GDF8 gene modified non-human animal

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) GDF8 protein and methods of use thereof.

Description

Technical Field

[0001] The present invention provides a non-human animal expressing a human or chimeric (eg, humanized) GDF8 protein and methods of using the same. Background Art

[0002] Traditional drug development usually uses in vitro screening methods, but these screening methods cannot provide the body environment (such as tumor microenvironment, stromal cells, extracellular matrix components and immune cell interactions, etc.), resulting in a high failure rate of drug development. In addition, given the differences between humans and animals, the test results obtained from in vivo pharmacology tests using conventional experimental animals may not reflect the actual disease state and the interaction of the target site, resulting in significant differences between the results of many clinical trials and the results of animal experiments.

[0003] Therefore, developing a humanized animal model suitable for screening and evaluation of human antibodies will significantly improve the efficiency of new drug development and reduce the cost of drug development.

[0004] However, the construction of humanized animal models is very difficult. For example, the non-patent literature: Generation and utility of genetically humanized mouse models (Scheer N, Snaith M, Wolf CR, Seibler J., Drug Discovery Today; 18(23-24): 1200-11, 2013) discloses that the humanization of any gene carries certain risks, and even with carefully designed strategies, the expression and function of human genes in animals cannot be ensured. Summary of the invention

[0005] The present application provides an animal model with human or chimeric GDF8 protein. The animal model can express human or chimeric GDF8 (e.g., humanized GDF8) protein. It can be used for the study of GDF8 gene function, and can also be used for the screening and evaluation of GDF8 signaling pathway regulators (e.g., anti-human GDF8 antibodies, oligonucleotide drugs and / or polypeptide drugs). In addition, the animal model prepared by the construction method described in the present application can be used for drug screening, pharmacodynamics research, treatment of immune diseases and disease treatment of human GDF8 target sites; the animal model can also be used to promote new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of GDF8 protein and provides a platform for screening related drugs.

[0006] On the one hand, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human or chimeric myostatin (GDF8) protein. In some embodiments, the nucleotide sequence encoding a human or chimeric GDF8 protein is operably linked to an endogenous regulatory element or a human regulatory element (such as a 5'UTR and / or a promoter) of an endogenous GDF8 locus of at least one chromosome. In some embodiments, the expression of the nucleotide sequence encoding a human or chimeric GDF8 protein is regulated by a regulatory element, such as a 5'UTR of an endogenous GDF8 gene of a non-human animal and / or a promoter of an endogenous GDF8 gene of a non-human animal. In some embodiments, the regulatory element is such as a 3'UTR of a human GDF8 gene. In some embodiments, the chimeric GDF8 protein is a humanized GDF8 protein, and the humanized GDF8 protein comprises at least 50 to at least 375 consecutive amino acid sequences consistent with a human GDF8 protein. In some embodiments, the amino acid sequence of the human or chimeric GDF8 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375; or, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375; or, comprises an amino acid sequence that differs from SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or comprises an amino acid sequence that is substituted, deleted and / or inserted with one or more amino acids as shown in SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent comprises a mouse or a rat. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal does not express endogenous GDF8 protein or expresses reduced levels of GDF8 compared to wild-type animals. In some embodiments, one or more cells of the non-human animal express human or chimeric GDF8 protein.

[0007] On the one hand, the present invention provides a genetically modified non-human animal or a method for constructing it, wherein the endogenous GDF8 gene of the non-human animal is replaced with a nucleotide sequence comprising human GDF8 at the endogenous GDF8 locus of the non-human animal. In some embodiments, the corresponding region of the endogenous GDF8 gene of the non-human animal is replaced with a nucleotide sequence comprising human GDF8 at the endogenous GDF8 locus of the non-human animal. In some embodiments, the nucleotide sequence of human GDF8 is operably linked to an endogenous regulatory element of the endogenous GDF8 locus of the non-human animal, such as a 5'UTR and / or a promoter. In some embodiments, one or more cells of the non-human animal express a human or humanized GDF8 protein. In some embodiments, the endogenous GDF8 protein of the non-human animal is not expressed or the expression level is reduced compared to GDF8 in wild-type animals. In some embodiments, the nucleotide sequence of human GDF8 comprises a portion of exon 1 to all of exon 3 of the human GDF8 gene; wherein the portion of exon 1 of the human GDF8 gene preferably comprises at least 50bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence of human GDF8 comprises the nucleotide sequence of the coding region of the human GDF8 gene. In some embodiments, the nucleotide sequence of human GDF8 comprises the nucleotide sequence from the start codon to the stop codon of the human GDF8 gene. In some embodiments, the nucleotide sequence of human GDF8 comprises the 3'UTR of the human GDF8 gene. In some embodiments, the nucleotide sequence of human GDF8 comprises at least 50bp of continuous nucleotides downstream of the 3'UTR of the human GDF8 gene. In some embodiments, the nucleotide sequence of human GDF8 comprises the nucleotide sequence from the start codon to the stop codon of the human GDF8 gene and the 3'UTR of the human GDF8 gene and at least 50bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human GDF8 comprises at least 50bp of continuous nucleotides from part of exon 1 of the human GDF8 gene to the downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human GDF8 comprises SEQ ID NO: 5; or, comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the nucleotide sequence shown in SEQ ID NO: 5; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 5 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 bp; or, comprises a nucleotide sequence that substitutes, deletes and / or inserts one or more nucleotides shown in SEQ ID NO: 5. In some embodiments, part of exon 1 to all of exon 3 of the endogenous GDF8 gene of a non-human animal is replaced; wherein, part of exon 1 of the endogenous GDF8 gene of a non-human animal preferably comprises at least 50 bp of continuous nucleotides.In some embodiments, the replaced non-human animal endogenous GDF8 gene also comprises at least 50bp of continuous nucleotides downstream of the 3'UTR of the non-human animal endogenous GDF8 gene. In some embodiments, part of exon 1 of the non-human animal endogenous GDF8 gene is replaced to at least 50bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the modified GDF8 gene in the non-human animal genome is homozygous or heterozygous for the endogenous replaced locus. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent includes a rat or a mouse. In some embodiments, the mRNA transcribed from the modified GDF8 gene in the genome of the non-human animal comprises SEQ ID NO: 6; or, comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the nucleotide sequence shown in SEQ ID NO: 6; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 6 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 bp; or, comprises a nucleotide sequence that replaces, deletes and / or inserts one or more nucleotides shown in SEQ ID NO: 6. In some embodiments, the non-human animal comprises nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins preferably comprise at least one of ANGPTL3, GPR75, NPR1, IL36R, NKP46, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0008] In one aspect, the present invention provides a non-human animal or a method for constructing the same, wherein the non-human animal comprises at least one cell encoding a nucleotide sequence of a human or humanized GDF8 protein, wherein the humanized GDF8 protein comprises at least 50, 100, 200, 300, 310, 320, 330, 340, 350, 351, 352, 353, 354, 355, 360, 370, 371, 372, 373, 374 or 375 consecutive amino acid sequences that are identical to human GDF8 protein. In some embodiments, the non-human animal expresses a human or humanized GDF8 protein. In some embodiments, the nucleotide sequence encoding a human or humanized GDF8 protein is operably linked to an endogenous GDF8 regulatory element. In some embodiments, the nucleotide sequence encoding a human or humanized GDF8 protein can be integrated into the endogenous GDF8 locus of the non-human animal. In some embodiments, the human or humanized GDF8 protein has at least one activity, such as a non-human animal GDF8 activity and / or a human GDF8 activity.

[0009] On the one hand, the present invention provides a method for constructing a genetically modified non-human animal, in at least one cell of the non-human animal, at the endogenous GDF8 locus of the non-human animal, the nucleotide sequence encoding the endogenous GDF8 region of the non-human animal is replaced by the nucleotide sequence encoding the corresponding region of human GDF8. In some embodiments, the endogenous GDF8 protein of the non-human animal is not expressed or the expression level is reduced compared with GDF8 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human GDF8 comprises all or part of exon 1, exon 2 and / or exon 3 of the human GDF8 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human GDF8 comprises at least 50bp of continuous nucleotides from part of exon 1 of the human GDF8 gene to the downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human GDF8 comprises at least 300bp of continuous nucleotides from part of exon 1 of the human GDF8 gene to the downstream of the 3'UTR. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human GDF8 comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375. In some embodiments, the nucleotide sequence encoding the corresponding region of human GDF8 comprises SEQ ID NO: 5, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous GDF8 region of a non-human animal comprises all or part of exon 1, exon 2 and / or exon 3 of an endogenous GDF8 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding the endogenous GDF8 region of a non-human animal comprises a portion of exon 1 of an endogenous GDF8 gene of a non-human animal to at least 50bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous GDF8 region of a non-human animal comprises a portion of exon 1 of an endogenous GDF8 gene of a non-human animal to 350bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human GDF8 is operably linked to an endogenous GDF8 regulatory element, such as a 5'UTR and / or a promoter. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent comprises a mouse or a rat.In some embodiments, the non-human animal further comprises nucleotide sequences of human or chimeric proteins encoded by other genes, wherein the other genes comprise at least one of ANGPTL3, GPR75, NPR1, IL36R, NKP46, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0010] On the one hand, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric GDF8, the construction method comprising replacing a nucleotide sequence encoding an endogenous GDF8 region of a non-human animal with a nucleotide sequence encoding a corresponding region of human GDF8 at an endogenous non-human animal GDF8 locus, producing a genetically modified non-human animal cell, the genetically modified non-human animal cell expressing a human or chimeric GDF8 protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human GDF8 comprises all or part of exon 1, exon 2, and / or exon 3 of the human GDF8 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human GDF8 comprises at least 50bp of continuous nucleotides from a portion of exon 1 of the human GDF8 gene to the downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human GDF8 comprises at least 300bp of continuous nucleotides from a portion of exon 1 of the human GDF8 gene to the downstream of the 3'UTR. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human GDF8 comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375. In some embodiments, the nucleotide sequence encoding the corresponding region of human GDF8 comprises SEQ ID NO: 5, or comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, the nucleotide sequence encoding the endogenous GDF8 region of a non-human animal comprises all or part of exon 1, exon 2 and / or exon 3 of an endogenous GDF8 gene of a non-human animal. In some embodiments, the nucleotide sequence encoding the endogenous GDF8 region of a non-human animal comprises a portion of exon 1 of an endogenous GDF8 gene of a non-human animal to at least 50bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous GDF8 region of a non-human animal comprises a portion of exon 1 of an endogenous GDF8 gene of a non-human animal to 350bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding a human or chimeric GDF8 protein is operably linked to a regulatory element of endogenous GDF8, such as a 5'UTR and / or a promoter. In some embodiments, the non-human animal is a mouse.

[0011] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating a disease, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the above-mentioned construction method, wherein the non-human animal suffers from a disease; 2) determining the inhibitory effect of the therapeutic agent on the disease. In some embodiments, the therapeutic agent comprises a GDF8 therapeutic agent, and the GDF8 therapeutic agent comprises one or more of an anti-GDF8 antibody, an oligonucleotide drug, or a polypeptide drug. In some embodiments, the therapeutic agent further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the disease comprises a tumor, a metabolic disease, a neuronal disease, an inflammation, or an immune disease. In some embodiments, the tumor comprises one or more of endocrine cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, lung cancer, or pancreatic cancer. In some embodiments, the metabolic disease comprises one or more of diabetes, obesity, hyperthyroidism, hypoglycemia, hyperlipidemia, or hypercalcemia. In certain embodiments, the neuronal disease includes one or more of muscular dystrophy, amyotrophic lateral sclerosis or spinal muscular atrophy. In certain embodiments, the inflammation includes one or more of rheumatoid arthritis, atopic dermatitis, myocarditis. In certain embodiments, the immune disease includes osteoporosis, myasthenia gravis, graft-versus-host disease (GVHD), psoriasis, allergy, asthma, myocarditis, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune liver disease, pain or nervous system disease, rheumatoid arthritis, atopic dermatitis, Crohn's disease, systemic lupus erythematosus or scleroderma. In certain embodiments, the tumor includes one or more tumor cells injected into non-human animals. In certain embodiments, the inhibitory effect of therapeutic agents on tumors is determined by measuring the tumor volume of non-human animals.

[0012] In one aspect, the present invention provides a method for determining the toxicity of a GDF8 therapeutic agent, the method comprising: 1) administering a GDF8 therapeutic agent to the non-human animal or the non-human animal obtained by the above-mentioned construction method; 2) determining the effect of the GDF8 therapeutic agent on the non-human animal. In some embodiments, the determination of the effect of the GDF8 therapeutic agent on the non-human animal comprises measuring the weight of the non-human animal or a blood test. In some embodiments, the blood test includes one or more of red blood cell count, hematocrit, or hemoglobin content.

[0013] In one aspect, the present invention provides a humanized GDF8 gene, which comprises any of the following nucleotide sequences: A) encoding SEQ ID NO: 2 or a nucleotide sequence encoding positions 24-375 of SEQ ID NO: 2; B) SEQ ID NO: 3, 4, 5, 6, 7, 9, 10, 25 or 26; C) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, 4, 5, 6, 7, 9, 10, 25 or 26; D) a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 4, 5, 6, 7, 9, 10, 25 or 26.

[0014] In one aspect, the present invention provides a cell, tissue or organ, wherein the cell, tissue or organ comprises the above-mentioned humanized GDF8 gene.

[0015] In one aspect, the present invention provides an animal model, wherein the animal model comprises the above-mentioned humanized GDF8 gene.

[0016] In one aspect, the present invention provides an application of the above-mentioned non-human animal, the non-human animal obtained by the above-mentioned construction method, the cell obtained by the above-mentioned construction method, the above-mentioned humanized GDF8 gene, the above-mentioned cell, tissue or organ or the above-mentioned animal model. In some embodiments, the application includes: A) application in product development involving GDF8-related immune processes in human cells; B) application in a model system related to GDF8 for pharmacology, immunology, microbiology and medical research; C) application involving the production and use of animal experimental disease models for GDF8-related etiology research and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) application in the screening, efficacy detection, evaluation of efficacy, verification or evaluation of human GDF8 signaling pathway regulators in vivo; or, E) study of GDF8 gene function, study of drugs and efficacy targeting human GDF8 target sites, and study of GDF8-related tumors, metabolic diseases, neuronal diseases, inflammation or immune diseases.

[0017] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. This application describes methods and materials for use in the present invention; other suitable methods and materials known in the art may be used. The materials, methods, and examples are exemplary and non-limiting only. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0018] The term "all or part" in the present invention, "all" refers to the whole, and "part" refers to a part of the whole, or an individual part that constitutes the whole.

[0019] The term "locus" of the present invention refers to the position of a gene on a chromosome in a broad sense, and refers to a DNA fragment on a gene in a narrow sense, which can be a gene or a part of a gene. For example, the "GDF8 locus" refers to a DNA fragment of any section of the GDF8 gene. In some embodiments, the replaced non-human animal endogenous GDF8 locus can be a DNA fragment of any section of exons 1-3 of the non-human animal endogenous GDF8 gene.

[0020] The term "portion of exon XX" of the present invention means that continuous or intermittent several, dozens or hundreds of nucleotide sequences are consistent with the entire exon nucleotide sequence, for example, a portion of exon 1 of the human GDF8 gene, including continuous or intermittent 5-506bp, for example 5, 10, 50, 100, 150, 200, 250, 300, 350, 370, 371, 372, 373, 374, 375, 400, 450, 500, 505 or 506bp nucleotide sequences are consistent with the nucleotide sequence of exon 1 of the human GDF8 gene.

[0021] The term "exon XX-XXX" of the present invention refers to exons and introns therebetween, for example, exons 1-3 include the entire nucleotide sequence of exon 1, intron 1, exon 2, intron 2 and exon 3.

[0022] The term "part of exon XX to all of exon XXX" in the present invention refers to the nucleotide sequence from part of one exon to all of another exon and the introns in between, for example, the nucleotide sequence from part of exon 1 to all of exon 3 includes part of exon 1, all of intron 1, all of exon 2, all of intron 2 and all of exon 3.

[0023] The term "intron xx" in the present invention refers to an intron between two exons, for example, intron 1 refers to an intron between exon 1 and exon 2.

[0024] The term "comprising" or "including" in the present invention is an open-ended writing method, which contains the specified components or steps described, as well as other specified components or steps that will not be substantially affected. When used to describe a protein or nucleic acid sequence, the protein or nucleotide may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleotide, but still have the same or similar activity as the original sequence.

[0025] The term "and / or" of the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in the present application. For example, "A and / or B" includes "A", "B" and "A and B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0026] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description as described below.

[0027] Detailed description

[0028] GDF8

[0029] In the human genome, the GDF8 gene (Gene ID: 2660) contains three exons, namely exon 1, exon 2 and exon 3 ( Figure 1 ). The nucleotide sequence of human GDF8 mRNA is NM_005259.3, and the amino acid sequence of human GDF8 is NP_005250.1 (SEQ ID NO: 2). The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_005259.3 and its encoded protein NP_005250.1 is shown in Table 1:

[0030] Table 1

[0031]

[0032]

[0033] In the mouse genome, the GDF8 gene (Gene ID: 17700) contains three exons, namely exon 1, exon 2 and exon 3 ( Figure 1 ). The nucleotide sequence of mouse GDF8 mRNA is NM_010834.3, and the amino acid sequence of mouse GDF8 is NP_034964.1 (SEQ ID NO: 1). The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_010834.3 and its encoded protein NP_034964.1 is shown in Table 2:

[0034] Table 2

[0035]

[0036] GDF8 genes, proteins and gene loci of other species are also known in the art. For example, Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog), Sus scrofa (pig). The relevant information of these genes (such as intron sequence, exon sequence and amino acid sequence) can be found in NCBI, the entire contents of which are incorporated into this application by reference.

[0037] The present invention provides a human or chimeric (e.g., humanized) GDF8 protein, or a human or chimeric (e.g., humanized) GDF8 gene. In some embodiments, the amino acid sequence of the humanized GDF8 protein is identical to that of the human GDF8 protein in at least 50 to at least 375, such as at least 50, 100, 200, 300, 310, 320, 330, 340, 350, 351, 352, 353, 354, 355, 360, 370, 371, 372, 373, 374 or 375 consecutive amino acid sequences. In some embodiments, the amino acid sequence of the human or humanized GDF8 protein includes SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375; or, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375; or, comprises an amino acid sequence that differs from SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or comprises an amino acid sequence that substitutes, deletes and / or inserts one or more amino acids as shown in SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375. In some embodiments, the endogenous GDF8 gene of a non-human animal or the amino acid sequence encoded thereof is replaced by the corresponding nucleotide sequence of the human GDF8 gene or the amino acid sequence encoded thereof. In some embodiments, a "part" of exon 1, exon 2 and / or exon 3 of the endogenous GDF8 gene of a non-human animal is replaced by a corresponding nucleotide sequence or amino acid sequence of a human GDF8 gene. The "part" refers to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, , 2500, 2579, 2600, 2700, 2705, 3000, 3055, 3500, 4000, 4500, 5000, 5500, 6000, 6400, 6500, 6440, 6500, 6600, 6662, 6663, 6664, 6665 or 6790 bp of continuous nucleotide sequence, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 350 or 376 continuous amino acid sequence.In some embodiments, the "part" is identical or identical to all or part of exon 1, exon 2 and / or exon 3 of the endogenous GDF8 gene of a non-human animal by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99%. In some embodiments, the "part" or "whole" sequence of exon 1, exon 2 and / or exon 3 of the endogenous GDF8 gene of a non-human animal (e.g., a part of exon 1 of the endogenous GDF8 gene of a non-human animal to at least 50 bp of continuous nucleotide sequence downstream of 3'UTR) is replaced by the "part" or "whole" sequence of the corresponding exon 1, exon 2 and / or exon 3 of the human GDF8 gene (e.g., a part of exon 1 of the human GDF8 gene to at least 50 bp of continuous nucleotide sequence downstream of 3'UTR). In some embodiments, the corresponding nucleotide sequence or amino acid sequence of the human GDF8 gene refers to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500, 3000, 4000, 4378, 5000, 6000, 6500, 6800, 40, 50, 60, 70, 80, 90, 100, 200, 300, 350, 357, 360, 370 or 375 consecutive amino acid sequences.

[0038] In some embodiments, the present invention provides a genetically modified non-human animal, the genome of which comprises a human or chimeric (e.g., humanized) GDF8 nucleotide sequence. In some embodiments, the protein encoded by the human or chimeric (e.g., humanized) GDF8 nucleotide sequence comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375; or, comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95% or 99% identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375; or, comprises an amino acid sequence that differs from SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or comprises an amino acid sequence that is substituted, deleted and / or inserted with one or more amino acids shown in SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375. In some embodiments, the non-human animal genome comprises a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 95% or 99% identical to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 9, 10, 25 or 26.

[0039] In some embodiments, the non-human animal comprises a human or humanized GDF8 gene. In some embodiments, the humanized GDF8 gene comprises 3 exons. In some embodiments, the humanized GDF8 gene comprises humanized exon 1, human exon 2, and human exon 3. In some embodiments, the humanized GDF8 gene comprises a non-human animal endogenous or human or humanized 5'UTR. In some embodiments, the humanized GDF8 gene comprises a non-human animal endogenous or human or humanized 3'UTR. In some embodiments, the humanized GDF8 gene comprises a non-human animal endogenous 5'UTR. In some embodiments, the humanized GDF8 gene comprises a human 3'UTR.

[0040] In some embodiments, the genetically modified non-human animal can express human GDF8 and / or chimeric (e.g., humanized) GDF8 protein. In some embodiments, the endogenous GDF8 gene sequence of the non-human animal is replaced by the human GDF8 gene and / or nucleotide sequence, so that it can express human GDF8 and / or chimeric (e.g., humanized) GDF8 protein. In some embodiments, the amino acid sequence of human GDF8 encoded by the human GDF8 gene and / or nucleotide sequence is consistent or identical to the amino acid sequence shown in human GDF8 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the nucleotide sequence of the endogenous GDF8 gene of the non-human animal is replaced in whole or in part by the nucleotide sequence encoding the mature human GDF8 protein.

[0041] In some embodiments, the genetically modified non-human animal expresses human GDF8 and / or chimeric GDF8 protein (e.g., humanized GDF8) under endogenous regulatory elements (such as promoters and / or 5'UTR) of the non-human animal. Replacement of the endogenous GDF8 locus of the non-human animal provides a non-human animal that expresses human or chimeric GDF8 protein (e.g., humanized GDF8) in the same type of cells. The genetically modified non-human animal (e.g., mouse) does not show potential diseases observed in certain other transgenic non-human animals (e.g., mice) known in the art. The human GDF8 or chimeric GDF8 protein expressed in the non-human animal can maintain the function of one or more wild-type and / or human GDF8 proteins. In some embodiments, the genetically modified non-human animal does not express endogenous GDF8 protein. In some embodiments, the endogenous GDF8 protein expression of the genetically modified non-human animal is reduced. The "endogenous GDF8 protein" refers to the GDF8 protein encoded by the endogenous GDF8 nucleotide sequence of the non-human animal (e.g., mouse) before genetic modification.

[0042] In some embodiments, the genome of the genetically modified non-human animal comprises a nucleotide sequence encoding an amino acid that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the amino acid sequence of all or part of human GDF8 protein (e.g., SEQ ID NO: 2 or SEQ ID NO: 2, positions 24-375). In some embodiments, the genome of the genetically modified non-human animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the nucleotide sequence of SEQ ID NO: 5.

[0043] In some embodiments, one or more cells of the genetically modified non-human animal express a human or chimeric GDF8 protein (e.g., a humanized GDF8 protein). In some embodiments, the human or chimeric GDF8 protein comprises at least 50, 100, 200, 300, 310, 320, 330, 340, 350, 351, 352, 353, 354, 355, 360, 370, 371, 372, 373, 374, or 375 consecutive amino acids identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 2, positions 24-375.

[0044] In some embodiments, the genome of the genetically modified non-human animal comprises all or part of exon 1, exon 2, and exon 3 of the human GDF8 gene, or all or part of the nucleotide sequence shown in SEQ ID NO:5.

[0045] In some embodiments, the modified GDF8 gene in the genome of the genetically modified non-human animal is heterozygous or homozygous for the endogenous modified locus.

[0046] In some embodiments, a humanized GDF8 genome is provided. In some embodiments, the humanized GDF8 genome comprises the 5'UTR of the human GDF8 gene. In some embodiments, the humanized GDF8 genome comprises the (e.g., mouse) 5'UTR of the endogenous GDF8 gene of a non-human animal. In some embodiments, the humanized GDF8 genome comprises the 3'UTR of the human GDF8 gene. In some embodiments, the humanized GDF8 genome comprises the (e.g., mouse) 3'UTR of the endogenous GDF8 gene of a non-human animal. Where appropriate, based on the similarity of the GDF8 sequences of humans and non-human animals, it can be reasonably inferred that the non-human animal and human GDF8 genes are subject to similar regulation. As described herein, the non-human animal with humanized GDF8 gene comprises a replacement of the endogenous GDF8 locus of a non-human animal, which retains the endogenous regulatory elements of the non-human animal, but comprises a human or humanized GDF8 coding sequence. The expression of GDF8 in a genetically modified heterozygous non-human animal or homozygous non-human animal is completely normal.

[0047] In some embodiments, the present invention provides a genetically modified non-human animal, the genome of which comprises a deletion of an endogenous GDF8 gene of the non-human animal. In some embodiments, the deletion of an endogenous GDF8 gene of the non-human animal comprises exon 1, exon 2 and / or exon 3, or a portion of an endogenous GDF8 locus.

[0048] In some embodiments, the deletion of the endogenous GDF8 gene comprises one or more exons or portions of exons selected from exon 1, exon 2, and / or exon 3.

[0049] In some embodiments, the endogenous GDF8 gene deletion further comprises one or more introns or portions of introns, wherein the introns are selected from intron 1 and / or intron 2 of the GDF8 gene.

[0050] The present invention provides a humanized GDF8 genomic DNA sequence; a construct expressing the amino acid sequence of the humanized GDF8 protein; a cell comprising the construct; and a tissue or organ comprising the cell.

[0051] In some embodiments, the present invention provides a chimeric (e.g., humanized) GDF8 nucleotide sequence and / or amino acid sequence. In some embodiments, the chimeric GDF8 nucleotide sequence and / or amino acid sequence is homologous to an endogenous GDF8 mRNA (e.g., NM_010834.3) or an endogenous GDF8 amino acid sequence (e.g., NP_034964.1, SEQ ID NO: 1). ID NO: 1), or a portion thereof (e.g., a portion of exon 1, such as a 5'UTR) having a sequence identity or identity of 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% or 99%. In some embodiments, the chimeric GDF8 nucleotide sequence and / or amino acid sequence 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% or 99% identical to a human GDF8 mRNA sequence (e.g., NM_005259.3), a human GDF8 amino acid sequence (e.g., NP_005250.1, SEQ ID NO: 2), or a portion thereof (e.g., a portion of exon 1 to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR).

[0052] In some embodiments, the nucleotide sequence encoding amino acids 1-376 or 25-376 of non-human animal GDF8 (eg, SEQ ID NO: 1) is replaced by a nucleotide sequence encoding amino acids 1-375 or 24-375 of human GDF8 (eg, SEQ ID NO: 2).

[0053] In some embodiments, the above nucleotides are operably linked to endogenous regulatory elements of non-human animals, such as endogenous GDF8 promoters, inducible promoters, and enhancers of non-human animals.

[0054] In some embodiments, at least a portion of the chimeric GDF8 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, for example, a continuous or non-continuous nucleotide sequence) is different from all or part of the non-human animal GDF8 nucleotide sequence (e.g., part of exon 1 to all of exon 3 of the mouse GDF8 gene transcript NM_010834.3, preferably also including at least 50bp of continuous nucleotide sequence downstream of the 3'UTR of the mouse GDF8 gene).

[0055] In some embodiments, at least a portion of the chimeric GDF8 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., a continuous or non-contiguous nucleotide sequence) is identical to all or part of a non-human animal GDF8 nucleotide sequence (e.g., a portion of exon 1 of the mouse GDF8 gene transcript NM_010834.3, e.g., the 5'UTR of the mouse GDF8 gene).

[0056] In some embodiments, at least a portion of the chimeric GDF8 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, e.g., a continuous or non-contiguous nucleotide sequence) is different from all or a portion of a human GDF8 nucleotide sequence (e.g., a portion of exon 1 of human GDF8 gene transcript NM_005259.3, e.g., human GDF8 gene 5'UTR).

[0057] In some embodiments, at least a portion of the chimeric GDF8 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides, for example, a continuous or non-continuous nucleotide sequence) is identical to all or part of the human GDF8 nucleotide sequence (e.g., part of exon 1 to all of exon 3 of the human GDF8 gene transcript NM_005259.3, preferably also including at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR of the human GDF8 gene).

[0058] In some embodiments, at least a portion of the amino acid sequence encoded by the chimeric GDF8 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, such as continuous or non-contiguous amino acid residues) is different from all or part of the amino acid sequence of the non-human animal GDF8 protein (e.g., amino acids 1-376 or 25-376 of the mouse GDF8 protein sequence NP_034964.1 (SEQ ID NO: 1)).

[0059] In some embodiments, at least a portion of the amino acid sequence encoded by the chimeric GDF8 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, consecutive or non-consecutive amino acid residues) is identical to all or part of the amino acid sequence of a non-human animal GDF8 protein (e.g., amino acids 1-24 of the mouse GDF8 protein sequence NP_034964.1 (SEQ ID NO: 1)).

[0060] In some embodiments, the amino acid sequence encoded by the chimeric GDF8 nucleotide sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from all or part of the amino acid sequence of the human GDF8 protein.

[0061] In some embodiments, at least a portion of the amino acid sequence encoded by the chimeric GDF8 nucleotide sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acid residues, for example, consecutive or non-consecutive amino acid residues) is identical to all or part of the amino acid sequence of the human GDF8 protein (e.g., amino acids 1-375 or 24-375 of the human GDF8 protein sequence NP_005250.1 (SEQ ID NO: 2)).

[0062] The present invention also provides a humanized GDF8 amino acid sequence, wherein the amino acid sequence comprises any one of the following groups:

[0063] A) the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 2, positions 24 to 375;

[0064] B) is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO:2 or the amino acid sequence set forth in positions 24-375 of SEQ ID NO:2;

[0065] C) an amino acid sequence encoded by a nucleotide sequence, wherein the nucleotide sequence is capable of hybridizing under low stringency conditions or stringent conditions with a nucleotide sequence encoding the amino acids set forth in SEQ ID NO: 2 or SEQ ID NO: 2 at positions 24 to 375;

[0066] D) differs from SEQ ID NO:2 or the amino acid sequence set forth in positions 24-375 of 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

[0067] E) an amino acid sequence comprising a substitution, deletion and / or insertion of one or more amino acid residues as shown in SEQ ID NO: 2 or SEQ ID NO: 2 at positions 24-375.

[0068] The present invention also provides a humanized GDF8 nucleotide (eg, DNA or RNA) sequence, wherein the nucleotide sequence comprises any one of the following groups:

[0069] A) a nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 9, 10, 25 and / or 26, or a nucleotide sequence encoding a human GDF8 homologous amino acid sequence;

[0070] B) a nucleotide sequence that can hybridize to the nucleotide sequence shown in SEQ ID NO: 3, 4, 5, 6, 7, 9, 10, 25 and / or 26 under low stringency conditions or stringent conditions;

[0071] C) a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO: 3, 4, 5, 6, 7, 9, 10, 25 and / or 26;

[0072] D) the amino acid sequence encoded by it is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:2 at positions 24-375;

[0073] E) the encoded amino acid sequence differs from SEQ ID NO:2 or the amino acid sequence set forth in positions 24-375 of 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

[0074] F) The amino acid sequence encoded by the amino acid sequence is the same as that shown in SEQ ID NO: 2 or SEQ ID NO: 2 from position 24 to position 375, including substitution, deletion and / or insertion of one or more amino acid residues.

[0075] The present invention further provides a humanized GDF8 genomic DNA sequence, which is obtained by reverse transcription of the mRNA transcribed from the humanized GDF8 and is consistent with or complementary to a DNA sequence homologous to the sequence shown in SEQ ID NO: 5 or 6.

[0076] In order to determine the percent identity of two amino acid sequences or two nucleotide sequences, the sequences are compared for the purpose of optimal comparison (e.g., for optimal comparison, gaps can be introduced in one or both of the first and second amino acid or nucleotide sequences, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides at the 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, 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, which needs to be introduced to achieve the optimal comparison of the two sequences, taking into account the number of gaps and the length of each gap. For example, the comparison of the sequences and the determination of the percent identity between the two sequences can be completed using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0077] The percentage (homology percentage) of conservative residues with similar physicochemical properties, such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage is higher than the identity percentage.

[0078] The present invention also provides cells, tissues, organs or non-human animals (e.g., mice) comprising a nucleotide sequence of human GDF8, as well as cells, tissues, organs or non-human animals (e.g., mice) expressing human or chimeric (e.g., humanized) GDF8 proteins at the endogenous GDF8 locus of the non-human animal.

[0079] Genetically modified non-human animals

[0080] The "genetically modified non-human animal" of the present invention refers to a non-human animal in which at least one chromosome in the genome has exogenous DNA. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal have exogenous DNA. The cells with exogenous DNA can be various cells, for example, somatic cells, immune cells (including T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells), germ cells, blastocysts or tumor cells. In some embodiments, a genetically modified non-human animal is provided, the non-human animal comprises a modified endogenous GDF8 locus, the modified endogenous GDF8 locus comprises an exogenous sequence (e.g., a human sequence), for example, one or more non-human sequences are replaced with one or more human sequences, or one or more human and / or non-human sequences are inserted. Non-human animals are generally able to pass genetic modifications to offspring through germline transmission.

[0081] The "chimeric (x) gene" or "chimeric (x) nucleic acid" of the present invention refers to a gene or nucleic acid, wherein two or more parts of the gene or nucleic acid are from different species, or at least a portion of the nucleotide sequence of the gene or nucleic acid is different from the nucleotides in the wild-type animal. In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid has at least a portion of the nucleotide sequence having two or more different species sources, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid refers to a humanized (x) gene or a humanized (x) nucleic acid.

[0082] The "chimeric (x) protein" or "chimeric (x) polypeptide" of the present invention refers to a protein or polypeptide, wherein two or more parts of the polypeptide or protein are from different species, or at least a portion of the amino acid sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the amino acid sequence of a chimeric (x) protein or chimeric (x) polypeptide has two or more different species sources, for example, the same (or homologous) proteins of different species. In some embodiments, a chimeric (x) protein or chimeric (x) polypeptide refers to a humanized (x) protein or humanized (x) polypeptide.

[0083] The "humanized (x) protein" or "humanized (x) polypeptide" of the present invention refers to a protein or polypeptide, wherein at least a portion of the protein or polypeptide is derived from a human (x) protein or human (x) polypeptide. In some embodiments, the humanized (x) protein or humanized (x) polypeptide refers to a human (x) protein or human (x) polypeptide.

[0084] The "humanized (x) nucleic acid" or "humanized (x) gene" of the present invention refers to a nucleic acid, wherein at least a portion of the nucleic acid is derived from a human. In some embodiments, at least a portion of the nucleic acid is derived from a non-human animal. In some embodiments, the humanized (x) nucleic acid or humanized (x) gene refers to a humanized exon, and the humanized exon can be a human exon or a human / non-human animal chimeric exon.

[0085] In some embodiments, the chimeric (x) gene or chimeric (x) nucleic acid is a humanized GDF8 gene or a humanized GDF8 nucleic acid. In some embodiments, at least a portion of the humanized GDF8 gene or the humanized GDF8 nucleic acid is derived from a human GDF8 gene, and at least a portion of the humanized GDF8 gene or the humanized GDF8 nucleic acid is derived from an endogenous GDF8 gene of a non-human animal. In some embodiments, the humanized GDF8 gene or the humanized GDF8 nucleic acid comprises a sequence encoding a GDF8 protein. The encoded GDF8 protein has at least one activity of a human GDF8 protein and / or a non-human animal GDF8 protein.

[0086] In some embodiments, the chimeric (x) protein or chimeric (x) polypeptide is a humanized GDF8 protein or a humanized GDF8 polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the humanized GDF8 protein or humanized GDF8 polypeptide are from human GDF8 protein. In some embodiments, at least one or more portions of the amino acid sequence of the humanized GDF8 protein or humanized GDF8 polypeptide are from non-human animal GDF8 protein. In some embodiments, the humanized GDF8 protein or humanized GDF8 polypeptide is functional, or has at least one activity of a human GDF8 protein and / or a non-human animal GDF8 protein.

[0087] The non-human animal of genetic modification can be various animals, for example, mouse, rat, rabbit, pig, cattle (for example, cattle, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (for example, marmoset, rhesus monkey).For the non-human animal of suitable genetically modified embryonic stem cell (ES) that is not easy to obtain, adopt alternative methods to construct the non-human animal comprising genetic modification.Such method includes, for example, modifying non-ES cell genome (for example, fibroblast or induced pluripotent stem cell) and adopting nuclear transplantation to transfer the modified genome to suitable cell, for example oocyte, and incubating the modified cell (for example, modified oocyte) in non-human animal under appropriate conditions to form embryo.The above-mentioned construction method is known in the art, and is described in "A.Nagy, et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition), " Cold Spring Harbor Laboratory Press, 2006 ", the entire contents of which are incorporated into the present application by reference.

[0088] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. Rodents can be selected from mice, rats and hamsters. In one embodiment, the rodent is selected from the family Muridae. In one embodiment, the genetically modified animal is from a family selected from the family Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (true mice and rats, gerbils, spiny mice, crested rats), Isla Muscidae (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Spiny Dormouse (e.g., spiny dormouse) and Muridae (e.g., mole rats, bamboo rats and zokors). In a specific embodiment, the genetically modified rodent is selected from true mice or rats (Muroidea), gerbils, spiny mice and crested rats. In one embodiment, the genetically modified mouse is from a member of the Muridae family. In one embodiment, the animal is a rodent. In a specific embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0089] In some embodiments, the non-human animal may be an immunodeficient non-human mammal, such as an immunodeficient rodent, an immunodeficient rabbit, an immunodeficient pig, an immunodeficient monkey, etc.

[0090] In some embodiments, the non-human animal is a mouse of the C57BL strain, wherein the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described in, for example, 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), the relevant contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the C57BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as a BALB / c strain. In some embodiments, the mouse is a hybrid 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 certain embodiments, non-human animals are rodents. In certain embodiments, non-human animals are mice with BALB / c, BALB / cHeAn, BALB / cJ, BALB / cR1, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st or CBA / H strains. In certain embodiments, non-human animals are rats. Rats can be selected from Wistar rats, LEA strains, Sprague-Dawley strains, Fischer strains, F344, F6 and Dark Agouti rats. In certain embodiments, rat strains are hybrid species of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6 and Dark Agouti.

[0091] Genetically modified non-human animals include modifications of endogenous GDF8 gene sites (locus) in non-human animals. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of a mature human GDF8 protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to a mature human GDF8 protein amino acid sequence). Although cells (e.g., ES cells, somatic cells) that may contain the genetic modification are provided in the present invention, in many embodiments, genetically modified non-human animals include modifications of endogenous GDF8 gene sites (locus) in non-human animals.

[0092] Genetically modified non-human animals can express human GDF8 and / or chimeric (e.g., humanized) GDF8 proteins at the non-human animal endogenous GDF8 locus, wherein the non-human animal endogenous GDF8 gene has been replaced (substituted) or inserted with a human GDF8 gene and / or a nucleotide sequence encoding a human GDF8 protein or a nucleotide sequence encoding an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% or 99% identical to a human GDF8 protein. In some embodiments, the non-human animal endogenous GDF8 locus is modified by comprising all or part of a nucleotide sequence encoding a mature human GDF8 protein.

[0093] In some embodiments, genetically modified non-human animals can express human GDF8 and / or chimeric GDF8 (e.g., humanized GDF8) under the control of endogenous regulatory elements (e.g., promoters and / or 5'UTR) of non-human animals. Insertion or replacement at the endogenous GDF8 locus of non-human animals provides non-human animals that express human GDF8 or chimeric GDF8 (e.g., humanized GDF8) in suitable cells and are obtained in a manner that does not cause potential pathologies observed in some other transgenic non-human animals known in the art. In some embodiments, human GDF8 or chimeric GDF8 (e.g., humanized GDF8) expressed in non-human animals can maintain one or more functions of wild-type non-human animals or human GDF8 in non-human animals. In addition, in some embodiments, genetically modified non-human animals do not express endogenous GDF8. In some embodiments, compared with the GDF8 expression level in wild-type animals, the expression level of endogenous GDF8 in genetically modified non-human animals is reduced. As used herein, the term "endogenous GDF8" refers to the GDF8 protein expressed by the endogenous GDF8 nucleotide sequence of a non-human animal (eg, mouse) prior to any genetic modification.

[0094] The genome of the genetically modified non-human animal comprises a nucleotide sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the amino acid sequence of human GDF8 (SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375). In some embodiments, the genome of the genetically modified non-human animal comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 5. In some embodiments, the genome comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to positions 134-2819 of NM_005259.3.

[0095] In some embodiments, the genome of the genetically modified non-human animal includes: all or part of exon 1, exon 2 and / or exon 3 of the human GDF8 gene, preferably also including at least 50bp of continuous nucleotide sequence downstream of the 3'UTR; or a nucleotide sequence encoding all or part of the amino acid sequence of human GDF8; or a nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375.

[0096] In some embodiments, the genome of the genetically modified non-human animal includes at least 50-1000bp, such as at least 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000bp of continuous nucleotide sequence from part of exon 1 of the human GDF8 gene to the 3'UTR downstream. In some embodiments, the 3'UTR downstream of the human GDF8 gene includes at least 200-400bp of continuous nucleotide sequence.

[0097] In some embodiments, the genetically modified non-human animal has a nucleotide sequence encoding a chimeric human / non-human GDF8 polypeptide at an endogenous GDF8 locus. In some embodiments, the genetically modified non-human animal expresses functional GDF8 on the cell surface. In some embodiments, the human portion of the chimeric human / non-human GDF8 polypeptide may comprise an amino acid sequence encoded by exon 1 to exon 3 of a human GDF8 gene. In some embodiments, the human portion of the chimeric human / non-human GDF8 polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 2 or SEQ ID NO: 2, positions 24-375.

[0098] In addition, the modified gene in the genome of the genetically modified non-human animal is homozygous or heterozygous for the endogenous replaced locus. In a specific embodiment, the modified GDF8 gene in the genome of the genetically modified non-human animal is homozygous or heterozygous for the endogenous replaced locus.

[0099] In some embodiments, the humanized GDF8 gene comprises the 5'UTR of the human GDF8 gene. In some embodiments, the humanized GDF8 gene comprises the 5'UTR of the endogenous GDF8 gene of a non-human animal (e.g., a mouse). In some embodiments, the humanized GDF8 gene comprises the 3'UTR of the human GDF8 gene. In some embodiments, the humanized GDF8 gene comprises the 3'UTR of the endogenous GDF8 gene of a non-human animal (e.g., a mouse). Where appropriate, it can be reasonably assumed that, based on the similarity of the non-human animal and human GDF8 gene sequences, they may be similarly regulated. As shown in the present application, the humanized GDF8 gene comprising an insertion or replacement in the endogenous GDF8 locus of a non-human animal retains the endogenous regulatory elements of the non-human animal but comprises the humanization of the GDF8 coding sequence, and does not exhibit pathological phenomena. Both genetically modified mice of humanized GDF8 heterozygous or homozygous are normal.

[0100] In one aspect, the present invention also provides a genetically modified non-human animal, whose genome comprises a disruption of an endogenous GDF8 gene of the non-human animal, wherein the disruption of the endogenous GDF8 gene comprises a deletion of exon 1, exon 2 and / or exon 3, or a partial deletion thereof. In some embodiments, the disruption of the endogenous GDF8 gene of the non-human animal comprises a deletion of at least 50 bp of nucleotides of exon 1, intron 1, exon 2, intron 2 and / or exon 3.

[0101] In some embodiments, the disruption of the endogenous GDF8 gene of a non-human animal comprises a portion of exon 1 of an endogenous GDF8 gene of a non-human animal to a deletion of at least 50 bp of nucleotides downstream of the 3'UTR of the endogenous GDF8 gene of a non-human animal. In some embodiments, the portion of exon 1 of an endogenous GDF8 gene of a non-human animal that is deleted comprises at least 50-502 bp, for example, at least 50, 100, 150, 200, 250, 300, 350, 370, 375, 376, 377, 378, 379, 380, 400, 405, 500 or 502 bp of continuous nucleotide sequence. In some embodiments, the portion of exon 1 of an endogenous GDF8 gene of a non-human animal that is deleted comprises the nucleotide sequence of the coding region. In some embodiments, the nucleotide sequence downstream of the 3'UTR of the non-human animal endogenous GDF8 gene deleted includes at least 50-1000bp, such as at least 50, 60, 70, 80, 90, 100, 200, 300, 350, 400, 500, 600, 700, 800, 900 or 1000bp of continuous nucleotide sequence. In some embodiments, the nucleotide sequence downstream of the 3'UTR of the non-human animal endogenous GDF8 gene deleted includes 300-400bp of continuous nucleotide sequence.

[0102] The present invention further relates to a humanized GDF8 genomic DNA sequence, a DNA sequence obtained by reverse transcription of mRNA that is identical to or complementary to the DNA sequence; or a construct expressing the amino acid sequence thereof; or a cell comprising the construct thereof; or a tissue or organ comprising the cell thereof.

[0103] The present invention further relates to a non-human mammal produced by the above construction method. In some embodiments, its genome comprises a human or humanized GDF8 gene.

[0104] In some embodiments, the non-human mammal is a rodent, and in some embodiments, the non-human mammal is a mouse.

[0105] In some embodiments, the non-human mammal expresses a protein encoded by a humanized GDF8 gene.

[0106] In addition, the present invention also provides a non-human mammal model carrying a tumor, wherein the non-human mammal model is obtained by the method described in the present application. In some embodiments, the non-human mammal is a rodent (eg, mouse).

[0107] The present invention also provides a cell or cell line derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a primary cell culture, which is derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a tissue, organ, or culture thereof derived from a non-human mammal or its offspring. When it carries a tumor, it is derived from a tumor tissue of a non-human mammal or its offspring, or a non-human mammal carrying a tumor.

[0108] The present invention provides a non-human mammal produced by any of the above construction methods. In some embodiments, a non-human mammal or a genetically modified non-human animal is provided, wherein the genome of the genetically modified non-human animal comprises DNA of human or humanized GDF8.

[0109] In some embodiments, the non-human mammal or genetically modified non-human animal comprises a genetic construct (e.g., Figure 2 or Figure 5 In some embodiments, a non-human mammal expressing a human or humanized GDF8 protein is provided. In some embodiments, a cell, tissue or organ that specifically expresses a human or humanized GDF8 protein is provided.

[0110] In some embodiments, the expression of human or humanized GDF8 protein in the genetically modified non-human animal is controllable. For example, by adding a specific inducer or repressor. In some embodiments, the specific inducer is selected from the tetracycline system (Tet-Off System / Tet-On System) or the tamoxifen system (Tamoxifen System).

[0111] The non-human mammal can be any non-human animal known in the art that can be used in the methods. In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0112] The non-human mammals are subjected to genetic, molecular and behavioral analysis. The present invention provides offspring produced by mating with non-human mammals of the same genotype or other genotypes.

[0113] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its progeny. For example, a cell culture-based model can be prepared by the following method. The cell culture can be obtained by isolation from a non-human mammal, or cells can be obtained from a cell culture established using the same construct and standard cell transfection techniques. The integration of a genetic construct comprising a DNA sequence encoding a human GDF8 protein can be detected by a variety of methods.

[0114] There are many analytical methods that can be used to detect exogenous DNA, including nucleic acid level methods (including the use of reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and protein level methods (including histochemical analysis, immunoblot analysis and in vitro binding studies). In addition, the expression level of the target gene can be quantified by the ELSA method well known to those skilled in the art. Many standard analytical methods can be used to perform quantitative detection. For example, RT-PCR and hybridization methods can be used to detect transcription levels, including RNase protection assays, Southern Blots, and RNA dot hybridization assays (RNAdot). Immunohistochemical staining, flow cytometry, and Western blots can also be used to detect the presence of human or humanized GDF8 protein.

[0115] In some embodiments, the genetically modified non-human animal (eg, mouse) can express human or humanized GDF8 in one or more cells (eg, liver tissue cells).

[0116] Carrier

[0117] The present invention provides a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of the conversion region to be changed (5' homology arm or 5' arm), which is selected from the genomic DNA of the GDF8 gene and has a length of 100 to 10,000 nucleotides; b) a donor sequence; and c) a DNA fragment homologous to the 3' end of the conversion region to be changed (3' homology arm or 3' arm), which is selected from the genomic DNA of the GDF8 gene and has a length of 100 to 10,000 nucleotides.

[0118] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence with at least 90% identity to NCBI Accession No. NC_000067.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence with at least 90% identity to NCBI Accession No. NC_000067.7.

[0119] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence of positions 53096758 to 53100924 of NCBI Accession No. NC_000067.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence of positions 53107589 to 53111867 of NCBI Accession No. NC_000067.7.

[0120] In some embodiments, a) a DNA fragment homologous to the 5' end of the switch region to be altered is selected from the nucleotide sequence of positions 53099440 to 53100924 of NCBI Accession No. NC_000067.7; c) a DNA fragment homologous to the 3' end of the switch region to be altered is selected from the nucleotide sequence of positions 53107589 to 53109070 of NCBI Accession No. NC_000067.7.

[0121] In some embodiments, the length of the genomic nucleotide sequence selected by the targeting vector may exceed about 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 5.8 kb, or 6 kb.

[0122] In some embodiments, the switch region to be altered is located from exon 1 to exon 3 of the endogenous GDF8 gene of a non-human animal. In some embodiments, or from exon 1 of the endogenous GDF8 gene of a non-human animal to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR of the endogenous GDF8 gene of a non-human animal (e.g., at least 350 bp of continuous nucleotide sequence).

[0123] In some embodiments, the targeting vector further comprises one or more marker genes. For example, a resistance gene for positive clone screening or a coding gene for a negative screening marker. In some embodiments, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo. In some embodiments, the coding gene for a negative screening marker is a coding gene for a diphtheria toxin A subunit (DTA).

[0124] In some embodiments, the 5' arm is a nucleotide having at least 90% identity with NCBI Accession No. NC_000067.7, and further preferably, the 5' arm sequence comprises the nucleotide sequence shown in SEQ ID NOs: 3 and 25. In some embodiments, the 3' arm is a nucleotide having at least 90% identity with NCBI Accession No. NC_000067.7, and further preferably, the 3' arm sequence comprises the nucleotide sequence shown in SEQ ID NOs: 4 and 26.

[0125] In some embodiments, the targeting vector comprises a donor sequence. In some embodiments, the donor sequence comprises a human sequence (e.g., positions 190055400 to 190062596 of NC_000002.12). For example, the human sequence in the targeting vector includes: part or all of the nucleotide sequence of the human GDF8 gene, preferably all or part of exon 1, exon 2 and / or exon 3 of the human GDF8 gene, preferably also including at least 50bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the human sequence in the targeting vector includes: part of exon 1 of the human GDF8 gene to at least 50bp of continuous nucleotide sequence downstream of the 3'UTR (e.g., at least 300bp). In some embodiments, the human sequence encodes all or part of the human GDF8 protein, such as SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375.

[0126] In some embodiments, the targeting vector comprises, from the 5' end to the 3' end, a 5' arm, a portion of exon 1 of the human GDF8 gene to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR, and a 3' arm.

[0127] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence, wherein the sgRNA sequence targets a non-human animal endogenous GDF8 gene, and the sgRNA is unique on the target sequence (target site) of the conversion region to be changed, and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'; and in some embodiments, the target site of the sgRNA in the non-human animal endogenous GDF8 gene is located on exon 1, exon 2 and / or exon 3 of the non-human animal GDF8 gene, or on the nucleotide sequence upstream of the 5'UTR and / or on the nucleotide sequence downstream of the 3'UTR. In some embodiments, the target site of the sgRNA in the non-human animal endogenous GDF8 gene is located on exon 1 of the non-human animal GDF8 gene, and / or, on the nucleotide sequence downstream of the 3'UTR of the non-human animal GDF8 gene.

[0128] In some embodiments, the target sites are shown as SEQ ID NOs: 15 and 16. Therefore, the present invention provides sgRNA sequences for constructing genetically modified animal models. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NOs: 17 and 19. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NOs: 21 and 23. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NOs: 18 and 20. In some embodiments, the oligonucleotide sgRNA sequences are listed in SEQ ID NOs: 22 and 24.

[0129] In some embodiments, the present disclosure relates to a plasmid construct (sgRNA vector, e.g., pT7-sgRNA) comprising an sgRNA sequence and / or a cell comprising the sgRNA vector.

[0130] The present invention also relates to a cell comprising a targeting vector as described above.

[0131] In addition, the present invention also provides a non-human mammalian cell having any one of the above-mentioned targeting vectors and one or more in vitro transcripts of the constructs described herein (e.g., gene constructs or sgRNA vectors). In some embodiments, the cell comprises Cas9mRNA or its in vitro transcript.

[0132] In some embodiments, the cell is heterozygous for the gene. In some embodiments, the cell is homozygous for the gene.

[0133] 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 an embryonic stem cell. In some embodiments, the cell can be any cell that expresses the GDF8 protein.

[0134] Methods for constructing genetically modified non-human animals

[0135] Genetically modified non-human animals can be prepared by several gene editing techniques known in the art, including homologous recombination technology using embryonic stem cells, gene targeting technology, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology techniques. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals. In some embodiments, CRISPR / Cas9 gene editing technology is used to produce genetically modified non-human animals. Many of these genome editing technologies are known in the art and are described in Yin et al. "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated herein by reference. The present invention also provides many other methods for genome editing, for example, microinjecting transgenic cells into enucleated oocytes and fusing the enucleated oocytes with another transgenic cell.

[0136] In some embodiments, the nucleotide sequence encoding the endogenous GDF8 region in the endogenous genome of at least one cell of the non-human animal is replaced by the nucleotide sequence encoding the corresponding region of human GDF8. In some embodiments, the expression of endogenous GDF8 protein in the non-human animal is reduced or absent compared to the wild type. In some embodiments, the replacement occurs in cells such as germ cells, somatic cells, blastocysts or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0137] Figure 2 or Figure 5 The humanization targeting strategy of the mouse GDF8 locus is shown. The targeting vector comprises a vector consisting of a 5' homology arm, a human or humanized GDF8 gene fragment and a 3' homology arm. The process involves replacing the corresponding non-human animal endogenous GDF8 sequence with a human or humanized GDF8 gene fragment using homologous recombination. In some embodiments, cutting upstream and downstream of the target site (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double-strand breaks, and homologous recombination is used to replace the non-human animal endogenous GDF8 sequence with a human or humanized GDF8 sequence.

[0138] Therefore, in some embodiments, the method for preparing a genetically modified humanized non-human animal includes replacing the nucleotide sequence encoding the endogenous GDF8 region with the nucleotide sequence encoding the corresponding region of human GDF8 at the endogenous GDF8 locus (or site) of the non-human animal. The replaced sequence may include regions (e.g., part or all of the regions) of exon 1, exon 2, and / or exon 3 of the human GDF8 gene. In some embodiments, the replaced sequence includes a portion of exon 1 of the human GDF8 gene to the nucleotide sequence downstream of the 3'UTR (e.g., the nucleotide sequence of positions 134-2819 of NM_005259.3). In some embodiments, the portion of exon 1 of the human GDF8 gene comprises at least 50-506bp, for example, at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 351, 352, 353, 354, 355, 370, 371, 372, 373, 374, 375, 380, 400, 450, 500 or 506bp of continuous nucleotide sequence. In some embodiments, the portion of exon 1 of the human GDF8 gene comprises the nucleotide sequence of the coding region. In some embodiments, the 3'UTR downstream nucleotide sequence of the human GDF8 gene comprises at least 50-1000bp, for example, at least 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000bp of continuous nucleotide sequence. In some embodiments, the 3'UTR downstream nucleotide sequence of the human GDF8 gene includes 200-400 bp of continuous nucleotide sequence.

[0139] The present invention also provides a method for establishing a GDF8 gene humanized animal model, comprising the following steps:

[0140] (a) providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0141] (b) culturing the cells (preferably in a liquid culture medium);

[0142] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal;

[0143] (d) identifying germline transmission in offspring of the non-human mammal with humanized GDF8 gene of the pregnant female in step (c).

[0144] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0145] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy (or pregnancy).

[0146] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0147] The fertilized egg can be from any non-human animal, such as any non-human animal described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can be introduced into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human animal gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0148] In some embodiments, the method for preparing a genetically modified non-human animal includes modifying the coding frame of the GDF8 gene of the non-human animal, for example, by replacing the nucleotide sequence encoding the endogenous GDF8 region with the nucleotide sequence encoding the corresponding region of human GDF8 under the control of the endogenous regulatory element of the GDF8 gene of the non-human animal (for example, genomic DNA sequence, CDS sequence or cDNA sequence). For example, one or more functional region sequences of the GDF8 gene of the non-human animal can be knocked out or inserted into a sequence so that the endogenous GDF8 protein of the non-human animal cannot be expressed or the expression level is reduced. In some embodiments, the coding frame of the GDF8 gene of the modified non-human animal can be all or part of the nucleotide sequence of exon 1, exon 2 and / or exon 3 of the GDF8 gene of the non-human animal.

[0149] In some embodiments, the method for preparing a genetically modified non-human animal comprises inserting a nucleotide sequence and / or an auxiliary sequence encoding a human or humanized GDF8 protein after the endogenous regulatory element of the GDF8 gene of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon so that the GDF8 gene humanized animal model can express a human or humanized GDF8 protein in vivo, but does not express the GDF8 protein of the non-human animal. In some embodiments, the auxiliary sequence includes WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA.

[0150] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0151] (1) providing a plasmid comprising a human GDF8 gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target endogenous GDF8 of a non-human animal;

[0152] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous GDF8 gene of a non-human animal;

[0153] (3) modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2) and Cas9;

[0154] (4) Transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express humanized GDF8 protein.

[0155] In some embodiments, the method further comprises (5) mating the offspring mice obtained in step (4) to obtain homozygous mice.

[0156] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting a 5'-terminal target site and a 3'-terminal target site.

[0157] In some embodiments, the nucleotide sequence encoding the human or humanized GDF8 protein is operably linked to an endogenous regulatory element at the endogenous GDF8 locus of the non-human animal.

[0158] In some embodiments, the genetically modified non-human animal does not express endogenous GDF8 protein.

[0159] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0160] (1) providing a plasmid comprising a human or chimeric GDF8 gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target endogenous GDF8 of a non-human animal;

[0161] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous GDF8 gene of a non-human animal;

[0162] (3) Modifying the genome of fertilized eggs or embryonic stem cells by inserting the human or chimeric GDF8 gene fragment into the endogenous GDF8 gene of a non-human animal.

[0163] In some embodiments, the method comprises replacing a portion of exon 1 to a portion of the 3'UTR downstream nucleotide sequence of an endogenous GDF8 gene of a non-human animal with a portion of exon 1 to a portion of the 3'UTR downstream nucleotide sequence of a human GDF8 gene.

[0164] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 pp. 25-376 in the genome of a non-human animal with a nucleotide sequence encoding a human or humanized GDF8 protein or a human or humanized GDF8 gene.

[0165] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1, 25-376 in the genome of a non-human animal with the genomic DNA sequence, cDNA sequence or CDS sequence of human GDF8.

[0166] In some embodiments, the construction method includes replacing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 1, positions 25-376 in the genome of a non-human animal with a nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 2, positions 24-375.

[0167] In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1, 25-376 in the genome of a non-human animal with SEQ ID NO: 5.

[0168] Use of genetically modified non-human animals

[0169] Replacing a non-human animal gene with a homologous or orthologous human gene or human sequence or inserting a homologous or orthologous human gene or human sequence into a non-human animal at the endogenous GDF8 locus of the non-human animal and under the control of endogenous regulatory elements (e.g., promoter and / or 5'UTR) can produce a non-human animal with qualities and characteristics that may be significantly different from typical knockout plus transgenic animals. In typical knockout plus transgenic animals, the endogenous locus is removed or destroyed, and the full human transgene is inserted into the genome of the animal and may be randomly integrated into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of human gene and / or protein assays and / or functional assays. In human transgenes, the upstream and / or downstream of the human sequence provide suitable support for expression and / or regulation of the transgene.

[0170] In some cases, transgenes with human regulatory elements are expressed in a non-physiological or otherwise unsatisfactory manner, and may actually be harmful to the non-human animal. The present invention demonstrates that the replacement or insertion of human sequences at endogenous loci under the control of endogenous regulatory elements produces humanized non-human animals, providing physiologically appropriate expression patterns and levels that are meaningful and appropriate in the context of the physiology of the humanized animal with respect to the physiology of the replaced gene.

[0171] Genetically modified non-human animals that express human or humanized GDF8 protein, e.g., in a physiologically appropriate manner, provide a variety of uses, including but not limited to developing treatments for human diseases and disorders, and evaluating the toxicity and / or efficacy of these human treatments in animal models.

[0172] The present invention also provides a use of the GDF8 gene-modified non-human animal and the non-human animal obtained by any of the above construction methods.

[0173] In some embodiments, the application comprises:

[0174] A) Application in product development involving GDF8-related immune processes in human cells;

[0175] B) Application as a model system related to GDF8 for pharmacology, immunology, microbiology and medical research;

[0176] C) Applications involving the production and use of animal experimental disease models for the study of GDF8-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0177] D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human GDF8 signaling pathway modulators; or,

[0178] E) Study the function of the GDF8 gene, study the drugs and efficacy targeting the human GDF8 target site, and study the application of GDF8-related drugs in the treatment of tumors, metabolic diseases, neuronal diseases, inflammation or immune diseases.

[0179] The present invention provides a non-human animal expressing human or humanized GDF8 protein, which can be used for screening of human GDF8 specific regulators. In some embodiments, the non-human animal is a human disease animal model. For example, the disease is genetically induced (knock-in or knock-out). In some embodiments, the genetically modified non-human animal also comprises an impaired immune system, such as a genetically modified human-derived tissue xenograft, including a human solid tumor (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor).

[0180] In some embodiments, the anti-GDF8 antibody blocks or inhibits a GDF8-mediated signaling pathway. In some embodiments, the anti-GDF8 antibody described herein can block the interaction between GDF8 complexes, thereby inhibiting the GDF8 signaling pathway.

[0181] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-GDF8 antibodies, oligonucleotide drugs, or polypeptide drugs) in treating tumors. In some embodiments, a therapeutic agent is administered to the above-mentioned non-human animal or the non-human animal obtained by the above-mentioned construction method, wherein the non-human animal has a tumor, and the inhibitory effect of the therapeutic agent on the tumor is detected. In some embodiments, the detection includes determining the size and / or proliferation rate of tumor cells. In some embodiments, the detection method includes vernier caliper measurement, flow cytometry, and / or animal in vivo imaging detection. In some embodiments, the detection includes assessing individual body weight, fat mass, activation pathways, neuroprotective activity, or metabolic changes, and the metabolic changes include changes in food consumption or water consumption.

[0182] In some embodiments, the tumor cells include one or more cancer cells injected into a non-human animal (e.g., cancer cells derived from a human or non-human animal). In some embodiments, the therapeutic agent inhibits the GDF8 signaling pathway. In some embodiments, the therapeutic agent does not inhibit the GDF8 signaling pathway.

[0183] In some embodiments, genetically modified non-human animals can be used to detect whether a therapeutic agent is an agonist or an antagonist. In some embodiments, the methods described herein can be used to detect the function of a therapeutic agent (e.g., an anti-GDF8 antibody, an oligonucleotide drug, or a polypeptide drug), for example, whether the therapeutic agent can upregulate an immune response or downregulate an immune response, and / or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent for treating a subject's disease (e.g., a tumor, a metabolic disease, inflammation, a neuronal disease, or an immune disease). The inhibitory effect on tumors can also be determined by methods known in the art, for example, by measuring the tumor volume in an animal, and / or determining the 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 average tumor volumes (or weights) of the treatment and control groups.

[0184] In some embodiments, therapeutic agents (e.g., anti-GDF8 antibodies) can be used to treat various cancers. "Cancer" as used herein refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapid cell growth and proliferation. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or invasive stage. "Tumor" as used herein includes, but is not limited to, skin cancer, lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, brain glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In some embodiments, the tumor is one or more of breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer, neuroendocrine tumor, mesothelial tumor, oropharyngeal tumor, female reproductive system cancer or meningioma.

[0185] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-GDF8 antibodies, oligonucleotide drugs, or polypeptide drugs) in treating various metabolic diseases. In some embodiments, the metabolic diseases include one or more of diabetes, obesity, hyperthyroidism, hypoglycemia, hyperlipidemia, or hypercalcemia.

[0186] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-GDF8 antibodies, oligonucleotide drugs, or polypeptide drugs) in treating various neuronal diseases. In some embodiments, the neuronal diseases include one or more of muscular dystrophy, amyotrophic lateral sclerosis, or spinal muscular atrophy.

[0187] Genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., anti-GDF8 antibodies, oligonucleotide drugs, or polypeptide drugs) in treating inflammation. In some embodiments, the inflammation includes one or more of rheumatoid arthritis, atopic dermatitis, and myocarditis.

[0188] In some embodiments, therapeutic agents (e.g., antibodies targeting GDF8; or drugs targeting the GDF8 signaling pathway) are designed to treat various immune diseases, including osteoporosis, myasthenia gravis, graft-versus-host disease (GVHD), psoriasis, allergies, asthma, myocarditis, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune liver disease, pain or nervous system diseases, rheumatoid arthritis, Crohn's disease, systemic lupus erythematosus or scleroderma. One or more of.

[0189] Therefore, the methods described herein can be used to determine the effectiveness of therapeutic agents in suppressing an immune response.

[0190] The present invention also provides a detection method for determining the toxicity of a therapeutic agent (e.g., an anti-GDF8 antibody, an oligonucleotide drug, or a polypeptide drug). The detection method comprises administering a therapeutic agent to the above-mentioned non-human animal or the non-human animal obtained by the above-mentioned construction method, and evaluating the weight change or blood test of the non-human animal. In some embodiments, the blood test includes one or more of red blood cell count, hematocrit, or hemoglobin content. In some embodiments, the therapeutic agent can reduce red blood cells (RBC), hematocrit, or hemoglobin content by 20%, 30%, 40%, or more than 50%. In some embodiments, the body weight of the non-human animal is at least 5%, 10%, 20%, 30%, or 40% less than that of a control group (e.g., the average body weight of an animal not treated with an antibody).

[0191] The present invention also provides an animal model obtained by the construction method described in the present application for developing products related to human cellular immune processes, producing human antibodies, or for use in a model system for pharmacology, immunology, microbiology and medical research.

[0192] In some embodiments, an animal model obtained by the construction method described in the present application is provided for producing and utilizing animal experimental disease models of human cell immune processes, studying pathogens, or formulating new diagnostic strategies and / or therapeutic strategies.

[0193] The present invention also provides an animal model obtained by the construction method described in the present application to screen, verify, evaluate or study the GDF8 gene function, human GDF8 antibodies, therapeutic drugs or effectiveness of human GDF8-related diseases, such as therapeutic drugs for tumors, metabolic diseases, neuronal diseases, inflammation or immune diseases.

[0194] In some embodiments, the present application provides a method for verifying the in vivo efficacy of TCR-T, CAR-T and / or other immunotherapies (e.g., T cell adoptive transfer therapy). For example, the method includes transplanting human tumor cells into the non-human animal, and applying human CAR-T to the non-human animal with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animal is selected from a GDF8 gene humanized non-human animal prepared by the construction method, a double gene or multi-gene humanized non-human animal (or its offspring) produced by the construction method, a non-human animal expressing a human or humanized GDF8 protein, or a tumor-bearing or inflammatory animal model described in the present application. In some embodiments, TCR-T, CAR-T and / or other immunotherapies can treat GDF8-related diseases (e.g., tumors, metabolic diseases, neuronal diseases or immune diseases) described in the present application. In some embodiments, TCA-T, CAR-T and / or other immunotherapies provide an evaluation method for treating GDF8-related diseases described in the present application.

[0195] Non-human animal models with two or more human or chimeric genes

[0196] The present invention also provides a transgenic animal model or non-human animal having two or more human or chimeric genes. The animal model or non-human animal may contain a human or chimeric GDF8 gene and a sequence encoding an additional human or chimeric protein.

[0197] In some embodiments, the additional human or chimeric protein comprises at least one of ANGPTL3, GPR75, NPR1, IL36R, NKP46, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0198] In some embodiments, the animal model or non-human animal also expresses at least one of human or humanized ANGPTL3, GPR75, NPR1, IL36R, NKP46, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 proteins.

[0199] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the method comprising:

[0200] (1) Providing the above-mentioned construction method to obtain a non-human animal;

[0201] (ii) mating, in vitro fertilization or direct gene editing of the non-human animals provided in step (i) with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0202] In some embodiments, the other genetically modified non-human animals include non-human animals modified with one or a combination of two or more of the genes ANGPTL3, GPR75, NPR1, IL36R, NKP46, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4.

[0203] In some embodiments, GDF8 gene humanization is performed directly on a non-human animal with modifications of one or more of the human or chimeric ANGPTL3, GPR75, NPR1, IL36R, NKP46, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 genes.

[0204] Since these proteins may involve different mechanisms, a combination therapy targeting two or more of these proteins may be a more effective treatment method. In fact, many related clinical trials are ongoing and have shown good results. Multigene modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins, for example, anti-GDF8 antibodies, and additional therapeutic agents for treating tumors or immune diseases (e.g., asthma or atopic dermatitis). The method includes administering an anti-GDF8 antibody and an additional therapeutic agent to a non-human animal, wherein the non-human animal has a tumor or immune disease, and determining the effect of the combined therapy on the immune tumor or immune disease. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to one or more of ANGPTL3, GPR75, NPR1, IL36R, NKP46, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., nivolumab), or an anti-PD-L1 antibody. In some embodiments, the non-human animal described above further comprises a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the tumor described above comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0205] In some embodiments, the combination therapy is used to treat various cancers described in the present application, for example, breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, lung cancer. In some embodiments, the combination therapy is designed to treat immune diseases described in the present application, such as psoriasis. In some embodiments, the method described in the present application can be used to evaluate the combination therapy with some other methods. The method for treating cancer that can be used alone or in combination with the method described in the present application includes, for example, treating the subject with chemotherapy, for example, camphor alkali, doxorubicin, cisplatin, carboplatin, procarbazine, methylclorazepate, cyclophosphamide, doxorubicin, ifosamide, melphalan, chloramphenicol, thiophene, nitrosourea, dacrynic acid, daunorubicin, bleomycin, primycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatinum, 5-fluorouric acid, vincristine, vinblastine and / or methotrexate. Alternatively, or in addition, the method can include performing surgery on the subject to remove at least a portion of the tumor, for example, removing a portion or all of the tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0206] Figure 1 : Schematic diagram comparing the mouse GDF8 locus and the human GDF8 locus (not to scale);

[0207] Figure 2 : Schematic diagram of GDF8 gene targeting strategy and targeting vector V1 design (not to scale);

[0208] Figure 3 : Southern blot identification results of F1 generation of GDF8 gene humanized mice, where WT is the wild-type control;

[0209] Figure 4 : ELISA test results, where + / + refers to wild-type C57BL / 6 mice, and H / H refers to homozygous GDF8 gene humanized mice;

[0210] Figure 5 : Schematic diagram of GDF8 gene targeting strategy and targeting vector V2 design (not to scale);

[0211] Figure 6 : RT-PCR test results, where + / + refers to wild-type C57BL / 6 mice, H / H refers to humanized homozygous GDF8 mice, and H2O refers to water control. DETAILED DESCRIPTION

[0212] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0213] In each of the following examples, equipment and materials were obtained from the following companies:

[0214] C57BL / 6 mice and Flp transgenic mice were purchased from the National Rodent Laboratory Animal Seed Center, China Food and Drug Inspection Institutes;

[0215] NcoI and BsrGI were purchased from NEB with catalog numbers: R3193S and R3575S, respectively;

[0216] GDF-8 / Myostatin Quantikine ELISA Kit was purchased from R&D Systems, catalog number: DGDF80.

[0217] Example 1 GDF8 gene humanized mice

[0218] A schematic diagram of the comparison of the mouse GDF8 gene (NCBI Gene ID: 17700, UniProt ID: O08689, located at positions 53100799 to 53107238 of chromosome 1 NC_000067.7, based on transcript NM_010834.3 and its encoded protein NP_034964.1 (SEQ ID NO: 1)) and the human GDF8 gene (NCBI Gene ID: 2660, UniProt ID: O14793, located at positions 190055700 to 190062729 of chromosome 2 NC_000002.12, based on transcript NM_005259.3 and its encoded protein NP_005250.1 (SEQ ID NO: 2)) is shown in FIG. Figure 1 shown.

[0219] In order to achieve the purpose of the present invention, a nucleotide sequence encoding human GDF8 protein can be introduced into the mouse endogenous GDF8 locus, so that the mouse expresses human or humanized GDF8 protein. Specifically, using gene editing technology, under the control of the mouse GDF8 gene regulatory element, the partial sequence of mouse exon 1 to the downstream of 3'UTR (such as the start codon to 300bp downstream of 3'UTR) of about 7.2kb is replaced with the partial sequence of mouse exon 1 to the downstream of 3'UTR (such as the start codon ATG to 350bp downstream of 3'UTR) of about 6.7kb, to obtain a humanized GDF8 locus, and realize the humanization transformation of the mouse GDF8 gene.

[0220] Design as Figure 2 The targeting strategy shown, Figure 2 The homology arm sequences upstream and downstream of the mouse GDF8 gene and the A fragment containing the human GDF8 fragment are shown in the targeting vector V1. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 3) is identical to the nucleotide sequence of positions 53096758 to 53100924 of NCBI accession number NC_000067.7, and the downstream 3' homology arm sequence (SEQ ID NO: 4) is identical to the nucleotide sequence of positions 53107589 to 53111867 of NCBI accession number NC_000067.7. The nucleotide sequence of the human GDF8 gene fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 190055400 to 190062596 of NCBI accession number NC_000002.12; the connection design of the upstream of the human GDF8 fragment sequence and the mouse is: 5'-

[0221]

[0222] The sequence " TTAAA The last "A" in the sequence is the last nucleotide in the mouse. The first "A" in is the first nucleotide of the human sequence.

[0223] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the human GDF8 gene is designed as follows: 5'-

[0224]

[0225] The sequence " GTGCTThe last "T" in " is the last nucleotide of the human GDF8 gene, the sequence The first "G" in the Neo box is the first nucleotide; the connection between the 3' end of the Neo box and the mouse GDF8 gene is designed as: 5'-

[0226]

[0227] The sequence " ACTTC The last "C" in the sequence is the last nucleotide of the Neo box. The "C" in is the first nucleotide of the mouse GDF8 gene. The mRNA sequence transcribed from the humanized GDF8 gene contained in the transformed mouse is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0228] The construction of the targeting vector can be carried out by conventional methods, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified by sequencing is electroporated and transfected into the embryonic stem cells of C57BL / 6 mice, and the obtained cells are screened using the positive clone screening marker gene to screen the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the separated blastocysts (white mice) according to the technology known in the art, and the obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white) can be produced. F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. Positive mice can also be mated with Flp tool mice to remove the positive clone screening marker gene, and then mated with each other to obtain GDF8 gene humanized homozygous mice.

[0229] In addition, CRISPR / Cas9 technology can be used for gene editing to further design Figure 5 The targeting strategy shown, Figure 5The homology arm sequences upstream and downstream of the mouse GDF8 gene and the human GDF8 fragment are shown in the targeting vector V2. Among them, the upstream 5' homology arm sequence (SEQ ID NO: 25) is identical to the nucleotide sequence of positions 53099440 to 53100924 of NCBI accession number NC_000067.7, and the downstream 3' homology arm sequence (SEQ ID NO: 26) is identical to the nucleotide sequence of positions 53107589 to 53109070 of NCBI accession number NC_000067.7. The nucleotide sequence of the human GDF8 fragment (SEQ ID NO: 5) is identical to the nucleotide sequence of positions 190055400 to 190062596 of NCBI accession number NC_000002.12. The connection design of the upstream of the human GDF8 fragment sequence and the mouse is: 5'-

[0230]

[0231] The sequence " TTAAA The last "A" in the sequence is the last nucleotide in the mouse. The first "A" in the sequence is the first nucleotide of the human sequence. The downstream connection between the human GDF8 fragment sequence and the mouse sequence is designed as: 5'-

[0232]

[0233] The sequence The last "T" in the sequence is the last nucleotide in humans. CTGA "C" in " is the first nucleotide of the mouse sequence. The mRNA sequence transcribed from the humanized GDF8 gene contained in the transformed mouse is shown in SEQ ID NO: 6, and the expressed protein sequence is shown in SEQ ID NO: 2.

[0234] Conventional methods can be used to construct the targeting vector, such as enzyme digestion, ligation, direct synthesis, etc. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector that is verified to be correct by sequencing is used for subsequent experiments.

[0235] The target sequence (target site) determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, efficient and specific target sequence selection and design are the prerequisites for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize target sites. The target sequence of exemplary sgRNA on the GDF8 gene is as follows:

[0236] sgRNA1 target site (SEQ ID NO: 15): 5′-GCCAAGAGCGCCTCCACTCCGGG-3′;

[0237] sgRNA2 target site (SEQ ID NO: 16): 5′-TGGGGCATTGAAATTTGACTGGG-3′;

[0238] The activity of sgRNA was detected by UCA kit. After confirming that it could mediate efficient cutting efficiency, restriction sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 3. After annealing, the annealing products were connected to pT7-sgRNA plasmid (the plasmid was linearized with BbsI first) to obtain expression vectors pT7-GDF8-1 and pT7-GDF8-2.

[0239] Table 3 sgRNA1 and sgRNA2 sequence list

[0240]

[0241] The pT7-sgRNA vector was synthesized by a plasmid synthesis company and contained a DNA fragment containing T7 promoter and sgRNA scaffold (SEQ ID NO:

[0242] 8) and connected to the backbone vector (source Takara, item number 3299) in turn by enzyme digestion (EcoRI and BamHI), and verified by sequencing by a professional sequencing company, the results show that the target plasmid is obtained. Take the pronuclear fertilized egg of the mouse, such as C57BL / 6 mouse, and use a microinjector to inject the in vitro transcription products of pT7-GDF8-1 and pT7-GDF8-2 plasmids (using Ambion in vitro transcription kit, transcribed according to the instruction manual), the targeting vector and Cas9 mRNA into the mouse fertilized egg cytoplasm or nucleus after premixing. According to the method in the Mouse Embryo Operation Experiment Manual (Third Edition) (Andras Nagy, Chemical Industry Press, 2006), the fertilized egg is microinjected, the injected fertilized egg is transferred to the culture medium for short-term culture, and then transplanted into the oviduct of the recipient mother mouse for development, and the obtained mice (F0 generation) are hybridized and self-fertilized to expand the population and establish a stable GDF8 gene humanized mouse strain.

[0243] The PCR method can be used to identify the somatic cell genotype of F1 generation mice, and the F1 generation PCR-positive mice are subjected to Southern blot detection to confirm whether there is random insertion. The mouse tail is cut to extract genomic DNA, and the genome is digested with NcoI enzyme or BsrGI enzyme, respectively, transferred to a membrane, and hybridized. The 5' probe (5'Probe) and the 3' probe (3'Probe) are located on the 5' homology arm and the human GDF8 gene fragment, respectively. The specific probe and target fragment length are shown in Table 4 below, and the exemplary results are shown in Table 4 below. Figure 3 As shown, combined with the PCR and sequencing results, 8 mice numbered F1-07 to F1-14 were positive mice. This shows that the present method can be used to construct GDF8 gene humanized mice that can be stably propagated and have no random insertions.

[0244] Table 4 Length of specific probes and target fragments

[0245] Restriction enzymes Probe Wild-type fragment size Recombinant sequence fragment size BnB 3'Probe — 17.4kb NcoI 5'Probe 8.2kb 7.3kb

[0246] 5'Probe-F (SEQ ID NO: 11): 5'-GTCTAATGTGTGTGTTTCTCTCTGAG-3',

[0247] 5'Probe-R (SEQ ID NO: 12): 5'-GCTTTTAACTGCCCGTGCTGT-3';

[0248] 3'Probe-F (SEQ ID NO: 13): 5'-GTGTTCCTTTTTATAGGTAGAATTTT-3',

[0249] 3'Probe-R (SEQ ID NO: 14): 5'-AAATATGCCTGGTAACTCCTGGAGG-3';

[0250] The expression of mRNA in humanized GDF8 mice can be detected by RT-PCR. Specifically, 10-week-old male C57BL / 6 mice (+ / +) and 10-week-old male homozygous GDF8 humanized mice (H / H) prepared in this example were selected, skeletal muscle tissue was taken, and RT-PCR was performed using the primer sequences shown in Table 5 below. The test results are shown in FIG. Figure 6 As shown. Figure 6 As can be seen in the figure, only mouse GDF8 mRNA was detected in wild-type C57BL / 6 mice, and no human GDF8 mRNA was detected. Only human GDF8 mRNA was detected in mice homozygous for the humanized GDF8 gene.

[0251] Table 5 RT-PCR primer sequences and target fragment sizes

[0252]

[0253] In addition, the expression of humanized GDF8 (hGDF8) protein in GDF8 gene humanized mice can be detected by conventional methods such as ELISA. Specifically, 3 10-week-old male C57BL / 6 mice (+ / +) and 3 8-week-old male GDF8 gene humanized homozygous (H / H) mice prepared in this example were taken, and mouse plasma was taken, and ELISA detection was performed using the human-mouse cross antibody GDF-8 / Myostatin Quantikine ELISA Kit. The test results are shown in Figure 2. Figure 4 As shown in the figure, the expression of GDF8 protein can be detected in the serum of C57BL / 6 mice and GDF8 gene humanized homozygous mice using the human-mouse cross GDF8 kit. This result shows that the transformed humanized mice can normally express GDF8 protein.

[0254] Example 2 Model and efficacy

[0255] The humanized GDF8 mouse prepared in Example 1 can be used to evaluate the safety and efficacy of modulators targeting human GDF8 in the treatment of spinal muscular atrophy. For example, a number of homozygous humanized GDF8 mice are taken and randomly divided into a control group or a treatment group. Chemical substances (such as dexamethasone) are injected into the mice to induce muscle atrophy or muscle damage in the mice. The treatment group randomly selects drugs targeting human GDF8, and the control group is injected with an equal volume of normal saline. The mice were euthanized on days 1, 2, 3, 7, 10 and 28, and the tissues were collected. The in vivo safety and in vivo efficacy of the compound can be effectively evaluated by comparing the changes in mouse body weight and muscle weight.

[0256] Example 3 Preparation of double-gene or multi-gene humanized mice

[0257] The GDF8 gene humanized mouse prepared in the present application can also be used to prepare a multi-gene humanized mouse model. For example, in the above-mentioned Example 1, the embryonic stem cells used for microinjection can be selected from mice containing at least one gene modification of ANGPTL3, GPR75, NPR1, IL36R, NKP46, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4, or, on the basis of humanized GDF8 mice, the separation of mouse ES embryonic stem cells and gene recombination targeting technology can be used to obtain a double gene humanized or multi-gene humanized mouse model. The homozygous or heterozygous humanized mouse of the GDF8 gene obtained in the present application can also be mated with other gene-modified mice, and the offspring can be screened. According to the Mendel's law of inheritance, there is a certain probability of obtaining a multi-gene mouse modified with the humanized GDF8 gene and other genes, and then the heterozygotes can be mated with each other to obtain a homozygous modified by double genes or multiple genes.

[0258] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0259] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0260] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric myostatin (GDF8) protein.

2. The construction method according to claim 1, characterized in that: The chimeric GDF8 protein is a humanized GDF8 protein, and the humanized GDF8 protein comprises at least 50 to at least 375 consecutive amino acid sequences identical to human GDF8 protein; Preferably, the human or chimeric GDF8 protein comprises SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375; or, comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375; or, comprises an amino acid sequence that differs from SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or comprises an amino acid sequence that substitutes, deletes and / or inserts one or more amino acids as shown in SEQ ID NO: 2 or SEQ ID NO: 2 positions 24-375.

3. A method for constructing a genetically modified non-human animal, characterized in that: At the non-human animal endogenous GDF8 gene locus, the non-human animal endogenous GDF8 gene is replaced with a nucleotide sequence comprising human GDF8.

4. The construction method according to claim 3, characterized in that: The nucleotide sequence of human GDF8 includes a portion of exon 1 to the entirety of exon 3 of the human GDF8 gene; wherein the portion of exon 1 of the human GDF8 gene preferably includes at least 50 bp of continuous nucleotide sequence; Preferably, the nucleotide sequence of human GDF8 further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR of the human GDF8 gene; Further preferably, the nucleotide sequence of human GDF8 comprises the nucleotide sequence from the start codon to the stop codon of the human GDF8 gene and the 3'UTR of the human GDF8 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR.

5. The construction method according to any one of claims 3-4, characterized in that: The nucleotide sequence of human GDF8 comprises SEQ ID NO: 5; or, comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the nucleotide sequence shown in SEQ ID NO: 5; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 5 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 bp; or, comprises a nucleotide sequence that replaces, deletes and / or inserts one or more nucleotides shown in SEQ ID NO:

5.

6. The construction method according to any one of claims 3 to 5, characterized in that: Part of exon 1 to all of exon 3 of the endogenous GDF8 gene of the non-human animal is replaced; Preferably, the replaced non-human animal endogenous GDF8 gene further comprises at least 50 bp of continuous nucleotides downstream of the 3'UTR of the non-human animal endogenous GDF8 gene; Among them, the portion of exon 1 of the endogenous GDF8 gene of a non-human animal preferably contains at least 50 bp of continuous nucleotides.

7. The construction method according to any one of claims 1 to 6, characterized in that: The nucleotide sequence of human GDF8 or the nucleotide sequence encoding human or chimeric GDF8 protein is operably linked to the endogenous regulatory elements of the endogenous GDF8 locus of the non-human animal, such as the 5'UTR and / or promoter; Preferably, the endogenous GDF8 protein of the non-human animal is not expressed or is expressed at a reduced level compared to GDF8 in wild-type animals; Preferably, the modified GDF8 gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus.

8. The construction method according to any one of claims 1 to 7, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent; preferably, the rodent includes a rat or a mouse.

9. The construction method according to any one of claims 1 to 8, characterized in that: The mRNA transcribed from the modified GDF8 gene in the genome of the non-human animal comprises SEQ ID NO: 6; or, comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the nucleotide sequence shown in SEQ ID NO: 6; or, comprises a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 6 by no more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 bp; or, comprises a nucleotide sequence that replaces, deletes and / or inserts one or more nucleotides shown in SEQ ID NO:

6.

10. The construction method according to any one of claims 1 to 9, characterized in that: The non-human animal comprises nucleotide sequences of human or chimeric proteins encoded by other genes, and the human or chimeric proteins preferably comprise at least one of ANGPTL3, GPR75, NPR1, IL36R, NKP46, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

11. A method for determining the effectiveness of a therapeutic agent in treating a disease, characterized in that: The method comprises: 1) administering a therapeutic agent to a non-human animal obtained by the construction method according to any one of claims 1 to 10, wherein the non-human animal suffers from a disease; 2) Determine the inhibitory effect of the therapeutic agent on the disease.

12. The method according to claim 11, characterized in that The therapeutic agent includes a GDF8 therapeutic agent, and the GDF8 therapeutic agent includes one or more of an anti-GDF8 antibody, an oligonucleotide drug, or a polypeptide drug; Preferably, the therapeutic agent further comprises an additional therapeutic agent, and the additional therapeutic agent preferably comprises one or more of anti-PD-1 antibody, anti-PD-L1 antibody or anti-CTLA4 antibody.

13. The method according to any one of claims 11-12, characterized in that: The diseases include tumors, metabolic diseases, neuronal diseases, inflammation or immune diseases; Preferably, the tumor includes one or more of endocrine cancer, breast cancer, ovarian cancer, endometrial cancer, melanoma, renal cancer, lung cancer or pancreatic cancer; Preferably, the metabolic disease includes one or more of diabetes, obesity, hyperthyroidism, hypoglycemia, hyperlipidemia or hypercalcemia; Preferably, the neuronal disease comprises one or more of muscular dystrophy, amyotrophic lateral sclerosis or spinal muscular atrophy; Preferably, the inflammation includes one or more of rheumatoid arthritis, atopic dermatitis, and myocarditis; Preferably, the immune disease includes one or more of osteoporosis, myasthenia gravis, graft-versus-host disease (GVHD), psoriasis, allergy, asthma, myocarditis, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune liver disease, pain or nervous system disease, rheumatoid arthritis, atopic dermatitis, Crohn's disease, systemic lupus erythematosus or scleroderma.

14. A method for determining the toxicity of a GDF8 therapeutic agent, characterized in that: The method comprises: 1) administering a GDF8 therapeutic agent to a non-human animal obtained by the construction method according to any one of claims 1 to 10; 2) Determine the effects of GDF8 therapeutics in non-human animals; Preferably, the determining of the effect of the GDF8 therapeutic agent on the non-human animal comprises measuring the body weight or performing a blood test on the non-human animal; preferably, the blood test comprises one or more of red blood cell count, hematocrit or hemoglobin content.

15. A humanized GDF8 gene, characterized in that: The humanized GDF8 gene comprises any of the following nucleotide sequences: A) a nucleotide sequence encoding SEQ ID NO: 2 or SEQ ID NO: 2, positions 24 to 375; B) SEQ ID NO: 3, 4, 5, 6, 7, 9, 10, 25 or 26; C) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 3, 4, 5, 6, 7, 9, 10, 25 or 26; D) a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3, 4, 5, 6, 7, 9, 10, 25 or 26.

16. A cell, tissue or organ, characterized in that: The cells, tissues or organs contain the humanized GDF8 gene according to claim 15.

17. A use of a non-human animal obtained by the construction method of claims 1-10, the humanized GDF8 gene of claim 15, or the cell, tissue or organ of claim 16, characterized in that: The applications described include: A) Application in product development involving GDF8-related immune processes in human cells; B) Application as a model system related to GDF8 for pharmacology, immunology, microbiology and medical research; C) Applications involving the production and use of animal experimental disease models for the study of GDF8-related etiology and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human GDF8 signaling pathway modulators; or, E) Study the function of the GDF8 gene, study the drugs and efficacy targeting the human GDF8 target site, and study the application of GDF8-related drugs in the treatment of tumors, metabolic diseases, neuronal diseases, inflammation or immune diseases.

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