Transformed replicating pigs from which heterologous antigens GGTA1, CMAH, iGb3s, β4GalNT2, and β2M genes have been removed from a PERV Envelope C-negative basis, and a method for producing the same.

Abstract

This invention provides transformed cells for the production of transformed replica pigs for xenotransplantation. [Solution] This invention relates to a transformed replica pig from which the heterologous antigens GGTA1, CMAH, iGb3s, β4GalNT2, and β2M genes have been removed from a PERV Envelope C-negative base, and a method for producing the same. The transformed replica pig according to the present invention can overcome hyperacute and antigen-antibody-mediated immune rejection reactions and T-cell-mediated immune rejection reactions without causing the transfer of porcine endogenous retroviruses that occur in xenologous organ transplantation, and can be usefully utilized as a donor animal for interspecies organ and cell transplantation.

Description

Technical Field

[0001] The present invention relates to a transformed and replicated pig in which genes of heterogeneous antigens GGTA1, CMAH, iGb3s, β4GalNT2, and β2M of a PERV Envelope C-negative substrate have been removed, and a method for producing the same.

Background Art

[0002] Every year, the number of organ transplant candidates increases, while the number of organ donors tends to decrease. Efforts have been continuously made to improve the organ donation system not only in Korea but also worldwide, but the gap between donors and candidates is accelerating. Pig-to-human xenotransplantation is one of the solutions expected to completely solve the organ transplant supply problem when pig organs are activated by completely replacing human organs.

[0003] Among the raw animals for xenogeneic organs, pigs are highly morphologically and genetically similar to humans. Unlike primates, they have low zoonotic infections and ethical problems. In addition, they have the advantage of being prolific and can sufficiently overcome the insufficient supply. They are widely used as non-clinical models and are utilized in many basic researches. In particular, the Yucatan miniature pig is phenotypically and anatomically similar to humans, and the sizes of organs such as 94% of the heart, 66% of the liver, 81% of the pancreas, and 91% of the kidneys are similar, and it has been selected as the most suitable pig species for raw materials of xenogeneic organs compared to other species. Despite such similarities, antigen-antibody-mediated immune rejection reactions and zoonotic infections due to differences between species still remain as issues that must be overcome in replacing human-to-human allotransplantation in xenogeneic organ transplantation.

[0004] To address this, genetically modified pigs have been developed using gene editing technology. In 2003, the first genetically modified pigs for xenotransplantation (GGTA1 knock-out; GTKO) were developed using gene editing technology to eliminate the GGTA1 (α1,3-galactosyltransferase) gene, preventing the production of α-gal epitopes (Carol J Phelps et al., Science, 2003). When organs derived from GTKO-transformed pigs were transplanted into monkeys, hyperacute immune rejection, where tissue necrosis occurs within seconds to minutes, was controlled, and the monkeys survived. However, problems arose where tissue necrosis continued for several days to several years, and it is known that this is caused by antigen-antibody reactions other than GGTA1. Among these, CMAH (Cytidine monophosphate-N-acetylneuraminic acid hydroxylase), iGb3s (Isogloboside 3 synthease), and β4GalNT2 (Beta-1,4-N-Acetyl-Galactosaminyl Transferase 2) are the most representative genes that synthesize Neu5Gc, iGb3, and SD(a) heteroantigens, respectively, and are known to induce immune rejection (Mohamed Ezzelarab et al., Immunology & Cell Biology, 2005). Therefore, multiple transgenic pigs in which multiple carbohydrate-derived heteroantigens have been removed have been developed worldwide to regulate immune rejection, and excellent results have been derived using them (David KC Cooper et al., Journal of Pathology, 2016).

[0005] While excellent results have been achieved with in-vivo models utilizing diverse genetic mutations, the issue of zoonotic diseases has recently emerged. PERV (Porcine Endogenous Retrovirus) is a naturally occurring endogenous virus in pigs, and there have been no clear reports of its transmissibility within the body. However, if transmissibility is confirmed at the cellular level and PERV is transmissible into the human body over the long term, various problems could arise. In particular, the A / C recombinant form, in which PERV Envelope A and Envelope C are recombined, has been reported to have a 500 times higher probability of transmissibility. Since this cannot be resolved with vaccines or treatments, it is recommended to use PERV Envelope C-negative individuals as raw material animals for xenotransplantation.

[0006] Major histocompatibility complexes (MHCs) are crucial for organ survival in allogeneic organ transplantation, and this also applies to xenotransplantation. Porcine MHCs are divided into Swine Leukocyte Antigen (SLA) class I and II. Class I is expressed on the surface of all cells with a nucleus, while class II is expressed on the surface of B cells and macrophages. When cells, organs, tissues, or blood containing SLA class I are transplanted, infiltration and lysis occur by the recipient's T cells, resulting in necrosis. SLA class I is expressed on the surface of β2M (Beta-2-microglobulin) cells and is a gene essential for maintaining the stability of peptide bonds. Therefore, it has been reported that if the β2M gene is deficient, SLA class I protein is not expressed, resulting in reduced necrosis in porcine-mouse xenotransplantation (Yong Wang et al., Scientific Reports, 2016).

[0007] Therefore, there is a need to develop transformed and replicated pigs that can be used for xenotransplantation by removing the factors involved in the aforementioned immune rejection reaction.

[0008] Against the aforementioned background, the inventors continued their efforts to develop a transformed replicating pig that could be used for xenotransplantation. As a result, they developed a transformed cell line in which five genes, GGTA1, CMAH, iGb3s, β4GalNT2, and β2M, were removed from a base cell line that is negative for the porcine endogenous retrovirus Envelope C. Using the developed transformed cell line, they produced a transformed replicating pig and completed the present invention by verifying the function of the said transformed replicating pig. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] KR 10-2176161 B1 [Non-patent literature]

[0010] [Non-Patent Document 1] Wang, Y., Du, Y., Zhou, X. et al. Efficient generation of B2m-null pigs via injection of zygote with TALENs. Sci Rep 6,38854(2016). [Non-Patent Document 2] Cooper DK, Ekser B, Ramsoondar J, Phelps C, Ayares D. The role of genetically engineered pigs in xenotransplantation research.J Pathol.2016 Jan;238(2):288-99. [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The object of the present invention is to provide transformed cells for the production of transformed replicating pigs for xenotransplantation, into which a recombinant vector for GGTA1 knockout, a recombinant vector for CMAH knockout, a recombinant vector for iGb3s knockout, a recombinant vector for β4GalNT2 knockout, and a recombinant expression vector for β2M gene knockout have been introduced, and which are negative for PERV EnvC.

[0012] Another object of the present invention is to provide a method for producing a transformed replica pig for xenotransplantation, comprising the steps of: transplanting the transformed cells into an enucleated oocyte to form a nuclear transplant egg; and transplanting the nuclear transplant egg into the fallopian tube of a surrogate mother.

[0013] Another object of the present invention is to provide transformed replica pigs for xenotransplantation produced by the method described above. [Means for solving the problem]

[0014] To achieve the above objective, the present invention provides transformed cells for the production of transformed replicating pigs for xenotransplantation, into which recombinant vectors for GGTA1 knockout, CMAH knockout, iGb3s knockout, β4GalNT2 knockout, and β2M gene knockout have been introduced and which are negative for PERV EnvC.

[0015] Furthermore, the present invention provides a method for producing a transformed replica pig for xenotransplantation, comprising the steps of: transplanting the transformed cells into an enucleated oocyte to form a nuclear transplant egg; and transplanting the nuclear transplant egg into the fallopian tube of a surrogate mother.

[0016] Furthermore, the present invention provides transformed replica pigs for xenoorgan transplantation produced by the method described above. [Effects of the Invention]

[0017] The present invention relates to a transgenic cloned pig in which the genes of heterologous antigens GGTA1, CMAH, iGb3s, β4GalNT2 and β2M of a PERV Envelope C-negative substrate have been removed, and a method for producing the same. The transgenic cloned pig according to the present invention does not cause the transfer of porcine endogenous retrovirus occurring in xenotransplantation, but can overcome hyperacute and antigen-antibody-mediated immune rejection reactions and immune rejection reactions by T-cells, and can be usefully utilized as a donor animal for xenotransplantation of organs and cells.

Brief Description of Drawings

[0018] [Figure 1] It is a figure showing the PCR result for verification of porcine endogenous retrovirus Envelope C. [Figure 2] It is a figure showing the vector produced for targeting heterologous antigen genes. [Figure 3] It is a figure showing the gene analysis result of a transgenic cloned pig from which four kinds of heterologous antigens of PERV Envelope C-negative, GGTA1, CMAH, iGb3s and β4GalNT2 produced using the constructed cell line have been removed. [Figure 4] It is a figure showing the protein expression analysis using PBMCs of a transgenic cloned pig from which four kinds of heterologous antigens of PERV Envelope C-negative, GGTA1, CMAH, iGb3s and β4GalNT2 produced using the constructed cell line have been removed. [Figure 5] It is a figure showing a transgenic cloned pig from which four kinds of heterologous antigens of PERV enlvope C-negative, GGTA1, CMAH, iGb3s and β4GalNT2 produced using the constructed cell line have been removed. [Figure 6] It is a figure showing the T7E1 result using four candidate guide RNA sequences for the β2M gene target. [Figure 7] It is a figure showing the FACS result of the separated cells after transducing the completed vector into the cells. [Figure 8]This figure shows the sequencing results of the constructed cell line β2M gene. [Figure 9] This figure shows the SLA class I protein expression in the constructed cell line. [Figure 10] This figure shows the identification of endogenous porcine retroviruses in transformed replicated pigs produced using the constructed cell line. [Figure 11] This figure shows the nucleotide sequences of five targeted heterologous antigen genes analyzed in transformed replicating pigs produced using the constructed cell line. [Figure 12] This figure shows a transformed replicating pig that is PERV Envelope C negative and has had five heterologous antigens removed: GGTA1, CMAH, iGb3s, β4GalNT2, and β2M. [Figure 13] This figure shows the experimental results for confirming SLA class I protein expression using transformed pig-derived PBMCs produced. [Figure 14] This figure shows experimental results confirming a human T-cell-mediated immune response using transformed pig-derived vascular endothelial cells produced. [Modes for carrying out the invention]

[0019] The present invention will be described in detail below.

[0020] The present invention provides transformed cells for the production of transformed replicating pigs for xenotransplantation, into which recombinant vectors for GGTA1 knockout, CMAH knockout, iGb3s knockout, β4GalNT2 knockout, and β2M gene knockout have been introduced, and which are negative for PERV EnvC.

[0021] In the present invention, “vector” means a gene construct comprising a gene sequence operably linked to a suitable regulatory sequence so as to enable the expression of a target gene in a suitable host, wherein the regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, and sequences for regulating the termination of transcription and detoxification. The vector of the present invention is not particularly limited as long as it is replicable within a cell, and any vector known in the art may be used, for example, plasmids, cosmids, phage particles, or viral vectors.

[0022] In the present invention, the knockout recombinant vector may be in a form in which the nucleotide sequences encoding sgRNAs for GGTA1, CMAH, iGb3s, β4GalNT2, and β2M are all contained in a single vector, or it may be in a form consisting of a group of multiple vectors in which one or more nucleotide sequences encoding each of the sgRNAs are contained in separate vectors. In other words, as long as the target sequence can be contained, the form and number of vectors are not limited.

[0023] In the present invention, the GGTA1 knockout recombinant vector may recognize exon 4 of porcine chromosome 1 to knock out the GGTA1 gene and may contain a nucleotide sequence that encodes the gRNA (guide RNA) represented by Sequence ID No. 1. The GGTA1 gene (GenBank accession No. AH010595.2) is known to be responsible for α-gal biosynthesis.

[0024] In the present invention, the CMAH knockout recombinant vector may recognize exon 9 of porcine chromosome 7 to knock out the CMAH gene and may contain a nucleotide sequence that encodes the gRNA (guide RNA) represented by Sequence ID No. 2. The CMAH gene (GenBank accession No. NM_001113015.1) is known to biosynthesize Neu5Ac to Neu5Gc.

[0025] In the present invention, the recombinant vector for iGb3s knockout may recognize exon 4 of porcine chromosome 6 to knock out the iGb3s gene and may contain a nucleotide sequence that encodes the gRNA (guide RNA) represented by Sequence ID No. 3. The iGb3s gene (GenBank accession No. XM_021095855) is known to be responsible for the biosynthesis of the glycosphingolipid iGb3.

[0026] In the present invention, the recombinant vector for β4GalNT2 knockout may recognize exon 1 of porcine chromosome 12 to knock out the β4GalNT2 gene and may contain a nucleotide sequence that encodes the gRNA (guide RNA) represented by Sequence ID No. 4. a It is known to synthesize antigens.

[0027] In the present invention, the recombinant vector for β2M knockout may recognize exon 2 of porcine chromosome 1 to knock out the β2M gene and may contain a nucleotide sequence that encodes the gRNA (guide RNA) represented by Sequence ID No. 5. The β2M gene is known to have an α-chain of SLA class I (Swine Leukocyte antigen class I) that is responsible for stable expression on the cell surface.

[0028] In the present invention, the gRNA is an RNA that can form a complex with the Cas9 protein and guides the Cas protein to target DNA, and can be transcribed, for example, from the DNA represented by SEQ ID NOs: 1 to 5.

[0029] In this invention, "CRISPR-Cas9" is a type of gene scissors used for cloning to remove genes. In this invention, the Cas9 protein refers to an essential protein element in the CRISPR / Cas9 system, and when it forms a complex with two RNAs called CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), it forms an active endonuclease or nickase. The gene that encodes the Cas9 protein is generally associated with a CRISPR repeat-spacer array, and there are more than 40 distinct Cas9 protein families. There are three representative CRISPR-Cas systems, among which the type II CRISPR / Cas system with the Cas9 protein is representative.

[0030] In this invention, "gene scissors" refers to a technique for cutting DNA from a desired location in a gene, and means a gene editing technique that recognizes a specific base sequence in a gene and then precisely cuts the DNA from that location.

[0031] The recombinant knockout vector of the present invention contains a gRNA (guide RNA) associated with DNA binding, as well as a Cas9 gene for DNA cleavage. Preferably, the Cas9 is SpCas9 derived from Streptococcus pyogenes, but is not limited thereto. The gRNA domain has a cloning site that can bind to any sequence in DNA, and can bind to a specific sequence of genomic DNA of choice. DNA cleavage is induced through the induction of gRNA bound to a specific site and the activation of the Cas9 protein.

[0032] In this invention, "transformation" means introducing DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or through the completion of chromosome integration. Transformation includes, but is not limited to, any method of introducing nucleic acid molecules into an organism, cell, tissue, or organ, and can be carried out by selecting and performing standard techniques suitable for the host cell, as has been known in the art, such as electrophoresis, 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 term "transfection" is sometimes used to distinguish the transformation of eukaryotic cells by plasmid or non-plasmidal exposure (naked DNA) from transformation in the sense of cell tumorigenesis, but in this invention, the same meaning is used.

[0033] The transformed cells are preferably fibroblasts, more preferably porcine fibroblasts, but are not limited thereto.

[0034] In the present invention, the transformed cell line was deposited with the Korea Cell Line Bank on July 7, 2021, under the name PKO, and was assigned accession number KCLRF-BP-00515.

[0035] Furthermore, the present invention provides a method for producing a transformed replica pig for xenotransplantation, comprising the steps of: transplanting the transformed cells into an enucleated oocyte to form a nuclear transplant egg; and transplanting the nuclear transplant egg into the fallopian tube of a surrogate mother.

[0036] 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 it is possible to use methods that are publicly known in this field.

[0037] In this invention, "nuclear transplanted egg" refers to an egg cell into which donor nucleogenetic cells have been introduced or fused.

[0038] In this invention, "denucleated oocyte" refers to an oocyte from which the nucleus has been removed.

[0039] The transformed replicating pigs according to the present invention are negative for the porcine endogenous retrovirus Envelope C and have two loci removed by the CRISPR-Cas9 system, which acts as a genetic scissors. As such, they can overcome hyperacute and antigen-antibody-mediated immune rejection, as well as T-cell-mediated immune rejection, without causing the transfer of porcine endogenous retroviruses that occur in xenotransplantation.

[0040] Therefore, the transformed and replicated pigs according to the present invention can be usefully utilized as donor animals for interspecies organ and cell transplantation.

[0041] Furthermore, the present invention provides transformed replica pigs for xenoorgan transplantation produced by the method described above.

[0042] In this invention, the transformed and replicated pigs are significant in disease research due to their physiological or genetic similarities to humans. In disease research, transformed and replicated pigs can provide research material for understanding the diverse causes, pathogenesis, and diagnosis of diseases. Through research on transformed and replicated pigs, it is possible to identify disease-related genes, understand the interactions between genes, and obtain basic data to determine the feasibility of practical application through actual efficacy and toxicity tests of developed new drug candidates.

[0043] In the transformed replicating pig of the present invention, any part that overlaps with the above description may be used in the same sense as described above.

[0044] The present invention will be described in detail below with reference to examples.

[0045] The following examples are for illustrative purposes only, and the present invention is not limited to these examples.

[0046] <Example 1> Selection of porcine individuals negative for the endogenous retrovirus Envelope C We conducted experiments to select individuals that were negative for the endogenous retrovirus Envelope C in pigs.

[0047] Specifically, ear tissue was obtained from 106 Yucatan miniature pigs, and genomic DNA was isolated. PCR was performed using the isolated genomic DNA as a template strand and the primer pairs shown in Table 3. More specifically, after thoroughly disinfecting the ears of the individuals to be analyzed with ethanol, ear tissue was obtained using an ear engraver. After transporting the ear tissue to the laboratory, genomic DNA was extracted according to the Dneasy Blood & tissue kit (Qiagen, Germany) protocol. Approximately 100 ng of extracted genomic DNA was mixed with 10 pmole forward primer and 10 pmole reverse primer, and PCR was performed using Profi PCR taq premix (Bioneer). Initially, the mixture was reacted at 95°C for 5 minutes to denaturate it, followed by 35 cycles of 40 seconds at 95°C, 40 seconds at 61°C, and 1 minute at 72°C, and finally reacted at 72°C for 7 minutes. The PCR products were loaded onto 2% TAE agarose gels, and the results are shown in Figure 1. As a result, as shown in Figure 1, it was confirmed that wild-type W16-172 individuals were negative for the porcine endogenous retrovirus Envelope C. Ear fibroblasts were isolated from the aforementioned W16-172 individuals and subsequently used as somatic cells for the production of transformed replicating pigs.

[0048] [Table 1]

[0049] <Example 2> Construction of transformed cell lines with the GGTA1, CMAH, iGb3s, and β4GalNT2 genes removed, and production of transformed replicating pigs <2-1> Production of GGTA1, CMAH, iGb3s, and β4GalNT2 gene targeting vectors For knockout of the porcine GGTA1, CMAH, iGb3s, and β4GalNT2 genes, the gene sequences were analyzed, and the exon sequences to which gRNAs could bind were determined. Oligonucleotides for gRNA synthesis were synthesized by a bionector. Table 1 shows the gRNA sequences, NCBI registry numbers, chromosome locations, and exon locations for each gene.

[0050] [Table 2]

[0051] Two primers for each gene, as shown in Table 2, were hybridized to contain gRNA sequences capable of binding to the porcine GGTA1, CMAH, iGb3s, and β4GalNT2 exon sequences disclosed in Table 1. The resulting product was then inserted into a Cas9-GFP vector. More specifically, 100 pmole of each of the two gene-specific primers was mixed, and hybridization was carried out at 95°C for 10 minutes, then at 95°C for 10 minutes, decreasing the temperature by 0.1°C per second to 12°C. The hybridized product was then ligated and transformed into a Cas9-GFP vector cleaved with the restriction enzyme BbsI, using T4 DNA ligase (NEB) to insert each target gene-specific gRNA (Figure 2). The completed vector was confirmed to have guide sequences introduced by sequencing analysis (Solgent).

[0052] [Table 3]

[0053] <2-2> Construction of transformed cell lines in which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes have been removed. Ear fibroblasts derived from wild-type W16-172 individuals that were negative for the endogenous porcine retrovirus Envelope C isolated in <Example 1> were introduced using Lipofectamine 3000 (Invitrogen) to introduce the GGTA1, CMAH, iGb3s, and β4GalNT2 targeting recombinant vectors prepared in <Example 2-1>. After introduction of the targeting vectors, only GFP gene-positive cells inserted into the Cas9 vector were selected using FACS AriaIII equipment. The selected cells were subjected to single-cell colony culture, followed by colony gene analysis. More specifically, genomic DNA was extracted from each colony using the Dneasy Blood & tissue kit, and then PCR was performed using primers (Table 4) containing the target sequences of the targeting genes GGTA1, CMAH, iGb3s, and β4GalNT2. The obtained PCR products underwent base sequence analysis (using Solgent), and transformed cell lines from which heterologous antigens had been removed were selected and used to create nuclear transfer eggs.

[0054] [Table 4]

[0055] <2-3> Production of transformed pigs from which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes have been removed <2-3-1> Preparation of oocytes

[0056] Immature sow <2-3> Production of transformed pigs from which the GGTA1, CMAH, iGb3s, and β4GalNT2 genes have been removed <2-3-1> Preparation of oocytes After obtaining the ovaries of immature sows from the slaughterhouse, they were transported to the laboratory in 0.9% NaCl saline solution at 35°C. Cumulus-oocyte complexes (COCs) were aspirated from immature anthral folicles with a diameter of 2–6 mm using an 18-gauge needle fixed to a 10 mL disposable syringe. COCs were washed three times with TCM 199 (31100-035, Gibco) containing 0.1% polyvinyl alcohol, 3.05 nM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 μg / mL LH (L-5269, Sigma-Aldrich Corp.), 0.5 μg / mL FSH (F-2293, Sigma-Aldrich Corp.), 10 ng / mL epidermal growth factor (E-4127, Sigma-Aldrich Corp.), 75 μg / mL penicillin G, and 50 μg / mL streptomycin. Approximately 50-60 COCs were transferred to a 4-well multi-dish covered with mineral oil, then 500 μL of the same medium was added, and the mixture was incubated at 39°C in a 5% CO2 incubator.

[0057] <2-3-2>Nuclear transfer We conducted experiments to perform somatic cell nuclear transfer (SCNT) for the production of transformed pigs.

[0058] Specifically, 42-44 hours after culturing, oocytes were separated from cumulus cells by vigorously vortexing for 4 minutes with TL-HEPES containing 0.1% PVA and 0.2% hyaluronidase. The nucleus was removed from oocytes lacking cumulus cells by aspirating the first polar body and adjacent cytoplasm using a microglass pipette with TCM 199 containing 0.3% BSA (Sigma-Aldrich Corp., A-8022) and 7.5 μg / mL cytochalasin B. Prior to nuclear transfer, the donor cells prepared in Example 3-1 were cultured for 3 days in DMEM medium containing 0.5% FBS for serum starvation. A single donor cell was positioned in the periviteline 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 culture medium consisting of 0.3 M mannitol, 1.0 mM CaCl2H2O, 0.1 mM MgCl26H2O, and 0.5 mM HEPES. Fusion / activation was induced by applying two consecutive 1.1 kV / cm DC pulses for 30 μs (ECM2001; BTX Inc). Subsequently, 20–30 reconstructed embryos were transferred to a 4-well multi-dish lined with mineral oil and supplemented with NCSU (North Carolina State University)-23 medium supplemented with 500 mL of 0.4% BSA. After 1 or 2 days of culture, the NT embryos were surgically transplanted into the oviducts of sows on the first day of the standing estrus period. The pregnancy status was confirmed using an ultrasound scanner (Mysono 201, Medison Co., LTD).

[0059] <2-3-3> Analysis of Transgenic Replicated Pigs After obtaining tail tissue from transformed replicated pigs produced through the above-mentioned <Example 2-3-2>, genomic DNA was extracted using the Dneasy Blood & tissue kit. Using the extracted genomic DNA as a template, PCR and sequencing analyses were performed using the GGTA1, CMAH, iGb3s, and β4GalNT2 analytical primers shown in Table 4.

[0060] As a result, as shown in Figure 3, we confirmed the absence of heterologous antigens GGTA1, CMAH, iGb3s, and two loci of β4GalNT2.

[0061] FACS analysis was performed to confirm the presence or absence of expression of heterologous antigen proteins due to DNA knockout.

[0062] More specifically, blood was collected from the external jugular vein of the neck of wild-type and knockout individuals using a syringe, and then sampled into vacuum blood collection tubes containing EDTA. The separated blood was then used to separate the PBMC layer using ficoll gredient solution (GE Healthcare) and washed with DPBS. The PBMCs were stained with non-staining, IB4-lectin (Sigma), Neu5Gc (Biolegend), and DBA-lectin (Vector Labs), respectively, and then subjected to FACS (BD FACS Aria III) analysis.

[0063] As a result, as shown in Figure 4, it was confirmed that all three heterologous antigens were not expressed in the PBMCs of the knockout individuals compared to the wild type.

[0064] Furthermore, as shown in Figure 5, healthy individuals without abnormalities were secured, and ear tissue-derived fibroblasts were isolated and cultured to construct five heterologous antigen-transformed cell lines.

[0065] <Example 3> Construction of a transformed cell line from which the GGTA1, CMAH, iGb3s, β4GalNT2, and β2M genes have been removed, and production of transformed replicating pigs. <3-1> Production of β2M gene targeting vector Candidate target sites in the porcine β2M gene were selected and target vectors were constructed using the same method as described in <Example 2-1> above (Table 5). Four sequences from 20 guide RNAs were selected, and T7E1 analysis was performed to validate the recombinant vectors.

[0066] [Table 5]

[0067] More specifically, wild-type porcine fibroblasts were transduced using Lipofectamine 3000, cultured, and then genomic DNA was extracted. Using the isolated cell-derived genomic DNA and wild-type cell-derived genomic DNA as templates, PCR was performed using the β2M gene analysis primers shown in Table 3, and the resulting products were purified. The purified products were mixed with the wild-type β2M product and the β2M product from cells into which the target vector had been introduced, and then treated with T7 endonuclease I (NEB) for 1 hour. The PCR products were loaded onto a 2% agarose TBE gel, and the results are shown in Figure 6.

[0068] As a result, we confirmed that the band split at target number 4, as shown in Figure 6.

[0069] <3-2> Construction of transformed cell lines from which the GGTA1, CMAH, iGb3s, β4GalNT2, and β2M genes have been removed. Transgenic pig-derived ear fibroblasts produced in Example 2 were introduced with the β2M targeting recombinant vector selected in Example 3-1 using Lipofectamine 3000. 48 hours after targeting, the cells were sorted using FACS Aria III equipped with the GFP gene inserted into the Cas9 vector. Some of the sorted cells were cultured and then subjected to FACS analysis using SLA class I antibody, the results of which are shown in Figure 7.

[0070] As a result, as shown in Figure 7, fluorescence against the SLA class I antibody was expressed in wild-type cells, and it was confirmed that some of the selected cells did not express the protein.

[0071] The cells remaining after being used for analysis were subjected to single-cell colony culture, followed by individual colony-specific genetic analysis.

[0072] PCR and sequencing were performed using the β2M primers shown in Table 6, and the results are shown in Figure 8.

[0073] [Table 6]

[0074] As a result, as shown in Figure 8, we confirmed that the G sequence was deleted at both loci in cell line #14. Translation of the nucleotide sequence into a protein sequence revealed a sequence deformation (frame shift) compared to the wild-type normal protein sequence, and it was predicted that the β2M protein would not be produced.

[0075] To further verify protein expression, FACS validation was performed using knock-out cell line #14. Wild-type and cell line #14 were immunofluorescence stained using FITC-conjugated SLA class I antibody, and the stained cells were analyzed using FACS Aria III equipment. As a result, as shown in Figure 9, the #14 cell line showed the same level of fluorescence as the NC (unstained group) compared to the wild type, confirming that the β2M protein was not expressed.

[0076] <3-3> Production of transformed pigs from which the GGTA1, CMAH, iGb3s, β4GalNT2 and β2M genes have been removed Using the #14 cell line constructed in Example 3-2 described above, nuclear transfer eggs were prepared and transplanted into surrogate sows in the same manner as in Example 2-3 of this patent. Two replicated piglets were successfully produced (one stillborn, one surviving), and after biopsy of tail tissue from each individual, genomic DNA was extracted. Using the extracted genomic DNA as a template, PCR was performed using the PERV analysis primers shown in Table 1, and the PCR products were subjected to electrophoresis on a 1% agarose TAE gel.

[0077] As a result, as shown in Figure 10, both of the produced replicating piglets were confirmed to be negative for PERV Envelope C. Furthermore, PCR was performed using genomic DNA as a template strand and primers specific to each gene to analyze the five removed heterologous antigens. The amplified products were sequenced by Solgent Co., Ltd., and the analysis results are shown in Figure 11.

[0078] As a result, as shown in Figure 11, all two loci of the heterologous antigen genes GGTA1, CMAH, iGb3s, β4GalNT2, and β2M were removed.

[0079] The produced individuals are shown in Figure 12, and it was confirmed that they were produced in a healthy manner without any abnormalities.

[0080] <Example 4> Functional verification of transformed pigs from which the GGTA1, CMAH, iGb3s, β4GalNT2, and β2M genes have been removed. Immunofluorescence staining was performed on PBMC cells to confirm the presence or absence of SLA class I protein expression in transformed replicating pigs with β2M gene deficiency. The stained cells were analyzed using FACS AriaIII equipment, and the results are shown in Figure 13.

[0081] As a result, as shown in Figure 13, we confirmed that the transformed replicating pigs produced did not produce SLA class I protein.

[0082] CD8-positive T-cells are known to induce immune rejection after xenotransplantation and play a crucial role in the long-term survival of graft organs and recipients. To confirm this, 2 × 10⁻¹⁶ 5 After staining individual human PBMCs with cell trace violet reagent, vascular endothelial cells were divided into 1 × 10⁶ cells according to their respective gene types. 6 The cells were co-cultured with PBMCs for 3 weeks. Subsequently, the cells were stained with CD8 antibody to detect CD8 T cells in the PBMCs, and FACS analysis was performed. Simultaneously, the degree of cell division in the PBMCs was confirmed by checking the fluorescence at the cell trace violet wavelength.

[0083] As a result, as shown in Figure 14, the degree of cell division in the wild type was 34.575±1.424, and in the QKO it was 22.8±10.113, with no significant difference observed. In contrast, the PKO value was 5.56±0.708, similar to the control group (spontaneous) of 5.28±2.779, and a significant difference was observed compared to WT and QKO (P<0.05). Based on these results, it was confirmed that the PKO-transformed pigs produced showed a significant reduction in human T-cell-mediated immune rejection compared to WT and QKO.

[0084] Overall, through the aforementioned experiments, it was confirmed that the transformed replicating pigs according to the present invention are negative for the porcine endogenous retrovirus Envelope C and possess the characteristic of having two loci of the GGTA1, CMAH, iGb3s, β4GalNT2, and β2M genes knocked out by the gene scissors CRISPR-Cas9. As a result, the transformed replicating pigs according to the present invention can overcome hyperacute and antigen-antibody-mediated immune rejection reactions and T-cell-mediated immune rejection reactions without causing the transfer of porcine endogenous retroviruses that occur in xenotransplantation, and can be usefully utilized as donor animals for interspecies organ, tissue, and cell transplantation.

[0085] [Accession Number] Depository name: Korea Cell Line Research Foundation (KCLRF) Accession number: KCLRFBP00515BP Date of acceptance: 20210707

Claims

1. Recombinant vector for GGTA1 (Alpha 1,3-Galactosiltransferase) knockout; CMAH (CMP-N-acetylneuraminic acid hydroxylase) recombinant vector for knockout; Recombinant vector for iGb3s (Isoglobotrihexosylceramide synthase) knockout; Recombinant vector for β4GalNT2 (Beta-1,4-N-Acetyl-Galactosaminyl Transfer 2) knockout; and A recombinant vector for knocking out the β2M (Beta-2-microglobulin) gene was introduced. PERV (Porcine Endogenous Retrovirus) is a transformed cell line that is negative for EnvC (Envlope C) and is used for the production of transformed replicating pigs for xenotransplantation.

2. The aforementioned recombinant vector for knocking out the GGTA1 gene contains a base sequence that encodes the gRNA (guide RNA) represented by Sequence ID No.

1. The CMAH gene knockout recombinant vector contains a base sequence that encodes the gRNA (guide RNA) represented by Sequence ID No.

2. The aforementioned recombinant vector for iGb3s gene knockout contains a base sequence that encodes the gRNA (guide RNA) represented by Sequence ID No. 3, The aforementioned recombinant vector for knocking out the β4GalNT2 gene contains a base sequence that encodes the gRNA (guide RNA) represented by Sequence ID No.

4. The transformed cell according to claim 1, characterized in that the β2M gene knockout recombinant vector contains a base sequence that encodes the gRNA (guide RNA) represented by Sequence ID No.

5.

3. The aforementioned recombinant vector for GGTA1 knockout recognizes exon 4 of porcine chromosome 1 and knocks out the GGTA1 gene. The aforementioned CMAH knockout recombinant vector recognizes exon 9 of porcine chromosome 7 and knocks out the CMAH gene. The aforementioned recombinant vector for iGb3s knockout recognizes exon 4 of porcine chromosome 6 and knocks out the iGb3s gene. The aforementioned recombinant vector for β4GalNT2 knockout recognizes exon 1 of porcine chromosome 12 and knocks out the β4GalNT2 gene. The transformed cells according to claim 1, characterized in that the β2M knockout recombinant vector recognizes exon 2 of porcine chromosome 1 and knocks out the β2M gene.

4. The transformed cell according to claim 1, characterized in that the transformed cell has accession number KCLRF-BP-00515.

5. The step of transplanting the transformed cells according to any one of claims 1 to 4 into an enucleated oocyte to form a nuclear transfer egg; and A method for producing a transformed replica pig for xenotransplantation, comprising the step of transplanting the nuclear transplant egg into the fallopian tube of a surrogate mother.

6. A transformed replica pig for xenotransplantation, manufactured by the method described in claim 5.

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