Genetically modified cells, tissues, and organs for treating disease

Abstract

To provide genetically modified cells, tissues, and organs for treating or preventing diseases.SOLUTION: A genetically modified human induced pluripotent stem cell includes genomic disruption in a NOD-like receptor family CARD domain containing 5 (NLRC5) gene, where the genomic disruption in the NLRC5 gene is a nuclease mediated double-strand break at a target sequence in an exon of the NLRC5 gene. The genetically modified human induced pluripotent stem cell includes genomic disruption and is reduced in expression of one or more MHC class I proteins compared to a not genetically modified human induced pluripotent stem cell. The cell further includes an exogenous nucleic acid sequence having a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 350,048, filed June 14, 2016, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Background to the disclosure There is a shortage of organs, tissues, or cells available for transplantation into recipients, such as humans. Xenotransplantation or allotransplantation of organs, tissues, or cells into humans has the potential to fill this need and help hundreds of thousands of people each year. Nonhuman animals can be selected as organ donors based on their anatomical and physiological similarities to humans. Furthermore, xenotransplantation is relevant not only to humans but also to veterinary applications.

[0003] However, unmodified wild-type non-human animal tissues can be rejected by the immune system of recipients, such as humans. Rejection is thought to be caused, at least in part, by antibody binding to the tissue and cell-mediated immunity, leading to graft loss. For example, pig grafts can be rejected by cellular mechanisms mediated by adaptive immune cells.

[0004] Incorporation by Reference All publications, patents, and patent applications herein are incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between the terms of this specification and the terms of the incorporated references, the terms of this specification shall control. Summary of the Invention [Means for solving the problem]

[0005] Abstract In a first aspect, disclosed herein is a genetically modified non-human animal comprising an exogenous nucleic acid sequence that is at least 95% identical to SEQ ID NO:359 or SEQ ID NO:502.

[0006] In some embodiments of the first aspect, the exogenous nucleic acid is at least 96% identical to SEQ ID NO: 359 or SEQ ID NO: 502. In some embodiments, the exogenous nucleic acid is at least 97% identical to SEQ ID NO: 359 or SEQ ID NO: 502. In some embodiments, the exogenous nucleic acid is at least 98% identical to SEQ ID NO: 359 or SEQ ID NO: 502. In some embodiments, the exogenous nucleic acid is at least 99% identical to SEQ ID NO: 359 or SEQ ID NO: 502. In some embodiments, the exogenous nucleic acid is 100% identical to SEQ ID NO: 359 or SEQ ID NO: 502.

[0007] In a second aspect, disclosed herein is a genetically modified non-human animal comprising an exogenous nucleic acid that is transcribed as human leukocyte antigen G (HLA-G) mRNA having a modified 3' untranslated region.

[0008] In some embodiments of the second aspect, the modified 3' untranslated region comprises one or more deletions. In some embodiments, the modified 3' untranslated region increases mRNA stability compared to unmodified HLA-G mRNA. In some embodiments, the HLA-G is HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7. In some embodiments, the HLA-G is HLA-G1. In some embodiments, the HLA-G is HLA-G2.

[0009] In some embodiments of the first or second aspect, at least one cell of the genetically modified non-human animal expresses an HLA-G protein. In some embodiments, the HLA-G protein is HLA-G1.

[0010] Some embodiments of the first or second aspect further comprise a second exogenous nucleic acid encoding a beta-2-microglobulin (B2M) protein. In some embodiments, the B2M protein is a human B2M protein.

[0011] In a third aspect, disclosed herein is a genetically modified non-human animal comprising an exogenous nucleic acid sequence that is at least 75% identical to SEQ ID NO:240.

[0012] In some embodiments of the third aspect, the exogenous nucleic acid sequence is at least 80% identical to SEQ ID NO: 240. In some embodiments, the exogenous nucleic acid sequence is at least 85% identical to SEQ ID NO: 240. In some embodiments, the exogenous nucleic acid sequence is at least 90% identical to SEQ ID NO: 240. In some embodiments, the exogenous nucleic acid sequence is at least 95% identical to SEQ ID NO: 240. In some embodiments, the exogenous nucleic acid sequence is identical to SEQ ID NO: 240.

[0013] In some embodiments of the third aspect, at least one cell of the genetically modified non-human animal expresses a human CD47 protein.

[0014] Some embodiments of the third aspect further include a second exogenous nucleic acid sequence transcribed as a human leukocyte antigen G (HLA-G) mRNA having a modified 3' untranslated region. In some embodiments, the modified 3' untranslated region comprises one or more deletions. In some embodiments, the modified 3' untranslated region increases mRNA stability compared to unmodified HLA-G mRNA. In some embodiments, the HLA-G is HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7. In some embodiments, the HLA-G is HLA-G1. In some embodiments, the HLA-G is HLA-G2.

[0015] In some embodiments of the third aspect, the second exogenous nucleic acid sequence is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:359 or SEQ ID NO:502.

[0016] In some embodiments of the first, second, or third aspect, the exogenous nucleic acid sequence is operably linked to a constitutively active endogenous promoter.

[0017] In some embodiments of the first, second, or third aspect, the exogenous nucleic acid sequence is inserted into the genome of the genetically modified non-human animal at the ROSA26 gene site.

[0018] In some embodiments of the first, second, or third aspect, the exogenous nucleic acid sequence is inserted into the genome of the genetically modified non-human animal at a site effective to reduce expression of glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH), β1,4 N-acetylgalactosaminyltransferase (B4GALNT2), C-X-C motif chemokine 10 (CXCL10), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB), transporter associated with antigen processing 1 (TAP1), NOD-like receptor family CARD domain containing 5 (NLRC5), or a combination thereof, compared to an animal of the same species without the exogenous nucleic acid sequence or an animal of the same species with the exogenous nucleic acid inserted at a different site.

[0019] In some embodiments of the first, second, or third aspect, the exogenous nucleic acid sequence is inserted into the genome of the genetically modified non-human animal at a site effective to reduce expression of glycoprotein galactosyltransferase alpha 1,3 (GGTA1).

[0020] In some embodiments of the first, second, or third aspect, the genetically modified non-human animal further comprises a genomic disruption in one or more genes selected from the list consisting of glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH), β1,4 N-acetylgalactosaminyltransferase (B4GALNT2), C-X-C motif chemokine 10 (CXCL10), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB), transporter associated with antigen processing 1 (TAP1), NOD-like receptor family CARD domain containing 5 (NLRC5), and any combination thereof.

[0021] In some embodiments of the first, second, or third aspect, the genetically modified non-human animal further comprises a genomic disruption in one or more genes selected from the list consisting of a component of the MHC I-specific enhanceosome, a transporter of MHC I-binding peptides, a natural killer (NK) group 2D ligand, a CXC chemokine receptor (CXCR)3 ligand, an MHC II transactivator (CIITA), C3, an endogenous gene not expressed in humans, and any combination thereof. Some embodiments comprise a genomic disruption of a component of the MHC I-specific enhanceosome, wherein the component of the MHC I-specific enhanceosome is NOD-like receptor family CARD domain-containing 5 (NLRC5). Some embodiments comprise a genomic disruption of a transporter of MHC I-binding peptides, wherein the transporter is transporter associated with antigen processing 1 (TAP1). Some embodiments comprise a genomic disruption of C3. Some embodiments include a genomic disruption of an NK group 2D ligand, wherein the NK group 2D ligand is MHC class I polypeptide-related sequence A (MICA) or MHC class I polypeptide-related sequence B (MICB). Some embodiments include a genomic disruption of an endogenous gene not expressed in humans, wherein the endogenous gene not expressed in humans is glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH), or β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). Some embodiments include a genomic disruption of a CXCR3 ligand, wherein the CXCR3 ligand is C-X-C motif chemokine 10 (CXCL10).

[0022] In some embodiments, the genome disruption reduces expression of the disrupted gene compared to an animal of the same species that does not have the genome disruption.

[0023] In some embodiments, the genome disruption reduces expression of a protein from the disrupted gene compared to an animal of the same species that does not have the genome disruption.

[0024] Some embodiments of the first, second, or third aspect further comprise an additional exogenous nucleic acid sequence encoding infected cell protein 47 (ICP47).

[0025] In some embodiments of the first, second, or third aspect, the genetically modified non-human animal is a member of the superorder Laurasiatheria.

[0026] In some embodiments of the first, second, or third aspect, the genetically modified non-human animal is an ungulate.

[0027] In some embodiments of the first, second, or third aspect, the genetically modified non-human animal is a pig.

[0028] In some embodiments of the first, second, or third aspect, the genetically modified non-human animal is a non-human primate animal.

[0029] In some embodiments of the first, second, or third aspect, the genetically modified non-human animal is a fetus.

[0030] Also disclosed herein are cells isolated from the genetically modified non-human animals of any embodiment of the first, second, or third aspects. In some embodiments, the cells are islet cells. In some embodiments, the cells are stem cells.

[0031] Also disclosed herein is a tissue isolated from any embodiment of the genetically modified non-human animal of the first, second, or third aspect. In some embodiments, the tissue is a solid organ transplant. In some embodiments, the tissue is the whole or part of a liver. In some embodiments, the tissue is the whole or part of a kidney.

[0032] In a fourth aspect, disclosed herein is a non-human cell comprising an exogenous nucleic acid sequence that is at least 95% identical to SEQ ID NO:359 or SEQ ID NO:502.

[0033] In some embodiments of the fourth aspect, the exogenous nucleic acid is at least 96% identical to SEQ ID NO: 359 or SEQ ID NO: 502. In some embodiments, the exogenous nucleic acid is at least 97% identical to SEQ ID NO: 359 or SEQ ID NO: 502. In some embodiments, the exogenous nucleic acid is at least 98% identical to SEQ ID NO: 359 or SEQ ID NO: 502. In some embodiments, the exogenous nucleic acid is at least 90% identical to SEQ ID NO: 359 or SEQ ID NO: 502. In some embodiments, the exogenous nucleic acid is 100% identical to SEQ ID NO: 359 or SEQ ID NO: 502.

[0034] In some embodiments of the fourth aspect, the non-human cell expresses human leukocyte antigen G1 (HLA-G1) on the cell surface.

[0035] In a fifth aspect, disclosed herein is a non-human cell comprising an exogenous nucleic acid that is transcribed as a human leukocyte antigen G (HLA-G) mRNA having a modified 3' untranslated region.

[0036] In some embodiments of the fifth aspect, the modified 3' untranslated region comprises one or more deletions. In some embodiments, the modified 3' untranslated region increases the stability of the mRNA compared to an unmodified HLA-G mRNA.

[0037] In some embodiments of the fifth aspect, the HLA-G is HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7. In some embodiments, the HLA-G is HLA-G1. In some embodiments, the HLA-G is HLA-G2.

[0038] In some embodiments of the fourth or fifth aspect, the non-human cell further comprises a second exogenous nucleic acid encoding a beta-2-microglobulin (B2M) protein. In some embodiments, the B2M protein is a human B2M protein.

[0039] In a sixth aspect, disclosed herein is a non-human cell comprising an exogenous nucleic acid that is at least 75% identical to SEQ ID NO:240.

[0040] In some embodiments of the sixth aspect, the exogenous nucleic acid sequence is at least 80% identical to SEQ ID NO:240.

[0041] In some embodiments, the exogenous nucleic acid sequence is at least 85% identical to SEQ ID NO: 240. In some embodiments, the exogenous nucleic acid sequence is at least 90% identical to SEQ ID NO: 240. In some embodiments, the exogenous nucleic acid sequence is at least 95% identical to SEQ ID NO: 240. In some embodiments, the exogenous nucleic acid sequence is 100% identical to SEQ ID NO: 240.

[0042] In some embodiments of the sixth aspect, at least one non-human cell expresses a human CD47 protein.

[0043] In some embodiments of the sixth aspect, the non-human cell further comprises a second exogenous nucleic acid sequence transcribed as a human leukocyte antigen G (HLA-G) mRNA having a modified 3' untranslated region. In some embodiments, the modified 3' untranslated region comprises one or more deletions. In some embodiments, the modified 3' untranslated region increases mRNA stability compared to unmodified HLA-G mRNA. In some embodiments, the HLA-G is HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7. In some embodiments, the HLA-G is HLA-G1. In some embodiments, the HLA-G is HLA-G2. In some embodiments, the second exogenous nucleic acid sequence is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 359 or SEQ ID NO: 502.

[0044] In some embodiments of the fourth, fifth, or sixth aspect, the exogenous nucleic acid sequence is operably linked to a constitutively active endogenous promoter.

[0045] In some embodiments of the fourth, fifth, or sixth aspect, the exogenous nucleic acid sequence is inserted into the genome of the non-human cell at the ROSA26 gene site.

[0046] In some embodiments of the fourth, fifth, or sixth aspect, the exogenous nucleic acid sequence is inserted into the genome of the non-human cell at a site effective to reduce expression of glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH), β1,4 N-acetylgalactosaminyltransferase (B4GALNT2), C-X-C motif chemokine 10 (CXCL10), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB), transporter associated with antigen processing 1 (TAP1), NOD-like receptor family CARD domain containing 5 (NLRC5), or a combination thereof, compared to a cell of the same species lacking the exogenous nucleic acid sequence or a cell of the same species in which the exogenous nucleic acid has been inserted at a different site.

[0047] In some embodiments of the fourth, fifth, or sixth aspect, the exogenous nucleic acid sequence is inserted into the genome of the non-human cell at a site that reduces expression of glycoprotein galactosyltransferase alpha 1,3 (GGTA1).

[0048] In some embodiments of the fourth, fifth, or sixth aspect, the non-human cell further comprises a genomic disruption in one or more genes selected from the list consisting of glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH), β1,4 N-acetylgalactosaminyltransferase (B4GALNT2), C-X-C motif chemokine 10 (CXCL10), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB), transporter associated with antigen processing 1 (TAP1), NOD-like receptor family CARD domain-containing 5 (NLRC5), and any combination thereof.

[0049] In some embodiments of the fourth, fifth, or sixth aspect, the non-human cell further comprises a genomic disruption in one or more genes selected from the list consisting of a component of an MHC I-specific enhanceosome, a transporter of MHC I-bound peptides, a natural killer (NK) group 2D ligand, a C-X-C chemokine receptor (CXCR)3 ligand, an MHC II transactivator (CIITA), C3, an endogenous gene not expressed in humans, and any combination thereof. In some embodiments, the non-human cell comprises a genomic disruption in a component of an MHC I-specific enhanceosome, wherein the component of the MHC I-specific enhanceosome is NOD-like receptor family CARD domain-containing 5 (NLRC5). In some embodiments, the non-human cell comprises a genomic disruption in a transporter of MHC I-bound peptides, wherein the transporter is transporter associated with antigen processing 1 (TAP1).

[0050] In some embodiments, the non-human cell comprises a genomic disruption of C3.

[0051] In some embodiments, the non-human cell comprises a genomic disruption of an NK group 2D ligand, wherein the NK group 2D ligand is MHC class I polypeptide-related sequence A (MICA) or MHC class I polypeptide-related sequence B (MICB).

[0052] In some embodiments, the non-human cell comprises a genomic disruption of an endogenous gene that is not expressed in humans, wherein the endogenous gene that is not expressed in humans is glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH), or β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). In some embodiments, the non-human cell comprises a genomic disruption of a CXCR3 ligand, wherein the CXCR3 ligand is C-X-C motif chemokine 10 (CXCL10). In some embodiments, the genomic disruption reduces expression of the disrupted gene compared to cells of the same species that do not have the genomic disruption.

[0053] In some embodiments, the genome disruption reduces protein expression from the disrupted gene compared to a cell of the same species that does not have the genome disruption.

[0054] Some embodiments of the fourth, fifth, or sixth aspect further comprise an additional exogenous nucleic acid sequence encoding infected cell protein 47 (ICP47).

[0055] In some embodiments of the fourth, fifth, or sixth aspect, the non-human cell is a cell of the superorder Laurasiatheria.

[0056] In some embodiments of the fourth, fifth, or sixth aspect, the non-human cell is an ungulate cell.

[0057] In some embodiments of the fourth, fifth, or sixth aspect, the non-human cell is a porcine cell.

[0058] In some embodiments of the fourth, fifth, or sixth aspect, the non-human cell is a cell of a non-human primate.

[0059] In some embodiments of the fourth, fifth, or sixth aspect, the non-human cell is a fetal cell.

[0060] In some embodiments of the fourth, fifth, or sixth aspect, the non-human cell is a stem cell.

[0061] In some embodiments of the fourth, fifth, or sixth aspect, the non-human cells are islet cells.

[0062] Also disclosed herein is a solid organ graft comprising the non-human cells of any embodiment of the fourth, fifth, or sixth aspects.

[0063] Also disclosed herein is an embryo comprising a non-human cell of any embodiment of the fourth, fifth, or sixth aspect.

[0064] In a seventh aspect, disclosed herein is a method comprising providing at least one non-human cell of any embodiment of the fourth, fifth, or sixth aspects to a subject. In some embodiments, the at least one non-human cell is a solid organ transplant. In some embodiments, the at least one non-human cell is a stem cell transplant. In some embodiments, the at least one non-human cell is an islet cell transplant.

[0065] Some embodiments of the seventh aspect include providing a tolerization vaccine to a subject. In some embodiments, the tolerization vaccine is provided before, simultaneously with, or after providing at least one non-human cell to the subject. In some embodiments, the tolerization vaccine comprises apoptotic cells. In some embodiments, the tolerization vaccine comprises cells derived from the same species as the at least one non-human cell provided to the subject. In some embodiments, the tolerization vaccine comprises cells that are genetically identical to the at least one non-human cell provided to the subject.

[0066] Some embodiments of the seventh aspect include providing an anti-CD40 antibody to a subject. In some embodiments, the anti-CD40 antibody is provided before, simultaneously with, or after providing at least one non-human cell to the subject. In some embodiments, the anti-CD40 antibody specifically binds to an epitope within the amino acid sequence of SEQ ID NO: 487. In some embodiments, the anti-CD40 antibody specifically binds to an epitope within the amino acid sequence of SEQ ID NO: 488.

[0067] In an eighth aspect, disclosed herein is a xenotransplantation system comprising: a) at least one cell isolated from the genetically modified non-human animal of any embodiment of the first, second, or third aspect; and b) a tolerizing vaccine, an anti-CD40 antibody, or a combination thereof. In some embodiments, the at least one cell comprises an islet cell, a stem cell, or a combination thereof. In some embodiments, the at least one cell is a solid organ graft. In some embodiments, the at least one cell is a whole or partial liver. In some embodiments, the at least one cell is a whole or partial kidney.

[0068] Some embodiments of the eighth aspect include a tolerization vaccine. In some embodiments, the tolerization vaccine comprises apoptotic cells. In some embodiments, the tolerization vaccine comprises cells derived from the same species as the at least one cell. In some embodiments, the tolerization vaccine comprises cells that are genetically identical to the at least one cell.

[0069] Some embodiments of the eighth aspect comprise or further comprise an anti-CD40 antibody. In some embodiments, the anti-CD40 antibody specifically binds to an epitope within the amino acid sequence of SEQ ID NO: 487. In some embodiments, the anti-CD40 antibody specifically binds to an epitope within the amino acid sequence of SEQ ID NO: 488.

[0070] In a ninth aspect, disclosed herein is a xenotransplantation system comprising: a) at least one non-human cell according to any one of claims 58 to 108; and b) a tolerizing vaccine, an anti-CD40 antibody, or a combination thereof. In some embodiments, the at least one cell comprises an islet cell, a stem cell, or a combination thereof. In some embodiments, the at least one cell is a solid organ graft. In some embodiments, the at least one cell is a whole or partial liver. In some embodiments, the at least one cell is a whole or partial kidney.

[0071] Some embodiments of the ninth aspect include a tolerization vaccine. In some embodiments, the tolerization vaccine comprises apoptotic cells. In some embodiments, the tolerization vaccine comprises cells derived from the same species as at least one cell. In some embodiments, the tolerization vaccine comprises cells that are genetically identical to at least one cell.

[0072] Some embodiments of the ninth aspect comprise or further comprise an anti-CD40 antibody. In some embodiments, the anti-CD40 antibody specifically binds to an epitope within the amino acid sequence of SEQ ID NO: 487. In some embodiments, the anti-CD40 antibody specifically binds to an epitope within the amino acid sequence of SEQ ID NO: 488.

[0073]

[0074] Provided herein are methods comprising providing at least one engineered cell to an individual, the engineered cell comprising at least two genomic modifications that result in an inhibition of the individual's immune response to the at least one engineered cell, the inhibition measured by reduced effector function of at least one endogenous cell selected from the group consisting of T cells, B cells, monocytes, macrophages, natural killer (NK) cells, dendritic cells, and combinations thereof, and / or increased immune cell regulation of at least one endogenous cell selected from the group including, but not limited to, CD4+ regulatory T cells, CD8+ regulatory T cells, CD8+ natural suppressor cells, Tr1 cells, regulatory B cells, B10 cells, myeloid-derived suppressor cells, and any combination thereof, compared to the individual's immune response contacted with corresponding unengineered cells. In some cases, the at least one engineered cell can be a solid organ transplant. In other cases, the at least one engineered cell can be a stem cell transplant. In some cases, the at least one engineered cell can be an islet cell transplant. The individual may be tolerized to the at least one engineered cell. Optionally, tolerization may occur before, during, or after the at least one engineered cell is provided to the individual.

[0075] In some cases, tolerization can be promoted by administering a vaccine. In some cases, tolerization can be administration of at least one engineered cell. In some cases, tolerization can be administration of a vaccine and administration of at least one engineered cell. The vaccine can include apoptotic cells. The vaccine can also include live cells. In some cases, the reduced effector function can be selected from the group consisting of reduced proliferation, reduced cytokine expression, reduced expression of cytolytic effector molecules, reduced persistence, loss, induction of anergy, increased immune cell regulation, and any combination thereof in response to exposure to the at least one engineered cell.

[0076] It may also be disclosed herein that at least one additional treatment step is administered to the individual. In some cases, the at least one additional treatment step may be an immunosuppressive therapy. The immunosuppressive therapy may include an anti-CD40 antibody, an anti-CD20 antibody, an anti-IL6 receptor antibody, a C 51 H 79 NO 13 (rapamycin), soluble tumor necrosis factor receptor (sTNFR), C 66 H 99 N 23 O 17S2 (compstatin), and any combination thereof. The individual may not be sensitized to the major histocompatibility complex (MHC). The anti-CD40 antibody may be an antagonistic antibody. The anti-CD40 antibody may be an anti-CD40 antibody that specifically binds to an epitope within the amino acid sequence: EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCV (SEQ ID NO: 487). The anti-CD40 antibody may be an anti-CD40 antibody that specifically binds to an epitope within the amino acid sequence: EKQYLINSQCCSLCQPGQKLVSDCTEFTETECL (SEQ ID NO: 488). The anti-CD40 antibody may be the Fab' anti-CD40L monoclonal antibody fragment CDP7657. The anti-CD40 antibody can be an FcR-engineered, Fc-silent anti-CD40L monoclonal domain antibody. Optionally, the individual can be sensitized to a major histocompatibility complex (MHC). The MHC can be a human leukocyte antigen (HLA). The individual can be sensitized to a major histocompatibility complex (MHC), as determined by a positive response to a panel reactive antibody (PRA) screening assay.

[0077] In some cases, the individual may have a calculated panel reactive antibody (cPRA) score of 0.1 to 100%. In some cases, the reduced effector function may be reduced effector function of at least two endogenous cell types selected from the group consisting of T cells, B cells, monocytes, macrophages, natural killer (NK) cells, dendritic cells, and any combination thereof. The genomic modification may be gene disruption, deletion, induction of anergy, increased immune cell regulation, or a combination thereof. The gene may be selected from the group consisting of C-X-C motif chemokine 10 (CXCL10), antigen processing-associated transporter 1 (TAP1), NOD-like receptor family CARD domain-containing 5 (NLRC5), and any combination thereof. In some cases, at least one engineered cell is a xenograft.

[0078] Disclosed herein is an engineered polynucleic acid comprising at least two sequences encoding a targeting oligonucleotide, wherein the targeting oligonucleotide comprises a complementary sequence to at least one non-human genomic sequence flanked by a protospacer adjacent motif (PAM) sequence. Optionally, the targeting oligonucleotide may be a guide RNA (gRNA). The gRNA may comprise a complementary sequence to a gene selected from the group consisting of GGTA1, Gal2-2, NLRC5, and any combination thereof. Optionally, the gRNA may comprise a complementary sequence to GGTA1 and / or Gal2. The gRNA may comprise a complementary sequence to NLRC5 and Gal2. Optionally, the targeting oligonucleotide may bind to the first exon of the gene. The non-human genome may be derived from a Laurasiatheria superorder or a non-human primate. The Laurasiatheria superorder may be an ungulate. Optionally, the ungulate may be a pig. The PAM sequence may be 5'-NGG-3' (SEQ ID NO: 265).

[0079] In some cases, the guide RNA may include at least one modification, such as a 5' adenylate, a 5' guanosine triphosphate cap, a 5' N 7-Methylguanosine triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate, Cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3'-3' modification, 5'-5' modification, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, double biotin, PC-biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, Black Hole Quencher 1, Black Hole Quencher 2, DABCYL SE, dT-DABCYL, IRDye The nucleic acid may be selected from the group consisting of QC-1, QSY-21, QSY-35, QSY-7, QSY-9, a carboxyl linker, a thiol linker, a 2' deoxyribonucleoside analog purine, a 2' deoxyribonucleoside analog pyrimidine, a ribonucleoside analog, a 2'-O-methylribonucleoside analog, a sugar-modified analog, a wobble / universal base, a fluorescent dye label, a 2' fluoroRNA, a 2' O-methylRNA, a methylphosphonate, a phosphodiester DNA, a phosphodiester RNA, a phosphothioate DNA, a phosphorothioate RNA, a UNA, a pseudouridine-5'-triphosphate, a 5-methylcytidine-5'-triphosphate, and any combination thereof.

[0080] Disclosed herein are xenograft grafts comprising at least one genomic disruption of SEQ ID NO: 261.

[0081] Disclosed herein are xenograft grafts comprising at least one genomic disruption of SEQ ID NO: 262.

[0082] In some cases, the xenotransplant graft may further comprise at least one transgene. The transgene may be endogenous. The transgene may be engineered. The transgene may encode a human leukocyte antigen (HLA). The HLA may be HLA-G. The transgene may be CD47.

[0083] Provided herein are genetically modified animals having genomic disruptions in two or more genes selected from the group consisting of components of the MHC I-specific enhanceosome, transporters of MHC I-binding peptides, natural killer (NK) group 2D ligands, CXC chemokine receptor (CXCR)3 ligands, MHC II transactivator (CIITA), C3, endogenous genes not expressed in humans, and any combination thereof, wherein the genetically modified animals have reduced expression of the genes compared to corresponding non-genetically modified animals. Optionally, the genetically modified animals may be members of the superorder Laurasiatheria, and the members of the superorder Laurasiatheria are ungulates. The ungulates may be pigs.

[0084] Optionally, the protein expression of the two or more genes may be absent in the genetically modified animal. Optionally, the reduction in protein expression inactivates the function of the two or more genes. Optionally, the genetically modified animal may have reduced protein expression of three or more genes. The genetically modified animal may have reduced protein expression of a component of the MHC I-specific enhanceosome, which may be NOD-like receptor family CARD domain-containing 5 (NLRC5). The genetically modified animal may include reduced protein expression of a transporter of MHC I-bound peptides, which may be transporter associated with antigen processing 1 (TAP1).

[0085] In some cases, the genetically modified animal may comprise reduced protein expression of C3. In some cases, the reduced protein expression may inactivate the function of two or more genes. In some cases, the NK group 2D ligand with reduced protein expression may be MHC class I polypeptide-related sequence A (MICA) or MHC class I polypeptide-related sequence B (MICB). In some cases, the endogenous gene with reduced protein expression may not be expressed in humans, and the endogenous gene that may not be expressed in humans may be glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH), or β1,4 N-acetylgalactosaminyltransferase (B4GALNT2).

[0086] In some genetically modified animals described herein, at least two genomic disruptions result in reduced protein expression of a CXCR3 ligand, which may be C-X-C motif chemokine 10 (CXCL10).

[0087] Provided herein is at least one genetically modified animal further comprising one or more exogenous transgenes encoding at least one protein or functional fragment thereof, wherein said at least one protein is selected from a suppressor of MHC I formation, a regulator of complement activation, an inhibitory ligand of NK cells, a member of the B7 family, CD47, a serine protease inhibitor, a galectin, and any combination thereof.

[0088] In some cases, the at least one protein may be at least one human protein. The one or more exogenous transgenes encoding an MHC I formation suppressor may be infected cell protein 47 (ICP47). In some cases, the one or more exogenous transgenes encoding a regulator of complement activation may be cluster of differentiation 46 (CD46), cluster of differentiation 55 (CD55), or cluster of differentiation 59 (CD59). In some cases, the one or more exogenous transgenes encoding an inhibitory ligand for NK cells may be leukocyte antigen E (HLA-E), human leukocyte antigen G (HLA-G), or beta-2-microglobulin (B2M). In other cases, the one or more exogenous transgenes encode HLA-G, which may be HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7. Optionally, the HLA-G can be HLA-G1.

[0089] In some genetically modified animals provided herein, one or more exogenous transgenes encoding a B7 family member are provided, and the B7 family member may be a programmed death-ligand. The programmed death-ligand may be programmed death-ligand 1 (PD-L1) or programmed death-ligand 2 (PD-L2). Optionally, the one or more exogenous transgenes may encode both PD-L1 and PD-L2. Optionally, the one or more exogenous transgenes may encode a serine protease inhibitor, and the serine protease inhibitor may be serine protease inhibitor 9 (Spi9). Optionally, the one or more exogenous transgenes may encode a galectin, and the galectin may be galectin 9. Optionally, the one or more exogenous transgenes may be inserted adjacent to a ubiquitous promoter, and the ubiquitous promoter may be the Rosa26 promoter.

[0090] In some cases, one or more exogenous transgenes can be inserted adjacent to the promoter of the targeted gene, within the targeted gene, or adjacent to a protospacer adjacent motif (PAM) sequence. In some cases, protein expression of two or more genes can be reduced using the CRISPR / Cas system.

[0091] Provided herein are genetically modified animals having a genomic disruption in at least one gene selected from the group consisting of a component of the MHC I-specific enhanceosome, a transporter of MHC I-binding peptides, a natural killer (NK) group 2D ligand, a CXC chemokine receptor (CXCR)3 ligand, an MHC II transactivator (CIITA), C3, an endogenous gene not expressed in humans, and any combination thereof, wherein the genetically modified animals have reduced expression of the gene compared to a corresponding animal that is not genetically modified, and the genetically modified animals survive for at least 22 days after birth. In some cases, the genetically modified animals may survive for at least 23, 30, 35, 50, 70, 100, 150, 200, 250, 300, 350, or 400 days after birth.

[0092] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0093] [Figure 1]Figure 1 is a diagram supporting an immunotherapy strategy centered around the use of genetically modified cell and organ transplants lacking functional expression of MHC class I. The need for maintenance immunosuppression required to prevent transplant rejection is diminished (or the applicability of cell and organ xenograft transplants and stem cell-derived cell allografts and xenograft transplants is increased) when genetically modified cell and organ transplants are combined with the transient use of antagonistic anti-CD40 antibodies, and even more so when combined with the administration of a tolerizing vaccine comprising apoptotic donor cells under the auspices of anti-CD40 antibodies.

[0094] [Figure 2] Figure 2 is a diagram supporting one strategy for generating genetically modified pig islet cells and tolerization vaccines. Two clonal populations of pigs are created. One population with at least GGTA1 knocked out can be used to create a tolerization vaccine. The other clonal population of pigs with at least GGTA1 and MHC I genes (e.g., NRLC5) knocked out can be used for cell, tissue, and / or organ donors.

[0095] [Figure 3] FIG. 3 is a diagram supporting the use of positive and tolerizing vaccines (also called negative vaccines).

[0096] [Figure 4] FIG. 4 is a diagram supporting an exemplary approach to prolonging xenograft survival in subjects using infusion of apoptotic donor splenocytes for tolerizing vaccination under the aid of transient immunosuppression.

[0097] [Figure 5]FIG. 5 shows an exemplary approach to prevent rejection of a xenograft in a recipient or prolong survival of the xenograft in the absence of chronic and systemic immunosuppression of the xenograft recipient. This exemplary approach includes and integrates three components: i) genetically engineered islets lacking and / or reducing the expression of αGal, MHC class I, complement C3, and CXCL10, and transgenic expression of HLA-G; ii) genetically engineered donor apoptotic and non-apoptotic mononuclear cells (e.g., splenocytes) lacking and / or reducing the expression of αGal, Neu5Gc, and Sda / CAD, and transgenic expression of HLA-G with or without human CD47, human PD-L1, and human PD-L2 (e.g., genetically engineered vaccines); and iii) administration of transient immunosuppression (including antagonistic anti-CD40 mAb, anti-CD20 mAb, rapamycin) and transient anti-inflammatory therapy (including compstatin (e.g., compstatin-inducible APL-2), anti-IL-6 receptor mAb, and soluble TNF receptor).

[0098] [Figure 6] Figure 6 is a diagram supporting an exemplary protocol for preventing transplant rejection during pig-to-cynomolgus islet xenotransplantation. IE: islet equivalent; sTNFR: soluble TNF receptor (e.g., etanercept); α-IL-6R: anti-interleukin 6 receptor; Tx'd: transplanted.

[0099] [Figure 7A]Figures 7A-7E are diagrams supporting the strategy for cloning the px330-Gal2-1 plasmid targeting GGTA1. Figure 7A shows the cloning strategy and oligonucleotides (SEQ ID NOs: 266-267, respectively, in order of appearance) for generating guide RNAs targeting GGTA1. Figure 7B shows the insertion site on the px330 plasmid (SEQ ID NO: 268). Figure 7C shows a flowchart supporting the cloning and validation strategy. Figure 7D shows the cloning site (SEQ ID NO: 270) and sequencing primers (SEQ ID NOs: 269 and 271, respectively, in order of appearance). Figure 7E shows the sequencing results (SEQ ID NOs: 272-274, respectively, in order of appearance). [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above.

[0100] [Figure 8A] Figures 8A-8E are diagrams supporting the strategy for cloning the px330-CM1F plasmid targeting CMAH. Figure 8A shows the cloning strategy and oligonucleotides (SEQ ID NOs: 275 and 276, respectively, in order of appearance) for generating guide RNAs targeting CMAH1. Figure 8B shows the insertion site on the px330 plasmid (SEQ ID NO: 277). Figure 8C shows a flowchart supporting the cloning and validation strategy. Figure 8D shows the cloning site (SEQ ID NO: 279) and sequencing primers (SEQ ID NOs: 278 and 280, respectively, in order of appearance). Figure 8E shows the sequencing results (SEQ ID NOs: 281-283, respectively, in order of appearance). [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above.

[0101] [Figure 9A]Figures 9A-9E are diagrams supporting the strategy for cloning the px330-NL1_1 plasmid targeting NLRC5. Figure 9A shows the cloning strategy and oligonucleotides (SEQ ID NOs: 284 and 285, respectively, in order of appearance) for generating guide RNAs targeting NLRC5. Figure 9B shows the insertion site on the px330 plasmid (SEQ ID NO: 286). Figure 9C shows a flowchart supporting the cloning and validation strategy. Figure 9D shows the cloning site (SEQ ID NO: 288) and sequencing primers (SEQ ID NOs: 287 and 289, respectively, in order of appearance). Figure 9E shows the sequencing results (SEQ ID NOs: 290-292, respectively, in order of appearance). [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 9E] Same as above.

[0102] [Figure 10A] Figures 10A-10E support the strategy for cloning the px330 / C3-5 plasmid targeting C3. Figure 10A shows the cloning strategy and oligonucleotides (SEQ ID NOs: 293 and 294, respectively, in order of appearance) for generating guide RNAs targeting C3. Figure 10B shows the insertion site on the px330 plasmid (SEQ ID NO: 295). Figure 10C shows a flowchart supporting the cloning and validation strategy. Figure 10D shows the cloning site (SEQ ID NO: 297) and sequencing primers (SEQ ID NOs: 296 and 298, respectively, in order of appearance). Figure 10E shows the sequencing results (SEQ ID NOs: 299-301, respectively, in order of appearance). [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 10E] Same as above.

[0103] [Figure 11A] Figures 11A-11E are diagrams supporting the strategy for cloning the px330 / B41_2 plasmid targeting B4GALNT2. Figure 11A shows the cloning strategy and oligonucleotides (SEQ ID NOs: 302 and 303, respectively, in order of appearance) for generating guide RNAs targeting B4GALNT2. Figure 11B shows the insertion site on the px330 plasmid (SEQ ID NO: 304). Figure 11C shows a flowchart supporting the cloning and validation strategy. Figure 11D shows the cloning site (SEQ ID NO: 306) and sequencing primers (SEQ ID NOs: 305 and 307, respectively, in order of appearance). Figure 11E shows the sequencing results (SEQ ID NOs: 308-310, respectively, in order of appearance). [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above.

[0104] [Figure 12] FIG. 12 confirms the map of the Rosa26 locus sequenced in Example 2.

[0105] [Figure 13A] Figures 13A-13E are diagrams supporting the strategy for cloning the px330 / Rosa exon 1 plasmid targeting Rosa26. Figure 13A shows the cloning strategy and oligonucleotides (SEQ ID NOs: 311-312, respectively, in order of appearance) for generating guide RNAs targeting Rosa26. Figure 13B shows the insertion site on the px330 plasmid (SEQ ID NO: 313). Figure 13C shows a flowchart supporting the cloning and validation strategy. Figure 13D shows the cloning site (SEQ ID NO: 315) and sequencing primers (SEQ ID NOs: 314 and 316, respectively, in order of appearance). Figure 13E shows the sequencing results (SEQ ID NOs: 317-319, respectively, in order of appearance). [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E] Same as above.

[0106] [Figure 14A] Figure 14A shows a map of the genomic sequence of HLA-G, and Figure 14B shows a map of the cDNA sequence of HLA-G. [Figure 14B] Same as above.

[0107] [Figure 15] FIG. 15 shows an exemplary photomicrograph of porcine fetal fibroblasts transfected with pSpCas9(BB)-2A-GFP.

[0108] [Figure 16] FIG. 16 shows fluorescence in situ hybridization (FISH) with a specific probe to the GGTA1 gene revealing its location on chromosome 1.

[0109] [Figure 17] Figures 17A-17B provide evidence of an example of phenotypic selection of cells using cas9 / sgRNA-mediated GGTA1 / NLCR5 disruption. Figure 17A shows genetically modified cells that do not express alpha-galactosidase. Figure 17B shows non-genetically modified cells that express alpha-galactosidase, labeled with ferrous beads conjugated with isolectin B4 (IB).

[0110] [Figure 18A]Figures 18A-18B show the sequencing of DNA isolated from fetal cells from two separate litters (Gestation 1: Figure 18A or Gestation 2: Figure 18B) that were subjected to PCR amplification of the GGTA1 (Sus scrofa mixed breed chromosome 1, Sscrofa10.2 compared to NCBI reference sequence: NC_010443.4) target region. The resulting amplicons were separated on a 1% agarose gel. Amplicons were also analyzed by Sanger sequencing using the forward primer from each reaction alone. Figure 18A shows the results for fetuses 1-7 (SEQ ID NOS: 322-328, respectively) from fetuses in Gestation 1, aligned to the reference and target gene sequences (SEQ ID NOS: 320-321, respectively). Fetuses 1, 2, 4, 5, 6, and 7 showed a 6-nucleotide truncation after the GGTA1 target site. Fetus 3 had a 17-nucleotide truncation after the cleavage site, followed by a 2,511 (668-3179) nucleotide deletion, followed by a single base substitution. Truncation, deletion, and substitution from a single sequencing experiment including alleles from both copies of the target gene can only suggest that a genetic modification has occurred, but cannot reveal the exact sequence for each allele. From this analysis, all seven fetuses appear to have a single allele modification for GGTA1. Figure 18B shows results for fetuses 1-5 (SEQ ID NOS: 331-335, respectively) from fetal DNA samples from pregnancy 2, aligned to the reference and target gene sequences (SEQ ID NOS: 329-330, respectively). Fetuses 1, 3, 4, and 5 had a 3-nucleotide truncation from the GGTA1 target site. Fetus 2 had variability in Sanger sequencing, suggesting complex variability in DNA mutations or poor sample quality. However, the quality of the fetal DNA template was sufficient to generate the GGTA1 genetic screening experiments described above. [Figure 18B] Same as above.

[0111] [Figure 19A]Figures 19A-19B show the sequencing of DNA isolated from fetal cells from two separate litters (pregnancy 1: Figure 19A or pregnancy 2: Figure 19B) subjected to PCR amplification of the NLRC5 (consensus sequence) target region. The resulting amplicons were separated on a 1% agarose gel. The amplicons were also analyzed by Sanger sequencing using the forward primer from each reaction alone. Figure 19A shows the results for fetuses 1, 3, 5, 6, and 7 from pregnancy 1 fetuses (SEQ ID NOS: 338-342, respectively), aligned to the reference and target gene sequences (SEQ ID NOS: 336-337, respectively). Sequence analysis of the NLRC5 target site failed to demonstrate consistent alignment. This suggests unknown complexities in the sequencing reaction or differential DNA alterations between NLRC5 alleles complicating the Sanger sequencing reaction and analysis. Figure 19B shows the results for fetuses 1 to 5 (SEQ ID NOS: 345 to 349, respectively) from fetuses at trimester 2, aligned to the reference and target gene sequences (SEQ ID NOS: 343 to 344, respectively). The NLRC5 gene amplicons from fetuses 1 to 5 were all cleaved 120 nucleotides downstream of the NLRC5 gene cleavage site. [Figure 19B] Same as above.

[0112] [Figure 20]Figures 20A-20B show data from fetal DNA (wt and 1-7 (Figure 20A: pregnancy 1) or 1-5 (Figure 20B: pregnancy 2)) isolated from hindlimb biopsies. The target gene was amplified by PCR, and the PCR products were separated on a 1% agarose gel and visualized with a fluorescent DNA stain. The amplicon band present in the wt lane represents the unaltered DNA sequence. An increase or decrease in amplicon size suggests an insertion or deletion within that amplicon, respectively. Bands may appear more diffuse due to variation in DNA alterations between alleles within a single sample. Pregnancy 1 (Figure 20A) yielded seven fetuses, while pregnancy 2 (Figure 20B) yielded five fetuses, which were harvested on days 45 and 43, respectively. The absence of a band (bottom gel) as in the NLRC5 gel in fetuses 1, 3, and 4 in Figure 20A suggests that alterations to the target region disrupted binding of the DNA amplification primers. The presence of all GGTA1 bands in Figure 20A (top gel) suggests that the DNA quality was sufficient to generate DNA amplicons during the NLRC5-targeting PCR reaction. Fetuses 1, 2, 4, and 5 of pregnancy 1 (Figure 20A) have larger GGTA1 amplicons than the WT control, suggesting an insertion within the target region. In fetus 3 of pregnancy 1 (Figure 20A), the GGTA1 amplicon migrated faster than the WT control, suggesting a deletion within the target region. In fetuses 6 and 7 of pregnancy 1 (Figure 20A), the NLRC5 amplicon migrated faster than the WT control, suggesting a deletion within the target region. In fetuses 1-5 (Figure 20B), the GGTA1 amplicons were difficult to interpret due to their size and were diffuse compared to the WT control. In fetuses 1-5 (Figure 20B), the NLRC5 amplicons were uniform in size and density compared to the wild-type control.

[0113] [Figure 21A]Figures 21A-21E show phenotypic analysis of fetuses from two separate litters of pigs (Figures 21A, 21B, and 21C: pregnancy 1, or Figures 21D-21E: pregnancy 2). Fetuses were harvested on day 45 (pregnancy 1) or day 43 (pregnancy 2) and processed for DNA and culture cell isolation. Tissue fragments and cells were seeded in culture medium for two days to allow fetal cells to adhere and proliferate. Wild-type cells (unmodified fetal cells) and fetal cells from pregnancies 1 and 2 were removed from the culture plates and labeled with IB4 lectin conjugated with Alexa fluor 488 or anti-pig MHC class I antibody conjugated with FITC. Flow cytometry analysis is shown as histograms depicting the labeling intensity of the tested cells. A histogram for WT cells is included in each panel to highlight the overall intensity reduction for each group of fetal cells. There is a decrease in alpha-Gal and MHC class I labeling in trimester 1 (Figure 21A), shown as a decrease in peak intensity. In trimester 2 (Figure 21B), there is a large decrease in alpha-gal labeling and a significant reduction in MHC class I labeling in fetuses 1 and 3 compared to WT fetal cells. [Figure 21B] Same as above. [Figure 21C] Same as above. [Figure 21D] Same as above. [Figure 21E] Same as above.

[0114] [Figure 22A]Figures 22A-22C show the effect of reduced MHC class I expression in cells from fetus 3 (trimester 1) compared to wild-type fetal cells from a genetic clone. The proliferative responses of human CD8+ cells and CD4+ T cells to porcine control fibroblasts and NLRC5 knockout fetal cells were measured. Figure 22A. Cells were gated as CD4+ or CD8+ before assessment of proliferation. Figure 22B. CD8+ T cell proliferation was reduced after stimulation with porcine fetal GGTA1 / NLRC5 knockout cells compared to unmodified control porcine fibroblasts. When human responders were treated with porcine fetal GGTA1 / NLRC5 knockout cells at a 1:1 ratio, an approximately 55% reduction in CD8+ T cell proliferation was observed. Wild-type fetal cells induced a 17.2% proliferation of human CD8+ T cells, whereas MHC class I-deficient cells from fetus 3 (trimester 1) induced only a 7.6% proliferation. Figure 22C. At ratios of 1:5 and 1:10, no differences in CD8 T cell proliferation responses were observed compared to unmodified fetal cells.At all ratios studied, no changes in CD4 T cell proliferation were observed in response to NLRC5 knockout porcine fetal cells and unmodified control porcine fetal cells. [Figure 22B] Same as above. [Figure 22C] Same as above.

[0115] [Figure 23] FIG. 23 shows live births of GGTA1 / NLRC5 knockout piglets generated using CRISPR / Cas technology.

[0116] [Figure 24A] 24A-24C show DNA gel analyses of the genotypes of the piglets produced in Example 6. FIG. 24A shows the results of the first PCR experiment in Example 6. FIG. 24B shows the results of the second PCR experiment in Example 6. FIG. 24C shows the results of the third PCR experiment in Example 6. [Figure 24B] Same as above. [Figure 24C] Same as above.

[0117] [Figure 25A] Figure 25A shows the sequencing data and sequence call for a portion of the NLRC5 gene from piglet #1 (SEQ ID NO: 350). Figure 25B shows the sequencing data and sequence call for a portion of the NLRC5 gene from piglet #2 (SEQ ID NO: 351). Figure 25C shows the sequencing data and sequence call for a portion of the NLRC5 gene from piglet #4 (SEQ ID NO: 352). Figure 25D shows the sequencing data and sequence call for a portion of the NLRC5 gene from piglet #5 (SEQ ID NO: 353). Figure 25E shows the sequencing data and sequence call for a portion of the NLRC5 gene from piglet #6 (SEQ ID NO: 354). Figure 25F shows the sequencing data and sequence call for a portion of the NLRC5 gene from piglet #7 (SEQ ID NO: 355). [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D] Same as above. [Figure 25E] Same as above. [Figure 25F] Same as above.

[0118] [Figure 26A] Figure 26A shows the left arm of Rosa26 (SEQ ID NO: 356) in Example 8. Figure 26B shows DNA gel analysis of the homologous recombination construct in Example 8. Figure 26C shows the consensus sequence of the amplicon (SEQ ID NO: 357) based on paired read analysis in Example 8. Figure 26D (SEQ ID NO: 358), Figure 26E (SEQ ID NO: 359), and Figure 26F (SEQ ID NO: 360) show the homology-guided recombination construct for HLA-G1 insertion at the Rosa26 locus in Example 8. [Figure 26B] Same as above. [Figure 26C] Same as above. [Figure 26D] Same as above. [Figure 26E] Same as above. [Figure 26F] Same as above.

[0119] [Figure 27A] Figure 27A shows the sequence of the correct px330 plasmid (SEQ ID NO: 362) containing the Rosa26 targeting oligo generated in Example 8, and the sequencing primers (SEQ ID NOs: 361 and 363, respectively, in order of appearance). Figure 27B shows the sequencing results of the constructed px330 plasmid containing the Rosa26 targeting oligo in Example 8. SEQ ID NOs: 364-366 are disclosed, respectively, in order of appearance. Figure 27C shows the restriction digest of the constructed px330 plasmid containing the Rosa26 targeting oligo in Example 8. [Figure 27B] Same as above. [Figure 27C] Same as above.

[0120] [Figure 28] FIG. 28 shows a map of the GalMet plasmid and oligos (SEQ ID NOs: 367 to 368, respectively, in order of appearance) used in Example 8.

[0121] [Figure 29] Figure 29 shows the in vitro Cas9-mediated cleavage reaction of in vitro transcribed gRNA. Lane 1: Uncleaved porcine Rosa26 (2000 bp). Lane 2: Cas9 cleavage of porcine Rosa26 guided by the designed gRNA; Lane 3: Uncleaved porcine GGTA1; Lane 4: Cas9 cleavage of the GGTA1 template guided by the designed gRNA.

[0122] [Figure 30] FIG. 30 shows flow cytometric sorting of genetically modified cells generated in Example 8.

[0123] [Figure 31]FIG. 31 shows the construct (SEQ ID NO: 369) for homologous recombination of CD47 into the GGTA1 locus, generated in Example 9.

[0124] [Figure 32] FIG. 32 shows the sequences of the right arm (FIG. 32A; SEQ ID NO: 370) and left arm (FIG. 32B; SEQ ID NO: 371) of the GGTA1 locus in Example 9.

[0125] [Figure 33A] 33A, 33B and 33C show the sorting of unstained cells in Example 9. [Figure 33B] Same as above. [Figure 33C] Same as above.

[0126] [Figure 34A] 34A, 34B and 34C show the sorting of px330 stained cells in Example 9. [Figure 34B] Same as above. [Figure 34C] Same as above.

[0127] [Figure 35A] 35A, 35B and 35C show the sorting of IB4 stained cells in Example 9. [Figure 35B] Same as above. [Figure 35C] Same as above.

[0128] [Figure 36A] 36A, 36B and 36C show the sorting of CD47 / IB4 stained cells in Example 9. [Figure 36B] Same as above. [Figure 36C] Same as above.

[0129] [Figure 37A] 37A, 37B, and 37C are diagrams showing sorted IB4-stained cells and CD47 / IB4-stained cells in Example 9. [Figure 37B]Same as above. [Figure 37C] Same as above.

[0130] [Figure 38A] 38A, 38B, and 38C are diagrams showing sorted CD47 / IB4 stained cells in Example 9. [Figure 38B] Same as above. [Figure 38C] Same as above.

[0131] [Figure 39] Figure 39 shows the gating strategy used to select single and live cells for analysis. Total CD3+ cells were observed, and CD4+ and CD8+ cells within that population were selected and counted for experimental parameters.

[0132] [Figure 40] Figure 40A shows that unstimulated cells in quadrant 2 showed only slight expansion when in identical culture conditions as the same cells stimulated with PHA. Figure 40B shows that PHA stimulation induced 20.7% (CD3), 24.7% (CD4), 18.4% (CD8), and 21% (CD20) proliferation in lymphocyte samples, suggesting the maximum stimulation possible in this assay.

[0133] [Figure 41] Figure 41 shows flow cytometry results from a coculture assay in which CD8+ T cells were added to cultures of adherent WT or genetically engineered porcine fibroblasts at dilutions of 100:1, 50:1, 10:1, or 1:1. WT cells stimulated T cells to proliferate at ratios of 50:1, 10:1, and 1:1. GM cells #3 and #4 were largely ineffective at stimulating T cells at ratios of 100:1, 50:1, and 10:1, suggesting a complete loss of T cell proliferative responses.

[0134] [Figure 42]Figure 42 shows flow cytometry results from a coculture assay in which CD4+ T cells were added to cultures of adherent WT or genetically engineered porcine fibroblasts at dilutions of 100:1, 50:1, 10:1, and 1:1. GM cells #3 and #4 were largely ineffective in stimulating T cells at the 100:1, 50:1, and 10:1 ratios, suggesting a complete loss of T cell proliferative responses.

[0135] [Figure 43] Figure 43 shows flow cytometry results of a coculture assay in which CD3+ T cells (total CD4 and CD8) were added to cultures of adherent WT or genetically engineered porcine fibroblasts at dilutions of 100:1, 50:1, 10:1, and 1:1. GM cells #3 and #4 were largely ineffective in stimulating T cells at the 100:1, 50:1, and 10:1 ratios, suggesting a complete loss of T cell proliferative responses.

[0136] [Figure 44] FIG. 44 shows approximately 50% inhibition of B cell proliferation when incubated with GGTA1 / NLRC5 knockout cells compared to wild-type cells.

[0137] [Figure 45] Figure 45 shows flow cytometry results of a coculture assay in which human lymphocytes were incubated with WT or GM cells, followed by cytokine measurement by introducing brefeldin A to block endocytosis, resulting in intracellular accumulation of the four cytokines in endosomes. Fixation and permeabilization of the cells allowed for intracellular measurement of cytokine accumulation. Within the CD8 T cell population, no IL2 stimulation was observed at a 100:1 ratio, and moderate reductions in CD107a, perforin, and granzyme B were observed at a 100:1 ratio. Perforin and granzyme B double-positive cells were significantly inhibited at both the 100:1 and 10:1 ratios.

[0138] [Figure 46] Figure 46 shows flow cytometry results of co-culture assays of human lymphocytes with WT or genetically modified porcine fibroblasts at a T cell to Fc ratio of 10:1. Within the CD8 T cell population, IL2 expression was reduced by approximately 40% upon stimulation at a 10:1 ratio and culture with genetically modified porcine cells. CD107a expression was reduced by approximately 25%. Perforin expression was reduced by approximately 40%, and granzymes were unaffected at this incubation ratio.

[0139] [Figure 47] Figure 47 shows flow cytometry results of co-culture assays of human lymphocytes with WT or genetically modified porcine fibroblasts at a T cell to Fc ratio of 10:1. In CD3 cells, CD107a was reduced by approximately 50%. Perforin and granzyme B were also reduced after incubation with genetically modified cells, as represented by double-positive cells receding from the second quadrant in comparison.

[0140] [Figure 48] Figure 48 shows flow cytometry results of co-culture assays of human lymphocytes with WT or genetically modified porcine fibroblasts at a T cell to FC ratio of 10:1. CD4+ T cells were less activated to produce cytokines in the presence of GM cells. IL2 expression was reduced by 40%. CD107a was reduced by approximately 50%. Perforin and granzyme B were reduced by approximately 50% and 30%, respectively.

[0141] [Figure 49]Figure 49 shows flow cytometry results of a co-culture assay of human lymphocytes with WT or genetically modified porcine fibroblasts at a T cell to Fc ratio of 10:1. Among CD3 cells, IFNγ expression was significantly reduced when lymphocytes were cultured with GM porcine fibroblasts at a 10:1 ratio. TNFα expression was low in cultures with WT cells but was reduced when cultured with GM cells. In this experiment, granzyme B was also dramatically reduced when incubated with GM cells compared to WT cells.

[0142] [Figure 50] Figure 50 shows flow cytometry results of a co-culture assay of human lymphocytes with WT or genetically modified porcine fibroblasts at a T cell to Fc ratio of 10:1. Among CD4 cells, IFNγ expression was significantly reduced when lymphocytes were cultured with GM porcine fibroblasts at a 10:1 ratio. TNFα expression was low in cultures with WT cells but was reduced when cultured with GM cells. In this experiment, granzyme B was also dramatically reduced when incubated with GM cells compared to WT cells.

[0143] [Figure 51] Figure 51 shows flow cytometry results of a co-culture assay of human lymphocytes with WT or genetically modified porcine fibroblasts at a T cell to Fc ratio of 10:1. Among CD8 cells, IFNγ expression was significantly reduced when lymphocytes were cultured with GM porcine fibroblasts at a 10:1 ratio. TNFα expression was low in cultures with WT cells but was reduced when cultured with GM cells. In this experiment, granzyme B was also dramatically reduced when incubated with GM cells compared to WT cells.

[0144] [Figure 52]Figure 52 shows flow cytometry results of co-culture assays of human lymphocytes with WT or genetically modified porcine fibroblasts at a T cell to Fc ratio of 10:1. NK cells (CD56+) are shown to be activated in the absence of MHC class I expression on the cells. IFNγ (y-axis) and granzyme B (x-axis) were expressed in co-cultures with WT cells but significantly reduced in co-cultures with GGTA1 / NLRC5 knockout cells. Regarding TNFα expression, no expression or no change was observed using GM cells compared to WT cells.

[0145] [Figure 53] Figure 53 shows that human PBMCs incubated with WT porcine fibroblasts had a normal background percentage (11%) of CD4+ T cells expressing IL10. GGTA1 / NLRC5 knockdown cells (13.3 and 20.2%), designated #3 and #4, respectively, had only a minor effect on IL10 expression. Pig fibroblasts expressing human inhaled HLAG1 protein optimized for expression in pigs induced 60.7% of human CD4+ T cells to produce IL10.

[0146] [Figure 54] Figure 54 shows that soluble HLA-G (100 ng / ml) blocks proliferation of CD8+, CD8- and PBMCs in culture with WT pig islets. Q1 and Q2 represent the proliferating (CFSE lo) and non-proliferating (CFSE hi) fractions, respectively.

[0147] [Figure 55] Figure 55 shows the flow cytometry gating strategy used to analyze CD3, CD4, or CD8 populations for cytokine and effector function molecule analysis of human T cells cultured with genetically modified porcine fibroblasts (HLAG1 expressing), WT, or WT plus PT85 antibody.

[0148] [Figure 56] Figure 56 shows cytometry data for CD4+ populations cocultured with wild-type porcine fibroblasts, WT porcine fibroblasts with PT85 antibody, or HLAG1-expressing porcine fibroblasts. Substantial reductions in cytokine levels (IL-2) and effector molecule secretion were observed with PT85-blocked or HLAG1-expressing cells at 10:1 and 1:1 ratios in MLR cultures. The PT85-blocking antibody was used to determine how much of the observed immunosuppressive effect was attributable to NLRC5 knockout (MHC class 1 null) or GGTA1 knockout. The PT85 antibody mimicked the effects of NLRC5 knockout in the presence of normal WT alphaGal surface expression. HLAG1 protein expression on the cell surface had a significant inhibitory effect on CD4+ and CD8+ T cell cytokine production and effector function.

[0149] [Figure 57] Figure 57 shows cytometry data for CD8+ populations cocultured with either wild-type porcine fibroblasts, WT porcine fibroblasts with PT85 antibody, or HLAG1-expressing porcine fibroblasts. Substantial decreases in cytokine levels (IL-2) and effector molecule secretion were observed with PT85-blocked or HLAG1-expressing cells at 10:1 and 1:1 ratios in MLR cultures. The PT85-blocking antibody was used to determine the extent to which the observed immunosuppressive effects were attributable to NLRC5 knockout (MHC class 1 null) or GGTA1 knockout. The PT85 antibody mimicked the effects of NLRC5 knockout in the presence of normal WT alphaGal surface expression within the CD8+ population. Cell surface expression of HLAG1 protein had a significant inhibitory effect on CD4+ and CD8+ T cell cytokine production and effector function. Cell surface expression of HLAG1 protein had a significant inhibitory effect on CD4+ and CD8+ T cell cytokine production and effector function.

[0150] [Figure 58]Figure 58 shows cytometry data for CD4+ populations cocultured with wild-type porcine fibroblasts, WT porcine fibroblasts with PT85 antibody, or HLAG1-expressing porcine fibroblasts. Substantial reductions in cytokine levels (TNF-α, IFN-γ) and effector molecule secretion were observed with PT85-blocked or HLAG1-expressing cells at 10:1 and 1:1 ratios in MLR cultures. The PT85-blocking antibody was used to determine how much of the observed immunosuppressive effect was attributable to NLRC5 knockout (MHC class 1 null) or GGTA1 knockout. The PT85 antibody mimicked the effects of NLRC5 knockout in the presence of normal WT alphaGal surface expression. HLAG1 protein expression on the cell surface had a significant inhibitory effect on CD4+ and CD8+ T cell cytokine production and effector function.

[0151] [Figure 59] Figure 59 shows cytometry data for CD8+ populations cocultured with wild-type porcine fibroblasts, WT porcine fibroblasts with PT85 antibody, or HLAG1-expressing porcine fibroblasts. Substantial reductions in cytokine levels (TNF-α, IFN-γ) and effector molecule secretion were observed with PT85-blocked or HLAG1-expressing cells at 10:1 and 1:1 ratios in MLR cultures. The PT85-blocking antibody was used to determine how much of the observed immunosuppressive effect was attributable to NLRC5 knockout (MHC class 1 null) or GGTA1 knockout. The PT85 antibody mimicked the effects of NLRC5 knockout in the presence of normal WT alphaGal surface expression. HLAG1 protein expression on the cell surface had a significant inhibitory effect on CD4+ and CD8+ T cell cytokine production and effector function.

[0152] [Figure 60] FIG. 60 shows the flow gating scheme for cell proliferation / CFSE hypoproliferation analysis.

[0153] [Figure 61]Figures 61A and B show flow cytometry analysis of cell proliferation (CFSE dilution) experiments of CD3, CD4 or CD8 populations in A. unstimulated cells or B. PHA stimulated cells (positive control or highest dilution).

[0154] [Figure 62] Figure 62 shows that T cell proliferation was reduced after stimulation with PT-85 blocking Ab-treated porcine fibroblasts at a human PBMC to FC ratio of 10:1 compared to unmodified control porcine fibroblasts / WT. A substantial reduction in T cell (CD3) proliferation was observed when human responders were treated with SLA-I-blocking PT-85 Ab or HLA-G expression at 10:1 and 1:1 ratios. Ratios of 100:1 and 50:1 showed little difference in T cell proliferation responses compared to unmodified / WT porcine fibroblasts.

[0155] [Figure 63] Figure 63 shows that T cell proliferation was reduced after stimulation with PT-85 blocking Ab-treated porcine fibroblasts at a human PBMC to FC ratio of 10:1 compared to unmodified control porcine fibroblasts / WT. A substantial reduction in T cell (CD4) proliferation was observed when human responders were treated with SLA-I-blocking PT-85 Ab or HLA-G expression at 10:1 and 1:1 ratios. Ratios of 100:1 and 50:1 showed little difference in T cell proliferation responses compared to unmodified / WT porcine fibroblasts.

[0156] [Figure 64]Figure 64 shows the reduction in T cell proliferation after stimulation with PT-85 blocking Ab-treated porcine fibroblasts at a 10:1 human PBMC to FC ratio compared to unmodified control porcine fibroblasts / WT. A substantial reduction in T cell (CD8) proliferation was observed when human responders were treated with SLA-I-blocking PT-85 Ab or HLA-G expression at 10:1 and 1:1 ratios. Ratios of 100:1 and 50:1 showed little difference in T cell proliferation responses compared to unmodified / WT porcine fibroblasts.

[0157] [Figure 65] Figure 65 shows the reduction in T cell proliferation after stimulation with porcine fibroblasts treated with PT-85 blocking Abs compared to unmodified control porcine fibroblasts / WT at a ratio of 10:1 human PBMC to FC, respectively. There was no substantial reduction in B cell proliferation with either SLA-I blockade with PT-85 or HLA-G expression.

[0158] [Figure 66] Figure 66 shows that IFNγ is primarily produced by natural killer (NK) cells and natural killer T (NKT) cells as part of the innate immune response. DKO#3 and #4 are independently generated genetic and phenotypic GGTA1 / NLRC5 knockout cells. IFNγ is also produced by CD4 Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells after antigen-specific immune development.

[0159] [Figure 67] Figure 67 shows the GM-CSF population in genetically modified cells cultured with human immune cells and controls. Double knockout (DKO) cells were unable to stimulate the GM-CSF population. HLAG1 expression was significantly reduced. DKO#3 and #4 are genetic and phenotypic GGTA1 / NLRC5 knockout cells generated separately.

[0160] [Figure 68] Figure 68 shows IL-17A expression among genetically modified cells cultured with human immune cells. Neither DKO nor HLAG1 transgenic cells were able to induce a pro-inflammatory response from human PMBCs.

[0161] [Figure 69] Figure 69 shows fractalkine expression in genetically modified cells cultured with human immune cells. HLAG1 expression remains a significant inhibitor of T cell activation and fractalkine production, even when expressed at a logarithmic scale.

[0162] [Figure 70] FIG. 70 shows TNF-alpha expression in genetically modified cells cultured with human immune cells.

[0163] [Figure 71] FIG. 71 shows IL-6 production in genetically modified cells cultured with human immune cells.

[0164] [Figure 72] FIG. 72 shows IL-4 production in genetically modified cells cultured with human immune cells.

[0165] [Figure 73] Figure 73 shows MIP 1 alpha production in genetically modified cells cultured with human immune cells.

[0166] [Figure 74] Figure 74 shows MIP 1beta production in genetically modified cells cultured with human immune cells.

[0167] [Figure 75]Figure 75 shows that T cell proliferation was reduced after stimulation with PT-85 blocking Ab-treated porcine fibroblasts at a human PBMC to FC ratio of 10:1 compared to unmodified control porcine fibroblasts / WT. A substantial reduction in T cell (CD3) proliferation was observed when human responders were treated with SLA-I-blocking PT-85 Ab or HLA-G expression at 10:1 and 1:1 ratios. Ratios of 100:1 and 50:1 showed little difference in T cell proliferation responses compared to unmodified / WT porcine fibroblasts.

[0168] [Figure 76] Figure 76 shows that T cell proliferation was reduced after stimulation with PT-85 blocking Ab-treated porcine fibroblasts at a 10:1 human PBMC to FC ratio compared to unmodified control porcine fibroblasts / WT. A substantial reduction in T cell (CD4) proliferation was observed when human responders were treated with SLA-I-blocking PT-85 Ab or HLA-G expression at 10:1 and 1:1 ratios. Ratios of 100:1 and 50:1 showed little difference in T cell proliferation responses compared to unmodified / WT porcine fibroblasts.

[0169] [Figure 77] Figure 77 shows the reduction in T cell proliferation after stimulation with PT-85 blocking Ab-treated porcine fibroblasts at a 10:1 human PBMC to FC ratio compared to unmodified control porcine fibroblasts / WT. A substantial reduction in T cell (CD8) proliferation was observed when human responders were treated with SLA-I-blocking PT-85 Ab or HLA-G expression at 10:1 and 1:1 ratios. Ratios of 100:1 and 50:1 showed little difference in T cell proliferation responses compared to unmodified / WT porcine fibroblasts.

[0170] [Figure 78]Figure 78 shows the reduction in T cell proliferation after stimulation with porcine fibroblasts treated with PT-85 blocking Abs compared to unmodified control porcine fibroblasts / WT at a ratio of 10:1 human PBMC to FC, respectively. There was no substantial reduction in B cell proliferation with either SLA-1 blockade with PT-85 or HLA-G expression.

[0171] [Figure 79] Figure 79 shows IFN-gamma expression after coculture of human mixed lymphocytes with genetically modified pig cells. Double knockout (DKO) #3 and #4 are independently generated genetic and phenotypic GGTA1 / NLRC5 knockout cells. HLA-G1 transgenic cells were treated in a separate experiment for human donor #2 (Figure 76B) (but not for donor #1; Figure 79A), so corresponding unstimulated and wild-type cell controls are included.

[0172] [Figure 80] Figure 80 shows GM-CSF gamma expression after co-culture of human mixed lymphocytes with genetically modified porcine cells. Double knockout (DKO) #3 and #4 are independently generated genetic and phenotypic GGTA1 / NLRC5 knockout cells. HLA-G1 transgenic cells were treated in a separate experiment for human donor #2 (Figure 80B) (but not for donor #1; Figure 80A), so corresponding unstimulated and wild-type cell controls are included.

[0173] [Figure 81] Figure 81 shows IL-2 expression after co-culture of human donor #1 mixed lymphocytes with genetically modified pig cells. Double knockout (DKO) #3 and #4 are independently generated genetic and phenotypic GGTA1 / NLRC5 knockout cells.

[0174] [Figure 82]Figure 82 shows IL-17alpha expression after co-culture of human mixed lymphocytes (Figures 82A and B) from two donors with genetically modified pig cells. Double knockout (DKO) #3 and #4 are genetically and phenotypically GGTA1 / NLRC5 knockout cells generated separately. Neither DKO nor HLA-G1 transgenic cells were capable of inducing a pro-inflammatory response from human PMBCs.

[0175] [Figure 83] Figure 83 shows fractalkine expression after coculture of human mixed lymphocytes with genetically modified pig cells. Double knockout (DKO) #3 and #4 are genetically and phenotypically generated GGTA1 / NLRC5 knockout cells. HLA-G1 transgenic cells were treated in a separate experiment for human donor #2 (Figure 83B) (but not for donor #1; Figure 83A), so corresponding unstimulated and wild-type cell controls are included. HLA-G1 expression remains a significant inhibitor of T cell activation and fractalkine production, even when expressed on a logarithmic scale.

[0176] [Figure 84] Figure 84 shows TNF-alpha expression after co-culture of human mixed lymphocytes with genetically modified porcine cells. Double knockout (DKO) #3 and #4 are independently generated genetic and phenotypic GGTA1 / NLRC5 knockout cells. HLA-G1 transgenic cells were treated in a separate experiment for human donor #2 (Figure 84B) (but not for donor #1; Figure 84A), so corresponding unstimulated and wild-type cell controls are included.

[0177] [Figure 85]Figure 85 shows IL-6 expression after coculture of human mixed lymphocytes with genetically modified pig cells. Double knockout (DKO) #3 and #4 are independently generated genetic and phenotypic GGTA1 / NLRC5 knockout cells. HLA-G1 transgenic cells were treated in a separate experiment for human donor #2 (Figure 85B) (but not for donor #1; Figure 85A), so corresponding unstimulated and wild-type cell controls are included.

[0178] [Figure 86] Figure 86 shows IL-4 expression after co-culture of human mixed lymphocytes with genetically modified pig cells. Double knockout (DKO) #3 and #4 are independently generated genetic and phenotypic GGTA1 / NLRC5 knockout cells. HLA-G1 transgenic cells were treated in a separate experiment for human donor #2 (Figure 86B) (but not for donor #1; Figure 86A), so corresponding unstimulated and wild-type cell controls are included.

[0179] [Figure 87] Figure 87 shows MIP-1 alpha expression after coculture of human mixed lymphocytes with genetically modified porcine cells. Double knockout (DKO) #3 and #4 are independently generated genetic and phenotypic GGTA1 / NLRC5 knockout cells. HLA-G1 transgenic cells were treated in a separate experiment for human donor #2 (Figure 87B) (but not for donor #1; Figure 87A), so corresponding unstimulated and wild-type cell controls are included.

[0180] [Figure 88]Figure 88 shows MIP-1 beta expression after coculture of human mixed lymphocytes with genetically modified porcine cells. Double knockout (DKO) #3 and #4 are independently generated genetic and phenotypic GGTA1 / NLRC5 knockout cells. HLA-G1 transgenic cells were treated in a separate experiment for human donor #2 (Figure 88B) (but not for donor #1; Figure 88A), so corresponding unstimulated and wild-type cell controls are included.

[0181] [Figure 89] Figure 89 is a diagram showing the CRISPR / Cas construct in the PX333 vector.

[0182] [Figure 90] Figure 90 shows a schematic diagram of transfection of primary porcine fibroblasts using the constructs: GGTA1-10 / B4GALNT2 (condition 2), NLRC5-6 / B4GALNT2 (condition 3), GGTA1-10 / B4GALNT2 and NLRC5-6 / B4GALNT2 (condition 4), condition 1 (WT): cells only.

[0183] [Figure 91] Figure 91 shows genetic modification selection using magnetic bead sorting.

[0184] [Figure 92] Figure 92 shows genetic modification selection using cell sorting for SLA I+ / IB4+ (top right); SLA I+ / IB4- (bottom right); SLA I- / IB4+ (top left); and SLA I- / IB4 (bottom left).

[0185] [Figure 93] FIG. 93 shows flow cytometry analysis of condition 2: GGTA1-10 / B4GALNT2.

[0186] [Figure 94]FIG. 94 shows flow cytometry analysis of condition 3: NLRC5-6 / B4GALNT2.

[0187] [Figure 95] Figure 95 shows flow cytometry analysis of condition 4: GGTA1-10 / B4GALNT2+NLRC5-6 / B4GALNT2.

[0188] [Figure 96] Figure 96 shows flow cytometry analysis of condition 2: GGTA1-10 / B4GALNT2 after sorting. Each population was sorted to verify that the correct population was obtained after sorting and that there were no crossover samples from other gates.

[0189] [Figure 97] Figure 97 shows flow cytometry analysis of condition 3: NLRC5-6 / B4GALNT2. Each population was sorted to verify that the correct population was obtained after sorting and that there were no crossover samples from other gates.

[0190] [Figure 98] Figure 98 shows flow cytometry analysis of condition 4: GGTA1-10 / B4GALNT2+NLRC5-6 / B4GALNT2. Each population was sorted to verify that the correct population was obtained after sorting and that there were no crossover samples from other gates.

[0191] [Figure 99] Figure 99A shows flow cytometry analysis of IB4 lectin in WT unstained, whole cell unstained, WT negative, and condition #2 Gal negative fractions cultured with WT or PFF1. Figure 99B shows flow cytometry analysis of IB4 lectin in side scatter vs. forward scatter of condition #4 Gal negative fraction, WT positive, condition #2 Gal positive fraction, condition #3 Gal positive fraction, or condition #4 Gal positive fraction cultured with WT or PFF1.

[0192] [Figure 100] FIG. 100 shows flow cytometry quantification for genetically modified cells in conditions 1 (WT), 2, 3 and 4 (left to right, respectively).

[0193] [Figure 101] Figure 101A shows flow cytometry analysis of SLAI in WT unstained, whole cell unstained, WT negative, and condition #2 Gal negative fractions cultured with WT or PFF1. Figure 101B shows flow cytometry analysis of SLAI in side scatter vs. forward scatter of condition #4 Gal negative fraction, WT positive, condition #2 Gal positive fraction, condition #3 Gal positive fraction, or condition #4 Gal positive fraction cultured with WT or PFF1.

[0194] [Figure 102] FIG. 102 shows flow cytometry quantification of SLA1 (FITC) in A. condition 3 cells and B. condition 4 cells.

[0195] [Figure 103] Figures 103A and 103B show confocal microscopy observations: A. Imaging results of WT porcine cells and cells genetically modified under conditions 2, 3 and 4; B. Slides of the images produced.

[0196] [Figure 104] Figure 104 is a diagram showing the results of sequencing NLRC5 of the cell line under condition 4. SEQ ID NOs: 372 to 376 are disclosed in order of appearance.

[0197] [Figure 105]Figure 105 shows a table of PCR oligos and target sequences (column 2) for GG1, Gal2-1, Gal2-2, Gal2-3, Gal2-4, Gal2-5, GGTA1-10, GGTA1-11, GGTA1-16, NL1, NLRC5-6, NLRC5-7, and NLRC5-8. SEQ ID NOs: 377-404, respectively, are disclosed in column 2 in order of appearance. SEQ ID NOs: 405-413, respectively, are disclosed in column 4 in order of appearance. SEQ ID NOs: 414-422, respectively, are disclosed in column 6 in order of appearance.

[0198] [Figure 106] Figure 106 shows a table of PCR oligos and target sequences (column 2) for CM1F, CM2RS, CM3RS, and CM4RS. SEQ ID NOs: 423-430, respectively, are disclosed in column 2 in order of appearance. SEQ ID NOs: 431-434, respectively, are disclosed in column 4 in order of appearance. SEQ ID NOs: 435-437, respectively, are disclosed in column 6 in order of appearance.

[0199] [Figure 107] Figure 107A shows a table of gRNA target sequences for B41, C3-9_1, C3-9_2, C3-5_1, C3-5_2, C3-15RS_1, and C3-15RS_2. SEQ ID NOs: 438-447, respectively, are disclosed in the second column in order of appearance. Figure 107B shows a table of detection screening primers for Gal1 and their respective sequences. SEQ ID NOs: 448-453, respectively, are disclosed in the second column in order of appearance.

[0200] [Figure 108] Figure 108 shows an overview of the Gal2-2 (B4GALNT2) vector and cloning strategy. The nucleotide sequence for a portion of the vector (SEQ ID NO: 454) and two oligos: Gal2-2_forward (SEQ ID NO: 455) and Gal2-2_reverse (SEQ ID NO: 456) are disclosed.

[0201] [Figure 109] Figure 109 shows the predicted Gal2-2 (B4GALNT2) clone sequence (top panel) upon correct insertion based on the vector and cloning strategy of Figure 113. SEQ ID NOs: 457-459, respectively, are disclosed in order of appearance. In the bottom panel, the sequencing results of the constructed plasmid (SEQ ID NO: 462) are aligned to the predicted sequence (SEQ ID NOs: 460-461).

[0202] [Figure 110] Figures 110A and B. A shows the Gal2-1 (B4GALNT2) target site within the GGTA1 gene (SEQ ID NO: 464) and two oligos (Gal2-1_screen_forward_1, SEQ ID NO: 463; and Gal2-1_screen_reverse_1, SEQ ID NO: 465). B shows the Gal2-1_screen_1 primer set, Gal2-1_screen primer set PCR product, and expected amplicon size of 303 bp observed on a gel. The strong single band observed in the expected amplicon size product was sequence verified and shown to contain the Gal2-1 target cleavage site desired for screening.

[0203] [Figure 111] Figures 111A and 111B. A shows the CM1F target site within the CMAH gene (SEQ ID NO: 467) and two oligos (CM1F-1_screen_forward_1, SEQ ID NO: 466; and CM1F-1_screen_reverse_1, SEQ ID NO: 468). B shows the CM1F_screen_1 primer set expected amplicon size of 309 bp, and the CM1F_screen primer set PCR product observed on a gel. A strong band was observed at the expected amplicon size, and a faint band at approximately 600 bp was also observed. The product at approximately 300 bp was sequence verified and shown to contain the desired target cleavage site for screening.

[0204] [Figure 112]Figures 112A and 112B. (A) Illustrates the NL1_first target site in the NLRC5 gene (SEQ ID NO: 470) and two oligos (NLR amp2 forward, SEQ ID NO: 469; and NLR amp2 reverse, SEQ ID NO: 471). (B) Expected amplicon size for the NLR amp2 primer: 217 bp. NLR amp2 primer set PCR product observed on gel, with a strong single band observed at the expected amplicon size. The product was sequence verified and shown to contain the NL1_first target cleavage site, which is desirable for screening.

[0205] [Figure 113A] Figures 113A-113I are diagrams depicting exon 1 genomic modifications of the Gal2-2 and NLRC5 genes. A shows the location of Gal screening primers. B. Gal2-2 PCR screen using Gal2-2 Screen 1 primers. C. Gal2-2 sequencing results. SEQ ID NOs: 472-478, respectively, are disclosed in order of appearance. D. GGTA1-10 PCR screen using GGTA1-10, 11 screen primers. E. NLRC5-6 screen primer locations. F. NLRC5-6 Set A (NLRC5-678 screen primers). G. NLRC5-6 sequencing results from Set A. SEQ ID NOs: 479-486, respectively, are disclosed in order of appearance. H. NLRC5-6 Set B (NLRC5-678 forward and NLR1st Screen 2 reverse screen primers). I. NLRC5-6 Set C (NLRC5-678 forward and NLR1st Screen 2 reverse screen primers). [Figure 113B] Same as above. [Figure 113C] Same as above. [Figure 113D] Same as above. [Figure 113E] Same as above. [Figure 113F] Same as above. [Figure 113G] Same as above. [Figure 113H] Same as above. [Figure 113I] Same as above.

[0206] [Figure 114] Figures 114A-C show live births of GGTA1 / NLRC5 knockout / HLA-G1 knockin piglets generated using CRISPR / Cas technology.

[0207] [Figure 115] Figure 115 shows the sequencing results confirming insertion into the ROSA gene site of HLA-G1. SEQ ID NO: 499 is disclosed.

[0208] [Figure 116] Figure 116 shows the sequencing results confirming correct construction of the homology-guided recombinant construct for HLA-G1 insertion at the Rosa26 locus in Example 8. SEQ ID NO: 500 is disclosed.

[0209] [Figure 117] Figure 117 shows the sequence of the left arm corresponding to the Rosa26 locus, which can be used in constructing a homology targeting vector for insertion of HLA-G1 or another sequence into the Rosa26 locus. SEQ ID NO: 501 is disclosed.

[0210] [Figure 118] Figure 118 shows the sequence of a modified HLA-G coding sequence that can be used in the construction of a homology targeting vector for insertion of HLA-G1 into a locus such as the Rosa26 locus. SEQ ID NO: 502 is disclosed.

[0211] [Figure 119] Figure 119 shows the sequence of the right arm corresponding to the Rosa26 locus, which can be used in constructing a homology targeting vector for insertion of HLA-G1 or another sequence into the Rosa26 locus. SEQ ID NO: 503 is disclosed. DETAILED DESCRIPTION OF THE INVENTION

[0212] Detailed Description of Disclosure The following description and examples illustrate in detail the embodiments of the present invention. It is understood that the present invention is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that there are numerous variations and modifications of the present invention that fall within its scope.

[0213] Transplant rejection can be prevented by methods that alleviate immune response, including those described herein.For example, by one method described herein for preventing transplant rejection or prolonging the time to transplant rejection without or with minimal immunosuppressive drugs, animals, such as donor non-human animals, can be modified, for example, genetically.The cells, organs, and / or tissues of the modified animals, such as donor non-human animals, can then be collected and used in allografts or xenografts.Alternatively, cells can be extracted from animals, such as humans or non-human animals (including, but not limited to, primary cells), or cells can be previously extracted animal cells, such as cell lines.These cells can be used to create genetically modified cells.

[0214] Graft rejection (e.g., T cell-mediated graft rejection) can be prevented by chronic immunosuppression. However, immunosuppression is costly and carries the risk of serious side effects. To avoid the need for chronic immunosuppression, multifaceted T cell-targeted rejection prevention has been developed (Figure 1), which i) Utilizing genetically modified grafts lacking functional expression of MHC class I, thereby providing direct specificity for CD8 + interfere with T cell activation and inhibit these CD8 + interferes with the cytolytic effector function of T cells, ii) using induction immunotherapy including antagonistic anti-CD40 mAbs (and depleting anti-CD20 mAbs and mTOR inhibitors) to interfere with B cell (and other APC)-mediated priming and memory generation of anti-donor T cells; and / or iii) Peritransplant injection of an apoptotic donor cell vaccine to deplete anti-donor T cells with indirect specificity.

[0215] Described herein are methods for treating or preventing disease in a recipient in need thereof by transplantation of a genetically modified non-human animal (such as a non-human primate or a member of the superorder Laurasiatheria, e.g., a genetically modified animal that is an ungulate), an organ, tissue, or cell isolated therefrom, a tolerization vaccine, and an organ, tissue, or cell isolated from the non-human animal. The organ, tissue, or cell isolated from the non-human animal (such as a non-human primate or a genetically modified animal that is a member of the superorder Laurasiatheria, e.g., an ungulate) can be transplanted into a recipient in need thereof of the same species (allograft) or a different species (xenotransplant). The recipient may be tolerized using a tolerization vaccine and / or one or more immune modulating agents (e.g., antibodies). In embodiments involving xenotransplantation, the recipient may be human. Suitable diseases that can be treated are any diseases in which the recipient's organ, tissue, or cells are defective or damaged (e.g., heart, lung, liver, vein, skin, or pancreatic islet cells) and in which the recipient can be treated by transplantation of an organ, tissue, or cells isolated from a non-human animal.

[0216] Human leukocyte antigen G (HLA-G) HLA-G can be a potent immunosuppressive and tolerogenic molecule.Therefore, in one aspect, genetically modified non-human animals and cells are disclosed herein that comprise an exogenous nucleic acid sequence encoding HLA-G protein.The genetically modified non-human animals and cells can also comprise one or more additional genetic modifications, such as any of the genetic modifications (e.g., knock-in, knock-out, gene disruption, etc.) disclosed herein.

[0217] definition As used herein, the term "about" and its grammatical equivalents in reference to a reference numerical value can include the numerical value itself and values ​​within a range of plus or minus 10% from that numerical value. For example, the quantity "about 10" includes 10 and any quantity from 9 to 11. For example, the term "about" in reference to a reference numerical value can also include values ​​within a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

[0218] As used herein, the term "non-human animal" and its grammatical equivalents include all animal species other than humans, including non-human mammals, which may be naturally occurring or genetically modified. Non-human mammals include ungulates, such as ungulates (e.g., pigs, peccaries, hippos, camels, llamas, mouse deer (mouse deer), deer, giraffes, pronghorns, antelopes, caprine-antelopes (including sheep, goats, etc.), or cattle), or perissodactyls (e.g., horses, tapirs, and rhinoceroses), non-human primates (e.g., monkeys, or chimpanzees), canines (e.g., dogs), or cats. The non-human animal may be a member of the superorder Laurasiatheria. The superorder Laurasiatheria may include the mammalian groups described in Waddell et al., Towards Resolving the Interordinal Relationships of Placental Mammals, Systemic Biology 48(1):1-5 (1999). Members of the superorder Laurasiatheria may include Eulipotyphla (hedgehogs, shrews, and moles), Perissodactyla (rhinoceroses, horses, and tapirs), Carnivora (carnivores), Cetariodactyla (artiodactyla and cetaceans), Chiroptera (bats), and Pholidota (pangolins). Members of the superorder Laurasiatheria may be ungulates, such as perissodactyla or even-ungulates, as described herein. The ungulate may be a pig. Members may be members of Carnivora, such as cats, or dogs. In some cases, members of the superorder Laurasiatheria may be pigs.

[0219] As used herein, the term "pig" and its grammatical equivalents may refer to animals of the genus Sus, which are part of the Suidae family of cloven-hoofed animals. For example, the pig may be a wild pig, a domestic pig, a miniature pig, a Sus scrofa pig, a Sus scrofa domesticus pig, or an inbred pig.

[0220] As used herein, the term "transgene" and its grammatical equivalents may refer to a gene or genetic material that can be transferred into an organism. For example, a transgene can be a stretch or segment of DNA containing a gene that is introduced into an organism. The gene or genetic material can be from a different species. The gene or genetic material can be synthetic. When a transgene is transferred into an organism, the organism may be referred to as a transgenic organism. The transgene can retain its ability to produce RNA or a polypeptide (e.g., a protein) in the transgenic organism. A transgene can comprise a polynucleotide that encodes a protein or a fragment (e.g., a functional fragment) thereof. The transgene polynucleotide can be an exogenous polynucleotide. A fragment (e.g., a functional fragment) of a protein can comprise at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or at least about these proportions, of the amino acid sequence of the protein. A fragment of a protein can be a functional fragment of the protein. A functional fragment of a protein may retain some or all of the function of the protein.

[0221] The term "exogenous nucleic acid sequence" can refer to a gene or genetic material that originates outside the cell or animal and is transferred into a cell or animal. An exogenous nucleic acid sequence can be produced synthetically. An exogenous nucleic acid sequence can be derived from a different species or from a different member of the same species. An exogenous nucleic acid sequence can be another copy of an endogenous nucleic acid sequence.

[0222] As used herein, the term "genetically modified" and its grammatical equivalents may refer to one or more modifications of a nucleic acid, for example, a nucleic acid in the genome of an organism. For example, genetic modification may refer to the modification, addition, and / or deletion of a gene. A genetically modified cell may also refer to a cell having an added, deleted, and / or modified gene. A genetically modified cell may be derived from a genetically modified non-human animal. A genetically modified cell derived from a genetically modified non-human animal may be a cell isolated from such a genetically modified non-human animal. A genetically modified cell derived from a genetically modified non-human animal may be a cell derived from such a genetically modified non-human animal.

[0223] The term "gene knockout" or "knockout" can refer to any genetic modification that reduces expression of the gene being "knocked out." Reduced expression can include no expression. The genetic modification can include a genome disruption.

[0224] As used herein, the terms "islet" or "islet cell" and their grammatical equivalents may refer to endocrine (e.g., hormone-producing) cells present in the pancreas of an organism. For example, islet cells may include different types of cells, including, but not limited to, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic F cells, and / or pancreatic epsilon cells. Islet cells may also refer to groups of cells, cell clusters, and the like.

[0225] As used herein, the term "condition" and its grammatical equivalents may refer to a disease, an event, or a change in health status.

[0226] The term "diabetes" and its grammatical equivalents used herein can refer to the disease characterized by prolonged high blood sugar levels.For example, the term "diabetes" and its grammatical equivalents used herein can refer to all or any type of diabetes, including but not limited to type 1, type 2, cystic fibrosis-related, surgical, gestational diabetes, and mitochondrial diabetes.In some cases, diabetes can be a form of hereditary diabetes.

[0227] As used herein, the term "phenotype" and its grammatical equivalents may refer to the composite of observable characteristics or traits of an organism, such as its shape, development, biochemical or physiological properties, phenology, behavior, and the consequences of behavior. Depending on the context, the term "phenotype" may in some cases refer to the composite of observable characteristics or traits of a population.

[0228] As used herein, the term "disrupting" and its grammatical equivalents may refer to the process of modifying a gene, for example, by deletion, insertion, mutation, rearrangement, or any combination thereof. For example, a gene can be disrupted by knockout. Disruption of a gene can be a partial reduction or complete suppression of gene expression (e.g., mRNA and / or protein expression). Disruption can also include inhibitory techniques, such as shRNA, siRNA, microRNA, dominant negative, or any other means of inhibiting the functionality or expression of a gene or protein.

[0229] As used herein, the term " gene editing " and its grammatical equivalents may refer to the genetic manipulation of inserting, replacing, or removing one or more nucleotides in a genome.For example, gene editing can be performed using nuclease (for example, naturally occurring nuclease or artificially engineered nuclease).

[0230] As used herein, the term "graft rejection" and its grammatical equivalents may refer to one or more processes in which the immune response of an organ transplant recipient mounts a reaction against the transplanted material (e.g., cells, tissues, and / or organs) sufficient to impair or destroy the function of the transplanted material.

[0231] As used herein, the term "hyperacute rejection" and its grammatical equivalents may refer to rejection of transplanted material or tissue that occurs or begins within the first 24 hours after transplantation. For example, hyperacute rejection may include, but is not limited to, "acute humoral rejection" and "antibody-mediated rejection."

[0232] As used herein, the terms "negative vaccine," "tolerizing vaccine," and their grammatical equivalents may be used interchangeably. Tolerizing vaccines, when used under the auspices of appropriate immunotherapy, may cause or contribute to the recipient's tolerance to a transplant. This may help prevent transplant rejection.

[0233] As used herein, the terms "recipient," "subject," and their grammatical equivalents may be used interchangeably. The recipient or subject may be a human or a non-human animal. The recipient or subject may be a human or a non-human animal that will receive, is receiving, or has received a transplant, a tolerization vaccine, and / or other compositions disclosed in the present application. The recipient or subject may also be in need of a transplant, a tolerization vaccine, and / or other compositions disclosed in the present application. In some cases, the recipient may be a human or a non-human animal that will receive, is receiving, or has received a transplant.

[0234] Some numerical values ​​disclosed throughout are referred to as, for example, "X is at least 100; or 200 [or any number], or at least about these numbers." This number is used both by itself and by the following: i) X is at least 100; ii) X is at least 200; iii) X is at least about 100; and iv) X is at least about 200 Includes all of the above. All these different combinations are contemplated by the numerical values ​​disclosed throughout. All disclosed numerical values, whether referring to administration of therapeutic agents, or to days, months, years, weights, dosages, etc., should be construed in this manner unless specifically indicated otherwise.

[0235] Ranges disclosed throughout are sometimes referred to, for example, as "X is administered on days 1-2, or days 2-3 [or any range of values], or within about these time periods." The ranges may be expressed in terms of the numbers themselves (e.g., the endpoints of the range) and the following: i) X is administered between days 1 and 2; ii) X is administered between days 2 and 3; iii) X is administered between about days 1 and 2; iv) X is administered between about days 2 and 3; v) X is administered between day 1 and about day 2; vi) X is administered between day 2 and about day 3; vii) X is administered between about day 1 and about day 2; and viii) X is administered between about day 2 and about day 3 Includes all of the above. All these different combinations are contemplated by the ranges disclosed throughout. All disclosed ranges, whether referring to administration of therapeutic agents, or to days, months, years, weights, dosages, etc., should be construed in this manner unless specifically indicated otherwise.

[0236] As used herein, the terms "and / or" and "any combination thereof," and their grammatical equivalents, may be used interchangeably. These terms may convey that any combination is specifically contemplated. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C."

[0237] The term "or" may be used conjunctively or disjunctively unless the context specifically dictates disjunctive use.

[0238] I. Genetically Modified Non-Human Animals Provided herein are genetically modified non-human animals that can be donors of cells, tissues, and / or organs for transplantation. The genetically modified non-human animals can be of any desired species. For example, the genetically modified non-human animals described herein can be genetically modified non-human mammals. The genetically modified non-human mammals can be genetically modified ungulates, including genetically modified ungulates (e.g., pigs, peccaries, hippopotamuses, camels, llamas, mouse deer (mouse deer), deer, giraffes, pronghorns, antelopes, capricorn-antelopes (including sheep, goats, etc.), or cattle) or genetically modified perissodactyls (e.g., horses, tapirs, and rhinos), genetically modified non-human primates (e.g., monkeys, or chimpanzees), or genetically modified Canidae (e.g., dogs). The genetically modified non-human animals can be members of the superorder Laurasiatheria. The genetically modified non-human animal can be a non-human primate animal, such as a monkey or a chimpanzee.When the non-human animal is a pig, the pig can be at least 1, 5, 50, 100, or 300 pounds, or at least approximately these weights, for example, the pig can be between 5 pounds and 50 pounds; between 25 pounds and 100 pounds; or between 75 pounds and 300 pounds, or between about 5 pounds and 50 pounds; between about 25 pounds and 100 pounds; or between about 75 pounds and 300 pounds.In some cases, the non-human animal is a pig that has given birth at least once.

[0239] The genetically modified non-human animal can be of any age. For example, the genetically modified non-human animal can be a fetus; 1 day or about 1 day to 1 month or about 1 month; 1 month or about 1 month to 3 months or about 3 months; 3 months or about 3 months to 6 months or about 6 months; 6 months or about 6 months to 9 months or about 9 months; 9 months or about 9 months to 1 year or about 1 year; 1 year or about 1 year to 2 years or about 2 years. The genetically modified non-human animal can be a non-human fetal animal, a perinatal non-human animal, a newborn non-human animal, a pre-weaning non-human animal, a juvenile non-human animal, or an adult non-human animal.

[0240] Genetically modified non-human animals can survive for at least a certain period after birth. For example, genetically modified non-human animals can survive for at least 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 4 months, 8 months, 1 year, 2 years, 5 years, or 10 years after birth. Multiple genetically modified animals (e.g., pigs) can be born as a litter. A litter of genetically modified animals can have a survival rate of at least 30%, 50%, 60%, 80%, or 90%, where the survival rate is, for example, the number of animals in a litter that survive after birth divided by the total number of animals in the litter.

[0241] Genetically modified non-human animals can comprise reduced expression of one or more genes compared with corresponding non-genetically modified animals.The reduced expression of genes can be due to mutations in one or more alleles of genes.For example, genetically modified animals can comprise mutations in two or more alleles of genes.In some cases, such genetically modified animals can be diploid animals.

[0242] The genetically modified non-human animal may comprise reduced expression of one or more genes compared to a non-genetically modified counterpart animal. The genetically modified non-human animal may comprise reduced expression of two or more genes compared to a non-genetically modified counterpart animal. The genetically modified animal may have a genomic disruption in at least one gene selected from the group consisting of a component of the MHC I-specific enhanceosome, a transporter of MHC I-binding peptides, a natural killer (NK) group 2D ligand, a CXC chemokine receptor (CXCR)3 ligand, an MHC II transactivator (CIITA), C3, an endogenous gene not expressed in humans, and any combination thereof.

[0243] In some cases, the genetically modified animal has reduced expression of the gene compared to a corresponding animal that is not genetically modified. In some cases, the genetically modified animal survives for at least 22 days after birth. In other cases, the genetically modified animal survives for at least 23-30 days, 25-35 days, 35-45 days, 45-55 days, 55-65 days, 65-75 days, 75-85 days, 85-95 days, 95-105 days, 105-115 days, 115-225 days, 225-235 days, 235-245 days, 245-255 days, 255-265 days, 265-275 days, 285-295 days, 305-315 days, 315-325 days, 325-335 days, 335-345 days, 345-355 days, 355-365 days, 365-375 days, 375-385 days, 385-395 days, 395-405 days, 405-415 days, 415-425 days, 425-435 days, 435-445 days, 445-455 days, 455-465 days, 465-475 days, 485-495 days, 495-505 days, 505-515 days, 515-525 days, 525-535 days, 535-545 days, 545-555 days, 555-565 days, 565-575 days, 575-585 days, 585-595 days, 595-605 days, 605-6 They may survive for 75 days, 275-285 days, 285-295 days, 295-305 days, 305-315 days, 315-325 days, 325-335 days, 335-345 days, 345-355 days, 355-365 days, 365-375 days, 375-385 days, 385-395 days, or 395-400 days, or at least approximately these numbers of days.

[0244] The non-genetically modified counterpart animal can be an animal that is substantially identical to the genetically modified animal but does not have the genetic modification in its genome. For example, the non-genetically modified counterpart animal can be a wild-type animal of the same species as the genetically modified animal.

[0245] Genetically modified non-human animals can provide cells, tissues, or organs for transplantation into a recipient or subject in need thereof. The recipient or subject in need thereof can be a recipient or subject known to have or suspected of having a condition. The condition can be treated, prevented, reduced, eliminated, or augmented by the methods and compositions disclosed herein. The recipient may exhibit a low or no immune response to the transplanted cells, tissue, or organ. The transplanted cells, tissue, or organ may not be recognized by CD8+ T cells, NK cells, or CD4+ T cells of the recipient (e.g., a human or another animal). Genes with reduced expression can include MHC molecules, expression regulators of MHC molecules, and genes that are differentially expressed between the donor non-human animal and the recipient (e.g., a human or another animal). Reduced expression can be mRNA expression or protein expression of one or more genes. For example, reduced expression can be protein expression of one or more genes. Reduced expression can also include no expression. For example, an animal, cell, tissue, or organ with reduced gene expression may not have gene expression (e.g., mRNA and / or protein expression). Reduced gene expression can inactivate the function of the gene. In some cases, when gene expression is reduced in a genetically modified animal, gene expression is absent in the genetically modified animal.

[0246] The genetically modified non-human animal can comprise reduced expression of one or more MHC molecules compared to a corresponding animal that is not genetically modified. For example, the non-human animal can be an ungulate, such as a pig, that has reduced expression of one or more swine leukocyte antigen (SLA) class I and / or SLA class II molecules.

[0247] A genetically modified non-human animal can comprise reduced expression of any gene regulating major histocompatibility complex (MHC) molecules (e.g., MHC I molecules and / or MHC II molecules) compared to a non-genetically modified counterpart. Reduced expression of such genes can result in reduced expression and / or function of MHC molecules (e.g., MHC I molecules and / or MHC II molecules). In some cases, the one or more genes whose expression is reduced in the non-human animal can comprise one or more of the following: a component of the MHC I-specific enhanceosome, a transporter of MHC I-bound peptides, a natural killer group 2D ligand, a CXC chemoreceptor (CXCR)3 ligand, complement component 3 (C3), and a major histocompatibility complex II transactivator (CIITA). In some cases, the component of the MHC I-specific enhanceosome can be NLRC5. In some cases, components of the MHC I-specific enhanceosome may also include regulatory factor X (RFX) (e.g., RFX1), nuclear transcription factor Y (NFY), and cAMP response element-binding protein (CREB). In some examples, the transporter of MHC I-binding peptides may be antigen processing-associated transporter 1 (TAP1). In some cases, natural killer (NK) group 2D ligands may include MICA and MICB. For example, the genetically modified non-human animal may include reduced expression of one or more of the following genes: NOD-like receptor family CARD domain-containing 5 (NLRC5), antigen processing-associated transporter 1 (TAP1), C-X-C motif chemokine 10 (CXCL10), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB), complement component 3 (C3), and CIITA. The genetically modified animal may comprise reduced expression of one or more of the following genes: a component of the MHC I-specific enhanceosome (e.g., NLRC5), a transporter of MHC I-bound peptides (TAP1), and C3.

[0248] A genetically modified non-human animal may comprise one or more genes that are expressed at different levels between the non-human animal and the recipient of cells, tissues, or organs derived from the non-human animal, and the expression level of these genes may be reduced compared to their non-genetically modified counterparts. For example, one or more genes may be expressed at lower levels in humans than in non-human animals. In some cases, one or more genes may be endogenous genes of the non-human animal. An endogenous gene may be a gene that is not expressed in another species. For example, an endogenous gene of a non-human animal may be a gene that is not expressed in humans. For example, in some cases, homologs (e.g., orthologs) of one or more genes do not exist in humans. In another example, homologs (e.g., orthologs) of one or more genes may exist in humans but not be expressed.

[0249] In some cases, the non-human animal may be a pig, and the recipient may be a human. In these cases, the one or more genes may be any gene that is expressed in pigs but not in humans. For example, the one or more genes may include glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH), and β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). The genetically modified non-human animal may have reduced expression of B4GALNT2, GGTA1, or CMAH, compared to the corresponding non-genetically modified animal. The genetically modified non-human animal may have reduced expression of B4GALNT2 and GGTA1, compared to the corresponding non-genetically modified animal. The genetically modified non-human animal may comprise reduced expression of B4GALNT2 and CMAH, where the reduced expression is compared to a corresponding animal that is not genetically modified. The genetically modified non-human animal may comprise reduced expression of B4GALNT2, GGTA1, and CMAH, where the reduced expression is compared to a corresponding animal that is not genetically modified.

[0250] The genetically modified non-human animal may comprise reduced expression, relative to the non-genetically modified counterpart, of any one or more of the genes disclosed herein, including NLRC5, TAP1, CXCL10, MICA, MICB, C3, CIITA, GGTA1, CMAH, and B4GALNT2.

[0251] The genetically modified non-human animal may comprise one or more genes with reduced expression, for example, reduced gene expression. The one or more genes with reduced expression include, but are not limited to, NOD-like receptor family CARD domain-containing 5 (NLRC5), transporter associated with antigen processing 1 (TAP1), glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH), C-X-C motif chemokine 10 (CXCL10), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB), class II major histocompatibility complex transactivator (CIITA), beta-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2), complement component 3 (C3), and / or any combination thereof.

[0252] A genetically modified non-human animal may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more genes whose expression is disrupted. For illustrative purposes, and not to limit the various combinations, one skilled in the art can envision a genetically modified non-human animal having NLRC5 and TAP1 disrupted individually. A genetically modified non-human animal may also have both NLRC5 and TAP1 disrupted. A genetically modified non-human animal may also have NLRC5 and TAP1 in addition to one or more of the following: disrupted GGTAl, CMAH, CXCL10, MICA, MICB, B4GALNT2, or CIITA genes; for example, "NLRC5, TAP1, and GGTAl" or "NLRC5, TAP1, and CMAH" may be disrupted. The genetically modified non-human animal may also have disrupted NLRC5, TAP1, GGTA1, and CMAH. Alternatively, the genetically modified non-human animal may have disrupted NLRC5, TAP1, GGTA1, B4GALNT2, and CMAH. Optionally, the genetically modified non-human animal may have disrupted C3 and GGTA1. Optionally, the genetically modified non-human animal may have reduced expression of NLRC5, C3, GGTA1, B4GALNT2, CMAH, and CXCL10. Optionally, the genetically modified non-human animal may have reduced expression of TAP1, C3, GGTA1, B4GALNT2, CMAH, and CXCL10. Optionally, the genetically modified non-human animal may have reduced expression of NLRC5, TAP1, C3, GGTA1, B4GALNT2, CMAH, and CXCL10. The B4GALNT2 gene can be Gal2-2 or Gal2-1.

[0253] Lack of MHC class I expression on transplanted human cells can result in passive activation of natural killer (NK) cells (Ohlen et al., 1989). This lack of MHC class I expression may be due to deletion of the NLRC5, TAP1, or B2M genes. NK cell cytotoxicity can be overcome by expression of the human MHC class I gene HLA-E, which can stimulate the inhibitory receptor CD94 / NKG2A on NK cells to prevent cell killing (Weiss et al., 2009; Lilienfeld et al., 2007; Sasaki et al., 1999). Successful expression of the HLA-E gene may depend on coexpression of the human B2M (beta-2 microglobulin) gene and its cognate peptide (Weiss et al., 2009; Lilienfeld et al., 2007; Sasaki et al., 1999; Pascasova et al., 1999). Nuclease-mediated breaks in the stem cell DNA can allow for the insertion of one or more genes by homology-guided repair. HLA-E and hB2M genes can be consecutively integrated into the region of nuclease-mediated DNA breaks while inserting a transgene, preventing expression of the target gene (e.g., NLRC5).

[0254] The expression level of a gene can be reduced to various degrees. For example, the expression of one or more genes can be reduced by 100% or about 100%. In some cases, the expression of one or more genes can be reduced by 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% of normal expression, or by about these percentages, for example, compared to the expression of an unmodified control. In some cases, the expression of one or more genes can be reduced by at least 99%-90%, 89%-80%, 79%-70%, 69%-60%, 59%-50%, or by at least about these percentages, for example, compared to the expression of an unmodified control. For example, the expression of one or more genes can be reduced by at least or at least about 90% of normal expression, or by at least 90%-99%, or by at least about these percentages.

[0255] Expression can be measured by any known method, such as quantitative PCR (qPCR), including, but not limited to, PCR, real-time PCR (e.g., Sybr-green), and / or hot PCR. In some cases, expression of one or more genes can be measured by detecting the level of gene transcripts. For example, expression of one or more genes can be measured by Northern blotting, nuclease protection assay (e.g., RNase protection assay), reverse transcription PCR, quantitative PCR (e.g., real-time PCR, such as real-time quantitative reverse transcription PCR), in situ hybridization (e.g., fluorescent in situ hybridization (FISH)), dot blot analysis, differential display, serial analysis of gene expression, subtractive hybridization, microarray, nanostring, and / or sequencing (e.g., next-generation sequencing). In some cases, expression of one or more genes can be measured by detecting the level of proteins encoded by the genes. For example, the expression of one or more genes can be measured by protein immunostaining, protein immunoprecipitation, electrophoresis (e.g., SDS-PAGE), Western blotting, bicinchoninic acid assay, spectrophotometry, mass spectrometry, enzyme assay (e.g., enzyme-linked immunosorbent assay), immunohistochemistry, flow cytometry, and / or immunocytochemistry. The expression of one or more genes can also be measured by microscopy. Microscopy can be optical, electron, or scanning probe microscopy. Optical microscopy can include the use of brightfield, oblique illumination, cross-polarized light, dispersed staining, darkfield, phase contrast, differential interference contrast, interference reflectance microscopy, fluorescence (e.g., when particles, e.g., cells, are immunostained), confocal, single-plane illumination microscopy, light-sheet fluorescence microscopy, deconvolution, or continuous time-encoded amplitude microscopy. The expression of MHC I molecules can also be detected by any of the methods for testing expression described herein.

[0256] Disrupted genes Cells, organs, and / or tissues with different combinations of disrupted genes as described herein can result in cells, organs, and / or tissues that are less likely to be rejected when transplanted into recipients.For example, the present inventors have found that disruption (e.g., reduction in expression) of certain genes, such as NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10, MICA, MICB, C3, and / or CIITA, can increase the survival rate of transplants.In some cases, at least two genes are disrupted.For example, GGTA1-10 and Gal2-2 can be disrupted.In some cases, GGTA1-10, Gal2-2, and NLRC5-6 can be disrupted.In other cases, NLRC5-6 and Gal2-2 can be disrupted.

[0257] In some cases, disruption is not limited to these genes. It is contemplated that gene homologs of the genes in the present application (e.g., any mammalian form of the gene) may also be targeted. For example, the disrupted gene may exhibit a certain degree of identity and / or homology with the genes disclosed herein, such as NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10, MICA, MICB, C3, and / or CIITA. Thus, it is contemplated that genes exhibiting at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or at least approximately these percentages of homology, for example, genes exhibiting at least 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 99%, or at least approximately these percentages of homology (at the nucleic acid or protein level) can be disrupted. It is also envisioned that genes exhibiting at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 99%, or 100%, or at least approximately these percentages of identity, for example, genes exhibiting at least 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 99%, or at least approximately these percentages of identity (at the nucleic acid or protein level), can be disrupted. Some gene homologs are known in the art, but in some cases, homologs are not known. However, homologous genes between mammals can be found by comparing nucleic acid (DNA or RNA) sequences or protein sequences using publicly available databases, such as NCBI's BLAST. The genomic, cDNA, and protein sequences of exemplary genes are shown in Table 1.

[0258] Gene suppression can also be carried out in many ways. For example, gene expression can be reduced by knockout, by modifying the promoter of the gene, and / or by administering interfering RNA (knockdown). This can be done at the organism level, tissue level, organ level, and / or cellular level. When knocking down one or more genes in a non-human animal, in a cell, tissue, and / or organ, one or more genes can be reduced by administering an RNA interference reagent, such as siRNA, shRNA, or microRNA. For example, a nucleic acid capable of expressing shRNA can be stably transfected into cells to knock down expression. Furthermore, a nucleic acid capable of expressing shRNA can be inserted into the genome of a non-human animal, thereby knocking down a gene in a non-human animal.

[0259] Disruption methods can also include overexpressing a dominant-negative protein, which can result in a total loss of function of the functional wild-type gene. In addition, expressing a dominant-negative gene can result in a phenotype similar to that of a knockout and / or knockdown.

[0260] In some cases, a stop codon can be inserted or created (e.g., by nucleotide substitution) within one or more genes, which can result in a non-functional transcript or a non-functional protein (sometimes referred to as a knockout). For example, if a stop codon is created in the middle of one or more genes, the resulting transcript and / or protein may be truncated and may be non-functional. However, in some cases, truncation can result in an active (partially active or overactive) protein. In some cases, if the protein is overactive, this can result in a dominant-negative protein, for example, a mutant polypeptide that disrupts the activity of the wild-type protein.

[0261] The dominant-negative protein can be expressed in the nucleic acid under the control of any promoter. For example, the promoter can be a ubiquitous promoter. The promoter can also be an inducible promoter, a tissue-specific promoter, and / or a development-specific promoter.

[0262] Then, the nucleic acid encoding the dominant negative protein can be inserted into a cell or a non-human animal. Any known method can be used. For example, stable transfection can be used. In addition, the nucleic acid encoding the dominant negative protein can also be inserted into the genome of a non-human animal.

[0263] One or more genes in a non-human animal can be knocked out using any method known in the art. For example, knocking out one or more genes can include deleting one or more genes from the genome of the non-human animal. Knockout can also include removing all or part of a gene sequence from the non-human animal. It is also contemplated that knockout can include replacing all or part of a gene in the genome of the non-human animal with one or more nucleotides. Knockout of one or more genes can also include inserting a sequence into one or more genes, thereby disrupting the expression of one or more genes. For example, inserting a sequence can create a stop codon in the middle of one or more genes. Inserting a sequence can also shift the open reading frame of one or more genes. In some cases, knockout can be performed in the first exon of a gene. In other cases, knockout can be performed in the second exon of a gene.

[0264] Knockout can be performed in any cell, organ, and / or tissue of a non-human animal. For example, knockout can be a systemic knockout, e.g., the expression of one or more genes is reduced in all cells of a non-human animal. Knockout can also be specific to one or more cells, tissues, and / or organs of a non-human animal. This can be achieved by conditional knockout, which selectively reduces the expression of one or more genes in one or more organs, tissues, or cell types. Conditional knockout can be performed using the Cre-lox system, in which cre is expressed under the control of a cell-specific promoter, a tissue-specific promoter, and / or an organ-specific promoter. For example, one or more genes may be knocked out (or expression may be reduced) in one or more tissues or organs, where the one or more tissues or organs may include the brain, lung, liver, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, smooth muscle, skin, bone, adipose tissue, hair, thyroid, trachea, gallbladder, kidney, ureter, urinary bladder, aorta, vein, esophagus, diaphragm, stomach, rectum, adrenal gland, bronchi, ear, eye, retina, reproductive organs, hypothalamus, larynx, nose, tongue, spinal cord, or ureter, uterus, ovaries, testes, and / or any combination thereof.One or more genes may also be knocked out (or expression reduced) within a cell type, where the cell type or cells are hair follicles, keratinocytes, gonadotropes, corticotropes, thyrotropes, somatotropes, prolactinocytes, chromaffin cells, parafollicular cells, glomus cells, melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts, keratocytes, retinal Müller cells, retinal pigment epithelial cells, neurons, glia (e.g., oligodendrocytes, astrocytes), ependymal cells, pineal cells, lung cells (e.g., type I and type II pneumocytes), Clara cells, goblet cells, G cells, D cells, enterochromaffin cells, Cells, gastric chief cells, parietal cells, gastric pit cells, K cells, D cells, I cells, goblet cells, Paneth cells, enterocytes, M cells, hepatocytes, hepatic stellate cells (e.g., Kupffer cells derived from mesoderm), gallbladder cells, central acinar cells, pancreatic stellate cells, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells, pancreatic ε cells, thyroid gland (e.g., follicular cells), parathyroid gland (e.g., parathyroid chief cells), eosinophilic cells, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, myosatellite cells, tendon cells, cardiac myocytes, lipoblasts, adipocytes, interstitial cells of Cajal, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, interstitial cells cell), interstitial cell, telocyte, simple epithelial cell, podocyte, renal proximal tubule brush border cell, Sertoli cell, Leydig cell, granulosa cell, non-ciliated epithelial cell, germ cell, sperm, egg, lymphocyte, myeloid cell, endothelial progenitor cell, endothelial stem cell, hemangioblast, mesoangioblast, pericyte, mural cell, and / or any combination thereof.

[0265] A conditional knockout can be an inducible knockout, for example, by using a tetracycline-inducible promoter or a developmentally specific promoter. This can eliminate or suppress gene / protein expression at any time or specific time. For example, in the case of a tetracycline-inducible promoter, tetracycline can be given to a non-human animal at any time after birth. When a non-human animal is developing in the uterus, the promoter can be induced by giving tetracycline to the pregnant mother. When a non-human animal is developing in an egg, the promoter can be induced by injecting or incubating in tetracycline. When tetracycline is given to a non-human animal, it results in the expression of cre, which results in the excision of the gene of interest.

[0266] The cre / lox system can also be under the control of developmentally specific promoters. For example, some promoters are turned on after birth or after the onset of puberty. These promoters can be used to control cre expression and therefore can be used in developmentally specific knockouts.

[0267] It is also envisioned that any combination of knockout techniques may be implemented. For example, a tissue-specific knockout may be combined with an induction technique to create a tissue-specific, inducible knockout. Additionally, other systems, such as developmentally specific promoters, may be used in combination with tissue-specific promoters and / or inducible knockouts.

[0268] In some cases, gene editing can be useful for designing knockout.For example, gene editing can be carried out using nuclease, including CRISPR-associated protein (Cas protein, for example, Cas9), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and meganuclease.Nuclease can be naturally occurring nuclease, genetically modified nuclease, and / or recombinant nuclease.For example, CRISPR / Cas system can be suitable as gene editing system.

[0269] It is also contemplated that less than all alleles of one or more genes in a non-human animal can be knocked out. For example, in a diploid non-human animal, it is contemplated that one of the two alleles can be knocked out. This can result in reduced expression of the gene and reduced protein levels. The overall reduction in expression can be less than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20%, or less than approximately these percentages, for example, 99% to 90%, 90% to 80%, 80% to 70%, 70% to 60%, 60% to 50%, 50% to 40%, 40% to 30%, or 30% to 20%, or approximately these percentages, compared to when both alleles are functional, e.g., when not knocked out and / or knocked down. Furthermore, the overall reduction in protein levels can be the same as the overall reduction in expression. The overall reduction in protein levels can be about 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20%, or less than about these percentages, for example, 99% to 90%, 90% to 80%, 80% to 70%, 70% to 60%, 60% to 50%, 50% to 40%, 40% to 30%, or 30% to 20%, or less than these percentages, compared to when both alleles are functional, e.g., not knocked out and / or knocked down. However, it is also contemplated that all alleles of one or more genes can be knocked out in a non-human animal.

[0270] The knockout of one or more genes can be verified by genotyping. Genotyping methods can include sequencing, restriction fragment length polymorphism identification (RFLPI), random amplified polymorphism detection (RAPD), amplified fragment length polymorphism detection (AFLPD), PCR (e.g., long-range PCR or stepwise PCR), allele-specific oligonucleotide (ASO) probes, and hybridization to DNA microarrays or beads. For example, genotyping can be performed by sequencing. In some cases, sequencing can be high-fidelity sequencing. Sequencing methods can include Maxam-Gilbert sequencing, chain-termination methods (e.g., Sanger sequencing), shotgun sequencing, and bridge PCR. In some cases, genotyping can be performed by next-generation sequencing. Next-generation sequencing methods can include massively parallel signature sequencing, colony sequencing, pyrosequencing (e.g., pyrosequencing developed by 454 Life Sciences), single-molecule real-time sequencing (e.g., by Pacific Biosciences), ion semiconductor sequencing (e.g., by Ion Torrent semiconductor sequencing), sequencing-by-synthesis (e.g., by Illumina's Solexa sequencing), sequencing by ligation (e.g., by Applied Biosystems' SOLiD sequencing), DNA nanoball sequencing, and heliscope single-molecule sequencing. In some cases, genotyping of non-human animals in the present invention can involve whole-genome sequencing. In some cases, gene knockout in animals can be verified by sequencing a portion of the gene or the entire gene (e.g., next-generation sequencing). For example, knockout of the NLRC5 gene in pigs may be validated by next-generation sequencing of the entire NLRC5.Next-generation sequencing of NLRC5 can be performed, for example, using the forward primer 5'-gctgtggcatatggcagttc-3' (SEQ ID NO: 1) and the reverse primer 5'-tccatgtataagtctttta-3' (SEQ ID NO: 2), or the forward primer 5'-ggcaatgccagatcctcaac-3' (SEQ ID NO: 3) and the reverse primer 5'-tgtctgatgtctttctcatg-3' (SEQ ID NO: 4). [Table 1]

[0271] transgene Transgenes, or exogenous nucleic acid sequences, can be useful for overexpressing endogenous genes at levels higher than would be the case without the transgene. Additionally, exogenous nucleic acid sequences may be used to express exogenous genes. Transgenes can also include other types of genes, such as dominant-negative genes.

[0272] An X protein transgene may refer to a transgene comprising an exogenous nucleic acid sequence encoding the X protein. In some cases, the X protein-encoding transgene, as used herein, may encode 100% or approximately 100% of the amino acid sequence of the X protein. In some cases, the X protein-encoding transgene may encode the full-length or partial amino acid sequence of the X protein. For example, the transgene may encode at least 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%, or at least approximately these percentages, e.g., 99%-90%, 90%-80%, 80%-70%, 70%-60%, or 60%-50%, or approximately these percentages, of the amino acid sequence of the X protein. Expression of the transgene may ultimately result in a functional protein, e.g., a partially or fully functional protein. As discussed above, when a partial sequence is expressed, the final result may be a non-functional protein or a dominant-negative protein in some cases. A non-functional protein or a dominant-negative protein may also compete with a functional (endogenous or exogenous) protein. A transgene may also encode RNA (e.g., mRNA, shRNA, siRNA, or microRNA). In some cases, if a transgene encodes mRNA, it can be translated into a polypeptide (e.g., a protein). Thus, it is envisioned that a transgene may encode a protein. In some cases, a transgene may encode a protein or a portion of a protein. In addition, a protein may have one or more mutations (e.g., deletions, insertions, amino acid substitutions, or rearrangements) compared to a wild-type polypeptide. A protein may be a natural polypeptide or an artificial polypeptide (e.g., a recombinant polypeptide). A transgene may encode a fusion protein formed by two or more polypeptides.

[0273] When a transgene or exogenous nucleic acid sequence encodes an mRNA based on a naturally occurring mRNA (e.g., an mRNA normally found in another species), the mRNA may contain one or more modifications in the 5' or 3' untranslated region. The one or more modifications may include one or more insertions, one or more deletions, or one or more nucleotide changes, or a combination thereof. The one or more modifications may increase the stability of the mRNA. The one or more modifications may remove a binding site for an miRNA molecule, such as an miRNA molecule, that can inhibit translation or stimulate mRNA degradation. For example, an mRNA encoding an HLA-G protein can be modified to remove a binding site for an miRNA of the miR148 family. Removal of this binding site may increase the stability of the mRNA.

[0274] A transgene can be introduced into an organism, cell, tissue, or organ to produce a transgene product. For example, non-human animals containing one or more transgenes are disclosed herein. One or more transgenes can be combined with one or more disruptions described herein. A transgene can be integrated into a cell. For example, a transgene can be integrated into the germline of an organism. When inserted into a cell, a transgene can be a complementary DNA (cDNA) segment, which is a copy of messenger RNA (mRNA), or it can be a gene itself that exists in its original region of genomic DNA (with or without introns).

[0275] A transgene may comprise a polynucleotide that encodes a protein of one species and expresses the protein in an animal of a different species. For example, a transgene may comprise a polynucleotide that encodes a human protein. Such a polynucleotide may be used to express a human protein (e.g., CD47) in a non-human animal (e.g., a pig). In some cases, the polynucleotide may be synthetic, e.g., differing in sequence and / or chemical characteristics from any naturally occurring polynucleotide.

[0276] A polynucleotide encoding a protein of species X may be optimized to express the protein in an animal of species Y. Codon usage bias (e.g., differences in the frequency of occurrence of synonymous codons in the encoding DNA) may be present. A codon may be a stretch of nucleotides (e.g., a stretch of three nucleotides) that encodes a specific amino acid residue in a polypeptide chain or that terminates translation (a stop codon). Different species may have different DNA codon preferences. An optimized polynucleotide may encode a protein of species X using codons of species Y, such that the polypeptide expresses the protein more efficiently in species Y compared to a native gene encoding the protein of species X. In some cases, the optimized polynucleotide may express a protein at least 5%, 10%, 20%, 40%, 80%, 90%, 1.5-fold, 2-fold, 5-fold, or 10-fold more efficiently in species Y than a native gene of species X encoding the same protein.

[0277] Human leukocyte antigen G (HLA-G)

[0278] HLA-G may be a potent immunoinhibitory and tolerogenic molecule. Expression of HLA-G in human fetuses may allow them to escape maternal immune responses. Neither stimulatory function nor responses to allogeneic HLA-G have been reported. HLA-G may be a non-classical HLA class I molecule. It may differ from classical MHC class I molecules by its genetic diversity, expression, structure, and function. HLA-G may be characterized by low allelic polymorphism. HLA-G expression may be restricted to trophoblast cells, adult thymic medulla, and stem cells. However, neo-expression of HLA-G may be induced in pathological conditions such as cancer, multiple sclerosis, inflammatory diseases, or viral infections.

[0279] Seven isoforms of HLA-G have been identified. Different isoforms may be the product of alternative splicing. Four of these may be membrane-bound (HLA-G1 to -G4), and three may be soluble isoforms (HLA-G5 to -G7). HLA-G1 and HLA-G5 isoforms exhibit the typical structure of classical HLA class I molecules, formed by a three-globular domain (α1 to α3) heavy chain noncovalently associated with β-2-microglobulin (B2M) and a nonapeptide. Truncated isoforms lack one or two domains, but they all contain the α1 domain and are all B2M-less isoforms.

[0280] HLA-G can exert its immunoinhibitory function through direct binding to inhibitory receptors, for example, ILT2 / CD85j / LILRB1, ILT4 / CD85d / LILRB2, or KIR2DL4 / CD158d.

[0281] ILT2 can be expressed by B cells, some T cells, some NK cells, and monocytes / dendritic cells. ILT4 may be myeloid-specific, and its expression may be restricted to monocytes / dendritic cells. KIR2DL4 may be a specific receptor for HLA-G. It binds to CD56 on NK cells.bright The ILT2 and ILT4 receptors can bind to a wide range of classical HLA molecules through the α3 domain and B2M, but HLA-G may be their highest affinity ligand.

[0282] ILT2-HLA-G interactions can mediate, for example, the inhibition of: i) the cytolytic function of NK and antigen-specific CD8+ T cells, ii) the alloproliferative response of CD4+ T cells, and iii) the maturation and function of dendritic cells. ILT2-HLA-G interactions can interfere with the function of both naive and memory B cells in vitro and in vivo. HLA-G can inhibit B cell proliferation, differentiation, and Ig secretion in both T cell-dependent and -independent models of B cell activation. HLA-G can act as a negative B cell regulator in modulating B cell Ab secretion. HLA-G can also induce the differentiation of regulatory T cells, which in turn can inhibit alloresponses and may themselves be involved in allograft tolerance.

[0283] Expression of HLA-G by tumor cells can enable them to evade immune surveillance mediated by host T lymphocytes and NK cells. Thus, expression of HLA-G by malignant cells may prevent immune elimination of tumors by inhibiting the activity of tumor-infiltrating NK cells, cytotoxic T lymphocytes (CTLs), and antigen-presenting cells (APCs).

[0284] Variations in HLA-G structure, particularly its monomeric / multimeric state and association with B2M, may play a role in the biological function of HLA-G, its regulation, and its interaction with the inhibitory receptors ILT2 and ILT4.

[0285] The ILT2 and ILT4 inhibitory receptors may have higher affinity for HLA-G multimers than for monomeric structures. HLA-G1 and HLA-G5 (HLA-G1 / 5) can form dimers through a disulfide bond (Cys42-Cys42) between unique cysteine ​​residues at position 42 within the α1 domain. Dimers of B2M-associated HLA-G1 may bind ILT2 and ILT4 with higher affinity than monomers. This increased affinity of the dimers may be due to an inclined orientation that exposes the ILT2 and ILT4 binding sites in the α3 domain, making them more accessible to the receptors. Both ILT2 and ILT4 bind to HLA-G at the level of residues F195 and Y197. It can bind to the α3 domain.

[0286] ILT2 and ILT4 bind differently to their HLA-G isoforms. ILT2 may recognize only B2M-associated HLA-G structures, whereas ILT4 may recognize both B2M-associated and B2M-free HLA-G heavy chains. B2M-free heavy chains have been detected on the cell surface in the supernatant of cultures of HLA-G-expressing cells. Furthermore, B2M-free HLA-G heavy chains may be the predominant structure produced by human villous trophoblast cells. The presence of (B2M-free) α1-α3 structures (HLA-G2 and G-6 isoforms) has been shown in the circulation of human heart transplant recipients, which may be associated with better allograft acceptance. α1-α3 structures may bind only ILT4, but not ILT2. However, α1-α3 dimers (dimerization of α1-α3 monomers is achieved through a disulfide bond between the two free cysteines at position 42) can be tolerogenic in vivo in an allogeneic murine skin transplant model. A synthetic (α1-α3) × 2 molecule could inhibit the growth of tumor cell lines that did not express ILT4. This may indicate the existence of an unknown receptor for HLA-G.

[0287] Thus, in one aspect, the present specification discloses genetically modified non-human animals and cells that comprise an exogenous nucleic acid sequence encoding HLA-G protein.The genetically modified non-human animals and cells can also comprise one or more additional genetic modifications, such as any of the genetic modifications (e.g., knock-in, knock-out, gene disruption, etc.) disclosed herein.For example, the genetically modified non-human animals and cells can also comprise another exogenous nucleic acid sequence encoding B2M protein.

[0288] The non-human animal may comprise one or more transgenes containing one or more polynucleotide inserts. The polynucleotide inserts may encode one or more proteins or functional fragments thereof. For example, the genetically modified non-human animal may comprise one or more exogenous nucleic acid sequences encoding one or more proteins or functional fragments thereof. In some cases, the non-human animal may comprise one or more transgenes containing one or more polynucleotide inserts encoding proteins capable of reducing the expression and / or function of MHC molecules (e.g., MHC I molecules and / or MHC II molecules). The one or more transgenes may comprise one or more polynucleotide inserts encoding an MHC I formation suppressor, a regulator of complement activation, an inhibitory ligand for NK cells, a member of the B7 family, CD47, a serine protease inhibitor, a galectin, and / or any fragment thereof. In some cases, the MHC I formation suppressor may be infected cell protein 47 (ICP47). In some cases, regulators of complement activation may include cluster of differentiation 46 (CD46), cluster of differentiation 55 (CD55), and cluster of differentiation 59 (CD59). In some cases, inhibitory ligands for NK cells may include leukocyte antigen E (HLA-E), human leukocyte antigen G (HLA-G), and beta-2-microglobulin (B2M). Inhibitory ligands for NK cells may be isoforms of HLA-G, such as HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7. For example, the inhibitory ligand for NK cells may be HLA-G1. An HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7) transgene may refer to a transgene that includes a nucleotide sequence encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7).As used herein, in some cases, a transgene encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7) can be a transgene that encodes 100% or about 100% of the amino acid sequence of HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7). In other cases, the transgene encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7) may be a transgene encoding the full-length or partial sequence of HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7). For example, the transgene may encode at least 99%, 95%, 90%, 80%, 70%, 60%, or 50%, or at least approximately these proportions, of the amino acid sequence of HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7). For example, the transgene may encode 90% of the amino acid sequence of HLA-G. The transgene may include a polynucleotide encoding a functional (e.g., partially or fully functional) HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7). In some cases, the transgene(s) may include one or more polynucleotide inserts encoding one or more of ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), and B2M. The genomic DNA sequence of HLA-G may have eight exons, resulting in seven isoforms through alternative splicing. The HLA-G1 isoform may exclude exon 7. The HLA-G2 isoform may exclude exons 3 and 7.Translation of intron 2 or intron 4 can result in secreted isoforms due to loss of expression of the transmembrane domain. The genomic sequence and cDNA map of HLA-G are shown in Figures 14A-14B. In some cases, B7 family members can include CD80, CD86, programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), CD275, CD276, V-set domain containing T-cell activation inhibitor 1 (VTCN1), platelet receptor Gi24, natural cytotoxicity-inducing receptor 3 ligand 1 (NR3L1), and HERV-H LTR binding 2 (HHLA2). For example, the B7 family member can be PD-L1 or PD-L2. In some cases, the serine protease inhibitor can be serine protease inhibitor 9 (Spi9). In some cases, the galectin can include galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14, and galectin-15. For example, the galectin can be galectin 9.

[0289] The genetically modified non-human animal may comprise reduced expression of one or more genes and one or more transgenes disclosed herein. In some cases, the genetically modified non-human animal may comprise reduced expression of one or more of NLRC5, TAP1, CXCL10, MICA, MICB, C3, CIITA, GGTA1, CMAH, and B4GALNT2, and one or more transgenes comprising one or more polynucleotide inserts encoding one or more of ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, PD-L1, PD-L2, CD47, Spi9, and galectin-9. In some cases, the genetically modified non-human animal may comprise reduced expression of GGTA1, CMAH, and B4GALNT2, as well as exogenous polynucleotides encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), CD47 (e.g., human CD47), PD-L1 (e.g., human PD-L1), and PD-L2 (e.g., human PD-L2). In some cases, the genetically modified non-human animal may comprise reduced expression of GGTA1, CMAH, and B4GALNT2, as well as exogenous polynucleotides encoding HLA-E, CD47 (e.g., human CD47), PD-L1 (e.g., human PD-L1), and PD-L2 (e.g., human PD-L2). In some cases, the genetically modified non-human animal may comprise reduced expression of NLRC5, C3, CXC10, GGTA1, CMAH, and B4GALNT2, as well as exogenous polynucleotides encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), CD47 (e.g., human CD47), PD-L1 (e.g., human PD-L1), and PD-L2 (e.g., human PD-L2).In some cases, the genetically modified non-human animal may comprise reduced expression of TAP1, C3, CXC10GGTA1, CMAH, and B4GALNT2, and exogenous polynucleotides encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), CD47 (e.g., human CD47), PD-L1 (e.g., human PD-L1), and PD-L2 (e.g., human PD-L2). In some cases, the genetically modified non-human animal may comprise reduced expression of NLRC5, C3, CXC10, GGTA1, CMAH, and B4GALNT2, and exogenous polynucleotides encoding HLA-E, CD47 (e.g., human CD47), PD-L1 (e.g., human PD-L1), and PD-L2 (e.g., human PD-L2). In some cases, the genetically modified non-human animal may comprise reduced expression of TAP1, C3, CXC10, GGTA1, CMAH, and B4GALNT2, and an exogenous polynucleotide encoding HLA-E. In some cases, the genetically modified non-human animal may comprise reduced expression of GGTA1, and a transgene comprising one or more polynucleotide inserts encoding HLA-E. In some cases, the genetically modified non-human animal may comprise reduced expression of GGTA1, and a transgene comprising one or more polynucleotide inserts encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7). In some cases, the genetically modified non-human animal may comprise a transgene comprising one or more polynucleotide inserts encoding HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7) inserted adjacent to a Rosa26 promoter, e.g., a porcine Rosa26 promoter.In some cases, the genetically modified non-human animal may comprise reduced expression of NLRC5, C3, GGTA1, CMAH, and B4GALNT2, and a transgene comprising a polynucleotide encoding a protein or functional fragment thereof, wherein the protein comprises HLA-G1, Spi9, PD-L1, PD-L2, CD47, and Galectin 9. In some cases, the genetically modified non-human animal may comprise reduced expression of TAP1, C3, GGTA1, CMAH, and B4GALNT2, and a transgene comprising a polynucleotide encoding a protein or functional fragment thereof, wherein the protein comprises HLA-G1, Spi9, PD-L1, PD-L2, CD47, and Galectin 9. In some cases, the genetically modified non-human animal may comprise reduced expression of NLRC5, TAP1, C3, GGTA1, CMAH, and B4GALNT2, and a transgene comprising a polynucleotide encoding a protein or a functional fragment thereof, wherein the protein comprises HLA-G1, Spi9, PD-L1, PD-L2, CD47, and galectin 9. In some cases, the genetically modified non-human animal may comprise reduced protein expression of NLRC5, C3, GGTA1, and CXCL10, and a transgene comprising a polynucleotide encoding a protein or a functional fragment thereof, wherein the protein comprises HLA-G1 or HLA-E. In some cases, the genetically modified non-human animal may comprise reduced protein expression of TAP1, C3, GGTA1, and CXCL10, and a transgene comprising a polynucleotide encoding a protein or a functional fragment thereof, wherein the protein comprises HLA-G1 or HLA-E. In some cases, the genetically modified non-human animal may comprise a transgene comprising reduced protein expression of NLRC5, TAP1, C3, GGTA1, and CXCL10, and a polynucleotide encoding a protein or a functional fragment thereof, wherein the protein comprises HLA-G1 or HLA-E. In some cases, the CD47, PD-L1, and PD-L2 encoded by the transgene in the present invention may be human CD47, human PD-L1, and human PD-L2.

[0290] A genetically modified non-human animal may contain a transgene inserted into a locus in the animal's genome. In some cases, the transgene may be inserted adjacent to or inside the promoter of the targeted gene. In some cases, the insertion of the transgene may reduce the expression of the targeted gene. The targeted gene may be a gene whose expression is reduced as disclosed herein. For example, the transgene may be inserted adjacent to or inside one or more promoters of NLRC5, TAP1, CXCL10, MICA, MICB, C3, CIITA, GGTA1, CMAH, and B4GALNT2. In some cases, the transgene may be inserted adjacent to or inside the promoter of GGTA1. In some cases, the transgene (e.g., CD47 transgene) may be inserted adjacent to a promoter that allows the transgene to be selectively expressed in a certain type of cell. For example, the CD47 transgene can be inserted adjacent to a promoter that allows for selective expression of the CD47 transgene in blood cells and spleen cells. One such promoter can be the GGTA1 promoter.

[0291] For example, the non-human animal can include one or more transgenes (e.g., exogenous nucleic acid sequences) that include one or more polynucleotide inserts of infected cell protein 47 (ICP47), cluster of differentiation 46 (CD46), cluster of differentiation 55 (CD55), cluster of differentiation 59 (CD59), HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, Spi9, PD-L1, PD-L2, CD47, galectin 9, any functional fragment thereof, or any combination thereof. A polynucleotide encoding ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), or B2M may encode one or more of the following human proteins: ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, Spi9, PD-L1, PD-L2, CD47, or galectin 9. A non-human animal may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more transgenes. For example, a non-human animal may contain one or more transgenes including ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, Spi9, PD-L1, PD-L2, CD47, galectin 9, any functional fragment thereof, or any combination thereof. A non-human animal may also contain a single transgene encoding ICP47. A non-human animal may, in some cases, contain a single transgene encoding CD59. The non-human animal may, in some cases, contain a single transgene encoding an HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7). The non-human animal may, in some cases, contain a single transgene encoding an HLA-E.In some cases, the non-human animal may contain a single transgene encoding B2M. The non-human animal may also contain two or more transgenes, where the two or more transgenes are ICP47, CD46, CD55, CD59, and / or any combination thereof. For example, the two or more transgenes may include CD59 and CD46 or CD59 and CD55. The non-human animal may also contain three or more transgenes, where the three or more transgenes may include ICP47, CD46, CD55, CD59, or any combination thereof. For example, the three or more transgenes may include CD59, CD46, and CD55. The non-human animal may also contain four or more transgenes, where the four or more transgenes may include ICP47, CD46, CD55, and CD59. The non-human animal may contain four or more transgenes, including ICP47, CD46, CD55, and CD59.

[0292] A combination of transgenes and gene disruption can be used. A non-human animal can include one or more reduced genes and one or more transgenes. For example, one or more genes whose expression is reduced can include any one of NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10, MICA, MICB, C3, CIITA, and / or any combination thereof, and one or more transgenes can include ICP47, CD46, CD55, CD59, any functional fragment thereof, and / or any combination thereof. For example, just to illustrate various combinations, one or more genes whose expression is disrupted can include NLRC5, and one or more transgenes can include ICP47. One or more genes whose expression is disrupted can also include TAP1, and one or more transgenes can include ICP47. The one or more genes whose expression is disrupted may also include NLRC5 and TAP1, and the one or more transgenes include ICP47. The one or more genes whose expression is disrupted may also include NLRC5, TAP1, and GGTA1, and the one or more transgenes include ICP47. The one or more genes whose expression is disrupted may also include NLRC5, TAP1, B4GALNT2, and CMAH, and the one or more transgenes include ICP47. The one or more genes whose expression is disrupted may also include NLRC5, TAP1, GGTA1, B4GALNT2, and CMAH, and the one or more transgenes include ICP47. The one or more genes whose expression is disrupted may also include NLRC5, and the one or more transgenes include CD59. The one or more genes whose expression is disrupted may also include TAP1, and the one or more transgenes include CD59. The one or more genes whose expression is disrupted may also include NLRC5 and TAP1, and the one or more transgenes include CD59.The one or more genes whose expression is disrupted may also include NLRC5, TAP1, and GGTA1, and the one or more transgenes include CD59. The one or more genes whose expression is disrupted may also include NLRC5, TAP1, B4GALNT2, and CMAH, and the one or more transgenes include CD59. The one or more genes whose expression is disrupted may also include NLRC5, TAP1, GGTA1, B4GALNT2, and CMAH, and the one or more transgenes include CD59.

[0293] In some cases, the first exon of a gene is genetically modified.For example, one or more first exons of the gene that can be genetically modified can be selected from the group consisting of NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10, MICA, MICB, C3, CIITA, and any combination thereof.For example, Figure 112A shows the guide RNA that targets the first exon of the NLCR5 gene.In other cases, the second exon of a gene is targeted.For example, Figure 105, Figure 106, and Figure 107 show the relevant sequences of primer pairs for creating guide RNAs that target the first and second exons, and the primer sequences for determining genetic modification by sequencing.

[0294] Transgenes that can be used and are specifically contemplated can include genes exhibiting a certain identity and / or homology to the genes disclosed herein, such as ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, Spi9, PD-L1, PD-L2, CD47, Galectin 9, any functional fragment thereof, and / or any combination thereof. Thus, it is contemplated that genes can be used as transgenes if they exhibit (at the nucleic acid or protein level) at least 60%, 70%, 80%, 90%, 95%, 98%, or 99%, or at least approximately these percentages of homology, e.g., at least 99%-90%, 90%-80%, 80%-70%, 70%-60%, or at least approximately these percentages of homology. It is also contemplated that genes exhibiting (at the nucleic acid or protein level) at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, or at least approximately these percentages of identity, e.g., at least 99%-90%, 90%-80%, 80%-70%, 70%-60%, or at least approximately these percentages of identity, can be used as transgenes.

[0295] The non-human animal may also contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more dominant-negative transgenes. Expression of a dominant-negative transgene may suppress the expression and / or function of the wild-type counterpart of the dominant-negative transgene. Thus, for example, a non-human animal containing a dominant-negative transgene X may have a similar phenotype compared to a different non-human animal containing an X gene with reduced expression. The one or more dominant-negative transgenes may be a dominant-negative NLRC5, a dominant-negative TAP1, a dominant-negative GGTA1, a dominant-negative CMAH, a dominant-negative B4GALNT2, a dominant-negative CXCL10, a dominant-negative MICA, a dominant-negative MICB, a dominant-negative CIITA, a dominant-negative C3, or any combination thereof.

[0296] Also provided are non-human animals comprising one or more transgenes encoding one or more nucleic acids capable of suppressing gene expression, e.g., knocking down a gene. RNAs that suppress gene expression can include, but are not limited to, shRNA, siRNA, RNAi, and microRNA. For example, siRNA, RNAi, and / or microRNA can be administered to non-human animals to suppress gene expression. Furthermore, non-human animals can comprise one or more transgenes encoding shRNA. The shRNA can be specific to a particular gene. For example, the shRNA can be specific to any gene described herein, including, but not limited to, NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10, MICA, MICB, B4GALNT2, CIITA, C3, and / or any combination thereof.

[0297] When transplanted into a subject, cells, tissues, or organs derived from a genetically modified non-human animal may induce a lower immune response (e.g., graft rejection) in the subject compared to cells, tissues, or organs derived from a non-genetically modified counterpart. In some cases, the immune response may include activation, proliferation, and cytotoxicity of T cells (e.g., CD8+ T cells and / or CD4+ T cells) and NK cells. Thus, the phenotype of the genetically modified cells disclosed herein can be measured by co-culturing the cells with NK cells, T cells (e.g., CD8+ T cells or CD4+ T cells) and testing the activation, proliferation, and cytotoxicity of NK cells or T cells. In some cases, the activation, proliferation, and cytotoxicity of T cells or NK cells induced by the genetically modified cells may be lower than that induced by non-genetically modified cells. In some cases, the phenotype of the genetically modified cells of the present invention can be measured by enzyme-linked immunospot (ELISPOT) assay.

[0298] One or more transgenes can be derived from different species.For example, one or more transgenes can include human genes, mouse genes, rat genes, pig genes, cow genes, dog genes, cat genes, monkey genes, chimpanzee genes, or any combination thereof.For example, transgenes can be derived from humans with human genetic sequences.One or more transgenes can include human genes.In some cases, one or more transgenes are not adenovirus genes.

[0299] Transgenes can be inserted randomly or site-specifically into the genome of non-human animals. For example, transgenes can be inserted into random loci in the genome of non-human animals. These transgenes can be fully functional regardless of where they are inserted in the genome. For example, transgenes can encode their own promoters or can be inserted into a position under the control of an endogenous promoter. Alternatively, transgenes can be inserted into genes, such as introns or exons, promoters, or non-coding regions of genes. Transgenes can be integrated into the first exon of genes.

[0300] In some cases, more than one copy of a transgene can be inserted into more than one random locus in a genome. For example, multiple copies can be inserted into random loci in a genome. This can result in increased overall expression compared to when a transgene is inserted randomly once. Alternatively, one copy of a transgene can be inserted into one gene, and another copy of the transgene can be inserted into a different gene. A transgene can be targeted to be inserted into a specific locus in the genome of a non-human animal.

[0301] The expression of a transgene can be controlled by one or more promoters. The promoter can be a ubiquitous promoter, a tissue-specific promoter, or an inducible promoter. The expression of a transgene inserted adjacent to a promoter can be regulated. For example, if a transgene is inserted near or next to a ubiquitous promoter, the transgene will be expressed in all cells of a non-human animal. Some ubiquitous promoters can be the CAGGS promoter, the hCMV promoter, the PGK promoter, the SV40 promoter, or the Rosa26 promoter.

[0302] The promoter may be an endogenous promoter or an exogenous promoter. For example, one or more transgenes may be inserted adjacent to an endogenous or exogenous Rosa26 promoter. Furthermore, the promoter may be specific to a non-human animal. For example, one or more transgenes may be inserted adjacent to a porcine Rosa26 promoter.

[0303] A tissue-specific promoter (which may be synonymous with a cell-specific promoter) can be used to control the location of expression. For example, one or more transgenes can be inserted adjacent to a tissue-specific promoter. The tissue-specific promoter can be a FABP promoter, an Lck promoter, a CamKII promoter, a CD19 promoter, a keratin promoter, an albumin promoter, an aP2 promoter, an insulin promoter, an MCK promoter, a MyHC promoter, a WAP promoter, or a Col2A promoter. For example, the promoter can be a pancreatic-specific promoter, such as an insulin promoter.

[0304] Inducible promoters can also be used. These inducible promoters can be turned on or off as desired by adding or removing an inducer. It is contemplated that the inducible promoter can be Lac, tac, trc, trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx, and / or Trex.

[0305] The non-human animals or cells described herein can contain a transgene encoding insulin.The transgene encoding insulin can be a human gene, a mouse gene, a rat gene, a pig gene, a cow gene, a dog gene, a cat gene, a monkey gene, a chimpanzee gene, or any other mammalian gene.For example, the transgene encoding insulin can be a human gene.The transgene encoding insulin can also be a chimeric gene, for example, a partially human gene.

[0306] Transgene expression can be measured by detecting the level of transgene transcripts. For example, transgene expression can be measured by Northern blotting, nuclease protection assays (e.g., RNase protection assays), reverse transcription PCR, quantitative PCR (e.g., real-time PCR, such as real-time quantitative reverse transcription PCR), in situ hybridization (e.g., fluorescent in situ hybridization (FISH)), dot blot analysis, differential display, serial analysis of gene expression, subtractive hybridization, microarrays, nanostrings, and / or sequencing (e.g., next-generation sequencing). In some cases, transgene expression can be measured by detecting the protein encoded by the gene. For example, expression of one or more genes can be measured by protein immunostaining, protein immunoprecipitation, electrophoresis (e.g., SDS-PAGE), Western blotting, bicinchoninic acid assay, spectrophotometry, mass spectrometry, enzyme assays (e.g., enzyme-linked immunosorbent assays), immunohistochemistry, flow cytometry, and / or immunocytochemistry. In some cases, the expression of transgene can be measured by microscopy. Microscopy can be optical, electron, or scanning probe microscopy. In some cases, optical microscopy includes bright field, oblique illumination, cross-polarized light, dispersed staining, dark field, phase contrast, differential interference contrast, interference reflection microscopy, fluorescence (for example, when particles, for example, cells, are immunostained), confocal, single-plane illumination microscopy, light sheet fluorescence microscopy, deconvolution, or sequential time-encoded amplitude microscopy.

[0307] Transgene insertion can be verified by genotyping. Genotyping methods can include sequencing, restriction fragment length polymorphism identification (RFLPI), random amplified polymorphism detection (RAPD), amplified fragment length polymorphism detection (AFLPD), PCR (e.g., long-range PCR or stepwise PCR), allele-specific oligonucleotide (ASO) probes, and hybridization to DNA microarrays or beads. In some cases, genotyping can be performed by sequencing. In some cases, sequencing can be high-fidelity sequencing. Sequencing methods can include Maxam-Gilbert sequencing, chain termination methods (e.g., Sanger sequencing), shotgun sequencing, and bridge PCR. In some cases, genotyping can be performed by next-generation sequencing. Next-generation sequencing methods include massively parallel signature sequencing, colony sequencing, pyrosequencing (e.g., pyrosequencing developed by 454 Life Sciences), single-molecule real-time sequencing (e.g., by Pacific Biosciences), ion semiconductor sequencing (e.g., by Ion Torrent semiconductor sequencing), sequencing-by-synthesis (e.g., by Illumina's Solexa sequencing), sequencing by ligation (e.g., by Applied Biosystems' SOLiD sequencing), DNA nanoball sequencing, and heliscope single-molecule sequencing. In some cases, genotyping of non-human animals in the present invention may involve whole-genome sequencing analysis.

[0308] In some cases, transgene insertion in an animal can be verified by sequencing (e.g., next-generation sequencing) a portion of the transgene or the entire transgene. For example, in pigs, insertion of a transgene flanked by the Rosa26 promoter can be verified by next-generation sequencing of exons 1-4 of Rosa using, for example, the forward primer 5'-cgcctagagaagaggctgtg-3' (SEQ ID NO: 35) and the reverse primer 5'-ctgctgtggctgtggtgtag-3' (SEQ ID NO: 36). [Table 2] [Table 3]

[0309] Non-human animal populations Single non-human animals, as well as populations of non-human animals, are provided herein. The population of non-human animals can be genetically identical. The population of non-human animals can also be phenotypically identical. The population of non-human animals can be both phenotypically identical and genetically identical.

[0310] Further provided herein is a population of non-human animals that can be genetically modified. For example, the population can include at least 2, 5, 10, 50, 100, or 200, or at least about these numbers, of the non-human animals disclosed herein. The non-human animals of the population can have the same phenotype. For example, the non-human animals of the population can be clones. The population of non-human animals can have the same physical characteristics. The non-human animals of the population with the same phenotype can contain the same transgene(s). The non-human animals of the population with the same phenotype can also contain the same gene(s) with reduced expression. The non-human animals of the population with the same phenotype can also contain the same gene(s) with reduced expression and the same transgene(s). The population of non-human animals can include at least 2, 5, 10, 50, 100, or 200, or at least about these numbers, of non-human animals with the same phenotype. For example, the phenotype of any particular litter can have the same phenotype (e.g., in one example, anywhere from 1 to about 20 non-human animals). The non-human animals of the population can be pigs with the same phenotype.

[0311] The non-human animals of the population may have the same genotype. For example, all nucleic acid sequences in the chromosomes of the non-human animals in the population may be identical. The non-human animals of the population with the same genotype may contain the same transgene(s). The non-human animals of the population with the same genotype may also contain the same gene(s) with reduced expression. The non-human animals of the population with the same genotype may also contain the same gene(s) with reduced expression and the same transgene(s). The population of non-human animals may contain at least 2, 5, 50, 100, or 200, or at least approximately these numbers of non-human animals with the same genotype. The non-human animals of the population may be pigs with the same genotype.

[0312] Cells derived from two or more non-human animals with the same genotype and / or phenotype can be used in tolerization vaccines. In some cases, the tolerization vaccines disclosed herein can contain a plurality of cells (e.g., genetically modified cells) derived from two or more non-human animals (e.g., pigs) with the same genotype and / or phenotype. A method for immunotolerizing a recipient to a transplant can include administering to the recipient a tolerization vaccine containing a plurality of cells (e.g., genetically modified cells) derived from two or more non-human animals with the same genotype or phenotype.

[0313] Cells from two or more non-human animals with the same genotype and / or phenotype can be used in transplantation.In some cases, a transplant (e.g., a xenograft or allograft) can include multiple cells from two or more non-human animals with the same genotype and / or phenotype.In embodiments of the methods described herein, for example, a method for treating a disease in a subject in need thereof can include transplanting multiple cells (e.g., genetically modified cells) from two or more non-human animals with the same genotype and / or phenotype.

[0314] A population of non-human animals can be generated using any method known in the art. In some cases, a population of non-human animals can be generated by breeding. For example, inbreeding can be used to generate phenotypically or genetically identical non-human animals or populations of non-human animals. For example, sibling-to-sibling, parent-to-child, grandchild-to-grandparent, or great-grandchild-to-great-grandparent inbreeding can be used. Successive rounds of inbreeding can eventually produce phenotypically or genetically identical non-human animals. For example, at least 2, 3, 4, 5, 10, 20, 30, 40, or 50 generations of inbreeding, or at least approximately these numbers of generations, can produce phenotypically and / or genetically identical non-human animals. After 10 to 20 generations of inbreeding, the genetic makeup of the non-human animals is considered to be at least 99% pure. Continual inbreeding can lead to non-human animals that are essentially homogeneous, or nearly homogeneous, as non-human animals may not have identical twins.

[0315] Mating can be performed using non-human animals with the same genotype. For example, the non-human animals have the same gene(s) with reduced expression and / or the same transgene(s). Mating can also be performed using non-human animals with different genotypes. Mating can be performed using genetically modified non-human animals and non-genetically modified non-human animals, for example, genetically modified sows and wild-type boars, or genetically modified boars and wild-type sows. All these combinations of mating can be used to produce the desired non-human animals.

[0316] Populations of genetically modified non-human animals can also be produced by cloning. For example, a population of genetically modified non-human animal cells can asexually produce a similar population of genetically or phenotypically identical individual non-human animals. Cloning can be performed by various methods, such as twinning (e.g., isolating one or more cells from an embryo and developing them into new embryos), somatic cell nuclear transfer, or artificial insemination. Further details of methods are provided throughout this disclosure.

[0317] II. Genetically modified cells Disclosed herein are one or more genetically modified cells that can be used to treat or prevent disease. These genetically modified cells can be derived from a genetically modified non-human animal. For example, the genetically modified non-human animal disclosed above can be processed to isolate one or more cells to produce isolated genetically modified cells. These isolated cells can also optionally be further genetically modified cells. However, cells can also be modified ex vivo outside of an animal, for example, using modified or unmodified human or non-human animal cells. For example, cells (including human and non-human animal cells) can be modified in culture. It is also contemplated that genetically modified cells can be used to generate the genetically modified non-human animals described herein. In some cases, genetically modified cells can be isolated from a genetically modified animal. In some cases, genetically modified cells can be derived from cells derived from an animal that is not genetically modified. Cell isolation can be performed by methods known in the art, including methods for isolating and culturing primary cells. It is specifically contemplated that the genetically modified cells are not extracted from a human.

[0318] Therefore, all that can be applied to genetically modified non-human animals, including the various production methods described throughout, can also be applied to the present invention.For example, all genes that are disrupted and transgenes that are overexpressed can be applied to the production of genetically modified cells used in the present invention.In addition, any method that tests the genotype and expression of genes in genetically modified non-human animals described throughout can be used to test the genetic modification of cells.

[0319] The genetically modified cells can be derived from a member of the superorder Laurasiatheria or a non-human primate. Such genetically modified cells can be isolated from a member of the superorder Laurasiatheria or a non-human primate. Alternatively, such genetically modified cells can be derived from a member of the superorder Laurasiatheria or a non-human primate. For example, the genetically modified cells can be produced from cells isolated from a member of the superorder Laurasiatheria or a non-human primate, for example, using cell culture or genetic modification methods.

[0320] Genetically modified cells, for example, cells derived from genetically modified animals or cells produced ex vivo, can be analyzed and sorted.In some cases, genetically modified cells can be analyzed and sorted by flow cytometry, for example, fluorescence-activated cell sorting.For example, genetically modified cells that express transgenes can be detected and purified from other cells by flow cytometry based on the label (for example, fluorescent label) that recognizes the polypeptide encoded by transgene.

[0321] In some cases, genetically modified cells can reduce, inhibit, or eliminate immune response.For example, genetic modification can reduce the effector function of cells, reduce proliferation, reduce persistence, and / or reduce the expression of cytolytic effector molecules such as granzyme B and CD107 alpha in immune cells.Immune cells can be monocytes and / or macrophages.In some cases, T cell-derived cytokines such as IFN-g can activate macrophages through the secretion of IFN-gamma.In some cases, T cell activation can be inhibited, and also cause the inhibition of macrophages.

[0322] Stem cells can be used, including non-human animal and human stem cells.Stem cells do not have the ability to generate living humans.For example, stem cells can be irreversibly differentiated so that they cannot generate living humans.Stem cells can be pluripotent as long as they cannot generate living humans.

[0323] As discussed above in the section on genetically modified non-human animals, genetically modified cells can contain one or more genes whose expression is reduced.The same genes as those disclosed above for genetically modified non-human animals can be disrupted.For example, genetically modified cells can contain one or more genes whose expression is disrupted (e.g., reduced), and the one or more genes can include NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10, MICA, MICB, C3, CIITA, and / or any combination thereof.In addition, genetically modified cells can contain one or more transgenes containing one or more polynucleotide insertions. For example, the genetically modified cells can comprise one or more transgenes comprising one or more polynucleotide inserts of ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, Spi9, PD-L1, PD-L2, CD47, Galectin 9, any functional fragment thereof, or any combination thereof. The genetically modified cells can comprise one or more reduced genes and one or more transgenes. For example, the one or more genes with reduced expression may include any one of NLRC5, TAP1, GGTA1, B4GALNT2, CMAH, CXCL10, MICA, MICB, CIITA, and / or any combination thereof, and the one or more transgenes may include ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, Spi9, PD-L1, PD-L2, CD47, galectin 9, any functional fragment thereof, and / or any combination thereof.In some cases, the genetically modified cells may comprise reduced expression of NLRC5, C3, GGTA1, CMAH, and B4GALNT2, and a transgene comprising a polynucleotide encoding a protein or functional fragment thereof, wherein the protein comprises HLA-G1, Spi9, PD-L1, PD-L2, CD47, and Galectin 9. In some cases, the genetically modified cells may comprise reduced expression of TAP1, C3, GGTA1, CMAH, and B4GALNT2, and a transgene comprising a polynucleotide encoding a protein or functional fragment thereof, wherein the protein comprises HLA-G1, Spi9, PD-L1, PD-L2, CD47, and Galectin 9. In some cases, the genetically modified cells may contain transgenes containing reduced expression of NLRC5, TAP1, C3, GGTA1, CMAH, and B4GALNT2, as well as polynucleotides encoding proteins or functional fragments thereof, including HLA-G1, Spi9, PD-L1, PD-L2, CD47, and galectin-9. In some cases, the CD47, PD-L1, and PD-L2 encoded by the transgenes in the present invention may be human CD47, human PD-L1, and human PD0-L2. In some cases, the genetically modified cells may have their surface coated with CD47. Coating of CD47 on the cell surface can be achieved by biotinylating the cell surface and then incubating the biotinylated cells with streptavidin-CD47 chimeric protein. The coated CD47 may be human CD47.

[0324] As discussed above in the section on genetically modified non-human animals, genetically modified cells can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more disrupted genes. Genetically modified cells can also contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more transgenes.

[0325] As discussed in detail above, genetically modified cells, e.g., porcine cells, can also contain dominant-negative transgenes and / or transgenes that express one or more knockdown genes. As also discussed above, expression of the transgenes can be controlled by one or more promoters.

[0326] The genetically modified cells may be one or more cells derived from a tissue or organ, including the brain, lung, liver, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, smooth muscle, skin, bone, adipose tissue, hair, thyroid, trachea, gallbladder, kidney, ureter, bladder, aorta, vein, esophagus, diaphragm, stomach, rectum, adrenal gland, bronchi, ear, eye, retina, reproductive organs, hypothalamus, larynx, nose, tongue, spinal cord, or ureter, uterus, ovary, and testis. For example, the genetically modified cells, such as pig cells, may be derived from the brain, heart, liver, skin, intestine, lung, kidney, eye, small intestine, or pancreas. In some cases, the genetically modified cells may be derived from the pancreas. More specifically, the pancreatic cells may be islet cells. Furthermore, the one or more cells may be pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic F cells (e.g., PP cells), or pancreatic epsilon cells. For example, the genetically modified cell can be a pancreatic β cell. The tissue or organ disclosed herein can comprise one or more genetically modified cells. The tissue or organ can be derived from one or more genetically modified animals described herein, for example, pancreatic tissue, such as pancreatic islets derived from one or more genetically modified pigs.

[0327] The genetically modified cells, e.g., porcine cells, may comprise one or more types of cells, including hair follicles, keratinocytes, gonadotropin-producing cells, corticotropes, thyrotropes, somatotropes, prolactin-producing cells, chromaffin cells, parafollicular cells, glomus cells, melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts, keratocytes, retinal Müller cells, retinal pigment epithelial cells, neurons, glia (e.g., oligodendrocytes, astrocytes), ependymal cells, pineal cells, alveolar epithelial cells (e.g., type I and type II pneumocytes), Clara cells, goblet cells, G cells, D cells, ECL cells, and chief cells of the stomach. , parietal cells, gastric pit cells, K cells, D cells, I cells, goblet cells, Paneth cells, enterocytes, M cells, hepatocytes, hepatic stellate cells (e.g., Kupffer cells derived from the mesoderm), gallbladder cells, central acinar cells, pancreatic stellate cells, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells (e.g., PP cells), pancreatic ε cells, thyroid (e.g., follicular cells), parathyroid (e.g., parathyroid chief cells), eosinophilic cells, urothelial cells, osteoblasts, osteocytes, chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, myosatellite cells, tendon cells, cardiac myocytes, lipoblasts, adipocytes, interstitial cells of Cajal, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, interstitial cells These include cells, interstitial cells, telocytes, simple epithelial cells, podocytes, renal proximal tubule brush border cells, Sertoli cells, Leydig cells, granulosa cells, non-ciliated epithelial cells, germ cells, sperm, eggs, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, hemangioblasts, mesoangioblasts, and pericytes, mural cells. The genetically modified cells can potentially be any cells used in cell therapy. For example, cell therapy can be the replacement or replacement of pancreatic beta cells for diseases such as diabetes.

[0328] The genetically modified cells, e.g., pig cells, can be derived (e.g., extracted) from a non-human animal. One or more cells can be derived from a mature adult non-human animal. However, one or more cells can also be derived from fetal or neonatal tissue.

[0329] Depending on the disease, one or more cells can be derived from a transgenic non-human animal that has grown to a size sufficient to be useful as an adult donor, e.g., an islet cell donor. In some cases, the non-human animal can be an animal past the age of weaning. For example, the non-human animal can be at least or at least about 6 months old. In some cases, the non-human animal can be at least or at least about 18 months old. In some cases, the non-human animal has reached the age of survival and mating. For example, islets for xenotransplantation can be derived from a neonatal (e.g., 3-7 days old) or pre-weaned (e.g., 14-21 days old) donor pig. One or more genetically modified cells, e.g., pig cells, can be cultured cells. For example, the cultured cells can be derived from wild-type cells or genetically modified cells (as described herein). Furthermore, the cultured cells can be primary cells. The primary cells can be extracted and frozen, e.g., in liquid nitrogen or at -20°C to -80°C. Cultured cells may also be immortalized by known methods and frozen and stored, for example, in liquid nitrogen or at -20°C to -80°C.

[0330] The genetically modified cells described herein, e.g., porcine cells, may have a lower risk of rejection compared to wild-type, non-genetically modified cells when transplanted.

[0331] Disclosed herein are vectors comprising the polynucleotide sequence of ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, Spi9, PD-L1, PD-L2, CD47, Galectin 9, any functional fragment thereof, or any combination thereof. These vectors can be inserted into the genome of a cell (by transfection, transformation, viral delivery, or any other known method). These vectors may encode ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M Spi9, PD-L1, PD-L2, CD47, and / or Galectin 9 proteins or functional fragments thereof.

[0332] Contemplated vectors include, but are not limited to, plasmid vectors, artificial / minichromosomes, transposons, and viral vectors. Also disclosed herein are isolated or synthetic nucleic acids, including RNA, wherein the RNA is encoded by any of the sequences in Table 2. The RNA can also encode any sequence that exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, or at least about these percentages of homology to any of the sequences in Table 2. The RNA can also encode any sequence that exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, or at least about these percentages of identity to any of the sequences in Table 2.

[0333] The RNA may be a single-stranded guide RNA. The present disclosure also provides isolated or synthetic nucleic acids comprising any of the sequences in Table 1. The RNA may also be isolated or synthetic nucleic acids that exhibit at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology to any of the sequences in Table 1, or at least about these percentages. The RNA may also be isolated or synthetic nucleic acids that exhibit at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology to any of the sequences in Table 1, or at least about these percentages.

[0334] Guide RNA sequence can be used to target one or more genes in the genome of non-human animals.For example, guide RNA sequence can target a single gene in the genome of non-human animals.In some cases, guide RNA sequence can target one or more target sites of one or more genes in the genome of non-human animals.

[0335] The genetically modified cells can also be leukocytes, lymphocytes, B lymphocytes, or any other cells, such as islet cells, islet beta cells, or hepatocytes. These cells can be fixed or made apoptotic by any of the methods disclosed herein, for example, ECDI fixation.

[0336] Genetically modified cells can be derived (e.g., recovered) from non-human fetal animals, perinatal non-human animals, newborn non-human animals, pre-weaning non-human animals, juvenile non-human animals, adult non-human animals, or any combination thereof.In some cases, genetically modified non-human animal cells can be derived from embryonic tissue, such as embryonic pancreatic tissue.For example, genetically modified cells can be derived (e.g., recovered) from embryonic pig pancreatic tissue from 42-day embryo (E42).

[0337] The term "fetal animal" and its grammatical equivalents may refer to any unborn animal offspring. The term "perinatal animal" and its grammatical equivalents may refer to an animal just before or just after birth. For example, the perinatal period may begin between 20 and 28 weeks of gestation and end between 1 and 4 weeks after birth. The term "neonatal animal" and its grammatical equivalents may refer to any newly born animal. For example, a newborn animal may be an animal that is less than one month old. The term "preweaned non-human animal" and its grammatical equivalents may refer to any animal before it is weaned from its mother's milk.

[0338] Genetically modified non-human animal cells can be formulated into pharmaceutical compositions.For example, genetically modified non-human animal cells can be combined with a pharmaceutically acceptable excipient.A usable excipient is saline.The pharmaceutical composition can be used to treat patients who need transplantation.

[0339] Genetically modified cells can comprise any gene expression reduction and / or any transgene disclosed herein.The genetic modification of cells can be carried out using any of the same methods as those described herein for producing genetically modified animals.In some cases, the method for producing genetically modified cells derived from non-human animals can include reducing the expression of one or more genes and / or inserting one or more transgenes.Reducing gene expression and / or inserting transgenes can be carried out using any method described in this application, for example, gene editing.

[0340] Genetically modified cells derived from stem cells The genetically modified cells can be stem cells. These genetically modified stem cells can be used to generate a potentially unlimited supply of cells that can then be processed into fixed or apoptotic cells by the methods disclosed herein. As discussed above, stem cells cannot generate living humans.

[0341] The generation of hundreds of millions of insulin-producing, glucose-responsive pancreatic beta cells from human pluripotent stem cells provides an unprecedented cell source for cell transplantation therapy of diabetes (Pagliuca et al., 2014). Other human stem cell (embryonic, pluripotent, placental, induced pluripotent, etc.) derived cell sources for cell transplantation therapy of diabetes and other diseases are under development.

[0342] These stem cell-derived cell transplants are subject to rejection, which may be mediated by CD8+ T cells. In recipients with type 1 diabetes, functional beta cells derived from human stem cells are subject to rejection and recurrence of autoimmunity, both of which are thought to be mediated by CD8+ T cells.

[0343] To interfere with the activation and effector function of these alloreactive and autoreactive CD8+ T cells, established molecular methods of genetic modification, including CRISP / Cas9 gene targeting, can be used to mutate the NLRC5, TAP1, and / or B2M genes in human stem cells to prevent cell surface expression of functional MHC class I in stem cell-derived partially or fully differentiated cell grafts. Thus, transplantation of human stem cell-derived cell grafts lacking functional expression of MHC class I may minimize the need for immunosuppression that would otherwise be required to prevent rejection and autoimmune relapse.

[0344] However, the lack of MHC class I expression on transplanted human cells can lead to passive activation of natural killer (NK) cells (Ohlen et al., 1989). NK cell cytotoxicity can be overcome by expression of the human MHC class I gene HLA-E, which stimulates the inhibitory receptor CD94 / NKG2A on NK cells to prevent cell killing (Weiss et al., 2009; Lilienfeld et al., 2007; Sasaki et al., 1999). Successful expression of the HLA-E gene depended on coexpression of the human B2M (beta-2 microglobulin) gene and its cognate peptide (Weiss et al., 2009; Lilienfeld et al., 2007; Sasaki et al., 1999; Pascasova et al., 1999). Nuclease-mediated breaks in stem cell DNA allow for the insertion of one or more genes via homology-directed repair. During transgene insertion, the HLA-E and hB2M genes can be consecutively integrated into regions of nuclease-mediated DNA cleavage to prevent expression of the target gene (eg, NLRC5).

[0345] To further minimize, if not eliminate, the need for maintenance immunosuppression in recipients of stem cell-derived cell transplants lacking functional expression of MHC class I, recipients of these transplants can also be treated with the tolerizing apoptotic donor cells disclosed herein.

[0346] The method for producing insulin-producing pancreatic beta cells (Pagliuca et al., 2014) may be applicable to primary isolated pluripotent embryonic stem cells or stem-like cells from non-human (e.g., porcine) animals (Goncalves et al., 2014; Hall et al., 2008). However, recipients of these insulin-producing pancreatic beta cells may have an active immune response that threatens the success of the transplant. To overcome antibody-mediated and CD8+ T cell immune attack, donor animals can be genetically modified prior to isolating primary non-human pluripotent embryonic stem cells or stem-like cells to prevent expression of the GGTA1, CMAH, B4GalNT2, or MHC class I-related genes disclosed throughout this application. The pluripotent embryonic stem cells or stem-like cells isolated from the genetically modified animals can then potentially be differentiated into millions of insulin-producing pancreatic beta cells.

[0347] Transplantation of cells derived from heterologous stem cells may be desirable in some cases. For example, the use of human embryonic stem cells may be ethically objectionable to the recipient. Therefore, human recipients may be more comfortable receiving cell transplants derived from non-human sources of embryonic stem cells.

[0348] Non-human stem cells may include porcine stem cells. These stem cells may be derived from wild-type pigs or genetically engineered pigs. If derived from wild-type pigs, genetic manipulation using established molecular methods of gene modification, including CRISP / Cas9 gene targeting, may be best performed at the stem cell stage. Genetic manipulation may target disruption of the expression of NLRC5, TAP1, and / or B2M genes to prevent functional expression of MHC class I. Disruption of genes such as NLRC5, TAP1, and B2M in the graft may interfere with the post-transplant activation of autoreactive CD8+ T cells by causing a lack of functional expression of MHC class I on graft cells, including islet beta cells. This may therefore protect the transplant, e.g., transplanted islet beta cells, from the cytolytic effector function of autoreactive CD8+ T cells.

[0349] However, because genetic manipulation of stem cells can alter their differentiation capacity, approaches can be taken to generate stem cell lines from genetically engineered pigs, including pigs in which expression of the NLRC5, TAP1, and / or B2M genes has been disrupted.

[0350] The generation of porcine-derived stem cells that have also been genetically modified to prevent expression of the GGTA1, CMAH, B4GalNT2 genes, or modified to express transgenes encoding the complement regulatory proteins CD46, CD55, or CD59 as disclosed throughout this application, may further improve the therapeutic use of insulin-producing pancreatic beta cells or other cell therapy products. Similarly, the same strategies described herein can be used in other methods and compositions described throughout.

[0351] Similar to recipients of human stem cell-derived cell grafts lacking functional expression of MHC class I, the need for maintenance immunosuppression in recipients of porcine stem cell-derived grafts can be further minimized by peritransplant treatment with tolerized apoptotic donor cells.

[0352] III. Tolerizing Vaccines Traditionally, vaccines are used to confer immunity to a host. For example, injecting an inactivated virus with an adjuvant under the skin can lead to transient or permanent immunity against an active and / or virulent version of the virus. This is sometimes referred to as a positive vaccine (Figure 3). However, inactivated cells (e.g., cells from a donor or a genetically distinct animal) injected intravenously can result in tolerance of donor cells or cells with similar cell markers. This is sometimes referred to as a tolerizing vaccine (or sometimes a negative vaccine) (Figure 3). Inactivated cells can be injected without an adjuvant. Alternatively, inactivated cells may be injected with an adjuvant. These tolerizing vaccines can be advantageous in transplants, such as xenotransplants, by tolerizing the recipient and preventing rejection. Tolerization can be conferred on the recipient without the use of immunosuppressive therapy. However, in some cases, other immunosuppressive therapies combined with tolerizing vaccines can reduce transplant rejection.

[0353] Figure 4 demonstrates an exemplary approach for extending the survival of a transplanted graft (e.g., a xenograft) in a subject (e.g., a human or non-human primate) by injecting (e.g., intravenously) donor-derived apoptotic cells for tolerizing vaccination assisted by transient immunosuppression. The donor may provide cells (e.g., spleen cells) as a tolerizing vaccine along with the xenograft (e.g., islets) for transplantation. The tolerizing vaccine cells may be apoptotic cells (e.g., by ECDI fixation) that can be administered to the recipient before (e.g., a first vaccine on day 7 pre-transplant) and after (e.g., a booster vaccine on day 1 post-transplant). The tolerizing vaccine may provide transient immunosuppression that extends the survival time of the transplanted graft (e.g., islets).

[0354] The tolerizing vaccine may comprise one or more of the following types of cells: i) apoptotic cells, including genotypically identical cells with reduced expression of GGTA1 alone, or GGTA1 and CMAH, or GGTA1, CMAH, and B4GALNT2. This can minimize or eliminate cell-mediated immunity and cell-dependent antibody-mediated immunity against organ, tissue, cell, and cell line transplants (e.g., xenografts) from animals that are genotypically identical to the donor animal of the apoptotic cell vaccine, or that have undergone further genetic modification (e.g., suppression of the NLRC5, TAP1, MICA, MICB, CXCL10, C3, CIITA genes, or expression of transgenes comprising two or more polynucleotide inserts of ICP47, CD46, CD55, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, CD59, or functional fragments of any of these), but are genotypically similar to the donor animal from which the apoptotic cell vaccine is derived.ii) derived from animals genotypically identical to the donor animals of the apoptotic cell vaccine or with further genetic modifications (e.g., suppression of NLRC5, TAP1, MICA, MICB, CXCL10, C3, CIITA genes, or two or more of ICP47, CD46, CD55, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, CD59, or functional fragments of any of these); apoptotic stem cell (e.g., embryonic, pluripotent, placental, induced pluripotent, etc.) derived donor cells (e.g., leukocytes, lymphocytes, T lymphocytes, B lymphocytes, red blood cells, graft cells, or any other donor cells; iii) apoptotic stem cell (e.g., embryonic, pluripotent, placental, induced pluripotent, etc.) derived donor cells (e.g., leukocytes, lymphocytes, T lymphocytes, B lymphocytes, red blood cells, functional islet beta cells, or any other donor cells) to minimize or eliminate cell-mediated immunity and cell-dependent antibody-mediated immunity for organ, tissue, cell, and cell grafts (e.g., allografts) that are genotypically identical to human stem cell lines or for grafts (e.g., allografts) derived from the same stem cell line that has undergone genetic modification (e.g., suppression of NLRC5, TAP1, MICA, MICB, CXCL10, C3, CIITA genes) but is otherwise genotypically similar to the apoptotic human stem cell-derived donor cell vaccine; iv) apoptotic donor cells that have been rendered apoptotic by UV irradiation, gamma irradiation, or other methods that do not involve incubation in the presence of ECDI. Optionally, the tolerizing vaccine cells can be administered, e.g., injected (optionally, repeatedly injected), to a subject in need thereof. Tolerizing vaccines can be produced by disrupting (e.g., reducing expression of) one or more genes from cells.For example, the genetically modified cells described throughout this application can be used to create tolerization vaccines. For example, the cells can have one or more genes that can be disrupted (e.g., have reduced expression), including glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH), B4GALNT2, and / or any combination thereof. For example, the cells can have disrupted GGTA1 alone, or disrupted CMAH alone, or disrupted B4GALNT2 alone. The cells can also have disrupted GGTA1 and CMAH, disrupted GGTA1 and B4GALNT2, or disrupted CMAH and B4GALNT2. The cells can have disrupted GGTA1, CMAH, and B4GALNT2. In some cases, the disrupted genes do not include GGTA1. The cells can also express NLRC5 (endogenously or exogenously) but have disrupted GGTA1 and / or CMAH. The cells can also have disrupted C3.

[0355] Tolerization vaccines can be produced using cells that additionally express one or more transgenes, e.g., as described throughout this application. For example, a tolerization vaccine can include cells that contain one or more transgenes that include one or more polynucleotide inserts of infected cell protein 47 (ICP47), cluster of differentiation 46 (CD46), cluster of differentiation 55 (CD55), cluster of differentiation 59 (CD59), HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, PD-L1, PD-L2, CD47, any functional fragment thereof, or any combination thereof. In some cases, the tolerization vaccine may comprise genetically modified cells containing transgenes with reduced protein expression of GGTA1, CMAH, and B4GALNT2 and polynucleotides encoding proteins or functional fragments thereof, where the proteins include HLA-G1, PD-L1, PD-L2, and CD47. In some cases, the tolerization vaccine may comprise genetically modified cells containing transgenes with reduced protein expression of GGTA1, CMAH, and B4GALNT2 and polynucleotides encoding proteins or functional fragments thereof, where the proteins include HLA-E, PD-L1, PD-L2, and CD47. In some cases, the tolerization vaccine may comprise cells whose surfaces are coated with CD47. Coating of CD47 on the surface of cells can be achieved by biotinylating the cell surface and then incubating these biotinylated cells with streptavidin-CD47 chimeric protein. For example, a tolerization vaccine may comprise cells whose surface is coated with CD47, the cells having reduced protein expression of GGTA1, CMAH, and B4GALNT2, and transgenes comprising polynucleotides encoding proteins or functional fragments thereof, the proteins including HLA-G1, PD-L1, and PD-L2. The CD47-coated cells may be non-apoptotic cells. Alternatively, the CD47-coated cells may be apoptotic cells.

[0356] In some cases, tolerization may involve the administration of a genetically modified graft. The graft may be a cell, tissue, organ, or a combination. In some cases, immunosuppression is combined with a vaccine or a tolerizing graft. In some cases, the expression of HLA-G1 in the graft and the deficiency of MHC or HLA class I in the graft may have tolerogenic activity independent of the administration of a vaccine.

[0357] When administered to a subject, the cells of the tolerization vaccine can have a circulating half-life of at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, 24, 36, 48, 60, or 72 hours, or at least approximately these hours. For example, the circulating half-life of the tolerization vaccine can be 0.1 to 0.5, 0.5 to 1.0, 1.0 to 2.0, 1.0 to 3.0, 1.0 to 4.0, 1.0 to 5.0, 5 to 10, 10 to 15, 15 to 24, 24 to 36, 36 to 48, 48 to 60, or 60 to 72 hours, or approximately these hours. The cells in the tolerization vaccine can be treated to enhance their circulating half-life. Such treatment may include coating the cells with a protein, such as CD47. Cells treated to enhance their circulating half-life may be non-apoptotic cells. Cells treated to enhance their circulating half-life may be apoptotic cells. Alternatively, cells in the tolerization vaccine may be genetically modified (e.g., by inserting a transgene such as CD47 into their genome) to enhance their circulating half-life. Cells genetically modified to enhance their circulating half-life may be non-apoptotic cells. Cells genetically modified to enhance their circulating half-life may be apoptotic cells.

[0358] A tolerization vaccine can have both one or more disrupted genes (e.g., reduced expression) and one or more transgenes. Any of the genes and / or transgenes described herein can be used.

[0359] Cells containing one or more disrupted genes (e.g., reduced expression) can be used as or part of a tolerizing vaccine, i.e., cells containing one or more disrupted genes can be or be made into a tolerizing vaccine.

[0360] The tolerization vaccine may have the same genotype and / or phenotype as the cells, organs, and / or tissues used in the transplant. In some cases, the genotype and / or phenotype of the tolerization vaccine differs from the genotype and / or phenotype of the graft. The tolerization vaccine used for the transplant recipient may contain cells derived from the transplant graft donor. The tolerization vaccine used for the transplant recipient may contain cells that are genetically and / or phenotypically different from the transplant graft. In some cases, the tolerization vaccine used for the transplant recipient may contain cells derived from the transplant graft donor and cells that are genetically and / or phenotypically different from the transplant graft. The cells that are genetically and / or phenotypically different from the transplant graft may be derived from an animal of the same species as the transplant graft donor.

[0361] The source of cells for a tolerizing vaccine can be from a human or non-human animal.

[0362] The cells disclosed throughout the present application can be used as a tolerizing vaccine. For example, the tolerizing vaccine can be made from one or more of the transplanted cells disclosed herein. Alternatively, the tolerizing vaccine can be made from one or more cells different from any of the transplanted cells. For example, the cells used as a tolerizing vaccine can be genotypically and / or phenotypically different from any of the transplanted cells. However, in some cases, the tolerizing vaccine expresses NLRC5 (endogenously or exogenously). The tolerizing vaccine can promote the survival of cells, organs, and / or tissues in transplantation. The tolerizing vaccine can be derived from a non-human animal that is genotypically identical or similar to the donor cells, organs, and / or tissues. For example, the tolerizing vaccine can be cells derived from a pig (e.g., apoptotic pig cells) that is genotypically identical or similar to the donor pig cells, organs, and / or tissues. The donor cells, organs, and / or tissues can then be used in allografts or xenografts. In some cases, cells for tolerizing vaccines can be derived from genetically modified animals (e.g., pigs) with reduced expression of GGTA1, CMAH, and B4GalNT2, and transgenes encoding HLA-G (or HLA-E-), human CD47, human PD-L1, and human PD-L2. Graft donor animals can be generated by further genetically modifying animals (e.g., pigs) for tolerizing vaccine cells. For example, graft donor animals can be generated by disrupting additional genes (e.g., NLRC5 (or TAP1), C3, and CXCL10) in the above-mentioned animals for tolerizing vaccine cells (Figure 5).

[0363] The tolerization vaccine may comprise non-human animal cells (e.g., non-human mammalian cells). For example, the non-human animal cells may be derived from pigs, cats, cows, deer, dogs, ferrets, gaurs, goats, horses, mice, mouflons, mules, rabbits, rats, sheep, or primates. Specifically, the non-human animal cells may be pig cells. The tolerization vaccine may also comprise genetically modified non-human animal cells. For example, the genetically modified non-human animal cells may be dead cells (e.g., apoptotic cells). The tolerization vaccine may also comprise any genetically modified cells disclosed herein.

[0364] Treatment of cells to generate a tolerizing vaccine Tolerization vaccines may contain cells that have been treated with chemicals. In some cases, the treatment may induce apoptosis of the cells. Without being bound by theory, apoptotic cells may be captured by host antigen-presenting cells (e.g., in the spleen) and presented to host immune cells (e.g., T cells) in a non-immunogenic manner, leading to the induction of anergy in the immune cells (e.g., T cells).

[0365] The tolerization vaccine may comprise apoptotic cells and non-apoptotic cells. The apoptotic cells in the tolerization vaccine may be genetically identical to the non-apoptotic cells in the tolerization vaccine. Alternatively, the apoptotic cells in the tolerization vaccine may be genetically different from the non-apoptotic cells in the tolerization vaccine. The tolerization vaccine may comprise fixed cells and non-fixed cells. The fixed cells in the tolerization vaccine may be genetically identical to the non-fixed cells in the tolerization vaccine. Alternatively, the fixed cells in the tolerization vaccine may be genetically different from the non-fixed cells in the tolerization vaccine. Optionally, the fixed cells may be cells fixed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI).

[0366] The cells in the tolerization vaccine can be fixed using a chemical, such as ECDI. Fixation can cause the cells to become apoptotic. The tolerization vaccines, cells, kits, and methods disclosed herein can include ECDI and / or ECDI treatment. For example, the tolerization vaccine can be cells, such as the genetically modified cells disclosed herein, treated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI). That is, the genetically modified cells described throughout can be treated with ECDI to create a tolerization vaccine. The tolerization vaccine can then be used in transplantation to promote the survival of transplanted cells, organs, and / or tissues. It is also envisioned that ECDI derivatives, functionalized ECDI, and / or substituted ECDI can also be used to treat cells for the tolerization vaccine. Optionally, cells for tolerization vaccines can be treated with any suitable carbodiimide derivative, such as ECDI, N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), and other carbodiimide derivatives understood by those skilled in the art.

[0367] Cells for tolerizing vaccines can also be made apoptotic by methods that do not involve incubation in the presence of ECDI, such as other chemicals or irradiation, such as UV or gamma irradiation.

[0368] ECDI can chemically crosslink free amine and carboxyl groups and can effectively induce apoptosis in cells, organs, and / or tissues derived from animals that produce both tolerizing vaccines and donor non-human animals. That is, the same genetically modified animal can produce tolerizing vaccines and cells, tissues, and / or organs used in transplantation. For example, the genetically modified cells disclosed herein can be treated with ECDI. This ECDI fixation can lead to the creation of tolerizing vaccines.

[0369] The genetically modified cells that can be used to produce tolerization vaccines can be derived from the spleen (including splenic B cells), liver, peripheral blood (including peripheral blood B cells), lymph nodes, thymus, bone marrow, or any combination thereof. For example, the cells can be spleen cells, such as porcine spleen cells. In some cases, the cells can be expanded ex vivo. In some cases, the cells can be derived from fetal, perinatal, newborn, pre-weaning, and / or young adult non-human animals. In some cases, the cells can be derived from the embryos of non-human animals.

[0370] The cells in the tolerization vaccine may also contain two or more disrupted (e.g., reduced expression) genes, and the two or more disrupted genes may be glycoprotein galactosyltransferase alpha 1,3 (GGTA1), putative cytidine monophosphate-N-acetylneuraminic acid hydroxylase-like protein (CMAH), HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, and B4GALNT2, any functional fragment thereof, or any combination thereof. In some cases, the two or more disrupted genes do not include GGTA1. As described above, the disruption may be a knockout or suppression of gene expression. The knockout can be achieved by gene editing, for example, using the CRISPR / Cas system. Alternatively, gene expression can be suppressed by knockdown, for example, using RNA interference, shRNA, one or more dominant negative transgenes.In some cases, cell can further comprise one or more transgenes disclosed herein.For example, one or more transgenes can be CD46, CD55, CD59, or any combination thereof.

[0371] The cells in the tolerization vaccine can also be derived from one or more donor non-human animals. In some cases, the cells can be derived from the same donor non-human animal. The cells can be derived from one or more recipient non-human animals. In some cases, the cells can be derived from two or more non-human animals (e.g., pigs).

[0372] A tolerization vaccine can contain 0.001 to 5.0 endotoxin units per kg body weight of the prospective recipient, or within about this range, e.g., 0.001 to 1.0, or within about this range. For example, a tolerization vaccine can contain 0.01 to 5.0, 0.01 to 4.5, 0.01 to 4.0, 0.01 to 3.5, 0.01 to 3.0, 0.01 to 2.5, 0.01 to 2.0, 0.01 to 1.5, 0.01 to 1.0, 0.01 to 0.9, 0.01 to 0.8, 0.01 to 0.7, 0.01 to 0.6, 0.01 to 0.5, 0.01 to 0.4, 0.01 to 0.3, 0.01 to 0.2, or 0.01 to 0.1 endotoxin units per kg body weight of the prospective recipient, or within about these ranges.

[0373] A tolerization vaccine may contain, or about, 1-100 aggregates per μl. For example, a tolerization vaccine may contain, or about, 1-5, 1-10, or 1-20 aggregates per μl. A tolerization vaccine may contain, or about, at least 1, 5, 10, 20, 50, or 100 aggregates.

[0374] The tolerization vaccine may induce the release of IL-1 beta at or about this level, e.g., 0.001 pg / ml to 10.0 pg / ml, when about 50,000 thawed human peripheral blood mononuclear cells are incubated with about 160,000 cells (e.g., porcine cells) of the tolerization vaccine. For example, the tolerization vaccine induces the release of IL-1 beta at or about these levels, e.g., 0.001 to 10.0, 0.001 to 5.0, 0.001 to 1.0, 0.001 to 0.8, 0.001 to 0.2, or 0.001 to 0.1 pg / ml, when about 50,000 thawed human peripheral blood mononuclear cells are incubated with about 160,000 cells (e.g., porcine cells) of the tolerization vaccine. The tolerization vaccine may induce the release of 0.001 to 2.0 pg / ml or about this level, e.g., 0.001 to 0.2 pg / ml or about this level, when about 50,000 thawed human peripheral blood mononuclear cells are incubated with about 160,000 cells (e.g., porcine cells) of the tolerization vaccine. For example, the tolerization vaccine may induce the release of 0.001 to 2.0, 0.001 to 1.0, 0.001 to 0.5, or 0.001 to 0.1 pg / ml or about these levels of IL-6 when about 50,000 thawed human peripheral blood mononuclear cells are incubated with about 160,000 cells (e.g., porcine cells) of the tolerization vaccine.

[0375] The tolerization vaccine may comprise greater than or about 60% Annexin V-positive apoptotic cells, e.g., greater than or about 85% Annexin V-positive apoptotic cells, after 4 hours or about 4 hours of release incubation at 37° C. For example, the tolerization vaccine comprises greater than 60%, 70%, 80%, 90%, or 99% Annexin V-positive apoptotic cells after about 4 hours of release incubation at 37° C.

[0376] The tolerization vaccine may contain 0.01% to 10% or about this ratio of necrotic cells, e.g., 0.01% to 2% or about this ratio. For example, the tolerization vaccine contains 0.01% to 10%, 0.01% to 7.5%, 0.01% to 5%, 0.01% to 2.5%, or 0.01% to 1%, or about these ratios of necrotic cells.

[0377] Administration of a tolerization vaccine comprising ECDI-treated cells, organs, and / or tissues before, during, and / or after administration of donor cells can induce tolerance to the cells, organs, and / or tissues in a recipient (e.g., a human or non-human animal). ECDI-treated cells can be administered by intravenous infusion.

[0378] Tolerance induced by injection of a tolerizing vaccine containing ECDI-treated splenocytes is mediated by an intact programmed death 1 receptor-programmed death ligand 1 signaling pathway and CD4 + CD25 + Foxp3 + This may depend on synergy between regulatory T cells.

[0379] Cells in a tolerizing vaccine can be made apoptotic (e.g., a tolerizing vaccine) through other methods as well as by ECDI fixation. For example, any of the genetically modified cells disclosed throughout, e.g., non-human animal cells or human cells (including stem cells), can be made apoptotic by exposing the genetically modified cells to UV irradiation. Genetically modified cells can also be made apoptotic by exposing them to gamma irradiation. Other methods not involving ECDI, such as EtOH fixation, are also contemplated.

[0380] Cells in a tolerization vaccine, such as ECDI-treated cells, antigen-coupled cells, and / or epitope-coupled cells, may include donor cells (e.g., cells derived from the donor of a transplant graft). Cells in a tolerization vaccine, such as ECDI-treated cells, antigen-coupled cells, and / or epitope-coupled cells, may include recipient cells (e.g., cells derived from the recipient of a transplant graft). Cells in a tolerization vaccine, such as ECDI-treated cells, antigen-coupled cells, and / or epitope-coupled cells, may include cells of a third party (e.g., neither the donor nor the recipient). In some cases, the third party cells are derived from a non-human animal of the same species as the recipient and / or donor. In other cases, the third party cells are derived from a non-human animal of a different species from the recipient and / or donor.

[0381] The ECDI treatment of cells can be performed in the presence of one or more antigens and / or epitopes. The ECDI-treated cells can include cells of the donor, recipient, and / or third party. Similarly, the antigens and / or epitopes can include antigens and / or epitopes of the donor, recipient, and / or third party. In some cases, donor cells are coupled to recipient antigens and / or epitopes (e.g., ECDI-induced coupling). For example, soluble donor antigens derived from genetically engineered and genotypically identical donor cells (e.g., pig cells) are coupled to recipient peripheral blood mononuclear cells using ECDI, and the ECDI-coupled cells are administered via intravenous infusion.

[0382] In some cases, recipient cells are coupled to donor antigens and / or epitopes (e.g., ECDI-induced coupling). In some cases, recipient cells are coupled to third party antigens and / or epitopes (e.g., ECDI-induced coupling). In some cases, donor cells are coupled to recipient antigens and / or epitopes (e.g., ECDI-induced coupling). In some cases, donor cells are coupled to third party antigens and / or epitopes (e.g., ECDI-induced coupling). In some cases, third party cells are coupled to donor antigens and / or epitopes (e.g., ECDI-induced coupling). In some cases, third party cells are coupled to recipient antigens and / or epitopes (e.g., ECDI-induced coupling). For example, soluble donor antigens derived from genetically engineered and genotypically identical donor cells (e.g., porcine cells) are coupled to polystyrene nanoparticles using ECDI, and the ECDI-coupled cells are administered via intravenous infusion.

[0383] The tolerogenic potential of any of these tolerizing cellular vaccines can be further optimized by coupling one or more of the following to the surface of the cells: IFN-g, NF-kB inhibitors (such as curcumin, triptolide, Bay-117085), vitamin D3, siCD40, cobalt protoporphyrin, insulin B9-23, or other immunomodulatory molecules that alter the function of host antigen-presenting cells and host lymphocytes.

[0384] These apoptotic cell vaccines can also be complemented by donor cells engineered to display molecules on their surface (such as FasL, PD-L1, galectin 9, CD8 alpha) that induce the apoptotic death of donor-reactive cells.

[0385] The tolerization vaccines disclosed herein can increase the survival time of transplants (e.g., xenografts or allografts) in recipients. The tolerization vaccines disclosed herein can also reduce or eliminate the need for post-transplant immunosuppression. The xenograft or allograft can be an organ, tissue, cell, or cell line. The xenograft and the tolerization vaccine can also be derived from different species. Alternatively, the xenograft and the tolerization vaccine can be derived from the same species. For example, the xenograft and the tolerization vaccine can be derived from substantially genetically identical individuals (e.g., the same individual).

[0386] In some cases, a tolerizing vaccine or a negative vaccine may produce a synergistic effect in a subject administered the tolerizing or negative vaccine. In other cases, a tolerizing or negative vaccine may produce an antagonistic effect in a subject administered the tolerizing or negative vaccine.

[0387] The cells fixed by ECDI can be formulated into a pharmaceutical composition. For example, the cells fixed by ECDI can be combined with a pharmaceutically acceptable excipient. A usable excipient is saline. A usable excipient is phosphate buffered saline (PBS). The pharmaceutical composition can then be used to treat patients who require transplantation.

[0388] IV. Methods for Producing Genetically Modified Non-Human Animals Various techniques can be used to create the above-mentioned genetically modified non-human animals.A few examples for creating genetically modified animals are disclosed herein.It should be understood that the methods disclosed herein are merely examples and do not mean any limitation.

[0389] Gene disruption Gene disruption can be performed by any of the methods described above, for example, knockout, knockdown, RNA interference, dominant negative, etc. A detailed description of the methods is disclosed above in the section on genetically modified non-human animals.

[0390] CRISPR / Cas system The method described herein can use a CRISPR / Cas system. For example, a CRISPR / Cas system, such as a type II CRISPR / Cas system, can be used to generate double-strand breaks (DSBs). The Cas enzyme used in the method disclosed herein can be Cas9, which catalyzes DNA cleavage. The Cas9 derived from Streptococcus pyogenes or any related Cas9 enzyme can hybridize to a 20-nucleotide guide sequence and generate a double-strand break at the target site sequence that has a protospacer adjacent motif (PAM) following the 20-nucleotide target sequence.

[0391] The vector can be operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Cas proteins that can be used in the present invention include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csx1, Csx2, Csx3, Csx4, Csx5, Csx1, Csx2 ...1, Csx2, C CRISPR enzymes include sm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, C2c1, C2c2, C2c3, Cpf1, CARF, DinG, homologs thereof, or modified forms thereof. Unmodified CRISPR enzymes can have DNA cleavage activity, such as Cas9. CRISPR enzymes can induce cleavage of one or both strands in a target sequence, such as within the target sequence and / or within the complement of the target sequence.For example, CRISPR enzymes can induce cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence, or within approximately these number of base pairs.A vector encoding a CRISPR enzyme that is mutated compared to the corresponding wild-type enzyme can be used, so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.Cas protein can be a high-fidelity cas protein, such as Cas9HiFi.

[0392] Cas9 may refer to a polypeptide with at least, or at least about, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or homology to an exemplary wild-type Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9 may refer to a polypeptide with at most, or at most about, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or homology to an exemplary wild-type Cas9 polypeptide (e.g., Cas9 from S. pyogenes). Cas9 may refer to a wild-type form of the Cas9 protein, or an engineered form of the Cas9 protein, which may include amino acid changes such as deletions, insertions, substitutions, mutants, mutations, fusions, chimeras, or any combination thereof.

[0393] S. pyogenes Cas9 (SpCas9) can be used as a CRISPR endonuclease for genome manipulation. However, others may also be used. In some cases, different endonucleases can be used to target specific genomic targets. In some cases, synthetic SpCas9-derived variants with PAM sequences other than NGG can be used. In addition, other Cas9 orthologs from various species have been identified; these "non-SpCas9" can bind to various PAM sequences and may also be useful in the present invention. For example, the relatively large size of SpCas9 (approximately 4 kb coding sequence) may result in plasmids carrying SpCas9 cDNA not being efficiently expressed in cells. Conversely, the coding sequence of Staphylococcus aureus Cas9 (SaCas9) is approximately 1 kilobase shorter than SpCas9, potentially allowing for efficient expression in cells. Like SpCas9, SaCas9 endonuclease can modify target genes in mammalian cells in vitro and in vivo in mice. In some cases, Cas proteins may target different PAM sequences. In some cases, target genes such as NLRC5 may be flanked by the Cas9 PAM, 5'-NGG, for example. In other cases, other Cas9 orthologs may have different PAM requirements. For example, other PAMs, such as those in S. thermophilus (5'-NNAGAA for CRISPR1 and 5'-NGGNG for CRISPR3) and Neisseria meningiditis (5'-NNNNGATT), may also be found adjacent to target genes such as NLRC5. Transgenes of the present invention can be inserted adjacent to any PAM sequence from any Cas or Cas derivative protein. In some cases, PAMs may be found every 8-12 base pairs in the genome, or approximately every base pair within this range. PAMs can be found every 1-15 base pairs in the genome. PAMs can also be found every 5-20 base pairs in the genome.In some cases, a PAM can be found every 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more base pairs in the genome. A PAM can be found every 5-100 base pairs, or between 5-100 base pairs in the genome.

[0394] For example, for the S. pyogenes system, the target gene sequence is 5'-NGG The PAM may precede (i.e., be 5' to) the guide RNA sequence, and a 20-nt guide RNA sequence may base-pair with the opposite strand to mediate Cas9 cleavage adjacent to the PAM. In some cases, the adjacent cleavage may be 3 base pairs or approximately 3 base pairs upstream of the PAM. In some cases, the adjacent cleavage may be 10 base pairs or approximately 10 base pairs upstream of the PAM. In some cases, the adjacent cleavage may be 0 to 20 base pairs upstream of the PAM or approximately within this range. For example, the adjacent cleavage may be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs upstream of the PAM. The adjacent cleavage may also be 1 to 30 base pairs downstream of the PAM.

[0395] An alternative to S. pyogenes Cas9 may include RNA-guided endonucleases from the Cpf1 family, which exhibit cleavage activity in mammalian cells. Unlike Cas9 nucleases, Cpf1-mediated DNA cleavage results in double-strand breaks with short 3' overhangs. The sticky-end cleavage pattern of Cpf1 may open up the possibility of directional gene transfer, similar to traditional restriction enzyme cloning, and thus increasing the efficiency of gene editing. Similar to the above-described Cas9 mutants and orthologs, Cpf1 can also expand the number of sites that can be targeted by CRISPR into AT-rich regions or AT-rich genomes that lack the NGG PAM site preferred by SpCas9.

[0396] The vector encoding the CRISPR enzyme can be used that contains one or more nuclear localization sequences (NLS).For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or approximately these number of NLSs can be used.The CRISPR enzyme can contain the NLS at or near the amino-terminus, or can contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more approximately these number of NLSs at or near the carboxy-terminus, or any combination thereof (for example, one or more NLSs at the amino-terminus and one or more NLSs at the carboxy-terminus).When more than one NLS is present, each NLS can be selected independently from other NLSs, so that a single NLS can exist in more than one copy and / or in combination with one or more other NLSs that exist in one or more copies.

[0397] The CRISPR enzyme used in the method can contain up to six NLSs.An NLS is considered to be near the N-terminus or C-terminus if the amino acid closest to the NLS is within about 50 amino acids, for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino acids along the polypeptide chain from the N-terminus or C-terminus.

[0398] guide RNA As used herein, the term "guide RNA," and its grammatical equivalents, may refer to an RNA that can be specific for a target DNA and can form a complex with a Cas protein. The RNA / Cas complex can help "guide" the Cas protein to the target DNA.

[0399] The methods disclosed herein can also include introducing into a cell or embryo at least one guide RNA or a nucleic acid, e.g., DNA, encoding at least one guide RNA, which can interact with an RNA-guided endonuclease to direct the endonuclease to a specific target site where the 5' end of the guide RNA base-pairs with a specific protospacer sequence within the chromosomal sequence.

[0400] The guide RNA may comprise two RNAs, such as a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). In some cases, the guide RNA may comprise a single-stranded RNA or a single guide RNA (sgRNA), which is formed by the fusion of portions (e.g., functional portions) of the crRNA and the tracrRNA. The guide RNA may also be a duplex RNA comprising a crRNA and a tracrRNA. Furthermore, the crRNA may hybridize with the target DNA.

[0401] As discussed above, guide RNA can be an expression product.For example, the DNA encoding guide RNA can be a vector comprising a sequence encoding guide RNA.Guide RNA can be transferred into cells or organisms by transfecting cells or organisms with isolated guide RNA or isolated plasmid DNA comprising a sequence encoding guide RNA and a promoter.Guide RNA can also be transferred into cells or organisms in other ways, such as by using virus-mediated gene delivery.

[0402] Guide RNA can be isolated guide RNA.For example, guide RNA can be transfected into cell or organism in the form of isolated RNA.Guide RNA can be prepared by in vitro transcription, using any in vitro transcription system known in the art.Guide RNA can be introduced into cell in the form of isolated RNA, not in the form of the plasmid that contains the coding sequence of guide RNA.

[0403] A guide RNA may comprise three regions: a first region at the 5' end that may be complementary to a target site in a chromosomal sequence, a second internal region that may form a stem-loop structure, and a third 3' region that may be single-stranded. The first region of each guide RNA may also be different so that each guide RNA guides the fusion protein to a specific target site. Furthermore, the second and third regions of each guide RNA may be identical in all guide RNAs.

[0404] The first region of the guide RNA can be complementary to the sequence of the target site in the chromosomal sequence so that the first region of the guide RNA can base-pair with the target site. In some cases, the first region of the guide RNA can comprise 10 to 25 nucleotides or approximately this range (i.e., 10 nt to 25 nt, or about 10 nt to about 25 nt, or 10 nt to about 25 nt, or about 10 nt to 25 nt), or more. For example, the base-pairing region between the first region of the guide RNA and the target site in the chromosomal sequence can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length, or approximately these numbers of nucleotides in length. In some cases, the first region of the guide RNA can be 19, 20, or 21 nucleotides in length, or approximately these numbers of nucleotides in length.

[0405] The guide RNA may also include a second region that forms a secondary structure. For example, the secondary structure formed by the guide RNA may include a stem (or hairpin) and a loop. The lengths of the loop and stem may vary. For example, the loop may be 3 to 10 nucleotides or approximately this length, and the stem may be 6 to 20 base pairs or approximately this length. The stem may include one or more bulges of 1 to 10 or approximately 10 nucleotides. The total length of the second region may be 16 to 60 nucleotides or approximately this length. For example, the loop may be 4 nucleotides or approximately this length, and the stem may be 12 base pairs or approximately this length.

[0406] The guide RNA may also include a third region at the 3' end, which may be essentially single-stranded. For example, in some cases, the third region is not complementary to any chromosomal sequence in the target cell, and in some cases, is not complementary to the remainder of the guide RNA. Furthermore, the length of the third region may vary. The third region may be greater than or approximately greater than 4 nucleotides in length. For example, the length of the third region may range from 5 to 60 nucleotides, or approximately this length.

[0407] Guide RNA can target any exon or intron of gene target.In some cases, guide RNA can target exon 1 or 2 of gene, and in other cases, guide RNA can target exon 3 or 4 of gene.Composition can contain multiple guide RNAs that all target the same exon, or multiple guide RNAs that can target different exons in some cases.Can target exons and introns of gene.

[0408] The guide RNA may target a nucleic acid sequence of 20 nucleotides or approximately this length. The target nucleic acid may be less than 20 nucleotides or less than approximately this length. The target nucleic acid may be at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or between 1 and 100 nucleotides in length, or at least approximately this length. The target nucleic acid may be up to 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or between 1 and 100 nucleotides in length, or up to approximately this length. The target nucleic acid sequence may be 20 bases immediately 5' to the first nucleotide of the PAM, or approximately this length. The guide RNA may target a nucleic acid sequence. The target nucleic acids can be at least 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100, or at least about these numbers.

[0409] A guide nucleic acid, e.g., a guide RNA, may refer to a nucleic acid that can hybridize to another nucleic acid, e.g., a target nucleic acid or a protospacer in a cellular genome. A guide nucleic acid can be RNA. A guide nucleic acid can be DNA. A guide nucleic acid can be programmed or designed to specifically bind to the sequence of a nucleic acid site. A guide nucleic acid can comprise a polynucleotide strand and be referred to as a single guide nucleic acid. A guide nucleic acid can comprise two polynucleotide strands and be referred to as a dual guide nucleic acid. A guide RNA can be introduced into a cell or embryo as an RNA molecule. For example, an RNA molecule can be transcribed in vitro and / or chemically synthesized. RNA can be transcribed from a synthetic DNA molecule, e.g., a gBlocks® gene fragment. The guide RNA can then be introduced into a cell or embryo as an RNA molecule. A guide RNA can also be introduced into a cell or embryo in the form of a nucleic acid molecule other than RNA, e.g., a DNA molecule. For example, DNA encoding the guide RNA can be operably linked to a promoter regulatory sequence for expression of the guide RNA in the cell or embryo of interest. The RNA coding sequence can be operably linked to a promoter sequence recognized by RNA polymerase III (PolIII). Plasmid vectors that can be used to express guide RNAs include, but are not limited to, px330 vectors and px333 vectors (Figures 11 and 89). In some cases, a plasmid vector (e.g., px333 vector) can contain DNA sequences encoding at least two guide RNAs. For example, a px333 vector can be used to introduce GGTA1-10 and Gal2-2, or GGTA1-10, Gal2-2, and NLRC5-6. In other cases, a px333 vector can be used to introduce NLRC5-6 and Gal2-2.

[0410] The DNA sequence encoding the guide RNA can also be part of a vector. In addition, the vector can contain additional expression control sequences (for example, enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selectable marker sequences (for example, antibiotic resistance genes), origins of replication, etc. The DNA molecule encoding the guide RNA can also be linear. The DNA molecule encoding the guide RNA can also be circular.

[0411] When DNA sequences encoding an RNA-guided endonuclease and a guide RNA are introduced into a cell, each DNA sequence can be part of separate molecules (e.g., one vector containing the coding sequence for the RNA-guided endonuclease and a second vector containing the coding sequence for the guide RNA) or can be part of the same molecule (e.g., one vector containing the coding (and regulatory) sequences for both the RNA-guided endonuclease and the guide RNA).

[0412] The guide RNA may target a gene in a pig or pig cell. In some cases, the guide RNA may target the pig NLRC5 gene, for example, the sequence listed in Table 4. In some cases, the guide RNA may be designed to target the pig NLRC5, GGTA1, or CMAH gene. Exemplary oligonucleotides for creating guide RNAs are listed in Table 5. In some cases, at least two guide RNAs are introduced. At least two guide RNAs may target two genes, respectively. For example, in some cases, the first guide RNA may target GGTA1, and the second guide RNA may target Gal2-2. In some cases, the first guide RNA may target NLRC5, and the second guide RNA may target Gal2-2. In other cases, the first guide RNA may target GGTA1-10, and the second guide RNA may target Gal2-2.

[0413] The guide nucleic acid may contain one or more modifications that confer new or enhanced features to the nucleic acid. The guide nucleic acid may contain a nucleic acid affinity tag. The guide nucleic acid may contain synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0414] In some cases, gRNA can contain modifications.Modifications can be made at any position of gRNA.A single gRNA can be made to more than one modification.After modification, gRNA can undergo quality control.In some cases, quality control can include PAGE, HPLC, MS, or any combination thereof.

[0415] The modification of the gRNA can be a substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof.

[0416] gRNAs can also be modified with any of the following: 5' adenylate, 5' guanosine triphosphate cap, 5' N7-methylguanosine triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate, Cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3'-3' modified, 5'-5' modified, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, double biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3' DABCYL, Black Hole Quencher 1, Black Hole Quencher 2, and DABCYL. The nucleic acid may be modified with SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, a carboxyl linker, a thiol linker, a 2' deoxyribonucleoside analog purine, a 2' deoxyribonucleoside analog pyrimidine, a ribonucleoside analog, a 2'-O-methylribonucleoside analog, a sugar-modified analog, a wobble / universal base, a fluorescent dye label, 2' fluoroRNA, 2' O-methylRNA, a methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, or any combination thereof.

[0417] In some cases, the modification is permanent. In other cases, the modification is transient. In some cases, multiple modifications are made to the gRNA. The modification of the gRNA can change the physicochemical properties of nucleotides, such as their conformation, polarity, hydrophobicity, chemical reactivity, base pairing interaction, or any combination thereof.

[0418] Modifications can also be phosphorothioate substitutions. In some cases, natural phosphodiester bonds are susceptible to rapid degradation by intracellular nucleases, and modifications of internucleotide linkages using phosphorothioate (PS) bond substitutions can be more stable to hydrolysis by intracellular degradation. Modifications can increase the stability of gRNAs. Modifications can also enhance biological activity. In some cases, phosphorothioate-enhanced gRNAs can inhibit RNase A, RNase T1, bovine serum nuclease, or any combination thereof. These properties may enable the use of PS-RNA gRNAs in in vivo or in vitro applications where exposure to nucleases is likely. For example, phosphorothioate (PS) bonds can be introduced within the last 3-5 nucleotides at the 5' or 3' end of the gRNA to inhibit exonucleolytic degradation. In some cases, phosphorothioate bonds can be added throughout the gRNA to reduce attack by endonucleases. [Table 4] [Table 5]

[0419] Homologous recombination Homologous recombination can also be used for any of the related genetic modifications disclosed herein.Homologous recombination can allow site-specific modification in endogenous gene, so that new modifications can be engineered into genome.For example, the ability of homologous recombination (gene conversion and classical strand breakage / recombination) to transfer gene sequence information between DNA molecules can provide targeted homologous recombination, and can be a powerful method in genetic engineering and gene manipulation.

[0420] Cells that have undergone homologous recombination can be identified by a number of methods. For example, selection methods can detect the absence of an immune response to the cells, such as with a human anti-gal antibody. Selection methods can also include assessing the level of clotting of human blood when exposed to the cells or tissue. Selection via antibiotic resistance can be used for screening.

[0421] Creation of transgenic non-human animals Random Insertion In the method described herein, one or more transgenes can be randomly inserted into any locus in the genome of a cell.These transgenes can be functional regardless of where they are inserted in the genome.For example, a transgene can encode its own promoter, or can be inserted into a position under the control of an endogenous promoter.Alternatively, a transgene can be inserted into a gene, such as an intron of a gene, an exon of a gene, a promoter, or a non-coding region.A transgene can be integrated into the first exon of a gene.

[0422] The DNA encoding transgene sequence can be randomly inserted into the chromosome of cell.Random integration can be obtained by any method known to those skilled in the art for introducing DNA into cells.This includes but is not limited to electroporation, sonoporation, using a gene gun, lipotransfection, calcium phosphate transfection, using dendrimer, microinjection, using viral vectors, including adenovirus vectors, AAV vectors and retrovirus vectors, and / or using group II ribozymes.

[0423] The DNA encoding the transgene can also be designed to contain a reporter gene so that the presence of the transgene or its expression product can be detected through the activation of the reporter gene. Any reporter gene known in the art can be used, such as those disclosed above. Cells containing the transgene can be selected in cell culture by selecting cells in which the reporter gene is activated.

[0424] DNA encoding a transgene can be introduced into cells via electroporation (Figure 90). DNA can also be introduced into cells via lipofection, infection, or transformation. Electroporation and / or lipofection can be used to transfect fibroblasts.

[0425] The expression of transgene can be verified by expression assay, for example, qPCR, or by measuring RNA level.Expression level can also indicate copy number.For example, if the expression level is extremely high, this can indicate that more than one copy of transgene is integrated into genome.Alternatively, high expression can indicate that transgene is integrated into highly transcriptional region, for example, near highly expressing promoter.Expression can also be verified by measuring protein level, for example, by Western blotting.

[0426] Site-specific insertion Insertion of one or more transgenes in any of the methods disclosed herein can be site-specific, for example, one or more transgenes can be inserted adjacent to a promoter, such as adjacent to or near the Rosa26 promoter.

[0427] Modification of a targeted locus in a cell can be achieved by introducing DNA having homology to the target locus into the cell. The DNA can contain a marker gene that allows for selection of cells containing integrated constructs. The homologous DNA in the target vector can recombine with chromosomal DNA at the target locus. The marker gene can be flanked by homologous DNA sequences, 3' recombination arms, and 5' recombination arms.

[0428] Various enzymes can catalyze the insertion of foreign DNA into the host genome. For example, site-specific recombinases can be divided into two protein families with significantly different biochemical properties: tyrosine recombinases (in which DNA is covalently attached to tyrosine residues) and serine recombinases (in which covalent attachment occurs at serine residues). In some cases, recombinases can include Cre, fC31 integrase (a serine recombinase derived from Streptomyces phage fC31), or bacteriophage-derived site-specific recombinases (including Flp, lambda integrase, bacteriophage HK022 recombinase, bacteriophage R4 integrase, and phage TP901-1 integrase).

[0429] Expression control sequences can also be used within the construct. For example, the expression control sequence can include a constitutive promoter that is expressed in various cell types. For example, suitable strong constitutive promoters and / or enhancers include expression control sequences derived from DNA viruses (e.g., SV40, polyomavirus, adenovirus, adeno-associated virus, poxvirus, CMV, HSV, etc.) or from retroviral long terminal repeats. Tissue-specific promoters can also be used to direct expression to specific cell lineages. Although the experiments discussed in the examples below are performed using the Rosa26 gene promoter, similar results can be obtained using other Rosa26-related promoters capable of directing gene expression, as will be apparent to those skilled in the art. Therefore, the description herein is not meant to be limiting, but rather to disclose one of many possible examples. In some cases, shorter 5' upstream sequences of Rosa26 can be used, which can achieve similar levels of expression despite being shorter. Minor DNA sequence variants of the Rosa26 promoter, such as point mutations, partial deletions, or chemical modifications, are also useful.

[0430] The Rosa26 promoter can be expressed in mammals.For example, a sequence similar to the 5' flanking sequence of the pig Rosa26 gene can also be used, and the sequence includes but is not limited to the promoter of the Rosa26 homologue of other species (such as human, cow, mouse, sheep, goat, rabbit and rat).The Rosa26 gene may be sufficiently conserved between different mammalian species, and the Rosa26 promoter of other mammals can also be used.

[0431] The CRISPR / Cas system can be used to perform site-specific insertion, for example, nicking of the genome at the insertion site can be performed by CRISPR / Cas to facilitate insertion of the transgene at the insertion site.

[0432] The methods described herein can employ techniques that can be used to allow DNA or RNA constructs to enter host cells, including, but not limited to, calcium phosphate / DNA co-precipitation, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, lipofection, infection, particle bombardment, sperm-mediated gene transfer, or any other technique known to those of skill in the art.

[0433] Certain embodiments disclosed herein may use vectors. Any plasmids and vectors may be used as long as they are replicable and viable in the selected host. Vectors known in the art and commercially available vectors (and their variants or derivatives) may be engineered to contain one or more recombination sites for use in the methods. The vectors were pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.), p3'SS, pXT1, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBa-cHis A, B, and C, pVL1392, pBlueBac111, pCDM8, pcDNA1, pZeoSV, and pcDNA3. These may include, but are not limited to, expression vectors for eukaryotic cells such as pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.), and variants or derivatives thereof.

[0434] These vectors can be used to express a gene of interest, e.g., a transgene, or a portion of a gene of interest, which can be inserted by using known methods such as restriction enzyme-based techniques.

[0435] Creation of similar genetically modified non-human animals using cell nuclear transfer An alternative method for producing genetically modified non-human animals can be by cell nucleus transfer.The method for producing genetically modified non-human animals can include: a) producing cells that have reduced expression of one or more genes and / or contain the exogenous polynucleotide disclosed herein; b) providing a second cell, and transferring the nucleus of the cell resulting from a) into the second cell to produce an embryo; c) growing the embryo into a genetically modified non-human animal.The cell in this method can be enucleated cell.The cell in a) can be produced by any method, for example, gene disruption and / or insertion as described herein or known in the art.

[0436] This method can be used to produce genetically modified non-human animals similar to those disclosed herein.For example, the method for producing genetically modified non-human animals can include: a) producing cells with reduced expression of NLRC5, TAP1 and / or C3; b) providing a second cell, and transferring the nucleus of the cell resulting from a) into the second cell to produce embryos; and c) growing the embryos into genetically modified non-human animals.The cell in this method can be enucleated cell.

[0437] The cell used in this method can be derived from any disclosed genetically modified cell described herein.For example, the gene that is disrupted is not limited to NRLC5, TAP1, and / or C3.Other combinations of gene disruption and transgene can be found throughout the disclosure of this application.For example, this method can include: providing a first cell from any non-human animal disclosed herein; providing a second cell; transferring the nucleus of the first cell of a) into the second cell of b); creating an embryo from the product of c); and growing the embryo into a genetically modified non-human animal.

[0438] The cell in a) in the method disclosed herein can be a zygote. The zygote can be formed by the union of i) wild-type non-human animal sperm and wild-type non-human animal egg; ii) wild-type non-human animal sperm and genetically modified non-human animal egg; iii) genetically modified non-human animal sperm and wild-type non-human animal egg; and / or iv) genetically modified non-human animal sperm and genetically modified non-human animal egg. The non-human animal can be a pig.

[0439] In the method disclosed herein, one or more genes in the cell of a) can be disrupted by creating a break at a desired location in the genome. For example, the break can be a double-strand break (DSB). The DSB can be created using a nuclease, including Cas (e.g., Cas9), ZFN, TALEN, and meganuclease. The nuclease can be a naturally occurring nuclease or a modified nuclease. A nucleic acid encoding the nuclease can be delivered to a cell where the nuclease is expressed. Cas9 and a guide RNA that targets a gene in the cell can be delivered to the cell. In some cases, an mRNA molecule encoding Cas9 and a guide RNA can be injected into the cell. In some cases, a plasmid encoding Cas9 and a different plasmid encoding the guide RNA can be delivered to the cell (e.g., by infection). In some cases, a plasmid encoding both Cas9 and a guide RNA can be delivered to the cell (e.g., by infection).

[0440] As described above, after DSB, one or more genes can be destroyed by DNA repair mechanisms such as homologous recombination (HR) and / or non-homologous end joining (NHEJ). The method can include inserting one or more transgenes into the genome of the cell of a). The one or more transgenes can include ICP47, CD46, CD55, CD59, HLA-E, HLA-G (e.g., HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7), B2M, any functional fragment thereof, and / or any combination thereof.

[0441] The methods provided herein can include inserting one or more transgenes, which can be any of the transgenes in any non-human animal or genetically modified cell disclosed herein.

[0442] Also disclosed herein is a method for producing non-human animals using cells derived from genetically modified non-human animals.Cells can be derived from any genetically modified non-human animals disclosed herein.The method can include: a) providing cells derived from genetically identified non-human animals; b) providing cells; c) transferring the nucleus of the cell of a) into the cell of b); c) producing embryos from the product of c); and d) growing the embryos into genetically modified non-human animals.The cells of this method can be enucleated cells.

[0443] Furthermore, the cell in a) in the method can be any cell derived from a genetically modified non-human animal. For example, the cell in a) in the method disclosed herein can be a somatic cell such as a fibroblast or fetal fibroblast.

[0444] The enucleated cell in the method can be any cell from an organism. For example, the enucleated cell can be a pig cell. The enucleated cell can be an ovum, for example, an enucleated unfertilized egg.

[0445] The genetically modified non-human animals disclosed herein can be produced using any suitable technique known in the art, including, but not limited to, microinjection (e.g., of pronuclei), sperm-mediated gene transfer, egg or zygote electroporation, and / or nuclear transfer.

[0446] Methods for producing similar genetically modified non-human animals may include: a) disrupting one or more genes in a cell; b) using the cell resulting from a) to produce an embryo; and c) developing the embryo into a genetically modified non-human animal.

[0447] The cell in a) in the method disclosed herein can be a somatic cell. There is no limitation on the type or source of the somatic cell. For example, it can be derived from a pig, or from a cultured cell line or any other living cell. The cell can also be a skin cell, a neural cell, a cumulus cell, an oviduct epithelial cell, a fibroblast (e.g., fetal fibroblast), or a liver cell. The cell in a) in the method disclosed herein can be derived from a wild-type non-human animal, a genetically modified non-human animal, or a genetically modified cell. Furthermore, the cell in b) can be an enucleated egg (e.g., an enucleated unfertilized egg).

[0448] Enucleation can also be performed by known methods. For example, metaphase II oocytes can be placed in HECM, optionally containing 7-10 micrograms or approximately 7 micrograms of cytochalasin B per milliliter, for immediate enucleation, or can be enucleated after placement in a suitable medium (e.g., embryo culture medium such as CR1a / aa supplemented with 10% fetal bovine serum) (e.g., within 24 hours or 16-18 hours). Enucleation can also be achieved by microsurgery by using a micropipette to remove the polar body and adjacent cytoplasm. Oocytes can then be screened to identify those that have been successfully enucleated. One method for screening oocytes is to stain them with 3-10 micrograms or approximately 33342 Hoechst dye per milliliter in a suitable holding medium, followed by viewing the oocytes under ultraviolet light for less than 10 seconds. Successfully enucleated oocytes can then be placed in a suitable culture medium, e.g., CR1a / aa supplemented with 10% serum. Oocyte processing can also be optimized for nuclear transfer.

[0449] The embryos produced in the present invention can be transferred into a surrogate non-human animal (e.g., a pig) to produce offspring (e.g., piglets). For example, the embryos can be transferred into the oviduct of a recipient sow on the day of estrus or one day thereafter, for example, after midline laparotomy under general anesthesia. Pregnancy can be diagnosed, for example, by ultrasound. Pregnancy can be diagnosed 28 days or about 28 days after transfer. Pregnancy can then be confirmed at 2-week intervals or about 2-week intervals by ultrasound examination. All of the microinjected offspring (e.g., piglets) can be delivered by natural parturition. Gestation and birth information (e.g., time of pregnancy, pregnancy rate, number of offspring, survival rate, etc.) can be recorded. The genotype and phenotype of the offspring can be measured using any method described throughout this application, such as sequencing (e.g., next-generation sequencing). Sequencing can also be performed using Zas as shown in Figures 109 and 110A. 258 sequencing. The sequencing product can also be verified by electrophoresis of the amplification product (Figure 110B). For example, CM1F sequencing is shown in Figure 111A, and the electrophoresis product is shown in Figure 111B.

[0450] The cultured cells can be immediately used for nuclear transfer (e.g., somatic cell nuclear transfer), embryo transfer, and / or induction of pregnancy, allowing embryos derived from the stable genetic modification to give rise to offspring (e.g., piglets). Such procedures can reduce the time and expense, for example, months of costly cell screening that can result in failure to produce live and / or healthy piglets from the genetically modified cells.

[0451] Embryo development and transfer can be performed using standard procedures used in the embryo development and transfer industry. For example, a surrogate mother can be used. Embryos can also be developed and transferred in culture, for example, by using an incubator. In some cases, the embryo can be transferred to an animal, such as a surrogate animal, to establish a pregnancy.

[0452] It may be desirable to replicate or generate multiple genetically modified non-human animals having the same genotype and / or phenotype disclosed herein. For example, genetically modified non-human animals can be replicated by breeding (e.g., selective breeding). Genetically modified non-human animals can be replicated by nuclear transfer (e.g., somatic cell nuclear transfer) or by introducing DNA into cells (e.g., oocytes, sperm, zygotes, or embryonic stem cells). These methods can be replicated multiple times to replicate or generate multiple genetically modified non-human animals disclosed herein. In some cases, cells can be isolated from the fetus of a pregnant genetically modified non-human animal. The isolated cells (e.g., fetal cells) can be used to generate multiple genetically modified non-human animals similar to or identical to the pregnant animal. For example, isolated fetal cells can provide a donor nucleus for generating genetically modified animals by nuclear transfer (e.g., somatic cell nuclear transfer).

[0453] V. How to use The cells, organs, and / or tissues can be derived from non-human animals as described herein. The cells, organs, and / or tissues can be genetically modified ex vivo and used as appropriate. These cells, organs, and / or tissues can be used for cell-based therapy. These cells, organs, and / or tissues can be used to treat or prevent disease in recipients (e.g., humans or non-human animals). Surprisingly, the genetic modifications described herein can help prevent rejection. Furthermore, the cells, organs, and / or tissues can be made into tolerization vaccines to help tolerize the immune system to transplantation. Furthermore, tolerization vaccines can moderate the immune system, including inhibiting autoimmune responses.

[0454] Disclosed herein are methods for treating a disease in a subject in need thereof, which may include administering a tolerizing vaccine to the subject; administering a pharmaceutical agent that inhibits T cell activation to the subject; and transplanting genetically modified cells into the subject. The pharmaceutical agent that inhibits T cell activation may be an antibody. The antibody may be an anti-CD40 antibody disclosed herein. The anti-CD40 antibody may be an antagonistic antibody. The anti-CD40 antibody may be an anti-CD40 antibody that specifically binds to an epitope within the amino acid sequence: EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCV (SEQ ID NO: 487). The anti-CD40 antibody may be an anti-CD40 antibody that specifically binds to an epitope within the amino acid sequence: EKQYLINSQCCSLCQPGQKLVSDCTEFTETECL (SEQ ID NO: 488). The anti-CD40 antibody can be the Fab' anti-CD40L monoclonal antibody fragment CDP7657. The anti-CD40 antibody can be an FcR-engineered, Fc-silent anti-CD40L monoclonal domain antibody. The cells transplanted into the subject can be any of the genetically modified cells described throughout this application. The tissue or organ transplanted into the subject can contain one or more of the genetically modified cells. In some cases, the method can further include administering one or more immunosuppressants described herein, such as providing the recipient with one or more of a B-cell-depleting antibody, an mTOR inhibitor, a TNF-alpha inhibitor, an IL-6 inhibitor, a nitrogen mustard alkylating agent (e.g., cyclophosphamide), and a complement C3 or C5 inhibitor.

[0455] Also disclosed herein are methods for treating diseases, which include transplanting one or more cells into a subject in need thereof. The one or more cells may be any of the genetically modified cells disclosed herein. In some cases, the method may include transplanting a tissue or organ containing one or more cells (e.g., genetically modified cells) into a subject in need thereof.

[0456] Described herein are methods of treating or preventing disease in a recipient (e.g., a human or non-human animal) that involve transplantation into the recipient (e.g., a human or non-human animal) of one or more cells (including organs and / or tissues) derived from a genetically modified non-human animal that contains one or more genes with reduced expression. The one or more cells may be derived from the genetically modified non-human animals described throughout.

[0457] The methods disclosed herein can be used to treat or prevent diseases, including, but not limited to, diabetes, cardiovascular disease, pulmonary disease, liver disease, skin disease, or neurological disorders. For example, the methods can be used to treat or prevent Parkinson's disease or Alzheimer's disease. The methods can also be used to treat or prevent diabetes, including type 1, type 2, cystic fibrosis-related, surgical diabetes, gestational diabetes, mitochondrial diabetes, or a combination thereof. In some cases, the methods can be used to treat or prevent inherited diabetes or genetic forms of diabetes. Furthermore, the methods can be used to treat or prevent type 1 diabetes. The methods can also be used to treat or prevent type 2 diabetes. The methods can be used to treat or prevent prediabetes.

[0458] For example, when treating diabetes, genetically modified spleen cells can be fixed by ECDI and given to recipients.Furthermore, genetically modified pancreatic islet cells can be transplanted into the same recipient to produce insulin.The genetically modified spleen cells and pancreatic islet cells can be genetically identical and can be derived from the same genetically modified non-human animal.

[0459] Provided herein are: i) genetically modified cells, tissues, or organs for use in administration to a subject to treat a condition in the subject in need thereof; ii) a tolerization vaccine comprising genetically modified cells, tissues, or organs for use in immune tolerization of a subject to a transplant; iii) one or more pharmaceutical agents for use in inhibiting T cell activation, B cell activation, dendritic cell activation, or a combination thereof in a subject; or iv) any combination thereof.

[0460] The present invention also includes genetically modified cells, tissues, or organs for use in administering to a subject in need thereof to treat a condition in the subject.The subject may be tolerized or tolerized to the genetically modified cells, tissues, or organs by using a tolerization vaccine.In addition, the subject may be administered one or more pharmaceutical agents that inhibit T cell activation, B cell activation, dendritic cell activation, or a combination thereof. transplant

[0461] The methods disclosed herein may include transplantation. The transplantation may be an autologous transplant, an allograft, a xenograft, or any other transplantation. For example, the transplantation may be a xenograft. The transplantation may also be an allograft.

[0462] As used herein, "xenotransplantation" and its grammatical equivalents may include any procedure involving the transplantation, implantation, or transfusion of cells, tissues, or organs into a recipient, where the recipient and donor are of different species. The cell, organ, and / or tissue transplants described herein can be used for xenotransplantation into humans. Xenotransplantation includes, but is not limited to, vascularized xenotransplants, partially vascularized xenotransplants, non-vascularized xenotransplants, xenogeneic dressings, xenogeneic bandages, and nanostructures.

[0463] As used herein, "allotransplantation" and its grammatical equivalents can include any procedure involving the transplantation, implantation, or transfusion of cells, tissues, or organs into a recipient, where the recipient and donor are of the same species. The cell, organ, and / or tissue transplants described herein can be used for allotransplantation into humans. Allotransplantation includes, but is not limited to, vascularized allotransplants, partially vascularized allotransplants, non-vascularized allotransplants, allogeneic dressings, allogeneic bandages, and allogeneic structures.

[0464] After treatment (e.g., any of the treatments disclosed herein), graft rejection may be ameliorated compared to transplanting one or more wild-type cells into the recipient. For example, the graft rejection may be hyperacute rejection. The graft rejection may also be acute rejection. Other types of rejection may include chronic rejection. The graft rejection may also be cell-mediated rejection or T-cell-mediated rejection. The graft rejection may also be natural killer cell-mediated rejection.

[0465] In some cases, the subject is sensitized to a major histocompatibility complex (MHC) or human leukocyte antigen (HLA). For example, the subject may have a positive result in a panel reactive antibody (PRA) screen. In some cases, the subject may have a calculated PRA (cPRA) score of 0.1 to 100%. The cPRA score may be 0.1 to 10%, 5% to 30%, 10% to 50%, 20% to 80%, 40% to 90%, 50% to 100%, or approximately these ratios. In some cases, subjects with a positive PRA screen may be transplanted with genetically modified cells of the present invention.

[0466] In some cases, the subject may have had PRA levels quantified by a single antigen bead (SAB) test, which may identify the MHC or HLA to which the subject has antibodies.

[0467] As used herein, "improving" and its grammatical equivalents can mean any improvement recognized by one of ordinary skill in the art. For example, improving transplantation can mean a reduction in hyperacute rejection, which can include attenuation, reduction, or attenuation of undesirable effects or symptoms.

[0468] The present disclosure describes a method for treating or preventing diabetes or prediabetes. For example, the method includes, but is not limited to, administering one or more pancreatic islet cells from a donor non-human animal described herein to a recipient or a recipient in need thereof. The method can be transplantation or, in some cases, xenotransplantation. The donor animal can be a non-human animal. The recipient can be a primate, such as a non-human primate, including, but not limited to, a monkey. The recipient can be a human, and in some cases, a human with diabetes or prediabetes. In some cases, whether a patient with diabetes or prediabetes can be treated with transplantation can be determined using, for example, the algorithm described in Diabetes Care 2015;38:1016-1029 (which is incorporated herein by reference in its entirety).

[0469] The methods may also include xenotransplantation methods in which the transgenic cells, tissues, and / or organs provided herein, such as pancreatic tissues or cells, are transplanted into a primate, such as a human, and the primate requires less or no immunosuppressive therapy after transplantation. Requiring less or no immunosuppressive therapy includes, but is not limited to, a reduction (or complete elimination) of the dose of immunosuppressant drug(s) / agent(s) compared to that required by other methods; a reduction (or complete elimination) of the number of immunosuppressant drug(s) / agent(s) compared to that required by other methods; a reduction (or complete elimination) of the duration of immunosuppressive treatment compared to that required by other methods; and / or a reduction (or complete elimination) of maintenance immunosuppression compared to that required by other methods.

[0470] The methods disclosed herein can be used to treat or prevent disease in a recipient (e.g., a human or non-human animal). The recipient can be any non-human animal or human. For example, the recipient can be a mammal. Other examples of recipients include, but are not limited to, primates, such as monkeys, chimpanzees, monkeys, or humans. If the recipient is a human, the recipient can be a human in need thereof. The methods described herein can also be used with non-primate, non-human recipients; for example, the recipient can be a pet animal, including, but not limited to, a dog, cat, horse, wolf, rabbit, ferret, gerbil, hamster, chinchilla, fancy rat, guinea pig, canary, parakeet, or parrot. If the recipient is a pet animal, the pet animal can be in need thereof. For example, the recipient can be a dog in need thereof or a cat in need thereof.

[0471] The transplant may be any transplant known in the art. The graft may be transplanted into various sites in the recipient. Sites may include, but are not limited to, the liver subcapsular space, the splenic subcapsular space, the kidney subcapsular space, the omentum, the omental bursa, the stomach or intestinal submucosa, a vascular segment of the small intestine, the venous sac, the testis, the brain, the spleen, or the cornea. For example, the transplant may be a subcapsular transplant. The transplant may also be an intramuscular transplant. The transplant may be an intraportal vein transplant.

[0472] The transplant can be of one or more cells, tissues, and / or organs from a human or non-human animal. For example, the tissue and / or organ can be an organ that can be the brain, heart, lung, eye, stomach, pancreas, kidney, liver, intestine, uterus, bladder, skin, hair, nails, ear, gland, nose, oral cavity, lip, spleen, gums, teeth, tongue, salivary gland, tonsil, pharynx, esophagus, large intestine, small intestine, rectum, anus, thyroid, thymus, bone, cartilage, tendon, ligament, epirenal gland, skeletal muscle, smooth muscle, blood vessel, blood, spinal cord, trachea, ureter, urethra, hypothalamus, pituitary gland, pylorus, adrenal gland, ovary, fallopian tube, uterus, vagina, mammary gland, testis, seminal vesicle, penis, lymph, lymph node, or lymphatic vessel. Alternatively, the one or more cells may be derived from an organ, which may be the brain, heart, lung, eye, stomach, pancreas, kidney, liver, intestine, uterus, bladder, skin, hair, nails, ear, gland, nose, oral cavity, lip, spleen, gums, teeth, tongue, salivary gland, tonsil, pharynx, esophagus, large intestine, small intestine, rectum, anus, thyroid, thymus, bone, cartilage, tendon, ligament, epirenal gland, skeletal muscle, smooth muscle, blood vessel, blood, spinal cord, trachea, ureter, urethra, hypothalamus, pituitary gland, pylorus, adrenal gland, ovary, fallopian tube, uterus, vagina, mammary gland, testis, seminal vesicle, penis, lymph, lymph node, or lymphatic vessel. The one or more cells may also be derived from the brain, heart, liver, skin, intestine, lung, kidney, eye, small intestine, or pancreas. The one or more cells may be derived from the pancreas, kidney, eye, liver, small intestine, lung, or heart. The one or more cells may be derived from the pancreas, kidney, eye, liver, small intestine, lung, or heart. The one or more cells may be derived from the pancreas. The one or more cells may be pancreatic islet cells, such as pancreatic beta cells. Furthermore, the one or more cells may be pancreatic islet cells and / or cell clusters, including, but not limited to, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic F cells (e.g., PP cells), or pancreatic epsilon cells. In one example, the one or more cells may be pancreatic alpha cells. In another example, the one or more cells may be pancreatic beta cells.

[0473] As discussed above, genetically modified non-human animals can be used to provide xenografts (e.g., cells, tissues, and / or organs). For illustrative purposes only, genetically modified non-human animals, such as pigs, can be used as donors of pancreatic tissue, including, but not limited to, pancreatic islets and / or islet cells. Pancreatic tissue or cells derived from such tissue can include pancreatic islet cells, islets, or islet cell clusters. For example, the cells can be transplantable pancreatic islets. More specifically, the cells can be pancreatic beta cells. The cells can also be insulin-producing. Alternatively, the cells can be islet-like cells. The islet cell clusters can include any one or more of alpha, beta, delta, PP, or epsilon cells. The disease treated by the methods and compositions of the present invention can be diabetes. The transplantable graft can be pancreatic islets and / or islet-derived cells. The modification to the transgenic animal can be to pancreatic islets or islet-derived cells. In some cases, the pancreatic islets or islet-derived cells can be porcine. In some cases, the islet-derived cells include pancreatic beta cells.

[0474] The donor non-human animal can be at any developmental stage, including, but not limited to, embryonic, fetal, newborn, juvenile, and adult. For example, donor islet cells can be isolated from an adult non-human animal. Donor cells, e.g., islet cells, can also be isolated from a fetal or newborn non-human animal. The donor non-human animal can be less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year old. For example, islet cells can be isolated from a non-human animal less than 6 years old. Islet cells can also be isolated from a non-human animal less than 3 years old. The donor can be a non-human animal and can be 0 (including fetal) to 2 years, 2 to 4 years, 4 to 6 years, 6 to 8 years, or 8 to 10 years old, or any age between these ages. The non-human animal can be greater than 10 years old or greater than about 10 years old. The donor cells can be derived from a human.

[0475] Islet cells can be isolated from non-human animals of various ages. For example, islet cells can be isolated from non-human animals that are newborn to 2 years old or approximately this age. Islet cells can also be isolated from non-human animals that are fetal to 2 years old or approximately this age. Islet cells can be isolated from non-human animals that are 6 months old to 2 years old or approximately this age. Islet cells can also be isolated from non-human animals that are 7 months old to 1 year old or approximately this age. Islet cells can be isolated from non-human animals that are 2 to 3 years old or approximately this age. In some cases, the non-human animal can be younger than 0 years old (e.g., a fetus or embryo). In some cases, newborn islets can be more robust and more persistent after isolation than adult islets, can be more resistant to oxidative stress, and can exhibit significant growth potential (presumably derived from a nascent islet stem cell subpopulation) such that they have the ability to proliferate and engraft at the transplant site after transplantation.

[0476] With regard to the treatment of diabetes, neonatal islets may have the disadvantage that they may take up to 4-6 weeks or even approximately this period to mature sufficiently to produce significant levels of insulin, but this can be overcome by treating the neonatal islets with exogenous insulin for a period sufficient for maturation. In xenograft transplantation, neonatal islet survival and functional engraftment can be determined by measuring donor-specific c-peptide levels, which are easily distinguished from any recipient's endogenous c-peptide.

[0477] As discussed above, adult cells can be isolated. For example, islets from adult non-human animals, such as adult pig cells, can be isolated. The islets can then be cultured for 1-3 days or approximately this period before transplantation to deplete the preparation of contaminating exocrine tissue. Prior to treatment, islets can be counted and viability assessed by double staining with fluorescent calcein-AM and propidium iodide. Islet cell viabilities greater than 75% can be used. However, cell viabilities greater than or equal to 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% can also be used. For example, cells exhibiting viabilities of 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, or 90%-100%, or approximately these percentages, can be used. Furthermore, purity can be greater than or equal to 80% islets per total tissue. Purity can also be at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% islets per total tissue, or at least about these percentages of islets, for example, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-100%, 90%-95%, or 95%-100%, or about these purities.

[0478] Functional properties of islets, including glucose-stimulated insulin secretion and viability assessed by dynamic perifusion, can be determined in vitro prior to treatment (Balamurugan, 2006). For example, islet cells from non-human animals, such as transgenic porcine islets, can be cultured in vitro to expand, mature, and / or purify them so that they are suitable for transplantation.

[0479] Islet cells can also be isolated by standard collagenase digestion of minced pancreas. For example, using sterile techniques, glands are expanded with tissue-dissociating enzymes (a mixture of purified enzymes formulated for rapid dissociation of the pancreas and maximal recovery of healthy, intact, and functional islets of Langerhans; the target substrates of these enzymes have not been fully identified but are presumed to be collagen and non-collagen proteins comprising the extracellular matrix of pancreatic acinar tissue) (1.5 mg / ml), removed of excess fat, blood vessels, and connective tissue, minced, and digested in a shaking water bath at 120 rpm for 15 minutes at 37°C. Digestion can be accomplished using lignocaine mixed with the tissue-dissociating enzymes to avoid cell damage during digestion. After digestion, the cells may be passed through a sterile 50 mm to 1000 mm mesh, e.g., 100 mm, 200 mm, 300 mm, 400 mm, 500 mm, 600 mm, 700 mm, 800 mm, 900 mm, or 1000 mm mesh, in a sterile beaker. Additionally, a second digestion step may be used on any undigested tissue.

[0480] Islets can also be isolated from the pancreas of adult pigs (Brandhorst et al., 1999). The pancreas is harvested from a suitable source pig, peripancreatic tissue is removed, and the pancreas is divided into splenic and duodenal / connecting lobes. The ducts of each lobe are cannulated and the lobes are distended with tissue-dissociating enzymes. The pancreatic lobes are placed in a Ricordi chamber, the temperature is gradually increased to 28-32°C, and the pancreatic lobes are dissociated by enzymatic activity and mechanical force. The liberated islets are separated from the acini and ductal tissue using a continuous density gradient. The purified islets are cultured for 2-7 days or approximately for this period, and are subjected to characterization. Islet products meeting all specifications are shipped for transplantation (Korbutt et al., 2009).

[0481] Donor cells, organs, and / or tissues can be functional before transplantation, after transplantation, and / or at the time of transplantation.For example, transplanted cells, organs, and / or tissues can be functional for at least 1, 5, 10, 20, 30 days, or at least approximately these periods, after transplantation. Transplanted cells, organs, and / or tissues can be functional for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least approximately these periods, after transplantation. Transplanted cells, organs, and / or tissues can be functional for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 years, or at least approximately these periods, after transplantation.In some cases, transplanted cells, organs, and / or tissues can be functional for up to the lifetime of the recipient.This can indicate that the transplantation is successful. This may also indicate a lack of rejection of the transplanted cells, tissues, and / or organs.

[0482] Furthermore, the transplanted cells, organs, and / or tissues may function at 100% of their intended normal function. The transplanted cells, organs, and / or tissues may also function at at least 50, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% of their intended normal function, or at least about these percentages, e.g., 50-60, 60-70, 70-80, 80-90, 90-100%, or about these percentages. In certain instances, the transplanted cells, organs, and / or tissues may function at greater than 100% of their intended normal function (when compared to non-transplanted cells, organs, or tissues of normal function as determined by the American Medical Association). For example, the transplanted cells, organs, and / or tissues may function at or near 110, 120, 130, 140, 150, 175, 200% or more of their intended normal function, e.g., 100-125, 125-150, 150-175, 175-200%, or near these ratios.

[0483] In certain cases, the transplanted cells may be functional for at least or at least about 1 day. The transplanted cells may also be functional for at least or at least about 7 days. The transplanted cells may be functional for at least or at least about 14 days. The transplanted cells may be functional for at least or at least about 21 days. The transplanted cells may be functional for at least or at least about 28 days. The transplanted cells may be functional for at least or at least about 60 days.

[0484] Another indicator of the success of transplantation can be the number of days that recipient does not require immunosuppressive therapy.For example, after the treatment (e.g., transplantation) provided herein, recipient may not require immunosuppressive therapy for at least 1, 5, 10, 100, 365, 500, 800, 1000, 2000, 4000 or more periods, or at least approximately these periods.This can indicate that transplantation is successful.This can also indicate that there is no rejection of transplanted cells, tissues and / or organs.

[0485] In some cases, the recipient may be free of immunosuppressive therapy for at least one day or at least about one day. The recipient may also be free of immunosuppressive therapy for at least or at least about seven days. The recipient may be free of immunosuppressive therapy for at least or at least about 14 days. The recipient may be free of immunosuppressive therapy for at least or at least about 21 days. The recipient may be free of immunosuppressive therapy for at least or at least about 28 days. The recipient may be free of immunosuppressive therapy for at least or at least about 60 days. Furthermore, the recipient may not require immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 years, or at least about these periods, e.g., at least 1-2, 2-3, 3-4, 4-5, 1-5, 5-10, 10-15, 15-20, 20-25, 25-50 years, or at least about these periods.

[0486] Another indicator of successful transplantation can be the number of days that the recipient requires reduced immunosuppressive therapy.For example, after the treatment provided herein, the recipient may require reduced immunosuppressive therapy for at least 1, 5, 10, 50, 100, 200, 300, 365, 400, 500 days, or at least approximately these periods, for example, at least 1-30, 30-120, 120-365, 365-500 days, or at least approximately these periods.This can indicate that the transplantation is successful.It can also indicate that the rejection of the transplanted cells, tissues, and / or organs is not observed or is minimal.

[0487] For example, the recipient may require reduced immunosuppressive therapy for at least or at least about 1 day. The recipient may also require reduced immunosuppressive therapy for at least 7 days. The recipient may require reduced immunosuppressive therapy for at least or at least about 14 days. The recipient may require reduced immunosuppressive therapy for at least or at least about 21 days. The recipient may require reduced immunosuppressive therapy for at least or at least about 28 days. The recipient may require reduced immunosuppressive therapy for at least or at least about 60 days. Furthermore, the recipient may require reduced immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 years, or at least approximately these periods, e.g., at least 1-2, 2-3, 3-4, 4-5, 1-5, 5-10, 10-15, 15-20, 20-25, 25-50 years, or at least approximately these periods.

[0488] As used herein, "reduced" and its grammatical equivalents may refer to a lower dose of immunosuppressive therapy compared to that required when transplanting one or more wild-type cells into a recipient.

[0489] The donor (e.g., the donor of the transplant graft and / or the cells in the tolerization vaccine) can be a mammal. The donor of an allograft can be unmodified human cells, tissues, and / or organs, including, but not limited to, pluripotent stem cells. The donor of a xenograft can be any cells, tissues, and / or organs derived from a non-human animal, such as a mammal. In some cases, the mammal can be a pig.

[0490] The methods of the present invention may further include treating the disease by transplanting one or more donor cells into a tolerized recipient (eg, a human or non-human animal). [Example]

[0491] Example 1 Generation of plasmids expressing guide RNAs to disrupt the GGTA1, CMAH, NLRC5, B4GALNT2, and / or C3 genes in pigs Genetically modified pigs can be used to produce transplants that induce low or no immune rejection in recipients, and / or cells that serve as tolerizing vaccines to enhance immune tolerization in recipients. Compared to non-genetically modified counterparts, such pigs will have reduced expression of any gene regulating MHC molecules (e.g., MHC I and / or MHC II molecules). Reducing the expression of such genes will result in reduced expression and / or function of MHC molecules. These genes may be one or more of the following: components of the MHC I-specific enhanceosome, transporters of MHC I-binding peptides, natural killer group 2D ligands, CXCR3 ligands, C3, and CIITA. Additionally or alternatively, such pigs will have reduced protein expression of endogenous genes not expressed in humans (e.g., CMAH, GGTA1, and / or B4GALNT2). For example, the pig will have reduced protein expression of one or more of NLRC5, TAP1, C3, CXCL10, MICA, MICB, CIITA, CMAH, GGTA1, and / or B4GALNT2. In some cases, the pig will have reduced protein expression of NLRC5, C3, CXCL10, CMAH, GGTA1, and / or B4GALNT2.

[0492] This example shows an exemplary method for generating a plasmid for disrupting the GGTA1, CMAH, NLRC5, B4GALNT2, and / or C3 genes in pigs using the CRISPR / cas9 system. The plasmid was generated using the px330 vector, which simultaneously expresses the Cas9 DNA endonuclease and guide RNA.

[0493] The px330-U6-chimera_BB-CBh-hSpCas9 (#42230) plasmid was obtained from Addgene in bacterial stab culture format. The stab culture was streaked onto prewarmed LB agar plates containing ampicillin and incubated overnight at 37°C. The next day, a single colony was selected and inoculated into an overnight liquid LB culture containing ampicillin (5 mL for minipreps or 80-100 mL for maxipreps). Minipreps were performed using a Qiagen kit according to the manufacturer's instructions. Plasmids were eluted in nuclease-free water, and stocks were stored at -20°C. Oligonucleotides designed to target GGTA1, CMAH, NLRC5, C3, and B4GALNT2 are listed in Table 6. Oligonucleotides were synthesized by IDT. Figures 7A-7E, 8A-8E, 9A-9E, 10A-10E, and 11A-11E show the cloning strategy for cloning plasmids targeting GGTA1 (i.e., px330 / Gal2-1) (Figures 7A-7E), CMAH (i.e., px330 / CM1F) (Figures 8A-8E), NLRC5 (i.e., px330 / NL1_1) (Figures 9A-9E), C3 (i.e., px330 / C3-5) (Figures 10A-10E), and B4GALNT2 (i.e., px330 / B41_2) (Figures 11A-11E). The constructed px330 plasmids were verified by sequencing using the sequencing primers shown in Table 7 and by sequencing as shown in Figure 109. Oligonucleotides were resuspended at 100 μM in nuclease-free water and stored in a −20° C. freezer.

[0494] Vector digest: 5 μg of px330 stock and 5 μL of 10x FastDigest The px330 vector was digested in a reaction solution containing Reaction Buffer, 35 μL of nuclease-free water, and 5 μL of FastDigest BbsI enzyme (cutting site: GAAGAC). The reaction solution was incubated at 37°C for 15 minutes and heat-inactivated at 65°C for 15 minutes. To dephosphorylate the vector, 0.2 μL (2 U...

Claims

1. A genetically modified human induced pluripotent stem cell, comprising: a genomic disruption in a NOD-like receptor family CARD domain containing 5 (NLRC5) gene, wherein the genomic disruption in the NLRC5 gene is a nuclease-mediated double-stranded break in a target sequence of an exon of the NLRC5 gene; The genetically modified human induced pluripotent stem cells have reduced expression of at least one MHC class I protein compared to non-genetically modified human induced pluripotent stem cells. Genetically modified human induced pluripotent stem cells.

2. The genetically modified human induced pluripotent stem cell of claim 1, further comprising a genomic disruption in one or more genes encoding a protein selected from the group consisting of C-X-C motif chemokine 10 (CXCL10), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB), MHC II transactivator (CIITA), transporter associated with antigen processing 1 (TAP1), beta-2-microglobulin (B2M), and combinations thereof.

3. The genetically modified human induced pluripotent stem cells are characterized in that they express cluster of differentiation 47 (CD47) protein, infected cell protein 47 (ICP47), cluster of differentiation 46 (CD46), cluster of differentiation 55 (CD55), cluster of differentiation 59 (CD59), human leukocyte antigen E (HLA-E), human leukocyte antigen G (HLA-G), beta-2-microglobulin (B2M), CD80, CD86, programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), CD275, CD276, V-set domain-containing inhibitor of T-cell activation 1 (VTCN1), platelet receptor Gi24, natural receptor for cytotoxicity 3 ligand 1 (NR3L1), and HERV-H.

2. The genetically modified human induced pluripotent stem cell of claim 1, further comprising one or more exogenous nucleic acids encoding a protein selected from the group consisting of: long-term repeat binding 2 (HHLA2), serine protease inhibitor 9 (Spi9), galectin-1, galectin-2, galectin-3, galectin-4, galectin-5, galectin-6, galectin-7, galectin-8, galectin-9, galectin-10, galectin-11, galectin-12, galectin-13, galectin-14, and galectin-15, alternative splicing isoforms and functional fragments thereof.

4. A genetically modified human induced pluripotent stem cell as described in claim 1, wherein the exon is exon 1.

5. The genetically modified human induced pluripotent stem cell described in claim 1, wherein the target sequence includes sequence number 261.

6. The genetically modified human induced pluripotent stem cell of claim 1, wherein the target sequence is within 20 nucleotides of the PAM.

7. The genetically modified human induced pluripotent stem cell of claim 1, wherein the target sequence is adjacent to a PAM containing 5'-NGG.

8. The genetically modified human induced pluripotent stem cell of claim 1, wherein contacting a population of human T cells with the genetically modified human induced pluripotent stem cells results in a reduced population of human T cells compared to a corresponding population of human T cells not contacted with the composition.

9. The genetically modified human induced pluripotent stem cell of claim 1, wherein the genetically modified human induced pluripotent stem cell is an apoptotic genetically modified human induced pluripotent stem cell. (a) a genetically modified human induced pluripotent stem cell according to any one of claims 1 to 9; and (b) Carbodiimide derivative A composition comprising:

11. The composition of claim 10, wherein the carbodiimide derivative is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ECDI).

12. (a) a first container containing a genetically modified human cell comprising a genomic disruption in a NOD-like receptor family CARD domain containing 5 (NLRC5) gene, wherein said genomic disruption in the NLRC5 gene is a nuclease-mediated double-stranded break in a target sequence of an exon of the NLRC5 gene; (b) a second container containing an anti-CD40 antibody; Including the kit.

13. The kit of claim 12, wherein the anti-CD40 antibody comprises Fab' anti-CD40L monoclonal antibody fragment CDP7657, an FcR engineered antibody, or an Fc-silent anti-CD40L monoclonal domain antibody.

14. The kit of claim 12, wherein the anti-CD40 antibody binds to an epitope within the amino acid sequence of SEQ ID NO:487 or SEQ ID NO:

488.

15. The kit of claim 12, further comprising an immunosuppressant.

16. A population of human cells, the population of human cells comprising differentiated progeny of a genetically modified human induced pluripotent stem cell described in any one of claims 1 to 8.

17. The population of human cells described in claim 16, wherein the population of human cells produces insulin.

18. The population of human cells described in claim 16, wherein the differentiated progeny include white blood cells, lymphocytes, B lymphocytes, islet cells, islet beta cells, or hepatocytes. (a) a first container comprising a population of human cells according to any one of claims 16 to 18; and (b) a second container containing an anti-CD40 antibody. Including the kit.

20. A composition for use in treating a condition in a subject in need thereof, the composition comprising: a genetically modified human induced pluripotent stem cell according to any one of claims 1 to 9; a composition according to any one of claims 10 or claim 11; a kit according to any one of claims 12 to 15 or claim 19; a population of human cells according to any one of claims 16 to 18; or a combination thereof.

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