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EP-4739109-A1 - TRANSGENIC PIG COMPRISING A TRANSGENE FOR XENOTRANSPLANTATION

EP4739109A1EP 4739109 A1EP4739109 A1EP 4739109A1EP-4739109-A1

Abstract

The present invention relates to a transgenic pig comprising at least one transgene for xenotransplantation within its genome, wherein said at least one transgene is incorporated and expressed at a single locus 10448 (NCBI Reference Sequence: NC_010448.4). Further, the present invention relates to an organ, an organ fragment, a tissue, a scaffold or a cell obtained from said transgenic pig as defined in the present invention. The present invention also relates to the organ, the organ fragment, the tissue, the scaffold or the cell for use in a method of treating a disease in a subject comprising replacing a diseased or failed organ, organ fragment, tissue or cell in a subject by implanting into said subject at least one organ, organ fragment, tissue, scaffold or cell as defined by the present invention and an in vitro method of producing such transgenic pig of the present invention.

Inventors

  • SCHNIEKE, Angelika
  • FISCHER, Konrad Josef

Assignees

  • Technische Universität München, in Vertretung des Freistaats Bayern

Dates

Publication Date
20260513
Application Date
20240705

Claims (20)

  1. 1. A transgenic pig comprising at least one transgene for xenotransplantation within its genome, wherein said at least one transgene is incorporated and expressed at a single locus 10448 having the NCBI Reference Sequence: NC_010448.4.
  2. 2. The transgenic pig of claim 1, wherein said at least one transgene encodes: i a complement inhibitor; ii a cytoprotectant; iii an anticoagulant; iv an immunomodulator; v a regulator protein of at least any one of reactive oxygen (ROS) formation, superoxide formation, or of caspase activity; or vi an alpha 1 ,2 fucosyltransferase, or a combination thereof.
  3. 3 The transgenic pig of claim 2, wherein i said complement inhibitor is any one of Cluster of Differentiation 55 (CD55), Cluster of Differentiation 46 (CD46) or Cluster of Differentiation (CD59), or a combination thereof; ii said cytoprotectant is any one of Tumor necrosis factor-alpha induced protein 3 (A20/TNFAIP3), Heme oxygenase 1 (HO-1) or Gluthatione peroxidase 4 (GPX4), or a combination thereof; iii said anticoagulant is any one of endothelial protein C receptor (EPCR), Thrombomodulin (THBD), tissue factor pathway inhibitor (TFPI), or Cluster of Differentiation 39 (CD39), Van Willebrand factor, or Cobra Venom factor, or a combination thereof; iv said immunomodulator is an immunosuppressant; v said immunomodulator is an immunosuppressant selected from the group consisting of Cluster of Differentiation 47 (CD47), dominant-negative class II transactivator (CIITA- DN), a programmed cell death 1 ligand 1 (PD-L1), cytotoxic t-lymphocyte-associated antigen (CTLA4/LEA29Y), IL-1 receptor antagonist (IL1RA), and IL-27 alpha (IL27a), or a combination thereof; and/or vi said regulator protein is A Kinase interacting Protein 1 (AKIP1).
  4. 4 The transgenic pig of any one of the preceding claims, wherein said pig expresses at least any one of: i a complement inhibitor; ii a cytoprotectant; iii an anticoagulant; iv an immunomodulator; v) a regulator protein of at least any one of reactive oxygen (ROS) formation, superoxide formation, or of caspase activity; or vi) an alpha 1,2 fucosyltransferase; or a combination thereof which is encoded by said at least one transgene.
  5. 5. The transgenic pig of claim 4, wherein the expression of at least any one of i)-vi) as defined by claim 4 is above the corresponding expression of at least any one of i)-vi) as defined by claim 4 in a subject from which said at least one transgene encoding at least any one of i)-vi) as defined by claim 4 is derived from.
  6. 6. The transgenic pig of any one of the preceding claims, further comprising at least one additional genetic modification.
  7. 7. The transgenic pig of claim 6, wherein said at least one additional genetic modification is selected from the group consisting of a gene knock-out, a gene knock-in, a gene replacement, a point mutation, and a deletion, insertion or substitution of a gene, a gene fragment or a nucleotide, or a combination thereof.
  8. 8. The transgenic pig of claim 7, wherein said gene knock-out comprises the knock-out of at least one gene selected from the group consisting of a gene encoding: i alpha 1,3 galactosyltransferase 1 (GGTA1); ii alpha 1,3 galactosyltransferase 2 (GGTA2); iii cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH); iv p-1 ,4-N-acetylgalactosaminyl transferase 2 (B4GALNT2); v swine leucocyte antigen I (SLAI); vi UL16 binding protein 1 (LILBP1); vii class II major histocompatibility complex transactivator (CIITA); viii growth hormone receptor (GHR); ix asialoglycoprotein receptor 1 (ASGR1); x toll-like receptor-4 (TLR4), xi swine leucocyte antigen II (SLAII); xii MHC class I polypeptide-related sequence A (MICA); xiii MHC class I polypeptide-related sequence B (MICB); xiv CC-chemokine ligand 2 (CCL2); xv CC-motif-chemokine receptor 5 (CCR5); xvi CX3C-motif chemokine receptor 1 (CX3CR1), and xvii isoglobotriosylceramide synthase (iGB3), or a combination thereof.
  9. 9. The transgenic pig of any one of the preceding claims, wherein said pig additionally lacks expression of at least any one of GGTA1, GGTA2, CMAH, B4GALNT2, SLAI, LILBP1, CIITA, GHR, ASGR1, TLR4, SLAII, MICA, MICB, CCL2, CCR5, CX3CR1, or iGB3, or a combination thereof.
  10. 10. The transgenic pig of any one of the preceding claims, wherein said at least one transgene is a gene sequence, a minigene or a cDNA construct, preferably a gene sequence.
  11. 11. The transgenic pig of any one of the preceding claims, wherein said at least one transgene is a human or porcine gene sequence, a human or porcine minigene or a human or porcine cDNA construct, preferably a human or porcine gene sequence.
  12. 12. The transgenic pig of any one of the preceding claims, wherein said pig is free of porcine endogenous retrovirus C (PERV-C) and/or free of porcine Cytomegalovirus (pCMV).
  13. 13. The transgenic pig of any one of the preceding claims, wherein said pig has a weight in the range between 1kg and 450 kg.
  14. 14. The transgenic pig of claim 13, wherein said pig has a weight in the range between 1kg and 150kg.
  15. 15. An organ, an organ fragment, a tissue, a scaffold or a cell obtained from said transgenic pig of any one of claims 1-14.
  16. 16. The organ of claim 15, wherein said organ is any one of a lung, a heart, a kidney, a liver, a pancreas, or a spleen.
  17. 17. The organ of claim 16, wherein said organ is a liver or a kidney.
  18. 18. The organ, the organ fragment, the tissue, the scaffold or the cell of any one of claims 15 to 17 for use in a method of treating a disease in a subject, the method comprising replacing a diseased or failed organ, organ fragment, tissue or cell in a subject by implanting into said subject at least one organ, organ fragment, tissue, scaffold or cell of any one of claims 15 to 17.
  19. 19. A method of producing a transgenic pig of any one of claims 1-14, the method comprising i incorporating at least one transgene at a single locus 10448 having the NCBI Reference Sequence: NC_010448.4 within a pig genome to provide a polygenic pig genome in vitro', ii) permitting a cell comprising the polygenic pig genome to mature into the transgenic pig.
  20. 20. The method of claim 19, wherein the pig genome is a somatic cell and the cell is a pig zygote, wherein the pig zygote is provided by somatic cell nuclear transfer (SCNT).

Description

TRANSGENIC PIG COMPRISING A TRANSGENE FOR XENOTRANSPLANTATION CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims the benefit of priority of EP Patent Application No. 23184107.3 filed 07 July 2023, the content of which is hereby incorporated by reference in its entirety for all purposes. TECHNICAL FIELD OF THE INVENTION [001] The present invention relates to a transgenic pig comprising at least one transgene for xenotransplantation within its genome, wherein said at least one transgene is incorporated and expressed at a single locus 10448 (NCBI Reference Sequence: NC_010448.4). Further, the present invention relates to an organ, an organ fragment, a tissue, a scaffold or a cell obtained from said transgenic pig as defined in the present invention. The present invention also relates to the organ, the organ fragment, the tissue, the scaffold or the cell for use in a method of treating a disease in a subject comprising replacing a diseased or failed organ, organ fragment, tissue or cell in a subject by implanting into said subject at least one organ, organ fragment, tissue, scaffold or cell as defined by the present invention and an in vitro method of producing such transgenic pig of the present invention. BACKGROUND OF THE INVENTION [002] Xenotransplantation which describes the transfer of cells, tissues or organs between different species could effectively address the shortage of human donors. While advantageous in many ways, xenotransplantation creates a more complex immunological scenario than allotransplantation. The most profound barrier to xenotransplantation is the rejection of the grafted organ by a cascade of immune mechanisms, divided into three phases: hyperacute rejection (HAR), acute vascular rejection (AVR), and cellular rejection. [003] Considerable effort has been directed at addressing the immune barrier posed by xenotransplantation through genetic modification of the donor animal. The most commonly used donor animals are pigs. Pigs have been the focus of most research in xenotransplantation because pigs share many anatomical and physiological characteristics with human. Furthermore, pigs have relatively short gestation periods and can be bred in pathogen-free environments. Pigs also do not present the same ethical issues associated with most animal research (e.g., primates) because pigs are commonly used as a food source by human. Due to the phylogenetic distance and the faster and more severe interspecies rejection reaction compared to allogeneic transplantation, genetically modified pigs are used to obtain organs for xenotransplantation. Transplantation of porcine organs into recipients such as human recipients however leads to severe immunological rejection, which can be classified as antibody-mediated rejection including HAR and AVR as mentioned above and immune-cell-mediated rejection mechanisms. HAR is a very rapid event that results in irreversible graft damage and loss within minutes to hours following graft reperfusion. If HAR is overcome, a xenograft is then subjected to AVR which is initiated days to weeks after xenotransplantation and is caused by binding of preformed and elicited antibodies to xenoreactive non-Gal antigens on the graft endothelium. Cellular rejection mechanisms are another obstacle to the function and survival of porcine xenografts in human recipients. Particularly, in acute cellular xenograft rejection (ACXR) circulating mononuclear cells recognise the xenograft vascular endothelium and migrate into the xenogeneic tissue. This rejection process is thus characterised by cellular infiltrates of T- and B- lymphocytes, macrophages and natural killer (NK) cells and is associated with direct tissue damage. [004] Effective inhibition of xenograft rejection requires a number of genetic modifications that make porcine organs suitable for such transplantation and enable graft acceptance. [005] Therefore, there is a need to provide new transgenic pigs comprising genetic modifications exhibiting a stable expression pattern to protect xenografts against the powerful rejection mechanisms mounted by the recipient. [006] The solution of the present invention is described in the following, exemplified in the appended examples, illustrated in the figures and reflected in the claims. SUMMARY OF THE INVENTION [007] As outlined above, genetically modified pigs are thought to be a potential organ source for patients in end-stage organ failures unable to receive a timely allograft. However, in recent years, many failures in xenograft transplants have been linked to high rejection responses and therefore did not support long-term survival of the transplant due to antibody-mediated rejection including HAR and AVR as well as immune-cell-mediated rejection mechanisms. [008] Now, the inventors found out that by incorporating and expressing at least one transgene which is important in xenotransplantation such as a complement inhibitor, a cytoprotectant, an anticoagulan