Non-human animal modified by LPA and / or APOB genes

Abstract

The present invention provides a non-human animal expressing human or chimeric LPA and / or human or chimeric APOB proteins and methods of use thereof.

Description

Technical Field

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

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

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

[0004] The present application provides an animal model with human or chimeric LPA and / or human or chimeric APOB protein. The animal model can express human or chimeric LPA and / or human or chimeric APOB protein. It can be used for the study of LPA and / or APOB gene function, and can also be used for the screening and evaluation of LPA and / or APOB signaling pathway regulators (e.g., anti-human LPA and / or APOB antibodies, oligonucleotide drugs and / or polypeptide drugs). In addition, the animal model prepared by the method described in the present application can be used for drug screening, pharmacodynamics research, and treatment of diseases (e.g., diseases of human LPA and / or APOB targets, including tumors, inflammation, metabolic diseases or immune diseases); the animal model can also be used to promote new drug development and design, saving time and cost. In summary, the present invention provides a powerful tool for studying the function of LPA and / or APOB proteins and provides a platform for screening related drugs.

[0005] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises at least one chromosome, the chromosome comprising a nucleotide sequence encoding a human lipoprotein a (LPA) protein or a chimeric LPA protein, and the chimeric LPA protein comprises at least one Kringle domain of a human LPA protein. In some embodiments, the human LPA protein comprises at least 1 to 16 Kringle domains, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, the sequences of multiple Kringle domains are the same or different. In some embodiments, the Kringle domain comprises one or more of KIV-1, KIV-2, KIV-3, KIV-4, KIV-5, KIV-6, KIV-7, KIV-8, KIV-9, KIV-10 or KV. In some embodiments, the KIV-2 comprises KIV-2I, KIV-2II, KIV-2III, KIV-2IV, KIV-2v and / or KIV-2VI. In some embodiments, the amino acid sequence of KIV-1 comprises SEQ ID NO: 1, positions 27-105, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 27-105. In some embodiments, the amino acid sequence of KIV-2I comprises SEQ ID NO: 1, positions 141-219, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 141-219.

[0006] In some embodiments, the amino acid sequence of KIV-2II comprises SEQ ID NO: 1, positions 255-333, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 255-333. In some embodiments, the amino acid sequence of KIV-2III comprises SEQ ID NO: 1, positions 369-447, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 369-447. In some embodiments, the amino acid sequence of KIV-2IV comprises SEQ ID NO: 1, positions 483-561, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 483-561. In some embodiments, the amino acid sequence of KIV-2v comprises SEQ ID NO: 1, positions 597-675, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 597-675. In some embodiments, the amino acid sequence of KIV-2VI comprises SEQ ID NO: 1, positions 711-789, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 711-789. In some embodiments, the amino acid sequence of KIV-3 comprises SEQ ID NO: 1, positions 825-903, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 825-903. In some embodiments, the amino acid sequence of KIV-4 comprises SEQ ID NO: 1, positions 939-1017, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 939-1017. In some embodiments, the amino acid sequence of KIV-5 comprises SEQ ID NO: 1, positions 1053-1131, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 1053-1131.In some embodiments, the amino acid sequence of KIV-6 comprises SEQ ID NO: 1, positions 1167-1245, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 1167-1245. In some embodiments, the amino acid sequence of KIV-7 comprises SEQ ID NO: 1, positions 1273-1351, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 1273-1351. In some embodiments, the amino acid sequence of KIV-8 comprises SEQ ID NO: 1, positions 1387-1465, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 1387-1465. In some embodiments, the amino acid sequence of KIV-9 comprises SEQ ID NO: 1, positions 1501-1579, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 1501-1579. In some embodiments, the amino acid sequence of KIV-10 comprises SEQ ID NO: 1, positions 1615-1693, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 1615-1693. In some embodiments, the amino acid sequence of KV comprises SEQ ID NO: 1, positions 1719-1799, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1, positions 1719-1799. In some embodiments, the amino acid sequence of human LPA protein comprises SEQ ID NO: 1, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the chimeric LPA protein comprises SEQ ID NO: 7, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 7. In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein is regulated by an exogenous regulatory element, preferably, the exogenous regulatory element comprises an Alb promoter (i.e., Albumin promoter, albumin promoter) or a human LPA promoter.In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (e.g., a mouse or a rat). In some embodiments, the non-human animal is a mouse. In some embodiments, one or more cells of the non-human animal express a human or chimeric LPA protein.

[0007] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing it, the construction method comprising inserting a nucleotide sequence of human LPA into a safe site in the genome of a non-human animal, preferably, the safe site comprises a ROSA26 site or a Hipp11 site. In some embodiments, the nucleotide sequence of human LPA is operably linked to a regulatory element. In some embodiments, the regulatory element is an exogenous regulatory element (e.g., an Alb promoter or a human LPA promoter). In some embodiments, the nucleotide sequence of human LPA comprises a nucleotide sequence encoding a human or chimeric LPA protein. In some embodiments, the nucleotide sequence of human LPA comprises at least 1 to 16 Kringle domains encoding a human LPA protein, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 Kringle domains, wherein the multiple Kringle domains may be continuous or non-continuous. In some embodiments, the nucleotide sequence of human LPA comprises a nucleotide sequence encoding KIV-1, KIV-3, KIV-4, KIV-5, KIV-6, KIV-7, KIV-8, KIV-9, KIV-10, KV domains of human LPA protein, and any one or more of the six KIV-2 domains. In some embodiments, the nucleotide sequence of human LPA comprises a nucleotide sequence encoding KIV-1, KIV-2I, KIV-3, KIV-4, KIV-5, KIV-6, KIV-7, KIV-8, KIV-9, KIV-10 and KV domains of human LPA protein. In some embodiments, the nucleotide sequence of human LPA comprises a nucleotide sequence encoding SEQ ID NO: 1 or 7, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to a nucleotide sequence encoding SEQ ID NO: 1 or 7. In some embodiments, the nucleotide sequence of human LPA comprises all or part of exon 1 to exon 39 of the human LPA gene.In some embodiments, the nucleotide sequence of human LPA comprises any one of the following groups: A) part of exon 1 to all of exon 5 of the human LPA gene, and all of exon 16 to part of exon 39, wherein the part of exon 1 of the human LPA gene preferably comprises at least 5 bp nucleotide sequence, preferably a nucleotide sequence of the coding region; the part of exon 39 of the human LPA gene preferably comprises at least 5 bp nucleotide sequence, preferably a nucleotide sequence of the coding region; B) a nucleotide sequence from the start codon to the stop codon of the human LPA gene, preferably further comprising 5'UTR and / or 3'UTR, further preferably comprising all of exon 1 to exon 39 of the human LPA gene; more preferably further comprising at least 50 consecutive nucleotides upstream of the 5'UTR of the human LPA gene and / or at least 50 consecutive nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence of human LPA comprises the coding region of exons 1-5 of the human LPA gene. In some embodiments, the nucleotide sequence of human LPA comprises the coding region of exons 16-39 of the human LPA gene. In some embodiments, the nucleotide sequence of human LPA comprises the coding region of exons 1-5 and exons 16-39 of the human LPA gene. In some embodiments, the nucleotide sequence of human LPA comprises the nucleotide sequence of SEQ ID NO: 5 or NCBI accession number NC_000006.12, positions 160528474 to 160686025; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 5 or NCBI accession number NC_000006.12, positions 160528474 to 160686025. In some embodiments, the safe site of insertion into the non-human animal genome includes insertion between exon 1 and exon 2 of the ROSA26 gene in the non-human animal genome. In some embodiments, the nucleotide sequence inserted into the genome of a non-human animal further comprises an auxiliary sequence, and the auxiliary sequence preferably comprises one or more of a stop codon, WPRE, Insulator, loxP, STOP sequence or PolμA. In some embodiments, the modified LPA gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous modified gene site. In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent. In some embodiments, the rodent comprises a mouse or a rat.In some embodiments, the mRNA transcribed from the modified LPA gene in the genome of the non-human animal comprises a nucleotide sequence of SEQ ID NO: 6 or NCBI accession number NM_005577.4, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to a nucleotide sequence of SEQ ID NO: 6 or NCBI accession number NM_005577.4. In some embodiments, the genome of the non-human animal further comprises a nucleotide sequence encoding other human or humanized proteins, the other human or humanized proteins comprising at least one of APOB, PCSK9, APOC3, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the other human or humanized proteins are human or chimeric APOB proteins. In some embodiments, the amino acid sequence of the human or chimeric APOB protein comprises SEQ ID NO:33 or 38, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO:33 or 38.

[0008] In one aspect, the present invention provides a non-human animal or a method for constructing the same, wherein the non-human animal comprises at least one cell encoding a nucleotide sequence of a human or chimeric LPA protein. In some embodiments, the genetically modified non-human animal expresses a human or chimeric LPA protein. In some embodiments, the LPA protein expressed by the genetically modified non-human animal is consistent with at least 50 to 2040, for example, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1470, 1500, 1600, 1700, 1800, 1900, 2000 or 2040 consecutive amino acid sequences of the human LPA protein. In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein is regulated by an exogenous regulatory element, preferably, the exogenous regulatory element is an Alb promoter or a human LPA promoter. In some embodiments, the nucleotide sequence encoding human or chimeric LPA protein can be integrated into the endogenous gene site of the non-human animal. In some embodiments, the nucleotide sequence encoding human or chimeric LPA protein can be inserted into a safe site of the endogenous genome of a non-human animal, preferably, the safe site includes the ROSA26 site or the Hipp11 site.

[0009] On the one hand, the present invention provides a method for constructing a genetically modified non-human animal, in which at least one cell of the non-human animal is introduced into the genome of the non-human animal, and preferably, the introduction is insertion or replacement. In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein is all or part of the genomic DNA sequence of human LPA, all or part of the CDS sequence, or all or part of the cDNA sequence. In some embodiments, the introduction is insertion. In some embodiments, the introduction into the non-human animal genome is inserted into a safe site of the non-human animal genome (e.g., ROSA26 site or Hipp11 site). In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein comprises all or part of exon 1 to exon 39 of the human LPA gene, preferably at least 100-1000, 1000-5000, 5000-10000 or 10000-160000 bp of exon 1 to exon 39 of the human LPA gene. In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein is any one of the following groups: A) part of exon 1 to all of exon 5 of the human LPA gene, and all of exon 16 to part of exon 39, wherein the part of exon 1 of the human LPA gene preferably comprises at least 5-110 bp, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 74, 75, 80, 85, 90, 95, 100, 105 or 110 bp of nucleotide sequence, and further preferably comprises a nucleotide sequence of the coding region; the part of exon 39 of the human LPA gene preferably comprises at least 5-409 bp, such as 5, 10, 50, 100, 150, 160, 162, 165, 200, 250, 300, 350, 400 or 409 bp of nucleotide sequence, and further preferably comprises a nucleotide sequence of the coding region. B) all of exon 1 to exon 39 of the human LPA gene; preferably further comprising at least 50 bp of continuous nucleotides upstream of the 5'UTR of the human LPA gene, for example 50, 100, 500, 1000, 5000, 10000, 20000, 21750, 25000 or 30000 bp, preferably at least 50 bp to 30000 bp of continuous nucleotides, further preferably at least 20000 to 25000 bp of continuous nucleotides, and / or, at least 50 bp of continuous nucleotides downstream of the 3'UTR, for example 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3008, 3500 or 4000 bp, preferably at least 50 bp to 5000 bp, further preferably at least 3000 bp to 4000 bp of continuous nucleotides.In some embodiments, the amino acid sequence of the human or chimeric LPA protein comprises SEQ ID NO: 1 or 7; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence shown in SEQ ID NO: 1 or 7. In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein comprises SEQ ID NO: 5 or the nucleotide sequence of NCBI accession number NC_000006.12 at positions 160528474 to 160686025; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 5 or the nucleotide sequence of NCBI accession number NC_000006.12 at positions 160528474 to 160686025. In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein is operably linked to an exogenous regulatory element (eg, A1b promoter or human LPA promoter). In some embodiments, the non-human animal is a mammal, such as a monkey or a rodent (eg, a mouse or a rat).

[0010] In one aspect, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric LPA, the construction method comprising inserting a nucleotide sequence encoding a human or chimeric LPA protein into a safe site of the endogenous genome of a non-human animal, preferably, the safe site is a ROSA26 site or a Hipp11 site, to produce a genetically modified non-human animal cell expressing a human or chimeric LPA protein. In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein comprises all or part of exon 1 to exon 39 of the human LPA gene. Preferably, the nucleotide sequence encoding the human or chimeric LPA protein comprises any one of the following groups: A) part of exon 1 to all of exon 5 of the human LPA gene, and all of exon 16 to part of exon 39, wherein the part of exon 1 of the human LPA gene preferably comprises at least 5 bp of nucleotide sequence, preferably comprising the nucleotide sequence of the coding region; the part of exon 39 of the human LPA gene preferably comprises at least 5 bp of nucleotide sequence, preferably comprising the nucleotide sequence of the coding region; B) all of exon 1 to exon 39 of the human LPA gene; preferably, it also comprises at least 50 to 30,000 bp of continuous nucleotides upstream of the 5'UTR of the human LPA gene and / or at least 50 to 5,000 bp of continuous nucleotides downstream of the 3'UTR; more preferably, it also comprises at least 21,750 bp of continuous nucleotides upstream of the 5'UTR of the human LPA gene and / or at least 3,008 bp of continuous nucleotides downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein comprises the coding region of exons 1-5 of the human LPA gene. In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein comprises the coding region of exons 16-39 of the human LPA gene. In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein comprises the coding region of exons 1-5 of the human LPA gene, and, the coding region of exons 16-39. In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein comprises the nucleotide sequence of SEQ ID NO: 5 or NCBI accession number NC_000006.12 at positions 160528474 to 160686025; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in SEQ ID NO: 5 or NCBI accession number NC_000006.12 at positions 160528474 to 160686025. In some embodiments, the amino acid sequence of the human or chimeric LPA protein comprises SEQ ID NO: 1 or 7; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence shown in SEQ ID NO: 1 or 7.In some embodiments, the nucleotide sequence encoding the human or chimeric LPA protein is operably linked to an exogenous regulatory element (eg, Alb promoter or human LPA promoter). In some embodiments, the non-human animal is a mouse.

[0011] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the genome of the non-human animal comprises at least one chromosome, wherein the chromosome comprises a nucleotide sequence encoding a human or chimeric apolipoprotein B (APOB) protein. In some embodiments, the amino acid sequence of the human or chimeric APOB protein comprises SEQ ID NO: 33 or 38; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO: 33 or 38. In some embodiments, the nucleotide sequence encoding the human or chimeric APOB protein is regulated by a regulatory element, wherein the regulatory element comprises an endogenous regulatory element or an exogenous regulatory element, and preferably, the exogenous regulatory element is a human APOB promoter, 5'UTR and / or 3'UTR. In some embodiments, the non-human animal comprises a mammal, such as a monkey or a rodent (e.g., a mouse or a rat). In some embodiments, the non-human animal is a mouse. In some embodiments, the endogenous APOB protein of the non-human animal is not expressed or is expressed at a reduced level compared to APOB in wild-type animals. In some embodiments, one or more cells of the non-human animal express human or chimeric APOB protein.

[0012] In one aspect, the present invention provides a genetically modified non-human animal or a method for constructing the same, wherein the endogenous APOB gene of the non-human animal is replaced with a nucleotide sequence comprising human APOB at the endogenous APOB locus of the non-human animal. In some embodiments, the nucleotide sequence of human APOB is operably linked to a regulatory element, and the regulatory element comprises an endogenous regulatory element of the non-human animal or an exogenous regulatory element; wherein the exogenous regulatory element comprises a human APOB promoter, a 5'UTR and / or a 3'UTR. In some embodiments, the endogenous APOB protein of the non-human animal is not expressed or the expression level is reduced compared with APOB in wild-type animals. In some embodiments, the nucleotide sequence of human APOB comprises a nucleotide sequence encoding human APOB protein. In some embodiments, the nucleotide sequence of human APOB comprises a nucleotide sequence from the start codon to the stop codon of the human APOB gene, preferably further comprising a 5'UTR and / or a 3'UTR. In some embodiments, the nucleotide sequence of human APOB comprises all of exon 1 to exon 29 of the human APOB gene. In some embodiments, the nucleotide sequence of human APOB further comprises at least 50 bp of continuous nucleotides upstream of the 5'UTR and, or at least 50 bp of continuous nucleotides downstream of the 3'UTR of the APOB gene. Preferably, the nucleotide sequence of human APOB further comprises at least 19100 bp of continuous nucleotides upstream of the 5'UTR and / or at least 17454 bp of continuous nucleotides downstream of the 3'UTR of the APOB gene. In some embodiments, the nucleotide sequence of human APOB comprises a nucleotide sequence encoding SEQ ID NO: 33 or 38. In some embodiments, the nucleotide sequence of human APOB comprises the nucleotide sequence of NCBI Accession No. NC_000002.12, 20983975 to 21063173, or a variant thereof, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in NCBI Accession No. NC_000002.12, 20983975 to 21063173, or a variant thereof.In some embodiments, the variants include mutating G at position 21042269 to T, mutating A at position 21040767 to C, mutating C at position 21037729 to T; inserting A between 21037656-21037657, mutating G at position 21032803 to A, inserting AGATG between 21032701-21032702, and inserting AGATG between 21029494-21029 497 deletion, mutation of A at position 21028168 to C, mutation of C at position 21028042 to T, mutation of A at position 21016795 to G, mutation of T at position 21013610 to C, mutation of A at position 21009931 to G, mutation of C at position 21009932 to T, mutation of C at position 21009323 to T, or mutation of G at position 21002409 to A. In some embodiments, the modified APOB gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus. In some embodiments, the replaced endogenous APOB gene of a non-human animal comprises all of exon 1 to exon 29 of the endogenous APOB gene of the non-human animal, and more preferably comprises at least 50 bp of continuous nucleotides upstream of the 5'UTR and / or at least 50 bp of continuous nucleotides downstream of the 3'UTR of the endogenous APOB gene of the non-human animal, and even more preferably comprises at least 19997 bp of continuous nucleotides upstream of the 5'UTR and / or at least 15433 bp of continuous nucleotides downstream of the 3'UTR of the endogenous APOB gene of the non-human animal. In some embodiments, the mRNA transcribed from the modified APOB gene in the genome of the non-human animal comprises SEQ ID NO: 37, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 37. In some embodiments, the genome of the non-human animal further comprises nucleotide sequences encoding other human or humanized proteins, including at least one of LPA, PCSK9, APOC3, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the other human or humanized proteins are human or chimeric LPA proteins. In some embodiments, the amino acid sequence of the human LPA protein comprises SEQ ID NO: 1, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.5% identical to SEQ ID NO: 1.In some embodiments, the amino acid sequence of the chimeric LPA protein comprises SEQ ID NO:7, or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:7.

[0013] In one aspect, the present invention provides a non-human animal or a method for constructing the same, wherein the non-human animal comprises at least one cell comprising a nucleotide sequence encoding a human or chimeric APOB protein, wherein the chimeric APOB protein comprises an amino acid sequence that is identical to at least 100 to 4563, such as 100, 1000, 2000, 3000, 4000, 4200, 4400, 4500, 4560 or 4563 consecutive or non-contiguous amino acid sequences of a human APOB protein. In some embodiments, the nucleotide sequence encoding a human or chimeric APOB protein is regulated by a regulatory element, preferably, the regulatory element comprises an endogenous regulatory element of the non-human animal or an exogenous regulatory element (e.g., a human APOB promoter, 5'UTR and / or 3'UTR). In some embodiments, the nucleotide sequence encoding a human or chimeric APOB protein is introduced into an endogenous APOB locus of a non-human animal, preferably, the introduction is a replacement or insertion. In some embodiments, the chimeric APOB protein has at least one activity, such as a non-human animal APOB activity and / or a human APOB activity.

[0014] On the one hand, the present invention provides a method for constructing a genetically modified non-human animal, wherein a nucleotide sequence encoding a human APOB corresponding region is introduced into the endogenous APOB locus of the non-human animal, and preferably, the introduction is an insertion or replacement. In one embodiment, the construction method includes replacing the endogenous APOB gene of the non-human animal with a nucleotide sequence comprising a human APOB corresponding region at the endogenous APOB locus of the non-human animal. In some embodiments, the endogenous APOB protein of the non-human animal is not expressed or the expression level is reduced compared with APOB in wild-type animals. In some embodiments, the nucleotide sequence encoding the human APOB corresponding region is a genomic DNA sequence, CDS sequence or cDNA sequence of human APOB. In some embodiments, the nucleotide sequence encoding the human APOB corresponding region comprises all or part of exon 1 to exon 29 of the human APOB gene, and preferably, the nucleotide sequence encoding the human APOB corresponding region comprises a nucleotide sequence from the start codon to the stop codon of the human APOB gene, and preferably also comprises a 5'UTR and / or a 3'UTR. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOB includes all of exon 1 to exon 29 of the human APOB gene, and preferably also includes at least 50bp upstream of the 5'UTR, for example 50, 100, 500, 1000, 5000, 10000, 15000, 19100, 20000, 25000 or 30000bp of consecutive nucleotides, preferably at least 50bp to 30000bp of consecutive nucleotides, and / or, at least 50bp downstream of the 3'UTR, for example 50, 100, 500, 1000, 5000, 10000, 15000, 17000, 17454, 17500 or 20000bp of consecutive nucleotides, preferably at least 50bp to 20000bp of consecutive nucleotides. In some embodiments, the amino acid sequence of the human APOB corresponding region comprises SEQ ID NO: 33 or 38; or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence shown in SEQ ID NO: 33 or 38. In some embodiments, the nucleotide sequence encoding the human APOB corresponding region comprises the nucleotide sequence of NCBI Accession No. NC_000002.12, nucleotides 20983975 to 21063173, or a variant thereof, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence shown in NCBI Accession No. NC_000002.12, nucleotides 20983975 to 21063173, or a variant thereof.In some embodiments, the nucleotide sequence encoding the human APOB corresponding region is operably linked to a regulatory element, preferably, the regulatory element includes a non-human animal endogenous or exogenous regulatory element (e.g., human APOB promoter, 5'UTR and, or 3'UTR). In some embodiments, the non-human animal includes a non-human mammal, such as a monkey or a rodent (e.g., a mouse or a rat).

[0015] On the one hand, the present invention provides a method for constructing a genetically modified non-human animal cell expressing human or chimeric APOB, the construction method comprising introducing a nucleotide sequence encoding a corresponding region of human APOB at the endogenous APOB locus of the non-human animal, preferably, the introduction is an insertion or a replacement. In some embodiments, in the non-human animal cells, the endogenous APOB protein is not expressed or the expression level is reduced compared to APOB in wild-type animals. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOB is a genomic DNA sequence, CDS sequence or eDNA sequence of human APOB. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOB includes all of exon 1 to exon 29 of the human APOB gene, preferably also includes at least 50bp of continuous nucleotide sequence upstream of the 5'UTR and / or at least 50bp of continuous nucleotide sequence downstream of the 3'UTR. In some embodiments, the nucleotide sequence encoding the human APOB corresponding region is at least 100-50675, such as 100, 1000, 5000, 10000, 12000, 13000, 14000, 14121, 15000, 20000, 25000, 30000, 31575, 33221, 35000, 40000, 45000, 50000 or 50675 bp nucleotides identical to the human APOB gene. In some embodiments, the amino acid sequence of the human APOB corresponding region comprises SEQ ID NO: 33 or 38, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence of SEQ ID NO: 33 or 38. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOB comprises the nucleotide sequence of NCBI accession number NC_000002.12, 20983975 to 21063173, or a variant thereof, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in NCBI accession number NC_000002.12, 20983975 to 21063173, or a variant thereof. In some embodiments, the nucleotide sequence encoding the corresponding region of human APOB is operably linked to an exogenous regulatory element, preferably, the exogenous regulatory element is a human APOB promoter, 5'UTR and / or 3'UTR. In some embodiments, the non-human animal is a mouse.

[0016] In one aspect, the present invention provides a method for determining the effectiveness of a therapeutic agent in treating a disease, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method, wherein the non-human animal has a disease; 2) determining the effect of the therapeutic agent on the disease, preferably an inhibitory effect. In some embodiments, the therapeutic agent comprises an LPA and / or APOB therapeutic agent, preferably comprising one or more of an anti-LPA and / or APOB antibody, an LPA and / or APOB inhibitor, or an LPA and / or APOB nucleic acid drug. In some embodiments, the therapeutic agent further comprises an additional therapeutic agent, and the additional therapeutic agent preferably comprises one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA4 antibody. In some embodiments, the disease comprises one or more of a tumor, an immune disease, a metabolic disease, or inflammation. In some embodiments, the tumor comprises one or more tumor cells injected into an animal. In some embodiments, determining the inhibitory effect of the therapeutic agent on the disease involves measuring the tumor volume in a non-human animal. In some embodiments, the tumor comprises a solid tumor and / or a hematological tumor. In some implementations, the tumor comprises one or more of head and neck cancer, liver cancer, or lung cancer. In some embodiments, the immune disease includes one or more of asthma, psoriasis, atopic dermatitis, rheumatoid arthritis or multiple sclerosis. In some embodiments, the inflammation includes inflammatory bowel disease (IBD). In some embodiments, the metabolic disease includes hyperlipidemia or atherosclerosis.

[0017] In one aspect, the present invention provides a method for determining the toxicity of a therapeutic agent, the method comprising: 1) administering a therapeutic agent to the non-human animal or the non-human animal obtained by the construction method; 2) determining the effect of the therapeutic agent on the non-human animal. In some embodiments, the determination of the effect of the therapeutic agent on the non-human animal comprises measuring the body weight of the non-human animal or performing a blood test, preferably, the blood test includes but is not limited to red blood cell count, hematocrit and / or hemoglobin content.

[0018] In one aspect, the present invention provides a chimeric LPA protein. In some embodiments, the chimeric LPA protein comprises at least one Kringle domain of a human LPA protein. In some embodiments, the amino acid sequence of the chimeric LPA protein comprises SEQ ID NO: 7, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 7.

[0019] In one aspect, the present invention provides a chimeric LPA gene, the chimeric LPA gene comprising any of the following nucleotide sequences: A) a nucleotide sequence encoding a human LPA protein, preferably comprising a nucleotide sequence encoding SEQ ID NO: 1; B) a nucleotide sequence encoding a chimeric LPA protein, preferably comprising a nucleotide sequence encoding SEQ ID NO: 7; C) a nucleotide sequence of SEQ ID NO: 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 23, 24, 25, NCBI accession number NM_005577.4 or NCBI accession number NC_000006.12 at positions 160528474 to 160686025; D) a nucleotide sequence encoding a chimeric LPA protein, preferably comprising a nucleotide sequence encoding SEQ ID NO: 7; NO: 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 23, 24, 25, a nucleotide sequence with at least 90% identity to the nucleotide sequence at positions 160528474 to 160686025 of NCBI accession number NM_005577.4 or NCBI accession number NC_000006.12; E) a nucleotide sequence with at least 90% identity to the nucleotide sequence at positions 160528474 to 160686025 of SEQ ID NO: 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 23, 24, 25, a nucleotide sequence with NCBI accession number NM_005577.4 or NCBI accession number NC_000006.12 and nucleotide sequence identity from positions 160528474 to 160686025 of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%.

[0020] In one aspect, the present invention provides a chimeric APOB gene, wherein the chimeric APOB gene comprises any of the following nucleotide sequences: A) a nucleotide sequence encoding a human or chimeric APOB protein, preferably comprising a nucleotide sequence encoding SEQ ID NO: 33 or 38; B) a nucleotide sequence of SEQ ID NO: 34, 35, 37, 39, 40, 41, 42 or NC_000002.12 nucleotides 20983975 to 21063173 or a variant thereof; C) a nucleotide sequence having at least 90% identity to a nucleotide sequence of SEQ ID NO: 34, 35, 37, 39, 40, 41, 42 or NC_000002.12 nucleotides 20983975 to 21063173 or a variant thereof; D) a nucleotide sequence having at least 90% identity to a nucleotide sequence of SEQ ID NO: 34, 35, 37, 39, 40, 41, 42 or NC_000002.12 nucleotides 20983975 to 21063173 or a variant thereof; NO: 34, 35, 37, 39, 40, 41, 42 or NC_000002.12 nucleotide sequence 20983975 to 21063173 or its variants thereof have an identity of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% of the nucleotide sequence; preferably, the variants include mutating G at position 21042269 to T, mutating A at position 21040767 to C, mutating C at position 21037729 to T; inserting A between 21037656-21037657, and mutating 21042269 to T; The G at position 032803 is mutated to A, AGATG is inserted between 21032701-21032702, 21029494-21029497 is deleted, A at position 21028168 is mutated to C, C at position 21028042 is mutated to T, A at position 21016795 is mutated to G, T at position 21013610 is mutated to C, A at position 21009931 is mutated to G, C at position 21009932 is mutated to T, C at position 21009323 is mutated to T, or G at position 21002409 is mutated to A, one or more of the following.

[0021] In one aspect, the present invention provides a chimeric APOB protein. In some embodiments, the chimeric APOB protein is a humanized APOB protein. In some embodiments, the amino acid sequence of the humanized APOB protein comprises an amino acid sequence that is identical to at least 100 to 4563, such as 100, 1000, 2000, 3000, 4000, 4200, 4400, 4500, 4560 or 4563 consecutive or non-contiguous amino acid sequences of a human APOB protein. In some embodiments, the humanized APOB protein has at least one amino acid mutation compared to the human APOB protein, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid mutations. In some embodiments, the humanized APOB protein has one, two or three of the mutations from position 618 from A to v, from position 2313 from I to V, or from position 4338 from S to N compared to the human APOB protein. In some embodiments, compared with SEQ ID NO: 33, the mutations of the humanized APOB protein include one, two or three of the mutations of position 618 from A to V, position 2313 from I to V, and position 4338 from S to N. In some embodiments, the amino acid sequence of the humanized APOB protein includes SEQ ID NO: 33 or 38, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 33 or 38.

[0022] In one aspect, the present invention provides a cell, tissue or organ, which expresses one or more of human LPA protein, human APOB protein, the above-mentioned chimeric LPA protein or the above-mentioned chimeric APOB protein; and / or the genome of the cell, tissue or organ contains the above-mentioned chimeric LPA gene and / or the above-mentioned chimeric APOB gene.

[0023] In one aspect, the present invention provides an animal model, which expresses one or more of human LPA protein, human APOB protein, the above-mentioned chimeric LPA protein or the above-mentioned chimeric APOB protein; and / or the genome of the animal model contains the above-mentioned chimeric LPA gene and / or the above-mentioned chimeric APOB gene.

[0024] In one aspect, the use of the non-human animal, the non-human animal obtained by the construction method, the cell obtained by the construction method, the chimeric LPA protein, the chimeric LPA gene, the chimeric APOB protein, the chimeric APOB gene, the cell, tissue or organ or the animal model, the use comprises: A) application in the development of products involving immune processes related to LPA and / or APOB in human cells; B) application as a model system related to human LPA and / or human APOB in pharmacology, immunology, microbiology and medical research; C) Involving the production and use of animal experimental disease models for the study of etiology related to human LPA and / or human APOB and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo research on the screening, efficacy testing, efficacy assessment, verification or evaluation of human LPA and / or human APOB signaling pathway regulators; or, E) studying the gene function of human LPA and / or human APOB, studying drugs and efficacy targeting human LPA and / or human APOB targets, and studying the application of drugs for tumors, inflammation, immunity, and metabolic diseases related to human LPA and / or human APOB.

[0025] The term "all or part" in the present invention, "all" refers to the whole, and "part" refers to a part of the whole, or an individual part that constitutes the whole.

[0026] The term "locus" in the present invention refers to the position of a gene on a chromosome in a broad sense, and refers to a DNA fragment on a gene in a narrow sense, which can be a gene, a part of a gene, or a regulatory region of a gene, etc. For example, the "LPA locus" includes a DNA fragment selected from exons 1 to 39 of the LPA gene. For example, the "APOB locus" includes a DNA fragment selected from exons 1 to 29 of the APOB gene.

[0027] The term "portion of exon XX" of the present invention means that the continuous or interval of several bp, tens of bp or hundreds of bp nucleotide sequence is consistent with the entire exon nucleotide sequence. For example, the portion of exon 1 of the human LPA gene includes continuous 5-110bp, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 74, 75, 80, 85, 90, 95, 100, 105 or 110bp nucleotide sequence.

[0028] The term "all of exon XX to exon XXX" or "exon XX to exon XXX" or "exon XX--XXX" or "all of exon XX to all of exon XXX" of the present invention refers to exons and introns therebetween, for example, all of exon 1 to exon 29 includes exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, exon 5, intron 5, exon 6, intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, intron 9, exon 10, intron 10, exon 11, intron 11, The entire nucleotide sequence of exon 12, intron 12, exon 13, intron 13, exon 14, intron 14, exon 15, intron 15, exon 16, intron 16, exon 17, intron 17, exon 18, intron 18, exon 19, intron 19, exon 20, intron 20, exon 21, intron 21, exon 22, intron 22, exon 23, intron 23, exon 24, intron 24, exon 25, intron 25, exon 26, intron 26, exon 27, intron 27, exon 28, intron 28 and exon 29.

[0029] The term "part of exon XX to all of exon XXX" or "all of exon XX to part of exon XXX" of the present invention includes all or part of one exon to all or part of another exon and introns therebetween. For example, part of exon 1 to all of exon 5 includes part of exon 1, all of intron 1, all of exon 2, all of intron 2, all of exon 3, all of intron 3, all of exon 4, all of intron 4, and all of exon 5. For example, the entirety of exon 16 to the portion of exon 39 includes the entirety of exon 16, the entirety of intron 16, the entirety of exon 17, the entirety of intron 17, the entirety of exon 18, the entirety of intron 18, the entirety of exon 19, the entirety of intron 19, the entirety of exon 20, the entirety of intron 20, the entirety of exon 21, the entirety of intron 21, the entirety of exon 22, the entirety of intron 22, the entirety of exon 23, the entirety of intron 23, the entirety of exon 24, the entirety of intron 24, the entirety of exon 25, the entirety of intron 25, the entirety of exon 26, the entirety of intron 26, the entirety of exon 27. All of intron 27, all of intron 27, all of exon 28, all of intron 28, all of exon 29, all of intron 29, all of exon 30, all of intron 30, all of exon 31, all of intron 31, all of exon 32, all of intron 32, all of exon 33, all of intron 33, all of exon 34, all of intron 34, all of exon 35, all of intron 35, all of exon 36, all of intron 36, all of exon 37, all of intron 37, all of exon 38, all of intron 38, and part of exon 39.

[0030] The term "intron xx" of the present invention refers to an intron between two exons, for example, intron 1 is an intron between exon 1 and exon 2.

[0031] The term "safety site" in the present invention refers to a part of a chromosome that will not have adverse effects on host cells or organisms, is used for safe gene knock-in and can ensure the normal and stable expression of the transferred gene.

[0032] The term "comprising" or "including" in the present invention is an open-ended writing method, which contains the specified components or steps described, as well as other specified components or steps that will not be substantially affected. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0033] The term "and / or" of the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately in the present application. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and, or C" includes "A", "B", "C", "A and C", "B and C", "A and B" and "A and B and C".

[0034] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. This application describes methods and materials for use in the present invention; other suitable methods and materials known in the art may be used. The materials, methods, and examples are exemplary and non-limiting only. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0035] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description.

[0036] LPA

[0037] In the human genome, the LPA gene (NCBI Gene ID: 4018, UniProt ID: P08519, located at positions 160531482 to 160664275 of chromosome 6 NC_000006.12) contains 39 exons. The corresponding positions of the domains in the nucleotide sequence and amino acid sequence of the transcript NM_005577.4 and its encoded protein NP_005568.2 (SEQ ID NO: 1) are shown in Table 1:

[0038] Table 1

[0039]

[0040]

[0041] APOB

[0042] In the human genome, the APOB gene (NCBI Gene ID: 338, UniProt ID: P04114, located at 21001429 to 21044073 of chromosome 2 NC_000002.12) contains 29 exons. The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_000384.3 and its encoded protein NP_000375.3 (SEQ ID NO: 33) is shown in Table 2:

[0043] Table 2

[0044]

[0045] In the mouse genome, the APOB gene (NCBI Gene ID: 238055, UniProt ID: E9Q414, located at 8027629 to 8066839 of chromosome 12 NC_000078.7) contains 29 exons. The corresponding position of each exon in the nucleotide sequence and amino acid sequence of the transcript NM_009693.2 and its encoded protein NP_033823.2 (SEQ ID NO: 32) is shown in Table 3:

[0046] Table 3

[0047]

[0048] LPA and APOB genes, proteins and gene loci of other species are also known in the art. For example, the relevant information (e.g., intron sequence, exon sequence and amino acid sequence) of these genes of Rattus norvegicus (rat), Macaca mulatta (rhesus monkey), Canis lupus familiaris (dog), Sus scrofa (pig) can be found in NCBI, the entire contents of which are incorporated into this application by reference.

[0049] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are compared for the purpose of optimal comparison (e.g., for optimal comparison, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides on the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, which needs to be introduced to achieve the optimal comparison of the two sequences, taking into account the number of gaps and the length of each gap. For example, the comparison of the sequences and the determination of the percent identity between the two sequences can be completed using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0050] The percentage (homology percentage) of conservative residues with similar physicochemical properties, such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage is higher than the identity percentage.

[0051] Carrier

[0052] The present invention provides a targeting vector comprising: a) a DNA fragment homologous to the 5' end of the switch region to be changed (5' homology arm or 5' arm), which is selected from the genomic DNA of the ROSA26 gene and has a length of 100-10000 bp nucleotides; b) a donor region; and c) a DNA fragment homologous to the 3' end of the switch region to be changed (3' homology arm or 3' arm), which is selected from the genomic DNA of the ROSA26 gene and has a length of 100-10000 bp nucleotides.

[0053] In some embodiments, the switch region to be altered is located between exon 1 and exon 2 (on intron 1) of the ROSA26 gene of a non-human animal.

[0054] In some embodiments, the 5' homology arm sequence comprises SEQ ID NO: 2, 12 or 15. In some embodiments, the 3' homology arm sequence comprises SEQ ID NO: 3, 13 or 16.

[0055] In some embodiments, the donor sequence in the targeting vector comprises a human sequence (e.g., SEQ ID NO: 5 or the nucleotide sequence shown in NCBI accession number NC_000006.12, positions 160528474 to 160686025). In some embodiments, the nucleotide sequence contained in the targeting vector encodes a human or chimeric LPA protein.

[0056] The present invention provides a targeting vector comprising: a) a DNA fragment homologous to the 5' end of the conversion region to be changed (5' homology arm or 5' arm), which is selected from the genomic DNA of the APOB gene and has a length of 100-10000 bp nucleotides; b) a donor region; and c) a DNA fragment homologous to the 3' end of the conversion region to be changed (3' homology arm or 3' arm), which is selected from the genomic DNA of the APOB gene and has a length of 100-10000 bp nucleotides.

[0057] In some embodiments, a) the DNA fragment homologous to the 5' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% identity with NCBI Accession No. NC_000078.7; c) the DNA fragment homologous to the 3' end of the switch region to be altered is selected from a nucleotide sequence having at least 90% identity with NCBI Accession No. NC_000078.7.

[0058] In some embodiments, the switch region to be changed is located from at least 50 bp of continuous nucleotide sequence upstream of the 5'UTR to at least 50 bp of continuous nucleotide sequence downstream of the 3'UTR of the APOB gene of the non-human animal.

[0059] In some embodiments, the 5' homology arm sequence comprises SEQ ID NO: 34. In some embodiments, the 3' homology arm sequence comprises SEQ ID NO: 35.

[0060] In some embodiments, the donor sequence in the targeting vector comprises a human sequence (e.g., NCBI accession number NC_000002.12 nucleotide sequence 20983975 to 21063173 or a variant thereof, wherein the variant preferably comprises mutating G at position 21042269 to T, mutating A at position 21040767 to C, mutating C at position 21037729 to T; inserting A between 21037656-21037657, mutating G at position 21032803 to A, and mutating C at position 21032804 to C. In some embodiments, the nucleotide sequence contained in the targeting vector encodes all or part of the human APOB protein. In some embodiments, the NCBI protein number of the human APOB protein is NP_000375.3 (SEQ ID NO: 33). In some embodiments, the amino acid sequence of the human APOB protein is SEQ ID NO: 38.

[0061] In some embodiments, the length of the genomic nucleotide sequence selected for the targeting vector can exceed about 0.8 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb or 20 kb.

[0062] In some embodiments, the targeting vector further comprises one or more marker genes (or resistance genes). For example, a resistance gene for positive clone screening or a coding gene for a negative screening marker. In some embodiments, the resistance gene for positive clone screening is a neomycin phosphotransferase coding sequence Neo. Preferably, the targeting vector further comprises two Frt recombination sites arranged in the same direction on both sides of the marker gene. In some implementations, the coding gene for the negative screening marker is a coding gene (DTA) for the diphtheria toxin A subunit.

[0063] In some embodiments, the targeting vector further comprises an auxiliary sequence, wherein the auxiliary sequence comprises a stop codon, WPRE (WHP post-transcriptional response element), Insulator, loxP, STOP sequence and / or Poly A.

[0064] In some embodiments, the targeting vector comprises a STOP sequence, a 2xInsulator sequence, an A1b promoter or a human LPA promoter, a human or chimeric LPA sequence, and a PolyA sequence from 5' to 3', preferably followed by a Neo sequence and two Frt sites in the same direction on both sides of the PolyA sequence.

[0065] In some embodiments, in order to prevent the influence of the endogenous locus of the non-human animal, the nucleotide sequence encoding human LPA can be placed in reverse, such as Figure 3 shown.

[0066] The present invention also provides a vector for constructing a humanized animal model or a knockout model. In some embodiments, the vector comprises an sgRNA sequence. In some embodiments, the sgRNA sequence targets the ROSA26 gene, and the target sequence (target site) of the sgRNA on the conversion region to be changed is unique and satisfies the sequence arrangement rule of 5'-NNN(20)-NGG3' or 5'-CCN-N(20)-3'. In some embodiments, the target sequence (target site) on the non-human animal ROSA26 gene is located between exon 1 and exon 2 (on intron 1).

[0067] In some embodiments, the target site is shown as SEQ ID NO: 17. Therefore, the present invention provides sgRNA sequences for constructing genetically modified animal models. In some implementations, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 18 and 20. In some implementations, the oligonucleotide sgRNA sequences are listed in SEQ ID NO: 19 and 21.

[0068] In some embodiments, the present application relates to a plasmid construct (sgRNA vector, such as pT7-sgRNA) comprising an sgRNA and / or a cell comprising the construct.

[0069] The present invention also relates to a cell comprising the targeting vector as described above or comprising the sgRNA vector as described above.

[0070] In addition, the present invention also provides a non-human mammalian cell having any one of the above-mentioned vectors and one or more in vitro transcripts of the construct described in the present application. In some embodiments, the cell comprises Cas9 mRNA or its in vitro transcript.

[0071] In some embodiments, the gene in the cell is heterozygous. In some embodiments, the gene in the cell is homozygous.

[0072] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell. In some embodiments, the cell is an embryonic stem cell. In some embodiments, the cell is any cell capable of expressing LPA and / or APOB.

[0073] Genetically modified non-human animals

[0074] The "genetically modified non-human animal" or "genetically modified non-human animal" described in the present invention refers to a non-human animal in which at least one chromosome in the genome of the non-human animal has exogenous DNA. In some embodiments, at least one or more cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% or 50% of the cells in the genetically modified non-human animal have exogenous DNA. The cells with exogenous DNA can be various cells, for example, somatic cells, immune cells (including T cells, B cells, NK cells, antigen presenting cells, macrophages, dendritic cells), germ cells, blastocysts or tumor cells. In some embodiments, a genetically modified non-human animal is provided, the non-human animal comprising a modified endogenous locus, comprising an exogenous sequence (e.g., a human sequence), 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 into the genome (e.g., inserting at a safe site). Non-human animals are generally able to pass genetic modifications to offspring through germline transmission.

[0075] The "humanized (x) gene" or "humanized (x) nucleic acid" described in the present invention refers to a gene or nucleic acid, wherein two or more parts of the gene or nucleic acid are from different species, or at least a portion of the sequence of the gene or nucleic acid is different from the nucleic acid in a wild-type animal. In some embodiments, the humanized (x) gene or humanized (x) nucleic acid has at least a portion of the sequence having two or more different species sources, for example, sequences encoding different proteins or sequences encoding the same (or homologous) proteins of two or more different species. In some embodiments, the gene or nucleic acid comprises a sequence encoding LPA and / or APOB protein. The encoded LPA protein has human LPA activity. The encoded APOB protein has at least one activity, such as human APOB protein activity and / or non-human animal APOB protein activity. In some embodiments, the "humanized (x) gene" or "humanized (x) nucleic acid" refers to a humanized exon, and the humanized exon can be a human exon or a humanized exon.

[0076] The "humanized (x) protein" or "humanized (x) polypeptide" of the present invention refers to a protein or polypeptide, wherein two or more parts of the polypeptide or protein are from different species, or at least a portion of the sequence of the protein or polypeptide is different from the amino acid sequence in a wild-type animal. In some embodiments, at least a portion of the sequence of the humanized (x) protein or humanized (x) polypeptide has two or more different species sources, for example, the same (or homologous) proteins of different species. For example, the chimeric APOB protein is a humanized APOB protein, and the humanized APOB protein is functional, or has at least one activity, such as the activity of a human APOB protein, and / or, the activity of an APOB protein of a non-human animal.

[0077] The "chimeric (x) protein" or "chimeric (x) polypeptide" of the present invention refers to a protein or polypeptide. In some embodiments, at least a portion or all of the protein or polypeptide is derived from a human (x) protein or a human (x) polypeptide. In some embodiments, a chimeric (x) protein or a chimeric (x) polypeptide refers to one or more domains of a human (x) protein or a human (x) polypeptide connected together to function. In some embodiments, the two or more domains may be continuous or non-continuous. In some embodiments, the "chimeric (x) protein" is a chimeric LPA protein, and the chimeric LPA protein comprises at least 1 to 16 Kringle domains of an LPA protein, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 Kringle domains. In some embodiments, the chimeric LPA protein comprises the KIV-1, KIV-3, KIV-4, KIV-5, KIV-6, KIV-7, KIV-8, KIV-9, KIV-10, KV domains of the LPA protein, and any one or more of the six KIV-2 domains. In some embodiments, the chimeric LPA protein comprises the KIV-1, KIV-2I, KIV-3, KIV-4, KIV-5, KIV-6, KIV-7, KIV-8, KIV-9, KIV-10 and KV domains of the LPA protein. In some implementations, the "chimeric (x) protein" is a chimeric APOB protein.

[0078] The "chimeric (x) nucleic acid" or "chimeric (x) gene" of the present invention refers to a gene or a nucleic acid. In some embodiments, at least a portion or all of the gene or nucleic acid is from a human. In some embodiments, the nucleic acids in the chimeric (x) nucleic acid or chimeric (x) gene are all derived from humans. In some embodiments, the "chimeric (x) nucleic acid" or "chimeric (x) gene" is a chimeric LPA gene, and the chimeric LPA gene comprises a nucleotide sequence encoding at least 1 to 16 Kringle domains in the LPA gene, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 Kringle domains. In some embodiments, the chimeric LPA gene comprises a nucleotide sequence encoding any one or more of the KIV-1, KIV-3, KIV-4, KIV-5, KIV-6, KIV-7, KIV-8, KIV-9, KIV-10, KV domains, and 6 KIV-2 domains in the LPA gene. In some embodiments, the chimeric LPA gene comprises nucleotide sequences encoding KIV-1, KIV-2I, KIV-3, KIV-4, KIV-5, KIV-6, KIV-7, KIV-8, KIV-9, KIV-10, and KV domains in the LPA gene.

[0079] The "domain" mentioned in the present invention refers to a region that performs a specific function in a protein and is an independent functional unit. In some embodiments, the domain refers to a Kringle domain, including KIV-1, KIV-2, KIV-3, KIV-4, KIV-5, KIV-6, KIV-7, KIV-8, KIV-9, KIV-10 and KV, etc. The amino acid positions corresponding to exemplary functional regions include but are not limited to those shown in Table 1.

[0080] The genetically modified non-human animal can be various animals, for example, mice, rats, rabbits, pigs, cattle (e.g., cattle, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For non-human animals that are not easy to obtain suitable genetically modified embryonic stem cells (Es), other methods are used to construct non-human animals comprising genetic modifications. Such methods include, for example, modifying non-ES cell genomes (e.g., fibroblasts or induced pluripotent stem cells) and using nuclear transplantation to transfer the modified genome to suitable cells, such as oocytes, and incubating modified cells (e.g., modified oocytes) in non-human animals under appropriate conditions to form embryos. The above-mentioned construction methods 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 into this application by reference.

[0081] In one aspect, the non-human animal is a mammal. In some embodiments, the genetically modified non-human animal is a rodent. Rodents can be selected from mice, rats and hamsters. In some embodiments, the rodent is selected from the family of Muridae. In some embodiments, the genetically modified non-human animal is from a family selected from the family of Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muroidea (mice and rats, gerbils, spiny mice, crested rats), Isla Muscidae (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Spiny Dormouse (e.g., spiny dormouse) and Muridae (e.g., mole rats, bamboo rats and zokors). In some embodiments, the genetically modified rodent is selected from mice or rats (Muroidea), gerbils, spiny mice and crested rats. In some embodiments, the genetically modified mouse is from a member of the Muridae family. In some embodiments, the non-human animal is a rodent. In some embodiments, the rodent is selected from mice and rats. In some embodiments, the non-human animal is a mouse.

[0082] In some embodiments, the non-human animal can be an immunodeficient non-human mammal. For example, an immunodeficient rodent, an immunodeficient rabbit, an immunodeficient pig, an immunodeficient monkey, etc. In some embodiments, the non-human animal is a mouse of the C57BL strain, and the C57BL strain is selected from C57BL / a, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL6, C57BL / 10, C57BL10ScSn, C57BL / 10Cr and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from 129 / J, 129 / ReJ, 129 / OlaHsd, 129 / Sv, 129 / SvJ, 129 / Re, 129 / RrJ, 129 / Sv-ter / +. These mice are described in, for example, Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv-and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), the relevant contents of which are incorporated herein by reference in their entirety. In some embodiments, the genetically modified mouse is a hybrid of the 129 strain and the C57BL / 6 strain. In some embodiments, the mouse is a hybrid of the 129 strain, or a hybrid of the C57BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as a BALB / c strain. In some embodiments, the mouse is a hybrid of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129). In certain embodiments, non-human animals are rodents. In certain embodiments, non-human animals are mice with BALB / c, BALB / cHeAn, BALB / cJ, BALB / cR1, BALB / cWt, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st or CBA / H strains. In certain embodiments, non-human animals are rats. Rats can be selected from Wistar rats, LEA strains, Sprague-Dawley strains, Fischer strains, F344, F6 and DarkAgouti.In some embodiments, the rat strain is a hybrid of two or more strains selected from Wistar, LEA, Sprague-Dawley, Fischer, F344, F6, and Dark Agouti.

[0083] The non-human animal may have one or more other genetic modifications and / or other modifications that are suitable for the specific purpose of preparing a humanized non-human animal. For example, a suitable mouse for maintaining a xenograft (e.g., a human cancer or tumor) may have one or more modifications that damage, inactivate, or destroy all or part of the immune system of the non-human animal. Damage, inactivation, or destruction of the immune system of a non-human animal may include, for example, chemical means (e.g., administration of toxins), physical means (e.g., irradiation of the animal), and / or genetic modification (e.g., knocking out one or more genes). Non-limiting examples of such mice include, for example, NOD mice, SCID mice, NOD / SCID mice, IL2Rγ knockout mice, NOD / SCID / γe nullMice (Ito, M. et al., NOD / SCID / γcnull mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100(9): 3175-3182, 2002), nude mice, and Rag1 and / or Rag2 knockout mice. These mice can be optionally irradiated or otherwise treated to destroy one or more immune cell types. Therefore, in various embodiments, a genetically modified mouse is provided, which can include humanization of at least a portion of an endogenous locus or gene site (e.g., an endogenous APOB locus of a non-human animal, or an endogenous gene site of a non-human animal, such as a safe site, and the safe site is preferably a ROSA26 site or a Hipp11 site), and also includes modifications that damage, inactivate or partially destroy the immune system (or one or more cell types of the immune system) of a non-human animal. In some embodiments, the mouse modification type is selected from NOD mice, SCID mice, NOD / SCID mice, IL-2Rγ knockout mice, NOD / SCID / γcnull mice, nude mice, Rag1 and / or Rag2 knockout mice, NOD Prkdcscid IL-2Rγnull mice, NODRag 1- / -IL2rg- / -(NRG) mice, Rag2- / -IL2rg- / -(RG) mice and modifications of combinations thereof. These transgenic animals are described, for example, in US10820580B2, which is incorporated herein by reference in its entirety. In some embodiments, the mouse may include replacing all or part of the mouse endogenous mature LPA and, or all or part of the APOB coding sequence with all or part of the human mature LPA and / or APOB coding sequence, respectively. In some embodiments, the mouse may include inserting human mature LPA or chimeric LPA protein in a safe site in the genome.

[0084] The present invention also provides a non-human mammal model carrying a tumor, wherein the non-human mammal model is obtained by the method described in the present application. In some embodiments, the non-human mammal is a rodent (eg, mouse).

[0085] The present invention also provides a cell or cell line derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a primary cell culture, which is derived from a non-human mammal or its offspring, or a non-human mammal carrying a tumor, or a tissue, organ, or culture thereof derived from a non-human mammal or its offspring. When it carries a tumor, it is derived from a tumor tissue of a non-human mammal or its offspring, or a non-human mammal carrying a tumor.

[0086] The present invention provides a non-human mammal produced by any of the methods described herein. In some embodiments, a non-human mammal, a genetically modified non-human animal, wherein the genome of the genetically modified non-human animal comprises DNA encoding human or chimeric LPA and / or human or chimeric APOB is provided.

[0087] In some embodiments, a non-human mammal comprises a gene construct as described herein. In some embodiments, a non-human mammal expressing a human or chimeric LPA protein and / or a human or chimeric APOB protein is provided. In some embodiments, a cell, tissue or organ that specifically expresses a human or chimeric LPA protein and / or a human or chimeric APOB protein is provided.

[0088] In some embodiments, the expression of human or chimeric LPA protein and / or human or chimeric APOB protein in the genetically modified non-human animal 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 (Tamoxifen System).

[0089] 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 rodents. In some embodiments, the non-human mammal is a mouse.

[0090] The non-human mammals described above are subjected to genetic, molecular and behavioral analyses. The present invention provides offspring produced by mating with non-human mammals of the same genotype or other genotypes.

[0091] The present invention provides a cell line or primary cell culture derived from a non-human mammal or its progeny. For example, a cell culture-based model can be prepared by the following method. The primary cell culture can be obtained by isolation from a non-human mammal, or cells can be obtained from a primary cell culture established using the same construct and standard cell transfection techniques. The integration of a genetic construct comprising a DNA sequence encoding a human or chimeric LPA protein and / or a human or chimeric APOB protein can be detected by a variety of methods.

[0092] There are many analytical methods that can be used to detect exogenous DNA, including nucleic acid level methods (including the use of reverse transcription-polymerase chain reaction (RT-PCR) or Southern Blot and in situ hybridization) and protein level methods (including histochemical analysis, immunoblot analysis and in vitro binding studies). In addition, the expression level of the target gene can be quantified by ELISA methods well known to those skilled in the art. Many standard analytical methods can be used to achieve quantitative detection. For example, RT-PCR and hybridization methods can be used to detect transcription levels, including RNase protection analysis, Southern Blot, RNA dot hybridization analysis (RNAdot). Immunohistochemical staining, flow cytometry, Western blot can also be used to detect the presence of human or chimeric LPA and / or human or chimeric APOB protein.

[0093] In some embodiments, the genetically modified non-human animals described herein (eg, LPA and / or APOB gene homozygous humanized mice) can express human or chimeric LPA protein and / or human or chimeric APOB protein in one or more cells.

[0094] Non-human animals having endogenous gene loci containing human or chimeric LPA gene sequence modifications

[0095] The non-human animal includes modification of endogenous gene sites of the non-human animal. In some embodiments, the modification comprises a nucleotide sequence encoding all or at least a portion of a mature LPA protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to the amino acid sequence of a mature LPA protein). Although cells (e.g., ES cells, somatic cells) that may contain the genetic modifications described in the present application are provided in the present invention, in many embodiments, the genetically modified non-human animal includes modification of a safe site in the endogenous genome of the non-human animal, such as modification of the endogenous ROSA26 site or the Hipp11 site.

[0096] The genetically modified non-human animal can express a human or chimeric LPA protein, wherein a safe site (e.g., ROSA26 site or Hipp11 site) of the endogenous genome of the non-human animal has been replaced or inserted with all or part of a human or chimeric LPA gene and / or a nucleotide sequence encoding all or part of a human or chimeric LPA or a nucleotide sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with a human LPA. In some embodiments, the endogenous ROSA26 or Hipp11 locus of the non-human animal is modified by a nucleic acid sequence comprising a human mature LPA protein or a chimeric LPA protein.

[0097] In some embodiments, the genetically modified non-human animal can express human or chimeric LPA protein under the control of endogenous or exogenous regulatory elements in the non-human animal. Insertion or replacement in the endogenous genome of the non-human animal provides a non-human animal that expresses human or chimeric LPA protein in suitable cells and in a manner that does not cause potential pathologies observed in some other transgenic non-human animals known in the art. Human or chimeric LPA protein expressed in non-human animals can maintain one or more functions of LPA in non-human animals. As shown in the present application, LPA gene humanized mice containing insertions or replacements at the endogenous ROSA26 site or Hipp11 site of non-human animals do not show pathological phenomena. Both genetically modified mice that are heterozygous or homozygous for humanization of the LPA gene are normal.

[0098] The present invention further relates to a genomic DNA sequence of a non-human animal modified by the LPA gene, a DNA sequence obtained by reverse transcription of mRNA that is identical to or complementary to the DNA sequence; a construct expressing the amino acid sequence thereof; a cell comprising the construct; and a tissue or organ comprising the cell.

[0099] The present invention further relates to a non-human mammal produced by the above construction method. In some embodiments, its genome comprises all or part of a human or chimeric LPA gene.

[0100] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0101] In some embodiments, the non-human mammal expresses a protein encoded by a human or chimeric LPA gene, eg, a human LPA or chimeric LPA protein.

[0102] In some embodiments, the non-human animal further comprises other gene modifications, preferably, the other genes include at least one of APOB, PCSK9, APOC3, ANGPTL3, GPR75, ANGPTLA, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0103] In some embodiments, the non-human animal further comprises an APOB gene modification. In some embodiments, the non-human animal also expresses a human or chimeric APOB protein.

[0104] Non-human animals with a chimeric APOB locus

[0105] The non-human animal includes a modification of an endogenous locus of the non-human animal. In some embodiments, the modification comprises a nucleotide sequence encoding all or at least a portion of a mature APOB protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the amino acid sequence of a mature APOB protein). Although cells (e.g., ES cells, somatic cells) that may include the genetic modifications described herein are provided in the present invention, in many embodiments, the genetically modified non-human animal includes a modification of an endogenous APOB locus in the non-human animal.

[0106] The genetically modified non-human animal can express human or chimeric APOB at the non-human animal endogenous APOB locus, wherein the non-human animal endogenous APOB locus is replaced or inserted with a human or chimeric APOB gene and / or a nucleotide sequence encoding a human or chimeric APOB protein or a nucleotide sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with a human APOB. In various embodiments, the non-human animal endogenous APOB locus is modified by comprising all or part of a nucleic acid sequence encoding a mature human APOB protein.

[0107] In some embodiments, the genetically modified non-human animal can express human or chimeric APOB proteins under the control of endogenous or exogenous regulatory elements. Insertion or substitution at the endogenous locus of the non-human animal provides a non-human animal that expresses human or chimeric APOB proteins in suitable cells and in a manner that does not cause potential pathology observed in some other transgenic mice known in the art. The human or chimeric APOB proteins expressed in the non-human animal can maintain one or more functions of APOB in the non-human animal.

[0108] In some embodiments, the chimeric APOB gene comprises the 5'UTR of the human APOB gene. In some embodiments, the chimeric APOB gene comprises the 5'UTR of an endogenous APOB gene of a non-human animal (e.g., a mouse). In some embodiments, the chimeric APOB gene comprises the 3'UTR of an endogenous APOB gene of a non-human animal (e.g., a mouse). In appropriate cases, it can be reasonably assumed that, based on the similarity of the APOB gene sequences of non-human animals and humans, they appear to be similarly regulated. As shown in the present application, humanized mice containing an APOB gene inserted or replaced in the endogenous APOB locus of a non-human animal, which contains the humanization of the APOB coding sequence, do not exhibit pathological phenomena. Both heterozygous or homozygous genetically modified mice of the humanized APOB gene are normal.

[0109] The present invention further relates to the genomic DNA sequence of non-human animals with humanized APOB gene, the DNA sequence obtained by mRNA reverse transcription is consistent with or complementary to the DNA sequence; a construct expressing its amino acid sequence; a cell containing the construct; and a tissue or organ including the cell.

[0110] The present invention further relates to a non-human mammal produced by the above construction method. In some embodiments, its genome comprises all or part of a human or chimeric APOB gene.

[0111] In some embodiments, the non-human mammal is a rodent, preferably, the non-human mammal is a mouse.

[0112] In some embodiments, the non-human mammal expresses a protein encoded by a human or chimeric APOB gene, eg, a human or chimeric APOB protein.

[0113] In some embodiments, the non-human animal further comprises other gene modifications, preferably, the other genes include at least one of LPA, PCSK9, APOC3, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4.

[0114] In some embodiments, the non-human animal further comprises an LPA gene modification. In some embodiments, the non-human animal also expresses a human or chimeric LPA protein.

[0115] Methods for constructing genetically modified non-human animals

[0116] Genetically modified non-human animals can be prepared by several techniques known in the art, including gene targeting technology using embryonic stem cells, homologous recombination technology, CRISPR / Cas9 technology, zinc finger nuclease technology, transcription activator-like effector nuclease technology, homing endonuclease or other molecular biology technology. In some embodiments, homologous recombination technology is preferably used. In some embodiments, CRISPR / Cas9 gene editing technology can construct genetically modified non-human animals. In some embodiments, CRISPR / Cas9 gene editing technology is used to produce genetically modified non-human animals. Many of these genome editing technologies are known in the art and are described in Yin et al. "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399", which is incorporated by reference into this application. The present invention also provides many other methods for genome editing, for example, microinjecting transgenic cells into enucleated oocytes and fusing the enucleated oocytes with another transgenic cell.

[0117] In some embodiments, the endogenous genome of at least one cell of the non-human animal comprises a nucleotide sequence encoding a human or chimeric LPA. In some embodiments, the nucleotide sequence encoding a human or chimeric LPA is inserted into a non-human animal endogenous safe site, preferably an endogenous ROSA26 or Hipp11 site of a non-human animal. In some embodiments, the insertion occurs in cells such as germ cells, somatic cells, blastocysts or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0118] The present invention provides a targeting vector. The targeting vector comprises a vector consisting of a 5' homology arm, a human LPA gene fragment and a 3' homology arm. The process involves introducing the human LPA gene fragment into an endogenous locus of a non-human animal using homologous recombination. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nuclease, TALEN or CRISPR) can result in a double-stranded DNA break, and homologous recombination is used to insert the human LPA gene fragment into the ROSA26 or Hippl1 site of a non-human animal.

[0119] Thus, in some embodiments, a method of making a genetically modified non-human animal comprises introducing a nucleotide sequence encoding a human or chimeric LPA protein into an endogenous ROSA26 or Hipp11 locus.

[0120] The present invention also provides a method for establishing an LPA gene-modified animal model, comprising the following steps:

[0121] (a) providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0122] (b) culturing the cells, preferably in a liquid culture medium;

[0123] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal;

[0124] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0125] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0126] In some embodiments, the non-human mammal in step (c) is a pregnant (or pseudo-pregnant) female.

[0127] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, or DBA / 2 fertilized eggs.

[0128] The fertilized egg can be from any non-human animal, such as any non-human animal described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can be introduced into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human mammal gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0129] In some embodiments, the method for preparing a genetically modified non-human animal includes modifying a safe site in the endogenous genome of the non-human animal, for example, by introducing a nucleotide sequence encoding a human or chimeric LPA (e.g., all or part of a genomic DNA sequence, a CDS sequence, or a cDNA sequence) into a safe site in the endogenous genome of the non-human animal under the control of an exogenous regulatory element (e.g., a human LPA promoter or an Alb promoter). For example, the ROSA26 or Hipp11 site of a non-human animal can be inserted with a sequence so that human LPA or chimeric LPA is expressed in the non-human animal. In some embodiments, in order to prevent the influence of the endogenous locus of the non-human animal, the nucleotide sequence encoding human LPA can be placed in reverse, such as Figure 3 shown.

[0130] In some embodiments, the method for preparing a genetically modified non-human animal comprises inserting a nucleotide sequence and / or an auxiliary sequence encoding a human or chimeric LPA protein at the ROSA26 or Hipp11 site of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon or an insulator so that the LPA gene modified animal model can express a human or chimeric LPA protein in vivo. In some embodiments, the auxiliary sequence includes Insulator, WPRE (WHP post-transcriptional response element), loxP, STOP and / or polyA.

[0131] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0132] (1) providing a plasmid comprising a human LPA gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target a safe site of a non-human endogenous genome, such as the ROSA26 or Hipp11 site;

[0133] (2) providing one or more guide RNAs (sgRNAs) targeting safe sites of the non-human endogenous genome (e.g., ROSA26 or Hipp11 sites);

[0134] (3) modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2) and Cas9;

[0135] (4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express human or chimeric LPA protein. Preferably, the method further comprises:

[0136] (5) The offspring mice obtained in step (4) are mated to obtain homozygous mice.

[0137] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting a 5'-terminal targeting site and a 3'-terminal targeting site.

[0138] In some embodiments, the sequence encoding the human or chimeric LPA protein is operably linked to an exogenous regulatory element. Preferably, the exogenous regulatory element includes an Alb promoter or a human LPA promoter.

[0139] In some embodiments, the sequence encoding the human or chimeric LPA protein is operably linked to endogenous regulatory elements of the non-human animal.

[0140] In some embodiments, the APOB locus in the endogenous genome of at least one cell of the non-human animal comprises a nucleotide sequence encoding a human or chimeric APOB protein. In some embodiments, the nucleotide sequence encoding the endogenous APOB protein of the non-human animal is replaced by a nucleotide sequence encoding a human or chimeric APOB protein. In some embodiments, the expression level of the endogenous APOB protein of the non-human animal is reduced or absent compared to the wild type. In some embodiments, the insertion occurs in cells such as germ cells, somatic cells, blastocysts or fibroblasts. The nucleus of a somatic cell or fibroblast can be inserted into an enucleated oocyte.

[0141] The present invention provides a targeting vector. The targeting vector comprises a vector consisting of a 5' homology arm, a human or chimeric APOB gene fragment and a 3' homology arm. The process involves the use of homologous recombination to introduce the human or chimeric APOB gene fragment into the endogenous APOB locus of a non-human animal. In some embodiments, cleavage upstream and downstream of the target site (e.g., by zinc finger nuclease, TALEN or CRISPR) can result in a double-stranded DNA break, and homologous recombination is used to replace or insert the human or chimeric APOB gene fragment into the endogenous APOB locus of a non-human animal.

[0142] Thus, in some embodiments, a method of making a genetically modified humanized animal comprises introducing a nucleotide sequence encoding a human or chimeric APOB protein into an endogenous APOB locus.

[0143] The present invention also provides a method for establishing an APOB gene humanized animal model, comprising the following steps:

[0144] (a) providing cells (e.g., fertilized egg cells) based on the method described in the present application;

[0145] (b) culturing the cells, preferably culturing the cells in a liquid culture medium;

[0146] (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal;

[0147] (d) identifying germline transmission in offspring of the genetically modified humanized non-human mammal of the pregnant female in step (c).

[0148] In some embodiments, the non-human mammal in the above methods is a mouse (eg, a C57BL / 6 mouse).

[0149] In some embodiments, the non-human mammal in step (c) is a pregnant (pseudo-pregnant) female.

[0150] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0151] The fertilized egg can be from any non-human animal, such as any non-human animal described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can be introduced into the fertilized egg by microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, and then the pseudopregnant non-human mammal gives birth to a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0152] In some embodiments, a method for preparing a genetically modified non-human animal comprises modifying the endogenous APOB locus of the non-human animal, for example, by replacing or inserting a nucleotide sequence (e.g., genomic DNA, CDS sequence, or cDNA sequence) encoding a human or chimeric APOB protein into the endogenous APOB locus of the non-human animal under the control of regulatory elements of the human APOB gene.

[0153] In some embodiments, the method for preparing a genetically modified non-human animal comprises inserting a nucleotide sequence and / or an auxiliary sequence encoding a human or chimeric APOB protein into the APOB locus of the non-human animal. In some embodiments, the auxiliary sequence can be a stop codon or an insulator, so that the APOB gene humanized animal model can express a human or chimeric APOB protein in vivo without expressing endogenous APOB protein. In some embodiments, the auxiliary sequence comprises an Insulator, a WPRE (WHP post-transcriptional response element), loxP, a STOP and / or a polyA.

[0154] In some embodiments, a method for preparing a genetically modified non-human animal comprises:

[0155] (1) providing a plasmid comprising a human APOB gene fragment, wherein the plasmid is flanked by a 5' homology arm and a 3' homology arm, wherein the 5' homology arm and the 3' homology arm target an endogenous APOB gene locus of a non-human animal;

[0156] (2) providing one or more guide RNAs (sgRNAs) targeting the endogenous APOB locus of a non-human animal;

[0157] (3) modifying the genome of a fertilized egg or embryonic stem cell by using the plasmid of step (1), the sgRNA of step (2) and Cas9;

[0158] (4) transplanting the fertilized egg obtained in step (3) into the oviduct of a pseudo-pregnant female mouse, or transplanting the embryonic stem cells obtained in step (3) into a blastocyst, and then transplanting the blastocyst into the oviduct of a pseudo-pregnant female mouse to produce offspring mice that functionally express human or chimeric APOB protein. Preferably, the method further comprises:

[0159] (5) The offspring mice obtained in step (4) are mated to obtain homozygous mice.

[0160] In some embodiments, the fertilized egg is modified by CRISPR with sgRNAs targeting a 5'-terminal targeting site and a 3'-terminal targeting site.

[0161] In some embodiments, the sequence encoding a human or chimeric APOB protein is operably linked to human regulatory elements.

[0162] In some embodiments, the sequence encoding the human or chimeric APOB protein is operably linked to endogenous regulatory elements of the non-human animal.

[0163] In some embodiments, at least one safe site in the endogenous genome of at least one cell of a non-human animal is modified by a human sequence, for example, the ROSA26 site or the Hipp11 site of the endogenous genome of at least one cell of a non-human animal is inserted with a nucleotide sequence encoding a human or chimeric LPA protein.

[0164] In some embodiments, the nucleotide sequence encoding the APOB region in the endogenous genome of at least one cell of the non-human animal is replaced with a nucleotide sequence encoding a human or chimeric APOB protein.

[0165] In some embodiments, a safe site (e.g., a ROSA26 site or a Hipp11 site) in the endogenous genome of at least one cell of a non-human animal, and / or, a nucleotide sequence encoding an endogenous APOB region is inserted or replaced by a nucleotide sequence encoding a human or chimeric LPA protein, and, or, a nucleotide sequence encoding a human or chimeric APOB protein.

[0166] In some embodiments, in the endogenous genome of at least one cell of the non-human animal, the nucleotide sequence encoding the endogenous APOB region is replaced by a nucleotide sequence encoding human or chimeric APOB, and a nucleotide sequence encoding human or chimeric LPA protein is inserted into a safe site (e.g., ROSA26 site or Hipp11 site) in the endogenous genome of the non-human animal.

[0167] In some embodiments, the non-human animal endogenous nucleotide sequence encoding APOB protein is deleted. In some embodiments, all or part of exons 1-29 of the non-human animal endogenous APOB gene are deleted. In some embodiments, the nucleotide sequence from the start codon to the stop codon of the non-human animal endogenous APOB gene is deleted. In some embodiments, the deleted portion further comprises 5'UTR and / or 3'UTR, preferably further comprises at least 50bp continuous nucleotide sequence upstream of 5'UTR and / or at least 50bp continuous nucleotide sequence downstream of 3'UTR. In some embodiments, the deleted portion further comprises an intron, such as intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 7, intron 18, intron 19, intron 20, intron 21, intron 22, intron 23, intron 24, intron 25, intron 26, intron 27 and, or intron 28.

[0168] In some embodiments, the construction method comprises replacing the nucleotide sequence of the endogenous APOB protein of the non-human animal with a nucleotide sequence encoding a human or chimeric APOB protein (e.g., a genomic DNA sequence, a CDS, or a cDNA sequence). In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 32 in the genome of the non-human animal with a nucleotide sequence encoding SEQ ID NO: 33 or 38. In some embodiments, the construction method comprises replacing the nucleotide sequence from the start codon to the stop codon of the endogenous APOB gene of the non-human animal with a nucleotide sequence from the start codon to the stop codon of the human APOB gene. In some embodiments, the construction method comprises replacing all of exons 1-29 of the endogenous APOB gene of the non-human animal with all of exons 1-29 of the human APOB gene. In some embodiments, the construction method comprises replacing the nucleotide sequence from at least 50 bp upstream of the 5'UTR to at least 50 bp downstream of the 3'UTR of the endogenous APOB gene of the non-human animal with a nucleotide sequence from at least 50 bp upstream of the 5'UTR to at least 50 bp downstream of the 3'UTR of the human APOB gene. In some embodiments, the construction method comprises replacing the nucleotide sequence encoding SEQ ID NO: 32 in the genome of a non-human animal with SEQ ID NO: 37 or the nucleotide sequence 20983975 to 21063173 of NCBI accession number NC_000002.12 or a variant thereof.

[0169] In some embodiments, the construction method includes inserting a nucleotide sequence encoding a human or chimeric LPA protein into the endogenous genome of a non-human animal, for example, into a safe site of the endogenous genome (including the ROSA26 site or the Hipp11 site). In some embodiments, the construction method includes inserting a nucleotide sequence encoding a human or chimeric LPA protein (such as a genomic DNA sequence, a CDS sequence, or an eDNA sequence) between exon 1 and exon 2 of an endogenous ROSA26 gene of a non-human animal. In some embodiments, the construction method includes inserting the start codon to the stop codon of the human LPA gene between exon 1 and exon 2 of an endogenous ROSA26 gene of a non-human animal. In some embodiments, the construction method includes inserting all of exons 1-39 of the human LPA gene between exon 1 and exon 2 of an endogenous ROSA26 gene of a non-human animal. In some embodiments, the construction method includes inserting a continuous nucleotide sequence of at least 50 bp upstream of the 5'UTR of the human LPA gene to at least 50 bp downstream of the 3'UTR between exon 1 and exon 2 of an endogenous ROSA26 gene of a non-human animal. In some embodiments, the construction method comprises inserting a portion of exon 1 to all of exon 5 of the human LPA gene, and a portion of all of exon 16 to exon 39 between exon 1 and exon 2 of an endogenous ROSA26 gene of a non-human animal. In some embodiments, the construction method comprises inserting a nucleotide sequence of SEQ ID NO: 5, NCBI accession number NC_000006.12 at positions 160528474 to 160686025, SEQ ID NO: 6, or a nucleotide sequence of NCBI accession number NM_005577.4 between exon 1 and exon 2 of an endogenous ROSA26 gene of a non-human animal.

[0170] Use of genetically modified non-human animals

[0171] In the endogenous locus (or genome) of the non-human animal, and under the control of endogenous and / or exogenous regulatory elements, the non-human animal gene is replaced with a homologous or orthologous human gene or human sequence, or a homologous or orthologous human gene or human sequence is inserted into the non-human animal, and a non-human animal with qualities and characteristics that may be significantly different from a typical knockout plus transgenic non-human animal can be produced. In a typical knockout plus transgenic non-human animal, the endogenous locus is removed or destroyed, and the full human transgene is inserted into the genome of the animal and may be randomly integrated into the genome. Typically, the location of the integrated transgene is unknown; the expression of human proteins is measured by transcription of human gene and / or protein assays and / or functional assays. In human transgenes, the upstream and / or downstream of the human sequence provide suitable support for the expression and / or regulation of the transgene.

[0172] Genetically modified non-human animals that express human or chimeric LPA protein and / or human or chimeric APOB protein, e.g., in a physiologically appropriate manner, provide a variety of uses, including but not limited to developing treatments for human diseases and disorders, and evaluating the toxicity and / or efficacy of these human treatments in animal models.

[0173] The present invention also provides a use of the above-mentioned LPA and / or APOB gene-modified non-human animal, or a non-human animal obtained by any of the above-mentioned construction methods.

[0174] In some embodiments, the application comprises:

[0175] A) Application in the development of products involving immunological processes related to LPA and / or APOB in human cells;

[0176] B) Application as a model system related to human LPA and / or human APOB for pharmacological, immunological, microbiological and medical research;

[0177] C) Applications involving the production and use of animal experimental disease models for etiological studies related to human LPA and / or human APOB and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies;

[0178] D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human LPA and / or human APOB signaling pathway modulators; or,

[0179] E) To study the gene functions of human LPA and / or human APOB, to study the drugs and drug efficacy targeting human LPA and / or human APOB, and to study the application of drugs for treating tumors, inflammation, immune diseases, and metabolic diseases related to human LPA and / or human APOB.

[0180] The present invention provides a non-human animal expressing human or chimeric LPA and / or human or chimeric APOB protein, which can be used for screening of human LPA and / or human APOB specific modulators. In some embodiments, the non-human animal is a human disease animal model. For example, the disease is genetically induced (knock-in or knock-out). In different embodiments, the genetically modified non-human animal also comprises a compromised immune system, such as a genetically modified human-derived tissue xenograft, including a human solid tumor (e.g., breast cancer) or a blood cell tumor (e.g., a lymphocyte tumor, a B or T cell tumor).

[0181] In some embodiments, the anti-LPA antibody or anti-APOB antibody blocks or inhibits the signaling pathway mediated by LPA and / or APOB. In some embodiments, the anti-LPA antibody or anti-APOB antibody can block the interaction between LPA complexes, thereby inhibiting the LPA signaling pathway.

[0182] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of LPA and / or APOB therapeutic agents (e.g., anti-LPA and / or APOB antibodies, LPA and / or APOB inhibitors, or LPA and, or APOB nucleic acid drugs) 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, atopic dermatitis, myocarditis, nephritis, hepatitis (preferably non-alcoholic fatty hepatitis), systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders. In some embodiments, the immune disease is one or more of asthma, psoriasis, atopic dermatitis, rheumatoid arthritis, or multiple sclerosis.

[0183] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of LPA and, or APOB therapeutic agents (e.g., anti-LPA and / or APOB antibodies, LPA and / or APOB inhibitors, or LPA and / or APOB nucleic acid drugs) in treating various inflammations. In some embodiments, the inflammation includes acute inflammation and chronic inflammation. Specifically, it includes but is not limited to degenerative inflammation, exudative inflammation (serous inflammation, fibrinous inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.). In some embodiments, the inflammation is inflammatory bowel disease (IBD).

[0184] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of LPA and / or APOB therapeutic agents (e.g., anti-LPA and / or APOB antibodies, LPA and / or APOB inhibitors, or LPA and / or APOB nucleic acid drugs) in treating various metabolic diseases. In some embodiments, the metabolic disease is hyperlipidemia or atherosclerosis.

[0185] In some embodiments, genetically modified non-human animals can be used to determine the effectiveness of LPA and / or APOB therapeutic agents (e.g., anti-LPA and / or APOB antibodies, LPA and / or APOB inhibitors, or LPA and / or APOB nucleic acid drugs) in treating cancer (tumors). In some embodiments, LPA and / or APOB therapeutic agents (e.g., anti-LPA and / or APOB antibodies, LPA and / or APOB inhibitors, or LPA and / or APOB nucleic acid drugs) are administered to non-human animals, wherein the non-human animals have cancer or tumors, and the effect of LPA and / or APOB therapeutic agents on cancer or tumors is detected, preferably an inhibitory effect. In some embodiments, the detection includes determining the size and / or proliferation rate of tumor cells. In some embodiments, the detection method includes vernier caliper measurement, flow cytometry, and / or in vivo animal imaging detection. 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.

[0186] In some embodiments, the tumor cells include one or more cancer cells injected into an animal (e.g., cancer cells derived from a human or non-human animal). In some embodiments, the LPA and / or APOB therapeutic agent inhibits the LPA and / or APOB signaling pathway. In some embodiments, the LPA and / or APOB therapeutic agent does not inhibit the LPA and / or APOB signaling pathway.

[0187] In some embodiments, genetically modified non-human animals can be used to detect whether LPA and / or APOB therapeutic agents are agonists or antagonists. In some embodiments, the methods described herein can be used to detect the function of LPA and / or APOB therapeutic agents (e.g., anti-LPA and / or APOB antibodies, LPA and / or APOB inhibitors, or LPA and / or APOB nucleic acid drugs), for example, whether the therapeutic agent can upregulate the immune response or downregulate the immune response, and / or whether the therapeutic agent can induce complement-mediated cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, genetically modified non-human animals can be used to determine the effective dose of a therapeutic agent for treating a disease in a subject (e.g., a tumor, an immune disease, an inflammatory disease, or a metabolic disease). The inhibitory effect on tumors can also be determined by methods known in the art, for example, measuring the tumor volume in the 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-Tv t / Tv c ) x 100, where Tv t and TV c is the average tumor volume (or weight) of the treatment group and the control group.

[0188] In some embodiments, LPA and / or APOB therapeutic agents (e.g., anti-LPA and / or APOB antibodies, LPA and / or APOB inhibitors, or LPA and / or APOB nucleic acid drugs) can be used to treat various cancers. The "cancer" of the present invention refers to cells with autonomous growth ability, that is, an abnormal state or condition characterized by rapid cell growth and proliferation. The term is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or invasive stage. The "tumor" of the present invention includes, but is not limited to, lymphoma, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, brain glioma, lung cancer (e.g., non-small cell lung cancer), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia; the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenstrom's macroglobulinemia; the sarcoma is selected from osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In a specific embodiment of the present invention, the tumor is breast cancer, pancreatic cancer, endocrine cancer, head and neck cancer, gastrointestinal cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, glioblastoma, prostate cancer, neuroendocrine tumor, mesothelial tumor, oropharyngeal tumor, female reproductive system cancer or meningioma. In some embodiments, the tumor includes solid tumors and / or hematological tumors. In some embodiments, the tumor includes one or more of head and neck cancer, liver cancer or lung cancer.

[0189] The present invention also provides a detection method for determining the toxicity of LPA and / or APOB therapeutic agents (e.g., anti-LPA and / or APOB antibodies, LPA and / or APOB inhibitors, or LPA and / or APOB nucleic acid drugs). The method comprises administering antibodies to the non-human animals described above, evaluating the weight changes or blood tests of the animals, and preferably the blood tests include but are not limited to red blood cell counts, hematocrits, and / or hemoglobin content. In some embodiments, LPA and / or APOB therapeutic agents can reduce red blood cells (RBC), hematocrit, or hemoglobin by 20%, 30%, 40%, or more than 50%. In some embodiments, the body weight of non-human animals treated with LPA and / or APOB therapeutic agents is at least 5%, 10%, 20%, 30%, or 40% less than the average body weight of non-human animals not treated with LPA and / or APOB therapeutic agents.

[0190] The present invention also provides an animal model constructed by the method, which is used in developing products related to human cellular immune processes, producing human antibodies, or as a model system for pharmacology, immunology, microbiology and medical research.

[0191] In some embodiments, an animal model generated by the method is provided for producing and utilizing animal experimental disease models of immune processes of human cells, studying pathogens, or developing new diagnostic strategies and / or therapeutic strategies.

[0192] The present invention also provides an animal model generated by the method to screen, verify, evaluate or study LPA and / or APOB gene functions, human LPA and / or APOB therapeutic agents, drugs or effectiveness against human LPA and / or APOB targets, and therapeutic drugs for LPA and / or APOB-related diseases (such as immune diseases, tumors, inflammation or metabolic diseases).

[0193] In some embodiments, the present application provides a method for verifying the in vivo efficacy of TCR-T, CAR-T and / or other immunotherapies (e.g., T cell adoptive transfer therapy). For example, the method includes transplanting human tumor cells into non-human animals described in the present application, and applying human CAR-T to non-human animals with human tumor cells. The effectiveness of CAR-T treatment can be determined and evaluated. In some embodiments, the non-human animal is selected from LPA and / or APOB gene humanized non-human animals prepared by the methods described in the present application, double-gene or multi-gene humanized non-human animals (or their offspring) produced by the methods described in the present application, non-human animals expressing human or chimeric LPA proteins and / or human or chimeric APOB proteins, or animal models carrying tumors, immune disease animal models, metabolic disease animal models, or inflammatory animal models described in the present application. In some embodiments, TCR-T, CAR-T and / or other immunotherapies can treat LPA and / or APOB-related diseases (e.g., tumors, immune diseases, metabolic diseases, or inflammation) described in the present application. In some embodiments, TCA-T, CAR-T and / or other immunotherapies provide evaluation methods for treating LPA and / or APOB-related diseases (e.g., tumors, immune diseases, metabolic diseases, or inflammation) described herein.

[0194] Non-human animal models with two or more human or humanized genes

[0195] The present invention also provides an animal model or non-human animal having two or more human or humanized genes. The animal model or non-human animal may comprise a human or chimeric LPA gene and / or a human or chimeric APOB gene, and a sequence encoding an additional human or chimeric protein.

[0196] In some embodiments, the additional human or chimeric protein includes at least one of PCSK9, APOC3, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4. In some embodiments, the non-human animal described above also expresses at least one of human or chimeric PCSK9, APOC3, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4.

[0197] The present invention also provides a method for constructing a non-human animal with two or more human or humanized genes, the method comprising:

[0198] (1) Providing the above-mentioned construction method to obtain a non-human animal;

[0199] (ii) mating, in vitro fertilization or direct gene editing of the non-human animals provided in step (i) with other genetically modified non-human animals, and screening to obtain multi-gene modified non-human animals.

[0200] In some embodiments, the other genetically modified non-human animals include non-human animals with one or a combination of two or more of the genes PCSK9, APOC3, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4 modified (e.g., humanized).

[0201] In some embodiments, LPA and / or APOB genetic modifications are performed directly on non-human animals having PCSK9, APOC3, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1, or CTLA4 genetic modifications.

[0202] Since these proteins may involve different mechanisms, a combination therapy targeting two or more of these proteins may be a more effective treatment method. In fact, many related clinical trials are ongoing and show good results. Multigene modified non-human animal models can be used to determine the effectiveness of combination therapies targeting two or more proteins, for example, LPA and / or APOB therapeutic agents, and additional therapeutic agents for treating cancer, metabolic diseases, inflammation or immune diseases (e.g., asthma or atopic dermatitis). The combination therapy includes administering LPA and / or APOB therapeutic agents and additional therapeutic agents to non-human animals, wherein the non-human animals suffer from diseases (e.g., tumors, immune diseases, metabolic diseases or inflammation), and determining the effect of the combination therapy on the disease. In some embodiments, the additional therapeutic agent includes an antibody that specifically binds to PCSK9, APOC3, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab), an anti-PD-1 antibody (e.g., nivolumab), or an anti-PD-L1 antibody. In some embodiments, the non-human animal described above further comprises a sequence encoding human or humanized PD-1, a sequence encoding human or humanized PD-L1, or a sequence encoding human or humanized CTLA-4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab), an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the tumor described above comprises one or more tumor cells expressing PD-L1 and / or PD-L2.

[0203] In some embodiments, the combination therapy is used to treat various cancers described in the present application, for example, breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer or cancer. In some embodiments, the combination therapy is designed to treat immune diseases described in the present application, such as psoriasis. In some embodiments, the method described in the present application can be used to evaluate the combination therapy with some other methods. The method for treating cancer that can be used alone or in combination with the method described in the present application includes, for example, treating the subject with chemotherapy, for example, camphor alkali, doxorubicin, cisplatin, carboplatin, procarbazine, methylclorazepate, cyclophosphamide, doxorubicin, ifosamide, melphalan, chlorbutiazine, thiophene, nitrosourea, dacrynic acid, daunorubicin, bleomycin, primycin, mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, paclitaxel, transplatinum, 5-fluorouric acid, vincristine, vinblastine and / or methotrexate. Alternatively, or in addition, the method can include performing surgery on the subject to remove at least a portion of the cancer, for example, removing a portion or all of a tumor from the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0204] Figure 1 : Schematic diagram of LPA gene targeting strategy and targeting vector v1 design (not to scale), where Alb is the Alb promoter;

[0205] Figure 2 : Schematic diagram of LPA gene targeting strategy and targeting vector V2 design (not to scale), where Alb is the Alb promoter;

[0206] Figure 3 : Schematic diagram of LPA gene targeting strategy and targeting vector V3 design (not to scale);

[0207] Figure 4 : Southern blot identification results of F1 generation of LPA gene humanized mice, where WT is the wild-type control;

[0208] Figure 5 : ELISA test results, where + / + is wild-type C57BL / 6 mice, and H / + is LPA gene humanized heterozygous mice;

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

[0210] Figure 7 : Schematic diagram of dosing and sampling time for LPA humanized heterozygous mice;

[0211] Figure 8: ELISA was used to detect the concentration of chimeric LPA in the plasma of LPA humanized heterozygous mice before and after administration;

[0212] Fig. 9 :Changes of chimeric LPA in plasma of LPA humanized heterozygous mice before and after administration;

[0213] Fig.10 : ELISA test results, H / + is the LPA gene humanized heterozygous mouse;

[0214] Fig.11 : Schematic diagram of APOB gene targeting strategy and targeting vector V4 design (not to scale);

[0215] Fig.12 : PCR identification results of APOB gene humanized mouse F1 generation, where WT is the wild-type control; H2O is the water control, and M is the marker;

[0216] Fig.13 : ELISA test results, where + / +, M are male wild-type C57BL / 6 mice, H / +, F are female APOB gene humanized heterozygous mice, and H / +, M are male APOB gene humanized heterozygous mice;

[0217] Fig.14 : Human LPA protein concentration in plasma of LPA humanized heterozygous mice before and after administration. DETAILED DESCRIPTION

[0218] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0219] In each of the following examples, equipment and materials were obtained from the following companies:

[0220] C57BL / 6 mice and F1p transgenic mice were purchased from the National Rodent Laboratory Animal Seed Center, China Food and Drug Inspection Institutes;

[0221] BglII was purchased from NEB, catalog number R0144S;

[0222] SpeI was purchased from NEB, catalog number R3133S;

[0223] Human Lipoprotein A ELISA kit was purchased from Abcam, catalog number ab212165;

[0224] Human Apolipoprotein B ELISA kit was purchased from Abcam with the catalog number ab108807.

[0225] Example 1 LPA gene humanized mice (I)

[0226] In order to achieve the purpose of the present invention, the nucleotide sequence of the human LPA gene can be introduced into the mouse endogenous ROSA26 site so that the mouse expresses human or chimeric LPA protein. Specifically, using gene editing technology, under the control of the Alb promoter, a CDS sequence containing a portion of the human LPA gene is inserted into the ROSA26 site of the mouse.

[0227] Design as Figure 1 The targeting strategy shown, Figure 1 The targeting vector V1 contains homology arm sequences upstream and downstream of the mouse endogenous ROSA26 gene, and the A1 fragment containing the human LPA gene fragment (the A1 fragment contains the A fragment and the Neo box, wherein the A fragment is shown in SEQ ID NO.4). Among them, the upstream 5' homology arm sequence is shown in SEQ ID NO: 2, and the downstream 3' homology arm sequence is shown in SEQ ID NO: 3. The human LPA gene fragment containing one KIV-2 domain (e.g., KIV-2I) is shown in SEQ ID NO: 5; the connection design of the upstream of the A1 fragment sequence and the mouse gene is: The sequence " TTCTC The last "C" in the sequence is the last nucleotide of the mouse gene. The first "T" in is the first nucleotide of the A1 fragment. The connection between the A1 fragment sequence downstream and the mouse gene is designed as: The sequence " GGGGA "A" in the sequence is the last nucleotide of the A1 fragment. The first "A" in is the first nucleotide of the mouse gene.

[0228] The targeting vector V1 also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the A fragment is designed as follows: The sequence " GGGGA "A" in the sequence is the last nucleotide of the A segment. The first "A" in is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the mouse gene is designed as: The sequence " ACTTC The last "C" in the sequence is the last nucleotide of the Neo box. The "C" in is the first nucleotide of the mouse gene. The mRNA sequence of the LPA gene transcribed in the transformed humanized mouse is shown in SEQ ID NO: 6, and the expressed protein is shown in SEQ ID NO: 7.

[0229] The construction of the targeting vector can be carried out by conventional methods, such as enzyme digestion and ligation. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector verified by sequencing is electroporated and transfected into the embryonic stem cells of C57BL / 6 mice, and the obtained cells are screened using the positive clone screening marker gene to screen the correct positive clone cells. The screened correct positive clone cells (black mice) are introduced into the separated blastocysts (white mice) according to the technology known in the art, and the obtained chimeric blastocysts are transferred to the culture medium for short-term culture and then transplanted into the oviduct of the recipient mother mouse (white mouse), and F0 generation chimeric mice (black and white) can be produced. F0 generation chimeric mice are backcrossed with wild-type mice to obtain F1 generation mice, and then F1 generation heterozygous mice are mated with each other to obtain F2 generation homozygous mice. Positive mice can also be mated with Flp tool mice (Flp transgenic mice) to remove the positive clone screening marker gene, and then mated with each other to obtain LPA gene humanized homozygous mice.

[0230] In addition, CRISPR / Cas9 technology can be used for gene editing, Figure 2 The targeting strategy shown in the figure, the targeting vector V2 contains homology arm sequences upstream and downstream of the mouse endogenous ROSA26 site, and the A fragment containing the human LPA fragment (SEQ ID NO: 4). Among them, the upstream 5' homology arm sequence is shown in SEQ ID NO: 12, and the downstream 3' homology arm sequence is shown in SEQ ID NO: 13. The nucleotide sequence of the human LPA fragment is shown in SEQ ID NO: 5. The mRNA sequence transcribed from the LPA gene in the transformed humanized mouse is shown in SEQ ID NO: 6, and the expressed protein is shown in SEQ ID NO: 7.

[0231] Conventional methods can be used to construct the targeting vector, such as enzyme digestion, ligation, direct synthesis, etc. After the constructed targeting vector is initially verified by enzyme digestion, it is sent to a sequencing company for sequencing verification. The targeting vector that is verified to be correct by sequencing is used for subsequent experiments.

[0232] The target sequence (target site) determines the targeting specificity of sgRNA and the efficiency of inducing Cas9 to cut the target gene. Therefore, the selection and design of efficient and specific target sequence (target site) is a prerequisite for constructing sgRNA expression vectors. Design and synthesize sgRNA sequences that recognize target sites. The target sequence (target site) of exemplary sgRNA at the ROSA26 site is as follows:

[0233] sgRNA1 target site (SEQ ID NO: 17): 5'-AAGGCCGCACCCTTCTCCGGAGG-3';

[0234] The activity of sgRNA was detected by UCA kit. After confirming that it could mediate efficient cutting efficiency, restriction sites were added to its 5' end and complementary chain to obtain forward oligonucleotide and reverse oligonucleotide sequences as shown in Table 4. After annealing, the annealed product was connected to pT7-sgRNA plasmid (the plasmid was linearized with BbsI first) to obtain the expression vector pT7-LPA-1.

[0235] Table 4 sgRNA1 sequence list

[0236]

[0237] The pT7-sgRNA vector was synthesized by a plasmid synthesis company to contain a fragment DNA (SEQ ID NO: 22) of the T7 promoter and sgRNA scaffold and connected to the backbone vector (source: Takara, item number 3299) by restriction digestion (EcoRI and BamHI) in sequence. After sequencing verification by a professional sequencing company, the results showed that the target plasmid was obtained. Take the pronuclear fertilized egg of the mouse, such as C57BL / 6 mouse, and use a microinjector to pre-mix the in vitro transcription product of the pT7-LPA-1 plasmid (using the Ambion in vitro transcription kit, according to the instructions for transcription), the targeting vector and Cas9 mRNA and inject them into the cytoplasm or nucleus of the mouse fertilized egg. Microinjection of fertilized eggs was performed according to the method in the Mouse Embryo Manipulation Experiment Manual (3rd Edition) (Andras Nagy, Chemical Industry Press, 2006). The injected fertilized eggs were transferred to culture medium for a short period of culture and then transplanted into the oviduct of the recipient mother mouse for development. The obtained mice (F0 generation) were hybridized and self-fertilized to expand the population and establish a stable LPA gene humanized mouse strain.

[0238] The PCR method can be used to identify the somatic cell genotype of F1 generation mice, and the F1 generation PCR-positive mice are subjected to Southern blot detection to confirm whether there is random insertion. The mouse tail is cut to extract genomic DNA, and the genome is digested with BglII enzyme or SpeI enzyme, respectively, transferred to a membrane, and hybridized. The 5' probe (5'Probe) and the A probe (A Probe) are located upstream of the 5' homology arm and on the human LPA gene fragment, respectively. The specific probe and target fragment length are shown in Table 5 below), and the exemplary results are shown in Table 5 below. Figure 4 As shown, combined with the PCR and sequencing results, 10 mice numbered F1-01 to F1-05 and F1-07 to F1-11 were positive mice. This shows that the present method can be used to construct LPA gene humanized mice that can be stably propagated and have no random insertions.

[0239] Table 5 Length of specific probes and target fragments

[0240] Restriction enzymes Probe Wild-type fragment size Recombinant sequence fragment size BglII AProbe 7.9kb SpeI 5'Probe 8.9kb 16.4kb

[0241] 5'Probe-F (SEQ ID NO: 26): 5'-CCTCCTAGCGACCTAAACCATTAT-3',

[0242] 5'Probe-R (SEQ ID NO: 27): 5'-CCTGGTTTTAGTAGGTAATCGCTCA-3';

[0243] A Probe-F (SEQ ID NO: 28): 5'-GGTGTCCTAGAGACTCCCACTG-3',

[0244] A Probe-R (SEQ ID NO: 29): 5'-GCCTGTATCGGCATCTGGATT-3';

[0245] In addition, the expression of human or chimeric LPA protein in LPA humanized mice can be detected by conventional methods such as ELISA. Specifically, serum was collected from C57BL / 6 mice (+ / +) and the humanized LPA gene heterozygotes (H / +) prepared in this example and tested using a human Lipoprotein A ELISA kit. The test results are shown in FIG. Figure 5 shown.

[0246] from Figure 5 As can be seen in the figure, when using the human Lipoprotein A ELISA kit, chimeric LPA protein was only detected in LPA gene humanized heterozygous mice. This proves that chimeric LPA protein can be successfully expressed in LPA gene humanized heterozygous mice.

[0247] The expression of chimeric LPA protein in LPA gene humanized homozygous mice can be detected by ELISA. Specifically, 3 female and 3 male C57BL / 6 mice (+ / +) and 3 female and 3 male LPA gene humanized homozygous (H / H) mice prepared in this example were taken, and serum was collected and tested using a human Lipoprotein A ELISA kit. The test results are as follows: Figure 6 shown.

[0248] from Figure 6 It can be seen that when using the human Lipoprotein A ELISA kit, chimeric LPA protein was only detected in LPA gene humanized homozygous mice, and the LPA expression level in male mice was higher than that in female mice. This proves that chimeric LPA protein can be successfully expressed in LPA gene humanized homozygous mice.

[0249] Example 2 LPA gene humanized mice (II)

[0250] In order to achieve the purpose of the present invention, the nucleotide sequence of the human LPA gene can also be introduced into the mouse endogenous ROSA26 site so that the mouse expresses the human LPA protein. Specifically, using gene editing technology, under the control of the human LPA gene regulatory element, a nucleotide sequence of about 157.6 kb from the 5'UTR upstream nucleotide sequence to the 3'UTR downstream nucleotide sequence of the human LPA gene is inserted into the mouse ROSA26 site.

[0251] Design as Figure 3 The targeting strategy shown, Figure 3 The targeting vector V3 contains homology arm sequences upstream and downstream of the mouse endogenous ROSA26 gene, and the A2 fragment containing the human LPA gene fragment. The upstream 5' homology arm sequence is shown in SEQ ID NO: 15, and the downstream 3' homology arm sequence is shown in SEQ ID NO: 16. The human LPA gene fragment is identical to the nucleotide sequence of positions 160528474 to 160686025 with NCBI accession number NC_000006.12; the connection design of the downstream sequence of the human LPA gene fragment and the mouse gene is: The sequence " ACCTG "G" in the sequence is the last nucleotide of the human LPA gene fragment. The "C" in is the first nucleotide of the mouse gene.

[0252] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the mouse gene is designed as follows: The sequence " TTCTC The last "C" in the sequence is the last nucleotide of the mouse gene. The first "G" in is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the human LPA gene fragment is designed as follows: The sequence " ATCGC The last "C" in the sequence is the last nucleotide of the Neo box. The first "A" in is the first nucleotide of the human LPA gene fragment. The mRNA sequence of the LPA gene transcription in the transformed humanized mouse is shown in NM_005577.4, and the expressed protein is shown in SEQ ID NO:1.

[0253] The expression of human LPA protein in LPA gene humanized heterozygous mice can be detected by ELISA. Specifically, three female LPA gene humanized heterozygous mice prepared in this example were taken, serum was taken, and diluted 10× and 100×. The protein in serum of different concentrations was detected using a human Lipoprotein A ELISA kit. The detection results are as follows: Fig.10 shown.

[0254] from Fig.10 It can be seen that when the human Lipoprotein A ELISA kit was used for detection, human LPA protein was detected in the LPA gene humanized heterozygous mice, proving that the LPA gene humanized heterozygous mice prepared in this example can successfully express human LPA protein.

[0255] Example 3 APOB gene humanized mice

[0256] In order to achieve the purpose of the present invention, a nucleotide sequence encoding human APOB protein can be introduced into the mouse endogenous APOB locus, so that the mouse expresses human APOB protein. Specifically, using gene editing technology, the mouse 5'UTR upstream nucleotide sequence to the 3'UTR downstream nucleotide sequence of about 79.20 kb is replaced with the mouse 5'UTR upstream nucleotide sequence to the 3'UTR downstream nucleotide sequence of about 74.59 kb, to obtain a humanized APOB locus, thereby achieving humanization of the mouse APOB gene.

[0257] In order to realize the targeting strategy of the present invention, the following Fig.11 The targeting vector V4 shown contains homology arm sequences upstream and downstream of the mouse APOB gene, and an A3 fragment containing a human APOB gene fragment. The upstream 5' homology arm sequence is SEQ ID NO: 34, the downstream 3' homology arm sequence is SEQ ID NO: 35, and the nucleotide sequence of the human APOB gene fragment is a variant of the nucleotide sequence from 20983975 to 21063173 of NC_000002.12 (wherein, the variant is a mutation of G at position 21042269 to T, A at position 21040767 to C, C at position 21037729 to T; A is inserted between 21037656-21037657, G is mutated to A at position 21032803, and AG is inserted between 21032701-21032702 ATG, deleting 21029494-21029497, mutating A at position 21028168 to C, mutating C at position 21028042 to T, mutating A at position 21016795 to G, mutating T at position 21013610 to C, mutating A at position 21009931 to G, mutating C at position 21009932 to T, mutating C at position 21009323 to T, and mutating G at position 21002409 to A, one or more of the following), the connection between the upstream of the A3 fragment and the mouse gene is designed as follows: The sequence The last "A" in the sequence is the last nucleotide of the mouse gene. AATC The first "A" in " is the first nucleotide of the A3 fragment. The connection between the downstream of the A3 fragment and the mouse gene is designed as follows: The sequence The "G" in the A3 fragment is the last nucleotide, and the sequence " AGAGG The first "A" in " is the first nucleotide of the mouse gene.

[0258] The targeting vector also includes a resistance gene for positive clone screening, namely the neomycin phosphotransferase coding sequence Neo, and two site-specific recombination system Frt recombination sites arranged in the same direction are installed on both sides of the resistance gene to form a Neo cassette. The connection between the 5' end of the Neo cassette and the human APOB gene is designed as follows: The sequence The last "G" in the sequence is the last nucleotide at the junction of the human APOB gene and the 5' end of the Neo box. GTTC "G" in " is the first nucleotide of the Neo box; the connection between the 3' end of the Neo box and the human APOB gene is designed as: The sequence The last "C" in the sequence is the last nucleotide of the Neo box. CAACT The first "C" in " is the first nucleotide at the junction of the human APOB gene and the 3' end of the Neo box. The mRNA sequence transcribed from the modified humanized APOB gene is shown in SEQ ID NO: 37, and the expressed protein sequence is shown in SEQ ID NO: 38.

[0259] The genotype of somatic cells of F1 mice can be identified by PCR, using the primers shown in Table 6 below for detection. The exemplary results are shown in Fig.12 As shown, two mice numbered F1-01 and F1-02 were positive mice.

[0260] Table 6 Primer sequences and recombinant fragment sizes for PCR detection of F1 genotypes

[0261]

[0262] The expression of human APOB protein in APOB humanized mice can be detected by conventional methods such as ELISA. Specifically, a 7-week-old male C57BL / 6 mouse (+ / +, M) and three 8-week-old APOB gene humanized heterozygous male mice (H / +, M) and two 8-week-old APOB gene humanized heterozygous female mice (H / +, F) prepared in this example were selected, plasma was collected, and human APOB ELISA kit (human Apolipoprotein B ELISA kit, purchased from Abeam, catalog number ab108807) was used for detection. The test results are shown in FIG. Fig.13 shown.

[0263] from Fig.13It can be seen that human APOB protein cannot be detected in the plasma of wild-type mice; human APOB protein is only detected in the plasma of APOB gene humanized heterozygous mice, indicating that human APOB protein can be successfully expressed in APOB gene humanized heterozygous mice.

[0264] Example 4 Pharmacodynamic Model

[0265] The LPA and / or APOB humanized mice prepared by Examples 1-3 of the present application can be used to evaluate the efficacy of targeted human LPA and / or APOB regulators in hyperlipidemia or atherosclerotic diseases.

[0266] For example, Olpasiran is a siRNA developed by Amgen, which is a double-stranded small interfering RNA targeting the LPA gene, and its sense strand and antisense strand are shown in SEQ ID NO: 30 and 31, respectively. The LPA gene humanized mice prepared by Example 1 or 2 of the present application can be used to evaluate the efficacy of regulators targeting human LPA in hyperlipidemia or atherosclerotic diseases. Specifically, two 8-week-old LPA gene humanized heterozygous mice obtained in Example 1 were subcutaneously injected with Olpasiran analog (3 mg / kg). The specific dosing and sampling time are as follows: Figure 7 As shown in Figure 2, peripheral blood was collected 7 days and 3 days before administration, and 7, 14 and 28 days after administration, and the LPA concentration in plasma was detected by ELISA. Figure 8 As shown in the figure, Olpasiran analog can significantly reduce the LPA concentration in LPA gene humanized heterozygous mice, and LPA expression can be reduced by more than 80% after a single dose ( Fig. 9 ). For another example, specifically, 6 LPA gene humanized heterozygous male mice obtained in Example 2 were randomly divided into a control group (G1) and a treatment group (G2), with 3 mice in each group. On the 0th day of administration, the mice in the treatment group were subcutaneously injected with Olpasiran analog at a dose of 2 mg / rg, and the mice in the control group were subcutaneously injected with an equal volume of saline. Peripheral blood was collected from all mice 7 days before administration and 7, 14, 28 and 42 days after administration, and the LPA concentration in plasma was detected by ELISA. The results are shown in Fig.14 As shown, compared with the control group, the plasma LPA concentration of mice in the drug administration group was significantly reduced, indicating that Olpasiran analog can significantly reduce the plasma LPA concentration of LPA gene humanized heterozygous mice. This indicates that LPA gene humanized heterozygous mice can be used for preclinical research on drugs targeting human LPA.

[0267] Example 5 Preparation of double-gene or multi-gene humanized mice

[0268] The LPA and / or APOB gene humanized mice prepared in Examples 1-3 of the present application can also be used to prepare a multi-gene humanized mouse model. For example, in the aforementioned Examples 1-3, the embryonic stem cells used for microinjection can be selected from mice modified with at least one gene containing PCSK9, APOC3, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 and CTLA4, or, on the basis of LPA and / or APOB gene humanized mice, mouse ES embryonic stem cells can be isolated and gene recombination targeting technology can be used to obtain a double-gene humanized or multi-gene humanized mouse model. The homozygous or heterozygous LPA and / or APOB mice obtained in Examples 1-3 of the present application can also be mated with other genetically modified mice, and their offspring can be screened. According to Mendel's law of inheritance, there is a certain probability of obtaining multigenic mice with humanized LPA and / or APOB genes and other genetic modifications, and then the heterozygotes can be mated with each other to obtain homozygous mice with double or multiple genes modified.

[0269] Taking LPA / APOB dual-gene humanized mice as an example, the LPA gene humanized mice prepared in Example 1 or Example 2 and the APOB gene humanized homozygous mice prepared in Example 3 were mated, and after offspring screening, LPA / APOB dual-gene humanized mice were obtained.

[0270] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0271] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0272] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

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

2. The construction method according to claim 1, characterized in that: The human LPA protein comprises at least 1 to 16 Kringle domains; Preferably, the amino acid sequence of the human LPA protein comprises SEQ ID NO: 1, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1; Preferably, the amino acid sequence of the chimeric LPA protein comprises SEQ ID NO: 7, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

7.

3. A method for constructing a genetically modified non-human animal, characterized in that: The construction method comprises inserting the nucleotide sequence of human LPA into a safe site of the non-human animal genome, preferably, the safe site comprises the ROSA26 site or the Hipp11 site; Preferably, the nucleotide sequence of human LPA comprises all or part of exon 1 to exon 39 of the human LPA gene; Preferably, the nucleotide sequence of human LPA comprises any one of the following groups: A) a portion of exon 1 to all of exon 5, and a portion of all of exon 16 to a portion of exon 39 of a human LPA gene, wherein the portion of exon 1 of a human LPA gene preferably comprises at least 5 bp of nucleotide sequence; the portion of exon 39 of a human LPA gene preferably comprises at least 5 bp of nucleotide sequence; B) a nucleotide sequence from the start codon to the stop codon of the human LPA gene, preferably further comprising 5'UTR and / or 3'UTR, more preferably comprising all of exon 1 to exon 39 of the human LPA gene; more preferably further comprising at least 50 bp of continuous nucleotides upstream of the 5'UTR and / or at least 50 bp of continuous nucleotides downstream of the 3'UTR of the human LPA gene; Preferably, the nucleotide sequence of human LPA comprises a nucleotide sequence encoding SEQ ID NO: 1 or 7; Preferably, the nucleotide sequence of human LPA comprises the nucleotide sequence of SEQ ID NO: 5 or NCBI accession number NC_000006.12 at positions 160528474 to 160686025; or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the nucleotide sequence of SEQ ID NO: 5 or NCBI accession number NC_000006.12 at positions 160528474 to 160686025; Preferably, the safe site of insertion into the non-human animal genome includes insertion between exon 1 and exon 2 of the ROSA26 gene in the non-human animal genome; Preferably, the nucleotide sequence inserted into the genome of a non-human animal further comprises an auxiliary sequence, and the auxiliary sequence preferably comprises one or more of a stop codon, WPRE, Insulator, loxP, STOP sequence or Poly A.

4. The construction method according to any one of claims 1 to 3, characterized in that: The nucleotide sequence encoding the human or chimeric LPA protein or the nucleotide sequence of human LPA is operably linked to a regulatory element; Preferably, the regulatory element comprises an exogenous regulatory element (eg, Alb promoter or human LPA promoter); Preferably, the modified LPA gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous modified gene site.

5. The construction method according to any one of claim 14, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent; preferably, the rodent includes a mouse or a rat; Preferably, the mRNA transcribed from the modified LPA gene in the genome of a non-human animal comprises a nucleotide sequence of SEQ ID NO: 6 or NCBI Accession No. NM_005577.4, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to a nucleotide sequence of SEQ ID NO: 6 or NCBI Accession No. NM_005577.

4.

6. The construction method according to any one of claims 1 to 5, characterized in that: The genome of the non-human animal further includes nucleotide sequences encoding other human or humanized proteins, wherein the other human or humanized proteins include at least one of apolipoprotein B (APOB), PCSK9, APOC3, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4; Preferably, the other human or humanized protein is a human or chimeric APOB protein; Preferably, the amino acid sequence of the human or chimeric APOB protein comprises SEQ ID NO: 33 or 38, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence shown in SEQ ID NO: 33 or 38.

7. A method for constructing a genetically modified non-human animal, characterized in that: The genome of the non-human animal comprises at least one chromosome comprising a nucleotide sequence encoding a human or chimeric APOB protein; Preferably, the amino acid sequence of the human or chimeric APOB protein comprises SEQ ID NO: 33 or 38, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to the amino acid sequence shown in SEQ ID NO: 33 or 38.

8. A method for constructing a genetically modified non-human animal, characterized in that: At the non-human animal endogenous APOB gene locus, replacing the non-human animal endogenous APOB gene with a nucleotide sequence comprising human APOB; Preferably, the nucleotide sequence of human APOB comprises the nucleotide sequence from the start codon to the stop codon of the human APOB gene, and preferably further comprises 5'UTR and / or 3'UTR; Preferably, the nucleotide sequence of human APOB includes all of exon 1 to exon 29 of the human APOB gene; more preferably, it also includes at least 50 bp of continuous nucleotides upstream of the 5'UTR and / or at least 50 bp of continuous nucleotides downstream of the 3'UTR of the human APOB gene; Preferably, the nucleotide sequence of human APOB comprises a nucleotide sequence encoding SEQ ID NO: 33 or 38; Preferably, the nucleotide sequence of human APOB comprises the nucleotide sequence of NCBI accession number NC_000002.12 from 20983975 to 21063173 or a variant thereof, or comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identity to the nucleotide sequence shown in NCBI accession number NC_000002.12 from 20983975 to 21063173 or a variant thereof; wherein the variant preferably comprises a mutation of G at position 21042269 to T, a mutation of A at position 21040767 to C, a mutation of C at position 21037729 to T; Insert A between 21037656 and 21037657, mutate G at position 21032803 to A, insert AGATG between 21032701 and 21032702, delete 21029494 and 21029497, mutate A at position 21028168 to C, mutate C at position 21028042 to T, mutate A at position 21016795 to G, mutate T at position 21013610 to C, mutate A at position 21009931 to G, mutate C at position 21009932 to T, mutate C at position 21009323 to T, or mutate G at position 21002409 to A, or more than one of the following; Preferably, the endogenous APOB gene of a non-human animal comprises all of exon 1 to exon 29 of the endogenous APOB gene of a non-human animal, and further preferably comprises at least 50 bp of continuous nucleotides upstream of the 5'UTR and / or at least 50 bp of continuous nucleotides downstream of the 3'UTR of the endogenous APOB gene of a non-human animal.

9. The construction method according to claim 7 or 8, characterized in that: The nucleotide sequence of human APOB or the nucleotide sequence encoding human or chimeric APOB protein is operably linked to a regulatory element, wherein the regulatory element includes an endogenous regulatory element of a non-human animal or an exogenous regulatory element; wherein the exogenous regulatory element includes a human APOB promoter, 5'UTR and / or 3'UTR; Preferably, the modified APOB gene in the genome of the non-human animal is homozygous or heterozygous for the endogenous replaced locus; Preferably, the endogenous APOB protein in the non-human animal is not expressed or is expressed at a reduced level compared to APOB in wild-type animals.

10. The construction method according to any one of claims 8-9, characterized in that: The non-human animal is a mammal, such as a monkey or a rodent; preferably, the rodent includes a mouse or a rat; Preferably, the mRNA transcribed from the modified APOB gene in the genome of the non-human animal comprises SEQ ID NO:37, or comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

37.

11. The construction method according to any one of claims 8 to 10, characterized in that: The genome of the non-human animal further includes nucleotide sequences encoding other human or humanized proteins, wherein the other human or humanized proteins include at least one of LPA, PCSK9, APOC3, ANGPTL3, GPR75, ANGPTL4, LAG3, 4-1BB, CD40, TIGIT, CD27, CD28, B7H3, OX40, PD-1, PD-L1 or CTLA4; Preferably, the other human or humanized protein is a human or chimeric LPA protein; Preferably, the amino acid sequence of the human LPA protein comprises SEQ ID NO: 1, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO: 1; Preferably, the amino acid sequence of the chimeric LPA protein comprises SEQ ID NO: 7, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

7.

12. A method for determining the effectiveness or toxicity of a therapeutic agent in treating a disease, characterized in that: The method comprises: 1) administering a therapeutic agent to a non-human animal obtained by the construction method according to any one of claims 1 to 11; 2) determine the effects of therapeutic agents on non-human animals; Preferably, the non-human animal suffers from a disease; Preferably, the disease includes one or more of tumor, immune disease, metabolic disease or inflammation; Preferably, the therapeutic agent comprises an LPA and / or APOB therapeutic agent, preferably comprising one or more of an anti-LPA and / or APOB antibody, an LPA and / or APOB inhibitor, or an LPA and / or APOB nucleic acid drug; Further preferably, the therapeutic agent further comprises an additional therapeutic agent, and the additional therapeutic agent preferably comprises one or more of anti-PD-1 antibody, anti-PD-L1 antibody or anti-CTLA4 antibody.

13. A chimeric LPA protein, characterized in that The chimeric LPA protein comprises at least one Kringle domain of a human LPA protein; Preferably, the amino acid sequence of the chimeric LPA protein comprises SEQ ID NO: 7, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:

7.

14. A chimeric LPA gene, characterized in that The chimeric LPA gene comprises any of the following nucleotide sequences: A) a nucleotide sequence encoding human LPA protein, preferably comprising a nucleotide sequence encoding SEQ ID NO: 1; B) a nucleotide sequence encoding a chimeric LPA protein, preferably comprising a nucleotide sequence encoding SEQ ID NO: 7; C) SEQ ID NO: 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 23, 24, 25, NCBI accession number NM_005577.4 or NCBI accession number NC_000006.12 nucleotide sequence at positions 160528474 to 160686025; D) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 23, 24, 25, NCBI Accession No. NM_005577.4, or NCBI Accession No. NC_000006.12 at positions 160528474 to 160686025; E) a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the nucleotide sequence of positions 160528474 to 160686025 of SEQ ID NO: 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 23, 24, 25, NCBI Accession No. NM_005577.4 or NCBI Accession No. NC_000006.

12.

15. A chimeric APOB protein, characterized in that The amino acid sequence of the chimeric APOB protein comprises SEQ ID NO:33 or 38, or comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.5% identical to SEQ ID NO:33 or 38.

16. A chimeric APOB gene, characterized in that The chimeric APOB gene comprises any of the following nucleotide sequences: A) a nucleotide sequence encoding a human or chimeric APOB protein, preferably comprising a nucleotide sequence encoding SEQ ID NO: 33 or 38; B) SEQ ID NO: 34, 35, 37, 39, 40, 41, 42 or NC_000002.12 nucleotide sequence 20983975 to 21063173 or a variant thereof; C) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 34, 35, 37, 39, 40, 41, 42 or NC_000002.12, nucleotide sequence 20983975 to 21063173, or a variant thereof; D) a nucleotide sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO: 34, 35, 37, 39, 40, 41, 42 or NC_000002.12, nucleotide sequence 20983975 to 21063173, or a variant thereof; Preferably, the variant comprises mutating G at position 21042269 to T, mutating A at position 21040767 to C, mutating C at position 21037729 to T; inserting A between 21037656-21037657, mutating G at position 21032803 to A, inserting AGATG between 21032701-21032702, and inserting AGATG between 21029494-2102949 7 deletion, mutation of A at position 21028168 to C, mutation of C at position 21028042 to T, mutation of A at position 21016795 to G, mutation of T at position 21013610 to C, mutation of A at position 21009931 to G, mutation of C at position 21009932 to T, mutation of C at position 21009323 to T or mutation of G at position 21002409 to A, one or more of the following.

17. A cell, tissue or organ, characterized in that: The cells, tissues or organs express one or more of human LPA protein, human APOB protein, the chimeric LPA protein according to claim 13 or the chimeric APOB protein according to claim 15, and / or the genome of the cells, tissues or organs contains the chimeric LPA gene according to claim 14 and, or the chimeric APOB gene according to claim 16.

18. Use of the non-human animal obtained by the construction method of any one of claims 1 to 11, the chimeric LPA protein of claim 13, the chimeric LPA gene of claim 14, the chimeric APOB protein of claim 15, the chimeric APOB gene of claim 16, or the cell, tissue or organ of claim 17, characterized in that: The application includes: A) Application in the development of products involving immunological processes related to LPA and / or APOB in human cells; B) Application as a model system related to LPA and / or APOB for pharmacological, immunological, microbiological and medical research; C) Applications involving the production and use of animal experimental disease models for the study of the etiology of LPA and / or APOB and / or for the development of diagnostic strategies and / or for the development of therapeutic strategies; D) in vivo studies on the screening, efficacy testing, efficacy assessment, validation or evaluation of human LPA and / or APOB signaling pathway modulators; or, E) Study the function of LPA and / or APOB genes, study the drugs and efficacy targeting human LPA and / or APOB targets, and study the application of LPA and / or APOB-related therapeutic drugs in tumors, inflammation, immune diseases, and metabolic diseases.

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