-cleaving peptides with similar functions to Furin-GSG-P2A can also achieve similar effects in this application.
  • This example also utilizes hCD68 to express cytokines in mouse myeloid cells.
  • the advantage of expressing human cytokines in myeloid cells is that it can reduce genotoxicity and reduce the excessive activation of the human immune system by excess exogenous cytokines.
  • the present application also helps to express other cytokines that are related to maintaining the mouse's own health and lack cross-activity with human homologous cytokines, such as human TPO, M-CSF, etc.
  • a severe immunodeficiency mouse expressing human cytokines can be constructed more efficiently and conveniently, saving manpower and material costs.
  • the mice constructed by the method described in the present application are more conducive to the colonization and function of the human immune system and its components, and provide better experimental materials for immunology-related animal experiments. For example, it is used to detect the differentiation and colonization function of human hematopoietic stem cells and therapeutic cells based on human hematopoietic stem cells; to simulate the human immune system and tumor immune microenvironment, in the development of antibody drugs, cellular immune drugs, etc., infectious diseases, autoimmune diseases It plays an important role in many fields such as oncology research.
  • this work obtained severe immunodeficiency mice (NVGhIL5) expressing humanized IL15, which can be used for the evaluation of NK cell-related immunotherapy (ADCC, CAR-NK, etc.).

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Abstract

涉及一种对非人动物进行基因改造的方法，其包括：对非人动物细胞的受精卵进行基因改造，基因改造包括利用CRISPR/Cas9对非人动物的受精卵进行基因编辑，或利用Piggy转座酶依赖的转基因系统对非人动物受精卵进行基因编辑。还涉及一种构建免疫缺陷动物模型的方法。

Description

对非人动物进行基因改造和构建免疫缺陷动物模型的方法 技术领域
本申请涉及一种对非人动物进行基因改造和构建免疫缺陷动物模型的方法。
背景技术
小鼠免疫系统人源化通常是指在免疫缺陷型小鼠体内植入人外周血白细胞(hPBMC)或人造血干细胞(CD34+HSC)以后，在小鼠体内重建一种或几种人免疫细胞。一方面，小鼠免疫系统人源化过程可以用于检测人类造血干细胞及基于人类造血干细胞的治疗细胞的分化和定植功能，另一方面人源化的小鼠还能够更好地模拟人类免疫系统及肿瘤免疫微环境，在传染性疾病、抗体药物开发，自身免疫病以及肿瘤学研究等多领域的有不可替代的作用。
免疫系统人源化通常需要使用重度免疫缺陷小鼠。在重度免疫缺陷小鼠表达某些人源化的细胞因子，有助于在小鼠重建完善的人免疫系统。重度免疫缺陷小鼠通常是指NOD scid IL2RγKO小鼠(SCID突变及IL2Rγ敲除的NOD小鼠)，BALB/c Rag1KO.IL2RγKO(IL2Rγ及Rag1敲除的BALB/c小鼠)或BALB/c Rag2KO.IL2RγKO小鼠(IL2Rγ及Rag2敲除的BALB/c小鼠。相较于仅有SCID、Rag1或Rag2敲除的普通的免疫缺陷小鼠，重度免疫缺陷小鼠进一步敲除了IL2Rγ，不仅缺少T细胞，B细胞，而且缺少NK细胞功能，并且巨噬细胞、树突状细胞的数量也大量减少，进一步降低了免疫功能的小鼠，更加有利于人源化免疫系统的构建。
二十年前，日本的实验动物中央研究所(CLEA)发展了以NOD为遗传背景的重度免疫缺陷型小鼠(NOD scid IL2RγKO，
Figure PCTCN2021106058-appb-000001
)。随后，美国的Jackson Lab也开发了类似的小鼠，命名为
Figure PCTCN2021106058-appb-000002
很多中国公司也开发了以NOD为背景的重度免疫缺陷型小鼠，分别命名为NPG，NCG，NSI，B-NDG，M-NSG等。除了以NOD为遗传背景的重度免疫缺陷小鼠，还有以BALB/c为遗传背景的BALB/c Rag1/2KO IL2RγKO小鼠。目前，广泛用于重建人免疫系统的重度免疫缺陷小鼠是NOD scid IL2RγKO小鼠，而BALB/c Rag1/2KO IL2RγKO小鼠天然免疫活性比NOD scid IL2RγKO小鼠要高，重建人免疫系统能力也比NOD scid IL2RγKO小鼠要弱，如果要提高BALB/c Rag1or2KO IL2RγKO小鼠重建人免疫系统的程度，需要经过更多的基因修饰。因此，BALB/c Rag1or2KO IL2RγKO小鼠在应用上不如NOD scid IL2RγKO小鼠广泛。以上所述重度免疫缺陷小鼠被统称为第一代重度免疫缺陷小鼠。
在重度免疫缺陷型小鼠体内重建人源化免疫系统通常是通过向小鼠体内植入人的造血干细胞(CD34+HSC)来实现的。一段时间后，人造血干细胞可在小鼠体内重建出各种免疫细胞，例如人T细胞、人B细胞、人髓系细胞等。人的免疫系统通常需要人细胞因子的支持才能分化成熟。但由于第一代重度免疫缺陷小鼠缺少人MHC分子，而且部分小鼠细胞因子和人细胞因子缺少交叉反应，因此在一代重度免疫缺陷小鼠体内重建的人免疫系统存在功能缺陷，例如髓系细胞比较少，淋巴组织发育不全，T细胞和B细胞功能不完善。
因此，不断有人尝试在重症免疫缺陷小鼠体内表达人细胞因子或可发挥与人细胞因子同等作用的工程化细胞因子。然而目前广泛用于重建人免疫系统的NOD scid IL2RγKO小鼠由于遗传背景的特性，其胚胎干细胞(ES细胞)不适于进行基因改造。而且这种小鼠的受精卵受孕率低，细胞核发育不成熟，也很难直接用于制作基因敲入或者过表达的转基因小鼠。由于对这种小鼠进行基因修饰的难度大，因此制作转基因重度免疫缺陷小鼠的方法通常是先对C57BL小鼠进行基因修饰，然后和NOD scid IL2RγKO小鼠连续回交10代。经过约两年后，C57BL遗传背景的转基因小鼠可转变为NOD scid IL2RγKO遗传背景的转基因小鼠。
例如，近几年来，CLEA和Jackson Lab也开发了以NOD scid IL2RγKO为遗传背景的转基因小鼠，如NSG-Tg(hu-IL-15)、NSG-SGM3、hIL2NOG、hIL-6NOG、hIL-15NOG、NOG-EXL等，以获得更加适宜于重建人造血干细胞系统的小鼠。日本的实验动物中央研究所(CLEA)开发的hIL-15NOG，利用CMV启动子在NOG小鼠过表达了人的IL-15，血浆表达量达到80-120pg/ml。但是因为细胞因子表达量略高，亦或细胞因子非特异性表达导致的细胞毒性，小鼠在重建人免疫系统之后容易获得消耗性疾病(waiting disease)，减少实验的窗口期。不过试验证明在hIL-15NOG小鼠中纯化的人外周NK细胞可以长时间维持生长。为了使人IL-15达到生理性表达，美国Jackson Lab利用BAC转基因技术，将完整人IL5基因座转入NSG小鼠获得NSG-Tg(hu-IL-15)。人IL-15在血浆表达量为7.1pg/ml。无论NSG-Tg(hu-IL-15)小鼠或是hIL-15NOG小鼠，都是利用转基因技术在小鼠表达外源的人IL-15，并不能替换NOG或NSG小鼠内源的IL-15。因此内源的IL-15也会对人免疫系统的功能，特别是人NK细胞的功能产生干扰。另外，由于遗传背景的特异性，利用同源重组原理对NOD scid IL2RγKO小鼠的受精卵或ES细胞直接进行基因编辑的技术难度高，也没有成功报道。因此这些小鼠都是以经典的受精卵显微注射方式建立C57BL为遗传背景的转基因小鼠，然后和NOD scid IL2RγKO小鼠连续回交10代以上，得到过表达人细胞因子的重度免疫缺陷小鼠。整个回交过程历时约2年。
综上所述，现有技术中对NOD scid IL2RγKO小鼠直接进行基因改造的成功率低，难度大，因此需要利用其它遗传背景的小鼠进行多代回交，导致消耗大量的时间和人力成本。并且现有技术中小鼠体内的人源细胞因子过表达，或与小鼠自身细胞因子的互相干扰，对小鼠本身发育成长和寿命产生不良影响，同时也会影响到在小鼠体内构建的人免疫系统的功能。对于与维持小鼠自身健康相关且与人同源细胞因子缺乏交叉活性的细 胞因子，在小鼠体内进行人源化替代小鼠原有细胞因子或是使用现有技术进行过表达均会影响小鼠的健康和寿命，以及在其体内构建人免疫系统的功能。
发明内容
技术问题
为更方便高效地引入人细胞因子或人源化细胞因子，构建更加适宜于重建人造血干细胞系统的小鼠，同时降低时间和人力成本，本申请利用优化的基因工程学方法，同时在一些步骤上进行创造性的改进，直接改造非人动物，例如NOD scid IL2RγKO重度免疫缺陷型小鼠的受精卵细胞以避免回交，得到更加适宜于重建多功能人免疫系统的重度免疫缺陷型小鼠。
本申请解决了NOD scid IL2RγKO等重度免疫缺陷小鼠不适于基因改造和/或受精卵受孕率低，细胞核发育不成熟，只能借助其他遗传背景的小鼠进行长时间的多代回交来获得遗传稳定的基因工程小鼠的问题。
技术方案
具体来说，本申请涉及如下技术方案：
1.一种对非人动物，进行基因改造的方法，其包括：
对非人动物细胞的受精卵进行基因改造，
基因改造包括利用CRISPR/Cas9对非人动物的受精卵进行基因编辑；
优选地，所述受精卵是体外受精的受精卵；
进一步优选地，所述受精卵是通过体外受精得到的，并且在体外培养一定时间的受精卵，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
2.根据项1所述的方法，其中，对非人动物细胞的受精卵进行基因改造是通过对受精卵的细胞核或细胞质进行注射来完成的；
优选地，利用CRISPR/Cas9方法，需要注射用于同源重组的线性DNA模板大于等于2.5kb，对非人动物的受精卵进行基因编辑是对非人动物细胞的受精卵的细胞核进行显微注射。
3.根据项1或2所述的方法，其中，
所述非人动物是以NOD为遗传背景的小鼠，
所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠，
所述小鼠再进一步优选是进一步敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。
4.一种对非人动物进行基因改造的方法，其包括：
通过对非人动物的受精卵进行基因改造使得细胞因子基因或免疫相关基因在原位进行人源化，而在这些细胞因子基因或免疫相关基因人源化后免疫缺陷型小鼠的健康和繁殖效率不会受到影响；
所述细胞因子包括以下任意一种或两种以上：白细胞介素-15(IL-15)、白细胞介素-7(IL-7)、白细胞介素-6(IL-6)、B细胞活化因子(BAFF)、FMS样酪氨酸激酶3配体(Flt3L)重组蛋白；
所述免疫相关基因包括主要组织相容性复合体(MHC)；
优选地，所述细胞因子为白细胞介素-15(IL-15)。
5.根据项4所述的方法，其中，
对非人动物进行基因改造的方法是对非人动物的受精卵进行基因改造，
优选利用CRISPR/Cas9对非人动物的受精卵进行基因编辑，
进一步优选，对受精卵的细胞核进行显微注射从而利用CRISPR/Cas9对非人动物的受精卵进行基因编辑以实现对细胞因子进行原位人源化。
6.根据项5所述的方法，其中，对非人动物的受精卵进行基因改造是在所述非人动物的IL-15第四个外显子的后面插入兼并的人IL-15第五到第八个外显子的编码序列；
优选地，对非人动物的受精卵进行基因改造还包括在所述非人动物的IL-15第四个外显子后面进一步插入bGHpolyA(bovine growth hormone polyadenylation)序列；
优选地，所述bGHpolyA序列如SEQ ID No.2所示；
优选地，在所述非人动物的IL-15第四个外显子后面插入兼并的人IL15第五到第八个外显子的编码序列如SEQ ID No.1所示。
7.根据项4～6中任一项所述的方法，其中，所述非人动物是以NOD为遗传背景的小鼠，
所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠，
所述小鼠再进一步优选是进一步敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠；
优选地，所述小鼠的IL-15第四个外显子的序列如SEQ ID No.4所示。
8.根据项5～7中任一项所述的方法，其中，所述非人动物的受精卵是通过体外受精获得的；
优选地，对所述非人动物的受精卵进行基因改造之前对体外受精得到的受精卵在体外培养一定时间，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
9.根据项5所述的方法，其中，所述对所述非人动物的受精卵进行基因改造是对受精卵的细胞核进行显微注射，显微注射物质包括：
同源重组DNA；
gRNA1，
gRNA2，以及
mRNACas9；
优选地，所述同源重组DNA包括如SEQ ID No.3所示的人IL-15同源重组DNA序列；
优选地，所述gRNA1的识别靶序列为：
CATAGCTATTATCAAGTTAGTGG(SEQ ID No.5)，
所述gRNA2的识别靶序列为：
GAAACACAAGTAGCACGAGATGG(SEQ ID No.6)。
10.一种构建重度免疫缺陷的动物模型的方法，该方法包括：
通过对非人动物的受精卵进行基因改造使得细胞因子基因或免疫相关基因在原位进行人源化，而这些细胞因子基因或免疫相关基因人源化改造后的不会影响免疫缺陷型小鼠的健康和繁殖效率的人源化；
所述细胞因子包括以下任意一种或两种以上：白细胞介素-15(IL-15)、白细胞介素-7(IL-7)、白细胞介素-6(IL-6)、B细胞活化因子(BAFF)、FMS样酪氨酸激酶3配体(Flt3L)重组蛋白；
所述免疫相关基因包括主要组织相容性复合体(MHC)；
优选地，所述细胞因子为白细胞介素-15(IL-15)。
11.根据项10所述的方法，其中，
对非人动物进行基因改造的方法是对非人动物的受精卵进行基因改造，
优选利用CRISPR/Cas9对非人动物的受精卵进行基因编辑，
进一步优选，对受精卵的细胞核进行显微注射从而利用CRISPR/Cas9对非人动物的受精卵进行基因编辑以实现对细胞因子进行原位人源化。
12.根据项11所述的方法，其中，对非人动物的受精卵进行基因改造是在所述非人动物的IL-15第四个外显子的后面插入兼并的人IL-15第五到第八个外显子的编码序列；
优选地，对非人动物的受精卵进行基因改造还包括在所述非人动物的IL-15第四个外显子后面进一步插入bGHpolyA(bovine growth hormone polyadenylation)序列；
优选地，所述bGHpolyA序列如SEQ ID No.2所示；
优选地，在所述非人动物的IL-15第四个外显子后面插入兼并的人IL第五到第八个外显子的编码序列如SEQ ID No.1所示。
13.根据项10～12中任一项所述的方法，其中，所述非人动物是以NOD为遗传背景的小鼠，
所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠，
所述小鼠再进一步优选是进一步敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠；
优选地，所述小鼠的IL-15第四个外显子的序列如SEQ ID No.4所示。
14.根据项11～13中任一项所述的方法，其中，所述非人动物的受精卵是通过体外受精获得的；
优选地，对所述非人动物的受精卵进行基因改造之前对体外受精得到的受精卵在体外培养一定时间，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
15.根据项11所述的方法，其中，所述对所述非人动物的受精卵进行基因改造是对受精卵的细胞核进行显微注射，显微注射物质包括：
同源重组DNA；
gRNA1，
gRNA2，以及
mRNACas9；
优选地，所述同源重组DNA包括如SEQ ID No.3所示的人IL-15同源重组DNA序列；
优选地，所述gRNA1的识别靶序列为：
CATAGCTATTATCAAGTTAGTGG(SEQ ID No.5)，
所述gRNA2的识别靶序列为：
GAAACACAAGTAGCACGAGATGG(SEQ ID No.6)。
16.线性DNA，其包括如SEQ ID No.3所示的人IL-15同源重组序列。
17.一种对非人动物进行基因改造的方法，其包括：
对非人动物细胞的受精卵进行基因改造，
基因改造包括利用Piggy转座酶依赖的转基因系统对非人动物的受精卵进行基因编辑；
优选地，所述受精卵是体外受精的受精卵；
进一步优选地，所述受精卵是通过体外受精得到的，并且在体外培养一定时间的受精卵，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
18.根据项17所述的方法，其中，对非人动物细胞的受精卵进行基因改造是通过对受精卵的细胞核或细胞质进行注射来完成的；
优选地，需要进行基因编辑的基因片段为环形质粒，对非人动物的受精卵进行基因编辑是对非人动物细胞的受精卵的细胞质进行细胞浆或细胞核注射。
19.根据项17或18所述的方法，其中，
所述非人动物是以NOD为遗传背景的小鼠，
所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠，
所述小鼠再进一步优选是进一步敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。
20.根据项17～19中任一项所述的方法，其中，其包括：
在非人动物中过表达目标蛋白一和/或目标蛋白二；
其中，所述目标蛋白一主要在T细胞、B细胞和NK细胞中表达；所述目标蛋白一的基因原位人源化后，在重度免疫缺陷非人动物中检测不到所述目标蛋白一的基因的表达；优选所述目标蛋白一包括以下任意一种或两种：人白细胞介素-3(人IL3)、人白细胞介素-2(人IL2)；进一步优选所述目标蛋白一为人白细胞介素-3(人IL3)；
所述目标蛋白二可能会影响免疫缺陷非人动物的健康；优选所述目标蛋白二包括以下任意一种或两种以上：人血小板生成素(人TPO)、人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)、人巨噬细胞集落刺激因子(人MCSF)；进一步优选所述目标蛋白二为人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)。
21.根据项20所述的方法，其中，
编码人白细胞介素-3(人IL3)的人IL3cDNA的序列如SEQ ID No.14所示；
编码人粒细胞巨噬细胞集落刺激因子(人GM-CSF)的人GM-CSFcDNA的序列如SEQ ID No.15所示。
22.根据项20或21所述的方法，其中，
对非人动物进行基因改造的方法是对非人动物的受精卵进行基因改造使得在非人动物中过表达目标蛋白是指在非人动物的髓系细胞中过表达目标蛋白；
优选利用特异性在髓系细胞表达的启动子来在非人动物的髓系细胞中过表达目标蛋白；
优选所述髓系细胞表达的启动子选自人CD68启动子、CSF1R启动子、CD11c启动子、CX3CR1启动子、Langerin/CD207启动子、MMLV LTR启动子、Visna virus LTR启动子、DC-STAMP启动子、Human MSR启动子、MSR-A启动子、CD4启动子、CD2启动子、Iba-AIF-1启动子、CD11b启动子、c-fms启动子、scavenger receptor A(SR-A)启动子、lysozyme启动子和MHC class II启动子(MHC-II)；
进一步优选利用人CD68启动子在非人动物的髓系细胞中过表达目标蛋白；
进一步优选利用piggyBac转座子系统质粒和piggyBac转座酶将细胞因子表达盒导入非人动物受精卵。
23.根据项20或21所述的方法，其中，在非人动物中过表达两个以上目标蛋白时，利用双重自我剪切短肽RAKR-GSG-P2A(RAKR-GSG-ATNFSLLKQAGDVEENPGP)、RAKR-GSG-T2A(RAKR-GSG-EGRGSLLTCGDVEENPGP)、RAKR-GSG-E2A(RAKR-GSG-QCTNYALLKLAGDVESNPGP)或RAKR-GSG-F2A(RAKR-GSG-VKQTLNFDLLKLAGDVESNPGP)介导不同蛋白的过表达，其中，RAKR是Furin剪切短肽的序列，GSG是linker；
优选地，所述双重自我剪切短肽为RAKR-GSG-P2A，编码双重自我剪切短肽RAKR-GSG-P2A的基因的序列如SEQ ID No.13所示。
24.根据项22所述的方法，其中，利用piggyBac转座子系统质粒来在非人动物的髓系细胞中过表达目标蛋白，在piggyBac转座子系统质粒中还插入有bGH polyA序列，优选其序列如SEQ ID No.2所示；
优选地，在piggyBac转座子系统质粒中还插入有反响重复序列，优选其序列如SEQ ID No.16或SEQ ID No.17所示。
25.根据项20～24中任一项所述的方法，其中，
所述非人动物是以NOD为遗传背景的小鼠；
所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠；
所述小鼠再进一步优选是敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。
26.根据项20～25中任一项所述的方法，其中，所述非人动物的受精卵是通过体外受精获得的；
优选地，对所述非人动物的受精卵进行基因改造之前对体外受精得到的受精卵在体外培养一定时间，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
27.根据项22所述的方法，其中，所述对所述非人动物的受精卵进行基因改造是对受精卵的细胞质进行胞浆注射，胞浆注射的物质包括：
PiggyBac转座子系统质粒和piggyBac转座酶；
优选地，PiggyBac转座子系统质粒包括SEQ ID No.11所示的序列。
28.一种构建重度免疫缺陷的动物模型的方法，该方法包括：
在非人动物中过表达目标蛋白，所述目标蛋白为目标蛋白一和/或目标蛋白二；
其中，所述目标蛋白一主要在T细胞、B细胞和NK细胞中表达；所述目标蛋白二可能会影响免疫缺陷小鼠的健康；
优选地，在非人动物中过表达的目标蛋白包括以下任意一种或两种以上：人白细胞介素-3(人IL3)、人白细胞介素-2(人IL2)、人血小板生成素(人TPO)、人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)、人巨噬细胞集落刺激因子(人MCSF)；
优选地，在非人动物中过表达人白细胞介素-3(人IL3)和人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)；
优选地，编码人白细胞介素-3(人IL3)的人IL3cDNA的序列如SEQ ID No.14所示；编码人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)的人GM-CSFcDNA的序列如SEQ ID No.15所示。
29.根据项28所述的方法，其中，
对非人动物进行基因改造的方法是对非人动物的受精卵进行基因改造使得在非人动物中过表达目标蛋白是指在非人动物的髓系细胞中过表达目标蛋白；
优选利用特异性在髓系细胞表达的启动子来在非人动物的髓系细胞中过表达目标蛋白；
优选所述髓系细胞表达的启动子选自人CD68启动子、CSF1R启动子、CD11c启动子、CX3CR1启动子、Langerin/CD207启动子、MMLV LTR启动子、Visna virus LTR启动子、DC-STAMP启动子、Human MSR启动子、MSR-A启动子、CD4启动子、CD2启动子、Iba-AIF-1启动子、CD11b启动子、c-fms启动子、scavenger receptor A(SR-A)启动子、lysozyme启动子和MHC class II启动子(MHC-II)，启动子在非人动物的髓系细胞中过表达目标蛋白；
进一步优选利用人CD68启动子在非人动物的髓系细胞中过表达目标蛋白；
进一步优选利用piggyBac转座子系统质粒和piggyBac转座酶来在非人动物的髓系细胞中过表达目标蛋白。
30.根据项29所述的方法，其中，在非人动物中过表达两个以上目标蛋白时，利用双重自我剪切短肽RAKR-GSG-P2A(RAKR-GSG-ATNFSLLKQAGDVEENPGP)、RAKR-GSG-T2A(RAKR-GSG-EGRGSLLTCGDVEENPGP)、RAKR-GSG-E2A(RAKR-GSG-QCTNYALLKLAGDVESNPGP)或RAKR-GSG-F2A(RAKR-GSG-VKQTLNFDLLKLAGDVESNPGP)介导不同目标蛋白的过表达，其中RAKR是Furin剪切短肽的序列，GSG是linker；
优选地，所述双重自我剪切短肽为RAKR-GSG-P2A，编码双重自我剪切短肽PAKR-GSG-P2A的基因的序列如SEQ ID No.13所示。
31.根据项29所述的方法，其中，利用piggyBac转座子系统质粒来在非人动物的髓系细胞中过表达目标蛋白，在piggyBac转座子系统质粒中还插入有pGH polyA序列，优选其序列如SEQ ID No.2所示；
优选地，在piggyBac转座子系统质粒中还插入有反响重复序列，优选其序列如SEQ ID No.16或SEQ ID No.17所示。
32.根据项28～31中任一项所述的方法，其中，
所述非人动物是以NOD为遗传背景的小鼠；
所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠；
所述小鼠再进一步优选是进一步敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。
33.根据项28～32中任一项所述的方法，其中，所述非人动物的受精卵是通过体外受精获得的；
优选地，对所述非人动物的受精卵进行基因改造之前对体外受精得到的受精卵在体外培养一定时间，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
34.根据项29所述的方法，其中，所述对所述非人动物的受精卵进行基因改造是对受精卵的细胞质进行胞浆注射，胞浆注射的物质包括：
PiggyBac转座子系统质粒和piggyBac转座酶；
优选地，PiggyBac转座子系统质粒包括SEQ ID No.11所示的序列。
35.一种PiggyBac转座子系统质粒，其序列如SEQ ID No.11所示。
发明的效果
一方面，本申请解决了NOD scid IL2RγKO小鼠受精活性低，细胞核发育不成熟，不适于直接进行大片段的基因编辑的问题，可以直接对NOD scid IL2RγKO小鼠受精卵进行基因改造，得到表达人源化细胞因子的NOD scid IL2RγKO小鼠，从而避免多代回交，从而节省时间和人力成本。另外，本申请保留了部分人鼠源同源基因的片段，与截短的人源基因重组，获得嵌合的人源化基因。这样，既减小了插入片段的大小，提升了重组效率；又以人源化基因替代了鼠源基因，相较于现有技术中常用的过表达方式，避免了人与小鼠基因共同存在且互相干扰的情况，减少了在该小鼠体内构建人免疫系统时对人免疫系统分化定植及功能的影响，同时避免了过表达的人细胞因子对小鼠发育及生理功能的影响，延长了小鼠寿命及实验窗口期。
本申请涉及的一种工程化重度免疫缺陷小鼠的方法，所述工程化重度免疫缺陷小鼠可用于更加完整高效地在小鼠体内重建人免疫系统。相较于现有技术，使用本申请所述方法获取工程化小鼠更加省时高效，并且所获得的小鼠具有更长的试验窗口期，利用所述小鼠重建的人免疫系统功能也更加完备。
本申请解决了NOD scid IL2RγKO小鼠受精活性低，细胞核发育不成熟不适于直接进行大片段的基因编辑的问题，实现了直接对NOD scid IL2RγKO小鼠受精卵进行基因改造，得到表达人源化细胞因子的NOD scid IL2RγKO小鼠，避免多代回交，从而节省时间和人力成本。另外，对于与维持小鼠自身健康相关且与人同源细胞因子缺乏交叉活性的细胞因子，本申请做到了适时适度的表达，一方面减少了在该小鼠体内构建人免疫系统时对人免疫系统分化定植及功能的影响，另一方面又避免了过度表达的人细胞因子对小鼠发育及生理功能的影响，从而维持了小鼠的健康，延长了小鼠寿命及实验窗口期。
另一方面，本申请直接对NOD scid IL2RγKO小鼠受精卵进行基因改造，得到表达人源化细胞因子的NOD scid IL2RγKO小鼠，避免多代回交，从而节省时间和人力成本。另外，对于与维持小鼠自身健康相关且与人同源细胞因子缺乏交叉活性的细胞因子，本申请做到了适时适度的表达，既减少了在该小鼠体内构建人免疫系统时对人免疫系统分化定植及功能的影响，又避免了过度表达的人细胞因子对小鼠发育及生理功能的影响，从而维持了小鼠的健康，延长了小鼠寿命及实验窗口期。此外，普通DNA显微注射需要大片段进入受精卵细胞核，而piggybac技术可以进行受精卵染色质注射，因此特别适合细胞发育不全或迟缓的受精卵。
本申请涉及的另一种工程化重度免疫缺陷小鼠的方法，所述工程化重度免疫缺陷小鼠可用于更加完整高效地在小鼠体内重建人免疫系统。相较于现有技术，使用本申请所述方法获取工程化小鼠更加省时高效，并且所获得的小鼠健康状况更好，具有更长的试验窗口期，利用所述小鼠重建的人免疫系统功能也更加完备。
附图说明
图1小鼠IL-15基因座人源化设计示意图；
图2小鼠IL-15基因人源化后的cDNA序列和蛋白质氨基酸序列。图2A为IL-15人源化后的cDNA序列(5’-3’，5’utl\鼠源4+\人源5-8\)；图2B为IL-15人源化后的氨基酸序列和鼠源IL-15氨基酸序列比较；
图3 hIL-15同源重组质粒图谱；
图4通过ELISA方法检测到的NVG小鼠和NVG-hIL15小鼠血浆内人IL-15的平均表达浓度；
图5(A)～(D)分别为注射人PBMC后，人CD45细胞、人T细胞、人NK细胞、表达CD16的细胞毒性的人NK细胞在小鼠血液中的频率；
图6小鼠注射人PBMC后的存活率；
图7小鼠先注射人PBMC再注射K562-Luc后的K562-Luc的IVIS成像结果图；
图8向小鼠体内先后注射人外周血分离的NK细胞和K562-Luc，并进行体内荷瘤试验的流程图；
图9注射人外周血分离的NK细胞后，小鼠活体内NK细胞百分比随时间的变化情况；
图10小鼠先注射人外周血分离的NK细胞再注射K562-Luc后的K562-Luc的IVIS成像结果图；
图11向小鼠体内先后注射MIA PaCa-2和CAR-NK92，并进行体内荷瘤试验的流程图；
图12小鼠先注射MIA PaCa-2再注射CAR-NK92后的MIA PaCa-2的IVIS成像结果图；
图13小鼠先注射MIA PaCa-2，再注射CAR-NK92后，体内平均荧光素酶含量变化图。
图14为piggyBAC CD68Pro-Intron-hGM-CSF-PAKR-GSG-P2A-hIL3-pA重组质粒。
图15为piggyBAC转基因小鼠PCR鉴定电泳图，数字标号为阳性小鼠，M为1kb DNA标记物。其中3、4、5、6、10为F0代NOD-SCID IL2RγKO-Tg(hCD68-hIL3/GMCSF)小鼠，12、13、14为F0代NOD-SCID IL2RγKO-Tg(SV40-hIL3/GMCSF)小鼠，WT为阴性转基因小鼠。
图16为反向PCR的工作原理图。
图17为反向PCR产物电泳结果图。
图18为人IL3和人GM-CSF在转基因小鼠组织内表达特异性鉴定结果图。
图19(A)～(D)为注射人CD34干细胞第12周后，人免疫系统在小鼠体内的表型特征。
图20为移植人CD34干细胞后小鼠的存活比例。
图21为注射人CD34干细胞第12周后小鼠血浆中细胞因子的表达水平。
图22为注射人CD34干细胞第16周后小鼠血红蛋白数。
具体实施方案
定义
如本文中使用的术语“后代”包括子孙后代，并且包括自亲本细胞衍生的分化的或未分化的后代细胞。在一种用法中，术语后代包括与亲本遗传相同的后代细胞。在另一种使用中，术语后代包括与亲本遗传且表型相同的后代细胞。在又一种用法中，术语后代包括已经自亲本细胞分化的后代细胞。
如本文中使用的术语“启动子”包括与要转录的核酸序列(诸如编码期望分子的核酸序列)可操作连接的DNA序列。启动子一般位于要转录的核酸序列的上游，并且提供RNA聚合酶和其它转录因子特异性结合的位点。在特定的实施方案中，启动子一般位于转录的核酸序列的上游以生成期望的分子，并且提供RNA聚合酶和其它转录因子特异性结合的位点。
如本文中使用的术语“免疫缺陷的”意指非人动物在其天然免疫系统的一个或多个方面是缺陷的，例如动物在一种或多种类型的发挥功能的宿主免疫细胞方面是缺陷的，例如在非人B细胞数目和/或功能、非人T细胞数目和/或功能、非人NK细胞数目和/或功能等方面是缺陷的。
在本文中使用的术语“非人动物”例如实验室动物、家畜、牲畜等，例如诸如鼠、啮齿类、犬、猫、猪、马、牛、绵羊、非人灵长类等物种；例如小鼠、大鼠、家兔、仓鼠、豚鼠、牛、猪、绵羊、山羊、和其它转基因动物物种，特别是哺乳动物物种，如本领域中已知的。在某些实施方案中，主题经遗传修饰的动物是小鼠、大鼠或家兔。
在一个实施方案中，非人动物是哺乳动物。在一些此类实施方案中，非人动物是小型哺乳动物，例如跳鼠总科(Dipodoidea)或鼠总科(Muroidea)的。在一个实施方案中，经遗传修饰的动物是啮齿类。在一个实施方案中，啮齿类选自小鼠、大鼠、和仓鼠。在一个实施方案中，啮齿类选自鼠总科。在一个实施方案中，经遗传修饰的动物来自选自下述的科：丽仓鼠科(Calomyscidae)(例如小鼠样仓鼠)、仓鼠科(Cricetidae)(例如仓鼠、新世界(NewWorld)大鼠和小鼠、田鼠)、鼠科(Muridae)(真小鼠和大鼠、沙鼠、多刺小鼠(spiny mice)、鬃毛大鼠(crested rats))、马岛鼠科(Nesomyidae)(攀爬小鼠(climbing mice)、岩石小鼠(rock mice)、有尾大鼠(with-tailed rat)、马达加斯加(Malagasy)大鼠和小鼠)、刺山鼠科(Platacanthomyidae)(例如多刺睡鼠(spiny dormice))、和鼢鼠科(Spalacidae)(例如鼹鼠(mole rates)、竹鼠(bamboo rat)、和鼢鼠(zokors))。在一个具体的实施方案中，经遗传修饰的啮 齿类选自真小鼠或大鼠(鼠科)、沙鼠、多刺小鼠、和鬃毛大鼠。在一个实施方案中，经遗传修饰的小鼠来自鼠科的成员。
如本文中使用的术语，NOD小鼠是指no obesity diabetes即非肥胖糖尿病小鼠。NOD小鼠有很多衍生品系，包括NOD/Scid NOD/Ltj小鼠等。
如本文中使用的术语“免疫缺陷小鼠”是指先天性遗传突变或用人工方法造成一种或多种免疫系统组成成分缺陷的小鼠，其常见突变基因型是，SCID突变，Rag1敲除，Rag2敲除，主要表型是缺少T细胞和B细胞。
如本文中使用的术语“重度免疫缺陷鼠”通常是指进一步敲除了IL2Rγ基因功能的免疫缺陷型小鼠。IL2Rγ为很多细胞因子的共有受体。IL2Rγ的敲除可进一步降低小鼠免疫系统的功能。重度免疫缺陷鼠主要表型是缺少T细胞，B细胞，NK细胞功能，而且巨噬细胞、树突状细胞的数量也大量减少。重度免疫缺陷鼠的主要用途是在小鼠重建人的免疫系统。
如本文中使用的术语“免疫系统人源化小鼠”通常是指在重度免疫缺陷型小鼠植入了人外周血白细胞(hPBMC)或人造血干细胞(CD34+HSC)以后，在小鼠体内重建一种或几种人免疫细胞的小鼠。
如本文中使用的术语“CRISPR/Cas9”是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御，可用来对抗入侵的病毒及外源DNA。CRISPR/Cas9基因编辑技术，则是对靶向基因进行特定DNA修饰的技术。以CRISPR/Cas9基础的基因编辑技术在一系列基因治疗的应用领域都展现出极大的应用前景，例如血液病、肿瘤和其他遗传疾病。该技术成果已应用于人类细胞、斑马鱼、小鼠以及细菌的基因组精确修饰。
如本文中使用的术语“Piggy转座系统”也称为sleeping beauty转座系统(或PiggyBac系统)，PiggyBac(PB)转座子来源于鳞翅目昆虫，在分类上属于真核生物的第二类转座子，是一个自主因子，长2476bp，有短的末端反向重复序列(ITR)和一个开放编码框(ORF)。Piggy转座子主要采取“cut-paste”机制发生转座。Piggy转座系统转座效率高，宿主范围广，广泛应用于昆虫等低等生物的基因转移及突变筛选。利用Piggy转座子构成的载体-辅助质粒系统已成功地获得了转基因地中海果蝇、黑腹果蝇和家蚕。转座时转座酶通过与转座子末端结合形成短暂的发夹结构，转座子完全切离后，通过其3'OH末端攻击靶DNA序列中TTAA位点的5'末端，转座子5'序列的TTAA悬垂与靶DNA打开的单链TTAA相配对，与整合位置两侧的空隙重新连接，连接过程不需要合成DNA。
在本文中使用的术语“白细胞介素”是由多种细胞产生并作用于多种细胞的一类细胞因子。指一类分子结构和生物学功能已基本明确，具有重要调节作用而统一命名的细胞因子，它和血细胞生长因子同属细胞因子。两者相互协调，相互作用，共同完成造血和免疫调节功能。白细胞介素在传递信息，激活与调节免疫细胞，介导T、B细胞活化、增殖与分化及在炎症反应中起重要作用。白细胞介素interleukin缩写为IL，功能关系免疫反应的表达和调节，这种调节有来源于淋巴细胞或巨噬细胞等的许多因子参与。来源于淋巴细胞的有淋巴细胞活素，来源于巨噬细胞的总称为monokine，其中的各个因子的 生物活性各有不同(例如巨噬细胞活化，促进T细胞繁殖等)，因子自身的物理化学性质多不清楚。
白细胞介素-15(IL-15)可由活化的单核-巨噬细胞、表皮细胞和成纤维细胞等多种细胞产生。IL-15的分子结构与IL-2有许多相似之外，因此可以利用IL-2受体的β链和γ链与靶细胞结合，发挥类似IL-2的生物学活性。IL-15可诱导B细胞增殖和分化，是唯一能部分取代IL-2诱导初期抗体产生的细胞因子；IL-15能够刺激T细胞和NK细胞增殖，诱导LAK细胞活性，还能与IL-12协同刺激NK细胞产生IFN-γ。
白细胞介素-7(IL-7)是由骨髓基质细胞分泌的糖蛋白，分子量为25KD；其基因位于第8号染色体。IL-7的靶细胞主要为淋巴细胞，对来自人或小鼠骨髓的B祖细胞、胸腺细胞及外周成熟的T细胞等均有促生长活性。
白细胞介素-6(IL-6)的作用的靶细胞很多，包括巨噬细胞、肝细胞、静止的T细胞、活化的B细胞和浆细胞等；其生物效应也十分复杂，曾称为B细胞刺激因子2(bsf-2)、26KD蛋白、B细胞分化因子(bCDf)、肝细胞刺激因子(hsf)等。IL-6不能刺激相应细胞分泌其它细胞因子，在生理浓度下对免疫细胞的自分泌作用亦比较弱，提示其主要免疫学功能是加强其它细胞因子的效果。IL-6可由多种细胞合成，包括活化的T细胞和B细胞、单核-巨噬细胞、内皮细胞、上皮细胞以及成纤维细胞等。人类IL-6基因位于第7号染色体上；IL-6分子量在21～30KD之间，其差异是由于肽链的糖基化和磷酸程度不同所致。IL-6由2条糖蛋白链组成；1条为α链，分子量80KD；另1条为β链，分子量130KD。α链缺少胞内区，只能以低亲合性与IL-6结合，所形成的复合物迅即与高亲和性的β链结合，通过β链向细胞内传递信息。
白细胞介素-3(IL3)是白细胞介素家族重要成员之一，又名多向性集落刺激因子(multi-CSF)，是一种细胞因子，属于白细胞介素的一种。它是由T淋巴细胞所产生，能够刺激参与免疫反应的细胞增殖、分化并提高其功能。在本申请中涉及的编码人白细胞介素-3(人IL3)的人IL3cDNA的序列如SEQ ID No.5所示。
白细胞介素-2(IL2)是趋化因子家族的一种细胞因子。它是由多细胞来源(主要由活化T细胞产生)，又具有多向性作用的细胞因子(主要促进淋巴细胞生长、增殖、分化)；对机体的免疫应答和抗病毒感染等有重要作用，能刺激已被特异性抗原或致丝裂因数启动的T细胞增殖；能活化T细胞，促进细胞因子产生；刺激NK细胞增殖，增强NK杀伤活性及产生细胞因子，诱导LAK细胞产生；促进B细胞增殖和分泌抗体；激活巨噬细胞。可用于临床研究和肿瘤治疗。
细胞活化因子(BAFF)属肿瘤坏死因子超家族成员(TNFSF13B)，主要由单核细胞、树突状细胞及T细胞产生，可促进B细胞成熟和分化，在免疫应答中起重要作用，并与自身免疫密切相关。膜型BAFF主要表达于抗原提呈细胞，蛋白酶可使之水解而转变为可溶性BAFF。
FMS样酪氨酸激酶3配体(Flt3L)是一种能够调节早期造血的关键性细胞因子，与IL-3、IL-6、G-CSF和SCF等细胞因子联用对原始造血干/祖细胞产生强烈的增殖效应，Flt3L 是主要表达于造血干细胞/祖细胞上的III型酪氨酸激酶受体FMS样酪氨酸激酶3(Flt3)的配体，是一种与早起造血调控有关的生长因子。Flt3L联合其它细胞因子，能够在体内外显著地扩增DC，刺激T细胞核NK细胞增殖，为肿瘤免疫治疗提供了良好的基础。
FMS样酪氨酸激酶3配体(Flt3L)重组蛋白是指FMS样酪氨酸激酶3配体在宿主细胞中进行重组表达的蛋白质。
白细胞抗原(HLA)是一群存在于细胞表面的糖蛋白分子，曾认为引起器官移植排斥反应的主要抗原。检测HLA有助于对某些疾病的诊断、分型、推测预后，可采用血清法测定A、B、C位点抗原，以混合淋巴细胞培养法测定D、DR抗原。
血小板生成素(TPO)是催化甲状腺激素的重要酶。TPO由甲状腺滤泡细胞合成，它是由933个氨基酸残基组成的分子量为103kD的10％糖化的血色素样蛋白质，在滤泡腔面的微绒毛处分布最为丰富。TPO以过氧化氢为氧化剂。试验中摘除大鼠垂体48h后，TPO活性消失，注入促甲状腺激素后，TPO活性即恢复，可见TPO的生成和活性受TSH调节。硫脲类药物能抑制TPO活性，因而可抑制甲状腺激素的合成，是临床上用于治疗甲状腺功能亢进(甲亢)的常用药物。
粒细胞-巨噬细胞集落刺激因子(GM-CSF)是造血生长因子之一，它参与造血调节过程。GM-CSF在体内体外维持着造血细胞的生存、增殖和分化，并且也影响着成熟粒细胞、单核-巨噬细胞的生物功能。GM-CSF临床应用方面发展很快，例如用于恶性肿瘤化疗、骨髓移植、骨髓增生异常综合征、中性粒细胞减少等等。在本申请中涉及的编码人粒细胞巨噬细胞集落刺激因子(人GM-CSF)的人GM-CSFcDNA的序列如SEQ ID No.6所示
巨噬细胞集落刺激因子(MCSF)也称为集落刺激因子-1(CSF-1)，是一种具有谱系特异性的细胞因子。M-CSF为链间二硫键连接而成的二聚体糖蛋白，主要存在于骨髓腔内，对单核细胞的增殖、分化及维持其活性有重要作用。其受体为c-Fms。
组织相容性复合体(major histocompatibility complex，MHC)是一组编码动物主要组织相容性抗原的基因群的统称。人类的MHC被称为HLA(human leukocyte antigen，HLA)，即人白细胞抗原；小鼠MHC则被称为H-2。HLA位于人的6号染色体短臂上，H-2位于小鼠的17号染色体上。根据基因的位置和功能，主要组织相容性复合体分为三类，分别为MHC class I、MHC class II、MHC class III。
MHC class I(MHC I)：位于一般细胞表面上，可以提供一般细胞内的一些状况，比如该细胞遭受病毒感染，则将病外膜碎片之氨基酸链(peptide)透过MHC提示在细胞外侧，可以供杀手CD8+T细胞等辨识，以进行扑杀。
MHC class II(MHC II)：大多位于抗原提呈细胞(APC)上，如巨噬细胞等。这类提供则是细胞外部的情况，像是组织中有细菌侵入，则巨噬细胞进行吞食后，把细菌碎片利用MHC提示给辅助T细胞，启动免疫反应。
为了使在小鼠体内重建的人类免疫系统更加完善，需要在小鼠体内过表达人细胞因子或者将小鼠细胞因子人源化。本申请提供一种对非人动物，尤其是小鼠，尤其是以NOD 为遗传背景的小鼠进行基因改造的方法，其包括：对非人动物细胞的受精卵进行基因改造，基因改造包括利用CRISPR/Cas9对非人动物的受精卵进行基因编辑。
更具体来说，小鼠是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD、敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。
在本申请一个具体实施方式中，所述非人动物的受精卵是上述重度免疫缺陷NOD小鼠的受精卵，尤其是体外受精的受精卵。本申请的结果显示跟B6小鼠比较，NOD小鼠体内受精的成胎率差。而体外受精的受精卵比体内受精的受精卵活性强。所以对于NOD遗传背景的小鼠体外受精制备受精卵是首选。
在本申请一个具体方式中，进行基因改造的受精卵是对体外受精得到的，并且在体外培养一定时间的受精卵，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上得到的受精卵。有文献报道，Crispr技术可以在NOD小鼠进行基因敲除和基因突变，但是对于大片段基因的定点整合没有成功案例。其差别在于这类小鼠的细胞核不成熟，小片段DNA或gRNA可以通过注射细胞质然后计入细胞核，而大片段线性DNA从细胞质进入细胞核的效率低，只有通过细胞核注射才可以。研究发现，在体外培养一定时间可以等待细胞核的成熟，有助于对于小鼠的基因改造。
在本申请一个具体方式中，对非人动物细胞的受精卵进行基因改造是通过对受精卵的细胞核或细胞质进行注射来完成的。这猜测是由于NOD小鼠细胞核不成熟，对于染色体随机整合大片段DNA，很难进行要细胞核注射，几率也比较低。具体来说，利用CRISPR/Cas9方法，需要进行基因编辑的基因片段为长度在500bp以上的线性DNA，对非人动物的受精卵进行基因编辑是对非人动物细胞的受精卵的细胞核进行显微注射。本申请将超过500bpcDNA和polyA定点插入基因组，而两侧的同源序列总共超过2.5kb。因此，在本申请具体的实施方式中，对体外受精得到的，并且在体外培养一定时间的受精卵，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上得到的受精卵进行细胞核显微注射来完成的。
本申请还涉及一种对非人动物进行基因改造的方法，其包括：通过对非人动物的受精卵进行基因改造，使得细胞因子基因或免疫相关基因原位人源化，而原位人源化改造后的细胞因子基因或免疫相关基因不会影响免疫缺陷型小鼠的健康和繁殖效率的人源化。这些细胞因子基因或免疫相关基因人源化后不会影响免疫缺陷型小鼠的健康和繁殖效率。例如人和小鼠的同源性较高的细胞因子基因或免疫相关基因，即使将这些细胞因子基因或免疫相关基因同源化也不会改变其功能，因此不会显著改变免疫缺陷型小鼠的健康和繁殖效率。例如人和小鼠的同源性在50％以上的细胞因子基因或免疫相关基因，优选同源性在60％以上的细胞因子基因或免疫相关基因，进一步优选同源性在70％以上的细胞因子基因或免疫相关基因，进一步优选同源性在80％以上的细胞因子基因或免疫相关基 因，进一步优选同源性在90％以上的细胞因子基因或免疫相关基因，进一步优选同源性在95％以上的细胞因子基因或免疫相关基因。
在本文中，原位人源化是指将小鼠基因或基因的一部分在同一基因位置替换成同源的人的基因或者人的基因一部分，例如在本文中在所述非人动物的IL-15第四个外显子的后面，原本是非人动物的IL-15的第五到第八个外显子及其内含子位置，替换为兼并的人IL第五到第八个外显子的编码序列，这样的人源化即为原位人源化。
在本申请一个具体方式中，细胞因子包括但不限于：白细胞介素-15(IL-15)、白细胞介素-7(IL-7)、白细胞介素-6(IL-6)、B细胞活化因子(BAFF)、FMS样酪氨酸激酶3配体(Flt3L)重组蛋白和白细胞抗原(HLA)。在本申请一个具体方式中，所述细胞因子为白细胞介素-15(IL-15)。
在本申请一个具体方式中，免疫相关基因是组织相容性复合体(MHC)。
在本申请一个具体方式中，对非人动物进行基因改造的方法是对非人动物的受精卵进行基因改造。具体来说，利用CRISPR/Cas9对非人动物的受精卵进行基因编辑，对受精卵的细胞核进行显微注射从而利用CRISPR/Cas9对非人动物的受精卵进行基因编辑以实现对细胞因子进行原位人源化。具体来说，对体外受精的、在体外培养一定时间的受精卵，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上得到的受精卵的细胞核进行显微注射从而利用CRISPR/Cas9对非人动物的受精卵进行基因编辑以实现对细胞因子进行原位人源化。
在一个具体的实施方式中，对非人动物的受精卵进行基因改造是在所述非人动物的IL-15第四个外显子的后面插入兼并的人IL第五到第八个外显子的编码序列。在本文中，在所述非人动物的IL-15第四个外显子的后面是指非人动物的IL-15第四个外显子的下一个核苷酸的位置，即紧挨着第四个外显子的后面。在一个具体的实施方式中，非人动物是小鼠，小鼠的IL-15第四个外显子的序列如SEQ ID No.4所示，在小鼠的IL-15第四个外显子即在SEQ ID No.4的最后一个核苷酸位置后面插入兼并的人IL第五到第八个外显子的编码序列。
具体来说，对非人动物的受精卵进行基因改造还包括在所述非人动物的IL-15第四个外显子后面进一步插入bGHpolyA(bovine growth hormone polyadenylation)序列。具体来说，bGHpolyA序列如SEQ ID No.2所示。
在具体的实施方式中，在所述非人动物的IL-15第四个外显子后面插入兼并的人IL第五到第八个外显子的编码序列如SEQ ID No.1所示。
在具体的实施方式中，非人动物是以NOD为遗传背景的小鼠。具体来说，小鼠是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD、且敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。如上所述的小鼠的IL-15第四个外显子后面用于插入兼并的人IL第五到第八个外显子的编码序列的靶序列如SEQ ID No.4所示。
在具体的实施方式中，对所述非人动物的受精卵进行基因改造是对受精卵的细胞核进行显微注射，显微注射物质包括：同源重组DNA；gRNA1，gRNA2，以及mRNACas9。具体来说，同源重组DNA包括如SEQ ID No.3所示的人IL-15同源重组DNA序列。所述gRNA1的识别靶序列为CATAGCTATTATCAAGTTAGTGG(SEQ ID No.5)，所述gRNA2的识别靶序列为GAAACACAAGTAGCACGAGATGG(SEQ ID No.6)。
在本申请中，还涉及一种构建重度免疫缺陷的动物模型的方法，通过对非人动物的受精卵进行基因改造从而使得非人动物中表达不会影响免疫缺陷型小鼠的健康和繁殖效率的细胞因子的基因人源化。具体来说，其中涉及的内容可以上述针对本申请所描述的全部内容。
在有些情况下，人和小鼠细胞因子序列相似功能相近或该鼠源细胞因子的缺失并不影响重度免疫缺陷小鼠的健康。因此这些小鼠细胞因子在人源化后，在有利于人免疫系统重建的同时又不会对小鼠已有免疫系统带来损害。例如IL-15可以促进T细胞和NK细胞的发育和成熟。但是重度免疫缺陷小鼠没有内源的T细胞和NK细胞。在这类小鼠人源化IL-15可以促进人T细胞和NK细胞的重建，但又不会影响重度免疫缺陷小鼠的健康。
本申请尝试利用优化的CRISPR/Cas9依赖的受精卵显微注射技术直接将NOD scid IL2RγKO小鼠的IL-15人源化。
本申请通过优化的显微注射方法直接对NOD scid IL2RγKO小鼠的受精卵进行基因修饰，得到原位人源化IL-15的重度免疫缺陷型小鼠。这样的人源化方法可以同样适用于改造被动免疫相关的细胞因子，如IL-6、IL-7、BAFF、HLA等。
本申请涉及一种线性DNA，其包括如SEQ ID No.3所示的人IL-15同源重组序列。
对重度免疫缺陷小鼠，特别是NOD scid IL2RγKO遗传背景的小鼠，进行基因修饰一直是世界性技术难题。主要是因为这种小鼠的受精卵成活率低，数量少。在本申请中，发明人利用了体外受精(IVF)的方法，提高了受精卵的胚胎成活率。此外，本申请的发明人保留了小鼠IL-15的前4个外显子和前3个内含子，在其后插入了合并的人第5到第8个外显子。这种设计不仅可以减少基因敲入的长度，还可以保留小鼠的表达调控元件，包括启动子和部分内含子，和小鼠的信号肽。因此IL-15基因人源化的NOD scid IL2RγKO小鼠血清表达水平为90pg/ml，近似于生理水平。本申请的方法也同样适用于在NOD scid IL2RγKO小鼠定点敲入其他人基因，如IL-7，IL-6，BAFF和HLA等被动免疫相关的细胞因子。
为了使在小鼠体内重建的人类免疫系统更加完善，需要在小鼠体内过表达人细胞因子或者将小鼠细胞因子人源化。本申请提供一种对非人动物，尤其是小鼠，尤其是以NOD为遗传背景的小鼠进行基因改造的方法，其包括：对非人动物细胞的受精卵进行基因改造，基因改造包括利用Piggy转座酶依赖的转基因系统对非人动物的受精卵进行基因编辑。
更具体来说，小鼠是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD、敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。
在本申请一个具体实施方式中，所述非人动物的受精卵是上述重度免疫缺陷NOD小鼠的受精卵，尤其是体外受精的受精卵。本申请的结果显示跟B6小鼠比较，NOD小鼠体内受精的成胎率差。而体外受精的受精卵比体内受精的受精卵活性强。所以对于NOD遗传背景的小鼠体外受精制备受精卵是首选。
在本申请一个具体方式中，进行基因改造的受精卵是对体外受精得到的，并且在体外培养一定时间的受精卵，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上得到的受精卵。研究发现，在体外培养一定时间可以等待细胞核的成熟，有助于对于小鼠的基因改造。
在本申请一个具体方式中，对非人动物细胞的受精卵进行基因改造是通过对受精卵的细胞核或细胞质进行注射来完成的。这猜测是由于NOD小鼠细胞核不成熟，对于染色体随机整合大片段DNA，很难进行要细胞核注射，几率也比较低。在本申请具体的实施方式中，对体外受精得到的，并且在体外培养一定时间的受精卵，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上得到的受精卵进行细胞核显微注射来完成的。
在本申请一个具体方式中，利用Piggy转座酶方法，需要进行基因编辑的基因片段为环形质粒，对非人动物的受精卵进行基因编辑是对非人动物细胞的受精卵的细胞质进行细胞浆或细胞核注射。在使用转座酶技术如，piggyBac可以以质粒的方式注入细胞质，这增加了成功率。
在本文中，原位人源化是指将小鼠基因或基因的一部分在同一基因位置替换成同源的人的基因或者人的基因一部分。
在具体的实施方式中，非人动物是以NOD为遗传背景的小鼠。具体来说，小鼠是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD、且敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。
在一个具体实施方式中，本申请的方法包括：在非人动物中过表达目标蛋白一和/或目标蛋白二；
其中，所述目标蛋白一主要在T细胞、B细胞和NK细胞中表达；所述目标蛋白一的基因原位人源化后，在重度免疫缺陷非人动物中检测不到所述目标蛋白一的基因的表达；
所述目标蛋白二可能会影响免疫缺陷非人动物的健康；
可能会影响免疫缺陷小鼠的健康的目标蛋白是指人和小鼠之间的同源性较低的目标蛋白，将这些目标蛋白人源化时，会影响免疫缺陷小鼠的健康。例如人和小鼠的同源性在50％以下的细胞因子，优选同源性在40％以下的细胞因子，进一步优选同源性在30％ 以下的细胞因子，进一步优选同源性在20％以下的细胞因子，进一步优选同源性在10％以下的细胞因子，进一步优选同源性在5％以下的细胞因子。
例如，GM-CSF对于保持巨噬细胞的成熟和发育至关重要。但是人和小鼠的GM-CSF同源性很低，没有交叉活性。当小鼠GM-CSF被人源化后，人GM-CSF并不能替代小鼠GM-CSF的功能。在人源化GM-CSF后，小鼠因巨噬细胞的活性降低，而产生肺炎。又如，血小板生成素(TPO)被人源化后，人源化的TPO并不能促进小鼠血小板的生长，因而会导致小鼠得贫血症。对于这类细胞因子需采取过表达策略。
在一个具体的实施方式中，所述目标蛋白一包括但不限于以下任意一种或两种：人白细胞介素-3(人IL3)、人白细胞介素-2(人IL2)；由于IL2和IL3主要在T细胞表达，免疫缺陷小鼠没有T细胞，因此人源化后，没有细胞因子表达。优选所述目标蛋白一为人白细胞介素-3(人IL3)；所述目标蛋白二包括但不限于以下任意一种或两种以上：人血小板生成素(人TPO)、人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)、人巨噬细胞集落刺激因子(人MCSF)；优选所述目标蛋白二为人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)。
在一个具体的实施方式中，在非人动物中过表达目标蛋白一和目标蛋白二。
在一个具体的实施方式中，在非人动物中过表达人白细胞介素-3(人IL3)和人粒细胞巨噬细胞集落刺激因子(人GM-CSF)。具体来说，对非人动物进行基因改造的方法是对非人动物的受精卵进行基因改造使得在非人动物中过表达目标蛋白是指在非人动物的髓系细胞中过表达目标蛋白，优选利用特异性在髓系细胞表达的启动子来在非人动物的髓系细胞中过表达目标蛋白。具体来说，在髓系细胞表达的启动子包括但不限于人CD68启动子、CSF1R启动子、CD11c启动子、CX3CR1启动子、Langerin/CD207启动子、MMLV LTR启动子、Visna virus LTR启动子、DC-STAMP启动子、Human MSR启动子、MSR-A启动子、CD4启动子、CD2启动子、Iba-AIF-1启动子、CD11b启动子、c-fms启动子、scavenger receptor A(SR-A)启动子、lysozyme启动子和MHC class II启动子(MHC-II)中等启动子。利用上述启动子在髓系细胞进行表达是因为通常观察到在免疫缺陷小鼠被打入人的造血干细胞之后，在人细胞因子的帮助下会在小鼠体内重建人原化的免疫系统，这时小鼠的髓系细胞会逐渐消失，这样过表达的外源表达基因也会降低或者消失，从而过表达的人的细胞因子不会因持续表达而过度激活已经完全人源化的免疫系统，从而保障小鼠的健康。
在一个具体实施方式中，进行基因改造的非人动物是以NOD为遗传背景的小鼠，所述小鼠是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠，且进一步敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。具体来说，非人动物的受精卵是通过体外受精获得的。对所述非人动物的受精卵进行基因改造之前对体外受精得到的受精卵在体外培养一定时间，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
在一个具体的实施方式中，利用人CD68启动子在非人动物的髓系细胞中过表达目标蛋白，即过表达人白细胞介素-3(人IL3)和人粒细胞巨噬细胞集落刺激因子(人GM-CSF)。
在一个具体的实施方式中，利用piggyBac转座子系统质粒和piggyBac转座酶来在非人动物的髓系细胞中过表达目标蛋白。
在一个具体的实施方式中，在非人动物中过表达两个以上目标蛋白时利用双重自我剪切短肽RAKR-GSG-P2A(RAKR-GSG-ATNFSLLKQAGDVEENPGP)、RAKR-GSG-T2A(RAKR-GSG-EGRGSLLTCGDVEENPGP)、RAKR-GSG-E2A(RAKR-GSG-QCTNYALLKLAGDVESNPGP)或RAKR-GSG-F2A(RAKR-GSG-VKQTLNFDLLKLAGDVESNPGP)介导不同目标蛋白的过表达，其中，RAKR是Furin剪切短肽的序列，GSG是linker。具体来说，双重自我剪切短肽为RAKR-GSG-P2A，编码双重自我剪切短肽RAKR-GSG-P2A的基因的序列如SEQ ID No.13所示。
在一个具体的实施方式中，利用piggyBac转座子系统质粒来在非人动物的髓系细胞中过表达目标蛋白，在piggyBac转座子系统质粒中还插入有bGH polyA序列，优选其序列如SEQ ID No.2所示。
在一个具体的实施方式中，在piggyBac转座子系统质粒中插入有反响重复序列，优选其序列如SEQ ID No.16或SEQ ID No.17所示。其中适合插入piggyBAC 5’端的ITR的如SEQ ID No.16所示，适合插入piggyBAC 3’端的ITR的如SEQ ID No.17所示。
在一个具体的实施方式中，对所述非人动物的受精卵进行基因改造是对受精卵的细胞质进行胞浆或细胞核注射，胞浆或细胞核注射的物质包括：PiggyBac转座子系统质粒和piggyBac转座酶。其中，PiggyBac转座子系统质粒包括SEQ ID No.11所示的序列。
在本申请中，还涉及一种构建重度免疫缺陷的动物模型的方法，该方法包括：在非人动物中过表达目标蛋白，所述目标蛋白为目标蛋白一和/或目标蛋白二；其中，所述目标蛋白一主要在T细胞、B细胞和NK细胞中表达；所述目标蛋白二可能会影响免疫缺陷小鼠的健康。具体来说，其中涉及的内容可以上述针对本申请所描述的全部内容。
本申请还涉及一种PiggyBac转座子系统质粒，其序列如SEQ ID No.11所示。
为了使在小鼠体内重建的人类免疫系统更加完善，需要在小鼠体内过表达人细胞因子或者将小鼠细胞因子人源化。
对于与维持小鼠自身健康相关且与人同源细胞因子缺乏交叉活性的细胞因子，在NOD scid IL2RγKO小鼠中保留其本身的鼠源IL-3和GM-CSF，同时探索如何适度表达人IL3和GM-CSF成为优化的选择。
本申请用PiggyBac技术，在NOD scid IL2RγKO小鼠巨噬细胞过表达单拷贝的人IL3和人GM-CSF。过表达的人IL3和人GM-CSF可以支持NOD scid IL2RγKO小鼠重建人髓系细胞，进而也可提高人T细胞和B细胞的生长成熟。另外，由于使用了组织特异性启动子，这些细胞因子会局限在小鼠髓系细胞表达。在小鼠免疫系统完全人源化的时候，小鼠髓系细胞会被人免疫细胞替换掉，所以小鼠体内过表达的外源人IL3和GM-CSF会 随着人免疫系统的成熟而逐渐减少。这时免疫系统人源化小鼠体内检测到的人IL3和人GM-CSF主要来自于人免疫系统的T细胞和巨噬细胞。这样的设计即提供了必要的人细胞因子支持人免疫系统在小鼠体内的成熟，又不会使过表达的外源人细胞因子过度激活成熟后的人免疫系统，从而影响小鼠的健康和以后的功能分析。类似的设计也可用于在小鼠体内过表达跟重建人天然免疫相关的细胞因子，如G-MCSF，TPO，M-CSF等。
本申请通过优化的显微注射方法直接对NOD scid IL2RγKO小鼠的受精卵进行基因修饰，得到过表达IL3/GMCSF双转基因的重度免疫缺陷型小鼠。
本申请利用PiggyBac转基因技术和优化的受精卵显微注射技术直接在NOD scid IL2RγKO小鼠过表达人IL3和人GM-CSF，节省了小鼠开发的时间。本申请利用了人CD68启动子将人细胞因子在小鼠的髓系细胞过表达。因为在重度免疫缺陷型小鼠重建人免疫系统的过程，小鼠内源的髓系细胞会逐渐被人免疫细胞替代，所以在髓系细胞表达的外源细胞因子随着人免疫系统重建水平的增高而降低。这种设计的优点即可加快人免疫系统的重建速度，也可以避免随后过度激活人免疫系统导致的小鼠寿命的降低。
过表达的人IL3和人GMCSF的NOD scid IL2RγKO小鼠，即NOD scid IL2RγKO Tg(hIL3/hGMCSF)小鼠，其优势除了可直接在重度免疫系统小鼠进行基因修饰外，还限制这些细胞因子在小鼠髓系细胞表达。因为小鼠髓系细胞在小鼠免疫系统人源化后会逐渐消失，过表达的人细胞因子也会随着小鼠髓系细胞的减少而降低，所以可以避免过表达人细胞因子给小鼠人源化的免疫系统带来的基因毒性。
经过优化的重度免疫缺陷小鼠在用于重建人免疫系统的过程中，其表达人细胞因子的内源髓系细胞会逐渐被外源的人免疫细胞替代，从而一方面加快人免疫系统的重建速度，另一方面避免随后过度激活人免疫系统导致的小鼠寿命的降低。
本申请使用的转基因方法与直接将小鼠自身同源基因人源化的方法相比较，优点在于本申请既可以保留小鼠同源基因的活性，也可以得到表达水平多样的转基因小鼠。外源基因不同的表达水平会赋予转基因小鼠不同的功能。为了同时表达多个细胞因子，本申请中还使用了Furin(RARK)-GSG-P2A双重自我剪切短肽介导人IL3和GM-CSF的共表达。除Furin-GSG-P2A以外，其他与Furin-GSG-P2A功能类似的自我剪切肽也可以达到本申请中类似的效果。本申请还利用了hCD68将细胞因子表达在小鼠的髓系细胞。在髓系细胞表达人细胞因子的优点在于可以减少基因毒性，减少过量外源细胞因子对人免疫系统的过度激活。本申请还有助于表达其他与维持小鼠自身健康相关且与人同源细胞因子缺乏交叉活性的细胞因子，例如：人TPO、M-CSF等。
实施例
实施例1 在NOD scid IL2RγKO小鼠中人源化细胞因子IL-15
为了加快转基因的NOD scid IL2RγKO小鼠的开发，省去回交过程，本申请的发明人尝试联合使用优化的受精卵体外受精技术和基因编辑工具依赖的同源重组技术对NOD scid IL2RγKO小鼠的受精卵进行基因编辑。本实施例的NOD scid IL2RγKO小鼠为 购自上海南方模型的M-NSG小鼠，其为应用CRISPR/Cas9技术，敲除NODscid小鼠的IL2Rγ基因，得到的重度免疫缺陷小鼠。
以人源化小鼠IL-15基因为例，人IL-15有8个外显子，长度达96kb。在本实施例中，发明人使用了CRISPR/Cas9依赖的同源重组技术进行小鼠IL-15基因的人源化。为克服CRISPR/Cas9依赖的同源重组技术对基因长度依赖的局限性，在基因设计上，发明人在小鼠IL-15第四个外显子处定点插入兼并的人IL-15第五到第八个外显子，见图1。新生成的基因不仅去除了鼠源IL-15的干扰，还利用了小鼠IL-15的全部启动子、部分内含子调控序列和鼠源IL-15的分泌信号肽，这种基因设计很大程度上实现了细胞因子生理性水平表达，又缩小了敲入基因的长度提高了基因敲入的成功率。为了优化NOD scid IL2RγKO小鼠的基因编辑流程，发明人以C57BL小鼠的受精卵作为对照。具体步骤如下。
1.人源化IL-15同源重组质粒的构建
根据小鼠IL-15-MGP_NODShiLtJ_T0089152.1转录本的信息进行分析：该转录本内含子、外显子信息均被NCBI收录，是唯一的转录本。该转录本结构内有8个外显子(exons)，蛋白翻译起始于外显子3，终止于外显子8。小鼠IL-15-MGP_NODShiLtJ_T0089152.1转录本信息见以下链接：http://useast.ensembl.org/Mus_musculus_NOD_ShiLtJ/Transcript/Exons？db＝core；g＝MGP_N ODShiLtJ_G0033628；r＝8:91916329-91999766；t＝MGP_NODShiLtJ_T0089 152。
人IL-15-202转录本(IL-15-202)同样含有8个exons，蛋白翻译起始于外显子3，终止于外显子8。人IL-15-202转录本的信息见以下链接：http://useast.ensembl.org/Homo_sapiens/Transcript/Exons？db＝core；g＝ENSG00000164136；r＝4:141636583-141733987；t＝ENST00000320650。
根据上述信息，在小鼠IL-15-MGP_NODShiLtJ_T0089152.1转录本第四个外显子3’端插入兼并的人IL-15-202转录本的第五到第八个外显子的编码序列(SEQ ID No.1)和pGHpolyA(SEQ ID No.2)。人源化后的IL-15核酸序列及蛋白序列与小鼠同源序列比较结果如图2A，2B所示。小鼠IL-15基因座第四个外显子末端上游约500bp的基因组DNA为5’同源臂，下游约500bp的基因组DNA为3’同源臂。按照本领域技术人员所熟知的分子生物学克隆方法，将上述同源臂序列和人IL-15部分外显子(合称为同源重组序列)重组到pBR322载体(sigma，cata:10481238001)(重组完成后称人IL-15同源重组质粒)。人IL-15同源重组质粒见图3。同源重组序列如SEQ ID No.3所示。
2.针对鼠IL-15基因外显子4的CRISPR gRNA序列设计
使用crispr.mit.edu来设计指导RNA(gRNA)，所述gRNA的靶向位点位于小鼠IL-15转录本第四个外显子。所述gRNA的靶向位点的序列如下：
Figure PCTCN2021106058-appb-000003
Figure PCTCN2021106058-appb-000004
纯化的Cas9mRNA购自Origene，商品编号为GE100054。
3.小鼠受精卵的制备和显微注射
NOD scid IL2RγKO小鼠(实验组)和C57BL小鼠(对照组)作为精子及卵子的供体。CD1小鼠为假孕母鼠。NOD scid IL2RγKO小鼠源自NOD scid小鼠，利用CRISPR/Cas9技术敲除了IL2Rγ基因第四到第八个外显子。以上小鼠购自上海模式生物公司。
3.1受精
自然受精(nature mating)制备受精卵:选择雌鼠注射孕马血清促性腺激素(PMSG)(Sigma Chemical Inc.，cat.no.G4877-2000IU)，46h～48h后注射人绒毛膜促性腺激素(HCG)(Sigma Chemical Inc.，cat.no.CG10-1VL)，注射后即与正常雄鼠按照1:1合笼。第2天，断颈处死成栓受精卵供体鼠，手术取出整个输卵管，放入透明质酸酶～0.3mg/M2液中。显微镜下，仔细观察放在透明质酸酶M2液(sigma chemical，cata:M 7167)中的受精卵，当受精卵周围的颗粒细胞脱离时，将受精卵吸出，放入M2液中洗涤，最后放在M16液(Sigma Chemical，cat.no.M7292)中放入5％CO 2，37℃培养箱培养。在显微镜下观察，挑选细胞饱满，透明带清晰，细胞核清晰可见的受精卵待用。
体外受精(IVF)制备受精卵:按照常规操作促排卵，选择适龄雌鼠(6周)注射孕马血清促性腺激素(PMSG)，46h～48h后注射HCG，第二天检验阴道成栓。收集精子：将符合要求的性成熟(8周以上)的雄鼠颈椎脱臼处死，取出附睾上体尾部，去除脂肪与血迹后，置于HTF液(Millipore，cata:MR-070-D)内。利用眼科剪在液滴内横断附睾尾，当精子充分游离出附睾尾后，将组织从液滴内取出，将培养皿置于培养箱。处死超排后的雌鼠，取出两侧的输卵管，用显微镊将卵母细胞团拨到受精滴中。用10μl移液器从精子滴吸取精子，加到卵子滴中，根据卵团大小情况以及精子的活力每滴加8μl左右的精子。将精卵混合后，最后放在M16液中放入5％CO 2，37℃培养箱培养。
3.2受精卵活性测定
制备假孕鼠:将正常雌鼠与结扎雄鼠合笼(3:1)，第2天见栓的雌鼠为假孕鼠。每只假孕鼠的输卵管被植入20个左右的受精卵。3周后，小鼠出生。以出生小鼠的数量和植入受精卵的比例衡量受精卵的活性。
3.3显微注射
在受精卵在M16培养一段时间后开始显微注射。在组织培养载玻片中央滴一滴M2培养液，用矿物油将液滴覆盖。用持卵针吸住受精卵，每次将含有线性化的同源重组质粒DNA(100ng)，上述gRNA(50ng each)，mRNACas9(100ng)注射针刺入受精卵的细胞核(Pronucleus)或者细胞质(Cytosol)中。注射使其细胞膨胀，退出注射针，再吸取下一枚受精卵注射。
4.胚胎移植
注射受精卵同一天，用移卵针吸取约20枚受精卵植入见栓假孕雌鼠的输卵管壶腹部。然后将卵巢和输卵管送回假孕鼠体内，缝合伤口。
5.转基因小鼠基因型和表型的测定
5.1小鼠基因型分析
手术后19～21天仔鼠分娩，待仔鼠3周后，剪耳，编号，剪尾。通过PCR扩增及测序对其进行基因型鉴定。设计PCR引物序列如下。
5’端同源臂重组克隆鉴定引物信息：
引物 序列5'-->3' 引物类型
I TCTGATGGGAACTGGATGA(SEQ ID No.7) 正向
II AGCCTGACTTACTGTTGTG(SEQ ID No.8) 反向
3’端同源臂重组克隆鉴定引物信息：
引物 序列5'-->3' 引物类型
III TGCAGCCCCATTCATCATC(SEQ ID No.9) 正向
IV GGCAGCTCCATTTTCCATCAT(SEQ ID No.10) 反向
按照经典分子生物学方法利用PCR鉴定子代小鼠的基因组DNA。PCR阳性克隆鉴定结果显示，5’臂同源重组阳性基因组应扩增出4.1kb片段，阴性基因组应扩增出6.9kb片段；3’臂同源重组阳性基因组应扩增出3.6kb片段，阴性基因组应扩增出6.9kb片段。测序结果与PCR结果一致。具体结果如表1中F1代所示。
由于受精卵早期卵裂速度很快，因此得到的F0代小鼠为嵌合体，不一定具备稳定遗传的能力，需要进行传代以获得可稳定遗传的F1代小鼠。按照如上方法，从5个F0小鼠的子代中鉴定了3个阳性的F1代小鼠。结果见表1和表2。
表1.F1代阳性小鼠编号和基本信息
Figure PCTCN2021106058-appb-000005
Figure PCTCN2021106058-appb-000006
表1中的He表示杂合子
表2 CRISPR/Cas9介导的在NOD-SCID IL2RγKO小鼠进行基因定点敲入的优化
Figure PCTCN2021106058-appb-000007
根据以上结果可知，使用体外受精、胞质内注射重组、及注射前培养等优化条件，NOD-SCID IL2RγKO重度免疫缺陷小鼠的成胎率及重组成功率均得到大幅提升，达到了与对照组C57BL小鼠相当的水平。
表型测定(人IL-15在转基因小鼠表达量)
从小鼠眼眶静脉取抗凝血，离心制备血清。使用Human IL-15 Quantikine ELISA Kit(R&D systems，Catalog#D1500)检测F1代小鼠血清中人IL-15细胞因子的表达水平。非转基因NOD scid IL2RγKO小鼠的血清作为阴性对照。Elisa结果见图4。结果显示本申请所述方法可使NOD scid IL2RγKO小鼠稳定表达人源化IL-15，并且其血清表达水平为90pg/ml，非转基因NOD scidIL2RγKO小鼠体内，人IL-15的平均血浆表达浓度为0。将上述成功构建的可稳定表达人源化IL-15的NOD scid IL2RγKO小鼠命名为NVG-hIL15小鼠非转基因NOD scid IL2RγKO小鼠命名为NVG小鼠。
对重度免疫缺陷小鼠，特别是NOD scid IL2RγKO遗传背景的小鼠，进行基因修饰一直是世界性技术难题。主要是因为这种小鼠的受精卵成活率低，数量少。如表2示自 然受精的受精卵胚胎成活率只有9.8％，远低于C57BL小鼠的48.9％。所以大多数转基因的NOD scid IL2RγKO小鼠是通经转基因的C57BL小鼠与NOD scid IL2RγKO小鼠连续回交得到的。因此育种周期长。在本申请中，发明人利用了体外受精(IVF)的方法，提高了受精卵的胚胎成活率。如表2所示，对体外受精后受精卵的细胞核进行显微注射后，也很难得到定点敲入IL-15基因的转基因小鼠。这有可能是由于NOD scid IL2RγKO小鼠的受精卵细胞核发育迟缓。在对细胞核进行经典的显微注射后，受精卵的生殖活性容易受到影响。利用最新的基因编辑技术，如CRISPR技术，也可以实现在受精卵水平敲除小鼠内源基因。但是，未见报道利用CRISPR技术将大片段的外源DNA，如人IL-15，定点敲入NOD scid IL2RγKO的受精卵并得到可遗传的转基因小鼠的案例。这主要区别在于利用CRISPR技术敲除基因的时候，只需要向受精卵的细胞质导入gRNA和Cas9mRNA。这些小分子很容易进入细胞核。而定点敲入大片段DNA的时候，需要提供大片段的DNA作为同源重组修复的模板。而大片段很难从细胞质进入细胞核。从实验对比中还发现，体外受精后6小时对细胞核进行显微注射的时候无阳性重组小鼠出生。而经过延长受精卵培养时间，体外受精16小时以后进行受精卵细胞核显微注射，则有5个阳性F0代小鼠出生。其中3个F0代小鼠得到阳性子代。以上结果显示NOD scid IL2RγKO小鼠受精卵的细胞核似乎发育比较慢，延长细胞培养时间可以促进受精卵细胞核在显微注射后的成活率。通过不同实验条件的比对，本申请优化了对NOD scid IL2RγKO小鼠受精卵进行基因修饰，例如对CRISPR介导的基因定点插入的方法。该实验方法还适用于其他基因编辑方法，例如ZFNs，TALENs等技术同样适用于对NOD scid IL2RγKO小鼠受精卵的基因定点整合。
虽然利用基因编辑的方法进行受精卵基因定点敲入比较高效和简洁。但是插入片段大小会受限。如果基因超过2KB，定点插入效率会大大降低。例如本实施例中的人IL-15基因，其完整的基因座有95kb。因此在基因设计的时候，本申请的发明人保留了小鼠IL-15的前4个外显子和前3个内含子，在其后插入了合并的人第5到第8个外显子。这种设计不仅可以减少基因敲入的长度，还可以保留小鼠的表达调控元件，包括启动子和部分内含子，和小鼠的信号肽。因此IL-15基因人源化的NOD scid IL2RγKO小鼠血清表达水平为18pg/ml，近似于生理水平。本申请的方法也同样适用于在NOD scid IL2RγKO小鼠定点敲入其他人基因，如IL-7，IL-6，BAFF和HLA等被动免疫相关的细胞因子。
综上所述，本申请的优点是可直接对NOD scid IL2RγKO小鼠受精卵进行基因改造，得到表达人源化细胞因子的NOD scid IL2RγKO小鼠，避免多代回交，从而节省时间和人力成本。另外，本申请保留了鼠源同源基因的部分片段，与截短的人源基因重组，获得人源化基因。例如人源化IL-15，利用了包含小鼠本身IL-15基因UTR在内的部分，减少了定点插入片段的大小，提高了同源重组的效率。同时，这一设计中，小鼠本身的IL-15由人源化的IL-15替代，避免了现有技术中常常出现的人与小鼠IL-15共同存在且互相干扰的情况，减少了对人NK细胞等人免疫细胞功能的影响。此外，由于人源化IL-15使用的是小鼠本身的启动子，因此避免了现有技术中经常出现的人IL-15过表达的情况， 从而更大程度地避免了人IL-15对小鼠本身发育的影响。人源化IL-15后，NOD scid IL2RγKO小鼠将支持人的NK细胞的生长和成熟，可以用于检测CAR-NK的活性和抗体的ADCC效应。
实施例2 应用实施例1构建的人源化IL-15免疫缺陷小鼠(NVG-hIL15)评估NK细胞相关的免疫疗法
由于NK细胞拥有的天然杀伤肿瘤的细胞毒性，所以以嵌合抗原受体NK细胞治(CAR-NK)和抗体依赖细胞介导的细胞毒性作用(ADCC)为代表的免疫治疗策略逐步引起了人们的开发兴趣。携带人类免疫系统的免疫缺陷小鼠已经被广泛用于评估新型人体免疫疗法。但是这些模型缺乏功能性自然杀伤细胞(NK细胞)限制了其在这方面的应用。因此，开发基于NK细胞介导的免疫疗法迫切需要一种改进型临床前动物模型用于临床转化研究。
1.NVG小鼠和实施例1构建的NVG-hIL15小鼠
NVG小鼠为购自上海南方模型的M-NSG小鼠，其为应用CRISPR/Cas9技术，敲除NODscid小鼠的IL2Rγ基因，得到的NOD scid IL2RγKO小鼠，即重度免疫缺陷小鼠，我们将其命名为NVG小鼠，作为对照组。
NVG-IL15小鼠及其它转基因小鼠皆是在NVG小鼠这个品系上的基因修饰。由于人IL-15是天然杀伤细胞(NK细胞)体内稳态和成熟的关键细胞因子，如图1所示，我们通过人源化NVG小鼠的IL-15，得到表达人IL-15的NVG-hIL15小鼠，作为实验组。
通过ELISA方法测定NVG小鼠和NVG-hIL15小鼠体内人IL-15的平均血浆表达浓度，测定结果如图5所示，在NVG-hIL15小鼠体内，人IL-15的平均血浆表达浓度为90pg/ml，在NVG小鼠体内，人IL-15的平均血浆表达浓度为0。
2.比较人PBMC在NVG小鼠和NVG-hIL15小鼠的移植效率和体内对K562-luc细胞的生长抑制
分别向NVG小鼠和NVG-hIL15小鼠中注射500万个人PBMC，检测人PBMC中各细胞在NVG小鼠和NVG-hIL15小鼠血液中的频率，测定结果如图5(A)～(D)所示，在注射人PBMC后，人CD45细胞、人NK细胞、表达CD16的细胞毒性的人NK细胞在NVG-hIL15小鼠血液中的频率显著高于其在对照组NVG小鼠血液中的频率。因高表达CD16的人NK细胞具有抗体依赖性细胞毒性(ADCC)的功能，这使人PBMC移植的NVG-hIL15小鼠有望应用于ADCC相关的免疫治疗的转化研究。
由于高度人源化的免疫系统可能导致NVG-hIL15小鼠在6周后表现移植物抗宿主病(GVHD)，如图6所示，NVG-hIL15小鼠在注射500万个人的PBMC约6周后存活率骤降，约8周后其存活率为0，NVG小鼠在注射人PBMC约10周后存活率开始逐渐降低(因为有了区别，10周以后没有再检测)。
向NVG小鼠和NVG-hIL15小鼠中注射人PBMC后，再注射人白血病荧光素酶标记细胞K562-Luc，高度人源化的免疫系统也使得NVG-hIL15小鼠展现抑制K562-Luc细胞 生长的特性，如图7所示，注射K562-Luc 6周后，与NVG小鼠相比，NVG-hIL15小鼠体内K562-Luc细胞的生长受到明显的抑制。
3.外周血来源-NK细胞在人源化小鼠模型中的增殖和细胞毒性
如图8所示，向NVG小鼠和NVG-hIL15小鼠体内分别注射10 7个来自健康供体的人外周血分离的NK细胞，3天后再注射5×10 6个K562-Luc，最后利用IVIS活体成像系统检测肿瘤细胞溶解情况。
注射来自健康供体的人外周血分离的NK细胞后，测定体内NK细胞的百分比，结果如图9所示，来自健康供体的人外周血分离的NK细胞可以在NVG-IL15小鼠持续生长6周，而其在NVG小鼠中只能维持2周生长。
利用IVIS活体成像系统进行的体内荷瘤试验结果如图10所示，人NK细胞可明显抑制K562-Luc细胞在NVG-IL15小鼠体内的生长。以上结果表明，NVG-hIL15小鼠也可以用于NK细胞相关免疫治疗的转化研究。
4.CAR-NK92细胞在小鼠体内对肿瘤细胞的杀伤毒性
如图11所示，用环磷酰胺(CTX)分别预处理NVG小鼠和NVG-hIL15小鼠，然后原位注射MIA PaCa-2(一种人类胰腺癌细胞系)，注射量为100万个细胞，注射MIA PaCa-2两周后，再每周注射10 7个经辐照过的CAR-NK92细胞直到对照组小鼠死亡，最后利用IVIS活体成像系统检测肿瘤细胞溶解情况。
利用IVIS活体成像系统进行的体内荷瘤试验结果如图12所示，表明经辐照过的CAR-NK92细胞可以明显抑制在NVG-hIL15小鼠体内原位移植的MIA PaCa-2的生长。以上结果表明，NVG-hIL15小鼠在研究基于NK疗法方面具有明显的优越性。
每周注射10 7个经辐照过的CAR-NK92细胞系后，再向小鼠注射荧光素酶底物荧光素(luciferin)，用高灵敏CCD相机检测NVG小鼠和NVG-hIL15小鼠体内荧光素酶含量变化情况，结果如图13所示，NVG-hIL15小鼠体内的荧光素酶含量骤降，而NVG小鼠体内的荧光素酶含量变化不大。结果显示在NVG-hIL15小鼠，CAR-NK92细胞可以杀死活肿瘤MIA PaCa-2细胞。
实施例3 在NOD scid IL2RγKO小鼠表达外源基因：人IL3和人GM-CSF
1.PiggyBac重组质粒(Piggy hCD68-GMCSF/IL3)的构建
本申请利用Piggy转座酶依赖的转基因系统在小鼠过表达人GM-CSF和人IL3。因PiggyBac转座酶倾向于将目的片段插入到转录活跃的区域，所以将大大提高获得阳性表达的转基因小鼠的几率。
为使基因过表达仅发生于在小鼠的单核细胞等髓系细胞，载体使用了3.1kb的人的CD68的启动子。为了同时表达不同的靶标基因，载体利用双重自我剪切短肽RAKR-GSG-P2A，连接人IL3和人GM-CSF基因。
在表达原件hCD68Pro-Intron-hGM-CSF-PAKR-GSG-P2A-hIL3-pA的两侧设计了Piggy转座酶依赖的反响向重复序列(ITR)。利用基因合成和经典分子克隆技术将含有ITR序列的表达元件克隆到pBR322载体(sigma，cata:10481238001)。重组质粒设计原理见图14。重组插入序列见SEQ ID No.11所示。为了显示在重建人免疫系统的过程中，本申请与现有技术的优势，发明人将前述表达原件中的人CD68启动子替换为通用的SV40启动子(SEQ ID No.12)构建了作为对照的piggyBac载体(Piggy SV40-GMCSF/IL3)，其它重组元件不变。
2.小鼠受精卵的制备和显微注射
NOD scid IL2RγKO小鼠(实验组)和C57BL小鼠(对照组)作为精子及卵子的供体。CD1小鼠为假孕母鼠。NOD scid IL2RγKO小鼠源自NOD scid小鼠为购自上海南方模型的M-NSG小鼠，其为利用CRISPR/Cas9技术敲除了IL2Rγ基因第四到第八个外显子得到的重度免疫缺陷小鼠，将其命名为NVG小鼠。
3.受精
自然受精(nature mating)制备受精卵:选择雌鼠注射PMSG，46h～48h后注射人绒毛膜促性腺激素HCG(Sigma Chemical Inc.，cat.no.CG10-1VL)，注射后即与正常雄鼠按照1:1合笼。第2天，断颈处死成栓受精卵供体鼠，手术取出整个输卵管，放入透明质酸酶～0.3mg/M2液(sigma chemical，cata:M 7167)中。显微镜下，仔细观察放在透明质酸酶M2液中的受精卵，当受精卵周围的颗粒细胞脱离时，将受精卵吸出，放入M2液中洗涤，最后放在M16液(Sigma Chemical，cat.no.M7292)中放入5％CO 2，37℃培养箱培养。在显微镜下观察，挑选细胞饱满，透明带清晰，细胞核清晰可见的受精卵待用。
体外受精(IVF)制备受精卵:按照常规操作促排卵，选择适龄雌鼠(6周)注射孕马血清促性腺激素(PMSG(Sigma Chemical Inc.，cat.no.G4877-2000IU))，46h～48h后注射HCG，第二天检验阴道成栓。收集精子：将符合要求的性成熟(8周以上)的雄鼠颈椎脱臼处死，取出附睾上体尾部，去除脂肪与血迹后，置于HTF液(Millipore，cata:MR-070-D)内。利用眼科剪在液滴内横断附睾尾，当精子充分游离出附睾尾后，将组织从液滴内取出，将培养皿置于培养箱。处死超排后的雌鼠，取出两侧的输卵管，用显微镊将卵母细胞团拨到受精滴中。用10μl移液器从精子滴吸取精子，加到卵子滴中，根据卵团大小情况以及精子的活力每滴加8μl左右的精子。将精卵混合后，最后放在M16液中放入5％CO 2，37℃培养箱培养。
4.PiggyBac转基因操作
将NOD scid IL2RγKO小鼠作为精子和卵子的供体。CD1小鼠为假孕母鼠。以上小鼠均购自上海南方模式生物公司。使用显微注射将携带有外源基因的piggyBac转座子系统质粒(Piggy hCD68-GMCSF/IL3或Piggy SV40-GMCSF/IL3)与piggyBac转座酶 mRNA(上海南方模式)共同通过胞浆注射法注入体外受精卵或体内受精卵，注射时机为体外受精完成后16～18h。注射时，piggyBac转座子系统质粒的浓度为50ng/μL，piggyBac转座酶mRNA(上海南方模式)50ng/μl，注射体积为15pL。当针穿透卵质膜后，控制注射针将DNA注入胞浆内。注射受精卵同一天，用移卵针吸取约20枚受精卵植入见栓假孕雌鼠的输卵管壶腹部。然后将卵巢和输卵管送回假孕鼠体内，缝合伤口。
5.F0代转基因小鼠基因型和表型的测定
5.1 PCR基因型鉴定
注射后20天左右出生的小鼠为首建鼠(F0代小鼠)。通过PCR方法对其进行基因型鉴定。设计引物进行PCR后，鉴定到阳性小鼠。引物序列如表3所示：
表3
引物 序列5'-->3' 引物类型
V GCGGGGCAGCCTCACCAA(SEQ ID No.22) 正向
VI AATTCATTCCAGTCACCGTCCTT(SEQ ID No.23) 反向
按照经典分子生物学的方法鉴定阳性PCR条带，大小为639bp。PCR鉴定电泳结果如图15所示。其中3、4、5、6、10为F0代NOD scid IL2RγKO-Tg(hCD68-hIL3/GMCSF)小鼠，12、13、14为F0代NOD scid IL2RγKO-Tg(SV40-hIL3/GMCSF)小鼠。
5.2 F0代转基因鼠基因组插入位点的鉴定
为了鉴定外源基因在小鼠基因组的插入位置，采用反向PCR鉴定插入基因两侧的基因序列。反向PCR参见经典分子生物学方法和试剂盒说明书。以Zymo Research的DNA提取试剂盒(Catalog#D3024)提取小鼠尾巴的基因组DNA。随后，基因组被DpnII限制性内切酶(NEB Biolabs，Catalog#R0543S)酶切3hr后，80℃20分钟灭活内切酶。酶切产物用T4连接酶(Enzymatics，Catalog#L6030-LC-L)进行环化链接1个小时，然后放入-20℃冰箱终止反应。建立嵌套PCR反应，扩增插入基因两侧的基因组DNA。DNA聚合酶选用Phusion DNA Polymerase(NEB Biolabs，Catalog#M0530S)。扩增引物如表4所示：
表4
引物 序列5'-->3' 引物类型
VII GCTCTATGGCTTCTGTTTGT(SEQ ID No.24) 正向_1 st
VIII GATAAAACACATGCGTCAATTT(SEQ ID No.25) 正向_2 nd
IX CCAATCCTCCCCCTTGCTGTCC(SEQ ID No.26) 反向_1 st
X AAACAACAGATGGCTGGCAACTA(SEQ ID No.27) 反向_2 nd
XI GTAAAACGACGGCCAG(SEQ ID No.28) M13正向
其中引物VII和IX用于扩增第一轮的连接产物，引物VIII和引物X用于扩增第一轮的PCR产物。反向PCR原理(图16)和第二次PCR扩展电泳结果(图17)如图所示。第二轮PCR产物克隆到pUC18质粒，用M13正向通用引物(XI)测序克隆产物。测序结果在NCBI网站进行序列比对。经过序列比对，各转基因鼠插入位置如表5所示。结果显示，使用piggyBAC转基因方法，多个小鼠存在多个转基因插入位点，且位于已知基因的内部。但是有些小鼠的插入位点为功能未知区域。
表5 F0代piggyBac转基因小鼠基因组插入位置分析
Figure PCTCN2021106058-appb-000008
5.3转基因鼠细胞因子表达水平的测定
由于部分F0代小鼠有多个插入位点，因此利用PCR引物筛选出插入位点不在已知功能基因内部的转基因小鼠。侧交F0代小鼠，在F2代筛选获得下列小鼠：SV40-12-6，SV40-14-X，CD68-6-13，CD68-10-12。检测血清中人细胞因子的表达水平。从各筛选出的小鼠眼眶静脉取抗凝血，离心制备血清。分别使用Human IL-3 Quantikine ELISA Kit(R&D systems，Catalog#D3000)和Human GM-CSF Quantikine ELISA Kit(R&D systems，Catalog#DGM00)检测F2代小鼠血清细胞因子表达量。检测结果见表6。实验 证明，不同的转基因小鼠系，插入位点不同，细胞因子表达量不同。将获得的CD68-10-12小鼠命名为NVG-hCD68-10-12小鼠，将获得的SV40-14-X小鼠命名为NVG-SV40-14-X小鼠。
表6 人IL3和人GM-CSF在小鼠血清的表达水平
F2代小鼠 基因组插入位置(GRCm38) hIL3(pg/ml) hGM-CSF(pg/ml)
WT小鼠 N/A 1.5±0.6 1.5±0.6
SV40-12-6 chr6:51，053，900-51，054，904 312±74 533±91
SV40-14-X ChrX:126586507-126585891 112±34 212±42
CD68-6-13 Chr13:51083363-51083460 35±7 29±4
CD68-10-12 chr6:51，053，900-51，054，904 195±26 245±42
5.4人细胞因子组织特异性表达鉴定
转基因小鼠分别选用人CD68启动子和SV40启动子表达人IL3和人GM-CSF细胞因子。为了鉴定这些启动子驱动的细胞因子表达的组织特异性，用Real-time RT-PCR分别检测人IL3和内源GAPDH在不同组织(骨髓，肌肉，肺，肾脏和脾脏)的mRNA的丰度。具体方案如下：以Trizol(Thermofisher，Catalog#15596026)提取小鼠骨髓，肌肉，肺，肾脏和脾脏的RNA。以PrimeScript RT Master Mix(Takara，Catalog#RR036A)反转录mRNA成cDNA。用TB
Figure PCTCN2021106058-appb-000009
qPCR Premix(Takara，Catalog#639676)对cDNA进行Realtime PCR反应。人IL3cDNA的扩增引物选自Sinobiological(catalog# HP104624)，鼠源GAPDH cDNA的扩增引物购自Sinobiological(catalog#MP200537)。人IL3mRNA相对于鼠GAPDH mRNA的相对量用2 -ΔΔCt公式计算。从结果显示，NOD scid IL2RγKO tg(SV40-IL3/GM-CSF)(表示以SV40启动子表达人IL3和人GMCSF)小鼠在多种组织表达高丰度的IL-3mRNA，而NOD scid IL2RγKO tg(hCD68-IL3/GM-CSF)(表示以人CD68启动子表达人IL3和人GMCSF)只是在髓系细胞富集的组织，如肺，脾脏，骨髓，表达高丰度的IL3mRNA，而在肌肉和肾脏的表达量很少(如图18所示)。
实施例4
分别向NVG小鼠(对照组)、对实施例3获得的NVG-hCD68-10-12小鼠(实验组1)和NVG-SV40-14-X小鼠(实验组2)进行全身γ照射(175cGy)，以降低鼠源免疫系统的活性，照射24小时后注射10万个个人CD34干细胞，12周后，检测人免疫系统在各对照组和实验组小鼠体内的表型，测定结果如图19(A)～(D)所示，在注射人CD34干细胞后，人淋巴细胞、人CD3T细胞、人CD33髓系来源细胞在实验组NVG-hCD68-10-12小鼠和NVG-SV40-14-X小鼠血液中的频率显著高于其在对照组NVG小鼠血液中的频率。
如图20所示，NVG-SV40-14-X小鼠在注射CD34干细胞120天后存活率骤降，约160天后存活率仅为10％；NVG-hCD68-10-12小鼠在130天左右存活率降低至80％且没有继续降低；NVG小鼠的存活率没有变化。
注射人CD34干细胞第12周后，通过ELISA方法测定对照组和实验组小鼠血浆中hGMCSF和hIL3的平均表达水平，测定结果如图21所示，NVG小鼠体内，hGMCSF平均血浆表达浓度为0，hIL3平均血浆表达浓度约为0；NVG-SV40-14-X小鼠体内，hGMCSF平均血浆表达浓度约为200pg/ml，hIL3平均血浆表达浓度约为150pg/ml；NVG-hCD68-10-12小鼠体内，hGMCSF平均血浆表达浓度约为25pg/ml，hIL3平均血浆表达浓度约为25pg/ml。
注射CD34干细胞8周后和16周后，分别测定对照组和实验组小鼠体内的血红蛋白数，结果如图22所示，注射16周后，NVG-SV40-14-X小鼠小鼠血细胞数目的减少最为明显，其次为NVG-hCD68-10-12小鼠，最后为NVG小鼠。
以上各实验结果表明，持续过表达的人细胞因子也会过度激活人源化的免疫系统，如NVG-SV40-14-X小鼠使用SV40启动子持续过表达GM-CSF和IL3，有可能会过度激活人的巨噬细胞，从而引起小鼠血细胞数目的减少，小鼠的生存率受到影响。使用hCD68启动子，可以局限人细胞因子在小鼠淋巴细胞(mCD45)表达。在小鼠免疫系统人源化后，小鼠的淋巴细胞得到了减少，因此，过表达的人细胞因子浓度也大为减少。从而避免人细胞因子过度激活人免疫系统。
本申请使用的是piggyBac技术在NOD scid IL2RγKO表达人IL3和人GM-CSF。由于实验采用了优化的体外受精和显微注射条件，因此其它具有类似性质的转基因技术，例如Tol2、Sleep Beauty等转座酶依赖的转基因技术，也可在NOD scid IL2RγKO遗传背景的小鼠中使用，以达到与本实施例中相似的发明效果。然而，跟其他转基因技术比较，piggyBac转基因技术的优点是只需要向细胞浆注射环状质粒，而不需要向细胞核注射线性DNA，从而提高了受精卵的生殖活性。piggyBac转基因技术还可将大片段外源基因以单拷贝的形式整合到高转录活性区域，保证了外源基因的表达率。因此本申请优选地使用piggyBac转基因技术。
本申请使用的转基因方法与直接将小鼠自身同源基因人源化的方法相比较，优点在于本申请既可以保留小鼠同源基因的活性，也可以得到表达水平多样的转基因小鼠。外源基因不同的表达水平会赋予转基因小鼠不同的功能。为了同时表达多个细胞因子，本实施例中还使用了Furin(RARK)-GSG-P2A双重自我剪切短肽介导人IL3和人GM-CSF的共表达。除Furin-GSG-P2A以外，其他与Furin-GSG-P2A功能类似的自我剪切肽也可以达到本申请中类似的效果。本实施例还利用了hCD68将细胞因子表达在小鼠的髓系细胞。在髓系细胞表达人细胞因子的优点在于可以减少基因毒性，减少过量外源细胞因子对人免疫系统的过度激活。本申请还有助于表达其他与维持小鼠自身健康相关且与人同源细胞因子缺乏交叉活性的细胞因子，例如：人TPO，M-CSF等。
工业实用性
利用本申请所述方法可以更高效便捷地构建表达人细胞因子的重度免疫缺陷小鼠，节省人力物力成本。并且，利用本申请所述方法构建的小鼠更加利于人类免疫系统及其组成部分的定植和发挥功能，为免疫学相关动物试验提供更加优质的实验材料。例如，用于检测人类造血干细胞及基于人类造血干细胞的治疗细胞的分化和定植功能；模拟人类免疫系统及肿瘤免疫微环境，在抗体药物、细胞免疫药物等的开发，传染性疾病、自身免疫病以及肿瘤学研究等多个领域发挥重要作用。另外，本工作得到表达人源化IL15的重度免疫缺陷型小鼠(NVGhIL5，可以用于NK细胞相关免疫疗法(ADCC,CAR-NK等)的评估。
序列表
Figure PCTCN2021106058-appb-000010
Figure PCTCN2021106058-appb-000011
Figure PCTCN2021106058-appb-000012
Figure PCTCN2021106058-appb-000013
Figure PCTCN2021106058-appb-000014

Claims (35)

  1. 一种对非人动物，进行基因改造的方法，其包括：
    对非人动物细胞的受精卵进行基因改造，
    基因改造包括利用CRISPR/Cas9对非人动物的受精卵进行基因编辑；
    优选地，所述受精卵是体外受精的受精卵；
    进一步优选地，所述受精卵是通过体外受精得到的，并且在体外培养一定时间的受精卵，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
  2. 根据权利要求1所述的方法，其中，对非人动物细胞的受精卵进行基因改造是通过对受精卵的细胞核或细胞质进行注射来完成的；
    优选地，利用CRISPR/Cas9方法，需要注射用于同源重组的线性DNA模板大于等于2.5kb，对非人动物的受精卵进行基因编辑是对非人动物细胞的受精卵的细胞核进行显微注射。
  3. 根据权利要求1或2所述的方法，其中，
    所述非人动物是以NOD为遗传背景的小鼠，
    所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠，
    所述小鼠再进一步优选是进一步敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。
  4. 一种对非人动物进行基因改造的方法，其包括：
    通过对非人动物的受精卵进行基因改造使得细胞因子基因或免疫相关基因在原位进行人源化，而在这些细胞因子基因或免疫相关基因人源化后免疫缺陷型小鼠的健康和繁殖效率不会受到影响；
    所述细胞因子包括以下任意一种或两种以上：白细胞介素-15(IL-15)、白细胞介素-7(IL-7)、白细胞介素-6(IL-6)、B细胞活化因子(BAFF)、FMS样酪氨酸激酶3配体(Flt3L)重组蛋白；
    所述免疫相关基因包括主要组织相容性复合体(MHC)；
    优选地，所述细胞因子为白细胞介素-15(IL-15)。
  5. 根据权利要求4所述的方法，其中，
    对非人动物进行基因改造的方法是对非人动物的受精卵进行基因改造，
    优选利用CRISPR/Cas9对非人动物的受精卵进行基因编辑，
    进一步优选，对受精卵的细胞核进行显微注射从而利用CRISPR/Cas9对非人动物的受精卵进行基因编辑以实现对细胞因子进行原位人源化。
  6. 根据权利要求5所述的方法，其中，对非人动物的受精卵进行基因改造是在所述非人动物的IL-15第四个外显子的后面插入兼并的人IL-15第五到第八个外显子的编码序列；
    优选地，对非人动物的受精卵进行基因改造还包括在所述非人动物的IL-15第四个外显子后面进一步插入bGHpolyA(bovine growth hormone polyadenylation)序列；
    优选地，所述bGHpolyA序列如SEQ ID No.2所示；
    优选地，在所述非人动物的IL-15第四个外显子后面插入兼并的人IL15第五到第八个外显子的编码序列如SEQ ID No.1所示。
  7. 根据权利要求4～6中任一权利要求所述的方法，其中，所述非人动物是以NOD为遗传背景的小鼠，
    所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠，
    所述小鼠再进一步优选是进一步敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠；
    优选地，所述小鼠的IL-15第四个外显子的序列如SEQ ID No.4所示。
  8. 根据权利要求5～7中任一权利要求所述的方法，其中，所述非人动物的受精卵是通过体外受精获得的；
    优选地，对所述非人动物的受精卵进行基因改造之前对体外受精得到的受精卵在体外培养一定时间，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
  9. 根据权利要求5所述的方法，其中，所述对所述非人动物的受精卵进行基因改造是对受精卵的细胞核进行显微注射，显微注射物质包括：
    同源重组DNA；
    gRNA1，
    gRNA2，以及
    mRNACas9；
    优选地，所述同源重组DNA包括如SEQ ID No.3所示的人IL-15同源重组DNA序列；
    优选地，所述gRNA1的识别靶序列为：
    CATAGCTATTATCAAGTTAGTGG(SEQ ID No.5)，
    所述gRNA2的识别靶序列为：
    GAAACACAAGTAGCACGAGATGG(SEQ ID No.6)。
  10. 一种构建重度免疫缺陷的动物模型的方法，该方法包括：
    通过对非人动物的受精卵进行基因改造使得细胞因子基因或免疫相关基因在原位进行人源化，而这些细胞因子基因或免疫相关基因人源化改造后的不会影响免疫缺陷型小鼠的健康和繁殖效率的人源化；
    所述细胞因子包括以下任意一种或两种以上：白细胞介素-15(IL-15)、白细胞介素-7(IL-7)、白细胞介素-6(IL-6)、B细胞活化因子(BAFF)、FMS样酪氨酸激酶3配体(Flt3L)重组蛋白；
    所述免疫相关基因包括主要组织相容性复合体(MHC)；
    优选地，所述细胞因子为白细胞介素-15(IL-15)。
  11. 根据权利要求10所述的方法，其中，
    对非人动物进行基因改造的方法是对非人动物的受精卵进行基因改造，
    优选利用CRISPR/Cas9对非人动物的受精卵进行基因编辑，
    进一步优选，对受精卵的细胞核进行显微注射从而利用CRISPR/Cas9对非人动物的受精卵进行基因编辑以实现对细胞因子进行原位人源化。
  12. 根据权利要求11所述的方法，其中，对非人动物的受精卵进行基因改造是在所述非人动物的IL-15第四个外显子的后面插入兼并的人IL-15第五到第八个外显子的编码序列；
    优选地，对非人动物的受精卵进行基因改造还包括在所述非人动物的IL-15第四个外显子后面进一步插入bGHpolyA(bovine growth hormone polyadenylation)序列；
    优选地，所述bGHpolyA序列如SEQ ID No.2所示；
    优选地，在所述非人动物的IL-15第四个外显子后面插入兼并的人IL第五到第八个外显子的编码序列如SEQ ID No.1所示。
  13. 根据权利要求10～12中任一权利要求所述的方法，其中，所述非人动物是以NOD为遗传背景的小鼠，
    所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠，
    所述小鼠再进一步优选是进一步敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠；
    优选地，所述小鼠的IL-15第四个外显子的序列如SEQ ID No.4所示。
  14. 根据权利要求11～13中任一权利要求所述的方法，其中，所述非人动物的受精卵是通过体外受精获得的；
    优选地，对所述非人动物的受精卵进行基因改造之前对体外受精得到的受精卵在体外培养一定时间，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
  15. 根据权利要求11所述的方法，其中，所述对所述非人动物的受精卵进行基因改造是对受精卵的细胞核进行显微注射，显微注射物质包括：
    同源重组DNA；
    gRNA1，
    gRNA2，以及
    mRNACas9；
    优选地，所述同源重组DNA包括如SEQ ID No.3所示的人IL-15同源重组DNA序列；
    优选地，所述gRNA1的识别靶序列为：
    CATAGCTATTATCAAGTTAGTGG(SEQ ID No.5)，
    所述gRNA2的识别靶序列为：
    GAAACACAAGTAGCACGAGATGG(SEQ ID No.6)。
  16. 线性DNA，其包括如SEQ ID No.3所示的人IL-15同源重组序列。
  17. 一种对非人动物进行基因改造的方法，其包括：
    对非人动物细胞的受精卵进行基因改造，
    基因改造包括利用Piggy转座酶依赖的转基因系统对非人动物的受精卵进行基因编辑；
    优选地，所述受精卵是体外受精的受精卵；
    进一步优选地，所述受精卵是通过体外受精得到的，并且在体外培养一定时间的受精卵，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
  18. 根据权利要求17所述的方法，其中，对非人动物细胞的受精卵进行基因改造是通过对受精卵的细胞核或细胞质进行注射来完成的；
    优选地，需要进行基因编辑的基因片段为环形质粒，对非人动物的受精卵进行基因编辑是对非人动物细胞的受精卵的细胞质进行细胞浆或细胞核注射。
  19. 根据权利要求17或18所述的方法，其中，
    所述非人动物是以NOD为遗传背景的小鼠，
    所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠，
    所述小鼠再进一步优选是进一步敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。
  20. 根据权利要求17～19中任一项所述的方法，其中，其包括：
    在非人动物中过表达目标蛋白一和/或目标蛋白二；
    其中，所述目标蛋白一主要在T细胞、B细胞和NK细胞中表达；所述目标蛋白一的基因原位人源化后，在重度免疫缺陷非人动物中检测不到所述目标蛋白一的基因的表达；优选所述目标蛋白一包括以下任意一种或两种：人白细胞介素-3(人IL3)、人白细胞介素-2(人IL2)；进一步优选所述目标蛋白一为人白细胞介素-3(人IL3)；
    所述目标蛋白二可能会影响免疫缺陷非人动物的健康；优选所述目标蛋白二包括以下任意一种或两种以上：人血小板生成素(人TPO)、人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)、人巨噬细胞集落刺激因子(人MCSF)；进一步优选所述目标蛋白二为人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)。
  21. 根据权利要求20所述的方法，其中，
    编码人白细胞介素-3(人IL3)的人IL3cDNA的序列如SEQ ID No.14所示；
    编码人粒细胞巨噬细胞集落刺激因子(人GM-CSF)的人GM-CSFcDNA的序列如SEQ ID No.15所示。
  22. 根据权利要求20或21所述的方法，其中，
    对非人动物进行基因改造的方法是对非人动物的受精卵进行基因改造使得在非人动物中过表达目标蛋白是指在非人动物的髓系细胞中过表达目标蛋白；
    优选利用特异性在髓系细胞表达的启动子来在非人动物的髓系细胞中过表达目标蛋白；
    优选所述髓系细胞表达的启动子选自人CD68启动子、CSF1R启动子、CD11c启动子、CX3CR1启动子、Langerin/CD207启动子、MMLV LTR启动子、Visna virus LTR启动子、DC-STAMP启动子、Human MSR启动子、MSR-A启动子、CD4启动子、CD2启动子、Iba-AIF-1启动子、CD11b启动子、c-fms启动子、scavenger receptor A(SR-A)启动子、lysozyme启动子和MHC class II启动子(MHC-II)；
    进一步优选利用人CD68启动子在非人动物的髓系细胞中过表达目标蛋白；
    进一步优选利用piggyBac转座子系统质粒和piggyBac转座酶将细胞因子表达盒导入非人动物受精卵。
  23. 根据权利要求20或21所述的方法，其中，在非人动物中过表达两个以上目标蛋白时，利用双重自我剪切短肽RAKR-GSG-P2A(RAKR-GSG-ATNFSLLKQAGDVEENPGP)、RAKR-GSG-T2A(RAKR-GSG-EGRGSLLTCGDVEENPGP)、RAKR-GSG-E2A(RAKR-GSG-QCTNYALLKLAGDVESNPGP)或RAKR-GSG-F2A(RAKR-GSG-VKQTLNFDLLKLAGDVESNPGP)介导不同蛋白的过表达，其中，RAKR是Furin剪切短肽的序列，GSG是linker；
    优选地，所述双重自我剪切短肽为RAKR-GSG-P2A，编码双重自我剪切短肽RAKR-GSG-P2A的基因的序列如SEQ ID No.13所示。
  24. 根据权利要求22所述的方法，其中，利用piggyBac转座子系统质粒来在非人动物的髓系细胞中过表达目标蛋白，在piggyBac转座子系统质粒中还插入有bGH polyA序列，优选其序列如SEQ ID No.2所示；
    优选地，在piggyBac转座子系统质粒中还插入有反响重复序列，优选其序列如SEQID No.16或SEQ ID No.17所示。
  25. 根据权利要求20～24中任一项所述的方法，其中，
    所述非人动物是以NOD为遗传背景的小鼠；
    所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠；
    所述小鼠再进一步优选是敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。
  26. 根据权利要求20～25中任一项所述的方法，其中，所述非人动物的受精卵是通过体外受精获得的；
    优选地，对所述非人动物的受精卵进行基因改造之前对体外受精得到的受精卵在体外培养一定时间，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
  27. 根据权利要求22所述的方法，其中，所述对所述非人动物的受精卵进行基因改造是对受精卵的细胞质进行胞浆注射，胞浆注射的物质包括：
    PiggyBac转座子系统质粒和piggyBac转座酶；
    优选地，PiggyBac转座子系统质粒包括SEQ ID No.11所示的序列。
  28. 一种构建重度免疫缺陷的动物模型的方法，该方法包括：
    在非人动物中过表达目标蛋白，所述目标蛋白为目标蛋白一和/或目标蛋白二；
    其中，所述目标蛋白一主要在T细胞、B细胞和NK细胞中表达；所述目标蛋白二可能会影响免疫缺陷小鼠的健康；
    优选地，在非人动物中过表达的目标蛋白包括以下任意一种或两种以上：人白细胞介素-3(人IL3)、人白细胞介素-2(人IL2)、人血小板生成素(人TPO)、人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)、人巨噬细胞集落刺激因子(人MCSF)；
    优选地，在非人动物中过表达人白细胞介素-3(人IL3)和人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)；
    优选地，编码人白细胞介素-3(人IL3)的人IL3cDNA的序列如SEQ ID No.14所示；编码人粒细胞-巨噬细胞集落刺激因子(人GM-CSF)的人GM-CSFcDNA的序列如SEQ ID No.15所示。
  29. 根据权利要求28所述的方法，其中，
    对非人动物进行基因改造的方法是对非人动物的受精卵进行基因改造使得在非人动物中过表达目标蛋白是指在非人动物的髓系细胞中过表达目标蛋白；
    优选利用特异性在髓系细胞表达的启动子来在非人动物的髓系细胞中过表达目标蛋白；
    优选所述髓系细胞表达的启动子选自人CD68启动子、CSF1R启动子、CD11c启动子、CX3CR1启动子、Langerin/CD207启动子、MMLV LTR启动子、Visna virus LTR启动子、DC-STAMP启动子、Human MSR启动子、MSR-A启动子、CD4启动子、CD2启动子、Iba-AIF-1启动子、CD11b启动子、c-fms启动子、scavenger receptor A(SR-A)启动子、lysozyme启动子和MHC class II启动子(MHC-II)，启动子在非人动物的髓系细胞中过表达目标蛋白；
    进一步优选利用人CD68启动子在非人动物的髓系细胞中过表达目标蛋白；
    进一步优选利用piggyBac转座子系统质粒和piggyBac转座酶来在非人动物的髓系细胞中过表达目标蛋白。
  30. 根据权利要求29所述的方法，其中，在非人动物中过表达两个以上目标蛋白时，利用双重自我剪切短肽RAKR-GSG-P2A(RAKR-GSG-ATNFSLLKQAGDVEENPGP)、RAKR-GSG-T2A(RAKR-GSG-EGRGSLLTCGDVEENPGP)、RAKR-GSG-E2A(RAKR-GSG-QCTNYALLKLAGDVESNPGP)或RAKR-GSG-F2A(RAKR-GSG-VKQTLNFDLLKLAGDVESNPGP)介导不同目标蛋白的过表达，其中RAKR是Furin剪切短肽的序列，GSG是linker；
    优选地，所述双重自我剪切短肽为RAKR-GSG-P2A，编码双重自我剪切短肽PAKR-GSG-P2A的基因的序列如SEQ ID No.13所示。
  31. 根据权利要求29所述的方法，其中，利用piggyBac转座子系统质粒来在非人动物的髓系细胞中过表达目标蛋白，在piggyBac转座子系统质粒中还插入有pGH polyA序列，优选其序列如SEQ ID No.2所示；
    优选地，在piggyBac转座子系统质粒中还插入有反响重复序列，优选其序列如SEQ ID No.16或SEQ ID No.17所示。
  32. 根据权利要求28～31中任一项所述的方法，其中，
    所述非人动物是以NOD为遗传背景的小鼠；
    所述小鼠优选是敲除了重组激活基因1(Rag1)或敲除了重组激活基因2(Rag2)或SCID突变的、缺失了T细胞和B细胞的免疫缺陷NOD小鼠；
    所述小鼠再进一步优选是进一步敲除了IL2受体γ链的缺失了NK细胞的重度免疫缺陷NOD小鼠。
  33. 根据权利要求28～32中任一项所述的方法，其中，所述非人动物的受精卵是通过体外受精获得的；
    优选地，对所述非人动物的受精卵进行基因改造之前对体外受精得到的受精卵在体外培养一定时间，优选一定时间为6小时以上，进一步优选为8小时以上、10小时以上、12小时以上、14小时以上，最优选16小时以上。
  34. 根据权利要求29所述的方法，其中，所述对所述非人动物的受精卵进行基因改造是对受精卵的细胞质进行胞浆注射，胞浆注射的物质包括：
    PiggyBac转座子系统质粒和piggyBac转座酶；
    优选地，PiggyBac转座子系统质粒包括SEQ ID No.11所示的序列。
  35. 一种PiggyBac转座子系统质粒，其序列如SEQ ID No.11所示。
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