A non-human animal genetically modified for leucine-rich repeat kinase 2 (LRRK2)

Abstract

The present invention provides a non-human animal expressing a human or chimeric (e.g., humanized) LRRK2 protein and methods of using the same.

Description

[0001] Priority Statement

[0002] This application claims the benefit of Chinese application No. 202511028430.9, filed on July 24, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention provides a non-human animal expressing human or chimeric (e.g., humanized) LRRK2 protein and a method of using the same. background

[0004] Traditional drug development typically employs in vitro screening methods. However, these methods cannot provide the in vivo environment (such as the tumor microenvironment, stromal cells, extracellular matrix components, and immune cell interactions), leading to a high failure rate in drug development. Furthermore, given the differences between humans and animals, in vivo pharmacological studies using conventional laboratory animals may not reflect the true disease state and target site interactions, resulting in significant discrepancies between clinical trial results and animal experimental results.

[0005] Therefore, developing humanized animal models suitable for screening and evaluating human antibodies will significantly improve the efficiency of new drug development and reduce drug research and development costs. Overview

[0006] This application provides an animal model containing human or chimeric leucine-rich repeat kinase 2 (LRRK2) and a method for preparing the same. This animal model can express human or chimeric LRRK2 (e.g., humanized LRRK2) protein. It can be used to study the function of the LRRK2 gene and to screen and evaluate LRRK2 signaling pathway regulators (e.g., small nucleic acid drugs targeting LRRK2). Furthermore, the animal model prepared by the method described herein can be used for drug screening, pharmacodynamic studies, treatment of immune-related diseases, and disease treatment targeting human LRRK2 sites; this model can also facilitate new drug development and design, saving time and costs. In summary, this invention provides a powerful tool for studying the function of the LRRK2 protein and a platform for screening related drugs.

[0007] On one hand, this application provides a method for preparing a genetically modified non-human animal, the method comprising: modifying the genome of the non-human animal such that the genome of the animal contains at least one chromosome, the chromosome containing a nucleotide sequence encoding a human or chimeric leucine-rich repeat kinase 2 (LRRK2). In some embodiments, the method comprises one or more of the following features: (i) the nucleotide sequence encoding the human or chimeric LRRK2 protein is operatively linked to an exogenous regulatory element (e.g., a human LRRK2 promoter, 5'UTR, and / or 3'UTR) of at least one chromosome; (ii) the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric LRRK2 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 10; (iii) the animal is a mammal, such as a monkey, rodent, mouse, or rat; preferably, the animal is a mouse; (iv) the modification causes one or more cells of the animal to express the human or chimeric LRRK2 protein.

[0008] On the other hand, this application provides a method for preparing a genetically modified non-human animal, the method comprising: modifying the genome of the non-human animal such that the genome of the animal contains a nucleotide sequence encoding an endogenous LRRK2 region at an endogenous LRRK2 locus replaced by a nucleotide sequence encoding a corresponding region of human LRRK2. In some embodiments, the method comprises one or more of the following features: (i) the nucleotide sequence encoding the corresponding region of human LRRK2 is operatively linked to an exogenous regulatory element (e.g., a human LRRK2 promoter, 5'UTR, and / or 3'UTR); (ii) the modification causes one or more cells of the animal to express human or humanized LRRK2 protein; (iii) the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all or part of exons 1 to 51 of the human LRRK2 gene; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR. p continuous nucleotides; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 contains all human exons 1 to 51 and at least 937 bp continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 contains all human exons 1 to 51, at least 937 bp continuous nucleotides downstream of the 3'UTR and at least 20165 bp continuous nucleotides upstream of the 5'UTR; (iv) the sequence identity of the nucleotide sequence encoding the corresponding region of human LRRK2 with the nucleotide sequence shown at positions 40204832-40370222 of NCBI Gene Accession Number NC_000012.12 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%; preferably, the coding region of the nucleotide sequence encoding the corresponding region of human LRRK2 contains NCBI Gene Accession Number NC_000012.12. The mutation occurs at position 40225280 from G to A, position 40237989 from T to C, position 40320032 from C to A, position 40320071 from A to G, position 40320099 from T to A, position 40322458 from T to C, position 40340400 from G to A, position 40348452 from G to A, and / or position 40364850. (v) The animal is a mammal, such as a monkey, rodent, mouse, or rat; preferably, the animal is a mouse; (vi) The animal is a mouse, and the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene. The nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and 801 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR, and at least 20125 bp of continuous nucleotides upstream of the 5'UTR; (vii) the modified LRRK2 gene in the animal genome is homozygous or heterozygous for the endogenously substituted locus.

[0009] In some embodiments, the modification occurs in at least one cell of the animal.

[0010] In another aspect, this application provides a method for preparing a genetically modified non-human animal, the method comprising: modifying at least one cell of the non-human animal, the modification causing the animal to contain at least one cell encoding a nucleotide sequence of a human or humanized LRRK2 protein, wherein the humanized LRRK2 protein comprises at least 50, 100, 500, 1000, 1500, 2000, 2500, 2520, 2525, 2526, or 2527 consecutive amino acid residues consistent with a corresponding region of a human LRRK2 protein. In some embodiments, the method comprises one or more of the following features: (i) the modification causes the cell to express a human or humanized LRRK2 protein; (ii) the nucleotide sequence encoding the human or humanized LRRK2 protein is operatively linked to an exogenous regulatory element (e.g., a human LRRK2 promoter, 5'UTR, and / or 3'UTR); and (iii) the nucleotide sequence encoding the human or humanized LRRK2 protein can be integrated into the animal's endogenous LRRK2 locus.

[0011] In some embodiments, the human or humanized LRRK2 protein or chimeric LRRK2 protein has at least one mouse LRRK2 activity and / or human LRRK2 activity.

[0012] In some embodiments, the modification causes the animal to not express endogenous LRRK2 protein or to express it at a lower level compared to wild-type animals.

[0013] In some embodiments, the non-human animal also includes nucleotide sequences of other genes encoding human or chimeric proteins selected from at least one of SNCA, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

[0014] In another embodiment, this application provides a genetically modified non-human animal genome comprising a nucleotide sequence encoding a human or chimeric leucine-rich repeat kinase 2 (LRRK2). In some embodiments, the genome has one or more of the following features: (i) the nucleotide sequence encoding the human or chimeric LRRK2 protein is operatively linked to an exogenous regulatory element (e.g., a human LRRK2 promoter, 5'UTR, and / or 3'UTR) on at least one chromosome; (ii) the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric LRRK2 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 10; and (iii) the animal is a mammal, such as a monkey, rodent, mouse, or rat; preferably, the animal is a mouse. In some embodiments, the genome includes a nucleotide sequence at the endogenous LRRK2 locus encoding the endogenous LRRK2 region replaced by a nucleotide sequence encoding the corresponding region of human LRRK2. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all or part of exons 1 to 51 of the human LRRK2 gene; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 937 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 937 bp of continuous nucleotides downstream of the 3'UTR and at least 20165 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the nucleotide sequence shown at positions 40204832-40370222 of NCBI Gene Accession Number NC_000012.12.In some embodiments, the coding region of the nucleotide sequence encoding the corresponding region of human LRRK2 contains the following mutations in NCBI accession number NC_000012.12: position 40225280 is mutated from G to A, position 40237989 is mutated from T to C, position 40320032 is mutated from C to A, position 40320071 is mutated from A to G, position 40320099 is mutated from T to A, position 40322458 is mutated from T to C, position 40340400 is mutated from G to A, position 40348452 is mutated from G to A, and / or position 40364850 is mutated from T to C. In some embodiments, the animal is a mouse, and the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene and 801 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR, and at least 20125 bp of continuous nucleotides upstream of the 5'UTR. In some implementations, the modified LRRK2 gene in the genome is homozygous or heterozygous for the endogenously replaced locus.

[0015] In another aspect, this application provides a method for constructing genetically modified non-human animal cells, the method comprising: modifying the genome of the non-human animal cell such that the genome of the cell contains at least one chromosome, the chromosome containing a nucleotide sequence encoding human or chimeric leucine-rich repeat kinase 2 (LRRK2). In some embodiments, the method comprises one or more of the following features: (i) the nucleotide sequence encoding the human or chimeric LRRK2 protein is operatively linked to an exogenous regulatory element (e.g., a human LRRK2 promoter, 5'UTR, and / or 3'UTR) of at least one chromosome; (ii) the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric LRRK2 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 10; (iii) the cell is a mammalian cell (e.g., a monkey, rodent, mouse, or rat); preferably, the cell is a mouse cell; (iv) the modification causes the cell to express the human or chimeric LRRK2 protein. In some embodiments, the modification causes the cell's genome to include a nucleotide sequence at the endogenous LRRK2 locus encoding the endogenous LRRK2 region replaced by a nucleotide sequence encoding the corresponding region of human LRRK2. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all or part of exons 1 to 51 of the human LRRK2 gene; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 937 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 937 bp of continuous nucleotides downstream of the 3'UTR and at least 20165 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the nucleotide sequence shown at positions 40204832-40370222 of NCBI Gene Accession Number NC_000012.12.In some embodiments, the coding region of the nucleotide sequence encoding the corresponding region of human LRRK2 contains the following mutations in NCBI accession number NC_000012.12: position 40225280 is mutated from G to A, position 40237989 is mutated from T to C, position 40320032 is mutated from C to A, position 40320071 is mutated from A to G, position 40320099 is mutated from T to A, position 40322458 is mutated from T to C, position 40340400 is mutated from G to A, position 40348452 is mutated from G to A, and / or position 40364850 is mutated from T to C. In some embodiments, the cell is a mouse cell, and the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and 801 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR, and at least 20125 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the modified LRRK2 gene in the cell genome is homozygous or heterozygous for the endogenously replaced locus.

[0016] On the other hand, this application provides a method for determining the effectiveness of a therapeutic agent in treating cancer, wherein the method includes: 1) Provide: a non-human animal obtained by the method described above, or a non-human animal containing the genome of a genetically modified non-human animal as described above; 2) Administering the LRRK2 therapeutic agent to the non-human animal, wherein the non-human animal has a tumor; 3) Determine the inhibitory effect of the therapeutic agent on the tumor.

[0017] In some embodiments, the method has one or more of the following features: (i) the therapeutic agent is a small nucleic acid drug targeting LRRK2; (ii) the tumor comprises one or more tumor cells injected into an animal; (iii) determining the inhibitory effect of the LRRK2 therapeutic agent on the tumor involves measuring the tumor volume in the animal; and (iv) the cancer is a solid tumor, hematologic malignancy, head and neck cancer, liver cancer, or lung cancer.

[0018] On the other hand, this application provides a method for determining the effectiveness of LRRK2 therapeutic agents and other therapeutic agents in treating cancer, wherein the method includes: 1) Provide: a non-human animal obtained by the method described above, or a non-human animal containing the genome of a genetically modified non-human animal as described above; 2) Administering LRRK2 therapeutic agent and other therapeutic agents to the non-human animal, the non-human animal having a tumor; 3) Determine its inhibitory effect on tumors.

[0019] In some embodiments, the method comprises one or more features selected from the following: (i) the animal further comprises a sequence encoding human or chimeric PD-1, human or chimeric PD-L1 and / or human or chimeric CTLA4; preferably, the other therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; (ii) the other therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; (iii) the tumor comprises one or more tumor cells expressing PD-L1; (iv) the tumor comprises one or more tumor cells injected into the animal; (v) the determination of tumor inhibition involves measuring the tumor volume in the animal; (vi) the animal has a solid tumor, hematologic malignancy, head and neck cancer, liver cancer or lung cancer.

[0020] On the other hand, this application provides a method for determining the effectiveness of LRRK2 therapeutic agents in treating immune diseases, wherein the method includes: 1) Provide: a non-human animal obtained by the method described above, or a non-human animal containing the genome of a genetically modified non-human animal as described above; 2) Administering the LRRK2 therapeutic agent to the non-human animal, wherein the non-human animal suffers from an immune disease; 3) Determine the therapeutic effect of LRRK2 therapeutic agents on immune diseases.

[0021] In some embodiments, the therapeutic agent is a small nucleic acid drug targeting LRRK2; and / or, the immune disease is asthma, rheumatoid arthritis, or multiple sclerosis.

[0022] On the other hand, this application provides a method for determining the effectiveness of LRRK2 therapeutic agents in treating inflammatory or neurodegenerative diseases, wherein the method includes: 1) Provide: a non-human animal obtained by the method described above, or a non-human animal containing the genome of a genetically modified non-human animal as described above; 2) Administering LRRK2 treatment to the animals, wherein the animals have inflammatory or neurodegenerative diseases; 3) Determine the effectiveness of LRRK2 therapeutic agents in treating inflammatory or neurodegenerative diseases.

[0023] In some embodiments, the therapeutic agent is a small nucleic acid drug targeting LRRK2; and / or, the inflammation is inflammatory bowel disease (IBD), and the neurodegenerative disease is Parkinson's syndrome.

[0024] On the other hand, this application provides a method for determining the toxicity of LRRK2 therapeutic agents, wherein the method includes: 1) Provide: a non-human animal obtained by the method described above, or a non-human animal containing the genome of a genetically modified non-human animal as described above; 2) Administer LRRK2 treatment agent to the animals; 3) Determine the effects of LRRK2 therapeutic agents on animals.

[0025] In some embodiments, the therapeutic agent is a small nucleic acid drug targeting LRRK2; and / or, the determination of the effect of the LRRK2 therapeutic agent on the animal involves measuring the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin.

[0026] On the other hand, this application provides a cell, tissue, or organ, wherein the cell or cells in the tissue or organ contain the genome described above. In some embodiments, the cell, tissue, or organ is a non-human cell, tissue, or organ.

[0027] On one hand, the present invention provides a genetically modified non-human animal whose genome contains at least one chromosome containing a nucleotide sequence encoding a human or chimeric LRRK2 protein. In some embodiments, the nucleotide sequence encoding the human or chimeric LRRK2 protein is operatively linked to an exogenous regulatory element (e.g., a human LRRK2 promoter, 5'UTR, and / or 3'UTR) on at least one chromosome. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric LRRK2 protein shares at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with human LRRK2 (NP_940980.4, SEQ ID NO: 2) or SEQ ID NO: 10. In some embodiments, the animal is a mammal, such as a monkey, rodent, mouse, or rat. In some embodiments, the animal is a mouse. In some embodiments, the animal does not express endogenous LRRK2 protein or expresses it at a reduced level compared to LRRK2 in wild-type animals. In some embodiments, one or more cells of the animal express the human or chimeric LRRK2 protein.

[0028] In one aspect, the present invention provides a genetically modified non-human animal whose genome contains a nucleotide sequence encoding an endogenous LRRK2 region at an endogenous LRRK2 locus replaced by a nucleotide sequence encoding a corresponding region of human LRRK2. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 is operatively linked to an exogenous regulatory element (e.g., a human LRRK2 promoter, 5'UTR, and / or 3'UTR), and one or more cells of the animal express human or humanized LRRK2 protein. In some embodiments, the endogenous LRRK2 protein of the animal is not expressed or is expressed at a reduced level compared to LRRK2 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all or part of exons 1 to 51 of the human LRRK2 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all human exons 1 to 51, and at least 937 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all human exons 1 to 51, at least 937 bp of continuous nucleotides downstream of the 3'UTR, and at least 20165 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the sequence identity of the nucleotide sequence encoding the corresponding region of human LRRK2 with the nucleotide sequence shown in positions 40204832-40370222 of NCBI Gene Accession Number NC_000012.12 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2, compared with the nucleotide sequence shown at positions 40204832-40370222 of NCBI accession number NC_000012.12, contains the following mutations in the coding region: position 40225280 is changed from G to A, position 40237989 from T to C, position 40320032 from C to A, position 40320071 from A to G, position 40320099 from T to A, position 40322458 from T to C, position 40340400 from G to A, position 40348452 from G to A, and / or position 40364850 from T to C. In some embodiments, the animal is a mammal, such as a monkey, rodent, mouse, or rat.In some embodiments, the animal is a mouse. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and at least 801 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR, and at least 20125 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the modified LRRK2 gene in the animal genome is homozygous or heterozygous for the endogenously replaced locus.

[0029] In one aspect, the present invention provides a non-human animal comprising at least one cell encoding a nucleotide sequence of a human or humanized LRRK2 protein, wherein the humanized LRRK2 protein comprises at least 50, 100, 500, 1000, 1500, 2000, 2500, 2520, 2525, 2526, or 2527 consecutive amino acid sequences consistent with the corresponding region of a human LRRK2 protein, and the animal expresses the human or humanized LRRK2 protein. In another aspect, the animal comprises at least one cell encoding a nucleotide sequence of a human or humanized LRRK2 protein, wherein the humanized LRRK2 protein comprises at least 50, 100, 500, 1000, 1500, 2000, 2500, 2520, 2525, 2526, or 2527 consecutive amino acid residues consistent with the corresponding region of a human LRRK2 protein, and the animal expresses the human or humanized LRRK2 protein. In some embodiments, the nucleotide sequence encoding the human or humanized LRRK2 protein is operatively linked to an exogenous regulatory element (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR). In some embodiments, the nucleotide sequence encoding the human or humanized LRRK2 may be integrated into the animal endogenous LRRK2 locus. In some embodiments, the human or humanized LRRK2 protein has at least one mouse LRRK2 activity and / or human LRRK2 activity.

[0030] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal, wherein in at least one cell of the animal, at the animal's endogenous LRRK2 gene locus, the nucleotide sequence encoding the endogenous LRRK2 region is replaced by the nucleotide sequence of the corresponding region of human LRRK2. In some embodiments, the endogenous LRRK2 protein of the animal is not expressed or its expression level is reduced compared to LRRK2 in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all or part of exons 1 to 51 of the human LRRK2 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all human exons 1 to 51 and at least 937 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all human exons 1 to 51, at least 937 bp of continuous nucleotides downstream of the 3'UTR, and at least 20165 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human LRRK2 has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the nucleotide sequence shown in positions 40204832-40370222 of NCBI Gene Accession Number NC_000012.12. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2, compared with the nucleotide sequence shown at positions 40204832-40370222 of NCBI gene accession number NC_000012.12, contains the following mutations in the coding region: position 40225280 is changed from G to A, position 40237989 is changed from T to C, position 40320032 is changed from C to A, position 40320071 is changed from A to G, position 40320099 is changed from T to A, position 40322458 is changed from T to C, position 40340400 is changed from G to A, position 40348452 is changed from G to A, and / or position 40364850 is changed from T to C.In some embodiments, the animal is a mammal, such as a monkey, rodent, mouse, or rat. In some embodiments, the animal is a mouse. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene and 801 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR, and at least 20,125 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 is operatively linked to an exogenous regulatory element (e.g., the human LRRK2 promoter, the 5'UTR, and / or the 3'UTR).

[0031] In some embodiments, the animal is a mammal, such as a monkey, rodent, mouse, or rat.

[0032] In one aspect, the present invention provides a method for constructing a gene-modified non-human animal cell expressing human or chimeric LRRK2. The method includes replacing the nucleotide sequence encoding an endogenous LRRK2 region at an endogenous LRRK2 gene locus with a nucleotide sequence representing a corresponding region of human LRRK2, thereby generating a gene-modified non-human animal cell expressing human or chimeric LRRK2 protein. In some embodiments, the method includes replacing the nucleotide sequence encoding an endogenous mouse LRRK2 region at an endogenous mouse LRRK2 gene locus with a nucleotide sequence representing a corresponding region of human LRRK2, thereby generating a gene-modified non-human animal cell expressing human or chimeric LRRK2 protein. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all or part of exons 1 to 51 of the human LRRK2 gene. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all human exons 1 to 51, and at least 937 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all human exons 1 to 51, at least 937 bp of continuous nucleotides downstream of the 3'UTR, and at least 20165 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the amino acid sequence encoded by the nucleotide sequence encoding the corresponding region of human LRRK2 has sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 10. In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2 has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the nucleotide sequence shown at positions 40204832-40370222 of NCBI Gene Accession Number NC_000012.12.In some embodiments, the nucleotide sequence encoding the corresponding region of human LRRK2, compared with the nucleotide sequence shown at positions 40204832-40370222 of NCBI gene accession number NC_000012.12, contains the following mutations in the coding region: position 40225280 is changed from G to A, position 40237989 is changed from T to C, position 40320032 is changed from C to A, position 40320071 is changed from A to G, position 40320099 is changed from T to A, position 40322458 is changed from T to C, position 40340400 is changed from G to A, position 40348452 is changed from G to A, and / or position 40364850 is changed from T to C. In some embodiments, the animal is a mammal, such as a monkey, rodent, mouse, or rat. In some embodiments, the animal is a mouse. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and 801 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR, and at least 20125 bp of continuous nucleotides upstream of the 5'UTR. In some embodiments, the encoding nucleotide sequence of the human or chimeric LRRK2 protein is operatively linked to an exogenous regulatory element (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR). In some embodiments, the non-human animal is a mouse.

[0033] In some embodiments, the non-human animal also includes nucleotide sequences of other genes encoding human or chimeric proteins selected from at least one of SNCA, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

[0034] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating cancer, the method comprising: 1) Administering the LRRK2 therapeutic agent to the non-human animal, wherein the non-human animal has a tumor; 2) Determine the inhibitory effect of the therapeutic agent on the tumor.

[0035] In some embodiments, the therapeutic agent is a small nucleic acid drug that targets LRRK2.

[0036] In some embodiments, the tumor comprises one or more tumor cells injected into an animal.

[0037] In some embodiments, determining the inhibitory effect of LRRK2 therapeutics on tumors involves measuring tumor volume in animals.

[0038] In some embodiments, the cancer is a solid tumor, hematologic malignancy, head and neck cancer, liver cancer, or lung cancer.

[0039] In one aspect, the present invention provides a method for determining the effectiveness of LRRK2 therapeutic agents and other therapeutic agents in treating cancer, the method comprising: 1) The non-human animal was given LRRK2 therapeutic agent and other therapeutic agents, the non-human animal having a tumor; 2) Determine its inhibitory effect on tumors.

[0040] In some embodiments, the animal further comprises sequences encoding human or chimeric PD-1, human or chimeric PD-L1, and / or human or chimeric CTLA4.

[0041] In some embodiments, the other therapeutic agents are anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA4 antibodies.

[0042] In some embodiments, the tumor comprises one or more tumor cells expressing PD-L1.

[0043] In some embodiments, the tumor comprises one or more tumor cells injected into an animal.

[0044] In some embodiments, determining the inhibitory effect on the tumor involves measuring the tumor volume in an animal.

[0045] In some embodiments, the animal has a solid tumor, hematoma, head and neck cancer, liver cancer, or lung cancer.

[0046] In some embodiments, the other therapeutic agents are anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA4 antibodies.

[0047] In one aspect, the present invention provides a method for determining the effectiveness of LRRK2 therapeutic agents in treating immune diseases, the method comprising: 1) Administering LRRK2 therapeutic agent to the non-human animal, wherein the non-human animal suffers from an immune disease; 2) Determine the therapeutic effect of LRRK2 therapeutic agents on immune diseases.

[0048] In some embodiments, the therapeutic agent is a small nucleic acid drug that targets LRRK2.

[0049] In some embodiments, the immune disease is asthma, rheumatoid arthritis, or multiple sclerosis.

[0050] In one aspect, the present invention provides a method for determining the effectiveness of LRRK2 therapeutic agents in treating inflammation, the method comprising: 1) Administering the LRRK2 treatment agent to the animals, wherein the animals have inflammation; 2) Determine the effectiveness of LRRK2 therapy in treating inflammation.

[0051] In some embodiments, the therapeutic agent is a small nucleic acid drug that targets LRRK2.

[0052] In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0053] In one aspect, the present invention provides a method for determining the effectiveness of LRRK2 therapeutic agents in treating neurodegenerative diseases, the method comprising: 1) Administering LRRK2 therapeutic agent to the animals, wherein the animals have neurodegenerative diseases; 2) To determine the effectiveness of LRRK2 therapy in treating neurodegenerative diseases.

[0054] In some embodiments, the therapeutic agent is a small nucleic acid drug that targets LRRK2.

[0055] In some embodiments, the neurodegenerative disease is Parkinson's syndrome.

[0056] In one aspect, the present invention provides a method for determining the toxicity of LRRK2 therapeutic agents, the method comprising: 1) Administer LRRK2 treatment agent to the animals described above; 2) Determine the effects of LRRK2 therapeutic agents on animals.

[0057] In some embodiments, the therapeutic agent is a small nucleic acid drug that targets LRRK2.

[0058] In some embodiments, determining the effect of the LRRK2 therapeutic agent on an animal involves measuring the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin.

[0059] In one aspect, the present invention provides a humanized LRRK2 gene, wherein the humanized LRRK2 gene comprises any of the following nucleotide sequences: A) The nucleotide sequence encoding the protein of SEQ ID NO: 2 or SEQ ID NO: 10; B) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or the nucleotide sequence shown at positions 40204832-40370222 of NCBI Gene Accession No. NC_000012.12, containing the following nucleotide sequences: position 40225280, where G is mutated to A; position 40237989, where T is mutated to C; position 40320032, where C is mutated to A; position 40320071, where A is mutated to G; position 40320099, where T is mutated to A; position 40322458, where T is mutated to C; position 40340400, where G is mutated to A; position 40348452, where G is mutated to A; and / or position 40364850, where T is mutated to C. C) Nucleotide sequences that are at least 90% identical to the following nucleotide sequences: SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or nucleotide sequences that, compared with the nucleotide sequences shown at positions 40204832-40370222 of NCBI Gene Accession No. NC_000012.12, contain a nucleotide sequence in which position 40225280 is changed from G to A, position 40237989 is changed from T to C, position 40320032 is changed from C to A, position 40320071 is changed from A to G, position 40320099 is changed from T to A, position 40322458 is changed from T to C, position 40340400 is changed from G to A, position 40348452 is changed from G to A and / or position 40364850 is changed from T to C; D) A nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the following nucleotide sequence: SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 or the nucleotide sequence shown at positions 40204832-40370222 of NCBI Gene Accession Number NC_000012.12, which contains the following nucleotide sequences: position 40225280, where G is mutated to A; position 40237989, where T is mutated to C; position 40320032, where C is mutated to A; position 40320071, where A is mutated to G; position 40320099, where T is mutated to A; position 40322458, where T is mutated to C; position 40340400, where G is mutated to A; position 40348452, where G is mutated to A; and / or position 40364850, where T is mutated to C.

[0060] In one aspect, the present invention provides seed cells containing the humanized LRRK2 gene. In some embodiments, the cells are non-human cells.

[0061] In one aspect, the present invention provides an animal model comprising the humanized LRRK2 gene. In some embodiments, the animal is a non-human animal.

[0062] On the other hand, this application provides the following exemplary implementation: Implementation Scheme 1: A method for preparing a genetically modified non-human animal, the method comprising: modifying the genome of the non-human animal such that the genome of the animal contains at least one chromosome, the chromosome containing a nucleotide sequence encoding human or chimeric leucine-rich repeat kinase 2 (LRRK2).

[0063] Implementation Scheme 2: The method according to Implementation Scheme 1, wherein the nucleotide sequence encoding the human or chimeric LRRK2 protein is operatively linked to at least one exogenous regulatory element of a chromosome (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR).

[0064] Implementation Scheme 3: The method according to Implementation Scheme 1 or 2, wherein the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric LRRK2 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 10.

[0065] Implementation Scheme 4: The method according to any one of Implementation Schemes 1-3, wherein the animal is a mammal, such as a monkey, rodent, mouse, or rat; preferably, the animal is a mouse.

[0066] Implementation Scheme 5: The method according to any one of Implementation Schemes 1-4, wherein the modification causes one or more cells of the animal to express human or chimeric LRRK2 protein.

[0067] Implementation Scheme 6: A method for preparing a genetically modified non-human animal, the method comprising: modifying the genome of the non-human animal, the modification causing the genome of the animal to contain a nucleotide sequence encoding an endogenous LRRK2 region at an endogenous LRRK2 locus replaced by a nucleotide sequence encoding a corresponding region of human LRRK2.

[0068] Implementation Scheme 7: The method according to Implementation Scheme 6, wherein the nucleotide sequence encoding the corresponding region of human LRRK2 is operatively linked to an exogenous regulatory element (e.g., human LRRK2 promoter, 5'UTR and / or 3'UTR).

[0069] Implementation Scheme 8: The method according to Implementation Scheme 6 or 7, wherein the modification causes one or more cells of the animal to express human or humanized LRRK2 protein.

[0070] Implementation Scheme 9: The method according to any one of Implementation Schemes 6-8, wherein the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all or part of exons 1 to 51 of the human LRRK2 gene; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 937 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 937 bp of continuous nucleotides downstream of the 3'UTR and at least 20165 bp of continuous nucleotides upstream of the 5'UTR.

[0071] Implementation Scheme 10: The method according to any one of Implementation Schemes 6-9, wherein the sequence identity between the nucleotide sequence encoding the corresponding region of human LRRK2 and the nucleotide sequence shown at positions 40204832-40370222 of NCBI Gene Accession Number NC_000012.12 is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%; preferably, the coding region of the nucleotide sequence encoding the corresponding region of human LRRK2 contains NCBI The gene accession number NC_000012.12 has the following mutations: position 40225280 from G to A, position 40237989 from T to C, position 40320032 from C to A, position 40320071 from A to G, position 40320099 from T to A, position 40322458 from T to C, position 40340400 from G to A, position 40348452 from G to A, and / or position 40364850 from T to C.

[0072] Implementation Scheme 11: The method according to any one of Implementation Schemes 6-10, wherein the animal is a mammal, such as a monkey, rodent, mouse, or rat; preferably, the animal is a mouse.

[0073] Implementation Scheme 12: The method according to any one of Implementation Schemes 6-11, wherein the animal is a mouse, and the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises exons 1 to 51 of the mouse LRRK2 gene. The sequence comprises all exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 20,125 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and at least 801 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR and at least 20,125 bp of continuous nucleotides upstream of the 5'UTR.

[0074] Implementation Scheme 13: The method according to any one of Implementation Schemes 6-12, wherein the modified LRRK2 gene in the animal genome is homozygous or heterozygous for the endogenously replaced locus.

[0075] Implementation Scheme 14: The method according to any one of Implementation Schemes 1-13, wherein the modification occurs in at least one cell of the animal.

[0076] Implementation Scheme 15: A method for preparing a genetically modified non-human animal, the method comprising: modifying at least one cell of the non-human animal, the modification causing the animal to contain at least one cell encoding a nucleotide sequence of a human or humanized LRRK2 protein, wherein the humanized LRRK2 protein contains at least 50, 100, 500, 1000, 1500, 2000, 2500, 2520, 2525, 2526, or 2527 consecutive amino acid residues consistent with the corresponding region of the human LRRK2 protein.

[0077] Implementation Scheme 16: The method according to Implementation Scheme 15, wherein the modification causes the cells to express human or humanized LRRK2 protein.

[0078] Implementation Scheme 17: The method according to Implementation Scheme 15 or 16, wherein the nucleotide sequence encoding the human or humanized LRRK2 protein is operatively linked to an exogenous regulatory element (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR).

[0079] Implementation Scheme 18: The method according to any one of Implementation Schemes 15-17, wherein the nucleotide sequence encoding human or humanized LRRK2 may be integrated into the animal endogenous LRRK2 locus.

[0080] Implementation Scheme 19: The method according to any one of Implementation Schemes 1-18, wherein the human or humanized LRRK2 protein or chimeric LRRK2 protein has at least one mouse LRRK2 activity and / or human LRRK2 activity.

[0081] Implementation Scheme 20: The method according to any one of Implementation Schemes 1-19, wherein the modification causes the endogenous LRRK2 protein in the animal to be not expressed or to have a reduced expression level compared to LRRK2 in wild-type animals.

[0082] Implementation Scheme 21: The method according to any one of Implementation Schemes 1-20, wherein the non-human animal further includes nucleotide sequences of human or chimeric proteins encoded by other genes, said human or chimeric proteins being selected from at least one of SNCA, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

[0083] Implementation scheme 22: A genetically modified non-human animal genome containing a nucleotide sequence encoding a human or chimeric leucine-rich repeat kinase 2 (LRRK2).

[0084] Implementation Scheme 23: The genome according to Implementation Scheme 22, wherein the nucleotide sequence encoding the human or chimeric LRRK2 protein is operatively linked to at least one exogenous regulatory element of a chromosome (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR).

[0085] Implementation Scheme 24: The genome according to Implementation Scheme 22 or 23, wherein the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric LRRK2 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 10.

[0086] Implementation Scheme 25: The genome according to any one of Implementation Schemes 22-24, wherein the animal is a mammal, such as a monkey, rodent, mouse or rat; preferably, the animal is a mouse.

[0087] Implementation Scheme 26: The genome according to any one of Implementation Schemes 22-25, wherein the nucleotide sequence containing the region encoding the endogenous LRRK2 at the endogenous LRRK2 locus is replaced by the nucleotide sequence encoding the corresponding region of human LRRK2.

[0088] Implementation Scheme 27: The genome according to Implementation Scheme 26, wherein the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all or part of exons 1 to 51 of the human LRRK2 gene; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 937 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 937 bp of continuous nucleotides downstream of the 3'UTR and at least 20165 bp of continuous nucleotides upstream of the 5'UTR.

[0089] Implementation Scheme 28: The genome according to Implementation Scheme 26 or 27, wherein the nucleotide sequence encoding the corresponding region of human LRRK2 has a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% with the nucleotide sequence shown at positions 40204832-40370222 of NCBI Gene Accession Number NC_000012.12; Preferably, the coding region of the nucleotide sequence encoding the corresponding region of human LRRK2 contains the following mutations in NCBI accession number NC_000012.12: position 40225280 is mutated from G to A, position 40237989 is mutated from T to C, position 40320032 is mutated from C to A, position 40320071 is mutated from A to G, position 40320099 is mutated from T to A, position 40322458 is mutated from T to C, position 40340400 is mutated from G to A, position 40348452 is mutated from G to A, and / or position 40364850 is mutated from T to C.

[0090] Implementation Scheme 29: A genome according to any one of Implementation Schemes 26-28, wherein the animal is a mouse, and the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises exons 1 to 51 of the mouse LRRK2 gene. The sequence comprises all of the exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 20,125 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of the exons 1 to 51 of the mouse LRRK2 gene and at least 801 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of the exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR and at least 20,125 bp of continuous nucleotides upstream of the 5'UTR.

[0091] Implementation Scheme 30: The genome according to any one of Implementation Schemes 26-29, wherein the modified LRRK2 gene in the genome is homozygous or heterozygous for the endogenously replaced locus.

[0092] Implementation Scheme 31: A method for constructing a genetically modified non-human animal cell, the method comprising: modifying the genome of the non-human animal cell such that the genome of the cell contains at least one chromosome, the chromosome containing a nucleotide sequence encoding human or chimeric leucine-rich repeat kinase 2 (LRRK2).

[0093] Implementation Scheme 32, the method according to Implementation Scheme 31, wherein the nucleotide sequence encoding the human or chimeric LRRK2 protein is operatively linked to at least one exogenous regulatory element of a chromosome (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR).

[0094] Implementation Scheme 33: The method according to Implementation Scheme 31 or 32, wherein the amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric LRRK2 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 10.

[0095] Implementation Scheme 34: The method according to any one of Implementation Schemes 31-33, wherein the cell is a mammalian cell (such as a monkey, rodent, mouse, or rat); preferably, the cell is a mouse cell.

[0096] Implementation Scheme 35: The method according to any one of Implementation Schemes 31-34, wherein the modification causes the cells to express human or chimeric LRRK2 protein.

[0097] Implementation Scheme 36: The method according to any one of Implementation Schemes 31-35, wherein the modification causes the nucleotide sequence encoding the endogenous LRRK2 region at the endogenous LRRK2 locus in the genome of the cell to be replaced by a nucleotide sequence encoding the corresponding region of human LRRK2.

[0098] Implementation Scheme 37: The method according to Implementation Scheme 36, wherein the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all or part of exons 1 to 51 of the human LRRK2 gene; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 937 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 937 bp of continuous nucleotides downstream of the 3'UTR and at least 20165 bp of continuous nucleotides upstream of the 5'UTR.

[0099] Implementation Scheme 38: The method according to Implementation Scheme 36 or 37, wherein the nucleotide sequence encoding the corresponding region of human LRRK2 has a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% with the nucleotide sequence shown in positions 40204832-40370222 of NCBI Gene Accession Number NC_000012.12; Preferably, the coding region of the nucleotide sequence encoding the corresponding region of human LRRK2 contains the following mutations in NCBI accession number NC_000012.12: position 40225280 is mutated from G to A, position 40237989 is mutated from T to C, position 40320032 is mutated from C to A, position 40320071 is mutated from A to G, position 40320099 is mutated from T to A, position 40322458 is mutated from T to C, position 40340400 is mutated from G to A, position 40348452 is mutated from G to A, and / or position 40364850 is mutated from T to C.

[0100] Implementation Scheme 39: The method according to any one of Implementation Schemes 36-38, wherein the cell is a mouse cell, and the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises exons 1 to 51 of the mouse LRRK2 gene. The sequence comprises all of the exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 20,125 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of the exons 1 to 51 of the mouse LRRK2 gene and at least 801 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of the exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR and at least 20,125 bp of continuous nucleotides upstream of the 5'UTR.

[0101] Implementation Scheme 40: The method according to any one of Implementation Schemes 36-39, wherein the modified LRRK2 gene in the cell genome is homozygous or heterozygous for the endogenously replaced locus.

[0102] Implementation Scheme 41: A method for determining the effectiveness of a therapeutic agent in treating cancer, wherein the method includes: 1) Provide: a non-human animal obtained by the method according to any one of embodiments 1-21, or a non-human animal containing the genome of a genetically modified non-human animal as described in any one of embodiments 22-30; 2) Administering the LRRK2 therapeutic agent to the non-human animal, wherein the non-human animal has a tumor; 3) Determine the inhibitory effect of the therapeutic agent on the tumor.

[0103] Implementation Scheme 42: The method according to Implementation Scheme 41, wherein the therapeutic agent is a small nucleic acid drug targeting LRRK2.

[0104] Implementation Scheme 43: The method according to Implementation Scheme 41 or 42, wherein the tumor comprises one or more tumor cells injected into the animal.

[0105] Implementation Scheme 44, the method according to any one of Implementation Schemes 41-43, wherein determining the inhibitory effect of the LRRK2 therapeutic agent on tumors involves measuring tumor volume in an animal.

[0106] Implementation Scheme 45: The method described according to any one of Implementation Schemes 41-44, wherein the cancer is a solid tumor, hematologic malignancy, head and neck cancer, liver cancer, or lung cancer.

[0107] Implementation Scheme 46: A method for determining the efficacy of LRRK2 therapeutic agents and other therapeutic agents in treating cancer, wherein the method comprises: 1) Provide: a non-human animal obtained by the method according to any one of embodiments 1-21, or a non-human animal containing the genome of a genetically modified non-human animal as described in any one of embodiments 22-30; 2) Administering LRRK2 therapeutic agent and other therapeutic agents to the non-human animal, the non-human animal having a tumor; 3) Determine its inhibitory effect on tumors.

[0108] Implementation Scheme 47, the method according to Implementation Scheme 46, wherein the animal further comprises sequences encoding human or chimeric PD-1, human or chimeric PD-L1 and / or human or chimeric CTLA4; preferably, the other therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody.

[0109] Implementation Scheme 48: The method according to Implementation Scheme 46 or 47, wherein the other therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody.

[0110] Implementation Scheme 49: The method according to any one of Implementation Schemes 46-48, wherein the tumor comprises one or more tumor cells expressing PD-L1.

[0111] Implementation Scheme 50: The method according to any one of Implementation Schemes 46-49, wherein the tumor comprises one or more tumor cells injected into the animal.

[0112] Implementation Scheme 51: The method according to any one of Implementation Schemes 46-50, wherein determining the inhibitory effect on the tumor involves measuring the tumor volume in an animal.

[0113] Implementation Scheme 52: The method according to any one of Implementation Schemes 46-51, wherein the animal suffers from a solid tumor, hematologic malignancy, head and neck cancer, liver cancer, or lung cancer.

[0114] Implementation Scheme 53: A method for determining the efficacy of LRRK2 therapeutic agents in treating immune diseases, wherein the method comprises: 1) Provide: a non-human animal obtained by the method according to any one of embodiments 1-21, or a non-human animal containing the genome of a genetically modified non-human animal as described in any one of embodiments 22-30; 2) Administering the LRRK2 therapeutic agent to the non-human animal, wherein the non-human animal suffers from an immune disease; 3) Determine the therapeutic effect of LRRK2 therapeutic agents on immune diseases.

[0115] Implementation Scheme 54: The method according to Implementation Scheme 53, wherein the therapeutic agent is a small nucleic acid drug targeting LRRK2.

[0116] Implementation Scheme 55: The method described in Implementation Scheme 53 or 54, wherein the immune disease is asthma, rheumatoid arthritis, or multiple sclerosis.

[0117] Implementation Scheme 56: A method for determining the effectiveness of LRRK2 therapeutic agents in treating inflammatory or neurodegenerative diseases, wherein the method comprises: 1) Provide: a non-human animal obtained by the method according to any one of embodiments 1-21, or a non-human animal containing the genome of a genetically modified non-human animal as described in any one of embodiments 22-30; 2) Administering LRRK2 treatment to the animals, wherein the animals have inflammatory or neurodegenerative diseases; 3) Determine the effectiveness of LRRK2 therapeutic agents in treating inflammatory or neurodegenerative diseases.

[0118] Implementation Scheme 57: The method according to Implementation Scheme 56, wherein the therapeutic agent is a small nucleic acid drug targeting LRRK2.

[0119] Implementation Scheme 58: The method according to Implementation Scheme 56 or 57, wherein the inflammation is inflammatory bowel disease (IBD) and the neurodegenerative disease is Parkinson's syndrome.

[0120] Implementation Scheme 59: A method for determining the toxicity of LRRK2 therapeutic agents, wherein the method comprises: 1) Provide: a non-human animal obtained by the method according to any one of embodiments 1-21, or a non-human animal containing the genome of a genetically modified non-human animal as described in any one of embodiments 22-30; 2) Administer LRRK2 treatment agent to the animals; 3) Determine the effects of LRRK2 therapeutic agents on animals.

[0121] Implementation Scheme 60: The method according to Implementation Scheme 59, wherein the therapeutic agent is a small nucleic acid drug targeting LRRK2.

[0122] Implementation Scheme 61: The method according to Implementation Scheme 59 or 60, wherein the determination of the effect of the LRRK2 therapeutic agent on the animal involves measuring the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin.

[0123] Implementation Scheme 62: A cell, tissue, or organ, wherein the cell or cells in the tissue or organ contain the genome described in any one of Implementation Schemes 22-30; preferably, the cell, tissue, or organ is a non-human cell, tissue, or organ.

[0124] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may be used. Materials, methods, and embodiments are exemplary and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0125] Other aspects and advantages of this application will be readily apparent to those skilled in the art from the detailed description below. Attached Figure Description

[0126] Figure 1 PCR identification results of humanized mice with LRRK2 gene, where WT is the wild-type control and H2O is the water control; Figure 2 RT-PCR results, where + / + represent wild-type C57BL / 6 mice, H / H represent LRRK2 gene humanized mouse homozygotes, and H2O represents water control; Figure 3 Western blot results, where + / + represent wild-type C57BL / 6 mice, H / H represent LRRK2 gene humanized mouse homozygotes, and GAPDH is an internal control; Figure 4 qPCR detection results of LRRK2 gene in the midbrain, striatum, cortex, and hippocampus of homozygous LRRK2 gene-humanized mice, where G1 is the control group and G2 is the drug-treated group. This means p < 0.001. Detailed description

[0127] I LRRK2

[0128] As used herein, “LRRK2 protein” refers to leucine-rich repeatkinase 2, a multidomain protein possessing both GTPase and protein kinase activities. It typically comprises an ankyrin repeat region, a leucine-rich repeat domain, ROC (GTPase) and COR (adapter) domains, a kinase domain, and a WD40 repeat region, and participates in multiple cellular signaling pathways and neuroinflammatory responses. Unless the context otherwise requires, this term should be interpreted in the broadest sense to encompass polypeptides, proteins composed of or substantially composed of reference sequences (such as the human LRRK2 sequence shown in SEQ ID NO: 1, 2, or 10) that possess at least 80%, 85%, 90%, 95%, or higher sequence identity and retain at least one characteristic LRRK2 activity, biologically active fragments thereof, natural or artificial variants, and orthologs from any species (preferably human), recombinant fusion proteins, and any of the aforementioned forms of proteins modified by one or more post-translational modifications.

[0129] In the human genome, the LRRK2 gene (NCBI Gene ID: 120892, UniProt ID: Q5S007, located on chromosome 12 NC_000012.12, vertices 40224997 to 40369285) contains 51 exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 2 ...28, exon 29, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 20, exon 21, exon 22, ex Exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, and exon 51. Based on the nucleotide and amino acid sequences of transcript NM_198578.4 and its encoded protein NP_940980.4 (SEQ ID NO: 2), the corresponding positions of some exons are as follows: Table 1

[0130] In the mouse genome, the LRRK2 gene (NCBI Gene ID: 66725, UniProt ID: Q5S006, located on chromosome 15, NC_000081.7, from 91556994 to 91700327) contains 51 exons: exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 2 ...2 Exon 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and 51. Based on the nucleotide and amino acid sequences of transcript NM_025730.3 and its encoded protein NP_080006.3 (SEQ ID NO: 1), the corresponding positions of some exons are as follows: Table 2

[0131] The LRRK2 gene, protein, and gene locus are also known in other species in this field. For example, Rattus norvegicus (Rat) Macaca mulatta (Rhesus monkey) Canis lupus familiaris (dog) and Sus scrofa (Pig). Relevant information about these genes (e.g., intron sequences, exon sequences, and amino acid sequences) can be found in NCBI, and the full content is incorporated herein by reference.

[0132] II. Genetically Modified Non-human Animals

[0133] The "genetically modified non-human animal" as described in this invention refers to a non-human animal whose genome contains exogenous DNA on at least one chromosome. 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, contain exogenous DNA. Cells containing exogenous DNA can be various types of cells, such as endogenous cells, somatic cells, immune cells, T cells, B cells, NK cells, antigen-presenting cells, macrophages, dendritic cells, germ cells, blastocysts, or endogenous tumor cells. In some embodiments, a genetically modified non-human animal is provided, which contains a modified endogenous LRRK2 locus containing exogenous sequences (e.g., human sequences), for example, replacing one or more non-human sequences with one or more human sequences, or inserting one or more human and / or non-human sequences. Animals are generally able to pass on genetic modifications to their offspring through germline transmission.

[0134] The term "chimeric gene" or "chimeric nucleic acid" as used in this invention refers to a gene or nucleic acid in which two or more portions of the gene or nucleic acid originate from different species, or where at least one sequence of the gene or nucleic acid differs from that of a nucleic acid in a wild-type animal. In some embodiments, a chimeric gene or chimeric nucleic acid has at least a portion of its sequence originating from two or more different species, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins from two or more different species. In some embodiments, a chimeric gene or chimeric nucleic acid refers to a humanized gene or humanized nucleic acid.

[0135] The term "chimeric protein" or "chimeric polypeptide" as used in this invention refers to a protein or polypeptide where two or more portions of the polypeptide or protein originate from different species, or where at least one sequence of the protein or polypeptide differs from the amino acid sequence of a wild-type animal. In some embodiments, at least a portion of the sequence of the chimeric protein or chimeric polypeptide has origins from two or more different species, for example, identical (or homologous) proteins from different species. In some embodiments, the chimeric protein or chimeric polypeptide refers to a humanized protein or humanized polypeptide.

[0136] The term "humanized protein" or "humanized polypeptide" as used in this invention refers to a protein or polypeptide, wherein at least a portion of the protein or polypeptide is derived from a human protein or human polypeptide. In some embodiments, the term "humanized protein" or "humanized polypeptide" refers to a human protein or polypeptide.

[0137] The "humanized nucleic acid" described in this invention refers to nucleic acid, wherein at least a portion of the nucleic acid is derived from humans. In some embodiments, all nucleic acids in the humanized nucleic acid are derived from humans. In some embodiments, humanized nucleic acid refers to humanized exons, which may be human exons or chimeric exons.

[0138] In some embodiments, the chimeric gene or chimeric nucleic acid is a humanized LRRK2 gene or a humanized LRRK2 nucleic acid. In some embodiments, at least a portion of the gene or nucleic acid is derived from a human LRRK2 gene, and at least a portion of the gene or nucleic acid is derived from a non-human LRRK2 gene. In some embodiments, the gene or nucleic acid comprises a sequence encoding an LRRK2 protein. The encoded LRRK2 protein has the activity of at least one human LRRK2 protein or a non-human animal LRRK2 protein.

[0139] In some embodiments, the chimeric protein or chimeric polypeptide is a humanized LRRK2 protein or a humanized LRRK2 polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a human LRRK2 protein, and at least one or more portions of the amino acid sequence of the protein or polypeptide are derived from a non-human animal LRRK2 protein. The humanized LRRK2 protein or humanized LRRK2 polypeptide is functional, or has at least the activity of a human LRRK2 protein or a non-human animal LRRK2 protein.

[0140] Genetically modified non-human animals can be a variety of animals, such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets, rhesus monkeys). For non-human animals from which suitable heritably modified embryonic stem cells (ES) are not readily available, other methods are employed to construct genetically modified non-human animals. Such methods include, for example, modifying the genome of non-ES cells (e.g., fibroblasts or induced pluripotent stem cells) and transferring the modified genome to suitable cells, such as oocytes, using nuclear transfer, and gestating the modified cells (e.g., modified oocytes) in a non-human animal under appropriate conditions to form an embryo. The methods described above are known in the art and are 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 herein by reference.

[0141] In one aspect, the animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. The rodent may be selected from mice, rats, and hamsters. In one embodiment, the rodent is selected from the rat family. In one embodiment, the genetically modified animal is selected from the families Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny rats, crested rats), Madagascar Muridae (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Dormicidae (e.g., spiny dormice), and Mole-like Muridae (e.g., mole rats, bamboo rats, and mole rats). In a particular embodiment, the genetically modified rodent is selected from true mice or rats (Muroidea), gerbils, spiny rats, and crested rats. In one embodiment, the genetically modified mouse is a member of the Muridae family. In one embodiment, the animal is a rodent. In a particular embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

[0142] In some embodiments, the animal is a mouse of the C57BL strain, wherein the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mice are selected from strains 129, including 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, and 129T2. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant content of which is incorporated herein by reference in its entirety. In some embodiments, the genetically modified mice are a cross between the 129 strain and the C57BL / 6 strain. In some embodiments, the mice are a cross between the 129 strain or the C57BL / 6 strain. In some embodiments, the mice are BALB strains, such as BALB / c, BALB / cHeAn, BALB / cJ, BALB / cRl, or BALB / cWt mice. In some embodiments, the mice are a cross between the BALB strain and another strain. In some embodiments, the mice are derived from a hybrid line (e.g., 50% BALB / c - 50% 129; or 50% C57BL / 6 - 50% 129). In some embodiments, the non-human animals are mice of the BLD, CM, DX, FX, HZ, JD, KM, PD, QP, SMX, SY, TW, TZ, YP, YX, ZC, ZZ1, or ZZ2 strains, and NOD, NOD / SCID, or NOD-Prkdc strains. scid IL-2rg null The mouse in the background.

[0143] Genetically modified nonhuman animals include modifications to the LRRK2 gene locus in endogenous nonhuman animals. In some embodiments, the modification comprises a nucleotide sequence encoding at least a portion of the mature LRRK2 protein (e.g., having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of the mature LRRK2 protein). While cells (e.g., ES cells, somatic cells) that may contain the genetic modifications described herein are provided in this invention, in many embodiments, genetically modified nonhuman animals include modifications to the endogenous LRRK2 gene locus in an animal.

[0144] Genetically modified animals can express human LRRK2 and / or chimeric (e.g., humanized) LRRK2 at endogenous mouse loci, wherein the endogenous mouse LRRK2 gene has been replaced or inserted with nucleotide sequences encoding the human LRRK2 gene and / or nucleotide sequences encoding the human LRRK2 sequence region or amino acid sequences having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 100% sequence identity with the human LRRK2 sequence. In various embodiments, the endogenous non-human animal LRRK2 locus is modified with all or part of a human nucleic acid sequence encoding the mature LRRK2 protein.

[0145] In some embodiments, genetically modified mice can express human LRRK2 and / or chimeric LRRK2 (e.g., humanized LRRK2) under the control of human promoters and / or human regulatory elements. Insertion or substitution at the mouse endogenous locus provides a nonhuman animal with expression of human LRRK2 or chimeric LRRK2 (e.g., humanized LRRK2) in suitable cells and in a manner that does not lead to the potential pathology observed in some other transgenic mice known in the art. Expression of human LRRK2 or chimeric LRRK2 (e.g., humanized LRRK2) in an animal can maintain one or more functions of wild-type mouse or human LRRK2 in the animal. Furthermore, in some embodiments, the animal does not express endogenous LRRK2. In some embodiments, the expression level of endogenous LRRK2 in the animal is reduced compared to the LRRK2 expression level in wild-type animals. As used herein, the term "endogenous LRRK2" refers to the LRRK2 protein expressed by the endogenous LRRK2 nucleotide sequence of a nonhuman animal (e.g., a mouse) prior to any genetic modification.

[0146] In some embodiments, the humanized LRRK2 locus contains a human 5' UTR. In some embodiments, the humanized LRRK2 locus contains an endogenous (e.g., mouse) 5' UTR. In some embodiments, the humanized LRRK2 locus contains a human 3' UTR. In some embodiments, the humanized locus contains an endogenous (e.g., mouse) 3' UTR. Where appropriate, it can be reasonably assumed that, based on the similarity of the 5' flanking sequences of the mouse and human LRRK2 genes, they appear to be subject to similar regulation. As shown in the application, mice with inserted or replaced humanized LRRK2 loci at the endogenous mouse LRRK2 locus do not exhibit pathological phenomena. Both heterozygous and homozygous humanized LRRK2 modified mice are normal.

[0147] The present invention further relates to the LRRK2 genomic DNA sequence of humanized mice, a DNA sequence obtained by reverse transcription of mRNA that is identical or complementary to the DNA sequence; a construct expressing the amino acid sequence thereon; a cell containing the construct thereon; and a tissue containing the cell thereon.

[0148] The present invention further relates to non-human mammals produced by the methods described above. In some embodiments, their genomes contain human genes.

[0149] In some embodiments, the non-human mammal is a rodent, preferably a mouse.

[0150] In some embodiments, non-human mammals express a protein encoded by the humanized LRRK2 gene.

[0151] Furthermore, the present invention provides a non-human mammalian model carrying a tumor, which is obtained by the method described herein. In some embodiments, the non-human mammal is a rodent (e.g., a mouse).

[0152] This invention also provides cells or cell lines, or primary cell cultures, derived from non-human mammals or their offspring, or non-human mammals carrying tumors, that are derived from non-human mammals or their offspring, or non-human mammals carrying tumors, or tissues, organs, or cultures derived from non-human mammals or their offspring. When carrying a tumor, it is derived from tumor tissue of a non-human mammal or its offspring, or from a non-human mammal carrying a tumor.

[0153] This invention provides a non-human mammal produced by any of the methods described herein. In some embodiments, a non-human mammal or a genetically modified non-human animal is provided, the genome of which contains human or humanized LRRK2 DNA.

[0154] In some embodiments, the non-human mammal includes the gene constructs described herein. In some embodiments, a non-human mammal expressing human or humanized LRRK2 protein is provided. In some embodiments, a tissue specifically expressing human or humanized LRRK2 protein is provided.

[0155] In some embodiments, the expression of human or humanized LRRK2 protein in non-human animals is controllable, such as 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.

[0156] The non-human mammal can be any non-human animal known in the art that can be used in the methods described herein. Preferred non-human mammals are mammals (e.g., rodents). In some embodiments, the non-human mammal is a mouse.

[0157] Genetic, molecular, and behavioral analyses were performed on the non-human mammals described above. This invention provides an offspring produced by mating with a non-human mammal of the same or other genotypes.

[0158] This invention provides a cell line or primary cell culture derived from a non-human mammal or its offspring. For example, cell culture-based models can be prepared by the following methods. Cell cultures can be obtained by isolating cells from non-human mammals, or cells can be obtained using the same construct and a cell culture established using standard cell transfection techniques. Integration of the genetic structure containing the DNA sequence encoding the human LRRK2 protein can be detected by various methods.

[0159] Numerous analytical methods are available for detecting exogenous DNA, including nucleic acid level methods (such as reverse transcription-polymerase chain reaction (RT-PCR) or Southern blotting and in situ hybridization) and protein level methods (such as histochemical analysis, immunoblotting, and in vitro binding studies). Furthermore, the expression level of the target gene can be quantified using ELISA methods well-known to those skilled in the art. Many standard analytical methods are available for quantitative detection. For example, transcriptional levels can be detected using RT-PCR and hybridization methods, including RNase protection assays, Southern blotting, and RNA dot hybridization (RNAdot). Immunohistochemical staining, flow cytometry, and Western blotting can also be used to detect the presence of human or humanized LRRK2 protein.

[0160] In some embodiments, the genome of the genetically modified non-human animal described herein comprises at least one chromosome containing a nucleotide sequence encoding a human or chimeric LRRK2 protein (e.g., a humanized LRRK2 protein). In some embodiments, the human or chimeric LRRK2 protein (e.g., a humanized LRRK2 protein) includes disease-associated pathogenic mutations, such as those associated with Parkinson's disease. In some embodiments, the human or chimeric LRRK2 protein (e.g., a humanized LRRK2 protein) includes mutations: R50H, G2019S, M2396T, and / or S1647T. In some embodiments, the human or chimeric LRRK2 protein (e.g., a humanized LRRK2 protein) includes the mutation G2019S.

[0161] In some embodiments, the genetically modified non-human animal described herein comprises a cell encoding at least one nucleotide sequence of a human or humanized LRRK2 protein. In some embodiments, the human or humanized LRRK2 protein includes disease-associated pathogenic mutations, such as those associated with Parkinson's disease. In some embodiments, the human or humanized LRRK2 protein includes mutations: R50H, G2019S, M2396T, and / or S1647T. In some embodiments, the human or humanized LRRK2 protein includes the mutation G2019S.

[0162] In some embodiments, the genome of the genetically modified non-human animal described herein includes a nucleotide sequence at the endogenous LRRK2 locus encoding the endogenous LRRK2 region replaced by a nucleotide sequence encoding the corresponding region of human LRRK2. In some embodiments, the genome containing the replacement encodes a human or humanized LRRK2 protein. In some embodiments, the human or humanized LRRK2 protein includes disease-associated pathogenic mutations, such as those associated with Parkinson's disease. In some embodiments, the human or humanized LRRK2 protein includes mutations: R50H, G2019S, M2396T, and / or S1647T. In some embodiments, the human or humanized LRRK2 protein includes the mutation G2019S.

[0163] In some embodiments, the genetically modified animals described herein (e.g., humanized homozygous mice with the LRRK2 gene) may express human or humanized LRRK2 in one or more liver tissue cells.

[0164] In some embodiments, the genetically modified animals described herein (e.g., humanized homozygous mice with the LRRK2 gene) may express human or humanized LRRK2 in one or more cerebral cortical cells.

[0165] In some embodiments, the genetically modified animals described herein (e.g., humanized homozygous mice with the LRRK2 gene) may express human or humanized LRRK2 in one or more hippocampal cells.

[0166] In some embodiments, the genetically modified animals described herein (e.g., humanized homozygous mice with the LRRK2 gene) may express human or humanized LRRK2 in one or more thalamic cells.

[0167] In some embodiments, the genetically modified animals described herein (e.g., humanized homozygous mice with the LRRK2 gene) may express human or humanized LRRK2 in one or more cerebellar cells.

[0168] III. Methods for constructing genetically modified non-human animals

[0169] Genetically modified nonhuman animals can be prepared using several techniques known in the art, including gene targeting techniques using embryonic stem cells, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonucleases, or other molecular biology techniques. In some embodiments, homologous recombination technology is preferred. In some embodiments, CRISPR / Cas9 gene editing technology can be used to construct genetically modified nonhuman animals. In some embodiments, CRISPR-Cas9 genome editing is used to produce genetically modified nonhuman animals. Many of these genome editing techniques 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, such as microinjecting transgenic cells into enucleated oocytes and fusing enucleated oocytes with another transgenic cell.

[0170] In some embodiments, the nucleotide sequence encoding the endogenous LRRK2 region in the endogenous genome of at least one cell of a non-human animal is replaced by the nucleotide sequence encoding the corresponding region of human LRRK2. In some embodiments, the expression level of the endogenous LRRK2 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 the somatic cell or fibroblast may be inserted into an enucleated oocyte.

[0171] This invention provides a targeting vector. The targeting vector comprises a 5' homologous arm, a human or humanized LRRK2 gene fragment, and a 3' homologous arm, or both. The process involves replacing the corresponding endogenous LRRK2 sequence with a human or humanized LRRK2 sequence using homologous recombination. In some embodiments, upstream and downstream cleavage at the target site (e.g., via zinc finger nucleases, TALEN, or CRISPR) can lead to DNA double-strand breaks, replacing the mouse endogenous LRRK2 sequence with a human or humanized LRRK2 sequence using homologous recombination.

[0172] Therefore, in some embodiments, the method for preparing genetically modified humanized animals includes replacing the nucleic acid sequence encoding the endogenous LRRK2 region at the endogenous LRRK2 locus (or site) with a nucleotide sequence encoding the corresponding region of human LRRK2.

[0173] This invention also provides a method for establishing a humanized animal model of the LRRK2 gene, comprising the following steps: (a) Providing cells (e.g., fertilized egg cells) based on the methods described herein; (b) Culture the cells in a liquid culture medium; (c) Transplanting 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. (d) Identify phylogenetic transmission in the offspring of genetically modified humanized non-human mammals of pregnant females in step (c).

[0174] In some embodiments, the non-human mammal in the above method is a mouse (e.g., a C57BL / 6 mouse).

[0175] In some embodiments, the non-human mammal in step (c) is a female with pseudopregnancy.

[0176] In some embodiments, the fertilized egg used in the above method is a C57BL / 6 fertilized egg. 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.

[0177] The fertilized egg can be derived 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 via microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, which then gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above methods.

[0178] In some embodiments, a method for preparing genetically modified animals includes modifying the coding frame of a non-human animal's LRRK2 gene, for example, by replacing the nucleic acid sequence (e.g., DNA or cDNA sequence) encoding the endogenous LRRK2 region with a nucleotide sequence encoding the corresponding region of human LRRK2 under the control of exogenous regulatory elements (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR). For example, one or more functional region sequences of the non-human animal's LRRK2 gene may be knocked out or inserted, such that the non-human animal's endogenous LRRK2 protein is not expressed or its expression level is reduced. In some embodiments, the coding frame of the modified non-human animal's LRRK2 gene may be all or part of the nucleotide sequence of exons 1 to 51 of the non-human animal's LRRK2 gene.

[0179] In some embodiments, the method of preparing genetically modified animals includes inserting a nucleotide sequence encoding a human or humanized LRRK2 protein and / or an auxiliary sequence into the LRRK2 locus of a non-human animal. In some embodiments, the auxiliary sequence may be a stop codon, enabling the LRRK2 gene humanized animal model to express the human or humanized LRRK2 protein in vivo, but not the non-human LRRK2 protein. In some embodiments, the auxiliary sequence includes WPRE (WHP post-transcriptional response element), loxP, STOP, and / or polyA.

[0180] In some embodiments, the method for establishing a humanized animal model of the LRRK2 gene is used to provide the genetically modified non-human animal described herein. In some embodiments, the LRRK2 gene humanized animal model provided by the method possesses the characteristics of the genetically modified non-human animal described herein. In some embodiments, the LRRK2 gene humanized animal model provided by the method possesses the characteristics of the genetically modified non-human animal as described in any one or more embodiments or examples as presented in the sections “Overview,” “II Genetically Modified Non-human Animals,” or “V Non-human Animal Models with Two or More Human or Chimeric Genes” herein.

[0181] In some embodiments, a method for preparing transgenic animals includes: (1) Provide a plasmid containing a human LRRK2 gene fragment, wherein the plasmid is flanked by a 5' homologous arm and a 3' homologous arm, wherein the 5' and 3' homologous arms target endogenous LRRK2; (2) Provide one or more guide RNAs (sgRNAs) that target the endogenous LRRK2 gene; (3) Modify the genome of fertilized eggs or embryonic stem cells by using the plasmid from step (1), the sgRNA from step (2), and Cas9; (4) The fertilized eggs obtained in step (3) are transplanted into the fallopian tubes of pseudopregnant female mice, or the embryonic stem cells obtained in step (3) are transplanted into blastocysts, and then the blastocysts are transplanted into the fallopian tubes of pseudopregnant female mice to produce offspring mice that functionally express humanized LRRK2 protein. (5) Mating the offspring mice obtained in step (4) to obtain homozygous mice.

[0182] In some embodiments, the fertilized egg is modified by CRISPR with sgRNA targeting the 5'-end target site and the 3'-end target site.

[0183] In some embodiments, the sequence encoding the humanized LRRK2 protein is operatively linked to exogenous regulatory elements (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR).

[0184] In some embodiments, the genetically modified animals do not express the endogenous LRRK2 protein.

[0185] In some embodiments, a method for preparing transgenic animals includes: (1) Provide a plasmid containing a human or chimeric LRRK2 gene fragment, wherein the plasmid is flanked by a 5' homologous arm and a 3' homologous arm, wherein the 5' and 3' homologous arms target endogenous LRRK2; (2) Provide one or more guide RNAs (sgRNAs) that target the endogenous LRRK2 gene; (3) Modifying the genome of a fertilized egg or embryonic stem cell by inserting the human or chimeric LRRK2 gene fragment into the genome.

[0186] In some embodiments, the method for preparing transgenic animals is used to provide the genetically modified nonhuman animals described herein. In some embodiments, the transgenic animals provided by the method possess the characteristics of the genetically modified nonhuman animals described herein. In some embodiments, the transgenic animals provided by the method possess the characteristics of the genetically modified nonhuman animals as described in any one or more embodiments or examples in the sections “Overview,” “II Genetically Modified Nonhuman Animals,” or “V Nonhuman Animal Models with Two or More Human or Chimeric Genes.”

[0187] IV. Application of Genetically Modified Non-Human Animals

[0188] Replacing a nonhuman animal gene with a homologous or orthologous human gene or human sequence at an endogenous nonhuman animal locus, or inserting a homologous or orthologous human gene or human sequence into a nonhuman animal, can produce nonhuman animals 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 a fully human transgene is inserted into the animal's genome, potentially integrating randomly. Typically, the location of the integrated transgene is unknown; human protein expression is measured through transcriptional assays and / or functional assays of human genes and / or proteins. In human transgenes, the upstream and / or downstream of the human sequence provides appropriate support for the expression and / or regulation of the transgene.

[0189] Genetically modified animals expressing human or humanized LRRK2 protein, for example in a physiologically suitable manner, provide a variety of uses, including but not limited to developing treatments for human diseases and conditions, and evaluating the toxicity and / or efficacy of these human treatments in animal models.

[0190] This invention also provides an application of the above-described LRRK2 gene-modified non-human animal and any of the above-described construction methods to non-human animals.

[0191] In some embodiments, the application includes: A) Applications in product development involving LRRK2-related immune processes in human cells; B) Applications as LRRK2-related model systems in pharmacological, immunological, microbiological, and medical research; C) Involving the production and use of animal experimental disease models for etiological studies related to LRRK2 and / or for the development of diagnostic and / or therapeutic strategies; D) Application in in vivo studies of the screening, efficacy testing, evaluation, validation, or assessment of human LRRK2 signaling pathway modulators; or, E) Research the function of the LRRK2 gene, study drugs and their efficacy targeting human LRRK2, and explore the application of drugs for LRRK2-related tumors, inflammation, and immune-related diseases.

[0192] This invention provides a non-human animal expressing human or humanized LRRK2 protein, which can be used for screening human LRRK2-specific regulators. In some embodiments, the non-human animal is a human disease animal model, such as a genetically induced disease (knock-in or knock-out). In different embodiments, the genetically modified non-human animal also includes a compromised immune system, such as a genetically modified human tissue xenograft, including human solid tumors (e.g., breast cancer) or hematologic malignancies (e.g., lymphocytic tumors, B-cell or T-cell tumors).

[0193] In some embodiments, the present invention provides the use of the genetically modified non-human animals described herein in the screening, efficacy testing, evaluation, verification, or assessment of LRRK2 therapeutics. In some embodiments, the LRRK2 therapeutics are believed to be able to intervene in (e.g., block or inhibit; or activate) LRRK2-mediated signaling pathways.

[0194] In some embodiments, LRRK2 therapeutic agents block or inhibit LRRK2-mediated signaling pathways. In some embodiments, the LRRK2 therapeutic agents described herein can block interactions between LRRK2 complexes, thereby inhibiting the LRRK2 signaling pathway.

[0195] In some embodiments, genetically modified nonhuman animals can be used to determine the effectiveness of therapeutic agents (e.g., LRRK2 therapeutic agents, such as small nucleic acid drugs targeting LRRK2) in treating various immune diseases. In some embodiments, the immune diseases include, but are not limited to, GVHD (graft-versus-host disease), psoriasis, allergies, asthma, myocarditis, nephritis, hepatitis (preferably non-alcoholic steatohepatitis), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders. In some embodiments, the immune disease is asthma, rheumatoid arthritis, or multiple sclerosis.

[0196] In some embodiments, genetically modified nonhuman animals can be used to determine the effectiveness of therapeutic agents (e.g., LRRK2 therapeutic agents, such as LRRK2-targeting small nucleic acid drugs) in treating various inflammatory infections. In some embodiments, the inflammation includes both acute and chronic inflammation. Specifically, this includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, purulent inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, and specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma venereum, etc.). In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0197] In some embodiments, genetically modified nonhuman animals can be used to determine the effectiveness of therapeutic agents (e.g., LRRK2 therapeutic agents, such as small nucleic acid drugs targeting LRRK2) in treating various neurodegenerative diseases. In some embodiments, the neurodegenerative diseases include, but are not limited to, Alzheimer's disease, frontotemporal dementia, Lewy body dementia, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis (ALS). In some embodiments, the neurodegenerative disease is Parkinson's disease.

[0198] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of therapeutic agents (e.g., LRRK2 therapeutic agents, such as LRRK2-targeting small nucleic acid drugs) in treating cancer. In some embodiments, a therapeutic agent (e.g., an LRRK2-targeting small nucleic acid drug) is administered to a non-human animal, wherein the non-human animal has cancer or a tumor, and the inhibitory effect of the therapeutic agent on the cancer or 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 caliper measurement, flow cytometry, and / or in vivo animal imaging. In some embodiments, the detection includes assessing individual body weight, fat mass, activation pathways, neuroprotective activity, or metabolic changes, wherein the metabolic changes include changes in food consumption or water consumption.

[0199] In some embodiments, the tumor cells comprise one or more cancer cells injected into an animal (e.g., cancer cells derived from humans or non-human animals). In some embodiments, the therapeutic agent inhibits the LRRK2 signaling pathway. In some embodiments, the therapeutic agent does not inhibit the LRRK2 signaling pathway.

[0200] In some embodiments, genetically modified non-human animals can be used to detect whether an LRRK2 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., a small nucleic acid drug targeting LRRK2), for example, whether the therapeutic agent upregulates or downregulates an immune response, and / or whether the therapeutic agent induces complement-mediated cytotoxicity (CMC) or antibody-dependent cytotoxicity (ADCC). In some embodiments, genetically modified non-human animals can be used to determine an effective dose of a therapeutic agent for treating a subject's disease (e.g., an immune disease). Tumor inhibition can also be determined by methods known in the art, for example, measuring 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 – T) Vt / T Vc ) x 100 to calculate, where T Vt and T Vc This is the average tumor volume (or weight) in the treatment group and the control group.

[0201] In some embodiments, therapeutic agents (e.g., LRRK2 therapeutic agents, such as small nucleic acid drugs targeting LRRK2) can be used to treat various cancers. The term "cancer" as used in this invention refers to cells with autonomous growth capacity, i.e., an abnormal state or condition characterized by rapid cell growth and proliferation. This term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. The term "tumor" as used in this invention includes, but is not limited to, lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal carcinoma, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, 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. The leukemia is selected from acute 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 Waldenström macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In one specific embodiment of the present invention, the tumor is breast cancer, pancreatic cancer, endocrine carcinoma, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer, neuroendocrine tumor, mesothelial tissue tumor, oropharyngeal tumor, female reproductive system cancer, or meningioma. In some embodiments, the tumor is a solid tumor, hematologic malignancy, head and neck cancer, or lung cancer.

[0202] This invention also provides a method for determining the toxicity of a therapeutic agent (e.g., an LRRK2 therapeutic agent, such as a small nucleic acid drug targeting LRRK2). The method includes administering an antibody to a non-human animal as described above and assessing changes in the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin. In some embodiments, the antibody may reduce red blood cells (RBCs), hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%. In some embodiments, the animal's body weight is at least 5%, 10%, 20%, 30%, or 40% less than that of a control group (e.g., the average body weight of animals not treated with the antibody).

[0203] The present invention also provides an animal model system constructed by the method described herein for developing products related to human cellular immune processes, manufacturing human antibodies, or for use in pharmacological, immunological, microbiological and medical research.

[0204] In some embodiments, an animal model generated by the methods described herein is provided for use in animal experimental disease models of immune processes of human cells, for studying pathogens, or for developing new diagnostic and / or therapeutic strategies.

[0205] This invention also provides animal models generated by the methods described herein for screening, validating, evaluating, or studying LRRK2 gene function, human LRRK2 antibodies, drugs or efficacy targeting human LRRK2 sites, drugs for immune-related diseases, and anti-tumor drugs.

[0206] In some embodiments, this disclosure provides a method for validating 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 animals described herein and applying human CAR-T to the animals having human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the animals are selected from LRRK2 gene-humanized non-human animals prepared by the methods described herein, dual or multiple humanized non-human animals (or their progeny) generated by the methods described herein, non-human animals expressing human or humanized LRRK2 protein, or tumor- or inflammatory animal models described herein. In some embodiments, TCR-T, CAR-T, and / or other immunotherapies can treat LRRK2-related diseases described herein (e.g., immune diseases, inflammatory infections, cancers; preferably cancers as described above). In some embodiments, TCR-T, CAR-T, and / or other immunotherapies provide methods for evaluating the treatment of LRRK2-related diseases described herein (e.g., immune diseases, inflammatory infections, cancers; preferably cancers as described above).

[0207] V. Non-human animal models containing two or more human or chimeric genes

[0208] The present invention also provides a method for generating a transgenic animal model having two or more human or chimeric genes. The animal may contain a human or chimeric LRRK2 gene and a sequence encoding an additional human or chimeric protein.

[0209] In some embodiments, the additional gene is a non-human animal modified with at least one of the genes selected from SNCA, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. In some embodiments, the aforementioned non-human animal also expresses at least one of the human or humanized SNCA, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4 proteins.

[0210] The present invention also provides a method for constructing a non-human animal with two or more human or chimeric genes, the method comprising: (i) Obtain non-human animals by providing the above construction method; (ii) The non-human animals provided in step (i) are mated with other genetically modified non-human animals, fertilized in vitro, or directly genetically edited, and then screened to obtain multi-gene-modified non-human animals.

[0211] In some embodiments, the other genetically modified non-human animals include humanized non-human animals with one or more of the genes SNCA, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

[0212] In some embodiments, LRRK2 humanization is performed directly on non-human animals with human or chimeric SNCA, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4 gene modifications.

[0213] Because these proteins may involve different mechanisms, combination therapies targeting two or more of them may be a more effective treatment approach. In fact, many relevant clinical trials are underway and have shown promising results. Multi-gene modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins, such as anti-LRRK2 antibodies, and adjunctive therapeutic agents for treating cancer or immune diseases (e.g., asthma or atopic dermatitis). The method involves administering an anti-LRRK2 antibody and an adjunctive therapeutic agent to an animal with a tumor or immune disease and determining the effect of the combination therapy on the immunotumor or immune disease. In some embodiments, the adjunctive therapeutic agent is an antibody that specifically binds to SNCA, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. In some embodiments, the adjunctive 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 includes 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 adjunctive 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.

[0214] In some embodiments, the combination therapy is used to treat various cancers described herein, such as breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, or other cancers. In some embodiments, the combination therapy is designed to treat immune disorders described herein, such as psoriasis. In some embodiments, the methods described herein can be used to evaluate combination therapy with some other methods. Methods for treating cancer that can be used alone or in combination with the methods described herein include, for example, treating a subject with chemotherapy, such as camphor, doxorubicin, cisplatin, carboplatin, procarbazine, meloratadine, cyclophosphamide, doxorubicin, ifosfamide, melphalan, chlorpromazine, bisthiophene, nitrosourea, daktarin, daunorubicin, bleomycin, prilocytic erythromycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatinum, 5-fluorouracil, vincristine, vinblastine, and / or methotrexate. Alternatively, the method may include performing surgery on the subject to remove at least a portion of the cancer, such as removing part or all of a tumor from the patient. Detailed Implementation

[0215] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0216] In each of the following embodiments, the equipment and materials are obtained from the following companies: C57BL / 6 mice were purchased from the National Rodent Experimental Animal Seed Center of the National Institutes for Food and Drug Control, China. Recombinant Anti-LRRK2 antibody [MJFF2 (c41-2)] was purchased from Abcam, catalog number ab133474.

[0217] Example 1: LRRK2 gene-humanized mice

[0218] To achieve the objectives of this invention, a nucleotide sequence encoding the human LRRK2 protein can be introduced into the mouse endogenous LRRK2 gene locus, enabling the mouse to express the human or humanized LRRK2 protein. Specifically, using gene editing technology, approximately 163.83 kb of the sequence from upstream of the 5'UTR to downstream of the 3'UTR of the mouse LRRK2 gene is replaced with approximately 165.39 kb of the sequence from upstream of the 5'UTR to downstream of the 3'UTR containing the human LRRK2 gene (including the G2019S mutation), resulting in a humanized LRRK2 gene locus and achieving humanization of the mouse LRRK2 gene.

[0219] To achieve the targeting strategy of this invention, a targeting vector was constructed. The targeting vector contains upstream and downstream homologous arm sequences of the mouse LRRK2 gene, as well as fragment A containing the human LRRK2 fragment. Specifically, the upstream 5' homologous arm sequence is SEQ ID NO: 3, and the downstream 3' homologous arm sequence is SEQ ID NO: 4. The human LRRK2 fragment is derived from BAC (CH17-436L16), and its nucleotide sequence shares greater than 99% identity with positions 40204832-40370222 of accession number NC_000012.12. It contains at least the following mutations: G to A at position 40225280, T to C at position 40237989, C to A at position 40320032, A to G at position 40320071, T to A at position 40320099, T to C at position 40322458, G to A at position 40340400, G to A at position 40348452, and T to C at position 40364850. The upstream linker of the human LRRK2 fragment to the mouse fragment is designed as follows:

[0220] The mRNA sequence of the modified humanized mouse LRRK2 is shown in SEQ ID NO: 9, and the expressed protein sequence is shown in SEQ ID NO: 10.

[0221] Targeted vector construction can be performed using conventional methods, such as enzyme digestion and ligation. After preliminary validation via enzyme digestion, the constructed targeted vector is sent to a sequencing company for sequencing verification. The sequenced and validated targeted vector is then electroporated into embryonic stem cells of C57BL / 6 mice. Positive clone selection marker genes are used to screen the resulting cells, identifying the correct positive clones. These selected positive clones (black mice) are then introduced into isolated blastocysts (white mice) using techniques known in the art. The resulting chimeric blastocysts are transferred to a culture medium for brief culture before being transplanted into the oviducts of recipient mother mice (white mice) to produce F0 generation chimeric mice (black and white). Backcrossing the F0 generation chimeric mice with wild-type mice yields the F1 generation. Mating the F1 generation heterozygous mice with each other yields the F2 generation homozygous mice.

[0222] The genotype of F1 generation mouse somatic cells can be identified using PCR. The primers shown in the table below are used for detection. Example results are shown below. Figure 1 As shown, the seven mice numbered F1-1 to F1-7 were positive mice.

[0223] Table 3 Primer sequences and recombinant fragment sizes for F1 generation genotype PCR detection

[0224] The expression of LRRK2 gene mRNA in humanized mice can be detected by RT-PCR. Specifically, one 6-7 week old wild-type C57BL / 6 mouse (+ / +) and one 6-7 week old female LRRK2 gene humanized mouse homozygous (H / H) prepared in this example were selected. Brain tissue was collected after euthanasia, and RT-PCR was performed using the primer sequences shown in the table below. The obtained mRNA fragments were then sequenced. The RT-PCR results are shown below. Figure 2 As shown in the figure, only mouse LRRK2 mRNA was detected in wild-type C57BL / 6 mice (+ / +), and human LRRK2 mRNA was not detected; human LRRK2 mRNA was detected in homozygous humanized mice (H / H), but mouse LRRK2 mRNA was not detected. Further sequencing confirmed the presence of the G6190A (p.G2019S) mutation on this human mRNA fragment, while the control mouse mRNA fragment showed a wild-type expression at the corresponding site, and this mutation was not detected.

[0225] Table 4 RT-PCR primer sequences and target fragment sizes

[0226] In addition, the expression of human LRRK2 protein in LRRK2 humanized mice can be detected by conventional methods such as Western blot. Specifically, one 6-7 week old male wild-type C57BL / 6 mouse (+ / +) and one 6-7 week old male LRRK2 gene humanized mouse homozygous (H / H) prepared in this study were selected. Cerebral cortex, hippocampus, thalamus, and cerebellum tissues were collected and detected using a human-mouse cross-antibody recombinant anti-LRRK2 antibody [MJFF2 (c41-2)] (Abcam, catalog number ab133474). The detection results are as follows: Figure 3 As shown.

[0227] The results showed that LRRK2 protein expression could be detected in the cerebral cortex, hippocampus, thalamus, and cerebellum of wild-type C57BL / 6 mice (+ / +), and in the cerebral cortex, hippocampus, thalamus, and cerebellum of LRRK2 gene-humanized mouse homozygotes (H / H). Combined with the above RT-PCR results, it was confirmed that human LRRK2 protein could be successfully expressed in LRRK2 gene-humanized mouse homozygotes (H / H).

[0228] Example 2: Pharmacodynamic Model

[0229] The humanized mice disclosed in this invention can be used to induce various human disease models, such as Parkinson's disease models, which can be used to test the in vivo efficacy of human-specific drugs. For example, LRRK2 gene-humanized mice can be used to evaluate the efficacy, pharmacokinetics, and in vivo therapeutic efficacy of human-specific LRRK2 signaling pathway antagonists in various disease models known in the art.

[0230] For example, the inhibitory efficiency of nucleic acid drugs on human LRRK2 mRNA in LRRK2 gene-humanized mice can be detected. Specifically, eight 8-week-old homozygous LRRK2 gene-humanized mice prepared in Example 1 were randomly divided into a control group (G1, 4 mice) and a treatment group (G2, 4 mice). On day 0 of the grouping, mice in the treatment group (G2) were injected intracerebroventricularly with a human LRRK2-targeting nucleic acid drug (sequence mCesGeomCesAesmCesTdsTdsAdsAdsmCdsAdsAdsTdsAdsTdsmCesAeoTesAesTe (SEQ ID NO: 20), where A represents adenine nucleobase, mC represents 5-methylcytosine nucleobase, G represents guanine nucleobase, T represents thymine nucleobase, e represents 2'-MOE-modified sugar, d represents 2'-deoxyribose, s represents thiophosphate nucleoside internucleotide bond, and o represents phosphodiester nucleoside internucleotide bond). Mice in the control group (G1) were injected subcutaneously with an equal volume of artificial cerebrospinal fluid (Huazhong Haiwei, catalog number: SR3721). On day 14 of grouping, all mice were euthanized, and the midbrain, striatum, cortex, and hippocampus were collected. The expression level of human LRRK2 mRNA in each tissue was detected by qPCR. The qPCR primers are shown in Table 5 below, and the detection results are as follows. Figure 4 As shown.

[0231] Table 5. Primer sequences and recombinant fragment sizes for qPCR detection

[0232] like Figure 4 As shown, compared with the control group (G1), the expression levels of human LRRK2 mRNA in the brain, striatum, cortex, and hippocampus of the treated mice (G2) were significantly reduced, with expression inhibition rates of approximately 75.6%, 82.1%, 87.1%, and 83.4%, respectively. This indicates that the LRRK2 gene-humanized mice prepared in this application can be used to evaluate the efficacy of drugs targeting human LRRK2 nucleic acid in mice.

[0233] Taking the preparation of a Parkinson's disease model as an example, LRRK2 gene-humanized mice prepared according to this invention can be used. The mice are normally fed, and after spontaneous onset of the disease, they are randomly divided into a control group and a treatment group. Drugs targeting LRRK2 are randomly selected. The treatment group is administered the drug via various routes, including lateral ventricle injection, subcutaneous injection, gavage, intraperitoneal injection, or tail vein injection. The control group receives an equal volume of PBS solution. The in vivo efficacy of different human drugs can be evaluated through multiple detection indicators, including changes in body weight, behavioral tests such as the open field test, rotador test, grip strength test, and elevated plus maze test, as well as pathological examination of protein staining in different brain regions.

[0234] Example 3: Preparation of dual-gene or multi-gene humanized mice

[0235] The LRRK2 gene-humanized mice obtained using this method can also be used to prepare multi-gene mouse models. For example, in Example 1 above, the embryonic stem cells used for microinjection can be selected from mice modified with at least one gene: SNCA, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4. Alternatively, based on humanized LRRK2 mice, mouse ES embryonic stem cells can be isolated and gene recombination targeting technology can be used to obtain dual-humanized or multi-gene-humanized mouse models. The homozygous or heterozygous LRRK2 mice obtained by this method can also be mated with other gene-modified mice, and their offspring can be screened. According to Mendelian inheritance, there is a certain probability of obtaining multi-gene mice with humanized LRRK2 gene and other gene modifications. Furthermore, mating heterozygotes with each other can yield homozygotes with dual or multi-gene modifications.

[0236] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0237] 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 describe the various possible combinations separately.

[0238] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for preparing genetically modified non-human animals, the method comprising: The genome of a non-human animal is modified such that the genome of the animal contains at least one chromosome containing a nucleotide sequence encoding human or chimeric leucine-rich repeat kinase 2 (LRRK2).

2. The method according to claim 1, wherein it possesses one or more of the following features: (i) The nucleotide sequence encoding the human or chimeric LRRK2 protein is operatively linked to at least one exogenous regulatory element on a chromosome (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR). (ii) The amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric LRRK2 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 10; (iii) The animal is a mammal, such as a monkey, rodent, mouse, or rat; preferably, the animal is a mouse; (iv) The modification causes one or more cells of the animal to express human or chimeric LRRK2 protein.

3. A method for preparing genetically modified non-human animals, the method comprising: Modifying the genome of a non-human animal, wherein the modification causes the nucleotide sequence in the genome of the animal that encodes the endogenous LRRK2 region at the endogenous LRRK2 locus to be replaced by the nucleotide sequence that encodes the corresponding region of human LRRK2.

4. The method according to claim 3, wherein it possesses one or more of the following features: (i) The nucleotide sequence encoding the corresponding region of human LRRK2 is operatively linked to exogenous regulatory elements (e.g., human LRRK2 promoter, 5'UTR and / or 3'UTR). (ii) The modification causes one or more cells of the animal to express human or humanized LRRK2 protein; (iii) The nucleotide sequence encoding the corresponding region of human LRRK2 contains all or part of exons 1 to 51 of the human LRRK2 gene; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 contains all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 contains all of human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 contains all of human exons 1 to 51 and at least 937 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 contains all of human exons 1 to 51, at least 937 bp of continuous nucleotides downstream of the 3'UTR and at least 20165 bp of continuous nucleotides upstream of the 5'UTR; (iv) The nucleotide sequence encoding the corresponding region of human LRRK2 has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the nucleotide sequence shown at positions 40204832-40370222 of NCBI Gene Accession Number NC_000012.12; preferably, the coding region of the nucleotide sequence encoding the corresponding region of human LRRK2 includes NCBI Gene Accession Number NC_0000. 12.12 The 40225280th position mutates from G to A, the 40237989th position mutates from T to C, the 40320032th position mutates from C to A, the 40320071st position mutates from A to G, the 40320099th position mutates from T to A, the 40322458th position mutates from T to C, the 40340400th position mutates from G to A, the 40348452nd position mutates from G to A, and / or the 40364850th position mutates from T to C; (v) The animal is a mammal, such as a monkey, rodent, mouse, or rat; preferably, the animal is a mouse; (vi) The animal is a mouse, and the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene, at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene and 801 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all of exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR and at least 20125 bp of continuous nucleotides upstream of the 5'UTR. (vii) The modified LRRK2 gene in the animal genome is homozygous or heterozygous for the endogenously replaced locus.

5. The method according to any one of claims 1-4, wherein, The modification occurs in at least one cell of the animal.

6. A method for preparing genetically modified non-human animals, the method comprising: Modifying at least one cell of a non-human animal, said modification causing said animal to contain at least one cell encoding a nucleotide sequence of a human or humanized LRRK2 protein, said humanized LRRK2 protein comprising at least 50, 100, 500, 1000, 1500, 2000, 2500, 2520, 2525, 2526 or 2527 consecutive amino acid residues consistent with the corresponding region of human LRRK2 protein.

7. The method according to claim 6, wherein it possesses one or more of the following features: (i) The modification causes the cells to express human or humanized LRRK2 protein; (ii) The nucleotide sequence encoding the human or humanized LRRK2 protein is operatively linked to an exogenous regulatory element (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR). (iii) The nucleotide sequence encoding human or humanized LRRK2 may be integrated into the animal endogenous LRRK2 locus.

8. The method according to any one of claims 1-7, wherein, The human or humanized LRRK2 protein or chimeric LRRK2 protein has at least one mouse LRRK2 activity and / or human LRRK2 activity.

9. The method according to any one of claims 1-8, wherein, The modification results in the animal not expressing endogenous LRRK2 protein or having a reduced expression level of LRRK2 compared to wild-type animals.

10. The method according to any one of claims 1-9, wherein, The non-human animals also include nucleotide sequences of other genes encoding human or chimeric proteins selected from at least one of SNCA, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, and CTLA4.

11. A genetically modified nonhuman animal genome containing a nucleotide sequence encoding human or chimeric leucine-rich repeat kinase 2 (LRRK2).

12. The genome according to claim 11, wherein it possesses one or more of the following features: (i) The nucleotide sequence encoding the human or chimeric LRRK2 protein is operatively linked to at least one exogenous regulatory element on a chromosome (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR). (ii) The amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric LRRK2 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 10; (iii) The animal is a mammal, such as a monkey, rodent, mouse or rat; preferably, the animal is a mouse.

13. The genome according to claim 11 or 12, wherein the nucleotide sequence comprising encoding the endogenous LRRK2 region at the endogenous LRRK2 locus is replaced by the nucleotide sequence encoding the corresponding region of human LRRK2; Preferably, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all or part of exons 1 to 51 of the human LRRK2 gene; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 937 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 937 bp of continuous nucleotides downstream of the 3'UTR and at least 20165 bp of continuous nucleotides upstream of the 5'UTR. Preferably, the nucleotide sequence encoding the corresponding region of human LRRK2 has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the nucleotide sequence shown in positions 40204832-40370222 of NCBI gene accession number NC_000012.

12. Preferably, the coding region of the nucleotide sequence encoding the corresponding region of human LRRK2 includes the following mutations in NCBI accession number NC_000012.12: position 40225280 is mutated from G to A, position 40237989 is mutated from T to C, position 40320032 is mutated from C to A, position 40320071 is mutated from A to G, position 40320099 is mutated from T to A, position 40322458 is mutated from T to C, position 40340400 is mutated from G to A, position 40348452 is mutated from G to A, and / or position 40364850 is mutated from T to C; Preferably, the animal is a mouse, and the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and 801 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR, and at least 20125 bp of continuous nucleotides upstream of the 5'UTR. Preferably, the modified LRRK2 gene in the genome is homozygous or heterozygous for the endogenously replaced locus.

14. A method for constructing genetically modified non-human animal cells, the method comprising: The genome of a non-human animal cell is modified such that the genome of the cell contains at least one chromosome containing a nucleotide sequence encoding human or chimeric leucine-rich repeat kinase 2 (LRRK2).

15. The method of claim 14, wherein it comprises one or more of the following features: (i) The nucleotide sequence encoding the human or chimeric LRRK2 protein is operatively linked to at least one exogenous regulatory element on a chromosome (e.g., the human LRRK2 promoter, 5'UTR, and / or 3'UTR). (ii) The amino acid sequence encoded by the nucleotide sequence encoding the human or chimeric LRRK2 protein has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the sequence of SEQ ID NO: 2 or SEQ ID NO: 10; (iii) The cell is a mammalian cell (such as a monkey, rodent, mouse, or rat); preferably, the cell is a mouse cell; (iv) The modification causes the cells to express human or chimeric LRRK2 protein.

16. The method according to claim 14 or 15, wherein, The modification causes the cell's genome to include a nucleotide sequence at the endogenous LRRK2 locus encoding the endogenous LRRK2 region replaced by a nucleotide sequence encoding the corresponding region of human LRRK2. Preferably, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all or part of exons 1 to 51 of the human LRRK2 gene; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 50 bp of continuous nucleotides downstream of the 3'UTR and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51 and at least 937 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the corresponding region of human LRRK2 comprises all of human exons 1 to 51, at least 937 bp of continuous nucleotides downstream of the 3'UTR and at least 20165 bp of continuous nucleotides upstream of the 5'UTR. Preferably, the nucleotide sequence encoding the corresponding region of human LRRK2 has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with the nucleotide sequence shown in positions 40204832-40370222 of NCBI gene accession number NC_000012.

12. Preferably, the coding region of the nucleotide sequence encoding the corresponding region of human LRRK2 includes the following mutations in NCBI accession number NC_000012.12: position 40225280 is mutated from G to A, position 40237989 is mutated from T to C, position 40320032 is mutated from C to A, position 40320071 is mutated from A to G, position 40320099 is mutated from T to A, position 40322458 is mutated from T to C, position 40340400 is mutated from G to A, position 40348452 is mutated from G to A, and / or position 40364850 is mutated from T to C; Preferably, the cell is a mouse cell, and the nucleotide sequence encoding the endogenous LRRK2 region comprises all or part of exons 1 to 51 of the mouse LRRK2 gene; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and at least 50 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 50 bp of continuous nucleotides downstream of the 3'UTR, and at least 50 bp of continuous nucleotides upstream of the 5'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene and 801 bp of continuous nucleotides downstream of the 3'UTR; for example, the nucleotide sequence encoding the endogenous LRRK2 region comprises all exons 1 to 51 of the mouse LRRK2 gene, at least 801 bp of continuous nucleotides downstream of the 3'UTR, and at least 20125 bp of continuous nucleotides upstream of the 5'UTR. Preferably, the modified LRRK2 gene in the cell genome is homozygous or heterozygous for the endogenously replaced locus.

17. A method for determining the effectiveness of a therapeutic agent in treating cancer, wherein, The method includes: 1) Provide: a non-human animal obtained by the method according to any one of claims 1-10, or a non-human animal containing the genome of a genetically modified non-human animal as described in any one of claims 11-13; 2) Administering the LRRK2 therapeutic agent to the non-human animal, wherein the non-human animal has a tumor; 3) Determine the inhibitory effect of the therapeutic agent on the tumor; Preferably, the method has one or more features selected from the following: (i) The therapeutic agent is a small nucleic acid drug targeting LRRK2: (ii) The tumor comprises one or more tumor cells injected into the animal; (iii) The determination of the inhibitory effect of LRRK2 therapeutic agents on tumors involves measuring tumor volume in animals; (iv) The cancer mentioned is a solid tumor, hematologic malignancy, head and neck cancer, liver cancer or lung cancer.

18. A method for determining the effectiveness of LRRK2 therapeutic agents and other therapeutic agents in treating cancer, wherein, The method includes: 1) Provide: a non-human animal obtained by the method according to any one of claims 1-10, or a non-human animal containing the genome of a genetically modified non-human animal as described in any one of claims 11-13; 2) Administering LRRK2 therapeutic agent and other therapeutic agents to the non-human animal, the non-human animal having a tumor; 3) Determine its inhibitory effect on tumors; Preferably, the method has one or more features selected from the following: (i) The animal further comprises sequences encoding human or chimeric PD-1, human or chimeric PD-L1 and / or human or chimeric CTLA4; preferably, the other therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA4 antibody; (ii) The other therapeutic agents are anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA4 antibodies; (iii) The tumor contains one or more tumor cells expressing PD-L1; (iv) The tumor comprises one or more tumor cells injected into the animal; (v) The determination of tumor inhibition involves measuring tumor volume in animals; (vi) The animal suffers from solid tumors, hematomas, head and neck cancer, liver cancer, or lung cancer.

19. A method for determining the efficacy of LRRK2 therapeutic agents in treating immune diseases, wherein, The method includes: 1) Provide: a non-human animal obtained by the method according to any one of claims 1-10, or a non-human animal containing the genome of a genetically modified non-human animal as described in any one of claims 11-13; 2) Administering the LRRK2 therapeutic agent to the non-human animal, wherein the non-human animal suffers from an immune disease; 3) Determine the therapeutic effect of LRRK2 therapeutic agents on immune diseases; Preferably, the therapeutic agent is a small nucleic acid drug targeting LRRK2; and / or, the immune disease is asthma, rheumatoid arthritis, or multiple sclerosis.

20. A method for determining the effectiveness of LRRK2 therapeutic agents in treating inflammatory or neurodegenerative diseases, wherein, The method includes: 1) Provide: a non-human animal obtained by the method according to any one of claims 1-10, or a non-human animal containing the genome of a genetically modified non-human animal as described in any one of claims 11-13; 2) Administering LRRK2 treatment to the animals, wherein the animals have inflammatory or neurodegenerative diseases; 3) To determine the effectiveness of LRRK2 therapeutic agents in treating inflammatory or neurodegenerative diseases; Preferably, the therapeutic agent is a small nucleic acid drug targeting LRRK2; and / or, the inflammation is inflammatory bowel disease (IBD), and the neurodegenerative disease is Parkinson's syndrome.

21. A method for determining the toxicity of LRRK2 therapeutic agents, wherein, The method includes: 1) Provide: a non-human animal obtained by the method according to any one of claims 1-10, or a non-human animal containing the genome of a genetically modified non-human animal as described in any one of claims 11-13; 2) Administer LRRK2 treatment agent to the animals; 3) Determine the effects of LRRK2 therapeutic agents on animals; Preferably, the therapeutic agent is a small nucleic acid drug targeting LRRK2; and / or, the determination of the effect of the LRRK2 therapeutic agent on the animal involves measuring the animal's body weight, red blood cell count, hematocrit, and / or hemoglobin.

22. A cell, tissue, or organ, wherein, The cell or the cells in the tissue or organ contain the genome as described in any one of claims 11-13; preferably, the cell, tissue or organ is a non-human cell, tissue or organ.

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