WO2021043289A1

WO2021043289A1 - Genetically modified non-human animals with kit mutations

Info

Publication number: WO2021043289A1
Application number: PCT/CN2020/113608
Authority: WO
Inventors: Yuelei SHEN; Meiling Zhang; Rui Huang; Yanan GUO; yang BAI; Jiawei Yao; Chaoshe GUO
Original assignee: Biocytogen Pharmaceuticals (Beijing) Co., Ltd
Priority date: 2019-09-06
Filing date: 2020-09-04
Publication date: 2021-03-11
Also published as: CN112553194B; CN112553194A

Abstract

Provided genetically modified non-human animals with one or more mutations in KIT, and methods of use thereof.

Description

GENETICALLY MODIFIED NON-HUMAN ANIMALS WITH KIT MUTATIONS

CLAIM OF PRIORITY

This application claims the benefit of Chinese Patent Application App. No. 201910842268.2, filed on September 6, 2019. The entire contents of the foregoing are incorporated herein by reference.

ATECHNICAL FIELD

This disclosure relates to genetically modified animals with one or more mutations in KIT, and methods of use thereof.

BACKGROUND

Immunodeficient animals are very important for disease modeling and drug developments. In recent years, immunodeficient mice are routinely used as model organisms for research of the immune system, cell transplantation strategies, and the effects of disease on mammalian systems. They have also been extensively used as hosts for normal and malignant tissue transplants, and are widely used to test the safety and efficacy of therapeutic agents.

However, the engraftment capacity of these immunodeficient animals can vary. More immunodeficient animals with different genetic makeup and better engraftment capacities are needed.

SUMMARY

This disclosure is related to genetically modified animals that express mutated KIT protein, and methods of making and use thereof.

In one aspect, the disclosure is related to a genetically-modified, non-human animal expressing a KIT (CD117) protein. In some embodiments, the amino acid residue that corresponds to T660 of SEQ ID NO: 2 in the KIT protein is hydrophobic.

In some embodiments, the amino acid sequence of the KIT protein comprises a sequence is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 2 or 4.

In some embodiments, the amino acid sequence of the KIT protein comprises a sequence that is atleast 80%identical to SEQ ID NO: 4.

In some embodiments, the amino acid sequence of the KIT protein comprises a sequence that is identical to SEQ ID NO: 4.

In some embodiments, the amino acid that corresponds to T660 of SEQ ID NO: 2 is Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp. In some embodiments, the amino acid that corresponds to T660 of SEQ ID NO: 2 is Ala, Val, Ile, Leu, or Met. In some embodiments, the amino acid that corresponds to T660 of SEQ ID NO: 2 is Met.

In some embodiments, the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 3.

In some embodiments, the genome of the animal comprises a disruption in the animal’s endogenous CD132 gene.

In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, a rat, or a mouse. In some embodiments, the animal is a NOD/scid mouse, a NOD/scid nude mouse, or a B-NDG mouse. In some embodiments, the animal is a B-NDG mouse.

In some embodiments, the animal is heterozygous with respect to exon 13 of endogenous KIT gene. In some embodiments, the animal is homozygous with respect to exon 13 of endogenous KIT gene.

In some embodiments, the animal does not express a wild-type KIT protein.

In some embodiments, the animal after being engrafted with human hematopoietic stem cells to develop a human immune system has one or more of the following characteristics:

(a) the percentage of human CD45+ cells is greater than 50%or 60%oftotal live cells from blood (after lysis of red blood cells) in the animal (e.g., at or after week 8, 12, 16, 18, or 20after the animal is engrafted) ;

(b) the percentage of human CD3+cells is greater than 5%or 10%of human CD45+ cells in the animal (e.g., at or after

week

12, 16, 18, or 20 after the animal is engrafted) ;

(c) the percentage of human CD19+cells is greater than 50%or 60%of human CD45+ cells in the animal (e.g., at or after

week

4, 8, 12, 16, 18, or 20 after the animal is engrafted) ;

(d) the percentage of human CD56+ cells is greater than 2%or 5%of human CD45+ cells in the animal (e.g., at or after

week

16, 18, or 20 after the animal is engrafted) ;

(e) the percentage of human CD33+ cells is greater than 2%or 5%of human CD45+ cells in the animal (e.g., at or after

week

4, 8, 12, 16, 18, or 20 after the animal is engrafted) ;

(f) the percentage of human CD14+ cells is greater than 50%or 60%of human CD33+ cells in the animal (e.g., at or after

week

16, 18, or 20 after the animal is engrafted) ; and

(g) the percentage of human CD66b+ cells is greater than 5%or 10%of human CD33+ cells in the animal (e.g., at or after

week

16, 18, or 20 after the animal is engrafted) .

In some embodiments, the animal is not irradiated before being engrafted.

In some embodiments, the animal after being engrafted with human hematopoietic stem cells to develop a human immune system has better development of humanT cells, monocytes, and/or granulocytes relative to a B-NDG mouse. In some embodiments, the B-NDG mouse is irradiated before being engrafted.

In some embodiments, the animal after being engrafted with human hematopoietic stem cells to develop a human immune system has a higher percentage of leukocytes in total live cells from blood (after lysis of red blood cells) relative to a B-NDG mouse. In some embodiments, the B-NDG mouse is irradiated before being engrafted.

In some embodiments, the animal after being engrafted with human hematopoietic stem cells to develop a human immune system has a higher success rate of reconstruction relative to a B-NDG mouse. In some embodiments, the B-NDG mouse is irradiated before being engrafted.

In some embodiments, the animal has an enhanced engraftment capacity of exogenous cells relative to a B-NDG mouse.

In one aspect, the disclosure is related to a method of determining effectiveness of an agent or a combination of agents for treating cancer, comprising:

(a) engrafting tumor cells to the animal as described herein, thereby forming one or more tumors in the animal;

(b) administering the agent or the combination of agents to the animal; and

(c) determining the inhibitory effects on the tumors.

In some embodiments, before engrafting the tumor cells to the animal, human peripheral blood cells (hPBMC) or human hematopoietic stem cells are injected to the animal.

In some embodiments, the tumor cells are from cancer cell lines.

In some embodiments, the tumor cells are from a tumor sample obtained from a human patient.

In some embodiments, the inhibitory effects are determined by measuring the tumor volume in the animal.

In some embodiments, the tumor cells are melanoma cells, lung cancer cells, primary lung carcinoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, primary gastric carcinoma cells, bladder cancer cells, breast cancer cells, and/or prostate cancer cells.

In some embodiments, the agent is an anti-CD47 antibody or an anti-PD-1 antibody. In some embodiments, the agent is an IL6 antibody or an IL15 antibody.

In some embodiments, the combination of agents comprises one or more agents selected from the group consisting of paclitaxel, cisplatin, carboplatin, pemetrexed, 5-FU, gemcitabine, oxaliplatin, docetaxel, and capecitabine.

In one aspect, the disclosure is related to a method of producing an animal comprising a human hemato-lymphoid system, the method comprising: engrafting a population of cells comprising human hematopoietic cells or human peripheral blood cells into the animal as described herein.

In some embodiments, the human hemato-lymphoid system comprises human cells selected from the group consisting of hematopoietic stem cells, myeloid precursor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, lymphocytes, and platelets.

In one aspect, the disclosure is related to a method of producing a genetically-modified rodent, the method comprising

(a) providing a plasmid comprising a 5’ homologous arm and a 3’ homologous arm;

(b) providing a small guide RNA (sgRNA) that targets a sequence in exon 13 of the endogenous KIT gene;

(c) modifying genome of a rodent embryo by using the plasmid of step (1) , the sgRNA of step (2) , and Cas9; and

(d) transplanting the embryo to a receipt rodent to produce a genetically-modified rodent.

In some embodiments, the sgRNA targets SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the sgRNA targets SEQ ID NO: 6.

In some embodiments, the 5’ homologous arm is at least 80%identical to SEQ ID NO: 14 and the 3’ homologous arm is at least 80%identical to SEQ ID NO 15.

In some embodiments, the plasmid further comprises a nucleic acid sequence that is inserted between the 5’ homologous arm and the 3’ homologous arm. In some embodiments, the nucleic acid sequence is CAT.

In some embodiments, the rodent is a mouse.

In some embodiments, the method further comprises establishing a stable mouse line from progenies of the genetically-modified rodent. In some embodiments, the embryo has a NOD/scid background, a NOD/scid nude background, or a B-NDG background.

In one aspect, the disclosure is related to a method of producing a KIT gene mutant mouse, the method comprising the steps of:

(a) transforming a mouse embryonic stem cell with a gene editing system that targets endogenous KIT gene, thereby producing a transformed embryonic stem cell;

(b) introducing the transformed embryonic stem cell into a mouse blastocyst;

(c) implanting the mouse blastocyst into a pseudopregnant female mouse; and

(d) allowing the blastocyst to undergo fetal development to term, thereby obtaining the KIT gene mutant mouse.

(b) implanting the transformed embryonic cell into a pseudopregnant female mouse; and

(c) allowing the transformed embryonic cell to undergo fetal development to term, thereby obtaining the KIT gene mutant mouse.

In some embodiments, the gene editing system comprises a nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in exon 13 of the endogenous KIT gene.

In some embodiments, the nuclease is CRISPR associated protein 9 (Cas9) .

In some embodiments, the target sequence in exon 13 of the endogenous KIT gene is set forth in SEQ ID NO: 5, 6, 7, or 8.

In some embodiments, the target sequence in exon 13 of the endogenous KIT gene is set forth in SEQ ID NO: 6.

In some embodiments, the mouse embryonic stem cell has a NOD/scid background, a NOD/scid nude background, or a B-NDG background.

In one aspect, the disclosure is related to a genetically-modified, non-human animal or a progeny thereof. In some embodiments, the animal is produced by a method comprising: mutating one or more nucleotides of endogenous KIT gene by using a nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in exon 13 of the endogenous KIT gene.

In some embodiments, the nuclease is CRISPR associated protein 9 (Cas9) .

In some embodiments, the target sequence in exon 13 of the endogenous KIT gene is set forth in SEQ ID NO: 5, 6, 7, or 8. In some embodiments, the target sequence in exon 13 of the endogenous KIT gene is set forth in SEQ ID NO: 6.

In another aspect, the disclosure relates to a non-human mammalian cell, comprising a disruption, a deletion, or a genetic modification as described herein.

In some embodiments, the cell includes Cas9 mRNA or an in vitro transcript thereof.

In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is a germ cell. In some embodiments, the cell is a blastocyst.

In another aspect, the disclosure relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein.

The disclosure also relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.

The disclosure further relates to the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal.

In another aspect, the disclosure relates to a tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.

The disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the development of a product related to an immunization processes of human cells, the manufacture of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.

The disclosure also relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the method as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and /or a therapeutic strategy.

The disclosure further relates to the use of the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal, the animal model generated through the methods as described herein, in the screening, verifying, evaluating or studying the KIT gene function, and the drugs for immune-related diseases and antitumor drugs.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic diagram showing mouse KIT gene locus.

FIG. 1Bis a schematic diagram showing human KIT gene locus.

FIG. 2 shows activity testing results for kit-sgRNA1, kit-sgRNA2, kit-sgRNA3, and kit-sgRNA4, in which kit-NC and kit-PC are negative and positive controls, respectively. Y-axis shows the relative activity of sgRNAs.

FIG. 3 shows PCR identification results of F0 generation mice. Mouse tail genomic DNA of a randomly selected F0 generation mouse KIT-9 was amplified by PCR. M is marker. H ₂O is negative control.

FIG. 4 shows PCR identification results of F1 generation mice. M is marker. H ₂O is negative control. F1-1, F1-2, F1-3, F1-5, F1-6, and F1-9 are positive mouse numbers.

FIG. 5 shows survival curves of KIT B-NDG mice and B-NDG mice with reconstructed immune system. Y-axis indicates the percentage of survived mice in each group.

FIG. 6 shows percentages of human leukocytes (CD45+) in total live cells from blood (after lysis of red blood cells) in KIT B-NDG mice and B-NDG mice during the experimental period.

FIG. 7 shows success rate curves of immune system reconstruction in KIT B-NDG mice and B-NDG mice. The success rates are calculated by dividing number of mice with successfully reconstructedimmune system (hCD45+ cell percentage ≥25%of total live cells from blood after lysis of red blood cells) over total number of survived mice.

FIG. 8 shows percentages of human T cells (CD3+) in human CD45+ cells from peripheral blood of KIT B-NDG mice and B-NDG mice, as determined by flow cytometry.

FIG. 9 shows percentages of human B cells (CD19+) in human CD45+ cells from peripheral blood of KIT B-NDG mice and B-NDG mice, as determined by flow cytometry.

FIG. 10 shows percentages of human NK cells (CD56+) in human CD45+ cells from peripheral blood of KIT B-NDG mice and B-NDG mice, as determined by flow cytometry.

FIG. 11 shows percentages of human myeloid cells (CD33+) in human CD45+ cells from peripheral blood of KIT B-NDG mice and B-NDG mice, as determined by flow cytometry.

FIG. 12 shows percentages of human monocytes (CD14+) in human CD33+ cells from peripheral blood of KIT B-NDG mice and B-NDG mice, as determined by flow cytometry.

FIG. 13 shows percentages of human granulocytes (CD66b+) in human CD33+ cells from peripheral blood of KIT B-NDG mice and B-NDG mice, as determined by flow cytometry.

DETAILED DESCRIPTION

This disclosure relates to non-human animals expressing mutated KIT protein, and methods of use thereof.

KIT (CD117) , also known as proto-oncogene c-KIT, mast/stem cell growth factor receptor (SCFR) , or KIT proto-oncogene, receptor tyrosine kinase, is a cytokine receptor expressed on the surface of hematopoietic stem cells as well as other cell types. KIT is a receptor tyrosine kinase type III, which binds to stem cell factor (a substance that causes certain types of cells to grow) , also known as "steel factor" or "c-kit ligand" . When this receptor binds to stem cell factor (SCF) it forms a dimer that activates its intrinsic tyrosine kinase activity, that in turn phosphorylates and activates signal transduction molecules that propagate the signal in the cell. After activation, the receptor is ubiquitinated to mark it for transport to a lysosome and eventual destruction. Signaling through KIT plays a role in cell survival, proliferation, and differentiation. For instance, KIT signaling is required for melanocyte survival, and it is also involved in haematopoiesis and gametogenesis

Like other members of the receptor tyrosine kinase III family, KIT consists of an extracellular domain, a transmembrane domain, a juxtamembrane domain, and an intracellular tyrosine kinase domain. The extracellular domain is composed of five immunoglobulin-like domains, and the protein kinase domain is interrupted by a hydrophilic insert sequence of about 80 amino acids. The ligand stem cell factor binds via the second and third immunoglobulin domains.

The present disclose provides non-human animals expressing a mutated KIT protein. The animals can be used as a research tool for studying the etiology, pathogenesis of various diseases, as well as the development of therapeutic drugs for various diseases (e.g., cancers) .

The animals described herein provide several advantages. The animals do not require irradiation before being engrafted with human cells (e.g., hematopoietic stem cells) to develop a human immune system. Eliminating the irradiation step further improves the overall health of the animals after being engrafted. In some embodiments, the animals described herein have a higher survival rate as compared to irradiated B-NDG mice after engraftment. In some embodiments, the animals described herein promotes human leukocyte (e.g., T cells, monocytes, and/or granulocytes) development as compared to B-NDG mice after engraftment. In some embodiments, the animals described herein have higher success rate of reconstruction as compared to that of B-NDG mice.

Unless otherwise specified, the practice of the methods described herein can take advantage of the techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology. These techniques are explained in detail in the following literature, for examples: Molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) ; DNA Cloning, Volumes I and II (D.N. Glovered., 1985) ; Oligonucleotide Synthesis (M.J. Gaited., 1984) ; Mullisetal U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B.D. Hames&S.J. Higginseds. 1984) ; Transcription And Translation (B. D. Hames&S.J. Higginseds. 1984) ; Culture Of Animal Cell (R.I. Freshney, Alan R. Liss, Inc., 1987) ; Immobilized Cells And Enzymes (IRL Press, 1986) ; B. Perbal, A Practical Guide To Molecular Cloning (1984) , the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York) , specifically, Vols. 154 and 155 (Wuetal. eds. ) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed. ) ; Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Caloseds., 1987, Cold Spring Harbor Laboratory) ; Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987) ; Hand book Of Experimental Immunology, Volumes V (D.M. Weir and C.C. Blackwell, eds., 1986) ; and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986) ; each of which is incorporated herein by reference in its entirety.

KIT (tyrosine-protein kinase KIT or CD117)

The KIT gene, officially known as “KIT proto-oncogene receptor tyrosine kinase” (GenBank ID: 3815) , is also more commonly known as c-kit, kit, or stem cell factor receptor. CD117 was first identified as the cellular homolog of the feline sarcoma viral oncogene v-kit. The CD117 gene consists of a single copy located on chromosome 4 (4q12) encompassing ～88 kb (base pairs 54, 657, 927 to 54, 740, 714) and spanning twenty-one exons producing a transcript of 5.23 kb. The cDNA of CD117 encodes a protein of 145 kDa. The resultant CD117 protein is a member of the type III receptor tyrosine kinase family, which also includes CSF-1R, PDGFRβ, PDGFRα, and FLT3. This receptor tyrosine kinase family is defined by an extracellular domain with five immunoglobulin-like loops, a highly hydrophobic transmembrane domain (23 amino acids for CD117) , a juxtamembrane domain, and an intracellular domain with tyrosine kinase activity split by a kinase insert in an ATP-binding region and in the phosphotransferase domain. The CD117 protein contains ten known glycosylation sites and is largely conserved between species, with the human protein having about 83%homology to mouse and about 68%homology to chicken. CD117 and the other type III receptor tyrosine kinases are an important piece in cell signaling and are responsible for maintaining cell functions such as cell survival, metabolism, cell growth and progression, proliferation, apoptosis, cell migration, and cell differentiation. These are important in understanding the biology of cancer cells.

It has been demonstrated that CD117 of both mice and humans is expressed as two different isoforms, caused by alternative splicing, with only four amino acids differing (glycine, asparagine, asparagine, lysine, abbreviated as GNNK) . These amino acids are either present or absent upstream of CD117’s transmembrane domain (GNNK+ GenBank ID: NM_000222 and GNNK-GenBank ID: NM_00109372, with respective sizes of 5190 and 5178 bp) . Several studies demonstrated that these splice variants, depending on the cell type, can activate different signal transduction pathways and their effects on tumorigenicity, confer constitutive tyrosine phosphorylation, and stimulate association with phosphatidylinositol 3-kinase (PI3-K) . A study demonstrated that isoform GNNK-transformed NIH3T2 fibroblasts caused tumorigenicity in nude mice. Another study showed increased expression of the GNNK-isoform in testicular germline cell tumors, compared to the normal testis which had a higher expression of GNNK+CD117 receptor. While GNNK-has a higher affinity for SCF, CD117’s ligand, as well as faster phosphorylation kinetics, the GNNK-isoform is the dominant isoform in normal tissue, such as bone marrow and melanocytes. Other studies suggest the ratio of GNNK-/GNNK+ is what causes tumorgenicity, with a higher ratio of GNNK-/GNNK+ being the driving force when the D816V mutation is present.

A detailed description of KIT and its function can be found, e.g., in Foster et al., "CD117/c-kit in cancer stem cell-mediated progression and therapeutic resistance. " Biomedicines 6.1 (2018) : 31; Sperling, et al., "Expression of the stem cell factor receptor C-KIT (CD117) in acute leukemias. " Haematologica 82.5 (1997) : 617-621; each of which is incorporated herein by reference in its entirety.

In human genomes, KIT gene (Gene ID: 3815) is located on chromosome 5, and has twenty one exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, and exon 21 (FIG. 1B) .

In mice, KIT gene locus has twenty one exons, exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, and exon 21 (FIG. 1A) . The KIT protein also has an extracellular region, a transmembrane region, and a cytoplasmic region, and the signal peptide is located at the extracellular region of KIT. The nucleotide sequence for mouse KITmRNA is NM_021099.3 (SEQ ID NO: 1) , the amino acid sequence for mouse KIT is NP_066922.2 (SEQ ID NO: 2) . The location for each exon and each region in the mouse KIT nucleotide sequence and amino acid sequence is listed below:

Table 1

The mouse KIT gene (Gene ID: 16590) is located in Chromosome 5 of the mouse genome, which is located from 75, 574, 987 to 756, 567, 22 of NC_000071.6 (GRCm38. p4 (GCF_000001635.24) ) . The 5’ -UTR is from 75, 574, 916 to 75, 575, 051, exon 1 is from 75, 574, 916 to 75, 575, 118, the first intron is from 75, 575, 119 to 75, 607, 025, exon 2 is from 75, 607, 026 to 75, 607, 298, the second intron is from 75, 607, 299 to 75, 609, 269, exon 3 is from 75, 609, 270 to 75, 609, 551, the third intron is from 75, 609, 552 to 75, 610, 806, exon 4 is from 75, 610, 807 to 75, 610, 943, the fourth intron is from 75, 610, 944 to 75, 615, 318, exon 5 is from 75, 615, 319 to 75, 615, 493, the fifth intron is from 75, 615, 494 to 75, 620, 846, the exon 6 is from 75, 620, 847 to 75, 621, 036, the sixth intron is from 75, 621, 037 to 75, 622, 988, the exon 7 is from 75, 622, 989 to 75, 623, 104, the seventh intron is from 75, 623, 105 to 75, 637, 293, the exon 8 is from 75, 637, 294 to 75, 637, 408, the eighth intron is from 75, 637, 409 to 75, 638, 964, the exon 9 is from 75, 638, 965 to 75, 639, 146, the ninth intron is from 75, 639, 147 to 75, 640, 492, the exon 10 is from 75, 640, 493 to 75, 640, 599, the tenth intron is from 75, 640, 600 to 75, 640, 698, the exon 11 is from 75, 640, 699 to 75, 640, 825, the eleventh intron is from 75, 640, 826 to 75, 641, 109, the exon 12 is from 75, 641, 110 to 75, 641, 214, the twelfth intron is from 75, 641, 215 to 75, 641, 326, the exon 13 is from 75, 641, 327 to 75, 641, 437, the thirteenth intron is from 75, 641, 438 to 75, 645, 835, the exon 14 is from 75, 645, 836 to 75, 645, 986, the fourteenth intron is from 75, 645, 987 to 75, 647, 747, the exon 15 is from 75, 647, 748 to 75, 647, 836, the fifteenth intron is from 75, 647, 837 to 75, 648, 386, the exon 16 is from 75, 648, 387 to 75, 648, 514 , the sixteenth intron is from 75, 648, 515 to 75, 649, 546, the exon 17 is from 75, 649, 547 to 75, 649, 669, the seventeenth intron is from 75, 649, 670 to 75, 652, 549, the exon 18 is from 75, 652, 550 to 75, 652, 661, the eighteenth intron is from 75, 652, 662 to 75, 652, 756, the exon 19 is from 75, 652, 757 to 75, 652, 856, the nineteenth intron is from 75, 652, 857 to 75, 653, 219, the exon 20 is from 75, 653, 220 to 75, 653, 325, the twentieth intron is from 75, 653, 326 to 75, 654, 413, the exon 21 is from 75, 654, 414 to 75, 656, 722, and the 3’ -UTR is from 75, 654, 546 to 75, 656, 722, based on transcript NM_021099.3. All relevant information for mouse KIT locus can be found in the NCBI website with Gene ID 16590, which is incorporated by reference herein in its entirety.

KIT genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for KIT in Rattus norvegicus (rat) is 64030, the gene ID for KIT in Macaca mulatta (Rhesus monkey) is 696759, the gene ID for KIT in Canis lupus familiaris (dog) is 403811, the gene ID for KIT in Sus scrofa (pig) is 396810. The relevant information for these genes (e.g., intron sequences, exon sequences, amino acid residues of these proteins) can be found, e.g., in NCBI database.

In one aspect, the present disclosure provides a genetically-modified, non-human animal expressing a mutated KIT protein. In some embodiments, the mutated KIT protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid mutations relative to a wild-type KIT protein sequence (e.g., NP_066922.2 (SEQ ID NO: 2) or NP_001116205.1 (SEQ ID NO: 18) ) . In some embodiments, the mutated KIT protein is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 2, 4 or 18. In some embodiments, the mutated KIT protein comprises one or more mutations at a regioncorresponding to position 549-625 and/or position 663-975 of SEQ ID NO: 2. In some embodiments, the mutated KIT protein comprises one or more mutationsat a region corresponding to position 550-560 of SEQ ID NO: 2. In some embodiments, the mutated KIT protein comprises one or more mutations at a regioncorresponding to position 26-527 of SEQ ID NO: 2. In some embodiments, the mutated KIT protein comprises one or more mutations at a regioncorresponding to position 502-503 of SEQ ID NO: 2. In some embodiments, the mutated KIT protein comprises one or more mutations within the extracellular region. In some embodiments, the mutated KIT protein comprises one or more mutations within the transmembrane region. In some embodiments, the mutated KIT protein comprises one or more amino acidsat a region corresponding to position 528-548 of SEQ ID NO: 2. In some embodiments, mutated KIT protein comprises one or more mutations within the signal peptide. In some embodiments, the mutated KIT protein comprises one or more mutations at a region corresponding to position 1-25 of SEQ ID NO: 2.

In some embodiments, one or more amino acids corresponding to position 626-662 of SEQ ID NO: 2 are mutated. In some embodiments, an amino acid that corresponds to T660 of NP_066922.2 (SEQ ID NO: 2) in the KIT protein is mutated. In some embodiments, an amino acid that corresponds to T664 of NP_001116205.1 (SEQ ID NO: 18) in the KIT protein is mutated. In some embodiments, the amino acid in the KIT protein is mutated to a hydrophobic amino acid, e.g., alanine (Ala) , valine (Val) , isoleucine (Ile) , leucine (Leu) , methionine (Met) , phenylalanine (Phe) , tyrosine (Tyr) , or tryptophan (Trp) . In some embodiments, the amino acidis mutated to Met. In some embodiments, the amino acid is mutated to a non-natural amino acid. In some embodiments, the mutated amino acid is encoded by exon 13 of endogenous (e.g., mouse) KIT gene.

In some embodiments, the expression level of the mutated KIT protein is increased (e.g., by at least or about 10%, 20%, 30%, 40%, or 50%) as compared to the expression level of wild-type KIT protein. In some embodiments, the expression level of the mutated KIT protein is decreased (e.g., to less than or about 90%, 80%, 70%, 60%, or 50%) as compared to the expression level of wild-type KIT protein. In some embodiments, expression level of the mutated KIT protein is similar to the expression level of wild-type KIT protein.

In one aspect, the present disclosure provides a genetically-modified, non-human animal whose genome comprises a disruption (e.g., mutation) in the animal’s endogenous KIT gene locus. In some embodiments, the KIT gene comprises one or more point mutations in exon 13 of the endogenous KIT gene locus. In some embodiments, the genome of the animal comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 3. In some embodiments, the genome of the animal comprises a point mutationthat corresponds to position 75640429 of NCBI Reference Sequence NC_000071.6. In some embodiments, the point mutation is from cytosine (C) to thymine (T) . In some embodiments, the KIT gene comprises a point mutation (e.g., from cytosine (C) to thymine (T) ) that corresponds to position 2040 of SEQ ID NO: 1. In some embodiments, the KIT gene comprises mutations that corresponds to position 1946, 1963, 2023 and/or 2044 of SEQ ID NO: 1. In some embodiments, the KIT gene comprises mutations that corresponds to C1946T, G1963A, C2023T, and/or G2044T of SEQ ID NO: 1. In some embodiments, the genome of the animal comprises point mutations (e.g., within exon 13 of the endogenous KIT gene locus) that do not affect protein translation, e.g., identity of the translated amino acid.

In one aspect, the present disclosure provides a genetically-modified, non-human animal expressing a mutated KIT protein. In some embodiments, the amino acid sequence of the mutated KIT protein is selected from the group consisting of:

a) the entire or part of an amino acid sequence shown in SEQ ID NO: 4;

b) an amino acid sequence having a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 2;

c) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 4 under a low stringency condition or a strict stringency condition;

d) an amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 2;

e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

f) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 2.

In one aspect, the present disclosure provides a genetically-modified, non-human animal comprising a mutated KIT nucleic acid (e.g., DNA or RNA) sequence that encodes a mutated KIT protein. In some embodiments, the nucleic acid sequence is selected from the group consisting of:

a) a nucleic acid sequence encoding the mutated KIT protein amino acid sequence as described herein;

b) a nucleic acid sequence that is transcribed into the entire or part of the mRNA sequence shown in SEQ ID NO: 3;

c) a nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleotide sequence as shown in SEQ ID NO: 1 or 3;

d) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 3 under a low stringency condition or a strict stringency condition;

e) a nucleic acid sequence is different from the nucleic acid sequence shown in SEQ ID NO: 1 or 3 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleic acid;

f)a nucleic acid sequence that comprises a substitution, a deletion and/or insertion of one or more nucleic acids to the nucleic acid sequence shown in SEQ ID NO: 3;

g) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 4 or 2;

h) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 4 or 2;

i) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 4 or 2 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

j) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 4 or 2.

The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein (e.g., exon and intron sequences) , and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein (e.g., amino acid sequences encoded by exons) . In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, or 500 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 150 amino acid residues.

In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of illustration, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Cells, tissues, and animals (e.g., mice) are also provided that express a mutated KIT protein or comprise a disruption of the endogenous KIT gene as described herein, as well as cells, tissues, and animals (e.g., mice) that have any nucleic acid sequence as described herein.

Genetically modified animals

As used herein, the term “genetically-modified non-human animal” refers to a non-human animal having a modified sequence (e.g., mutated exon 13 of endogenous KIT gene) in at least one chromosome of the animal’s genome. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%of cells of the genetically-modified non-human animal have the modified sequence in its genome. The cell having the modified sequence can be various kinds of cells, e.g., an endogenous cell, a somatic cell, an immune cell, a T cell, a B cell, a germ cell, a blastocyst, or an endogenous tumor cell. In some embodiments, genetically-modified non-human animals are provided that comprise mutations (e.g., a point mutation) at the endogenous KIT locus. The animals are generally able to pass the modification to progeny, i.e., through germline transmission.

In some embodiments, the genetically-modified non-human animal does not express wild-typeKIT. In some embodiments, the genetically-modified non-human animal does not express functional KIT.

In some embodiments, the genetically-modified non-human animal described herein (e.g., mouse) has developmental defects of melanocytic, hematopoietic stem cells, and/or primordial germ cell lineages as compared to a wild-type animal. In some embodiments, the genetically-modified non-human animal (e.g., mouse) that expresses a mutated KIT protein (e.g., SEQ ID NO: 4) has a tyrosine kinase activity that is less than or about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%as compared to an animal (e.g., mouse) expressing a wild-type KIT protein (e.g., SEQ ID NO: 2) . In some embodiments, the mutated KIT protein (e.g., SEQ ID NO: 4) has a reduced (e.g., less than or about 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) binding capability with its ligands (e.g., phospholipase C, GTPase-activating protein, and/or phosphoinositide 3-kinase (PI3K) ) compared to a wild-type KIT protein (e.g., SEQ ID NO: 2) . In some embodiments, the genetically-modified non-human animal described herein is immunodeficient.

In some embodiments , the genetically-modified non-human animal described herein (e.g., mouse) have a deactivated (e.g., silenced) KIT gene. For example, the KIT gene is silenced by RNAi. In some embodiments, the genetically-modified non-human animal described herein (e.g., mouse) expresses a dysfunctional KIT protein. For example, one or more amino acid residues in the active site of the expressed KIT protein are modified (e.g., mutated) . In some embodiments, the mutated KIT protein described herein has a reduced (e.g., to less than or about 90%, 80%, 70%, 60%, or 50%) downstream signaling pathway activity. In some embodiments, the function of the expressed KIT protein is specifically inhibited, e.g., by inhibitors or KIT-specific antibodies.

As used herein, the term “leukocytes” or “white blood cells” include T cells (CD3+) , B cells (CD19+) , myeloid cells (CD33+) , NK cells (CD56+) , granulocytes (CD66b+) , and monocytes (CD14+) . All leukocytes have nuclei, which distinguishes them from the anucleated red blood cells (RBCs) and platelets. CD45, also known as leukocyte common antigen (LCA) , is a cell surface marker for leukocytes. Lymphocyte is a subtype of leukocyte. Lymphocytes include natural killer (NK) cells (which function in cell-mediated, cytotoxic innate immunity) , T cells, and B cells. Myeloid cell is a subtype of leukocyte. Myeloid cells include monocytes and granulocytes.

In some embodiments, the genetically-modified non-human animal is a mouse. In some embodiments, thegenetically-modified non-human animal is a B-NDG mouse. Details of B-NDG mice can be found, e.g., in PCT/CN2018/079365; each of which is incorporated herein by reference in its entirety.

In one aspect, the genetically-modified non-human animal (e.g., mouse) is engrafted with human hematopoietic stem cells to develop a human immune system.

In one aspect, the genetically-modified animal is engrafted with human hematopoietic stem cells to develop a human immune system. In some embodiments, the average percentage of human leukocytes (or CD45+ cells) in the animal is at least or about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold higher than that of an animal with B-NDG background (e.g., a B-NDG mouse) , wherein the animal with B-NDG background is irradiated and then engrafted with human hematopoietic stem cells to develop a human immune system. In some embodiments, the average percentage of human leukocytes (or CD45+ cells) isdetermined at least or about 8 weeks, at least or about 12 weeks, at least or about 16 weeks, at least or about 18 weeks, at least or about 20 weeks after being engrafted.

In some embodiments, the success rate of reconstruction in the genetically-modified animal (e.g., mouse) is at least or about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the success rate of reconstruction in the genetically-modified animal (e.g., mouse) is at least or about 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, or 1000-fold higher than that of an animal with B-NDG background (e.g., a B-NDG mouse) . The success rate is calculated by dividing number of mice with successfully reconstructed immune system (hCD45+ cell percentage ≥25%of total live cells from blood after lysis of red blood cells) over total number of survived mice. In some embodiments, the success rate is determined at least or about 8 weeks, at least or about 12 weeks, at least or about 16 weeks, at least or about 18 weeks, at least or about 20 weeks after the animal (e.g., mouse) is engrafted with human cells (e.g., hematopoietic stem cells) to develop a human immune system. In some embodiments, at least or about 8 weeks after engraftment, the success rate of reconstruction in the animal is at least or about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% (e.g., 50%) . In some embodiments, at least or about 12 weeks after engraftment, the success rate of reconstruction in the animal is at least or about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%or 95%. In some embodiments, at least or about 16 weeks after engraftment, the success rate of reconstruction in the animal is at least or about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, at least or about 18 weeks after engraftment, the success rate of reconstruction in the animal is at least or about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, at least or about 20 weeks after engraftment, the success rate of reconstruction in the animal is at least or about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

In some embodiments, the survival rate of the genetically-modified animal (e.g., mouse) is at least or about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, after about 100 days, about 120 days, about 140 days, about 160 days, or about 180 days of the engraftment. In some embodiments, the survival rate of the genetically-modified animal (e.g., mouse) is at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%higher than that of an animal with B-NDG background (e.g., a B-NDG mouse) , after about 100 days, about 120 days, about 140 days, about 160 days, or about 180 days of the engraftment.

The genetically modified non-human animal can also be various other animals, e.g., a rat, rabbit, pig, bovine (e.g., cow, bull, buffalo) , deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey) . For the non-human animals where suitable genetically modifiable ES cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo. These methods are known in the art, and are described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003, which is incorporated by reference herein in its entirety.

In one aspect, the animal is a mammal, e.g., of the superfamily Dipodoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In some embodiment, the rodent is selected from the superfamily Muroidea. In some embodiments, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters) , Cricetidae (e.g., hamster, New World rats and mice, voles) , Muridae (true mice and rats, gerbils, spiny mice, crested rats) , Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice) , Platacanthomyidae (e.g., spiny dormice) , and Spalacidae (e.g., mole rates, bamboo rats, and zokors) . In some embodiments, the genetically modified rodent is selected from a true mouse or rat (family Muridae) , a gerbil, a spiny mouse, and a crested rat. In one embodiment, the non-human animal is a mouse.

In some embodiments, the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In some embodiments, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2. These mice are described, e.g., in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999) ; Auerbach et al., Establishment and Chimera Analysis of 129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000) , both of which are incorporated herein by reference in the entirety. In some embodiments, the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a mix of the 129 strains, or a mix of the BL/6 strains. In some embodiment, the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129) .

In some embodiments, the animal is a rat. The rat can be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

The animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the animal expressing mutated KIT is made. For example, suitable mice for maintaining a xenograft (e.g., a human cancer or tumor) , can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part. Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin) , physical means (e.g., irradiating the animal) , and/or genetic modification (e.g., knocking out one or more genes) .

Non-limiting examples of such mice include, e.g., NOD mice, SCID mice, NOD/SCID mice, nude mice, NOD/SCID nude mice, NOD-Rag 1 ^-/--IL2rg ^-/- (NRG) mice, Rag 2 ^-/--IL2rg ^-/-(RG) mice, B-NDG (NOD-Prkdc ^scid IL-2rγ ^null) mice, and Rag1 and/or Rag2 knockout mice. In some embodiments, these mice can optionally be irradiated, or otherwise treated to destroy one or more immune cell types. Thus, in various embodiments, a genetically modified mouse is provided that can include one or more mutations at the endogenous non-human KIT locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part. In some embodiments, modification is, e.g., selected from the group consisting of a modification that results in NOD mice, SCID mice, NOD/SCID mice, B-NDG (NOD-Prkdc ^scid IL-2rγ ^null) mice, nude mice, Rag1 and/or Rag2 knockout mice, and a combination thereof. These genetically modified animals are described, e.g., in US20150106961 and PCT/CN2018/079365; each of which is incorporated herein by reference in its entirety.

In some embodiments, the genetically-modified non-human animal described herein does not require irradiation to destroy one or more immune cell types. In some embodiments, the KIT mutation renders developmental defects of melanocytic, hematopoietic stem cells, and/or primordial germ cell lineages as compared to a wild-type animal. Thus, the lack of irradiation improves the overall health condition of the animal expressing mutated KIT protein after being engrafted with human cells (e.g., hematopoietic stem cells) to develop a human immune system. The improvement of overall health condition can be increased mobility (e.g., by about 10%, 20%, 30%, 40%, 50%, or more) , decreased number of mice (e.g., to about 90%, 80%, 70%, 60%, 50%, or less) with hunched backs and/or sparse body hair.

Although genetically modified cells are also provided that can comprise the modifications (e.g., disruption, mutations) described herein (e.g., ES cells, somatic cells) , in many embodiments, the genetically modified non-human animals comprise the modification of the endogenous KIT locus in the germline of the animal.

Furthermore, the genetically modified animal can be homozygous with respect to the modifications (e.g., mutation) of the endogenous KIT gene. In some embodiments, the animal can be heterozygous with respect to the modification (e.g., mutation) of the endogenous KIT gene.

In one aspect, the disclosure relates to a genetically-modified, non-human animal whose genome comprise a disruption in the animal’s endogenous CD132 gene, wherein the disruption of the endogenous CD132 gene comprises deletion of exon 2 of the endogenous CD132 gene.

In some embodiments, the disruption of the endogenous CD132 gene further comprises deletion of exon 1 of the endogenous CD132 gene. In some embodiments, the disruption of the endogenous CD132 gene comprises deletion of part of exon 1 of the endogenous CD132 gene.

In some embodiments, the disruption of the endogenous CD132 gene further comprises deletion of one or more exons or part of exons selected from the group consisting of exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8 of the endogenous CD132 gene. In some embodiments, the disruption of the endogenous CD132 gene comprises deletion of exons 1-8 of the endogenous CD132 gene.

In some embodiments, the disruption of the endogenous CD132 gene further comprises deletion of one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7 of the endogenous CD132 gene.

In some embodiments, the disruption consists of deletion of more than 150 nucleotides in exon 1; deletion of the entirety of intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7; and deletion of more than 250 nucleotides in exon 8.

In some embodiments, the animal is homozygous with respect to the disruption of the endogenous CD132 gene. In some embodiments, the animal is heterozygous with respect to the disruption of the endogenous CD132 gene.

In some embodiments, the disruption prevents the expression of functional CD132 protein.

In some embodiments, the length of the remaining exon sequences at the endogenous CD132 gene locus is less than 30%of the total length of all exon sequences of the endogenous CD132 gene. In some embodiments, the length of the remaining sequences at that the endogenous CD132 gene locus is less than 15%of the full sequence of the endogenous CD132 gene.

In another aspect, the disclosure relates to a genetically-modified, non-human animal, wherein the genome of the animal does not have exon 2 of CD132 gene at the animal’s endogenous CD132 gene locus.

In some embodiments, the genome of the animal does not have one or more exons or part of exons selected from the group consisting of exon 1, exon 3, exon 4, exon 5, exon 6, exon 7, and exon 8. In some embodiments, the genome of the animal does not have one or more introns or part of introns selected from the group consisting of intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, and intron 7.

In one aspect, the disclosure also provides a CD132 knockout non-human animal, wherein the genome of the animal comprises from 5’ to 3’ at the endogenous CD132 gene locus, (a) a first DNA sequence; optionally (b) a second DNA sequence comprising an exogenous sequence; (c) a third DNA sequence, wherein the first DNA sequence, the optional second DNA sequence, and the third DNA sequence are linked, wherein the first DNA sequence comprises an endogenous CD132 gene sequence that is located upstream of intron 1, the second DNA sequence can have a length of 0 nucleotides to 300 nucleotides, and the third DNA sequence comprises an endogenous CD132 gene sequence that is located downstream of intron 7.

In some embodiments, the first DNA sequence comprises a sequence that has a length (5’ to 3’ ) of from 10 to 100 nucleotides (e.g., approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides) , wherein the length of the sequence refers to the length from the first nucleotide in exon 1 of the CD132 gene to the last nucleotide of the first DNA sequence.

In some embodiments, the first DNA sequence comprises at least 10 nucleotides from exon 1 of the endogenous CD132 gene. In some embodiments, the first DNA sequence has at most 100 nucleotides from exon 1 of the endogenous CD132 gene.

In some embodiments, the third DNA sequence comprises a sequence that has a length (5’ to 3’ ) of from 200 to 600 nucleotides (e.g., approximately 200, 250, 300, 350, 400, 450, 500, 550, 600 nucleotides) , wherein the length of the sequence refers to the length from the first nucleotide in the third DNA sequence to the last nucleotide in exon 8 of the endogenous CD132 gene.

In some embodiments, the third DNA sequence comprises at least 300 nucleotides from exon 8 of the endogenous CD132 gene. In some embodiments, the third DNA sequence has at most 400 nucleotides from exon 8 of the endogenous CD132 gene.

In one aspect, the disclosure also relates to a genetically-modified, non-human animal produced by a method comprising knocking out one or more exons of endogenous CD132 gene by using (1) a first nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in exon 1 of the endogenous CD132 gene or upstream of exon 1 of the endogenous CD132 gene, and (2) a second nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a sequence in exon 8 of the endogenous CD132 gene. In some embodiments, the nuclease is CRISPR associated protein 9 (Cas9) . In some embodiments, the animal does not express a functional CD132 protein. In some embodiments, the animal does not express a functional interleukin-2 receptor.

In one aspect, the disclosure relates to a genetically-modified mouse or a progeny thereof, whose genome comprises a disruption in the mouse’s endogenous CD132 gene, wherein the disruption of the endogenous CD132 gene comprisesdeletion of more than 150 nucleotides in exon 1; deletion of the entirety of intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7; and deletion of more than 250 nucleotides in exon 8. In some embodiments, the animal has an enhanced engraftment capacity of exogenous cells relative to a NSG mouse, a NOG mouse, or a NOD/scid mouse.

The present disclosure further relates to a non-human mammal generated through the methods as described herein. In some embodiments, the genome thereof contains human gene (s) .

In addition, the present disclosure also relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein. In some embodiments, the non-human mammal is a rodent (e.g., a mouse) .

The present disclosure further relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; and the tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.

The present disclosure also provides non-human mammals produced by any of the methods described herein. In some embodiments, a non-human mammal is provided; and the genetically modified animal contains a modification (e.g., mutation) of the KIT gene in the genome of the animal.

Genetic, molecular and behavioral analyses for the non-human mammals described above can be performed. The present disclosure also relates to the progeny produced by the non-human mammal provided by the present disclosure mated with the same or other genotypes.

The present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof. A model based on cell culture can be prepared, for example, by the following methods. Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cell can be obtained from the cell culture established using the same constructs and the cell transfection techniques. The modification of KIT gene can be detected by a variety of methods.

There are also many analytical methods that can be used to detect DNA expression, including methods at the level of RNA (including the mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies) . Analysis methods can be used to complete quantitative measurements. For example, transcription levels of wild-type KIT can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human proteins.

Vectors

The disclosure also provides vectors for constructing a KIT animal model. In some embodiments, the vectors comprise sgRNA sequence, wherein the sgRNA sequence target KIT gene, and the sgRNA is unique on the target sequence of the KIT gene to be altered, and meets the sequence arrangement rule of 5’ -NNN (20) -NGG3’ or 5’ -CCN-N (20) -3’ ; and in some embodiments, the targeting site of the sgRNA in the mouse KIT gene is located on the exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13,exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, intron 19, and intron 20, upstream of exon 1, or downstream of exon 21 of the mouse KIT gene. In some embodiments, the targeting site of the sgRNA in the mouse KIT gene is located on exon 13.

In some embodiments, the sgRNA sequence recognizes a targeting site within exon 13 of mouse KIT gene. In some embodiments, the targeting sites within exon 13 are set forth in SEQ ID NOS: 5-8.

In some embodiments, the disclosure relates to a plasmid construct (e.g., pT7-sgRNA) including the sgRNA sequence, and/or a cell including the construct.

In some embodiments, the disclosure relates to a targeting vector including a 5’ homologous arm and a 3’ homologous arm. In some embodiments, the 5’ homologous arm comprises a sequence spanning the entire or part of exon 10, exon 11, exon 12, and exon 13. In some embodiments, the 3’ homologous arm comprises a sequence spanning the entire or part of exon 13. In some embodiments, the 5’ homologous arm comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100 %identical to SEQ ID NO: 14. In some embodiments, the 3’ homologous arm comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100 %identical to SEQ ID NO: 15. In some embodiments, the 5’ homologous arm comprises a sequence that 80%, 85%, 90%, 95%, or 100 %identical to 75640475-75641426 of the NCBI Reference Sequence NC_000071.6. In some embodiments, the 3’ homologous arm comprises a sequence that is 80%, 85%, 90%, 95%, or 100 %identical to 75641430-75642376 of the NCBI Reference Sequence NC_000071.6. In some embodiments, the 5’ homologous arm comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations (e.g., at positions corresponding to C860, G878, and/or C938 of SEQ ID NO: 14) within the sequence. In some embodiments, the 3’ homologous arm comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations (e.g., at positions corresponding to G4 of SEQ ID NO: 15) within the sequence. In some embodiments, a nucleotide sequence is inserted between the 5’ and 3’ homologous arm. In some embodiments, the nucleotide sequence is CAT. In some embodiments, the 5’ homologous arm comprises one or more mutations as described herein. In some embodiments, the 3’ homologous arm comprises one or more mutations as described herein.

In addition, the present disclosure further relates to a non-human mammalian cell, having any one of the foregoing targeting vectors, and one or more in vitro transcripts of the sgRNA construct as described herein. In some embodiments, the cell includes Cas9 mRNA or an in vitro transcript thereof.

In some embodiments, the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.

In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.

Methods of making genetically modified animals

Genetically modified animals can be made by several techniques that are known in the art, including, e.g., nonhomologous end-joining (NHEJ) , homologous recombination (HR) , zinc finger nucleases (ZFNs) , transcription activator-like effector-based nucleases (TALEN) , and the clustered regularly interspaced short palindromic repeats (CRISPR) -Cas system. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these genome editing techniques are known in the art, and is described, e.g., in Yin et al., "Delivery technologies for genome editing, " Nature Reviews Drug Discovery 16.6 (2017) : 387-399, which is incorporated by reference in its entirety. Many other methods are also provided and can be used in genome editing, e.g., micro-injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.

Thus, in some embodiments, the disclosure provides mutating in at least one cell of the animal, at an endogenous KIT gene locus, one or more exons (e.g., exon 13) of the endogenous KIT gene. In some embodiments, the modification occurs in a germ cell, a fertilized egg cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can also be inserted into an enucleated oocyte.

In some embodiments, cleavages at regions close to the mutation site of the endogenous KIT gene locus by a nuclease (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double strands break, which triggers homologous recombination between the endogenous KIT gene locus and the targeting vector , thereby mutating the nucleotides of interest.

Zinc finger proteins, TAL-effector domains, or single guide RNA (sgRNA) DNA-binding domains can be designed to target a region close to the mutation site. SEQ ID NOs: 5-8 are exemplary target sequences for the modification. All of them are located in exon 13 of the endogenous KIT gene locus. After the zinc finger proteins, TAL-effector domains, or single guide RNA (sgRNA) DNA-binding domains bind to the target sequences, the nuclease cleaves the genomic DNA. In some embodiments, the nuclease is CRISPR associated protein 9 (Cas9) .

Thus, the methods of producing a mouse expressing mutated KIT can involve one or more of the following steps: transforming a mouse embryonic stem cell with a gene editing system that targets endogenous KIT gene, thereby producing a transformed embryonic stem cell; introducing the transformed embryonic stem cell into a mouse blastocyst; implanting the mouse blastocyst into a pseudopregnant female mouse; and allowing the blastocyst to undergo fetal development to term.

In some embodiments, the transformed embryonic cell is directly implanted into a pseudopregnant female mouse instead, and the embryonic cell undergoes fetal development.

In some embodiments, the gene editing system can involve Zinc finger proteins, TAL-effector domains, or single guide RNA (sgRNA) DNA-binding domains.

The present disclosure further provides a method for establishing an animal model expressing mutated KIT, involving the following steps:

(a) providing the cell (e.g. a fertilized egg cell) with the genetic modification based on the methods described herein;

(b) culturing the cell in a liquid culture medium;

(c) transplanting the cultured cell to the fallopian tube or uterus of the recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;

(d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c) .

In some embodiments, the non-human mammal in the foregoing method is a mouse (e.g., a C57BL/6 mouse, a NOD/scid mouse, a NOD/scid nude mouse, or a B-NDG mouse) . In some embodiments, the non-human mammal is a B-NDG (NOD-Prkdc ^scid IL-2rγ ^null) mouse. In some embodiments, the non-human mammal is a NOD/scid mouse.

In the B-NDG mouse, the Prkdc ^scid (commonly known as “SCID” or “severe combined immunodeficiency” ) mutation has been transferred onto a non-obese diabetic (NOD) background. Animals homozygous for the SCID mutation have impaired T and B cell lymphocyte development. The NOD background additionally results in deficient natural killer (NK) cell function. IL-2rγ ^null refers to a specific knock out modification in mouse CD132 gene. Details can be found, e.g., in PCT/CN2018/079365, which is incorporated herein by reference in its entirety. In some embodiments, the non-human mammal is a B-NDG mouse. The B-NDG mouse additionally has a disruption of FOXN1 gene on chromosome 11 in mice.

In some embodiments, the fertilized eggs for the methods described above are NOD/scid fertilized eggs, NOD/scid nude fertilized eggs, or B-NDG fertilized eggs. Other fertilized eggs that can also be used in the methods as described herein include, but are not limited to, C57BL/6fertilized eggs, FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs and DBA/2 fertilized eggs.

Fertilized eggs can come from any non-human animal, e.g., any non-human animal as described herein. In some embodiments, the fertilized egg cells are derived from rodents. The genetic construct can be introduced into a fertilized egg by microinjection of DNA. For example, by way of culturing a fertilized egg after microinjection, a cultured fertilized egg can be transferred to a false pregnant non-human animal, which then gives birth of a non-human mammal, so as to generate the non-human mammal mentioned in the method described above.

The genetically modified animals (e.g., mice) as described herein can have several advantages. For example, the genetically modified mice do not require backcrossing, and thus have a relatively purer background (e.g., B-NDG) as compared to some other immunodeficient mice known in the art. A pure background is beneficial to obtain consistent experiment results. In addition, because almost all sequences in CD132 have been knocked out, these mice are likely to have a higher degree of immunodeficiency and are likely to be better recipients for engraftment as compared to some other immunodeficient mice known in the art. Further, because of the loss-of-function mutation in KIT gene locus, the animals do not require irradiation before being engrafted with human cells (e.g., hematopoietic stem cells) to develop a human immune system, which improves the overall health condition of the animals after being engrafted. Despite the immunodeficiency, these mice are also relatively healthy, and have a relatively long life span (e.g., more than 1 year, 1.5 years, or 2 years) .

Methods of using genetically modified animals

Genetically modified animals that express mutated KIT proteins can provide a variety of uses that include, but are not limited to, establishing a human hemato-lymphoid animal model, developing therapeutics for human diseases and disorders, and assessing the efficacy of these therapeutics in the animal models.

In some embodiments, the genetically modified animals can be used for establishing a human hemato-lymphoid system. The methods involve engrafting a population of cells comprising human hematopoietic cells (CD34+ cells) or human peripheral blood cells into the genetically modified animal described herein. In some embodiments, the methods further include the step of irradiating the animal prior to the engrafting. In some embodiments, the step of irradiating is not required prior to the engrafting. The human hemato-lymphoid system in the genetically modified animals can include various human cells, e.g., hematopoietic stem cells, myeloid precursor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, lymphocytes, and platelets.

The genetically modified animals described herein (e.g., with one or more mutations in exon 13) are also an excellent animal model for establishing the human hemato-lymphoid system. In some embodiments, the animal after being engrafted with human hematopoietic stem cells or human peripheral blood cells to develop a human immune system has one or more of the following characteristics:

(a) the percentage of human leukocytes (or CD45+ cells) is at least or about 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%of total live cells from blood (after lysis of red blood cells) in the animal

(b) the percentage of human T cells (or CD3+ cells) is at least or about 1%, 2%, 3%, 4%, 5%, 8%, or 10%of human leukocytes (or CD45+ cells) in the animal;

(c) the percentage of human B cells (or CD19+ cells) is at least or about 50%, 55%, 60%, 65%, 70%, 75%, or 80%of human leukocytes (or CD45+ cells) in the animal;

(d) the percentage of human NK cells (or CD56+ cells) is at least or about 1%, 2%, 3%, 4%, 5%, 8%, or 10%of human leukocytes (or CD45+ cells) in the animal

(e) the percentage of human myeloid cells (or CD33+ cells) is at least or about 2%, 5%, 8%, 10%, 15%, or 20%of human leukocytes (or CD45+ cells) in the animal;

(f) the percentage of human monocytes (or CD14+ cells) is at least or about 50%, 55%, 60%, 65%, 70%, 75%, or 80%of human myeloid cells (or CD33+ cells) in the animal; and

(g) the percentage of humangranulocytes (or CD66b+ cells) is at least or about 1%, 2%, 3%, 4%, 5%, 8%, or 10%of human myeloid cells (or CD33+ cells) in the animal.

In some embodiments, the one or more characteristics are determined at least or about 4 weeks, at least or about 8 weeks, at least or about 12 weeks, at least or about 16 weeks, at least or about 18 weeks, at least or about 20 weeks after the animal (mouse) is engrafted with human hematopoietic stem cells to develop a human immune system.

In some embodiments, the animal has an enhanced engraftment capacity of exogenous cells relative to a NSG mouse, a NOG mouse, a NOD/scid mouse, or a B-NDG mouse. In some embodiments, the animal models described here are better animal models for establishing the human hemato-lymphoid system (e.g., having a higher percentage of human leukocytes, human T cells, human B cells, or human NK cells) . A detailed description of the NSG mice, NOD mice, and B-NDG can be found, e.g., in Ishikawa et al. "Development of functional human blood and immune systems in NOD/SCID/IL2 receptor γ chainnull mice. " Blood 106.5 (2005) : 1565-1573; Katano et al. "NOD-Rag2null IL-2Rγnull mice: an alternative to NOG mice for generation of humanized mice. " Experimental animals 63.3 (2014) : 321-330; US20190320631A1; each of which is incorporated herein by reference in the entirety.

In some embodiments, the genetically modified animals can be used to determine the effectiveness of an agent or a combination of agents for the treatment of cancer. The methods involve engrafting tumor cells to the animal as described herein, administering the agent or the combination of agents to the animal; and determining the inhibitory effects on the tumors.

In some embodiments, the tumor cells are from a tumor sample obtained from a human patient. These animal models are also known as Patient derived xenografts (PDX) models. PDX models are often used to create an environment that resembles the natural growth of cancer, for the study of cancer progression and treatment. Within PDX models, patient tumor samples grow in physiologically-relevant tumor microenvironments that mimic the oxygen, nutrient, and hormone levels that are found in the patient’s primary tumor site. Furthermore, implanted tumor tissue maintains the genetic and epigenetic abnormalities found in the patient and the xenograft tissue can be excised from the patient to include the surrounding human stroma. As a result, PDX models can often exhibit similar responses to anti-cancer agents as seen in the actual patient who provide the tumor sample.

While the genetically modified animals do not have functional T cells or B cells, the genetically modified animals still have functional phagocytic cells, e.g., neutrophils, eosinophils (acidophilus) , basophils, or monocytes. Macrophages can be derived from monocytes, and can engulf and digest cellular debris, foreign substances, microbes, cancer cells. Thus, the genetically modified animals described herein can be used to determine the effect of an agent (e.g., anti-CD47 antibodies, anti-IL6 antibodies, anti-IL15 antibodies, or anti-SIRPαantibodies) on phagocytosis, and the effects of the agent to inhibit the growth of tumor cells.

In some embodiments, human peripheral blood cells (hPBMC) or human hematopoietic stem cells are injected to the animal to develop human hematopoietic system. The genetically modified animals described herein can be used to determine the effect of an agent in human hematopoietic system, and the effects of the agent to inhibit tumor cell growth or tumor growth. Thus, in some embodiments, the methods as described herein are also designed to determine the effects of the agent on human immune cells (e.g., human T cells, B cells, or NK cells) , e.g., whether the agent can stimulate T cells or inhibit T cells, whether the agent can upregulate the immune response or downregulate immune response. In some embodiments, the genetically modified animals can be used for determining the effective dosage of a therapeutic agent for treating a disease in the subject, e.g., cancer, or autoimmune diseases.

In some embodiments, the tested agent or the combination of tested agents is designed for treating various cancers. As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

In some embodiments, the tested agent is designed for the treating melanoma, primary lung carcinoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , primary gastric carcinoma, bladder cancer, breast cancer, and/or prostate cancer.

In some embodiments, the injected tumor cells are human tumor cells. In some embodiments, the injected tumor cells are melanoma cells, primary lung carcinoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, primary gastric carcinoma cells, bladder cancer cells, breast cancer cells, and/or prostate cancer cells.

The inhibitory effects on tumors can also be determined by any methods known in the art. In some embodiments, the tumor cells can be labeled by a luciferase gene. Thus, the number of the tumor cells or the size of the tumor in the animal can be determined by an in vivo imaging system (e.g., the intensity of fluorescence) . In some embodiments, the inhibitory effects on tumors can also be determined by measuring the tumor volume in the animal, and/or determining tumor (volume) inhibition rate (TGI _TV) . The tumor growth inhibition rate can be calculated using the formula TGI _TV (%) = (1 –TVt/TVc) x 100, where TVt and TVc are the mean tumor volume (or weight) of treated and control groups.

In some embodiments, the tested agent can be one or more agents selected from the group consisting of paclitaxel, cisplatin, carboplatin, pemetrexed, 5-FU, gemcitabine, oxaliplatin, docetaxel, and capecitabine.

In some embodiments, the tested agent can be an antibody, for example, an antibody that binds to CD47, PD-1, CTLA-4, LAG-3, TIM-3, BTLA, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, CD27, GITR, or OX40. In some embodiments, the antibody is a human antibody.

The present disclosure also relates to the use of the animal model generated through the methods as described herein in the development of a product related to an immunization processes of human cells, the manufacturing of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.

In some embodiments, the disclosure provides the use of the animal model generated through the methods as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.

In some embodiments, the disclosure provides a method to verify in vivo efficacy of TCR-T , CAR-T, and/or other immunotherapies (e.g., T-cell adoptive transfer therapies) . For example, the methods include transplanting human tumor cells into the animal described herein, and applying human CAR-T therapy to the animal with human tumor cells. Effectiveness of the CAR-T therapy can be determined and evaluated. In some embodiments, the animal is selected from the non-human animal prepared by the methods described herein, the non-human animal described herein, the double-or multi-humanized non-human animal generated by the methods described herein (or progeny thereof) , a non-human animal expressing mutated KIT, or the tumor-bearing or inflammatory animal models described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies can treat the diseases described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies provides an evaluation method for treating the diseases (e.g., cancer) described herein.

Animal models with additional genetic modifications

The present disclosure further relates to methods for generating genetically modified animal models described herein with some additional modifications (e.g., human or chimeric genes or additional gene knockout) .

In some embodiments, the animal can comprise a modification (e.g., mutation) at the endogenous KIT gene and a sequence encoding a human or chimeric protein. In some embodiments, the human or chimeric protein can be programmed cell death protein 1 (PD-1) , TNF Receptor Superfamily Member 9 (4-1BB or CD137) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , LAG-3, T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD47, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , or TNF Receptor Superfamily Member 4 (TNFRSF4; or OX40) .

In some embodiments, the animal can comprise a modification (e.g., mutation) at the endogenous KIT gene and a disruption at some other endogenous genes (e.g., CD132, Beta-2-Microglobulin (B2m) or Forkhead Box N1 (Foxn1) ) .

The methods of KIT knockout animal model with additional genetic modifications (e.g., humanized genes or additional gene knockout) can include the following steps:

(a) using the methods as described herein to obtain an animal expressing mutated KIT;

(b) mating the animal with another genetically modified non-human animal with the desired genetic modifications, and then screening the progeny to obtain an animal with the desired genetic modifications.

In some embodiments, in step (b) of the method, the genetically modified animal can be mated with a genetically modified non-human animal with human or chimeric PD-1, CTLA-4, LAG-3, TIM-3, BTLA, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, GITR, or OX40. Some of these genetically modified non-human animals are described, e.g., in PCT/CN2017/090320, PCT/CN2017/099577, PCT/CN2017/099575, PCT/CN2017/099576, PCT/CN2017/099574, PCT/CN2017/106024; each of which is incorporated herein by reference in its entirety.

In some embodiments, the mutation of KIT gene can be directly performed on a genetically modified animal having a human or chimeric PD-1, CTLA-4, LAG-3, BTLA, TIM-3, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, GITR, or OX40 gene.

In some embodiments, the mutation of KIT gene can be directly performed on a B2m knockout mouse or a Foxn1 knockout mouse. In some embodiments, the mutation of KIT gene can be directly performed on a B-NDG mouse.

As these proteins may involve different mechanisms, a combination therapy that targets two or more of these proteins thereof may be a more effective treatment. In fact, many related clinical trials are in progress and have shown a good effect. The mutated KIT animal model, and/or the mutated KIT animal model with additional genetic modifications can be used for determining effectiveness of a combination therapy.

In some embodiments, the combination of agents can include one or more agents selected from the group consisting of paclitaxel, cisplatin, carboplatin, pemetrexed, 5-FU, gemcitabine, oxaliplatin, docetaxel, and capecitabine.

In some embodiments, the combination of agents can include one or more agents selected from the group consisting of campothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen, taxol, transplatinum, 5-flurouracil, vincristin, vinblastin, and methotrexate.

In some embodiments, the combination of agents can include one or more antibodies that bind to CD47, PD-1, CTLA-4, LAG-3, BTLA, TIM-3, PD-L1, 4-1BB, CD27, CD28, CD47, TIGIT, GITR, and/or OX40.

Alternatively or in addition, the methods can also include performing surgery on the subject to remove at least a portion of the cancer, e.g., to remove a portion of or all of a tumor (s) , from the subject.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

The following materials were used in the following examples.

NOD/scid mice were purchased from Beijing HFK Bioscience Co., Ltd.

NOD-Prkdc ^scid IL-2rg ^null (B-NDG) mice were obtained from Beijing Biocytogen Co., Ltd. The catalog number is B-CM-002.

Cas9 mRNA was obtained from SIGMA. The catalog number is CAS9MRNA-1EA.

UCA kit was obtained from Beijing Biocytogen Co., Ltd. The catalog number is BCG-DX-001.

Ambion ^TMin vitro transcription kit (MEGAshortscript ^TMKit) was purchased from Thermo Fisher Scientific. The catalog number is AM1354.

EcoRI, BamHI, and BbsI were purchased from NEB. The catalog numbers are R3101M, R3136M, and R0539L, respectively.

EXAMPLE 1: Generation of KIT gene mutant mice

The mouse KIT gene (NCBI Gene ID: 16590, Primary source: MGI: 96677, UniProt ID: P05532) is located at 75574987 to 75656722 of chromosome 5 (NC_000071.6) . The transcript sequence NM_021099.3 is set forth in SEQ ID NO: 1, and the corresponding protein sequence NP_066922.2 is set forth in SEQ ID NO: 2. Mouse and human KIT gene loci are shown in FIG. 1A and FIG. 1B, respectively.

One or more point mutations can be introduced into the coding sequence at the endogenous mouse KIT locus to generate mutated KIT genes. The encoded protein sequence has one or more amino acid mutations as compared to the wild-type protein. Specifically, gene editing technologies (e.g., CRISPR) can be used to introduce multiple point mutations in exon 13 of the KIT gene locus, such that the amino acid at position 660 of the KIT protein expressed in the mouse was mutated from threonine (T) to methionine (M) . The partial DNA sequence after the mutation is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

CRISPR/Cas gene editing technology was used to obtain the KIT gene mutant mice. The target sequences are important for the targeting specificity of sgRNAs and the efficiency of Cas9-induced cleavage. The sgRNA sequences were designed and synthesized to recognize the targeting sites, which are located within exon 13 of the KIT gene locus. The sequence of each sgRNA targeting site is as follows:

kit-sgRNA1 targeting site (SEQ ID NO: 5) : 5’ -CCACCGTGCATGCGCCAAGCAGG-3’

kit-sgRNA2 targeting site (SEQ ID NO: 6) : 5’ -CCTGCTTGGCGCATGCACGGTGG-3’

kit-sgRNA3 targeting site (SEQ ID NO: 7) : 5’ -CTGCTTGGCGCATGCACGGTGGG-3’

kit-sgRNA4 targeting site (SEQ ID NO: 8) : 5’ -GAACCTGCTTGGCGCATGCACGG-3’

The UCA kit was used to detect the activities of sgRNAs. The results showed that the sgRNAs had different activities (FIG. 2) . In particular, kit-sgRNA1 and kit-sgRNA3 exhibited relatively low activity, which may be caused by sequence variations. As shown inFIG. 2, the relative activities of kit-sgRNA1 and kit-sgRNA3 were still significantly higher than that of the negative control (NC) . Therefore, kit-sgRNA1 and kit-sgRNA3 were active and can be used for the gene editing experiment. Because of the relatively high activity, kit-sgRNA2 was selected for subsequent experiments. Oligonucleotides were added to the 5’ end and a complementary strand to obtain a forward oligonucleotide and a reverse oligonucleotide (see Table 2 for the sequence) . After annealing, the products were ligated to the pT7-sgRNA plasmid (the plasmid was first linearized with BbsI) , respectively, to obtain expression vector pT7-sgRNA2.

The pT7-sgRNA vector included a DNA fragment containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 9) , and was ligated to the backbone vector (Takara, Catalog number: 3299) by restriction enzyme digestion (EcoRI and BamHI) and ligation. The final plasmid was confirmed by sequencing.

Table 2

The targeting vector that targets mouse KIT gene was constructed as follows. The targeting vector includes a 5’ homologous arm (SEQ ID NO: 14) comprising part of exon 13 (with point mutations in KIT gene) , and exons 10-12 upstream of the mutation site; and a 3’ homologous arm (SEQ ID NO: 15) comprising downstream sequences of the mutation site. The 5' homologous arm includes a sequence that is identical to nucleic acids 75640475-75641426 of the NCBI Reference Sequence NC_000071.6 (corresponding to nucleic acids 1-952 of SEQ ID NO: 14) , except at three sites, i.e., C860T, G878A, and C938T, which did not affect protein translation and expression. The 3' homologous arm is identical to nucleic acids75641430-75642376 of the NCBI Reference SequenceNC_000071.6 (corresponding to nucleic acids 1-947 of SEQ ID NO: 15) , except at one site, i.e., G4T, which did not affect protein expression. A specific sequence “CAT” was inserted at the 3’ end of the 5’ homologous arm.

The targeting vector was constructed using standard methods, e.g., by restriction enzyme digestion and ligation, or direct synthesis. The constructed targeting vector sequence was preliminarily verified by restriction enzyme digestion, followed by verification by sequencing, and then used for subsequent experiments.

The pre-mixed Cas9 mRNA, the targeting vector, and in vitro transcription products of the pT7-sgRNA2 plasmid (using Ambion in vitro transcription kit to carry out the transcription according to the method provided in the product instruction) were injected into the cytoplasm or nucleus of NOD-Prkdc ^scid IL-2rγ ^null (B-NDG) mouse, or NOD/scid mouse fertilized eggs with a microinjection instrument. The embryo microinjection was carried out according to the method described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003. The injected fertilized eggs were then transferred to a culture medium to culture for a short time and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified mice (F0 generation) . The mouse population was further expanded by cross-mating and self-mating to establish stable mouse lines.

Because of the high immunodeficiency level of NOD-Prkdc ^scid IL-2rγ ^null (e.g., B-NDG) mice, when injected with its fertilized eggs, the resulting KIT gene mutant mice were highly immunodeficient with a clear genotypic background. The fertilized eggs of NOD/scid mice can also be selected for microinjection. The resulting KIT gene mutants can be further bred with NOD-Prkdc ^scid IL-2rγ ^null mice (or byin vitro fertilization) , and the offspring can be screened. According to Mendel’s law, there is a possibility to obtain heterozygous animal model (NOD/scid background) with KIT gene mutation and IL-2rg gene knockout. The heterozygous mice can then be bred with each other to produce highly immunodeficient KIT gene mutant mice.

Experiments were performed to identify somatic cell genotype of the F0 generation mice. For example, PCR analysis was performed using mouse tail genomic DNA of the F0 generation mice. DNA of the PCR bands with correct molecular weight in a gel were recovered and sequenced to confirm whether the mouse genome had the target point mutations. Next, the positive mice identified by sequencing were subjected to second round of PCR analysis on samples from mouse tails, and verified by re-sequencing. The positive F0 generation mice comprising the target point mutations were mated with wild-type C57BL/6 mice to generate F1 generation mice. Exemplary PCR results of the F0 mice are shown in FIG. 3. The same method (e.g., PCR) was used for genotypic identification of the F1 generation mice. As shown in FIG. 4, the results showed that mice numbered F1-1, F1-2, F1-3, F1-5, F1-6 and F1-9 were positive mice. Further sequencing confirmed that the 6 mice were positive heterozygotes and no random insertions were detected. This indicates that the method described above can be used to generate genetically engineered mice that can be stably passaged and comprises specific point mutations in the target gene. It was observed that the hair color of the genetically engineered mice and their offspring was white. As compared to wild-type mice, no obvious abnormalities of the genetically engineered mice were observed, except for a slightly smaller body size. The following primer pair was used for PCR analysis:

KIT-MUT-F1: 5’ -ACTGTTGGTTGGTCTTCCCACTGAC-3’ (SEQ ID NO: 16) ,

KIT-MUT-R1: 5’ -AGCCTAGTAGGGAAGTAACCAGGGA-3’ (SEQ IDNO: 17) .

EXAMPLE 2: Reconstruction of human immune system in immuno-deficient KIT gene mutant mice

6-week old mice with the KIT gene mutation (B-NDG background, n=13) and B-NDG mice (n=19) were selected. 1.5 × 10 ⁵ human hematopoietic stem cells (HSCs) were injected to the tail vein of irradiated (at 2.0 Gy) B-NDG mice and un-irradiated mice with the KIT gene mutation, to reconstruct the immune system. The reconstruction was regarded successful if proportion of hCD45+ cells were no less than 25%of the total viable cells after lysis of red blood cells. Peripheral blood (PB) was collected every four weeks after the injection and analyzed by flow cytometry. Mouse healthwas evaluated and overall survival was recorded.

The results showed that un-irradiated mice with KIT gene mutation (hereinafter referred to as "KIT B-NDG mice" ) and irradiated B-NDG mice (hereinafter referred to as "B-NDG mice" ) had good survival rate during the experimental period. At the end of the experiment (week 20, more specificallyday 136 after injection) , the survival rate of KIT B-NDG mice was 46.2%, while the survival rate of B-NDG mice was 31.6%. As shown in FIG. 5, asignificant difference of survival rates between the two groups was observed. In addition, it was observed that the overall health condition of KIT B-NDG mice was better than that of the B-NDG mice at the end of the experiment. For example, the B-NDG mice exhibited decreased mobility, and some hadhunched backs and sparse body hair.

Flow cytometry results showed that cells expressing human leukocyte surface marker (CD45+) can be detected in all mice from week 4. At week 12, the average percentage of human leukocytes observed in KIT B-NDG mice (61.13%±15.55) was 7 times higher than that of B-NDG mice (8.84%±4.00) . According to the dataof the entire experimental period (FIG. 6 and FIG. 7) , starting from week 8, the percentage of human leukocytes and the success rate of reconstruction in KIT B-NDG mice were significantly higher than those in B-NDG mice. The development of T cells (CD3+) , B cells (CD19+) and myeloid cells (CD33+) in the peripheral blood were further analyzed by flow cytometry. Starting from week 16, the development of NK cells (CD56+) , monocytes (CD14+) and granulocytes (CD66b+) were also analyzed (FIGS. 8-13) . The test results showed that the differentiation ratios of various types of cells in KIT B-NDG mice and B-NDG mice were similar, indicating that the KIT B-NDG mice allowed direct and stable transplantation of human hematopoietic stem cells. In KIT B-NDG mice, the percentage of T cells in human leukocytes and the percentages of monocytes and granulocytes in myeloid cells were all higher than those in B-NDG mice since week 16 (see Table 3, and FIGS. 8, 12, and 13) , indicating that the KIT B-NDG mice had better development of T cells, monocytes, granulocytes, etc. In addition, the following gating strategies were used in flow cytometry. Human leukocytes were gated as intact, single, live, hCD45+ and mCD45-cells. In the human leukocyte population, T cells (CD3+) were gated as intact, single, live, hCD45+, mCD45-, hCD3+, and hCD19-cells; B cells were gated as intact, single, live, hCD45+, mCD45 -, hCD3-, and hCD19+ cells; NK cells were gated as intact, single, live, hCD45+, mCD45-, hCD3-, and hCD56+ cells. Myeloid cells were gated as intact, single, live, hCD45+, mCD45-, and hCD33+ cells. In the myeloid cell population, monocytes were gated as intact, single, live, hCD45+, mCD45-, hCD33+, and hCD14+ cells; granulocytes were gated as intact, single, live, hCD45+, mCD45-, hCD33+ , and hCD66b+ cells.

Table 3Percentage of T cells in human leukocytes after immune system reconstruction

The above results showed that the mice with KIT gene mutations generated by the methods described herein can be directly used for immune system reconstruction (e.g., by injecting human hematopoietic stem cells (HSCs) ) without the treatment ofirradiation. In addition, the mice can effectively promote development of human cells in vivo, and increase the transplantation success rate of human tissues and cells.

The mice with reconstructed humanized immune system can be used to developtumor xenograft models, which are useful in drug screening, pharmacodynamic and clinical researches. Specifically, tumor tissues can be transplanted in KIT B-NDG mice about 8-16 weeks after CD34+ cells are injected. After the tumor grows to a certain size, the mice can be grouped and administered with anti-tumor drugs (e.g., antibodies) . Tumor volume, mouse body weight, and survival rate can be measured regularly, to evaluateefficacy and safetyofanti-tumor drugs or their combinations thereof.

EXAMPLE 3: Preparation of mice with two or more gene modifications

Mice with KIT gene mutations prepared using the methods as described in the present disclosure can also be used to prepare transgenic mice with double-or multi-gene modifications. For example, in Example 1, the fertilized eggs used in the microinjection can be selected from fertilized eggs of other genetically modified mice. Alternatively, fertilized eggs from the KIT gene mutant mice can be selected for gene editing, to obtain double-or multiple-gene modified mouse models. In addition, the KIT gene mutant homozygous or heterozygous mice can be bred with other genetically modified homozygous or heterozygous mice (or through in vitro fertilization) , and the progeny can be screened. According to Mendel’s law, it is possible to obtain double-gene or multiple-gene modified heterozygous mice with mutated KIT gene, and then the heterozygous mice can be bred with each other to obtain the double-gene or multiple-gene modified homozygous mice. These double-gene or multi-gene modified mice are useful for development of xenograft cell or tissue models, human pathology researches, drug screening, etc.

The non-human mammals described herein can also be prepared through other gene editing systems and approaches, including but not limited to: gene homologous recombination techniques based on embryonic stem cells (ES) , zinc finger nuclease (ZFN) techniques, transcriptional activator-like effector factor nuclease (TALEN) technique, homing endonuclease (megakable base ribozyme) , or other techniques. In the follow example, gene homologous recombination techniques based on embryonic stem cells (ES) are used to illustrate how to generate transgenic mice with KIT gene mutations.

Multiple point mutations in exon 13 of the KIT gene locus are introduced by the method described herein, such that the amino acid at position 660 of the KIT protein expressed in the mouse is mutated from threonine (T) to methionine (M) . A targeting vector that targets mouse KIT gene can be constructed to include a 5’ homologous arm, a 3’ homologous arm, and a mutant KIT gene fragment. The vector can also contain a resistance gene for positive clone screening, such as neomycin phosphotransferase coding sequence Neo. On both sides of the resistance gene, two site-specific recombination systems in the same orientation, such as Frt or LoxP, can be added. Furthermore, a coding gene with a negative screening marker, such as the diphtheria toxin A subunit coding gene (DTA) , can be constructed downstream of the recombinant vector 3’ homologous arm. Vector construction can be carried out using methods known in the art, such as enzyme digestion and ligation. Next, the recombinant vector with correct sequence can be transfected into mouse embryonic stem cells, such as C57BL/6 mouse embryonic stem cells. Next, the transfected cells are screened using the positive clone marker gene, and Southern Blot can be used for DNA recombination identification. The positive clone cells (black mice) are injected into the isolated blastocysts (white mice) by microinjection according to the method described in Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003. The resulting chimeric blastocysts formed following the injection are transferred to the culture medium for a short time culture and then transplanted into the fallopian tubes of the recipient mice (white mice) to produce F0 generation chimeric mice (black and white) . After PCR identification of the mouse tail genomic DNA, the F0 chimeric mice with correctly modified gene locus are selected for subsequent breeding and identification. The F1 generation mice are obtained by breeding the F0 chimeric mice with wild-type mice. By PCR identification of mouse tail genomic DNA, positive F1 generation heterozygous mice that can be stably passaged are selected, and then bred with each other to obtain F2 generation homozygous mice. In addition, the F1 generation heterozygous mice can also be mated with Flp or Cre mice to remove the positive clone screening marker gene (e.g., neo) , and homozygous mice can then be obtained by breeding these mice with each other. The genotypic and phenotypic verification methods of the F1 generation heterozygous mice or F2 generation homozygous mice are similar to the methods as described in the examples above.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

A genetically-modified, non-human animal expressing a KIT (CD117) protein, wherein the amino acid residuethat corresponds to T660 of SEQ ID NO: 2 in the KIT protein is hydrophobic.
The animal of claim 1, wherein the amino acid sequence of the KIT protein comprises a sequence is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 2 or 4.
The animal of claim 1, wherein the amino acid sequence of the KIT protein comprises a sequence that is atleast 80%identical to SEQ ID NO: 4.
The animal of claim 1, wherein the amino acid sequence of the KIT protein comprises a sequence that is identical to SEQ ID NO: 4.
The animal of any one of claims1-4, wherein the amino acid that corresponds to T660 of SEQ ID NO: 2 is Ala, Val, Ile, Leu, Met, Phe, Tyr, or Trp.
The animal of any one of claims 1-4, wherein the amino acid that corresponds to T660 of SEQ ID NO: 2 is Ala, Val, Ile, Leu, or Met.
The animal of any one of claims 1-4, wherein the amino acid that corresponds to T660 of SEQ ID NO: 2 is Met.
The animal of any one of claims 1-7, wherein the genome of the animal comprises a sequence that is at least 80%, 85%, 90%, 95%, or 100%identical to SEQ ID NO: 3.
The animal of any one of claims 1-8, wherein the genome of the animal comprises a disruption in the animal’s endogenous CD132 gene.
The animal of any one of claims 1-9, wherein the animal is a mammal, e.g., a monkey, a rodent, a rat, or a mouse.
The animal of any one of claims 1-9, wherein the animal is a NOD/scid mouse, a NOD/scid nude mouse, or a B-NDG mouse.
The animal of any one of claims 1-9, wherein the animal is a B-NDG mouse.
The animal of any one of claims 1-12, wherein the animal is heterozygous with respect to exon 13 of endogenous KIT gene.
The animal of any one of claims 1-12, wherein the animal is homozygous with respect to exon 13 of endogenous KIT gene.
The animal of any one of claims 1-14, wherein the animal does not express a wild-type KIT protein.
The animal of any one of claims 1-15, wherein the animal after being engrafted with human hematopoietic stem cells to develop a human immune system has one or more of the following characteristics:

(a) the percentage of human CD45+ cells is greater than 50%or 60%oftotal live cellsin the animal (e.g., at or after week 8, 12, 16, 18, or 20after the animal is engrafted) ;

(b) the percentage of human CD3+cells is greater than 5%or 10%of human CD45+cells in the animal (e.g., at or after week 12, 16, 18, or 20 after the animal is engrafted) ;

(c) the percentage of human CD19+cells is greater than 50%or 60%of human CD45+ cells in the animal (e.g., at or after week 4, 8, 12, 16, 18, or 20 after the animal is engrafted) ;

(d) the percentage of human CD56+ cells is greater than 2%or 5%of human CD45+cells in the animal (e.g., at or after week 16, 18, or 20 after the animal is engrafted) ;

(e) the percentage of human CD33+ cells is greater than 2%or 5%of human CD45+cells in the animal (e.g., at or after week 4, 8, 12, 16, 18, or 20 after the animal is engrafted) ;

(f) the percentage of human CD14+ cells is greater than 50%or 60%of human CD33+ cells in the animal (e.g., at or after week 16, 18, or 20 after the animal is engrafted) ; and

(g) the percentage of human CD66b+ cells is greater than 5%or 10%of human CD33+ cells in the animal (e.g., at or after week 16, 18, or 20 after the animal is engrafted) .
The animal of claim 16, wherein the animal is not irradiated before being engrafted.
The animal of claim 16 or 17, wherein the animal after being engrafted with human hematopoietic stem cells to develop a human immune system has better development of humanT cells, monocytes, and/or granulocytes relative to a B-NDG mouse, wherein the B-NDG mouse is irradiated before being engrafted.
The animal of claim 16 or 17, wherein the animal after being engrafted with human hematopoietic stem cells to develop a human immune system has a higher percentage of leukocytes in total live cells relative to a B-NDG mouse, wherein the B-NDG mouse is irradiated before being engrafted.
The animal of claim 16 or 17, wherein the animal after being engrafted with human hematopoietic stem cells to develop a human immune system has a higher success rate of reconstruction relative to a B-NDG mouse, wherein the B-NDG mouse is irradiated before being engrafted.
The animal of any one of claims1-20, wherein the animal has an enhanced engraftment capacity of exogenous cellsrelative to a B-NDG mouse.
A method of determining effectiveness of an agent or a combination of agents for treating cancer, comprising:

(a) engrafting tumor cells to the animal of any one of claims 1-18, thereby forming one or more tumors in the animal;

(b) administering the agent or the combination of agents to the animal; and

(c) determining the inhibitory effects on the tumors.
The method of claim 22, wherein before engrafting the tumor cells to the animal, human peripheral blood cells (hPBMC) or human hematopoietic stem cells are injected to the animal.
The method of claim 22 or 23, wherein the tumor cells are from cancer cell lines.
The method of claim 22 or 23, wherein the tumor cells are from a tumor sample obtained from a human patient.
The method of any one of claims22-25, wherein the inhibitory effects are determined by measuring the tumor volume in the animal.
The method of any one of claims22-26, wherein the tumor cells are melanoma cells, lung cancer cells, primary lung carcinoma cells, non-small cell lung carcinoma (NSCLC) cells, small cell lung cancer (SCLC) cells, primary gastric carcinoma cells, bladder cancer cells, breast cancer cells, and/or prostate cancer cells.
The method of any one of claims22-27, wherein the agent is an anti-CD47 antibody.
The method of any one of claims22-27, wherein the agent is an anti-PD-1 antibodyIL6 antibody or an IL15 antibody.
The method of any one of claims 22-29, wherein the combination of agents comprises one or more agents selected from the group consisting of paclitaxel, cisplatin, carboplatin, pemetrexed, 5-FU, gemcitabine, oxaliplatin, docetaxel, and capecitabine.
A method of producing an animal comprising a human hemato-lymphoid system, the method comprising:

engrafting a population of cells comprising human hematopoietic cells or human peripheral blood cells into the animal of any one of claims 1-21.
The method of claim 31, wherein the human hemato-lymphoid system comprises human cells selected from the group consisting of hematopoietic stem cells, myeloid precursor cells, myeloid cells, dendritic cells, monocytes, granulocytes, neutrophils, mast cells, lymphocytes, and platelets.
A method of producing a genetically-modified rodent, the method comprising

(a) providing a plasmid comprising a 5’ homologous arm and a 3’ homologous arm;

(b) providing a small guide RNA (sgRNA) that targets a sequence in exon 13 of the endogenous KIT gene;

(c) modifying genome of a rodent embryo by using the plasmid of step (1) , the sgRNA of step (2) , and Cas9; and

(d) transplanting the embryo to a receipt rodent to produce a genetically-modified rodent.
The method of claim 33, wherein the sgRNA targets SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
The method of claim 33, wherein the sgRNA targets SEQ ID NO: 6.
The method of any one of claims33-35, wherein the 5’ homologous arm is at least 80%identical to SEQ ID NO: 14 and the 3’ homologous arm is at least 80%identical to SEQ ID NO 15.
The method of any one of claims 33-36, wherein the plasmid further comprises a nucleic acid sequencethat is inserted between the 5’ homologous arm and the 3’ homologous arm, wherein the nucleic acid sequence is CAT.
The method of any one of claims 33-37, wherein the rodent is a mouse.
The method of any one of claims 33-38, wherein the method further comprises establishing a stable mouse line from progenies of the genetically-modified rodent.
The method of any one of claims 33-39, wherein the embryo has a NOD/scid background, a NOD/scid nude background, or a B-NDG background.
A method of producing a KIT gene mutant mouse, the method comprising the steps of:

(a) transforming a mouse embryonic stem cell with a gene editing system that targets endogenous KIT gene, thereby producing a transformed embryonic stem cell;

(b) introducing the transformed embryonic stem cell into a mouse blastocyst;

(c) implanting the mouse blastocyst into a pseudopregnant female mouse; and

(d) allowing the blastocyst to undergo fetal development to term, thereby obtaining the KIT gene mutant mouse.
A method of producing a KIT gene mutant mouse, the method comprising the steps of:

(a) transforming a mouse embryonic stem cell with a gene editing system that targets endogenous KIT gene, thereby producing a transformed embryonic stem cell;

(b) implanting the transformed embryonic cell into a pseudopregnant female mouse; and

(c) allowing the transformed embryonic cell to undergo fetal development to term, thereby obtaining the KIT gene mutant mouse.
The method of claim 41 or claim 42, wherein the gene editing system comprises a nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in exon 13 of the endogenous KIT gene.
The method of claim 43, wherein the nuclease is CRISPR associated protein 9 (Cas9) .
The method of claim 43, wherein the target sequence in exon 13 of the endogenous KIT gene is set forth in SEQ ID NO: 5, 6, 7, or 8.
The method of claim 43, wherein the target sequence in exon 13 of the endogenous KIT gene is set forth in SEQ ID NO: 6.
The method of any one of claims 41-46, wherein the mouse embryonic stem cell has a NOD/scid background, a NOD/scid nude background, or a B-NDG background.
A genetically-modified, non-human animal or a progeny thereof, wherein the animalis produced by a method comprising: mutating one or more nucleotides of endogenous KIT gene by using a nuclease comprising a zinc finger protein, a TAL-effector domain, or a single guide RNA (sgRNA) DNA-binding domain that binds to a target sequence in exon 13 of the endogenous KIT gene.
The animal of claim 48, wherein the nuclease is CRISPR associated protein 9 (Cas9) .
The animal of claim 48 or 49, wherein the target sequence in exon 13 of the endogenous KIT gene is set forth in SEQ ID NO: 5, 6, 7, or 8.
The animal of claim 48 or 49, wherein the target sequence in exon 13 of the endogenous KIT gene is set forth in SEQ ID NO: 6.