KR102363891B1 - Transgenic pigs expressing both shTNFRI-Fc and HA-HO-1 without Gal epitope and N-glycolylneuraminic acid epitope and the Use of thereof - Google Patents

Abstract

The present invention simultaneously expresses the HO-1 (Heme oxygenase-1) gene and the TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) gene, and the GGTA1 (α-1,3-galactosyltransferase) gene and CMAH (cytidine monophosphate-N-) The present invention relates to a transgenic pig in which the acetylneuraminic acid hydroxylase) gene is knocked out, immune rejection is suppressed during xenogeneic organ transplantation, and a method for manufacturing the same.
Transgenic pigs in which the human HO-1 and TNFR1-Fc fusion genes of the present invention are co-expressed and the GGTA1 gene and CMAH gene are knocked out during organ isolation and in vitro culture due to the antioxidant and cytoprotective functions of the HO-1 gene It can reduce oxidative stress, and can also reduce the TNF-α-mediated inflammatory response that occurs at the early stage of transplantation by TNFR1-Fc expression. Also, it inhibits the maturation of dendritic cells and regulates the proliferation and activation of T cells in the acute vasculature. It can promote early engraftment of transplanted organs by reducing rejection. Furthermore, it is possible to increase the survival rate of transplanted organs by suppressing the hyperacute immune rejection by GGTA1 and the acute immune response by CMAH. Accordingly, an organ in which immune rejection is suppressed during xenotransplantation can be produced using the transgenic pig.

Description

Transgenic pigs expressing both HO-1 and TNF1-F genes and knocking out GGTA1 and CMAH genes and their uses {Transgenic pigs expressing both shTNFRI-Fc and HA-HO-1 without Gal epitope and N-glycolylneuraminic acid epitope and the Use of them}

The present invention simultaneously expresses the HO-1 (Heme oxygenase-1) gene and the TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) gene, and the GGTA1 (α-1,3-galactosyltransferase) gene and CMAH (cytidine monophosphate-N-) The present invention relates to a transgenic pig in which the acetylneuraminic acid hydroxylase) gene is knocked out, immune rejection is suppressed during xenogeneic organ transplantation, and a method for manufacturing the same.

In the case of end-stage organ failure patients, when organ functions are completely lost and treatment such as drug treatment is no longer possible, the only treatment method is to transplant a new replacement organ. However, there is a problem in that the supply of transplanted organs to be donated is remarkably insufficient, and in various attempts to solve this problem, a method of transplanting an organ between different types of organs is utilized. can be considered.

Xenogeneic organ transplantation using animals capable of securing sufficient donor organs is emerging as an alternative to replace scarce human organs. However, primates are an endangered species, difficult to reproduce, socially repulsive to use as a source of heterogeneous organs, and have a high risk of infection, so it is difficult to use them as donors of heterogeneous organs. On the other hand, mini-pigs are similar in organ size to humans, anatomically and physiologically similar to humans, have short sexual maturity and gestation period, and are highly productive because they are multiple animals (6 to 12 live offspring produced per time). , since it is an animal that has been with humans for a long time, it is unlikely to carry a lethal source of infection to humans. In addition, it has the advantage that transformation and aseptic breeding are possible, so the possibility of using mini-pigs as a source of heterogeneous organs is highly evaluated.

Immune rejection is a serious problem in the case of heterogeneous organ transplantation using pigs and the like. Accordingly, the present inventors express a human HO-1 (Heme oxygenase-1) gene and a human TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) fusion gene and knock out the GGTA1 gene to protect cells from oxidative stress and A transgenic pig with suppressed hyperacute immune rejection capable of suppressing the inflammatory response after transplantation was prepared (Korean Patent Application Laid-Open No. 10-2011-0079485). However, the transgenic pig xenograft was not resistant to acute rejection (AR).

Natural antibodies that respond to xenoantigens possessed by pigs are present in humans and some primates, which is a major cause of the rapid antibody-mediated immune response that occurs after xenotransplantation. Therefore, production of pigs from which these xenogeneic antigens have been removed is an essential requirement for successful xenogeneic islet transplantation. The heterologous antigens that respond to natural antibodies include α-gal antigen and non-gal antigen. Among the non-gal antigens, the N-glycolylneuraminic acid antigen is representative. In order to remove it, the GGTA1 gene or N-glycolylneuraminic gene that synthesizes the α-gal antigen It is necessary to develop pigs in which the CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) gene, which synthesizes an acid antigen, has been removed. In order to reduce oxidative stress and inflammatory response during xenogeneic transplantation, and to solve hyperacute immune rejection, a transgenic pig in which human HO-1 gene and human TNFR1-Fc fusion gene was introduced and GGTA1 gene was knocked out was developed. did However, this pig cannot be expected to overcome the acute rejection reaction.

Accordingly, the present inventors made intensive efforts to develop a transgenic pig for organ transplantation, in which oxidative stress and inflammatory responses are reduced, and there is no hyperacute rejection or acute immune rejection by removing the gene of CMAH that synthesizes a representative non-Gal antigen. As a result, the present invention was completed by developing a transgenic pig in which the human HO-1 gene and the human TNFR1-Fc fusion gene were introduced and the GGTA1 and CMAH genes were knocked out.

One object of the present invention is to introduce a human HO-1 (Heme oxygenase-1) gene and a human TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) fusion gene and GGTA1 (α-1,3-galactosyltransferase) gene and CMAH To provide a transgenic pig in which a gene is knocked out, immune rejection is suppressed during xenogeneic organ transplantation, and a method for producing the same.

Another object of the present invention is to provide a somatic cell donor for producing the transgenic pig.

Another object of the present invention is to prepare the transgenic pig; And to provide a method for producing an organ in which immune rejection is suppressed during xenogeneic organ transplantation, comprising the step of isolating an organ for transplantation from the transgenic pig.

As an aspect for achieving the above object, the present invention provides a method comprising: a) isolating somatic cells from pigs; b) introducing a human HO-1 (Heme oxygenase-1) gene and a human TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) fusion gene into the somatic cells, GGTA1 (α-1,3-galactosyltransferase) gene and CMAH ( cytidine monophosphate-N-acetylneuraminic acid hydroxylase) gene knockout (knock-out); c) selecting and culturing somatic cells into which the human HO-1 gene and the human TNFR1-Fc fusion gene are introduced and the GGTA1 gene and the CMAH gene are knocked out; And d) removing the nucleus of the pig egg and fusion with the selected somatic cells to prepare a fertilized egg in which the somatic cell nucleus has been transplanted. .

An object of the present invention is to produce a transgenic pig having these characteristics in order to produce an organ in which immune rejection is suppressed during xenogeneic organ transplantation. Accordingly, in the present invention, the human HO-1 gene and the human TNFR1-Fc fusion gene were introduced and expressed in the somatic cells of the isolated pig, and the GGTA1 gene and the CMAH gene were knocked out to prepare a transgenic pig. Hereinafter, each step of the manufacturing method will be described in detail.

As used herein, the term "transgenic pig" refers to a pig in which a part of the trait is changed, such as by recombination of an externally introduced gene, artificially inserting it into the pig's chromosome, or deleting a gene inherently in the pig. The animal type is preferably a pig, but the method of the present invention can be applied to an animal capable of transplanting an organ into a human as a natural mammal to produce a transgenic animal in which immune rejection is suppressed.

Step a) of the present invention is a step of isolating somatic cells from a pig, wherein the pig becomes a cell donor (or nuclear donor) in the production of cloned pigs using nuclear transfer technology. Accordingly, a person skilled in the art may select and use a pig according to the purpose, and the type of pig is not particularly limited. That is, the pig may be a pig fetus or adult pig, and is a concept that includes not only a pig in a natural state but also an artificially transformed pig.

In the present invention, the term "somatic cell" is a cell that can be isolated from the pig, as long as it is a cell that can be transformed through gene introduction and knockout, and the type is not particularly limited, and may include both differentiated cells and undifferentiated cells. .

As a method for isolating somatic cells, a conventionally known method may be used without limitation.

Step b) of the present invention is a step of transforming the isolated pig somatic cells, specifically, fusion of human HO-1 (Heme oxygenase-1) gene and human TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) into the somatic cells. This is a step of introducing a gene and knocking out the GGTA1 (α-1,3-galactosyltransferase) gene and CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) gene.

Step c) of the present invention is a step of selecting the transformed cells and culturing the selected cells.

In the present invention, the term "HO-1 (Heme oxygenase-1) gene" is induced expression in cells by various stressors such as heavy metals, endotoxins, ultraviolet rays, heat shock, reactive oxygen species and hypoxic conditions. A gene that encodes an enzyme that HO-1 is an enzyme that finally decomposes heme into bilirubin and Fe ^{2 +} improve function. In the present invention, HO-1 suppresses CD4+ T-cell proliferation and at the same time activates endothelial cell proliferation to normalize immune cells in vivo, thereby suppressing heterogeneous immune rejection and resistance to oxidative stress during organ isolation and suppressing apoptosis. It protects the cells that make up the organs.

Specifically, the human HO-1 gene may have its sequence obtained from a known gene database, and more specifically, it may include the nucleotide sequence of SEQ ID NO: 1, but it may be introduced into a pig somatic cell to improve the function of HO-1 Anything that can be shown may be included without limitation in the scope of the present invention.

As used herein, the term "TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) fusion gene" refers to a fusion of the extracellular region of TNFR1 capable of binding to TNF-α and the Fc region of immunoglobulin. In the present invention, the terms "TNFR1-Fc fusion gene" and "TNFR1-Fc gene" are used without distinction. In the present invention, the form of TNFR1-Fc that binds to TNF-α and can inhibit TNF-α can be used without limitation, and in particular, a soluble form of soluble tumor necrosis factor receptor 1 (soluble TNFR1, sTNFR1) and The Fc region of an immunoglobulin may be fused.

As used herein, the term "immunoglobulin Fc region" refers to the heavy and light chain variable regions of immunoglobulin, heavy chain constant region 1 (CH1) and light chain constant region 1 (CL1), except for heavy chain constant region 2 (CH2) and heavy chain It refers to the constant region 3 (CH3) portion, and also includes a hinge portion in the heavy chain constant region. In addition, as long as the immunoglobulin Fc region of the present invention has substantially the same or improved effect as the native type, part or all of the heavy chain constant region 1 (CH1) and/or light chain constant region except for only the heavy and light chain variable regions of immunoglobulin 1 (CL1) may be an extended Fc region. Also, it may be a region in which some fairly long amino acid sequences corresponding to CH2 and/or CH3 have been removed. That is, the immunoglobulin Fc region of the present invention comprises 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) In combination with one or two or more domains and an immunoglobulin hinge region (or a part of the hinge region), 6) heavy chain constant region may be a dimer of each domain and light chain constant region. In addition, the immunoglobulin Fc region of the present invention includes a native amino acid sequence as well as a sequence derivative (mutant) thereof. An amino acid sequence derivative means that one or more amino acid residues in a natural amino acid sequence have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331 known to be important for binding may be used as suitable sites for modification. In addition, various types of derivatives are possible, such as a site capable of forming a disulfide bond is removed, some amino acids at the N-terminus of native Fc are removed, or a methionine residue may be added to the N-terminus of native Fc Do. In addition, in order to eliminate the effector function, the complement binding site, for example, the C1q binding site, may be removed, or the ADCC site may be removed.

Techniques for preparing such an immunoglobulin Fc region sequence derivative are disclosed in International Patent Publication No. 97/34631 and International Patent Publication No. 96/32478. Amino acid exchanges in proteins and peptides that do not entirely alter the activity of the molecule are known in the art. The most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly. In some cases, it is modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc. ( may be modified). The above-described Fc derivative exhibits the same biological activity as the Fc region of the present invention, but has increased structural stability to heat, pH, etc. of the Fc region.

Meanwhile, the immunoglobulin Fc region may be of human or animal origin, such as cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, preferably human origin. In addition, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, or IgM, a combination thereof, or a hybrid thereof. Preferably from IgG or IgM, which is most abundant in human blood, and most preferably from IgG known to enhance the half-life of ligand binding proteins.

Meanwhile, as used herein, the term “combination” means that when a dimer or multimer is formed, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin forms a bond with a single-chain polypeptide of a different origin. do. That is, dimers or multimers can be prepared from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc fragments.

As used herein, the term "hybrid" is a term meaning that sequences corresponding to immunoglobulin Fc fragments of two or more different origins exist in a single-chain immunoglobulin Fc region. In the case of the present invention, various types of hybrids are possible. That is, a hybrid of domains consisting of 1 to 4 domains from the group consisting of CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc is possible, and may include a hinge. On the other hand, IgG can also be divided into subclasses of IgG1, IgG2, IgG3 and IgG4, and in the present invention, a combination thereof or hybridization thereof is also possible.

In the present invention, the fusion of a cytokine soluble receptor and an immunoglobulin (hereinafter referred to as 'Ig') may have the following advantages compared to a monomer of the original molecule or a molecule not fused with Ig:

1) In the dimer form, the fusion protein becomes bivalency, so that the total affinity for the ligand increases (avidity);

2) an increase in the length of time it can exist in the serum without being destroyed, i.e. an increase in the stability of the molecule;

3) activation of effector cells through Fc (Fragment crystallizable) of immunoglobulin heavy chain; and

4) Convenience of protein separation and purification (eg, separation and purification using protein A).

The fusion of the present invention is manufactured in a form excluding the CH1 domain of the heavy chain region, and as a result, it can be prepared as a dimer that does not bind to the light chain of immunoglobulin.

Specifically, the sequences related to the human TNFR1 and Fc genes can be obtained from known databases, and more specifically, the TNFR1-Fc fusion gene described in SEQ ID NO: 8 can be used, but introduced into somatic cells of a pig, TNFR1 Any sequence capable of showing the function of the -Fc fusion protein may be used without limitation.

The gene introduction may be performed through a known method capable of expressing human HO-1 gene and human TNFR1-Fc in porcine somatic cells, but is not limited thereto, for example, introducing an expression vector containing the gene, It is possible to increase the copy number of the gene in the genome, or introduce or overexpress the promoter sequence of the gene by substitution or modification.

As a method of transducing the gene into cells, a biochemical method, a physical method, or a virus-mediated transduction method may be used, and preferably, the biochemical method is FuGene6 (Roche, USA), Lipofectamine (Lipofectamine TM2000). , Invitrogen, USA) or ExGen 500 (MBI Fermentas International Inc. CANADA) may be used, and more preferably, a lipid-mediated method using lipofectamine may be used. In addition, as the expression vector containing the gene, any expression vector that can be expressed in a pig somatic cell line can be used, and in a specific embodiment of the present invention, pcDNA 3.1 vector was used as an expression vector containing the human HO-1 gene. pcDNA6 was used as an expression vector containing the human TNFR1-Fc fusion gene.

Although not limited thereto, in a specific embodiment, a single vector including the human HO-1 gene of step b), in particular a vector having the cleavage map of FIG. 1, and a single vector including a human TNFR1-Fc fusion gene, particularly The vector having the cleavage map of FIG. 2 may be separately or simultaneously introduced into somatic cells. In a specific embodiment of the present invention, a vector having the cleavage map of FIG. 1 was prepared by inserting the human HO-1 gene into the pcDNA3.1 vector, which is an expression vector containing a neomycin resistance gene, and the expression of the inserted gene was checked. It was confirmed in the HEK293 cell line. In addition, the human TNFR1-Fc fusion gene was inserted into pcDNA6, a gene expression vector containing a blasticidine resistance gene, to prepare a vector having the cleavage map of FIG. 2, and the expression of the inserted gene was checked in HEK293 cell line. Confirmed.

As used herein, the term "vector" refers to an expression vector capable of expressing a target gene in a cell into which the vector is introduced, and a gene construct including essential regulatory elements operably linked so that the gene insert introduced into the vector is expressed. . As an example, the present invention can prepare a recombinant vector containing a human HO-1 gene or a human TNFR1-Fc fusion gene, and by introducing it into a somatic cell using the prepared recombinant vector, for producing a transformed fertilized egg A donor cell line can be generated.

Specifically, as the promoter used in the present invention, promoter sequences for preparing expression vectors commonly used in the art may be used without limitation. Examples of a promoter that can be used include a CMV promoter, an SV40 promoter, or a CAG promoter, but the promoter sequences that can be used in the present invention are not limited by the above examples. As such a promoter, if necessary, a specific promoter may be used for tissue-specific expression.

In addition, the polyadenylation sequence of the present invention is a commonly used polyadenylation sequence, for example, SV40 polyadenylation sequence, human growth hormone polyadenylation sequence, mouse protamine-1 gene polyadenylation sequence (protamine-1 poly A signal), polyadenylation sequence derived from large T antigen poly A region, polyadenylation sequence derived from rabbit β-globin or fetal bovine growth hormone polyadenylation Sequences and the like can be used without limitation.

In order to determine whether the human HO-1 gene or the human TNFR1-Fc fusion gene is expressed, the vector of the present invention may additionally include a tag sequence for protein isolation, purification or confirmation. Examples of the tag sequence include GFP, Glutathione Stransferase (GST)-tag, HA, His-tag, Myc-tag, T7-tag, and the like, but the tag sequence of the present invention is not limited by the above examples.

In a specific embodiment of the present invention, the HO-1 gene having the nucleotide sequence of SEQ ID NO: 1 was introduced into the pcDNA3.1 vector to prepare a HO-1 gene expression vector (FIG. 1), having the nucleotide sequence of SEQ ID NO: 8 The sTNFR1-Fc gene was introduced into the pcDNA6 vector to prepare a sTNFR1-Fc gene expression vector (FIG. 2). In another specific embodiment of the present invention, the two types of expression vectors are introduced into somatic cells isolated from pigs, and cells expressing the HO-1 gene and the sTNFR1-Fc gene simultaneously, and a transgenic pig using the cells. prepared.

As used herein, the term "GGTA1 (α-1,3-galactosyltransferase) gene" refers to an enzyme involved in the production of N-glycans from glycoproteins, and the enzyme is N- through α-1,3 glycosidic bonds. The galactose residues are linked from the glycan to the galactose to form a terminal Gal-α-1,3-Gal (ie, α-Gal) moiety. In the case of xenogeneic organ transplantation, in which pig organs are transplanted into another species, a hyperacute immune rejection caused by the pig GGTA1 gene occurs within minutes to hours of transplantation, resulting in death of the transplanted animal or human. Since this is very lethal for organ transplantation, the present inventors tried to prepare a pig in which the GGTA1 gene was completely knocked out in order to solve this problem.

Specifically, the GGTA1 gene may be sequenced from a known gene database, and any gene corresponding to a protein exhibiting α-1,3-galactosyltransferase activity in pigs may be included without limitation in the scope of the present invention.

As used herein, the term “cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene” refers to an enzyme involved in the production of N-glycolylneuraminic acid, and the enzyme converts N-acetylneuraminic acid to N-glycolylneuraminic acid. In the case of xenogeneic organ transplantation in which pig organs are transplanted into humans, the acute immune rejection caused by the swine CMAH gene is very lethal in xenogeneic organ transplantation. .

Specifically, the CMAH gene may be sequenced from a known gene database, and any gene corresponding to a protein exhibiting cytidine monophosphate-N-acetylneuraminic acid hydroxylase activity in pigs may be included without limitation in the scope of the present invention.

In the present invention, the term "gene knock-out" refers to an artificial manipulation for the purpose of causing a gene to lose its function, but is not limited thereto, but some nucleotides are inserted, replaced, deleted, etc. It may be caused by an event, or all or part of a gene may be removed from the genome.

The gene knockout is not particularly limited as long as it is a known knockout method and may be included in the scope of the present invention. For example, the gene knockout may be performed using TALEN (transcription activator-like effector nuclease).

As used herein, the term “TALEN” refers to a nuclease capable of recognizing and cleaving a target region of DNA, and specifically refers to a fusion protein comprising a TALE domain and a nucleotide cleavage domain. In the present invention, the terms "TAL effector nuclease" and "TALEN" are interchangeable. TAL effectors are known as proteins that are secreted through their type secretion system when Xanthomonas bacteria are infected with various plant species. The protein can bind to a promoter sequence in the host plant to activate the expression of a plant gene that aids in bacterial infection. The protein recognizes plant DNA sequences through a central repeat domain consisting of a variable number of repeats of up to 34 amino acids. Therefore, TALE is considered to be a novel platform for tools in genome engineering. However, in order to construct a functional TALEN with genome-editing activity, a few key parameters that have not been known so far should be defined as follows. i) the minimal DNA-binding domain of the TALE, ii) the length of the spacer between the two half-sites constituting one target region, and iii) a linker or fusion junction connecting the FokI nuclease domain to the dTALE ).

A TALE domain of the present invention refers to a protein domain that binds to nucleotides in a sequence-specific manner via one or more TALE-repeat modules. The TALE domain includes, but is not limited to, at least one TALE-repeat module, more specifically 1 to 30 TALE-repeat module. In the present invention, the terms "TAL effector domain" and "TALE domain" are interchangeable. The TALE domain may include half of a TALE-repeat module. The entire content disclosed in International Patent Publication No. WO/2012/093833 or US Patent Publication No. 2013-0217131 in relation to the TALEN is incorporated herein by reference.

In the present invention, the TALEN may be used to target the GGTA1 gene and knock out the gene, but is not limited thereto, and particularly, it may target the exon 9 region of the GGTA1 gene.

In a specific example of the present invention, a TALEN for knocking out the GGTA1 gene was prepared by targeting the site of exon 9, which plays a decisive role in the site for catalysis of the GGTA1 enzyme (FIG. 3). In another specific embodiment of the present invention, the prepared TALEN was transduced into fibroblasts expressing the human HO-1 gene and the human sTNFR1-Fc fusion gene at the same time to prepare a cell in which the GGTA1 gene was knocked out (FIG. 4) , to obtain a cell population deficient in the GGTA1 gene by separating them using magnetic beads (FIG. 5).

In the present invention, the TALEN may be used to target the CMAH gene and knock out the gene, but is not limited thereto, but may specifically target the exon 3 region of the CMAH gene.

In a specific example of the present invention, a TALEN for knocking out the CMAH gene was prepared by targeting the site of exon 3, which plays a decisive role in the site catalyzing the CMAH enzyme (FIG. 6). In another specific embodiment of the present invention, the prepared TALEN is transduced into fibroblasts in which the human HO-1 gene and the human sTNFR1-Fc fusion gene are simultaneously expressed and GGTA1 is knocked out to prepare a CMAH gene knockout cell and (Fig. 7), cells into which TALEN was introduced by the reporter gene (H2K) were obtained. In addition, the isolated cell line was selected using an anti-N-glyconeuraminic acid (Neu5Gc) antibody, and as a result of repeated repetition, a cell population lacking the CMAH gene was obtained. The obtained cell line was cultured to increase the number of cells, and it was confirmed whether or not Neu5Gc was deleted using the gene sequencing analysis of the cells ( FIG. 8 ) and flow cytometry ( FIG. 9 ).

In step b), introducing a single vector comprising a human HO-1 gene and a single vector comprising a human TNFR1-Fc fusion gene and knocking out a GGTA1 gene and a CMAH gene are performed simultaneously or separately It may be one, and the order is not particularly limited as long as cells of the same genotype can be produced.

In step c), for example, somatic cells into which the expression vector of step b) has been introduced can be easily selected by using an expression vector into which a selection marker has been introduced. An antibiotic resistance gene may be used as a selection marker. Examples of the antibiotic resistance gene include, but are not limited to, bsd ^r , neo ^r , pac ^r , bsr ^r , hph ^r and the like. In a specific embodiment of the present invention, neomycin was treated in the cell culture medium using neo ^r to select somatic cells into which the expression vector containing the human HO-1 gene was introduced, and bsd ^r was used to add blastisy to the cell culture medium. Somatic cells into which an expression vector containing a human TNFR1-Fc fusion gene was introduced were selected by treatment with Dean.

As another example, selecting a somatic cell in which the GGTA1 and CMAH genes are knocked out using TALEN may be, but is not limited to, using a TALEN plasmid including a reporter system. The reporter system may use, for example, the H2K ^K membrane protein gene, and in this case, magnetic beads may be used to select cells expressing the gene. After colonies are formed from the selected cells, for each colony, T7E1 analysis, targeted deep sequencing, etc. may be performed to further confirm whether the GGTA1 and CMAH genes are knocked out.

Step d) is a step of preparing a fertilized egg having the same genetic trait as the somatic cell using the selected somatic cells, and aims to produce cloned pigs with respect to the somatic cells. Specifically, step d) is a step of removing the nucleus of a pig egg and fusion with the selected somatic cells to prepare a fertilized egg in which a somatic cell nucleus has been transplanted, and is a step using somatic cell nuclear transfer technology.

In step d), the oocytes of the present invention may be used by culturing immature oocytes collected from the ovaries of untreated pigs.

As used herein, the term “nuclear transfer” refers to transferring the nucleus of a cell into an egg from which the nucleus has already been removed. An individual born by implanting such a nuclear-transferred fertilized egg is a cloned individual that is genetically identical to the nuclear donor individual because the genetic material of the nuclear donor cell is transferred to the nuclear donor cytoplasm as it is.

Methods for removing the genetic material of eggs include a physical method, a chemical method, and a centrifugation method using Cytochalasin B (Tatham et al., Hum Reprod., 11(7); 1499-1503, 1996). In the present invention, using a micromanipulator, a physical nucleation method was used. Transformed somatic cells are introduced into an egg from which the nucleus has been removed using a cell membrane fusion method, intracytoplasmic microinjection, or the like. The cell membrane fusion method has the advantage of being simple and suitable for large-scale production of fertilized eggs, and the intracytoplasmic microinjection method has the advantage of maximizing the contact between the nucleus and materials in the egg. Fusion of a somatic cell and an egg with the nucleus removed is recombined through a method of fusion by changing the viscosity of the cell membrane through electrical stimulation. In this case, it is convenient to use an electric fusion device that can freely adjust the microcurrent and voltage. In a specific embodiment of the present invention, the nucleus of the egg was physically removed by micromanipulation, and the nucleus from which the nucleus was removed and the selected somatic cell donor cell line were fused with electrical stimulation to prepare a fertilized egg.

The method for producing a transgenic pig of the present invention may further include the step of implanting the fertilized egg of step d) into a pig uterus. For this purpose, it is preferable to select a surrogate sow for transplantation of a fertilized egg into which the somatic cell nucleus has been transplanted.

The nuclear-transferred fertilized egg is activated and developed to a stage where it can be transplanted, and then implanted as a surrogate mother. Activation of a replicating fertilized egg means reactivating the cell cycle temporarily suspended during maturation so that the fertilized egg can divide. In order to activate the cloned fertilized egg, it is necessary to decrease the activity of cell signaling substances such as MPF and MAP kinase, which are cell cycle arresting factors. A method of rapidly increasing calcium influx from outside the cell by largely modifying cell membrane permeability by electrical stimulation, and a method of directly inhibiting the activity of cell cycle arresting factors using chemical substances such as ionomycin and 6-DMAP These are used alone or in combination.

In a specific embodiment of the present invention, PCR (FIG. 10), RT-PCR (FIG. 11), ELISA (FIG. 12) shows that HO-1 and sTNFR1-Fc genes are inserted and expressed in all the transgenic pigs produced by the above method. ) and western blot (Fig. 13). In addition, it was confirmed that the GGTA1 gene and CMAH were knocked out through nucleotide sequence analysis (FIG. 16) and flow cytometry analysis (FIG. 17).

In another specific embodiment of the present invention, as a result of analyzing cell viability against apoptosis stimulation and oxidative stress stimulation using fibroblasts isolated from the transgenic pig, the fibroblasts isolated from the transgenic pig are wild-type blood vessels. It was confirmed that cell viability was significantly increased compared to endothelial cells (FIG. 18). In addition, it was confirmed that the vascular endothelial cells and fibroblasts isolated from the transgenic pigs had significant resistance to human serum and complement compared to the wild-type cells (FIG. 19). In addition, it was confirmed that vascular endothelial cells and fibroblasts isolated from transgenic pigs also had significantly lower antibody binding affinities to IgG and IgM compared to wild-type cells with respect to human serum and complement ( FIG. 20 ).

Thus, using the method for producing a transgenic pig of the present invention, a transgenic pig in which the human HO-1 gene and the TNFR1-Fc fusion gene are stably expressed and the GGTA1 gene and the CMAH gene are knocked out can be prepared, the pig Organs isolated from oxidative stress have high viability, have resistance to human serum and complement, and it can be confirmed that antigens related to hyperacute immune rejection and acute immune rejection are removed. Accordingly, it can be seen that cells or tissues are protected from oxidative stress during organ transplantation isolated from the pig, and immune rejection will be suppressed.

Accordingly, the transgenic pig of the present invention simultaneously expresses the existing human HO-1 gene and the TNFR1-Fc fusion gene, and has additional characteristics in which CMAH is knocked out, compared to pigs in which GGTA1 is knocked out, so CMAH knockout resistant to acute immune rejection by Therefore, it can be very usefully used for xenogeneic organ transplantation without immune rejection.

As another aspect, the present invention is a human HO-1 (Heme oxygenase-1) gene and human TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) fusion gene is introduced, GGTA1 (α-1,3-galactosyltransferase) Provided is a transgenic pig for organ transplantation in which a gene and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) are knocked out and immune rejection is suppressed during xenotransplantation. The organ may specifically be islets, pancreas, heart, kidney, liver, lung, cornea, etc., but is not limited thereto, and may include all organs of a pig that can be transplanted into a human without limitation.

As another aspect, the present invention provides a somatic cell donor cell line for producing the transgenic pig.

The somatic donor cell line stably expresses the human HO-1 gene and the human TNFR1-Fc gene, and a cell line in which the GGTA1 gene and the CMAH gene are knocked out may be used without limitation.

As another aspect, the present invention comprises the steps of producing a transgenic pig by the method for producing the transgenic pig; And it provides a method for producing an organ in which immune rejection is suppressed during xenogeneic organ transplantation, comprising the step of isolating an organ for transplantation from the transgenic pig.

The organ may specifically be islets, pancreas, heart, kidney, liver, lung, cornea, etc., but is not limited thereto, and may include all organs of a pig that can be transplanted into a human without limitation. In addition, when the organ is transplanted into a human, an immune rejection reaction does not occur, so that a disease according to a type of a target organ can be treated.

Transgenic pigs in which the human HO-1 and TNFR1-Fc fusion genes of the present invention are co-expressed and the GGTA1 gene and CMAH gene are knocked out during organ isolation and in vitro culture due to the antioxidant and cytoprotective functions of the HO-1 gene It can reduce oxidative stress, and can also reduce the TNF-α-mediated inflammatory response that occurs at the early stage of transplantation by TNFR1-Fc expression. Also, it inhibits the maturation of dendritic cells and regulates the proliferation and activation of T cells in the acute vasculature. It can promote early engraftment of transplanted organs by reducing rejection. Furthermore, it is possible to increase the survival rate of transplanted organs by suppressing the hyperacute immune rejection by GGTA1 and the acute immune rejection by CMAH. Accordingly, an organ in which immune rejection is suppressed during xenotransplantation can be produced using the transgenic pig.

1 schematically shows a cleavage map of an expression vector into which a human HO-1 gene is inserted.
2 schematically shows a cleavage map of an expression vector into which a human sTNFR1-Fc fusion gene is inserted.
3 is a schematic diagram of a TALEN targeting the GGTA1 gene of the present invention.
FIG. 4 is a result of transforming the GGGT1 gene using TALEN and confirming whether or not the gene is deleted in the selected cells through cloning and sequencing.
5 is a result of magnetically activated cell separation method of TALEN-mediated mutant pig cells.
6 is a schematic diagram of TALEN targeting the CMAH gene in pigs.
7 is a result of transforming the CMAH gene using TALEN and confirming whether or not the gene is deleted in the selected cells through T7E1.
8 is a sequencing result of three cell colonies obtained by targeting the CMAH gene of the present invention.
9 is a result of confirming the deletion of the CMAH gene and whether the CMAH gene is deleted in the selected cells by performing flow cytometry with Anti-Neu5Gc.
10 is a result of confirming the expression of HO-1 gene and sTNFR1-Fc gene in transgenic pigs (16401, 16402, 16403) produced through somatic cell cloning technology by PCR.
11 is a result of confirming the expression of HO-1 gene and sTNFR1-Fc gene in transgenic pigs (16401, 16402, 16403) by RT-PCR.
12 is a result of confirming the expression of sTNFR1-Fc gene in transgenic pigs (16401, 16402, 16403) by culturing cells derived from the transformed pig and confirming the expression of shTNFRI-Fc in the culture medium by ELISA.
13 shows the results of confirming the protein expression of the HO-1 gene and the sTNFR1-Fc gene in transgenic pigs (16401, 16402, 16403) using a Western blot method.
14 shows the results of TALEN-mediated GGTA1 gene mutation produced through somatic replication on the nucleotide sequence.
15 is a result confirming the removal of a-1,3-Gal epitopes from the tail-derived fibroblasts of transgenic pigs using flow cytometry.
16 is a result of sequencing to confirm whether the CMAH and GGTA1 genes of three cloned pigs (16401, 16402, 16403) were deleted.
17 is a flow cytometry result to confirm the deletion of the CMAH and GGTA1 genes in the produced three cloned pigs (16401, 16402, 16403).
18 shows cells in fibroblasts of transgenic pigs (16401, 16402, 16403) using CCK-8 after treatment with apoptosis stimulation hTNF-α and CHX for 15 minutes and oxidative stress stimulation H ₂ O ₂ for 1 hour It is the result of confirming the viability.
19 shows antibody/complement mediation in vascular endothelial cells (pECs) and fibroblasts (pFbs) of transgenic pigs (16401, 16402, 16403) using fluorescence activated cell separation by 7-AAD staining after treatment with 40% human serum; It is the result of confirming the dissolution of sex.
20 is a result of verifying the removal of heterologous antigens by low antibody binding strength of vascular endothelial cells (pECs) and fibroblasts (pFbs) derived from transgenic pigs (16401, 16402, 16403) after treatment with human IgG and IgM.

Hereinafter, the present invention will be described in more detail by way of Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited by these examples.

Example 1: HO-1 gene and sTNFR1 - Fc Gene expression vector construction

An expression vector was prepared by cloning each of the HO-1 gene and the sTNFR1-Fc gene, and introduced into the cell to prepare a cell line expressing the gene at the same time. The cloning and transformation were performed in the same manner as described in Korean Patent Application Laid-Open No. 10-2011-0079485, specifically as follows.

1-1. Construction of HO-1 gene expression vector and confirmation of gene expression

Sequence of the human HO-1 (Heme oxygenase-1) gene using the NCBI website (http://www.ncbi.nlm.nih.gov/) and the ExPASy website (http://expasy.org/) , and using this, a forward primer (SEQ ID NO: 2) and a reverse primer (SEQ ID NO: 3) of HO-1 were prepared. The HO-1 gene (SEQ ID NO: 1) was obtained by polymerase chain reaction (PCR) using the primer set. In order to express the gene, pcDNA3.1 vector (Invitrogen, CA, USA), which is a gene expression vector containing a neomycin resistance gene, was treated with NheI and EcoRI restriction enzymes, and then the obtained HO- A HO-1 gene expression vector was prepared by inserting 1 gene ( FIG. 1 ).

In order to check the expression of the inserted gene, the HO-1 gene expression vector was transfected into HEK293 cell line using Lipofectamine TM2000 (Lipofectamine TM2000, Invitrogen, CA, USA). 3 X 10 ⁵ in a 35 mm plastic Petri dish (Becton Dickinson, NJ, USA) Dog HEK293 cells were seeded. The next morning, 1 μg HO-1 expression vector was diluted with 50 μl Opti-MEM I Reduced Serum Medium (Invitrogen, CA, USA), and Lipofectamine TM2000 in the same volume as the HO-1 expression vector was also diluted with 50 μl Opti- After dilution with MEM I medium, they were incubated at room temperature for 5 minutes. After incubation, the diluted HO-1 expression vector and the diluted Lipofectamine TM2000 were mixed and incubated at room temperature for 20 minutes. After the incubation, the mixture was added to a 35 mm cell culture dish inoculated with cells and cultured in a 37 ° C. CO ₂ incubator. After 4 hours, the culture medium was replaced with DMEM (Invitrogen, CA, USA) containing 10% FBS and penicillin/streptomycin, and cultured at 37 ° C. CO ₂ incubator.

After 48 hours, the cells were harvested with an elution buffer (lysis buffer: 1% Triton X-100, 50 mM TrisHCl, 20 mM NaF, 150 mM NaCl, protease inhibitors), 30 μg of the cell extract was electrophoresed, and the PVDF membrane was electrophoresed. The PVDF membrane was blocked with a blocking buffer (blocking buffer: 5% skim milk in TBST) for 1 hour, and an anti-HO-1 antibody (rabbit monoclonal antibody, Abcam, Cambridge, UK) was diluted 1: 2000 and reacted at room temperature for 1 hour. made it After the reaction, after washing 3 times for 30 minutes with TBST buffer, HRP-linked anti-rabbit IgG antibody (Santa Cruz Biotechnology, CA, USA) was diluted 1:5000 and reacted at room temperature for 1 hour. After the reaction, it was washed 3 times for 30 minutes with TBST buffer, and then treated with chemiluminescent substrates (WestSaveUp™, Abfrontier, Seoul, Korea) and reacted. It was developed by exposing it to an X-ray film, and it was confirmed that the human HO-1 gene was inserted into the vector and expressed.

1-2. sTNFR1 - Fc Construction of fusion gene expression vector and confirmation of gene expression

The sequence of the human tumor necrosis factor receptor 1 (TNFR1) gene was obtained from the NCBI website (http://www.ncbi.nlm.nih.gov/) and the ExPASy website (http://expasy.org/). analysis, and using this, a forward primer (SEQ ID NO: 4) and a reverse primer (SEQ ID NO: 5) of the extracellular domain of tumor necrosis factor receptor 1 were prepared. In addition, a forward primer (SEQ ID NO: 6) and a reverse primer (SEQ ID NO: 7) of the Fc region were prepared through gene sequence analysis of human immunoglobulin G1. Polymerase chain reaction (PCR) using each of the prepared primers was performed to obtain soluble tumor necrosis factor receptor 1 (soluble TNFR1) and immunoglobulin G1-Fc (IgG1-Fc) fusion genes (sTNFR1-Fc, sequence number 8). In order to express the secured gene, pcDNA6 (Invitrogen, CA, USA), a gene expression vector containing a blasticidine resistance gene, was cut by treatment with HindIII and XhoI restriction enzymes, and sTNFR1 secured at the cut site And an IgG1-Fc fusion gene was inserted to construct a sTNFR1-Fc gene expression vector (FIG. 2).

In order to check the expression of the inserted gene, the sTNFR1-Fc gene expression vector was transfected into HEK293 cell line using Lipofectamine TM2000 (Invitrogen, CA, USA). 3 X 10 ⁵ HEK293 cells were inoculated in a 35 mm plastic culture dish (Becton Dickinson, NJ, USA). The next morning, 1 μg sTNFR1-Fc expression vector was diluted with 50 μl Opti-MEM I Reduced Serum Medium (Invitrogen, CA, USA), and Lipofectamine TM2000 in the same volume as the sTNFR1-Fc expression vector was also diluted with 50 μl Opti-MEM I Reduced Serum Medium (Invitrogen, CA, USA). After dilution with MEM I medium, they were incubated at room temperature for 5 minutes. After incubation, the diluted sTNFR1-Fc expression vector and the diluted Lipofectamine TM2000 were mixed and incubated at room temperature for 20 minutes. After culturing, the mixture was added to a 35 mm cell culture dish inoculated with the cells, and cultured at 37° C. CO ₂ in an incubator. After 4 hours, the culture medium was replaced with serum-free DMEM (including penicillin/streptomycin) (Invitrogen, CA, USA) and cultured at 37° C. CO ₂ in an incubator.

After 48 hours, Western blotting was performed using an anti-hlgG antibody (Santa Cruz Biotechnology, CA, USA) as a culture medium and cell lysate. Perform electrophoresis with cell lysate and culture medium. After electrophoresis, it was transferred to a PVDF membrane. After electrophoresis, the PVDF membrane was blocked with blocking buffer (5% skim milk in TBST) for 1 hour. After blocking, HRP-linked anti-human IgG antibody (Santa Cruz Biotechnology, CA, USA) was diluted 1:5000 and reacted at room temperature for 1 hour. After the reaction, it was washed three times with TBST buffer, and then treated with a chemiluminescent substrate (WestSaveUp™, Abfrontier, Seoul, Korea) to react. It was developed by exposing it to an X-ray film to confirm the protein expression of water-soluble TNFR1-Fc.

Example 2: GGTA1 knockout (knock-out) for the production of cells TALEN (transcription activator-like effector nuclease) treatment and colony collect

The HO-1 gene and the sTNFR1-Fc gene were cloned, respectively, to produce an expression vector, and the produced transgenic pig cells were isolated. A knockout cell line was prepared. The cloning, transformation, and knockout of the GGTA1 gene were performed in the same manner as described below, specifically as follows.

A TALEN was prepared for knocking out the GGTA1 (α-1,3-galactosyltransferase) gene ( FIG. 3 ).

The target site of the TALEN is the site of exon 9, which plays a decisive role in the site for catalyzing the GGTA1 enzyme. A TALEN capable of recognizing and binding to a specific sequence of the gene was prepared and inserted into the plasmid. The TANEN-transformed cell line was identified and recovered using a reporter system in which the target gene sequence of TALEN and reporter genes such as GFP and H2K ^K were inserted.

Specifically, fibroblasts were isolated from the newly produced shTNFRI-Fc-F2A-HA-HO-1 (TNF/HAHO, #013) transgenic pigs (Korea Patent Publication No. 10-2011-0079485). Then, it was cultured using DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS. TALEN plasmid and reporter system for GGTA1 were transduced into fibroblasts using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 48 hours, the cells were incubated with IB4-FITC (isolectin B4-conjugated fluorescein isothiocyanate, Sigma) for 30 minutes and washed with PBS. IB4-negative cells were isolated using FACS. The number of cells was increased through cell culture and used for genomic DNA isolation and somatic cell nuclear transfer.

PCR was performed with swine α-gal-specific primers using the isolated DNA. The PCR product was inserted into pGEM T-easy vector (Promega Inc.), and the nucleotide removal efficiency by TALEN in the chromosome was analyzed. As an alternative method, the TALEN plasmid and reporter plasmid containing the H2K ^K membrane expression protein gene were transduced for 48 hours, and then cells were selected using MACS separation system using MACSelect H2K ^K microBeads (Miltenyi Biotec.). Selected cells were negatively isolated using IB4-biotin and streptavidin-conjugated microbeads. The isolated cells were cultured at a low density (200 cells/dish) in a 100 mm dish to form individual colonies. Each colony was transferred to a 48 well plate to increase the number of cells, and mutations were screened by T7E1 assay.

For T7E1 analysis, the target site, the GGTA1 gene, was amplified using the PCR method. The amplified sequence was denatured by heating and synthesized by treatment with T7 endonuclease I at 37° C. for 20 minutes to form a heterogeneous complex, and then confirmed on a 2% agarose gel. Two positive cell colonies were integrated and used as donor cells for somatic cell nuclear transfer. The knockout was confirmed by performing T7E1 analysis once again on the transgenic pigs produced from the cells.

Example 3: CMAH knockout (knock-out) for the production of cells TALEN (transcription activator-like effector nuclease) treatment and colony collect

A TALEN was prepared for knocking out the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene (FIG. 6).

The target site of the TALEN is the exon 3 site, which plays a decisive role in the site for catalyzing the CMAH enzyme. A TALEN capable of recognizing and binding to a specific sequence of the gene was prepared and inserted into the plasmid. A TANEN-transformed cell line was identified and recovered using a reporter system in which the target gene sequence of TALEN and a reporter gene such as H2K ^K were inserted.

Specifically, after separating fibroblasts from newborn pigs of GGTA1 KO/shTNFRI-Fc-F2A-HA-HO-1 transgenic pigs produced in Example 2 and Example 4 below, 10% FBS was added thereto It was cultured using the DMEM medium (Invitrogen, Carlsbad, CA, USA). The TALEN plasmid and reporter system for CMAH were transduced into fibroblasts using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 48 hours, the cells were incubated with Anti-Neu5Gc antibody (chicken anti-Neu5Gc, clone Poly21469, Biolegend) for 30 minutes and washed with PBS. Neu5Gc-negative cells were isolated using FACS. The number of cells was increased through cell culture and used for genomic DNA isolation and somatic cell nuclear transfer.

PCR was performed using the isolated DNA with swine cytidine monophosphate-N-acetylneuraminic acid hydroxylase-specific primers. The PCR product was inserted into pGEM T-easy vector (Promega Inc.), and the nucleotide removal efficiency by TALEN in the chromosome was analyzed. As an alternative method, the TALEN plasmid and reporter plasmid containing the H2K ^K membrane expression protein gene were transduced for 48 hours, and then cells were selected using MACS separation system using MACSelect H2K ^K microBeads (Miltenyi Biotec.). The selected cells were negatively isolated using Neu5Gc and streptavidin-conjugated microbeads. The isolated cells were cultured at a low density (200 cells/dish) in a 100 mm dish to form individual colonies. Each colony was transferred to a 48-well plate to increase the number of cells, mutations were screened by T7E1 assay, and three cell colonies usable for somatic cell nuclear transfer were obtained.

For the T7E1 analysis, the target region of the CMAH gene was amplified using the PCR method. The amplified sequence was denatured by heating and synthesized by treatment with T7 endonuclease I at 37° C. for 20 minutes to form a heterogeneous complex, and then confirmed on a 2% agarose gel. Two positive cell colonies were integrated and used as donor cells for somatic cell nuclear transfer. The knockout was confirmed by performing T7E1 analysis once again on the transgenic pigs produced from the cells.

Example 4: somatic cells nuclear transfer

For nuclear transfer of the transformed somatic cells in the above example, all cumulus cells of in vitro matured oocytes were removed, and the nucleus and pole nuclei of the oocytes were stained with 5 μg/mL Hoechst 33342. The stained eggs were fixed with a holding micropipette, and enucleation was performed using a suction pipette in TALP medium supplemented with 5 μg/mL cytochalasin B. The enucleated oocytes were placed in TALP medium and subsequently used for somatic cell nuclear transfer. One knockout transformed somatic cell was injected into the gastric cavity of the enucleated egg, and then precipitated in a fusion medium containing 0.28 M mannitol, 0.1 mM MgSO ₄ , and 0.5 mM HEPES for 4 minutes. Then, an electrical stimulation of 1.2 Kv/cm DC voltage and 30 µs duration was induced with the BTX Electro-Cell Manipulator to induce cytoplasmic fusion between the cytoplasm of the enucleated egg and the injected somatic cell. After 20 - 30 minutes of fusion, the reconstructed fertilized eggs were treated with a BTX electro-cell manipulator in an activated medium supplemented with 0.28 M mannitol, 0.5 mM HEPES, 0.1 mM CaCl ₂ , and 0.1 mM MgSO ₄ at 1.5 kv/cm DC voltage, once 60 The reconstructed fertilized egg was activated by applying electrical stimulation with a duration of μs. Activated cloned fertilized eggs are cultured in PZM-5 (porcine zygote medium-5; IFP0410P, Funakoshi, Tokyo, Japan) for 1-2 days in an incubator maintained with 5% carbon dioxide, 5% oxygen, and 90% nitrogen, and morphologically Normal fertilized eggs (1 cell stage on day 1; 2-8 cell stage on day 2) were selected for fertilized egg transplantation. Then, 90 to 120 fertilized eggs were implanted laparotomy into synchronized surrogates. The protocol for animal use was approved by the Animal Experimental Ethics Committee of Seoul National University in accordance with the Guide for the Care and Use of Laboratory Animals of Seoul National University (SNU-151019-4).

Example 5: transgenic pig fibroblast cells viability evaluation

In order to evaluate the cell viability of the fibroblasts of the transgenic pigs through the above Examples, wild-type, TNF/HAHO transgenic pigs, GGTA1 knockout pigs, GGTA1 KO/TNF/HAHO transgenic pigs, and three CMAH-GGTA1 pigs Fibroblasts (1 × 10 ⁵ cells/well) isolated from KO/TNF/HAHO transgenic pigs (16401, 16402, 16403) were prepared in a 24-well plate, and TNF-α (20 ng/mL) and It was treated with CHX (cycloheximide, 10 μg/mL), and H ₂ O ₂ was treated for 1 hour. Cell viability was measured using CCK-8 solution according to the manufacturer's manual (Dojindo Laboratories, Kumamoto, Japan). In this evaluation, fibroblasts derived from wild-type Yucatan pigs were used as controls. Absorbance was measured with a microplate reader (Tecan Sunrise, Hayward, CA, USA).

Example 6: PCR , rt- PCR , ELISA and western blot analyze

In order to confirm the expression of the HO-1 gene and sTNFR1-Fc gene of the transformed pig through the above example, DNA, RNA and protein of the produced transgenic pig tail-derived fibroblasts were extracted, and PCR, RT-PCR, ELISA and Western blot were performed.

For PCR and RT-PCR, genomic DNA (iNtRON Biotechnology, Inc.) and RNA (iNtRON Biotechnology, Inc.) were extracted, and RNA was amplified using reverse transcriptase (iNtRON Biotechnology, Inc.) to secure complementary DNA. The reaction was carried out. To confirm the insertion of the foreign gene hHO-1 and TNFRI-Fc in genomic DNA and complementary DNA, primers specific for each gene were synthesized and PCR was performed under constant conditions. Reactants derived from transgenic pig tail-derived fibroblasts Bands of 633 bp and 867 bp, respectively, were identified only in .

To analyze the protein expression level, the transgenic pig cells were cultured for two days using a shTNFRI-specific ELISA kit (R&D system, MN, USA), and then the culture medium was centrifuged to measure the amount of shTNFRI in the culture medium.

For western blot analysis, PRO-PREP was dissolved in protein extraction solution (iNtRON Biotechnology, Inc.), diluted in SDS buffer solution (GeneDepot), and incubated at 100° C. for 5 minutes. Equal amounts of protein were loaded onto 10% SDS-PAGE. Proteins were transferred to PVDF (polyvinylidene difluoride) membrane after electrophoresis. After electrophoresis, PVDF membranes were blocked with 5% skim milk, followed by HRP-conjugated anti-human IgG (1:2,000, Binding Site, Birmingham, UK), anti-hHO-1 (Abcam, MA, USA) or 1:4,000. The diluted anti-HA (Abcam, MA, USA) antibody was incubated at 4° C. for 24 hours. Blots were developed using a Pierce SuperSignal West Pico Chemiluminescent System (Thermo Fisher Scientific).

Example 7: Porcine fibroblasts and vascular endothelial cells complement Mediated dissolution evaluation

To evaluate natural antibodies to N-glycolylneuraminic acid antigen, complement-mediated cytotoxicity was measured. Vascular endothelial cells derived from wild-type pigs, CMAH-GGTA1 KO/TNF/HAHO transgenic pigs-derived vascular endothelial cells and fibroblasts (1 x 10 ⁵ ) were removed, transferred to a 1.5 mL tube, and human serum (Sigma Aldrich, MO, USA) and incubated for 1 h at room temperature. Cell viability was measured using a 7-AAD (BD Bioscience, CA, USA) in a FACS Canto (BD Bioscience, CA, USA).

Example 8: Evaluation of antibody binding affinity of porcine fibroblasts and vascular endothelial cells

To evaluate antibody binding, endothelial cells isolated from a wild-type porcine endothelial cell line (MPN3), CMAH-GGTA1 KO/TNF/HAHO transgenic pigs (16403) and three CMAH-GGTA1 KO/TNF/HAHO transgenic pigs ( Fibroblasts (3 × 10 ⁵ cells) isolated from each of 16401, 16402, and 16403) were prepared by washing twice with 1X PBS.

In order to fluoride the complement activity in the serum, human serum is heat-treated at 56°C for 30 minutes, diluted to 40% using 1X PBS, and then treated with 100 ul of each cell and incubated at 4°C for 30 minutes. do. After washing twice with 1X PBS, treat with anti-human IgM APC (eBioscience, USA) or anti-human IgG PE (eBioscience, USA) and incubate for 30 minutes at 4°C in the dark. After washing twice with 1X PBS, fluorescence expression is analyzed using flow cytometry equipment.

test example One: GGTA1 gene knockout Cell line establishment

To confirm the knockout of the GGTA1 gene, and to select these cells, the following experiment was performed.

The GGTA1 gene was knocked out using a TALEN targeting GGTA1, and the deletion and qualitative analysis of the GGTA1 gene in the cells selected using the MACS method were confirmed through cloning and sequencing (FIG. 4). As a result, it was confirmed that some nucleotides were deleted at the target position of the GGTA1 gene in the selected cells, and it was confirmed that the GGTA1 gene was knocked out.

The selection method for cells in which the GGTA1 gene is knocked out is a method using magnetic beads, and is a method of incubating an antibody to which a magnetic bead against H2K ^K is bound with the cells and then separating the cells using a column (Fig. 5). A cell population deficient in the GGTA1 gene was obtained through the above method.

test example 2: CMAH gene knockout Cell line establishment

The following experiment was performed to confirm the knockout of the CMAH gene and to select these cells.

The CMAH gene was knocked out using TALEN targeting CMAH, and the deletion and qualitative analysis of the CMAH gene in cells selected using the MACS method were confirmed through cloning and sequencing (FIG. 8). As a result, it was confirmed that some nucleotides were deleted at the target position of the CMAH gene in the selected cells, and it was confirmed that the CMAH gene was knocked out.

The CMAH gene was knocked out using TALEN targeting CMAH, and the deletion and qualitative analysis of the CMAH gene in cells selected using the MACS method were confirmed through cloning and sequencing (FIG. 8). As a result, it was confirmed that some nucleotides were deleted at the target position of the CMAH gene in the selected cells, and it was confirmed that the CMAH gene was knocked out.

test example 3: transformation cloned pig Gene Insertion Verification

In order to verify the insertion of the HO-1 gene and sTNFR1-Fc gene of the transformed pig through the above example, DNA was extracted from the fibroblasts of live transgenic pigs and the donor cell donor transgenic pig tissue to be specific for each gene. As a result of verification by PCR using a specific primer, it was confirmed that the HO-1 and sTNFR1-Fc genes were inserted in all the produced transgenic pigs (16401, 16402, 16403) (FIG. 10). RT-PCR (FIG. 11), ELISA (FIG. 12), and Western blot (FIG. 13) methods were performed for HO-1 and sTNFR1-Fc transcripts and proteins in transgenic pig-derived fibroblasts. A band of a size suitable for the cadaver and protein type and a significant concentration of shTNFRI-Fc were clearly observed, respectively.

test example 4: Transformation cloned pig GGTA1 Gene Removal Verification

To verify the deletion of the GGTA1 gene in transgenic cloned pigs, genetic mutation verification was performed. DNA was extracted from each individual produced and the nucleotide sequence of the GGTA1 gene exon 9 corresponding to the target of TALEN was analyzed. As a result of the analysis, it was confirmed that 18, 6, 29, etc. of the GGTA1 gene nucleotide sequences were deleted in the transgenic cloned pigs, respectively (FIG. 14). Due to the deletion of each nucleotide sequence, the amino acid sequence was also deleted in the order of 6, 2, 9, etc., so loss of function of the GGTA1 gene was also expected. It was verified that the GGTA1 gene was deleted through FACS analysis using an antibody (FIG. 15). This suggests that it is possible to overcome the hyperacute rejection when applying the xenotransplantation of organs derived from the transgenic cloned pigs.

test example 5: Transformation cloned pig CMAH Gene Removal Verification

Gene mutation verification was performed to verify the deletion of the CMAH gene in transgenic cloned pigs. DNA was extracted from each individual produced and the base sequence of the CMAH gene exon 3 corresponding to the target of TALEN was analyzed. As a result of the analysis, it was confirmed that 2 and 13 CMAH gene nucleotide sequences were deleted in the produced transgenic cloned pigs (16401, 16402, 16403) (FIG. 16). It was verified that the CMAH and GGTA1 genes were deleted through FACS analysis using the Neu5Gc and BS-IB4 antibodies (FIG. 17). This suggests that it is possible to overcome acute rejection when applying the xenotransplantation of organs derived from the transgenic cloned pigs.

test example 6: Cell viability Functional verification through analysis

For cell viability analysis of the transgenic pigs prepared in the above Examples, wild-type, TNF/HAHO transgenic pigs, GGTA1 knockout pigs, GGTA1 KO/TNF/HAHO transgenic pigs, and three CMAH-GGTA1 KO/TNF pigs Cell viability against apoptosis stimulation (hTNFα and CHX) and oxidative stress stimulation (H ₂ O ₂ ) using fibroblasts isolated from /HAHO transgenic pigs (16401, 16402, 16403), respectively, by CCK-8 assay Confirmed. As a result, fibroblasts derived from TNF/HAHO transgenic pigs, GGTA1 KO/TNF/HAHO transgenic pigs, and three CMAH-GGTA1 KO/TNF/HAHO transgenic pigs (16401, 16402, and 16403) were all inactivated in wild-type fibroblasts. Compared to that, cell viability was significantly increased ( FIG. 18 ). This suggests that the viability of the transplanted cells will be increased when the xenotransplantation of organs derived from the transgenic cloned pig is applied.

test example 7: Antibody/ of gene removal complement mediation Functional verification through dissolution reaction and evaluation of naturally occurring antibody binding force

In order to confirm the suppression of the hyperacute and acute immune responses of the transgenic pigs from which the GGTA1 and CMAH genes have been removed, vascular endothelial cells derived from wild-type pigs, CMAH-GGTA1 KO/TNF/HAHO transgenic pigs ( 7-AAD staining was performed using vascular endothelial cells (pECs) and fibroblasts (pFbs) derived from 16401, 16402, 16403), and antibody/complement-mediated lysis by human serum was confirmed from this. As a result, vascular endothelial cells and fibroblasts derived from CMAH-GGTA1 KO/TNF/HAHO transgenic pigs showed resistance to human serum and complement compared to wild-type pig-derived vascular endothelial cells ( FIG. 19 ). This suggests that the antibody/complement-mediated lysis response will be reduced in the application of xenotransplantation of organs derived from these transgenic cloned pigs.

In addition, vascular endothelial cells and fibroblasts derived from CMAH-GGTA1 KO/TNF/HAHO transgenic pigs showed significantly lower binding affinity to naturally occurring antibodies, IgG and IgM, compared to wild-type pig-derived vascular endothelial cells (FIG. 20). .

Summarizing the above results, the transgenic pigs of the present invention co-express the HO-1 and TNFR1-Fc genes to increase the viability of the transplanted organs, and to delete the GGTA1 gene and the CMAH gene to induce a hyperacute immune response and acute immunity during organ transplantation. reaction is suppressed.

Therefore, the transgenic pig of the present invention can produce organs in which immune rejection is suppressed during xenogeneic organ transplantation, and thus can be very usefully used for organ transplantation.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

<110> Seoul National University R&DB Foundation CHONG KUN DANG PHARMACEUTICAL CORP. <120> Transgenic pigs expressing both shTNFRI-Fc and HA-HO-1 without Gal epitope and N-glycolylneuraminic acid epitope and the Use of it <130> KPA170439-KR <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 867 <212> DNA <213> Artificial Sequence <220> <223> HO-1 <400> 1 atggagcgtc cgcaacccga cagcatgccc caggatttgt cagaggccct gaaggaggcc 60 accaaggagg tgcacaccca ggcagagaat gctgagttca tgaggaactt tcagaagggc 120 caggtgaccc gagacggctt caagctggtg atggcctccc tgtaccacat ctatgtggcc 180 ctggaggagg agattgagcg caacaaggag agcccagtct tcgcccctgt ctacttccca 240 gaagagctgc accgcaaggc tgccctggag caggacctgg ccttctggta cgggccccgc 300 tggcaggagg tcatccccta cacaccagcc atgcagcgct atgtgaagcg gctccacgag 360 gtggggcgca cagagcccga gctgctggtg gcccacgcct acacccgcta cctgggtgac 420 ctgtctgggg gccaggtgct caaaaagatt gcccagaaag ccctggacct gcccagctct 480 ggcgagggcc tggccttctt caccttcccc aacattgcca gtgccaccaa gttcaagcag 540 ctctaccgct cccgcatgaa ctccctggag atgactcccg cagtcaggca gagggtgata 600 gaagaggcca agactgcgtt cctgctcaac atccagctct ttgaggagtt gcaggagctg 660 ctgacccatg acaccaagga ccagagcccc tcaggggcac cagggcttcg ccagcgggcc 720 agcaacaaag tgcaagattc tgcccccgtg gagactccca gagggaagcc cccactcaac 780 acccgctccc aggctccgct tctccgatgg gtccttacac tcagctttct ggtggcgaca 840 gttgctgtag ggctttatgc catgtga 867 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for HO-1 <400> 2 cgggctagca ccatggagcg tccgcaaccc gac 33 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for HO-1 <400> 3 cgggaattct cacatggcat aaagccctac 30 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for sTNFR1 <400> 4 ataagcttat gggcctctcc accgtgc 27 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for sTNFR1 <400> 5 tgtggtgcct gagtcctcag tg 22 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for human IgG-Fc <400> 6 acatgcccac cgtgcccagc acc 23 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for human IgG-Fc <400> 7 atctcgagtc atttacccgg agacaggg 28 <210> 8 <211> 1305 <212> DNA <213> Artificial Sequence <220> <223> Human sTNFR1-Fc <400> 8 atgggcctct ccaccgtgcc tgacctgctg ctgccactgg tgctcctgga gctgttggtg 60 ggaatatacc cctcaggggt tattggactg gtccctcacc taggggacag ggagaagaga 120 gatagtgtgt gtccccaagg aaaatatatc caccctcaaa ataattcgat ttgctgtacc 180 aagtgccaca aaggaaccta cttgtacaat gactgtccag gcccggggca ggatacggac 240 tgcagggagt gtgagagcgg ctccttcacc gcttcagaaa accacctcag acactgcctc 300 agctgctcca aatgccgaaa ggaaatgggt caggtggaga tctcttcttg cacagtggac 360 cgggacaccg tgtgtggctg caggaagaac cagtaccggc attattggag tgaaaacctt 420 ttccagtgct tcaattgcag cctctgcctc aatgggaccg tgcacctctc ctgccaggag 480 aaacagaaca ccgtgtgcac ctgccatgca ggtttctttc taagagaaaa cgagtgtgtc 540 tcctgtagta actgtaagaa aagcctggag tgcacgaagt tgtgcctacc ccagattgag 600 aatgttaagg gcactgagga ctcaggcacc acaacatgcc caccgtgccc agcacctgaa 660 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 720 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 780 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 840 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 900 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 960 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1020 tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1080 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1140 acgcctcccg tgctggactc cgacggcccc ttcttcctct acagcaagct caccgtggac 1200 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1260 aaccactaca cgcagaagag cctctccctg tctccgggta aatga 1305

Claims (13)

a) isolating somatic cells from pigs;
b) introducing a human HO-1 (Heme oxygenase-1) gene and a human TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) fusion gene into the somatic cells, GGTA1 (α-1,3-galactosyltransferase) gene and CMAH ( cytidine monophosphate-N-acetylneuraminic acid hydroxylase) gene knockout (knock-out);
c) selecting and culturing somatic cells into which the human HO-1 gene and the human TNFR1-Fc fusion gene are introduced and the GGTA1 gene and the CMAH gene are knocked out; and
A method for producing a transgenic pig in which immune rejection is suppressed during xenogeneic organ transplantation, comprising the steps of d) removing the nucleus of a pig egg and fusion with the selected somatic cells to prepare a fertilized egg in which the somatic cell nucleus is transplanted,
The transgenic pig is a human HO-1 (Heme oxygenase-1) gene and human TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) fusion gene is introduced, GGTA1 (α-1,3-galactosyltransferase) gene and CMAH ( cytidine monophosphate-N-acetylneuraminic acid hydroxylase) gene is knocked out, a method for producing a transgenic pig in which immune rejection is suppressed during xenogeneic organ transplantation.
The method of claim 1, wherein the human HO-1 gene of step b) comprises the nucleotide sequence of SEQ ID NO: 1.
The method of claim 1, wherein the human TNFR1-Fc fusion gene of step b) comprises the nucleotide sequence of SEQ ID NO: 8.
The method of claim 1, wherein step b) introduces a single vector comprising a human HO-1 gene and a single vector comprising a human TNFR1-Fc fusion gene separately or simultaneously into a somatic cell.
The method of claim 1, wherein the GGTA1 gene knockout is performed using TALEN (transcription activator-like effector nuclease).
The method of claim 5, wherein the TALEN targets the exon 9 region of the GGTA1 gene.
The method of claim 1, wherein the CMAH gene knockout is performed using a transcription activator-like effector nuclease (TALEN).
The method of claim 7, wherein the TALEN targets the exon 3 region of the CMAH gene.
The method of claim 1, further comprising implanting the fertilized egg of step d) into a pig uterus.
Human HO-1 (Heme oxygenase-1) gene and human TNFR1-Fc (Tumor necrosis factor receptor 1-Fc) fusion gene were introduced, GGTA1 (α-1,3-galactosyltransferase) gene and CMAH (cytidine monophosphate-N) were introduced -Acetylneuraminic acid hydroxylase) gene is knocked out, transgenic pigs for transplantation of which immune rejection is suppressed during xenotransplantation.
A somatic cell donor cell line for producing the transgenic pig of claim 10 .
Preparing a transgenic pig by the method of claim 1; and
A method for producing an organ in which immune rejection is suppressed during xenogeneic organ transplantation, comprising the step of isolating an organ for transplantation from the transgenic pig.
The method of claim 12, wherein the organ is one or more selected from the group consisting of islets and pancreas, heart, kidney, liver, lung, cornea, and the like.

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