Transgenic cloned pig for xenotransfusion and xenotransplantation with porcine GGTA1, CMAH, igb3s, and Β4galnt2 genes deleted and human CD59 gene knocked-in, and method for producing same

Abstract

The present invention relates to a transgenic cloned pig for xenotransfusion and xenotransplantation in which the porcine GGTA1, CMAH, iGb3s, and β4GalNT2 genes are deleted and the human CD59 gene is knocked in, and a method for producing same. By inducing expression of the human CD59 gene in a transgenic pig, which improves the existing antigen-antibody mediated immune rejection response, the complement mediated immune rejection response was reduced, and expression of the human CD59 gene was confirmed in red blood cells, and therefore, the pig may be effectively used for xenotransfusion and xenotransplantation.

Description

Transgenic cloned pigs for xenotransfusion and xenotransplantation, in which the porcine GGTA1, CMAH, IGB3S, and β4GALNT2 genes are deleted and the human CD59 gene is knocked in, and a method for producing the same

[0001] The present invention relates to a transgenic cloned pig for xenotransfusion and xenotransplantation in which the 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) genes are deleted and the human CD59 gene is knocked in, and a method for producing the same.

[0002] While there are approximately 40,000 people waiting for organ transplants in Korea, the number of donors is woefully inadequate, with the average waiting time for a transplant reaching five years and four months. Despite ongoing improvements to the organ donation system, the gap between supply and demand for human-to-human allogeneic organ transplants continues to grow, not only in Korea but also globally. This has fueled illegal organ trafficking, raising social concerns.

[0003] This organ transplant supply problem can be solved through pig-to-human xenotransplantation, where pig organs completely replace human ones. Among the sources of xenotransplantation, pigs are highly morphologically and genetically similar to humans, and unlike primates, they pose fewer zoonotic disease and ethical concerns. Furthermore, their high fecundity can sufficiently overcome supply shortages. Furthermore, they have been used extensively as a non-clinical model, contributing to extensive basic research. Furthermore, they are used for food, resulting in relatively less rejection than other laboratory animals. In particular, the Yucatan miniature pig is phenotypically and anatomically similar to humans, and its organ sizes—94% of the heart, 66% of the liver, 81% of the pancreas, and 91% of the kidney—have been selected as the optimal pig species for xenotransplantation compared to other species.

[0004] In December 2021, the U.S. Food and Drug Administration (FDA) granted emergency approval for experimental therapeutic xenotransplantation in patients whose lives were at risk. On January 11, the University of Maryland medical team performed the world's first pig heart transplant surgery with the consent of a living terminally ill patient and their family. On September 20, 2023, the second transplant was performed in a patient with end-stage heart failure. Pig-human xenotransplantation has advanced so much that it is conducting clinical trials on living patients. However, due to differences between species, it can cause a stronger immune rejection response than human-to-human allotransplantation, so the development of a completely humanized pig is essential.

[0005] As a prior art, Korean Patent No. 10-2040203 discloses that after producing a transgenic cloned pig lacking GGTA1 (α1,3-galactosyltransferase), CMAH (CMP-N-acetylneuraminic acid hydroxylase), iGb3s (Isoglobotrihexosylceramide synthase), and β4GalNT2 (Beta-1,4-N-Acetyl-Galactosaminyl Transferase2) genes, an in vitro human serum reaction test was conducted, and it was confirmed that IgG and IgM binding was significantly reduced, suggesting the possibility of overcoming antigen-antibody mediated rejection that occurs in xenotransplantation. However, in addition to controlling hyperacute and acute immune rejection through antigen-antibody mediated immune rejection, when pig organs, tissues, cells, and blood are transplanted into humans, immune rejection due to human complement activation occurs.

[0006] Accordingly, the inventors of the present invention induced human CD59 gene expression using a porcine endogenous promoter to reduce immune rejection reactions occurring in xenotransfusion, and confirmed that the developed transgenic pigs reduced antigen-antibody mediated immune rejection reactions and complement mediated immune rejection reactions, thereby completing the present invention.

[0007] The purpose of the present invention is to provide a knock-in vector for producing transgenic cloned pigs.

[0008] Another object of the present invention is to provide a transgenic cell line produced by transforming the knock-in vector into a somatic cell and a transgenic cloned pig produced by nuclear transfer of the transgenic cell line.

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

[0010] Another object of the present invention is to provide a method for producing xenogeneic organs for transplantation, which includes breeding cloned pigs to extract organs or producing organs using their germ cells or somatic cells.

[0011] To achieve the above purpose,

[0012] The present invention provides a knock-in vector for producing a transgenic cloned pig comprising a human CD59 gene represented by SEQ ID NO: 1 linked to a porcine GGTA1 gene.

[0013] In addition, the present invention provides a transformed cell line produced by transforming the knock-in vector into a somatic cell.

[0014] In addition, the present invention provides a transgenic cloned pig produced by nuclear transfer of the above-mentioned transgenic cell line.

[0015] In addition, the present invention provides a method for producing a transgenic cloned pig, comprising the steps of producing the above-described transgenic cell line, transplanting the cell line into an enucleated egg to form a nuclear transfer embryo, and transplanting the nuclear transfer embryo into the oviduct of a surrogate mother.

[0016] In addition, the present invention provides a transgenic cloned pig produced by the above method.

[0017] In addition, the present invention provides a method for producing xenogeneic organs for transplantation, which includes breeding the cloned pig to extract organs or producing organs using germ cells or somatic cells thereof.

[0018] The present invention relates to a transgenic cloned pig for xenotransfusion and xenotransplantation in which the porcine GGTA1, CMAH, iGb3s, and β4GalNT2 genes are deleted and the human CD59 gene is knocked in, and a method for producing the same. By inducing expression of the human CD59 gene in the transgenic pig, which has improved the existing antigen-antibody-mediated immune rejection response, the complement-mediated immune rejection response is reduced, and expression of the human CD59 gene has been confirmed in red blood cells, so that the pig can be usefully used for xenotransfusion and xenotransplantation.

[0019] Figure 1 is a schematic diagram showing the acquisition of base cell lines lacking the GGTA1, CMAH, iGb3s, and β4GalNT2 genes used in development.

[0020] Figure 2a is a schematic diagram showing a human CD59 knockin vector.

[0021] Figure 2b is a diagram showing a human CD59 expression strategy through knock-in.

[0022] Figure 3 is a diagram showing the results of separating human CD59 positive cells through sorting after transduction.

[0023] Figure 4 is a diagram showing the positions of primers used in the invention and whether a human CD59 knock-in vector is introduced into a transformed cell line.

[0024] Figure 5 is a diagram showing the results of confirming protein expression through FACS analysis after immunofluorescence staining of selected transformed cell lines #21, #30, #35, and #39.

[0025] Figure 6 is a photograph of transgenic cloned pigs produced through somatic cell cloning using transgenic cell line #21.

[0026] Figure 7 is a diagram showing the human CD59 gene analysis of the produced transgenic cloned pigs.

[0027] Figure 8 is a diagram showing the results of FACS analysis of major cells of the produced transgenic cloned pigs.

[0028] Figure 9a is a diagram showing the results of a cytotoxicity test on human serum using produced transgenic cloned pig peripheral blood mononuclear cells (PBMC).

[0029] Figure 9b is a diagram showing the results of a cytotoxicity test on monkey serum using the produced transgenic cloned porcine peripheral blood mononuclear cells (PBMC).

[0030] Figure 10a is a diagram showing the results of a cytotoxicity test on human serum using the produced transgenic cloned porcine red blood cells (RBC).

[0031] Figure 10b is a diagram showing the results of a cytotoxicity test on monkey serum using the produced transgenic cloned porcine red blood cells (RBC).

[0032] Hereinafter, the present invention will be described in detail.

[0033] The present invention provides a knock-in vector for producing a transgenic cloned pig comprising a human CD59 gene represented by SEQ ID NO: 1 linked to a porcine GGTA1 gene.

[0034] In the present invention, the human CD59 (Human Membrane Attack complex-inhibitory protein; MAC-IP) gene is a complement activation inhibitory gene, and 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 C9 insertion and polymerization, and ultimately prevents the formation of the membrane attack complex (MAC). The base sequence of the human CD59 gene is indicated as Genebank number NM_203329.3, and the base sequence of the porcine CD59 gene is indicated as Genbank number AH010595.2.

[0035] In the present invention, the term 'knock-in' means inserting a foreign base sequence that did not exist in another species or originally in the organism into the genome of the organism or a DNA base sequence derived from the organism using genetic recombination technology.

[0036] In the present invention, the knock-in vector comprises a first region consisting of a left arm including a sequence of exon 3 or a portion of exon 4 of a porcine GGTA1 gene, a second region consisting of a region encoding a human CD59 gene, and a right arm including a sequence of exon 4 or a portion of exon 5 of a porcine GGTA1 gene.

[0037] In the present invention, the knock-in vector has a human CD59 CDS sequence (SEQ ID NO: 2) inserted between the left arm sequence of sequences 261535947 to 261536969 and the right arm sequence of sequences 261535101 to 261535922 of chromosome 1.

[0038] In the present invention, “left arm” and “right arm” refer to regions where homologous recombination occurs.

[0039] In the present invention, “homologous recombination” means genetic recombination that occurs through exchange at a genetic locus with homology, and is used to produce transgenic animals having an allele with a lost function.

[0040] In one specific embodiment 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 embodiment of the present invention, the left arm may have a size of 1,023 bp and may be composed of a base sequence represented by SEQ ID NO: 3.

[0041] In one specific embodiment of the present invention, the light arm comprises a portion of exon 4 or exon 5 of the porcine GGTA1 gene. According to one specific embodiment of the present invention, the size of the light arm may be 822 bp and may be composed of a base sequence represented by SEQ ID NO: 4.

[0042] According to one specific example of the present invention, the knock-in vector may be composed of a base sequence of SEQ ID NO: 5.

[0043] In the present invention, “vector” means a genetic construct including a base sequence of a gene operably linked to a suitable regulatory sequence so as to express a target gene in a suitable host, wherein the regulatory sequence may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, and a sequence for regulating the termination of transcription and translation. 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 capable of replicating in a cell, and examples thereof include plasmids, cosmids, phage particles, and viral vectors.

[0044] The knock-in gene recombinant vector of the present invention can preferably be represented by the vector map disclosed in Fig. 2a.

[0045] In addition, the present invention provides a transformed cell line produced by transforming the knock-in vector into a somatic cell.

[0046] In the present invention, "transformation" means introducing DNA into a host so that the DNA becomes replicable as an extrachromosomal element or by chromosomal integration completion. Transformation includes any method of introducing a nucleic acid molecule into an organism, cell, tissue, or organ, and can be performed by selecting a standard technique suitable for the host cell as is known in the art, and examples thereof 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. In order to distinguish the transformation of eukaryotic cells by plasmid or non-plasmid naked DNA from transformation in the sense of tumorigenicity of cells, it is also called 'transfection', and it is used with the same meaning in the present invention.

[0047] In the present invention, the somatic cell is preferably a fibroblast, and more preferably a porcine fibroblast.

[0048] In the present invention, the somatic cell may be a somatic cell in which the GGTA1, CMAH, iGb3s and β4GalNT2 genes are knocked out.

[0049] The above 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 produces sugar chains, producing GalNAcβ1-4, Galβ1-4GlcNAcβ1-3Gal, and Sd(a) (Sid blood group; CAD or CT) antigens. A transgenic cloned pig lacking the four carbohydrate-derived heterologous antigens mentioned above is disclosed in Korean Patent No. 10-2040203.

[0050] In one specific example of the present invention, a transformed cell line was produced in which the human CD59 gene was knocked in order to suppress the complement-mediated immune rejection that occurs when xenografted into fibroblasts derived from a transformed pig in which the four genes GGTA1, CMAH, iGb3s, and β4GalNT2, which suppress antigen-antibody-mediated immune rejection, were deleted. The transformed cell according to the present invention may be a cell with the accession number KCLRF-BP-00527 deposited with the Korea Cell Line Research Foundation (KCLRF) on November 24, 2023.

[0051] In addition, the present invention provides a transgenic cloned pig produced by nuclear transfer of the above-mentioned transgenic cell line.

[0052] In addition, the present invention provides a method for producing a transgenic cloned pig, comprising the steps of producing the above-described transgenic cell line, transplanting the cell line into an enucleated egg to form a nuclear transfer embryo, and transplanting the nuclear transfer embryo into the oviduct of a surrogate mother.

[0053] In the present invention, “nuclear transfer” refers to a genetic manipulation technique that artificially combines nuclear DNA from another cell into a cell without a nucleus to give it the same characteristics, and it is possible to use a method known in the art.

[0054] In the present invention, “nuclear transfer oocyte” means an oocyte into which a nuclear donor cell has been introduced or fused.

[0055] In the present invention, “enucleated oocyte” means an oocyte from which the nucleus has been removed.

[0056] In addition, the present invention provides a transgenic cloned pig produced by the above method.

[0057] In addition, the present invention provides a method for producing xenogeneic organs for transplantation, which includes breeding the cloned pig to extract organs or producing organs using germ cells or somatic cells thereof.

[0058] The above organs can be extracted through conventional surgical procedures by adjusting the breeding period in consideration of the sex, age, weight, height, etc. of the recipient, breeding the donor cloned animal, and then transplanting it directly into the recipient or quickly storing it in a refrigerator. In addition, gametes such as sperm or eggs can be extracted from the cloned animal and artificially or naturally fertilized to obtain offspring, and these offspring can be used for organ transplantation or the production of other by-products for organ transplantation. Likewise, it goes without saying that somatic cells from the cloned animal can be extracted and used for organ transplantation or the production of other by-products as described above.

[0059] The transgenic pigs of the present invention, in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes are deleted and the human CD59 gene is expressed at the GGTA1 locus, can overcome hyperacute and complement-mediated immune rejection reactions occurring in xenotransplantation. Therefore, the transgenic cloned pigs of the present invention can be usefully utilized as donor animals for organ and blood transfusion between xenotransplantation species.

[0060] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0061] <Example 1> Development of GGTA1, CMAH, iGb3s, and β4GalNT2 knockout transformed cell lines

[0062] <1-1> Production of transgenic pigs

[0063] Transgenic pigs were produced to establish pig GGTA1, CMAH, iGb3s, and β4GalNT2 gene knockout QKO (quadruple knockouts) transgenic cell lines.

[0064] Specifically, the QKO F0 individuals developed in Korean Patent Application No. 10-2018-0034466 were crossed with wild-type (WT) individuals to produce F1 individuals with QKO heterozygous genetic traits. The produced F1 individuals were raised to sexual maturity (8 months of age) and crossed with QKO F0 individuals to produce F2 individuals with QKO homozygous genetic traits. The produced individuals were subjected to PCR using the primers disclosed in Table 1 for GGTA1, CMAH, iGb3s, and β4GalNT2 base sequence analysis. The obtained PCR products were subjected to base sequence analysis by SolGent.

[0065]

[0066] As a result, as shown in Table 2, it was confirmed that two loci of the GGTA1, CMAH, and β4GalNT2 genes and one locus of iGb3s were deleted in the F2 individuals.

[0067]

[0068] <1-2> Construction of transformed cell lines

[0069] Ear fibroblasts were isolated and cultured from the above-mentioned transgenic pigs to establish GGTA1, CMAH, iGb3s, and β4GalNT2 gene knockout QKO transgenic cell lines.

[0070] More specifically, the individual ears were disinfected with 70% ethanol, and ear tissue measuring 1 cm in length x width was biopsied, stored in DPBS containing penicillin-streptomycin antibiotics, and then transported to the laboratory. The tissues were washed three times with DPBS, the epidermis was removed, minced, and attached to a 6-well multiplate. The cells were cultured in DMEM (Lonza, Switzerland) containing 10% FBS and 1X penicillin-streptomycin antibiotics for 7 days, and then porcine ear fibroblasts (PEFs) derived from ear tissue were obtained.

[0071] <1-2> Construction of transformed cell lines

[0072] Ear fibroblasts were isolated and cultured from the above-mentioned transgenic pigs to establish GGTA1, CMAH, iGb3s, and β4GalNT2 gene knockout QKO transgenic cell lines.

[0073] More specifically, the individual ears were disinfected with 70% ethanol, and ear tissue measuring 1 cm in length x width was biopsied, stored in DPBS containing penicillin-streptomycin antibiotics, and then transported to the laboratory. The tissues were washed three times with DPBS, the epidermis was removed, minced, and attached to a 6-well multiplate. The cells were cultured in DMEM (Lonza, Switzerland) containing 10% FBS and 1X penicillin-streptomycin antibiotics for 7 days, and then porcine ear fibroblasts (PEFs) derived from ear tissue were obtained.

[0074] <Example 3> Construction of a transformed cell line in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes were deleted and the human CD59 gene was knocked in.

[0075] <3-1> Production of transformed cell lines in which GGTA1, CMAH, iGb3s, and β4GalNT2 genes are deleted and human CD59 gene is knocked in.

[0076] Experiments were performed to generate transformed cell lines in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes were deleted and the human CD59 gene was knocked in.

[0077] Specifically, the CD59 knock-in vector constructed in Example 2 was introduced into the transgenic 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 and then treated with 0.25% trypsin-EDTA (Gibco) to recover the cells. After centrifugation at 1500 rpm for 3 minutes and washing with DPBS, the vector was mixed with the buffer in the P3 Primary Cell 4D-Nucleofector kit (Amaxa, Germany), and then electroporated using the EN-150 protocol of the 4D-Nucleofector system (amaxa). One week after introduction, to increase the selection efficiency, the cells were immunostained with CD59 antibody, and only CD59-positive cells were selected using a FACS AriaIII (BD bioscience, USA) device. The selected cells were cultured into 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 containing positions within the human CD59 knock-in vector and outside the cancer cell line. The obtained PCR products were loaded onto a 1% agarose gel and analyzed.

[0078]

[0079] As a result, as shown in Fig. 4, it was confirmed that the human CD59 knock-in vector was inserted in six colonies, #23, #26, #27, #28, #31, and #37, and that homologous recombination also occurred well.

[0080] <3-2> Protein expression analysis of selected transformed cell lines

[0081] Among the six colonies selected in Example 3-1, cell immunostaining and FACS analysis were performed for protein expression analysis from colonies #21, #30, #35, and #39, which were morphologically superior.

[0082] Specifically, the cell lines were washed with DPBS and treated with 0.25% trypsin-EDTA solution for 3 minutes to harvest the cells. Trypsin-EDTA was inactivated with fetal bovine serum (FBS), washed with DPBS, and incubated with human CD59 antibody at a concentration of 1:100 for 1 hour. Subsequently, the cells were washed three times with DPBS containing Tween-20, and incubated with FITC-conjugated secondary antibody at a concentration of 1:100 in a refrigerator 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, a transgenic cell line expressing the human CD59 gene by the EF1α promoter was used for the analysis.

[0083] As a result, as shown in Fig. 5, it was confirmed that all four colonies of candidate transformed cell lines #21, #30, #35, and #39 expressed human CD59 protein well.

[0084] The above-mentioned transformed cell line #21 was named QKO / hCD59 KI and deposited with the Korea Cell Line Research Foundation (KCLRF) on November 24, 2023, and was assigned accession number KCLRF-BP-00527.

[0085] <Example 4> Production of transgenic pigs in which GGTA1, CMAH, iGb3s, and β4GalNT2 genes were deleted and the human CD59 gene was knocked in.

[0086] <4-1> Preparation of oocytes

[0087] To produce transgenic pigs, oocytes were first prepared.

[0088] Specifically, ovaries from immature female pigs were obtained and 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 antral follicles with a diameter of 2-6 mm using an 18-gauge needle attached to a 10 mL disposable syringe. To select COCs, the follicular fluid was washed in TL-HEPES medium, and COCs were sorted in petri dishes and transferred to TCM 199 (Gibco, USA) containing 0.1 mM 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 placed in 4-well multidish (Nunc, Denmark) covered with mineral oil. Approximately 70-80 COCs were transferred to TCM 199 (Gibco, USA). The cells were cultured for 22 hours and then cultured in TCM 199 medium from which only FSH and hCG were removed for 42 to 44 hours under conditions of 5% CO2 and 39°C.

[0089] <4-2> Nuclear transplantation

[0090] Nuclear transfer was performed to produce transgenic pigs.

[0091] Specifically, after 42 to 44 hours of culture, COCs induced to mature in vitro were transferred to a 4-well multidish 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, treated with 0.1% hyaluronidase, and gently pipetted about 70 to 80 times to separate oocytes from cumulus cells. Metaphase II oocytes with protruding first polar bodies were selected and nuclear staining was performed for 15 minutes in a culture medium containing 5 μg / mL Hoechst 33342 in MM (MM-CB) containing 7.5 μg / mL cytochalasin B, and then the oocytes were transferred to MM-CB. To remove the nucleus from oocytes without cumulus cells, the stained first polar bodies and nuclei were aspirated using a fine glass pipette (enucleation). For serum starvation prior to SCNT, donor cells from the transformed cell line #21 prepared in Example 3 were cultured in DMEM medium containing 0.5% FBS for 3 days. A single donor cell was placed in the perivitelline space of the oocyte in contact with the oocyte membrane. Inoculated oocytes were placed between two 0.2 mm diameter platinum electrodes spaced 1 mm apart in a medium containing 0.3 M mannitol, 1.0 mM CaCl2H2O, 0.1 mM MgCl26H2O, and 0.5 mM HEPES. Fusion / activation was induced by two consecutive 1.1 kV / cm DC pulses for 30 μs (BTX, USA). Electrically stimulated recombinant oocytes were placed in a 0.3% BSA-supplemented PZM-3 (Biol Reprod.66:112-9, 2002) were cultured in vitro in 30 μl droplets at a density of 15. After 1 or 2 days of culture, NT embryos were surgically transferred into the oviducts of sows on the first day of standing estrus. Pregnancy status was confirmed using an ultrasound scanner (Medison Co., Korea).

[0092] <Example 5> Production and verification of transgenic pigs in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes were deleted and the human CD59 gene was knocked in.

[0093] <5-1> Production of transgenic cloned pigs

[0094] The external appearance of the transgenic pig manufactured in Example 4 above is shown in Fig. 6.

[0095] In addition, in order to confirm the base sequence of the transgenic pig, fibroblasts of the piglets were obtained and the base sequence thereof was analyzed, and it was confirmed that the target gene regions, GGTA1, CMAH, iGb3s, and β4GalNT2 genes, were successfully deleted in the fibroblasts of the transgenic pig prepared in Example 4.

[0096] Additionally, to confirm human CD59 gene knock-in, PCR amplification was performed using the 463F-464R primer and the 463F-873R primer in Table 3, respectively, and the products were loaded onto a 1% agarose TAE gel and analyzed.

[0097] As a result, as shown in Fig. 7, the two loci, wild type and CD59 knock-in, were amplified by the introduction of the human CD59 knock-in vector in the 463F-464R primer set (left) by the introduction of the knock-in vector, and it was confirmed that the two loci were also amplified by homologous recombination in the 463F-873R primer set (right).

[0098] <5-2> Verification of transgenic cloned pigs

[0099] After isolating peripheral blood mononuclear cells (PBMCs), red blood cells (RBCs), and splenocytes derived from the blood of transgenic cloned pig #1 verified in Example 5-1 above, the protein expression of the defective gene and knocked-in CD59 was analyzed by FACS.

[0100] Specifically, blood was collected from the subject using a syringe and diluted 1:1 in DPBS. 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. The middle buffy coat layer and the lower red blood cell layer were separated, washed separately with DPBS, and immunostained using antibodies specific to each gene. Spleen cells were biopsied from the subject's spleen and transported to the laboratory in DPBS containing penicillin-streptomycin. The transported spleen samples were washed five times with DPBS, placed on a 40μm cell strainer, and finely chopped with a syringe. The obtained splenocytes were depleted of red blood cells using RBC lysis buffer (Sigma-Aldrich), and immunostained using antibodies specific to each gene. Each cell was analyzed using a cytoflex device along with an unstained control.

[0101] As a result, as shown in Fig. 8, it was confirmed that all three types of heterologous antigens GGTA1, CMAH, and β4GalNT2 were not expressed in PBMCs, splenocytes, and RBCs derived from transgenic cloned pigs, but the knocked-in CD59 protein was expressed.

[0102] <5-3> Functional verification of transgenic cloned pigs

[0103] To evaluate the function of the transgenic pig manufactured in Example 5-1 above, cytotoxicity analysis using human and primate serum was performed.

[0104] Specifically, peripheral blood mononuclear cells and red blood cells separated using ficoll paque plus solution in the same manner as in Example 5-2 were treated with human and primate complement serum at concentrations of 3.125, 6.25, 12.5, 25, and 50% for 3 hours in a 96-well multiplate. After that, the human complement serum in all wells was removed, and 100 μl of DPBS and 10 μl of CCK-8 (Dojindo, Japan) were added to measure cell viability.

[0105] As a result, as shown in Figures 9a and 9b, in the case of peripheral mononuclear cells, the survival rate was higher in pigs with QKO gene deletion than in normal pigs (WT) in all treatment groups. In particular, it was confirmed that the transgenic cloned pigs of the present invention, in which four types of foreign antigens were knocked out and the human CD59 gene was knocked in, had a significantly higher survival rate with human and primate complement serum. In addition, as shown in Figures 10a and 10b, in the case of red blood cells, it was confirmed that the survival rate with human and primate complement serum was also significantly higher.

[0106] In summary, it was confirmed that the transgenic pigs produced by the method of the present invention, in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes are deleted and the human CD59 gene is expressed at the GGTA1 locus, can overcome hyperacute and complement-mediated immune rejection reactions occurring in xenotransplantation. In particular, since the expression of the human CD59 gene was confirmed in red blood cells, it can be used as artificial blood for xenotransfusion.

[0107] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0108]

[0109] [Accession number]

[0110] Name of depositor: Korea Cell Line Research Foundation

[0111] Accession number: KCLRF-BP-00527

[0112] Date of acceptance: 202311124

[0113] [Correction pursuant to Rule 91, April 29, 2024]

Claims

1. A knock-in vector for producing a transgenic cloned pig, comprising a human CD59 gene represented by sequence number 1 that is targeted to a porcine GGTA1 (Alpha 1,3-Galactosyltransferase) gene.

2. In paragraph 1, The above knock-in vector comprises a first region comprising a left arm comprising a sequence of exon 3 or a part of exon 4 of the porcine GGTA1 (Alpha 1,3-Galactosyltransferase) gene; a region encoding the human CD59 gene; and A second region comprising a right arm comprising a sequence of exon 4 or a portion of exon 5 of the porcine GGTA1 gene; A knock-in vector, characterized by including:

3. In paragraph 2, A knock-in vector, characterized in that the first region is composed of a base sequence represented by sequence number 2.

4. In paragraph 2, A knock-in vector, characterized in that the second region is composed of a base sequence represented by sequence number 3.

5. A transformed cell line produced by transforming the knock-in vector of clause 1 into a somatic cell.

6. In paragraph 5, The above somatic cells are transformed cell lines characterized in that the GGTA1 (Alpha 1,3-Galactosyltransferase), CMAH (CMP-N-acetylneuraminic acid hydroxylase), iGb3s (Isogloboside 3 synthease), and β4GalNT2 (Beta-1,4-N-Acetyl-Galactosaminyl Transferase2) genes are knocked out.

7. In paragraph 6, A transformed cell line, characterized in that the transformed cell line has the accession number KCLRF-BP-00527.

8. A transgenic cloned pig produced by nuclear transfer of a transformed cell line according to any one of clauses 5 to 7. 9.(1) A step for producing a transformed cell line according to any one of clauses 5 to 7. (2) a step of transplanting the above cell line into an enucleated oocyte to form a nuclear transfer oocyte; and (3) a step of transplanting the nuclear transfer embryo into the fallopian tube of a surrogate mother; Method for producing transgenic cloned pigs.

10. Transgenic cloned pigs produced by the method of Article 9.

11. A method for producing xenograft organs for transplantation, which includes breeding cloned pigs of Article 8 to extract organs or producing organs using their reproductive cells or somatic cells.

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