A method for producing a transformed cloned pig for xenotransfusion and xenotransplantation, characterized by a deletion of the porcine GGTA1, CMAH, iGb3s, and β4GalNT2 genes and a knock-in of the human CD59 gene.

Abstract

The present invention relates to a transformed cloned pig for xenotransfusion and xenotransplantation, characterized by a deficiency in the porcine GGTA1, CMAH, iGb3s, and β4GalNT2 genes and a knock-in of the human CD59 gene, and a method for producing the same. By inducing human CD59 gene expression in the transformed pig, which has improved conventional antigen-antibody-mediated immune rejection, complement-mediated immune rejection is reduced, and human CD59 gene expression is confirmed in red blood cells, making it useful for xenotransfusion and xenotransplantation.

Description

Technical Field

[0001] The present invention relates to a transgenic cloned pig for xenotransfusion and xenotransplantation, in which genes of porcine GGTA1 (Alpha 1,3-galactosyltransferase), CMAH (CMP-N-acetylneuraminic acid hydroxylase), iGb3s (Isogloboside 3 synthease) and β4GalNT2 (Beta-1,4-N-Acetyl-Galactosaminyl Transferase 2) are deficient and the human CD59 gene is knocked in, and a method for producing the same.

Background Art

[0002] The number of organ transplant candidates in Korea has reached approximately 40,000, but the number of organ donors is extremely insufficient compared to the candidates, and the average waiting period until transplantation is said to be 5 years and 4 months. Despite continuous improvement of the organ donation system, the gap between supply and demand in human-to-human allogeneic organ transplantation not only in Korea but also globally has been widening year by year. As a result, illegal organ trafficking is rampant, and social problems have been raised.

[0003] Such organ transplant supply problems can be solved through pig-to-human xenotransplantation, where pig organs completely replace human organs. Among the animals that donate xenotransplants, pigs have a high degree of morphological and genetic similarity to humans, and unlike primates, they pose fewer risks of zoonotic diseases and ethical issues. They also have the advantage of being highly prolific, which can adequately overcome the shortage of organs, and they are widely used as a non-clinical model, leading to much basic research. Furthermore, because they are also used for food, there is relatively less resistance to using them compared to other experimental animals. In particular, the Yucatan miniature pig is phenotypic, anatomically, and physiologically similar to humans, and its organ sizes are close to those of humans (heart 94%, liver 66%, pancreas 81%, kidney 91%), making it the optimal pig breed for xenotransplantation compared to other breeds.

[0004] In December 2021, the U.S. Food and Drug Administration (FDA) granted emergency authorization for therapeutic xenotransplantation for life-threatening patients for experimental purposes. On January 11, 2022, a medical team at the University of Maryland performed the world's first pig heart transplant, with the consent of a terminally ill patient and their family. Furthermore, on September 20, 2023, a second transplant was performed for a patient with end-stage heart failure. Thus, xenotransplantation between pigs and humans has progressed to the point of conducting clinical trials on living patients, but due to differences between species, stronger immune rejection occurs than in allotransplantation between humans, making the development of perfectly humanized pigs essential.

[0005] As prior art, Korean Patent No. 10-2040203 discloses that after producing transformed cloned pigs lacking the GGTA1 (α1,3-galactosyltransferase), CMAH (CMP-N-acetylneuraminic acid hydroxylase), iGb3s (Isoglobotrihexosylceramide synthase), and β4GalNT2 (Beta-1,4-N-Acetyl-Galactosaminyl Transferase 2) genes, in vitro human serum reactions were conducted, confirming a significant decrease in IgG and IgM binding, suggesting the possibility of overcoming antigen-antibody-mediated rejection reactions that occur in xenotransplantation. However, in addition to controlling hyperacute and acute immune rejection reactions by regulating antigen-antibody-mediated immune rejection reactions, when pig organs, tissues, cells, or blood are transplanted into humans, immune rejection reactions due to human complement activity will occur.

[0006] Therefore, in order to reduce immune rejection reactions occurring in xenotransfusions, the inventors induced the expression of the human CD59 gene using a porcine endogenous promoter, and completed the present invention by confirming that the developed transformed pigs reduced antigen-antibody-mediated immune rejection and complement-mediated immune rejection. [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a knock-in vector for the production of transformed cloned pigs.

[0008] Another object of the present invention is to provide a transformed cell line produced by transforming somatic cells with the knock-in vector, and a transformed cloned pig produced by nuclear transplantation of the transformed cell line.

[0009] Another object of the present invention is to provide a method for producing transformed cloned pigs and cloned pigs using the same.

[0010] Another object of the present invention is to provide a method for producing xenotransplantable organs, which includes raising the cloned pigs, extracting organs, and producing organs using their germ cells or somatic cells. [Means for solving the problem]

[0011] To achieve the above objective, the present invention provides a knock-in vector for producing transformed cloned pigs, comprising a human CD59 gene represented by Sequence ID No. 1 being matched to the porcine GGTA1 gene.

[0012] Furthermore, the present invention provides a transformed cell line produced by transforming somatic cells with the aforementioned knock-in vector.

[0013] Furthermore, the present invention provides a transformed cloned pig produced by nuclear transplantation of the transformed cell line.

[0014] Furthermore, the present invention provides a method for producing a transformed cloned pig, comprising the steps of: producing the transformed cell line; transplanting the cell line into an enucleated oocyte to form a nuclear transplant egg; and transplanting the nuclear transplant egg into the fallopian tube of a surrogate mother.

[0015] Furthermore, the present invention provides transformed cloned pigs produced by the above method.

[0016] Furthermore, the present invention provides a method for producing xenoorgans for transplantation, which includes raising the cloned pigs, extracting organs, and producing organs using their germ cells or somatic cells. [Effects of the Invention]

[0017] This invention relates to a transformed cloned pig for xenotransfusion and xenotransplantation, characterized by a deficiency in the porcine GGTA1, CMAH, iGb3s, and β4GalNT2 genes and a knock-in of the human CD59 gene. By inducing human CD59 gene expression in the transformed pig, which has improved conventional antigen-antibody-mediated immune rejection, complement-mediated immune rejection is reduced, and human CD59 gene expression is confirmed in red blood cells, making it useful for xenotransfusion and xenotransplantation. [Brief explanation of the drawing]

[0018] [Figure 1] This figure shows a schematic diagram of the acquisition of base cell lines lacking the GGTA1, CMAH, iGb3s, and β4GalNT2 genes used in development. [Figure 2] Figure 2a: A schematic diagram of a human CD59 knock-in vector. Figure 2b: A diagram showing a human CD59 expression strategy via knock-in. [Figure 3] This figure shows the results of separating human CD59-positive cells by sorting after transduction. [Figure 4] This figure shows the positions of the primers used in the invention and whether or not a human CD59 knock-in vector was introduced into the transformed cell line. [Figure 5] This figure shows the results of confirming protein expression through FACS analysis after immunofluorescence staining of selected transformed cell lines #21, #30, #35, and #39. [Figure 6] This is a photograph of a transformed cloned pig produced via somatic cell cloning using transformed cell line #21. [Figure 7] This figure shows the human CD59 gene analysis of transformed cloned pigs produced. [Figure 8] This figure shows the results of FACS analysis of major cell types in the transformed cloned pigs produced. [Figure 9]FIG. 9a: A diagram showing the results of a cytotoxicity test against human serum using the produced transformed cloned pig depleted blood mononuclear cells (PBMC). FIG. 9b: A diagram showing the results of a cytotoxicity test against monkey serum using the produced transformed cloned pig depleted blood mononuclear cells (PBMC). [Figure 10] FIG. 10a: A diagram showing the results of a cytotoxicity test against human serum using the produced transformed cloned pig red blood cells (RBC). FIG. 10b: A diagram showing the results of a cytotoxicity test against monkey serum using the produced transformed cloned pig red blood cells (RBC).

BEST MODE FOR CARRYING OUT THE INVENTION

[0019] Hereinafter, the present invention will be described in detail.

[0020] The present invention provides a knock-in vector for producing a transformed cloned pig, which includes that the human CD59 gene represented by SEQ ID NO: 1 is integrated into the pig GGTA1 gene.

[0021] In the present invention, the human CD59 (Human Membrane Attack complex-inhibitory protein; MAC-IP) gene is a complement activity inhibitory gene. CD59 binds to the α-chain of C8 and the b domain of C9 in the C5b-8 complex among the complement activation mechanisms, prevents the insertion and polymerization of C9, and ultimately prevents the formation of the Membrane Attack complex (MAC). The nucleotide sequence of the human CD59 gene is represented by Genebank number NM_203329.3, and the nucleotide sequence of the pig CD59 gene is represented by Genebank number AH010595.2.

[0022] The term "Knock-in" in the present invention means inserting a foreign nucleotide sequence that does not originally exist in that living body into the genome of that living body or the DNA nucleotide sequence derived from that living body using genetic recombination technology.

[0023] In the present invention, the knock-in vector comprises a first region consisting of a left arm containing a partial sequence of exon 3 or exon 4 of the porcine GGTA1 gene, a second region consisting of a region encoding the human CD59 gene and a right arm containing a partial sequence of exon 4 or exon 5 of the porcine GGTA1 gene.

[0024] In the present invention, the knock-in vector has the human CD59 CDS sequence (SEQ ID NO: 2) inserted between the left arm sequence from sequence 261535947 to sequence 261536969 and the right arm sequence from sequence 261535101 to sequence 261535922 on chromosome 1.

[0025] In this invention, "left arm" and "right arm" refer to regions where homologous recombination occurs.

[0026] In this invention, "homologous recombination" refers to gene recombination that occurs through exchange at homologous gene loci, and is used to create transformed animals that have alleles that have lost their function.

[0027] In one specific example of the present invention, the left arm comprises a portion of exon 3 or exon 4 of the porcine GGTA1 gene. According to one specific example of the present invention, the size of the left arm may be 1,023 bp and consist of the nucleotide sequence represented by Sequence ID No. 3.

[0028] In one specific example of the present invention, the right arm comprises a portion of exon 4 or exon 5 of the porcine GGTA1 gene. According to one specific example of the present invention, the size of the right arm may be 822 bp and consist of the nucleotide sequence represented by Sequence ID No. 4.

[0029] According to one specific example of the present invention, the knock-in vector may consist of the base sequence of Sequence ID No. 5.

[0030] In the present invention, "vector" means a gene construct comprising a gene sequence operably linked to a suitable control sequence so as to enable the expression of a target gene in a suitable host, wherein the control sequence may include a promoter capable of initiating transcription, an optional operator sequence for controlling such transcription, and sequences for controlling the termination of transcription and decoding. The vector of the present invention is not particularly limited, and any vector known in the art may be used, as long as it is clonal in a cell, for example, a plasmid, cosmid, phage particle, or viral vector.

[0031] The genetically modified vector for knock-in of the present invention can preferably be represented by the vector map disclosed in Figure 2a.

[0032] Furthermore, the present invention provides a transformed cell line produced by transforming somatic cells with the aforementioned knock-in vector.

[0033] In this invention, "transformation" means introducing DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or upon completion of integration into the chromosome. Transformation includes any method of introducing nucleic acid molecules into an organism, cell, tissue, or organ, and can be performed by selecting and executing a standard technique appropriate to the host cell, as is known in the art, including, but 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. The transformation of eukaryotic cells by plasmids or non-plasmidal naked DNA is sometimes referred to as "transfection" to distinguish it from transformation in the sense of cell tumorigenesis, but in this invention, the term is used in the same sense.

[0034] In the present invention, the somatic cells are preferably fibroblasts, and more preferably porcine fibroblasts.

[0035] In the present invention, the somatic cells may be somatic cells in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes have been knocked out.

[0036] The GGTA1 gene is responsible for the biosynthesis of α-Gal, the CMAH gene is responsible for the biosynthesis of Neu5Gc, the iGb3s gene synthesizes the glycosphingolipid iGb3, and the β4GalNT2 gene is a gene that generates sugar chains, producing GalNAcβ1-4, Galβ1-4GlcNAcβ1-3Gal, and Sd(a) (Sid blood group; CAD or CT) antigens. Transformed cloned pigs lacking the four aforementioned carbohydrate-derived heterologous antigens are disclosed in Registered Patent No. 10-2040203 of the Republic of Korea.

[0037] In one specific example of the present invention, a transformed cell line was produced in which the human CD59 gene was knocked into fibroblasts derived from transformed pigs from which four genes that suppress antigen-antibody-mediated immune rejection—GGTA1, CMAH, iGb3s, and β4GalNT2—were removed, in order to suppress complement-mediated immune rejection that occurs during xenotransplantation. The transformed cells according to the present invention may also be cells of accession number KCLRF-BP-00527, deposited with the Korea Cell Line Research Foundation (KCLRF) on November 24, 2023.

[0038] Furthermore, the present invention provides a transformed cloned pig produced by nuclear transplantation of the transformed cell line.

[0039] Furthermore, the present invention provides a method for producing a transformed cloned pig, comprising the steps of: producing the transformed cell line; transplanting the cell line into an enucleated oocyte to form a nuclear transplant egg; and transplanting the nuclear transplant egg into the fallopian tube of a surrogate mother.

[0040] In this invention, "nuclear transfer" refers to a genetic manipulation technique that artificially attaches the nuclear DNA of another cell to a cell without a nucleus to make it possess the same traits, and methods known in this field can be used.

[0041] In this invention, "nuclear-transferred egg" means an egg cell into which donor nuclear source cells have been introduced or fused.

[0042] In this invention, "enucleated oocyte" means an oocyte from which the nucleus has been removed.

[0043] Furthermore, the present invention provides transformed cloned pigs produced by the above method.

[0044] Furthermore, the present invention provides a method for producing xenoorgans for transplantation, which includes raising the cloned pigs, extracting organs, and producing organs using their germ cells or somatic cells.

[0045] The aforementioned organs can be extracted by conventional surgery after raising the donor cloned animal, taking into consideration the sex, age, weight, height, etc. of the recipient, and adjusting the rearing period. After extraction, they can be immediately transplanted into the recipient or rapidly stored under refrigeration. Furthermore, germ cells such as sperm or eggs can be extracted from the cloned animal, artificially or naturally inseminated to obtain offspring, and these offspring can be used for organ transplantation or the production of other by-products for organ transplantation. Similarly, somatic cells can be extracted from the cloned animal and used for organ transplantation or the production of other by-products as described above.

[0046] Transgenic pigs, in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes of the present invention are removed and the human CD59 gene is expressed at the GGTA1 locus, can overcome hyperacute and complement-mediated immune rejection reactions that occur in xenotransplantation. Therefore, transgenic cloned pigs according to the present invention can be usefully utilized as donor animals for interspecies organs and blood transfusions.

[0047] The present invention will be described in more detail below through examples. These examples are merely illustrative and should not be construed as limiting the scope of the invention to those with ordinary skill in the art. [Modes for carrying out the invention]

[0048] <Example 1> Development of GGTA1, CMAH, iGb3s and β4GalNT2 knockout transformed cell lines <1-1> Production of transformed pigs Transformed pigs were produced to construct quadruple knockout (QKO) transformed cell lines for the porcine GGTA1, CMAH, iGb3s, and β4GalNT2 genes.

[0049] Specifically, QKO individuals F0, developed under Korean patent application No. 10-2018-0034466, were crossed with wild-type (WT) individuals to produce F1 individuals with the QKO heterozygous genetic trait. These F1 individuals were reared until sexual maturity (8 months of age) and crossed with QKO F0 individuals to produce F2 individuals with the QKO homozygous genetic trait. The resulting individuals were subjected to PCR using the primers shown in Table 1 for sequencing analysis of GGTA1, CMAH, iGb3s, and β4GalNT2. The obtained PCR products were sent to Solgent for sequencing analysis.

[0050] [Table 1]

[0051] As a result, as shown in Table 2, it was confirmed that in the final F2 individuals, two loci of the GGTA1, CMAH, and β4GalNT2 genes were deleted, and one locus of iGb3s was deleted.

[0052] [Table 2] <1-2> Construction of transformed cell lines Ear fibroblasts were isolated and cultured from the transformed pigs, and GGTA1, CMAH, iGb3s, and β4GalNT2 gene knockout QKO transformed cell lines were constructed.

[0053] More specifically, individual ears were disinfected with 70% ethanol, and ear tissue samples measuring 1 cm x 1 cm were biopsied. These samples were then stored in DPBS containing the antibiotic penicillin-streptomycin and transported to the laboratory. The tissue was washed three times with DPBS, the epidermis was removed, and the samples were finely chopped and attached to 6-well multiplates. The cells were cultured for 7 days in DMEM (Lonza, Switzerland) containing 10% FBS and 1X Penicillin-streptomycin antibiotic to obtain ear tissue-derived fibroblasts (porcine ear fibroblasts; PEFs).

[0054] <1-2> Construction of transformed cell lines Ear fibroblasts were isolated and cultured from the transformed pigs, and GGTA1, CMAH, iGb3s, and β4GalNT2 gene knockout QKO transformed cell lines were constructed.

[0055] More specifically, individual ears were disinfected with 70% ethanol, and ear tissue samples measuring 1 cm x 1 cm were biopsied. These samples were then stored in DPBS containing the antibiotic penicillin-streptomycin and transported to the laboratory. The tissue was washed three times with DPBS, the epidermis was removed, and the samples were finely chopped and attached to 6-well multiplates. The cells were cultured for 7 days in DMEM (Lonza, Switzerland) containing 10% FBS and 1X Penicillin-streptomycin antibiotic to obtain ear tissue-derived fibroblasts (porcine ear fibroblasts; PEFs).

[0056] <Example 3> Construction of a transformed cell line in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes have been removed and the human CD59 gene has been knocked in. <3-1> Production of transformed cell lines in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes have been removed and the human CD59 gene has been knocked in. Experiments were conducted to produce transformed cell lines in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes were removed and the human CD59 gene was knocked in.

[0057] Specifically, the CD59 knock-in vector constructed in Example 2 was introduced into transformed pig-derived fibroblasts, from which the heterologous antigen obtained in Example 1 had been removed, using electroporation. More specifically, the cultured cells were washed with DPBS, treated with 0.25% trypsin-EDTA (Gibco), and harvested. After centrifugation at 1500 rpm for 3 minutes and washing with DPBS, the buffer and vector in the P3 Primary Cell 4D-Nucleofector kit (Amaxa, Germany) were mixed, and then electroshock was applied using the EN-150 protocol equipped with the 4D-Nucleofector system (amaxa). One week after introduction, the cells were immunostained with CD59 antibody to improve sorting efficiency, and only CD59-positive cells were sorted using FACS Aria III (BD bioscience, USA). The sorted cells were cultured as single-cell colonies, and then genetic analysis was performed on each colony. More specifically, genomic DNA was extracted from each transformed cell colony using the Dneasy Blood & Tissue Kit, and then PCR was performed using primers that included both the internal and external locations of the human CD59 knock-in vector. The resulting PCR products were loaded onto a 1% agarose gel and analyzed.

[0058] [Table 3]

[0059] As a result, as shown in Figure 4, we confirmed that the human CD59 knock-in vector was inserted into six colonies, #23, #26, #27, #28, #31, and #37, and that homologous recombination also occurred successfully.

[0060] <3-2> Analysis of protein expression in selected transformed cell lines Of the six colonies selected in Example 3-1, morphologically superior colonies #21, #30, #35, and #39 were subjected to cell immunostaining and FACS analysis for protein expression analysis.

[0061] Specifically, cell lines were washed with DPBS, treated with 0.25% trypsin-EDTA solution for 3 minutes, and then cells were obtained. Trypsin-EDTA was inactivated using fetal boivine serum (FBS), washed with DPBS, and then reacted with human CD59 antibody at a concentration of 1:100 for 1 hour. Then, the cells were washed three times with DPBS containing Tween-20, and reacted with a secondary antibody conjugated to FITC fluorescence at a concentration of 1:100 under refrigeration for 1 hour. Similarly, the cells were washed three times with DPBS containing Tween-20, fixed with 1% formalin, and analyzed using Cytoflex (Beckman Coulter, USA). As a positive control group, transformed cell lines expressing the human CD59 gene via the EF1α promoter were used for analysis.

[0062] As a result, as shown in Figure 5, it was confirmed that human CD59 protein was well expressed in all four colonies of candidate transformed cell lines #21, #30, #35, and #39.

[0063] The transformed cell line #21 was named QKO / hCD59 KI and deposited with the Korea Cell Line Research Foundation (KCLRF) on November 24, 2023, receiving accession number KCLRF-BP-00527.

[0064] <Example 4> Production of transformed pigs in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes have been removed and the human CD59 gene has been knocked in. <4-1> Preparation of oocytes To produce transformed pigs, oocytes were prepared first.

[0065] Specifically, after obtaining ovaries from immature cancerous pigs, they were transported to the laboratory in a 0.9% NaCl solution at 35°C. Cumulus-oocyte complexes (COCs) were selected from follicular fluid aspirated from immature oocytes (antral follicles) with a diameter of 2-6 mm using an 18-gauge needle fixed to a 10 mL disposable syringe. To select COCs, follicular fluid was washed with TL-HEPES culture medium, and COCs were sorted using a Petri dish. Approximately 70-80 COCs were then transferred to TCM 199 (Gibco, USA) containing 0.1 ml polyvinyl alcohol, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 IU / mL hCG (MSD Animal Health, USA), 0.5 μg / mL FSH (Sigma-Aldrich), 10 ng / mL epidermal growth factor (Sigma-Aldrich), 75 μg / mL penicillin G, and 50 μg / mL streptomycin, which were dispensed into a 4-well multi-dish (Nunc, Denmark) lined with mineral oil. After 22 hours of incubation, the cells were incubated for 42-44 hours in TCM 199 culture medium from which only FSH and hCG had been removed, under conditions of 5% CO2 and 39°C.

[0066] <4-2>Nuclear transfer Nuclear transfer was performed to produce transformed pigs.

[0067] Specifically, after 42-44 hours of culture, the COCs that had undergone in vitro maturation were transferred to a 4-well multi-dish containing a culture medium (Micromanipulation medium, MM) containing 0.3% BSA (Sigma-Aldrich), 0.6 mM NaHCO3, 2.9 mM HEPES, 30.0 mM NaCl, 0.05 g / mL penicillin G, and 0.06 g / mL streptomycin in TCM 199. The dish was then treated with 0.1% hyaluronidase, and the dish was gently pipetted approximately 70-80 times to separate oocytes from cumulus cells. Metaphase II oocytes with prominent first polar bodies were selected, and nuclear staining was carried out for 15 minutes in a culture medium containing 7.5 μg / mL cytochalasin B in MM (MM-CB) mixed with 5 μg / mL Hoechst 33342. The oocytes were then transferred to MM-CB. To remove the cell nucleus from oocytes lacking cumulus cells, the stained first polar body and nucleus were aspirated using a microglass pipette (enucleation). Prior to SCNT, transformed cell line #21 donor cells prepared in Example 3 were cultured for 3 days in DMEM medium containing 0.5% FBS for serum starvation. A single donor cell was positioned in the perivitelline space of the oocyte, in contact with the oocyte membrane. Inoculated oocytes were placed between two 0.2 mM platinum electrodes spaced 1 mM apart in a culture medium consisting of 0.3 mM M mannitol, 1.0 mM CaCl2H2O, 0.1 mM MgCl26H2O, and 0.5 mM HEPES. Fusion / activation was induced by adding two consecutive 1.1 kV / cm DC pulses over 30 μs (BTX, USA). The electrically stimulated recombinant oocytes were cultured in vitro in 30 μl drops of PZM-3 (Biol Reprod. 66:112-9, 2002) supplemented with 0.3% BSA coated with mineral oil, in groups of 15. On day 1 or 2 of culture, the NT embryos were surgically transplanted into the oviducts of sows on the first day of standing estrus. The pregnancy status was confirmed using an ultrasound scanner (Medison Co., Korea).

[0068] <Example 5> Production and validation of transformed pigs in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes were removed and the human CD59 gene was knocked in. <5-1> Production of transformed clonal pigs Figure 6 shows the external shape of the transformed pig produced in Example 4 described above.

[0069] Furthermore, in order to confirm the base sequence of the transformed pigs, fibroblasts were obtained from the offspring and their base sequences were analyzed. It was confirmed that the target gene regions, GGTA1, CMAH, iGb3s, and β4GalNT2 genes, were successfully removed from the fibroblasts of the transformed pigs produced in Example 4.

[0070] Furthermore, to confirm human CD59 gene knock-in, PCR amplification was performed using the 463F-464R primer and 463F-873R primer shown in Table 3, respectively. The resulting products were loaded onto a 1% agarose TAE gel and analyzed.

[0071] As a result, as shown in Figure 7, the introduction of the knock-in vector amplified both the wild-type and CD59 knock-in loci in the 463F-464R primer set (left), and it was confirmed that both loci were amplified by homologous recombination in the 463F-873R primer set (right).

[0072] <5-2> Verification of transformed cloned pigs Peripheral blood monocytic cells (PBMCs), red blood cells (RBCs), and splenocytes derived from the blood of transformed cloned pig #1, as verified in Example 5-1, were isolated, and the protein expression of the deleted gene and the knocked-in CD59 was analyzed by FACS.

[0073] Specifically, blood was collected from individuals using a syringe and diluted in DPBS at a 1:1 ratio. The diluted blood was added to ficoll-paque plus (GE healthcare) at a 1:1 (volume / volume) ratio and centrifuged at 500g for 40 minutes. After separating the intermediate buffy coat and the underlying erythrocyte layer, each was washed with DPBS and immunostained using antibodies for each gene. For spleen cells, the spleen was biopsyed in individuals and then transported to the laboratory in DPBS containing penicillin-streptomycin. The transported spleen samples were washed five times with DPBS, placed on a 40 μm cell strainer, and finely chopped with a syringe. The obtained spleen cells were de-stained using RBC lysis buffer (Sigma-aldrich) and immunostained using antibodies for each gene. Each cell group was analyzed using Cytoflex equipment along with an unstained control group.

[0074] As a result, as shown in Figure 8, it was confirmed that all three heterologous antigens, GGTA1, CMAH, and β4GalNT2, were not expressed in PBMCs, splenocytes, and RBCs derived from transformed cloned pigs, while the knocked-in CD59 protein was expressed.

[0075] <5-3> Verification of the function of transformed cloned pigs To evaluate the function of the transformed pigs produced in Example 5-1, cytotoxicity analyses were performed using human and primate serum.

[0076] Specifically, using the method described in Example 5-2 above, separated peripheral blood mononuclear cells and erythrocytes were treated in a 96-well multiplate with human and primate complement serum at concentrations of 3.125%, 6.25%, 12.5%, 25%, and 50% for 3 hours using ficoll paque plus solution. After removing the human complement serum from all wells, 100 μl of DPBS and 10 μl of CCK-8 (Dojindo, Japan) were added, and cell viability was measured.

[0077] As a result, as shown in Figures 9a and 9b, in the case of peripheral mononuclear cells, the survival rate was higher in pigs lacking the QKO gene than in normal pigs (WT) in all treatment groups. In particular, the transformed cloned pigs of the present invention, in which four heterologous antigens were knocked out and the human CD59 gene was knocked in, showed a remarkably high survival rate with human and primate complement serum. Furthermore, as shown in Figures 10a and 10b, a remarkably high survival rate with human and primate complement serum was also confirmed in the case of red blood cells.

[0078] Overall, it was confirmed that transformed pigs produced by the method according to the present invention, in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes were removed and the human CD59 gene was expressed at the GGTA1 locus, can overcome hyperacute and complement-mediated immune rejection reactions that occur in xenotransplantation. In particular, when the expression of the human CD59 gene was confirmed in red blood cells, it was found that these could be used as artificial blood for xenotransfusion.

[0079] Although specific parts of the present invention have been described in detail above, it will be clear to those with ordinary skill in the art that these specific descriptions are merely examples of preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents.

[0080] [Accession Number] Depository name: Korea Cell Line Research Foundation Accession number: KCLRF-BP-00527 Date of acceptance: 20231124 JPEG2026518707000005.jpg214149

[0081] JPEG2026518707000006.jpg186168

Claims

1. A knock-in vector for producing transformed cloned pigs, comprising the human CD59 gene represented by Sequence ID No. 1, which matches the porcine GGTA1 (Alpha 1,3-Galactic transferase) gene.

2. The knock-in vector comprises a first region consisting of a left arm containing a portion of exon 3 or exon 4 of the porcine GGTA1 (Alpha 1,3-Galactosiltransferase) gene; The region encoding the human CD59 gene; and The second region consists of a right arm containing a portion of exon 4 or exon 5 of the porcine GGTA1 gene; The knock-in vector according to claim 1, characterized by containing the following:

3. The knock-in vector according to claim 2, characterized in that the first region consists of the base sequence represented by Sequence ID No.

2.

4. The knock-in vector according to claim 2, characterized in that the second region consists of the base sequence represented by Sequence ID No.

3.

5. A transformed cell line produced by transforming somatic cells with the knock-in vector described in claim 1.

6. The transformed cell line according to claim 5, characterized in that the somatic cells have the genes GGTA1 (Alpha 1,3-Galactosyltransferase), CMAH (CMP-N-acetylneuraminic acid hydroxylase), iGb3s (Isogloboside 3 synthesise), and β4GalNT2 (Beta-1,4-N-Acetyl-GalactosaminylTransferase2) knocked out.

7. The transformed cell line according to claim 6, characterized in that the transformed cell line has accession number KCLRF-BP-00527.

8. A transformed cloned pig produced by nuclear transplantation of a transformed cell line according to any one of claims 5 to 7.

9. (1) The step of producing a transformed cell line according to any one of claims 5 to 7. (2) The step of transplanting the cell line into an enucleated oocyte to form a nuclear transplant egg; and (3) The step of implanting the nuclear transplant egg into the fallopian tube of the surrogate mother, Method for producing transformed cloned pigs.

10. A transformed clonal pig produced by the method described in claim 9.

11. A method for producing xenotransplantable organs, comprising raising cloned pigs according to claim 8, extracting organs, or producing organs using their germ cells or somatic cells.

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