[go: up one dir, main page]

WO2025127214A1 - Porc cloné transgénique pour xénotransfusion et xénogreffe avec des gènes ggta1, cmah, igb3s et β4galnt2 porcins délétés et le gène cd59 humain inactivé, et son procédé de production - Google Patents

Porc cloné transgénique pour xénotransfusion et xénogreffe avec des gènes ggta1, cmah, igb3s et β4galnt2 porcins délétés et le gène cd59 humain inactivé, et son procédé de production Download PDF

Info

Publication number
WO2025127214A1
WO2025127214A1 PCT/KR2023/020782 KR2023020782W WO2025127214A1 WO 2025127214 A1 WO2025127214 A1 WO 2025127214A1 KR 2023020782 W KR2023020782 W KR 2023020782W WO 2025127214 A1 WO2025127214 A1 WO 2025127214A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
human
knock
transgenic
vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/KR2023/020782
Other languages
English (en)
Korean (ko)
Inventor
최기명
심주현
고나영
김형주
이용진
박재경
곽경민
김준형
강풀잎
김성훈
최재훈
김현일
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Optipharm Co Ltd
Original Assignee
Optipharm Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Optipharm Co Ltd filed Critical Optipharm Co Ltd
Publication of WO2025127214A1 publication Critical patent/WO2025127214A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates to a transgenic cloned pig for xenotransfusion and xenotransplantation in which the porcine GGTA1 (Alpha 1,3-galactosyltransferase), CMAH (CMP-N-acetylneuraminic acid hydroxylase), iGb3s (Isogloboside 3 synthease) and ⁇ 4GalNT2 (Beta-1,4-N-Acetyl-Galactosaminyl Transferase 2) genes are deleted and the human CD59 gene is knocked in, and a method for producing the same.
  • porcine GGTA1 Alpha 1,3-galactosyltransferase
  • CMAH CMP-N-acetylneuraminic acid hydroxylase
  • iGb3s Isogloboside 3 synthease
  • ⁇ 4GalNT2 Beta-1,4-N-Acetyl-Galactosaminyl Transfer
  • This organ transplant supply problem can be solved by using pig-to-human xenotransplantation, where pig organs completely replace human organs.
  • pigs are highly similar to humans in terms of morphology and genetics, and unlike primates, they have fewer zoonotic infectious diseases and ethical issues.
  • they have the advantage of high fertility, which can sufficiently overcome the shortage of supply, and they have been used as a non-clinical model for many basic studies, and they are used for food, so there is relatively less rejection compared to other experimental animals.
  • the Yucatan miniature pig was selected as the optimal pig species for xenotransplantation because it is phenotypically and anatomically similar to humans, and has similar organ sizes (94% heart, 66% liver, 81% pancreas, and 91% kidney) compared to other species.
  • Korean Patent No. 10-2040203 discloses that after producing a transgenic cloned pig lacking GGTA1 ( ⁇ 1,3-galactosyltransferase), CMAH (CMP-N-acetylneuraminic acid hydroxylase), iGb3s (Isoglobotrihexosylceramide synthase), and ⁇ 4GalNT2 (Beta-1,4-N-Acetyl-Galactosaminyl Transferase2) genes, an in vitro human serum response experiment was conducted, and it was confirmed that IgG and IgM binding was significantly reduced, suggesting the possibility of overcoming antigen-antibody mediated rejection in xenograft transplantation.
  • GGTA1 ⁇ 1,3-galactosyltransferase
  • CMAH CMP-N-acetylneuraminic acid hydroxylase
  • iGb3s Isoglobotrihexosylceramide synthas
  • Another object of the present invention is to provide a method for producing xenograft organs for transplantation, which includes breeding cloned pigs to extract organs or producing organs using their germ cells or somatic cells.
  • the present invention provides a knock-in vector for producing a transgenic cloned pig comprising a human CD59 gene represented by sequence number 1 that is targeted to a porcine GGTA1 gene.
  • the present invention provides a transformed cell line produced by transforming the knock-in vector into a somatic cell.
  • the present invention provides a transgenic cloned pig produced by nuclear transfer of the above-mentioned transgenic cell line.
  • the present invention provides a method for producing a transgenic cloned pig, comprising the steps of producing the above-described transgenic cell line, the step of transplanting the cell line into an enucleated egg to form a nuclear transfer embryo, and the step of transplanting the nuclear transfer embryo into the oviduct of a surrogate mother.
  • the present invention provides a transgenic cloned pig produced by the above method.
  • the present invention provides a method for producing xenograft organs for transplantation, which includes breeding the cloned pigs to extract organs or producing organs using their germ cells or somatic cells.
  • the present invention relates to a transgenic cloned pig for xenogeneic blood transfusion and xenogeneic organ transplantation in which the porcine GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 genes are deleted and the human CD59 gene is knocked in, and a method for producing the same.
  • Figure 1 is a schematic diagram showing the acquisition of base cell lines lacking the GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 genes used in development.
  • Figure 2a is a schematic diagram showing a human CD59 knock-in vector.
  • Figure 2b is a diagram showing a human CD59 expression strategy through knock-in.
  • Figure 3 is a diagram showing the results of separating human CD59 positive cells through sorting after transduction.
  • Figure 4 is a diagram showing the positions of primers used in the invention and whether the human CD59 knock-in vector was introduced into the transformed cell line.
  • Figure 5 shows the results of confirming protein expression through FACS analysis after immunofluorescence staining of selected transformed cell lines #21, #30, #35, and #39.
  • Figure 6 is a photograph of transgenic cloned pigs produced through somatic cell cloning using transgenic cell line #21.
  • Figure 7 is a diagram showing the human CD59 gene analysis of the produced transgenic cloned pigs.
  • Figure 8 is a diagram showing the results of FACS analysis of major cells of the produced transgenic cloned pigs.
  • Figure 9a is a diagram showing the results of a cytotoxicity test on human serum using produced transgenic cloned porcine peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • Figure 9b is a diagram showing the results of a cytotoxicity test on monkey serum using the produced transgenic cloned porcine peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • Figure 10a is a diagram showing the results of a cytotoxicity test on human serum using produced transgenic cloned porcine red blood cells (RBCs).
  • Figure 10b is a diagram showing the results of a cytotoxicity test on monkey serum using the produced transgenic cloned porcine red blood cells (RBC).
  • the present invention provides a knock-in vector for producing a transgenic cloned pig comprising a human CD59 gene represented by sequence number 1 that is targeted to a porcine GGTA1 gene.
  • the human CD59 Human Membrane Attack complex-inhibitory protein; MAC-IP
  • MAC-IP Human Membrane Attack complex-inhibitory protein
  • the base sequence of the human CD59 gene is indicated by Genebank number NM_203329.3, and the base sequence of the porcine CD59 gene is indicated by Genbank number AH010595.2.
  • the term 'knock-in' means inserting a foreign base sequence that did not exist in another species or originally in the organism, into the genome of the organism or a DNA base sequence derived from the organism, using genetic recombination technology.
  • the knock-in vector comprises a first region comprising a left arm including a partial sequence of exon 3 or exon 4 of a porcine GGTA1 gene, a second region comprising a region encoding a human CD59 gene and a right arm including a partial sequence of exon 4 or exon 5 of a porcine GGTA1 gene.
  • the knock-in vector has a human CD59 CDS sequence (SEQ ID NO: 2) inserted between the left arm sequence from sequences 261535947 to 261536969 and the right arm sequence from sequences 261535101 to 261535922 of chromosome 1.
  • left arm and right arm refer to regions where homologous recombination occurs.
  • homologous recombination means genetic recombination that occurs through exchange at a genetic locus having homology, and is used to produce transgenic animals having an allele with lost function.
  • the left arm comprises a part of exon 3 or exon 4 of the porcine GGTA1 gene.
  • the size of the left arm may be 1,023 bp and may be composed of a base sequence represented by SEQ ID NO: 3.
  • the light arm comprises a portion of exon 4 or exon 5 of the porcine GGTA1 gene.
  • the size of the light arm may be 822 bp and may be composed of a base sequence represented by SEQ ID NO: 4.
  • the knock-in vector may be composed of a base sequence of SEQ ID NO: 5.
  • the “vector” means a genetic construct including a base sequence of a gene operably linked to a suitable regulatory sequence so as to be able to express a target gene in a suitable host, wherein the regulatory sequence may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, and a sequence for regulating the termination of transcription and translation.
  • the vector of the present invention is not particularly limited and any vector known in the art may be used as long as it is capable of replicating in a cell, and examples thereof may be plasmids, cosmids, phage particles, and viral vectors.
  • the knock-in gene recombinant vector of the present invention can preferably be represented by the vector map disclosed in Fig. 2a.
  • the present invention provides a transformed cell line produced by transforming the knock-in vector into a somatic cell.
  • transformation means introducing DNA into a host so that the DNA becomes replicable as an extrachromosomal element or by chromosomal integration completion. Transformation includes any method of introducing a nucleic acid molecule into an organism, cell, tissue or organ, and can be performed by selecting a standard technique suitable for the host cell as is known in the art, and examples thereof include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.
  • a standard technique suitable for the host cell as is known in the art, and examples thereof include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate
  • the somatic cell is preferably a fibroblast, and more preferably a porcine fibroblast.
  • the somatic cell may be a somatic cell in which the GGTA1, CMAH, iGb3s and ⁇ 4GalNT2 genes are knocked out.
  • the above GGTA1 gene is responsible for the biosynthesis of ⁇ -Gal
  • the CMAH gene is responsible for the biosynthesis of Neu5Gc
  • the iGb3s gene synthesizes iGb3, a glycosphingolipid
  • the ⁇ 4GalNT2 gene is a gene that produces sugar chains, which produce GalNAc ⁇ 1-4, Gal ⁇ 1-4GlcNAc ⁇ 1-3Gal, and Sd(a) (Sid blood group; CAD or CT) antigens.
  • a transgenic cloned pig lacking the above-mentioned four carbohydrate-derived heterologous antigens is disclosed in Korean Patent No. 10-2040203.
  • a transformed cell line was produced in which the human CD59 gene was knocked in to suppress the complement-mediated immune rejection response that occurs when xenografted into fibroblasts derived from a transformed pig in which four genes, GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2, which suppress antigen-antibody-mediated immune rejection response, were deleted.
  • the transformed cell according to the present invention may be a cell having the accession number KCLRF-BP-00527 deposited with the Korea Cell Line Research Foundation (KCLRF) on November 24, 2023.
  • the present invention provides a transgenic cloned pig produced by nuclear transfer of the above-mentioned transgenic cell line.
  • the present invention provides a method for producing a transgenic cloned pig, comprising the steps of producing the above-described transgenic cell line, the step of transplanting the cell line into an enucleated egg to form a nuclear transfer embryo, and the step of transplanting the nuclear transfer embryo into the oviduct of a surrogate mother.
  • nuclear transfer refers to a genetic manipulation technique that artificially combines nuclear DNA from another cell into a cell without a nucleus to give it the same characteristics, and it is possible to use a method known in the art.
  • nuclear transfer oocyte means an oocyte into which a nuclear donor cell has been introduced or fused.
  • nucleated oocyte means an oocyte from which the nucleus has been removed.
  • the present invention provides a transgenic cloned pig produced by the above method.
  • the present invention provides a method for producing xenograft organs for transplantation, which includes breeding the cloned pigs to extract organs or producing organs using their germ cells or somatic cells.
  • the above organs can be extracted through a normal surgical operation after breeding a donor cloned animal by adjusting the breeding period considering the sex, age, weight, height, etc. of the recipient, and can be transplanted directly to the recipient after extraction or quickly refrigerated.
  • gametes such as sperm or eggs can be extracted from the cloned animal and artificially or naturally fertilized to obtain offspring, and these offspring can be used for organ transplantation or the production of other by-products for organ transplantation.
  • somatic cells of the cloned animal can be extracted and used for organ transplantation or the production of other by-products as described above.
  • the transgenic pig in which the GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 genes of the present invention are deleted and the human CD59 gene is expressed at the GGTA1 locus can overcome the hyperacute and complement-mediated immune rejection reactions occurring in xenograft organ transplantation. Therefore, the transgenic cloned pig according to the present invention can be usefully utilized as a donor animal for organ and blood transfusion between xenografts.
  • Transgenic pigs were produced to establish QKO (quadruple knockouts) transgenic cell lines of porcine GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 genes.
  • the QKO F0 individual developed in Korean Patent Application No. 10-2018-0034466 was crossed with the wild type (WT) to produce an F1 individual with QKO heterozygous genetic traits.
  • the produced F1 individuals were raised until sexual maturity (8 months of age) and crossed with the QKO F0 to produce an F2 individual with QKO homozygous genetic traits.
  • the produced individuals were subjected to PCR using the primers disclosed in Table 1 for GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 base sequence analysis.
  • the obtained PCR products were subjected to base sequence analysis by Solgent.
  • the individual ears were disinfected with 70% ethanol, and ear tissue measuring 1 cm in width ⁇ length was biopsied, stored in DPBS containing penicillin-streptomycin antibiotics, and then transported to the laboratory.
  • the tissues were washed three times with DPBS, the epidermis was removed, minced, and attached to a 6-well multiplate. After culturing the cells in DMEM (Lonza, Switzerland) containing 10% FBS and 1X Penicillin-streptomycin antibiotics for 7 days, porcine ear fibroblasts (PEF) derived from ear tissue were obtained.
  • DMEM Nonza, Switzerland
  • Ear fibroblasts were isolated from the above-mentioned transgenic pigs and cultured to establish GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 gene knockout QKO transgenic cell lines.
  • the individual ears were disinfected with 70% ethanol, and ear tissue measuring 1 cm in width ⁇ length was biopsied, stored in DPBS containing penicillin-streptomycin antibiotics, and then transported to the laboratory.
  • the tissues were washed three times with DPBS, the epidermis was removed, minced, and attached to a 6-well multiplate. After culturing the cells in DMEM (Lonza, Switzerland) containing 10% FBS and 1X Penicillin-streptomycin antibiotics for 7 days, porcine ear fibroblasts (PEF) derived from ear tissue were obtained.
  • DMEM Nonza, Switzerland
  • Example 3 Construction of transformed cell lines in which GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 genes are deleted and human CD59 gene is knocked in
  • GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 genes are deleted and human CD59 gene is knocked in
  • the CD59 knock-in vector constructed in Example 2 was introduced into the transgenic pig-derived fibroblasts from which the heterologous antigen obtained in Example 1 had been removed using electroporation. More specifically, the cultured cells were washed with DPBS and then treated with 0.25% trypsin-EDTA (Gibco) to recover the cells. After centrifugation at 1500 rpm for 3 minutes and washing with DPBS, the vector was mixed with the buffer in the P3 Primary Cell 4D-Nucleofector kit (Amaxa, Germany), and then electroporated using the EN-150 protocol of the 4D-Nucleofector system (amaxa).
  • P3 Primary Cell 4D-Nucleofector kit Amaxa, Germany
  • the cells were immunostained with CD59 antibody to increase the selection efficiency, and then only CD59 positive cells were selected using FACS AriaIII (BD bioscience, USA).
  • the selected cells were subjected to single cell colony culture, and then genetic analysis was performed on each colony. More specifically, genomic DNA was extracted from each transformed cell colony using the Dneasy Blood & tissue kit, and PCR was performed using primers containing each location inside the human CD59 knock-in vector and outside the cancer. The obtained PCR products were loaded onto a 1% agarose gel and analyzed.
  • Example 3-1 cell immunostaining and FACS analysis were performed for protein expression analysis from morphologically superior colonies #21, #30, #35, and #39.
  • the cell line was washed with DPBS, treated with 0.25% trypsin-EDTA solution for 3 minutes, and then the cells were harvested. Trypsin-EDTA was inactivated with fetal bovine serum (FBS), washed with DPBS, and then reacted with human CD59 antibody at a concentration of 1:100 for 1 hour. Thereafter, the cells were washed three times with DPBS containing Tween-20, and reacted with a secondary antibody conjugated with FITC fluorescence at a concentration of 1:100 in a refrigerator for 1 hour. Similarly, the cells were washed three times with DPBS containing Tween-20, fixed with 1% formalin, and analyzed using Cytoflex (Beckman Coulter, USA). As a positive control, a transformed cell line expressing the human CD59 gene by the EF1 ⁇ promoter was used for the analysis.
  • FBS fetal bovine serum
  • DPBS fetal bovine serum
  • the above-mentioned transformed cell line #21 was named QKO/hCD59 KI and deposited with the Korea Cell Line Research Foundation (KCLRF) on November 24, 2023, and was assigned the accession number KCLRF-BP-00527.
  • Example 4 Production of transgenic pigs in which GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 genes were deleted and human CD59 gene was knocked in
  • oocytes were first prepared.
  • COCs Cumulus-oocyte complexes
  • the follicular fluid was washed in TL-HEPES medium, and COCs were selected from petri dishes and transferred to TCM 199 (Gibco, USA) containing 0.1 mM PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 IU/mL hCG (MSD Animal Health, USA), 0.5 ⁇ g/mL FSH (Sigma-Aldrich), 10 ng/mL epidermal growth factor (Sigma-Aldrich), 75 ⁇ g/mL penicillin G, and 50 ⁇ g/mL streptomycin, which were covered with mineral oil and approximately 70-80 COCs were transferred to 4-well multidish (Nunc, Denmark). The cells were cultured for 22 hours and incubated in TCM 199 medium from which only FSH and hCG were removed for 42 to 44 hours under conditions of 5% CO2 and 39°C.
  • Nuclear transfer was performed to produce transgenic pigs.
  • COCs induced to mature in vitro were transferred to a 4-well multidish containing culture medium (Micromanipulation medium, MM) containing TCM 199, 0.3% BSA (Sigma-Aldrich), 0.6 mM NaHCO3, 2.9 mM HEPES, 30.0 mM NaCl, 0.05 g/mL penicillin G, and 0.06 g/mL streptomycin, treated with 0.1% hyaluronidase, and gently pipetted approximately 70 to 80 times to separate oocytes from cumulus cells.
  • culture medium Meromanipulation medium, MM
  • BSA Sigma-Aldrich
  • Metaphase II oocytes with protruding first polar body were selected and nuclear staining was performed for 15 minutes in a culture medium containing 5 ⁇ g/mL Hoechst 33342 in MM (MM-CB) containing 7.5 ⁇ g/mL cytochalasin B, and then the oocytes were transferred to MM-CB.
  • the stained first polar body and nucleus were aspirated using a fine glass pipette to remove the nucleus from oocytes without cumulus cells (enucleation).
  • donor cells of the transformed cell line #21 prepared in Example 3 were cultured in DMEM medium containing 0.5% FBS for 3 days.
  • a single donor cell was placed in the perivitelline space of the oocyte in contact with the oocyte membrane.
  • Inoculated oocytes were placed between two 0.2 mm diameter platinum electrodes spaced 1 mm apart in a medium consisting of 0.3 M mannitol, 1.0 mM CaCl2H2O, 0.1 mM MgCl26H2O, and 0.5 mM HEPES. Fusion/activation was induced by two consecutive 30 ⁇ s (BTX, USA) DC pulses of 1.1 kV/cm. Electrically stimulated recombinant oocytes were cultured (15 cells per group) in 30 ⁇ l drops of PZM-3 (Biol Reprod.
  • NT embryos were surgically transferred into the oviduct of sows on the first day of standing estrus. Pregnancy status was confirmed using an ultrasound scanner (Medison Co., Korea).
  • Example 5 Production and verification of transgenic pigs in which GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 genes were deleted and human CD59 gene was knocked in
  • fibroblasts from the piglets were obtained and their base sequences were analyzed, and it was confirmed that the target gene regions, GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 genes, were successfully deleted in the fibroblasts from the transgenic pigs produced in Example 4.
  • PCR amplification was performed using the 463F-464R primer and 463F-873R primer in Table 3, respectively, and the products were loaded onto a 1% agarose TAE gel and analyzed.
  • the two loci, wild type and CD59 knock-in were amplified by the introduction of the human CD59 knock-in vector in the 463F-464R primer set (left) by the introduction of the knock-in vector, and it was confirmed that the two loci were also amplified by homologous recombination in the 463F-873R primer set (right).
  • PBMCs peripheral blood mononuclear cells
  • RBCs red blood cells
  • splenocytes derived from the blood of the transgenic cloned pig #1 verified in Example 5-1 above protein expression of the defective gene and knocked-in CD59 was analyzed by FACS.
  • blood was collected from the subject using a syringe and diluted 1:1 in DPBS.
  • the diluted blood was added 1:1 (volume/volume) to ficoll-paque plus (GE healthcare) and centrifuged at 500g for 40 minutes.
  • the middle buffy coat layer and the lower red blood cell layer were separated, washed with DPBS, and immunostaining using antibodies for each gene was performed.
  • Spleen cells were biopsied from the subject's spleen and transported to the laboratory in DPBS containing penicillin-streptomycin.
  • the transported spleen sample was washed 5 times with DPBS, placed on a 40 ⁇ m cell strainer, and finely chopped with a syringe.
  • the obtained spleen cells were removed from the red blood cells using RBC lysis buffer (Sigma-aldrich) and immunostaining using antibodies for each gene was performed.
  • Each cell was analyzed using cytoflex equipment along with an unstained control.
  • Example 5-1 To evaluate the function of the transgenic pigs produced in Example 5-1 above, cytotoxicity analysis using human and primate serum was performed.
  • peripheral blood mononuclear cells and red blood cells separated using ficoll paque plus solution in the same manner as in Example 5-2 were treated with human and primate complement serum at concentrations of 3.125, 6.25, 12.5, 25, and 50% for 3 hours in a 96-well multiplate. After that, the human complement serum in all wells was removed, and 100 ⁇ l of DPBS and 10 ⁇ l of CCK-8 (Dojindo, Japan) were added to measure cell viability.
  • the survival rate was higher in pigs with a deletion of the QKO gene than in normal pigs (WT) in all treatment groups.
  • WT normal pigs
  • the transgenic cloned pigs of the present invention in which four types of foreign antigens were knocked out and the human CD59 gene was knocked in, had a significantly higher survival rate with human and primate complement serum.
  • FIGS. 10a and 10b in the case of red blood cells, it was confirmed that the survival rate with human and primate complement serum was also significantly higher.
  • the transgenic pigs in which the GGTA1, CMAH, iGb3s, and ⁇ 4GalNT2 genes were deleted and the human CD59 gene was expressed at the GGTA1 locus, manufactured by the method according to the present invention can overcome the hyperacute and complement-mediated immune rejection reactions occurring in xenograft organ transplantation.
  • the expression of the human CD59 gene was confirmed in red blood cells, it can be used as artificial blood for xenograft transfusion.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Environmental Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un porc cloné transgénique pour xénotransfusion et xénogreffe dans lequel les gènes GGTA1, CMAH, iGb3s et β4GalNT2 porcins sont délétés et le gène CD59 humain est inactivé, et son procédé de production. En induisant l'expression du gène CD59 humain dans un porc transgénique, ce qui améliore la réponse de rejet immunitaire à médiation par anticorps-anticorps existante, la réponse de rejet immunitaire médiée par le complément a été réduite, et l'expression du gène CD59 humain a été confirmée dans des globules rouges, et par conséquent, le porc peut être utilisé efficacement pour la xénotransfusion et la xénogreffe.
PCT/KR2023/020782 2023-12-15 2023-12-15 Porc cloné transgénique pour xénotransfusion et xénogreffe avec des gènes ggta1, cmah, igb3s et β4galnt2 porcins délétés et le gène cd59 humain inactivé, et son procédé de production Pending WO2025127214A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020230183055A KR102722231B1 (ko) 2023-12-15 2023-12-15 돼지 GGTA1, CMAH, iGb3s, β4GalNT2 유전자가 결손되고 인간 CD59 유전자가 넉인된 이종수혈 및 이종장기이식을 위한 형질전환 복제돼지 및 이의 제조방법
KR10-2023-0183055 2023-12-15

Publications (1)

Publication Number Publication Date
WO2025127214A1 true WO2025127214A1 (fr) 2025-06-19

Family

ID=93287799

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2023/020782 Pending WO2025127214A1 (fr) 2023-12-15 2023-12-15 Porc cloné transgénique pour xénotransfusion et xénogreffe avec des gènes ggta1, cmah, igb3s et β4galnt2 porcins délétés et le gène cd59 humain inactivé, et son procédé de production

Country Status (2)

Country Link
KR (1) KR102722231B1 (fr)
WO (1) WO2025127214A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180056419A (ko) * 2015-09-09 2018-05-28 레비비코르 인코포레이션 이종 장기이식을 위한 멀티-형질전환 피그
KR20190112453A (ko) * 2018-03-26 2019-10-07 주식회사 옵티팜 돼지 GGTA1, CMAH, iGb3s 및 β4GalNT2 유전자가 결손된 이종장기이식을 위한 형질전환 복제돼지 및 이의 제조방법

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101763196B1 (ko) * 2015-06-29 2017-07-31 주식회사 옵티팜 이종장기이식을 위한 형질전환 복제돼지 및 이의 제조방법
KR102363891B1 (ko) * 2017-04-28 2022-02-17 서울대학교산학협력단 ＨＯ-1 유전자 및 ＴＮＦＲ1-Ｆｃ 유전자를 동시에 발현하며 GGTA1 유전자 및 CMAH 유전자가 넉아웃된 형질전환 돼지 및 이의 용도
KR102738812B1 (ko) * 2021-12-16 2024-12-09 주식회사 옵티팜 돼지 GGTA1, CMAH, β4GalNT2 및 CD40 유전자가 결손된 이종장기이식을 위한 형질전환 복제돼지 및 이의 제조방법

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180056419A (ko) * 2015-09-09 2018-05-28 레비비코르 인코포레이션 이종 장기이식을 위한 멀티-형질전환 피그
KR20190112453A (ko) * 2018-03-26 2019-10-07 주식회사 옵티팜 돼지 GGTA1, CMAH, iGb3s 및 β4GalNT2 유전자가 결손된 이종장기이식을 위한 형질전환 복제돼지 및 이의 제조방법

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 22 September 2023 (2023-09-22), ANONYMOUS TOTH, LUQUE-FERNANDEZ ANGEL, O' HALPERIN J, PM SHEA: "Homo sapiens CD59 molecule (CD59 blood group) (CD59), transcript varia - Nucleotide - NCBI", XP093323274, Database accession no. NM_203329.3 *
KLYMIUK NIKOLAI; AIGNER BERNHARD; BREM GOTTFRIED; WOLF ECKHARD: "Genetic Modification of Pigs as Organ Donors for Xenotransplantation", MOLECULAR REPRODUCTION AND DEVELOPMENT, WILEY-LISS, INC, NEW YORK, NY, US, vol. 77, no. 3, 1 March 2010 (2010-03-01), NEW YORK, NY, US , pages 209 - 221, XP009135636, ISSN: 1040-452X, DOI: 10.1002/mrd.21127 *
KO NAYOUNG, SHIM JOOHYUN, KIM HYOUNG-JOO, LEE YONGJIN, PARK JAE-KYUNG, KWAK KYUNGMIN, LEE JEONG-WOONG, JIN DONG-IL, KIM HYUNIL, CH: "A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model", SCIENTIFIC REPORTS, NATURE PUBLISHING GROUP, US, vol. 12, no. 1, US , pages 1 - 11, XP093323271, ISSN: 2045-2322, DOI: 10.1038/s41598-022-13536-z *

Also Published As

Publication number Publication date
KR102722231B1 (ko) 2024-10-28

Similar Documents

Publication Publication Date Title
Cibelli et al. Trasgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells
CN104263754B (zh) 白化病模型猪的重构卵及其构建方法和模型猪的构建方法
US11180763B2 (en) CRISPR/Cas9 vector combination and application thereof in gene knockout
US6194635B1 (en) Embryonic germ cells, method for making same, and using the cells to produce a chimeric porcine
WO2021015571A1 (fr) Porc cloné transgénique pour xénogreffe exprimant des gènes cd46 et tbm humains, dans lequel l'enveloppe c de rétrovirus endogène porcine est négative et les gènes ggta1, cmah, igb3s et β4galnt2 sont inactivés, et leur procédé de préparation
US6545199B1 (en) Embryonic germ cells, method for making same, and using the cells to produce a chimeric porcine
WO2002077175A2 (fr) Procede de generation d'animaux clones par rearrangement de chromosomes
US20190032086A1 (en) Method for preparing a gene knock-out canine with somatic cell cloning technology
KR102040203B1 (ko) 돼지 GGTA1, CMAH, iGb3s 및 β4GalNT2 유전자가 결손된 이종장기이식을 위한 형질전환 복제돼지 및 이의 제조방법
CN104894163B (zh) 一种制备GGTA1和iGb3S双基因敲除的非人哺乳动物的方法及应用
KR100807644B1 (ko) 체외수정 수정란 및 체세포 복제 수정란의 동시 이식에의한 돼지 생산 방법
KR102738812B1 (ko) 돼지 GGTA1, CMAH, β4GalNT2 및 CD40 유전자가 결손된 이종장기이식을 위한 형질전환 복제돼지 및 이의 제조방법
WO2000039294A1 (fr) Cellules porcines incapables d'exprimer l'antigene cd40, et destinees a des xenogreffes
RU2391817C2 (ru) Способ получения клонированного представителя семейства псовых
US7071372B2 (en) Method for cloning animals with targetted genetic alterations by transfer of long-term cultured male or female somatic cell nuclei, comprising artificially-induced genetic alterations, to enucleated recipient cells
WO2025127214A1 (fr) Porc cloné transgénique pour xénotransfusion et xénogreffe avec des gènes ggta1, cmah, igb3s et β4galnt2 porcins délétés et le gène cd59 humain inactivé, et son procédé de production
CN1424870A (zh) 用挑选的供体细胞实施核移植
CN115161342B (zh) 一种具有新遗传性状的细胞系和动物的构建方法
Waghmare et al. Gene targeting and cloning in pigs using fetal liver derived cells
WO2021225384A1 (fr) Porc transgénique modèle animal présentant une sensibilité accrue au coronavirus du syndrome respiratoire aigu sévère 2, et son procédé de production
US20040031069A1 (en) Cloning endangered and extinct species
CN104630277A (zh) 一种采用手工克隆技术构建水牛转基因克隆胚胎的方法
Reyes et al. Efficient selection of Gal-knockout pig cells for somatic cell nuclear transfer
KR20120112966A (ko) Ｈｏ-1 유전자를 발현하는 형질전환 돼지 및 이의 용도
KR20090115081A (ko) 개과 동물의 복제 생산 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23961615

Country of ref document: EP

Kind code of ref document: A1