A method for constructing a donor pig for eight-gene-edited xenotransplantation

Abstract

The present invention relates to a method for constructing an eight-gene-edited xenogeneic organ transplantation donor pig, belonging to the field of animal biotechnology. In the wild-type porcine fetal fibroblast cell line, the GGTA1, β4GalNT2, and CMAH genes are knocked out by using the CRISPR / Cas9 gene editing technology, and the humanized genes of hCD39, hCD46, hCD55, hCD59, and hTBM are transfected. Combining with somatic cell cloning technology, GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene-edited cloned pigs are constructed. Further, through genotype, mRNA, protein expression identification and functional analysis, eight-gene-edited xenogeneic organ transplantation donor pigs are obtained. The present invention solves the technical problems of high production difficulty, low efficiency, and low survival rate of donor pigs for multi-gene-edited xenogeneic organ transplantation, and maximally solves the common problems of immune rejection reaction and complement dysregulation faced during xenogeneic organ transplantation, laying a foundation for more targeted development of donor pigs suitable for different tissue and organ xenotransplantation, and having important value for promoting the clinical transformation of xenogeneic organ transplantation.

Description

Technical Field

[0001] The present invention belongs to the field of animal biotechnology, and specifically relates to a method for constructing an eight-gene-edited donor pig for xenotransplantation. Background Art

[0002] Organ transplantation is a recognized major means for treating patients with organ failure in the international biomedical field. Approximately 200,000 people worldwide wait for organ transplantation every year, and the average organ supply-demand ratio is less than 1:20. The shortage of organ donors has become a major medical problem that attracts attention and urgently needs to be solved in the world's public health field. Xenotransplantation has been recognized as an effective way to address the global shortage of human organ transplantation supply and demand. In January 2023, the University of Maryland School of Medicine in the United States transplanted a 10-gene-edited pig heart into a 57-year-old heart disease patient for the first time, and the patient survived for 2 months after transplantation. In August 2023, the University of Alabama at Birmingham transplanted a 10-gene-edited pig kidney into a human body, and it functioned normally for 1 week. In September 2023, the University of Maryland School of Medicine in the United States successfully transplanted a 10-gene-edited pig heart into a patient with peripheral vascular disease and internal bleeding complications for the second time. After transplantation, the patient could breathe independently and survived successfully for 6 weeks.

[0003] Xenotransplantation still needs to overcome problems such as rejection reaction, control of inflammation, and regulation of coagulation disorders. To overcome these obstacles, gene manipulation for xenotransplantation mainly includes two purposes: (I) inactivating pig-derived xenoantigens; (II) transgenic expression of human protective proteins, including coagulation regulators, complement regulators, cellular immune response factors, anti-apoptosis and anti-inflammatory factors, etc. In recent years, with the progress of gene editing technology, various gene-edited donor pigs have been successively reported. The current results of subclinical studies on pig-to-human xenotransplantation also effectively prove that adding knockout of some other xenoantigen genes and humanized gene expression on the basis of GGTA1 gene knockout helps to reduce the immune rejection reaction generated by xenotransplantation. For the modification of these major xenoantigen genes and a large number of humanized gene expressions, the existing gene editing technologies generally have common problems such as difficult simultaneous editing of multiple gene combinations, low efficiency, low survival rate of multi-gene-edited cloned pigs, and uneven expression of target genes. Summary of the Invention

[0004] To address the above problems, the present invention provides a method for constructing an eight-gene edited xenotransplantation donor pig. This method aims at the common problem of immune rejection during xenotransplantation. By using the CRISPR / Cas9 gene editing technology, the piggyBac transposon technology, and combining with somatic cell cloning technology, a GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited xenotransplantation donor pig is constructed, which is of great significance for carrying out preclinical research on pig-non-human primate xenotransplantation and the clinical application of pig-human xenotransplantation.

[0005] To achieve the above object, the present invention is realized through the following technical solutions:

[0006] In the first aspect of the present invention, sgRNA targeting vectors for GGTA1, β4GalNT2, and CMAH genes are provided. Using the CRISPR / Cas9 gene editing technology, sgRNA targeting vectors for GGTA1, β4GalNT2, and CMAH genes are constructed, including: GGTA1-sgRNA1, GGTA1-sgRNA2, β4GalNT2-sgRNA1, β4GalNT2-sgRNA2, β4GalNT2-sgRNA3, CMAH-sgRNA1, and CMAH-sgRNA2.

[0007] Furthermore, the action sites of the above sgRNAs are located in the 3rd exon of the porcine GGTA1 gene, the 2nd exon of the β4GalNT2 gene, and the 4th exon of the CMAH gene.

[0008] Furthermore, the nucleotide sequence of the above GGTA1-sgRNA1 is as shown in SEQ ID NO:1; the nucleotide sequence of GGTA1-sgRNA2 is as shown in SEQ ID NO:2; the nucleotide sequence of β4GalNT2-sgRNA1 is as shown in SEQ ID NO:3; the nucleotide sequence of β4GalNT2-sgRNA2 is as shown in SEQ ID NO:4; the nucleotide sequence of β4GalNT2-sgRNA3 is as shown in SEQ ID NO:5; the nucleotide sequence of CMAH-sgRNA1 is as shown in SEQ ID NO:6; the nucleotide sequence of CMAH-sgRNA2 is as shown in SEQ ID NO:7.

[0009] Furthermore, for the above sgRNA targeting vectors of GGTA1, β4GalNT2, and CMAH genes, the backbone vector they are connected to is PX458-sgRNA-EGFP (Addgene no: 112220).

[0010] In the second aspect of the present invention, there is provided a recombinant plasmid for knocking out the GGTA1, β4GalNT2, and CMAH genes encoded in the pig genome, and its nucleotide sequence is as shown in SEQ ID NO:8.

[0011] In the third aspect of the present invention, there is provided an expression vector into which the humanized genes of hCD39, hCD46, hCD55, hCD59, and hTBM are transferred.

[0012] Furthermore, the nucleotide sequence of hCD39 is as shown in SEQ ID NO:9; the nucleotide sequence of hCD46 is as shown in SEQ ID NO:10; the nucleotide sequence of hCD55 is as shown in SEQ ID NO:11; the nucleotide sequence of hCD59 is as shown in SEQ ID NO:12; the nucleotide sequence of hTBM is as shown in SEQ ID NO:13.

[0013] Furthermore, the backbone vector to which the humanized genes of hCD39, hCD46, hCD55, hCD59, and hTBM are transferred into the expression vector is the PiggyBac transposon.

[0014] Furthermore, the nucleotide sequence of the expression vector into which the humanized genes of hCD39, hCD46, hCD55, hCD59, and hTBM are transferred and constructed based on the PiggyBac transposon system is as shown in SEQ ID NO:14.

[0015] In the fourth aspect of the present invention, there is provided a GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line. The sgRNA targeting vectors of the GGTA1, β4GalNT2, and CMAH genes and the recombinant plasmid for knocking out the GGTA1, β4GalNT2, and CMAH genes in the pig genome are co-transfected into wild-type porcine fetal fibroblasts under the action of transposase, and the obtained eight-gene edited positive monoclonal cell line is the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line.

[0016] Furthermore, the blood type of the wild-type porcine fetal fibroblast cell line without gene editing is type O, and the good compatibility of type O blood can reduce the immune rejection reaction caused by blood type incompatibility in xenotransplantation.

[0017] In the fifth aspect of the present invention, there is provided a method for constructing an eight-gene edited donor pig for xenotransplantation, and the specific steps are as follows:

[0018] (1)Based on the CRISPR / Cas9 gene editing system, construct the GTKO / β4GalNT2KO / CMAHKO gene targeting vector

[0019] Design sgRNA targeting sequences for exon 3 of the GGTA1 gene, exon 2 of the β4GalNT2 gene, and exon 4 of the CMAH gene in the pig genome, and ligate them to the backbone vector to obtain the GTKO, β4GalNT2KO, and CMAHKO gene sgRNA targeting vectors; among them, the nucleotide sequence of GGTA1-sgRNA1 is shown in SEQ ID NO:1; the nucleotide sequence of GGTA1-sgRNA2 is shown in SEQ ID NO:2; the nucleotide sequence of β4GalNT2-sgRNA1 is shown in SEQ ID NO:3; the nucleotide sequence of β4GalNT2-sgRNA2 is shown in SEQ ID NO:4; the nucleotide sequence of β4GalNT2-sgRNA3 is shown in SEQ ID NO:5; the nucleotide sequence of CMAH-sgRNA1 is shown in SEQ ID NO:6; the nucleotide sequence of CMAH-sgRNA2 is shown in SEQ ID NO:7; the nucleotide sequence of the targeting sgRNA recombinant plasmid for knocking out the GGTA1, β4GalNT2, and CMAH genes is shown in SEQ ID NO:8.

[0020] (2)Based on the piggyBac transposon system, construct the hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized gene transfer expression vector

[0021] The hCD39, hCD46, hCD55, hCD59, and hTBM humanized gene transfer expression vectors constructed based on the piggyBac transposon system, the nucleotide sequence of hCD39 is shown in SEQ ID NO:9; the nucleotide sequence of hCD46 is shown in SEQ ID NO:10; the nucleotide sequence of hCD55 is shown in SEQ ID NO:11; the nucleotide sequence of hCD59 is shown in SEQ ID NO:12; the nucleotide sequence of hTBM is shown in SEQ ID NO:13; the nucleotide sequence of the hCD39, hCD46, hCD55, hCD59, and hTBM humanized gene transfer expression vector is shown in SEQ ID NO:14.

[0022] (3)Construct the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line

[0023] The GGTA1, β4GalNT2, and CMAH gene sgRNA targeting vectors and the hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized gene were transferred into an expression vector and co-transfected into a wild-type porcine fetal fibroblast cell line under the action of a transposase. The transfection method includes liposome transfection or electroporation, preferably electroporation. After monoclonal cell genotype identification, the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line was obtained.

[0024] (4) Somatic cell nuclear transfer and embryo transfer

[0025] Using the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited positive monoclonal fibroblasts or cloned fetal fibroblasts or cloned porcine fibroblasts for somatic cell nuclear transfer, preferably cloned porcine fetal fibroblasts, to construct GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited cloned embryos. Further, the eight-gene edited cloned embryos were transferred into the body of an estrous surrogate sow for development. After 114 days of pregnancy, GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited cloned pigs were obtained by natural delivery, or the fetuses were taken out during pregnancy for genotype identification and a fetal fibroblast cell line was established.

[0026] (5) Genotype identification and phenotypic analysis of cloned pigs

[0027] Genomic DNA of cloned piglets was extracted, and the genotypes of cloned pigs were identified by PCR, T7ENI, and Sanger sequencing techniques to obtain GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited cloned pig individuals. Further, the mRNA and protein expression levels in different tissues and organs of the eight-gene edited cloned pigs were detected by Western blotting, immunofluorescence staining, flow cytometry, HE staining, and cross-matching molecular biology techniques.

[0028] 6) Using continuous cloning technology, perform somatic cell nuclear transfer on the eight-gene edited porcine fetal fibroblast cell line obtained in step 3) above or the eight-gene edited porcine fibroblast cell line obtained in step 5), and transplant the cloned embryos into the body of a surrogate sow. After protein identification, mass-produce GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited donor pigs for xenotransplantation.

[0029] The above GGTA1, β4GalNT2, CMAH gene sgRNA targeting vectors, recombinant plasmids for knocking out GGTA1, β4GalNT2, CMAH genes in the porcine genome, hCD39, hCD46, hCD55, hCD59, hTBM humanized gene transfer expression vectors, GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line, and / or the construction method of eight-gene edited donor pigs for xenotransplantation in the application of biomedical research in the field of xenotransplantation.

[0030] In order to overcome obstacles such as rejection, inflammation, and coagulation disorders in xenogeneic organs, gene manipulation of donor pigs for xenotransplantation mainly includes two purposes: (I) inactivating porcine-derived xenoantigens; (II) transgenic expression of human protective proteins, including coagulation regulators, complement regulators, cellular immune response factors, anti-apoptotic and anti-inflammatory factors, etc.

[0031] Inactivation of GGTA1 (producing α-Gal antigen), CMAH (producing Neu5Gc antigen), and β4GalNT2 (producing Sda antigen) in pigs can effectively reduce the occurrence of antigen-antibody-mediated hyperacute and acute rejection reactions in xenogeneic kidney transplantation. Knocking out CMAH and β4GalNT2 genes on the basis of knocking out the GGTA1 gene will more effectively alleviate the immune rejection reaction after xenotransplantation. Compared with GGTA1 single-gene and GGTA1 / CMAH two-gene deletion cells, the binding of GGTA1 / CMAH / β4GALNT2 triple-gene knockout porcine cells to xenogeneic IgM and IgG in vitro is significantly reduced, indicating that GGTA1 / CMAH / β4GalNT2 triple-gene knockout pigs have lower immunogenicity and are more capable of ensuring the long-term survival of xenografts.

[0032] Expressing human complement regulatory proteins (CD46, CD55, CD59) and thrombomodulin (CD39, TBM) in donor pigs for xenotransplantation can correct the disorders of complement and coagulation caused by species incompatibility. Compared with GTKO single-gene edited pigs, the survival time of kidneys transplanted from GTKO / hCD55 two-gene edited pigs into non-human primates can reach 499 days, suggesting that while knocking out xenogeneic antigen genes, transferring human complement regulatory proteins such as CD46, CD55, CD59, etc. into pigs can effectively alleviate the rejection reaction caused by complement activation and extend the survival period of xenogeneic kidney transplantation.

[0033] The results effectively prove that adding the knockout of other xenogeneic antigen genes and the expression of humanized genes on the basis of GGTA1 knockout helps to reduce the immune rejection reaction generated by xenotransplantation. Therefore, the GTKO / CMAHKO / β4GalNT2KO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited donor pigs for xenotransplantation constructed in the present invention contribute to solving the antigen-antibody mediated immune rejection reaction faced by pig-to-human xenotransplantation, and have important significance for carrying out preclinical research on pig-to-non-human primate xenotransplantation and clinical trials of pig-to-human xenotransplantation.

[0034] The beneficial effects of the present invention:

[0035] The present invention designs multiple different sgRNA nucleotide sequences for the targeting vector of gene knockout, and at the same time constructs expression vectors for simultaneous editing of multiple genes. While improving the gene targeting success rate, it overcomes the technical problems commonly existing in the CRISPR / Cas9 technology, such as low efficiency of multi-gene editing, low birth efficiency of cloned pigs after multi-gene modification, and difficulty in survival.

[0036] The present invention discloses a method for constructing an eight-gene edited donor pig for xenotransplantation. Through different gene combination modifications, the inactivation of three carbohydrate antigen genes in the pig genome and the overexpression of humanized genes in the pig genome are achieved, which solves the common problems such as hyperacute immune rejection reaction and antigen-antibody mediated immune rejection reaction existing in the process of xenotransplantation to a certain extent, and lays a foundation for further developing donor pigs suitable for xenotransplantation of tissues or organs such as heart, kidney, liver, skin, etc. more specifically, and has important prospects for carrying out preclinical research on pig-to-non-human primate xenotransplantation and clinical application of pig-to-human xenotransplantation. Brief Description of the Drawings

[0037] Figure 1 Schematic diagram of the construction of GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited cloned pigs and vectors;

[0038] Figure 1 In A, it is a schematic diagram for constructing GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM octa-gene edited cloned pigs;

[0039] Figure 1 In B, it is a schematic diagram for gene knockout targeting of GTKO / β4GalNT2KO / CMAHKO;

[0040] Figure 1 In C, it is a schematic diagram for constructing gene editing vectors of CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM.

[0041] Figure 2 It is the PCR identification result of gene editing of hCD39 / hCD46 / hCD55 / hCD59 / hTBM in monoclonal porcine fibroblast cell lines.

[0042] Figure 3 It is the PCR and Sanger sequencing identification results of gene editing of GTKO / β4GalNT2KO / CMAHKO in monoclonal porcine fibroblast cell lines;

[0043] Figure 3 In A, it is the PCR identification result of GGTA1, β4GalNT2, and CMAH genes in monoclonal porcine fibroblast cell lines;

[0044] Figure 3 In B, it is the Sanger sequencing identification result of PCR products of GGTA1, β4GalNT2, and CMAH genes in monoclonal porcine fibroblast cell lines.

[0045] Figure 4 It is the genotype identification result of GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM octa-gene edited cloned pig fetuses;

[0046] Figure 4 In A, it is the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM octa-gene edited cloned pig fetuses;

[0047] Figure 4 In B, it is the PCR identification result of GGTA1, β4GalNT2, and CMAH genes in the genome of cloned pig fetuses;

[0048] Figure 4 In C, it is the Sanger sequencing identification result of PCR products of GGTA1, β4GalNT2, and CMAH genes in the genome of cloned pig fetuses.

[0049] Figure 5 Identification results of mRNA and protein expression levels of GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited cloned pigs.

[0050] Figure 5 In A, it is GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited cloned pigs;

[0051] Figure 5 In B, it is the mRNA expression level of hCD46 gene in the heart, liver, kidney and lung of cloned pigs;

[0052] Figure 5 In C, it is the mRNA expression level of hCD55 gene in the heart, liver, kidney and lung of cloned pigs;

[0053] Figure 5 In D, it is the mRNA expression level of hCD59 gene in the heart, liver, kidney and lung of cloned pigs;

[0054] Figure 5 In E, it is the mRNA expression level of hTBM gene in the heart, liver, kidney and lung of cloned pigs;

[0055] Figure 5 In F, it is the mRNA expression level of hCD39 gene in the heart, liver, kidney and lung of cloned pigs;

[0056] Figure 5 In G, it is the immunofluorescence detection results of GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM gene expression in cloned pig tissues;

[0057] Figure 5 In H, it is the binding ability of peripheral blood mononuclear cells of eight-gene edited cloned pigs to monkey IgG;

[0058] Figure 5 In I, it is the binding ability of peripheral blood mononuclear cells of eight-gene edited cloned pigs to monkey IgM;

[0059] Figure 5 In J, it is the analysis of the cell survival level of eight-gene edited cloned pigs by monkey complement-dependent cytotoxicity assay. Specific embodiments

[0060] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below to facilitate understanding by those skilled in the art.

[0061] Example 1: Construction of an eight-gene editing targeting vector

[0062] (1) Construction of GTKO / β4GalNT2KO / CMAHKO gene targeting vector based on CRISPR / Cas9 gene editing technology

[0063] The nucleotide sequences of the GGTA1 (Gene ID: 396733), β4GalNT2 (Gene ID: 100621328), and CMAH (Gene ID: 396918) genes of pigs were obtained by searching in the NCBI database. For the 3rd exon of the porcine GGTA1 gene, the 2nd exon of the β4GalNT2 gene, and the 4th exon of the CMAH gene, online software (http: / / crispor.tefor.net / ) was used to design and screen the targeting sgRNA sites, and the sgRNA was ligated to the backbone vector PX458-sgRNA-EGFP (Addgene no: 112220) to obtain the targeting vectors GGTA1-sgRNA1 (SEQ ID NO: 1), GGTA1-sgRNA2 (SEQ ID NO: 2), β4GalNT2-sgRNA1 (SEQ ID NO: 3), β4GalNT2-sgRNA2 (SEQ ID NO: 4), β4GalNT2-sgRNA3 (SEQ ID NO: 5), CMAH-sgRNA1 (SEQ ID NO: 6), and CMAH-sgRNA2 (SEQ ID NO: 7) ( Figure 1 in B).

[0064] (2) Construction of hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized gene transfer expression vector based on the piggyBac system

[0065] Based on the piggyBac system, the nucleotide sequences of the hCD39 (SEQ ID NO: 9), hCD46 (SEQ ID NO: 10), hCD55 (SEQ ID NO: 11), hCD59 (SEQ ID NO: 12), and hTBM (SEQ ID NO: 13) genes were ligated to the piggyBac transposon vector to obtain the hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized gene transfer expression vector (SEQ ID NO: 14) ( Figure 1 in C).

[0066] Example 2: Construction of eight-gene editing cloned pigs

[0067] For the 3rd exon of the porcine GGTA1 gene (Gene ID: 396733), the 2nd exon of the β4GalNT2 gene (Gene ID: 100621328), and the 4th exon of the CMAH gene (Gene ID: 396918), online software (http: / / crispor.tefor.net / ) was used to design and screen target sgRNA sites, and the sgRNA sequences were ligated to the backbone vector (PX458-sgRNA-EGFP) to obtain the GGTA1-sgRNA1 (SEQ ID NO:1), GGTA1-sgRNA2 (SEQ ID NO:2), β4GalNT2-sgRNA1 (SEQ ID NO:3), β4GalNT2-sgRNA2 (SEQ ID NO:4), β4GalNT2-sgRNA3 (SEQ ID NO:5), CMAH-sgRNA1 (SEQ ID NO:6), and CMAH–sgRNA2 (SEQ ID NO:7) targeting vectors ( Figure 1 in B). Based on the piggyBac system, the nucleotide sequences of the hCD39 (SEQ ID NO:9), hCD46 (SEQ ID NO:10), hCD55 (SEQ IDNO:11), hCD59 (SEQ ID NO:12), and hTBM (SEQ ID NO:13) genes were ligated to the piggyBac transposon to obtain the hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized gene transfer expression vector (SEQ ID NO:10) ( Figure 1 in C). The GGTA1, β4GalNT2, and CMAH gene sgRNA targeting vectors and the hCD39, hCD46, hCD55, hCD59, and hTBM humanized gene transfer expression vectors were co-transfected into wild-type porcine fetal fibroblast cell lines under the action of transposase. After 48 hours of screening with puromycin and extreme dilution culture, a total of 25 single-cell clones were obtained. After genotype identification, the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line was successfully obtained. Using the C12# positive monoclonal cell line as the donor cell, somatic cell nuclear transfer was performed to construct cloned embryos, which were transplanted into the uteri of estrus surrogate sows. After 114 days of pregnancy, natural delivery occurred, and eight-gene edited cloned pigs were obtained ( Figure 5 in A). When the surrogate sows were 33 days pregnant, 1 live fetus was obtained by cesarean section ( Figure 4In A), the fibroblast cell line was isolated and the gene editing status of GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM was identified. The results showed that GGTA1, β4GalNT2, and CMAH were both knocked out at the biallelic level ( Figure 4 In B-C), the success rate of octa-gene editing in cloned pig fetuses was 100%. Further, using these as donor cells, somatic cell nuclear transfer was performed to produce octa-gene edited cloned pigs.

[0068] Example 3: Genotype identification and phenotype analysis of octa-gene edited cloned pigs

[0069] Using octa-gene edited fetal fibroblasts as donor cells for somatic cell nuclear transfer, the cloned embryos were respectively transferred into 17 surrogate sows, and 6 became pregnant. After 114 days of pregnancy, a total of 28 cloned pigs were obtained by natural delivery, and 24 of them survived ( Figure 5 In A), all were octa-gene edited cloned pigs, and the individual survival rate was 85.71%. Further, using human umbilical vein endothelial cells as a positive control, the expression of 5 humanized genes in the heart, kidney, liver, and lung tissues of octa-gene edited piglets was detected by q-PCR. Compared with the wild type, in octa-gene edited cloned piglets, hCD46 ( Figure 5 In B), hCD55 ( Figure 5 In C), hCD59 ( Figure 5 In D), hTBM ( Figure 5 In E), hCD39 ( Figure 5 In F), the mRNA of the genes showed different expression levels in different tissues of the heart, liver, kidney, and lung. By immunofluorescence staining, the knockout of 3 xenoantigens and the expression of 5 humanized proteins in the kidney tissue of octa-gene edited cloned pigs were verified. Compared with wild-type pigs, GGTA1, CMAH, and β4GalNT2 were successfully inactivated in the kidneys of octa-gene edited cloned pigs, and hTBM, hCD39, hCD46, hCD55, and hCD59 showed different expression patterns ( Figure 5 In G), at the same time, cross-matching experiments were performed using monkey serum and PBMC cells of octa-gene edited cloned pigs. Compared with wild-type pigs, the ability of peripheral blood mononuclear cells (PBMC) of octa-gene edited cloned pigs to bind monkey IgG and IgM was significantly reduced ( Figure 5 In H-I), the complement-dependent cytotoxicity experiment further confirmed that octa-gene edited cloned pigs helped protect pig cells from complement attack, thus increasing the survival rate of pig cells ( Figure 5 In J), indicating that the construction of octa-gene edited donor pigs for xenotransplantation was successful.

[0070] In the embodiments, for the common problem of immune rejection in xenotransplantation, the eight-gene-edited xenotransplantation donor pigs constructed by the present invention can accelerate the research and development process of xenotransplantation donor pigs, and maximize the solution of the common problems in the development of xenotransplantation donor pigs and the process of xenotransplantation. At the same time, based on the eight-gene-edited xenotransplantation donor pigs, different gene editing strategies can be further adopted to perform relevant gene modifications for the scientific problems faced by different cell, tissue, and organ xenotransplantations, which has important application value for the development of effective xenotransplantation donor pigs using multi-gene combination modifications.

[0071] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line, characterized in that: The sgRNA targeting vectors for GGTA1, β4GalNT2, and CMAH genes, or the recombinant plasmids used to knockout GGTA1, β4GalNT2, and CMAH genes in the pig genome, and the expression vectors into which the hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized genes are transferred are co-transfected into wild-type porcine fetal fibroblasts under the action of transposase. The obtained eight-gene-edited positive monoclonal cell line is the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene-edited porcine fetal fibroblast cell line; The sgRNA targeting vectors for GGTA1, β4GalNT2, and CMAH genes are constructed using the CRISPR / Cas9 gene editing technology; the sgRNA action sites are located in the 3rd exon of the porcine GGTA1 gene, the 2nd exon of the β4GalNT2 gene, and the 4th exon of the CMAH gene; The sgRNA targeting vectors for GGTA1, β4GalNT2, and CMAH genes include: GGTA1-sgRNA1, GGTA1-sgRNA2, β4GalNT2-sgRNA1, β4GalNT2-sgRNA2, β4GalNT2-sgRNA3, CMAH-sgRNA1, CMAH-sgRNA2; The nucleotide sequence of GGTA1-sgRNA1 is shown in SEQ ID NO:1; The nucleotide sequence of GGTA1-sgRNA2 is shown in SEQ ID NO:2; The nucleotide sequence of β4GalNT2-sgRNA1 is shown in SEQ ID NO:3; The nucleotide sequence of β4GalNT2-sgRNA2 is shown in SEQ ID NO:4; The nucleotide sequence of β4GalNT2-sgRNA3 is shown in SEQ ID NO:5; The nucleotide sequence of CMAH-sgRNA1 is shown in SEQ ID NO:6; The nucleotide sequence of CMAH-sgRNA2 is shown in SEQ ID NO:7; The backbone vector of the sgRNA targeting vectors for GGTA1, β4GalNT2, and CMAH genes is PX458-sgRNA-EGFP, and its gene number is: Addgene no: 112220; The nucleotide sequence of the recombinant plasmid used to knockout GGTA1, β4GalNT2, and CMAH genes in the pig genome is shown in SEQID NO:

8.

2. A method for constructing an eight-gene-edited xenotransplantation donor pig, characterized in that: The specific steps are as follows: (1) Based on the CRISPR / Cas9 gene editing system, construct a recombinant plasmid for knocking out GGTA1, β4GalNT2, and CMAH genes in the pig genome Design sgRNA targeting vectors for exon 3 of the GGTA1 gene, exon 2 of the β4GalNT2 gene, and exon 4 of the CMAH gene in the porcine genome, and ligate them to the backbone vector to obtain GTKO, β4GalNT2KO, and CMAHKO gene sgRNA targeting vectors, including: GGTA1-sgRNA1, GGTA1-sgRNA2, β4GalNT2-sgRNA1, β4GalNT2-sgRNA2, β4GalNT2-sgRNA3, CMAH-sgRNA1, CMAH-sgRNA2; The nucleotide sequence of GGTA1-sgRNA1 is shown in SEQ ID NO:1; The nucleotide sequence of GGTA1-sgRNA2 is shown in SEQ ID NO:2; The nucleotide sequence of β4GalNT2-sgRNA1 is shown in SEQ ID NO:3; The nucleotide sequence of β4GalNT2-sgRNA2 is shown in SEQ ID NO:4; The nucleotide sequence of β4GalNT2-sgRNA3 is shown in SEQ ID NO:5; The nucleotide sequence of CMAH-sgRNA1 is shown in SEQ ID NO:6; The nucleotide sequence of CMAH-sgRNA2 is shown in SEQ IDNO:7; The nucleotide sequence of the recombinant plasmid for knocking out GGTA1, β4GalNT2, and CMAH genes in the porcine genome is shown in SEQID NO:8; (2) Based on the piggyBac transposon system, construct an expression vector for the transfer of hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized genes Expression vectors for the transfer of hCD39, hCD46, hCD55, hCD59, and hTBM humanized genes constructed based on the piggyBac transposon system. The nucleotide sequence of hCD39 is shown in SEQ ID NO:9; the nucleotide sequence of hCD46 is shown in SEQ ID NO:10; the nucleotide sequence of hCD55 is shown in SEQ ID NO:11; the nucleotide sequence of hCD59 is shown in SEQ ID NO:12; the nucleotide sequence of hTBM is shown in SEQ ID NO:13; (3) Construct a GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line The recombinant plasmids for knocking out GGTA1, β4GalNT2, and CMAH genes in the pig genome and the hCD39 / hCD46 / hCD55 / hCD59 / hTBM humanized genes were transferred into an expression vector and co-transfected into wild-type porcine fetal fibroblast cells under the action of a transposase. The transfection methods include liposome transfection or electroporation, preferably electroporation. After monoclonal cell genotype identification, the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line was obtained; (4)Somatic cell nuclear transfer and embryo transfer Using GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited positive monoclonal fibroblasts or cloned fetal fibroblasts or cloned porcine fibroblasts as donor cells for somatic cell nuclear transfer, preferably cloned fetal fibroblasts, to construct GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine cloned embryos. Further, the cloned embryos were transferred into the body of estrus surrogate sows for development, and GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited cloned pigs were obtained by natural delivery, or the fetuses were taken out during pregnancy for genotype identification and fibroblast cell lines were established; (5)Genotype identification and phenotype analysis of cloned pigs Genomic DNA of cloned piglets was extracted, and the genotypes of cloned piglets were identified by PCR, T7ENI, and Sanger sequencing technologies. By Western blotting, immunofluorescence staining, flow cytometry, HE staining, cross-matching experiment molecular biology technologies, the mRNA and protein expression levels of different tissues and organs of eight-gene edited cloned pigs were detected to obtain GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited cloned pigs; (6)Using continuous cloning technology, the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line obtained in step 3) or the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fibroblast cell line obtained in step 5) was used for somatic cell nuclear transfer, and the cloned embryos were transferred into the body of surrogate sows. After protein function identification, GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited donor pigs for xenotransplantation were mass-produced.

3. The construction method of an eight-gene-edited xenotransplantation donor pig according to claim 2, characterized in that: The nucleotide sequence of the hCD39, hCD46, hCD55, hCD59, hTBM humanized gene transfer expression vector constructed based on the PiggyBac transposon system in step 2) is shown in SEQ ID NO:

14.

4. Use of the GTKO / β4GalNT2KO / CMAHKO / hCD39 / hCD46 / hCD55 / hCD59 / hTBM eight-gene edited porcine fetal fibroblast cell line according to claim 1, and / or use of a method for constructing an eight-gene edited xenotransplantation donor pig according to any one of claims 2-3 in the field of biomedical research on xenotransplantation, wherein the use is a non-diagnostic or therapeutic method for diseases.

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