Multi-transgenic pigs containing 10 gene modifications for xenotransplantation

Abstract

The present invention provides transgenic animals (e.g., transgenic pigs), organs, tissues, and cells derived from the transgenic animals, which are particularly useful for xenograft therapy. In particular, the present invention provides transgenic pigs that are completely devoid of expression of a functional α-1,3-galactosyltransferase (GTKO) gene, and contain at least six transgenes under the control of at least three promoters in a single multigene expression vector, and further contain at least four additional genetic modifications, as well as organs, tissues, and cells derived from the transgenic pigs. Also provided are methods of making transgenic animals (e.g., transgenic pigs), and methods of using the transgenic animals, organs, tissues, and cells derived from the transgenic animals in xenograft therapy to treat diseases or conditions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 261,393, filed September 20, 2021, which is incorporated herein by reference in its entirety for all purposes.

[0002] The present disclosure relates generally to xenotransplantation therapy, and more specifically to multi-transgenic pigs comprising at least 10 genetic modifications that make them suitable donors for xenotransplantation, as well as tissues and / or cells derived from the pigs. [Background technology]

[0003] The following discussion of the background of the present technology is provided merely to aid in the understanding of the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Xenotransplantation (transplantation of organs, tissues, and cells from donors of a different species) has the potential to effectively address the shortage of human donors. While xenotransplantation is beneficial in many ways, it presents a more complex immunological scenario than allotransplantation. The most profound barrier to xenotransplantation is the rejection of the transplanted organ by a cascade of immune mechanisms, which can be divided into three phases: hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), and T cell-mediated cellular rejection. HAR is a very rapid event, causing irreversible graft damage and loss within minutes to hours after graft reperfusion.

[0005] Considerable effort has been directed toward addressing this immune barrier posed by xenotransplantation through genetic modification of donor animals. The most commonly used donor animal is the pig. Pigs have been the focus of most xenotransplantation research because they share many anatomical and physiological characteristics with humans. Furthermore, pigs have a relatively short gestation period and can be bred in a pathogen-free environment. Furthermore, pigs are commonly used by humans as a food source, so they do not pose the typical ethical issues associated with most animal research (e.g., primates).

[0006] Significant progress has been made in overcoming biological barriers to the use of porcine organs in preclinical models, with several heart and kidney series demonstrating sustained organ function and recipient survival for months to years. Genetically modified pigs lacking the α-1,3-Gal epitope (the major xenoantigen that triggers HAR in pig-to-primate xenografts) are likely to provide a foundation for further genetic modifications to address other rejection mechanisms and incompatibilities between pig and primate blood coagulation systems. Successful xenotransplantation requires multiple genetic modifications, which present challenges, including production-related issues. Therefore, transgenic pigs stably expressing multiple immunomodulatory transgenes to overcome xenograft rejection are needed. Traditional breeding of multitransgenic pigs containing a single transgene has been used with considerable success. However, breeding is time-consuming and expensive, and consistency of transgene expression levels over time can be problematic.

[0007] The development of polycistronic expression systems for inserting multiple transgenes into a single locus and into various cell types and animals is a promising technology for generating multitransgenic pigs. Although still in its infancy, polycistronic expression systems offer an alternative to the traditional approaches typically employed to generate multitransgenic pigs. This technology has several drawbacks and is not yet efficient. For example, a multicistronic system containing three genes may result in transgenic pigs that express only two of the genes. Furthermore, efficient expression of downstream genes from a multicistronic system depends on the expression of the upstream gene.

[0008] Therefore, there is a need for new uses of polycistronic expression systems that result in stable integration and sufficient transgene expression in multi-transgenic donor animals (i.e., pigs) for xenotransplantation therapy. The present disclosure addresses this need. Summary of the Invention

[0009] Some embodiments relate to transgenic animals (e.g., transgenic pigs), organs, tissues, and cells derived from such transgenic animals, which are particularly useful for xenotransplantation therapy. In particular, some embodiments provide transgenic pigs that are completely devoid of expression of a functional α-1,3-galactosyltransferase (GTKO) gene and contain at least six transgenes under the control of at least three promoters in a single multigene expression vector, and further contain at least four additional genetic modifications, as well as organs, tissues, and cells derived from such transgenic pigs. Another embodiment relates to improved methods for producing transgenic animals (e.g., transgenic pigs), as well as methods for treating diseases or conditions using such transgenic animals, organs, tissues, and cells derived from such transgenic animals in xenotransplantation therapy. These methods provided herein facilitate efficient production of founder multi-transgenic animals (e.g., pigs), further facilitating breeding and expansion of production herds. This new, improved, and efficient method for generating multi-transgenic founder animals is based on the identification and insertion of six human transgenes in a single multigene expression vector and the targeted insertion of this multigene expression vector (a polycistronic vector or multicistronic vector (MCV)) into known loci or landing pads to achieve reliable and consistent transgene expression in transgenic animals and transplanted organs, tissues, and / or cells.

[0010] In one aspect, the present disclosure provides transgenic pigs comprising genetic modifications resulting in (i) the absence of expression of a functional α-1,3-galactosyltransferase; and (ii) the integration and expression, at a single genomic locus, of: (a) at least two complement inhibitor transgenes; (b) at least one immunosuppressant transgene; (c) at least one cytoprotective factor transgene; and (d) at least two anticoagulant transgenes. In some embodiments, the transgenes are human cDNAs. In some embodiments, the transgenic animal comprises at least six, at least seven, or at least eight transgenes. In some embodiments, the transgenic animal comprises at least six transgenes. In some embodiments, the at least six transgenes are encoded by a polycistronic vector (multicistronic vector (MCV)). In some embodiments, the polycistronic vector comprises at least three bicistronic units. In some embodiments, each bicistronic unit comprises a promoter driving a first transgene linked to a second transgene via a self-cleaving peptide. In some embodiments, the first bicistronic unit comprises the at least two anticoagulant factor transgenes, the second bicistronic unit comprises at least two complement inhibitor transgenes; and the third bicistronic unit comprises the at least one cytoprotective factor transgene and the at least one immunosuppressant factor transgene. In some embodiments, the self-cleaving peptide is selected from the group consisting of T2A, P2A, F2A, and E2A.

[0011] In some embodiments, the transgene is encoded by a polycistronic vector comprising: (i) at least two bicistronic units, each bicistronic unit comprising a promoter driving a first transgene linked to a second transgene via a self-cleaving peptide; and (ii) at least two non-polycistronic transgenes, each non-polycistronic transgene driven by its own promoter. In one embodiment, at least one bicistronic unit is driven by a tissue-specific promoter; at least one bicistronic unit is driven by an inducible promoter; and / or at least two bicistronic units are driven by dedicated constitutive promoters. In one embodiment, the at least two bicistronic units are each driven by a constitutive promoter, and the at least two non-polycistronic transgenes are each driven by a tissue-specific promoter.

[0012] In some embodiments, the transgene is under the control of at least three promoters in a polycistronic vector (MCV). In some embodiments, the promoters are constitutive promoters or tissue-specific promoters. In some embodiments, the constitutive promoter is selected from the group consisting of a CAG promoter, a Tie-2 promoter, and an ICAM-2 promoter. In some embodiments, the tissue-specific promoter is an endothelial cell-specific promoter. In some embodiments, the inducible promoter is a tetracycline / doxycycline-regulated promoter. In some embodiments, the tissue-specific promoter is selected from a porcine thrombomodulin promoter (pTBMpr), a human thrombomodulin promoter, a porcine EPCR promoter, or a human EPCR promoter.

[0013] In some embodiments, the transgenic pig comprises the integration and expression at a single genomic locus of at least two complement inhibitors selected from the group consisting of CD46, DAF (CD55), CD59, CR1, and combinations thereof, hi some embodiments, the at least two complement inhibitors are expressed ubiquitously and / or under the control of a constitutive promoter or under the control of an inducible promoter.

[0014] In some embodiments, the transgenic pig comprises integration and expression at a single genomic locus of at least one immunosuppressant transgene selected from the group consisting of cytotoxic T-lymphocyte antigen 4 (CTLA4), cluster of differentiation 47 (CD47), and class II transactivator-DN (CIITA-DN). In some embodiments, the at least one immunosuppressant transgene is under the control of a constitutive promoter.

[0015] In some embodiments, the transgenic pig comprises the integration and expression at a single genomic locus of at least one cytoprotective factor transgene selected from the group consisting of heme oxygenase 1 (HO-1), A20, FAT-1, soluble tumor necrosis factor alpha (TNF-α), and combinations thereof. In some embodiments, the at least one cytoprotective factor is under the control of a constitutive promoter or under the control of an endothelial-specific promoter.

[0016] In some embodiments, the transgenic pig comprises integration and expression at a single genomic locus of at least two anticoagulant transgenes selected from the group consisting of endothelial cell protein C receptor (EPCR), thrombomodulin, CD39, hirudin, tissue factor pathway inhibitor (TFPI), and combinations thereof, hi some embodiments, the at least two anticoagulant transgenes are under the control of an endothelial-specific promoter.

[0017] In some embodiments, the at least six transgenes are encoded by a polycistronic vector, the polycistronic vector comprising a bicistronic unit selected from the group consisting of: (i) a human TBM transgene linked to a human EPCR transgene via a 2A peptide and driven by a porcine TBM promoter (pTBMpr[hTBM-2A-hEPCR]); (ii) a human CD47 transgene linked to a human HO-1 transgene via a 2A peptide and driven by a CAG promoter. (iii) a human CD46 transgene linked to a human DAF transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD46-2A-hDAF]); (iv) a human TBM transgene linked to a human EPCR transgene via a 2A peptide and driven by a polyA signal-fused porcine TBM promoter (polyA / pTBMpr[hTBM-2A-hEPCR]); (v) a human HO-1 transgene linked to a human HO-1 transgene via a 2A peptide. (vi) a human TBM transgene linked to a human EPCR transgene via a 2A peptide and driven by a porcine EPCR promoter (pEPCRpr[hTBM-2A-hEPCR]); (vii) a human CD46 transgene linked to a human CD47 transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD46-2A-hCD47]); (viii) a first GHRgRNA and a first U6 promoter driving it, and a second GHRgRNA and a second U6 promoter driving it linked thereto (the first gRNA and second gRNA may be the same or different) (U6p[GHRgRNA-1]; U6p[GHRgRNA-2]); (xix) a Cas endonuclease and a TRE3G promoter driving it, and tTA and a CAG promoter driving it linked thereto via an insulator (TRE3Gp[CAS9]; CAGpr[tTA]); and (x) combinations thereof.

[0018] In some embodiments, the polycistronic vector comprises: (i) pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD47-2A-hH01]; and CAGpr[hCD46-2A-hDAF]; (ii) polyA-pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD47-2A-hH01]; and CAGpr[hCD46-2A-hDAF]; (iii) pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD59-P2A-hH01]; and CAGpr[hCD46-P2A-hDAF]; (iv) polyA-pTBMpr[hTBM]; CAGpr[hCD47-P2A-hH01]; pEPCRpr[hEPCR]; and CAGpr[h CD46P-2A-hDAF]; (v) pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD59-P2A-hHO1]; CAGpr[hCD46-2A-hCD47]; (vi) pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD59-2A-hHO1]; CAGpr[hCD46-2A-hCD55]; (vii) U6p[GHRgRNA-1]; U6p[GHRgRNA-2]; TRE3Gp[CAS9]; CAGpr[tTA]; CAGpr[hCD46-2A-hCD55]; (viii) SEQ ID NO: 7; (xix) SEQ ID NO: 8; (x) SEQ ID NO: 9; (xi) SEQ ID NO: 11; (xii) SEQ ID NO: 12; (xiii) SEQ ID NO: 13; or (xiv) SEQ ID NO: 14.

[0019] In some embodiments, the transgenic pig comprises genetic modifications that result in: (i) the absence of expression of a functional α-1,3-galactosyltransferase; (ii) integration and expression at a single genomic locus, which is a native locus or a modified native locus. In some embodiments, the modified native locus comprises: (i) an insertion, deletion, or substitution via gene editing; (ii) transgenic DNA; (iii) a selectable gene maker; and / or (iv) a landing pad. In some embodiments, the single genomic locus is selected from the group consisting of AAVS1, GHR, ROSA26, CMAH, B4GalNT2, and GGTA1.

[0020] In some embodiments, the polycistronic vector encoding the transgene further comprises a nucleotide sequence for homology recombination and / or a nucleotide sequence for homology directed repair (HDR) at a locus selected from the group consisting of AAVS1, GHR, ROSA26, CMAH, B4GalNT2, and GGTA1.

[0021] In one aspect, the disclosure provides a transgenic pig comprising genetic modifications that result in (i) the absence of expression of a functional alpha-1,3-galactosyltransferase, and (ii) the integration and expression at a single genomic locus of (a) at least two complement inhibitor transgenes, (b) at least one immunosuppressant transgene, (c) at least one cytoprotective factor transgene, and (d) at least two anticoagulant transgenes, and further comprising (iii) at least one additional genetic modification selected from the group consisting of a gene knockout; a gene knockin; a gene replacement; a point mutation; a deletion, insertion, or substitution of a gene, gene fragment, or nucleotide; a large genomic insertion; or a combination thereof.

[0022] In some embodiments, the transgenic pig further comprises a knockout of a gene selected from the group consisting of AAVS1, GHR, ROSA26, CMAH, B4GalNT2, GGTA1, and growth hormone receptor (GHR). In some embodiments, the gene knockout comprises: (i) an insertion, deletion, or replacement via gene editing; (ii) a gene editing via CRISPR-Cas9; (iii) an insertion via homologous recombination; and / or (iv) a gene knockout via NeoR insertion.

[0023] In some embodiments, the single genomic locus is CMAH and the additional modifications comprise knockouts of β4GalNT2, GGTA1, and GHR. In some embodiments, the single genomic locus is β4GalNT2 and the additional modifications comprise knockouts of CMAH, GGTA1, and GHR. In some embodiments, the single genomic locus is GGTA1 and the additional modifications comprise knockouts of β4GalNT2, CMAH, and GHR.

[0024] In one aspect, the present disclosure provides cells derived from the transgenic pig of the present invention. In one aspect, the present disclosure provides organs derived from the transgenic pig of the present invention. In some embodiments, the organ is selected from the group consisting of heart, lung, liver, pancreas, and kidney. In one aspect, the present disclosure provides tissue derived from the transgenic pig of the present invention. In some embodiments, the tissue is selected from the group consisting of vascular tissue, heart valve, retinal tissue, neural tissue, and corneal tissue. In some embodiments, the vascular tissue is a vascular graft.

[0025] In one aspect, the present disclosure provides a method for performing a xenotransplantation, comprising administering to a subject in need thereof an organ, tissue, or cells derived from a transgenic pig of the present invention. In some embodiments, the subject is a non-human primate or a human. In some embodiments, the organ is selected from the group consisting of heart, lung, liver, pancreas, and kidney. In some embodiments, the tissue is selected from the group consisting of vascular tissue, retinal tissue, nerve tissue, and corneal tissue.

[0026] In one aspect, the present disclosure provides a method for performing a xenotransplantation comprising administering to a subject in need thereof an organ, tissue, or cells derived from a transgenic pig of the present invention, and further comprising administering to the subject a clinically relevant immunosuppressant drug regimen following xenotransplantation of the organ, tissue, or cells.

[0027] In one embodiment, the present disclosure provides a method for producing a transgenic pig of the present invention. In one embodiment, the present disclosure provides a method for producing a transgenic pig containing at least six transgenes, the method comprising: (i) transfecting a pig cell with a single polycistronic vector containing (a) at least two complement inhibitor transgenes, (b) at least one immunosuppressant transgene, (c) at least one cytoprotective factor transgene, and (d) at least two anticoagulant transgenes; (ii) integrating and expressing the polycistronic vector at a single genomic locus to produce a multi-transgenic pig cell containing at least six transgenes; (iii) injecting the nucleus of the multi-transgenic pig cell into a reconstituted somatic cell nuclear transfer (SCNT) to produce a multi-transgenic pig zygote; and (iv) maturing the multi-transgenic pig zygote into a multi-transgenic pig (e.g., a transgenic pig). In some embodiments, the pig cells and the multi-transgenic pig (eg, transgenic pig) lack expression of α-1,3-galactosyltransferase (GTKO).

[0028] In some embodiments, the porcine cell is a somatic cell. In some embodiments, the multi-transgenic porcine cell comprises at least seven, at least eight, at least nine, or at least ten transgenes. In some embodiments, the at least six transgenes are encoded by a polycistronic vector, and optionally, the polycistronic vector comprises at least three bicistronic units. In some embodiments, each bicistronic unit comprises a promoter driving a first transgene linked to a second transgene via a self-cleaving peptide. In some embodiments, a first bicistronic unit comprises the at least two anticoagulant factor transgenes, a second bicistronic unit comprises at least two complement inhibitor transgenes, and a third bicistronic unit comprises the at least one cytoprotective factor transgene and the at least one immunosuppressive factor transgene.

[0029] In some embodiments, the self-cleaving peptide is selected from the group consisting of T2A, P2A, F2A, and E2A. In some embodiments, the at least six transgenes are encoded by a polycistronic vector comprising: (i) at least two bicistronic units, each bicistronic unit comprising a promoter driving a first transgene linked to a second transgene via a self-cleaving peptide; and (ii) at least two non-polycistronic transgenes, each non-polycistronic transgene driven by its own promoter.

[0030] In some embodiments, at least one bicistronic unit is driven by a tissue-specific promoter; at least one bicistronic unit is driven by an inducible promoter; and / or at least two bicistronic units are driven by dedicated constitutive promoters. In some embodiments, the at least two bicistronic units are each driven by a constitutive promoter, and the at least two non-polycistronic transgenes are each driven by a tissue-specific promoter. In some embodiments, the transgene is under the control of at least three promoters in a polycistronic vector (e.g., MCV). In some embodiments, the promoters are constitutive promoters or tissue-specific promoters. In some embodiments, the constitutive promoter is selected from the group consisting of CAG promoter, Tie-2 promoter, and ICAM-2 promoter. In some embodiments, the inducible promoter is a tetracycline / doxycycline-regulated promoter. In some embodiments, the tissue-specific promoter is an endothelial cell-specific promoter selected from the porcine thrombomodulin promoter (pTBMpr), human thrombomodulin promoter, porcine EPCR promoter, and human EPCR promoter.

[0031] In some embodiments, the method for producing a transgenic pig comprising at least six transgenes described above comprises transfecting pig cells with a single polycistronic vector comprising at least two complement inhibitors. In some embodiments, the at least two complement inhibitors are selected from the group consisting of CD46, DAF (CD55), CD59, CR1, and combinations thereof. In some embodiments, the at least two complement inhibitors are ubiquitously expressed; and / or are under the control of a constitutive promoter or an inducible promoter.

[0032] In some embodiments, the method for producing a transgenic pig comprising at least six transgenes comprises transfecting pig cells with a single polycistronic vector comprising at least one immunosuppressant transgene. In some embodiments, the at least one immunosuppressant transgene is selected from the group consisting of cytotoxic T-lymphocyte antigen 4 (CTLA4), cluster of differentiation 47 (CD47), and class II transactivator-DN (CIITA-DN). In some embodiments, the at least one immunosuppressant transgene is under the control of a constitutive promoter.

[0033] In some embodiments, the method for producing a transgenic pig comprising at least six transgenes comprises transfecting a pig cell with a single polycistronic vector comprising at least one cytoprotective factor transgene. In some embodiments, the at least one cytoprotective factor transgene is under the control of a constitutive promoter or an endothelial-specific promoter. In some embodiments, the at least one cytoprotective factor transgene is selected from the group consisting of heme oxygenase 1 (HO-1), A20, FAT-1, soluble tumor necrosis factor alpha (TNF-α), and combinations thereof.

[0034] In some embodiments, the method for producing a transgenic pig comprising at least six transgenes comprises transfecting a pig cell with a single polycistronic vector comprising at least two anticoagulant transgenes. In some embodiments, the at least two anticoagulant transgenes are under the control of an endothelial-specific promoter. In some embodiments, the at least two anticoagulant transgenes are selected from the group consisting of endothelial cell protein C receptor (EPCR), thrombomodulin, CD39, hirudin, tissue factor pathway inhibitor (TFPI), and combinations thereof.

[0035] In some embodiments, the method of producing a transgenic pig comprising at least six transgenes described above comprises transfecting a single polycistronic vector into pig cells.In some embodiments, the polycistronic vector comprises a bicistronic unit selected from the group consisting of: (i) a human TBM transgene linked to a human EPCR transgene via a 2A peptide and driven by a porcine TBM promoter (pTBMpr[hTBM-2A-hEPCR]); (ii) a human CD47 transgene linked to a human HO-1 transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD47-2A-hHO1]); (iii) a human (iv) a human CD46 transgene linked to a porcine DAF transgene via a 2A peptide, driven by a CAG promoter (CAGpr[hCD46-2A-hDAF]); (v) a human TBM transgene linked to a porcine EPCR transgene via a 2A peptide, driven by a polyA signal-fused porcine TBM promoter (polyA / pTBMpr[hTBM-2A-hEPCR]); (v) a human CD59 transgene linked to a porcine HO-1 transgene via a 2A peptide, driven by a polyA signal-fused porcine TBM promoter (polyA / pTBMpr[hTBM-2A-hEPCR]). (vi) a human TBM transgene linked to a human EPCR transgene via a 2A peptide and driven by a porcine EPCR promoter (pEPCRpr[hTBM-2A-hEPCR]); (vii) a human CD46 transgene linked to a human CD47 transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD46-2A-hCD47]); (viii) a first GHR gRNA and a first GHR gRNA driven by a porcine EPCR promoter (pEPCRpr[hTBM-2A-hEPCR]); A U6 promoter and a second GHRgRNA and a second U6 promoter driving it linked thereto (the first gRNA and the second gRNA may be the same or different) (U6p[GHRgRNA-1]; U6p[GHRgRNA-2]); (xix) a Cas endonuclease and a TRE3G promoter driving it, and tTA and a CAG promoter driving it linked thereto via an insulator (TRE3Gp[CAS9]; CAGpr[tTA]); and (x) combinations thereof.

[0036] In some embodiments, the polycistronic vector comprises: (i) pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD47-2A-hH01]; and CAGpr[hCD46-2A-hDAF]; (ii) polyA-pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD47-2A-hH01]; and CAGpr[hCD46-2A-hDAF]; (iii) pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD59-P2A-hH01]; and CAGpr[hCD46-P2A-hDAF]; (iv) polyA-pTBMpr[hTBM]; CAGpr[hCD47-P2A-hH01]; pEPCRpr[hEPCR]; and CAGpr[h CD46P-2A-hDAF]; (v) pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD59-P2A-hHO1]; CAGpr[hCD46-2A-hCD47]; (vi) pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD59-2A-hHO1]; CAGpr[hCD46-2A-hCD55]; (vii) U6p[GHRgRNA-1]; U6p[GHRgRNA-2]; TRE3Gp[CAS9]; CAGpr[tTA]; CAGpr[hCD46-2A-hCD55]; (viii) SEQ ID NO: 7; (xix) SEQ ID NO: 8; (x) SEQ ID NO: 9; (xi) SEQ ID NO: 11; (xii) SEQ ID NO: 12; (xiii) SEQ ID NO: 13; or (xiv) SEQ ID NO: 14.

[0037] In some embodiments, the method for producing a transgenic pig comprising at least six transgenes comprises integrating and expressing the polycistronic vector into a single genomic locus to produce a multi-transgenic pig cell comprising at least six transgenes. In some embodiments, the single genomic locus is a native locus or a modified native locus. In some embodiments, the modified native locus comprises: (i) an insertion, deletion, or substitution via gene editing; (ii) transgenic DNA; (iii) a selectable gene maker; and / or (iv) a landing pad. In some embodiments, the single genomic locus is selected from the group consisting of AAVS1, ROSA26, CMAH, GHR, B4GalNT2, and GGTA1. In some embodiments, the polycistronic vector encoding the transgene further comprises a nucleotide sequence for homology recombination and / or a nucleotide sequence for homology directed repair (HDR) at a locus selected from the group consisting of AAVS1, GHR, ROSA26, CMAH, B4GalNT2, and GGTA1.

[0038] In one aspect, the present disclosure provides a method for producing a transgenic pig comprising at least six transgenes, the method comprising: (i) transfecting a pig cell with a single polycistronic vector comprising: (a) at least two complement inhibitor transgenes; (b) at least one immunosuppressant transgene; (c) at least one cytoprotective factor transgene; and (d) at least two anticoagulant transgenes; (ii) integrating and expressing the polycistronic vector at a single genomic locus to produce a multi-transgenic pig cell comprising at least six transgenes, wherein the multi-transgenic pig cell further comprises at least one additional genetic modification; (iii) injecting the nucleus of the multi-transgenic pig cell into a reconstituted somatic cell nuclear transfer (SCNT) to produce a multi-transgenic pig zygote; and (iv) maturing the multi-transgenic pig zygote into a multi-transgenic pig. In some embodiments, the pig cell and the multi-transgenic pig lack expression of α-1,3-galactosyltransferase (GTKO), and the multi-transgenic (e.g., transgenic) pig further comprises at least one additional genetic modification. In some embodiments, the at least one additional genetic modification is selected from the group consisting of gene knockout; gene knockin; gene replacement; point mutation; gene, gene fragment, or nucleotide deletion, insertion, or substitution; large genomic insertion; or a combination thereof. In some embodiments, the transgenic pig comprises a knockout of a gene selected from the group consisting of AAVS1, ROSA26, CMAH, B4GalNT2, GGTA1, and growth hormone receptor (GHR). In some embodiments, the gene knockout comprises: (i) insertion, deletion, or replacement via gene editing; (ii) gene editing via CRISPR-Cas9; (iii) insertion via homologous recombination; and / or (iv) gene knockout via NeoR insertion.

[0039] In some embodiments, the single genomic locus is CMAH and the additional modifications comprise knockouts of β4GalNT2, GGTA1, and GHR. In some embodiments, the single genomic locus is β4GalNT2 and the additional modifications comprise knockouts of CMAH, GGTA1, and GHR. In some embodiments, the single genomic locus is GGTA1 and the additional modifications comprise knockouts of β4GalNT2, CMAH, and GHR. [Brief explanation of the drawings]

[0040] [Figure 1A]1A schematically illustrates several embodiments of the multigene vectors of the present disclosure. In particular, the B200 multicistronic vector (SEQ ID NO: 11), B201 multicistronic vector (SEQ ID NO: 12), B202 multicistronic vector (SEQ ID NO: 13), B209 multicistronic vector (SEQ ID NO: 14), B212 multicistronic vector (SEQ ID NO: 7), B214 multicistronic vector (SEQ ID NO: 8), and B217 multicistronic vector (SEQ ID NO: 9) are shown. The B200 vector is a multicistronic vector (MCV) containing three bicistronic units (pTBMpr[hTBM-2A-hEPCR] / CAGpr[hCD47-2A-hHO1] / CAGpr[hCD46-2A-hDAF]) flanked by targeting arms for homology-directed repair (HDR) at the CMAH locus. The B201 vector is an MCV containing three bicistronic units (polyA / pTBMpr[hTBM-2A-hEPCR] / CAGpr[hCD47-2A-hHO1] / CAGpr[hCD46-2A-hDAF]) flanked by targeting arms for HDR at the GGTA1 / Neo locus. The B202 vector is an MCV containing two monocistronic units (polyA / pTBMpr[hTBM]; pEPCRpr[hEPCR]) and two bicistronic units flanked by targeting arms for HDR at the GGTA1 / Neo locus, arranged in the following order: (polyA / pTBMpr[hTBM]; CAGpr[hCD47-2A-hH01]; pEPCRpr[hEPCR] / CAGpr[hCD46P-2A-hDAF]. The B209 vector is an MCV containing three bicistronic units (pTBMpr[hTBM-2A-hEPCR] / CAGpr[hCD47-2A-hH01] / CAGpr[hCD46-2A-hDAF]) flanked by targeting arms for HDR at the CMAH locus.The B212 vector is an MCV containing three bicistronic units (pTBMpr[hTBM-2A-hEPCR] / CAGpr[hCD59-2A-hHO1] / CAGpr[hCD46-2A-hCD47]) flanked by targeting arms for HDR at the GGTA1 / Neo locus. The B214 vector is an MCV containing three bicistronic units (pTBMpr[hTBM-2A-hEPCR] / CAGpr[hCD59-2A-hHO1] / CAGpr[hCD46-2A-hCD55]) flanked by targeting arms for HDR at the GGTA1 / Neo locus. The B217 vector is an MCV containing five expression units (U6 promoter [GHRgRNA-1], U6 promoter [GHRgRNA-2], TRE3G [CAS9], CAGpr [tTA], and CAGpr [hCD46-2A-hCD55]) flanked by targeting arms for HDR at the GGTA1 / Neo locus. [Figure 1B]Figure IB schematically illustrates several embodiments of the multigene vectors of the present disclosure. In particular, the B200 multicistronic vector (SEQ ID NO: 11), B201 multicistronic vector (SEQ ID NO: 12), B202 multicistronic vector (SEQ ID NO: 13), B209 multicistronic vector (SEQ ID NO: 14), B212 multicistronic vector (SEQ ID NO: 7), B214 multicistronic vector (SEQ ID NO: 8), and B217 multicistronic vector (SEQ ID NO: 9) are shown. The B200 vector is a multicistronic vector (MCV) containing three bicistronic units (pTBMpr[hTBM-2A-hEPCR] / CAGpr[hCD47-2A-hHO1] / CAGpr[hCD46-2A-hDAF]) flanked by targeting arms for homology-directed repair (HDR) at the CMAH locus. The B201 vector is an MCV containing three bicistronic units (polyA / pTBMpr[hTBM-2A-hEPCR] / CAGpr[hCD47-2A-hHO1] / CAGpr[hCD46-2A-hDAF]) flanked by targeting arms for HDR at the GGTA1 / Neo locus. The B202 vector is an MCV containing two monocistronic units (polyA / pTBMpr[hTBM]; pEPCRpr[hEPCR]) and two bicistronic units flanked by targeting arms for HDR at the GGTA1 / Neo locus, arranged in the following order: (polyA / pTBMpr[hTBM]; CAGpr[hCD47-2A-hH01]; pEPCRpr[hEPCR] / CAGpr[hCD46P-2A-hDAF]. The B209 vector is an MCV containing three bicistronic units (pTBMpr[hTBM-2A-hEPCR] / CAGpr[hCD47-2A-hH01] / CAGpr[hCD46-2A-hDAF]) flanked by targeting arms for HDR at the CMAH locus.The B212 vector is an MCV containing three bicistronic units (pTBMpr[hTBM-2A-hEPCR] / CAGpr[hCD59-2A-hHO1] / CAGpr[hCD46-2A-hCD47]) flanked by targeting arms for HDR at the GGTA1 / Neo locus. The B214 vector is an MCV containing three bicistronic units (pTBMpr[hTBM-2A-hEPCR] / CAGpr[hCD59-2A-hHO1] / CAGpr[hCD46-2A-hCD55]) flanked by targeting arms for HDR at the GGTA1 / Neo locus. The B217 vector is an MCV containing five expression units (U6 promoter [GHRgRNA-1], U6 promoter [GHRgRNA-2], TRE3G [CAS9], CAGpr [tTA], and CAGpr [hCD46-2A-hCD55]) flanked by targeting arms for HDR at the GGTA1 / Neo locus. [Figure 2A] Figures 2A-2H schematically illustrate preferred embodiments of the vectors described herein for producing multi-transgenic animals (i.e., 10GE pigs) containing at least 10 modifications, which may include functional knockout of four porcine genes (GGTA1, CMAH, B4GalNT2, and growth hormone receptor (GHR)) and targeted integration of a multicistronic vector containing six human transgenes (6-gene vector). Figure 2A illustrates the insertion of the B200 vector (SEQ ID NO: 11) into the CMAH locus. [Figure 2B]Figures 2A-2H schematically illustrate preferred embodiments of the vectors described herein for producing multi-transgenic animals (i.e., 10GE pigs) containing at least 10 modifications, which may include functional knockout of four pig genes (GGTA1, CMAH, B4GalNT2, and growth hormone receptor (GHR)) and targeted integration of a multicistronic vector containing six human transgenes (6-gene vector). Figure 2B illustrates insertion of the B201 vector (SEQ ID NO: 12) into the GGTA1 / Neo locus. [Figure 2C] Figures 2A-2H schematically illustrate preferred embodiments of the vectors described herein for producing multi-transgenic animals (i.e., 10GE pigs) containing at least 10 modifications, which may include functional knockout of four porcine genes (GGTA1, CMAH, B4GalNT2, and growth hormone receptor (GHR)) and targeted integration of a multicistronic vector containing six human transgenes (6-gene vector). Figure 2C illustrates insertion of the B202 vector (SEQ ID NO: 13) into the GGTA1 / Neo locus. [Figure 2D] Figures 2A-2H schematically illustrate preferred embodiments of the vectors described herein for producing multi-transgenic animals containing at least 10 modifications (i.e., 10GE pigs), which may include functional knockout of four porcine genes (GGTA1, CMAH, B4GalNT2, and growth hormone receptor (GHR)) and targeted integration of a multicistronic vector containing six human transgenes (6-gene vector). Figure 2D illustrates the insertion of the B209 vector (SEQ ID NO: 14) into CMAH. [Figure 2E]Figures 2A-2H schematically illustrate preferred embodiments of the vectors described herein for producing multi-transgenic animals containing at least 10 modifications (i.e., 10GE pigs), which may include functional knockout of four pig genes (GGTA1, CMAH, B4GalNT2, and growth hormone receptor (GHR)) and targeted integration of a multicistronic vector containing six human transgenes (6-gene vector). Figure 2E illustrates the insertion of B212 (SEQ ID NO: 7) into the GGTA1 / Neo locus. [Figure 2F] Figures 2A-2H schematically illustrate preferred embodiments of the vectors described herein for producing multi-transgenic animals (i.e., 10GE pigs) containing at least 10 modifications, which may include functional knockout of four pig genes (GGTA1, CMAH, B4GalNT2, and growth hormone receptor (GHR)) and targeted integration of a multicistronic vector containing six human transgenes (6-gene vector). Figure 2F illustrates the insertion of B214 (SEQ ID NO: 8) into the GGTA1 / Neo locus. [Figure 2G] Figures 2A-2H schematically illustrate preferred embodiments of the vectors described herein for producing multi-transgenic animals containing at least 10 modifications (i.e., 10GE pigs), which may include functional knockout of four pig genes (GGTA1, CMAH, B4GalNT2, and growth hormone receptor (GHR)) and targeted integration of a multicistronic vector containing six human transgenes (6-gene vector). Figure 2G illustrates the insertion of B217 (SEQ ID NO: 9) into the GGTA1 / Neo locus. [Figure 2H]2A-2H schematically illustrate preferred embodiments of vectors described herein for producing multi-transgenic animals containing at least 10 modifications (i.e., (ei), 10GE pigs), which may include functional knockout of four porcine genes (GGTA1, CMAH, B4GalNT2, and growth hormone receptor (GHR)) and targeted integration of a multicistronic vector containing six human transgenes (6-gene vector). FIG. 2H shows a schematic diagram of the B217 vector and a schematic diagram of preferred embodiments of vectors described herein for producing multi-transgenic animals containing at least 10 modifications (i.e., (ei), 10GE pigs). [Figure 3A] Figure 3A shows Western blot analysis of tissues from transgenic animals expressing the vectors of the present invention, demonstrating protein expression of each transgene encoded by B200, B201, B202, and B209 in the heart (Figure 3A), lung (Figure 3B), and kidney (Figure 3C). By Western blot, each transgenic protein from the transgenic tissue samples had the expected molecular weight. [Figure 3B] Figure 3B shows Western blot analysis of tissues from transgenic animals expressing the vectors of the present invention, demonstrating protein expression of each transgene encoded by B200, B201, B202, and B209 in the heart (Figure 3A), lung (Figure 3B), and kidney (Figure 3C). By Western blot, each transgenic protein from the transgenic tissue samples had the expected molecular weight. [Figure 3C] Figure 3C shows Western blot analysis of tissues from transgenic animals expressing the vectors of the present invention, demonstrating protein expression of each transgene encoded by B200, B201, B202, and B209 in the heart (Figure 3A), lung (Figure 3B), and kidney (Figure 3C). By Western blot, each transgenic protein from the transgenic tissue samples had the expected molecular weight. [Figure 4] Figure 4 shows a bar graph depicting the results of a complement-dependent cytotoxicity (CDC) assay performed on transgenic porcine aortic endothelial cells (pAECs) expressing the B200 vector (n = 3), the B201 vector (n = 4), and the B202 vector (n = 4). Significantly more serum-treated GTKO pAECs were lysed (87%) compared with pAECs expressing the B200 vector, the B201 vector, and the B202 vector (3.7%, 3.4%, and 2.2%, respectively) (P < .01), demonstrating the functionality of the complement inhibitor transgenes, hCD46 and hDAF. Quantification of this CDC assay is shown in Table 1. The bars indicate the percent of lysed cells 60 minutes after addition of complement. The left bar represents serum-treated wells, and the right bar represents untreated wells. GTKO pAEC (GGTA1 knockout (KO) only; lacking expression of human complement inhibitors; n=1) served as a control. Three replicate wells were used per treatment. Data were analyzed using GraphPad Prism software. [Figure 5] Figure 5 shows a bar graph depicting quantification of activated protein C (APC) assays performed on pAECs expressing the B200 vector (n=3), B201 vector (n=4), and B202 vector (n=2), demonstrating that B200 pAECs, B201 pAECs, and B202 pAECs produced more APCs than the GTKO negative control (P<0.05), demonstrating the anticoagulant functionality of the hTBM and hEPCR transgenes. [Figure 6] Figure 6 is a bar graph quantifying the results of an apoptosis assay performed on B201 pAECs and GTKO pAECs, showing that while both GTKO pAECs and B201 pAECs underwent apoptosis in response to hemin treatment, B201 cells were significantly less apoptotic than GTKO cells. Figure 6 demonstrates the protective function of the expressed HO-1 transgene in these cells. GTKO pAECs did not contain the HO-1 transgene and served as a control. [Figure 7A] Figures 7A to 7E show representative immunohistochemical staining images of pig lung tissue, pig heart tissue, and pig kidney tissue expressing human transgenes encoded by the B200 vector (Figure 7A), B201 vector (Figure 7B), and B202 vector (Figure 7C), and of pig heart tissue and pig kidney tissue expressing human transgenes encoded by the B209 vector (Figure 7D) and B212 vector (Figure 7E). [Figure 7B] Figures 7A to 7E show representative immunohistochemical staining images of pig lung tissue, pig heart tissue, and pig kidney tissue expressing human transgenes encoded by the B200 vector (Figure 7A), B201 vector (Figure 7B), and B202 vector (Figure 7C), and of pig heart tissue and pig kidney tissue expressing human transgenes encoded by the B209 vector (Figure 7D) and B212 vector (Figure 7E). [Figure 7C] Figures 7A to 7E show representative immunohistochemical staining images of pig lung tissue, pig heart tissue, and pig kidney tissue expressing human transgenes encoded by the B200 vector (Figure 7A), B201 vector (Figure 7B), and B202 vector (Figure 7C), and of pig heart tissue and pig kidney tissue expressing human transgenes encoded by the B209 vector (Figure 7D) and B212 vector (Figure 7E). [Figure 7D] Figures 7A to 7E show representative immunohistochemical staining images of pig lung tissue, pig heart tissue, and pig kidney tissue expressing human transgenes encoded by the B200 vector (Figure 7A), B201 vector (Figure 7B), and B202 vector (Figure 7C), and of pig heart tissue and pig kidney tissue expressing human transgenes encoded by the B209 vector (Figure 7D) and B212 vector (Figure 7E). [Figure 7E]Figures 7A to 7E show representative immunohistochemical staining images of pig lung tissue, pig heart tissue, and pig kidney tissue expressing human transgenes encoded by the B200 vector (Figure 7A), B201 vector (Figure 7B), and B202 vector (Figure 7C), and of pig heart tissue and pig kidney tissue expressing human transgenes encoded by the B209 vector (Figure 7D) and B212 vector (Figure 7E). [Figure 8A] Figures 8A-B show representative immunohistochemical staining images of pig hearts expressing the B200 vector transgenes in baboon and human recipients. Figure 8A shows immunohistochemical staining of a pig heart obtained immediately postmortem from a baboon recipient 126 days after transplantation, demonstrating expression of all six human transgenes encoded by the B200 vector. [Figure 8B] Figures 8A-B show representative immunohistochemical staining images of pig hearts expressing the B200 vector transgenes in baboon and human patients, and Figure 8B shows immunohistochemical staining of a pig heart obtained immediately post-mortem from a human graft 60 days after transplantation, demonstrating expression of all six human transgenes encoded in the B200 vector. [Figure 9] Figure 9 shows a representative immunohistochemical staining image of a pig kidney expressing the B201 transgene in a pig kidney transplanted into a baboon; expression of all human transgenes encoded by the B201 vector is shown. The pig kidney was obtained immediately post-mortem from the baboon 120 days after transplantation and stained. [Figure 10] Figure 10 shows representative immunohistochemical staining images of two pig heart samples (donor #1 and donor #2) expressing the B209 transgene in pig hearts transplanted into brain-dead human recipients, demonstrating expression of all six human transgenes encoded in the B209 vector. These two pig heart samples (donor #1 and donor #2) were obtained and stained immediately after death. DETAILED DESCRIPTION OF THE INVENTION

[0041] I. Overview A. Multi-transgenic animals for improving xenotransplantation It should be understood that certain aspects, modes, embodiments, variations, and features of the present methods are described below in varying levels of detail to provide a solid understanding of the present technology.

[0042] Xenotransplantation has the potential to alleviate the critical shortage of human organs for transplantation. Acceptance of pig organs by human recipients requires multiple genetic modifications, including silencing of key pig xenoantigens and expression of important human proteins, to mitigate immune rejection, thrombosis, and inflammation. The present disclosure provides six human transgenes that, when expressed in pig organs, are crucial for the long-term survival of pig organs in non-human primates and even human patients.

[0043] The present invention relates to transgenic animals that are particularly useful as a source of organs, organ fragments, tissues, or cells for xenotransplantation. In particular, the invention is directed to transgenic ungulates, particularly transgenic pigs, that are particularly useful 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 disorders.

[0044] The donor animals are advantageous in that they provide organs, organ fragments, tissues, and cells that are functionally superior in the context of transplantation (tx) 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 the present invention have improved survival and / or functionality due to a significant reduction in the consumptive coagulopathy (also known as disseminated intravascular coagulation (DIC)) and thrombotic microangiopathy currently observed after incompatible xenotransplantation.

[0045] The organ or organ fragment may be any suitable organ, such as lung, heart, liver, kidney, or pancreas. The tissue may be any suitable tissue, such as epithelial tissue or connective tissue. The cell may be any suitable cell. The cell may be an islet cell, pancreatic cell, kidney cell, cardiac cell, liver cell, or lung cell.

[0046] In preferred embodiments, the present invention provides transgenic animals (e.g., ungulates, pigs) that are particularly useful as sources of organs (i.e., heart, kidney, liver, pancreas, and lung), organ fragments, tissues, or cells for lung xenotransplantation, and extends to organs (i.e., heart, kidney, liver, pancreas, and lung), organ fragments, tissues, and cells derived from said transgenic animals, as well as methods for producing said transgenic animals and methods for using organs, tissues, and cells derived from said transgenic animals for lung xenotransplantation.

[0047] B. Transgenic animals containing at least 10 genetic modifications To facilitate efficient production of transgenic founder pigs and breeding and production herd expansion, the inventors of the present disclosure included all six transgenes in a single multigene expression vector. Insertion of the vector into the pig genome was targeted to specific loci known to be permissive for transgene expression, ensuring reliable and consistent expression in the transplanted organ. Targeting the multigene vector to a known locus, or landing pad, offered several advantages. First, it avoided off-target integration events by suppressing potentially deleterious random insertions. Second, it facilitated genotypic evaluation of the targeted gene vector and its genomic environment to confirm the intended design. Third, integrating all transgenes into a single multigene vector allowed for the transfer of these transgenes at a single locus. Transferring the transgenes at this single locus was particularly important because it simplified and enhanced the breeding of pigs carrying all the modifications required for successful xenotransplantation (e.g., 4, 4, 7, 9, 10, or 15 modifications). Fourth, a single multigene vector allows for the addition of up to 10 modifications (e.g., 6 transgenes + 4 gene knockouts) in one step rather than multiple sequential steps. Thus, the disclosed method saves weeks of time and valuable laboratory and animal resources for generating multi-transgenic animals (e.g., pigs).

[0048] To generate multi-transgenic animals (e.g., pigs) containing at least 10 modifications (e.g., six transgenes + four gene knockouts), a single multigene vector was created encoding at least six transgenes. Those skilled in the art will understand that the aggregate size of a vector with six transgenes, each driven by its own promoter, would exceed the feasible size for standard plasmid-based construction methods. Therefore, the inventors minimized the number of promoters by using a self-cleaving peptide derived from a virus (e.g., a viral 2A sequence).

[0049] Linking transgenes to viral 2A sequences enabled translation of each transgene-encoded polypeptide / protein from a single multi-transgenic transcript driven by a single promoter. This approach halved the number of promoters required and reduced the vector size to a viable size.

[0050] A multicistronic vector (MCV) consisting of three bicistronic units was then designed and constructed. Each bicistronic unit contained two human genes (e.g., cDNAs of two human genes) linked by a viral T2A or P2A peptide sequence, as defined herein, driven by an endothelial-specific promoter. In some embodiments, the endothelial-specific promoter was selected from the group consisting of the porcine thrombomodulin (TBM) promoter (pTBMpr) or the porcine vascular endothelial cell protein C receptor (EPCR) promoter (pEPCRpr). In some embodiments, each bicistronic unit contained two human genes (cDNAs) linked by a viral T2A or P2A peptide sequence, as defined herein, driven by a constitutively active CAG promoter. In some embodiments, the constitutive promoter was selected from the group consisting of the CMV enhancer, chicken β-actin promoter, and rabbit β-globulin intron. In some embodiments, the bicistronic unit is selected from the group consisting of hTBM-2A-hEPCR driven by the pTBM promoter; hCD47-2A-hHO1 driven by the CAG promoter; hCD46-2A-hDAF driven by the CAG promoter, and hCD59-2A-hHO1 driven by the CAG promoter.

[0051] In some embodiments, the polycistronic vector (MCV) contained various combinations of human genes (e.g., hTBM, hEPCR, hCD47, hH01, hCD46, hDAF, hCD59), with each bicistron driven by a specific promoter. In some embodiments, the MCV contained four bicistronic units. In some embodiments, the MCV contained hTBM-2A-hEPCR driven by the pTBM promoter; and hCD47-2A-hH01, hCD46-2A-hDAF, and hCD59-2A-hH01, each driven by a CAG promoter. In some embodiments, the MCVs containing hTBM and hEPCR were separately driven by the pTBM and pEPCR promoters, respectively. The bicistronic unit contained an hTBM cDNA (pTBMpr-hTBM-2A-pEPCRpr-hTBM) driven by the pTBM promoter (pTBMpr) linked via the 2A peptide to an hEPCR cDNA driven by the pEPCR promoter (pEPCRpr). MCVs containing pTBMpr-hTBM-2A-pEPCRpr-hTBM, as well as hCD47-2A-hHO1, hCD46-2A-hDAF, and hCD59-2A-hHO1, each driven by the CAG promoter, were generated.

[0052] Several bicistronic vectors were constructed to evaluate the effects of 1) promoter, 2) transgene combination, and 3) transgene order within the bicistronic vector on transgene expression, biological function, and efficacy in supporting post-transplant organ survival. These bicistronic vectors were used as building blocks for constructing larger and more complex multicistronic vectors (MCVs). The bicistronic combinations were selected based on their ability to appropriately express the transgenes to match the viability of the transgenic pigs and to enhance post-transplant organ and recipient survival. These unique, novel, and proven bicistronic designs enhanced the ability to efficiently integrate multiple transgenes into MCVs, both targeted and targetable for integration into permissive loci (e.g., CMAH, GGTA1, B4GalNT2), thereby enabling consistent, predictable, and appropriate expression of multiple transgenes from a single genomic locus, thus representing an advancement in the technology.

[0053] To generate multitransgenic animals using novel and unique bicistronic combinations, all vectors were introduced into porcine fetal fibroblasts in which the GGTA1 locus had previously been knocked out by NeoR-mediated insertional mutagenesis. See Dai, Y. et al., Nat Biotechnol. 20:251-5 (2002). For insertion into GGTA1 (B201, B202), the vectors were equipped with homology arms targeted to NeoR.

[0054] C. Pigs lacking functional α-1,3-galactosyltransferase and at least one additional genetic modification Organs, organ fragments, tissues, or cells derived from the transgenic animals are advantageous in that, following xenotransplantation, there is a low level or no incidence of one or more of the following: hyperacute rejection (HAR), acute humoral rejection (AHXR / DXR), and / or acute cellular xenograft rejection (ACXR).

[0055] In one embodiment, organs, organ fragments, tissues, or cells derived from the transgenic animal produce low levels or no HAR and AHXR after xenotransplantation. In another embodiment, organs, organ fragments, tissues, or cells derived from the transgenic animal produce low levels or no HAR, AHXR, and ACXR after xenotransplantation.

[0056] In a preferred embodiment, the transgenic animal is a pig that does not express any functional α-1,3-galactosyltransferase (α-Gal) (as a result of genetic modification or otherwise) and incorporates at least some additional genetic modifications (e.g., gene knockouts, gene knock-ins, gene replacements, point mutations, deletions, insertions, or substitutions (i.e., of genes, gene fragments, or nucleotides), large genomic insertions, or combinations thereof). These genetic modifications may be made via any suitable technique, including, for example, homologous recombination or gene editing.

[0057] In a preferred embodiment, the transgenic animal is a pig that does not express any functional α-1,3-galactosyltransferase (α-Gal) (as a result of genetic modification or otherwise) and incorporates and expresses at least six transgenes under the control of at least three promoters at a single locus. In one embodiment, one promoter controls the expression of at least two transgenes. For example, the expression of each of the at least six transgenes is controlled by a single (dedicated) promoter. In another embodiment, one promoter controls the expression of more than one transgene, for example, one promoter controls the expression of two transgenes.

[0058] Advantageously, these six or more transgenes co-integrate, co-express, and co-segregate upon mating. The single locus can vary. In some embodiments, the single locus is a native locus or a modified native locus. The modified native locus may be modified by any suitable technique, including, but not limited to, CRISPR-induced insertions or deletions (indels), the introduction of a selectable marker gene (e.g., neo), or the introduction of a large genomic insert (e.g., landing pad) intended to facilitate the integration of one or more transgenes. In certain embodiments, the single locus is a native locus or a modified GGTA1 gene. The GGTA1 locus is inactivated by the integration and expression of the at least six transgenes, for example, by the application of homologous recombination, gene editing, or recombinase technology. The single locus may be, for example, AAVS1, GHR, ROSA26, CMAH, or β4GalNT2. Optionally, the transgenic animal may have one or more additional genetic modifications and / or the expression of one or more additional porcine genes may be modified by mechanisms other than genetic modification.

[0059] In a preferred embodiment, the transgenic animal is a pig that does not express any functional α-1,3-galactosyltransferase (α-Gal) (as a result of genetic modification or otherwise) and incorporates and expresses 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 transgenes at a single locus. In one embodiment, expression of the 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 transgenes is regulated 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 the transgene, i.e., one promoter regulates the expression of one transgene, while in other embodiments, one promoter regulates the expression of two or more transgenes, e.g., one promoter regulates the expression of two transgenes. Advantageously, these two or more additional transgenes are co-integrated, co-expressed, and co-segregated upon mating. The single locus can vary. In some embodiments, the single locus is a native locus or a modified native locus. The modified native locus may be modified by any suitable technique, including, but not limited to, CRISPR-introduced insertions or deletions (indels), the introduction of a selectable marker gene (e.g., Neo), or the introduction of a large genomic insert (e.g., landing pad) intended to facilitate the integration of one or more transgenes. In certain embodiments, the single locus is a native locus or a modified GGTA1 gene.

[0060] The GGTA1 locus is inactivated by the integration and expression of the at least six transgenes, e.g., by homologous recombination, gene editing, or application of recombinase technology. The single locus may be, e.g., AAVS1, GHR, ROSA26, CMAH, or β4GalNT2. Optionally, the donor animal may have additional genetic modifications and / or the expression of one or more additional pig genes may be modified by mechanisms other than genetic modification.

[0061] In a preferred embodiment, the transgenic animal is a pig that does not express any functional α-1,3-galactosyltransferase (α-Gal) (as a result of genetic modification or otherwise), incorporates and expresses at least six transgenes at a single locus (i.e., Locus 1), and also incorporates and expresses one or more additional transgenes at a second single locus (i.e., Locus 2) and a third single locus (i.e., Locus 2). In one embodiment, a single promoter regulates expression of a single transgene, e.g., expression of each of the at least six transgenes at Locus 1, Locus 2, or Locus 3 is regulated by a single (dedicated) promoter. In another embodiment, a single promoter regulates expression of two or more transgenes, e.g., one promoter regulates expression of two transgenes at Locus 1. These specific loci can vary. In certain embodiments, the first single locus is GGTA1 and the second and third single loci are, e.g., CMAH, B4GalNT2, or growth hormone receptor (GHR). In certain embodiments, at least six transgenes are integrated and expressed at each single locus, i.e., Locus 1, Locus 2, and Locus 3, to produce an animal in which 12 or more transgenes are expressed at two separate and independent loci.

[0062] In one embodiment, the single locus is a native locus or a modified native locus. The modified native locus may be modified by any suitable technique, including, but not limited to, CRISPR-induced insertions or deletions (indels), the introduction of a selectable marker gene (e.g., neo), or the introduction of a large genomic insert (e.g., landing pad) intended to facilitate the integration of one or more transgenes. Optionally, the donor animal may have additional genetic modifications, and / or the expression of one or more additional pig genes may be modified by mechanisms other than genetic modification. Advantageously, these two or more additional transgenes co-integrate, co-express, and co-segregate upon mating.

[0063] The at least three promoters can vary. The promoters can be exogenous or native. In preferred embodiments, the promoters are constitutive or regulatable (e.g., tissue-specific, inducible) promoters. In one embodiment, all three promoters can be constitutively or ubiquitously expressed in the donor animal (e.g., from the CAG promoter, Tie-2 promoter, ICAM-2 promoter, or similar promoters). In another embodiment involving three promoters, one promoter allows tissue-specific expression of the transgene (e.g., endothelial-specific expression, such as the TBM promoter or EPCR promoter), and the second and third promoters allow constitutive or ubiquitous expression of one or more transgenes (at the same integration site) (e.g., from the CAG promoter or similar promoters).

[0064] In some embodiments, the promoter is a controllable promoter. The controllable promoter can be part of an inducible system. In some embodiments, the transgene and / or CRISPR / Cas system is under the control of an inducible promoter. For example, the inducible system can include a tetracycline-inducible promoter selected from Tet-On or Tet-Off, a small molecule two-hybrid transcription activation system (such as FKBP or ABA), or a light-inducible system (phytochrome, LOV domain, or cryptochrome). Examples of inducible DNA binding proteins and their uses are provided in U.S. Patent Application Nos. 61 / 736,465 and 61 / 721,283, and International Publication No. WO 2014 / 018423, as well as U.S. Patent Nos. 8,889,418, 8,895,308, U.S. Patent Application Publication Nos. 20140186919, 20140242700, 20140273234, 20140335620, and International Publication No. WO 2014093635. In some embodiments, the inducible system is a Light Inducible Transcriptional Effector (LITE) system, which directs changes in transcriptional activity in a sequence-specific manner. Light components can include enzymes or transgenes, light-reactive cytochrome heterodimers (e.g., from Arabidopsis thaliana), and transcriptional activation / repression domains.

[0065] In some embodiments, the CRISPR / Cas9-mediated gene editing comprises an inducible promoter or system, a tetracycline / doxycycline-regulated system, U6p[GHRgRNA-1], U6p[GHRgRNA-2], TRE3Gp[CAS9], CAGpr[tTA], CAGpr[hCD46-2A-hCD55], or a polycistronic vector comprising the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the inducible promoter regulates expression of a growth hormone receptor gene.

[0066] In some embodiments, the additional genetic modifications (i.e., in addition to the integration and expression of the multiple transgenes described above) may result in the inactivation of specific porcine genes, including, but not limited to, porcine growth factor genes, or the replacement of part or all of the porcine GHR gene with an equivalent counterpart from the human GHR gene. In some embodiments, growth hormone receptor gene expression is regulated by an inducible system comprising SEQ ID NO: 9. In some embodiments, the additional genetic modifications in the GHR gene include U6p[GHRgRNA-1], U6p[GHRgRNA-2], TRE3Gp[CAS9], CAGpr[tTA], CAGpr[hCD46-2A-hCD55], or a polycistronic vector comprising the nucleotide sequence of SEQ ID NO: 9. Other genes that may be inactivated along with the additional genetic modifications include, for example, CMP-NeuAc hydroxylase (CMAH), isogloboside 3 synthase, β4Gal, NT2-Forssmann synthase, or a combination thereof. In some embodiments, the single locus for transgene integration is not GGTA1, and the additional genetic modification comprises inactivation of GGTA1.

[0067] In some embodiments, the additional genetic modification is, for example, a deletion / insertion or gene replacement (INDEL) introduced by gene editing. In some embodiments, the additional genetic modification (i.e., in addition to the integration and expression of the multiple transgenes described above) can result in the integration and expression of one or more transgenes at a second locus.

[0068] In one embodiment, the invention is a pig that does not express any functional α-1,3-galactosyltransferase (α-Gal) (as a result of genetic modification or otherwise) and further comprises inactivation of the porcine growth hormone receptor (GHR) gene or replacement of part or all of the porcine GHR gene with an equivalent counterpart from the human GHR gene. Optionally, the pig comprises one or more additional genetic modifications. In some embodiments, the animal may be bred with a second animal that comprises one or more genetic modifications.

[0069] The present invention also extends to methods of producing and using such transgenic animals (or organs, tissues, or cells derived therefrom). In a preferred embodiment, the present invention provides a method of producing a transgenic pig that expresses at least six transgenic genes but lacks expression of α-1,3-galactosyltransferase, the method comprising: (i) integrating at least six transgenes under the control of at least three promoters at a single locus within the pig genome to obtain a multigenic pig genome; and (ii) maturing a cell containing the multigenic pig genome into a transgenic pig. In some embodiments, the pig genome is a somatic pig genome and the cell is a pig zygote. In some embodiments, the pig genome is selected from the group consisting of a gamete pig genome, a zygote pig genome, an embryo pig genome, or a blastocyst pig genome. In a preferred embodiment, the integration comprises a method selected from the group consisting of biological transfection, chemical transfection, physical transfection, viral-mediated transduction or transformation, or a combination thereof. In some embodiments, integration comprises cytoplasmic microinjection and pronuclear microinjection.

[0070] D. Multicistronic Vector Systems for the Generation of Multitransgenic Pigs The use of polycistronic expression systems has been developed to insert multiple transgenes into various cell types and animals. Using a 2A peptide bicistronic system and nuclear transfer via random integration of transgenes, transgenic pigs expressing four fluorescent proteins were produced (Deng et al. Plos One, 6 (5): e19986).

[0071] Furthermore, transgenic pigs expressing the complement regulatory factor CD59 gene and H-transferase gene were produced using an IRES-mediated tricistronic vector system and nuclear transfer. Attempts were made to express three genes using this tricistronic system, but expression of the third gene in the tricistronic system was not detected in the transgenic pigs, despite it being present in an IRES vector. (Jeong et al., Plos One, 8 (5): e63241) Furthermore, other publications have reported that efficient expression of downstream genes in multicistronic systems is impossible, but that it could be achieved if expression of the upstream gene was also efficient. (Hurh et al., Plos One, 8:(7) e70486)

[0072] In preferred embodiments, the methods involve the use of bicistronic or polycistronic vectors, including multicistronic vectors utilizing 2A technology, that allow transgenes to be cointegrated and coexpressed, providing functional and / or productive advantages. In preferred embodiments, each bicistron in a multicistronic vector containing at least six transgenes is under the control of its own promoter, where one or more promoters may provide constitutive expression of two or more genes and a second promoter may provide tissue-specific expression of two or more genes. These vectors are utilized in combination with gene editing tools, including editing nucleases and / or site-specific integrases.

[0073] In one embodiment, the method involves the use of a single polycistronic vector that allows six or more transgenes to be co-integrated and co-expressed, facilitating breeding in which all transgenes co-segregate and are passed on to offspring / progeny as a single unit.

[0074] 1. Generation of transgenic pigs containing at least six transgenes The present invention provides methods for producing a transgenic pig containing at least six transgenes, the method comprising: (i) transfecting a pig cell with a single polycistronic vector containing (a) at least two complement inhibitor transgenes, (b) at least one immunosuppressant transgene, (c) at least one cytoprotective factor transgene, and (d) at least two anticoagulant transgenes; (ii) integrating and expressing the polycistronic vector at a single genomic locus to produce a multi-transgenic pig cell containing at least six transgenes; (iii) injecting the nucleus of the multi-transgenic pig cell into a reconstituted somatic cell nuclear transfer (SCNT) to produce a multi-transgenic pig zygote; and (iv) maturing the multi-transgenic pig zygote into a multi-transgenic pig. In some embodiments, the pig cell and the multi-transgenic pig lack expression of α-1,3-galactosyltransferase.In some embodiments, the polycistronic vector comprises a bicistronic unit selected from the group consisting of: a human TBM transgene linked to a human EPCR transgene via a 2A peptide and driven by a porcine TBM promoter (pTBMpr[hTBM-2A-hEPCR]); a human CD47 transgene linked to a human HO-1 transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD47-2A-hHO1]); a human CD46 transgene linked to a human DAF transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD46-2A-hDAF]); a human TBM transgene linked to a human EPCR transgene via a 2A peptide and driven by a polyA signal-fused porcine TBM promoter (polyA / pTBMpr[hTBM-2A-hEPCR]); or a human CD59 transgene linked to a human HO-1 transgene via a 2A peptide and driven by a CAG promoter. (vii) a human CD46 transgene linked to a human EPCR transgene via a 2A peptide and driven by a porcine EPCR promoter (pEPCRpr[hTBM-2A-hEPCR]); (vii) a human CD47 transgene linked to a human CD46 transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD46-2A-hCD47]); (viii) a first GHR gRNA and a first U6 promoter driving it. (xix) a Cas endonuclease and a TRE3G promoter driving it, and a tTA and a CAG promoter driving it, linked to the first and second gRNAs via an insulator (TRE3Gp[CAS9]; CAGpr[tTA]); and combinations thereof.In some embodiments, the polycistronic vector comprises: pTBMpr[hTBM-2A-hEPCR], CAGpr[hCD47-2A-hH01], and CAGpr[hCD46-2A-hDAF]; polyA-pTBMpr[hTBM-2A-hEPCR], CAGpr[hCD47-2A-hH01], and CAGpr[hCD46-2A-hDAF]; pTBMpr[hTBM-2A-hEPCR], CAGpr[hCD59-P2A-hH01], and CAGpr[hCD46-P2A-hDAF]; polyA-pTBMpr[hTBM], CAGpr[hCD47-P2A-hH01], pEPCRpr[hEPCR ] and CAGpr[hCD46P-2A-hDAF]; pTBMpr[hTBM-2A-hEPCR], CAGpr[hCD59-P2A-hHO1], and CAGpr[hCD46-2A-hCD47]; pTBMpr[hTBM-2A-hEPCR], CAGpr[hCD59-2A-hHO1], and CAGpr[hCD46-2A-hCD55]; U6p[GHRgRNA-1], U6p[GHRgRNA-2], TRE3Gp[CAS9], CAGpr[tTA], and CAGpr[hCD46-2A-hCD55]; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; or SEQ ID NO: 14.

[0075] In some embodiments, the porcine cell is a somatic cell. In some embodiments, the multi-transgenic porcine cell comprises at least seven, at least eight, at least nine, or at least ten transgenes. In some embodiments, at least six transgenes are encoded by a polycistronic vector, and optionally, the polycistronic vector comprises at least three bicistronic units. In some embodiments, each bicistronic unit comprises a promoter driving a first transgene linked to a second transgene via a self-cleaving peptide. In some embodiments, a first bicistronic unit comprises the at least two anticoagulant factor transgenes, a second bicistronic unit comprises at least two complement inhibitor transgenes, and a third bicistronic unit comprises the at least one cytoprotective factor transgene and the at least one immunosuppressant transgene. In some embodiments, the self-cleaving peptide is selected from the group consisting of T2A, P2A, F2A, and E2A. In some embodiments, the at least six transgenes are encoded by a polycistronic vector comprising: (i) at least two bicistronic units, each bicistronic unit comprising a promoter driving a first transgene linked to a second transgene via a self-cleaving peptide; and (ii) at least two non-polycistronic transgenes, each non-polycistronic transgene driven by its own promoter. In some embodiments, at least one bicistronic unit is driven by a tissue-specific promoter; and / or at least two bicistronic units are driven by dedicated constitutive promoters. In some embodiments, at least one bicistronic unit is driven by an inducible promoter. In some embodiments, the at least two bicistronic units are each driven by a constitutive promoter, and the at least two non-polycistronic transgenes are each driven by a tissue-specific promoter.

[0076] 2. Generation of transgenic pigs containing at least six transgenes The present invention provides transgenic pigs comprising genetic modifications resulting in the absence of expression of a functional α-1,3-galactosyltransferase gene and the integration and expression, at a single genomic locus, of (a) at least two complement inhibitor transgenes; (b) at least one immunosuppressant transgene; (c) at least one cytoprotective factor transgene; and (d) at least two anticoagulant transgenes. In some embodiments, the transgenic pig comprises at least six, at least seven, or at least eight, at least nine, at least ten, at least 11, or at least 12 transgenes. The transgenic animal comprises at least six transgenes. In some embodiments, the at least six transgenes are encoded by a polycistronic vector, and optionally, the polycistronic vector comprises at least three bicistronic units. In some embodiments, each bicistronic unit comprises a promoter driving a first transgene linked to a second transgene via a self-cleaving peptide. In some embodiments, the first bicistronic unit comprises the at least two anticoagulant factor transgenes, the second bicistronic unit comprises at least two complement inhibitor transgenes, and the third bicistronic unit comprises the at least one cytoprotective factor transgene and the at least one immunosuppressive factor transgene. In some embodiments, the self-cleaving peptide is selected from the group consisting of T2A, P2A, F2A, and E2A.

[0077] As used herein, a "self-cleaving peptide" or "2A peptide" or "viral 2A sequence" refers to an oligopeptide that allows multiple proteins to be encoded as a polyprotein that post-translationally dissociates into its constituent proteins. Use of the term "self-cleaving" is not intended to imply a proteolytic reaction. Various "self-cleaving peptides," "2A peptides," or "viral 2A sequences" are known to those of skill in the art, including, but not limited to, those present in the Picomaviridae taxon, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAVO, Thosea asigna virus (TaV), and porcine teschovirus (PTV-1); and cardioviruses, such as tylovirus and encephalomyocarditis virus. Viral 2A sequences from FMDV, ERAVO, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. Those of skill in the art will be able to select an appropriate self-cleaving peptide for use in the present invention.

[0078] In some embodiments, the transgene is encoded by a polycistronic vector comprising: (i) at least two bicistronic units, each bicistronic unit comprising a promoter driving a first transgene linked to a second transgene via a self-cleaving peptide; and (ii) at least two non-polycistronic transgenes. Each non-polycistronic transgene is driven by its own promoter. In some embodiments, at least one bicistronic unit is driven by a tissue-specific promoter; and / or at least two bicistronic units are driven by dedicated constitutive promoters. In some embodiments, the at least two bicistronic units are each driven by a constitutive promoter, and the at least two non-polycistronic transgenes are each driven by a tissue-specific promoter.

[0079] In some embodiments, the polycistronic vector comprises a bicistronic unit selected from the group consisting of: a human TBM transgene linked to a human EPCR transgene via a 2A peptide and driven by a porcine TBM promoter (pTBMpr[hTBM-2A-hEPCR]); a human CD47 transgene linked to a human HO-1 transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD47-2A-hHO1]); a human CD46 transgene linked to a human DAF transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD46-2A-hDAF]); a human TBM transgene linked to a human EPCR transgene via a 2A peptide and driven by a polyA signal-fused porcine TBM promoter (polyA / pTBMpr[hTBM-2A-hEPCR]); or a human CD59 transgene linked to a human HO-1 transgene via a 2A peptide and driven by a CAG promoter. The CAG promoter driving the first GHRgRNA (CAGpr[hCD59-2A-hHO1]); the porcine EPCR promoter driving the human TBM transgene linked to the human EPCR transgene via a 2A peptide (pEPCRpr[hTBM-2A-hEPCR]); the CAG promoter driving the human CD46 transgene linked to the human CD47 transgene via a 2A peptide (CAGpr[hCD46-2A-hCD47]); and the first GHRgRNA driving the first U6 transgene. a promoter and a second GHRgRNA and a second U6 promoter driving it (the first gRNA and the second gRNA may be the same or different) linked thereto (U6p[GHRgRNA-1]; U6p[GHRgRNA-2]); a Cas endonuclease and a TRE3G promoter driving it, and a tTA and a CAG promoter driving it linked thereto via an insulator (TRE3Gp[CAS9]; CAGprtTA]); and combinations thereof. In some embodiments, the porcine promoter may be an endogenous promoter or an exogenous promoter.

[0080] In some embodiments, the polycistronic vector comprises: pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD47-2A-hH01]; and CAGpr[hCD46-2A-hDAF]; polyA-pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD47-2A-hH01]; and CAGpr[hCD46-2A-hDAF]; pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD59-P2A-hH01]; and CAGpr[hCD46-P2A-hDAF]; polyA-pTBMpr[hTBM]; CAGpr[hCD47-P2A-hH01]; pEPCRpr[hEPCR ]; and CAGpr[hCD46P-2A-hDAF]; pTBMpr[hTBM-2A-hEPCR], CAGpr[hCD59-P2A-hHO1], and CAGpr[hCD46-2A-hCD47]; pTBMpr[hTBM-2A-hEPCR], CAGpr[hCD59-2A-hHO1], and CAGpr[hCD46-2A-hCD55]; U6p[GHRgRNA-1], U6p[GHRgRNA-2], TRE3Gp[CAS9], CAGpr[tTA], and CAGpr[hCD46-2A-hCD55]; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; or SEQ ID NO: 14.

[0081] E. Treatment The present invention also encompasses methods of treating a subject in need thereof with one or more organs, organ fragments, tissues, or cells derived from the transgenic animals of the present invention. In preferred embodiments, the organ is a solid organ, such as the liver, lung, heart, or pancreas. Examples of tissues contemplated by the present invention include, but are not limited to, epithelial tissue and connective tissue. Transplants involving two or more organs or organ fragments are also contemplated by the present invention. For example, transplants involving the lung, pancreas, liver, retina, heart, kidney, or fragments thereof are contemplated by the present invention.

[0082] The present invention provides methods for performing xenotransplantation, comprising administering to a subject in need thereof an organ, tissue, or cells derived from the transgenic pig of the present invention. In some embodiments, the subject is a non-human primate or a human. In some embodiments, the organ is selected from the group consisting of heart, lung, liver, and kidney. In some embodiments, the tissue is selected from the group consisting of vascular tissue, retinal tissue, neural tissue, and corneal tissue. In some embodiments, the method further comprises administering to the subject a clinically relevant immunosuppressant regimen following xenotransplantation of the organ, tissue, or cells derived from the transgenic pig of the present invention.

[0083] II. Definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. For example, a reference to a "cell" includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry, and hybridization described below, are well known and commonly used in the art.

[0084] As used herein, the term "adverse event" refers to any untoward or unintended sign (including, for example, an abnormal laboratory finding), symptom, or disease that temporarily accompanies the use of a pharmaceutical product (e.g., a xenograft), whether or not considered to be related to the pharmaceutical product.

[0085] As used herein, the term "animal" refers to a mammal. In certain embodiments, the animal is at least 6 months old. In some embodiments, the animal is post-weaning. In some embodiments, the animal survives until mating age. The animals of the present invention are "genetically modified" or "transgenic," meaning that they have added or incorporated a transgene or other foreign DNA, or an endogenous gene that has been modified, including targeted, recombined, interrupted, deleted, disrupted, substituted, suppressed, enhanced, or otherwise altered, to mediate a genotypic or phenotypic effect, in at least one cell of the animal, and typically in at least one germline cell of the animal. In some embodiments, the animal may have a transgene integrated on one allele of its genome (heterozygous transgenic). In other embodiments, the animal may have a transgene on two alleles (homozygous transgenic).

[0086] As used herein, the terms "breeding" or "breeded," or derivatives thereof, refer to any means of reproduction, including both natural and artificial means.

[0087] As used herein, the terms "breeding herd" or "production herd" refer to a group of transgenic animals produced by the methods of the present invention. In some embodiments, genetic modifications may be confirmed in animals, which may then be bred to form a herd of animals with the desired set of genetic modifications (or a single genetic modification). See International Publication No. WO 2012 / 112586; International Application No. PCT / US2012 / 025097. These offspring may be further bred to produce different or the same set of genetic modifications (or a single genetic modification) in the offspring. This breeding cycle of animals with the desired genetic modifications may continue as long as desired. A "herd" in this context may include multiple generations of animals produced over time with the same or different genetic modifications. A "herd" may also refer to a single generation of animals with the same or different genetic modifications.

[0088] As used herein, the terms "CRISPR" or "Clustered Regularly Interspaced Short Palindromic Repeats" or "SPIDR" or "Spacer Interspersed Direct Repeats" refer to a family of DNA loci typically unique to specific bacterial species. CRISPR loci contain a distinct class of interspersed short sequence repeats (SSRs) recognized in Escherichia coli (Ishino et al., J. Bacteriol., 169:5429-5433

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

[1989] ) and associated genes. CRISPR / Cas molecules are components of the prokaryotic adaptive immune system that use RNA base pairing to direct DNA or RNA cleavage, functionally similar to eukaryotic RNA interference. Directing a DNA DSB requires two components: the Cas9 protein, which functions as an endonuclease, and CRISPR RNA (crRNA) and tracer RNA (tracrRNA) sequences, which direct the Cas9 / RNA complex to target the DNA sequence (Makarova et al., Nat Rev Microbiol, 9(6):467-477, 2011). Modification of a single targeting RNA can be sufficient to alter the nucleotide target of the Cas protein. In some cases, the crRNA and tracrRNA can be engineered to direct Cas9 cleavage activity as a single cr / tracrRNA hybrid (Jinek et al., Science, 337(6096):816-821, 2012).CRISPR / Cas systems can be used in bacteria, yeast, humans, and zebrafish, as described elsewhere (see, e.g., 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).

[0089] As used herein, the term "clinically relevant immunosuppressive regimen" refers to a clinically acceptable regimen of immunosuppressant drugs provided to a patient following transplantation of genetically modified porcine organs, tissues, or cells as disclosed herein. A determination of clinical significance generally requires a judgment by the FDA balancing acceptable risks and potential benefits such that efficacy of the drug or treatment is maintained while human safety is maintained.

[0090] As used herein, the term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes that gene product to be produced in a cell under most or all physiological conditions of the cell.

[0091] As used herein, the term "donor" is intended to encompass any non-human animal that can serve as a source of donor organs, tissues, or cells for xenotransplantation. The donor may be at any stage of development, including, but not limited to, fetal, neonatal, juvenile, or adult.

[0092] As used herein, the term "endogenous" as used herein with respect to nucleic acid sequences and animals refers to any nucleic acid sequence that naturally occurs within the genome of that animal. An endogenous nucleic acid sequence may include one or more gene sequences, intergenic sequences, portions of gene sequences or intergenic sequences, or combinations thereof.

[0093] As used herein, "endothelial-specific," "specific transgene expression in endothelial tissue," "specifically expressing at least one transgene in endothelial tissue," and like terms are understood to refer to a transgene under the control of endothelial-specific control elements that allow for restricted expression of the transgene in endothelial tissue and / or endothelial cells, such that the function and expression of the transgene is restricted to endothelial tissue and / or endothelial cells.

[0094] As used herein, the term "endothelium" refers to mesodermally derived epithelium composed of a single layer of thin, flat cells that lines the interior of body cavities. For example, serous cavities or the interior of the heart contain lining endothelial cells, and "vascular endothelium" refers to the endothelium that lines blood vessels.

[0095] As used herein, "endothelial-specific control element" and like terms refer to a promoter, enhancer, or combination thereof that drives the restricted expression of a transgene in endothelial tissue and / or endothelial cells. Such control elements result in transgene function and expression restricted to endothelial tissue and / or endothelial cells.

[0096] As used herein, the term "enhancer" refers to an element in a nucleic acid construct intended to promote increased expression of a transgene in a tissue-specific manner. Enhancers are external elements that dramatically alter the efficiency of gene transcription (Molecular Biology of the Gene, Fourth Edition, pp. 708-710, Benjamin Cummings Publishing Company, Menlo Park, CA (C) 1987). In some embodiments, the animal expresses a transgene under the control of a promoter combined with an enhancer element. In some embodiments, the promoter is used in combination with an enhancer element, which is a non-coding or intronic DNA region inherently associated with or co-localized with the promoter.

[0097] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as the "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0098] The term "gene" is used broadly herein to refer to any segment of DNA associated with a biological function. That is, a gene includes a coding sequence and / or regulatory sequences required for its expression. A gene may also include non-expressed DNA segments, such as those that form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a desired source or synthesis from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0099] As used herein, the term "gene editing" refers to a type of genetic engineering that uses gene editing tools to insert, replace, or remove DNA from a genome. Examples of gene editing tools include, but are not limited to, zinc finger nucleases, TALENs, and CRISPRs.

[0100] As used herein, the term "mediated by gene editing" or similar terms refers to genetic modification (e.g., deletion, substitution, rearrangement) involving gene editing / use of gene editing tools.

[0101] As used herein, the term "gene knockout" refers to a genetic modification that results from the disruption of genetic information encoded at a chromosomal locus.

[0102] As used herein, the term "gene knock-in" refers to a genetic modification that results from the replacement of genetic information encoded at a chromosomal locus with a different DNA sequence.

[0103] The term "genetic modification," as used herein, refers to one or more changes in a nucleic acid, e.g., a nucleic acid within the genome of an organism. For example, a genetic modification can refer to a genetic change, addition (e.g., gene knock-in), and / or deletion (e.g., gene knock-out).

[0104] As used herein, the term "high" with respect to expression level refers to an expression level that is considered sufficient to provide a phenotype (detectable expression or therapeutic effect). Typically, a "high" level of expression is sufficient to reduce graft rejection, including hyperacute rejection (HAR), acute humoral xenograft rejection (AHXR), T-cell-mediated cellular rejection, and immediate blood-mediated inflammatory response (IBMIR).

[0105] As used herein, the terms "homology-driven recombination" or "homology-directed repair" or "HDR" refer to homologous recombination events initiated by the presence of double-strand breaks (DSBs) in DNA (Liang et al. 1998); the specificity of HDR can be controlled by combining it with genome editing methods (e.g., CRISPR / Cas9 systems) known to create highly efficient, targeted double-strand breaks, allowing precise editing of the genome of target cells (Findlay et al. 2014; Mali et al. February 2014; and Ran et al. 2013).

[0106] As used herein, the term "enhanced homology-driven insertion or knock-in" describes the insertion of a DNA construct, more specifically, the insertion of a large DNA fragment or construct flanked by homology arms, i.e., a DNA segment homologous to the double-strand break, utilizing homology-driven recombination in combination with any genome editing method known to create highly efficient targeted double-strand breaks, allowing precise editing of the genome of a target cell (e.g., the CRISPR / Cas9 system) (Mali et al. Feb 2013).

[0107] As used herein, the term "humanized" refers to a nucleic acid or protein structure (i.e., nucleotide sequence or amino acid sequence) that contains portions that correspond substantially or identically to the structure of a particular gene or protein naturally occurring in a non-human animal, and also refers to the inclusion of portions that differ from those present in a related particular non-human gene or protein and instead correspond more closely to the equivalent structure present in the corresponding human gene or protein. In some embodiments, a "humanized" gene is one that encodes a polypeptide having an amino acid sequence substantially similar to that of a human polypeptide (e.g., a human protein or portion thereof—e.g., a characteristic portion thereof). The term "hyperacute rejection" refers to rejection of transplanted material or tissue that occurs or begins within the first 24 hours after transplantation.

[0108] The terms "implant" or "transplant" or "graft," as used herein, shall be understood to refer to the act of inserting a tissue or organ into a subject under conditions that allow the tissue or organ to become vascularized; and shall also refer to the tissue or organ so inserted (i.e., "implanted" or "transplanted" or "grafted"). Conditions favorable for vascularization of a graft in a mammal include the presence of a local tissue layer at the site of the graft that has an extensive blood supply.

[0109] As used herein, the term "immunomodulator" refers to a transgene capable of modulating an immune response. In a preferred embodiment, the immunomodulator of the present invention 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 inhibitors may be CD55 and CD59. In another embodiment, the immunomodulator may be a type II transactivator or a mutant thereof. In one embodiment, the immunomodulator may be a type II transactivator dominant-negative mutant (CIITA-DN). In another specific embodiment, the immunomodulator is an immunosuppressant. The immunosuppressant may be CTLA4-Ig. Other immunomodulatory factors may be selected from, but are not limited to, CIITA-DN, PDL1, PDL2, or tumor necrosis factor-α related-inducing ligand (TRAIL), Fas Ligand (FasL, CD95L), CD47, also known as integrin-associated protein (CD47), HLA-E, HLA-DP, HLA-DQ, and / or HLA-DR groups.

[0110] As used herein, an "inducible" promoter is a promoter under environmental or developmental control. Examples of promoters that are inducible and allow for gene editing or spatiotemporal regulation of gene expression may utilize forms of energy, including, but not limited to, sound energy, electromagnetic radiation, chemical energy, and / or thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activation systems (FKBP, ABA, etc.), or light-inducible systems (phytochromes, LOV domains, or cryptochromes) such as Light Inducible Transcriptional Effectors (LITEs) that direct changes in transcriptional activity in a sequence-specific manner. Components of light-inducible systems may include enzymes or transgenes, light-responsive cytochrome heterodimers (e.g., from Arabidopsis thaliana), and transcriptional activation / repression domains. Further examples of inducible DNA binding proteins and methods for their use are provided in US Patent Application Nos. 61 / 736,465 and 61 / 721,283.

[0111] Transient or inducible expression can be achieved, for example, by using a chemically regulated promoter, which can induce gene expression upon application of an exogenous chemical. Regulation of gene expression can also be achieved by a chemically repressible promoter, which represses gene expression upon application of a chemical. Chemically inducible promoters include, but are not limited to, the maize 1n2-2 promoter, which is activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-11-27, WO 93 / 01294), which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7). Antibiotic-regulated promoters, such as tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156), can also be used herein.

[0112] As used herein, the term "landing pad" or "engineered landing pad" refers to a nucleotide sequence containing at least one recognition sequence selectively bound and modified by a specific polynucleotide modification enzyme, such as a site-specific recombinase and / or a targeting endonuclease. Typically, the recognition sequence within the landing pad sequence is not endogenously present in the genome of the cell to be modified. Selecting a recognition sequence for a highly efficient nucleotide modification enzyme that is not endogenously present in the genome of the target cell may improve the rate of targeted integration. Selecting a recognition sequence that is not endogenously present also reduces the possibility of off-target integration. In other embodiments, it may be desirable to use a recognition sequence that is native to the cell to be modified. For example, when multiple recognition sequences are used in a landing pad sequence, one or more may be exogenous and one or more may be native.

[0113] The presence of multiple recognition sequences in a landing pad may allow two or more polynucleotide modifying enzymes to sequentially target the landing pad, allowing for the insertion of two or more unique sequences. Alternatively, the presence of multiple recognition sequences in a landing pad may allow multiple copies of the same sequence to be inserted into the landing pad. A landing pad may contain at least one recognition sequence. For example, an 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 two or more recognition sequences, the recognition sequences may be unique to each other (i.e., recognized by different polynucleotide modifying enzymes), may be the same repetitive sequence, or may be a combination of repetitive and unique sequences. Optionally, a landing pad may contain one or more sequences encoding a selectable marker, such as an antibiotic resistance gene, a metabolic selection marker, or a fluorescent protein. Other sequences may also be present, such as transcriptional control elements and transcriptional regulatory elements (ie, promoters, partial promoters, promoter traps, start codons, enhancers, introns, insulators, and other expression elements).

[0114] As used herein, the term "large targeting vector" or "LTVEC" includes large targeting vectors for eukaryotic cells that are derived from larger fragments of cloned genomic DNA than are typically used in other approaches intended for homologous gene targeting in eukaryotic cells. Examples of LTVEC include, but are not limited to, bacterial artificial chromosomes (BACs), human artificial chromosomes (HACs), and yeast artificial chromosomes (YACs).

[0115] As used herein, the term "genomic locus" or "locus" (plural: loci) refers to the specific location of a gene or DNA sequence on a chromosome and may include both intronic and exon sequences of a particular gene. A "gene" refers to a stretch of DNA or RNA that encodes a polypeptide or RNA chain that plays a functional role in an organism and is therefore the molecular unit of heredity in an organism. For purposes of the present invention, a gene is considered to include regions that control the production of a gene product, regardless of whether such control sequences are adjacent to the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, introns, exons, promoter sequences, terminators, translation control sequences such as 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.

[0116] As used herein, the term "lung transplant" refers to a surgical procedure in which a patient's diseased lung is partially or completely replaced with a lung from a donor. Lung transplantation can be a "single" lung transplant, in which only one of two lungs is removed in the recipient and replaced with one lung from a donor, or it can be a "double" lung transplant, in which both lungs, one on each side, are removed and replaced with one from a donor. In some embodiments, the lungs are transplanted along with the heart.

[0117] As used herein, the term "lung preservation" refers to the process of maintaining and protecting donor lungs from the time of lung procurement until transplantation in a recipient.

[0118] As used herein, the term "heart transplant" refers to a surgical procedure in which a patient's diseased heart is partially or completely replaced with a heart from a donor. In certain embodiments, the heart is transplanted along with the lungs.

[0119] As used herein, the term "heart valve transplantation" refers to a surgical procedure in which a patient's diseased heart valve is replaced with a heart valve from a donor.

[0120] As used herein, the term "heart preservation" refers to the process of maintaining and protecting the donor heart from the time of lung procurement until transplantation in the recipient.

[0121] As used herein, the term "renal transplant" refers to a surgical procedure in which a patient receives a donor kidney, with or without the removal of one or both diseased kidneys. In one embodiment, the kidney is transplanted into the recipient's lower abdominal cavity. As used herein, the phrase "loss of transplant function" refers to any physiological breakdown or impairment of the normal processes that an organ or tissue exhibits in the donor animal.

[0122] As used herein, the term "mammal" refers to any mammal other than a human, including, but not limited to, a pig, sheep, goat, cattle (bovine), deer, mule, horse, monkey, dog, cat, rat, and mouse. In certain embodiments, the animal is a pig. The pig can be of any size. For example, the pig can weigh from about 10 pounds (about 4.536 kg) to about 500 pounds (about 226.8 kg). In some embodiments, the pig can weigh more than 500 pounds (about 226.8 kg). The weight of the pig can depend on the weight and size of the xenotransplant recipient. In certain embodiments, the mammal is a sow and has given birth at least once. In certain embodiments, the mammal is a non-human primate, such as a monkey or baboon.

[0123] As used herein, a "marker" or "selectable marker" is a selectable marker that allows for the isolation of rare transfected cells that express 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 such as GFP.

[0124] As used herein, the terms "nucleotide," "polynucleotide," "nucleotide sequence," "nucleic acid," and "oligonucleotide" are used interchangeably. These terms refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown.

[0125] The following are non-limiting examples of polynucleotides: genes or gene fragments defined by linkage analysis, coding or non-coding regions of multiple loci (single locus), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The term also encompasses nucleic acid-like structures with synthetic backbones, see, e.g., Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97 / 03211; WO 96 / 39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides, and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0126] As used herein, the phrase "operably linked" includes a relationship in which each operably linked component functions in its intended manner. In one example, a nucleic acid sequence encoding a protein can be operably linked to a regulatory sequence (e.g., a promoter sequence, an enhancer sequence, a silencer sequence, etc.) to maintain appropriate transcriptional control.

[0127] The term "organ," as used herein, refers to a collection of tissues joined together in a structural unit to perform a common function. An organ may be a solid organ. A solid organ is an internal organ that has a solid tissue consistency and is neither hollow (such as the organs of the gastrointestinal tract) nor liquid (such as blood). Examples of solid organs include the heart, kidneys, liver, lungs, pancreas, spleen, and adrenal glands.

[0128] As used herein, the term "primate" refers to various mammals of the order Primates, consisting of lemurs, lorises, tarsiers, New World monkeys, Old World monkeys, and apes, including humans, characterized by claws on their fingers and toes, short snouts, and large brains. In some embodiments, the primate is a non-human primate. In other embodiments, the primate is a human.

[0129] As used herein, the term "promoter" refers to a region of DNA that is typically located upstream (5') of a coding region and that at least partially regulates the initiation and level of transcription. The term "promoter" is used in the broadest context to include classical genomic gene transcription control sequences, including TATA box and non-TATA box promoters, as well as additional control elements (i.e., activating sequences, enhancers, and silencers) that alter gene expression in response to developmental and / or environmental stimuli or in a tissue- or cell-type-specific manner. Promoters are usually, but not necessarily, located upstream or 5' of the structural gene whose expression they control. Furthermore, control elements, including promoters, are typically located within 2 kb of the transcription start site of the gene, although they may be located many kb away. Promoters may also contain additional specific control elements located more distal to the start site to further enhance expression in a cell and / or alter the timing or inducibility of expression of the structural gene to which they are operably linked.

[0130] As used herein, the terms "porcine," "porcine animal," "pig," and "swine" are generic terms referring to the same type of animal regardless of sex, size, or breed.

[0131] As used herein, the term "recognition site" or "recognition sequence" refers to a specific DNA sequence that binds to a DNA backbone and is recognized by an enzyme, such as a nuclease, which directs site-specific cleavage of the DNA backbone.

[0132] As used herein, the term "recombination site" refers to a nucleotide sequence that can be recognized by a site-specific recombinase and serve as a substrate for a recombination event.

[0133] As used herein, the terms "control element" and "expression control element" are used interchangeably and refer to nucleic acid molecules that, in a particular environment, can affect the transcription and / or translation of an operably linked coding sequence. These terms are used broadly to encompass all elements that promote or control transcription, including promoters, core elements required for basic interaction of RNA polymerase and transcription factors, upstream elements, enhancers, and response elements (see Lewin, "Genes V" (Oxford University Press, Oxford) pages 847-873). Exemplary control elements in prokaryotes include promoters, operator sequences, and ribosome binding sites. Control elements used in eukaryotic cells may include, but are not limited to, promoters, enhancers, splicing signals, and polyadenylation signals.

[0134] As used herein, the term "controllable promoter" refers to a promoter that can be used to control whether the peptide is expressed in an animal, tissue, or organ. A controllable promoter may be tissue-specific, such that it is only expressed in a particular tissue, or it may be temporally controllable (turned on at a specific time (driven by the developmental stage)), or it may be inducible, such that it is only turned on or off (expressed or not) as controlled by an inducible element (it may also be an immune-inducible promoter and a cytokine-responsive promoter induced by, for example, interferon-γ, TNF-α, IL-1, IL-6, or TGF-β). For example, expression may be blocked while the organ or tissue is part of a pig, and then induced for a period of time to overcome a cellular immune response after the pig is transplanted into a human. Furthermore, the expression level may be controlled by the controllable promoter system to prevent immunosuppression of the recipient's immune system.

[0135] As used herein, the terms "control sequence," "control element," and "regulatory element" are used interchangeably and refer to polynucleotide sequences that are upstream (5' non-coding sequences), within, or downstream (3' untranslated sequences) of a polynucleotide target to be expressed. Control sequences affect, for example, the timing of transcription, the amount or level of transcription, RNA processing or stability, and / or translation of associated structural nucleotide sequences. Control sequences can include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor binding sequences, stem-loop structures, translational termination sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, and the like.

[0136] The term "safe harbor" locus, as used herein, refers to a site within a genome to which transgenic DNA (e.g., a construct) can be added harmlessly and result in consistent levels of expression. In one embodiment, the invention encompasses the integration and expression of transgenic DNA comprising a transgene within a safe harbor locus.

[0137] 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, φC31, att, Bxb1, R4 (integrase), and / or Cre, Flp, and Dre recombinases.

[0138] As used herein, the term "subject" refers to any animal (e.g., mammal), including, but not limited to, humans, non-human primates, rodents, and allogeneic animals (e.g., that will be the recipient of a particular treatment (e.g., a transplant graft) or that will be the donor of a graft). The terms "subject" and "patient" are used interchangeably herein with respect to a human subject (e.g., when the subject is a graft donor), unless otherwise indicated. In one embodiment, the subject may be a donor. In another embodiment, the subject may be a recipient.

[0139] As used herein, the term "targeting vector" refers to a recombinant DNA construct that typically contains controlled DNA arms homologous to genomic DNA that flank key elements of a target gene or sequence. When introduced into a cell, the targeting vector is integrated into the cell's genome via homologous recombination. A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only if the cell is of the tissue type corresponding to the promoter.

[0140] As used herein, the term "tissue" refers to a level of cellular organization intermediate between a cell and an entire organ. A tissue is a collection of similar cells derived from the same source that together perform a specific function. Organs, in turn, are formed by functional groupings of multiple tissues. Examples of tissues contemplated by the present invention include, but are not limited to, connective tissue, muscle tissue, nervous tissue, epithelial tissue, and calcified tissue. Blood, bone tissue, tendon tissue, ligament tissue, adipose tissue, and loose connective tissue are examples of connective tissue, which may also be classified as fibrous connective tissue, skeletal connective tissue, and liquid connective tissue. Muscle tissue is divided into three distinct categories: visceral or smooth muscle, which resides within the lining of organs; skeletal muscle, which typically attaches to bones and generates whole-body movement; and cardiac muscle, which resides in the heart and contracts to pump blood throughout the organism. Cells that comprise the central and peripheral nervous systems are classified as nervous (or neural) tissue. In the central nervous system, nervous tissue forms the brain and spinal cord. In the peripheral nervous system, nerve tissue forms the cranial and spinal nerves and includes motor neurons.

[0141] The term "transcription activator-like effector nuclease" or "TALEN," as used herein, refers to an artificial restriction enzyme generated by fusing a TAL effector DNA-binding domain to a DNA-cleavage domain. These reagents enable efficient, programmable, and specific DNA cleavage, making them powerful tools for in situ genome editing. Transcription activator-like effectors (TALEs) can be readily engineered to bind to virtually any DNA sequence. The term TALEN, as used herein, is broad and includes monomeric TALENs that can cleave double-stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both components of a TALEN pair engineered to work together to cleave DNA at the same site. TALENs that work together are sometimes referred to as left- and right-handed TALENs, which refers to their DNA handedness. See U.S. Patent Application Nos. 12 / 965,590; 13 / 426,991 (U.S. Patent No. 8,450,471); 13 / 427,040 (U.S. Patent No. 8,440,431); 13 / 427,137 (U.S. Patent No. 8,440,432); and 13 / 738,381, all of which are incorporated herein by reference in their entireties.

[0142] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0143] A "transgene" is a gene or genetic material transferred from one organism to another. When a transgene is transferred into an organism, the organism is sometimes called a transgenic organism. Typically, the term refers to a DNA segment containing a genetic sequence isolated from one organism and introduced into another. This non-native DNA segment may retain the transgenic organism's ability to produce RNA or protein, or it may alter the normal function of the transgenic organism's genetic code. Typically, the DNA is integrated into the organism's germline. For example, in higher vertebrates, this can be achieved by injecting foreign DNA into the nucleus of a fertilized egg, or by somatic cell nuclear transfer, in which a somatic cell incorporating the desired transgene into the host genome is transferred into a nucleated oocyte, resulting in the production of live offspring after implantation into a surrogate mother. Once inserted into a cell, the transgene can be a cDNA (complementary DNA) segment, a copy of the mRNA (messenger RNA), or it can be the entire gene present in its original genomic DNA region. Transgenes can be genomic sequences, particularly when introduced as large clones in BACs (bacterial artificial chromosomes) or cosmids, or they can be in the form of "minigenes," which are often characterized by a combination of both genomic DNA (including intronic regions, e.g., intron 1), 5' or 3' regulatory regions, along with cDNA regions.

[0144] In the context of this specification, transgene "expression" means, unless otherwise specified, that a peptide sequence derived from a non-native nucleic acid is expressed in at least one cell within a host. The peptide may be expressed from a transgene integrated into the host genome. The transgene may comprise a polynucleotide encoding a protein or a fragment thereof (e.g., a functional fragment). A fragment (e.g., a functional fragment) of a protein may comprise at least, or at least about 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the amino acid sequence of the protein. A fragment of a protein may be a functional fragment of the protein. A functional fragment of a protein may retain some or all of the function of the protein.

[0145] As used herein, the term "transplant tolerance" is defined as a state of donor-specific unresponsiveness that does not require sustained pharmacological immunosuppression. Transplant tolerance can eliminate many of the adverse events associated with immunosuppressants. Thus, tolerance induction can lead to improved xenograft acceptance. In one embodiment, tolerance induction can be confirmed by a reduction in clinical symptoms of xenograft rejection. In another embodiment, tolerance induction can improve or prevent metabolic, inflammatory, and proliferative pathological conditions or diseases associated with xenograft transplantation. In yet another embodiment, tolerance induction can improve, reduce, or prevent adverse clinical conditions or diseases associated with the administration of immunosuppressive therapy used to prevent xenograft rejection. In yet another embodiment, tolerance induction can promote xenograft survival. In another embodiment, tolerance induction can prevent relapse in patients exhibiting these diseases or conditions.

[0146] The term "ungulate" refers to a mammal with hoofs. Artiodactyls are ungulates with an even number of toes (cloven hooves) and include antelopes, camels, cows, deer, goats, pigs, and sheep. Perissodactyls are ungulates with an odd number of toes and include horses, zebras, rhinos, and tapirs. The term ungulate, as used herein, refers to an adult, embryonic, or fetal ungulate.

[0147] The term "vector," as used herein, refers to a moiety capable of transferring a polynucleotide into a host cell. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules containing one or more free ends, nucleic acid molecules without free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and various other 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, such as by standard molecular cloning methods. Another type of vector is a viral vector, in which viral-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus).

[0148] Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of host cells after introduction and are replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Common expression vectors useful in recombinant DNA technology are often in the form of plasmids. A recombinant expression vector can contain a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, meaning that the recombinant expression vector contains one or more control elements operably linked to the nucleic acid sequence to be expressed, which can be selected based on the host cell to be used for expression.

[0149] Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to control elements in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). Regarding recombinant and cloning methods, reference is made to U.S. Patent Application No. 10 / 815,730, the contents of which are incorporated herein by reference in their entirety. Preferably, the vector is a DNA vector, more preferably capable of expressing RNA encoding the protein of the present invention. Many suitable vectors have been described in the art; examples may 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 D. M. Glover (IRL Press, 1995).

[0150] As used herein, the term "Zn finger nuclease" or "ZFN" refers to an artificial (engineered) DNA-binding protein containing a Zn finger DNA-binding domain and a DNA-cleavage domain. The Zn finger domain can be designed to target specific desired DNA sequences, allowing the Zn finger nuclease to target unique sequences within complex genomes. The Zn finger domain facilitates 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 a chain of two finger modules, each recognizing a unique hexameric (6 bp) DNA sequence. The two finger modules are stitched together to form a Zn finger protein, each with a specificity of 24 bp or more. b) a DNA-cleavage domain composed of the nuclease domain of FokI. When the DNA-binding domain and DNA-cleavage domain are fused, highly specific "genomic scissors" are generated. ZFNs are gene editing tools.

[0151] III. Transgenic Animals The present invention provides transgenic animals (e.g., transgenic pigs) that serve as sources of organs, organ fragments, tissues, or cells for use in xenotransplantation. The invention extends to organs, tissues, and cells derived from the transgenic animals, and to herds of such animals, e.g., production herds.

[0152] The animal may be any suitable animal. In a preferred embodiment, the animal is an ungulate, particularly a porcine animal or pig. The transgenic donor animal (e.g., an ungulate, porcine animal or pig) is genetically modified, particularly to include multiple transgenes, e.g., multiple transgenes at a single locus. In one embodiment, the transgenic donor animal is genetically modified to express multiple transgenes that are divided between a first locus (i.e., locus 1) and a second locus (i.e., locus 2). The loci can be native loci or modified native loci. Various strategies for modifying native loci to facilitate targeting are described herein.

[0153] In a preferred embodiment, the present invention provides a transgenic animal (e.g., a transgenic pig) that comprises the integration and expression of at least six transgenes at a single locus under the control of at least three promoters (e.g., exogenous promoters, or a combination of exogenous and native promoters), and lacks expression of α-1,3-galactosyltransferase. Optionally, the transgenic animal comprises one or more additional genetic modifications, including, but not limited to, gene additions and / or deletions, including knockouts and knockins, and gene replacements and rearrangements.

[0154] In certain embodiments, the present invention provides transgenic pigs comprising at least six transgenes integrated and expressed at a single locus, wherein expression of the at least six transgenes is regulated by dedicated promoters, i.e., one promoter drives expression of each individual transgene. For example, if the transgenic animal incorporates and expresses six transgenes at a single locus, expression of these transgenes is driven by four promoters, each promoter specific to a particular transgene. In another embodiment, a given promoter regulates expression of two or more transgenes (e.g., two transgenes, three transgenes). For example, if the transgenic animal incorporates and expresses six transgenes, two of the six transgenes are expressed as a polycistronic regulated by a first promoter, two of the six transgenes are expressed as a polycistronic regulated by a second promoter, and two of the six transgenes are expressed as a polycistronic regulated by a third promoter. In some embodiments, the first, second, and third promoters are the same. In some embodiments, the first, second, and third promoters are different. In some embodiments, at least one promoter is different.

[0155] In some embodiments, the at least six transgenes are selected from the group consisting of immunomodulatory (e.g., immunosuppressant) transgenes, anticoagulant transgenes, complement inhibitor transgenes, and cryoprotective transgenes. In some embodiments, the single locus is a native locus. In other embodiments, the single locus is a modified native locus, such as a transgenic locus. The transgenic locus may be, for example, a locus comprising a selectable marker gene or a locus comprising a landing pad. In some embodiments, the at least six transgenes are provided in a polycistronic vector (MCV) and are integrated by random integration or by utilizing gene editing tools.

[0156] Optionally, the transgenic animal may have one or more additional genetic modifications. The additional genetic modifications may be, for example, gene knockouts or gene knock-ins. In certain embodiments, the additional genetic modification comprises a chimeric porcine-human vWF.

[0157] In another embodiment, the present invention provides a transgenic animal (e.g., a pig) comprising at least five genetic modifications that result in: (i) the absence of expression of alpha 1, galactosyltransferase (i.e., alpha-Gal null), and (ii) the integration 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 at a single locus. Expression of the transgenes is driven by a promoter, either a dedicated promoter or a promoter that regulates the expression of two or more transgenes. The promoters may be exogenous or a combination of exogenous and native promoters.

[0158] In some embodiments, when more than six transgenes are added, the transgene may include integration of transgenes at more than one locus (e.g., integration of at least six transgenes under the control of at least three promoters integrated into GGTA1 and a second multicistronic integration at a second locus (e.g., CMAH, B4GalNT2, AAVS1, GHR, or Rosa26) to better regulate expression of the transgene combination. In some embodiments in which a second locus is genetically modified, such second locus may be modified (e.g., through the application of gene editing and / or homologous recombination techniques) to inactivate expression of another pig gene. In a preferred embodiment, the multiple transgenes integrated and expressed at the second locus are selected from the group consisting of immunomodulatory factor transgenes, complement inhibitor transgenes, anticoagulant factor transgenes, and cryoprotective transgenes. In a preferred embodiment, the second locus is a native locus, a modified native locus, or a transgenic locus (e.g., a landing pad). In a preferred embodiment, at least two transgenes at the second locus are provided in an MCV and integrated using gene editing tools. Optionally, the transgenic animal may have one or more additional genetic modifications.

[0159] In one embodiment, the present invention provides a transgenic animal (e.g., a pig) comprising at least six genetic modifications resulting in (i) reduced expression of alpha 1, galactosyltransferase, and (ii) the integration and expression of at least six transgenes at a single locus, wherein the six transgenes are expressed under the control of at least three promoters (e.g., exogenous promoters, or a combination of exogenous and native promoters). In a preferred embodiment, the transgenes are selected from the group consisting of an immunomodulatory factor transgene, an anticoagulant factor transgene, a complement inhibitor transgene, and a cryoprotective factor transgene. In a preferred embodiment, the single locus is a native locus, a modified native locus, or a transgenic locus (e.g., a landing pad). In a preferred embodiment, the at least two transgenes are provided in an MCV and are integrated 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 genetic modifications.

[0160] In some embodiments, the CRISPR / Cas9-mediated gene editing comprises an inducible promoter or system, a tetracycline / doxycycline-regulated system, U6p[GHRgRNA-1], U6p[GHRgRNA-2], TRE3Gp[CAS9], CAGpr[tTA], CAGpr[hCD46-2A-hCD55], or a polycistronic vector comprising the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the inducible promoter regulates expression of a growth hormone receptor gene.

[0161] In another embodiment, the present invention provides a transgenic animal (e.g., a pig) comprising at least five genetic modifications that result in: (i) reduced expression of alpha 1, galactosyltransferase, and (ii) integration 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 at a single locus or split between two loci. In preferred embodiments, the transgenes are selected from the group consisting of immunomodulatory factor transgenes, complement inhibitor transgenes, anticoagulant factor transgenes, and cryoprotective factor transgenes. In preferred embodiments, the single locus is a native locus, a modified native locus, or a transgenic locus (e.g., a landing pad). In a preferred embodiment, the at least two transgenes are provided in an MCV and are integrated 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 genetic modifications.

[0162] In a preferred embodiment, the transgenic animal lacks expression of alpha 1, galactosyltransferase (i.e., is alpha-Gal null) 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 genetic modifications. Optionally, in addition to the transgene integration, additional knockouts include knockout of the β4GalNT2 gene or the CMAH gene (both genes implicated in the innate immunity and rejection of xenografts).

[0163] In preferred embodiments, the transgenic animal has reduced expression of alpha 1, galactosyltransferase and comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven additional genetic modifications. In some embodiments, expression of alpha 1, 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%.

[0164] In a preferred embodiment, the transgenic animal comprises (i) a genetic modification resulting in the absence of expression of α-1,3-galactosyltransferase, and (ii) at least 10 additional genetic modifications, or in particular, 6 additional transgenes. These additional genetic modifications may be any suitable genetic modifications, including, but not limited to, CRISPR-induced deletions / insertions or gene replacements (INDELs), including knockouts or knockins, at other genetic loci (e.g., B4GalNT2, CMAH, vWF, or GHR).

[0165] In a preferred embodiment, the transgenic animal comprises (i) a genetic modification that results in reduced expression of α-1,3-galactosyltransferase and (ii) at least six additional genetic modifications, or in particular ten additional genetic modifications. In a preferred embodiment, the transgenic animal comprises (i) a genetic modification that results in the absence of expression of α-1,3-galactosyltransferase and (ii) at least five additional genetic modifications, or in particular five additional genetic modifications. In a preferred embodiment, the transgenic animal comprises (i) a genetic modification that results in the absence of expression of α-1,3-galactosyltransferase and (ii) at least six additional genetic modifications, or in particular six additional genetic modifications.

[0166] In certain embodiments, the donor animal (e.g., an ungulate, porcine animal or pig) comprises genetic modifications that result in (i) the absence of expression of α-1,3-galactosyltransferase and the integration and expression of at least five, or at least six, or more transgenes. In preferred embodiments, the donor animal (e.g., an ungulate, porcine animal or pig) comprises genetic modifications that result in (i) reduced expression of α-1,3-galactosyltransferase and (ii) the integration and expression of six additional transgenes. Optionally, the donor animal may comprise five or more additional genetic modifications.

[0167] In a preferred embodiment, the donor animal (e.g., an ungulate, porcine animal, or pig) comprises genetic modifications that result in (i) reduced expression of α-1,3-galactosyltransferase and (ii) the integration and expression of six additional transgenes. Optionally, the donor animal may also comprise one or more additional genetic modifications (knockout, knockin, INDEL, modification of porcine vWF, or porcine GHR).

[0168] A. Transgene Expression Expression of the transgene can be at any level, although in certain embodiments, the expression is at a high level. Depending on the desired level and tissue-specific expression, various promoter / enhancer elements can be used. The promoter / enhancer can be a constitutive promoter / enhancer or an inducible promoter / enhancer, depending on the desired expression pattern. The promoter can be an exogenous promoter or a native promoter, or a combination of an exogenous promoter and a native promoter.

[0169] In certain embodiments, the transgene is expressed from a constitutive or ubiquitous promoter. In certain other embodiments, the transgene is expressed from a tissue- or cell-type-specific promoter, or an inducible promoter, and may include additional regulatory elements such as enhancers, insulators, matrix attachment regions (MARs), and the like.

[0170] In preferred embodiments, the six or more transgenes are co-expressed. In preferred embodiments, the six or more transgenes are expressed in approximately equimolar amounts. In some embodiments, the at least six transgenes are driven by constitutive promoters or tissue-specific promoters. In some embodiments, the constitutive promoter is selected from the group consisting of a CAG promoter, a Tie-2 promoter, and an ICAM-2 promoter. In some embodiments, the inducible promoter is a tetracycline / doxycycline-regulated promoter. In some embodiments, the tissue-specific promoter is an endothelial cell-specific promoter; and / or the tissue-specific promoter is selected from the porcine thrombomodulin promoter (pTBMpr), a human thrombomodulin promoter, a porcine EPCR promoter, and a human EPCR promoter. In some embodiments, the porcine thrombomodulin promoter is an exogenous promoter or an endogenous promoter. In preferred embodiments, the transgenes are expressed by a promoter that is primarily active in endothelial cells. In some embodiments, expression of the transgenes is regulated by the porcine Icam-2 enhancer / promoter. In one embodiment, expression of the transgene is regulated by a constitutive CAG promoter.

[0171] In one embodiment, the transgenic animal is genetically modified to result in the integration and expression of six or more transgenes, at least one transgene being regulated by a constitutive promoter and at least one transgene being regulated by a tissue-specific promoter, particularly a promoter that is primarily active in endothelial cells.

[0172] In a preferred embodiment, the transgenic animal is genetically modified to result in the integration and expression of six or more transgenes at a single locus, with at least two transgenes regulated by constitutive promoters and at least two transgenes regulated by tissue-specific promoters, particularly promoters that are primarily active in endothelial cells.

[0173] The transgene can be any transgene suitable for use in modifying a donor animal (e.g., a pig) for use in xenotransplantation. In preferred embodiments, the transgene is selected from an immunomodulatory factor (e.g., a complement regulator, a complement inhibitor, an immunosuppressant) gene, an anticoagulant factor gene, a cryoprotective factor gene, or a combination thereof. In one embodiment, the sequence of the transgene in a human.

[0174] In one embodiment, the transgene is an immunomodulator. In one embodiment, the transgene is a complement regulator, 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 a human sequence. In one embodiment, the transgene is a complement pathway inhibitor (i.e., complement inhibitor) inhibitor. The complement inhibitor may include, but is not limited to, CD55 (DAF), CD59, CR1, and CD46 (MCP). The sequence of the complement inhibitor may be a human sequence. In some embodiments, the at least six transgenes include at least two complement inhibitors selected from the group consisting of CD46, DAF (CD55), CD59, CR1, and combinations thereof. In some embodiments, the at least two complement inhibitors are ubiquitously expressed; and / or are under the control of a constitutive promoter or an inducible promoter.

[0175] In certain embodiments, the at least one transgene is an immunosuppressant. In some embodiments, the at least one immunosuppressant transgene is selected from the group consisting of cytotoxic T-lymphocyte antigen 4 (CTLA4), cluster of differentiation 47 (CD47), and class II transactivator-DN (CIITA-DN). In some embodiments, the at least one immunosuppressant transgene is under the control of a constitutive promoter.

[0176] In some embodiments, the transgene is an immunosuppressant gene with T cell regulatory activity, such as CTLA4-Ig or a dominant-negative inhibitor of MHC class II molecules (CIITA), or other genes that regulate the expression of B cell- or T cell-mediated immune function. In other embodiments, such animals may be further modified to eliminate expression of genes that affect immune function. In some embodiments, the immunosuppressant is CD47. In some embodiments, at least two transgenes are anticoagulants. In some embodiments, the at least two anticoagulant transgenes are under the control of an endothelial-specific promoter. In some embodiments, the at least two anticoagulant transgenes are selected from the group consisting of endothelial cell protein C receptor (EPCR), thrombomodulin, CD39, hirudin, tissue factor pathway inhibitor (TFPI), and combinations thereof. In some embodiments, the sequences of the anticoagulants may be human sequences.

[0177] The transgenic animal may further comprise one or more additional genetic modifications. In one embodiment, the animal may be genetically modified to inhibit expression of the CMP-Neu5Ac hydroxylase gene (CMAH) (see, e.g., U.S. Patent Application Publication No. 2005-0223418), the iGb3 synthase gene (see, e.g., U.S. Patent Application Publication No. 2005-0155095), and / or the Forssmann synthase gene (see, e.g., U.S. Patent Application Publication No. 2006-0068479). In addition, the animal may be genetically modified to reduce expression of a procoagulant. In particular, in one embodiment, the animal is genetically modified to reduce or eliminate expression of a procoagulant gene, such as FGL2 (fibrinogen-like protein 2). In another embodiment, the animal may be genetically modified to inhibit expression of β-1,4N-acetylgalactosaminyltransferase 2 (β4GalNT2).

[0178] B. Specific Genetic Modifications 1. α-1,3-galactosyltransferase (α-Gal) In one embodiment, the present invention provides transgenic animals suitable for use as a source of organs, tissues, and cells for xenotransplantation, which lack or have reduced expression of α-Gal. Transgenic animals lacking α-Gal expression (i.e., α-Gal null) have one or more additional genetic modifications, and in some embodiments, at least four additional genetic modifications, at least five additional genetic modifications, or at least six additional genetic modifications. These genetic modifications may be, for example, the integration or expression of transgenes. In certain embodiments, the transgenic animals have at least three genetic modifications that result in (i) the lack of α-Gal expression; and (ii) the integration and expression of at least two transgenes at a single locus. In some embodiments, the single locus is a modified α-Gal.

[0179] Various strategies have been implemented to eliminate or modulate the anti-Gal humoral response induced by xenotransplantation, including enzymatic removal of the epitope with α-galactosidase (Stone et al., Transplantation 63:640-645, 1997), specific anti-Gal antibody removal (Ye et al., Transplantation 58:330-337, 1994), and capping of the epitope with other carbohydrate moieties. However, these strategies failed to eliminate α-GT expression, and the introduction of complement inhibitory proteins has been reported to only partially reduce the number of Gal epitopes in transgenic pigs. Similarly, attempts to block the expression of Gal epitopes in N-acetylglucosaminyltransferase III transgenic pigs also resulted in only a partial reduction in the number of Gal epitopes and failed to significantly prolong graft survival in primate recipients. Single-allelic knockout of the α-Gal locus in pig cells and live animals is known in the art. A major breakthrough in the field of xenotransplantation was the production of the first live pig lacking functional expression of α-Gal (Phelps et al. Science 299:411-414 (2003); see also PCT Publication No. WO 04 / 028243 to Revivicor, Inc. and PCT Publication No. WO 04 / 016742 to Immerge Biotherapeutics, Inc.).

[0180] In one embodiment, the animal (and organs, tissues, and cells derived from the animal) is provided from a transgenic animal (e.g., a transgenic pig) that contains at least six transgenes, the six transgenes being integrated and expressed at a single locus under the control of at least three promoters, and that lacks expression of α-1,3-galactosyltransferase. In a preferred embodiment, the transgenes are integrated and expressed at a modified α-Gal locus. In one embodiment, the at least three promoters are exogenous promoters, native promoters, or a combination of exogenous and native promoters.

[0181] In one embodiment, animals, and organs, tissues, and cells derived from the animals, are provided that (i) lack expression of functional α-Gal and (ii) incorporate and express 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 some embodiments, the transgenes are integrated and expressed at an altered α-Gal locus. In certain embodiments, the animals may contain one or more additional genetic modifications. These genetic modifications may result in the integration and expression of one or more additional transgenes at the same locus or at a different locus.

[0182] In another embodiment, animals, organs, tissues, and cells are provided that have reduced levels of expression of functional α-Gal and incorporate and express at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 additional transgenes. Expression of functional α-Gal may be reduced, for example, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%.

[0183] The absence of expression or a reduction in the expression level of functional α-GT may be achieved by any suitable means known to those of skill in the art. In some embodiments, an animal (e.g., an ungulate, a pig) is provided in which one allele of the α-Gal gene has been inactivated via a gene targeting event. In another embodiment, a pig is provided in which both alleles of the α-Gal gene have been inactivated via a gene targeting event. In one embodiment, the gene may be targeted via homologous recombination. In other embodiments, the gene may be disrupted, i.e., by altering a portion of the genetic code, thereby affecting the transcription and / or translation of that segment of the gene. For example, gene disruption may occur through replacement, deletion ("knock-out"), or insertion ("knock-in") methods, including targeted insertion of a selectable marker gene (e.g., neo) that interrupts the coding region of the α-Gal gene. Additional genes for desired proteins or regulatory sequences that regulate transcription of existing sequences may also be inserted.

[0184] In one embodiment, an allele of the α-Gal gene is inactivated so that the resulting α-Gal enzyme is unable to produce Gal on the cell surface. In one embodiment, the α-Gal gene may be transcribed into RNA but not translated into protein. In another embodiment, the α-Gal gene may be transcribed in a truncated form. Such truncated RNA may not be translated or may be translated into a non-functional protein. In another embodiment, the α-Gal gene may be inactivated so that transcription of the gene does not occur. In another embodiment, the α-Gal gene may be transcribed and then translated into a non-functional protein.

[0185] In some embodiments, expression of the active α-Gal gene may be reduced by using other methods, such as targeting transcription or translation of the gene. For example, the expression may be reduced by using antisense RNA or siRNA that targets the native α-GT gene or its mRNA. In other embodiments, site-specific recombinases are used to target regions of the genome for recombination. Examples of such systems are the CRE-lox system and the Flp-Frt system.

[0186] Pigs with two inactive alleles of the α-Gal gene do not occur naturally. Previous discoveries have identified a point mutation that prevents the second allele from producing functional α-Gal enzyme during attempts to knock out the second allele of the α-Gal gene through gene targeting.

[0187] That is, in another aspect of the present invention, α-Gal may be inactivated through at least one point mutation. In one embodiment, one allele of the α-Gal gene may be inactivated through at least one point mutation. In another embodiment, both alleles of the α-Gal gene may be inactivated through at least one point mutation. In one embodiment, the point mutation may occur via a gene targeting event. In another embodiment, the point mutation may be spontaneous. In another embodiment, the mutation may be introduced into the α-Gal gene via a mutagen.

[0188] 2.β4GaINT2 In one embodiment, the present invention provides transgenic animals suitable for use as a source of organs, tissues, and cells for xenotransplantation, lacking or having reduced expression of β-1,4-N-acetyl-galactosaminyltransferase 2 (β4GALNT2). Transgenic animals lacking expression of β4GALNT2 (i.e., being β4GALNT2 null) have one or more additional genetic modifications. These genetic modifications may be, for example, the integration or expression of a transgene. In certain embodiments, transgenic animals lacking or having reduced expression of β-1,4-N-acetyl-galactosaminyltransferase 2 (β4GALNT2) are also characterized by (i) the absence of α-Gal expression; and (ii) the integration and expression of at least six transgenes at a single locus under the control of at least three promoters.

[0189] Glycans produced by β4Gal-NT2 are xenoantigens in many humans. 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 to generate the Sda blood group antigen, GalNAc b1-4(Neu5Ac a2-3)Gal b1-4GlcNAc b1-3Gal. This gene is functional in transplantable organs (kidney, heart, liver, lung, and pancreas) and endothelial cells in pigs. Approximately 5% of humans have inactive β4GalNT2, resulting in the development of antibodies against the SDa and CAD carbohydrates produced by this gene.

[0190] Any suitable method can be used to generate pigs with a genome lacking or reduced expression of endogenous β4GALNT2. Disruptions can be located at many sites within the endogenous porcine β4GALNT2 nucleic acid sequence. Examples of disruptions include, but are not limited to, deletions within the native gene sequence and insertions of heterologous nucleic acid sequences into the native gene sequence. Examples of insertions include, but are not limited to, artificial splice acceptors linked to stop codons or splice donors linked to fusion partners such as GFP. Knockout constructs can contain sequences homologous to the endogenous β4GALNT2 nucleic acid sequence or to sequences adjacent to the endogenous β4GALNT2 nucleic acid sequence. In some cases, knockout constructs can contain a nucleic acid sequence encoding a selectable marker (e.g., antibiotic resistance, a fluorescent reporter (e.g., GFP or YFP), or an enzyme (e.g., β-galactosidase)) operably linked to a regulatory sequence (e.g., a promoter). The knockout construct may contain other nucleic acid sequences such as recombination sequences (e.g., loxP sequences; see Sendai, et al., Transplantation, 81(5):760-766 (2006)), splice acceptor sequences, splice donor sequences, transcription start sequences, and transcription termination sequences. Disruptions within the endogenous β4GALNT2 nucleic acid sequence can result in decreased expression of the gene or non-functional truncations or fusions of the encoded polypeptide.

[0191] In one embodiment, the present invention provides a transgenic animal (e.g., a pig) that has reduced or no expression of β4GALNT2. Optionally, the animal comprises one or more additional genetic modifications. In a preferred embodiment, the present invention provides a transgenic animal (e.g., a pig) that incorporates and expresses at least six transgenes under the control of at least three promoters and that has absent or reduced expression of β4GALNT2. Optionally, the animal comprises one or more additional genetic modifications. In one embodiment, the present invention provides a transgenic animal (e.g., a pig) that has reduced or absent expression of Sda or SDa-like glycans produced by porcine β4GALNT2. Optionally, the animal comprises one or more additional genetic modifications.

[0192] In a preferred embodiment, the present invention provides a transgenic animal (e.g., a pig) that incorporates and expresses at least six transgenes under the control of at least three promoters and lacks or reduces expression of Sda or SDa-like glycans produced from porcine β4GALNT2. Optionally, the animal contains one or more additional genetic modifications.

[0193] 3. CMAH In one embodiment, the present invention provides transgenic animals suitable for use as a source of organs, tissues, and cells for xenotransplantation, lacking or having reduced expression of cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). Transgenic animals lacking expression of CMAH (CMAH null) have one or more additional genetic modifications. These genetic modifications may be, for example, the integration or expression of transgenes. In a specific embodiment, the transgenic animals have (i) the lack of expression of α-Gal; and (ii) at least four additional genetic modifications resulting in the integration and expression of at least six transgenes at a single locus.

[0194] Pig cells express cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), which is absent in human cells. CMAH converts the sialic acid N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc). Therefore, when pig tissue is transplanted into humans, these epitopes elicit antibody-mediated rejection in human patients immediately after transplantation.

[0195] Any suitable method can be used to generate pigs with a genome lacking or reduced expression of endogenous CMAH. Disruptions can be located at many sites within the endogenous porcine CMAH nucleic acid sequence. Examples of disruptions include, but are not limited to, deletions within the native gene sequence and insertions of heterologous nucleic acid sequences into the native gene sequence. Examples of insertions include, but are not limited to, artificial splice acceptors linked to stop codons or splice donors linked to fusion partners such as GFP. Knockout constructs can contain sequences that are homologous to the endogenous CMAH nucleic acid sequence or to sequences adjacent to the endogenous CMAH nucleic acid sequence. In some cases, knockout constructs can contain a nucleic acid sequence encoding a selectable marker (e.g., antibiotic resistance, a fluorescent reporter (e.g., GFP or YFP), or an enzyme (e.g., β-galactosidase)) operably linked to a regulatory sequence (e.g., a promoter). Knockout constructs may contain other nucleic acid sequences, such as recombination sequences (e.g., loxP sequences; see Sendai, et al., Transplantation, 81(5):760-766 (2006)), splice acceptor sequences, splice donor sequences, transcription start sequences, and transcription termination sequences. Disruptions within the endogenous CMAH nucleic acid sequence can result in decreased expression of the gene or in a non-functional truncation or fusion of the encoded polypeptide.

[0196] In one embodiment, a disruption in the endogenous CMAH nucleic acid sequence can result in reduced expression of the gene or a non-functional truncation or fusion of the encoded polypeptide. Optionally, the animal contains one or more additional genetic modifications. In a preferred embodiment, the invention provides a transgenic animal (e.g., a pig) that incorporates and expresses at least six transgenes under the control of at least three promoters and that results in absent or reduced expression of CMAH. Optionally, the animal contains one or more additional genetic modifications.

[0197] 4.vWF The von Willebrand factor (vWF) gene is a large, complex gene with multiple domains that encodes a multimeric glycoprotein. The primary functions of the multimeric glycoprotein vWF are platelet adhesion to connective tissue and the subendothelium and platelet aggregation in response to vWF binding to platelet glycoprotein Ib (GPIb). However, this phenomenon is less desirable in xenotransplantation, where the aggregation of recipient platelets can have a damaging effect on the survival of the donated organ. For example, transplantation of pig lungs (and other organs) into humans or nonhuman primates results in the spontaneous aggregation and sequestration of human platelets. This can be circumvented by "humanizing" the pig vWF gene, which aims to eliminate this spontaneous binding of pig vWF to human platelets. Typically, humanization or modification of the pig vWF gene requires the deletion of gene sequences associated with spontaneous aggregation of human platelets and their replacement with human gene counterparts that do not cause spontaneous aggregation. This may involve the deletion of all or part of the porcine vWF gene by replacement with all or part of the human vWF gene.

[0198] Modifications of porcine vWF aimed at eliminating spontaneous platelet aggregation could include regions within the D3 (partial), A1, A2, and A3 (partial) domains known to be involved in folding and trapping the GP1b binding site of hvWF (D3 domain), as well as regions associated with the GP1b receptor (A1 domain) and the ADAMTS13 cleavage site (A2 domain). Exons 22-28 contain these regions. Human platelets spontaneously aggregate under normal stress in the presence of porcine blood. To avoid this potential threat to the success of xenotransplantation, regions of the human vWF gene involved in vWF protein folding, as well as regions associated with GP1b binding, collagen binding (one of two regions), and ADAMTS13 cleavage, could be used to replace genomic homologs within the porcine vWF gene (resulting in chimeric human / porcine proteins), since human vWF does not induce spontaneous platelet aggregation under normal shear stress in blood. In this way, an alternate fold that can hide or mask the GP1b binding site on vWF, and a humanized receptor site within the A domain, can be provided by a single cDNA or genomic fragment from the human vWF gene. This can be achieved through homologous recombination or gene targeting, including mechanisms enhanced using gene editing methods; for example, CRISPR-assisted homologous recombination can be used to integrate a human vWF fragment into the porcine vWF locus. This human fragment replaces the region associated with spontaneous platelet aggregation described above and can take the form of a cDNA or genomic fragment from the human vWF gene.

[0199] In a preferred embodiment, the insertion of the relevant human vWF gene sequence can be carried out by any current method used in genome editing, such as, but not limited to, CRISPR / CAS9, TALEN nuclease, etc. The modification of porcine vWF can be carried out by replacing only the relevant region of the porcine vWF gene, or by replacing the entire porcine vWF gene with human vWF.

[0200] In one embodiment, a region of the porcine vWF gene may be replaced with the human counterpart (regions E22-E28). Alternatively, transgenic animals may be completely knocked out of the vWF gene using a site-specific recombination system (i.e., a CRE-LOX recombination system) and / or specific nucleic acid base pair changes to replace nucleotides in the porcine vWF genomic sequence with their human counterparts, resulting in a complete replacement of the gene with a synthetic sequence of the human vWF gene.

[0201] In one embodiment, the present invention relates to a transgenic animal (e.g., a porcine transgenic animal) lacking expression of α-Gal and having a genetic modification to the porcine vWF gene. The modification can be, for example, knockout of the porcine vWF gene and replacement with a humanized or chimeric vWF gene. The transgenic animal can also include one or more additional genetic modifications. In one embodiment, the transgenic animal further includes incorporation and expression of CD46.

[0202] The transgenic animal may be further bred with a second transgenic animal containing one or more genetic modifications. For example, in one embodiment, a transgenic animal (e.g., a porcine transgenic animal) lacking expression of α-Gal and containing a genetic modification for the porcine vWF gene may be bred with a second transgenic animal containing at least six transgenes at a single locus, or at least six transgenes at a single locus and at least two transgenes at a second locus, to provide an animal containing multiple genetic modifications.

[0203] In one embodiment, the present invention provides a transgenic animal (e.g., a porcine transgenic animal) lacking expression of α-Gal and comprising a genetic modification to the porcine vWF gene (e.g., a chimeric human-porcine vWF) and at least four genetic modifications at a single locus under the control of at least three promoters. The loci can vary. In a preferred embodiment, the loci are native loci or modified native loci. The loci can be, for example, AAVS1, GHR, ROSA26, CMAH, β4GalNT2, and GGTA1. The at least six transgenes can be integrated by homologous recombination or gene editing tools.

[0204] 5. Growth Factor Receptors The present invention provides transgenic animals, such as transgenic pigs, with 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 is usually caused by mutations in the growth hormone receptor. Patients with Laron syndrome may be short in stature and resistant to certain conditions, such as type II diabetes and certain cancers. The transgenic animals may have genetic alterations that result in reduced expression of the growth human receptor (GHR) or that cause mutations in the GHR that impair GHR function. In some embodiments, the transgenic animals have a GHR knockout genetic alteration. Examples of GHRKO alterations are described, for example, in Yu et al., "Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs / Cas9 system and somatic cell nuclear transfer," J Transl Med 16:41 (2018). The transgenic animal may have a 30% or more, 40% or more, 50% or more, 75% or more, or 90% or more reduction in GHR expression compared to an animal not having the genetic alteration. The transgenic animal may produce 30% or less, 40% or less, 50% or less, 75% or less, or 90% or less IGF-1 compared to an animal not having the genetic alteration. The genetic alteration may be made alone or in combination with other genetic alterations. For example, the genetic alteration may be included with other genetic alterations described herein (Figures 2A-2D).

[0205] The transgene introduced into the genome of the transgenic animals of the present invention may be any suitable transgene.

[0206] C. Complement Regulators In one embodiment, the transgene is an immunomodulator. In a preferred embodiment, the donor animal has been genetically modified so that (i) expression of α-Gal is absent or reduced, and (ii) at least six transgenes are integrated and expressed at a single locus, and at least one of the at least two transgenes is an immunomodulator. The immunomodulator may be any suitable immunomodulator. In a preferred embodiment, the immunomodulator is a complement regulator (e.g., a complement inhibitor) or an immunosuppressant.

[0207] In one embodiment, the present invention provides transgenic animals (e.g., pigs) suitable for use as a source of organs, tissues, and cells for xenotransplantation, which have been genetically modified to incorporate and express at least one complement regulator, e.g., a complement inhibitor. In a preferred embodiment, the donor animal has been genetically modified such that (i) expression of α-Gal is absent or reduced, and (ii) at least six transgenes have been integrated and expressed at a single locus, at least one of the transgenes being a complement regulator, more specifically a complement inhibitor.

[0208] Complement is a group of blood proteins that is 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 destruction through the generation of potent inflammatory regulators and the recruitment and activation of immune effector cells. Complement activation products that mediate tissue injury are generated at various points within the complement pathway. Inappropriate complement activation against host tissues plays an important role in the pathology of many autoimmune and inflammatory diseases and is also involved in many conditions associated with bioincompatibility, such as post-cardiopulmonary inflammation and transplant rejection. Complement deposition on host cell membranes is prevented by complement inhibitory proteins expressed on the cell surface.

[0209] The complement system, comprising a collection of approximately 30 proteins, is one of the major effector mechanisms of the immune system. The complement cascade is primarily activated via either the classical pathway (usually antibody-dependent) or an alternative pathway (usually antibody-independent). Activation via either pathway results in the production of C3 convertase, the central enzyme complex of the cascade. C3 convertase cleaves serum C3 into C3a and C3b, which covalently bind to the activation site of C3 convertase, leading to its further production (the amplification loop). This activation generates C3b (and, via the classical pathway only, C4b), whose degradation products are important opsonins involved in promoting cell-mediated lysis of target cells (by phagocytes and NK cells) and transport and solubilization 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 cleaves C5 to generate C5a and C5b. C5a has potent proinflammatory and chemotactic properties and can recruit and activate immune effector cells. 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). Formation of the MAC at the target cell membrane can result in direct cell lysis but can also trigger cell activation and the expression / release of various inflammatory mediators.

[0210] There are two widespread classes of membrane complement inhibitors: inhibitors of the complement activation pathway (which inhibit C3 convertase formation) and inhibitors of the terminal complement pathway (which inhibit MAC formation). Membrane inhibitors of complement activation include complement receptor 1 (CR1), decay-accelerating factor (DAF or CD55), and complement regulatory protein (MCP or CD46). All of these share a protein structure consisting of a variable number of repeating units of approximately 60-70 amino acids, termed short consensus repeats (SCRs), a common feature 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 that performs functions similar to both DAF and MCP. Although rodents also express DAF and MCP, Cr1 appears to be the functionally most important regulator of complement activation in rodents. Although no homologues of Cr1 have been found in humans, the study of Cr1 and its use in animal models has clinical implications.

[0211] Regulation of the terminal complement pathway and MAC formation at host cell membranes occurs primarily through the activity of CD59, a widely distributed 20 kD glycoprotein attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. CD59 binds C8 and C9 during MAC assembly, preventing membrane insertion.

[0212] Host cells are protected from their own complement by membrane-bound complement regulatory proteins such as DAF, MCP, and CD59. When organs are transplanted into another species, natural antibodies in the recipient bind to the endothelium of the donor organ and activate complement, leading to rapid rejection. It has previously been suggested that, in contrast to human cells, pig cells are highly susceptible to human complement, which was thought to be due to the ineffectiveness of pig cell surface complement regulatory proteins against human complement. When organs are transplanted into another species, natural antibodies in the recipient bind to the endothelium of the donor organ and activate complement, leading to rapid rejection. Several strategies have been shown to prevent or delay rejection, including removal of natural IgM antibodies and systemic complement removal or inhibition using sCR1, heparin, or C1 inhibitor.

[0213] Another approach to the rejection problem is to express human membrane-bound complement regulatory molecules in transgenic pigs. Transgenic pigs expressing the decay acceleration factor DAF (CD55), the membrane co-factor protein MCP (CD46), and the membrane inhibitor of reactive lysis MIRL (CD59) have been generated (see Klymium et al. Mol Reprod Dev (2010) 77:209-221). These human inhibitors have been shown to be abundantly expressed on the vascular endothelium of pigs. Ex vivo perfusion of hearts from control animals with human blood resulted in complement-mediated destruction of the organ within minutes, whereas hearts from transgenic animals were resistant to complement and survived for several hours.

[0214] As outlined above, the rationale for "humanizing" pig organs by expressing human complement regulatory proteins 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 xenotransplantation setting. In addition, soluble complement inhibitors can prevent complement-mediated islet lysis in vitro.

[0215] Several porcine analogs of human complement regulatory proteins (CRPs) have been isolated and characterized. Pig organs expressing human CRP molecules were resistant to complement injury, not because the organs expressed human CRP molecules, but because the expression of functional CRP molecules was significantly increased. Increased expression of porcine CRP, like donor organs expressing human CRPs, may also be effective in protecting donor organs from complement injury, which can lead to hyperacute rejection.

[0216] CD46 has been characterized as a protein with regulatory properties that can protect host cells from attack mediated by complement activated via both the classical and alternative pathways, complement lysis during inflammation, and humoral rejection mediated by low levels of natural or induced anti-Gal or anti-non-Gal antibodies. As a result, more islets can engraft and subsequently receive better protection from rejection, reducing the need for immunosuppression.

[0217] In one embodiment of the present invention, animals (and organs, tissues, and cells derived therefrom) are provided that lack functional α-Gal expression (or have reduced α-Gal expression) and have been genetically modified to incorporate and express at least one, at least two, at least three, or at least four or more complement inhibitors, whose expression can be ubiquitous or under the control of a tissue-specific promoter.

[0218] In a preferred embodiment, the complement inhibitor is a membrane complement inhibitor. The membrane complement inhibitor can be either 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-accelerating factor (DAF or CD55), complement regulatory protein (MCP or CD46), and the like. Membrane inhibitors of the terminal complement pathway can include CD59 and the like.

[0219] In a preferred embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) comprising genetic modifications resulting in (i) the absence of α-Gal expression and (ii) the integration and expression of at least six transgenes at a single locus under the control of at least three promoters, wherein at least one of the at least two transgenes is a complement regulator, more specifically a complement inhibitor, and even more specifically a membrane complement inhibitor. The single locus can be selected from a native locus, a modified native locus, or a transgenic locus. In a preferred embodiment, the at least six transgenes are provided as MCVs, and integration can be by random integration or facilitated by gene targeting tools. Optionally, the transgenic animal comprises one or more additional genetic modifications, including, but not limited to, modifications of the native porcine vWF gene, the native porcine B4GalNT2 gene, the native porcine CMAH gene, or the native porcine Forsmann gene.

[0220] In a preferred embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that comprises at least six transgenes, the six transgenes being integrated and expressed at a single locus under the control of at least three promoters, the animal lacking expression of α-1,3-galactosyltransferase, and the at least six transgenes comprising at least two complement regulatory factors, more specifically, two complement inhibitors. The additional transgenes may be, for example, immunosuppressant genes, cytoprotective factor genes, or a combination thereof. The single locus may be selected from a native locus, a modified native locus, or a transgenic locus. In a preferred embodiment, the at least six transgenes are provided as MCVs, and integration is random or facilitated by gene targeting tools. Optionally, the transgenic animal comprises one or more additional genetic modifications.

[0221] In a preferred embodiment, animals (and organs, tissues, and cells derived therefrom) are provided that lack (or have reduced) expression of functional α-Gal and are genetically modified to incorporate and express at least four additional transgenes, at least one of at least two of said at least four additional transgenes being a complement inhibitor, particularly at least two membrane complement inhibitors.

[0222] In a preferred embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional α-Gal and has been genetically modified to (i) incorporate and express at least two complement inhibitors, particularly at least two membrane complement inhibitors, and (ii) incorporate and express at least two additional transgenes selected from anticoagulant genes, immunosuppressant genes, cytoprotective factor genes, or combinations thereof.

[0223] In one embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional α-Gal and has been genetically modified to incorporate and express (i) CD46 and CD55, and (ii) at least two additional transgenes. In certain embodiments, the additional transgenes are selected from an anticoagulant gene, an immunosuppressant gene, a cytoprotective gene, or a combination thereof.

[0224] In certain embodiments, animals (and organs, tissues, and cells derived therefrom) are provided that lack (or have reduced) expression of functional α-Gal and have been genetically modified to incorporate and express at least six transgenes under the control of at least three promoters, at least one of which is CD46, the expression of which is regulated by an endogenous promoter.

[0225] In another embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional α-Gal and has been genetically modified to incorporate and express (i) CD46 and CD55, and (ii) at least three additional transgenes. In certain embodiments, the additional transgenes are selected from an anticoagulant gene, an immunosuppressant gene, a cytoprotective factor gene, or a combination thereof. In a preferred embodiment, the at least three additional transgenes include at least two anticoagulants. In a preferred embodiment, the at least three additional transgenes include at least two anticoagulants and an immunosuppressant.

[0226] In another embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional α-Gal and has been genetically modified to incorporate and express (i) CD46 and CD55, and (ii) at least four additional transgenes. In certain embodiments, the additional transgenes are selected from anticoagulant genes, immunosuppressant genes, cytoprotective factor genes, or combinations thereof. In preferred embodiments, the at least four additional transgenes include at least two anticoagulant factors. In preferred embodiments, the at least four additional transgenes include at least two anticoagulant factors and an immunosuppressant. In preferred embodiments, the at least four additional transgenes include at least three anticoagulant factors.

[0227] In another embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional α-Gal and has been genetically modified to incorporate and express (i) CD46 and CD55, and (ii) at least five additional transgenes. In certain embodiments, the additional transgenes are selected from anticoagulant genes, immunosuppressant genes, cytoprotective factor genes, or combinations thereof. In preferred embodiments, the at least five additional transgenes include at least two anticoagulant factors and at least one immunosuppressant. In preferred embodiments, the at least five additional transgenes include at least three anticoagulant factors and at least one immunosuppressant. In preferred embodiments, the at least five additional transgenes include at least two anticoagulant factors and at least two immunosuppressants. In one embodiment, the animal can be modified to express a complement regulator peptide, a biologically active fragment, or a derivative thereof. In one embodiment, the complement regulator peptide is a full-length complement regulator. In another embodiment, the complement regulator peptide may comprise less than the full-length complement regulator protein.

[0228] Any human or porcine complement regulator sequence, or biologically active portion or fragment thereof, known to those of skill in the art may be used in accordance with the compositions and methods of the present invention. In a further embodiment, any consensus complement regulator peptide may be used in accordance with the present invention. In another embodiment, nucleic acid and / or peptide sequences that are at least 80%, at least 85%, at least 90%, or at least 95% homologous to the complement regulator peptides and nucleotide sequences described herein. In another embodiment, any fragment or homologous sequence that exhibits activity similar to a complement regulator may be used. Optionally, the animal expressing at least one complement regulator (e.g., a complement inhibitor) of the at least six transgenes described above and lacking expression of α-1,3-gal has at least one additional genetic modification.

[0229] D. Immunosuppressive factors In one embodiment, the present invention provides transgenic animals suitable for use as a source of organs, tissues, and cells for xenotransplantation, which have been genetically modified to incorporate and express at least one immunosuppressive factor. The transgenic animals typically have one or more additional genetic modifications, more particularly five or more additional genetic modifications, and even more particularly six or more additional genetic modifications.

[0230] "Immunosuppressant" transgenes can downregulate immune responses. The balance between efficacy and toxicity is a key factor in clinically validating any type of transplantation procedure. A further concern with islet transplantation is that many of the current immunosuppressants, particularly glucocorticoids or calcineurin inhibitors such as tacrolimus, damage beta cells and induce insulin resistance in peripheral nerves (Zeng et al. Surgery (1993) 113: 98-102). A steroid-free immunosuppressive protocol (the "Edmonton protocol") including sirolimus, low-dose tacrolimus, and a monoclonal antibody (mAb) against the IL-2 receptor has been used in a clinical trial of islet transplantation alone for patients with type 1 diabetes (Shapiro, AMJ et al., (2000), N. Eng. J. Med., 343: 230-238). Recent success with the "Edmonton protocol" has renewed enthusiasm for the treatment of diabetes with islet transplantation. However, concerns about the toxicity of tacrolimus may limit the application of this therapy in humans.

[0231] Biological agents that block important T cell costimulatory signals, particularly the CD28 pathway, are potential alternatives for protecting pancreatic islets. Examples of agents that block the CD28 pathway include, but are not limited to, soluble CTLA4, including mutant CTLA4 molecules.

[0232] T cell activation is implicated in the pathogenesis of transplant rejection. T cell activation requires at least two sets of signaling events. The first is triggered by specific recognition of antigenic peptides bound to major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs) via the T cell receptor. The second set of signals is antigen-nonspecific and is delivered by T cell costimulatory receptors (TCRs) interacting with their ligands on APCs. In the absence of costimulation, T cell activation is impaired or interrupted, resulting in a state of antigen-specific unresponsiveness known as clonal paralysis or elimination by apoptotic death. Therefore, blocking T cell costimulation may provide an approach to suppress antigen-specific undesired immune responses while preserving normal immune function (Dumont, FJ 2004 Therapy 1, 289-304).

[0233] 1.CTLA4 Among the several T cell costimulatory pathways identified to date, the most prominent is the CD28 pathway. CD28, a cell surface molecule expressed on T cells, and its counterreceptors, B7.1 (CD8O) and B7.2 (CD86), present on dendritic cells, macrophages, and B cells, have been characterized and are considered attractive targets for interrupting T cell costimulatory signals. A second T cell surface molecule similar to CD28 is known as cytotoxic T-lymphocyte-associated protein (CTLA4). CTLA4 is a cell surface signaling molecule, but unlike CD28, CTLA4 negatively regulates T cell function. CTLA4 has a 20-fold higher affinity for the B7 ligand than CD28.

[0234] The CD28 / B7 pathway has become an attractive target for disrupting T cell costimulatory signals. The design of CD28 / B7 inhibitors exploits CTLA4, an endogenous negative regulator of this pathway. CTLA4-immunoglobulin (CTLA4-Ig) fusion proteins have been widely investigated as a means of inhibiting T cell costimulation. A difficult balance must be achieved with any immunosuppressive treatment: sufficient suppression must be provided to overcome disease and rejection, while excessive immunosuppression can inhibit the entire immune system. The immunosuppressive activity of CTLA4-Ig has been demonstrated in preclinical animal models of organ transplantation and autoimmune disease. Soluble CTLA4 has recently been tested in human patients with renal failure, psoriasis, and rheumatoid arthritis and has been formulated as a drug (abatacept, soluble CTLA4-Ig), developed by Bristol-Myers Squibb, approved for the treatment of rheumatoid arthritis. This drug is the first in a class of novel selective T cell costimulation modulators. Bristol-Myers Squibb is also conducting a Phase II clinical trial using belatacept (LEA29Y) in renal allografts. LEA29Y is a mutant form of CTLA4 engineered to have higher affinity for the B7 receptor than wild-type CTLA4 fused to immunoglobulin. Repligen Corporation is also conducting a clinical trial using CTLA4-Ig in idiopathic thrombocytopenic purpura. U.S. Patent No. 5,730,403, entitled "Methods for protecting allogeneic islet transplants using soluble CTLA4 mutant molecules," describes the use of soluble CTLA4-Ig molecules and CTLA4 mutant molecules to protect allogeneic islet transplants.

[0235] CTLA-4 from one organism can bind to B7 from another organism, but it has been found to bind most strongly to the B7 of the same species. That is, soluble CTLA-4 from a donor organism can bind to both recipient B7 (on normal cells) and donor B7 (on xenograft cells), but preferentially binds to B7 on the xenograft. Therefore, in embodiments of the present invention involving pigs or cells for xenotransplantation, porcine CTLA4 is exemplary. International Publication No. 99 / 57266 by Imperial College describes the porcine CTLA4 sequence and the administration of soluble CTLA4-Ig for xenotransplantation 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 B7 (but not human B7) and blocks CD28 on recipient T cells, rendering these local T cells anergic without causing global T cell immunosuppression (see Mirenda et al., Diabetes 54:1048-1055, 2005).

[0236] Much of the research on CTLA4-Ig as an immunosuppressant has focused on administering soluble forms of CTLA4-Ig to patients. Transgenic mice engineered to express CTLA4-Ig have been generated and used in several experimental lines. Ronchese et al. broadly investigated immune system function after CTLA4 expression in mice (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) reported the protective effect of CTLA4-Ig secreted by transgenic fetal pancreatic allografts in mice to test the effect of transgenic expression of 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 expression of soluble CTLA4-Ig in the milk of the transgenic animals for use as bioreactors.

[0237] International Publication No. WO 01 / 30966 by Alexion Pharmaceuticals Inc. describes a chimeric DNA construct containing the T cell inhibitor CTLA-4 bound to the complement protein CD59, as well as transgenic pig cells, tissues, and organs containing the same. International Publication No. WO 2007035213 (Revivicore Inc.) describes transgenic pigs genetically modified to express CTLA4-Ig.

[0238] Additional immunosuppressive factors can be expressed in the animals, tissues, or cells. For example, genes inactivated in mice to create an immunocompromised phenotype can be cloned and disrupted in pigs by gene targeting. Some genes that can be targeted in mice to generate immunocompromised pigs include β2-microglobulin. In one embodiment, donor animals are engineered to transgenically express cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4). Animals or cells can be engineered to express a CTLA4 peptide or a biologically active fragment (e.g., a truncated version of the peptide in which the extracellular domain and at least the transmembrane domain have been removed) or derivative thereof. The peptide can be, for example, a human or porcine peptide. The CTLA4 peptide can be mutated.

[0239] 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 be expressed intracellularly. Other modifications of the CTLA4 peptide include the addition of an endoplasmic reticulum retention signal to the N- or C-terminus. The endoplasmic reticulum retention signal may be, for example, the sequence KDEL. The CTLA4 peptide may be fused to a peptide dimerization domain or an immunoglobulin (Ig) molecule. The CTLA4 fusion peptide may include a linker sequence that can connect these two peptides. In another embodiment, an animal produced according to the present invention lacking expression of functional immunoglobulin may be administered a CTLA4 peptide or variant thereof (pCTLA4-Ig, or hCTLA4-Ig (abatacept / Orencia, or belatacept) as a drug to suppress its T cell responses. As used herein, CTLA4 is used to refer to any of these variants or those known in the art, e.g., CTLA4-Ig.

[0240] In one embodiment, the CTLA4 peptide is full-length CTLA4. In another embodiment, the CTLA4 peptide may comprise less than the full-length CTLA4 protein. In one embodiment, the CTLA4 peptide may comprise the extracellular domain of a CTLA-4 peptide. In a specific embodiment, the CTLA4 peptide is the extracellular domain of CTLA4. In yet a further embodiment, the present invention 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 form. In a specific embodiment, the mutant CTLA4 may be human CTLA4 (Glu104, Tyr29).

[0241] In one embodiment, the CTLA4 may be a truncated form of CTLA4, with at least the transmembrane domain of the protein removed. In another embodiment, the CTLA4 peptide may be modified to be expressed intracellularly. 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 a sequence called KDEL, which may be added to the C-terminus or N-terminus of the CTLA4 peptide. In another embodiment, the CTLA4 peptide may be fused to a peptide dimerization domain. In one embodiment, the CTLA4 peptide may be fused to an immunoglobulin (Ig). In another embodiment, the CTLA4 fusion peptide may include a linker sequence capable of connecting these two peptides.

[0242] Any human or porcine CTLA4 sequence, or biologically active portion or fragment thereof, known to those of skill in the art may be amenable to the compositions and methods of the present invention. Non-limiting examples include, but are not limited to, the following GenBank accession numbers setting forth 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. Additional nucleotide sequences encoding CTLA4 peptides may be selected from the EST database, including, but not limited to, the following GenBank accession numbers: 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.

[0243] In a further embodiment, any consensus CTLA4 peptide may be used in accordance with the present invention. In another embodiment, nucleic acid and / or peptide sequences that are at least 80%, at least 85%, at least 90%, or at least 95% homologous to the native CTLA4 peptide and nucleotide sequence. In another embodiment, any fragment or homologous sequence that exhibits activity similar to CTLA4 may be used. In another embodiment, the amino acid sequence that exhibits T cell inhibitory activity may be amino acids 38-162 of the porcine CTLA4 sequence or amino acids 38-161 of the human CTLA4 sequence (see, e.g., WO 01 / 30966). In one embodiment, the portion used should have at least about 25%, preferably at least about 50%, of the activity of the parent molecule.

[0244] In other embodiments, the CTLA4 nucleic acids and peptides of the present invention can be fused to immunoglobulin genes and molecules, or fragments or regions thereof. References to the CTLA4 sequences of the present invention also encompass sequences fused to immunoglobulins. In one embodiment, the Ig can be human Ig. In another embodiment, the Ig can be IgG, particularly IgG1. In another embodiment, the Ig can be the constant region of IgG. In a specific embodiment, the constant region can be the C.gamma.1 chain of IgG1. In one specific embodiment of the present invention, the extracellular domain of porcine CTLA4 can be fused to human C.gamma.1 Ig. In another specific embodiment, the extracellular domain of human CTLA4 can be fused to IgG1 or IgG4. In a further specific embodiment, the extracellular domain of mutant CTLA4 (Glu104, Tyr29) can be fused to IgG1. In one embodiment, at least one of the transgenes is B7-H4, also known as B7x. B7-4H was identified in 2003 and belongs to the B7 family of immunoglobulins.

[0245] 2. CIITA In one embodiment, the donor animal is modified to transgenic express type II transactivator of leukemia (CIITA) and its variants PDL1, PDL2, tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL), Fas ligand (FasL, CD95L), integrin-related protein (CD47), HLA-E, HLA-DP, HLA-DQ, or HLA-DR.

[0246] Type II transactivator (CIITA) is a bi- or multi-domain protein that acts as a transcriptional activator and plays a crucial role in the expression of MHC class II genes. A mutant form of the human CIITA gene, encoding a protein lacking the amino-terminal 151 amino acid, has previously been shown to function as a potent dominant-negative suppressor of HLA class II expression (Yun et al., Int Immunol. 1997 October; 9(10):1545-53). Pig MHC class II antigens are potent stimulators of direct T cell recognition by human CD4+ T cells and therefore likely play an important role in the rejection of transgenic pig donors in clinical xenotransplantation. A mutant human CIITA construct was reported to be effective in porcine cells, significantly suppressing IFN-γ-induced and constitutive porcine MHC class II expression. Furthermore, stably transfected porcine vascular endothelial cell lines with mutant human CIITA constructs failed to stimulate direct T cell xenorecognition by 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 significantly reduce T cell rejection in human recipients. In combination with other transgenes, transgenic expression of mutant CIITA may enable long-term xenograft survival with clinically acceptable levels of immunosuppression.

[0247] In one embodiment, the present invention provides a transgenic animal (e.g., a pig) comprising genetic modifications that result in (i) the absence of expression of α-Gal and (ii) the integration and expression of at least two transgenes at a single locus, wherein the at least six transgenes comprise at least one immunosuppressant. The single locus can be selected from a native locus, a modified native locus, or a transgenic locus. Optionally, the transgenic animal comprises one or more additional genetic modifications.

[0248] In a preferred embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) comprising a genetic modification resulting in (i) the absence of expression of α-Gal and (ii) the integration and expression of at least six transgenes at a single locus, wherein at least two of the at least two transgenes comprise an immunosuppressant. The single locus can be selected from a native locus, a modified native locus, or a transgenic locus. The at least six transgenes can be provided as MCVs and integrated into the locus using gene editing tools. Optionally, the transgenic animal comprises one or more additional genetic modifications.

[0249] In a preferred embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional α-Gal and has been genetically modified to incorporate and express at least six transgenes at a single locus, wherein the at least six transgenes comprise at least one immunosuppressive factor. The immunosuppressive factor may be, for example, CIITA-DN or CLTA4-IG. The at least six transgenes may include an additional transgene selected from a complement inhibitor, an anticoagulant, or a combination thereof. The single locus may be selected from a native locus, a modified native locus, or a transgenic locus. The at least three transgenes may be provided as MCVs and integrated into the locus using gene editing tools. Optionally, the transgenic animal comprises one or more additional genetic modifications.

[0250] In a preferred embodiment, an animal (and organs, tissues, and cells derived therefrom) is provided that lacks (or has reduced) expression of functional α-Gal and has been genetically modified to incorporate and express at least six transgenes at a single locus, wherein the at least six transgenes include at least two immunosuppressive factors. The immunosuppressive factors may be, for example, CIITA-DN or CLTA4-IG. The at least six transgenes may also include at least one complement inhibitor, at least two anticoagulants, at least two complement inhibitors, or a combination thereof. The single locus may be selected from a native locus, a modified native locus, or a transgenic locus. The at least six transgenes may be provided as MCVs and integrated into the locus using gene editing tools. Optionally, the transgenic animal comprises one or more additional genetic modifications.

[0251] E. Other immunomodulatory factors 1. PDL1, PDL2 Typical costimulatory molecules for T cell activation are CD80 / 86 or CD40. In addition to these positive costimulatory pathways, new costimulatory pathways have been discovered over the past few years that mediate negative signals and are important in regulating T cell activation. One of these newer pathways is the pathway consisting of the programmed death 1 (PD-1) receptor and its ligands, PD-L1 and PD-L2. The PD-1 receptor is not expressed on resting cells but is upregulated after T cell and B cell activation. PD-1 contains an immunoreceptor tyrosine-based switch motif on its cytoplasmic side, and binding of PD-L1 or PD-L2 to PD-1 generates an inhibitory signal in T cells. Recent data suggest that the PD1 / PD-ligand pathway may be involved in regulating T cell subsets that exhibit regulatory activity. PD-1 signaling has been shown to be required for the suppressive activity of regulatory T cells (Tregs) and the generation of adaptive Tregs in mice. These findings suggest that PD-1 / PD-ligand interactions may not only inhibit T cell responses but also induce immune regulation.

[0252] Several lines of evidence indicate that the PD-1 / PD-ligand pathway can regulate allograft engraftment and rejection, suggesting that these molecules are interesting targets for immunomodulation after organ transplantation. Indeed, PDL1Ig gene transfer into donor hearts in a rat transplant model resulted in prolonged allograft survival. Furthermore, it has been reported that enhancing PD-1 signaling by injection of PD-L1 Ig protects grafts from rejection in mice. Recent data also indicate that overexpression of PD-L1 IG on pancreatic islet grafts in mice can partially prolong islet graft survival. Transgenic expression of human PD-L1 or human PD-L2 in pig cells and tissues should reduce early human anti-pig T cell responses elicited via a direct sensitization pathway (Plege et al., Transplantation. 2009 Apr. 15; 87(7):975-82). Induction of Tregs may also allow regulation of xenograft-primed T cells through an indirect pathway, which is necessary to achieve durable tolerance.

[0253] In certain embodiments, the transgenic animal lacks expression of α-Gal and incorporates and expresses at least six transgenes under the control of at least three promoters, including the incorporation and expression of PDL1 or PDL2.

[0254] 2.TRAIL / Fas L Expression of apoptosis-inducing ligands, such as Fas ligand (FasL, CD95L) or tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL, Apo-2L), can eliminate T cells attacking xenografts. TRAIL is a type II membrane protein with an extracellular domain similar to that of other tumor necrosis factor family members, showing the highest amino acid identity (28%) to FasL. TRAIL exerts its apoptosis-inducing effects preferentially on tumor cells. In normal cells, TRAIL receptor engagement does not result in cell death. Recent studies have shown that the cytotoxic effects of immune cells, including T cells, natural killer cells, macrophages, and dendritic cells, are mediated, at least in part, by TRAIL. Expression of human TRAIL in transgenic pigs may provide a rational strategy for protecting pig tissue from T cell-mediated rejection after xenotransplantation into primates. Stable expression of human TRAIL has been achieved in transgenic pigs, and the expressed TRAIL has been shown to be biologically functional in vitro (Klose et al., Transplantation. 2005 Jul. 27; 80(2):222-30). In some embodiments, the transgenic animals lack expression of α-Gal and incorporate and express at least six transgenes under the control of at least three promoters, including the incorporation and expression of TRAIL or Fas L.

[0255] 3. NK cell response - HLA-E / β2-microglobulin and HLA-DP, HLA-DQ, HLA-DR Human natural killer (NK) cells are a potential hurdle to successful pig-to-human xenotransplantation because they directly infiltrate pig organs perfused with human blood ex vivo, directly lyse pig cells in vitro, and lyse pig cells by antibody-dependent cell-mediated cytotoxicity in the presence of human serum. NK cell autoreactivity is prevented by the expression of major histocompatibility complex (MHC) class I ligands for inhibitory NK receptors on normal autologous cells. The inhibitory receptor CD94 / NKG2A, expressed on the majority of activated human NK cells, specifically binds to human leukocyte antigen (HLA)-E. The nonclassical human MHC molecule HLA-E is a potent inhibitory ligand for CD94 / NKG2A-bearing NK cells and, unlike classical MHC molecules, does not induce allogeneic T cell responses. HLA-E is assembled in the endoplasmic reticulum and transported to the cell surface as a stable trimeric complex consisting of the HLA-E heavy chain, β2-microglobulin (β2m), and a peptide derived from the leader sequence of several MHC class 1 molecules. Expression of HLA-E has been shown to provide partial protection from xenogeneic human NK cell cytotoxicity (Weiss et al., Transplantation. 2009 Jan. 15; 87(1):35-43). Transgenic expression of HLA-E on pig organs may substantially alleviate human NK cell-mediated rejection of pig xenografts without the risk of alloimmune reactions. Furthermore, transgenic pigs carrying other HLA genes have been successfully produced to "humanize" pig organs, tissues, and cells (Huang et al., Proteomics. 2006 Nov; 6(21):5815-25; see also U.S. Patent No. 6,639,122).

[0256] In a specific embodiment, the transgenic animal lacks expression of α-Gal and incorporates and expresses at least six transgenes under the control of at least three promoters, including the incorporation and expression of HLA-3.

[0257] 4.CD47 CD47 (cluster of differentiation 47), also known as an integrin-associated protein (IAP), is a transmembrane protein encoded by the CD47 gene in humans. CD47 is known to be an immunosuppressive and immunomodulatory factor and tolerogenic in SIRPα signaling. CD47 is a ubiquitously expressed 50 kDa cell surface glycoprotein and acts as a ligand for the immunoinhibitory receptor signaling regulator (SIRP)-α (also known as CD172a, SHPS-1, or SIRP-α), on macrophages. CD47 and SIRP-α constitute an intercellular communication system (CD47-SIRP-α axis) that plays an important role in various cellular processes, including cell migration, B cell adhesion, and T cell activation. Furthermore, the CD47-SIRP-α axis is also involved in the negative regulation of phagocytosis by macrophages. CD47 on the surface of several cell types (i.e., erythrocytes, platelets, or leukocytes) can prevent phagocytosis by macrophages by binding to the inhibitory macrophage receptor SIRPα. The role of the CD47-SIRP-α interaction in self-recognition and inhibition of phagocytosis is demonstrated by the finding that primary wild-type mouse macrophages rapidly phagocytose non-opsonized erythrocytes from CD47-deficient mice but not from wild-type mice.

[0258] Through the SIRPα receptor, CD47 inhibits both Fcγ receptor- and complement receptor-mediated phagocytosis. Porcine CD47 does not induce SIRPα-tyrosine phosphorylation in a human macrophage-like cell line, and soluble human CD47-Fc fusion protein inhibits the phagocytic activity of human macrophages toward porcine cells. Thus, engineering porcine cells to express human CD47 dramatically reduces their susceptibility to phagocytosis by human macrophages (Ide et al., Proc Natl Acad Sci USA. 2007 Mar. 20;104(12):5062-6). Expression of human CD47 on porcine cells may confer inhibitory signaling to SIRPα on human macrophages, providing an approach to prevent macrophage-mediated xenograft rejection.

[0259] In some embodiments, transgenic animals lacking expression of α-Gal and incorporating and expressing at least six transgenes under the control of at least three promoters include the incorporation and expression of CD47. In preferred embodiments, animals (and organs, tissues, and cells derived therefrom) are provided that lack (or have reduced) expression of functional α-Gal and have been genetically modified to (i) incorporate and express at least six transgenes at a single locus, one of the at least six transgenes being CD47. In some embodiments, the at least six transgenes may include an additional transgene selected from a complement inhibitor, an anticoagulant, or a combination thereof. In some embodiments, the single locus may be selected from a native locus, a modified native locus, or a transgenic locus. The at least three transgenes may be provided as MCVs and integrated into the locus using gene editing tools. Optionally, the transgenic animal comprises one or more additional genetic modifications.

[0260] F. Anticoagulant factors In one embodiment, the present invention provides transgenic animals suitable for use as a source of organs, tissues, and cells for xenotransplantation, genetically modified to incorporate and express at least one anticoagulant. In some embodiments, the animals contain additional genetic modifications. In some embodiments, the transgenic animals contain at least 10 genetic modifications, even more specifically, . In some embodiments, the 10 genetic modifications include at least six transgenic insertions and at least four knockout genes. In a preferred embodiment, the present invention provides transgenic animals containing at least 10 genetic modifications that result in (i) the absence of expression of α-Gal, and (ii) the integration and expression of at least six transgenes at a single locus under the control of at least three promoters, at least two of which are anticoagulants. In some embodiments, the at least two anticoagulant transgenes are under the control of an endothelial-specific promoter.

[0261] In some embodiments, the anticoagulant can be any suitable anticoagulant. Representative, non-limiting examples of suitable anticoagulant transgenes include tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor (EPCR), CD39, and combinations thereof. In some embodiments, the anticoagulant is selected from the group consisting of endothelial cell protein C receptor (EPCR), thrombomodulin, CD39, hirudin, tissue factor pathway inhibitor (TFPI), and combinations thereof. In some embodiments, expression of the anticoagulant can be ubiquitous or tissue-specific. In some embodiments, expression of the anticoagulant is regulated by a promoter that is primarily active in the endothelium.

[0262] 1.TFPI Tissue factor pathway inhibitor (TFPI) is a single-chain polypeptide that reversibly inhibits factor Xa (Xa) and thrombin (factor IIa), thereby inhibiting TF-dependent coagulation. For a review of TFPI, see Crawley and Lane (Arterioscler Thromb Vasc Biol. 2008, 28(2):233-42). Dorling et al. 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 to Weibel-Palade intracellular storage granules (Chen D, et al. Am J Transplant 2004; 4: 1958-1963). The resulting activation-dependent endothelial presentation of TFPI was sufficient to completely inhibit acute humoral rejection of mouse cardiac xenografts mediated by thrombosis in cyclosporine-treated rats, suggesting that effective coagulation control may also prevent chronic rejection. Similar results were obtained in transgenic mouse hearts expressing a hirudin / CD4 / P-selectin fusion protein, demonstrating that inhibition of thrombin generation or activity was key to protection in this model.

[0263] 2. Hirudin Hirudin, a naturally occurring peptide in the salivary glands of medicinal leeches (e.g., Hirudo medicinalis), 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 membrane-tethered hirudin fusion protein and transplanted their hearts into rats (mouse-rat Xeno-Tx). In contrast to control nontransgenic mouse hearts, which were all rejected within 3 days, 100% of organs from both lines of transgenic mice completely resisted humoral rejection and survived for over 100 days when T cell-mediated rejection was inhibited by administration of cyclosporine A. Riesbeck et al. (Circulation. 1998 December 15; 98(24):2744-52) also investigated the expression of a hirudin fusion protein in mammalian cells as a strategy to prevent intravascular thrombosis. Intracellular expression reduced local thrombin levels and inhibited fibrin formation, making hirudin another attractive anticoagulant transgene for preventing the thrombotic effects seen in xenotransplantation.

[0264] 3. Thrombomodulin Thrombomodulin (TBM) functions as a cofactor in thrombin-induced protein C activation in the anticoagulation pathway by forming a complex with thrombin in a 1:1 stoichiometric ratio. The endothelial cell protein C receptor (EPCR) is an N-glycosylated type I membrane protein that enhances protein C activation. The role of these proteins in the protein C anticoagulation system is reviewed in Van de Wouwer et al., Arterioscler Thromb Vasc Biol. 2004 August; 24(8):1374-83. Expression of these and other anticoagulant transgenes has been investigated by various groups as a potential solution to coagulation disorders in xenotransplantation (reviewed by Cowan and D'Apice, Cur Opin Organ Transplant. 2008 April; 13(2):178-83). Esmon et al. (Li et al., J Thromb Haemost. 2005 July; 3(7):1351-9) overexpressed EPCR on the endothelium of transgenic mice and showed that such expression protected the mice from thrombus induction. Iino et al. (J Thromb Haemost. 2004 May; 2(5):833-4) proposed ex vivo gene therapy-mediated overexpression of TBM in donor islets as a means of preventing thrombotic complications in islet transplantation.

[0265] 4.CD39 CD39 is the major intravascular nucleoside triphosphate diphosphohydrolase (NTPDase), converting ATP and ADP to AMP and ultimately to adenosine. Extracellular adenosine plays an important role in thrombosis and inflammation, and its beneficial role in transplantation has been investigated (reviewed by Robson et al. Semin Thromb Hemost. 2005 April; 31(2):217-33). Recent studies have demonstrated that CD39 has a significant impact in reducing inflammatory responses (Beldi et al., Front Biosci. 2008, 13:2588-2603). Transgenic mice expressing hCD39 exhibited impaired platelet aggregation, prolonged bleeding times, and resistance to systemic thromboembolism in a cardiac transplant model (Dwyer et al., J Clin Invest. 2004 May; 113(10):1440-6). We also demonstrated that the transgenic mice express CD39 in their pancreatic islets, and that when incubated with human blood, these islets significantly delayed clotting times compared with wild-type islets (Dwyer et al., Transplantation. 2006 Aug. 15; 82(3):428-32). Preliminary efforts to express high levels of hCD39 from a constitutive promoter system in transgenic pigs resulted in high postnatal mortality (Revivicore, unpublished data). However, endothelial cell-specific expression of CD39 has been shown to be better tolerated by transgenic pigs. Thus, there is a need to express specific anticoagulant transgenes in pigs in a manner that does not compromise animal welfare while providing sufficient levels of expression to be useful in clinical xenotransplantation.

[0266] In a preferred embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) having genetic modifications that result in (i) the absence (or reduced expression) of α-Gal, and (ii) the integration and expression of at least six transgenes at a single locus under the control of three promoters, at least one of the two transgenes being an anticoagulant factor.

[0267] In one embodiment, the anticoagulant is selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor (EPCR), CD39, and combinations thereof. In some embodiments, the single locus can be a native locus, a modified native locus, or a transgenic locus. In some embodiments, the native locus can be GGTA1, B4GalNT2, GHR, CMAH, Rosa26, AAVS1, or other endogenous locus that can confer advantageous expression characteristics to the inserted transgene.

[0268] In some embodiments, at least six transgenes under the control of the at least three promoters may be provided as an MCV, and integration may include gene editing tools. Such editing may include targeted insertion into predetermined sites (e.g., landing pads) that serve as "safe harbors" (so as not to disrupt any essential genes in the genome) and / or provide desirable characteristics specific to the integration site. In the case of insertions at loci important for preventing xenograft rejection, insertion of multiple transgenes may also result in inactivation of porcine genes involved in inducing xenotransfer reactions in primates (i.e., inactivation of α-Gal, CMAH, or B4GalNT2, or others (iGB3, Forssman)). Optionally, the animals may contain one or more additional genetic modifications at two or more loci, where the at least six transgenes are inserted at one locus and another set of two or more transgenes (under the control of at least three promoters) are co-integrated at a second site. Another embodiment allows for MCV insertion at one locus and targeted inactivation at a different locus, where such inactivation may be facilitated by gene editing tools.

[0269] In a preferred embodiment, the present invention provides transgenic animals (e.g., ungulates, pigs) having genetic modifications resulting in (i) the absence (or reduced expression) of α-Gal and (ii) the integration 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. In one embodiment, the anticoagulants are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In some embodiments, at least six transgenes may be provided as MCVs, and integration may require gene editing tools. In some embodiments, the single locus may be a native locus, a modified native locus, or a transgenic locus. In some embodiments, the transgenic animal may include one or more additional genetic modifications.

[0270] The present invention provides transgenic animals (e.g., ungulates, pigs) that lack (or have reduced) expression of α-Gal and have been genetically modified to incorporate and express at least three anticoagulant factors. In one embodiment, the anticoagulant factors are selected from tissue factor pathway inhibitor (TFPI), hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, at least one of the at least three anticoagulant factors is regulated by expression of a promoter that is predominantly active in endothelial cells. In one embodiment, at least two of the at least three anticoagulant factors are regulated by expression of a promoter that is predominantly active in endothelial cells.

[0271] In a preferred embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) that lacks (or has reduced) expression of α-Gal and is genetically modified to incorporate and express at least three anticoagulant factors, one of which is EPCR.

[0272] In a preferred embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) that lacks (or has reduced) expression of α-Gal and has been genetically modified to incorporate and express at least three anticoagulant factors, wherein the at least three anticoagulant factors include EPCR and TBM.

[0273] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) lacking (or having reduced) expression of α-Gal and genetically modified to incorporate and express at least four additional transgenes, wherein the at least four additional transgenes comprise at least one anticoagulant. In one embodiment, the at least one anticoagulant is selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least one anticoagulant is EPCR.

[0274] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) that lacks (or has reduced) expression of α-Gal and has been genetically modified to incorporate and express at least four additional transgenes, wherein the at least four additional transgenes comprise at least two anticoagulant factors. In one embodiment, the at least two anticoagulant factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least two anticoagulant factors comprise EPCR and TBM. In another embodiment, the at least two anticoagulant factors comprise EPCR and TFPI.

[0275] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) that lacks (or has reduced) expression of α-Gal and has been genetically modified to incorporate and express at least four additional transgenes, wherein the at least four additional transgenes comprise at least three anticoagulant factors. In one embodiment, the at least three anticoagulant factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least three anticoagulant factors comprise EPCR, TBM, and TFPI. In another embodiment, the at least three anticoagulant factors comprise EPCR, TBM, and CD39.

[0276] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) that lacks (or has reduced) expression of α-Gal and has been genetically modified to incorporate and express at least five additional transgenes, wherein the at least five additional transgenes comprise at least two anticoagulant factors. In one embodiment, the at least two anticoagulant factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least two anticoagulant factors comprise EPCR and TBM. In another embodiment, the at least two anticoagulant factors comprise EPCR and TFPI.

[0277] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) that lacks (or has reduced) expression of α-Gal and has been genetically modified to incorporate and express at least five additional transgenes, wherein the at least five additional transgenes comprise at least three anticoagulant factors. In one embodiment, the at least three anticoagulant factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least three anticoagulant factors comprise EPCR, TBM, and TFPI. In another embodiment, the at least three anticoagulant factors comprise EPCR, TBM, and CD39.

[0278] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) that lacks (or has reduced) expression of α-Gal and has been genetically modified to incorporate and express at least six additional transgenes, wherein the at least six additional transgenes comprise at least two anticoagulant factors. In one embodiment, the at least two anticoagulant factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least two anticoagulant factors comprise EPCR and TBM. In another embodiment, the at least two anticoagulant factors comprise EPCR and TFPI. Optionally, the at least six additional transgenes also comprise at least one immunosuppressant factor.

[0279] In one embodiment, the present invention provides a transgenic animal (e.g., an ungulate, a pig) that lacks (or has reduced) expression of α-Gal and has been genetically modified to incorporate and express at least six additional transgenes, wherein the at least six additional transgenes comprise at least three anticoagulant factors. In one embodiment, the at least three anticoagulant factors are selected from tissue factor pathway inhibitor, hirudin, thrombomodulin, endothelial cell protein C receptor, CD39, and combinations thereof. In one embodiment, the at least three anticoagulant factors comprise EPCR, TBM, and TFPI. In another embodiment, the at least three anticoagulant factors comprise EPCR, TBM, and CD39.

[0280] G. Cytoprotective Factor Transgene The present invention provides transgenic animals suitable for use as a source of organs, tissues, and cells for xenotransplantation, which have been genetically modified to incorporate and express at least one cryoprotective factor transgene ("cytoprotective factor"). In some embodiments, the at least one cytoprotective factor transgene is under the control of a constitutive promoter or an endothelial-specific promoter. In some embodiments, the at least one cytoprotective factor transgene is selected from the group consisting of heme oxygenase 1 (HO-1), A20, FAT-1, soluble tumor necrosis factor alpha (TNF-α), and combinations thereof.

[0281] The present invention provides transgenic animals (e.g., pigs) comprising genetic modifications that result in (i) the absence of expression of α-Gal; and (ii) the integration and expression of at least six transgenes at a single locus under the control of at least three promoters, wherein at least one of the at least six transgenes is a cytoprotective factor transgene.

[0282] Cytoprotective factor transgenes are considered to include anti-apoptotic factors, antioxidant factors, and anti-inflammatory factors, such as A20, heme oxygenase 1 (HO-1), FAT-1, and soluble tumor necrosis factor alpha (TNF-α).

[0283] 1.A20 A20 confers anti-inflammatory and anti-apoptotic activity. Vascularized transplanted organs can be protected from endothelial cell activation and cytotoxicity by anti-inflammatory, anticoagulant, and / or anti-apoptotic molecules. Among genes likely 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 by protecting endothelial cells from TNF-mediated apoptosis and inflammation through blockade of several caspases and the transcription factor nuclear factor κB, respectively. Live A20 transgenic piglets were produced, and hA20 expression was restricted to skeletal muscle, heart, and PAECs. These animals were protected from TNF-mediated apoptosis and, at least partially, from CD95(Fas)L-mediated cell death by hA20 expression. Additionally, cardiomyocytes derived from hA20 transgenic cloned pigs were partially protected from heart attack (Oropeza et al., Xenotransplantation. 2009 November; 16(6):522-34).

[0284] 2.HO-1 HO confers anti-inflammatory, anti-apoptotic, and antioxidant activities. Heme oxygenase (HO), also known as HSP32 and a member of the heat shock protein family, is the rate-limiting enzyme in heme catabolism, in which the heme ring is cleaved to yield ferrous iron, carbon monoxide (CO), and biliverdin, which is then converted to bilirubin by biliverdin reductase. Three isoforms of HO, including HO-1, HO-2, and HO-3, have been cloned. Expression of HO-1 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 AM et al., American Journal of Respiratory Cell & Molecular Biology 1996; 15:9-19). HO-1 - / - Analysis in mice suggests that the gene encoding HO-1 regulates iron homeostasis and acts as a cytoprotective gene with potent antioxidant, anti-inflammatory, and anti-apoptotic properties. Similar findings have recently been reported in human case reports of HO-1 deficiency.

[0285] The molecular mechanisms involved in 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 various metal-containing protoporphyrins. Cobalt protoporphyrin (CoPP) and iron protoporphyrin (FePP) can upregulate HO-1 expression. In contrast, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP) inhibit HO-1 activity at the protein level. HO-1 expression suppresses rejection of mouse-to-rat heart transplants, protects islet cells from apoptosis, and improves in vivo islet cell function after transplantation. Furthermore, transgenic administration of HO-1 protects against hyperoxia-induced lung injury, elevated HO-1 expression protects the liver of genetically obese Zucker rats from ischemia-reperfusion injury, and ablation or expression of the HO-1 gene modulates cisplatin-induced renal tubular apoptosis. In transgenic animal models, overexpression of HO-1 has been shown to prevent pulmonary inflammatory and vascular responses to hypoxia and protect the heart from ischemia and reperfusion injury. Although pigs carrying the HO-1 transgene have been produced, no clinical benefit associated with their use in xenotransplantation has been reported (U.S. Patent No. 7,378,569).

[0286] 3.FAT-1 FAT-1 exerts anti-inflammatory activity. Polyunsaturated fatty acids (PUFAs) (n-3 class) are involved in the inhibition of inflammation. Mammalian cells lack the desaturases required to convert n-6 PUFAs to n-3 PUFAs. Therefore, essential n-3 fatty acids must be provided through the diet. However, unlike mammals, the free-living nematode Caenorhabditis elegans expresses an n-3 fatty acid desaturase, which introduces a double bond at the n-3 position of the hydrocarbon chain of n-6 fatty acids to form n-3 PUFAs. Transgenic mice expressing the C. elegans fat-1 gene have been generated, which can efficiently convert six series of dietary PUFAs into three series of PUFAs, including EPA (20:5 n-3) and DHA (22:6 n-3). Another group further optimized the codons of the fat-1 cDNA for efficient translation in mammalian systems and generated a transgenic mouse model in which endogenous production of n-3 PUFAs was achieved through overexpression of the C. elegans n-3 fatty acid desaturase gene mfat-1. This group demonstrated that increasing n-3 PUFAs and decreasing n-6 PUFAs in cells through transgenic expression of mfat-1 enhanced glucose-, amino acid-, and GLP-1-stimulated insulin secretion in isolated mouse pancreatic islets and rendered the islets highly resistant to cytokine-induced cell death (Wei et al., Diabetes. 2010 February; 59(2):471-8).

[0287] 4.Soluble TNF-α receptor (sTNFR1) Tumor necrosis factor (TNF, cachexin, or cachectin, formerly known as tumor necrosis factor α) is a cytokine involved in systemic inflammation and a member of a group of cytokines that stimulate the acute phase response. The main role of TNF is to regulate immune cells. TNF can induce apoptotic cell death and induce inflammation. Soluble TNFα receptor 1 (sTNFR1) is the extracellular domain of TNFR1 and is an antagonist of TNFα (Su et al., 1998. Arthritis Rheum. 41, 139-149). Transgenic expression of sTNFR1 in xenografts may have beneficial anti-inflammatory effects.

[0288] Other cytoprotective factors 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 key role in ATP production, i.e., oxidative phosphorylation. During this process, reactive oxygen species (ROS), including superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), are generated as by-products. 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 to hydrogen peroxide, which is converted to water by catalase and glutathione peroxidase. Cell damage resulting from ROS generation can occur in transplant settings. Due to their reduced antioxidant defenses, pancreatic beta cells are particularly vulnerable to free radical and inflammatory injury. Commonly used anti-rejection drugs are effective at inhibiting adaptive immune responses; however, most are toxic to pancreatic islets and offer limited protection from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. Therefore, there has been growing interest in treating pancreatic islets with antioxidants ex vivo or expressing antioxidant genes in donor tissue via gene therapy or transgenic expression. Ex vivo gene transfer of EC-SOD and catalase was anti-inflammatory in a rat model of antigen-induced arthritis (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 attenuated hepatic I / R injury in a mouse model (He et al., Liver Transpl. 2006 December; 12(12):1869-79). In a recent mouse study, islets treated with catalytic antioxidants prior to syngeneic, suboptimal syngeneic, or xenotransplantation demonstrated superior function compared with untreated controls. In this same study, diabetic mouse recipients of allografted islets treated with catalytic antioxidants demonstrated improved glycemic control and delayed allograft rejection after transplantation.Furthermore, MnSOD-overexpressing islet grafts functioned approximately 50% longer than control grafts. Furthermore, certain anticoagulant factors also exert anti-inflammatory activity, including thrombomodulin, EPCR, and CD39.

[0289] In a preferred embodiment, the present invention provides a transgenic animal (e.g., a pig) comprising a genetic modification resulting in (i) the absence of expression of α-Gal; and (ii) the integration and expression of at least six transgenes at a single locus (under the control of at least three promoters), wherein at least one of the at least six transgenes is a cytoprotective factor transgene. The single locus can be a native locus, a modified native locus, or a transgenic locus. The at least two transgenes may be provided as MCVs, and the integration may include a gene editing tool. Optionally, the animal may have one or more additional genetic modifications.

[0290] In a preferred embodiment, the present invention provides a transgenic animal (e.g., a pig) comprising genetic modifications that result in (i) the absence of α-Gal expression; and (ii) the integration and expression of at least five, at least six, at least seven, or at least eight transgenes at a single locus, or at least six transgenes at one locus and one or more transgenes at a second locus, wherein at least one of the transgenes is a cytoprotective factor transgene, and the at least six transgenes are under the control of at least three promoters, which may be various combinations of constitutive, ubiquitous, tissue-specific, or inducible regulated promoter systems. The transgenes may be provided as MCVs, and the integration may include gene editing tools. The single locus may be a native locus, a modified native locus, or a transgenic locus. Optionally, the animal may have one or more additional genetic modifications.

[0291] IV. Production of Transgenic Animals The present invention provides methods for producing a transgenic pig containing at least six transgenes, the method comprising: (i) transfecting a pig cell with a single polycistronic vector containing (a) at least two complement inhibitor transgenes, (b) at least one immunosuppressant transgene, (c) at least one cytoprotective factor transgene, and (d) at least two anticoagulant transgenes; (ii) integrating and expressing the polycistronic vector at a single genomic locus to produce a multi-transgenic pig cell containing at least six transgenes; (iii) injecting the nucleus of the multi-transgenic pig cell into a reconstituted somatic cell nuclear transfer (SCNT) to produce a multi-transgenic pig zygote; and (iv) maturing the multi-transgenic pig zygote into a multi-transgenic pig. In some embodiments, the pig cell and the multi-transgenic pig lack expression of α-1,3-galactosyltransferase.

[0292] The transgenic animals of the present invention 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 the use of embryonic stem cells. Gene editing tools may also be utilized, as further described herein.

[0293] In some embodiments, genetic modifications may be identified in animals and then bred to form a herd of animals with the desired set of genetic modifications (or single genetic modification). These offspring may be further bred to produce different or the same set of genetic modifications (or single genetic modification) in their offspring. This breeding cycle of animals with the desired genetic modifications may continue as long as desired. A "herd" in this context may include multiple generations of animals produced over time with the same or different genetic modifications. A "herd" may also refer to a single generation of animals with the same or different genetic modifications.

[0294] Cells useful for genetic modification (e.g., via homologous recombination, random insertion / integration, nuclease editing, zinc finger + TALEN nucleases, CRISPR / Cas9 nucleases, etc., but are not limited to) include epithelial cells, neurons, 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 (e.g., via nuclear transfer, etc., but are not limited to) can be obtained from various organs, such as skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra, and other urinary organs. Cells can be obtained from any cell or organ of the body, including all somatic or germ cells.

[0295] Additionally, animal cells that can be genetically modified can be obtained from a variety of organs and tissues, including, but not limited to, skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder, blood vessels, kidney, urethra, reproductive organs, and isolated preparations of whole or part of embryonic, fetal, or adult animals. In one embodiment of the present invention, the cells are selected from the group consisting of epithelial cells, fibroblasts, neurons, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), macrophages, monocytes, mononuclear cells, cardiomyocytes, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, islet cells of Langerhans, blood cells, blood progenitor cells, bone cells, osteoprogenitor cells, neural stem cells, primordial 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, red blood cells, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, epithelial microtubules, and the like. The cell may be selected from the group consisting of, but is not limited to, somatic cells, parotid gland cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, 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, egg cells, Leydig cells, peritubular cells, Sertoli cells, lutein cells, cervical cells, endometrial cells, mammary gland cells, follicular cells, mucous cells, ciliated cells, non-keratinizing epithelial cells, keratinizing epithelial cells, lung cells, goblet cells, columnar epithelial cells, squamous epithelial cells, osteocytes, osteoblasts, and osteoclasts. In another embodiment, embryonic stem cells may be used. Embryonic stem cell lines may be used, or embryonic stem cells may be obtained de novo from a host such as a pig. These cells may be grown on an appropriate fibroblast-feeder cell layer or in the presence of leukemia inhibitory factor (LIF).

[0296] Embryonic stem cells are the preferred germ cell type, and embryonic stem cell lines may be used, or embryonic stem cells may be obtained de novo from a host such as a pig. These cells may be grown on an appropriate fibroblast-feeder cell layer or in the presence of leukemia inhibitory factor (LIF).

[0297] Cells of particular interest include stem cells, such as hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, islets of Langerhans, adrenal medullary cells capable of secreting dopamine, osteoblasts, osteoclasts, epithelial cells, endothelial cells, leukocytes, such as B lymphocytes and T lymphocytes, myelomonocytic cells, neurons, glial cells, ganglion cells, retinal cells, liver cells, such as hepatocytes, bone marrow cells, keratinocytes, hair follicle cells, and myoblast (muscle) cells, among other lineages.

[0298] In certain embodiments, the cells may be fibroblasts or fibroblast-like cells with a morphology or phenotype indistinguishable from fibroblasts, or with a pre-senescent lifespan of at least 10 days, or at least 12 days, or at least 14 days, or at least 18 days, or at least 20 days, or with a lifespan sufficient to allow homologous recombination and nuclear transfer of non-senescent nuclei; in a particular embodiment, the cells may be fetal fibroblasts. Fibroblasts are a preferred somatic cell type because they can be obtained in large quantities from developing fetuses and adult animals. These cells can be easily propagated in vitro with a rapid doubling time and can be clonally propagated for use in gene targeting methods. The cells to be used may be derived from fetal, neonatal, or adult animals. The cells may be mature or immature, differentiated or undifferentiated.

[0299] A. Homologous recombination Homologous recombination allows for site-specific modifications in endogenous genes, allowing new changes to be introduced into the genome. The first step in homologous recombination is DNA strand exchange, which involves pairing of two DNA strands with at least one DNA strand containing a complementary sequence to form an intermediate recombinant structure containing heteroduplex DNA (see, e.g., Radding, CM (1982) Ann. Rev. Genet. 16: 405; U.S. Pat. No. 4,888,274). This heteroduplex DNA can take several forms, including triplex structures containing three DNA strands, where one complementary strand invades the DNA duplex (Hsieh et al. (1990) Genes and Development 4: 1951; Rao et al., (1991) PNAS 88:2984), or, when two complementary DNA strands pair with the DNA duplex, forming a classical Holliday recombination junction or Chi structure (Holliday, R. (1964) Genet. Res. 5: 282), or a double-D loop ("Diagnostic Applications of Double-D Loop Formation," U.S. Patent Application Publication No. 07 / 755,462, filed September 4, 1991). After formation, the heteroduplex structure can be separated by strand breakage and exchange, which results in all or part of the invading DNA strand being spliced ​​into the recipient DNA duplex, adding or replacing a segment of the recipient DNA duplex.

[0300] Alternatively, heteroduplex structures can result in gene conversion, where the sequence of the invading strand is introduced into the recipient DNA duplex by repair of the mismatched bases using the invading strand as a template (Genes, 3rd Ed. (1987) Lewin, B., John Wiley, New York, NY; Lopez et al. (1987) Nucleic Acids Res. 15: 5643). Whether by a mechanism of breakage and rejoining or gene conversion, the formation of heteroduplex DNA at homologously paired junctions can serve to transfer genetic sequence information from one DNA molecule to another.

[0301] The ability of homologous recombination (gene conversion and classical strand breakage / rejoining) to transfer genetic sequence information between DNA molecules makes targeted homologous recombination a powerful method in genetic engineering and genetic manipulation.

[0302] In homologous recombination, incoming DNA interacts with and integrates into a site in the genome containing a substantially homologous DNA sequence. In non-homologous ("random" or "illicit") integration, the incoming DNA is not found in a homologous sequence within the genome but is instead integrated elsewhere, at one of many possible locations. Studies with higher eukaryotic cells have generally demonstrated that the frequency of homologous recombination is much lower than that of random integration. The ratio of these frequencies has direct implications for "gene targeting," which relies on integration via homologous recombination (i.e., recombination between an exogenous "targeting DNA" and the corresponding "target DNA" within the genome). The present invention may use homologous recombination to inactivate genes or inserts and to upregulate or activate genes in cells, such as those described above. The DNA may contain at least a portion of a gene at a specific locus, where an alteration has been introduced into at least one, and optionally both, copies of the native gene to prevent expression of a functional gene product. The alteration may be an insertion, deletion, substitution, mutation, or a combination thereof. If an alteration is introduced into only one copy of the inactivated gene, cells with a single, unmutated copy of the target gene may be amplified and subjected to a second targeting step, which may be the same or different from the first, but is usually different and includes a deletion or substitution, and may overlap at least a portion of the first introduced alteration. This second targeting step may use a targeting vector with the same homology arms but containing a different mammalian selectable marker. The resulting transformants may be screened for the absence of a functional target antigen, and the DNA of these cells may be further screened to confirm the absence of the wild-type target gene. Alternatively, hosts heterozygous for the mutation may be crossed to achieve phenotypic homozygosity.

[0303] Several papers describe the use of homologous recombination in mammalian cells. Examples of these papers are: 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. (1985) Mol. Cell. Bio. 8:2080-2089; Ayares et al. (1985) Genetics 111:375-388; Ayares et al. (1986) Mol. Cell. Bio. 7:1656-1662; Song et al. (1987) Proc. Natl. Acad. Sci. USA 84:6820-6824; 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; and Mansour 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.

[0304] In one embodiment, at least six transgenes that are integrated and expressed in the transgenic animals of the invention are introduced by homologous recombination. In another embodiment, at least one of the six transgenes that are integrated and expressed in the transgenic animals of the invention is introduced by homologous recombination.

[0305] B. Random Insertion In one embodiment, DNA encoding a transgene sequence may be randomly integrated into a cell's chromosome. This random integration may result from any method known to those skilled in the art for introducing DNA into cells. These methods may include, but are not limited to, electroporation, sonoporation, use of a gene gun, lipotransfection, calcium phosphate transfection, use of dendrimers, microinjection, use of viral vectors, including adenoviral, AAV, and retroviral vectors, and Group II ribozymes. In one embodiment, DNA encoding may be engineered to contain a reporter gene, allowing the presence of the transgene or its expression product to be detected via activation of the reporter gene. Any reporter gene known in the art, such as those disclosed above, may be used. A reporter gene may be added to the cells as one of the transgenes, such that cell surface expression of the transgene (e.g., DAF or CD46 or EPCR or CD47) can be used in combination with flow cytometry (and fluorescent antibodies specific for the transgene) as a means of enriching for transgene (and co-inserted transgene combinations) for transfection and subsequent expression. Cells containing the transgene can be selected in cell culture by selecting for cells in which the reporter gene is activated. In other embodiments, DNA encoding the transgene may be introduced into cells via electroporation. In other embodiments, DNA may be introduced into cells via lipofection, infection, or transformation. In one embodiment, electroporation and / or lipofection may be used to transfect fibroblasts. In certain embodiments, the transfected fibroblasts may be used as nuclear donors for nuclear transfer to generate transgenic animals, as known in the art and described below.

[0306] Cells stained for the presence of the reporter gene may then be FACS sorted to enrich the cell population for a higher proportion of cells containing DNA encoding the transgene of interest. In other embodiments, the FACS sorted cells may 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 a period of time that allows the DNA to integrate and result in a stable transfected cell population.

[0307] In one embodiment, the at least six transgenes integrated and expressed in the transgenic animals of the invention are introduced by random integration. In another embodiment, at least one of the six transgenes integrated and expressed in the transgenic animals of the invention is introduced by random integration. For example, a bicistronic vector containing at least two transgenes is integrated into the genome by random integration.

[0308] C. Targeted Genome Editing In a preferred embodiment, the transgene is integrated into the animal using genome editing tools, including but not limited to nucleases and site-specific recombinases. In a preferred embodiment, the insertion method is facilitated by genome editing techniques using gene editing tools, including but not limited to integrases (recombinases), CRISPR / CAS9 nucleases, TALAN nucleases, and zinc finger nucleases.

[0309] The transgene may be targeted to a single locus selected from a native locus, a modified native locus, or a transgenic locus (e.g., a landing pad). The native locus may be, for example, GGTA1, β4GalNT2, CMAH, GHR, ROSA26, AAVS1. The native locus may be modified, i.e., a modified native locus, such as (GGTA1, β4GalNT2, or CMAH).

[0310] In a preferred embodiment, the transgene may be targeted to a stable expression site, such as a landing pad and / or docking site. In one embodiment, the landing pad or docking vector may be inserted into any locus of interest, e.g., GGTA1, CMAH, β4Gal, ROSA26, GHR, AAVS1, or the transgene may be targeted to any known "safe harbor" locus or any predetermined locus that may confer a beneficial gene expression profile or whose simultaneous insertion and knockout may also inactivate a preferred gene that is beneficial to transplantation outcome. In another embodiment, gene editing may be used to generate small insertions, deletions, or nucleic acid substitutions (INDELs) that generate double-strand breaks that trigger DNA repair mechanisms, resulting in gene activation or knockout at the target site; in such cases, an indel at one given locus (e.g., GGTA1, CMAH, B4GalNT2) can be generated in cells or the resulting cloned pig simultaneously with gene-editing-enhanced knock-in of a multicistronic vector at another locus.

[0311] In certain 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 pig genome (e.g., one or all of GGTA1, CMAH, B4GalNT2, GHR), where one or more of these gene editing-augmented modifications also result in targeted insertion of a multicistronic vector bearing at least six transgenes under the control of at least three promoters in one or more of such native or modified native loci.

[0312] In some embodiments, the CRISPR / Cas9-mediated gene editing comprises an inducible promoter or system, a tetracycline / doxycycline-regulated system, U6p[GHRgRNA-1], U6p[GHRgRNA-2], TRE3Gp[CAS9], CAGpr[tTA], CAGpr[hCD46-2A-hCD55], or a polycistronic vector comprising the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the inducible promoter regulates expression of a growth hormone receptor gene.

[0313] 1. Zn finger nucleases / TALENs In one embodiment, the transgene is integrated using a zinc finger nuclease (ZFN). A zinc finger nuclease is a fusion of a nonspecific DNA cleavage motif and a sequence-specific zinc finger protein. The nuclease activity is a derivative of the FokI bacterial restriction endonuclease and can generate single-strand breaks. ZFNs function by dimerizing two DNA-binding domains and two FokI enzymes, generating a double-strand break with 18-bp specificity. In another embodiment, the transgene is integrated using a transcription activator-like effector nuclease (TALEN).

[0314] TALENs function similarly to ZFNs, generating double-strand breaks by tethering the FokI endonuclease to the DNA-binding domain. In this process, the targeting efficiency of TALEN-directed mutagenesis has been reported to reach 73.1%, with a 27.8% biallelic knockout rate. TALENs can be distinguished from ZFNs by their ease of gene design, reduced cost, and slightly improved targeting frequency. The present invention utilizes direct injection of ZFNs and TALENs into pig zygotes, which can introduce endogenous genes or small insertions, deletions, or nucleotide substitutions, to produce piglets with the desired genetic modifications.

[0315] 2. CRISPR / CAS9 nuclease In another embodiment, the transgene is integrated using CRISPR / CAS9 nuclease. CRISPR / Cas9 is derived from a bacterial defense mechanism that cleaves foreign DNA through RNA-guided targeting. In bacteria, foreign DNA is digested and inserted into the CRISPR locus, from which CRISPR RNA (crRNA) is produced. These short RNA sequences then bind to homologous (presumably foreign) sequences within the genome. If the homologous genomic sequence is followed by an appropriate "protospacer adjacent motif" (PAM) at its 3' end, the Cas9 endonuclease generates a double-stranded break. The PAM spacer serves to prevent the CRISPR locus from being targeted. The CRISPR / Cas9 system has proven useful outside of bacteria and was first used in 2013 to remove α-Gal from the pig genome. The most commonly used system is derived from Streptococcus pyogenes, which has a 3' PAM sequence of NGG (where N represents any nucleotide). This system makes it possible to generate mutation events in any pig genome sequence consisting of GN19NGG.

[0316] The CRISPR / Cas9 system is a natural nucleic acid repair system triggered by the presence of double-strand breaks (DSBs) in DNA (Liang et al. 1998) and may be used in combination with homology-directed repair (HDR). More specifically, the CRISPR / Cas9 system may be used to generate targeted double-strand breaks and to control the specificity of HDR genome engineering methods (Findlay et al. 2014; Mali et al. February 2014; Ran et al. 2013), making it useful for modifying genomes in many organisms, including mammals and humans (Sander and Young, 2014).

[0317] Specific sites in DNA can be subjected to RNA-guided cleavage to generate double-stranded breaks, after which a desired DNA fragment or construct can be inserted. This donor template, fragment, or construct contains the desired insertion or modification flanked by DNA segments homologous to the blunt ends of the cleaved DNA. In this way, the cell's natural DNA repair machinery can be utilized to insert the desired genetic material. Homology-driven recombination can be combined with any genome editing method known to generate highly targeted double-stranded breaks to precisely edit the genome of a target cell. This type of genome modification can be used to insert new genes, described as DNA insertions, and simultaneously knock out existing genes, a process known as "enhanced homology-driven insertion or knock-in" (Mali et al., February 2013).

[0318] The CRISPR / Cas system has several advantages over previous site-specific nucleases. Notably, the Cas9 endonuclease is the first untethered DNA cleavage method. Because the Cas9 endonuclease can freely bind multiple guide RNAs, it is possible to simultaneously target multiple loci in a single transfection. This allows for efficient combination of multiple gene knockouts in a single cell. In 2013, the generation of GGTA1, GGTA1 / iGb3S, GGTA1 / CMAH, and GGTA1 / iGb3S / CMAH homozygous knockout cells was achieved in a single reaction. Using the CRISPR / Cas9 system, transgenic animals could be generated 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] .

[0319] Targeting efficiency, i.e., the rate at which desired mutations are achieved, is one of the most important parameters for evaluating genome editing tools. The targeting efficiency of Cas9 compares favorably with more established methods, such as TALENs and ZFNs. For example, in human cells, custom-designed ZFNs and TALENs have only achieved efficiencies ranging from 1% to 50%. In contrast, the Cas9 system has been reported to have efficiencies exceeding 70% in zebrafish and plants and 2–5% in induced pluripotent stem cells.

[0320] In one embodiment, the present invention may utilize the CRISPR / Cas9 system to generate transgenic pigs (e.g., ungulates, swine) via microinjection of CRISPRs specifically designed to target genes of interest into "in vitro" derived zygotes.

[0321] In another embodiment, the present invention may utilize the CRISPR / Cas9 system to generate transgenic pigs (e.g., ungulates, swine) by modification of donor somatic cells with CRISPRs specifically designed to target genes of interest, followed by SCNT.

[0322] In another embodiment, the present invention may utilize the CRISPR / Cas9 system to generate transgenic pigs (e.g., ungulates, pigs) by targeting specific regions / sequences of existing genetic modifications. In a more specific embodiment, targeting sequences of the neomycin gene sequence.

[0323] In another embodiment, the present invention may utilize genome editing systems such as TALEN, Zn finger, or CRISPR / Cas9 systems to generate transgenic pigs (e.g., ungulates, pigs) by targeting specific regions / sequences of existing genetic modifications. In a more specific embodiment, targeting a single locus, which may be a native locus, a modified native locus, or a transgenic locus (e.g., a landing pad).

[0324] In another embodiment, the CRISPR / Cas9 system may be used to generate transgenic pigs (e.g., ungulates, swine) by utilizing homology-driven recombination to target specific regions / sequences of existing genetic modifications via insertion of large DNA fragments or constructs flanked by DNA arms or DNA segments homologous to double-stranded breaks.

[0325] In some embodiments, the CRISPR / Cas9-mediated gene editing comprises an inducible promoter or system, a tetracycline / doxycycline-regulated system, U6p[GHRgRNA-1], U6p[GHRgRNA-2], TRE3Gp[CAS9], CAGpr[tTA], CAGpr[hCD46-2A-hCD55], or a polycistronic vector comprising the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the inducible promoter regulates expression of a growth hormone receptor gene.

[0326] D. Site-specific recombinase In a preferred embodiment, the transgene is integrated using a site-specific recombinase. Specific recombinase technology is widely used to perform deletions, insertions, translocations, and inversions at specific sites within cellular DNA. This technology allows DNA modifications to be targeted to specific cell types or induced by specific external stimuli. This technology has been implemented in both eukaryotic and prokaryotic systems. Several recombination systems are available that efficiently implement genetic engineering strategies. The Flp-FRT recombinase system and the Cre-loxP recombinase system are reversible, facilitating both site-specific integration and excision. Integrases mediate the genome integration process by catalyzing highly site-specific recombination reactions that result in precise integration, excision, and / or inversion of DNA. Serine integrases (ΦC31, Bxb1, R4) and tyrosine integrases (λ, P22, HP1) are the two major integrase families currently applied in genome engineering. Broadly speaking, the process of site-specific recombination involves the binding and proximity of a recombinase to a recombinase substrate through protein-protein interactions. During this process, the substrate is cleaved and the DNA ends are reorganized in a strand exchange reaction, resulting in rejoining of the DNA backbone to generate the recombinant product. In most cases, serine integrases catalyze highly efficient irreversible recombination using simple att sites.

[0327] To take advantage of the high efficiency of site-specific recombinases, docking sites or landing pads may contain recombinase substrate binding sites, such as att sites; alternatively, recombination systems, such as Flp-FRT and Cre-loxP, may be introduced into desired loci of cell lines and / or animal strains. The docking vector is inserted into the target genome via random or homologous recombination. This allows for sequential plasmid integrations, where the plasmids or vectors may contain different transgenes and / or additional DNA sequences. Alternatively, recombination systems such as Flp / FRT may be used to remove unwanted vector and marker sequences.

[0328] E. Vectors for generating transgenic animals Nucleic acid targeting vector constructs may be designed to achieve homologous recombination in cells. In one embodiment, the targeting vector is designed with a promoter trap, allowing integration at the targeted locus to allow the inserted transgene open reading frame to utilize an endogenous or native promoter to drive expression of the inserted gene (or inserted selectable marker; e.g., Neo or Puro). In certain embodiments, the targeting vector is designed with a "poly(A) trap." Unlike promoter traps, poly(A) trap vectors capture a wider range of genes, including genes not expressed in target cells (i.e., fibroblasts or ES cells). Poly(A) trap vectors contain a constitutive promoter that drives expression of the selectable marker gene without a poly(A) signal. The alternative to the poly(A) signal is a splice donor site designed to splice into a downstream exon. In this strategy, the selectable marker gene mRNA can be stabilized upon capture of the endogenous gene's poly(A) signal, regardless of its expression status in the target cell. In one embodiment, a targeting vector is constructed that contains a selectable marker that is deficient in a polyadenylation signal.

[0329] These targeting vectors can be introduced into mammalian cells by any suitable method, including, but not limited to, transfection, transformation, virus-mediated transduction, or viral vector infection. In one embodiment, the targeting vector may contain 3' and 5' recombination arms (i.e., flanking sequences) homologous to a genomic sequence of interest. The 3' and 5' recombination arms may be designed to flank the 3' and 5' ends of at least one functional region of the genomic sequence. Targeting a functional region can render this functional region inactive, thereby preventing the cell from producing a functional protein. In another embodiment, the homologous DNA sequence may contain one or more intronic and / or exonic sequences. In addition to the nucleic acid sequence, the expression vector may contain a selectable marker sequence, such as an enhanced green fluorescent protein (eGFP) gene sequence, an initiation and / or enhancer sequence, a poly(A) tail sequence, and / or a nucleic acid sequence enabling expression of the construct in prokaryotic and / or eukaryotic host cells. The selectable marker may be located between the 5' and 3' recombination arm sequences.

[0330] Modification of a targeted locus in a cell can be produced by introducing into the cell DNA that is homologous to the target locus and contains a marker gene, allowing for selection of cells containing the integrated construct. The homologous DNA in the targeting vector recombines with chromosomal DNA at the target locus. The marker gene may be flanked on both sides by 3' and 5' recombination arms, which are homologous DNA sequences. Methods for constructing targeting vectors are described in the art; see, for example, Dai et al., Nature Biotechnology 20: 251-255, 2002; WO 00 / 51424. In such an example, the selectable marker gene may be a promoterless neomycin phosphotransferase (Neo) gene, which not only results in targeted insertion and expression of Neo (by capturing and utilizing the endogenous porcine α-Gal gene promoter), but also results in functional inactivation of the target locus (e.g., GGTA1) due to the targeted insertion and disruption of the GGTA1 catalytic domain.

[0331] Various enzymes can catalyze the insertion of foreign DNA into a host genome. Viral integrases, transposases, and site-specific recombinases mediate the integration of viral genomes, transposons, or bacteriophages into the host genome. A wide range of enzymes with these properties can be derived from a variety of sources. Retroviruses combine several useful features, including a relatively simple genome, ease of use, and the ability to integrate into the host cell genome, allowing long-term transgene expression in transduced cells and their progeny. Therefore, they are used in many gene therapy protocols. Lentiviral vectors have been attractive candidates for both gene therapy and transgenic applications, as have adeno-associated viruses (AAVs), which are small DNA viruses (parvoviruses) that co-replicate in mammalian cells with helper viruses such as adenovirus, herpes simplex virus, or human cytomegalovirus. The viral genome essentially consists of only two ORFs (the nonstructural protein rep and the structural protein cap), from which (at least) seven distinct polypeptides are derived by alternative splicing and the use of alternative promoters. In the presence of a helper virus, the rep protein mediates replication of the AAV genome. In the absence of a helper virus, integration, or latent viral infection, occurs.

[0332] Transposons have also attracted attention. These are mobile DNA segments that can be found in a variety of organisms. Active transposons exist in many prokaryotic systems and insects, but functional, naturally occurring transposons do not exist in vertebrates. Drosophila P-element transposons have been used as genome engineering tools for many years. The Sleeping Beauty transposon, established from a nonfunctional transposon copy present in salmonid fish, is significantly more active in mammalian cells than prokaryotic or insect transposons.

[0333] Site-specific recombinases are enzymes that catalyze DNA strand exchange between DNA segments with limited sequence homology. They bind to recognition sequences 30–200 nucleotides long, cleave the DNA backbone, exchange the two involved DNA double helices, and religate the DNA. In some site-specific recombination systems, a single polypeptide is sufficient to perform all of these reactions, while other recombinases require a variable number of accessory proteins to perform these tasks. Site-specific recombinases can be classified into two protein families with distinct biochemical properties: tyrosine recombinases (DNA is covalently bound at tyrosine residues) and serine recombinases (covalent binding occurs at serine residues). The most prominent enzymes used in genome engineering approaches are Cre (a tyrosine recombinase from Escherichia coli bacteriophage P1) and φC31 integrase (a serine recombinase from Streptomyces phage φC31). Several other bacteriophage-derived site-specific recombinases (including Flp, λ integrase, bacteriophage HK022 recombinase, bacteriophage R4 integrase, and phage TP901-1 integrase and bxb1 integrase) have also been successfully used to mediate stable gene insertion into mammalian genomes. Recently, a site-specific recombinase has been purified from Streptomyces bacteriophage. φC31 recombinase is a member of the resolvase family and mediates phage integration. During this process, the bacteriophage attP site recombines with the corresponding attB site in the bacterial genome. This crossover generates two sites, attL and attR, which are no longer accessible to recombinase in the absence of accessory proteins. This reaction also occurs in mammalian cells and can be used to mediate site-specific integration of therapeutic genes. The site specificity of tyrosine recombinases has been difficult to alter by direct protein engineering because the catalytic and DNA recognition domains are tightly intertwined, and therefore altered specificity is often accompanied by a loss of activity.Serine recombinases may be more amenable to engineering, and a highly active derivative of Tn3 resolvinase was engineered by exchanging the native DBD with the zinc finger domain of the human zinc finger transcription factor Zif268. The DNA site specificity of the resulting chimeric protein, termed a Z-resolvase, swaps with that of Zif268. Zinc finger proteins can be engineered to recognize arbitrary DNA sequences through in vitro protein evolution, and this approach may therefore enable the development of chimeric recombinases capable of integrating therapeutic genes into precise genomic locations. Methods for enhancing or mediating recombination include a combination of site-specific and homologous recombination, AAV vector-mediated recombination, and zinc finger nuclease-mediated recombination (Geurts et al., Science, 325: 433, 2009).

[0334] The term "vector," as used herein, refers to a nucleic acid molecule (preferably DNA) that confers useful biological or biochemical properties to an inserted nucleic acid. "Expression vectors" according to the present invention include vectors capable of enhancing expression of one or more molecules inserted or cloned into the vector after transformation of the vector into a cell. Examples of such expression vectors include phage, autonomously replicating sequences (ARS), centromeres, and other sequences capable of replicating or being replicated in vitro or intracellularly, or of delivering a desired nucleic acid segment to a desired location within an animal cell. Expression vectors useful in the present invention include chromosomally derived vectors, episomal vectors, and virus-derived vectors, such as vectors derived from bacterial plasmids or bacteriophages, and vectors derived from combinations thereof, such as cosmids and phagemids, or virus-based vectors such as adenoviruses, AAVs, and lentiviruses. A vector may have one or more restriction enzyme recognition sites at which sequences can be cleaved in a determinable manner without impairing the essential biological function of the vector, and to which nucleic acid fragments can be spliced ​​to trigger its replication and cloning.

[0335] Vectors may further provide primer sites (e.g., for PCR), transcription and / or translation initiation and / or control sites, recombination signals, replicons, selectable markers, etc. Obviously, methods for inserting desired nucleic acid fragments that do not require homologous recombination, transposition, or the use of restriction enzymes (e.g., but not limited to, UDG cloning of PCR fragments (U.S. Pat. No. 5,334,575), TA Cloning, RT-PCR, cloning (Invitrogen, Carlsbad, Calif.), etc.) may also be applied to clone nucleic acids into vectors to be used in accordance with the present invention.

[0336] Cells homozygous at the targeted locus can be generated by introducing DNA into cells that is homologous to the target locus and contains a marker gene, allowing for selection of cells containing the integrated construct. The homologous DNA in the targeting vector recombines with chromosomal DNA at the target locus. The marker gene may be flanked on both sides by 3' and 5' recombination arms, which are homologous DNA sequences. Methods for constructing targeting vectors have been reported in the art, see, for example, Dai et al. (2002) Nature Biotechnology 20: 251-255; WO 00 / 5142 Pamphlet 4, Figure 6; and Gene Targeting: A Practical Approach. Joyner, A., Oxford University Press, USA; 2nd ed., Feb. 15, 2000.

[0337] A variety of constructs can be prepared for homologous recombination at the target locus. Typically, the constructs may contain at least 25 bp, at least 50 bp, at least 100 bp, at least 500 bp, at least 10 kbp, at least 2 kbp, at least 4 kbp, at least 5 kbp, at least 10 kbp, at least 15 kbp, at least 20 kbp, or at least 50 kbp of sequence homologous to the target locus.

[0338] Determining the degree of homology of a target DNA sequence can involve various considerations, 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 targeting DNA can contain a sequence in which substantially isogenic DNA flanks the desired sequence modification to the corresponding target sequence in the genome to be modified. A substantially isogenic sequence can be at least about 95%, 97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identical to the corresponding target sequence (excluding the desired sequence modification). The targeting DNA and the target DNA preferably share a stretch of DNA that is 100% identical, preferably at least about 75 base pairs, at least about 150 base pairs, or at least about 500 base pairs. Thus, the targeting DNA can be derived from cells closely related to the cell line being targeted; alternatively, the targeting DNA can be derived from cells of the same cell line or animal as the cells being targeted.

[0339] Suitable selectable marker genes include, but are not limited to, genes that confer the ability to grow on a specific culture substrate, such as the tk gene (thymidine kinase) or hprt gene (hypoxanthine phosphoribosyltransferase), which confer the ability to grow on HAT medium (hypoxanthine, aminopterin, and thymidine); the bacterial gpt gene (guanine / xanthine phosphoribosyltransferase), which allows growth on MAX medium (mycophenolic acid, adenine, and xanthine). See Song et al. (1987) Proc. Nat'l Acad. Sci. USA 84:6820-6824. See also Chapter 16 of Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Other examples of selectable markers include genes that confer resistance to compounds such as antibiotics, genes that confer the ability to grow on a selected substrate, and genes that encode proteins that produce a detectable signal, such as luminescence, such as green fluorescent protein, enhanced green fluorescent protein (eGFP), etc. A wide variety of such markers are known and available, including 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).

[0340] Additional reporter genes useful in the methods of the invention include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), β-galactosidase (LacZ), β-glucoronidase (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), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, blasticidin, zeocin, methotrexate, phosphinothricin, puromycin, and tetracycline.

[0341] Methods for determining repression of reporter genes are well known in the art and include, but are not limited to, fluorometric methods (e.g., fluorescence spectroscopy, fluorescence activated cell sorting (FACS), fluorescence microscopy), antibiotic resistance determination methods, and the like.

[0342] Combinations of selectable markers may also be used. To use a combination of markers, the HSV-tk gene may be cloned outside the target DNA (with another selectable marker flanking it, if necessary). After introducing the DNA construct into the cells to be targeted, the cells can be selected on an appropriate antibiotic. Selectable markers may also be used for negative selection. Negative selection markers typically kill cells expressing the negative selectable marker because their expression is toxic or because they produce a catalyst that results in a toxic metabolite, such as herpes simplex virus type 1 thymidine kinase (HSV-tk) or diphtheria toxin A. Typically, the negative selectable marker is incorporated into the targeting vector so that it is lost after the correct recombination event. Similarly, conventional selectable markers such as GFP may be used for negative selection, for example, using FACS to sort selected transgene inserts; if expressed at significant levels on the cell surface, they can serve as gain-of-function or loss-of-function "selectable markers." The use of an inserted or targeted transgene as a selection tool allows for positive selection without the use of added fluorescent markers (e.g., GFP, RFP) or antibiotic selection genes. In certain cases, targeted insertion of a transgene may inactivate the target locus so that loss of function can be monitored or selected for. For example, inactivation of the GGTA1 locus eliminates or reduces binding of targeted cells to a lectin (IB4), or inactivation of B4GalNT2 eliminates or reduces binding of targeted cells by the DBA lectin; in each case, targeted integrants can be selected for or enriched in cells lacking such lectin binding.

[0343] Deletions may be at least about 50 bp, more typically at least about 100 bp, and usually no more than about 20 kbp, where the deletion may include at least part of the coding region, usually including part of an exon or one or more exons, part of an intron or one or more introns, and may or may not include part of the adjacent non-coding region, particularly the 5'-non-coding region (transcriptional regulatory region). Thus, the homologous region may extend beyond the coding region into the 5'-non-coding region or into the 3'-non-coding region. Insertions usually do not exceed 10 kbp, typically do not exceed 5 kbp, and are usually at least 50 bp, more typically at least 200 bp.

[0344] Homologous regions may contain mutations that may further inactivate the target gene by causing a frameshift or changing a critical amino acid, or they may correct a dysfunctional allele, etc. Mutations typically involve small changes of no more than about 5% of the homologous flanking sequence, or single nucleotide changes such as point mutations in the active site of an exon. If gene mutation is desired, a marker gene may be inserted into an intron so that it is excised from the target gene during transcription.

[0345] Determining the degree of homology of a target DNA sequence can involve various considerations, 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 targeting DNA can contain a sequence in which substantially isogenic DNA flanks the desired sequence modification to the corresponding target sequence in the genome to be modified. A substantially isogenic sequence can be at least about 95%, or at least about 97%, or at least about 98%, or at least about 99%, or 95-100%, 97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identical to the corresponding target sequence (excluding the desired sequence modification). In certain embodiments, the targeting DNA and the target DNA can share a stretch of DNA of at least about 75 base pairs, at least about 150 base pairs, or at least about 500 base pairs that is 100% identical. Thus, the targeting DNA may be derived from cells closely related to the cell line being targeted; alternatively, the targeting DNA may be derived from cells of the same cell line or animal as the cells being targeted.

[0346] Constructs can be prepared according to methods known in the art, and various fragments can be ligated, introduced into appropriate vectors, cloned, analyzed, and then further manipulated until the desired construct is achieved. Various modifications can be made to the sequence to enable restriction enzyme analysis, excision, probe identification, etc. Silent mutations can be introduced if desired. At various stages, restriction enzyme analysis, sequencing, polymerase chain reaction amplification, primer repair, in vitro mutagenesis, etc. can be employed.

[0347] The construct may be prepared using a bacterial vector containing a prokaryotic replication system, e.g., an origin recognizable by E. coli, and at each stage the construct may be subjected to cloning and analysis. A marker may be employed, which may be the same as or different from the marker to be used for insertion, and may be removed before introduction into the target cell. After the vector containing the construct is completed, further manipulations may be performed, such as deletion of bacterial sequences, linearization, or introduction of short deletions within homologous sequences. After final manipulation, the construct may be introduced into cells.

[0348] Techniques that can be used to allow DNA or RNA constructs to enter host cells include calcium phosphate / DNA coprecipitation, microinjection of DNA into nuclei, electroporation, bacterial protoplast fusion with intact cells, transfection, lipofection, infection, particle bombardment, or any other technique known to those skilled in the art. The DNA or RNA can 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).

[0349] The following vectors are provided as examples: Bacterial vectors: pBs, pQE-9 (Qiagen), PhageScript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR54O, pRIT5 (Pharmacia). Eukaryotic vectors: pWLneo, pSv2cat, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPv, pMSG, pSVL (Pharmacia). Any other plasmids and vectors may also be used as long as they are replicable and viable in the host. In accordance with the present invention, vectors known in the art and commercially available vectors (and variants or derivatives thereof) may be engineered to contain one or more recombination sites for use in the methods of the present invention. Such vectors are available from, for example, Vector Laboratories, Invitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim, Pharmacia, Epicentre, Origin Technologies, Stratagene, PerkinElmer, PharMingen, and Research Genetics. Other notable vectors include 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), p3'SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and pOG44 (Stratagene), and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBlueBacll, pCDM8, pcDNA1, pZeoSV, and pcDNA3. Examples include eukaryotic expression vectors such as pREP4, pCEP4, and pEBVHis (Invitrogen), and variants or derivatives thereof.

[0350] Other vectors include pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), P1 (E. coli phage), pQE70, pQE60, pQE9 (Qiagen), pBS vectors, PhageScript vectors, BlueScript vectors, 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 variants or derivatives thereof. Viral vectors, such as lentiviral vectors, may also be used (see, eg, WO 03 / 059923; Tiscornia et al. PNAS 100:1844-1848 (2003)).

[0351] The titles are as follows:pTrxFus pThioHis, pLEX, pTrcHis, pTrcHis2, p RSET, pBlueBacHis2, pcDNA3.1 / His, pcDNA3.1(-) / Myc-His, pSecTag, pEBVHis pPIC9K, pPIC3.5K, pAO81S, pPICZ, pPICZA, pPICZB, pPICZC, pGAPZA, pGAPZB, p GAPZC, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(S P1), pVgRXR, pcDNA2.1, pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt, pSE280, pSE3 80, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1 / Amp, pcDNA3.1, pcD NA3.1 / Zeo, pSe, SV2, pRc / CMV2, pRc / RSV, pREP4, pREP7, pREP8, pREP9, pREP10. pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac(protease scaffold);.lamda. ExCell、.ask. gt11, pTrc99A, 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, pCH11 0, pKK232-8, pSL1180, pNEO, and pUC4K(Carloid Antibody);pSCREEN-1b(+) pT7Blue(R) pT7Blue-2, pCITE-4-abc(+), pOCUS-2, pTAg, pET-32L1C, pET-30LIC, pBAC-2 cp LIC、pBACgus-2 cp LIC、pT7Blue-2 LIC、pT7Blue-2.lamda. SCREEN-1、.ask.BlueSTAR, pET-3abcd, pET-7abc, pET9abcd, pET11abcd, 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-3 cp, pBACgus-2 cp, pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg, and Selecta Vecta-Gpt (Novagen); pLexA, pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, 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 (Clontech); 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-Script Amp, pCR-Script 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 (Stratagene).

[0352] Additional 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 variants or derivatives thereof.

[0353] In a preferred embodiment, the vector is a bicistronic vector. The bicistronic vector comprises a promoter and two transgenes. In a specific embodiment, the bicistronic vector comprises a promoter and two transgenes linked by a 2A sequence. This embodiment allows for the co-expression of multiple functional transgenes from a single transcript. More specifically, this embodiment utilizes a short (18-24 aa) self-cleaving peptide, "2A," which allows linked open reading frames to be co-expressed to express functional transgenes from a single transcript 2A vector system.

[0354] In a preferred embodiment, the vector is a polycistronic vector (MCV). In one embodiment, the MCV comprises a promoter and at least six transgenes. In a specific embodiment, the MCV comprises six transgenes linked by 2A peptide sequences under the control of at least three promoters. This embodiment allows for the co-expression of multiple functional transgenes from a single transcript. More specifically, this embodiment utilizes a short (18-24 aa) self-cleaving peptide "2A" that allows linked open reading frames to be co-expressed to express functional transgenes from a single transcript 2A vector system.

[0355] In a preferred 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 regulated by a constitutive or ubiquitous promoter (e.g., a CAG promoter), and the second bicistronic unit is regulated by a tissue-specific or inducible promoter system, such as an endothelial-specific promoter. In certain embodiments, only at least six transgenes are inserted into a single locus, each regulated by its own promoter or a total of at least three promoters per single locus insertion. In a preferred embodiment, the vector is a six-gene MCV containing at least two anticoagulant factors, more specifically, at least three anticoagulant factors. In a preferred embodiment, the vector is a six-gene MCV vector containing at least two anticoagulant factors and one complement inhibitor, more specifically, three anticoagulant factors and one complement inhibitor. In a preferred embodiment, the vector is a six-gene MCV vector containing two anticoagulant factors, one complement inhibitor, and one immunosuppressant.

[0356] F. Control Sequence The vector constructs used to generate the animals of the present invention may include regulatory sequences, including but not limited to, promoter-enhancer sequences, "2A" peptide technology, and docking vectors, operably linked to the above sequences. In some embodiments, the self-cleaving peptide is selected from the group consisting of T2A, P2A, F2A, and E2A. Many suitable vectors and promoters are known to those of skill in the art and are commercially available.

[0357] In certain embodiments, the present invention provides animals, tissues, and cells that express at least one transgene in endothelial cells (in combination with at least one transgene under the control of a second, same or different promoter), specifically at least two, at least three, or at least six transgenes in endothelial cells. To target expression to specific tissues, animals are developed using vectors containing promoters specific for endothelial cell expression. In certain embodiments, expression is regulated primarily by promoter activity within the endothelium.

[0358] 1. Promoter In one embodiment, the nucleic acid construct includes a control sequence operably linked to the transgene sequence to be expressed. In one embodiment, the control sequence can be a promoter sequence. In one embodiment, the promoter can be a controllable promoter. In such a system, for example, a drug can be used to control whether the peptide is expressed in an animal, tissue, or organ. For example, expression can be suppressed while the organ or tissue is part of the pig, but can be induced for a period of time after the pig is transplanted into a human to overcome cellular immune responses. Furthermore, the expression level can be controlled by a controllable promoter system to avoid immunosuppression of the recipient's immune system. Furthermore, gene knockout can be selectively achieved by an inducible promoter system, such as tetracycline-inducible CAS9, in CRISPR-CAS9-mediated gene editing (see Zhang et al., Comput Struct Biotechnol J., 2019,(17):1171-1177). The regulatable promoter system may be selected from, but is not limited to, the following genetic systems: a metallothionein promoter inducible by metals such as copper (see Lichtlen and Schaffner, Swiss Med. Wkly., 2001, 131 (45-46):647-52); a tetracycline-regulated system (see Imhof et al., J Gene Med., 2000, 2(2):107-16); an ecdysone-regulated system (see Saez et al., Proc Natl Acad Sci USA., 2000, 97(26):14512-7); a cytochrome P450-inducible promoter such as the CYP1A1 promoter (see Fujii-Kuriyama et al., FASEB J., 1992, 6(2):706-10); a mifepristone-inducible system (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-inducible systems (responsive to macrolide antibiotics such as rapamycin, erythromycin, clarithromycin, and roxithromycin) (Weber et al., Nat Biotechnol., 2002, 20(9):901-7; Wang et al., Mol Ther., 2003, 7(6):790-800); ethanol-inducible systems (Garoosi et al., J Exp Bot., 2005, 56(416):163542; Roberts et al., Plant Physiol., 2005, 138(3):1259-67); streptogramin-inducible systems (Fussenegger et al., Nat Biotechnol., 2000 18(11):1203-8), electrophile-inducible systems (see Zhu and Fahl, Biochem Biophys Res Commun., 2001, 289(1):212-9); nicotine-inducible systems (see Malphettes et al., Nucleic Acids Res., 2005, 33(12):e107), immune-inducible promoters, cytokine-responsive promoters (e.g., promoters that are induced by IFN-γ, TNF-α, IL-1, IL-6, or TGF-β (or other secondary pathways) and thus can be turned on or upregulated in association with or in response to an immune or inflammatory response).

[0359] In certain embodiments, the animal comprises an endothelial-specific promoter, such as the porcine ICAM-2 promoter or the mouse Tie-2 promoter, and 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 further comprising an enhancer element. In certain elements, the ubiquitous promoter is CAG (CMV enhancer, chicken β-actin promoter, rabbit β-globin intron) (Tie2-CAG) used in combination with the endothelial-specific Tie-2 enhancer element. In the case of Tie2-CAG, the transgene is expected to be expressed both constitutively and ubiquitously, but at much higher levels in endothelial cells compared to other somatic cells. In some embodiments, the promoter is used in combination with an enhancer element, which is a non-coding or intronic DNA region inherently associated with or co-localized 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, viral promoters such as CMV and SV40, as well as chicken β-actin promoter and chicken γ-actin promoter, GAPDH promoter, H2K, CD46 promoter, GGTA1, ubiquitin, and ROSA promoter.

[0360] 2. Multicistronic systems In certain embodiments, the 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, the at least six transgenes in the multicistronic vector (MCV) are under the control of at least three promoters. These promoters may be exogenous promoters, native promoters, or a combination of both exogenous and native promoters. In some embodiments, the at least six transgenes are encoded by a polycistronic vector, which optionally comprises at least three bicistronic units. In some embodiments, each bicistronic unit comprises a promoter driving a first transgene linked to a second transgene via a self-cleaving peptide. In some embodiments, the first bicistronic unit comprises the at least two anticoagulant factor transgenes, the second bicistronic unit comprises at least two complement inhibitor transgenes, and the third bicistronic unit comprises the at least one cytoprotective factor transgene and the at least one immunosuppressant factor transgene. In some embodiments, the self-cleaving peptide is selected from the group consisting of T2A, P2A, F2A, and E2A.

[0361] In certain embodiments, the bicistronic vector comprises two transgenes and a constitutive promoter that expresses the transgenes ubiquitously in all organs, tissues, and cells. In certain embodiments, the bicistronic vector comprises two transgenes and a tissue-specific promoter that regulates expression in organs, tissues, and cells. In preferred embodiments, the vector is a six-gene MCV comprising at least two anticoagulants under the control of an endothelial-specific promoter. In preferred embodiments, the vector is a six-gene MCV comprising at least one complement inhibitor transgene under the control of a constitutive promoter and at least one anticoagulant transgene under the control of an endothelial cell-specific promoter. In preferred embodiments, the vector is a six-gene MCV comprising at least one complement inhibitor transgene under the control of a constitutive promoter and at least one anticoagulant gene under the control of a second constitutive promoter.

[0362] In a preferred embodiment, the vector is a six-gene MCV vector containing an anticoagulant transgene and an immunosuppressant transgene under the control of an endothelial cell promoter. In a preferred embodiment, the vector is a two-gene MCV vector containing a total of two genes under the control of at least two separate promoters; or, in select embodiments, a vector containing a series of multiple transgenes, each with its own promoter, all integrated into a single locus.

[0363] In some embodiments, the polycistronic (MCV) vector comprises a bicistronic unit selected from the group consisting of: a human TBM transgene linked to a human EPCR transgene via a 2A peptide and driven by a porcine TBM promoter (pTBMpr[hTBM-2A-hEPCR]); a human CD47 transgene linked to a human HO-1 transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD47-2A-hHO1]); a human CD46 transgene linked to a human DAF transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD46-2A-hDAF]); a human TBM transgene linked to a human EPCR transgene via a 2A peptide and driven by a polyA signal-fused porcine TBM promoter (polyA / pTBMpr[hTBM-2A-hEPCR]); a human CD59 transgene linked to a human HO-1 transgene via a 2A peptide and driven by a CAG promoter (CAGpr[hCD46-2A-hDAF]). The CAG promoter driving the human TBM transgene (CAGpr[hCD59-2A-hHO1]); the porcine EPCR promoter driving the human EPCR transgene linked to the human TBM transgene via a 2A peptide (pEPCRpr[hTBM-2A-hEPCR]); the CAG promoter driving the human CD46 transgene linked to the human CD47 transgene via a 2A peptide (CAGpr[hCD46-2A-hCD47]); and the first GHRgRNA driving the first U transgene. U6 promoter and a second GHRgRNA and a second U6 promoter driving it linked to them (the first gRNA and the second gRNA may be the same or different) (U6p[GHRgRNA-1]; U6p[GHRgRNA-2]); a Cas endonuclease and a TRE3G promoter driving it, and a tTA and a CAG promoter driving it linked to them via an insulator (TRE3Gp[CAS9]; CAGpr[tTA]); and combinations thereof.

[0364] In some embodiments, the polycistronic (MCV) vector comprises: pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD47-2A-hH01]; and CAGpr[hCD46-2A-hDAF]; polyA-pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD47-2A-hH01]; and CAGpr[hCD46-2A-hDAF]; pTBMpr[hTBM-2A-hEPCR]; CAGpr[hCD59-P2A-hH01]; and CAGpr[hCD46-P2A-hDAF]; or polyA-pTBMpr[hTBM]; CAGpr[h CD47-P2A-hHO1];pEPCRpr[hEPCR]; and CAGpr[hCD46P-2A-hDAF];pTBMpr[hTBM-2A-hEPCR], CAGpr[hCD59-P2A-hHO1]; and CAGpr[hCD46-2A-hCD47];pTBMpr[hTBM -2A-hEPCR], CAGpr[hCD59-2A-hHO1], and CAGpr[hCD46-2A-hCD55]; U6p[GHRgRNA-1], U6p[GHRgRNA-2], TRE3Gp[CAS9], CAGpr[tTA], and CAGpr[hCD46-2A-hCD55]. In some embodiments, the MCV comprises a nucleotide sequence selected from SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:11, SEQ ID NO:12, SEQ...

Claims

1. The following results: (i) lack of expression of functional α1,3-galactosyltransferase; (ii) knockout of a gene selected from growth hormone receptor (GHR), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), β1,4-N-acetylgalactosaminyltransferase (β4GalNT2), or a combination thereof; and (iii) At a single genomic locus: (a) at least two complement inhibitory transgenes; (b) at least one immunosuppressive transgene; (c) at least one cytoprotective transgene; and (d) at least two anticoagulant transgenes; Integration and expression of polycistronic vectors comprising: wherein the transgenic animal comprises at least six, at least seven, or at least eight transgenes; and wherein the polycistronic vector comprises a thrombomodulin promoter.

2. (a) the transgenic animal contains at least six transgenes; (b) the polycistronic vector contains at least three bicistronic units; and / or (c) The polycistronic vector is: (i) at least two bicistronic units, each bicistronic unit comprising a promoter driving a first transgene linked to a second transgene via a cleavage peptide; (ii) at least two non-polycistronic transgenes, where each non-polycistronic transgene is driven by its own promoter; (iii) at least two bicistronic units, wherein each of the at least two bicistronic units is driven by a constitutive promoter; and / or (iv) at least two bicistronic units, where at least two non-polycistronic transgenes are each driven by a tissue-specific promoter; 2. The transgenic pig of claim 1, comprising:

3. (a) each of the at least three bicistronic units comprises a promoter driving a first transgene gene linked to a second transgene gene via a cleavage peptide, wherein the cleavage peptide is selected from the group consisting of T2A, P2A, F2A, and E2A; (b) the first bicistronic unit comprises at least two anticoagulant transgenes, the second bicistronic unit comprises at least two complement inhibitory transgenes, and the third bicistronic unit comprises at least one cytoprotective transgene and at least one immunosuppressive transgene; (c) at least one bicistronic unit is driven by a tissue-specific promoter, and (i) the tissue-specific promoter is an endothelial cell-specific promoter, or (ii) The tissue-specific promoter is one of the following: selected from the group consisting of ICAM-2 promoter, porcine thrombocyte modulating promoter (pTBMpr), human thrombocyte modulating promoter, porcine EPCR promoter, and human EPCR promoter; (d) at least one bicistronic unit is driven by an inducible promoter, and wherein the inducible promoter is a tetracycline / doxycycline-regulated promoter; and / or (e) the at least two bicistronic units are driven by dedicated constitutive promoters, and wherein the constitutive promoters are selected from the group consisting of CAG promoter and Tie-2 promoter.

4. (a) at least two complement inhibitors (i) selected from the group consisting of CD46, DAF (CD55), CD59, CR1, and combinations thereof; (ii) whether it is widely expressed; (iii) is under the control of an inducible promoter; and / or (iv) under the control of a constitutive promoter (b) The at least one immunosuppressive transgene is: (i) selected from the group consisting of cytotoxic T-lymphocyte-associated protein 4 (CTLA4), cluster of differentiation 47 (CD47), and class II transactivator of transcription factor-dependent activator (CIITA-DN); (ii) is under the control of an inducible promoter; and / or (iii) under the control of a constitutive promoter; (c) The at least one cytoprotective transgene is: (i) Is it under the control of a constitutive promoter? (ii) is under the control of an inducible promoter; (iii) is under the control of an endothelial-specific promoter; and / or (iv) selected from the group consisting of heme oxygenase 1 (HO-1), A20, FAT-1, soluble tumor necrosis factor alpha (TNF-α), and combinations thereof; and / or (d) At least two anticoagulant transgenes: (i) under the control of an endothelial-specific promoter; (ii) under the control of an inducible promoter; and / or (iii) selected from the group consisting of endothelial protein C receptor (EPCR), thrombomodulin, CD39, hirudin, tissue factor pathway inhibitor (TFPI), and combinations thereof.

5. The polycistronic vector comprises: (i) a porcine TBM promoter, a human TBM transgene driven by the promoter, and a human EPCR transgene linked via the 2A peptide (pTBMpr [hTBM-2A-hEPCR]); (ii) a CAG promoter, a human CD47 transgene driven by the promoter, and a human HO-1 transgene linked via the 2A peptide (CAGpr [hCD47-2A-hHO1]); (iii) a CAG promoter, a human CD46 transgene driven by the promoter, and a human DAF transgene linked via a 2A peptide (CAGpr [hCD46-2A-hDAF]); (iv) a polyA signal fused to the porcine TBM promoter, a human TBM transgene driven by the promoter, and a human EPCR transgene linked via a 2A peptide (PolyA / pTBMpr [hTBM-2A-hEPCR]); (v) a CAG promoter, a human CD59 transgene driven by the promoter, and a human HO-1 transgene linked via the 2A peptide (CAGpr [hCD59-2A-hHO1]); (vi) a porcine EPCR promoter, a human TBM transgene driven by the promoter, and a human EPCR transgene linked via a 2A peptide (pEPCRpr [hTBM-2A-hEPCR]); (vii) a CAG promoter, a human CD46 transgene driven by the promoter, and a human CD47 transgene linked via a 2A peptide (CAGpr [hCD46-2A-hCD47]); (viii) a first U6 promoter and a first GHR gRNA driven by the promoter, and a second U6 promoter and a second GHR gRNA driven by the promoter (U6p [GHRgRNA-1]; U6p [GHRgRNA-2]), wherein the first and second gRNAs are the same or different; (ix) a TRE3G promoter, a Cas endonuclease driven by the promoter, a CAG promoter linked via an insulator, and a tTA driven by the promoter (TRE3Gp [CAS9]; CAGpr [tTA]); and (x) A combination of these 2. The transgenic pig of claim 1, comprising a bicistronic unit selected from the group consisting of:

6. The polycistronic vector comprises: (i) pTBMpr [hTBM-2A-hEPCR]; CAGpr [hCD47-2A-hHO1]; and CAGpr [hCD46-2A-hDAF]; (ii) PolyA-pTBMpr [hTBM-2A-hEPCR]; CAGpr [hCD47-2A-hHO1]; and CAGpr [hCD46-2A-hDAF]; (iii) pTBMpr [hTBM-2A-hEPCR]; CAGpr [hCD59-P2A-hHO1]; and CAGpr [hCD46-P2A-hDAF]; (iv) PolyA-pTBMpr [hTBM]; CAG pr [hCD47-P2A-hHO1]; pEPCRpr [hEPCR]; and CAGpr [hCD46P-2A-hDAF]; (v) pTBMpr [hTBM-2A-hEPCR]; CAGpr [hCD59-P2A-hHO1]; CAGpr [hCD46-2A-hCD47]; (vi) pTBMpr [hTBM-2A-hEPCR]; CAGpr [hCD59-2A- hHO1]; CAGpr [hCD46-2A-hCD55]; (vii) U6p [GHRgRNA-1];U6p [GHRgRNA-2]; TRE3Gp[CAS9]; CAGpr [tTA]; CAGpr [hCD46-2A-hCD55]; (viii) SEQ ID NO: 7; (ix) SEQ ID NO: 8; (x) SEQ ID NO: 9; (xi) SEQ ID NO: 11; (xii) SEQ ID NO: 12; (xiii) SEQ ID NO: 13; or (xiv) SEQ ID NO: 14 2. The transgenic pig of claim 1, comprising:

7. The transgenic pig of claim 1, wherein the polycistronic vector encoding the transgene further comprises a nucleotide sequence for homologous recombination and / or homology-directed repair (HDR) at a locus selected from the group consisting of AAVS1, ROSA26, GHR, CMAH, B4GalNT2, and GGTA1.

8. The knockout may be: (i) Insertion, deletion, or substitution via gene editing; (ii) CRISPR-Cas9-mediated gene editing; (iii) insertion via homologous recombination; and / or (iv) NeoR insertion-mediated gene knockout 2. The transgenic pig of claim 1, comprising:

9. The transgenic pig further comprises an additional genetic modification, and wherein: (a) whether the single genomic locus is CMAH and the additional genetic modifications include knockout of β4GalNT2, GGTA1, and GHR; (b) the single genomic locus is β4GalNT2 and additional genetic modifications include knockout of CMAH, GGTA1, and GHR; or (c) The transgenic pig of claim 1, wherein the single genomic locus is GGTA1 and the additional genetic modifications include knockout of β4GalNT2, CMAH, and GHR.

10. A cell, organ, or tissue derived from the transgenic pig according to any one of claims 1 to 9.

11. (a) the tissue is selected from the group consisting of vascular tissue, heart valve, retinal tissue, neural tissue, and corneal tissue; (b) the organ is selected from the group consisting of heart, lung, liver, pancreas, and kidney; or (c) The cell, organ, or tissue of claim 10, wherein the tissue is a vascular graft.

12. A pharmaceutical composition comprising an organ, tissue, or cell derived from a genetically modified pig according to any one of claims 1 to 9, for use in xenotransplantation in a subject in need thereof.

13. (a) whether the subject is a non-human primate or a human; (b) the organ is selected from the group consisting of heart, lung, pancreas, liver, and kidney; (c) the tissue is selected from the group consisting of vascular tissue, retinal tissue, neural tissue, and corneal tissue; (d) When an organ, tissue, or cells from a genetically modified pig is transplanted into a subject, the subject survives for at least 30 days longer than a subject transplanted with an organ, tissue, or cells from a wild-type pig; or (e) The pharmaceutical composition of claim 12, wherein when the organ, tissue, or cell derived from the transgenic pig is transplanted into a subject, the subject survives for at least 60 days.

14. A method for producing a transgenic pig animal according to any one of claims 1 to 9.

15. 1. A method for producing a transgenic pig containing at least six transgenes, comprising the steps of: (i) Porcine cells are isolated from one of the following: (a) at least two complement inhibitor transgenes; (b) at least one immunosuppressive transgenic gene; (c) at least one cytoprotective transgenic gene; and (d) at least two anticoagulant transgenic genes transfecting with a single polycistronic vector comprising: (ii) producing a multi-transgenic porcine cell containing at least six transgenic genes by integrating and expressing the polycistronic vector at a single genomic locus; (iii) generating multi-transgenic pig zygotes by injecting the nuclei of the multi-transgenic pig cells into reconstituted somatic cell nuclear transfer (SCNT); and (iv) maturing the polygenic pig zygote into a polygenic pig; Including, Here, the multi-genetically modified pig cells and the multi-genetically modified pigs lack the expression of α1,3 galactosyltransferase (GTKO), and The pharmaceutical composition of claim 12, wherein the pig cells and the multi-genetically modified pig further comprise at least one additional genetic modification, and the at least one additional genetic modification is a knockout of a gene selected from the group consisting of CMAH, B4GalNT2, and GHR.

16. 16. The method of claim 15, wherein the genetically modified porcine cell comprises at least 7, at least 8, at least 9, or at least 10 transgenes.

17. The single polycistronic vector comprises: (i) a porcine TBM promoter, a human TBM transgene driven by the promoter, and a human EPCR transgene linked via the 2A peptide (pTBMpr [hTBM-2A-hEPCR]); (i) a porcine TBM promoter, a human TBM transgene driven by the promoter, and a human EPCR transgene linked via the 2A peptide (pTBMpr [hTBM-2A-hEPCR]); (ii) a CAG promoter, a human CD47 transgene driven by the promoter, and a human HO-1 transgene linked via the 2A peptide (CAGpr [hCD47-2A-hHO1]); (iii) a CAG promoter, a human CD46 transgene driven by the promoter, and a human DAF transgene linked via a 2A peptide (CAGpr [hCD46-2A-hDAF]); (iv) a polyA signal fused to the porcine TBM promoter, a human TBM transgene driven by the promoter, and a human EPCR transgene linked via a 2A peptide (PolyA / pTBMpr [hTBM-2A-hEPCR]); (v) a CAG promoter, a human CD59 transgene driven by the promoter, and a human HO-1 transgene linked via the 2A peptide (CAGpr [hCD59-2A-hHO1]); (vi) a porcine EPCR promoter, a human TBM transgene driven by the promoter, and a human EPCR transgene linked via a 2A peptide (pEPCRpr [hTBM-2A-hEPCR]); (vii) a CAG promoter, a human CD46 transgene driven by the promoter, and a human CD47 transgene linked via a 2A peptide (CAGpr [hCD46-2A-hCD47]); (viii) a first U6 promoter and a first GHR gRNA driven by the promoter, and a second U6 promoter and a second GHR gRNA driven by the promoter (U6p [GHRgRNA-1]; U6p [GHRgRNA-2]), wherein the first and second gRNAs are the same or different; (ix) a TRE3G promoter, a Cas endonuclease driven by the promoter, a CAG promoter linked via an insulator, and a tTA driven by the promoter (TRE3Gp [CAS9]; CAGpr [tTA]); and (x) A combination of these 16. The method of claim 15, comprising a bicistronic unit selected from the group consisting of:

18. The polycistronic vector comprises: (i) pTBMpr [hTBM-2A-hEPCR]; CAGpr [hCD47-2A-hHO1]; and CAGpr [hCD46-2A-hDAF]; (ii) PolyA-pTBMpr [hTBM-2A-hEPCR]; CAGpr [hCD47-2A-hHO1]; and CAGpr [hCD46-2A-hDAF]; (iii) pTBMpr [hTBM-2A-hEPCR]; CAGpr [hCD59-P2A-hHO1]; and CAGpr [hCD46-P2A-hDAF]; (iv) PolyA-pTBMpr [hTBM]; CAG pr [hCD47-P2A-hHO1]; pEPCRpr [hEPCR]; and CAGpr [hCD46P-2A-hDAF]; (v) pTBMpr [hTBM-2A-hEPCR]; CAGpr [hCD59-P2A-hHO1]; CAGpr [hCD46-2A-hCD47]; (vi) pTBMpr [hTBM-2A-hEPCR]; CAGpr [hCD59-2A- hHO1]; CAGpr [hCD46-2A-hCD55]; (vii) U6p [GHRgRNA-1];U6p [GHRgRNA-2]; TRE3Gp[CAS9]; CAGpr [tTA]; CAGpr [hCD46-2A-hCD55]; (viii) SEQ ID NO: 7; (xix) SEQ ID NO:8; (x) SEQ ID NO: 9; (xi) SEQ ID NO: 11; (xii) SEQ ID NO: 12; (xiii) SEQ ID NO: 13; or (xiv) SEQ ID NO: 14 16. The method of claim 15, comprising:

19. Gene knockouts include: (i) Insertion, deletion, or substitution via gene editing; (ii) CRISPR / Cas9-mediated gene editing, wherein the CRISPR / Cas9-mediated gene editing is: (a) Inducible promoters or inducible systems; (b) tetracycline / doxycycline regulatory system; (c) U6p [GHRgRNA-1]; U6p [GHRgRNA-2]; TRE3Gp[CAS9]; CAGpr [tTA]; CAGpr [hCD46-2A-hCD55]; or (d) the nucleotide sequence of SEQ ID NO: 9 Includes: (iii) insertion by CRISPR / Cas9-mediated homologous recombination; and / or (iv) gene knockout by NeoR insertion; 16. The method of claim 15, comprising:

20. (a) whether the single genomic locus is CMAH and the additional modifications include knockout of β4GalNT2, GGTA1, and GHR; (b) the single genomic locus is β4GalNT2 and the additional modifications include knockout of CMAH, GGTA1, and GHR; or (c) The method of claim 15, wherein the single genomic locus is GGTA1 and the additional modifications include knockout of β4GalNT2, CMAH, and GHR.

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