Immunodeficient animal model lacking MHC gene and method for producing same

Abstract

The present invention relates to an immunodeficient animal model lacking the MHC gene and a method for producing same. The present invention is based on the finding that the occurrence of graft-versus-host disease is inhibited in mice lacking the B2M gene related to MHC class I, and in humanized mice lacking the B2M and H2-D1 genes related to MHC Class I and the H2-Ab1 gene related to MHC class II, and can thus be effectively used as a model capable of evaluating the efficacy and toxicity of various pharmaceutical products.

Description

MHC gene-deficient immunodeficient animal model and method for producing the same

[0001] The present invention relates to an immunodeficient animal model with a defective MHC gene and a method for producing the same.

[0002] Rodent animal models are useful tools for studying gene function, disease mechanisms, and novel therapeutics in vivo. However, the interpretation of these studies is limited due to species differences between rodents and humans. Non-human primates offer the best alternative model for human diseases, but their use is limited due to high costs and ethical concerns. Recently, humanized mice are being developed to replace primates.

[0003] Humanized mice are immunodeficient mice in which human cells or tissues have been transplanted, resulting in the cells retaining their natural functions. These immunodeficient mice lack T, B, and NK cells, resulting in a very low incidence of graft rejection. Therefore, they are known to readily accept CD34+ hematopoietic stem cell (HSC) or peripheral blood mononuclear cell (PBMC) transplants. However, when PBMCs are transplanted into immunodeficient mice, severe graft-versus-host disease (GvHD) occurs at 3-4 weeks of age due to graft rejection, leading to rapid debilitation, death, and weight loss, significantly disrupting animal experiments. This occurs when transplanted mature human T cells recognize mouse cells as non-self and attack mouse cells, such as host organs and tissues. This central recognition of self and non-self is known to occur through the interaction between human T cells and mouse MHC (major histocompatibility complex) proteins. MHC proteins on the surface of mouse cells are composed of Class I / II, and there is a research report that when both are deficient, the GvHD phenomenon is significantly reduced and the survival period is extended.

[0004] As a prior art, Korean Patent No. 10-2247977 discloses an immunodeficiency animal model with a mutation in the IL2Rg gene. The present invention has developed a mouse with a deletion of the B2M gene related to MHC Class I for the purpose of reducing the degree of abnormal clinical symptoms and mortality due to GvHD that appear when constructing a humanized mouse model using the mouse. In addition, a mouse with a deletion of the B2M gene and a deletion of both the H2-D1 gene related to MHC Class I and the H2-Ab1 gene corresponding to MHC Class II has been developed.

[0005] An object of the present invention is to provide a recombinant expression vector comprising a nucleotide sequence encoding a gRNA consisting of a base sequence of SEQ ID NO: 2 that hybridizes to DNA encoding a B2M gene; a nucleotide sequence encoding a Cas9 protein; and a promoter operably linked to the nucleotide sequence.

[0006] Another object of the present invention is to provide a transformed cell line for producing an immunodeficient animal model into which the recombinant expression vector is introduced.

[0007] Another object of the present invention is to provide a method for producing an immunodeficient animal model, which comprises a step of transplanting the transformed cell line into the oviduct of a surrogate mother, which is an animal other than a human.

[0008] Another object of the present invention is to provide an immunodeficiency animal model in which the B2M gene is knocked out.

[0009] To achieve the above purpose,

[0010] The present invention provides a recombinant expression vector comprising a nucleotide sequence encoding a gRNA comprising a base sequence of SEQ ID NO: 2 that hybridizes to DNA encoding a B2M gene; a nucleotide sequence encoding a Cas9 protein; and a promoter operably linked to the nucleotide sequence.

[0011] Another object of the present invention is to provide a transformed cell line for producing an immunodeficient animal model into which the recombinant expression vector is introduced.

[0012] Another object of the present invention is to provide a method for producing an immunodeficient animal model, which comprises a step of transplanting the transformed cell line into the oviduct of a surrogate mother, which is an animal other than a human.

[0013] Another object of the present invention is to provide an immunodeficiency animal model in which the B2M gene is knocked out.

[0014] The present invention relates to an immunodeficient animal model with a defective MHC gene and a method for producing the same. The present invention can be usefully utilized as a model capable of evaluating the efficacy and toxicity of various pharmaceuticals, as it has been confirmed that the occurrence of graft-versus-host disease is suppressed in a mouse with a defective B2M gene related to MHC Class I and a humanized mouse with a defective B2M and H2-D1 gene related to MHC Class I and the H2-Ab1 gene related to MHC Class II.

[0015] Figure 1 is a diagram showing the results of the G0 mouse T7E1 assay.

[0016] Figure 2 is a diagram showing the T7E1 Assay results of G1 mice.

[0017] Figure 3 is a graph showing the expression results of the B2M gene in B2M-NIG mice by qRT-PCR.

[0018] Figure 4 is a graph showing the results of expression of B2M protein in the spleen of B2M-NIG mice by FACS.

[0019] Figure 5 is a graph analyzing changes in mouse body weight following human PBMC transplantation into NIG and B2M KO-NIG mice.

[0020] Figure 6 is a graph analyzing the mouse survival rate following human PBMC transplantation into NIG and B2M KO-NIG mice.

[0021] Figure 7 is a graph showing the percentage of blood human CD3+ cells in the hPBMC-B2M-NIG model.

[0022] Figure 8 is a diagram showing the results of examining the expression of B2M, H2-D, and IA proteins in the spleen of B2M / dKO-NIG mice by FACS.

[0023] Figure 9 is a graph analyzing the change in mouse body weight following human PBMC transplantation into NIG, B2M-NIG, and B2M / dKO-NIG mice.

[0024] Figure 10 is a graph analyzing the mouse survival rate following human PBMC transplantation into NIG, B2M-NIG, and B2M / dKO-NIG mice.

[0025] Figure 11 shows photographs of GvHD responses observed in NIG, B2M-NIG, and B2M / dKO-NIG mice 6 weeks after PBMC transplantation.

[0026] Figure 12 is a graph analyzing the change in mouse body weight following human PBMC transplantation under the conditions of busulfan administration to NIG, B2M-NIG, and B2M / dKO-NIG mice.

[0027] Figure 13 is a graph analyzing the mouse survival rate according to human PBMC transplantation under the conditions of busulfan administration to NIG, B2M-NIG, and B2M / dKO-NIG mice.

[0028] Figure 14 is a graph analyzing the percentage of human CD45+ cells in the blood in the hPBMC-B2M-NIG model.

[0029] Figure 15 is a graph analyzing the percentage of human CD3+ cells in the blood in the hPBMC-B2M-NIG model.

[0030] Figure 16 is a graph analyzing the organ weight (%) by week of PBMC transplantation in the hPBMC-B2M-NIG model.

[0031] The present invention is described in detail below.

[0032] The present invention provides a recombinant expression vector comprising a nucleotide sequence encoding a gRNA comprising a base sequence of SEQ ID NO: 2 that hybridizes to DNA encoding a B2M gene; a nucleotide sequence encoding a Cas9 protein; and a promoter operably linked to the nucleotide sequence.

[0033] In this specification, a method is provided for producing a transgenic animal exhibiting an immunodeficiency phenotype by mutating the B2M gene in a mouse fertilized egg using a B2M-specific gene scissors utilizing the CRISPR / Cas9 system.

[0034] The term "CRISPR / Cas9 system" used in the present invention refers to a third-generation genetic scissors composed of Cas9 protein and guide RNA (gRNA), and refers to an artificial restriction enzyme designed to cut a desired genetic base sequence using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, known as the immune system of microorganisms.

[0035] The term "Cas9 protein" used in the present invention is an essential protein element in the CRISPR / Cas9 system, which can form a complex with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) to function as an active endonuclease or nickase. The Cas9 protein may be derived from the genus Staphylococcus, Streptococcus, Neisseria, Pasteurella, Francisella, or Campylobacter, but is not limited thereto.

[0036] The term "guide RNA (gRNA)" used in the present invention refers to an RNA specific to target DNA. The gRNA can form a complex with the Cas9 protein and bring the Cas9 protein to the target DNA. The gRNA may be a single guide RNA (sgRNA) in which a crRNA and a tracrRNA are linked together.

[0037] When a cell is transformed using the recombinant expression vector of the present invention, a guide RNA fragment can be delivered into the cell, and the delivered guide RNA fragment can recognize the IL2Rg gene. Therefore, when a cell is transformed using the recombinant expression vector, a guide RNA fragment and a tracrRNA fragment or an sgRNA in which crRNA and tracrRNA are linked can be delivered into the cell, and the delivered tracrRNA fragment or portion can form a complex or binding structure with the crRNA fragment or portion, thereby forming a structure that can be recognized by the Cas9 protein.

[0038] According to one specific example of the invention, the gRNA may be composed of a base sequence of SEQ ID NO: 2, and may be used to knock out exon 2 of the B2M gene.

[0039] The term "recombinant expression vector" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and the appropriate nucleic acid sequences essential for expressing the coding sequence operably linked to it in a specific host organism. Promoters, enhancers, termination signals, and polyadenylation signals available in eukaryotic cells are well known.

[0040] The term "operably linked" as used in the present invention refers to a functional linkage between a gene expression control sequence and another nucleotide sequence. The gene expression control sequence may be at least one selected from the group consisting of a replication origin, a promoter, and a transcription termination sequence. The transcription termination sequence may be a polyadenylation sequence (pA), and the replication origin may be, but is not limited to, an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, or a BBV replication origin.

[0041] The term "promoter" as used in the present invention means a region of DNA upstream from a structural gene, and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.

[0042] A promoter according to one specific embodiment of the present invention is one of the transcriptional regulatory sequences that regulates the initiation of transcription of a specific gene, and may be a polynucleotide fragment having a length of about 100 bp to about 2500 bp. The promoter may be used without limitation as long as it can regulate the initiation of transcription in a cell, for example, a eukaryotic cell (e.g., a plant cell, or an animal cell (e.g., a mammalian cell such as a human or mouse, etc.)). For example, the promoter may be a cytomegalovirus promoter (e.g., human or mouse CMV immediate-early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, SV40 promoter, adenovirus promoter (major late promoter), pL λ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, tk promoter of HSV, SV40E1 promoter, respiratory syncytial virus (RSV) promoter, metallothionin promoter, β-actin promoter, ubiquitin C promoter, human interleukin-2 gene promoter, human It may be selected from the group consisting of, but is not limited to, the human lymphotoxin gene promoter and the human granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter.

[0043] A recombinant expression vector according to one specific example of the present invention may be selected from the group consisting of a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. Vectors that can be used as recombinant expression vectors can be produced based on, but are not limited to, plasmids used in the art (e.g., pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, M13, etc.), or viral vectors (e.g., adeno-associated virus (AAV) vectors, etc.).

[0044] The recombinant expression vector of the present invention may further comprise one or more selectable markers. The markers are typically nucleic acid sequences having properties that can be selected by chemical methods, and include any gene that can distinguish transfected cells from non-transfected cells. For example, the markers may be, but are not limited to, herbicide resistance genes such as glyphosate, glufosinate ammonium, or phosphinothricin, or antibiotic resistance genes such as ampicillin, kanamycin, G418, bleomycin, hygromycin, or chloramphenicol.

[0045] The recombinant expression vector of the present invention can be produced using a genetic recombination technique well known in the art, and site-specific DNA cutting and ligation can be performed using enzymes generally known in the art.

[0046] Another aspect of the present invention provides a transformed cell line for producing an immunodeficient animal model into which the recombinant expression vector is introduced.

[0047] The term "immunodeficiency" as used in the present invention refers to a state in which an effective immune response ability is lacking, and specifically, may be caused by a defect in the development and function of T, B, and NK cells.

[0048] In order to produce a transformed cell line into which a recombinant expression vector according to one specific example of the present invention has been introduced, a method known in the art for introducing a nucleic acid molecule into an organism, cell, tissue, or organ can be used, and a standard technique suitable for the host cell can be selected and performed as known in the art. Such methods may include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0049] The type of cell to be used as the transformed cell line may be an animal cell or a cell derived from an animal cell, preferably a fertilized single-cell stage embryo derived from a mammal, most preferably a mouse.

[0050] According to one specific example of the present invention, the cell line may be derived from a NIG mouse lacking the IL2Rg gene. The NIG mouse is a NOD.CB17 / Prkdc SCID-This is a KO mouse with a 2 bp (base pair) deletion in exon 6 of the IL2Rg gene of the JKrb mouse, and is disclosed in Korean Patent No. 2247977.

[0051] Another aspect of the present invention provides a method for producing an immunodeficient animal model, comprising the step of transplanting the transformed cell line into the oviduct of a surrogate mother, which is an animal other than a human.

[0052] According to one specific example of the present invention, the animal may be a mouse. Mice are already being used in research on the pathological mechanisms and treatments of various diseases. In particular, their long-standing recognition as an economically valuable animal allows them to avoid ethical issues compared to using other animals as disease models. Furthermore, their stable breeding system facilitates maintenance and management during the development of experimental animal models.

[0053] According to one specific example of the present invention, the immunodeficiency may be due to a knock-out of the B2M gene.

[0054] The term "knockout" used in the present invention means modifying or removing a specific gene in the base sequence so that it cannot be expressed.

[0055] It is possible to produce an immunodeficiency animal model by B2M knockout by substitution, deletion, or insertion of some of the bases constituting the B2M gene, preferably deletion of some of the bases constituting the B2M gene, and most preferably deletion of the bases constituting exon 2 of the B2M gene.

[0056] According to one specific example of the present invention, the method for producing the animal model may further include a step of breeding the animal model with a mouse in which both the H2-D1 (H2-D1-histocompatibility 2, D region locus 1, Gene ID: 14964) and H2-Ab (H2-Ab1 histocompatibility 2, class II antigen A, beta 1, Gene ID: 14961) genes are deficient. The mouse in which both the H2-D1 and H2-Ab genes are deficient may be a NIG mouse.

[0057] Another aspect of the present invention provides an immunodeficient animal model in which the B2M gene is knocked out.

[0058] The term "animal model" used in the present invention refers to an animal with a disease that closely resembles a human disease. The significance of disease model animals in human disease research stems from the physiological and genetic similarities between humans and animals. Biomedical disease model animals can provide research materials for understanding the various causes, pathogenesis, and diagnosis of diseases. Therefore, through the study of disease model animals, genes related to diseases can be identified, the interactions between genes can be understood, and the potential for practical application can be determined through actual efficacy and toxicity testing of developed new drug candidates.

[0059] According to one specific example of the present invention, the knockout may be caused by a gRNA in which B2M gene exon 2 consists of the base sequence of SEQ ID NO: 2.

[0060] According to one specific example of the present invention, the animal model may further have a deficiency in the H2-D1 and H2-Ab1 genes.

[0061] According to one specific example of the present invention, the animal model may be a mouse, and may be a NIG mouse, which is a NOD / Scid mouse lacking the IL2Rg gene.

[0062] In the immunodeficiency animal model of the present invention, parts overlapping with the above-described contents may be used with the same meaning as the above-described meaning.

[0063] Hereinafter, the present invention will be described in more detail through the following examples.

[0064] However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0065] <Example 1> Design and synthesis of guide RNA for gene correction using CRISPR / Cas9

[0066] <1-1> B2M gene analysis

[0067] The base sequence of the B2M (beta 2 microglobulin) gene belonging to MHC class I was analyzed using Ensemble Genome Browser, UCSC genome browser, and National Center for Biotechnology Information (Table 1).

[0068]

[0069]

[0070] <1-2> Design of a single guide RNA (sgRNA) to target the B2M gene

[0071] To block transcription of the B2M gene belonging to MHC class I and prevent protein production, the sequence of exon 2 was entered into the ZiFiT Targeter website. Among the target sequences obtained, sequences with a low probability of off-targeting due to two or more mismatch bases were selected. The selected sequences are shown in Table 2 below.

[0072]

[0073] <1-3> Production of sgRNA vector for CRISPR / Cas9

[0074] The oligonucleotide sequences for each direction were obtained from the ZiFiT Targeter website to produce bidirectional DNA oligonucleotides. The oligonucleotides were heated to 90°C for 4 minutes to ensure complementary binding of each direction, and then slowly cooled. Then, to produce an expression vector for sgRNA containing the target sequences, the sgRNA was cloned into the pT7-gRNA vector cut with the Esp3I (BsmB1) restriction enzyme, thereby producing an sgRNA synthesis vector that expresses the sgRNA containing the target sequence under the T7 promoter.

[0075] <1-4> Synthesis and purification of sgRNA for microinjection

[0076] The pT7-gRNA vector containing the target guide RNA sequence was linearized and purified by digestion with BamHI restriction enzyme. A short sgRNA of approximately 100 bp was synthesized through an in vitro transcription reaction using the MEGA Shortscript T7 Kit (Ambion). After removing template DNA by DNase I treatment, the synthesized sgRNA was purified by ethanol precipitation.

[0077] <Example 2> Production of B2M KO founder mice by genetic editing

[0078] <2-1> Microinjection of gRNAs / Cas9 into fertilized eggs and transplantation into surrogate mothers

[0079] Sperm collected from NIG males and oocytes harvested from NIG females were used for in vitro fertilization (IVF) in HTF medium for 8 hours, and the fertilized eggs in HTF medium were transferred to M16 medium. After microinjecting sgRNAs targeting the B2M gene of MHC class I and Cas9 protein into the cytoplasm of the fertilized eggs, they were cultured overnight in a 5% CO2, 37°C incubator. The cultured 2-cell stage fertilized eggs were transferred to the oviducts of ICR surrogate mothers, producing 12 offspring (Table 3).

[0080]

[0081] <2-2> Production and genotyping of G0 (founder) mice

[0082] To analyze the G0 mouse genotype, genomic DNA was extracted from the toes. First, the toes were activated at 55℃ for 12 hours by adding 400 µl of lysis buffer (1 M TRIS, 0.5 M EDTA, 5 M NaCl, 20% SDS) and Protein Kinase, and DNA was extracted using the phenol / chloroform method. The extracted DNA was used as a template to amplify the B2M gene, an MHC class I gene, through PCR (conditions: 94℃ for 5 minutes, 60℃ for 30 seconds, 72℃ for 40 seconds - 35 cycles). The amplified gene was re-annealed with PCR, and then T7E1 (endonuclease) was added and activated at 37℃ for 30 minutes. The result was then electrophoresed on a 2.5% gel and confirmed by photographing.

[0083] As a result, as shown in Fig. 1, the DNA from which the bands were cut could be predicted to be mutants (2, 5, 10, 11). Through this, mice predicted to be mutants were secured and sequencing analysis was performed.

[0084] <2-3> G0 mouse sequencing analysis

[0085] Among 11 G0 mice, those predicted to be mutants were selected using the T7E1 assay, and their genotypes were confirmed by sequencing PCR products. For sequencing, the PCR-derived DNA was extracted and sent to a sequencing company (Macrogen), where the results were analyzed.

[0086] As a result, it was confirmed that both genes were deleted in G0 #5 and #11 mice. However, G0 #2 mouse died due to cannibalism of the surrogate mother two weeks after birth. In addition, G0 #10 mouse was analyzed to be more than 90% identical to the wild type B2M protein and was therefore not used for G1 production. Using G0 #11 and #5 mice, IVF was performed with wild type (NIG) female or male mice to produce germline transmitted G1 mice.

[0087] <Example 3> Production of homozygous B2M-NIG mice with gonadal-transferred genotypes

[0088] <3-1> G1 Production and Genotyping

[0089] As a result of IVF for G1 production, in case of G0 #11, the surrogate mother did not produce offspring, and G0 #5 produced G1 by the surrogate mother. Genomic DNA extracted from tissues (toes) of 13 G1 mice of line G0 #5 was subjected to PCR, and DNA was extracted. The B2M gene was amplified through PCR (conditions: 94℃ 5 min, 60℃ 30 sec, 72℃ 40 sec - 35 cycles) using the extracted DNA as a template. The amplified gene was re-annealed and PCR was performed, and then T7E1 (endonuclease) was added and activated at 37℃ for 30 min. Then, it was electrophoresed on a 2.5% gel and confirmed by a photograph.

[0090] As a result, as shown in Fig. 2, the DNA from which the band was cut could be predicted to be a mutant (Red). As a result of the T7E1 result, mice predicted to be mutants were secured and sequencing analysis was performed. As a result of sequencing, a B2M gene deletion (-10 bp) of the same genotype as G0 was confirmed in two G1 mice (G1 #8♂, 13♂). Using G1 mice with confirmed gonadal transition, IVF was performed with NIG female mice to produce G2 mice.

[0091] <3-2> G2 Production and Genotyping

[0092] IVF for G2 production resulted in 45 mice. Genomic DNA was extracted from tissues (toes) of 45 G2 mice, and then subjected to PCR to extract the B2M gene. The extracted DNA was used as a template to amplify the B2M gene. Sequencing analysis was performed using the amplified gene.

[0093] Sequencing results confirmed B2M gene deletion (-10 bp) in 10 G2 mice (G1 #2♂, 6♂, 7♂, 9♂, 14♂, 16♂, 26♂, 38♀, 43♂). To produce homozygous (Homo) KO mice, crosses were performed using G2 hetero #38♀ and #43♂ mice.

[0094] <3-3> B2M Homo KO G3 production and genotyping analysis

[0095] Four mice were produced as a result of crossbreeding for G3 production. Genomic DNA was extracted from tissues (toes) of the four G3 mice, subjected to PCR, and the DNA was sequenced and analyzed. As a result, one G3 mouse (G1 #1) was confirmed to be a B2M homo KO. A line was established using B2M homo KO-NIG mice, and mouse phenotypic analysis was performed.

[0096] <Example 4> B2M expression assay in spleen and blood cells of B2M KO-NIG mice

[0097] <4-1> Gene expression assay at the RNA level

[0098] To extract RNA from the spleen tissues of homozygous B2M KO-NIG, C57BL / 6 (control), and NIG (control) mice, 1 ml of TRIzol (Molecular Research Center) was added to the extracted spleen tissues and ground with a grinder. 200 μl of chloroform was added, mixed, and centrifuged at 4°C and 13,000 rpm for 20 minutes. After centrifugation, 500 μl of the supernatant was transferred to a new tube, 500 μl of isopropanol was added, mixed, and centrifuged at 4°C and 13,000 rpm for 15 minutes. The supernatant was discarded. After washing once with 70% ethanol, the supernatant was discarded, and only the RNA was dissolved in water for use. cDNA was synthesized from the extracted RNA using a first-strand cDNA synthesis kit (Fermentas). Real-time qRT-PCR was performed using Exicycler™96 Real-Time Quantitative Thermal Block and SYBR Premix Ex Taq (Takara).

[0099] As a result, as shown in Fig. 3, compared to the B2M gene expression level of wild type mice (C57BL / 6), the B2M KO homozygous mice were confirmed to be at the background level.

[0100] <4-2> Protein expression assay

[0101] Spleens of homozygous B2M KO-NIG, C57BL / 6 (control), and NIG (control) mice were ground to isolate cells. Small hepatocytes were passed through a 40 μm cell strainer, washed with RPMI, and dissociated into single cells. Red blood cell lysis buffer (1 ml) was added to remove red blood cells. The cell suspension was incubated at room temperature for 10 minutes, added to FACS buffer (7 ml), centrifuged at 1,200 rpm for 5 minutes, and the supernatant was aspirated. The above process (FACS buffer) was repeated twice, and FACS buffer (200 μl) was added to the final sample. The extracted cells were stained at 4°C for 20 minutes. FACS buffer (1 ml) was added to the stained cells, centrifuged at 1,200 rpm for 5 minutes, and the supernatant was aspirated. Fixation buffer (200 μl) was added to the final suspension cells and suspended. B2M protein was measured using a FACS instrument with the prepared cells.

[0102] As a result, as shown in Fig. 4, NIG mice had reduced B2M compared to C57BL / 6. In B2M KO mice, B2M protein was barely detectable compared to C57BL / 6 and NIG mice.

[0103] Through the above results, it was confirmed that B2M KO-NIG mice were developed in which B2M was knocked out (KO).

[0104] <Example 5> Confirmation of survival rate due to reduction in GvHD by PBMC transplantation in B2M-NIG mice

[0105] <5-1> Survival rate of NIG and B2M-NIG mice transplanted with human PBMCs

[0106] Experiments were performed to analyze the survival rate by xenogenic-GvHD in NIG and B2M-NIG mice.

[0107] Specifically, on day 0, human PBMCs (1 X 10 7 ) were transplanted. Busulfan was injected intraperitoneally into mice at a concentration of 25 mg / kg before transplantation of human PBMCs. Survival was measured for 6 weeks after human PBMC transplantation.

[0108] As a result, as shown in Figs. 5 and 6, NIG mice showed rapid weight loss after PBMC transplantation and died of xenogenic graft-versus-host disease (GvHD) within 2 to 3 weeks, but B2M-NIG mice maintained their weight or showed a slight decrease and showed a survival rate of more than 6 weeks after PBMC transplantation.

[0109] In conclusion, we confirmed that survival rate increased by reducing xenogenic-GvHD in B2M-NIG mice.

[0110] <5-2> Investigation of human immune cell expression in the B2M-NIG model with human PBMC transplantation

[0111] The level of engraftment of human T cells was determined in B2M-NIG mice transplanted with human PBMC (hPBMC-B2M-NIG).

[0112] As a result, as shown in Fig. 7, more than 25% of human CD3+ T cells were detected in the blood of hPBMC-B2M-NIG mice at 5-6 weeks.

[0113] In conclusion, we were able to confirm that B2M-NIG mice effectively induced engraftment of human immune cells, especially T cells, by human PBMC transplantation.

[0114] <Example 6> Development of B2M / dKO-NIG mice

[0115] In Example 3, the B2M-NIG produced was crossed with dKO-NIG mice (GH Bio) lacking both MHC class I (B2M, H2-D1) and II (H2-Ab1) genes, to produce eight B2M / dKO-NIG offspring with MHC triple KO (B2M, H2-D1, H2-Ab1). Genomic DNA extracted from tissues (toes) of eight mice was subjected to PCR, and then DNA was extracted. Using the extracted DNA as a template, each H2-D1, H2-Ab1, and B2M gene was amplified through PCR (conditions: 94°C for 5 minutes, 60°C for 30 seconds, 72°C for 40 seconds - 35 cycles). Sequencing analysis was performed using the amplified genes. Sequencing results confirmed that five mice (G1 #1, 3, 4, 6, and 7) had the same genotype as MHC dKO-NIG and B2M-NIG mice, with 10 bp deletion (B2M), 16 bp deletion (H2-D1), and 4 bp deletion (H2-Ab1).

[0116] <Example 7> Test for expression of B2M, H2-D, and IA proteins in the spleen of B2M / dKO-NIG mice

[0117] Spleens of B2M / dKO-NIG, C57BL / 6 (control), and NOD / SCID (control) mice were ground to isolate cells. Small hepatocytes were passed through a 40 μm cell strainer, washed with RPMI, and dissociated into single cells. Red blood cell lysis buffer (1 ml) was added to remove red blood cells. The cell suspension was incubated at room temperature for 10 minutes, added to FACS buffer (7 ml), centrifuged at 1,200 rpm for 5 minutes, and the supernatant was aspirated. The above process (FACS buffer) was repeated twice, and FACS buffer (200 μl) was added to the final sample. The extracted cells were stained at 4°C for 20 minutes. FACS buffer (1 ml) was added to the stained cells, centrifuged at 1,200 rpm for 5 minutes, and the supernatant was aspirated. Fixation buffer (200 μl) was added to the final suspension cells and suspended. The prepared cells were used for measuring B2M, H2-D (expressed by the H2-D1 gene), and IA proteins (expressed by the H2-Ab1 gene) using a FACS instrument.

[0118] As a result, as shown in Fig. 8, it was confirmed that B2M, H2-D, and IA proteins were not detected in B2M / dKO-NIG mice compared to control mice.

[0119] <Example 8> Survival rate investigation according to GvHD reduction by PBMC transplantation in B2M / dKO-NIG mice

[0120] <8-1> Survival study of B2M / dKO-NIG mice transplanted with human PBMCs

[0121] To investigate the increased survival rate by attenuating GvHD response in B2M / dKO-NIG mice transplanted with human PBMCs, human PBMCs were transplanted and survival was monitored for a long period of time. To analyze the survival rate by xenogenic GvHD in NIG, B2M-NIG, and B2M / dKO-NIG mice, human PBMCs (5 × 106) were transplanted intravenously on day 0. Survival was measured for 100 days after human PBMC transplantation.

[0122] As a result, as shown in Figures 9 and 10, NIG mice showed weight loss 4 weeks after PBMC transplantation, and more than 70% of them died from xenogenic graft-versus-host disease (GvHD). B2M-NIG mice maintained or slightly decreased their body weights, and showed a 30% decrease in survival rate from 7 weeks after PBMC transplantation. On the other hand, all B2M / dKO-NIG mice survived for 100 days.

[0123] Additionally, GvHD response was monitored 6 weeks after PBMC transplantation.

[0124] As a result, as shown in Fig. 11, it was confirmed that GvHD clinical symptoms such as hunched posture and hair loss appeared in NIG and B2M-NIG mice. In conclusion, it was confirmed that the survival rate was significantly increased due to the reduction of xenogenic-GvHD in B2M / dKO-NIG mice.

[0125] <8-2> Survival of B2M / dKO-NIG mice under GvHD-induced conditions by co-administration of busulfan and human PBMCs

[0126] To compare the survival rates between NIG, B2M-NIG, and B2M / dKO-NIG mice under conditions of GvHD induction by co-administration of busulfan and human PBMCs, human PBMCs (5 × 106) were transplanted intravenously on day 0. Mice were intraperitoneally injected with busulfan at a concentration of 25 mg / kg 48 h and 24 h before human PBMC transplantation, respectively. Survival was measured for 21 days after human PBMC transplantation.

[0127] As a result, as shown in Figs. 12 and 13, NIG mice showed an increase in body weight after PBMC transplantation (day 0) and then showed a decrease in body weight from day 10, and more than 50% of the mice died from xenogenic graft-versus-host disease (GvHD) within 3 weeks. In the case of B2M-NIG mice, the body weight increased after PBMC transplantation (day 0) and then showed a decrease in body weight from day 13, and showed a 65% decrease in survival rate within 3 weeks of PBMC transplantation. On the other hand, in the case of B2M / dKO-NIG mice, there was no significant change in body weight, and more than 80% of the mice survived for 3 weeks after PBMC transplantation.

[0128] <8-3> Investigation of the human immune cell engraftment efficiency in B2M / dKO-NIG mice transplanted with human PBMC under conditions of GvHD induction by combined administration of busulfan and human PBMC

[0129] To compare the engraftment levels of human immune cells between NIG, B2M-NIG, and B2M / dKO-NIG mice under conditions of GvHD induction by co-administration of busulfan and human PBMCs, mice were transplanted intravenously with human PBMCs (5 × 106) on day 0. Mice were injected intraperitoneally with busulfan at a concentration of 25 mg / kg 48 and 24 hours before human PBMC transplantation, respectively. Analyses were performed 2, 3, and 4 weeks after PBMC transplantation.

[0130] As a result, as shown in Fig. 14, higher levels of human CD45+ cells were detected in the blood of B2M / dKO-NIG mice compared to the NIG and B2M-NIG mouse groups. In addition, as shown in Fig. 15, a higher expression of human CD3+ T cells of more than 10% was confirmed in B2M / dKO-NIG mice compared to NIG mice. As a result of measuring the organ weights of all mice used in the analysis, no significant differences were confirmed between the groups, as shown in Fig. 16.

[0131] In conclusion, we confirmed that B2M / dKO-NIG mice showed high survival rates by reducing GvHD induction by PBMC transplantation and induced effective engraftment of human CD45+ immune cells, especially CD3+ T cells.

Claims

1. A nucleotide sequence encoding a gRNA consisting of a base sequence of SEQ ID NO: 2 that hybridizes to DNA encoding the B2M (beta 2 microglobulin) gene; a nucleotide sequence encoding the Cas9 protein; and A recombinant expression vector comprising a promoter operably linked to the above nucleotide sequence.

2. A transformed cell line for producing an immunodeficient animal model, into which the recombinant expression vector of paragraph 1 has been introduced.

3. In paragraph 2, The above cell line is a transformed cell line characterized by a deletion of the IL2Rg (interleukin 2 receptor gamma) gene.

4. A method for producing an immunodeficient animal model, comprising the step of transplanting the transformed cell line of paragraph 2 or 3 into the oviduct of a surrogate mother, which is an animal other than a human.

5. In paragraph 4, A method for producing an immunodeficiency animal model, characterized in that it further comprises a step of crossing the animal model with an animal other than a human that has both the H2-D1 and H2-Ab genes deficient.

6. In paragraph 3, A method for producing an immunodeficiency animal model, characterized in that the animal is a mouse.

7. B2M (beta 2 microglobulin) gene knockout, immunodeficient animal model.

8. In paragraph 7, The above animal model is an immunodeficiency animal model characterized in that B2M gene exon 2 is knocked out by gRNA consisting of the base sequence of sequence number 2.

9. In paragraph 7, The above animal model is an immunodeficiency animal model characterized in that it is a NIG mouse, which is a NOD / Scid mouse with a defective IL2Rg gene.

10. In paragraph 7, The above animal model is an immunodeficiency animal model characterized by further deletion of the H2-D1 and H2-Ab1 genes.

11. In paragraph 7, An immunodeficiency animal model, characterized in that the animal is a mouse.

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