Growth hormone receptor knockout, multi-transgenic pigs for xenotransplantation

Abstract

The present disclosure relates to transgenic animals (e.g., transgenic porcine animals) comprising a plurality of genetic modifications that advantageously make these animals suitable donors for xenotransplantation. The present disclosure extends to organs, organ segments, tissues, and cells derived from these animals and therapeutic uses thereof. The present disclosure further extends to methods of making such animals. In certain embodiments, the growth hormone receptor (GHR) gene expression of the transgenic animal (e.g., transgenic porcine animal) is reduced or the GHR protein function is impaired.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 116,718, filed November 20, 2020, which is incorporated herein by reference in its entirety for any and all purposes. Technical Field

[0003] This disclosure generally relates to donor animals, donor tissues and donor cells particularly suitable for xenotransplantation therapy, and more specifically to multi-transgenic pig animals containing at least six genetic modifications that make them suitable donors for xenotransplantation, as well as tissues and cells derived from these pig animals. Background Technology

[0004] Xenotransplantation (transplantation of organs, tissues, and cells from donors of different species) can effectively address the shortage of human donors. While xenotransplantation has advantages in many ways, it generates a more complex immunological situation compared to allogeneic transplantation. The most serious obstacle to xenotransplantation is the rejection of the transplanted organ by the immune cascade mechanism, which occurs in three phases: hyperacute rejection (HAR), acute humoral xenotransplantation rejection (AHXR), and T-cell-mediated cellular rejection. HAR is a very rapid event that leads to irreversible graft damage and loss within minutes to hours after graft reperfusion.

[0005] Significant efforts have been made to overcome the immune barriers caused by xenotransplantation by genetically modifying donor animals. Pigs are the most commonly used donor animal. Pigs have been the focus of most xenotransplantation research because they share many anatomical and physiological characteristics with humans. Furthermore, pigs have relatively short gestation periods and can be raised in pathogen-free environments. Pigs also do not present the ethical issues associated with most animal research (e.g., primates) because they are commonly used as a food source for humans.

[0006] Xenografting has made significant progress due to the increased availability of pigs with multiple genetic modifications, combined with effective immunosuppression and anti-inflammatory therapies to protect pig tissues after xenografting. However, novel physiological incompatibilities have been observed in recipients of pig-derived xenografts. Pig-derived xenografts exhibit an intrinsic growth phenotype that impairs long-term function of the graft after orthotopic transplantation in non-human primate models. In particular, pig-derived kidney and heart xenografts exhibit rapid growth after transplantation into non-human primates. For example, ventricular hypertrophy has been observed after orthotopic transplantation of pig hearts into non-human primates. Recipients of pig-derived heart xenografts ultimately die within less than a month from early hypertrophic cardiomyopathy and diastolic heart failure. Life support function of these pig-derived heart xenografts has been prolonged for up to 6 months after administration of temsirolimus and other afterload-reducing agents.

[0007] The reasons for the intrinsic growth phenotypes in pig-derived xenografts are unclear. Growth hormone (GH) is a major stimulant of growth after birth in many animals. Growth hormone stimulates growth by binding to its receptor. Activation of the growth hormone signaling pathway is initiated by the binding of growth hormone to its receptor (GHR). This signaling event leads to the production of insulin-like growth factor I (IGF-I), which promotes growth, development, and immune function in the organism. Excessive production of growth hormone can lead to acromegaly or gigantism. Defects in the growth hormone gene (including nonsense mutations, splice site mutations, frameshifts, deletions, and missense mutations) impair the GHR signaling pathway and lead to dwarfism. Naturally occurring mutations in the human GHR that cause GHR dysfunction are associated with Laron syndrome, characterized by a growth-retarding phenotype, delayed puberty, and short stature at maturity. GHR mutations in Laron syndrome prevent GHR from binding to GH or from activating intracellular signaling pathways, both of which result in severely reduced IGF-1 production and secretion. Experimental mutations in GHR in mouse models reproduced the growth retardation phenotype observed in patients with Laron syndrome. In summary, these observations suggest that intentionally mutating porcine GHR will produce a growth retardation phenotype in pigs, which could help limit excessive organ growth after xenotransplantation.

[0008] Therefore, there is a need for multi-transgenic donor animals lacking GHR expression (e.g., pigs) for xenograft therapy to prevent rapid growth of the intrinsic xenograft and improve xenograft survival without the use of chemical adjuvants. This disclosure addresses this need. Summary of the Invention

[0009] This disclosure relates to transgenic animals (e.g., transgenic pigs) containing multiple genetic modifications that advantageously make them suitable donors for xenotransplantation. This disclosure extends to organs, organ fragments, tissues, and cells derived from such animals and their therapeutic uses. This disclosure further extends to methods for preparing such transgenic animals.

[0010] One aspect of this disclosure provides transgenic pigs comprising: a genetic alteration that results in decreased expression of the growth hormone receptor (GHR) gene; or a genetic alteration that causes a mutation in at least one allele of the GHR gene, thereby impairing GHR function. In some embodiments, the genetic alteration is a GHR knockout genetic alteration. In some embodiments, compared with pigs without the genetic alteration, the transgenic pigs exhibit a reduction in GHR expression of at least about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, compared with pigs without the genetic alteration, the transgenic pigs produce at least about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% less insulin-like growth factor 1 (IGF-1).

[0011] One aspect of this disclosure provides a transgenic pig comprising: a gene alteration that causes decreased expression of the GHR gene; or a gene alteration that causes a mutation in at least one allele of the GHR gene, thereby impairing GHR function; and further comprising one or more additional gene alterations. In some embodiments, one or more additional gene alterations result in (i) decreased expression of one or more genes, (ii) impaired function of one or more genes, and / or (iii) expression of one or more transgenes. In some embodiments, one or more transgenes are independently selected from anticoagulants, complement regulators, immunomodulators, or cell-protective transgenes.

[0012] In some embodiments, the anticoagulant is selected from TBM, TFPI, EPCR, or CD39. In some embodiments, the complement modulator is a complement inhibitor. In some embodiments, the complement inhibitor is selected from CD46, CD55, or CD59. In some embodiments, the immunomodulator is an immunosuppressant. In some embodiments, the immunosuppressant is selected from porcine CLTA4-IG, CIITA-DN, or CD47. In some embodiments, one or more transgenes are selected from CD47, CD46, DAF / CD55, TBM, EPCR, or HO1. In some embodiments, one or more gene alterations include decreased expression of α1,3-galactosyltransferase, β-1,4-N-acetyl-galactosyltransferase 2 (β4GalNT2), and cytidine monophosphate-N-acetylneuraminidase (CMAH).

[0013] In one aspect, the present invention provides transgenic pigs comprising genetic alterations that result in reduced expression of the insulin-like growth factor 1 (IGF-1) gene. In some embodiments, the transgenic pigs produce at least about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% less IGF-1 compared to pigs without the genetic alteration.

[0014] In one aspect, this disclosure provides a transgenic pig comprising at least four transgenes. In some embodiments, the at least four transgenes are incorporated and expressed at a single locus and under the control of at least two promoters, and the pig lacks the expression of α1,3-galactosyltransferase and growth hormone receptor. In some embodiments, the at least four transgenes are incorporated and expressed at a single locus and under the control of at least two promoters, and the pig lacks the expression of α1,3-galactosyltransferase, growth hormone receptor, β-1,4-N-acetylgalactosamine aminotransferase 2 (β4GalNT2), and cytidine monophosphate-N-acetylneuraminidase (CMAH).

[0015] In some implementations, a single locus is: (i) a natural locus; (ii) a modified natural locus; (iii) selected from the group consisting of AAVS1, ROSA26, CMAH, β4GalNT2, and GGTA1; (iv) a natural GGTA1 locus; (v) a modified GGTA1 locus; (vi) a transgenic GGTA1 locus; (vii) a natural CMAH locus; (viii) a modified CMAH locus; (ix) a transgenic CMAH locus; (x) not a GGTA1 locus; (xi) a natural β4GalNT2 locus; (xii) a modified β4GalNT2 locus; or (xiii) a transgenic β4GalNT2 locus.

[0016] In some embodiments, the modified natural locus comprises a gene-editing-mediated insertion, deletion, or substitution; or transgenic DNA. In one embodiment, the transgenic DNA comprises an optional marker gene or a landing pad. In some embodiments, at least one of the promoters is a foreign promoter, a constitutive promoter, a modifiable promoter, an inducible promoter, or a tissue-specific promoter. In one embodiment, the modifiable promoter is a tissue-specific promoter or an inducible promoter.

[0017] In some embodiments, at least four transgenes are expressed as first polycistronic and second polycistronic. In some embodiments, at least two promoters comprise a first promoter controlling first polycistronic expression and a second promoter controlling second polycistronic expression. In some embodiments, the transgenic pig comprises at least four promoters, and each of the at least four transgenes is controlled by a specific promoter. In one embodiment, the first promoter and the second promoter are different. In one embodiment, the first promoter is a constitutive promoter and the second promoter is a tissue-specific promoter. In one embodiment, both the first and second promoters are constitutive promoters. In one embodiment, at least two promoters comprise CAG and TBM promoters.

[0018] In some embodiments, the tissue-specific promoter is an endothelial cell-specific promoter. In alternative embodiments, the tissue-specific promoter is an endothelial cell-specific promoter selected from the TBM promoter, EPCR promoter, ICAM-2 promoter, and / or Tie-2 promoter.

[0019] In some embodiments, at least four transgenes are selected from the group consisting of anticoagulants, complement inhibitors, immunomodulators, cell-protective transgenes, and combinations thereof. In some embodiments, (i) the anticoagulant is selected from the group consisting of TBM, TFPI, EPCR, CD39, and combinations thereof; (ii) the complement inhibitor is selected from the group consisting of CD46, CD55, CD59, and combinations thereof; (iii) the immunomodulator is an immunomodulator selected from the group consisting of CD47, HLA-E, CLTA4-IG, CIITA-DN, and combinations thereof; (iv) the immunomodulator is CD47; or (v) the cell-protective transgene is selected from the group consisting of HO-1, A20, and combinations thereof. In some embodiments, at least two of the transgenes are anticoagulants; at least one of the transgenes is a cell-protective transgene; at least one of the transgenes is immunomodulatory; or at least one of the transgenes is a complement inhibitor.

[0020] In some embodiments, the transgenic pigs described herein further comprise at least one additional genetic modification. In some embodiments, at least one additional genetic modification is selected from: (i) gene knockout; gene knock-in; gene substitution; point mutation; deletion, insertion, or substitution of a gene, gene segment, or nucleotide; large genome insertion; or a combination thereof; (ii) incorporation and expression of human CD46; (iii) incorporation and expression of human HLA-E; (iv) knockout of the B4GalNT2 gene; (v) knockout of the CMAH gene; or (vi) resulting in the elimination or reduction of expression of at least one natural gene.

[0021] In some embodiments, at least one additional genetic modification comprises at least the following incorporation and expression: at least two additional transgenes; two additional transgenes at a second single locus; or four additional transgenes at a second single locus. In one embodiment, the single locus is GGTA1 and the second single locus is CMAH; the single locus is β4GalNT2 and the second single locus is CMAH; the single locus is CMAH and the second single locus is β4GalNT2; or the single locus is GGTA1 and the second single locus is β4GalNT2. In some embodiments, at least one natural gene is selected from the group consisting of CMAH, isoerythrocyte glycoside 3 synthase, β4GalNT2, Forrsman synthase, or combinations thereof.

[0022] In some implementations, the transgenic pigs described herein express CD46; and combinations of at least four transgenic organisms selected from: (i) EPCR, HO-1, TBM, and CD47; (ii) EPCR, HO1, TBM, and TFPI; (iii) EPCR, CD55, TFPI, and CD47; (iv) EPCR, DAF, TFPI, and CD47; or (v) EPCR, CD55, TBM, and CD39.

[0023] In some implementations, two of the four transgenes expressed in the first or second polycistronic cell are selected from the group consisting of TBM, EPCR, DAF, CD39, TFPI, CTLA4-Ig, CIITA-DN, HOI, A20, and CD47. In some implementations, at least one pair of transgenes expressed in the polycistronic group is selected from the group consisting of: (a) TBM and CD39; (b) EPCR and DAF; (c) A20 and CD47; (d) TFPI and CD47; (e) CIITAKD and HO-1; (f) TBM and CD47; (g) CTLA4Ig and TFPI; (h) CIITAKD and A20; (i) TBM and A20; (j) EPCR and DAF; (k) TBM and HO-1; (l) TBM and TFPI; (m) CBTA and TFPI; (n) EPCR and HO-1; (o) TBM and CD47; (p) EPCR and TFPI; (q) TBM and EPCR; (r) CD47 and HO-1; (s) CD46 and CD47; (t) CD46 and HO-1; and (u) CD46 and TBM.

[0024] In some embodiments, the transgenic pigs described herein lack expression of the growth hormone receptor and contain genotypes selected from the following: (i) GTKO.CD46.pTBMpr-TBM.CD39-cag-A20.CD47; (ii) GTKO.CD46.Icam-2-TFPI.CD47-tiecag-A20.CD47; (iii) GTKO.CD46.pTBMpr-TBM.CD39-tiecag-CIITAKD.HO-1; (iv) GTKO.CD46.Icam-2-CTL A4Ig.TFPI-tiecag-CIITAKD.A20-1; (v) GTKO.CD46.Icam-2-CTLA4Ig.TFPI-tiecag-CIITAKD.HO-1; (vi) GTKO.CD46.pTBMp r-TBM.CD39-cag-EPCR.CD55; (vii) GTKO.CD46.pTBMpr-TBM.A20-cag-EPCR.DAF; (viii) GTKO.CD46.pTBMpr-TBM.HO-1-cag- EPCR.DAF; (ix) GTKO.CD46.pTBMpr-TBM.TFPI-cag-EPCR.DAF; (x) GTKO.CD46.Icam-2-CIITA.TFPI-cag-EPCR.DAF; (xi) GTK O.CD46.Icam-2-TFPI.CD47-cag-EPCR.DAF; (xii) GTKO.CD46.Icam-2-TFPI.CD47-cag-EPCR.HO-1; (xiii) GTKO.CD46.pTBM pr-TBM.HO-1-cag-TFPI.CD47; (xiv)GTKO.CD46.pTBMpr-TBM.CD47-cag-EPCR.TFPI; (xv)GTKO.CD46.pTBMpr-TBM.TFPI-ca g-EPCR.CD47; (xvi) GTKO.CD46.pTBMpr-TBM.EPCR-cag-CD47.HO-1; or (xvii) GTKO.CD46.cag-EPCR.DAF-tiecag-TFPI.CD47.

[0025] One aspect of this disclosure provides organs derived from the transgenic pigs described herein. Another aspect of this disclosure provides lungs or lung fragments, hearts or heart fragments, kidneys or kidney fragments, livers or liver fragments derived from the transgenic pigs described herein. Yet another aspect of this disclosure provides tissues or cells derived from the transgenic pigs described herein.

[0026] One aspect of the present invention provides a method for preparing a transgenic pig that expresses at least four transgenes but lacks expression of α1,3-galactosyltransferase and / or growth hormone receptor, the method comprising (i) incorporating at least four transgenes under the control of at least two promoters at a single locus within the pig genome to provide a polygenic pig genome; and (ii) maturing a cell containing the polygenic pig genome into a transgenic pig.

[0027] In some embodiments, the pig genome is a somatic pig genome and the cell is a pig zygote, which is provided via somatic cell nuclear transfer (SCNT). In some embodiments, a multigene pig genome is transferred into a reconstructed SCNT zygote via microinjection. In one embodiment, the somatic pig genome contains at least one additional gene modification. In one embodiment, the at least one additional gene modification is selected from the group consisting of gene knockout; gene knock-in; gene substitution; point mutation; deletion, insertion, or substitution of a gene, gene fragment, or nucleotide; large genome insertion; or combinations thereof.

[0028] In some embodiments, the methods described herein further include introducing at least one additional genetic modification into a polygenic pig genome. In one embodiment, the at least one additional genetic modification is selected from the group consisting of gene knockout; gene knock-in; gene substitution; point mutation; deletion, insertion, or substitution of a gene, gene fragment, or nucleotide; large genomic insertion; or combinations thereof. In some embodiments, the pig genome is selected from the group consisting of gamete pig genome, zygotic pig genome, embryonic pig genome, or blastocyst pig genome; or the pig genome contains at least one additional genetic modification.

[0029] In some embodiments, as described herein, the incorporation step further includes introducing at least one additional genetic modification into a polygenic pig genome. In some embodiments, the incorporation step includes (i) a method selected from the group consisting of biological transfection, chemical transfection, physical transfection, virus-mediated transduction or transformation, or combinations thereof; or (ii) cytoplasmic microinjection and prokaryotic microinjection.

[0030] In some implementations, a single locus is: (i) a natural locus; (ii) a modified natural locus; (iii) a modified natural locus containing gene editing-mediated insertions, deletions, or substitutions; (iv) a modified natural locus containing transgenic DNA selected from a selectable marker gene or a landing pad; (v) a natural GGTA1 locus; (vi) a modified GGTA1 locus; (vii) a transgenic GGTA1 locus; (viii) a transgenic GGTA1 locus containing a selectable marker gene or a transgenic pad; (xix) a natural β4GalNT2 locus; (x) a modified β4GalNT2 locus; (xi) a transgenic β4GalNT2 locus. (xii) A transgenic β4GalNT2 locus containing a selectable marker gene or a transgenic pad; (xiii) A single locus that is a natural locus selected from the group consisting of CMAH, β4GalNT2, AAVS1, and Rosa26; (xiv) A modified locus selected from the group consisting of CMAH, β4GalNT2, AAVS1, and Rosa26; (xv) Not a GGTA1 locus; (xvi) A natural CMAH locus; (xvii) A modified CMAH locus; (xviii) A transgenic CMAH locus; or (xix) A transgenic CMAH locus containing a selectable marker gene or a transgenic pad.

[0031] In some embodiments, the transgenic DNA contains recognition sequences for one or more polynucleotide modifying enzymes. In some embodiments, the polynucleotide modifying enzyme is selected from the group consisting of engineered endonucleases, site-specific recombinases, integrases, or combinations thereof. In one embodiment, the engineered endonuclease is selected from the group consisting of zinc finger nucleases, transcription activator-like effector nucleases, and regularly clustered short palindromic repeat (CRISPR) / Cas9 nucleases. In one embodiment, the site-specific recombinase is selected from the group consisting of λ integrase, Cre recombinase, FLP recombinase, γ-δ dissociation enzyme, Tn3 degradase, OC31 integrase, Bxb1-integrase, R4 integrase, or combinations thereof. In some embodiments, the gene editing-mediated insertion, deletion, or substitution comprises the deletion of one or more nucleotides of a defined sequence; or wherein the gene editing-mediated insertion, deletion, or substitution is mediated by a CRISPR / Cas system.

[0032] In some embodiments of the methods described herein, at least one additional gene modification includes (i) incorporation and expression of CD46; (ii) incorporation and expression of human HLA-E; or (iii) gene knockout, said gene being selected from the group consisting of the β4GalNT2 gene, the CMAH gene, and the GGTA1 gene.

[0033] One aspect of this disclosure provides transgenic animals or production herds produced by the methods described herein. In some embodiments, the method further includes breeding a transgenic pig into a second transgenic pig. In one embodiment, the second transgenic pig contains at least one gene modification. In some embodiments, the at least one gene modification includes: incorporating and expressing at least one transgene selected from the group consisting of anticoagulants, complement inhibitors, immunomodulators, cell-protective transgenes, and combinations thereof; or knocking out at least one pig gene.

[0034] One aspect of this disclosure provides for transgenic animals or production herds produced by any of the methods described herein.

[0035] One aspect of this disclosure provides a method for treating a subject in need, comprising implanting at least one organ, organ fragment, tissue, or cell derived from a transgenic pig as described herein into the subject in need. In some embodiments, the at least one organ or organ fragment is selected from the group consisting of the lung, heart, kidney, liver, pancreas, or combinations thereof. In one embodiment, the at least one organ or organ fragment is a lung. In some embodiments, the organ is used to replace or enhance a diseased or failing organ of the subject in need, performed by implanting the organ into the subject, wherein the organ transplant is: i) a kidney transplant; ii) a lung transplant; iii) a heart transplant; iv) a liver transplant; or v) a pancreas transplant. In some embodiments, the subject is a mammal, a non-human primate, or a human.

[0036] In some implementations, the recipient has advanced lung disease and a lung or lung fragment is implanted. In some implementations, advanced lung disease is associated with chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPD), cystic fibrosis (CF), alpha-1-antitrypsinopathy, or primary pulmonary hypertension.

[0037] In some implementations, the method for treating a subject in need further includes administering one or more therapeutic agents to the subject, the therapeutic agents being selected from anti-rejection agents, anti-inflammatory agents, immunosuppressants, immunomodulators, antimicrobial agents, antiviral agents, and combinations thereof. Attached Figure Description

[0038] Figure 1A A bicistronic unit for use in vectors disclosed herein is described, which consists of two transgenes linked by a 2A peptide sequence.

[0039] Figure 1B Expression vectors that can be used in this disclosure are described, including globin insulators located on the flanks of two bicistronic units driven by independent promoters / enhancers, and separating their insertion sites.

[0040] Figure 2 Gene expression in pigs with six gene editing (GE) / modifications (6GE pigs; GTKO.CD46.TBM.CD39.EPCR.DAF) was depicted by flow cytometry, which showed a lack of α-Gal expression and robust expression of five (5) human transgenes (including CD46, CD55(DAF), EPCR, TFPI and CD47).

[0041] Figure 3 Immunohistochemical staining of lung sections in 6GE pigs (GTKO.CD46.TBM.CD39.EPCR.DAF) using fluorescently labeled anti-EPCR, DAF, TFPI, and CD47 antibodies was described.

[0042] Figure 4A and 4B A polycistronic vector (MCV) designed and generated according to this disclosure is described. Pigs with six gene modifications are generated, including expression cassettes of the complement regulatory genes hCD46 and CD55, combined with endothelial-specifically or universally expressed anticoagulant genes thrombomodulin (TBM), endothelial protein C receptor (EPCR), CD39, and tissue factor pathway inhibitor (TFPI), the immunosuppressive gene porcine cytotoxic T-lymphocyte-associated protein 4 (pCTLA4Ig), major histocompatibility complex class II dominant inactivation (CIITA-DN), and / or anti-inflammatory transgenic heme oxygenase 1 (HO1), A20, and CD47.

[0043] Figure 5 The expression analysis of the pREV941 transgene in the lungs was described.

[0044] Figure 6 The expression analysis of the pREV971 transgene in the lungs was described.

[0045] Figure 7 The expression analysis of the pREV967 transgene in the lungs was described.

[0046] Figure 8 The 941HDR vector (MCV vector pREV941 containing human transgenic EPCR, DAF, TBM, and CD39; 500bp homologous arm specifically targeting the modified αGal locus in GTKO cells) was depicted.

[0047] Figure 9Immunohistochemical staining of EPCR, DAF, TBM, and CD39 transgenes was depicted in lung sections from negative control wild-type pigs and 941HDR-targeted pigs. Expression of all four human transgenes was observed. Transgene expression (EPCR and DAF) from the strongly constitutive CAG promoter was stronger in this MCV than transgene expression (TBM and CD39) observed under the control of the endothelial-specific porcine ICAM-2 (pICAM2) promoter.

[0048] Figure 10 Western blot analysis was performed on lysates of heart, liver, lung, and kidney tissues from pigs targeted with 941HDR. Anti-human monoclonal antibodies specific to TBM (controlled by the endogenous pICAM2 promoter) and EPCR and DAF (sharing the CAG promoter) were optimized for detecting transgene expression in MCV transgenic pig tissues (specifically, 941HDR in this case). For EPCR and DAF, expression at the αGal locus integration environment was observed in all tissues, while expression was weaker in TBM (except for high expression in the lungs), indicating good expression of multiple transgenes at this predetermined site in the genome, and importantly, in live pigs.

[0049] Figure 11A An ELISA assay was performed to characterize the expression of human thrombotic regulatory protein in multiline TBM transgenic MCV pigs (including 941HDR targeting the αGal locus) (Pig 875-5).

[0050] Figure 11B Flow cytometry expression of all transgenes from pREV971 targeting the αGal locus was depicted in fetal MVEC cells.

[0051] Figure 12 The humanization of the porcine vWF locus was described, involving CRISPR-enhanced knock-in and replacement of porcine exons 22-28 with human equivalent exons 22-28 as cDNA. In step 1, porcine fibroblasts were transfected with a targeting vector containing two porcine homologous arms flanking human exons 22-28 and an internal selection cassette with GFP-Puro. Subsequently, in step 2, CRISPR-induced double-strand breaks initiated strand exchange and homology-dependent repair at the junction of porcine exons 22 and 28, inserting the human vWF sequence. Then, fetal cells with confirmed biallelic replacements were treated with site-specific transposons (step 3) to remove the selection cassette, leaving an in-frame fusion sequence of the porcine-human sequence.

[0052] Figure 13 Sequence analysis of the junctions (5' and 3') revealed perfect alignment between the porcine VWF and human VWF sequences after knock-in and insertion of human exons 22-28.

[0053] Figure 14 The normal function of porcine vWF-edited whole blood was described by platelet aggregation assay.

[0054] Figure 15 This study depicts the absence of spontaneous platelet aggregation in human platelets exposed to vWF-edited pig platelet-deficient plasma. Porcine platelet-deficient plasma (PPP) was prepared from citrate-anticoagulated porcine blood samples using a two-step centrifugation protocol. Human platelet-rich plasma (PRP) was prepared from freshly drawn human blood samples (citrate-anticoagulated). Human PRP and porcine PPP were mixed 1:1 in test tubes, and platelet aggregation was immediately recorded using a Chrono-log whole blood aggregometer.

[0055] Figure 16 A bicistronic CD46 / CD55 (DAF) vector according to this disclosure is described.

[0056] Figure 17 The pig vWF modification by replacing human vWF is described.

[0057] Figure 18 The high-level expression of multiple transgenes in transgenic pigs according to this disclosure is shown, and more specifically, the incorporation expression of six gene modifications (GTKO.CD46.EPCR.CD55.TBM.CD39) and five transgenes (CD46.EPCR.CD55.TBM.CD39).

[0058] Figure 19A A schematic diagram is shown of bicistronic (B118) and polycistronic (B167) vectors used to generate multi-transgenic pigs containing at least 10 modifications and lacking GHR expression.

[0059] Figure 19B A schematic diagram is shown for generating a polycistronic (B200) vector containing at least 10 modifications in a single step in a multi-transgenic pig. The multi-transgenic animal contains six or more transgenes integrated into a single locus and lacks expression of the α1,3-galactosyltransferase (αGal; GTKO) gene, the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMP-NeuAc hydroxylase (CMAH)) gene, the β-1,4-N-acetylgalactosyltransferase 2 (β4GalNT2) gene, and the growth hormone receptor (GHR).

[0060] Figure 20A , Figure 20B and Figure 20C A schematic diagram of GHR knockout is shown. Figure 20A The sequence of the GHR CRISPR guide RNA targeting exon 3 of the porcine GHR gene is shown. Figure 20B and Figure 20C The cleavage efficiency of four GHR CRISPR guide RNA sequences, individually or in combination, was shown.

[0061] Figure 21 A schematic diagram of a two-step process for generating 10 gene-edited / modified (10GE) pigs is shown.

[0062] Figure 22A The diagram shows a line graph illustrating changes in the septal wall thickness of a 10GE porcine heart, measured by transthoracic echocardiography (TTE), after transplantation into baboons. Specifically, there was no difference in intrinsic growth between GHRKO and non-GHRKO grafts one month post-transplantation. B33130 and B32863 refer to baboons receiving GHRKO grafts, while B33121 and B32988 refer to baboons receiving non-GHRKO grafts. Figure 22B This is a line graph showing the changes in posterior wall thickness of a 10GE porcine heart, measured by TTE, after transplantation into a baboon. The dashed line represents 28 days post-surgery, corresponding to the mean value in previous studies before xenograft failure due to hypertrophy. Each data point corresponds to the mean of three measurements of the septal or posterior wall. Bars indicate the + / - standard deviation.

[0063] Figure 23A and 23B The strategy for generating GHR knockout using CRISPR / Cas9 is shown. Figure 23A The study showed that a single guide RNA (sgRNA) was designed to cleave at a site 37 bp within the GHR exon 3 to produce a frameshift deletion and premature termination coding, resulting in a truncated, nonfunctional GHR protein. Figure 23B Gel electrophoresis images of the RT-PCR reaction are shown, illustrating the relative migration of nucleic acids amplified from GHRKO pigs and wild-type pigs.

[0064] Figure 24 Growth charts for GHRKO pigs are displayed, showing that GHRKO pigs exhibit reduced growth rate and weight loss compared to wild-type control pigs. The curves are best-fit lines for both males and females. The mean of GHRKO pigs is represented by boxes and circles, and the standard error is represented by vertical lines. Data for GHR-WT pigs is displayed as a single data point.

[0065] Figure 25Growth charts show the growth rates of three 6GE GHRKO female pigs compared to the upper / lower limits of standard pigs predicted by the Gompertz equation, and indicate that the growth rates of the three 6GE female pigs from birth to ~250 days are in line with expectations and continue to approach the lower limit of physiological growth of commercial pigs determined by the established mathematical model (Gompertz growth equation; Wellock et al., Animal Science 78:370-388 (2004)).

[0066] Figure 26 Growth charts showing the growth rates of two 10-GE (GHRKO) pigs compared to the upper / lower limits of standard pigs predicted by the Gompertz equation are presented. The charts demonstrate that the growth rates of the two 10-GE male pigs from birth to ~130 days are in line with expectations and continue to approach the lower limit of physiological growth for commercial pigs as determined by the established mathematical model (Gompertz growth equation; Wellock et al., Animal Science 78:370-388 (2004)).

[0067] Figure 27 The images show photographs of GHRKO animals and wild-type pigs, illustrating the differential growth between the two. Both pigs shown are multi-transgenic litters, differing only in the GHR locus. The pig on the left is GHR-KO; the pig on the right is wild-type.

[0068] Figure 28 The bar chart shows that GHRKO pigs have lower serum IGF-1 levels compared to wild-type pigs.

[0069] Figure 29A and Figure 29B The results of the western blot analysis show that, based on tail and ear biopsies, transgene expression was present in each individual pig in 10 GE pigs. Figure 29A The results of tail biopsies from 10GE pigs are shown, and transgene expression in the tails of 524D1-8 and 525D-1 pigs is illustrated. Figure 29B Ear biopsy results from 10GE pigs are shown, and transgene expression in ear-punched samples from 525D-1 and 525D-15 is illustrated. Specifically, TBM, EPCR, HO-1, CD46, and DAF were expressed in all samples. Actin was used as a loading control, demonstrating its presence in all strains.

[0070] Figure 30A and Figure 30B Flow cytometry analysis confirmed all gene modifications in peripheral blood mononuclear cells (PBMCs) of 10GE pigs. Figure 30AThis indicates that GGTA1(αgal) knockout, β4GalNT2 knockout, and CMAH knockout are inactivated in 10GE pigs. Figure 30B This indicates the expression of CD46, CD55 (DAF), CD201 (EPCR), CD47, and CD141 (TBM) in 10GE pigs.

[0071] Figure 31A , Figure 31B and Figure 31C Immunohistochemical images are shown, indicating human (h)EPCR, hTBM ( Figure 31A hHO-1, hCD47 Figure 31B hDAF (CD55) and hCD46 ( Figure 31B The expression of the transgene in the heart, kidney, and lung tissues of 10GE pigs.

[0072] Figure 32A and Figure 32B Western blot and immunohistochemical images are shown, illustrating post-transplant analysis of transgene expression in deceased humans. Figure 32A The expression of human transgenic proteins in kidney biopsies is shown by Western blot analysis. The expected molecular weights of hTBM, hEPCR, hCD47, hHO1, hCD46, and hDAF were detected by Simple WES capillary electrophoresis. Kidney lysates from unengineered pigs (WT) served as a negative control for transgenic expression. Porcine actin was used as an endogenous control, showing the presence of the protein in both samples. Figure 32B Human transgene expression as detected by immunohistochemistry is shown, indicating the detection of hTBM, hEPCR, hCD47, hHO1, hCD46, and hDAF expression in kidney sections, as indicated by dark precipitates at antibody binding sites. No staining was observed in negative control sections from unengineered pigs (WT). All images were taken at 200×.

[0073] Figure 33 Gel electrophoresis images are shown, demonstrating PERV transfer and microchimerism analysis in PBMCs, and indicating that PERV or microchimerism (pig-specific RPL4) was not detected by RT-PCR using mRNA at different post-transplantation time intervals. Pigs (+) are PERVC-positive pig controls, and pigs (-) are PERVC-negative transplanted pigs. GAPDH is an endogenous control, showing the presence of mRNA in all samples. NC is a negative PCR control / water. Results were confirmed by qRT-PCR (data not shown). Detailed Implementation

[0074] Transgenic pigs are considered a potential organ source for patients with advanced organ failure who cannot receive allogeneic transplants in a timely manner. However, in recent years, many xenotransplant failures have been associated with graft overgrowth. In particular, when xenotransplants from xenogeneic donor organisms (e.g., pigs) are transplanted into recipient primates, the transplanted xenotransplants overgrow, ultimately leading to the death of the recipient primates. For example, non-human primates that received pig-derived heart xenotransplants eventually died within a month from early hypertrophic cardiomyopathy and diastolic heart failure. In some cases, life support for these xenotransplants can be extended to 6 months after administration of tesimolimus and other afterload-reducing agents. In addition to heart xenotransplants, rapid overgrowth of kidney xenotransplants has also been observed in the first three months after pig-to-baboon xenotransplants. While rapid kidney growth does not pose an imminent danger to the recipient animal (e.g., non-human primates) because the kidney can be accommodated within the abdominal cavity, rapid growth of the heart within the relatively limited thoracic cavity is dangerous for the animal. The cause of the overgrowth phenotype is unclear. The rapid growth phenotype may be caused by growth differences between, for example, the growth patterns of pigs and primates.

[0075] This disclosure provides an alternative solution to the problem of excessive growth that does not require the use of chemical adjuvants (e.g., tesimolimus). The inventors unexpectedly discovered that genetically engineering transgenic animals lacking growth hormone receptors can improve intrinsic excessive growth in xenografts and / or the survival of xenograft recipients. The goal was to reduce or slow growth in pigs (i.e., pig tissue growth). Transgenic animals lacking GHR knockout (GHRKO) exhibited all the phenotypes associated with Laron syndrome. In particular, GHRKO animals reproduced normally. However, they were short in stature, weighed more than 50% less than control animals, and had a proportionally reduced weight in most organs. Furthermore, serum insulin-like growth factor 1 (IGF1) levels were significantly reduced in GHRKO animals. Moreover, administration of IGF1 to GHRKO animals promoted their growth, suggesting that IGF1 may be the cause of the excessive growth phenotype.

[0076] This disclosure relates to transgenic animals that can be used as a source of organs, organ fragments, tissues, or cells, particularly suitable for xenotransplantation. In particular, the invention relates to transgenic ungulates, and more particularly, transgenic swine animals (pigs), which can be used as a source of organs, organ fragments, tissues, or cells for xenotransplantation. The invention also extends to organs, organ fragments, tissues, or cells derived from such donor animals, methods for producing such donor animals, and the use of organs, organ fragments, tissues, or cells derived from such animals in the treatment of diseases and conditions.

[0077] Advantageously, the donor animal provides organs, organ fragments, tissues, and cells that function better in transplantation than organs, organ fragments, tissues, and cells known in the art. Without wishing to be bound by any particular theory, it is believed that the organs, organ fragments, tissues, and cells of this disclosure have improved survival and / or function due to a significant reduction in consumptive coagulopathy (also known as disseminated intravascular coagulation (DIC)), thrombotic microangiopathy, HAR, and excessive growth of porcine xenografts currently observed after disharmony xenografts.

[0078] An organ or organ fragment can be any suitable organ, such as a lung, heart, kidney, liver, or pancreas. A tissue can be any suitable tissue, such as epithelial tissue or connective tissue. A cell can be any suitable cell, such as pancreatic islet cells.

[0079] In exemplary embodiments, this disclosure provides transgenic animals (e.g., ungulates, swine) that are particularly suitable as a source of organs (i.e., lungs, hearts, and kidneys), organ fragments, tissues, or cells for lung xenotransplantation, and extends to organs (i.e., lungs, hearts, and kidneys), organ fragments, tissues, and cells derived from the transgenic animal, as well as methods for producing the transgenic animal and methods for performing lung xenotransplantation using organs, tissues, and cells derived from the transgenic animal.

[0080] Advantageously, organs, organ fragments, tissues or cells derived from transgenic animals may produce low or no levels of one or more of the following conditions after xenografting: hyperacute rejection (HAR), acute humoral rejection (AHXR / DXR), acute cellular xenograft rejection (ACXR) and / or xenograft overgrowth.

[0081] In one embodiment, organs, organ fragments, tissues, or cells derived from a transgenic animal produce low to no levels of xenograft overgrowth (e.g., reduced serum levels of IGF-I and glucose) after xenograft transplantation. In one embodiment, organs, organ fragments, tissues, or cells derived from a transgenic animal produce low to no levels of HAR and AHXR after xenograft transplantation. In another embodiment, organs, organ fragments, tissues, or cells derived from a transgenic animal produce low to no levels of HAR, AHXR, and ACXR after xenograft transplantation. In yet another embodiment, organs, organ fragments, tissues, or cells derived from a transgenic animal produce low to no levels of HAR, AHXR, overgrowth, and ACXR after xenograft transplantation.

[0082] In an exemplary embodiment, the transgenic animal is a pig that lacks any expression of a functional GHR caused by genetic modification and incorporates at least several other genetic modifications (e.g., gene knockout, gene knock-in, gene substitution, point mutation, deletion, insertion, or substitution (i.e., of a gene, gene fragment, or nucleotide), large genomic insertion, or combinations thereof). Gene modifications can be mediated by any suitable technology, including, for example, homologous recombination or gene editing methods.

[0083] In an exemplary embodiment, the transgenic animal is a swine animal that lacks any expression of functional α1,3-galactosyltransferase (αGal) and GHR (as a result of gene modification or other reasons) and incorporates at least several other gene modifications (e.g., gene knockout, gene knock-in, gene substitution, point mutation, deletion, insertion, or substitution (i.e., of genes, gene fragments, or nucleotides), large genomic insertion, or combinations thereof). Gene modifications can be mediated by any suitable technology, including, for example, homologous recombination or gene editing methods.

[0084] In an exemplary embodiment, the transgenic animal is a swine animal that lacks any expression of functional α1,3-galactosyltransferase (αGal) and / or GRH (as a result of gene modification), and incorporates and expresses at least four transgenes at a single locus under the control of at least two promoters. In some embodiments, one promoter controls the expression of one transgene; for example, the expression of each of the at least four transgenes is controlled by a single (specific) promoter. In alternative embodiments, one promoter controls the expression of more than one transgene; for example, one promoter controls the expression of two transgenes.

[0085] Advantageously, four or more transgenes are co-integrated, co-expressed, and co-segregated during cultivation. Individual loci may differ. In some embodiments, a single locus is a natural locus or a modified natural locus. Modified natural loci can be modified by any suitable technique, including but not limited to CRISPR-induced insertions or deletions (indels), the introduction of selective marker genes (e.g., Neo), or the introduction of large genomic inserts (e.g., landing pads) designed to facilitate the incorporation of one or more transgenes. In a specific embodiment, the single locus is a natural GGTA1 locus or a modified GGTA1 locus. The GGTA1 locus is inactivated by the incorporation and expression of at least four transgenes, for example, through homologous recombination, gene editing, or the application of recombinase techniques. A single locus may also be, for example, AAVS1, ROSA26, CMAH, or β4GalNT2. Optionally, the transgenic animal may have one or more additional gene modifications and / or the expression of one or more additional porcine genes may be modified by mechanisms other than gene modification.

[0086] In exemplary embodiments, the transgenic animal is a swine animal lacking any expression of functional α1,3-galactosyltransferase (αGal) and / or GHR (as a result of gene modification or other reasons), and incorporating and expressing at least three, four, five, six, seven, eight, nine, ten, or more transgenic genes at a single locus. In some embodiments, at least one of the transgenic genes is TBM, HO1, TFPI, A20, EPCR, DAF, CD39, CTLA4-Ig, CIITA-DN, HLA-E, and CD47. In some embodiments, the expression of at least three, four, five, six, seven, eight, nine, ten, or more transgenic genes is controlled by at least two, three, four, five, six, seven, eight, nine, or ten promoters or more. In some embodiments, the promoter is dedicated to a transgene, i.e., one promoter controls the expression of one transgene, while in alternative embodiments, one promoter controls the expression of more than one transgene, for example, one promoter controls the expression of two transgenes. Advantageously, two or more additional transgenes are co-integrated, co-expressed, and co-segregated during cultivation. Individual loci can be different. In some embodiments, the individual locus is a natural locus or a modified natural locus. Modified natural loci can be modified by any suitable technique, including but not limited to CRISPR-induced insertions or deletions (indels), the introduction of selective marker genes (e.g., neo), or the introduction of large genomic inserts (e.g., landing pads), designed to facilitate the incorporation of one or more transgenes. In a specific embodiment, the individual locus is a natural GGTA1 locus or a modified GGTA1 locus.

[0087] The GGTA1 locus is inactivated by the incorporation and expression of at least four transgenes, for example, through homologous recombination, gene editing, or the application of recombinase technology. A single locus may also be, for example, AAVS1, ROSA26, CMAH, GRH, or β4GalNT2. Optionally, the donor animal may have additional gene modifications and / or the expression of one or more additional porcine genes may be modified by mechanisms other than gene modification.

[0088] In exemplary embodiments, the transgenic animal is a swine animal lacking any expression of functional α1,3-galactosyltransferase (αGal) and / or GHR (as a result of gene modification or other reasons), and at least four transgenes are incorporated and expressed at a single locus (i.e., locus 1), and one or more additional transgenes are incorporated and expressed at a second single locus (i.e., locus 2). In some embodiments, one promoter controls the expression of one transgene. In some embodiments, the expression of each of the at least four transgenes at locus 1 or locus 2 is controlled by a single (specific) promoter. In alternative embodiments, one promoter controls the expression of more than one transgene; for example, one promoter controls the expression of two transgenes at locus 1. The specific loci may vary. In a particular embodiment, the first single locus is GGTA1, and the second single locus is, for example, CMAH, B4GalNT2, GHR, or vWF. In certain embodiments, at least four transgenes are incorporated and expressed at each individual locus (i.e., locus 1 and locus 2) to produce animals expressing eight or more transgenes at two distinct independent loci. In some embodiments, the individual loci are natural loci or modified natural loci. Modified natural loci can be modified by any suitable technique, including but not limited to CRISPR-induced insertions or deletions (indels), the introduction of selective marker genes (e.g., Neo), or the introduction of large genomic inserts (e.g., landing pads) designed to facilitate the incorporation of one or more transgenes. Optionally, the donor animal may have additional gene modifications and / or the expression of one or more additional porcine genes may be modified by mechanisms other than gene modification. Advantageously, two or more additional transgenes are co-integrated, co-expressed, and co-segregated during culture.

[0089] At least two promoters can be different. Promoters can be exogenous or natural. In exemplary embodiments, the promoters are constitutive or modulotropic (e.g., tissue-specific, inducible). In one embodiment, both promoters can be constitutively or ubiquitously expressed in a donor animal (e.g., from CAG or similar promoters). In another embodiment with two promoters, one promoter allows transgene expression in a tissue-specific manner (e.g., endothelial-specific expression), while the second promoter allows one or more transgenes (at the same integration site) to be expressed constitutively or ubiquitously (e.g., from CAG or similar promoters).

[0090] In some embodiments, additional gene modifications (i.e., in addition to the incorporation and expression of the multiple transgenes described above) may result in the inactivation of specific porcine genes, including but not limited to the porcine von Willebrand factor (vWF) gene, or the replacement of part or all of the porcine vWF gene with an equivalent from the human vWF gene. Other genes that may be inactivated by additional gene modifications include, for example, CMP-NeuAc hydroxylase (CMAH), growth hormone receptor, isoerythroid glycoside 3 synthase, β4Gal, NT2 Forrsman synthase, or combinations thereof. In some embodiments, the single locus used for transgene incorporation is not the GGTA1 locus, and the additional gene modification includes GGTA1 inactivation. In some embodiments, the additional gene modification is, for example, gene editing-induced deletion / insertion or gene substitution (INDEL).

[0091] In some implementations, additional gene modifications (i.e., in addition to the incorporation and expression of the multiple transgenes mentioned above) may result in the incorporation and expression of one or more transgenes at the second locus.

[0092] In one embodiment, this disclosure pertains to a swine animal lacking any expression of functional α1,3-galactosyltransferase (αGal) and / or GHR (due to genetic modification or other reasons), and further comprising the inactivation of the porcine von Willebrand factor (vWF) gene, or the replacement of part or all of the porcine vWF gene with an equivalent counterpart from the human vWF gene. Optionally, the swine animal contains one or more additional genetic modifications. In some embodiments, the animal may be bred together with a second animal containing one or more genetic modifications.

[0093] This disclosure provides a transgenic pig lacking expression of α1,3-galactosyltransferase and / or growth hormone receptor, expressing CD46, and containing at least four transgenes. In some embodiments, at least four transgenes are incorporated and expressed at a single locus under the control of at least two promoters, and the at least four transgenes are selected from the following combinations: EPCR, DAF, TFPI, and CD47; EPCR, CD55, TBM, and CD39; EPCR, HO-1, TBM, and CD47; EPCR, HO-1, TBM, and TFPI; or EPCR, CD55, TFPI, and CD47.

[0094] In some implementations, the transgenic pigs lack growth hormone receptor expression and contain genotypes selected from the following: GTKO.CD46.Icam-2-TBM.CD39-cag-A20.CD47; GTKO.CD46.Icam-2-TFPI.CD47-tiecag-A20.CD47; GTKO.CD46.Icam-2-TBM.CD39-tiecag-CIITAKD.HO-1; GTKO.CD46.Icam-2-CTL A4Ig.TFPI-tiecag-CIITAKD.A20-1; GTKO.CD46.Icam-2-CTLA4Ig.TFPI-tiecag-CIITAKD.HO-1; GTKO.CD46.Ic am-2-TBM.CD39-cag-EPCR.CD55; GTKO.CD46.Icam-2-TBM.A20-cag-EPCR.DAF; GTKO.CD46.Icam-2-TBM.HO-1-ca g-EPCR.DAF;GTKO.CD46.Icam-2-TBM.TFPI-cag-EPCR.DAF;GTKO.CD46.Icam-2-CIITA.TFPI-cag-EPCR.DAF;GTKO.CD46.Icam-2-CIITA.TFPI-cag-EPCR.DAF; KO.CD46.Icam-2-TFPI.CD47-cag-EPCR.DAF; GTKO.CD46.Icam-2-TFPI.CD47-cag-EPCR.HO-1; GTKO.CD46.Icam -2-TBM.HO-1-cag-TFPI.CD47;GTKO.CD46.Icam-2-TBM.CD47-cag-EPCR.TFPI;GTKO.CD46.Icam-2-TBM.TFPI-c ag-EPCR.CD47; GTKO.CD46.Icam-2-TBM.EPCR-cag-CD47.HO-1; or GTKO.CD46.cag-EPCR.DAF-tiecag-TFPI.CD47.

[0095] In some implementations, the transgenic pigs lack growth hormone receptor expression and contain a gene selected from GTKO.CD46.pTBMpr-TBM.CD39-cag-A20.CD47; GTKO.CD46.Icam-2-TFPI.CD47-tiecag-A20.CD47; GTKO.CD46.pTBMpr-TBM.CD39-tiecag-CIITAKD.HO-1; GTKO.CD46.Icam-2-CTLA4Ig.T FPI-tiecag-CIITAKD.A20-1; GTKO.CD46.Icam-2-CTLA4Ig.TFPI-tiecag-CIITAKD.HO-1; GTKO.CD46.pTBMpr-T BM.CD39-cag-EPCR.CD55; GTKO.CD46.pTBMpr-TBM.A20-cag-EPCR.DAF; GTKO.CD46.pTBMpr-TBM.HO-1-cag-EPC R.DAF; GTKO.CD46.pTBMpr-TBM.TFPI-cag-EPCR.DAF; GTKO.CD46.Icam-2-CIITA.TFPI-cag-EPCR.DAF; GTKO.CD 46.Icam-2-TFPI.CD47-cag-EPCR.DAF; GTKO.CD46.Icam-2-TFPI.CD47-cag-EPCR.HO-1; GTKO.CD46.pTBMpr-TB The genotypes of M.HO-1-cag-TFPI.CD47; GTKO.CD46.pTBMpr-TBM.CD47-cag-EPCR.TFPI; GTKO.CD46.pTBMpr-TBM.TFPI-cag-EPCR.CD47; GTKO.CD46.pTBMpr-TBM.EPCR-cag-CD47.HO-1; or GTKO.CD46.cag-EPCR.DAF-tiecag-TFPI.CD47.

[0096] This disclosure also extends to methods for preparing and using such transgenic animals (or organs, tissues, or cells derived therefrom). In an exemplary embodiment, this disclosure provides a method for preparing a transgenic pig expressing at least four transgenes but lacking the expression of α1,3-galactosyltransferase and / or GHR, the method comprising (i) incorporating at least four transgenes under the control of at least two promoters into a single locus within a pig genome to provide a polygenic pig genome; and (ii) allowing a cell containing the polygenic pig genome to mature into a transgenic pig. In some embodiments, the pig genome is a somatic cell pig genome, and the cell is a zygote of a pig. In some embodiments, the pig genome is selected from the group consisting of gamete pig genomes, zygotic pig genomes, embryonic pig genomes, or blastocyst pig genomes. In exemplary embodiments, incorporation includes methods selected from the group consisting of biological transfection, chemical transfection, physical transfection, virus-mediated transduction or transformation, or combinations thereof. In some embodiments, incorporation includes cytoplasmic microinjection and prokaryotic microinjection.

[0097] In exemplary embodiments, the method involves using bicistronic or polycistronic vectors that allow for co-integration and co-expression of transgenes, possessing functional and / or manufacturing advantages, including polycistronic vectors utilizing 2A technology. In a preferred embodiment, within a polycistronic vector containing at least four transgenes, each bicistronic vector is controlled by its own promoter, with one or two promoters potentially leading to constitutive expression of two or more genes, and a second promoter potentially leading to tissue-specific expression of two or more genes. These vectors are used in conjunction with gene-editing tools, including editing nucleases and / or site-specific integrases.

[0098] In some implementations, the method involves using a single polycistronic vector that allows six or more transgenes to co-integrate and co-express to facilitate breeding, in which all transgenes co-segregate together and are passed on as a single unit to offspring / progeny.

[0099] This disclosure also extends to methods of treating a subject in need with one or more organs, organ fragments, tissues, or cells derived from transgenic animals of this disclosure. In exemplary embodiments, the organ is a kidney, liver, lung, heart, pancreas, or other solid organ. Examples of tissues considered in this disclosure include, but are not limited to, epithelial tissue and connective tissue.

[0100] The present invention also contemplates transplantation of more than one organ or organ segment. For example, this disclosure contemplates transplantation of the lung (or lung segment), kidney (or kidney segment), and heart (or segment thereof).

[0101] I. Definition

[0102] As used herein, the term “adverse event” means any adverse or unexpected sign (e.g., including abnormal laboratory findings), symptom, or illness that is temporarily associated with the use of a medical product (e.g., xenotransplantation), whether or not it is considered to be related to the medical product.

[0103] As used herein, the term "animal" refers to a mammal. In specific embodiments, the animal is at least six months old. In some embodiments, the animal has passed weaning age. In some embodiments, the animal survives to breeding age. The animals of the present invention are "genetically modified" or "transgenic," meaning they have added or incorporated transgenic or other foreign DNA, or modified endogenous genes, including targeted, recombinant, interrupted, deleted, disrupted, substituted, repressed, enhanced, or otherwise altered genes, to mediate genotypic or phenotypic effects in at least one cell of the animal, and generally in at least one germline cell of the animal. In some embodiments, the animal may have a transgene integrated into one allele of its genome (heterozygous transgene). In other embodiments, the animal may have a transgene on two alleles (homozygous transgene).

[0104] As used herein, the term “breeding” or its derivatives refer to any method of reproduction, including natural and artificial methods.

[0105] As used herein, the terms "breeding herd" or "production herd" refer to a group of transgenic animals produced by the methods of this disclosure. In some embodiments, genetic modifications in animals can be identified, and these animals can then be bred together to form a herd of animals with the desired set of genetic modifications (or a single genetic modification). See WO 2012 / 112586; PCT / US2012 / 025097. These offspring can be further bred to produce different or identical sets of genetic modifications (or single genetic modifications) in their offspring. This cycle of breeding animals with the desired genetic modifications can be continued as long as desired. "Herd" as used herein can include multiple generations of animals produced over a period of time with the same or different genetic modifications. "Herd" can also refer to a single generation of animals with the same or different genetic modifications.

[0106] As used herein, the terms “CRISPR” or “regularly clustered interspaced short palindromic repeats” or “SPIDR” or “interspaced sequence scattered in the same direction” refer to a family of DNA loci that are typically specific to a particular bacterial species. CRISPR loci comprise a distinct class of interspersed short sequence repeats (SSRs) (Ishino et al., J. Bacteriol., 169:5429-5433

[1987] ; and Nakata et al., J. Bacteriol., 171:3553-3556

[1989] ) and associated genes recognized in Escherichia coli. CRISPR / Cas molecules are components of the prokaryotic adaptive immune system, functioning similarly to RNA interference in eukaryotes, using RNA base pairing to guide DNA or RNA cleavage. The guidance of DNA DSB requires two components: the Cas9 protein, which acts as an endonuclease, and CRISPR RNA (crRNA) and tracer RNA (tracrRNA) sequences, which help guide the Cas9 / RNA complex to the target DNA sequence (Makarova et al., NatRev Microbiol, 9(6):467-477, 2011). Modification of a single target RNA is sufficient to alter the nucleotide target of the Cas protein. In some cases, crRNA and tracrRNA can be engineered into a single cr / tracrRNA hybrid to guide Cas9 cleavage activity (Jinek et al., Science, 337(6096):816-821, 2012). The CRISPR / Cas system can be used in bacteria, yeast, humans, and zebrafish, as described elsewhere (see, for example, Jiang et al., Nat Biotechnol, 31(3):233-239, 2013; Dicarlo et al., Nucleic Acids Res, doi:10.1093 / nar / gkt135, 2013; Cong et al., Science, 339(6121):819-823, 2013; Mali et al., Science, 339(6121):823-826, 2013; Cho et al., Nat Biotechnol, 31(3):230-232, 2013; and Hwang et al., Nat Biotechnol, 31(3):227-229, 2013).

[0107] As used herein, the term "clinically relevant immunosuppressive regimen" refers to a clinically acceptable immunosuppressive drug regimen administered to a patient following transplantation of an organ, tissue, or cell from a genetically modified pig, as disclosed herein. Determining clinical relevance typically requires FDA judgment to weigh acceptable risks against potential benefits, thereby maintaining human safety while preserving the efficacy of the drug or treatment.

[0108] As used herein, the term “constitutive” promoter refers to a nucleotide sequence that, when operatively linked to a polynucleotide encoding or specifying a gene product, results in the production of that gene product in the cell under most or all physiological conditions.

[0109] As used herein, the term "donor" is intended to include any non-human animal that can be used as a source of donor organs, tissues, or cells for xenotransplantation. Donors can be at any developmental stage, including but not limited to fetuses, newborns, juveniles, and adults.

[0110] As used herein, the term "endogenous" in relation to nucleic acid sequences and animals refers to any nucleic acid sequence that is naturally present in the genome of that animal. Endogenous nucleic acid sequences may comprise one or more gene sequences, intergenetic sequences, portions of gene sequences or intergenetic sequences, or combinations thereof.

[0111] As used herein, the terms "endothelium-specific," "specific transgene expression in endothelial tissue," and "specific expression of at least one transgene in endothelial tissue," etc., should be understood to refer to transgenes controlled by endothelial-specific regulatory elements that allow restricted expression of the transgene in endothelial tissue and / or cells. The function and expression of the transgene are limited to endothelial tissue and / or cells.

[0112] As used herein, the term "endothelium" refers to epithelium of mesoderm origin, consisting of a single layer of thin, flattened cells lining internal body cavities. For example, serous cavities or the interior of the heart contain an endothelial cell lining, while "vascular endothelium" refers to the endothelium lining blood vessels.

[0113] As used herein, the term "endothelial-specific regulatory element" refers to a promoter, enhancer, or combination thereof that drives the restricted expression of transgenes in endothelial tissues and / or cells. Regulatory elements provide transgene function and expression that are limited to endothelial tissues and / or cells.

[0114] As used herein, the term "enhancer" refers to an element in a nucleic acid construct designed to promote increased transgene expression in a tissue-specific manner. Enhancers are external elements that can significantly alter gene transcription efficiency (Molecular Biology of the Gene, Fourth Edition, pp. 708-710, Benjamin Cummings Publishing Company, Menlo Park, CA). (1987). In some embodiments, animals express transgenes under the control of a combination of promoter and enhancer elements. In some embodiments, a combination of promoter and enhancer elements is used, where the enhancer element is a non-coding or intronic region of DNA intrinsically associated with or co-located with the promoter.

[0115] As used herein, “expression” refers to the process by which polynucleotides are transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as “gene products.” If the polynucleotides originate from genomic DNA, expression can include the splicing of mRNA in eukaryotic cells.

[0116] The term "gene" in this document refers broadly to any segment of DNA that is associated with a biological function. Therefore, a gene includes the coding and / or regulatory sequences required for its expression. A gene may also include non-expressed DNA segments, such as recognition sequences that form other proteins. Genes can be obtained from a variety of sources, including cloning from sources of interest or synthesis from known or predicted sequence information, and may include sequences designed with desired parameters.

[0117] As used herein, the term "gene editing" refers to a form of genetic engineering in which DNA is inserted into, replaced, or removed from the genome using gene-editing tools. Examples of gene-editing tools include, but are not limited to, zinc finger nucleases, TALEN, and CRISPR.

[0118] As used herein, the term "gene editing-mediated" or similar terms refer to gene modifications (e.g., deletions, substitutions, rearrangements) that involve the use of gene editing / gene editing tools.

[0119] As used in this article, the term "gene knockout" refers to gene modification caused by the disruption of gene information encoded at a chromosomal locus.

[0120] As used in this article, the term "gene knock-in" refers to a gene modification that involves replacing the genetic information encoded at a chromosomal locus with a different DNA sequence.

[0121] As used herein, the term "genetic modification" refers to one or more alterations to nucleic acids, such as those within an organism's genome. For example, genetic modification can refer to changes in genes, additions (e.g., gene knock-in), and / or deletions (e.g., gene knockout).

[0122] As used in this article, the term "high" in relation to expression levels refers to an expression level considered sufficient to provide a phenotype (detectable expression or therapeutic benefit). Generally, "high" expression levels are sufficient to reduce transplant rejection, including hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), T-cell-mediated cellular rejection, and immediate blood-mediated inflammatory response (IBMIR).

[0123] As used herein, the terms “homology-driven recombination” or “homology direct repair” or “HDR” refer to homologous recombination events triggered by the presence of double-strand breaks (DSBs) in DNA (Liang et al. 1998); when used in conjunction with any known genome editing technology, the specificity of HDR can be controlled to create efficient and targeted double-strand breaks and allow for precise editing of the genome of the target cell; for example, the CRISPR / Cas9 system (Findlay et al. 2014; Mali et al. February 2014; and Ran et al. 2013).

[0124] As used herein, the term “enhanced homology-driven insertion or knock-in” is used to describe inserting DNA constructs, more specifically, inserting large DNA fragments or constructs with homologous arms or DNA segments flanking the double-strand breaks, utilizing homology-driven recombination in conjunction with any known genome editing technology to produce efficient and targeted double-strand breaks and allow for precise editing of the genome of targeted cells; for example, the CRISPR / Cas9 system (Mali et al. Feb 2013).

[0125] As used herein, the term "humanization" refers to a nucleic acid or protein whose structure (i.e., nucleotide or amino acid sequence) includes portions that substantially correspond to or are identical to the structure of a specific gene or protein found in non-human animals in nature, and also includes portions that differ from the structure found in the relevant specific non-human gene or protein, but are closer to a comparable structure found in the corresponding human gene or protein. In some embodiments, a "humanized" gene is a gene encoding a polypeptide having a sequence of amino acids substantially identical to that of a human polypeptide (e.g., a human protein or a portion thereof (e.g., its characteristic portion)). The term "hyperacute rejection" refers to a rejection reaction to transplanted material or tissue that occurs or begins within the first 24 hours after transplantation.

[0126] As used herein, the terms “implantation,” “transplantation,” or “graft” should be understood to refer to the act of inserting tissue or organ into a subject's body under conditions that allow for vascularization; and also to the tissue or organ thus inserted (i.e., “implanted,” “transplanted,” or “graft”). Conditions conducive to graft angiogenesis in mammals include a broad blood supply network in the local tissue bed at the graft site.

[0127] As used herein, the term "immunomodulator" refers to a transgenic substance capable of modulating an immune response. In exemplary embodiments, the immunomodulator according to this disclosure may be a complement inhibitor or an immunosuppressant. In a specific embodiment, the immunomodulator is a complement inhibitor. The complement inhibitor may be CD46 (or MCP), CD55, CD59, and / or CRI. In a specific embodiment, at least two complement inhibitors may be expressed. In one embodiment, the complement inhibitor may be CD55 and CD59. In another embodiment, the immunomodulator may be a class II transactivator or a mutant thereof. In a particular embodiment, the immunomodulator may be a class II transactivator dominant inactivating mutant (CIITA-DN). In another specific embodiment, the immunomodulator is an immunosuppressant. The immunosuppressant may be CTLA4-Ig. Other immunomodulators may be selected from, but are not limited to, CIITA-DN, PDL I, PDL2, or tumor necrosis factor-α-associated inducible ligand (TRAIL), Fas ligand (FasL, CD95L), CD47 (called integrin-associated protein (CD47)), HLA-E, HLA-DP, HLA-DQ and / or HLA-DR.

[0128] As used in this article, "inducible" promoters are promoters regulated by environment or development.

[0129] As used herein, the term "landing pad" or "engineered landing pad" refers to a nucleotide sequence containing at least one recognition sequence that is selectively bound to and modified by a specific polynucleotide modifying enzyme (e.g., site-specific recombinases and / or targeted endonucleases). Typically, the recognition sequence in the landing pad sequence is not endogenously present in the genome of the cell to be modified. Targeted integration rates can be improved by selecting recognition sequences from highly efficient nucleotide modifying enzymes that are not endogenously present in the genome of the target cell. Selecting non-endogenously present recognition sequences also reduces potential off-target integration. In other respects, using recognition sequences that are naturally present in the cell to be modified is also desirable. For example, in the case of multiple recognition sequences in the landing pad sequence, one or more recognition sequences may be exogenous, and one or more recognition sequences may be natural.

[0130] Multiple recognition sequences may be present in a single landing pad, allowing the landing pad to be sequentially targeted by two or more polynucleotide modifying enzymes, enabling the insertion of two or more unique sequences. Alternatively, multiple recognition sequences may be present in the landing pad, allowing multiple copies of the same sequence to be inserted into the landing pad. The landing pad may contain at least one recognition sequence. For example, the exogenous nucleic acid may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more recognition sequences. In embodiments containing more than one recognition sequence, the recognition sequences may be unique to each other (i.e., recognized by different polynucleotide modifying enzymes), identical repetitive sequences, or a combination of repetitive and unique sequences. Optionally, the landing pad may include one or more sequences encoding selectable markers (e.g., antibiotic resistance genes, metabolic selection markers, or fluorescent proteins). Other sequences may also be present, such as transcriptional regulatory and control elements (i.e., promoters, partial promoters, promoter captures, start codons, enhancers, introns, insulators, and other expression elements).

[0131] As used herein, the term "large targeting vector" or "LTVEC" refers to large targeting vectors for eukaryotic cells that are derived from cloned genomic DNA fragments that are larger than those typically used in other methods of homologous gene targeting in eukaryotic cells. Examples of LTVECs include, but are not limited to, bacterial artificial chromosomes (BACs), human artificial chromosomes (HACs), and yeast artificial chromosomes (YACs).

[0132] As used herein, the term "genomic locus" or "locus" (multiple loci) refers to a specific location on a chromosome of a gene or DNA sequence, which may include intron or exon sequences of a particular gene. A "gene" is a segment of DNA or RNA encoding a polypeptide or RNA chain that functions in an organism and is therefore a molecular unit of heredity in a living organism. For the purposes of this invention, a gene may be considered to include regions that regulate the production of gene products, regardless of whether such regulatory sequences are adjacent to coding and / or transcriptional sequences. Therefore, genes include, but are not limited to, introns, exons, promoter sequences, terminators, translation regulatory sequences (e.g., ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, boundary elements, 5' or 3' regulatory sequences, origins of replication, matrix attachment sites, and locus control regions.

[0133] As used herein, the term "lung transplant" refers to a surgical procedure in which a patient's diseased lung is partially or entirely replaced by a lung from a donor. A lung transplant can be a "single-lung transplant," in which only one of the recipient's two lungs is removed and replaced with a single lung from the donor, or a "bilateral lung transplant," which involves the removal of both lungs, one from each side, and their replacement with both lungs from the donor. In some implementations, the lungs are transplanted along with the heart.

[0134] As used in this article, the term "lung preservation" refers to the process of maintaining and protecting the donor lung from the time it is obtained until it is implanted in the recipient.

[0135] As used in this article, the phrase “loss of transplant function” refers to any physiological disruption or dysfunction of the normal processes exhibited by an organ or tissue in a donor animal.

[0136] As used herein, the term "mammal" means any non-human mammal, including but not limited to pigs, sheep, goats, cattle (bovines), deer, mules, horses, monkeys, dogs, cats, rats, and mice. In some embodiments, the animal is a pig weighing at least 300 pounds. In specific embodiments, the mammal is a sow that has given birth at least once. In some embodiments, the mammal is a non-human primate, such as a monkey or a baboon.

[0137] As used herein, a “marker” or “selectable marker” is a selective marker that allows the isolation of a minority of transfected cells expressing the marker from the majority of treated cells in a population. Such marker genes include, but are not limited to, neomycin phosphotransferase and hygromycin B phosphotransferase, or fluorescent proteins (e.g., GFP).

[0138] As used herein, the terms “nucleotide,” “polynucleotide,” “nucleotide sequence,” “nucleic acid,” and “oligonucleotide” are used interchangeably. They refer to nucleotides, deoxyribonucleotides, or ribonucleotides of any length in a polynucleotide form, or analogues thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function.

[0139] The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis (locus), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The term also includes nucleic acid-like structures with a synthetic backbone; see, for example, Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97 / 03211; WO 96 / 39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure can be modified before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeled components.

[0140] As used herein, the phrase “operably linked” encompasses a relationship in which operably linked components function in their intended manner. In one instance, a nucleic acid sequence encoding a protein can be operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.) to maintain proper transcriptional regulation.

[0141] As used herein, the term "organ" refers to a collection of tissues connected in structural units to provide a common function. Organs can be solid organs. Solid organs are internal organs with strong tissue consistency and are neither hollow (e.g., gastrointestinal organs) nor fluid (e.g., blood). Examples of solid organs include the heart, kidneys, liver, lungs, pancreas, spleen, and adrenal glands.

[0142] As used herein, the term "primate" refers to the various mammals of the order Primates, comprising lemurs, slow lorises, tarsiers, New World monkeys, Old World monkeys, and apes (including humans), characterized by having nails on their hands and feet, short noses, and large brains. In some embodiments, primates are non-human primates. In other embodiments, primates are humans.

[0143] As used herein, the term “promoter” refers to a DNA region, typically located upstream (5') of a coding region, that at least partially controls the initiation and level of transcription. The term “promoter” as used herein should be used in its broadest context, including transcriptional regulatory sequences of classical genomic genes, including TATA-box promoters or non-TATA-box promoters, and additional regulatory elements (i.e., activating sequences, enhancers, and silencers) that respond to developmental and / or environmental stimuli or alter gene expression in a tissue-specific or cell-type-specific manner. Promoters are typically, but not necessarily, located upstream or 5' of the structural gene whose expression they regulate. Furthermore, regulatory elements containing promoters are typically located within 2 kb of the gene transcription start site, although they may also be many kb apart. Promoters may contain additional specific regulatory elements located further from the start site to further enhance expression in the cell and / or alter the timing or inducibility of expression of the structural gene to which they are operatively linked.

[0144] As used in this article, the terms “porcine,” “pig,” and “swine” are general terms for the same type of animal, regardless of sex, size, or breed.

[0145] As used herein, the term “recognition site” or “recognition sequence” refers to a specific DNA sequence that is recognized by nucleases or other enzymes to bind to and guide site-specific cleavage of the DNA backbone.

[0146] As used in this article, the term "recombination site" refers to a nucleotide sequence that is recognized by site-specific recombinases and can serve as a substrate for recombination events.

[0147] As used herein, the terms “regulatory element” and “expression control element” are used interchangeably and refer to nucleic acid molecules that, in a given context, can influence the transcription and / or translation of operably linked coding sequences. These terms are widely used and encompass all elements that promote or regulate transcription, including promoters, core elements required for the fundamental interactions of RNA polymerases and transcription factors, upstream elements, enhancers, and reactive elements (see, for example, Lewin, “Genes V” (Oxford University Press, Oxford), pages 847–873). Exemplary regulatory elements in prokaryotes include promoters, operon sequences, and ribosome-binding sites. Regulatory elements used in eukaryotic cells may include, but are not limited to, promoters, enhancers, splicing signals, and polyadenylation signals.

[0148] As used herein, the term "regulatory promoter" refers to a promoter that can be used to regulate whether a peptide is expressed in an animal, tissue, or organ. A regulatory promoter can be tissue-specific and expressed only in a particular tissue, or time-regulatory (turning on at a specific time (driven by developmental stage)), or inducible, turning on or off only when controlled by an inducible element (expressing or not expressing) (it can also be an inducible promoter, such as an immune-inducible promoter and a cytokine response promoter, for example, induced by interferon-γ, TNF-α, IL-1, IL-6, or TGF-β). For example, expression may be blocked when the organ or tissue is part of a pig, but once the pig is transplanted into a human, expression is induced to overcome a cellular immune response after a period of time. Furthermore, expression levels can be controlled by a regulatory promoter system to ensure that immunosuppression of the recipient's immune system does not occur.

[0149] As used herein, the terms “regulatory sequence,” “regulatory element,” and “control element” are used interchangeably and refer to a polynucleotide sequence located upstream (5' non-coding), inside, or downstream (3' non-translation) of the polynucleotide target to be expressed. Regulatory sequences affect, for example, the timing, amount, or level of transcription, RNA processing or stability, and / or translation of the associated structural nucleotide sequence. Regulatory sequences may include activator-binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor-binding sequences, stem-loop structures, translation initiation sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, etc.

[0150] As used herein, the term "safe harbor" locus refers to a site in the genome where transgenic DNA (e.g., a construct) can be harmlessly added and produce consistent levels of expression. In some embodiments, this disclosure relates to the incorporation and expression of transgenic DNA, including transgenes within safe harbor loci.

[0151] As used herein, the term “site-specific recombinase” refers to a group of enzymes that can promote recombination between “recombination sites”, where the two recombination sites are physically separated within a single nucleic acid molecule or on separate nucleic acid molecules. Examples of “site-specific recombinases” include, but are not limited to, phiC31, att, Bxb1, R4 (integrase), and / or Cre, Flp, and Dre recombinases.

[0152] As used herein, the term “subject” means any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, etc. (e.g., a recipient or donor of a particular treatment (e.g., a transplanted graft)). Unless otherwise stated herein, the terms “subject” and “patient” are used interchangeably when referring to a human subject (e.g., where the subject is a graft donor).

[0153] As used herein, the term "targeting vector" refers to a recombinant DNA construct that typically contains custom-designed DNA arms homologous to genomic DNA, located flanking key elements of a target gene or sequence. When introduced into cells, the targeting vector integrates into the cell's genome via homologous recombination. A "tissue-specific" promoter is a nucleotide sequence that, when operatively linked to a polynucleotide encoding or specifying a gene product, essentially results in the production of the gene product in the cell only if the cell is a cell of the tissue type corresponding to the promoter.

[0154] As used herein, the term "tissue" refers to the cellular organizational hierarchy between cells and complete organs. A tissue is a collection of similar cells from the same origin that together perform a specific function. Organs, in turn, are functional clusters of multiple tissues. Examples of tissues considered in this disclosure include, but are not limited to, connective tissue, muscle tissue, nervous tissue, epithelial tissue, and mineralized tissue. Blood, bone, tendons, ligaments, fat, and cellular tissue are examples of connective tissue—they can also be classified as fibrous connective tissue, skeletal connective tissue, and fluid connective tissue. Muscle tissue is divided into three distinct categories: visceral or smooth muscle, which exists as the lining of organs; skeletal muscle, which typically attaches to bones and produces large-amplitude movements; and cardiac muscle, which exists in the heart and contracts to pump blood throughout the organism. Cells containing the central and peripheral nervous systems are classified as neural (or nerve) tissue. In the central nervous system, neural tissue forms the brain and spinal cord. In the peripheral nervous system, neural tissue forms cranial and spinal nerves, including motor neurons.

[0155] As used herein, the term "transcription activator-like effector nuclease" or "TALEN" refers to an artificial restriction enzyme created by fusing a TAL effector DNA-binding domain with a DNA-cutting domain. These agents enable efficient, programmable, and specific DNA cleavage, representing a powerful tool for in situ genome editing. Transcription activator-like effectors (TALEs) can be rapidly engineered to bind to virtually any DNA sequence. As used herein, the term TALEN is used broadly to include monomeric TALENs, which can cleave double-stranded DNA without the assistance of another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to work together to cleave DNA at the same site. TALENs working together can be referred to as the left TALEN and right TALEN, referencing the chirality of the DNA. See U.S. Serial No. 12 / 965,590; U.S. Serial No. 13 / 426,991 (U.S. Patent No. 8,450,471); U.S. Serial No. 13 / 427,040 (U.S. Patent No. 8,440,431); U.S. Serial No. 13 / 427,137 (U.S. Patent No. 8,440,432); and U.S. Serial No. 13 / 738,381, all of which are incorporated herein by reference in their entirety.

[0156] As used herein, the terms “transfection,” “transformation,” or “transduction” refer to the process of transferring or introducing exogenous nucleic acids into host cells. Cells that are “transfected,” “transformed,” or “transduced” are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. Cells include primary test cells and their progeny.

[0157] "Transgenic" refers to a gene or genetic material that has been transferred from one organism to another. When a transgene is transferred into an organism, that organism can be called a transgenic organism. Typically, the term describes a segment of DNA containing a gene sequence that has been isolated from one organism and introduced into another. This non-natural DNA segment may retain the ability to produce RNA or protein in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code. Typically, DNA is incorporated into the reproductive system of an organism. For example, in higher vertebrates, this can be achieved by injecting foreign DNA into the nucleus of a fertilized egg or through somatic cell nuclear transfer, where a somatic cell containing the transgene is incorporated into the host genome, transferred to an enucleated oocyte, and then transplanted into a surrogate mother to produce offspring. When inserted into a cell, the transgene can be a segment of cDNA (complementary DNA), which is a copy of mRNA (messenger RNA), or the gene itself located in the original region of the genomic DNA.

[0158] A transgene can be a genomic sequence, particularly when introduced as a large clone into a BAC (bacterial artificial chromosome) or spore, or it can be in the form of a “small gene,” typically characterized by a combination of genomic DNA (including intronic regions, such as intron 1) and 5' or 3' regulatory regions, together with cDNA regions. Unless otherwise stated, in the context of this specification, “expression” of a transgene means the expression of a peptide sequence derived from a non-natural nucleic acid in at least one cell of a host. The peptide can be expressed from a transgene incorporated into the host genome. A transgene can contain a polynucleotide encoding a protein or a fragment thereof (e.g., a functional fragment). A protein fragment (e.g., a functional fragment) can contain at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the amino acid sequence of the protein. A protein fragment can be a functional fragment of the protein. A functional fragment of the protein can retain some or all of the protein's function.

[0159] As used herein, the term "transplant tolerance" is defined as a donor-specific unresponsive state that does not require sustained pharmacological immunosuppression. Transplant tolerance can eliminate many adverse events associated with immunosuppressants. Therefore, induction of tolerance can improve xenograft reception. In one embodiment, induction of tolerance can be identified by a reduction in clinical symptoms of xenograft rejection. In another embodiment, induction of tolerance can improve or prevent metabolic, inflammatory, and proliferative pathological conditions or diseases associated with xenograft transplantation. In yet another embodiment, induction of tolerance can improve, reduce, or prevent adverse clinical conditions or diseases associated with the administration of immunosuppressive therapy for the prevention of xenograft rejection. In yet another embodiment, induction of tolerance can promote xenograft survival. In various embodiments, induction of tolerance can prevent relapse in patients exhibiting these diseases or conditions.

[0160] The term "ungulate" refers to ungulate mammals. Even-toed ungulates are even-toed (split-hoofed) ungulates, including antelope, camel, cattle, deer, goats, pigs, and sheep. Perissodactyls are odd-toed ungulates, including horses, zebras, rhinoceroses, and tapirs. As used herein, the term ungulate refers to an adult, embryonic, or fetal ungulate.

[0161] As used herein, the term "vector" refers to a portion capable of transferring polynucleotides into a host cell. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends; nucleic acid molecules without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which a virally derived DNA or RNA sequence is present in the vector for packaging into a virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by the virus for transfection into host cells. Some vectors are capable of autonomous replication in the host cells in which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) are integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes operably linked to them. Such vectors are referred to herein as "expression vectors." Expression vectors commonly used in recombinant DNA technology are typically in plasmid form. Recombinant expression vectors may contain the nucleic acids of the present invention in a form suitable for expression in host cells, meaning that the recombinant expression vector includes one or more regulatory elements that can be selected based on the host cell used for expression and are operably linked to the nucleic acid sequence to be expressed. Within the recombinant expression vector, "operably linked" means that the nucleotide sequence of interest is linked to the regulatory element in a manner that allows the nucleotide sequence to be expressed (e.g., when the vector is introduced into a host cell, in an in vitro transcription / translation system or in the host cell). For information on recombination and cloning methods, see U.S. Patent Application Serial No. 10 / 815,730, the contents of which are incorporated herein by reference in their entirety. Preferably, the vector is a DNA vector, and more preferably an RNA vector capable of expressing a protein encoding the protein according to the present invention.

[0162] Many suitable vectors are described in the literature; examples can be found in Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press or DNA cloning: a practical approach, Volume II: Expression systems, edited by DM Glover (IRL Press, 1995).

[0163] As used herein, the term "zinc finger nuclease" or "ZFN" refers to an artificially engineered DNA-binding protein containing a zinc finger DNA-binding domain and a DNA-cutting domain. The zinc finger domain can be engineered to target specific desired DNA sequences, enabling zinc finger nucleases to target unique sequences within complex genomes. They facilitate targeted genome editing by generating double-strand breaks in DNA at user-specified locations. Each ZFN contains two functional domains: a.) a DNA-binding domain composed of twin-fingered modules, each recognizing a unique hexameric (6 bp) DNA sequence. The twin-fingered modules are spliced ​​together to form a zinc finger protein, each with a specificity ≥24 bp. b.) a DNA-cutting domain composed of the nuclease domain of Fok I. When the DNA-binding and DNA-cutting domains fuse together, a highly specific pair of "genomic scissors" is produced. ZFNs are gene-editing tools.

[0164] II. Transgenic animals

[0165] This disclosure provides transgenic animals (e.g., transgenic pigs) as sources of organs, organ fragments, tissues, or cells for xenotransplantation. This disclosure extends to organs, tissues, and cells derived from transgenic animals, and populations of such animals, such as livestock herds.

[0166] The animal can be any suitable animal. In an exemplary embodiment, the animal is an ungulate and more specifically a swine or pig.

[0167] The transgenic donor animal (e.g., an ungulate, a swine, or a pig) is genetically modified, more specifically, to contain multiple transgenes, for example, multiple transgenes at a single locus. In some embodiments, the transgenic donor animal is genetically modified to express multiple transgenes separated between a first locus (i.e., locus 1) and a second locus (i.e., locus 2).

[0168] Loci can be natural loci or modified natural loci. This article describes various strategies for modifying natural loci to facilitate targeting.

[0169] In exemplary embodiments, this disclosure provides transgenic animals (e.g., transgenic pigs) comprising the incorporation and expression of at least four transgenes at a single locus under the control of at least two promoters (e.g., exogenous promoters, or a combination of exogenous and natural promoters), wherein the pigs lack expression of α1,3-galactosyltransferase. Optionally, the transgenic animal comprises one or more additional genetic modifications, including but not limited to gene additions and / or deletions, including gene knockouts and knock-ins, as well as gene substitutions and rearrangements.

[0170] In specific embodiments, this disclosure provides transgenic pig animals comprising at least four transgenes incorporated and expressed at a single locus, wherein the expression of the at least four transgenes is controlled by specific promoters, i.e., promoters drive the expression of each individual transgene. For example, in the case where the transgenic animal incorporates and expresses four transgenes at a single locus, the expression of these transgenes is driven by four promoters, each promoter being specific to a particular transgene. In alternative embodiments, a given promoter controls the expression of more than one transgene (e.g., two transgenes, three transgenes). For example, in the case where the transgenic animal incorporates and expresses four transgenes, two of the four transgenes are expressed as polycistronic transgenes controlled by a first promoter, and two of the four transgenes are expressed as polycistronic transgenes controlled by a second promoter.

[0171] In some embodiments, two of the four transgenes expressed in the first or second polycistronic cell are selected from the group consisting of TBM, EPCR, DAF, CD39, TFPI, CTLA4-Ig, CIITA-DN, HOI, A20, and CD47. In some embodiments, at least one pair of transgenes is selected from the group consisting of: TBM and CD39; EPCR and DAF; A20 and CD47; TFPI and CD47; CIITA-DN and HO-1; TBM and CD47; CTLA4Ig and TFPI; CIITA-DN and TFPI; CIITA-DN and A20; TBM and A20; EPCR and DAF; TBM and HO-1; TBM and TFPI; CIITA and TFPI; EPCR and HO-1; TBM and CD47; EPCR and TFPI; TBM and EPCR; CD47 and HO-1; CD46 and CD47; CD46 and HO-1; CD46 and TBM; and HLA-E and CD47.

[0172] In an exemplary embodiment, at least four transgenes are selected from the group consisting of immunomodulators (e.g., immunosuppressants), anticoagulants, complement inhibitors, and cryoprotective transgenes. In an exemplary embodiment, the single locus is a natural locus. In other embodiments, the single locus is a modified natural locus, such as a transgenic locus. The transgenic locus may be, for example, a locus containing a selectable marker gene or a locus containing a landing pad.

[0173] In an exemplary embodiment, at least four transgenes are provided in a polycistronic vector (MCV) and incorporated either through random integration or by using a gene-editing tool. Optionally, the transgenic animal may have one or more additional gene modifications. These additional gene modifications may be, for example, gene knockout or gene knock-in. In a specific embodiment, the additional gene modification comprises a chimeric pig-human vWF.

[0174] In another embodiment, this disclosure provides a transgenic animal (e.g., a pig) comprising at least five gene modifications resulting in (i) the absence of α1,galactosyltransferase expression (i.e., αGal-free) and (ii) the incorporation and expression of at least four, five, six, seven, eight, nine, or ten transgenes at a single locus. The expression of the transgenes is driven by a promoter, which may be a specialized promoter or a promoter controlling the expression of two or more transgenes. The promoter may be a foreign promoter or a combination of a foreign promoter and a natural promoter.

[0175] In some embodiments, if more than four transgenes are added, transgenes may be incorporated at more than one locus to better regulate the expression of the transgene combination (e.g., integration of at least four transgenes under the control of at least two promoters at GGTA1, and integration of a second polycistronic at a second locus (e.g., CMAH, B4GalNT2, AAVS1, or Rosa26)). In some embodiments where the second locus is genetically modified, it may be modified to inactivate the expression of another porcine gene (e.g., by applying gene editing and / or homologous recombination techniques). In exemplary embodiments, the multiple transgenes incorporated and expressed at the second locus are selected from the group consisting of immunomodulators, complement inhibitors, anticoagulants, and cryoprotective transgenes. In exemplary embodiments, the second locus is a natural locus, a modified natural locus, or a transgenic locus (e.g., a landing pad). In exemplary embodiments, at least two transgenes at the second locus are provided in the MCV and incorporated using gene editing tools. Optionally, the transgenic animal may have one or more additional gene modifications.

[0176] In one embodiment, this disclosure provides a transgenic animal (e.g., a pig) comprising at least four gene modifications resulting in (i) decreased expression of α1, galactosyltransferase and (ii) incorporation and expression of at least four transgenes at a single locus, wherein these four transgenes are expressed under the control of at least two promoters (e.g., exogenous promoters, or a combination of exogenous and natural promoters). In an exemplary embodiment, the transgenes are selected from the group consisting of immunomodulators, anticoagulants, complement inhibitors, and cryoprotective transgenes. In an exemplary embodiment, the single locus is a natural locus, a modified natural locus, or a transgenic locus (e.g., a landing pad). In an exemplary embodiment, at least two transgenes are provided in an MCV and incorporated using gene editing tools (i.e., CRISPR / Cas9, TALEN, or ZFN) to improve the efficiency of homologous recombination or homology-dependent repair. Optionally, the transgenic animal may have one or more additional gene modifications.

[0177] In another embodiment, this disclosure provides a transgenic animal (e.g., a pig) comprising at least five gene modifications resulting in (i) reduced expression of α1, galactosyltransferase and (ii) incorporation and expression of at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten transgenes in a single locus or separately between two loci. In an exemplary embodiment, the transgenes are selected from the group consisting of immunomodulators, complement inhibitors, anticoagulants, and cryoprotective transgenes. In an exemplary embodiment, the single locus is a natural locus, a modified natural locus, or a transgenic locus (e.g., a landing pad). In an exemplary embodiment, at least two transgenes are provided in an MCV and incorporated using gene-editing tools (i.e., CRISPR / Cas9, TALEN, or ZFN) to enhance the efficiency of homologous recombination or homology-dependent repair. Optionally, the transgenic animal may have one or more additional gene modifications.

[0178] In an exemplary embodiment, the transgenic animal lacks the expression of α1,galactosyltransferase (i.e., αGal is absent) and contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven or more gene modifications. Optionally, in addition to transgenic integration, further knockouts include the knockout of the β4GalNT2 gene or the CMAH gene (both of which are associated with innate immunity and xenograft rejection).

[0179] In exemplary embodiments, the expression of α1-galactosyltransferase in the transgenic animal is reduced and includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven additional gene modifications. In some embodiments, the expression of α1-galactosyltransferase is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%.

[0180] In an exemplary embodiment, the transgenic animal comprises (i) a genetic modification resulting in the absence of α1,3-galactosyltransferase expression and (ii) at least four additional genetic modifications, or more specifically, four additional genetic modifications. These additional genetic modifications can be any suitable genetic modifications, including but not limited to CRISPR-induced deletion / insertion or gene substitution (INDEL), including knockout or knock-in at other loci (e.g., B4GalNT2, CMAH, vWF).

[0181] In an exemplary embodiment, the transgenic animal comprises (i) a genetic modification resulting in reduced expression of α1,3-galactosyltransferase and (ii) at least four additional genetic modifications, or more specifically, four additional genetic modifications. In an exemplary embodiment, the transgenic animal comprises (i) a genetic modification resulting in a lack of α1,3-galactosyltransferase and / or growth hormone receptor expression and (ii) at least five additional genetic modifications, or more specifically, five additional genetic modifications.

[0182] In an exemplary embodiment, the transgenic animal comprises (i) a gene modification that reduces the expression of α1,3-galactosyltransferase and / or growth hormone receptor and (ii) at least five additional gene modifications, or more specifically at least five additional gene modifications.

[0183] In an exemplary embodiment, the transgenic animal comprises (i) a genetic modification resulting in a lack of α1,3-galactosyltransferase and / or growth hormone receptor expression and (ii) at least six additional genetic modifications, or more specifically, six additional genetic modifications. In an exemplary embodiment, the transgenic animal comprises (i) a genetic modification resulting in reduced α1,3-galactosyltransferase and / or growth hormone receptor expression and (ii) at least six additional genetic modifications, or more specifically, six additional genetic modifications.

[0184] In specific embodiments, the donor animal (e.g., an ungulate, a swine, or a pig) contains genetic modifications that result in (i) the absence of expression of α1,3-galactosyltransferase and / or growth hormone receptor and (ii) the incorporation and expression of at least one, at least two, at least three, at least four, at least five, or at least six or more transgenes. In exemplary embodiments, this disclosure provides swine animals containing genetic modifications that result in (i) the absence of expression of α1,3-galactosyltransferase and / or growth hormone receptor and (ii) the incorporation and expression of at least four additional transgenes. In exemplary embodiments, this disclosure provides swine animals containing genetic modifications that result in (i) the absence of expression of α1,3-galactosyltransferase and / or growth hormone receptor and (ii) the incorporation and expression of at least five additional transgenes, or more specifically, five additional genetic modifications.

[0185] In exemplary embodiments, this disclosure provides swine animals comprising genetic modifications resulting in (i) a deficiency in the expression of α1,3-galactosyltransferase and / or growth hormone receptor and (ii) the incorporation and expression of at least six additional transgenes, or more specifically, six additional genetic modifications. In specific embodiments, donor animals (e.g., ungulates, swine animals, or pigs) comprise genetic modifications resulting in (i) reduced expression of α1,3-galactosyltransferase and / or growth hormone receptor and (ii) the incorporation and expression of at least four, at least five, or at least six or more transgenes, or more specifically, four, five, or at least six additional transgenes.

[0186] In an exemplary embodiment, the donor animal (e.g., an ungulate, a swine, or a pig) contains genetic modifications that result in (i) reduced expression of α1,3-galactosyltransferase and / or growth hormone receptor and (ii) incorporation and expression of five additional transgenes. Optionally, the donor animal may contain more than one additional genetic modification. In an exemplary embodiment, the donor animal (e.g., an ungulate, a swine, or a pig) contains genetic modifications that result in (i) reduced expression of α1,3-galactosyltransferase and / or growth hormone receptor and (ii) incorporation and expression of six additional transgenes. Optionally, the donor animal may contain one or more additional genetic modifications (knockout, knock-in, INDEL, porcine vWF modification).

[0187] III. Transgenic Expression

[0188] Transgenic expression can be at any level, but in this specific implementation, it is expressed at a high level. A variety of promoter / enhancer elements can be used, depending on the desired level and tissue-specific expression. The promoter / enhancer can be constitutive or inducible, depending on the desired expression pattern. The promoter can be exogenous or natural, or a combination of exogenous and natural promoters.

[0189] In some embodiments, the transgene is expressed by a constitutive promoter or a ubiquitous promoter. In some other embodiments, the transgene is expressed by a tissue-specific promoter, a cell type-specific promoter, or an inducible promoter, and may include additional regulatory elements such as enhancers, insulators, matrix attachment regions (MARs), etc. In an exemplary embodiment, four or more transgenes are co-expressed. In an exemplary embodiment, four or more transgenes are expressed at approximately molar equivalents.

[0190] In exemplary embodiments, the transgene is expressed by a promoter that is primarily active in endothelial cells. In some embodiments, transgene expression is controlled by a porcine Icam-2 enhancer / promoter. In some embodiments, transgene expression is controlled by a constitutive CAG promoter.

[0191] In some implementations, the transgenic animal is genetically modified to result in the incorporation and expression of two or more transgenes, wherein at least one transgene is controlled by a constitutive promoter and at least one transgene is controlled by a tissue-specific promoter, or more specifically, by a promoter that is primarily active in endothelial cells.

[0192] In an exemplary embodiment, the transgenic animal is genetically modified to result in the incorporation and expression of four or more transgenes at a single locus, wherein at least one transgene is controlled by a constitutive promoter and at least one transgene is controlled by a tissue-specific promoter, or more specifically, by a promoter that is primarily active in endothelial cells.

[0193] The transgene can be any transgene suitable for modifying a donor animal (e.g., a swine animal) for xenotransplantation. In exemplary embodiments, the transgene is selected from immunomodulators (e.g., complement regulators, complement inhibitors, immunosuppressants), anticoagulants, cryoprotective genes, or combinations thereof. In some embodiments, the sequence of the transgene is human.

[0194] In some embodiments, the transgene is an immunomodulator. In some embodiments, the transgene is a complement regulator or more specifically a complement inhibitor. The complement inhibitor may include, but is not limited to, CD46 (MCP), CD59, or CR1. The sequence of the complement inhibitor may be human. In some embodiments, the transgene is a complement pathway inhibitor (i.e., a complement inhibitor). The complement inhibitor may include, but is not limited to, CD55, CD59, CR1, and CD46 (MCP). The sequence of the complement inhibitor may be human. The complement inhibitor may be human CD46 (hCD46), which is expressed via a mini-gene construct (see Loveland et al., Xenotransplantation, 11(2):171-183.2004).

[0195] In some embodiments, the transgene is an immunosuppressant. In some embodiments, the transgene is an immunosuppressive gene with T-cell regulatory function (e.g., CTLA4-Ig), or a class II MHC dominant inactivation inhibitor (CIITA), or other genes regulating B-cell or T-cell-mediated immune function expression. In further embodiments, such animals may be further modified to eliminate gene expression affecting immune function. In some embodiments, the immunosuppressant is CD47.

[0196] In some implementations, the transgene is an anticoagulant. Anticoagulants may include, but are not limited to, tissue factor pathway inhibitors (TFPIs), hirudin, thrombomodulin (TBM), endothelial protein C receptor (EPCR), and CD39. The sequence of the anticoagulant may be human.

[0197] The transgenic animals may also contain one or more additional gene modifications. In one embodiment, the animals may be genetically modified to suppress the expression of the CMP-Neu5Ac hydroxylase gene (CMAH) (see, for example, U.S. Patent Publication 2005-0223418), the iGb3 synthase gene (see, for example, U.S. Patent Publication 2005-0155095), and / or the Forssman synthase gene (see, for example, U.S. Patent Publication 2006-0068479). Furthermore, the animals may be genetically modified to reduce the expression of procoagulants. Specifically, in one embodiment, the animal is genetically modified to reduce or eliminate the expression of procoagulant genes such as FGL2 (fibrinogen-like protein 2) (see, for example, Marsden, et al. (2003) J din Invest. 112:58-66; Ghanekar, et al. (2004) J. Immunol. 172:5693-701; Mendicino, et al. (2005) Circulation. 112:248-56; Mu, et al. (2007) Physiol Genomics. 31(1):53-62). In another embodiment, the animal may be genetically modified to inhibit the expression of β-1,4N-acetylgalactosyltransferase 2 (β4GalNT2).

[0198] IV. Specific Genetics

[0199] 1. Growth hormone receptor

[0200] One aspect of this disclosure provides transgenic animals (e.g., pigs) containing gene alterations that result in decreased expression of the growth hormone receptor (GHR) gene; or gene alterations that cause mutations in at least one allele of the GHR gene, thereby impairing GHR function. Human GHR is a 638-amino acid transmembrane protein with an extracellular domain (primarily encoded by exons 3 to 7), a transmembrane domain (primarily encoded by exon 8), and an intracellular domain (primarily encoded by exons 9 and 10), belonging to the cytokine receptor family. Growth hormone (GH) binds to GHR, initiating the GH-GHR signaling pathway, leading to the production of IGF-I and promoting the organism's growth, development, and immune function. Therefore, mutations in the GHR gene have a destructive effect on the organism's growth and development.

[0201] In fact, mutations in the human growth hormone receptor (GHR) and various GHR defects, including nonsense mutations, splice site mutations, frameshifts, deletions, and missense mutations, all impair the GHR signaling pathway. These GHR defects are associated with Laron syndrome, an autosomal disorder characterized by dwarfism, prominent forehead, small midface, moderate obesity, and microgenitalia. In pigs, the GHR (GHRKO) mutation reproduces the phenotype of individuals with Laron syndrome (see Yu et al., J Transl. Med 16:41 (2018)).

[0202] In some embodiments, the GHR gene is inactivated via a gene-targeting event. In another embodiment, a swine animal is provided in which both alleles of the GHR gene are inactivated via a gene-targeting event. In one embodiment, the gene can be targeted via homologous recombination. In other embodiments, the GHR gene can be disrupted, i.e., a portion of the genetic code can be altered, thereby affecting the transcription and / or translation of that segment of the GHR gene. For example, gene disruption can occur via substitution, deletion (“knockout”), or insertion (“knock-in”) techniques, including targeted insertion of selectable marker genes (e.g., neo) that interrupt the coding region of the GHR gene.

[0203] In some embodiments, the GHR gene allele is inactivated, so that the resulting GHR can no longer respond to growth hormone stimulation to produce IGF-1. In one embodiment, the GHR gene can be transcribed into RNA but not translated into protein. In another embodiment, the GHR gene can be transcribed in a truncated form. This truncated GHR RNA cannot be translated or is translated into a nonfunctional GHR protein. In an alternative embodiment, the GHR gene can be inactivated in a manner that does not involve gene transcription. In a further embodiment, the GHR gene can be transcribed and then translated into a nonfunctional protein.

[0204] In some implementations, the expression of the active GHR gene can be reduced by using alternative methods, such as those targeting the transcription or translation of the GHR gene. For example, antisense RNA or siRNA targeting the natural GHR gene or its mRNA can be used to reduce expression. In other implementations, site-specific recombinases are used to target genomic regions for recombination. Examples of such systems are the CRE-lox system and the Flp-Frt system.

[0205] In some embodiments, transgenic animals, such as transgenic pigs, are produced having genetic alterations that confer one or more characteristics of Laron syndrome. Laron syndrome is characterized by a lack of IGF-1 production in response to growth hormone, and low serum IGF-1 and glucose levels. The transgenic animals may have genetic alterations that result in reduced expression of the human growth receptor (GHR), or alterations that induce GHR mutations that impair GHR function. In some embodiments, the transgenic animals have GHR knockout gene alterations. In some embodiments, the genetic alteration is a GHR knockout gene alteration. In some embodiments, GHR knockout (GHRKO) transgenic pigs exhibit at least about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% or more of reduced GHR expression compared to pigs without the GHRKO gene alteration.

[0206] Patients with Laron syndrome have extremely high levels of circulating growth hormone (GH) and extremely low levels of insulin-like growth factor I (IGF-I), and they do not respond to GH administration. Patients with Laron syndrome may also exhibit resistance to certain conditions, such as type II diabetes and certain cancers. To date, the only treatment for Laron syndrome is the administration of recombinant IGF-I. In some implementations, transgenic GHRKO pigs produce at least approximately 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% less insulin-like growth factor 1 (IGF-1) compared to pigs without the GHRKO gene alteration.

[0207] One aspect of this disclosure provides a transgenic animal (e.g., a pig) comprising a genetic alteration that results in decreased expression of the growth hormone receptor (GHR) gene; or a genetic alteration that causes at least one allelic mutation in the GHR gene, thereby impairing GHR function, and further comprising one or more additional genetic alterations. In some embodiments, one or more additional genetic alterations result in (i) decreased expression of one or more genes, (ii) impaired function of one or more genes, and / or (iii) expression of one or more transgenes. In some embodiments, one or more transgenes are independently selected anticoagulants, complement regulators, immunomodulators, and cell-protective transgenes. In some embodiments, the anticoagulant is selected from TBM, TFPI, EPCR, and CD39. In some embodiments, the complement regulator is a complement inhibitor selected from CD46, CD55, and CD59. In some embodiments, the immunomodulator is an immunosuppressant selected from porcine CLTA4-Ig, CIITA-DN, or CD47. In some embodiments, one or more transgenes are selected from CD47, CD46, DAF / CD55, TBM, EPCR, and HO1. In some implementations, one or more gene alterations involve reduced expression of α1,3-galactosyltransferase.

[0208] In some implementations, transgenic pigs lacking the growth hormone gene express CD46 and at least four combinations of transgenes selected from the following: (i) EPCR, HO-1, TBM, and CD47; (ii) EPCR, HO1, TBM, and TFPI; (iii) EPCR, CD55, TFPI, and CD47; (iv) EPCR, DAF, TFPI, and CD47; or (v) EPCR, CD55, TBM, and CD39.

[0209] In some implementations, two of the four transgenes are expressed in either the first or second polycistronic group and are selected from the group consisting of: TBM, EPCR, DAF, CD39, TFPI, CTLA4-Ig, CIITA-DN, HOI, A20, and CD47. In some embodiments, at least one pair of transgenes expressed in the polycistronic group is selected from the group consisting of: (a) TBM and CD39; (b) EPCR and DAF; (c) A20 and CD47; (d) TFPI and CD47; (e) CIITAKD and HO-1; (f) TBM and CD47; (g) CTLA4Ig and TFPI; (h) CIITAKD and A20; (i) TBM and A20; (j) EPCR and DAF; (k) TBM and HO-1; (l) TBM and TFPI; (m) CBTA and TFPI; (n) EPCR and HO-1; (o) TBM and CD47; (p) EPCR and TFPI; (q) TBM and EPCR; (r) CD47 and HO-1; (s) CD46 and CD47; (t) CD46 and HO-1; (u) CD46 and TBM; and HLA-E and CD47.

[0210] In some implementations, the transgenic pigs lack growth hormone receptor expression and contain genotypes selected from the following: (i) GTKO.CD46.Icam-2-TBM.CD39-cag-A20.CD47; (ii) GTKO.CD46.Icam-2-TFPI.CD47-tiecag-A20.CD47; (iii) GTKO.CD46.Icam-2-TBM.CD39-tiecag-CIITAKD.HO-1; (iv) GTKO.CD46.Icam-2-CTLA4Ig .TFPI-tiecag-CIITAKD.A20-1; (v) GTKO.CD46.Icam-2-CTLA4Ig.TFPI-tiecag-CIITAKD.HO-1; (vi) GTKO.CD46.Icam-2-T BM.CD39-cag-EPCR.CD55; (vii) GTKO.CD46.Icam-2-TBM.A20-cag-EPCR.DAF; (viii) GTKO.CD46.Icam-2-TBM.HO-1-cag-EP CR.DAF; (ix)GTKO.CD46.Icam-2-TBM.TFPI-cag-EPCR.DAF; (x)GTKO.CD46.Icam-2-CIITA.TFPI-cag-EPCR.DAF; (xi)GTKO .CD46.Icam-2-TFPI.CD47-cag-EPCR.DAF; (xii) GTKO.CD46.Icam-2-TFPI.CD47-cag-EPCR.HO-1; (xiii) GTKO.CD46.Icam- 2-TBM.HO-1-cag-TFPI.CD47; (xiv)GTKO.CD46.Icam-2-TBM.CD47-cag-EPCR.TFPI; (xv)GTKO.CD46.Icam-2-TBM.TFPI-cag -EPCR.CD47; (xvi) GTKO.CD46.Icam-2-TBM.EPCR-cag-CD47.HO-1; or (xvii) GTKO.CD46.cag-EPCR.DAF-tiecag-TFPI.CD47.

[0211] In some implementations, the transgenic pigs lack growth hormone receptor expression and contain genotypes selected from the following: GTKO.CD46.pTBMpr-TBM.CD39-cag-A20.CD47; GTKO.CD46.Icam-2-TFPI.CD47-tiecag-A20.CD47; GTKO.CD46.pTBMpr-TBM.CD39-tiecag-CIITAKD.HO-1; GTKO.CD46.Icam-2-CTLA 4Ig.TFPI-tiecag-CIITAKD.A20-1; GTKO.CD46.Icam-2-CTLA4Ig.TFPI-tiecag-CIITAKD.HO-1; GTKO.CD46.pTB Mpr-TBM.CD39-cag-EPCR.CD55; GTKO.CD46.pTBMpr-TBM.A20-cag-EPCR.DAF; GTKO.CD46.pTBMpr-TBM.HO-1-ca g-EPCR.DAF; GTKO.CD46.pTBMpr-TBM.TFPI-cag-EPCR.DAF; GTKO.CD46.Icam-2-CIITA.TFPI-cag-EPCR.DAF; GTKO.CD46.Icam-2-CIITA.TFPI-cag-EPCR.DAF; KO.CD46.Icam-2-TFPI.CD47-cag-EPCR.DAF; GTKO.CD46.Icam-2-TFPI.CD47-cag-EPCR.HO-1; GTKO.CD46.pTBM pr-TBM.HO-1-cag-TFPI.CD47; GTKO.CD46.pTBMpr-TBM.CD47-cag-EPCR.TFPI; GTKO.CD46.pTBMpr-TBM.TFPI-c ag-EPCR.CD47; GTKO.CD46.pTBMpr-TBM.EPCR-cag-CD47.HO-1; or GTKO.CD46.cag-EPCR.DAF-tiecag-TFPI.CD47.

[0212] In exemplary embodiments, the transgenic animal is a swine animal lacking any expression of functional α1,3-galactosyltransferase (αGal) and / or growth hormone receptor (GHR) (as a result of gene modification or other reasons), and incorporating and expressing at least three, four, five, six, seven, eight, nine, or ten or more transgenic genes at a single locus. In some embodiments, at least one of the transgenic genes is TBM, HO1, TFPI, A20, EPCR, DAF, CD39, CTLA4-Ig, CIITA-DN, HLA-E, and CD47. In some embodiments, the expression of at least three, four, five, six, seven, eight, nine, or ten or more transgenic genes is controlled by at least two, three, four, five, six, seven, eight, nine, or ten promoters or more. In some implementations, the promoter is dedicated to the expression of one transgene, i.e., one promoter controls the expression of one transgene, while in alternative implementations, one promoter controls the expression of more than one transgene, for example, one promoter controls the expression of two transgenes.

[0213] Advantageously, during cultivation, two or more additional transgenes are co-integrated, co-expressed, and co-segregated. Individual loci can be different. In some embodiments, the individual locus is a natural locus or a modified natural locus. Modified natural loci can be modified by any suitable technique, including but not limited to CRISPR-induced insertions or deletions (indels), the introduction of selective marker genes (e.g., neo), or the introduction of large genomic insertions (e.g., landing pads), designed to facilitate the incorporation of one or more transgenes. In a specific embodiment, the individual locus is a natural GGTA1 locus or a modified GGTA1 locus. The GGTA1 locus is inactivated by incorporation and expression of at least four transgenes, for example, through homologous recombination, gene editing, or the application of recombinase technology. The individual locus can also be, for example, AAVS1, ROSA26, CMAH, GRH, or β4GalNT2. Optionally, the donor animal may have additional gene modifications and / or the expression of one or more additional porcine genes may be modified by mechanisms other than gene modification.

[0214] 2. Insulin-like growth factor-1 (IGF-1)

[0215] One aspect of the present invention provides a transgenic pig comprising gene alterations that result in reduced expression of the insulin-like growth factor 1 (IGF-1) gene. The insulin-like growth factor (IGF) system represents a family comprising two ligands (IGF-1 (accession number DQ784687) and IGF-2 (accession number NM213883)), two transmembrane receptors (IGF-1R (accession number AB003362)) and IGF-2R (accession number AF339885), and at least six high-affinity IGF-binding proteins (IGFBPs 1-6; (IGFBP-3; accession number AF085482)) that specifically bind to IGF-1 and IGF-2. This complex system plays a crucial role in normal development in humans and animals, including embryogenesis, prenatal and postnatal growth, and maintaining tissue homeostasis. In some embodiments, the transgenic pigs contain genetic alterations that result in reduced expression of the insulin-like growth factor gene selected from IGF-1, IGF-2, IGF-1R, IGF-2R, or IGFBP-3. In some embodiments, the transgenic pigs contain genetic alterations that result in reduced expression of the insulin-like growth factor 1 receptor (IGF-1R) gene.

[0216] In some embodiments, the IGF-1 or IGF-1R gene is inactivated via a gene-targeting event. In another embodiment, a swine animal is provided in which both alleles of the IGF-1 or IGF-1R gene are inactivated via a gene-targeting event. In one embodiment, the gene can be targeted via homologous recombination. In other embodiments, the IGF-1 or IGF-1R gene can be disrupted, i.e., a portion of the genetic code can be altered, thereby affecting the transcription and / or translation of that segment of the IGF-1 or IGF-1R gene. For example, gene disruption can occur via substitution, deletion (“knockout”), or insertion (“knock-in”) techniques, including targeted insertion of a selectable marker gene (e.g., neo) that interrupts the coding region of the IGF-1 or IGF-1R gene.

[0217] In some embodiments, the alleles of the IGF-1 or IGF-1R gene are inactivated, so that the resulting IGF-1R no longer responds to IGF-1 stimulation to promote organ growth. In one embodiment, the IGF-1 or IGF-1R gene can be transcribed into RNA but not translated into protein. In another embodiment, the IGF-1 or IGF-1R gene can be transcribed in a truncated form. This truncated IGF-1 or IGF-1R RNA cannot be translated or is translated into nonfunctional IGF-1 or IGF-1R protein. In another embodiment, the IGF-1 or IGF-1R gene can be inactivated in a manner that does not involve gene transcription. In a further embodiment, the IGF-1 gene can be transcribed and then translated into a nonfunctional protein.

[0218] In some embodiments, the expression of active IGF-1 or IGF-1R genes can be reduced by using alternative methods, such as those targeting transcription or translation of the IGF-1 or IGF-1R genes. For example, expression can be reduced by using antisense RNA or siRNA targeting the native IGF-1 or IGF-1R genes or their mRNA. In other embodiments, site-specific recombinases are used to target genomic regions for recombination. Examples of such systems are the CRE-lox system and the Flp-Frt system. In some embodiments, transgenic pigs produce at least about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% less insulin-like growth factor 1 (IGF-1) compared to pigs without IGF-1 gene alterations.

[0219] In some embodiments, the transgenic pig contains gene alterations that result in decreased expression of the insulin-like growth factor 1 (IGF-1) or IGF-1R gene, and further contains one or more additional gene alterations. In some embodiments, one or more additional gene alterations result in (i) decreased expression of one or more genes, (ii) impaired function of one or more genes, and / or (iii) expression of one or more transgenes. In some embodiments, one or more transgenes are independently selected anticoagulants, complement regulators, immunomodulators, and cell-protective transgenes.

[0220] In some embodiments, the anticoagulant is selected from TBM, TFPI, EPCR, and CD39. In some embodiments, the complement regulator is a complement inhibitor selected from CD46, CD55, and CD59. In some embodiments, the immunomodulator is an immunosuppressant selected from porcine CLTA4-IG, CIITA-DN, or CD47. In some embodiments, one or more transgenes are selected from CD47, CD46, DAF / CD55, TBM, EPCR, and HO1. In some embodiments, one or more gene alterations involve decreased expression of α1,3-galactosyltransferase.

[0221] In some embodiments, IGF-1 or IGF-1R gene modification may be performed alone or in combination with other gene modifications. In some embodiments, the gene alteration includes one or more gene alterations described herein, including the 6GE or 10GE pigs disclosed in the examples.

[0222] In exemplary embodiments, the transgenic animal is a swine animal lacking any expression of functional α1,3-galactosyltransferase (αGal) and / or IGF-1 or IGF-1R (as a result of gene modification or other reasons), and incorporating and expressing at least three, four, five, six, seven, eight, nine, or ten or more transgenic genes at a single locus. In some embodiments, at least one of the transgenic genes is TBM, HO1, TFPI, A20, EPCR, DAF, CD39, CTLA4-Ig, CIITA-DN, HLA-E, and CD47.

[0223] In some embodiments, the expression of at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more transgenes is controlled by at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten promoters or more. In some embodiments, the promoter is dedicated to a single transgene, i.e., one promoter controls the expression of one transgene, while in alternative embodiments, one promoter controls the expression of more than one transgene, for example, one promoter controls the expression of two transgenes.

[0224] Advantageously, during cultivation, two or more additional transgenes are co-integrated, co-expressed, and co-segregated. Individual loci may differ. In some embodiments, the individual locus is a natural locus or a modified natural locus. Modified natural loci can be modified by any suitable technique, including but not limited to CRISPR-induced insertions or deletions (indels), the introduction of selective marker genes (e.g., neo), or the introduction of large genomic insertions (e.g., landing pads), designed to facilitate the incorporation of one or more transgenes. In a specific embodiment, the individual locus is a natural GGTA1 locus or a modified GGTA1 locus.

[0225] In some implementations, the GGTA1 locus is inactivated by incorporation and expression of at least four transgenes, for example, through homologous recombination, gene editing, or the application of recombinase technology. A single locus may also be, for example, AAVS1, ROSA26, CMAH, GRH, or β4GalNT2. Optionally, the donor animal may have additional gene modifications and / or the expression of one or more additional porcine genes may be modified by mechanisms other than gene modification.

[0226] 3. α1,3-galactosyltransferase (αGal)

[0227] In one embodiment, this disclosure provides transgenic animals suitable for use as sources of xenotransplant organs, tissues, and cells, wherein the donor animal lacks αGal expression or its expression has been reduced. The transgenic animal lacking αGal expression (i.e., αGal-free) has one or more additional genetic modifications, and in some embodiments, at least four, at least five, or at least six additional genetic modifications. These genetic modifications may be, for example, incorporation or expression of transgenes. In a specific embodiment, the transgenic animal has at least three genetic modifications resulting in (i) a lack of αGal expression; and (ii) incorporation and expression of at least two transgenes at a single locus. In some embodiments, the single locus is a modified αGal.

[0228] Several strategies have been implemented to eliminate or modulate anti-Gal humoral responses induced by xenograft, including epitope removal via α-galactosidase digestion (Stone et al., Transplantation 63:640-645, 1997), specific anti-gal antibody removal (Ye et al., Transplantation 58:330-337, 1994), and epitope capping with other carbohydrate fractions, which failed to eliminate α.GT expression (Tanemura et al., J. Biol. Chem. 27321:16421-16425, 1998 and Koike et al., Xenotransplantation 4:147-153, 1997), and the introduction of complement inhibitory proteins (Dalmasso et al., Clin. Exp. Immunol. 86:31-35, 1991, Dalmasso et al. Transplantation). 52:530-533 (1991)). C. Costa et al. (FASEB J 13, 1762 (1999)) reported that competitive inhibition of α.GT in transgenic pigs only resulted in a partial reduction in the number of epitopes. Similarly, S. Miyagaawa et al. (J. Biol. Chem. 276, 39310 (2000)) reported that blocking the expression of the gal epitope in N-acetylglucosamine transferase III transgenic pigs also resulted in a partial reduction in the number of gal epitopes and failed to significantly prolong the graft survival time of primate recipients.

[0229] Single allele knockout of the αGal locus has been reported in porcine cells and live animals. Denning et al. (Nature Biotechnology 19:559-562, 2001) reported the deletion of the target gene of one allele of the α.GT gene in sheep. Harrison et al. (Transgenics Research 11:143-150, 2002) reported the generation of heterozygous α.GT knockout somatic porcine fetal fibroblasts. In 2002, Lai et al. (Science 295:1089-1092, 2002) and Dai et al. (Nature Biotechnology 20:251-255, 2002) reported the creation of pigs in which one allele of the .α.GT gene was successfully inactivated, and αGal inactivation was achieved by targeting the neomycin phosphotransferase (Neo), a marker gene that interrupts the coding region of the αGal gene (Ramsoondar et al. (Biol of Reproduc 69, 437-445 (2003)) reported the creation of heterozygous .α.GT knockout pigs that also expressed human α-1,2-fucosyltransferase (HT) and both HT and αGal epitopes. The PCT publication number WO, headed by the University of Missouri, is also relevant. 03 / 055302 confirmed the creation of heterozygous αGal knockout miniature pigs for xenotransplantation, in which functional α.GT expression was reduced compared to wild-type pigs.

[0230] The production of α.GT-negative porcine cells based on cDNA from the .α.GT gene is discussed in PCT publication WO 94 / 21799 and US Patent No. 5,821,117 from the Austin Institute; PCT publication WO 95 / 20661 from Bresatec; and PCT publication WO 95 / 28412, US Patent No. 6,153,428, US Patent No. 6,413,769, and US Publication No. 2003 / 001477 from BioTransplant, Inc. and The General Hospital Corporation. A major breakthrough in xenotransplantation was the production of the first live pigs lacking any functional αGal expression (Phelps et al. Science 299:411-414 (2003); see also PCT publication WO 04 / 028243 from Revivicor, Inc. and PCT publication WO 04 / 016742 from Immerge Biotherapeutics, Inc.).

[0231] In one embodiment, the animal (and organs, tissues, and cells derived therefrom) is provided by a transgenic animal (e.g., a transgenic pig) containing at least four transgenes, wherein the four transgenes are incorporated and expressed at a single locus under the control of at least two promoters, and wherein the pig lacks expression of α1,3-galactosyltransferase. In an exemplary embodiment, the transgenes are incorporated and expressed at a modified αGal locus. In some embodiments, the at least two promoters are exogenous, native, or a combination of exogenous and native.

[0232] In one embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that (i) lacks any expression of functional αGal and (ii) incorporates and expresses at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more transgenes at a single locus. In an exemplary embodiment, the transgene is incorporated and expressed at a modified αGal locus.

[0233] In some embodiments, the animal may include one or more additional genetic modifications. These genetic modifications may result in the incorporation and expression of one or more additional transgenes at the same or different loci. In one embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks any expression of functional αGal and is incorporated and expresses at least one, at least two, at least three, at least four, at least five, or at least six additional transgenes. In another embodiment, an animal, organ, tissue, or cell is provided that has a reduced level of functional αGal expression and is incorporated and expresses at least one, at least two, at least three, at least four, at least five, or at least six additional transgenes. The expression of functional αGal may be reduced, for example, by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%.

[0234] The lack or reduction of functional αGal expression levels can be achieved through any suitable means. In one embodiment, an animal (e.g., an ungulate, a pig) is provided in which one allele of the αGal gene is inactivated via a gene-targeting event. In another embodiment, a pig is provided in which both alleles of the αGal gene are inactivated via a gene-targeting event. In one embodiment, the gene can be targeted via homologous recombination. In other embodiments, the gene can be disrupted, i.e., a portion of the genetic code can be altered, thereby affecting the transcription and / or translation of that gene segment. For example, gene disruption can occur through substitution, deletion (“knockout”), or insertion (“knock-in”) techniques, including targeted insertion of a selectable marker gene (e.g., neo) that interrupts the coding region of the αGal gene. A gene for another desired protein or a regulatory sequence that modulates the transcription of an existing sequence can be inserted.

[0235] In some embodiments, the alleles of the αGal gene are inactivated, so that the resulting αGal enzyme can no longer produce Gal on the cell surface. In one embodiment, the αGal gene can be transcribed into RNA but cannot be translated into protein. In another embodiment, the αGal gene can be transcribed in a truncated form. This truncated RNA cannot be translated or can be translated into a nonfunctional protein. In yet another embodiment, the αGal gene can be inactivated in a manner that does not involve gene transcription. In a further embodiment, the αGal gene can be transcribed and then translated into a nonfunctional protein.

[0236] In some implementations, the expression of the active αGal gene can be reduced by using alternative methods, such as those targeting gene transcription or translation. For example, antisense RNA or siRNA targeting the natural αGT gene or its mRNA can be used to reduce expression. In other implementations, site-specific recombinases are used to target genomic regions for recombination. Examples of such systems are the CRE-lox system and the Flp-Frt system.

[0237] Pigs with two inactive αGal gene alleles are not naturally occurring. Previous studies have identified a point mutation that prevents the second allele from producing the functional αGal enzyme when attempting to knock out the second allele of the αGal gene via a gene-targeting event.

[0238] Therefore, in another aspect of this disclosure, αGal can be inactivated by at least one point mutation. In one embodiment, one allele of the αGal gene can be inactivated by at least one point mutation. In another embodiment, both alleles of the αGal gene can be inactivated by at least one point mutation. In one embodiment, the point mutation can occur via a gene-targeting event. In another embodiment, the point mutation can occur naturally. In a further embodiment, the αGal gene mutation can be induced by a mutagen. Optionally, the animal contains one or more additional gene modifications. In some embodiments, the additional modifications are growth hormone receptor knockout, IGF-1 knockout, or IGF-1R knockout. In some embodiments, the transgenic animal exhibits 30%, 40%, 50%, 75%, or 90% or more reduced GHR expression compared to an animal without GHR gene alteration. In some embodiments, the transgenic animal can produce 30%, 40%, 50%, 75%, or 90% or less IGF-1 compared to an animal without GHR gene alteration.

[0239] 4.β4GaINT2

[0240] In one embodiment, this disclosure provides transgenic animals suitable for use as sources of organs, tissues, and cells for xenotransplantation, wherein the donor animal lacks or has reduced expression of β1,4N-acetylgalactosyltransferase 2 (β4GALNT2). The transgenic animal lacking β4GALNT2 expression (i.e., β4GALNT2-free) has one or more additional genetic modifications. These modifications may be, for example, incorporation or expression of transgenes. In a specific embodiment, the transgenic animal lacking or with reduced expression of β1,4N-acetylgalactosyltransferase 2 (β4GALNT2) is further characterized by (i) lack of αGal expression; and (ii) incorporation and expression of at least four transgenes at a single locus, these transgenes being controlled by at least two promoters.

[0241] The glycans produced by β4Gal-NT2 are xenoantigens for many people. Estrada JL et al, Xenotransplantation 2015:22:194–202. In humans and mice, β4GALNT2 catalyzes the addition of N-acetylgalactosamine to sialic acid-modified lactosamine, producing GalNAc b1-4(Neu5Ac a2-3)Gal b1-4GlcNAc b1-3Gal, the Sda blood group antigen. This gene functions in transplantable organs (kidney, heart, liver, lung, and pancreas) and endothelial cells in pigs. Approximately 5% of humans have inactive β4GalNT2, thus producing antibodies against the SDa and CAD carbohydrates produced by this gene. See Byrne GW et al. Transplantation 2011;91:287–292; Byrne GW, et al., Xenotransplantation 2014;21:543–554.

[0242] Any suitable method can be used to generate pigs whose genome lacks or has reduced expression of endogenous β4GALNT2. Disruptions can be located at numerous sites in the endogenous porcine β4GALNT2 nucleic acid sequence. Examples of disruptions include, but are not limited to, deletions in the natural gene sequence and insertions of heterologous nucleic acid sequences into the natural gene sequence. Examples of insertions can include, but are not limited to, artificial splice acceptors coupled to stop codons or splice donors coupled to fusion couplers (e.g., GFP). Knockout constructs can contain sequences homologous to or adjacent to the endogenous β4GALNT2 nucleic acid sequence. In some cases, knockout constructs can contain nucleic acid sequences encoding selection markers (e.g., antibiotic resistance, fluorescent reporter genes (e.g., GFP or YFP), or enzymes (e.g., β-galactosidase)) operably linked to regulatory sequences (e.g., promoters). Knockout constructs can include other nucleic acid sequences, such as recombinant sequences (e.g., loxP sequences, see Sendai, et al., Transplantation, 81(5):760-766(2006)), splice acceptor sequences, splice donor sequences, transcription initiation sequences, and transcription termination sequences. Disruption of the endogenous β4GalNT2 nucleic acid sequence can lead to decreased gene expression or nonfunctional truncation or fusion of the encoded polypeptide.

[0243] In one embodiment, this disclosure provides a transgenic animal (e.g., a swine animal) whose β4GALNT2 expression is reduced or absent. Optionally, the animal contains one or more additional gene modifications.

[0244] In one exemplary embodiment, this disclosure provides a transgenic animal (e.g., a swine) incorporating and expressing at least four transgenes controlled by at least two promoters, wherein the animal lacks β4GALNT2 or has reduced expression of β4GALNT2. Optionally, the animal contains one or more additional genetic modifications. In some embodiments, the additional modifications are growth hormone receptor knockout, IGF-1 knockout, or IGF-1R knockout. In some embodiments, the transgenic animal exhibits 30%, 40%, 50%, 75%, or 90% or more of reduced GHR expression compared to an animal without GHR gene alterations. In some embodiments, the transgenic animal may produce 30%, 40%, 50%, 75%, or 90% or less of IGF-1 compared to an animal without GHR gene alterations.

[0245] In one embodiment, this disclosure provides a transgenic animal (e.g., a swine animal) whose expression of Sda or SDa-like glycans produced by porcine β4GALNT2 is reduced or absent. Optionally, the animal contains one or more additional genetic modifications. In some embodiments, the additional modifications are growth hormone receptor knockout, IGF-1 knockout, or IGF-1R knockout. In some embodiments, the transgenic animal exhibits 30%, 40%, 50%, 75%, or 90% or more reduced GHR expression compared to animals without GHR gene alterations. In some embodiments, the transgenic animal may produce 30%, 40%, 50%, 75%, or 90% or less IGF-1 compared to animals without GHR gene alterations.

[0246] In exemplary embodiments, this disclosure provides a transgenic animal (e.g., a swine) incorporating and expressing at least four transgenes controlled by at least two promoters, wherein the animal lacks or reduces the expression of Sda or SDa-like glycans produced by porcine β4GalNT2. Optionally, the animal contains one or more additional genetic modifications.

[0247] 5. CMAH

[0248] In one embodiment, this disclosure provides transgenic animals suitable for use as sources of organs, tissues, and cells for xenotransplantation, wherein the donor animal lacks or has reduced expression of cytidine monophosphate-N-acetylneuraminidase (CMAH). The transgenic animal lacking CMAH expression (CMAH blank) has one or more additional genetic modifications. These genetic modifications may be, for example, incorporation or expression of transgenes. In a specific embodiment, the transgenic animal has at least four additional genetic modifications resulting in (i) a lack of αGal expression; and (ii) incorporation and expression of at least four transgenes at a single locus.

[0249] Porcine cells express cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), which is absent in human cells. CMAH converts sialic acid N-acetylneuraminic acid (Neu5Ac) to N-hydroxyacetylneuraminic acid (Neu5Gc). Therefore, when porcine tissue is transplanted into humans, this epitope immediately induces an antibody-mediated rejection response in human patients after implantation. See Varki A. Am J Phys Anthropol 2001; (Suppl. 33): 54–69; Zhu A. Xenotransplantation, 2002; 9: 376–381; Miwa Y. Xenotransplantation 2004; 11: 247–253; Tahara HJ Immunol 2010; 184: 3269–3275.

[0250] Any suitable method may be used to generate pigs whose genome lacks endogenous CMAH expression or has reduced endogenous CMAH expression. Disruptions may be located at numerous sites in the endogenous porcine CMAH nucleic acid sequence. Examples of disruptions include, but are not limited to, deletions in the natural gene sequence and insertions of heterologous nucleic acid sequences into the natural gene sequence. Examples of insertions may include, but are not limited to, artificial splice acceptors coupled to stop codons or splice donors coupled to fusion couplers (e.g., GFP). Knockout constructs may contain sequences homologous to or adjacent to the endogenous CMAH nucleic acid sequence. In some cases, knockout constructs may contain nucleic acid sequences encoding selection markers (e.g., antibiotic resistance, fluorescent reporter genes (e.g., GFP or YFP), or enzymes (e.g., β-galactosidase)) operably linked to regulatory sequences (e.g., promoters). Knockout constructs can include other nucleic acid sequences, such as recombinant sequences (e.g., loxP sequences, see Sendai, et al., Transplantation, 81(5):760-766(2006)), splice acceptor sequences, splice donor sequences, transcription initiation sequences, and transcription termination sequences. Disruption of endogenous CMAH nucleic acid sequences can lead to decreased gene expression or nonfunctional truncation or fusion of encoded peptides.

[0251] In one embodiment, this disclosure provides a transgenic animal (e.g., a swine animal) whose CMAH glycosyltransferase expression is reduced or absent. Optionally, the animal contains one or more additional genetic modifications. In some embodiments, the additional modifications are growth hormone receptor knockout, IGF-1 knockout, or IGF-1R knockout. In some embodiments, the transgenic animal exhibits 30%, 40%, 50%, 75%, or 90% or more reduced GHR expression compared to animals without GHR gene alterations. In some embodiments, the transgenic animal may produce 30%, 40%, 50%, 75%, or 90% or less IGF-1 compared to animals without GHR gene alterations.

[0252] In exemplary embodiments, this disclosure provides a transgenic animal (e.g., a swine animal) incorporating and expressing at least four transgenes controlled by at least two promoters, wherein the animal lacks or has reduced expression of CMAH and / or growth hormone receptors. Optionally, the animal contains one or more additional gene modifications.

[0253] 6.vWF

[0254] The von Willebrand factor (vWF) gene is a large and complex gene with multiple domains that encode a polysaccharide protein. (Ulrichts, H, Udvardy M, Lenting PJ, Pareyn I et al. Shielding of the A1 domain by the D'D3 domains of von Willebrand Factor Modulates Its interaction with Platelet Glycoprotein 1b-IX-V. (2006) JBC 281, 4699-4707.; Zhou YF, Eng ET, Zhu J, Lu C et all. Sequence and structure relations within von Willebrand factor. (2012) Blood 120, 449-458). The primary function of the polyglycoprotein von Willebrand factor (vWF) is to facilitate platelet adhesion to connective tissue and the subendothelial layer, as well as platelet aggregation, through its binding to platelet glycoprotein Ib (GPIb). However, this phenomenon is less desirable during xenotransplantation when the recipient's platelet aggregation has a detrimental effect on the survival of the donor organ. For example, transplanting pig lungs (and other organs) into humans or non-human primates results in spontaneous aggregation and fixation of human platelets. This can be avoided by "humanizing" the porcine vWF gene to eliminate this spontaneous binding of porcine vWF to human platelets.

[0255] Typically, humanization or modification of the porcine vWF gene requires deleting gene sequences associated with spontaneous human platelet aggregation and replacing them with human gene counterparts that do not produce spontaneous aggregation. This may involve deleting all or part of the porcine vWF gene and replacing it with all or part of the human vWF gene.

[0256] Modifications to porcine vWF designed to eliminate spontaneous platelet aggregation may include regions within the D3 (partial), A1, A2, and A3 (partial) domains known to be associated with folding and fixation of the GP1b binding site (D3 domain) in hvWF, as well as regions associated with the GP1b receptor (A1 domain) and the ADAMTS13 cleavage site (A2 domain). Exons 22-28 contain these regions. Under normal stress, human platelets spontaneously aggregate in the presence of porcine blood. To avoid this potential threat to successful xenotransplantation, and since human vWF does not induce spontaneous platelet aggregation under normal shear stress conditions in blood, genomic homologs in the porcine vWF gene (and thus the resulting chimeric human / porcine protein) can be replaced with regions in the human vWF gene associated with vWF protein folding, as well as regions associated with GPib binding, collagen binding (one of the two regions), and ADAMTS13 cleavage. In this way, a single cDNA or genomic fragment from the human vWF gene can provide alternative folds, thereby hiding or masking the GP1b binding site on vWF and the humanized receptor site within the A domain. This can be achieved through homologous recombination or gene targeting, including enhancing this mechanism using gene editing methods (e.g., CRISPR-assisted homologous recombination can be used to integrate a human vWF fragment into a porcine vWF locus). This human fragment replaces the region associated with the aforementioned spontaneous platelet aggregation and can be in the form of a cDNA or genomic fragment from the human vWF gene.

[0257] In an exemplary implementation, the insertion of the relevant human vWF gene sequence can be accomplished using any method currently used for genome editing, such as, but not limited to, CRISPR / CAS9 and TALEN nucleases. Modification of the porcine vWF can involve replacing only the relevant region of the porcine vWF gene, or replacing the entire pvWF gene with hvWF.

[0258] In one implementation, a region of the porcine vWF gene can be replaced with a human counterpart (E22-E28 region). Alternatively, the transgenic animal can completely knock out the vWF gene and completely replace the gene synthesis sequence of the human vWVF gene by using a site-specific recombination system (i.e., the CRE-LOX recombination system) and / or by changing specific nucleic acid base pairs to replace nucleotides in the porcine vWF genome sequence with a human counterpart.

[0259] In one embodiment, this disclosure pertains to a transgenic animal (e.g., a pig transgenic animal) lacking αGal expression and genetically modified with respect to the pig vWF gene. The modification may be, for example, knockout of the pig vWF gene and replacement with a humanized vWF gene or a chimeric vWF gene. The transgenic animal may contain one or more additional genetic modifications. In one embodiment, the transgenic animal further includes CD46 incorporation and expression. In some embodiments, additional modifications are growth hormone receptor knockout, IGF-1 knockout, or IGF-1R knockout. In some embodiments, the transgenic animal exhibits 30%, 40%, 50%, 75%, or 90% or more reduced GHR expression compared to animals without GHR gene alterations. In some embodiments, the transgenic animal may produce 30%, 40%, 50%, 75%, or 90% or less IGF-1 compared to animals without GHR gene alterations.

[0260] Transgenic animals can also be bred into second transgenic animals containing one or more gene modifications. In some embodiments, transgenic animals lacking expression of αGal and / or growth hormone receptors and genetically modified with the porcine vWF gene (e.g., pig transgenic animals) can be bred into second transgenic animals containing at least four transgenes at a single locus, or containing at least four transgenes at a single locus and at least two transgenes at a second locus, thereby providing animals with multiple gene modifications.

[0261] In one embodiment, this disclosure pertains to a transgenic animal (e.g., a pig transgenic animal) lacking expression of αGal and / or growth hormone receptors, and to a genetically modified pig vWF gene (e.g., chimeric human-pig vWF), with at least four genetic modifications at a single locus under the control of at least two promoters. In exemplary embodiments, the locus is a natural locus or a modified natural locus. In some embodiments, the locus may be, for example, AAVS1, ROSA26, CMAH, β4GalNT2, and GGTA1. In some embodiments, at least four transgenes may be incorporated via homologous recombination or gene editing tools.

[0262] V. Genetically Modified

[0263] The transgenic gene introduced into the genome of the transgenic animal disclosed herein can be any suitable transgenic gene.

[0264] 1. Immunomodulators

[0265] In one embodiment, the transgene is an immunomodulator. In an exemplary embodiment, the donor animal is genetically modified such that: (i) αGal and / or growth hormone receptor expression (e.g., lack of or reduced expression) and (ii) at least four transgenes are incorporated and expressed at a single locus, wherein at least two of the transgenes are immunomodulators. The immunomodulator can be any suitable immunomodulator. In an exemplary embodiment, the immunomodulator is a complement regulator (e.g., a complement inhibitor) or an immunosuppressant.

[0266] 2. Complement regulators

[0267] In one embodiment, this disclosure provides transgenic animals (e.g., swine animals) suitable for use as sources of organs, tissues, and cells for xenotransplantation, wherein the donor animal is genetically modified to incorporate and express at least one complement regulator, such as a complement inhibitor. In an exemplary embodiment, the donor animal is genetically modified such that (i) the expression (e.g., αGal) and / or GHR is absent or reduced, and (ii) at least four transgenes are incorporated and expressed at a single locus, wherein at least one of the transgenes is a complement regulator or more specifically a complement inhibitor.

[0268] Complement is a collective term for a series of blood proteins and a major effector mechanism of the immune system. Complement activation and its deposition on target structures can lead to direct complement-mediated cell lysis or indirectly to cell or tissue damage by producing potent inflammatory modulators and recruiting and activating immune effector cells. Complement activation products mediating tissue damage are produced at different points along the complement pathway. Inappropriate complement activation in host tissues plays a crucial role in the pathology of many autoimmune and inflammatory diseases and also contributes to numerous disease states associated with biocompatibility, such as postcardiopulmonary inflammation and transplant rejection. Complement inhibitory proteins expressed on cell surfaces prevent complement deposition on host cell membranes.

[0269] The complement system comprises a collection of approximately 30 proteins and is one of the main effector mechanisms of the immune system. The complement cascade is activated primarily through either the classical pathway (usually antibody-dependent) or the alternative pathway (usually antibody-independent). Regardless of the pathway, activation results in the production of C3 convertase, the core enzyme complex of the cascade. C3 convertase breaks down serum C3 into C3a and C3b, the latter covalently binding to the activation site, leading to the further production of C3 convertase (amplification loop). The activation product C3b (and C4b produced only through the classical pathway) and its breakdown products are important opsonins involved in promoting cell-mediated target cell lysis (via phagocytes and NK cells) and the transport and dissolution of immune complexes. C3 / C4 activation products and their receptors on various cells of the immune system are also important in regulating cellular immune responses. C3 convertase is involved in the formation of C5 convertase, a complex that breaks down C5 to produce C5a and C5b. C5a possesses potent pro-inflammatory and chemotactic properties, recruiting and activating immune effector cells. The formation of C5b initiates the terminal complement pathway, leading to the sequential assembly of complement proteins C6, C7, C8, and (C9)n to form the membrane attack complex (MAC or C5b-9). MAC formation in the target cell membrane can result in direct cell lysis, but it can also lead to cell activation and the expression / release of various inflammatory modulators.

[0270] There are two main classes of membrane complement inhibitors: complement activation pathway inhibitors (inhibiting C3 convertase formation) and terminal complement pathway inhibitors (inhibiting MAC formation). Membrane complement activation inhibitors include complement receptor 1 (CR1), decay accelerator factor (DAF or CD55), and membrane cofactor protein (MCP or CD46). Their protein structures consist of varying numbers of repeating units of approximately 60-70 amino acids, called short common repeats (SCRs), a common characteristic of C3 / C4 binding proteins. Rodent homologs of human complement activation inhibitors have been identified. The rodent protein Cr1 is a widely distributed complement activation inhibitor, functioning similarly to DAF and MCP. Rodents also express DAF and MCP, although Cr1 appears functionally to be the most important regulator of complement activation in rodents. Although no homologs of Cr1 have been found in humans, research on Cr1 and its application in animal models has clinical significance.

[0271] Control of the terminal complement pathway and MAC formation in the host cell membrane is primarily mediated by the activity of CD59, a widely distributed 20kD glycoprotein that is anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). CD59 binds to C8 and C9 in MAC assembly, preventing membrane insertion.

[0272] Host cells are protected from their own complement by membrane-bound complement regulatory proteins (such as DAF, MCP, and CD59). When an organ is transplanted into another species, the recipient's natural antibodies bind to the endothelial cells of the donor organ and activate complement, triggering a rapid rejection response. It has been previously proposed that, unlike human cells, pig cells are highly sensitive to human complement, and this is attributed to the ineffectiveness of complement regulatory proteins on the surface of pig cells against human complement. Several strategies exist to prevent or delay rejection, including removing IgM natural antibodies and / or using systemic decomplementation or complement inhibition with sCR1, heparin, or C1 inhibitors.

[0273] Another approach to addressing the rejection problem is to express human membrane-bound complement regulatory molecules in transgenic pigs. Transgenic pigs expressing the decay-accelerating factor DAF (CD55), the membrane cofactor protein MCP (CD46), and the reactive membrane-cleaving inhibitor MIRL (CD59) have already been produced (see Klymium et al. Mol Reprod Dev (2010) 77:209-221). These human inhibitors have been shown to be highly expressed in porcine vascular endothelium. While ex vivo perfusion of human blood into hearts from control animals causes complement-mediated organ damage within minutes, hearts obtained from transgenic animals are complement-tolerant and can survive for hours.

[0274] As mentioned above, the basic principle of expressing human complement regulatory proteins in porcine organs to "humanize" them is based on the assumption that endogenous porcine regulatory proteins are inefficient at inhibiting human complement and therefore contribute little to organ survival in the case of xenotransplantation. (Cantarovich et al., Xenotransplantation 9:25, 2002; Kirchhof et al., Xenotransplantation 11(5), 396, 2004; Tjernberg et al., Transplantation. 2008 Apr. 27; 85(8):1193-9). Furthermore, soluble complement inhibitors can prevent complement-mediated islet lysis in vitro (Bennet et al., Transplantation 69(5):711, 2000).

[0275] U.S. Patent No. 7,462,466 to Morgan et al. describes the isolation and characterization of several porcine analogues of human complement regulatory protein (CRP). The study showed that porcine organs expressing human complement regulatory protein molecules are resistant to complement damage, not because they express human CRP molecules, but because they express a significantly increased number of functional CRP molecules. Morgan et al. found that increased expression of porcine CRP is as effective as donor organs expressing human complement regulatory protein in protecting donor organs from complement damage that leads to hyperacute rejection.

[0276] CD46 has been characterized as a protein with regulatory properties that protect host cells from complement-mediated attack activated via both classical and alternative pathways (Barilla-LaBarca, ML et al., J. Immunol. 168, 6298-6304 (2002)). Human CD46 (hCD46) can provide protection against complement cleavage during inflammation and humoral rejection mediated by low levels of natural or induced anti-Gal or anti-non-Gal antibodies. Therefore, more islets can be transplanted and subsequently better protected from rejection, thus reducing the need for immunosuppression.

[0277] In one embodiment of this disclosure, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks functional αGal and / or GHR expression (or has reduced αGal and / or GHR expression) and is genetically modified to incorporate and express at least one, at least two, at least three, or at least four or more complement inhibitors. The expression of the complement inhibitors may be pervasive or controlled by tissue-specific promoters.

[0278] In an exemplary embodiment, the complement inhibitor is a membrane complement inhibitor. A membrane complement inhibitor can be an inhibitor of the complement activation pathway (inhibiting C3 convertase formation) or an inhibitor of the terminal complement pathway (inhibiting MAC formation). Membrane inhibitors of complement activation include complement receptor 1 (CR1), decay accelerator (DAF or CD55), membrane cofactor proteins (MCP or CD46), etc. Membrane inhibitors of the terminal complement pathway may include CD59, etc.

[0279] In exemplary embodiments, this disclosure provides transgenic animals (e.g., ungulates, swine) comprising genetic modifications resulting in (i) the absence of αGal and / or growth hormone receptor (GHR) expression and (ii) the incorporation and expression of at least four transgenes at a single locus, these transgenes being controlled by at least two promoters, wherein at least one of the at least two transgenes is a complement regulator, and more specifically a complement inhibitor, and even more specifically a membrane complement inhibitor. The single locus may be selected from natural loci, modified natural loci, or transgenic loci. In exemplary embodiments, at least four transgenes are provided as MCVs, and integration may be random integration or facilitated by gene-targeting tools. Optionally, the transgenic animal includes one or more additional genetic modifications, including but not limited to modifications of the natural porcine vWF, B4GalNT2, CMAH, or Forsmann genes.

[0280] In exemplary embodiments, an animal (and organs, tissues, and cells derived therefrom) is provided containing at least four transgenes, wherein the four transgenes are incorporated and expressed at a single locus, these transgenes are controlled by at least two promoters, and wherein the pig lacks expression of α1,3-galactosyltransferase and / or growth hormone receptor, wherein the at least four transgenes include at least one complement regulator, and more specifically at least one complement inhibitor. Additional transgenes may be, for example, immunosuppressants, cell-protective genes, or combinations thereof. The single locus may be selected from natural loci, modified natural loci, or transgenic loci. In exemplary embodiments, the at least four transgenes are provided in the form of MCVs, and integration is randomized or facilitated by gene-targeting tools. Optionally, the transgenic animal includes one or more additional gene modifications.

[0281] In an exemplary embodiment, an animal (and organs, tissues and cells derived therefrom) is provided that lacks (or has reduced) expression of functional αGal and / or growth hormone receptor (GHR) and is genetically modified to incorporate and express at least four additional transgenes, wherein at least two of the at least four additional transgenes are complement inhibitors, and more specifically at least two membrane complement inhibitors.

[0282] In an exemplary embodiment, an animal (and organs, tissues and cells derived therefrom) is provided that lacks (or has reduced) expression of functional αGal and / or growth hormone receptor (GHR) and is genetically modified to (i) incorporate and express at least two complement inhibitors, more specifically at least two membrane complement inhibitors, and (ii) incorporate and express at least two additional transgenes selected from anticoagulants, immunosuppressants, cell-protective genes or combinations thereof.

[0283] In one embodiment, a transgenic animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional αGal and / or growth hormone receptor (GHR) and is genetically modified to (i) incorporate and express CD46 and CD55, and (i) incorporate and express at least two additional transgenes. In one embodiment, the additional transgenes are selected from anticoagulants, immunosuppressants, cell-protective genes, or combinations thereof.

[0284] In a specific implementation, a transgenic animal (and organs, tissues and cells derived therefrom) is provided that lacks (or has reduced) expression of functional αGal and / or growth hormone receptor (GHR) and is genetically modified to incorporate and express at least four transgenes, which are controlled by at least two promoters, wherein at least one of the transgenes is CD46 and its expression is controlled by an endogenous promoter.

[0285] In another embodiment, a transgenic animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional αGal and / or growth hormone receptor (GHR) and is genetically modified to (i) incorporate and express CD46 and CD55, and (i) incorporate and express at least three additional transgenes. In one embodiment, the additional transgenes are selected from anticoagulants, immunosuppressants, cell-protective genes, or combinations thereof. In an exemplary embodiment, the at least three additional transgenes comprise at least two anticoagulants. In an exemplary embodiment, the at least three additional transgenes comprise at least two anticoagulants and immunosuppressants.

[0286] In another embodiment, a transgenic animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional αGal and / or growth hormone receptor (GHR) and is genetically modified to (i) incorporate and express CD46 and CD55, and (i) incorporate and express at least four additional transgenes. In one embodiment, the additional transgenes are selected from anticoagulants, immunosuppressants, cell-protective genes, or combinations thereof. In an exemplary embodiment, the at least four additional transgenes comprise at least two anticoagulants. In an exemplary embodiment, the at least four additional transgenes comprise at least two anticoagulants and immunosuppressants. In an exemplary embodiment, the at least four additional transgenes comprise at least three anticoagulants.

[0287] In another embodiment, a transgenic animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional αGal and / or growth hormone receptor (GHR) and is genetically modified to (i) incorporate and express CD46 and CD55, and (i) incorporate and express at least five additional transgenes. In one embodiment, the additional transgenes are selected from anticoagulants, immunosuppressants, cell-protective genes, or combinations thereof. In an exemplary embodiment, the at least five additional transgenes comprise at least two anticoagulants and at least one immunosuppressant. In an exemplary embodiment, the at least five additional transgenes comprise at least three anticoagulants and at least two immunosuppressants. In an exemplary embodiment, the at least five additional transgenes comprise at least two anticoagulants and at least two immunosuppressants. In one embodiment, the animal may have been modified to express a complement regulatory peptide, a biologically active fragment thereof, or a derivative thereof. In one embodiment, the complement regulatory peptide is a full-length complement regulator. In a further embodiment, the complement regulatory peptide may contain less than the full-length complement regulatory protein.

[0288] Any human or porcine complement regulatory sequence or its biologically active portion or fragment known to those skilled in the art can be used in the compositions and methods of this disclosure. In another embodiment, any common complement regulatory peptide can be used in this disclosure. In yet another embodiment, the nucleic acid and / or peptide sequence has at least 80%, 85%, 90%, or 95% homology to the complement regulatory peptide and nucleotide sequence described herein. In a further embodiment, any fragment or homologous sequence exhibiting similar activity to a complement regulator can be used. Optionally, animals expressing at least one complement regulator (e.g., a complement inhibitor) in at least four transgenes and lacking α1,3gal and / or growth hormone receptor (GHR) expression have at least one additional genetic modification.

[0289] 3. Immunosuppressants

[0290] In one embodiment, this disclosure provides transgenic animals suitable for use as sources of organs, tissues, and cells for xenotransplantation, wherein the donor animal is genetically modified to incorporate and express at least one immunosuppressant. The transgenic animals typically have one or more additional genetic modifications, more specifically, five or more additional genetic modifications, and even more specifically, six or more additional genetic modifications.

[0291] Immunosuppressant transgenic drugs can downregulate the immune response. For any type of transplant, the balance between efficacy and toxicity is a key factor in its clinical acceptance. Regarding islet transplantation, a further concern is that many current immunosuppressants, particularly glucocorticoids or calcineurin inhibitors (such as tacrolimus), can damage β-cells or induce peripheral insulin resistance (Zeng et al. Surgery (1993) 113:98-102). Steroid-free immunosuppressive regimens (“Edmonton regimens”), comprising sirolimus, low-dose tacrolimus, and an anti-IL-2 receptor monoclonal antibody (mAb), have been used in trials of islet transplantation alone in patients with type 1 diabetes (Shapiro, AMJ et al., (2000), N.Eng.J.Med., 343:230-238). Recent successes with the “Edmonton regimen” have reignited enthusiasm for islet transplantation in diabetes. However, concerns about tacrolimus toxicity may limit the application of this therapy in humans.

[0292] Biologics that block key T-cell co-stimulatory signals (particularly the CD28 pathway) are potential alternatives for protecting the pancreas. Examples of agents that block the CD28 pathway include, but are not limited to, soluble CTLA4, including mutated CTLA4 molecules.

[0293] T cell activation is involved in the pathogenesis of transplant rejection. T cell activation requires at least two sets of signaling events. The first set is initiated by the specific recognition of antigenic peptides on antigen-presenting cells (APC5) that bind to the major histocompatibility complex (MHC) molecule. The second set of signals is antigen-nonspecific and is delivered by the interaction of T cell co-stimulatory receptors with their ligands on APCs. In the absence of co-stimulation, T cell activation is impaired or terminated, which may result in a clonal incompetent state of antigen-specific unresponsiveness or loss due to apoptotic death. Therefore, blocking T cell co-stimulation provides a way to suppress unwanted immune responses in an antigen-specific manner while maintaining normal immune function (Dumont, FJ2004 Therapy 1, 289-304).

[0294] Of the several T cell co-stimulatory pathways identified to date, the CD28 pathway stands out. CD28 is a cell surface molecule expressed on T cells, and its anti-receptors B7.1 (CD80) and B7.2 (CD86) molecules are present on dendritic cells, macrophages, and B cells, and have been characterized and identified as attractive targets for disrupting T cell co-stimulatory signaling. A second T cell surface molecule homologous to CD28 is called cytotoxic T lymphocyte-associated protein (CTLA4). CTLA4 is a cell surface signaling molecule, but its role is opposite to that of CD28; CTLA4 negatively regulates T cell function. CTLA4 has a 20-fold higher affinity for B7 ligands than CD28. The human CTLA4 gene was cloned in 1988 and located on chromosomes in 1990 (Dariavach et al., Eur. J. Immunol. 18: 1901-1905 (1988); Lafage-Pochitaloff et al., Immunogenetics 31: 198-201 (1990); US Pat. No. 5, 977, 318).

[0295] The CD28 / B7 pathway has become an attractive target for disrupting T cell co-stimulatory signaling. CD28 / B7 inhibitors are designed to utilize the endogenous negative regulator of this system, CTLA4. CTLA4-immunoglobulin (CTLA4-Ig) fusion proteins have been extensively studied as a means of inhibiting T cell co-stimulation. Any immunosuppressive therapy must strike a difficult balance; sufficient inhibition must be provided to overcome disease or rejection, but excessive immunosuppression will suppress the entire immune system. The immunosuppressive activity of CTLA4-Ig has been demonstrated in preclinical studies in animal models of organ transplantation and autoimmune diseases. Soluble CTLA4 has recently been tested in patients with renal failure, psoriasis, and rheumatoid arthritis and has been formulated into a drug (Abatacept, soluble CTLA4-Ig) developed by Bristol-Myers Squibb, which has been approved for the treatment of rheumatoid arthritis. This drug is the first of its kind among novel selective T cell co-stimulatory modulators. Bristol-Myers Squibb is also conducting a Phase II clinical trial of Belatacept (LEA29Y) for allogeneic kidney transplantation. LEA29Y is a mutant form of CTLA4 that, compared to wild-type CTLA4, exhibits a higher affinity for the B7 receptor after fusion with immunoglobulin. Repligen Corporation is also conducting a clinical trial of its CTLA4-Ig for idiopathic thrombocytopenic purpura. US Patent No. 5,730,403, entitled "Method for Protecting Allogeneic Islet Transplants Using Soluble CTLA4 Mutant Molecules," describes the use of soluble CTLA4-Ig and CTLA4 mutant molecules to protect allogeneic islet grafts.

[0296] Although CTLA-4 from one organism can bind to B7 from another organism, allogeneic B7 has been found to have the highest affinity. Therefore, while soluble CTLA-4 from the donor organism can thus bind to both recipient B7 (on normal cells) and donor B7 (on xenograft cells), it preferentially binds to B7 on the xenograft. Thus, porcine CTLA4 is typical in embodiments of the invention that include porcine animals or cells for xenografting. Imperial College PCT Publication WO 99 / 5 7266 describes the porcine CTLA4 sequence and the administration of soluble CTLA4-Ig for xenograft therapy. Vaughn A. et al., J Immunol (2000) 3175-3181, describes the binding and function of soluble porcine CTLA4-Ig. Porcine CTLA4-Ig binds to porcine (not human) B7, blocks CD28 on receptor T cells, and renders these local T cells unresponsive without causing overall T cell immunosuppression (see Mirenda et al., Diabetes 54:1048-1055, 2005).

[0297] Many studies on CTLA4-Ig as an immunosuppressant have focused on administering soluble forms of CTLA4-Ig to patients. Transgenic mice engineered to express CTLA4-Ig have been established and numerous experiments conducted. Ronchese et al. examined overall immune system function in mice after CTLA4 expression (Ronchese et al. J Exp Med (1994) 179:809; Lane et al. J Exp Med. (1994) March 1; 179(3):819). Sutherland et al. (Transplantation. 2000 69(9):1806-12) described the protective effect of CTLA4-Ig secreted by transgenic fetal pancreas allogeneic grafts in mice to examine the effect of transgenic CTLA4-Ig on allogeneic islet transplantation. Lui et al. (J Immunol Methods 2003 277:171-183) reported the generation of transgenic mice expressing CTLA4-Ig under the control of a mammary gland-specific promoter to induce the expression of soluble CTLA4-Ig in the milk of transgenic animals for use as a bioreactor.

[0298] Alexion Pharmaceuticals Inc.'s PCT Publication WO 01 / 30966 describes chimeric DNA constructs containing the T-cell inhibitor CTLA-4, which is linked to the complement protein CD59, and transgenic pig cells, tissues, and organs containing them. PCT Publication WO2007035213 (Revivicor) describes genetically modified transgenic pigs expressing CTLA4-Ig.

[0299] Other immunosuppressants can be expressed in animals, tissues, or cells. For example, genes that are inactivated in mice to produce an immunocompromised phenotype can be cloned in pigs and disrupted through gene targeting. Some genes that have been targeted in mice and may be targeted to produce immunocompromised pigs include β2-microglobulin (MHC class I defect, Koller et al., Science, 248:1227-1230), TCRα, TCRβ (Mombaerts et al., Nature, 360:225-231), RAG-1 and RAG-2 (Mombaerts et al., (1992) Cell 68,869-877, Shinkai, et al., (1992) Cell 68,855-867, US Patent No. 5,859,307).

[0300] In one embodiment, the donor animal is modified to transgenic express cytotoxic T-lymphocyte-associated protein 4-immunoglobulin (CTLA4). The animal or cell may be modified to express the CTLA4 peptide or a biologically active fragment thereof (e.g., an extracellular domain, wherein at least the transmembrane domain has been removed from the truncated form of the peptide) or a derivative thereof. The peptide may be, for example, a human peptide or a porcine peptide. The CTLA4 peptide may be mutated.

[0301] The mutant peptide may have a higher affinity for porcine and / or human B7 molecules than the wild type. In one specific embodiment, the mutant CTLA4 may be CTLA4 (Glu104, Tyr29). The CTLA4 peptide may be modified to express intracellularly. Other modifications to the CTLA4 peptide include the addition of an endoplasmic reticulum (ER) retention signal to the N-terminus or C-terminus. The ER retention signal may be, for example, the sequence KDEL. The CTLA4 peptide may be fused with a peptide dimerization domain or an immunoglobulin (Ig) molecule. The CTLA4 fusion peptide may include a linker sequence that can connect the two peptides. In another embodiment, the CTLA4 peptide or a variant thereof (pCTLA4-Ig, or hCTLA4-Ig (Abatacept / Orencia or Belatacept)) may be administered to animals lacking functional immunoglobulin expression according to this disclosure as a drug to inhibit their T cell response. As used herein, CTLA4 is used to refer to any of these variants or variants known in the art, such as CTLA4-Ig.

[0302] In one embodiment, the CTLA4 peptide is the full-length CTLA4. In a further embodiment, the CTLA4 peptide may contain less than the full-length CTLA4 protein. In one embodiment, the CTLA4 peptide may contain the extracellular domain of the CTLA-4 peptide. In a specific embodiment, the CTLA4 peptide is the extracellular domain of CTLA4. In a further embodiment, this disclosure provides a mutant form of CTLA4. In one embodiment, the mutant form of CTLA4 may have a higher affinity for porcine B7 and / or human B7 than the wild type. In a specific embodiment, the mutant CTLA4 may be human CTLA4 (Glu104, Tyr29).

[0303] In one embodiment, CTLA4 may be a truncated form of CTLA4, wherein at least the transmembrane domain of the protein is removed. In another embodiment, the CTLA4 peptide may be modified to allow for intracellular expression. In one embodiment, a Golgi retention signal may be added to the N-terminus or C-terminus of the CTLA4 peptide. In one embodiment, the Golgi retention signal may be the sequence KDEL, which may be added to the C-terminus or N-terminus of the CTLA4 peptide. In a further embodiment, the CTLA4 peptide may be fused with a peptide dimerization domain. In one embodiment, the CTLA4 peptide may be fused with an immunoglobulin (Ig). In another embodiment, the CTLA4 fusion peptide may include a linker sequence capable of connecting the two peptides.

[0304] Anyone skilled in the art who knows the CTLA4 sequence or its biologically active portion or fragment may use it in the compositions and methods according to this disclosure.

[0305] Non-limiting examples include, but are not limited to, the following Genbank accession numbers describing human CTLA4 sequences: NM005214.2; BC074893.2; BC074842.2; AF414120.1; AF414120; AY402333; AY209009.1; BC070162.1; BC069566.1; L15006.1; AF486806.1; AC010138.6; AJ535718.1; AF225900.1; AF225900; AF411058.1; M37243.1; U90273.1; and / or AF316875.1. Other nucleotide sequences encoding the CTLA4 peptide may be selected from those including, but not limited to, the following Genbank accessions in the EST database: CD639535.1; A1733018.1; BM997840.1; BG536887.1; BG236211.1; BG058720.1; A1860i99.1; AW207094.1; AA210929.1; A1791416.1; BX113243.1; AW515943.1; BE837454.1; AA210902.1; BF329809.1; A1819438.1; BE837501.1; BE837537.1; and / or AA873138.1.

[0306] In another embodiment, any common CTLA4 peptide may be used according to this disclosure. In yet another embodiment, the nucleic acid and / or peptide sequence has at least 80%, 85%, 90%, or 95% homology to the native CTLA4 peptide and nucleotide sequence. In a further embodiment, any fragment or homologous sequence exhibiting similar activity to CTLA4 may be used. In other embodiments, the amino acid sequence exhibiting T-cell inhibitory activity may be amino acids 38 to 162 of the porcine CTLA4 sequence or amino acids 38 to 161 of the human CTLA4 sequence (see, for example, PCT Publication No. WO 01 / 30966). In one embodiment, the portion used should have at least about 25%, and preferably at least about 50%, of the activity of the parent molecule.

[0307] In other embodiments, the CTLA4 nucleic acid and peptide of this disclosure may be fused to immunoglobulin genes and molecules or fragments or regions thereof. The CTLA4 sequence of this disclosure includes those sequences fused to immunoglobulins. In one embodiment, the Ig may be human Ig. In another embodiment, the Ig may be IgG, particularly IgG1. In another embodiment, the Ig may be a constant region of IgG. In a specific embodiment, the constant region may be the C.γ.1 chain of IgG1. In a specific embodiment of this disclosure, the extracellular domain of porcine CTLA4 may be fused to human C.γ.1Ig. In another specific embodiment, the extracellular domain of human CTLA4 may be fused to IgG1 or IgG4. In a further specific embodiment, the extracellular domain of a mutant CTLA4 (Glu 104, Tyr 29) may be fused to IgG1. In one embodiment, at least one transgene is B7-H4, also known as B7x. B7-4H was identified in 2003 and belongs to the B7 immunoglobulin family. See Sica, GLImmunity, Vol. 18, 849–861, June, 2003.

[0308] In one implementation, the donor animal is modified to transgenerate the expression of class II transactivator (CIITA) and its mutants PDL1, PDL2, tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL), Fas ligand (FasL, CD95L), integrin-related protein (CD47), HLA-E, HLA-DP, HLA-DQ, or HLA-DR.

[0309] Class II transactivators (CIITA) are bifunctional or multifunctional domain proteins that act as transcriptional activators and play a crucial role in the expression of MHC class II genes. Previously, a mutant form of the human CIITA gene, encoding a protein lacking the 151 amino acids at its N-terminus, has been shown to be a potent dominant-inactivating repressor of HLA class II expression (Yun et al., Int Immunol. 1997 October; 9(10):1545-53). Porcine MHC class II antigens are potent stimuli for direct T cell recognition by human CD4+ T cells and may therefore play an important role in rejection of transgenic pig donors in clinical xenotransplantation. A mutant human CIITA construct has been reported to be effective in porcine cells, significantly inhibiting IFNγ-induced and constitutive porcine MHC class II expression. Furthermore, stable transfected porcine vascular endothelial cell lines carrying the mutant human CIITA construct failed to achieve direct T cell xenogeneic recognition via stimulation with purified human CD4+ T cells (Yun et al., Transplantation. 2000 Mar. 15; 69(5):940-4). Organs, tissues, and cells from CIITA-DN transgenic animals can induce significantly reduced T cell rejection responses in human recipients. In combination with other transgenes, transgenic expression of mutant CIITA can enable long-term xenograft survival with clinically acceptable levels of immunosuppression.

[0310] In one embodiment, this disclosure provides a transgenic animal (e.g., a pig) comprising genetic modifications resulting in (i) the absence of αGal and / or growth hormone receptor (GHR) expression and (ii) the incorporation and expression of at least two transgenes at a single locus, wherein at least four transgenes comprise at least one immunosuppressant. The single locus may be selected from natural loci, modified natural loci, or transgenic loci. Optionally, the transgenic animal includes one or more additional genetic modifications.

[0311] In exemplary embodiments, this disclosure provides transgenic animals (e.g., ungulates, swine) comprising genetic modifications resulting in (i) the absence of αGal and / or growth hormone receptor (GHR) expression and (ii) the incorporation and expression of at least four transgenes at a single locus, wherein at least two of the at least two transgenes are immunosuppressants. The single locus may be selected from natural loci, modified natural loci, or transgenic loci. The at least four transgenes may be provided as an MCV and incorporated into the locus using gene-editing tools. Optionally, the transgenic animal includes one or more additional genetic modifications.

[0312] In one exemplary embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional αGTαGal and / or growth hormone receptor (GHR) and is genetically modified to (i) incorporate and express at least four transgenes at a single locus, wherein the at least four transgenes include at least one immunosuppressant. The immunosuppressant may be, for example, CIITA-DN or CLTA4-IG. The at least four transgenes may include additional transgenes selected from complement inhibitors, anticoagulants, or combinations thereof. The single locus may be selected from a natural locus, a modified natural locus, or a transgenic locus. At least three transgenes may be provided in the form of an MCV and incorporated into the locus using a gene-editing tool. Optionally, the transgenic animal includes one or more additional gene modifications.

[0313] In an exemplary embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional αGT, αGal, and / or growth hormone receptor (GHR) and is genetically modified to (i) incorporate and express at least four transgenes at a single locus, wherein the at least four transgenes comprise at least two immunosuppressants. The immunosuppressants may be, for example, CIITA-DN or CLTA4-IG. The at least four transgenes may also comprise complement inhibitors, anticoagulants, or combinations thereof. The single locus may be selected from natural loci, modified natural loci, or transgenic loci. At least three transgenes may be provided in the form of MCVs and incorporated into the locus using gene-editing tools. Optionally, the transgenic animal includes one or more additional gene modifications.

[0314] 4. Other immunomodulators

[0315] PDL1, PDL2

[0316] Typical co-stimulatory molecules for T cell activation are CD80 / 86 or CD40. In recent years, in addition to these positive co-stimulatory pathways, new co-stimulatory pathways mediating negative signaling and important for regulating T cell activation have been discovered. One of these new pathways is composed of the programmed death 1 (PD-1) receptor and its ligands PD-L1 and PD-L2. The PD-1 receptor is not expressed in resting cells but is upregulated after T cell and B cell activation. PD-1 contains a tyrosine-based switch motif based on cytoplasmic immune receptors, and binding of PD-L1 or PD-L2 to PD-1 results in inhibitory signaling in T cells. Recent data suggest that the PD1 / PD ligand pathway may play a role in controlling T cell subsets exhibiting regulatory activity. In mice, PD-1 signaling has been demonstrated to be essential for the inhibitory activity of regulatory T cells (Tregs) and the generation of adaptive Tregs. These observations suggest that PD-1 / PD ligands and their interactions not only suppress T cell responses but may also trigger immune regulation. Multiple studies have demonstrated that the PD-1 / PD ligand pathway controls allogeneic graft implantation and rejection, suggesting that these molecules are targets of interest in post-transplant immunomodulation. Indeed, in rat transplantation models, the survival of allogeneic grafts has been prolonged by transferring the PDL1 Ig gene into the donor heart. Furthermore, enhanced PD-1 signaling by injecting PD-L1 Ig has been reported to protect grafts from rejection in mice. Recent data also indicate that overexpression of PD-L1IG in mouse islet grafts can partially prolong islet graft survival. Transgenic expression of human PD-L1 or PD-L2 in porcine cells and tissues should reduce early human anti-porcine T cell responses induced via direct sensitization (Plege et al., Transplantation. 2009 Apr. 15; 87(7):975-82). Induction of Tregs may also control xenograft-sensitive T cells through the indirect pathway required for durable tolerance.

[0317] In a specific implementation, a transgenic animal lacking αGal expression and incorporating and expressing at least four transgenes under the control of at least two promoters includes the incorporation and expression of PDL1 or PDL2.

[0318] TRAIL / Fas L

[0319] Expression of apoptosis-inducing ligands, such as Fas ligand (FasL, CD95L) or tumor necrosis factor-α-associated apoptosis-inducing ligand (TRAIL, Apo-2L), can eliminate T cells attacking xenografts. TRAIL is a type II membrane protein whose extracellular domain is homologous to the extracellular domains of other members of the tumor necrosis factor family, exhibiting the highest amino acid identity (28%) with FasL. TRAIL preferentially exerts its apoptosis-inducing effect on tumor cells. In normal cells, binding to the TRAIL receptor does not lead to cell death. Recent studies have shown that the cytotoxic effects of immune cells, including T cells, natural killer cells, macrophages, and dendritic cells, are at least partially mediated by TRAIL. Expression of human TRAIL in transgenic pigs could provide a rational strategy for protecting pig tissues from cell-mediated rejection after xenografting into primates. Stable expression of human TRAIL has been achieved in transgenic pigs, and the expressed TRAIL has been shown to have biological functions in vitro (Klose et al., Transplantation. 2005 Jul. 27; 80(2): 222-30). (d) CD47: CD47, known as integrin-associated protein, is a ubiquitously expressed 50-kDa cell surface glycoprotein that acts as a ligand for signal regulatory protein (SIRP.α.) (also known as CD172a, SHPS-1) and is an immunosuppressive receptor on macrophages. CD47 and SIRP.α. constitute a cell-cell communication system (CD47-SIRP.α. system) that plays an important role in a variety of cellular processes, including cell migration, B cell adhesion, and T cell activation. In addition, the CD47-SIRP.α. system is involved in the negative regulation of macrophage phagocytosis. CD47 on the surface of several cell types (i.e., erythrocytes, platelets, or leukocytes) can prevent macrophage phagocytosis by binding to the inhibitory macrophage receptor SIRP.α. The role of CD47-SIRP.α interaction in self-recognition and inhibition of phagocytosis has been demonstrated by the observation that primary wild-type mouse macrophages rapidly phagocytose unregulated RBCs obtained from CD47-deficient mice rather than wild-type mice. CD47 has also been reported to inhibit Fcγ and complement receptor-mediated phagocytosis via its SIRP.α receptor. It has been shown that porcine CD47 does not induce SIRP.α tyrosine phosphorylation in human macrophage-like cell lines, and that soluble human CD47-Fc fusion protein inhibits the phagocytic activity of human macrophages against porcine cells. Furthermore, it has been shown that manipulating porcine cell expression of human CD47 fundamentally reduces the cell's sensitivity to phagocytosis by human macrophages (Ide et al., Proc Natl Acad SciUSA. 2007 Mar. 20; 104(12): 5062-6).Expression of human CD47 on porcine cells can provide inhibitory signaling for SIRP.α on human macrophages, thereby providing a method to prevent macrophage-mediated xenograft rejection.

[0320] In a specific implementation, a transgenic animal lacking αGal and / or growth hormone receptor (GHR) expression and incorporating and expressing at least four transgenes under the control of at least two promoters includes TRAIL or Fas L.NK cell response, HLA-E / Beta 2 microglobulin, and the incorporation and expression of HLA-DP, HLA-DQ, and HLA-DR.

[0321] Human natural killer (NK) cells pose a potential obstacle to successful pig-to-human xenotransplantation because they infiltrate pig organs perfused with human blood in vitro and lyse pig cells directly and in the presence of human serum via antibody-dependent cell-mediated cytotoxicity. Expression of the major histocompatibility complex (MHC) class I ligand of the inhibitory NK receptor on normal autologous cells prevents NK cell autoreactivity. The inhibitory receptor CD94 / NKG2A, expressed on most activated human NK cells, specifically binds to human leukocyte antigen (HLA)-E. The non-classical human MHC molecule HLA-E is a potent inhibitory ligand for NK cells carrying CD94 / NKG2A, and unlike classical MHC molecules, it does not induce an allogeneic T cell response. HLA-E assembles in the endoplasmic reticulum and is transported to the cell surface as a stable trimeric complex composed of the HLA-E heavy chain, β.2-microglobulin (β.2m), and a peptide derived from a leader sequence of some MHC class I molecules. HLA-E expression has been shown to provide partial protection against xenogeneic human NK cell cytotoxicity (Weiss et al., Transplantation. 2009 Jan. 15; 87(1):35-43). Transgenic expression of HLA-E in pig organs has the potential to significantly reduce human NK cell-mediated pig xenograft rejection without the risk of allogeneic reactions. In addition, transgenic pigs carrying other HLA genes have been successfully generated with the aim of “humanizing” pig organs, tissues and cells (Huang et al., Proteomics. 2006 November; 6(21):5815-25, see also USPat. No. 6, 639, 122).

[0322] In a specific implementation, a transgenic animal lacking αGal expression and incorporating and expressing at least four transgenes under the control of at least two promoters contains HLA-3 incorporation and expression.

[0323] CD47

[0324] CD47 (differentiation cluster 47), also known as integrin-associated protein (IAP), is a transmembrane protein encoded by the CD47 gene in humans. CD47 is known to be an immunosuppressant and immunomodulator, and it plays a role in tolerogenicity in SIRPα signaling.

[0325] In an exemplary embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional αGTαGal and / or growth hormone receptor (GHR) and is genetically modified to (i) incorporate and express at least four transgenes at a single locus, wherein one of the at least four transgenes is CD47. The at least four transgenes may include additional transgenes selected from complement inhibitors, anticoagulants, or combinations thereof. The single locus may be selected from a natural locus, a modified natural locus, or a transgenic locus. At least three transgenes may be provided as an MCV and incorporated into the locus using a gene-editing tool. Optionally, the transgenic animal includes one or more additional gene modifications.

[0326] In an exemplary embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional αGTαGal and / or growth hormone receptor (GHR) and is genetically modified to (i) incorporate and express at least four transgenes at a single locus, wherein one of the at least four transgenes is CD7. The at least four transgenes may include additional transgenes selected from complement inhibitors, anticoagulants, or combinations thereof. The single locus may be selected from a natural locus, a modified natural locus, or a transgenic locus. At least three transgenes may be provided as an MCV and incorporated into the locus using a gene-editing tool. Optionally, the transgenic animal includes one or more additional gene modifications.

[0327] 5. Anticoagulants

[0328] In one embodiment, this disclosure provides transgenic donor animals suitable for use as sources of organs, tissues, and cells for xenotransplantation, wherein the donor animal is genetically modified to incorporate and express at least one anticoagulant. The animal typically has additional genetic modifications, more specifically, at least five additional genetic modifications, and even more specifically, at least six additional genetic modifications. In an exemplary embodiment, this disclosure is a transgenic animal containing genetic modifications that result in (i) a lack of αGal and / or growth hormone receptor (GHR) expression and (ii) the incorporation and expression of at least four transgenes at a single locus, these transgenes being controlled by at least two promoters, wherein at least one of the transgenes is an anticoagulant.

[0329] The anticoagulant can be any suitable anticoagulant. Expression can be ubiquitous or tissue-specific. In a specific implementation, expression is controlled by a promoter that is primarily active in the endothelium. Representative, non-limiting examples of suitable anticoagulant transgenes include tissue factor pathway inhibitors, hirudin, thrombomodulin, endothelial cell protein C receptor (EPCR), CD39, and combinations thereof.

[0330] Tissue factor pathway inhibitors (TFPIs) are single-chain peptides that reversibly inhibit factor Xa (Xa) and thrombin (factor IIa), thereby inhibiting TF-dependent coagulation. For a review of TFPIs, see Crawley and Lane (Arterioscler Thromb Vasc Biol. 2008, 28(2):233-42). Dorling and colleagues generated transgenic mice expressing a fusion protein consisting of three Kunitz domains of human TFPI linked to the transmembrane / cytoplasmic domain of human CD4, with a P-selectin tail for targeting intracellular storage granules in Weibel-Palade cells (Chen D, et al. Am J Transplant 2004; 4:1958-1963). The activation-dependent TFPIs produced on endothelial cells demonstrated sufficient to completely suppress thrombosis-mediated acute humoral rejection of mouse cardiac xenografts in rats treated with cyclosporine. It has also been suggested that effective regulation of coagulation may prevent chronic rejection. Similar results were obtained using the hearts of transgenic mice expressing the hirudin / CD4 / P-selectin fusion protein, indicating that inhibition of thrombin generation or activity is key to protection in this model.

[0331] Hirudin is a peptide naturally found in the salivary glands of medicinal leeches (such as the European medicinal leech, *Hirudo medicinalis*) and is a potent thrombin inhibitor. Dorling et al. (Chen et al., *J Transplant.* 2004, December; 4(12): 1958-63) also generated transgenic mice expressing a mesothelial chain hirudin fusion protein and transplanted their hearts into rats (mouse-rat Xeno-Tx). In contrast to the control non-transgenic mice, whose hearts were rejected within 3 days, the organs in both transgenic mouse strains were 100% completely resistant to humoral rejection and survived for over 100 days when T-cell-mediated rejection was suppressed by administration of cyclosporine A. Riesbeck et al. (*Circulation.* 1998, Dec. 15; 98(24): 2744-52)) also explored the expression of a hirudin fusion protein in mammalian cells as a strategy to prevent intravascular thrombosis. Expression in cells reduced local thrombin levels and inhibited fibrin formation. Therefore, hirudin is another anticoagulant transgene of interest that prevents thrombotic effects in xenotransplantation.

[0332] Thrombomotor protein (TBM) functions as a cofactor in the anticoagulation pathway by forming a 1:1 stoichiometric complex with thrombin, thus aiding in thrombin-induced protein C activation. Endothelial cell protein C receptor (EPCR) is an N-glycosylated type I membrane protein that enhances protein C activation. Van de Wouwer et al., Arterioscler Thromb VascBiol. 2004 August; 24(8):1374-83 reviewed the roles of these proteins in the protein C anticoagulation system. Various groups have been exploring the expression of these and other anticoagulant transgenes to potentially address coagulation disorders in xenografts (Cowanand D'Apice, Cur Opin Organ Transplant. 2008 April; 13(2):178-83 review). Esmon et al. (Li et al., J Thromb Haemost. 2005 July; 3(7): 1351-9) overexpressed EPCR in the endothelial cells of transgenic mice and showed that this expression protected mice from the challenge of thrombosis. Iino et al. (J Thromb Haemost. 2004 May; 2(5): 833-4) suggested that ex vivo overexpression of TBM in donor islets via gene therapy could be a means of preventing thrombotic complications during islet transplantation.

[0333] CD39 is a major angiotensin triphosphate diphosphate hydrolase (NTPDase) that converts ATP and ADP to AMP, which is ultimately converted to adenosine. Extracellular adenosine plays an important role in thrombosis and inflammation, and its beneficial effects in transplantation have been studied (Robson et al. Semin Thromb Hemost. 2005 April; 31(2):217-33 review). Recent studies have shown that CD39 plays an important role in reducing inflammatory responses (Beldi et al., Front Biosci, 2008, 13:2588-2603). Transgenic mice expressing hCD39 showed impaired platelet aggregation, prolonged bleeding time, and resistance to systemic thromboembolism in a heart transplantation model (Dwyer et al., J Clin Invest. 2004 May; 113(10):1440-6). They also showed expression of CD39 on the islets, and when incubated with human blood, these islets significantly delayed clotting time compared to wild-type islets (Dwyer et al., Transplantation. 2006 Aug. 15; 82(3):428-32). Preliminary studies of high-level expression of hCD39 from constitutive promoter systems in transgenic pigs have shown high postpartum lethality (Revivicor, Inc., unpublished data). However, endothelial cell-specific expression of CD39 has indicated better tolerability in transgenic pigs. Therefore, there is a need to express certain anticoagulant transgenes in pigs in a manner that does not harm animal health, but still provides sufficient levels of expression for clinical xenotransplantation.

[0334] In exemplary embodiments, this disclosure provides transgenic animals (e.g., ungulates, swine) that have been genetically modified to result in (i) a lack of (or reduced) expression of αGal and / or growth hormone receptor (GHR) and (ii) incorporation and expression of at least four transgenes at a single locus, controlled by two promoters, wherein at least one of the at least two transgenes is an anticoagulant. In one embodiment, the anticoagulant is selected from tissue factor pathway inhibitors, hirudin, thrombomodulin, endothelial cell protein C receptor (EPCR), CD39, and combinations thereof. The single locus may be a natural locus, a modified natural locus, or a transgenic locus. The natural locus may be GGTA1, B4GalNT2, CMAH, Rosa26, AAVS1, or other endogenous loci that may confer beneficial expression characteristics to the integrated transgene. The at least four transgenes controlled by at least two promoters may be provided as MCVs, and incorporation may be performed using gene editing tools. Such editing can involve targeted insertion at a predetermined site (e.g., a landing pad) that acts as a “safe harbor” (to avoid interrupting any essential genes in the genome) and / or providing desired characteristics specific to the integration site. If the insertion at the locus is important for preventing xenograft rejection, the insertion of multiple transgenes can also lead to the inactivation of porcine genes involved in inducing xenogeneic responses in primates (i.e., inactivation of αGal, GHR, CMAH, or B4GalNT2 or others (iGB3, Forssman)). Optionally, the animal may include one or more additional gene modifications and at more than one locus, wherein at least four transgenes are inserted at one locus, and two or more transgenes from another group can be co-integrated at a second site (under the control of at least two promoters). An alternative implementation involves inserting an MCV at one locus and targeting inactivation at different loci, wherein the gene-editing tool can facilitate this inactivation.

[0335] In exemplary embodiments, this disclosure provides transgenic animals (e.g., ungulates, swine) having genetic modifications that result in (i) a lack of expression (or reduced expression) of αGal and / or growth hormone receptor (GHR) and (ii) incorporation and expression of at least four, at least five, at least six, at least seven, or at least eight or more transgenes at a single locus, wherein at least one, at least two, or at least three of the transgenes are anticoagulants.

[0336] In one embodiment, the anticoagulant is selected from tissue factor pathway inhibitors, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. At least four transgenes can be provided as MCVs, and incorporation can be performed using gene editing tools. A single locus can be a natural locus, a modified natural locus, or a transgenic locus. Optionally, the animal may include one or more additional gene modifications.

[0337] In one embodiment, this disclosure provides transgenic animals (e.g., ungulates, swine) lacking (or with reduced) expression of αGal and / or growth hormone receptor (GHR) and genetically modified to incorporate and express at least three anticoagulants. In some embodiments, the anticoagulants are selected from tissue factor pathway inhibitors (TFPIs), hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In some embodiments, at least one of the at least three anticoagulants is controlled by the expression of a promoter primarily active in endothelial cells. In some embodiments, at least two of the at least three anticoagulants are controlled by the expression of a promoter primarily active in endothelial cells.

[0338] In exemplary embodiments, this disclosure provides transgenic animals (e.g., ungulates, swine) that lack (or have reduced) expression of αGal and / or growth hormone receptor (GHR) and are genetically modified to incorporate and express at least three anticoagulants, one of which is an EPCR.

[0339] In exemplary embodiments, this disclosure provides transgenic animals (e.g., ungulates, swine) that lack (or have reduced) expression of αGal and / or growth hormone receptor (GHR) and are genetically modified to incorporate and express at least three anticoagulants, wherein the at least three anticoagulants include EPCR and TBM.

[0340] In one embodiment, this disclosure provides a transgenic animal (e.g., an ungulate, a swine) lacking (or with reduced) expression of αGal and / or growth hormone receptor (GHR) and genetically modified to incorporate and express at least four additional transgenes, wherein the at least four additional transgenes include at least one anticoagulant. In some embodiments, the at least one anticoagulant is selected from tissue factor pathway inhibitors, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least one anticoagulant is an EPCR.

[0341] In one embodiment, this disclosure provides a transgenic animal (e.g., an ungulate, a swine) lacking (or with reduced) expression of αGal and / or growth hormone receptor (GHR) and genetically modified to incorporate and express at least four additional transgenes, wherein the at least four additional transgenes comprise at least two anticoagulants. In some embodiments, the at least two anticoagulants are selected from tissue factor pathway inhibitors, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least two anticoagulants comprise EPCR and TBM. In another embodiment, the at least two anticoagulants comprise EPCR and TFPI.

[0342] In one embodiment, this disclosure provides a transgenic animal (e.g., an ungulate, a swine) lacking (or with reduced) expression of αGal and / or growth hormone receptor (GHR) and genetically modified to incorporate and express at least four additional transgenes, wherein the at least four additional transgenes comprise at least three anticoagulants. In some embodiments, the at least three anticoagulants are selected from tissue factor pathway inhibitors, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least three anticoagulants comprise EPCR, TBM, and TFPI. In another embodiment, the at least three anticoagulants comprise EPCR, TBM, and CD39.

[0343] In one embodiment, this disclosure provides transgenic animals (e.g., ungulates, swine) lacking (or with reduced) expression of αGal and / or growth hormone receptor (GHR) and genetically modified to incorporate and express at least five additional transgenes, wherein the at least five additional transgenes comprise at least two anticoagulants. In some embodiments, the at least two anticoagulants are selected from tissue factor pathway inhibitors, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least two anticoagulants comprise EPCR and TBM. In another embodiment, the at least two anticoagulants comprise EPCR and TFPI.

[0344] In one embodiment, this disclosure provides a transgenic animal (e.g., an ungulate, a swine) lacking (or with reduced) expression of αGal and / or growth hormone receptor (GHR) and genetically modified to incorporate and express at least five additional transgenes, wherein the at least five additional transgenes comprise at least three anticoagulants. In some embodiments, the at least three anticoagulants are selected from tissue factor pathway inhibitors, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least three anticoagulants comprise EPCR, TBM, and TFPI. In another embodiment, the at least three anticoagulants comprise EPCR, TBM, and CD39.

[0345] In one embodiment, this disclosure provides transgenic animals (e.g., ungulates, swine) lacking (or with reduced) expression of αGal and / or growth hormone receptor (GHR) and genetically modified to incorporate and express at least six additional transgenes, wherein the at least six additional transgenes comprise at least two anticoagulants. In some embodiments, the at least two anticoagulants are selected from tissue factor pathway inhibitors, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least two anticoagulants comprise EPCR and TBM. In another embodiment, the at least two anticoagulants comprise EPCR and TFPI. Optionally, the at least six additional transgenes also comprise at least one immunosuppressant.

[0346] In one embodiment, this disclosure provides a transgenic animal (e.g., an ungulate, a swine) lacking (or with reduced) expression of αGal and / or growth hormone receptor (GHR) and genetically modified to incorporate and express at least six additional transgenes, wherein the at least six additional transgenes comprise at least three anticoagulants. In some embodiments, the at least three anticoagulants are selected from tissue factor pathway inhibitors, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least three anticoagulants comprise EPCR, TBM, and TFPI. In another embodiment, the at least three anticoagulants comprise EPCR, TBM, and CD39.

[0347] 6. Cell-protective transgenic

[0348] In one embodiment, this disclosure provides transgenic donor animals suitable for use as sources of organs, tissues, and cells for xenotransplantation, wherein the donor animal is genetically modified to incorporate and express at least one cryoprotective transgene (“cell protectant”). In an exemplary embodiment, this disclosure provides transgenic animals (e.g., pigs) comprising genetic modifications resulting in (i) a lack of expression of αGal and / or growth hormone receptor (GHR); and (ii) incorporation and expression of at least four transgenes at a single locus, these transgenes being controlled by at least two promoters, wherein at least one of the at least four transgenes is a cell protectant. Cell protectant transgenes are thought to include anti-apoptotic agents, antioxidants, and anti-inflammatory agents. Examples include:

[0349] A20

[0350] A20 provides anti-inflammatory and anti-apoptotic activity. It can protect vascularized transplanted organs from endothelial cell activation and cell damage through anti-inflammatory, anticoagulant, and / or anti-apoptotic molecules. Among genes with great potential for regulating acute vascular rejection (AVR), the human A20 gene (hA20) was first identified as a tumor necrosis factor (TNF)-α inducible factor in human umbilical vein endothelial cells. Human A20 has dual cytoprotective functions, protecting endothelial cells from TNF-mediated apoptosis and inflammation by blocking several caspases and the transcription factor nuclear factor-κB. Viable A20 transgenic piglets have been produced in which hA20 expression is limited to skeletal muscle, heart, and PAEC, protecting endothelial cells from TNF-mediated apoptosis and partially from CD95(Fas)L-mediated cell death. In addition, cardiomyocytes from hA20 transgenic cloned pigs were partially protected from heart damage (Oropeza et al., Xenotransplantation. 2009 November; 16(6): 522-34).

[0351] HO-1

[0352] Heme oxygenase (HO) provides anti-inflammatory, anti-apoptotic, and antioxidant activities. HO is the rate-limiting enzyme in heme catabolism, also known as HSP32, and belongs to the heat shock proteins family. In this process, the heme ring is cleaved into ferrous iron, carbon monoxide (CO), and biliverdin, which is then converted to bilirubin by biliverdin reductase. Three isoforms of HO have been cloned: HO-1, HO-2, and HO-3. HO-1 expression is highly inducible, while HO-2 and HO-3 are constitutively expressed (Maines MD et al., Annual Review of Pharmacology & Toxicology 1997;37:517-554, and Choi A Met al., American Journal of Respiratory Cell & Molecular Biology 1996;15:9-19). Analysis of HO-1- / - mice has shown that the gene encoding HO-1 regulates iron homeostasis and acts as a cytoprotective gene, possessing effective antioxidant, anti-inflammatory, and anti-apoptotic effects (Poss KD et al., Proceedings of the National Academy of Sciences of the United States of America 1997; 94:10925-10930, PossK D et al., Proceedings of the National Academy of Sciences of the United States of America 1997; 94:10919-10924, and Soares MP et al., Nature Medicine 1998; 4:1073-1077). Similar findings have recently been described in case reports of HO-1 deficiency in humans (Yachie Aetal., Journal of Clinical Investigation 1999; 103:129-135). The molecular mechanisms responsible for the cytoprotective effects of HO-1 (including anti-inflammatory, antioxidant, and anti-apoptotic effects) are mediated by its reaction products. HO-1 expression can be regulated in vitro and in vivo by protoporphyrins with different metals. Cobalt protoporphyrin (CoPP) and iron protoporphyrin (FePP) can upregulate HO-1 expression. Conversely, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP) inhibit HO-1 activity at the protein level.Recently, it has been demonstrated that HO-1 expression inhibits rejection in mouse-rat heart transplantation (Sato K et al., J. Immunol. 2001; 166:4185-4194), protects islet cells from apoptosis, and improves the in vivo function of transplanted islet cells (Pileggi A et al., Diabetes 2001; 50:1983-1991). It has also been demonstrated that HO-1 administration via gene transfer provides protection against hyperoxia-induced lung injury (Otterbein LE et al., J Clin Invest 1999; 103:1047-1054), upregulation of HO-1 protects the liver from ischemia / reperfusion injury in hereditary obese Zucker rats (Amersi F et al., J Clin Invest 1999; 104:1631-1639), and elimination or expression of the HO-1 gene regulates cisplatin-induced renal tubular cell apoptosis (Shiraishi F et al., Am J Physiol Renal Physiol 2000; 278:F726-F736). In transgenic animal models, overexpression of HO-1 has been shown to prevent lung inflammation and vascular responses to hypoxia (Minamino T et al., Proc. Natl. Acad. Sci. USA 2001; 98:8798-8803) and protect the heart from ischemia and reperfusion injury (Yet SF, et al., Cir Res 2001; 89:168-173). Pigs carrying the HO-1 transgene have been produced, but no clinical effects related to their use in xenotransplantation have been reported (US Patent No. 7,378,569).

[0353] FAT-1

[0354] FAT-1 provides anti-inflammatory activity. Polyunsaturated fatty acids (PUFAs) play a role in suppressing (n-3 class) inflammation. Mammalian cells lack desaturases that convert n-6 to n-3 polyunsaturated fatty acids. Therefore, essential n-3 fatty acids must be provided through diet. However, unlike mammals, the free-living nematode *C. elegans* expresses an n-3 fatty acid desaturase that introduces a double bond into the n-6-fatty acid at the n-3 position of the hydrocarbon chain to form n-3 PUFAs. Transgenic mice expressing the *C. elegans* fat-1 gene have been produced, thus enabling efficient conversion of 6-class dietary PUFAs into 3-class PUFAs, such as EPA (20:5n-3) and DHA (22:6n-3) (Kang et al., *Nature*. 2004, Feb. 5; 427(6974):504). Another team developed a transgenic mouse model in which the codons of fat-1 cDNA were further optimized for efficient translation in mammalian systems; endogenous production of n-3 PUFAs was achieved by overexpressing the n-3 fatty acid desaturase gene mfat-1 from Caenorhabditis elegans. This team demonstrated that transgenic expression of mfat-1 enhanced insulin secretion from isolated mouse islets in response to glucose, amino acids, and GLP-1 stimulation, and made the islets highly resistant to cytokine-induced cell death (Wei et al., Diabetes. 2010 February; 59(2):471-8).

[0355] Soluble TNF-α receptor (sTNFR1)

[0356] Tumor necrosis factor (TNF, cachexia, or cachectic factor-α) is a cytokine involved in systemic inflammation and a member of a group of cytokines that stimulate acute-phase responses. The main function of TNF is to regulate immune cells. TNF can induce apoptotic cell death, thereby triggering inflammation. Soluble TNF-α receptor 1 (sTNFR1) is an antagonist of TNF-α and its extracellular domain (Su et al., 1998. Arthritis Rheum. 41, 139-149). Transgenic expression of sTNFR1 in xenografts may have beneficial anti-inflammatory effects.

[0357] Other cytoprotective agents with relevant antioxidant properties include, but are not limited to, SOD and catalase. Oxygen is an essential molecule for all aerobic organisms and plays a major role in ATP production (i.e., oxidative phosphorylation). In this process, reactive oxygen species (ROS), including superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), are produced as byproducts. In humans, the antioxidant defense system balances ROS production. Superoxide dismutase (SOD) and catalase are two enzymes with antioxidant properties. SOD catalyzes the dismutation of superoxide radicals into hydrogen peroxide, which is then converted into water by catalase and glutathione peroxidase. Cell damage caused by ROS production can occur in the transplant environment. Pancreatic β-cells are particularly vulnerable to free radical and inflammatory damage due to reduced antioxidant defense capacity. Commonly used anti-rejection drugs are excellent at suppressing adaptive immune responses; however, most are detrimental to the islets and do not adequately protect them from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. Therefore, it is of interest to treat islets with antioxidants in vitro, or to express antioxidant genes via gene therapy or by expressing transgenes in donor tissue. In vitro gene transfer of EC-SOD and catalase has shown anti-inflammatory effects in an antigen-induced arthritis rat model (Dai et al., Gene Ther. 2003 April; 10(7):550-8). Furthermore, delivery of EC-SOD and / or catalase genes via the portal vein significantly reduced liver I / R injury in a mouse model (Heet al., Liver Transpl. 2006 December; 12(12):1869-79). In recent mouse studies, islets treated with catalytic antioxidants prior to syngeneic, suboptimal syngeneic, or xenogeneic transplantation exhibited better function compared to untreated controls. In the same study, diabetic mouse recipients of allogeneic islets treated with catalytic antioxidants showed improved glycemic control and delayed allogeneic graft rejection after transplantation (Sklavos et al., Diabetes. 2010 July; 59(7):1731-8. Epub 2010 Apr. 22). In another mouse study, islet grafts overexpressing MnSOD functioned approximately 50% longer than control grafts (Bertera et al., Diabetes. 2003 February; 52(2):387-93). Furthermore, some anticoagulants also provide anti-inflammatory activity, including thrombomodulin, EPCR, and CD39.

[0358] In exemplary embodiments, this disclosure provides transgenic animals (e.g., pigs) comprising gene modifications resulting in (i) a lack of expression of αGal and / or growth hormone receptor (GHR); and (ii) incorporation and expression of at least four transgenes at a single locus (controlled by at least two promoters), wherein at least one of the at least four transgenes is a cell-protective transgene. The single locus may be a natural locus, a modified natural locus, or a transgenic locus. At least two transgenes may be provided as MCVs, and incorporation may be performed using gene-editing tools. Optionally, the animal may have one or more additional gene modifications.

[0359] In exemplary embodiments, this disclosure provides transgenic animals (e.g., pigs) comprising gene modifications resulting in (i) a lack of expression of αGal and / or growth hormone receptor (GHR); and (ii) incorporation and expression of at least five, at least six, at least seven, or at least eight transgenes at a single locus, or incorporation and expression of at least four transgenes at one locus and incorporation and expression of one or more transgenes at a second locus, wherein at least one of the transgenes is a cell-protective transgene, and wherein at least four transgenes are controlled by at least two promoters, which may be different combinations of constitutive promoter systems, ubiquitous promoter systems, tissue-specific promoter systems, or inducible regulatory promoter systems. The transgenes may be provided as an MCV, and incorporation may be performed using gene editing tools. A single locus may be a natural locus, a modified natural locus, or a transgenic locus. Optionally, the animal may have one or more additional gene modifications.

[0360] VI. Production of transgenic animals

[0361] Transgenic animals can be produced by any method known to those skilled in the art, including but not limited to selective breeding, nuclear transfer, introduction of DNA into oocytes, sperm, zygotes, or blastomeres, or by using embryonic stem cells. Gene editing tools may also be used, as further described herein.

[0362] In some implementations, genetic modifications in animals can be identified, and these animals can then be bred together to form a herd with the desired set of genetic modifications (or a single genetic modification). These offspring can be further bred to produce different or identical sets of genetic modifications (or single genetic modifications) in their descendants. This cycle of breeding animals with the desired genetic modifications can continue as long as desired. The term "herd" as used herein can include multiple generations of animals with the same or different genetic modifications over time. "Herd" can also refer to a single generation of animals with the same or different genetic modifications.

[0363] Cells that can be used for gene modification (e.g., but not limited to homologous recombination, random insertion / integration, nuclease editing, zinc finger plus TALEN nucleases, CRISPR / Cas9 nucleases) include, for example, epithelial cells, nerve cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B lymphocytes and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiomyocytes, and other muscle cells. Furthermore, cells used to produce genetically modified animals (through, for example, but not limited to, nuclear transplantation) can be obtained from various organs, such as skin, lungs, pancreas, liver, stomach, intestines, heart, reproductive organs, bladder, kidneys, urethra, and other urinary organs. Cells can be obtained from any cell or organ in the body, including all somatic cells or germ cells.

[0364] In addition, genetically modified animal cells can be obtained from a variety of different organs and tissues, such as, but not limited to, skin, mesenchyme, lungs, pancreas, heart, intestines, stomach, bladder, blood vessels, kidneys, urethra, reproductive organs, as well as decomposition preparations of all or part of embryos, fetuses, or adult animals. In one embodiment of the invention, the cells may be selected from, but are not limited to, the group consisting of: epithelial cells, fibroblasts, nerve cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiomyocytes, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, pancreatic islet cells, blood cells, blood progenitor cells, osteocytes, bone progenitor cells, neuronal stem cells, primitive stem cells, adult stem cells, mesenchymal stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, hepatic stellate cells, aortic smooth muscle cells, cardiomyocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils Germ cells, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells, parotid gland cells, tumor cells, glial cells, astrocytes, erythrocytes, leukocytes, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, cardiac cells, pacemaker cells, spleen cells, antigen-presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testicular cells, germ cells, oocytes, interstitial cells, peritubular cells, sedolocytes, luteinized cells, cervical cells, endometrial cells, mammary cells, follicular cells, mucus cells, ciliated cells, non-keratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, squamous epithelial cells, osteocytes, osteoblasts, and osteoclasts. In an alternative implementation, embryonic stem cells may be used. Embryonic stem cell lines can be used, or embryonic stem cells can be obtained fresh from a host (e.g., swine). Cells can be grown on a suitable fibroblast feeder layer or in the presence of leukemia suppressor factor (LIF).

[0365] Embryonic stem cells are the preferred germ cell type, and can be obtained using embryonic stem cell lines or freshly from a host (e.g., swine). Cells can be grown on appropriate fibroblast feeder layers or in the presence of leukemia suppressor factor (LIF).

[0366] Cells of particular interest in other lineages include stem cells (such as hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, etc.), pancreatic islets, adrenal medullary cells that secrete dopamine, osteoblasts, osteoclasts, epithelial cells, endothelial cells, leukocytes (such as B lymphocytes and T lymphocytes), bone marrow monocytes, neurons, glial cells, ganglion cells, retinal cells, liver cells (such as hepatocytes), bone marrow cells, keratinocytes, hair follicle cells, and myoblasts (muscle cells).

[0367] In a specific embodiment, the cells may be fibroblasts or fibroblast-like cells having a morphology or phenotype indistinguishable from fibroblasts, or a pre-senescent lifespan of at least 10, 12, 14, 18, or 20 days, or a lifespan sufficient for homologous recombination and nuclear transfer of non-senescent nuclei; in one specific embodiment, the cells may be fetal fibroblasts. Fibroblasts are a suitable somatic cell type because they can be obtained in large quantities from developing fetuses and adult animals. These cells can be easily multiplied in vitro with a rapid doubling time and can be cloned for gene-targeting programs. The cells to be used may be derived from fetal animals, or may be neonatal or derived from adult animals. The cells may be mature or immature, and differentiated or undifferentiated.

[0368] 1. Homologous recombination

[0369] Homologous recombination allows for site-specific modifications of endogenous genes, thus enabling the engineering of new changes into the genome. The main step in homologous recombination is DNA strand exchange, which involves pairing a DNA duplex with at least one DNA strand containing a complementary sequence to form an intermediate recombination structure containing heteroduplex DNA (see, for example, Rading, CM (1982) Ann. Rev. Genet. 16:405; U.S. Patent No. 4,888,274). Heteroduplex DNA can take many forms, including three DNA strands in a triplet configuration, with one complementary strand invading the DNA double helix (Hsieh et al. (1990) Genes and Development 4:1951; Rao et al., (1991) PNAS 88:2984). When the two complementary DNA strands pair with the DNA double helix, they can form the classic Holliday recombination adapter or chi-type structure (Holliday, R. (1964) Genet. Res. 5:282), or a double D loop (“Diagnostic Applications of Double D Loop Formation” US Serial No. 07 / 755,462, submitted September 4, 1991). Once the heteroduplex structure is formed, it can be resolved through strand breakage and exchange, whereby all or part of the invading DNA strand is spliced ​​into a recipient DNA double helix, adding or replacing segments of the recipient DNA double helix.

[0370] Alternatively, heteroduplex structures can lead to gene conversion, where an invading strand is used as a template to repair mismatched bases, transferring the sequence of the invading strand to the recipient DNA duplex (Genes, 3rd Ed. (1987) Lewin, B., John Wiley, New York, NY; Lopez et al. (1987) Nucleic Acids Res. 15:5643). Whether through break-and-rejoint mechanisms or gene conversion mechanisms, the formation of heteroduplex DNA at homologous pairing adapters can transfer gene sequence information from one DNA molecule to another. The ability of homologous recombination (gene conversion and classical strand break / rejoint) to transfer gene sequence information between DNA molecules makes targeted homologous recombination a powerful method in genetic engineering and gene manipulation.

[0371] In homologous recombination, the introduced DNA interacts with and integrates into a site in the genome containing a basic homologous DNA sequence. In non-homologous (“random” or “illegal”) integration, the introduced DNA is not found in a homologous sequence in the genome but integrates elsewhere, at one of a large number of potential sites. Generally, studies of higher eukaryotic cells have shown that homologous recombination occurs much less frequently than random integration. The ratio of these frequencies has a direct impact on “gene targeting,” which depends on integration via homologous recombination (i.e., recombination between exogenous “target DNA” and the corresponding “target DNA” in the genome). This disclosure uses homologous recombination to inactivate or insert and upregulate or activate genes in cells (e.g., the cells described above). The DNA may be contained at least a portion of the gene at a specific locus, wherein at least one copy, optionally two copies, of the natural gene will be altered to prevent the expression of the functional gene product. The alteration may be an insertion, deletion, substitution, mutation, or a combination thereof. When only one copy of an inactivated gene is introduced as a change, cells with a single unmutated copy of the target gene are amplified and a second targeting step can be performed. This change may be the same as or different (usually different) from the first change, and when deletions or substitutions are involved, it may overlap with at least a portion of the initially introduced change. In this second targeting step, targeting vectors with the same homologous arms but containing different mammalian selectable markers can be used. Transformants lacking the functional target antigen are screened, and the cell's DNA can be further screened to ensure the absence of the wild-type target gene. Alternatively, phenotypic homozygosity can be achieved by breeding heterozygous mutant hosts.

[0372] Many articles describe the uses of homologous recombination in mammalian cells. These articles include Kucherlapati et al. (1984) Proc.Natl.Acad.Sci.USA 81:3153-3157; Kucherlapati et al. (1985) Mol.Cell.Bio.5:714-720; Smithies et al. (1985) Nature 317:230-234; Wake et al. al. (1985) Mol. Cell. Bio. 8: 2080-2089; Ayares et al. (1985) Genetics 111: 375-388; Ayareset al. (1986) Mol. Cell. Bio. 7: 1656-1662; Song et al. al. (1987) Proc.Natl.Acad.Sci.USA84:6820-6824; Thomas et al. (1986) Cell 44:419-428; Thomas and Capecchi, (1987) Cell 51:503-512; Nandi et al. (1988) Proc.Natl.Acad.Sci.USA 85:3845-3849; andMansour et al. (1988) Nature 336:348-352; Evans and Kaufman, (1981) Nature 294:146-154; Doetschman et al. (1987) Nature 330:576-578; Thoma and Capecchi, (1987) Cell 51:503-512; Thompson et al. (1989) Cell 56:316-321.

[0373] In one embodiment, at least four transgenes incorporated and expressed in the transgenic animal of this disclosure are introduced via homologous recombination. In another embodiment, at least one of the four transgenes incorporated and expressed in the transgenic animal of this disclosure is introduced via homologous recombination.

[0374] 2. Random insertion

[0375] In one embodiment, DNA encoding a transgenic sequence can be randomly inserted into the chromosome of a cell. Random integration can be achieved by any method known to those skilled in the art for introducing DNA into cells. This can include, but is not limited to, electroporation, sonication, using a gene gun, lipid transfection, calcium phosphate transfection, using dendritic polymers, microinjection, using viral vectors (including adenovirus vectors, AAV vectors, and retroviral vectors), and group II ribozymes. In one embodiment, the encoded DNA can be programmed to include a reporter gene, such that the presence of the transgene or its expression product can be detected by activation of the reporter gene. Any reporter gene known in the art can be used, such as those disclosed above. The reporter gene can also be one of the transgenes added to the cell, such that cell surface expression of the transgene (e.g., DAF or CD46 or EPCR or CD47) can be used in conjunction with flow cytometry (and fluorescent antibodies specific to said transgene) as a means of enriching the transgene (and co-inserted transgene combinations) for gene transfer and subsequent expression. Cells containing the transgene can be selected by selecting those cells in the cell culture where the reporter gene has been activated. In other embodiments, DNA encoding the transgene can be introduced into cells by electroporation. In other embodiments, DNA can be introduced into cells via lipid transfection, infection, or transformation. In one embodiment, electroporation and / or lipid transfection can be used to transfect fibroblasts. In a specific embodiment, the transfected fibroblasts can be used as nuclear donors for nuclear transfer to produce transgenic animals known in the art and described below.

[0376] Cells containing the reporter gene can then be sorted using FACS to enrich the cell population, resulting in a higher percentage of cells containing DNA encoding the transgene of interest. In other embodiments, the FACS-sorted cells can then be cultured for a period of time, such as 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, 96 hours, or more, or for a period of time to allow DNA integration to produce a stable transfected cell population.

[0377] In one embodiment, at least four transgenes incorporated and expressed in the transgenic animal of this disclosure are introduced via random integration. In another embodiment, at least one of the four transgenes incorporated and expressed in the transgenic animal of this disclosure is introduced via random integration. For example, a bicistronic vector containing at least two transgenes is incorporated into the genome via random integration. In some embodiments, the transgenic animal incorporates and expresses at least four transgenes. In some embodiments, two of the four transgenes are expressed as polycistronic transgenes controlled by a first promoter, and two of the four transgenes are expressed as polycistronic transgenes controlled by a second promoter.

[0378] In some embodiments, two of the four transgenes expressed in the first or second polycistronic cell are selected from the group consisting of TBM, EPCR, DAF, CD39, TFPI, CTLA4-Ig, CIITA-DN, HOI, A20, and CD47. In some embodiments, at least one pair of transgenes is selected from the group consisting of: TBM and CD39; EPCR and DAF; A20 and CD47; TFPI and CD47; CIITA-DN and HO-1; TBM and CD47; CTLA4Ig and TFPI; CIITA-DN and TFPI; CIITA-DN and A20; TBM and A20; EPCR and DAF; TBM and HO-1; TBM and TFPI; CIITA and TFPI; EPCR and HO-1; TBM and CD47; EPCR and TFPI; TBM and EPCR; CD47 and HO-1; CD46 and CD47; CD46 and HO-1; and CD46 and TBM.

[0379] 3. Targeted genome editing:

[0380] In exemplary embodiments, genome editing tools are used to incorporate transgenes into animals. These tools include, but are not limited to, nucleases and site-specific recombinases. In exemplary embodiments, genome editing tools (e.g., but not limited to, integrase (recombinase), CRISPR / CAS 9 nuclease, TALAN nuclease, zinc finger nuclease) are used to facilitate the insertion method via genome editing.

[0381] Transgenic loci can target a single locus selected from natural loci, modified natural loci, or transgenic loci (e.g., landing pads). Natural loci can be, for example, GGTA1, β4GalNT2, GRH, CMAH, ROSA26, AAVS1. Natural loci can be modified, i.e., modified natural loci, such as modified (GGTA1, β4GalNT2, GRH, or CMAH).

[0382] In exemplary embodiments, the transgene can target the landing pad and / or docking site or other stable expression sites. In one embodiment, the landing pad or docking vector can be inserted into any site of interest, such as GGTA1, GRH, CMAH, β4Gal, ROSA26, AAVS1, or the transgene can target any known “safe harbor” locus, or any predetermined locus that may provide a beneficial gene expression pattern, or wherein the predetermined locus can also inactivate a preferred gene, wherein simultaneous insertion and knockout are favorable for transplantation outcomes. In another embodiment, gene editing can be used to generate double-strand breaks, thereby initiating DNA repair mechanisms to produce small insertions, deletions, or nucleic acid substitutions (INDELs), resulting in activation or knockout of the gene at the target site; in this case, an INDEL at a predetermined locus (e.g., GGTA1, GRH, CMAH, B4GalNT2) can be generated in a cell or the resulting cloned pig, while a gene-edited enhanced polycistronic vector is knocked in at another locus.

[0383] In specific embodiments, gene editing is used to simultaneously (using multiple CRISPR-Cas9 guide RNAs, TALENs, or ZFNs (or combinations thereof)) inactivate one, two, or three endogenous loci in the porcine genome (e.g., one or all of GGTA1, GRH, CMAH, β4GalNT2), and one or more of these gene-editing enhancement modifications also result in the targeted insertion of a polycistronic vector, such that at least four transgenes are present at one or more such natural loci or modified natural loci under the control of at least two promoters. In some embodiments, the transgenic animal incorporates and expresses at least four transgenes. In some embodiments, two of the four transgenes are expressed as polycistronic vectors controlled by a first promoter, and two of the four transgenes are expressed as polycistronic vectors controlled by a second promoter.

[0384] In some embodiments, two of the four transgenes expressed in the first or second polycistronic cell are selected from the group consisting of TBM, EPCR, DAF, CD39, TFPI, CTLA4-Ig, CIITA-DN, HOI, A20, HLA-E, and CD47. In some embodiments, at least one pair of transgenes is selected from the group consisting of: TBM and CD39; EPCR and DAF; A20 and CD47; TFPI and CD47; CIITAKD and HO-1; TBM and CD47; CTLA4Ig and TFPI; CIITAKD and A20; TBM and A20; EPCR and DAF; TBM and HO-1; TBM and TFPI; CIITA and TFPI; EPCR and HO-1; TBM and CD47; EPCR and TFPI; TBM and EPCR; CD47 and HO-1; CD46 and CD47; CD46 and HO-1; CD46 and TBM; and HLA-E and CD47 .

[0385] 4. Zinc finger nucleases / TALENs

[0386] In one embodiment, the transgene is incorporated using a zinc finger nuclease (ZFN). A zinc finger nuclease is a fusion of a nonspecific DNA-cutting motif and a sequence-specific zinc finger protein. Its nuclease activity is a derivative of the FokI bacterial restriction endonuclease, capable of producing single-strand breaks. ZFNs produce 18 bp-specific double-strand breaks by dimerizing two DNA-binding domains and two FokI enzymes. In another embodiment, the transgene is incorporated via a transcription activator-like effector nuclease (TALEN).

[0387] TALENs function similarly to ZFNs, inducing double-strand breaks by attaching the FokI endonuclease chain to the DNA-binding domain. In this process, TALEN-induced mutagenesis has been reported to achieve a targeting efficiency of 73.1% and a biallelic knockout rate of 27.8%. TALENs differ from ZFNs in that they are easier to gene-design, less expensive, and have a slightly higher targeting frequency.

[0388] In one embodiment, the present invention utilizes the direct injection of ZFN and TALEN into pig zygotes, which can introduce endogenous genes or small insertions or deletions or nucleotide substitutions and produce piglets with the desired genetic modifications.

[0389] 5. CRISPR / CAS9 nuclease

[0390] In another implementation, a CRISPR / Cas9 nuclease is used to incorporate the transgene. CRISPR / Cas9 originates from a bacterial defense mechanism that involves RNA-guided targeting and cleavage of foreign DNA. In bacteria, the foreign DNA is digested and inserted into a CRISPR site, resulting in CRISPR RNA (crRNA). These short RNA sequences then associate with homologous sequences in the genome (which may be foreign sequences). When the homologous genomic sequence has an appropriate “protospacer adjacent motif (PAM)” after its 3' end, the Cas9 nuclease produces a double-strand break. The PAM spacer helps prevent the CRISPR site itself from being targeted. The CRISPR / Cas9 system has also been shown to be useful outside of bacteria and was first used in 2013 to remove αGal from the pig genome. In addition, the CRISPR / Cas9 system has been used to remove the pig growth hormone receptor (Yu et al., J Transl. Med. (2018)). The most commonly used system is derived from *Streptococcus pyogenes*, whose 3' PAM sequence is NGG, where N represents any nucleotide. This system allows for the generation of mutational events in any porcine genome sequence consisting of GN19NGG.

[0391] The CRISPR / Cas9 system can also be used in conjunction with homology-directed repair (HDR), a naturally occurring nucleic acid repair system initiated by double-strand breaks (DSBs) present in DNA (Liang et al. 1998). More specifically, the CRISPR / Cas9 system can be used to generate targeted double-strand breaks, which can be used to control the specificity of HDR genome engineering (Findlay et al. 2014; Mali et al. February 2014; Ran et al. 2013) and can be used to modify the genomes of many organisms, including mammals and humans (Sander and Joung, 2014).

[0392] After RNA-guided cleavage at specific sites in DNA to produce double-strand breaks, a DNA fragment or DNA construct of interest can be inserted. This donor template, fragment, or construct has the desired insertion or modification, flanked by DNA fragments homologous to the blunt ends of the cleaved DNA. Therefore, the cell's natural DNA repair mechanisms can be used to insert desired genetic material, precisely edit the genome of target cells, and utilize homology-driven recombination combined with any known genome editing technology to produce highly targeted double-strand breaks. Genome modifications performed in this manner can be used to insert new genes, termed "enhanced homology-driven insertion or knock-in," which is described as inserting DNA while simultaneously knocking out existing genes (Mali et al. Feb 2013).

[0393] Compared to previous site-specific nucleases, the CRISPR / Cas system offers several advantages. Most importantly, the Cas9 endonuclease represents the first non-stranded DNA cleavage method. It can freely associate with multiple guide RNAs, enabling simultaneous targeting of multiple sites in a single transfection. This allows for the efficient combination of multiple gene knockouts on a single cell. In 2013, homozygous knockout cells of GGTA1, GHR, GGTA1 / iGb3S, GGTA1 / CMAH, and GGTA1 / iGb3S / CMAH were generated in a single reaction. The CRISPR / Cas9 system has been successfully used to generate transgenic animals in various vertebrates, including zebrafish, monkeys, mice, rats, and pigs; see Withworth et al., Biol. Reprod. 91(3):78, pp.1–13 (2014) and Li et al., Xenotransplantation 22(1), pp.20–31 (2015).

[0394] Targeting efficiency, or the percentage of desired mutations achieved, is one of the most important parameters for evaluating genome editing tools. Cas9 exhibits superior targeting efficiency compared to more established methods such as TALEN or ZFN. For example, in human cells, custom-designed ZFNs and TALENs achieve efficiencies ranging from 1% to 50%. Conversely, the Cas9 system has been reported to achieve efficiencies of >70% in zebrafish and plants, and 2–5% in induced pluripotent stem cells.

[0395] In one embodiment, this disclosure can utilize the CRISPR / Cas9 system to generate transgenic pigs (e.g., ungulates, pigs) by microinjecting CRISPR specifically designed for a target gene of interest (e.g., GGTA1, GHR, CMAH, and B4GalNT2) into a zygote of "in vitro" origin. In another embodiment, this disclosure can utilize the CRISPR / Cas9 system to generate transgenic pigs (e.g., ungulates, pigs) by modifying donor somatic cells with CRISPR specifically designed for a gene of interest, followed by SCNT. In yet another embodiment, this disclosure can utilize the CRISPR / Cas9 system to generate transgenic pigs (e.g., ungulates, pigs) by targeting a specific region / sequence of an existing gene modification. More specifically, the sequence targets the neomycin gene sequence.

[0396] In another embodiment, this disclosure may utilize genome editing systems, such as the TALEN system, zinc finger system, or CRISPR / Cas9 system, to produce transgenic pigs (e.g., ungulates, pigs) by targeting specific regions / sequences of existing gene modifications. More specifically, the target may be a single locus of a natural locus, a modified natural locus, or a transgenic locus (e.g., a landing pad).

[0397] In another implementation, the CRISPR / Cas9 system can be used to generate transgenic pigs (e.g., ungulates, pigs) that utilize homology-driven recombination to target specific regions / sequences of existing genetic modifications by inserting large DNA fragments or constructs flanked by arms or segments of DNA homologous to double-strand breaks.

[0398] 6. Site-specific recombinase

[0399] In an exemplary embodiment, site-specific recombinases are used to incorporate transgenes. Site-specific recombinase technology is widely used for deletions, insertions, translocations, and inversions at specific sites in cellular DNA. It allows DNA modifications to be targeted to specific cell types or triggered by specific external stimuli. It has been implemented in both eukaryotic and prokaryotic systems. Several recombination systems can be effectively used for genetic engineering strategies; the Flp-FRT and Cre-loxP recombinase systems are reversible, thus facilitating site-specific integration and excision. Integrases mediate the genome integration process, catalyzing highly site-specific recombination reactions that result in precise DNA integration, excision, and / or inversion. Serine (ФC31, Bxb1, R4) and tyrosine integrases (… P22 and HP1 are two major families of integrases currently used in genome engineering. Broadly speaking, site-specific recombination involves the binding of a recombinase to its substrate, brought together by protein-protein interactions. During this process, the substrate is cleaved, and the DNA ends recombine in a strand exchange reaction, thus the rejoining of the DNA backbone produces the recombination product. In most cases, serine integrases catalyze efficient irreversible recombination using simple att sites.

[0400] To leverage the high efficiency of site-specific recombinases, docking sites or landing pads contain linker sites for recombinase substrate binding, such as the att site; or recombination systems, such as Flp-FRT and Cre-loxP, can be introduced into desired sites in cell lines and / or animal lines. Insertion of the docking vector into the target genome is random or achieved through homologous recombination. This allows for successive rounds of plasmid integration, where the plasmid or vector can contain different transgenes and / or additional DNA sequences. In turn, recombination systems such as Flp / FRT can be used to remove unwanted vector and marker sequences.

[0401] 7. Vectors for producing transgenic animals

[0402] Nucleic acid targeting vector constructs can be designed to achieve homologous recombination in cells. In one embodiment, a targeting vector is designed using promoter capture, wherein integration at the target locus allows the inserted transgenic open reading frame to drive the expression of the inserted gene (or an inserted alternative marker; such as Neo or Puro) using an endogenous or natural promoter. In a specific embodiment, a targeting vector is designed using “poly(A) capture.” Unlike promoter capture, poly(A) capture vectors can capture a wider range of genes, including those not expressed in the target cells (i.e., fibroblasts or ES cells). A polyA capture vector includes a constitutive promoter that drives the expression of an alternative marker gene lacking a polyA signal. The polyA signal is replaced by a splice donor site designed to splice into a downstream exon. In this strategy, the mRNA of the alternative marker gene can be stabilized after capturing the polyA signal of an endogenous gene, regardless of its expression status in the target cells. In one embodiment, a targeting vector is constructed that includes an alternative marker lacking a polyadenylation signal. These targeting vectors can be introduced into mammalian cells by any suitable method, including but not limited to transfection, transformation, virus-mediated transduction, or infection with viral vectors.

[0403] In one embodiment, the targeting vector may contain 3' and 5' recombinant arms (i.e., flanking sequences) homologous to the genomic sequence of interest. The 3' and 5' recombinant arms can be engineered such that they are located flanking the 3' and 5' ends of at least one functional region of the genomic sequence. Targeting the functional region can inactivate it, thereby preventing the cell from producing the functional protein. In another embodiment, the homologous DNA sequence may include one or more intron and / or exon sequences. In addition to the nucleic acid sequence, the expression vector may also contain selectable marker sequences, such as enhanced green fluorescent protein (eGFP) gene sequences, initiation and / or enhancer sequences, poly-A tail sequences, and / or nucleic acid sequences that provide for the expression of the construct in prokaryotic and / or eukaryotic host cells. The selectable marker may be located between the 5' and 3' recombinant arm sequences. Targeted cellular loci can be modified by introducing DNA into cells, wherein the DNA is homologous to the target locus and includes marker genes to allow selection of cells containing integrated constructs. The homologous DNA in the target vector will recombine with chromosomal DNA at the target locus. The marker gene may have homologous DNA sequences, 3' recombinant arms, and 5' recombinant arms on both sides. Methods for constructing targeting vectors have been described in the art; see, for example, Dai et al., Nature Biotechnology 20:251-255, 2002; WO 00 / 51424. In this example, the selectable marker gene may be a promoterless neomycin phosphotransferase (Neo) gene that not only leads to the targeted insertion and expression of Neo (by capturing and utilizing the endogenous porcine αGal gene promoter, or the endogenous porcine GHR promoter), but also leads to the functional inactivation of the target locus (e.g., GGTA1 or GHR) due to the targeted insertion and the disruption of the GGTA1 catalytic domain or GHR.

[0404] Multiple enzymes can catalyze the insertion of foreign DNA into the host genome. Viral integrase, transposase, and site-specific recombinase mediate the integration of viral genomes, transposons, or bacteriophages into the host genome. A large number of enzymes possessing these properties are available from a variety of sources. Retroviruses combine several useful features, including their relatively simple and easy-to-use genomes and their ability to integrate into the host cell genome, thus allowing for long-term expression of transgenes in transduced cells or their progeny. Therefore, they have been used in numerous gene therapy protocols. Lentiviral vectors have been attractive candidates for gene therapy and transgenic applications because adeno-associated viruses (AAVs) are small DNA viruses (parvoviruses) that co-replicate in mammalian cells with helper viruses such as adenoviruses, herpesviruses, or human cytomegaloviruses. The viral genome consists essentially of only two ORFs (rep, a non-structural protein, and cap, a structural protein), from which (at least) seven different polypeptides are derived through alternative splicing and the use of alternative promoters. In the presence of a helper virus, the rep protein mediates the replication of the AAV genome. Integration occurs in the absence of a helper virus, resulting in latent viral infection. Transposons are also of interest. Transposons are mobile DNA segments visible in a variety of organisms. Although active transposons have been found in many prokaryotic systems and insects, functional native transposons are absent in vertebrates. The Drosophila P-element transposon has been used as a genome engineering tool for many years. The Sleeping Beauty transposon was constructed based on a copy of a nonfunctional transposon found in salmon and is significantly more active in mammalian cells than prokaryotic or insect transposons. Site-specific recombinases are enzymes that catalyze the exchange of DNA strands between DNA segments with only a limited degree of sequence homology. They bind to recognition sequences ranging from 30 to 200 nucleotides in length, cleave the DNA backbone, exchange the two DNA double helices involved, and rejoin the DNA. In some site-specific recombination systems, a single polypeptide is sufficient to perform all these reactions, while other recombinases require varying numbers of accessory proteins to accomplish these tasks. Site-specific recombinases can be divided into two families of proteins with different biochemical properties: tyrosine recombinases (in which DNA is covalently linked to tyrosine residues) and serine recombinases (in which covalent linkage occurs at serine residues). The most commonly used enzymes in genome modification methods are Cre (a tyrosine recombinase derived from E. coli phage P1) and phiC31 integrase (a serine recombinase derived from Streptomyces phage phiC31).

[0405] Several other site-specific recombinases derived from bacteriophages (including Flp, λ integrase, bacteriophage HK022 recombinase, bacteriophage R4 integrase, bacteriophage TP901-1 integrase, and bxb1 integrase) have been successfully used to mediate stable gene insertion in mammalian genomes. Recently, a site-specific recombinase has been purified from Streptomyces phage. The phiC31 recombinase, a member of the degradase family, mediates phage integration. In this process, the phage attP site recombines with the corresponding attB site in the bacterial genome. Crossover produces two sites, attL and attR, which are no longer targets of the recombinase in the absence of accessory proteins. This reaction also occurs in mammalian cells and can therefore be used to mediate site-specific integration of therapeutic genes. The site specificity of tyrosine recombinases is difficult to alter through direct protein engineering because the catalytic domain and DNA recognition domain are tightly intertwined. Therefore, changes in specificity are often accompanied by a loss of activity. Serine recombinases may be more suitable for engineering; highly active derivatives of Tn3-degrading enzymes have been engineered by exchanging the natural DBD for the zinc finger domain of the human zinc finger transcription factor Zif268. The DNA site specificity of the resulting chimeric protein (called Z-degrading enzyme) has been converted to the site specificity of Zif268. Zinc finger proteins can be modified through in vitro protein evolution to recognize any DNA sequence; therefore, this approach can be used to develop chimeric recombinases that can integrate therapeutic genes to precise genomic locations. Methods for enhancing or mediating recombination include site-specific recombination and homologous recombination, and combinations of AAV vector-mediated and zinc finger nuclease-mediated recombination (reference: Geurts et al., Science, 325:433, 2009).

[0406] As used herein, the term "vector" refers to a nucleic acid molecule (preferably DNA) that provides useful biological or biochemical properties to an inserted nucleic acid. The "expression vector" according to the invention includes a vector capable of enhancing the expression of one or more molecules inserted into or cloned into the vector when transformed into cells. Examples of such expression vectors include bacteriophages, autonomously replicating sequences (ARS), centromeres, and other sequences capable of replicating or being replicated in vitro or intracellularly, or other sequences that deliver desired nucleic acid segments to desired locations within animal cells. Expression vectors that can be used in this disclosure include chromosomal, free-type, and viral-derived vectors, such as vectors derived from bacterial plasmids or bacteriophages, and vectors derived from combinations thereof, such as granules and phage particles, or virus-based vectors, such as adenoviruses, AAVs, and lentiviruses. The vector may have one or more restriction endonuclease recognition sites at which sequences can be cleaved in a deterministic manner without loss of the vector's essential biological function, and where nucleic acid fragments can be spliced ​​to achieve their replication and cloning.

[0407] The vector can further provide primer sites, such as sites for PCR, transcription and / or translation initiation and / or regulation, recombination signals, replicons, selectable markers, etc. Clearly, the method of inserting the desired nucleic acid fragment does not require the use of homologous recombination; transposable or restriction enzymes (e.g., but not limited to UDG clones of PCR fragments (US Patent No. 5,334,575), TA clone RT-PCR, and clones (Invitrogen Corp., Carlsbad, CA)) can also be used to clone nucleic acids into vectors used according to this disclosure.

[0408] Cells homozygous at a target locus can be generated by introducing DNA into cells, wherein the DNA is homologous to the target locus and includes a marker gene to allow selection of cells containing an integrated construct. The homologous DNA in the target vector will recombine with the chromosomal DNA at the target locus. The marker gene may have homologous DNA sequences, 3' recombination arms, and 5' recombination arms on either side. Methods for constructing targeting vectors have been described in the art; see, for example, Dai et al. (2002) Nature Biotechnology 20:251-255; WO 00 / 51424, FIG. 6; and Gene Targeting: A Practical Approach. Joyner, A. Oxford University Press, USA; 2. sup. nd ed. Feb. 15, 2000. Various constructs can be prepared for homologous recombination at the target locus. Typically, the construct may include a sequence homologous to the target locus of at least 25 bp, 50 bp, 100 bp, 500 bp, 1k bp, 2k bp, 4k bp, 5k bp, 10k bp, 15k bp, 20k bp, or 50k bp.

[0409] Various factors may be involved in determining the degree of homology of the target DNA sequence, such as the size of the target locus, sequence availability, the relative efficiency of double crossover events at the target locus, and the similarity of the target sequence to other sequences. Target DNA may include sequences in which the DNA is substantially homogeneous with the corresponding target sequence in the genome to be modified, flanking the desired gene modification. Substantially homogeneous sequences may have at least about 95%, 97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identity with the corresponding target sequence (excluding the desired sequence modification). Target DNA and target DNA preferably share at least about 75, 150, or 500 base pairs of 100% identical DNA fragments. Therefore, the target DNA may originate from cells closely related to the targeted cell line; or the target DNA may originate from cells of the same cell line or animal as the targeted cells.

[0410] Suitable alternative biomarker genes include, but are not limited to: genes that confer growth ability on certain culture substrates, such as the tk gene (thymidine kinase) or hprt gene (hypoxanthine phosphoribosyltransferase) that confer growth ability on HAT medium (hypoxanthine, aminopterin, and thymidine); and bacterial gpt genes (guanine / xanthine phosphoribosyltransferase) that allow growth on MAX medium (mycophenolate, adenine, and xanthine). See Song et al. (1987) Proc. Nat'l Acad. Sci. USA 84:6820-6824. See also Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, see chapter 16. Other examples of selectable biomarkers include genes that confer resistance to compounds (e.g., antibiotics), genes that confer the ability to grow on selected substrates, and genes that encode proteins that produce detectable signals (e.g., luminescence) (e.g., green fluorescent protein, enhanced green fluorescent protein (eGFP)). Many such biomarkers are known and available, including, for example, antibiotic resistance genes, such as the neomycin resistance gene (neo) (Southern, P., and P. Berg, (1982) J. Mol. Appl. Genet. 1:327-341); and the hygromycin resistance gene (hyg) (Nucleic Acids Research 11:6895-6911 (1983), and Te Riele et al. (1990) Nature 348:649-651).

[0411] Other reporter genes useful in the methods disclosed herein include acetylhydroxylase (AHAS), alkaline phosphatase (AP), β-galactosidase (LacZ), β-glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), carmine base synthase (NOS), octopus base synthase (OCS), and derivatives thereof. A variety of selective biomarkers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, blastomycin, zeocin, methotrexate, phosphatidylin, puromycin, and tetracycline. Methods for determining reporter gene repression are well known in the art, including but not limited to fluorescence methods (e.g., fluorescence spectroscopy, fluorescence-activated cell sorting (FACS), fluorescence microscopy), and antibiotic resistance assays.

[0412] Combinations of selectable markers can also be used. To use a combination of markers, the HSV-tk gene can be cloned and positioned outside the target DNA (another selectable marker can be placed on the opposite flanking side if needed). After the DNA construct is introduced into the cells to be targeted, cells can be selected on an appropriate antibiotic. Selectable markers can also be used for negative selection. Negative selection markers typically kill cells that express them because the expression itself is toxic or produces catalysts that lead to toxic metabolites, such as herpes simplex virus type I thymidine kinase (HSV-tk) or diphtheria toxin A.

[0413] Typically, negative selection markers are integrated into the targeting vector so that they are lost after a precise recombination event. Similarly, conventional selectable markers (e.g., GFP) can be used for negative selection; for example, if a transgene is expressed at a significant level on the cell surface, the insertion of the selected transgene can be sorted using FACS, which can serve as a “selectable marker” for gain or loss of function. Using inserted or targeted transgenes as selection tools allows for positive selection without the use of added fluorescent markers (e.g., GFP, RFP) or antibiotics to select genes. In some cases, targeted insertion of a transgene may inactivate the target locus, allowing for the monitoring or selection of loss of function. For example, inactivation of the GGTA1 locus will eliminate or reduce the binding of the target cell to the lectin (IB4), or inactivation of B4GalNT2 will eliminate or reduce the binding of the DBA lectin to the target cell; in each case, targeted integration can be sorted or enriched in cells lacking this lectin binding.

[0414] Deletions can be at least about 50 bp, more usually at least about 100 bp, and typically not more than about 20 kbp. Deletions can generally include at least a portion of a coding region, including some or more exons, some or more introns, and may or may not include a portion of flanking non-coding regions, particularly the 5' non-coding region (transcriptional regulatory region). Therefore, homologous regions can extend beyond the coding region into the 5' non-coding region or into the 3' non-coding region. Insertions are generally no more than 10 kbp, typically no more than 5 kbp, generally at least 50 bp, and more usually at least 200 bp.

[0415] Homologous regions can include mutations, which can further inactivate the target gene, provide a frameshift, alter key amino acids, or correct dysfunctional alleles. Typically, mutations can be subtle changes, not exceeding about 5% of the homologous flanking sequence, or even single nucleotide changes, such as point mutations at exon active sites. When gene mutation is needed, a marker gene can be inserted into an intron to be excised from the target gene during transcription.

[0416] Various factors may be involved in determining the degree of homology of the target DNA sequence, such as the size of the target locus, sequence availability, the relative efficiency of double crossover events at the target locus, and the similarity of the target sequence to other sequences. The target DNA may include sequences in which the DNA is substantially homogeneous with the corresponding target sequence in the genome to be modified, flanking the desired gene modification. The substantially homogeneous sequence may have at least about 95%, or at least about 97%, or at least about 98%, or at least about 99%, or 95% to 100%, 97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identity with the corresponding target sequence (other than the desired sequence modification). In a specific embodiment, the target DNA and the target DNA may share at least about 75 base pairs, 150 base pairs, or 500 base pairs of 100% identical DNA fragments. Therefore, the target DNA can originate from cells closely related to the targeted cell line; or the target DNA can originate from the same cell line or animal cells as the targeted cells. Constructs can be prepared using methods known in the art, where various fragments can be combined, introduced into a suitable vector, cloned, analyzed, and then further manipulated until the desired construct is obtained. Sequences can be modified in various ways to enable restriction analysis, excision, probe identification, etc. Silent mutations can be introduced as needed. At each stage, restriction analysis, sequencing, polymerase chain reaction amplification, primer repair, in vitro mutagenesis, etc., can be employed.

[0417] Constructs can be prepared using bacterial vectors, including prokaryotic replication systems such as origins recognizable by *E. coli*, and the constructs can be cloned and analyzed at each stage. The same or different markers used for insertion can be used, which can be removed before introduction into target cells. Once the vector containing the construct is complete, it can be further manipulated, such as by deleting bacterial sequences, linearizing, or introducing short deletions in homologous sequences. After final manipulation, the construct can be introduced into cells.

[0418] Techniques that can be used to introduce DNA or RNA constructs into host cells include calcium phosphate / DNA coprecipitation, DNA microinjection into the cell nucleus, electroporation, fusion of bacterial protoplasts with intact cells, transfection, lipid transfection, infection, particle bombardment, or any other technique known to those skilled in the art. The DNA or RNA may be single-stranded or double-stranded, linear or circular, relaxed or supercoiled. For various techniques for transfecting mammalian cells, see, for example, Keown et al., Methods in Enzymology Vol. 185, pp. 527-537 (1990).

[0419] The following vectors are provided by way of example. Bacteria: pBs, pQE-9 (Qiagen), phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR54O, pRIT5 (Pharmacia). Eukaryotes: pWLneo, pSv2cat, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPv, pMSG, pSVL (Pharmiacia). Furthermore, any other plasmids and vectors may be used, provided they can replicate and survive in the host. Vectors known in the art and commercially available vectors (and their variants or derivatives) can be engineered according to the invention to include one or more recombination sites for use in the methods of the invention.

[0420] Such vectors are available from, for example, Vector Laboratories Inc., Invitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter, OriGenes Technologies Inc., Stratagene, PerkinElmer, Pharmingen, and Research Genetics. Other vectors of interest include eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen); pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech); pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.); p3'SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and pOG44 (Stratagene, Inc.); and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBlueBaclll, pCDM8, pcDNA1, pZeoSV, and pcDNA3. pREP4, pCEP4 and pEBVHis (Invitrogen, Corp.) and their variants or derivatives.

[0421] Other vectors include pUC18, pUC19, pBlueScript, pSPORT, granules, phage particles, YAC (yeast artificial chromosome), BAC (bacterial artificial chromosome), P1 (Escherichia coli phage), pQE70, pQE60, pQE9 (quagan), pBS vector, PhageScript vector, BlueScript vector, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0, and pSY-SPORT1 (Invitrogen) and their variants or derivatives. Viral vectors, such as lentivirus vectors, can also be used (see, for example, WO 03 / 059923; Tiscornia et al. PNAS100:1844-1848(2003)).

[0422] Other vectors of interest include pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1 / His, pcDNA3.1(-) / Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO81S, pPICZ, pPICZA, pPICZB, pPICZC, pGAPZA, pGAPZB, pGAPZC, pBlueBac4.5, pBlueBacHis2, and pM from Invitrogen. elBac, pSinRep5, pSinHis, pIND, pIND(SP1), pVgRXR, pcDNA2.1, pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1 392. pVL1393, pCDM8, pcDNA1.1, pcDNA1.1 / Amp, pcDNA3.1, pcDNA3.1 / Zeo, pSe, SV2, pRc / CMV2, pRc / RSV, pREP4, pREP7, pREP8, pREP9, pREP 10. pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni and pCRBac; .lamda.ExCell, .lamda.gt11, pTrc99A from Pharmacia , pKK223-3, pGEX-1.lamda.T, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1, pGEX- 5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCH110, pKK232-8, pSL1180, pNEO, and pUC4K from Novagen; pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4-abc(+), pOCUS-2, pTAg, pET-32L1C, pET-30LIC, pBAC-2cp LIC, pBACgus-2cp LIC, pT7Blue-2 LIC, pT7Blue-2, .lamda.SCREEN-1, .lamda.BlueSTAR, pET-3abcd, pET-7abc, pET9abcd, pET11 abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb, pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+), pET-23abcd(+), pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+), pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+), pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3cp, pBACgus-2cp, pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg and Selecta Vecta-Gpt; pLexA, pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGADGH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1, pEGFP--N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2-Basic, pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, p.beta.gal-Basic, p.beta.gal-Control, p.beta.gal-Promoter, p.beta.gal-Enhancer, pCMV, pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1 / 2 / 3, pYEX-S1, pBacPAK-His, pBacPAK8 / 9, pAcUW31, BacPAK6, pTrip1Ex, 2.lamda.gt10, .lamda.gt11, pWE15 and .lamda.Trip1Ex; Lambda ZAP II, pBK-CMV, pBK-RSV, pBluescript II KS+ / -, pBluescript II SK+ / -, pAD-GAL4, pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script from Stratagene Cam, pCR-Script Direct, pBS+ / -, pBC KS+ / -, pBC SK+ / -, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI / MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44, pOG45, pFRT.beta.GAL, pNEO.beta.GAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415 and pRS416. .

[0423] Other vectors include, for example, pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp, and their variants or derivatives.

[0424] In an exemplary embodiment, the vector is a bicistronic vector. A bicistronic vector contains a promoter and two transgenes. In a specific embodiment, the bicistronic vector contains a promoter and two transgenes linked by a 2A sequence. This embodiment enables the co-expression of multiple functional transgenes from a single promoter. More specifically, this embodiment utilizes a short (18-24aa) cleavage peptide "2A," which allows for the co-expression of linked open reading frames to express functional transgenes from a single transcript 2A vector system.

[0425] In an exemplary embodiment, the vector is a polycistronic vector (MCV). In one embodiment, the MCV contains a promoter and at least four transgenes. In a specific embodiment, the MCV contains four transgenes linked by a 2A peptide sequence, under the control of at least two promoters. This embodiment allows for the co-expression of multiple functional transgenes from a single transcript. More specifically, this embodiment utilizes a short (18-24aa) cleaved peptide "2A," which allows for the co-expression of linked open reading frames to express functional transgenes from a single transcript 2A vector system.

[0426] In an exemplary embodiment, the vector is a 2A-peptide MCV vector containing at least two bicistronic units, each containing two transgenes. In a specific embodiment, one bicistronic unit is controlled by a constitutive or ubiquitous promoter (e.g., CAG), and the second bicistronic unit is controlled by an endothelial or other tissue-specific or inducible promoter system. In one embodiment, at least four transgenes are inserted at a single locus, but each transgene is controlled by its own promoter or the insertion at each single locus is controlled by at least two promoters in total. In some embodiments, a transgenic animal incorporates and expresses four transgenes, two of which are expressed as polycistronic (bicistronic units) controlled by a first promoter, and two of which are expressed as polycistronic (bicistronic units) controlled by a second promoter.

[0427] In some implementations, two of the four transgenes expressed in the first polycistronic or second polycistronic (bicistronic unit) are selected from the group consisting of TBM, EPCR, DAF, CD39, TFPI, CTLA4-Ig, CIITA-DN, HOI, A20, and CD47. In some implementations, at least one pair of transgenic polycistronic (bicistronic) units is selected from the group consisting of: TBM and CD39; EPCR and DAF; A20 and CD47; TFPI and CD47; CIITAKD and HO-1; TBM and CD47; CTLA4Ig and TFPI; CIITAKD and A20; TBM and A20; EPCR and DAF; TBM and HO-1; TBM and TFPI; CIITA and TFPI; EPCR and HO-1; TBM and CD47; EPCR and TFPI; TBM and EPCR; CD47 and HO-1; CD46 and CD47; CD46 and HO-1; and CD46 and TBM.

[0428] In an exemplary embodiment, the vector is a 4-gene MCV containing at least two anticoagulants, and more specifically at least three anticoagulants. In an exemplary embodiment, the vector is a 4-gene MCV vector containing at least two anticoagulants and a complement inhibitor, and more specifically, three anticoagulants and a complement inhibitor. In an exemplary embodiment, the vector is a 4-gene MCV vector containing two anticoagulants, a complement inhibitor, and an immunosuppressant.

[0429] 8. Promoter

[0430] Vector constructs used to generate animals according to the present invention may include regulatory sequences, including but not limited to promoter-enhancer sequences operatively linked to such sequences, "2A" peptide technologies, and docking vectors. A wide range of suitable vectors and promoters are known to those skilled in the art and are commercially available.

[0431] In specific embodiments, this disclosure provides animals, tissues, and cells that express at least one transgene (in combination with at least one transgene controlled by a second, identical or different promoter) in endothelial cells, and more specifically, animals that express at least two, three, or four transgenes in endothelial cells. To target expression to a specific tissue, animals are developed using vectors containing endothelial cell expression-specific promoters. In specific embodiments, expression is controlled by promoters that are primarily active in the endothelium. In one embodiment, the nucleic acid construct contains a regulatory sequence operatively linked to the transgene sequence to be expressed.

[0432] In one implementation, the regulatory sequence may be a promoter sequence. In another implementation, the promoter may be a tunable promoter. In such systems, for example, the expression of the peptide in an animal, tissue, or organ can be regulated using a drug. For example, expression can be blocked when the organ or tissue is part of a pig, but once the pig has been transplanted into a human for some time, expression can be induced to overcome the cellular immune response. Furthermore, the expression level can be controlled by a tunable promoter system to ensure that immunosuppression of the receptor's immune system does not occur.

[0433] Adjustable promoter systems can be selected from, but are not limited to, the following gene systems: metallothionein promoters, which can be induced by metals (e.g., copper) (see Lichtlen and Schaffner, Swiss Med. Wkly., 2001, 131(45-46): 647-52); tetracycline regulatory systems (see Imhof et al., J Gene Med., 2000, 2(2): 107-16); ecdysone regulatory systems (see Saez et al., Proc Natl Acad Sci USA., 2000, 97(26): 14512-7); cytochrome P450 inducible promoters, such as the CYP1A1 promoter (see Fujii-Kuriyama et al., FASEB J., 1992, 6(2): 706-10); mifepristone inducible systems (see Sirin and Park, Gene., 2003, 323:67-77); coumarin activation system (see Zhao et al., Hum Gene Ther., 2003, 14(17):1619-29); macrolide induction system (responsive to macrolide antibiotics (e.g., rapamycin, erythromycin, clarithromycin and roxithromycin)) (see Weber et al., Nat Biotechnol., 2002, 20(9):901-7; Wang et al., Mol Ther., 2003, 7(6):790-800); ethanol induction system (see Garoosi et al., J Exp Bot., 2005, 56(416):163542; Roberts et al., Plant Physiol., 2005, 138(3):1259-67); streptocin induction system (see Fussenegger et al., 2003, 14(17):1619-29); macrolide ...20(9):901-7; Wang et al., Mol Ther., 2003, 7(6):790-800); ethanol induction system (see Garoosi et al., J Exp Bot., 2005, 56(416):163542; Roberts et al., Plant Physiol., 2005, 138(3):1259-67); streptocin induction system (see Fussenegger et al., 2003, 138(3):1259-67); streptocin induction system (see al.,Nat Biotechnol.,2000 18(11):1203-8); electrophilic induction system (see Zhuand Fahl,Biochem Biophys Res Commun.,2001,289(1):212-9); nicotine induction system (see Malphettes et al.,Nucleic Acids Res.,2005,33(12):e107), immune induction promoter, cytokine response promoter (e.g. promoters induced by IFN-γ, TNF-α, IL-1, IL-6 or TGF-β (or other secondary pathways), and thus can be associated with or activated or upregulated in response to immune or inflammatory responses).

[0434] In specific embodiments, a bicistronic vector comprises two transgenes and a promoter that is primarily active in endothelial cells, or a constitutive promoter that expresses the transgenes ubiquitously in all organs, tissues, and cells. In other embodiments, at least four transgenes in a polycistronic vector (MCV) are controlled by at least two promoters. The promoters may be exogenous, natural, or a combination of exogenous and natural. In some embodiments, the first and second promoters are different.

[0435] In specific embodiments, a bicistronic vector includes two transgenes and a constitutive promoter that expresses the transgenes ubiquitously in all organs, tissues, and cells. In specific embodiments, a bicistronic vector includes two transgenes and a tissue-specific promoter that controls expression in organs, tissues, and cells. In an exemplary embodiment, the vector is a quadrigene MCV containing at least two anticoagulants controlled by an endothelial cell-specific promoter. In an exemplary embodiment, the vector is a quadrigene MCV containing at least one complement inhibitor transgene controlled by a constitutive promoter and at least one anticoagulant transgene controlled by an endothelial cell-specific promoter. In an exemplary embodiment, the vector is a quadrigene MCV containing at least one complement inhibitor transgene controlled by a constitutive promoter and at least one anticoagulant gene controlled by a second constitutive promoter. In an exemplary embodiment, the vector is a quadrigene MCV vector containing an anticoagulant transgene and an immunosuppressive transgene controlled by an endothelial cell promoter. In another exemplary embodiment, the vector is a six-gene MCV vector containing an anticoagulant transgene controlled by an endothelial cell promoter, an immunosuppressive transgene controlled by a constitutive promoter, a complement-regulating transgene controlled by a constitutive promoter, and a cell-protective transgene controlled by a constitutive promoter.

[0436] In an exemplary embodiment, the vector is a dual-gene MCV vector containing a total of two genes under the control of at least two independent promoters; or in a selected embodiment, the vector has a cascade of multiple transgenes, each having its own promoter, and all integrated into a single locus.

[0437] In other implementations, the nucleic acid construct uses enhancer elements to promote increased transgene expression in a tissue-specific manner. Enhancers are external elements that significantly alter gene transcription efficiency (Molecular Biology of the Gene, Fourth Edition, pp. 708-710, Benjamin Cummings Publishing Company, Menlo Park, Calif. COPYRGT. 1987). In specific implementation schemes, the pdx-1 enhancer (also known as IPF-1, STF-1, and IDX1) is used in the following implementation schemes: Gerrish K et al., Mol. Endocrinol., 2004, 18(3): 533; Ohlsson et al., EMBO J. 1993 Nov., 12(11): 4251-9; Leonard et al., Mol. Endocrinol., 1993, 7(10): 1275-83; Miller et al., EMBO J., 1994, 13(5): 1145-56; Serup et al., Proc Natl Acad SciUSA., 1996, 93(17): 9015-20; Meloul et al., Diabetes, 2002, 51 Suppl 3: S320-5; Glicket et al., J Biol Chem., 2000, 275(3): 2199-204; GenBank AF334615.)) is used in combination with the ins2 promoter for pancreas-specific expression of transgenes.

[0438] In some embodiments, animals express transgenes under the control of a combination of promoter and enhancer elements. In specific embodiments, animals express transgenes under the control of an endothelial-specific promoter selected from the TBM promoter, EPCR promoter, ICAM-2, and / or Tie-2. In specific embodiments, animals express transgenes under the control of an endothelial-specific promoter selected from the porcine TBM (pTBMpr) promoter, porcine EPCR promoter (pEPCRpr), porcine ICAM-2, and / or mouse Tie-2 promoter. In some embodiments, the endothelial promoter further comprises an enhancer element (e.g., a mouse Tie-2 enhancer or a CMV enhancer). In other embodiments, the promoter may be a ubiquitous promoter element that further comprises an enhancer element. In certain elements, the ubiquitous promoter is a combination of a CAG (CMV enhancer, chicken β-actin promoter, rabbit β-globin intron) and an endothelial-specific porcine TBM promoter (pTBMpr) and / or an endothelial-specific Tie-2 enhancer element (Tie2-CAG). For Tie2-CAG, the transgene is expected to be expressed constitutively and ubiquitously, but at higher levels in endothelial cells than in other somatic cells. In some embodiments, the promoter is used in combination with an enhancer element, which is a non-coding or intronic region of DNA intrinsically associated with or co-located with the promoter. In another specific embodiment, the enhancer element is ICAM-2 used in combination with the ICAM-2 promoter. Other ubiquitous promoters include, but are not limited to, the following: viral promoters (such as CMV and SV40), as well as chicken β-actin and γ-actin promoters, GAPDH promoters, H2K, CD46 promoters, GGTA1, ubiquitin, and ROSA promoters.

[0439] 9. Selection of Genetically Modified Cells

[0440] In some cases, the genetic modifications present in transgenic cells are the result of targeted insertion or integration (i.e., via homologous recombination) into the cell genome. In other cases, the genetic modifications present in transgenic cells are the result of non-targeted (random) integration into the cell genome. Cells can be grown in appropriately selected media to identify cells that provide appropriate integration. Those cells displaying the desired phenotype can then be further analyzed by restriction analysis, electrophoresis, Southern blotting, polymerase chain reaction, or another technique known in the art. By identifying fragments showing appropriate insertion at the target gene site (or, in non-targeted applications, where random integration techniques produce the desired results), cells in which homologous recombination (or strong non-targeted integration events) have occurred to inactivate or otherwise modify the target gene can be identified.

[0441] The presence of selectable marker genes or other positive selectors or transgenes confirms the integration of the target construct into the host genome. Cells displaying the desired phenotype can then be further analyzed using restriction digestion, electrophoresis, Southern blotting, polymerase chain reaction, etc., to determine whether homologous or non-homologous recombination has occurred. This can be determined by using probes of the insert and then sequencing the 5' and 3' regions flanking the insert to identify the presence of genes extending beyond the construct's flanking regions or identifying deletions (in the case of such deletions). Primers complementary to sequences inside the construct and to sequences outside the construct and at the target locus can also be used. Thus, a DNA duplex containing both the primer and the complementary strand can only be obtained if homologous recombination has occurred. For example, the presence of the primer sequence or the expected-size sequence can demonstrate that homologous recombination has occurred.

[0442] Polymerase chain reactions used for screening homologous recombination events are described in Kim and Smithies, (1988) Nucleic Acids Res. 16:8887-8903; and Joyner et al. (1989) Nature 338:153-156. Cell lines obtained in the first round of targeted (or untargeted (random) integration into the genome) may be heterozygous for the integrated allele. Homozygosity with both alleles modified can be achieved in several ways. One approach is to culture multiple cells with one copy already modified and then perform another round of targeted (or untargeted (random) integration) on these cells using different selectable markers. Alternatively, homozygosity can be obtained by breeding animals that are heterozygous for the modified allele. In some cases, two different modified alleles may be required. This can be achieved through successive rounds of gene targeting (or random integration) or by breeding heterozygotes (each carrying one of the desired modified alleles). Highly efficient genome editing events targeting double-strand breaks enable frequent biallelic targeting events, allowing for high-frequency homozygous knockout or knock-in events in a single transfection (or embryonic or zygotic targeting strategy). Such gene editing-enhanced (e.g., Crispr-CAS9 nuclease) gene targeting or homology-dependent repair events can include monoallelic or heterozygous and biallelic or homozygous knockouts (via small nucleotide insertions, deletions, substitutions, also known as INDELs), and gene insertions, including monoallelic and biallelic insertions / knock-ins of single transgenes, multiple transgene strings (transgene strings under their own promoters or bicistronic or polycistronic promoters), or polycistronic vectors (including 4-transgene polycistronic vectors controlled by at least two promoters), wherein the promoters can be constitutive or tissue-specific, such as CAG and Icam-2.

[0443] Alternatively, by using multiple gene-editing nucleases (e.g., CRISPR / Cas9), it is expected that cells (by transfection or infection) or zygotes (simultaneously by microinjection) combining basic genotypes (i.e., GHR knockout, GGTA1 knockout, GHR / CD46 gene knockout, GGTA1 / CD46, or GGTA1 / GHR / CD46) can be efficiently generated, where one gene modification may include knock-in (e.g., at GGTA1; GHR) or random insertion of 4 gene MCVs (under the control of at least two promoters), and simultaneously, INDELs mediated by any nuclease at another locus (monolithic or bialeic, e.g., at GGTA1, GHR, CMAH, or B4GalNT2). In a preferred embodiment, a multi-transgenic vector (bicistronic or 4-gene MCV) is targeted and inserted at two different loci (e.g., landing pad, safe harbor, or GGTA1, GHR, B4GalNT2, CMAH, ROSA26, AAVS1, or other predetermined loci, including natural loci or modified natural loci). In some embodiments, a 4-gene MCV is targeted and inserted at GGTA1, and a homologous recombination (or gene-edited enhanced) bicistronic or 4-gene MCV is targeted and inserted at a second locus (e.g., CMAH, GHR, or B4GalNT2). In some embodiments, ...

Claims

1. Organs, organ fragments, tissues, or cells derived from transgenic pigs, wherein the transgenic pigs comprise: (a) A genetic alteration that results in decreased expression of the growth hormone receptor (GHR) gene or impairs GHR function; wherein the genetic alteration causes a mutation in at least one allele of the GHR gene and the genetic alteration comprises a deletion in a target region of the GHR gene specified by two single-guide RNAs, and wherein the two single-guide RNAs are selected from SEQ ID NO:2 and SEQ ID NO:4, SEQ ID NO:2 and SEQ ID NO:11, or SEQ ID NO:2 and SEQ ID NO:13; and (b) One or more additional gene alterations, wherein the one or more additional gene alterations result in the expression of one or more transgenes, and wherein the transgenes are immunomodulatory transgenes selected from CD47, HLA-E, CTLA4-Ig or CIITA-DN and any combination thereof; The cells described therein are not stem cells, progenitor cells or germ cells; and the tissue does not contain embryonic tissue.

2. The organ, organ fragment, tissue, or cell according to claim 1, wherein the gene alteration is a GHR knockout gene alteration.

3. The organ, organ fragment, tissue, or cell according to claim 1, wherein the GHR expression of the transgenic pig is reduced by at least 30% compared to a pig without the genetic alteration.

4. The organ, organ fragment, tissue, or cell according to claim 1, wherein the GHR expression of the transgenic pig is reduced by at least 40% compared to a pig without the genetic alteration.

5. The organ, organ fragment, tissue, or cell according to claim 1, wherein the GHR expression of the transgenic pig is reduced by at least 50% compared to a pig without the genetic alteration.

6. The organ, organ fragment, tissue, or cell of claim 1, wherein the transgenic pig exhibits at least 60% reduced GHR expression compared to a pig without the genetic alteration.

7. The organ, organ fragment, tissue, or cell of claim 1, wherein the transgenic pig exhibits at least 70% reduced GHR expression compared to a pig without the genetic alteration.

8. The organ, organ fragment, tissue, or cell of claim 1, wherein the GHR expression of the transgenic pig is reduced by at least 75% compared to a pig without the genetic alteration.

9. The organ, organ fragment, tissue, or cell of claim 1, wherein the transgenic pig exhibits at least 80% reduced GHR expression compared to a pig without the genetic alteration.

10. The organ, organ fragment, tissue, or cell of claim 1, wherein the transgenic pig exhibits at least 85% reduced GHR expression compared to a pig without the genetic alteration.

11. The organ, organ fragment, tissue, or cell of claim 1, wherein the GHR expression of the transgenic pig is reduced by at least 90% compared to a pig without the genetic alteration.

12. The organ, organ fragment, tissue, or cell of claim 1, wherein the GHR expression of the transgenic pig is reduced by at least 95% compared to a pig without the genetic alteration.

13. The organ, organ fragment, tissue, or cell of claim 1, wherein the transgenic pig produces at least 30% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

14. The organ, organ fragment, tissue, or cell of claim 13, wherein the transgenic pig produces at least 40% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

15. The organ, organ fragment, tissue, or cell of claim 13, wherein the transgenic pig produces at least 50% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

16. The organ, organ fragment, tissue, or cell of claim 13, wherein the transgenic pig produces at least 60% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

17. The organ, organ fragment, tissue, or cell of claim 13, wherein the transgenic pig produces at least 70% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

18. The organ, organ fragment, tissue, or cell of claim 13, wherein the transgenic pig produces at least 75% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

19. The organ, organ fragment, tissue, or cell of claim 13, wherein the transgenic pig produces at least 80% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

20. The organ, organ fragment, tissue, or cell of claim 13, wherein the transgenic pig produces at least 85% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

21. The organ, organ fragment, tissue, or cell of claim 13, wherein the transgenic pig produces at least 90% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

22. The organ, organ fragment, tissue, or cell of claim 13, wherein the transgenic pig produces at least 95% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

23. The organ, organ fragment, tissue, or cell according to claim 1, wherein: (a) The two guide RNAs are SEQ ID NO: 2 and SEQ ID NO: 4; or (b) The transgenic pig contains the nucleic acid sequence of SEQ ID NO:

5.

24. The organ, organ fragment, tissue, or cell of claim 1, wherein the one or more additional gene alterations further result in (i) decreased expression of one or more genes, and / or (ii) impaired function of one or more genes.

25. The organ, organ fragment, tissue, or cell of claim 1, wherein one or more transgenes further comprises a transgene selected from the group consisting of anticoagulants, complement regulators, immunomodulators, and cell-protective transgenes.

26. The organ, organ fragment, tissue, or cell of claim 25, wherein the anticoagulant is selected from TBM, TFPI, EPCR, and CD39.

27. The organ, organ fragment, tissue, or cell of claim 25, wherein the complement regulator is a complement inhibitor.

28. The organ, organ fragment, tissue, or cell of claim 27, wherein the complement inhibitor is selected from CD46, CD55, or CD59.

29. The organ, organ fragment, tissue, or cell of claim 25, wherein one or more transgenes are encoded by a single vector.

30. The organ, organ fragment, tissue, or cell of claim 29, wherein the flanking arm of the single vector is a targeting arm for homology-directed repair (HDR) at the GGTA1 locus and comprises: (a) The porcine TBM promoter that drives TBM transgene and EPCR transgene expression; (b) The CAG promoter driving the expression of CD47 and HO-1 transgenes; and (c) The CAG promoter that drives the expression of CD46 and CD55 transgenes.

31. The organ, organ fragment, tissue, or cell of claim 24, wherein one or more transgenes are selected from CD47, CD46, DAF / CD55, TBM, EPCR, HLA-E, and HO1.

32. The organ, organ fragment, tissue, or cell according to claim 24, wherein the transgenic pig further lacks the expression of α1,3-galactosyltransferase, β-1,4-N-acetyl-galactosylaminotransferase 2 (β4GalNT2), and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH).

33. An organ, organ fragment, tissue, or cell derived from a transgenic pig, said transgenic pig comprising a genetic alteration that causes decreased expression of the insulin-like growth factor 1 (IGF-1) gene, wherein said genetic alteration causes a mutation in at least one allele of the growth hormone receptor (GHR) gene and said genetic alteration comprises a deletion in a target region of said GHR gene specified by two single-guide RNAs, and wherein said two single-guide RNAs are SEQ ID NO: 2 and SEQ ID NO: 4, SEQ ID NO: 2 and SEQ ID NO: 11, or SEQ ID NO: 2 and SEQ ID NO: 13; and The cells described therein are not stem cells, progenitor cells or germ cells; and the tissue does not contain embryonic tissue.

34. The organ, organ fragment, tissue, or cell of claim 33, wherein the transgenic pig produces at least 30% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

35. The organ, organ fragment, tissue, or cell of claim 33, wherein the transgenic pig produces at least 40% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

36. The organ, organ fragment, tissue, or cell of claim 33, wherein the transgenic pig produces at least 50% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

37. The organ, organ fragment, tissue, or cell of claim 33, wherein the transgenic pig produces at least 60% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

38. The organ, organ fragment, tissue, or cell of claim 33, wherein the transgenic pig produces at least 70% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

39. The organ, organ fragment, tissue, or cell of claim 33, wherein the transgenic pig produces at least 75% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

40. The organ, organ fragment, tissue, or cell of claim 33, wherein the transgenic pig produces at least 80% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

41. The organ, organ fragment, tissue, or cell of claim 33, wherein the transgenic pig produces at least 85% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

42. The organ, organ fragment, tissue, or cell of claim 33, wherein the transgenic pig produces at least 90% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

43. The organ, organ fragment, tissue, or cell of claim 33, wherein the transgenic pig produces at least 95% less insulin-like growth factor 1 (IGF-1) compared to a pig without the genetic alteration.

44. An organ, organ fragment, tissue, or cell derived from a transgenic pig, said transgenic pig comprising at least four transgenes, wherein said at least four transgenes are incorporated and expressed at a single locus and under the control of at least two promoters, and one of the promoters is an endothelial cell-specific promoter selected from the TBM promoter or the EPCR promoter, and wherein said pig lacks the expression of α1,3-galactosyltransferase, growth hormone receptor, β-1,4-N-acetyl-galactosylaminotransferase 2 (β4GalNT2), and cytidine monophosphate-N-acetylneuraminidase (CMAH); and The pigs described herein contain a deletion in the target region of the GHR gene specified by two single-guide RNAs selected from SEQ ID NO: 2 and SEQ ID NO: 4, SEQ ID NO: 2 and SEQ ID NO: 11, or SEQ ID NO: 2 and SEQ ID NO: 13; and The cells described therein are not stem cells, progenitor cells or germ cells; and the tissue does not contain embryonic tissue.

45. The organ, organ fragment, tissue, or cell according to claim 44, wherein the single gene locus is: (i) Natural gene locus; or (ii) Modified natural loci.

46. ​​The organ, organ fragment, tissue, or cell according to claim 44, wherein the single gene locus is: - Select the group consisting of AAVS1, ROSA26, CMAH, β4GalNT2, and GGTA1; -Natural GGTA1 locus; - Modified GGTA1 locus; -Natural CMAH locus; - Modified CMAH locus; -Not the GGTA1 locus; -Natural β4GalNT2 locus; or - Modified β4GalNT2 locus.

47. The organ, organ fragment, tissue, or cell according to claim 44, wherein the single gene locus is: -Transgenic GGTA1 locus; -Transgenic CMAH locus; or -Transgenic β4GalNT2 locus.

48. The organ, organ fragment, tissue, or cell of claim 45, wherein the modified natural locus comprises a gene editing-mediated insertion, deletion, or substitution.

49. The organ, organ fragment, tissue, or cell of claim 45, wherein the modified natural locus comprises transgenic DNA.

50. The organ, organ fragment, tissue, or cell of claim 49, wherein the transgenic DNA comprises: (i) a selectable marker gene; and (ii) a landing pad.

51. The organ, organ fragment, tissue, or cell according to claim 44, wherein at least one of the promoters is an exogenous promoter, a constitutive promoter, or a tunable promoter.

52. The organ, organ fragment, tissue, or cell according to claim 51, wherein the adjustable promoter is a tissue-specific promoter or an inducible promoter.

53. The organ, organ fragment, tissue, or cell of claim 44, wherein the at least four transgenes are expressed as a first polycistronic and a second polycistronic, and wherein the at least two promoters comprise a first promoter controlling the expression of the first polycistronic and a second promoter controlling the expression of the second polycistronic.

54. The organ, organ fragment, tissue, or cell of claim 44, wherein the transgenic pig comprises at least four promoters, and wherein each of the at least four transgenes is controlled by a specific promoter.

55. The organ, organ fragment, tissue, or cell according to claim 53, wherein: (i) The first promoter is different from the second promoter; (ii) The first promoter is a constitutive promoter, and the second promoter is a tissue-specific promoter; or (iii) The at least two promoters include the CAG and pTBM promoters.

56. The organ, organ fragment, tissue, or cell of claim 45, wherein the endothelial cell-specific promoter is the TBM promoter.

57. The organ, organ fragment, tissue, or cell of claim 44, wherein the at least four transgenes are selected from the group consisting of anticoagulants, complement inhibitors, immunomodulators, cell-protective transgenes, and combinations thereof.

58. The organ, organ fragment, tissue, or cell according to claim 57, wherein: (i) The anticoagulant is selected from the group consisting of TBM, TFPI, EPCR, CD39 and combinations thereof; (ii) The complement inhibitor is selected from the group consisting of CD46, CD55, CD59 and combinations thereof; (iii) The immunomodulatory agent is an immunosuppressant selected from CD47, HLA-E, CTLA4-Ig, CIITA-DN and combinations thereof; (iv) The immunomodulator is CD47; (v) The cell-protective transgenes are selected from the group consisting of HO-1, A20 and combinations thereof.

59. The organ, organ fragment, tissue, or cell according to claim 44, wherein: (i) At least two of the genetically modified organisms are anticoagulants; (ii) At least one of the genetically modified organisms is a cell-protective genetically modified organism; (iii) At least one of the genetically modified organisms is immunomodulatory; or (iv) At least one of the genetically modified organisms is a complement inhibitor.

60. The organ, organ fragment, tissue, or cell of claim 44, wherein the transgenic pig further comprises at least one additional genetic modification.

61. The organ, organ fragment, tissue, or cell of claim 60, wherein the at least one additional gene modification is selected from the group consisting of the deletion, insertion, or substitution of a gene, gene fragment, or nucleotide; or combinations thereof.

62. The organ, organ fragment, tissue, or cell of claim 60, wherein the at least one additional gene modification is selected from the group consisting of gene knockout; gene knock-in; gene substitution; point mutation; or combinations thereof.

63. The organ, organ fragment, tissue, or cell according to claim 60, wherein the at least one additional gene modification is: (i) is a large genome insertion; (ii) Incorporation and expression of human CD46; (iii) Incorporation and expression of human HLA-E; (iv) Knockout of the B4GalNT2 gene; or (v) Knockout of the CMAH gene.

64. The organ, organ fragment, tissue, or cell of claim 60, wherein the at least one additional gene modification results in the elimination or reduction of expression of at least one natural gene.

65. The organ, organ fragment, tissue, or cell of claim 60, wherein the at least one additional gene modification comprises the incorporation and expression of at least two additional transgenes.

66. The organ, organ fragment, tissue, or cell of claim 60, wherein the at least one additional gene modification comprises at least one of the following incorporation and expression: Two additional transgenes at the second single locus; or Four additional transgenes at the second single locus.

67. The organ, organ fragment, tissue, or cell according to claim 66, wherein: (i) The single locus is GGTA1, and the second single locus is CMAH; (ii) The single locus is β4GalNT2, and the second single locus is CMAH; (iii) The single locus is CMAH, and the second single locus is β4GalNT2; or (iv) The single locus is GGTA1, and the second single locus is β4GalNT2.

68. The organ, organ fragment, tissue, or cell according to claim 64, wherein the at least one natural gene is selected from the group consisting of CMAH, isoerythrocyte glycoside 3 synthase, β4GalNT2, Forrsman synthase, or combinations thereof.

69. The organ, organ fragment, tissue, or cell according to claim 44, wherein the transgenic pig expresses: (a) CD46; and (b) A combination of at least four genetically modified organisms, selected from: (i) EPCR, HO-1, TBM and CD47; (ii) EPCR, HO1, TBM, and TFPI; (iii) EPCR, CD55, TFPI and CD47; (iv) EPCR, DAF, TFPI, and CD47; or (v) EPCR, CD55, TBM and CD39.

70. The organ, organ fragment, tissue, or cell of claim 53, wherein two of the four transgenes expressed in the first or second polycistronic cell are selected from the group consisting of TBM, EPCR, DAF, CD39, TFPI, CTLA4-Ig, CIITA-DN, HOI, A20, HLA-E, and CD47.

71. The organ, organ fragment, tissue, or cell according to claim 53, wherein at least one pair of transgenes is selected from the group consisting of: (a) TBM and CD39; (b) EPCR and DAF; (c) A20 and CD47; (d) TFPI and CD47; (e) CIITAKD and HO-1 (f) TBM and CD47; (g) CTLA4Ig and TFPI; (h) CIITAKD and A20; (i) TBM and A20; (j) EPCR and DAF; (k) TBM and HO-1; (l) TBM and TFPI; (m) CBTA and TFPI; (n) EPCR and HO-1; (o) TBM and CD47; (p) EPCR and TFPI; (q) TBM and EPCR; (r)CD47 and HO-1; (s)CD46 and CD47; (t)CD46 and HO-1; (u)CD46 and TBM; and (v) HLA-E and CD47.

72. The organ, organ fragment, tissue, or cell of claim 44, wherein the transgenic pig lacks expression of growth hormone receptor and comprises a genotype selected from the following: (i) GTKO.CD46.pTBMpr-TBM.CD39-cag-A20.CD47; (ii) GTKO.CD46.pTBMpr-TBM.CD39-tiecag-CIITAKD.HO-1; (iii) GTKO.CD46.pTBMpr-TBM.CD39-cag-EPCR.CD55; (iv) GTKO.CD46.pTBMpr-TBM.A20-cag-EPCR.DAF; (v) GTKO.CD46.pTBMpr-TBM.HO-1-cag-EPCR.DAF; (vi) GTKO.CD46.pTBMpr-TBM.TFPI-cag-EPCR.DAF; (vii) GTKO.CD46.pTBMpr-TBM.HO-1-cag-TFPI.CD47; (viii) GTKO.CD46.pTBMpr-TBM.CD47-cag-EPCR.TFPI; (ix)GTKO.CD46. pTBMpr -TBM.TFPI-cag-EPCR.CD47; or (x) GTKO.CD46.pTBMpr-TBM.EPCR-cag-CD47.HO-1.

73. The organ, organ fragment, tissue, or cell according to any one of claims 1-72, wherein the organ is a lung, heart, kidney, or liver; and the organ fragment is a lung fragment, heart fragment, kidney fragment, or liver fragment.

74. A method for preparing transgenic pigs, said transgenic pigs expressing at least four transgenes but lacking the expression of α1,3-galactosyltransferase and growth hormone receptor, said method comprising: (i) Incorporating at least four transgenes under the control of at least two promoters at a single locus within the pig genome to provide a multigene pig genome; The porcine genome contains genetic alterations that result in decreased expression or impaired function of the growth hormone receptor (GHR) gene, wherein the genetic alterations cause a mutation in at least one allele of the GHR gene and the genetic alterations comprise deletions in target regions of the GHR gene specified by two single-guide RNAs; and The two guide RNAs mentioned above are selected from SEQ ID NO: 2 and SEQ ID NO: 4, SEQ ID NO: 2 and SEQ ID NO: 11, or SEQ ID NO: 2 and SEQ ID NO: 13; and (ii) Mature cells containing the multigene pig genome into transgenic pigs.

75. The method according to claim 74, wherein: (a) The pig genome is a somatic cell pig genome and the cell is a pig zygote, and the pig zygote is provided by somatic cell nuclear transfer (SCNT) and by microinjection of the multigene pig genome into a reconstructed SCNT zygote; or (b) The four transgenics are selected from the group consisting of TBM, EPCR, DAF, CD39, TFPI, CTLA4-Ig, CIITA-DN, HOI, A20, HLA-E and CD47 or combinations thereof.

76. The method according to claim 75, wherein: (a) The somatic cell pig genome contains at least one additional gene modification; or (b) The four genetically modified organisms are selected from the following combinations: (i) EPCR, HO1TBM and TFPI; (ii) EPCR, CD55, TBM, and CD39; or (iii) EPCR, CD55, TFPI, and CD47; or (iv) EPCR, HO-1, TBM and CD47.

77. The method of claim 76, wherein the at least one additional gene modification is selected from the group consisting of gene, gene fragment or nucleotide deletion, insertion or substitution; or combinations thereof.

78. The method of claim 76, wherein the at least one additional gene modification is selected from the group consisting of gene knockout; gene knock-in; gene substitution; point mutation; or combinations thereof.

79. The method of claim 76, wherein the at least one additional gene modification is a large genome insertion.

80. The method of claim 75, further comprising introducing at least one additional gene modification into the polygenic pig genome.

81. The method of claim 80, wherein the at least one additional gene modification is selected from the group consisting of gene, gene fragment or nucleotide deletion, insertion or substitution; or combinations thereof.

82. The method of claim 80, wherein the at least one additional gene modification is selected from the group consisting of gene knockout; gene knock-in; gene substitution; point mutation; or combinations thereof.

83. The method of claim 80, wherein the at least one additional gene modification is a large genome insertion.

84. The method of claim 76, wherein the porcine genome: (i) Select the group consisting of the gamete pig genome, zygote pig genome, embryonic pig genome, or blastocyst pig genome; or (ii) Contains at least one additional genetic modification.

85. The method of claim 84, wherein the at least one additional gene modification is selected from the group consisting of deletions, insertions, or substitutions of gene fragments or nucleotides; or combinations thereof.

86. The method of claim 84, wherein the at least one additional gene modification is selected from the group consisting of gene knockout; gene knock-in; gene substitution; point mutation; or combinations thereof.

87. The method of claim 84, wherein the at least one additional gene modification is a large genome insertion.

88. The method of claim 84, further comprising introducing at least one additional genetic modification into the polygenic pig genome.

89. The method of claim 88, wherein the at least one additional gene modification is selected from the group consisting of gene, gene fragment or nucleotide deletion, insertion or substitution; or combinations thereof.

90. The method of claim 88, wherein the at least one additional gene modification is selected from the group consisting of gene knockout; gene knock-in; gene substitution; point mutation; or combinations thereof.

91. The method of claim 88, wherein the at least one additional gene modification is a large genome insertion.

92. The method of claim 76, wherein the incorporation comprises a method selected from the group consisting of biological transfection, chemical transfection, physical transfection, or combinations thereof.

93. The method of claim 76, wherein the incorporation comprises virus-mediated transduction or transformation; or cytoplasmic microinjection or prokaryotic microinjection.

94. The method of claim 76, wherein the single locus is: (i) Natural gene locus; or (ii) Modified natural loci.

95. The method of claim 76, wherein the single locus is: a modified natural locus comprising a gene editing-mediated insertion, deletion, or substitution.

96. The method of claim 76, wherein the single locus is: a modified natural locus comprising transgenic DNA selected from optional marker genes or landing pads.

97. The method of claim 76, wherein the single locus is: -Natural GGTA1 locus; - Modified GGTA1 locus; -Natural CMAH locus; - Modified CMAH locus; - A single locus, which is a natural locus selected from the group consisting of CMAH, β4GalNT2, AAVS1 locus and Rosa26; - A single locus, which is a modified locus selected from the group consisting of CMAH, β4GalNT2, AAVS1 locus and Rosa26; -Not the GGTA1 locus; -Natural β4GalNT2 locus; or - Modified β4GalNT2 locus.

98. The method of claim 76, wherein the single locus is: -Transgenic GGTA1 locus; -Transgenic CMAH locus; or -Transgenic β4GalNT2 locus.

99. The method of claim 76, wherein the single locus is: - The transgenic GGTA1 locus contains selectable marker genes or transgenic pads; - A transgenic CMAH locus containing selectable marker genes or transgenic pads; or - The transgenic β4GalNT2 locus contains selectable marker genes or transgenic pads.

100. The method of claim 96, wherein the transgenic DNA comprises one or more recognition sequences of a polynucleotide modifying enzyme.

101. The method according to claim 100, wherein the polynucleotide modifying enzyme is selected from the group consisting of engineered endonucleases, site-specific recombinases, or combinations thereof.

102. The method according to claim 101, wherein: (i) The engineered endonuclease is selected from the group consisting of zinc finger nucleases, transcription activator-like effector nucleases, and regularly clustered short palindromic repeats (CRISPR) / Cas9 nucleases; or (ii) The site-specific recombinase is selected from the group consisting of λ integrase, Cre recombinase, FLP recombinase, γ-δ dissociation enzyme, Tn3 degradase, OC31 integrase, Bxb1-integrase, R4 integrase or a combination thereof.

103. The method according to any one of claims 76-102, wherein the at least one additional gene modification comprises: (i) Incorporation and expression of CD46; (ii) Incorporation and expression of human HLA-E; or (iii) Knockout of genes, wherein the genes are selected from the group consisting of the β4GalNT2 gene, the CMAH gene and the GGTA1 gene.

104. The method according to any one of claims 74-102, further comprising breeding the transgenic pig into a second transgenic pig, wherein the second transgenic pig contains at least one gene modification.

105. The method of claim 104, wherein the at least one gene modification comprises: (i) Incorporation and expression of at least one transgene, said transgene being selected from the group consisting of anticoagulants, complement inhibitors, immunomodulators, cell-protective transgenes, and combinations thereof; or (ii) Knockout of at least one pig gene.

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