Genetic modification for xenotransplantation

Abstract

Provided herein are recombinant minipigs that do not express endogenous porcine CD47 and SIRPA, but express human or humanized CD47 and human or humanized SIRPA under the same regulatory elements as the endogenous porcine CD47 and SIRPA. Also provided are cells, tissues, and organs derived from such recombinant minipigs. Additionally provided herein are methods of transplanting grafts derived from such recombinant minipigs of a first donor with or without bone marrow derived from such recombinant minipigs of a second donor.

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 304,220, filed January 28, 2022. The entire disclosure of that provisional application is incorporated herein by reference in its entirety.

[0002] 2. Reference to Electronically Submitted Sequence Listings This application contains a computer readable sequence listing that has been submitted herewith in XML file format. The entire contents of the sequence listing are incorporated herein by reference in their entirety. The sequence listing XML file submitted herewith is entitled "14648-006-228_SequenceListing.xml", was created on January 24, 2023, and is 16,653 bytes in size.

[0003] 3.Technology Field Provided herein is a recombinant minipig that does not express endogenous porcine CD47 or SIRPA, but expresses human or humanized CD47 and human or humanized SIRPA. In certain embodiments, the expression of human or humanized CD47 and human or humanized SIRPA in the recombinant minipig provided herein is under the same regulatory elements as endogenous porcine CD47 and SIRPA. Also provided are cells, tissues, and organs derived from such recombinant minipig. Also provided herein is a method of transplanting a graft derived from such recombinant minipig of a first donor with or without bone marrow derived from such recombinant minipig of a second donor. [Background technology]

[0004] 4.Background technology Due to a severe shortage of allogeneic donors, the number of organ transplants currently performed is limited. This imbalance between supply and demand may be corrected by the use of organs from other species (xenografts). Given the ethical issues and impracticalities associated with the use of non-human primates, pigs are considered the most suitable donor species for humans. Their organ size and physiological similarity to humans, plus the ability to rapidly breed and inbreed pigs, make them particularly suitable for genetic modification that may improve their ability to function as graft donors for humans. See, e.g., Sachs (1994), Path. Biol. 42:217-219 and Piedrahita et al. (2004), Am. J. Transplant, 4 Suppl. 6:43-50.

[0005] Although transplantation combined with nonspecific immunosuppressive therapy is associated with high early graft acceptance rates, the main limitation to the success of clinical organ transplantation is late graft loss, mainly due to chronic rejection of the transplant. Thus, immune tolerance, a major goal of transplantation, becomes even more important for successful clinical xenotransplantation, as the lifelong level of immunosuppression required to prevent xenograft rejection may be too toxic to tolerate. Furthermore, no markers have been identified that reliably indicate whether immune tolerance has been achieved in a patient, so there is no laboratory value on which to base the withdrawal of immunosuppression.

[0006] Thus, goals in xenotransplantation include achieving immune tolerance, which may be achieved by xenogeneic thymus transplantation or by optimizing the durability of mixed chimeric cells derived from a donor animal after they are transplanted into a xenogeneic recipient, and by maintaining the health and viability of the donor animal.

[0007] Mixed chimerism can induce tolerance to the donor at the recipient's T cell, B cell, and natural killer (NK) cell levels (see, e.g., Griesemer et al. (2014), Immunol. Rev. 258:241-258; Sachs et al. (2014), Cold Spring Harb. Perspect. Med. 4:a015529).

[0008] CD47, also known as integrin-associated protein (IAP), is a ubiquitously expressed 50-kDa cell surface glycoprotein that functions as a ligand for the signal regulatory protein SIRPα (also known as CD172a, and SHPS-1, whose gene is SIRPA). See, e.g., Brown (2002), Curr. Opin. Cell. Biol., 14:603-7, and Brown and Frazier (2001), Trends Cell Biol., 111130-5. CD47 and SIRPα constitute an intercellular communication system that plays an important role in a variety of cellular processes, including cell migration, B cell adhesion, and T cell activation. See, for example, Liu et al. (2002), J. Biol. Chem. 277:10028; Motegi et al. (2003), EMBO 122:2634; Yoshida et al. (2002), J. Immunol. 168:3213; and Latour et al. (2001), J. Immunol. 167:2547. Furthermore, the CD47-SIRPα system is involved in the negative regulation of phagocytosis by macrophages. CD47 on the surface of some cell types (i.e., erythrocytes, platelets, or leukocytes) inhibited phagocytosis by macrophages. The role of CD47-SIRPα interaction in inhibiting phagocytosis has been illustrated by the observation that primary wild-type mouse macrophages rapidly phagocytose non-opsonized red blood cells (RBCs) from CD47-deficient mice, but not from wild-type mice. See, e.g., Oldenborg et al. (2000), Science 288:2051. CD47 has also been reported to inhibit both Fcγ and complement receptor-mediated phagocytosis through its receptor SIRPα. See, e.g., Oldenborg et al. (2001), J. Exp. Med. 193:855. CD47KO cells are violently rejected by macrophages after injection into syngeneic wild-type (WT) mice, demonstrating that CD47 provides a "don't eat me" signal to macrophages.See, e.g., Oldenborg PA, et al. (2000), Science, 288:2051-4, and Wang et al. (2007), Proc Natl Acad Sci US A. 104:13744. Xenotransplantation using pigs as a graft source has the potential to solve the severe shortage of human organ donors, a major limiting factor in clinical transplantation. See, e.g., Yang et al. (2007), Nature Reviews Immunology. 7:519-31. The strong rejection of xenogeneic cells by macrophages (see, e.g., Abe (2002), The Journal of Immunology 168:621) is primarily caused by the lack of functional interaction between donor CD47 and recipient SIRPα (see, e.g., Wang et al. (2007), Blood;109:836-42, Ide et al. (2007), Proc Natl Acad Sci USA 104:5062-6, and Navarro-Alvarez (2014), Cell Transplantation,23:345-54), which has led to the development of human CD47 transgenic pigs (see, e.g., Tena et al. (2017), Transplantation 101:316-21, and Nomura et al. (2020), Xenotransplantation 2020;27:e12549). In addition to macrophages, a subpopulation of DCs also expresses SIRPα (see, e.g., Wang et al. (2007), Proc Natl Acad Sci US A. 104:13744-9, and Guilliams et al. (2016), Immunity. 45:669-84). CD47-SIRPα signaling also inhibits DC activation and their ability to sensitize T cells, playing an important role in the induction of T cell tolerance by donor-specific transfusion (DST) or hepatocyte transplantation.See, e.g., Wang et al. (2007), Proc Natl Acad Sci US A. 104:13744-9, Wang et al. (2014), Cell transplantation 23:355-63, and Zhang et al. (2016), Sci Rep. 6:26839. Thus, there is an unmet need to generate safe and well-tolerated donor materials (e.g., organs, tissues, cells, etc.) for xenotransplantation that are not subject to rejection via CD47-SIRPα signaling. Summary of the Invention

[0009] 5. Summary of the Invention In one aspect, provided herein are recombinant minipigs comprising: (a)(i) a deletion or functional inactivation of an endogenous gene encoding porcine CD47, wherein expression of the endogenous gene encoding the porcine CD47 is regulated by the porcine CD47 regulatory element, and (ii) a transgene encoding a human CD47 inserted into the pig genome, wherein expression of the transgene encoding the human CD47 is regulated by the porcine CD47 regulatory element; and (b)(i) a deletion or functional inactivation of an endogenous gene encoding porcine SIRPα, wherein expression of the endogenous gene encoding porcine SIRPα is regulated by the porcine SIRPA regulatory element, and (ii) a transgene encoding a human SIRPα inserted into the pig genome, wherein expression of the transgene encoding the human SIRPα is regulated by the porcine SIRPA regulatory element.

[0010] In some embodiments, the minipig is an alpha-1,3 galactosyltransferase deficient minipig.

[0011] In some embodiments, the alpha-1,3 galactosyltransferase deficient minipigs are major histocompatibility complex (MHC) inbred minipigs.

[0012] In some embodiments, the human CD47 comprises SEQ ID NO: 1, 2, or 3. In some embodiments, the human SIRPA comprises SEQ ID NO: 4 or 5.

[0013] In some embodiments, the transgenes encoding human SIRPα and human CD47 are inserted into the genome of the miniature pig by homologous recombination. In some embodiments, the transgenes encoding human SIRPα and human CD47 are inserted into the pig genome by non-homologous end joining. In some embodiments, the transgenes encoding human SIRPα and human CD47 are inserted into the pig genome by recombinase-mediated cassette exchange. In some embodiments, the transgenes encoding human SIRPα and human CD47 are inserted into the pig genome by a site-specific nuclease. In some embodiments, the site-specific nuclease is selected from the group consisting of zinc finger, ZFN dimer, ZF nickase, transcription activator-like effector nuclease (TALEN), and CRISPR / Cas9.

[0014] In some embodiments, the expression of the transgene encoding human CD47 is regulated by the porcine CD47 regulatory element of NCBI gene ID: 397042. The recombinant minipig of claim 1, wherein the expression of the transgene encoding human SIRPα is regulated by the porcine SIRPA regulatory element of NCBI gene ID: 494566.

[0015] In some embodiments, the expression of the human CD47 protein is substantially similar to the expression pattern of endogenous porcine CD47 as determined by immunohistochemistry. In some embodiments, the expression of the human SIRPα protein is substantially similar to the expression pattern of endogenous porcine SIRPα as determined by immunohistochemistry.

[0016] In another aspect, provided herein is a recombinant minipig comprising (a) a humanized CD47 gene, and / or (b) a humanized SIRPA gene. In some embodiments, the recombinant minipig comprises a humanized CD47 gene. In some embodiments, the recombinant minipig comprises a humanized SIRPA gene. In some embodiments, the recombinant minipig comprises a humanized CD47 gene and a humanized SIRPA gene. In some embodiments, the humanized CD47 gene comprises a porcine CD47 gene comprising exon 2 of human CD47.

[0017] In another aspect, provided herein are cells derived from the recombinant minipigs provided herein.

[0018] In another aspect, provided herein is an oocyte derived from a recombinant minipig provided herein.

[0019] In another aspect, provided herein is sperm derived from the recombinant minipigs provided herein.

[0020] In another aspect, provided herein is a tissue derived from a recombinant minipig provided herein.

[0021] In another aspect, provided herein are organs derived from the recombinant minipigs provided herein.

[0022] In yet another aspect, provided herein is a method of transplanting a graft from a first recombinant minipig into a primate, the method comprising: (a) harvesting a graft from the first recombinant minipig provided herein; and (b) transplanting the graft into the primate. In some embodiments, the primate is a human. In some embodiments, the graft comprises a cell, tissue, or organ. In some embodiments, the organ is selected from the group consisting of heart, kidney, pancreatic islet, liver, pancreas, lung, intestine, skin, trachea, and cornea, or a combination thereof.

[0023] In some embodiments, the method further comprises harvesting bone marrow from a second recombinant minipig provided herein and transplanting the bone marrow into the same primate, in some embodiments, the bone marrow is transplanted at least 28 days prior to transplantation from the first recombinant pig.

[0024] In some embodiments, the first recombinant minipig and the second recombinant minipig are the same recombinant pig. In some embodiments, the first recombinant minipig and the second recombinant minipig are from a group of highly inbred minipigs. In some embodiments, the first recombinant minipig and the second recombinant minipig are genetically compatible minipigs. In some embodiments, the first recombinant minipig and the second recombinant minipig are MHC-matched.

[0025] In some embodiments, the first recombinant minipig-derived graft survives within the recipient for at least 6 months, 1 year, 5 years, 10 years, 15 years, or 20 years. In some embodiments, the first recombinant minipig-derived graft functions within the recipient for at least 6 months, 1 year, 5 years, 10 years, 15 years, or 20 years.

[0026] In some embodiments, the recipient requires a 90%, 80%, 70%, 60%, or 50% reduction in immunosuppressive therapy. [Brief description of the drawings]

[0027] 6. Brief description of the drawings [Figure 1A] Expression levels of porcine CD47 without guide pairs are shown. [Figure 1B] Expression levels of porcine CD47 according to guide pair number 1 are shown. [Figure 1C] Expression levels of porcine CD47 according to guide pair number 2 are shown. [Figure 1D] Expression levels of porcine CD47 according to guide pair number 3 are shown. [Diagram 2]Shown are the results of CD47 staining for transfected cells sorted based on binding to anti-CD47 monoclonal antibody B6H12 (human only). When blocked with human-specific monoclonal B6H12, a very high percentage of the population was further demonstrated to be null for porcine CD47 by staining with anti-CD47 monoclonal antibody CC26, which binds both human and porcine CD47. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] 7. MODE FOR CARRYING OUT THEINVENTION Provided herein is a recombinant minipig that expresses human or humanized CD47 and human or humanized SIRPA, but does not express endogenous porcine CD47 or porcine SIRPA. In certain embodiments, the expression of human or humanized CD47 and / or human or humanized SIRPA is under the same regulatory elements as the respective endogenous porcine genes. The generation of the recombinant minipig is described in Section 7.1. Also provided are cells, tissues, and organs derived from such recombinant minipig, which are described in Section 7.2. Additionally provided herein is a method of transplanting a graft derived from such recombinant minipig of a first donor with or without bone marrow derived from such recombinant minipig of a second donor, which is described in Section 7.3.

[0029] 7.1 Generation of transgenic minipigs Provided herein is a genetically modified pig that expresses human or humanized CD47 and human or humanized SIRPA, but does not express endogenous porcine CD47 or porcine SIRPA. In certain embodiments, the expression of human or humanized CD47 and / or human or humanized SIRPA in the genetically modified pig is regulated by endogenous regulatory elements (i.e., porcine CD47 and porcine SIRPA regulatory elements, respectively). The cells, tissues, and organs of such genetically modified pigs can be used for transplantation into primates. Without being bound by any particular theory, such transplantation not only ensures the health of the donor pig and the transplanted graft, but also maximizes the long-term survival rate of the graft after transplantation into primates.

[0030] Deleting or inactivating endogenous porcine CD47 and SIRPA and inserting a human homolog or portion thereof can be accomplished using a variety of methods known in the art, such as those described in Section 7.1.1 or the Examples. In certain embodiments, modifying the porcine genome to delete the endogenous porcine CD47 and SIRPA genes or portions thereof and inserting the human CD47 and SIRPA genes (such as the human homologs described in Section 7.1.2) or portions thereof can be performed in a single step. In certain embodiments, modifying the porcine genome to delete the endogenous porcine CD47 and SIRPA genes or portions thereof and inserting the human CD47 and SIRPA genes or portions thereof can be performed in two or more steps. A table of the inserted human CD47 and SIRPA sequences can be found in Table 1. In some embodiments, exon 2 of porcine CD47 is replaced with exon 2 of human CD47. Methods for experimentally determining the success of the generation of the recombinant pig are described in Section 7.1.3. Additionally, the donor minipig may carry additional genetic modifications, such as those described in Section 7.1.4.

[0031] In certain embodiments, the donor minipig carries human CD47 and SIRPA transgenes that are expressed only if the donor animal's endogenous CD47 and SIRPA genes are knocked out. In some embodiments, the donor animal's endogenous CD47 and SIRPA genes are knocked out just before the graft is harvested for transplantation. This "gene switch" allows the donor minipig to remain healthy for most of its life while allowing expression of the human transgenes for optimal transplant tolerance in the human recipient.

[0032] In certain embodiments, the donor minipig expresses CD47 and SIRPA at levels comparable to the physiological levels of CD47 and SIRPA in humans. For example, the levels of CD47 and SIRPα protein expressed in the kidney of the donor minipig are comparable to the levels of CD47 and SIRPα protein expressed in the kidney of a healthy human subject. Protein expression can be measured by methods known in the art or described herein.

[0033] In a specific embodiment, only one kidney of the donor minipig expresses CD47 and SIRPα proteins.

[0034] 7.1.1 Deletion of endogenous porcine genes and insertion of human homologues 7.1.1.1 Deletion or inactivation of endogenous porcine genes In one aspect, provided herein is a method to delete or inactivate endogenous porcine CD47 and SIRPA and insert human or humanized CD47 and human or humanized SIRPA.

[0035] Homologous recombination (HR) is a genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules. In some embodiments, endogenous porcine CD47 and SIRPA are deleted or inactivated by HR-based methods, such that a portion of the endogenous porcine CD47 and SIRPA is replaced. In some specific embodiments, the replacement of the portion of the endogenous porcine CD47 and SIRPA disrupts the start codon of the endogenous porcine CD47 and SIRPA. In other specific embodiments, the replacement of the portion of the endogenous porcine CD47 and SIRPA disrupts its open reading frame. In other specific embodiments, the replacement of the portion of the endogenous porcine CD47 and SIRPA blocks, inhibits, or reduces transcription to negligible levels. In other specific embodiments, the replacement of the portion of the endogenous porcine CD47 and SIRPA results in no translation product or a non-functional transcript. In some embodiments, endogenous porcine CD47 and SIRPA are deleted or inactivated in an HR-based manner such that the entire length of the endogenous porcine CD47 and SIRPA are replaced with non-CD47 and non-SIRPA genes. In some embodiments, a portion (e.g., one or more exons) of porcine CD47 and / or SIRPA are replaced with homologous regions of human CD47 and / or SIRPA, respectively. In some specific embodiments, the non-CD47 and non-SIRPA genes are selection genes. In certain embodiments, the non-CD47 and non-SIRPA genes are drug resistance genes.

[0036] In certain embodiments, the open reading frames of porcine CD47 and SIRPA are deleted and replaced with the open reading frames of human CD47 and SIRPA, hi certain embodiments, the exons of porcine CD47 and SIRPA are deleted and replaced with the exons of human CD47 and SIRPA.

[0037] Without wishing to be bound by theory, by deleting the open reading frames of porcine CD47 and SIRPA, or the exons of porcine CD47 and SIRPA, and replacing them with their respective human counterparts, human CD47 and SIRPA are expressed under the control of the porcine regulatory elements of CD47 and SIRPA, respectively. Regulation of human CD47 and SIRPA under the control of the porcine regulatory elements of each porcine gene results in expression patterns and levels of human CD47 and SIRPA similar to those of endogenous porcine CD47 and SIRPA. Gene expression levels can be determined using any suitable method known in the art, including, for example, quantitative PCR, real-time PCR, Southern blotting, or RNA sequencing.

[0038] In some embodiments, the expression level of human or humanized CD47 under the control of porcine regulatory elements is substantially similar to the expression level of endogenous porcine CD47 (e.g., only about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% higher or lower than the expression of endogenous porcine CD47). In some embodiments, the expression level of human or humanized SIRPA under the control of the porcine regulatory elements is substantially similar to the expression level of endogenous porcine SIRPA (e.g., only about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% higher or lower than the expression of endogenous porcine SIRPA). In some embodiments, the expression levels of both human or humanized CD47 and human or humanized SIRPA under the control of the porcine regulatory elements are substantially similar to the expression levels of endogenous porcine CD47 and endogenous porcine SIRPA, respectively (e.g., only about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% higher or lower than the expression of endogenous porcine CD47 and endogenous porcine SIRPA).

[0039] In some embodiments, the spatial expression pattern of human or humanized CD47 under the control of porcine regulatory elements is substantially similar to the spatial expression pattern of endogenous porcine CD47. In some embodiments, the spatial expression pattern of human or humanized SIRPA under the control of porcine regulatory elements is substantially similar to the spatial expression pattern of endogenous porcine SIRPA. In some embodiments, the spatial expression patterns of both human or humanized CD47 and human or humanized SIRPA under the control of porcine regulatory elements are substantially similar to the spatial expression patterns of endogenous porcine CD47 and endogenous porcine SIRPA, respectively.

[0040] Spatial gene expression patterns can be identified using any suitable method known in the art, including, for example, single-cell sequencing, single-molecule fluorescence in situ hybridization, or spatial transcriptomics. Exemplary methods for analyzing special gene expression patterns are described, see, for example, Sun et al., Nat Methods. 2020 February; 17(2): 193-200 and Dries et al., Genome Biology (2021) 22: 78.

[0041] In some embodiments, the temporal expression pattern of human or humanized CD47 under the control of porcine regulatory elements is substantially similar to the temporal expression pattern of endogenous porcine CD47 (e.g., delayed or accelerated by no more than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% compared to the expression of endogenous porcine CD47). In some embodiments, the temporal expression pattern of human or humanized SIRPA under the control of porcine regulatory elements is substantially similar to the temporal expression pattern of endogenous porcine SIRPA (e.g., delayed or accelerated by no more than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% compared to expression of endogenous porcine SIRPA). In some embodiments, the temporal expression patterns of both human or humanized CD47 and human or humanized SIRPA under the control of porcine regulatory elements are substantially similar to the temporal expression patterns of endogenous porcine CD47 and endogenous porcine SIRPA, respectively (e.g., delayed or accelerated by no more than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% compared to the expression of endogenous porcine CD47 and endogenous porcine SIRPA).

[0042] Recombinases are enzymes that recognize specific polynucleotide sequences (recombinase recognition sites) flanking an intervening polynucleotide and catalyze interstrand exchange, resulting in inversion or excision of the intervening polynucleotide. See, e.g., Araki et al. (1995), Proc. Natl. Acad. Sci. USA 92:160-164. In some embodiments, endogenous porcine CD47 and SIRPA are deleted or inactivated using a recombinase-based method and recombinase recognition sites in the same orientation. In some specific embodiments, the location of the recombinase recognition sites in the same orientation is designed so that the entire full-length porcine gene is deleted. In some specific embodiments, the location of the recombinase recognition sites in the same orientation is designed so that a portion of the porcine gene is deleted, resulting in aberrant transcription or translation. In other embodiments, endogenous porcine CD47 and SIRPA are deleted or inactivated using a recombinase-based method and recombinase recognition sites in the opposite orientation. In some particular embodiments, the location of the inverted recombinase recognition site is designed such that the entire full-length porcine gene is inverted, resulting in aberrant transcription or translation. In some particular embodiments, the location of the inverted recombinase recognition site is designed such that a portion of the porcine gene is inverted, resulting in aberrant transcription or translation.

[0043] Sequence-specific endonucleases include, but are not limited to, RNA-guided DNA nucleases (e.g., CRISPR / Cas9 systems), ZFNs, ZFN dimers, ZF nickases, and TALENs. In some embodiments, endogenous porcine CD47 and SIRPA are deleted or inactivated by sequence-specific endonuclease-based methods. In some specific embodiments, the sequence-specific endonuclease is designed to disrupt the start codons of endogenous porcine CD47 and SIRPA. In other specific embodiments, the sequence-specific endonuclease is designed to disrupt the normal open reading frame of endogenous porcine CD47 and SIRPA. In other specific embodiments, the sequence-specific endonuclease is designed to result in no transcription product. In yet other specific embodiments, the sequence-specific endonuclease is designed to result in no translation product or a non-functional transcription product.

[0044] 7.1.1.2 Inserting human genes into the pig genome In some embodiments, the transgene used to replace porcine CD47 is the genomic sequence of human CD47 or a part thereof. By using genomic DNA, all human CD47 isoforms can be expressed. In other embodiments, the transgene used to replace porcine CD47 is the complementary DNA of the message RNA (mRNA) of a known splice variant of human CD47. In other embodiments, the transgene used to replace porcine CD47 is the coding sequence (CDS) of a known splice variant of human CD47. The nucleic acid sequence encoding human CD47 can be found at the following NCBI RefSeq accession number: NC_000003.12. The cDNA of the mRNA encoding human CD47 isoform 1 can be found at the following NCBI RefSeq accession number: NM_001777.4. The cDNA of the mRNA encoding human CD47 isoform 2 can be found at the following NCBI RefSeq accession number: NM_198793.3. A cDNA of an mRNA encoding human CD47 isoform 3 can be found at the following NCBI RefSeq accession number: NM_001382306.1. A CDS encoding human CD47 isoform 1 can be found at the following NCBI RefSeq accession number: CCDS43126.1 or SEQ ID NO: 1, which corresponds to the amino acid sequence NP_001768.1. A CDS encoding human CD47 isoform 2 can be found at the following NCBI RefSeq accession number: CCDS43125.1 or SEQ ID NO: 2, which corresponds to the amino acid sequence NP_942088.1. A CDS encoding human CD47 isoform 3 can be found at the following NCBI RefSeq accession number: SEQ ID NO: 3, which corresponds to the amino acid sequence NP_001369235.1. In some embodiments, the transgene used to replace the porcine CD47 is one or more human exons (e.g., exon 2 of human CD47).

[0045] In certain embodiments, the transgene encoding human CD47 used in the constructs described herein is a transgene listed in Table 1 below. In certain embodiments, the transgene encoding human CD47 comprises the nucleotide sequence of SEQ ID NO: 1. In other embodiments, the transgene encoding human CD47 comprises the nucleotide sequence of SEQ ID NO: 2. In other embodiments, the transgene encoding human CD47 comprises the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the transgene encoding human CD47 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 1. In other embodiments, the transgene encoding human CD47 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 2. In other embodiments, the transgene encoding human CD47 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:3.

[0046] In some embodiments, the transgene is a genomic sequence of human CD47, where the transgene replaces the genomic sequence of porcine CD47 extending from the first exon to the last exon. In other embodiments, the transgene is a cDNA of human CD47, where the transgene replaces the genomic sequence of porcine CD47 extending from the first exon to the last exon. In other embodiments, the transgene is a cDNA of human CD47, where the transgene replaces the genomic sequence of porcine CD47 with intact porcine 5'UTR and 3'UTR. In yet other embodiments, the transgene is a CDS of human CD47, where the transgene replaces the porcine CD47 with intact porcine 5'UTR and 3'UTR. In some embodiments, the transgene is a portion of human CD47 (e.g., exon 2 of human CD47).

[0047] As provided herein, in certain aspects, the CD47 transgene is a humanized CD47. In some embodiments, the humanized CD47 is an endogenous porcine CD47 comprising one or more exons replaced with one or more corresponding homologous exons of human CD47. For example, in some embodiments, the CD47 transgene is a humanized CD47 in which exon 1 of porcine CD47 is replaced with exon 1 of human CD47. In some embodiments, the CD47 transgene is a humanized CD47 in which exon 2 of porcine CD47 is replaced with exon 2 of human CD47. In some embodiments, the CD47 transgene is a humanized CD47 in which exon 3 of porcine CD47 is replaced with exon 3 of human CD47. In some embodiments, the CD47 transgene is a humanized CD47 in which exon 4 of porcine CD47 is replaced with exon 4 of human CD47. In some embodiments, the CD47 transgene is a humanized CD47 in which exon 5 of porcine CD47 is replaced with exon 5 of human CD47. In some embodiments, the CD47 transgene is a humanized CD47 in which exon 6 of porcine CD47 is replaced with exon 6 of human CD47. In some embodiments, the CD47 transgene is a humanized CD47 in which exon 7 of porcine CD47 is replaced with exon 7 of human CD47. In some embodiments, the CD47 transgene is a humanized CD47 in which exon 8 of porcine CD47 is replaced with exon 8 of human CD47. In some embodiments, the CD47 transgene is a humanized CD47 in which exon 9 of porcine CD47 is replaced with exon 9 of human CD47. In some embodiments, the CD47 transgene is a humanized CD47 in which exon 10 of porcine CD47 is replaced with exon 10 of human CD47. In some embodiments, a portion of the coding sequence of the porcine CD47 is replaced with a portion of the coding sequence of the human CD47. In some embodiments, about 10% of the coding sequence of the porcine CD47 is replaced with about 10% of the coding sequence of the corresponding homologous portion of the human CD47. In some embodiments, a portion of the coding sequence of the porcine CD47 is replaced with a portion of the coding sequence of the human CD47.In some embodiments, about 20% of the coding sequence of the porcine CD47 is replaced with about 20% of the coding sequence of the corresponding homologous portion of the human CD47. In some embodiments, a portion of the coding sequence of the porcine CD47 is replaced with a portion of the coding sequence of the human CD47. In some embodiments, about 30% of the coding sequence of the porcine CD47 is replaced with about 30% of the coding sequence of the corresponding homologous portion of the human CD47. In some embodiments, a portion of the coding sequence of the porcine CD47 is replaced with a portion of the coding sequence of the human CD47. In some embodiments, about 40% of the coding sequence of the porcine CD47 is replaced with about 40% of the coding sequence of the corresponding homologous portion of the human CD47. In some embodiments, a portion of the coding sequence of the porcine CD47 is replaced with a portion of the coding sequence of the human CD47. In some embodiments, about 50% of the coding sequence of the porcine CD47 is replaced with about 50% of the coding sequence of the corresponding homologous portion of the human CD47. In some embodiments, a portion of the coding sequence of the porcine CD47 is replaced with a portion of the coding sequence of the human CD47. In some embodiments, about 60% of the coding sequence of the porcine CD47 is replaced with about 60% of the coding sequence of the corresponding homologous portion of the human CD47. In some embodiments, a portion of the coding sequence of the porcine CD47 is replaced with a portion of the coding sequence of the human CD47. In some embodiments, about 70% of the coding sequence of the porcine CD47 is replaced with about 70% of the coding sequence of the corresponding homologous portion of the human CD47. In some embodiments, a portion of the coding sequence of the porcine CD47 is replaced with a portion of the coding sequence of the human CD47. In some embodiments, about 80% of the coding sequence of the porcine CD47 is replaced with about 80% of the coding sequence of the corresponding homologous portion of the human CD47. In some embodiments, a portion of the coding sequence of the porcine CD47 is replaced with a portion of the coding sequence of the human CD47. In some embodiments, about 90% of the coding sequence of the porcine CD47 is replaced with about 90% of the coding sequence of the corresponding homologous portion of the human CD47. In some embodiments, a portion of the coding sequence of the porcine CD47 is replaced with a portion of the coding sequence of the human CD47.In some embodiments, about 95% of the coding sequence of the porcine CD47 is replaced with about 95% of the coding sequence of the corresponding homologous portion of the human CD47.

[0048] In some embodiments, the transgene used to replace porcine SIRPA is the genomic sequence of human SIRPA. By using genomic DNA, all human SIRPA isoforms can be expressed. In other embodiments, the transgene used to replace porcine SIRPA is the complementary DNA of the message RNA (mRNA) of a known splice variant of human SIRPA. In other embodiments, the transgene used to replace porcine SIRPA is the coding sequence (CDS) of a known splice variant of human SIRPA. The nucleic acid sequence encoding human SIRPα can be found at the following NCBI RefSeq accession number: NC_000020.11. The cDNA of the mRNA encoding human SIRPα isoform 1 can be found at the following NCBI RefSeq accession number: NM_080792.3, NM_001040022.1 or NM_001040023.2. The cDNA of the mRNA encoding human SIRPα isoform 2 can be found at the following NCBI RefSeq accession number: NM_001330728.1. The CDS encoding human SIRPα isoform 1 can be found at the following NCBI RefSeq accession number: CCDS13022.1 or SEQ ID NO: 4, which corresponds to the amino acid sequence NP_001035111.1, NP_001035112.1, or NP_542970.1. The CDS encoding human SIRPα isoform 2 can be found at the following NCBI RefSeq accession number: CCDS82593.1 or SEQ ID NO: 5, which corresponds to the amino acid sequence NP_001317657.1. In some embodiments, the transgene is a portion of human SIRPA.

[0049] In certain embodiments, the transgene encoding human SIRPα used in the constructs described herein is a transgene listed in Table 1 below. In certain embodiments, the transgene encoding human SIRPα comprises the nucleotide sequence of SEQ ID NO: 4. In other embodiments, the transgene encoding human SIRPα comprises the nucleotide sequence of SEQ ID NO: 5. In certain embodiments, the transgene encoding human SIRPα comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 4. In other embodiments, the transgene encoding human SIRPα comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 5.

[0050] In some embodiments, when the transgene is a genomic sequence of human SIRPA, the transgene replaces the genomic sequence of porcine SIRPA extending from the first exon to the last exon. In other embodiments, when the transgene is a cDNA of human SIRPA, the transgene replaces the genomic sequence of porcine SIRPA extending from the first exon to the last exon. In other embodiments, when the transgene is a cDNA of human SIRPA, the transgene replaces porcine SIRPA with intact porcine 5'UTR and 3'UTR. In yet other embodiments, when the transgene is a CDS of human SIRPA, the transgene replaces porcine SIRPA with intact porcine 5'UTR and 3'UTR. In some embodiments, the transgene replaces the homologous exon of porcine SIRPA (e.g., exon 2 of human SIRPA replaces exon 2 of porcine SIRPA).

[0051] As provided herein, in certain aspects, the SIRPA transgene is humanized SIRPA. In some embodiments, the humanized SIRPA is endogenous porcine SIRPA, which comprises one or more exons replaced with one or more corresponding homologous exons of human SIRPA. For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 1 of porcine SIRPA is replaced with exon 1 of human SIRPA. For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 2 of porcine SIRPA is replaced with exon 2 of human SIRPA. For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 3 of porcine SIRPA is replaced with exon 3 of human SIRPA. For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 4 of porcine SIRPA is replaced with exon 4 of human SIRPA. For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 5 of porcine SIRPA is replaced with exon 5 of human SIRPA. For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 6 of porcine SIRPA is replaced with exon 6 of human SIRPA. For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 7 of porcine SIRPA is replaced with exon 7 of human SIRPA. For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 8 of porcine SIRPA is replaced with exon 8 of human SIRPA. For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 9 of porcine SIRPA is replaced with exon 9 of human SIRPA. For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 10 of porcine SIRPA is replaced with exon 10 of human SIRPA. For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 11 of porcine SIRPA is replaced with exon 11 of human SIRPA.For example, in some embodiments, the SIRPA transgene is humanized SIRPA, in which exon 12 of porcine SIRPA is replaced with exon 12 of human SIRPA. In some embodiments, a portion of the coding sequence of the porcine SIRPA is replaced with a portion of the coding sequence of the human SIRPA. In some embodiments, about 10% of the coding sequence of the porcine SIRPA is replaced with about 10% of the coding sequence of the corresponding homologous portion of the human SIRPA. In some embodiments, a portion of the coding sequence of the porcine SIRPA is replaced with a portion of the coding sequence of the human SIRPA. In some embodiments, about 20% of the coding sequence of the porcine SIRPA is replaced with about 20% of the coding sequence of the corresponding homologous portion of the human SIRPA. In some embodiments, a portion of the coding sequence of the porcine SIRPA is replaced with a portion of the coding sequence of the human SIRPA. In some embodiments, about 30% of the coding sequence of the porcine SIRPA is replaced with about 30% of the coding sequence of the corresponding homologous portion of the human SIRPA. In some embodiments, a portion of the coding sequence of the porcine SIRPA is replaced with a portion of the coding sequence of the human SIRPA. In some embodiments, about 40% of the coding sequence of the porcine SIRPA is replaced with about 40% of the coding sequence of the corresponding homologous portion of the human SIRPA. In some embodiments, a portion of the coding sequence of the porcine SIRPA is replaced with a portion of the coding sequence of the human SIRPA. In some embodiments, about 50% of the coding sequence of the porcine SIRPA is replaced with about 50% of the coding sequence of the corresponding homologous portion of the human SIRPA. In some embodiments, a portion of the coding sequence of the porcine SIRPA is replaced with a portion of the coding sequence of the human SIRPA. In some embodiments, about 60% of the coding sequence of the porcine SIRPA is replaced with about 60% of the coding sequence of the corresponding homologous portion of the human SIRPA. In some embodiments, a portion of the coding sequence of the porcine SIRPA is replaced with a portion of the coding sequence of the human SIRPA. In some embodiments, about 70% of the coding sequence of the porcine SIRPA is replaced with about 70% of the coding sequence of the corresponding homologous portion of the human SIRPA. In some embodiments, a portion of the coding sequence of the porcine SIRPA is replaced with a portion of the coding sequence of the human SIRPA.In some embodiments, about 80% of the coding sequence of the porcine SIRPA is replaced with about 80% of the coding sequence of the corresponding homologous part of the human SIRPA. In some embodiments, a part of the coding sequence of the porcine SIRPA is replaced with a part of the coding sequence of the human SIRPA. In some embodiments, about 90% of the coding sequence of the porcine SIRPA is replaced with about 90% of the coding sequence of the corresponding homologous part of the human SIRPA. In some embodiments, a part of the coding sequence of the porcine SIRPA is replaced with a part of the coding sequence of the human SIRPA. In some embodiments, about 95% of the coding sequence of the porcine SIRPA is replaced with about 95% of the coding sequence of the corresponding homologous part of the human SIRPA.

[0052] An expression cassette generally comprises a regulatory element and a transgene. The regulatory element can be, for example, a promoter. In some embodiments, the human or humanized CD47 promoter is a constitutively active promoter. In other embodiments, the human or humanized CD47 promoter is the endogenous porcine CD47 promoter.

[0053] In some embodiments, the human CD47 and SIRPA transgenes are randomly inserted into a locus in the pig genome. In some embodiments, the human CD47 and SIRPA transgenes are inserted into a safe harbor locus in the pig genome. In some embodiments, the human CD47 transgene is inserted near the porcine CD47 gene (NCBI gene ID: 397042) and the porcine CD47 gene is not deleted. In some embodiments, the human CD47 transgene is inserted into the endogenous porcine CD47 gene and the porcine CD47 gene is deleted. In some embodiments, the human SIRPA transgene is inserted near the porcine SIRPA gene (NCBI gene ID: 494566) and the porcine SIRPA gene is not deleted. In some embodiments, the human SIRPA transgene is inserted into the endogenous porcine SIRPA gene and the porcine SIRPA gene is deleted. In some embodiments, a portion of human CD47 or SIRPA replaces the homologous exon in porcine CD47 or SIRPA, respectively.

[0054] 7.1.1.3 Delivery vehicles and methods In some embodiments, the plasmid or vector carrying the components described in Sections 7.1.1.1 and 7.1.1.2 is delivered by a viral vector, including but not limited to adeno-associated virus (AAV), self-complementary adeno-associated virus (scAAV), adenovirus, retrovirus, lentivirus (e.g., simian immunodeficiency virus, human immunodeficiency virus, or modified human immunodeficiency virus), Newcastle disease virus (NDV), herpes virus (e.g., herpes simplex virus), alphavirus, and vaccinia virus. The viral vector may further include other elements, such as poly(A) sites, transcription termination sites, or virus-specific elements, such as terminal inverted repeats. See, e.g., Buard et al. (2009), British Journal of Pharmacology 157:153-165. In other embodiments, a portion of the plasmid or vector carrying the components described in Sections 7.1.1.1 and 7.1.1.2 is delivered via a transposase, including but not limited to, sleeping beauty and piggyback.

[0055] Methods for introducing the human or humanized CD47 and SIRPA genes into the germline of animals include, but are not limited to, somatic cell nuclear transfer (SCNT), pronuclear microinjection, sperm-mediated gene transfer (SMGT), oocyte transduction, and intracytoplasmic sperm injection.See, for example, Yum et al. (2016) J Vet Sci 2016,17:261-268; Whyte and Prather (2011), Mol Reprod Dev 78:879-891; Sachs and Gali (2009). Pronuclear microinjection involves direct injection of DNA into the pronucleus. Eggs for these purposes can be collected from superovulated females and then transferred to recipient pigs by embryo transfer.See, for example, Whyte and Prather (2011), Mol Reprod Dev 78:879-891. SMGT involves culturing a gene for a transgene of interest with sperm, which are then used for fertilization. See, e.g., Lavitrano et al., (2002), Proc Nat Acad Sci USA. 99:14230-14235. Oocyte transduction involves maturing porcine oocytes in vitro in a serum-free, chemically defined maturation medium, which are then infected in vitro with a replication-deficient pseudotyped retrovirus, fertilized, cultured, and then implanted into a recipient female. See, e.g., Cabot et al., (2001), Anim Biotechnol; 12(2):205-14. Genetic modification by intracytoplasmic sperm injection involves in vitro matured porcine oocytes that are fertilized by intracytoplasmic sperm injection. See, e.g., Lai et al., (2001) Zygote; 9(4):339-46.

[0056] 7.1.2 Replacement of endogenous porcine genes with human homologues Constructs for expression of a transgene generally include a nucleotide sequence encoding the transgene of interest, e.g., human or humanized CD47 or SIRPα, as described in Section 7.1.2.1. Additionally, constructs for expression of a transgene include other elements involved in the selected replacement technique, as described in Section 7.1.2.2, and potential selectable markers (positive and / or negative). Using the constructs of Section 7.1.2.1 and the replacement strategy of Section 7.1.2.2, human or humanized CD47 is inserted under the same regulatory elements of NCBI Gene ID: 397042 (endogenous porcine CD47) and human or humanized SIRPA is inserted under the same regulatory elements of NCBI Gene ID: 494566 (endogenous porcine SIRPA).

[0057] 7.1.2.1 Human CD47 and SIRPA sequences In some embodiments, the transgene used to replace porcine CD47 or a portion thereof is the genomic sequence of human CD47 or a portion thereof. By using genomic DNA, all human CD47 isoforms can be expressed. In other embodiments, the transgene used to replace porcine CD47 or a portion thereof is the complementary DNA of the message RNA (mRNA) of a known splice variant of human CD47 or a portion thereof. In other embodiments, the transgene used to replace porcine CD47 or a portion thereof is the coding sequence (CDS) of a known splice variant of human CD47 or a portion thereof. The nucleic acid sequence encoding human CD47 or a portion thereof can be found at the following NCBI RefSeq accession number: NC_000003.12. The cDNA of the mRNA encoding human CD47 isoform 1 can be found at the following NCBI RefSeq accession number: NM_001777.4. The cDNA of the mRNA encoding human CD47 isoform 2 can be found at the following NCBI RefSeq accession number: NM_198793.3. The cDNA of the mRNA encoding human CD47 isoform 3 can be found at the following NCBI RefSeq accession number: NM_001382306.1. The CDS encoding human CD47 isoform 1 can be found at the following NCBI RefSeq accession number: CCDS43126.1 or SEQ ID NO: 1, which corresponds to the amino acid sequence NP_001768.1. The CDS encoding human CD47 isoform 2 can be found at the following NCBI RefSeq accession number: CCDS43125.1 or SEQ ID NO: 2, which corresponds to the amino acid sequence NP_942088.1. The CDS encoding human CD47 isoform 3 can be found at the following NCBI RefSeq accession number: SEQ ID NO: 3, which corresponds to the amino acid sequence NP_001369235.1.

[0058] In certain embodiments, the transgene encoding human CD47 used in the constructs described herein is a transgene listed in Table 1 below. In certain embodiments, the transgene encoding human CD47 comprises the nucleotide sequence of SEQ ID NO: 1. In other embodiments, the transgene encoding human CD47 comprises the nucleotide sequence of SEQ ID NO: 2. In other embodiments, the transgene encoding human CD47 comprises the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the transgene encoding human CD47 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 1. In some embodiments, the transgene encoding human CD47 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 2. In some embodiments, the transgene encoding human CD47 comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:3.

[0059] In some embodiments, the transgene is a genomic sequence of human CD47, and the transgene replaces the genomic sequence of porcine CD47 extending from the first exon to the last exon. In other embodiments, the transgene is a cDNA of human CD47, and the transgene replaces the genomic sequence of porcine CD47 extending from the first exon to the last exon. In other embodiments, the transgene is a cDNA of human SIRPA, and the transgene replaces porcine CD47 with intact porcine 5'UTR and 3'UTR. In yet other embodiments, the transgene is a CDS of human CD47, and the transgene replaces porcine CD47 with intact porcine 5'UTR and 3'UTR. In some embodiments, the transgene replaces a portion of porcine CD47 (e.g., a single exon) with a portion of human CD47 (e.g., a homologous exon).

[0060] In other embodiments, the DNA sequence of the CD47 transgene is non-human. For example, the DNA sequence of CD47 corresponds to a DNA sequence selected from the group of non-human CD47 sequences, which group includes NCBI Gene ID Nos. 460569 (Pan troglodytes or chimpanzee), 704980 (Macaca mulatta or rhesus monkey), 478552 (Canis lupus familiaris or dog), 282661 (Bos taurus or cow), 16423 (Mus musculus or house mouse), 29364 (Rattus norvegicus or brown rat), 418408 (Gallus or chicken), 100926819 (Sarcophilus harrisii or Tasmanian devil), 102089340 (Columba livia or rock pigeon), 101681023 (Mustela putorius or chicken ... furo or ferret), 109691157 (Castor canadensis or American beaver), and 101836211 (Mesocricetus auratus or golden hamster).

[0061] In some embodiments, the transgene used to replace porcine SIRPA or a portion thereof is the genomic sequence of human SIRPA or a portion thereof. By using genomic DNA, all human SIRPα isoforms can be expressed. In other embodiments, the transgene used to replace porcine SIRPA or a portion thereof is the complementary DNA of the message RNA (mRNA) of a known splice variant of human SIRPα or a portion thereof. In other embodiments, the transgene used to replace porcine SIRPA or a portion thereof is the coding sequence (CDS) of a known splice variant of human SIRPα or a portion thereof. The nucleic acid sequence encoding human SIRPα can be found at the following NCBI RefSeq accession number: NC_000020.11. The cDNA of the mRNA encoding human SIRPα isoform 1 can be found at the following NCBI RefSeq accession number: NM_080792.3, NM_001040022.1 or NM_001040023.2. The cDNA of the mRNA encoding human SIRPα isoform 2 can be found at the following NCBI RefSeq accession number: NM_001330728.1. The CDS encoding human SIRPα isoform 1 can be found at the following NCBI RefSeq accession number: CCDS13022.1 or SEQ ID NO: 4, which corresponds to the amino acid sequence NP_001035111.1, NP_001035112.1, or NP_542970.1. The CDS encoding human SIRPα isoform 2 can be found at the following NCBI RefSeq accession number: CCDS82593.1 or SEQ ID NO: 5, which corresponds to the amino acid sequence NP_001317657.1.

[0062] In certain embodiments, the transgene encoding human SIRPα used in the constructs described herein is a transgene listed in Table 1 below. In certain embodiments, the transgene encoding human SIRPα comprises the nucleotide sequence of SEQ ID NO: 4. In other embodiments, the transgene encoding human SIRPα comprises the nucleotide sequence of SEQ ID NO: 5. In certain embodiments, the transgene encoding human SIRPα comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 4. In other embodiments, the transgene encoding human SIRPα comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 5.

[0063] In some embodiments, the transgene is a genomic sequence of human SIRPA, and the transgene replaces the genomic sequence of porcine SIRPA extending from the first exon to the last exon. In other embodiments, the transgene is a cDNA of human SIRPA, and the transgene replaces the genomic sequence of porcine SIRPA extending from the first exon to the last exon. In other embodiments, the transgene is a cDNA of human SIRPA, and the transgene replaces porcine SIRPA with intact porcine 5'UTR and 3'UTR. In yet other embodiments, the transgene is a CDS of human SIRPA, and the transgene replaces porcine SIRPA with intact porcine 5'UTR and 3'UTR. In some embodiments, the transgene replaces a portion of porcine SIRPA (e.g., a single exon) with a portion of human SIRPA (e.g., a homologous exon).

[0064] In other embodiments, the DNA sequence of the SIRPA transgene is non-human. For example, the DNA sequence of SIRPA corresponds to a DNA sequence selected from the group of non-human SIRPA sequences, which group includes NCBI Gene ID Nos. 458039 (Pan troglodytes or chimpanzee), 717811 (Macaca mulatta or rhesus monkey), 101926317 (Macaca fascicularis or cynomolgus monkey), 609452 (Canis lupus familiaris or dog), 327666 (Bos taurus or cow), 19261 (Mus musculus or house mouse), 25528 (Rattus norvegicus or brown rat), 118618252 (Molossus or Pallas's free-tailed bat), 109692903 (Castor canadensis or American beaver), 101677644 (Mustela putorius or guinea pig), 101677645 (Mustela putorius or guinea pig), 101677646 (Mustela putorius or guinea pig), 101677647 (Mustela putorius or guinea pig), 101677648 (Mustela putorius or guinea pig), 101677649 ... furo or ferret), 101839275 (Mesocricetus auratus or golden hamster), 100067339 (Equus caballus or horse), 118536470 (Halichoerus grypus or grey seal), 118011325 (Mirounga 23dminis or southern elephant seal), 116739680 (Phocoena sinus or vaquita), 116641565 (Phoca vitulina or harbor seal), 116563017 (Sapajus apella or tufted capuchin monkey), or 116461231 (Hylobates moloch or wah-wah gibbon).

[0065] 7.1.2.2 Replacement Strategies Gene replacement of porcine CD47 and SIRPA or a part thereof with human CD47 and SIRPA or a part thereof can be carried out by various methods known in the art.For example, one non-limiting exemplary technique includes homologous recombination.HR is widely used by researchers in gene targeting and transgenic animal production.HR is a genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules.Gene targeting by homologous recombination in embryonic stem cells allows unprecedented precision, thereby allowing genes to be manipulated and the effects of this manipulation to be investigated.

[0066] Another exemplary technique suitable for gene replacement of porcine CD47 and SIRPA or a portion thereof with human CD47 and SIRPA or a portion thereof includes non-homologous end joining (NHEJ). NHEJ ligates directly to the ends of DSBs and has little or no homologous sequences compared to HR. It has been utilized by some researchers to successfully integrate genes of interest in combination with site-specific endonucleases. See, for example, Schiermeyer et al. (2019) Plant Direct.;3(7):e00153.

[0067] Yet another exemplary technique suitable for gene replacement of porcine CD47 and SIRPA or a part thereof with human CD47 and SIRPA or a part thereof includes recombinase-mediated cassette exchange (RMCE). RCME technique allows swapping of large genomic regions. This technique is generally recommended for the generation of humanized models and several variants using the same parental embryonic stem cell clone. RMCE can easily and specifically adapt to the integration of genomic DNA, allowing the replacement of large genomic regions of the host genome with coexisting human genomic regions.

[0068] In some embodiments, the gene replacement of the porcine CD47 and SIRPA or a portion thereof with human CD47 and SIRPA or a portion thereof can be generated using an enzyme that promotes DNA DSBs. In some embodiments, the enzyme that promotes DNA DSBs comprises a sequence-specific endonuclease. In certain embodiments, the sequence-specific endonuclease comprises an RNA-guided DNA nuclease, such as the CRISPR / Cas9 system.

[0069] The CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats) system utilizes RNA-guided DNA binding and sequence-specific cleavage of target DNA. A guide RNA (gRNA) (e.g., containing 20 nucleotides) is complementary to the target genomic DNA sequence upstream of the genomic PAM (protospacer adjacent motif) site (NNG) and the constant RNA scaffold region. Cas (CRISPR-associated) protein binds to the gRNA and the target DNA to which the gRNA is bound, and introduces a double-stranded break at a predetermined location upstream of the PAM site. For example, Geurts et al. (2009), Science 325:433, Mashimo et al. (2010), PloS ONE 5, e8870, Carbery et al. (2010), Genetics 186:451-459, Tesson et al. (2011), Nat.Biotech.29:695-696, Wiedenheft See et al. (2012), Nature 482, 331-338, Jinek et al. (2012), Science 337:816-821, Mali et al. (2013), Science 339:823-826, Cong et al. (2013), Science 339:819-823. Further improvements to the CRISPR / Cas9 system include the use of truncated gRNAs with shorter target-complementary regions, less than 20 nucleotides in length, which can reduce unwanted mutagenesis at off-target sites by more than 5000-fold without sacrificing on-target genome editing efficiency. See, for example, Fu et al. (2014) Nat Biotechnol.;32(3):279-284. Modified CRISPR / Cas9-mediated HR uses single-stranded oligodeoxynucleotides (ssODNs) as repair templates (aka "easiCRISPR"), which is sufficient for efficient editing and replacement of host genes with their human counterparts. Its advantages include no need for selection, and therefore no scarring of the genome. See, for example, Quadros et al. (2017) Genome. Biol. 18, 92; Codner et al. (2018) BMC Biol. 16, 70.

[0070] In some embodiments, the sequence-specific endonuclease comprises a ZFN, a ZFN dimer, and / or a ZF nickase. A ZFN has separate DNA binding and DNA cleavage domains. The cleavage domain does not have obvious sequence specificity, and the cleavage may be redirected by the separate DNA binding domain. See, for example, Kim et al. (1994); Proc. Natl. Acad. Sci. USA 91:883-887, Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93:1156-1160, Kim et al. (1998) Biol. Chem. 379:489-495. Each unit of about 30 amino acids of a zinc finger (ZF) binds to a single atom of zinc. The crystal structure of a set of three fingers bound to DNA reveals that each finger mainly contacts 3 bp of DNA in a remarkably modular manner. See, e.g., Pavletich and Pabo (1991) Science 252:809-817. This suggests that many different sequences can be attacked by creating novel assemblies of ZFs. See, e.g., Carroll (2011), Genetics.;188(4):773-782. Meanwhile, ZF nickases are created by inactivating the catalytic activity of one ZFN monomer of a ZFN dimer required for double-stranded cleavage. ZF nickases exhibit strand-specific nicking activity in vitro, thus providing highly specific single-stranded cleavage in DNA. See, e.g., Ramirez et al. (2012); Nucleic Acids Research.40(7):5560-5568.

[0071] In some embodiments, the sequence-specific endonuclease comprises TALEN. TALEN comprises a non-specific DNA-cleaving nuclease fused to a DNA-binding domain that can be engineered so that TALEN can target essentially any sequence. The DNA-binding domain of TALEN is composed of highly conserved repeats derived from transcription activator-like effector (TALE). Researchers use a simple "protein-DNA code" that associates the DNA-binding TALE domain with individual bases in the target binding site. Thus, TALEN has gained popularity in targeted DSB introduction and genome editing due to its ease of design, high cleavage activity rate, and essentially unlimited targeting range. See, for example, Joung et al. (2013) Nat Rev Mol Cell Biol.; 14(1): 49-55.

[0072] In some embodiments, sequence-specific insertion (or knock-in) of human CD47 and SIRPA transgenes under the same regulatory elements as the endogenous porcine CD47 gene in the genome of the miniature pig can be achieved by HR, NHEJ, and / or RMCE. In a preferred embodiment, sequence-specific insertion (or knock-in) of human CD47 and SIRPA transgenes under the same regulatory elements as the endogenous porcine CD47 gene in the genome of the miniature pig can be achieved by HR facilitated by sequence-specific endonucleases. In a preferred embodiment, sequence-specific insertion (or knock-in) of human CD47 and SIRPA transgenes under the same regulatory elements as the endogenous porcine CD47 gene in the genome of the miniature pig can be achieved by NHEJ facilitated by sequence-specific endonucleases. In a preferred embodiment, sequence-specific insertion (or knock-in) of human CD47 and SIRPA transgenes under the same regulatory elements as the endogenous porcine CD47 gene in the genome of the miniature pig can be achieved by RMCE in combination with sequence-specific endonucleases. See, for example, Meyer et al. (2010), Proc. Natl. Acad. Sci. USA 107:15022-15026; Cui et al. (2010), Nat. Biotechnol. 29:64-67; Moehle et al. (2007), Proc Natl Acad Sci USA 104:3055-3060. This process relies on targeting specific gene sequences with endonucleases that recognize and bind to such sequences and induce double-strand breaks in the nucleic acid molecules of miniature pig cells. The double-strand breaks are then repaired. For example, if a template (e.g., a construct containing the human CD47 and another construct containing the human SIRPA) is provided in trans, the double-strand break can be repaired using the provided template. Thus, an expression cassette containing the human CD47 or SIRPA is integrated into the genome at a specific locus.Non-limiting examples of such endonucleases include zinc finger nucleases (ZFNs), ZFN dimers, ZF nickases, transcription activator-like effector nucleases (TALENs), or RNA-guided DNA endonucleases (e.g., CRISPR / Cas9).

[0073] In some embodiments, one endonuclease is used and its target site is upstream of the desired replacement region. In other embodiments, one endonuclease is used and its target site is downstream of the desired replacement region. In other embodiments, one endonuclease is used and its target site is within the desired replacement region. In some embodiments, two or more endonucleases are used and the target site of each endonuclease is upstream of the desired replacement region. In other embodiments, two or more endonucleases are used and the target site of each endonuclease is downstream of the desired replacement region. In other embodiments, two or more endonucleases are used and the target site of each endonuclease is within the desired replacement region. In still other embodiments, two or more endonucleases are used and at least one target site is upstream of the desired replacement region and at least one target site is downstream of the desired replacement. In some embodiments, two or more endonucleases are used and at least one target site is upstream of the desired replacement region and at least one target site is within the desired replacement. In some embodiments, two or more endonucleases are used, at least one target site is downstream of the desired replacement region and at least one target site is within the desired replacement. In some embodiments, three or more endonucleases are used, at least one target site is upstream of the desired replacement region, at least one target site is within the desired replacement, and at least one target site is downstream of the desired replacement region.

[0074] In some embodiments, after preparing the plasmids and / or vectors containing the elements described in the preceding paragraphs, the plasmids and / or vectors are transfected into cells, including, but not limited to, cultured porcine fetal fibroblast cells. Transfection techniques include, but are not limited to, physical transfection methods (e.g., electroporation, direct injection, biolistic particle delivery, laser irradiation, sonoporation, magnetic nanoparticles), chemical transfection methods (e.g., calcium phosphate, cationic polymers such as lipofectamine or lipofectin, cationic lipids), and biological transfection methods (e.g., virus-mediated delivery).

[0075] In some embodiments, after transfection of the cells, positive or / and negative selection is performed depending on the selectable marker included.

[0076] In certain aspects, provided herein are porcine cells that contain endogenous porcine CD47 and SIRPA deletion or inactivation, and human CD47 and SIRPA transgenes suitable for use in generating recombinant minipigs. Various methods of producing transgenic animals (e.g., minipigs) are known in the art. See, for example, Hryhorowicz et al. (2020), Genes 2020, 11, 670. Non-limiting exemplary methods are described herein below.

[0077] In certain embodiments, minipigs from a group of inbred minipigs are used. The transgenic animals can be produced by any suitable method known in the art. For example, the gene expression construct can be introduced into the germline of the animal, for example, using SCNT. SCNT involves transferring the nucleus of a donor cell into an oocyte or early embryo from which chromosomes have been removed. See, for example, Wilmut and Taylor (2015), Phil. Trans. R. Soc. B 370:20140366. The reconstructed embryo is surgically transferred into the oviduct of a surrogate pig in estrus. The recombinant minipigs are born from the surrogate pig. Methods for testing and verifying the expression of the transgene in the transgenic pig are discussed in Section 7.1.3.

[0078] In certain embodiments, a plasmid or vector is generated that contains a human CD47 sequence flanked by two homologous arms, one of which is upstream of the porcine CD47 gene sequence and the other is downstream of the porcine CD47 gene sequence. In some embodiments, a plasmid or vector is generated that contains a portion of a human CD47 sequence flanked by two homologous arms, one of which is upstream of the target portion of the porcine CD47 gene sequence and the other is downstream of the target portion of the porcine CD47 gene. One or more selection markers are included in the plasmid or vector. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced porcine CD47 with the human CD47 sequence are isolated, expanded, and verified using techniques well known in the art. SCNT or other equivalent techniques can be used to generate recombinant minipigs.

[0079] In other specific embodiments, a plasmid or vector is generated that contains a human CD47 sequence flanked by two homologous arms, one of which is upstream of the porcine CD47 gene sequence and the other is downstream of the porcine CD47 gene sequence. One or more selection markers are included in the plasmid or vector. A plasmid containing Cas9 and one or more plasmids containing gRNA are generated. In some embodiments, the one or more gRNAs target a site upstream of the desired replacement region. In other embodiments, the one or more gRNAs target a site downstream of the desired replacement region. In still other embodiments, the one or more gRNAs target a site upstream and downstream of the desired 28dminister28n region. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced porcine CD47 with the human CD47 sequence are isolated, expanded, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0080] In other specific embodiments, a plasmid or vector is generated that contains a human CD47 sequence flanked by two homologous arms, one of which is upstream of the porcine CD47 gene sequence and the other is downstream of the porcine CD47 gene. In some embodiments, one or more selectable markers are included in the plasmid or vector. A plasmid containing Cas9 and at least two plasmids containing gRNA are generated. Two ssODNs are generated. One ssODN contains (1) the region of the plasmid or vector upstream of the human CD47 sequence and (2) the beginning of the host CD47 gene. The other ssODN contains (1) the end of the host CD47 gene and (2) the region of the plasmid or vector downstream of the human CD47 sequence. In some embodiments, the two or more gRNAs target sites upstream and downstream of the desired 29dminister29n region. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells in which the porcine CD47 has been successfully replaced with the human CD47 sequence are isolated, expanded, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0081] In other specific embodiments, a plasmid or vector is generated that contains a human CD47 sequence flanked by two homologous arms, one of which is upstream of the porcine CD47 gene sequence and the other is downstream of the porcine CD47 gene sequence. One or more selectable markers are included in the plasmid or vector. One or more plasmids are generated that code for ZFNs, ZFN dimers, and ZF nickases. In some embodiments, the one or more ZFNs or ZF nickases target a site upstream of the desired replacement region. In other embodiments, the one or more ZFNs or ZF nickases target a site downstream of the desired replacement region. In yet other embodiments, the one or more ZFNs or ZF nickases target a site upstream and downstream of the desired replacement region. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells in which the porcine CD47 has been successfully replaced with the human CD47 sequence are isolated, expanded, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0082] In other specific embodiments, a plasmid or vector is generated that contains a human CD47 sequence flanked by two homologous arms, one of which is upstream of the porcine CD47 gene sequence and the other is downstream of the porcine CD47 gene sequence. One or more selection markers are included in the plasmid or vector. One or more plasmids are generated that encode TALENs. In some embodiments, the one or more TALENs target a site upstream of the desired replacement region. In other embodiments, the one or more TALENs target a site downstream of the desired replacement region. In yet other embodiments, the one or more TALENs target a site upstream and downstream of the desired replacement region. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced porcine CD47 with the human CD47 sequence are isolated, expanded, and verified using techniques well known in the art. SCNT or other equivalent techniques can be used to generate recombinant minipigs.

[0083] In other specific embodiments, a plasmid containing Cas9 and two or more plasmids containing gRNA are generated. In some embodiments, the two or more gRNAs target sites upstream and downstream of the porcine CD47 sequence such that the Cas9 endonuclease cleaves a specific base pair upstream of the beginning of the porcine CD47 sequence and the Cas9 endonuclease cleaves a specific base pair downstream of the end of the porcine CD47 sequence. In some embodiments, the number of base pairs is less than 50 bp. In other embodiments, the number of base pairs is 50-100 bp. In still other embodiments, the number of base pairs is 100-150 bp. In still other embodiments, the number of base pairs is 150-200 bp. In still other embodiments, the number of base pairs is more than 200 bp. In addition to the above base pairs, a plasmid or vector containing a human CD47 sequence flanked by two Cas9 cleavage sites. One or more selectable markers are included in the plasmid or vector. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts.Using a selection step that matches the selection marker(s), the cells that have successfully replaced the porcine CD47 with the human CD47 sequence are isolated, expanded and verified using techniques well known in the art.SCNT or other equivalent techniques can be used to generate recombinant minipigs.

[0084] In other specific embodiments, two or more plasmids encoding ZFNs or ZF nickases are generated. In some embodiments, the ZFNs or ZF nickases cleave at a site upstream of the beginning of the porcine CD47 sequence and the ZFNs or ZF nickases cleave at a site downstream of the end of the porcine CD47 sequence. In some embodiments, the number of base pairs upstream of the start site is less than 50 bp. In some embodiments, the number of base pairs downstream of the termination site is less than 50 bp. In some embodiments, the number of base pairs upstream of the start site is 50-200 bp. In some embodiments, the number of base pairs downstream of the termination site is 50-200 bp. In some embodiments, the number of base pairs upstream of the start site is 50-100 bp. In some embodiments, the number of base pairs downstream of the termination site is 50-100 bp. In some embodiments, the number of base pairs upstream of the start site is 100-150 bp. In some embodiments, the number of base pairs downstream of the termination site is 100-150 bp. In some embodiments, the number of base pairs upstream of the start site is 150-200 bp. In some embodiments, the number of base pairs downstream of the termination site is 150-200 bp. In some embodiments, the number of base pairs upstream of the start site is more than 200 bp. In some embodiments, the number of base pairs downstream of the termination site is more than 200 bp.

[0085] In addition to the above base pairing, a plasmid or vector contains the human CD47 sequence flanked by the two ZFN or ZF nickase cleavage sites. One or more selection markers are included in the plasmid or vector. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced porcine CD47 with the human CD47 sequence are isolated, expanded, and verified using techniques well known in the art. SCNT or other equivalent techniques can be used to generate recombinant minipigs.

[0086] In other specific embodiments, two or more plasmids encoding TALENs are generated. In some embodiments, the TALEN cleaves at a site upstream of the beginning of the porcine CD47 sequence and downstream of the end of the porcine CD47 sequence by a specific base pair. In some embodiments, the TALEN cleaves at a site less than 50 bp upstream from the start site. In some embodiments, the TALEN cleaves at a site less than 50 bp downstream from the termination site. In some embodiments, the TALEN cleaves at a site 50-100 bp upstream from the start site. In some embodiments, the TALEN cleaves at a site 50-100 bp downstream from the termination site. In some embodiments, the TALEN cleaves at a site 100-150 bp upstream from the start site. In some embodiments, the TALEN cleaves at a site 100-150 bp downstream from the termination site. In some embodiments, the TALEN cleaves at a site 150-200 bp upstream from the start site. In some embodiments, the TALEN cleaves at a site 150-200 bp downstream from the termination site. In some embodiments, the TALEN cleaves at a site more than 200 bp upstream from the start site. In some embodiments, the TALEN cleaves at a site more than 200 bp downstream from the termination site.

[0087] In addition to the above base pair, a plasmid or vector comprises the human CD47 sequence flanked by the two TALEN cleavage sites. One or more selection markers are included in the plasmid or vector. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced porcine CD47 with the human CD47 sequence are isolated, grown, and verified using techniques well known in the art. SCNT or other equivalent techniques can be used to generate recombinant minipigs.

[0088] In other specific embodiments, a pair of heterospecific recombinase recognition sites is inserted flanking the porcine CD47 sequence or a portion thereof. A plasmid or vector containing a human CD47 sequence flanked by an identical pair of recombinase recognition sites. One or more selection markers are included in the plasmid or vector. A plasmid encoding the corresponding recombinase is generated. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells in which the porcine CD47 has been successfully replaced with the human CD47 sequence are isolated, expanded, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0089] In other specific embodiments, a loading pad flanked by a pair of heterospecific recombinase recognition sites is first inserted near the porcine CD47 sequence or a portion thereof. A plasmid or vector containing a human CD47 sequence flanked by an identical pair of recombinase recognition sites. One or more selection markers are included in the plasmid or vector. A plasmid encoding a corresponding recombinase is generated. A plasmid containing Cas9 and two or more plasmids containing gRNAs are generated. In some embodiments, the two or more gRNAs target upstream and downstream of the porcine CD47 sequence such that the porcine CD47 sequence is deleted leaving the inserted human CD47 sequence. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells in which the porcine CD47 has been successfully replaced with the human CD47 sequence are isolated, expanded, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0090] In other specific embodiments, a loading pad flanked by a pair of heterospecific recombinase recognition sites is first inserted near the porcine CD47 sequence or a portion thereof. A plasmid or vector containing a human CD47 sequence flanked by an identical pair of recombinase recognition sites. One or more selection markers are included in the plasmid or vector. A plasmid encoding a corresponding recombinase is generated. A plasmid encoding a pair of ZEN or ZF nickases is generated. In some embodiments, the ZEN or ZF nickases target upstream and downstream of the porcine CD47 sequence such that the porcine CD47 sequence is deleted leaving the inserted human CD47 sequence. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells in which the porcine CD47 has been successfully replaced with the human CD47 sequence are isolated, expanded, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0091] In other specific embodiments, a loading pad flanked by a pair of heterospecific recombinase recognition sites is first inserted near the porcine CD47 sequence or a portion thereof. A plasmid or vector containing a human CD47 sequence flanked by an identical pair of recombinase recognition sites. One or more selection markers are included in the plasmid or vector. A plasmid encoding a corresponding recombinase is generated. A plasmid encoding a pair of TALENs is generated. In some embodiments, the TALENs target sites upstream and downstream of the porcine CD47 sequence such that the porcine CD47 sequence is deleted leaving the inserted human CD47 sequence. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced the porcine CD47 with the human CD47 sequence are isolated, expanded, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0092] In certain embodiments, a plasmid or vector is generated that contains a human SIRPA sequence flanked by two homologous arms, one of which is upstream of the porcine SIRPA gene sequence and the other is downstream of the porcine SIRPA gene sequence. In some embodiments, a plasmid or vector is generated that contains a portion of a human SIRPA sequence flanked by two homologous arms, one of which is upstream of the target portion of the porcine SIRPA gene sequence and the other is downstream of the target portion of the porcine SIRPA gene. One or more selection markers are included in the plasmid or vector. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced porcine SIRPA with the human SIRPA sequence are isolated, grown, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0093] In other specific embodiments, a plasmid or vector is generated that contains a human SIRPA sequence flanked by two homologous arms, one of which is upstream of the porcine SIRPA gene sequence and the other is downstream of the porcine SIRPA gene sequence. One or more selection markers are included in the plasmid or vector. A plasmid containing Cas9 and one or more plasmids containing gRNA are generated. In some embodiments, the one or more gRNAs target a site upstream of the desired replacement region. In some embodiments, the one or more gRNAs target a site downstream of the desired replacement region. In yet other embodiments, the one or more gRNAs target a site upstream and downstream of the desired 34dminister34n region. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced porcine SIRPA with the human SIRPA sequence are isolated, expanded, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0094] In other specific embodiments, a plasmid or vector is generated that contains a human SIRPA sequence flanked by two homologous arms, one of which is upstream of the porcine SIRPA gene sequence and the other is downstream of the porcine SIRPA gene sequence. One or more selectable markers are included in the plasmid or vector. A plasmid containing Cas9 and at least two plasmids containing gRNA are generated. Two ssODNs are generated. One ssODN contains (1) the region of the plasmid or vector upstream of the human SIRPA sequence and (2) the beginning of the host SIRPA gene. The other ssODN contains (1) the end of the host SIRPA gene and (2) the region of the plasmid or vector downstream of the human SIRPA sequence. In some embodiments, the two or more gRNAs target sites upstream and downstream of the desired 35dminister35n region. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells in which the porcine SIRPA has been successfully replaced with the human SIRPA sequence are isolated, expanded, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0095] In other specific embodiments, a plasmid or vector comprises a human SIRPA sequence flanked by two homologous arms, one of which is upstream of the porcine SIRPA gene sequence and the other is downstream of the porcine SIRPA gene sequence. One or more selectable markers are included in the plasmid or vector. One or more plasmids encoding ZFNs, ZFN dimers, ZF nickases are generated. In some embodiments, the one or more ZFNs or ZF nickases target a site upstream of the desired replacement region. In other embodiments, the one or more ZFNs or ZF nickases target a site downstream of the desired replacement region. In yet other embodiments, the one or more ZFNs or ZF nickases target a site upstream and downstream of the desired 35dminister35n region. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells in which the porcine SIRPA has been successfully replaced with the human SIRPA sequence are isolated, expanded, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0096] In other specific embodiments, a plasmid or vector containing a human SIRPA sequence flanked by two homologous arms, one of which is upstream of the porcine SIRPA gene sequence and the other is downstream of the porcine SIRPA gene sequence. One or more selection markers are included in the plasmid or vector. One or more plasmids encoding TALENs are generated. In some embodiments, the one or more TALENs target a site upstream of the desired replacement region. In other embodiments, the one or more TALENs target a site downstream of the desired replacement region. In yet other embodiments, the one or more TALENs target a site upstream and downstream of the desired 35dminister35n region. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced porcine SIRPA with the human SIRPA sequence are isolated, expanded, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0097] In other specific embodiments, a plasmid containing Cas9 and two or more plasmids containing gRNA are generated. In some embodiments, the two or more gRNAs are targeted upstream and downstream of the porcine SIRPA sequence such that the Cas9 endonuclease cleaves at a site upstream of the beginning of the porcine SIRPA sequence and the Cas9 endonuclease cleaves at a site downstream of the end of the porcine SIRPA sequence. In some embodiments, the Cas9 endonuclease cleaves at a site upstream of the beginning of the porcine SIRPA sequence and downstream of the end of the porcine SIRPA sequence. In some embodiments, the Cas9 endonuclease cleaves at a site less than 50 bp upstream from the start site. In some embodiments, the Cas9 endonuclease cleaves at a site less than 50 bp downstream from the termination site. In some embodiments, the Cas9 endonuclease cleaves at a site 50-100 bp upstream from the start site. In some embodiments, the Cas9 endonuclease cleaves at a site 50-100 bp downstream from the termination site. In some embodiments, the Cas9 endonuclease cleaves at a site 100-150 bp upstream from the start site. In some embodiments, the Cas9 endonuclease cleaves at a site 100-150 bp downstream from the termination site. In some embodiments, the Cas9 endonuclease cleaves at a site 150-200 bp upstream from the start site. In some embodiments, the Cas9 endonuclease cleaves at a site 150-200 bp downstream from the termination site. In some embodiments, the Cas9 endonuclease cleaves at a site more than 200 bp upstream from the start site. In some embodiments, the Cas9 endonuclease cleaves at a site more than 200 bp downstream from the termination site.

[0098] In addition to the above base pair, a plasmid or vector contains the human SIRPA sequence flanked by the two Cas9 cleavage sites. One or more selection markers are included in the plasmid or vector. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced the porcine SIRPA with the human SIRPA sequence are isolated, grown, and verified using techniques well known in the art. SCNT or other equivalent techniques can be used to generate recombinant minipigs.

[0099] In other particular embodiments, two or more plasmids encoding ZFN or ZF nickases are generated. In some embodiments, the ZFN or ZF nickases cleave at a site upstream of the beginning of the porcine SIRPA sequence and the ZFN or ZF nickases cleave at a site downstream of the end of the porcine SIRPA sequence. In some embodiments, the ZFN or ZF nickases cleave at a site less than 50 bp upstream from the start site. In some embodiments, the ZFN or ZF nickases cleave at a site less than 50 bp downstream from the termination site. In some embodiments, the ZFN or ZF nickases cleave at a site 50-100 bp upstream from the start site. In some embodiments, the ZFN or ZF nickases cleave at a site 50-100 bp downstream from the termination site. In some embodiments, the ZFN or ZF nickases cleave at a site 100-150 bp upstream from the start site. In some embodiments, the ZFN or ZF nickase cleaves at a site 100-150 bp downstream from the termination site. In some embodiments, the ZFN or ZF nickase cleaves at a site 150-200 bp upstream from the start site. In some embodiments, the ZFN or ZF nickase cleaves at a site 150-200 bp downstream from the termination site. In some embodiments, the ZFN or ZF nickase cleaves at a site more than 200 bp upstream from the start site. In some embodiments, the ZFN or ZF nickase cleaves at a site more than 200 bp downstream from the termination site.

[0100] In addition to the above base pairs, a plasmid or vector containing the human SIRPA sequence flanked by two ZEN or ZF nickase cleavage sites. One or more selection markers are included in the plasmid or vector. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced the porcine SIRPA with the human SIRPA sequence are isolated, grown, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0101] In other particular embodiments, two or more plasmids encoding TALENs are generated. In some embodiments, a TALEN cleaves at a site upstream of the beginning of the porcine SIRPA sequence, and a TALEN cleaves at a site downstream of the end of the porcine SIRPA sequence. In some embodiments, the Cas9 endonuclease cleaves at a site less than 50 bp upstream from the start site. In some embodiments, a TALEN cleaves at a site less than 50 bp downstream from the termination site. In some embodiments, a TALEN cleaves at a site 50-100 bp upstream from the start site. In some embodiments, a TALEN cleaves at a site 50-100 bp downstream from the termination site. In some embodiments, a TALEN cleaves at a site 100-150 bp upstream from the start site. In some embodiments, a TALEN cleaves at a site 100-150 bp downstream from the termination site. In some embodiments, a TALEN cleaves at a site 150-200 bp upstream from the start site. In some embodiments, the TALEN cleaves at a site 150-200 bp downstream from the termination site. In some embodiments, the TALEN cleaves at a site more than 200 bp upstream from the start site. In some embodiments, the TALEN cleaves at a site more than 200 bp downstream from the termination site.

[0102] In addition to the above base pair, a plasmid or vector comprises the human SIRPA sequence flanked by the two TALEN cleavage sites. One or more selection markers are included in the plasmid or vector. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells that have successfully replaced the porcine SIRPA with the human SIRPA sequence are isolated, grown, and verified using techniques well known in the art. SCNT or other equivalent techniques can be used to generate recombinant minipigs.

[0103] In other specific embodiments, a pair of heterospecific recombinase recognition sites is inserted flanking the porcine SIRPA sequence or a portion thereof. A plasmid or vector containing a human SIRPA sequence flanked by the same pair of recombinase recognition sites. One or more selection markers are included in the plasmid or vector. A plasmid encoding the corresponding recombinase is generated. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells in which the porcine SIRPA has been successfully replaced with the human SIRPA sequence are isolated, grown, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0104] In other specific embodiments, a loading pad flanked by a pair of heterospecific recombinase recognition sites is first inserted near the porcine SIRPA sequence or a portion thereof. A plasmid or vector containing a human SIRPA sequence flanked by an identical pair of recombinase recognition sites. One or more selection markers are included in the plasmid or vector. A plasmid encoding a corresponding recombinase is generated. A plasmid containing Cas9 and two or more plasmids containing gRNA are generated. In some embodiments, the two or more gRNAs target upstream and downstream of the porcine SIRPA sequence such that the porcine SIRPA sequence is deleted leaving the inserted human SIRPA sequence. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells in which the porcine SIRPA has been successfully replaced with the human SIRPA sequence are isolated, grown, and verified using techniques well known in the art. SCNT or other equivalent techniques can be used to generate recombinant minipigs.

[0105] In other specific embodiments, a loading pad flanked by a pair of heterospecific recombinase recognition sites is first inserted near the porcine SIRPA sequence or a portion thereof. A plasmid or vector containing a human SIRPA sequence flanked by an identical pair of recombinase recognition sites. One or more selection markers are included in the plasmid or vector. A plasmid encoding a corresponding recombinase is generated. A plasmid encoding a pair of ZEN or ZF nickases is generated. In some embodiments, the ZEN or ZF nickases target upstream and downstream of the porcine SIRPA sequence such that the porcine SIRPA sequence is deleted leaving the inserted human SIRPA sequence. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells in which the porcine SIRPA has been successfully replaced with the human SIRPA sequence are isolated, grown, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0106] In other specific embodiments, a loading pad flanked by a pair of heterospecific recombinase recognition sites is first inserted near the porcine SIRPA sequence or a portion thereof. A plasmid or vector containing a human SIRPA sequence flanked by an identical pair of recombinase recognition sites. One or more selection markers are included in the plasmid or vector. A plasmid encoding a corresponding recombinase is generated. A plasmid encoding a pair of TALENs is generated. In some embodiments, the TALENs target upstream and downstream of the porcine SIRPA sequence such that the porcine SIRPA sequence is deleted leaving the inserted human SIRPA sequence. The above plasmid or vector is transfected into cells, including but not limited to cultured porcine fetal fibroblasts. Using a selection step that matches the selection marker(s), cells in which the porcine SIRPA has been successfully replaced with the human SIRPA sequence are isolated, grown, and verified using techniques well known in the art. Recombinant minipigs can be generated using SCNT or other equivalent techniques.

[0107] In another aspect, provided herein is a method of generating a recombinant minipig, wherein endogenous porcine CD47, or a portion thereof, is genetically modified in such a way that its protein product can functionally bind to and activate both endogenous porcine SIRPA and human SIRPA, eliciting negative regulation of phagocytosis by macrophages.

[0108] 7.1.3 Methods for testing transgene expression in transgenic miniature pigs A variety of methods can be used to test and verify the expression of the transgene and are known in the art. In certain embodiments, the expression levels of human or humanized CD47 and SIRPA can be determined at the RNA (e.g., mRNA) level, for example, by the methods described in Section 7.1.3.1. In certain embodiments, the expression levels of human or humanized CD47 and SIRPA can be determined at the protein level, for example, by the methods described in Section 7.1.3.2.

[0109] In certain embodiments, the methods provided herein include methods for detecting and measuring differential gene expression in any cell, tissue, or organ of the donor minipig. In certain embodiments, the methods provided herein include methods for detecting and measuring differential mRNA levels of human or humanized CD47 and SIRPA in any cell, tissue, or organ of the donor minipig. In other embodiments, the methods provided herein include methods for detecting and measuring differential protein levels of human or humanized CD47 and SIRPA in any cell, tissue, or organ of the donor minipig.

[0110] Tissue-specific expression can be identified by measuring the levels of human or humanized CD47 and SIRPA protein or mRNA after physical isolation of the tissue of interest (e.g., biopsies of different tissues or organs, or flow cytometry of certain cell types), for example, applying the following method to measure the levels of human or humanized CD47 and SIRPA protein or mRNA in vitro. Alternatively, if a visualization label is engineered to be expressed simultaneously with the human or humanized CD47 and SIRPA genes, imaging techniques such as fluorescent microscopy may be used to visualize and measure human or humanized CD47 and SIRPA protein expression in a specific tissue. Single-cell qPCR may be used to measure the expression of human or humanized CD47 and SIRPA genes in a specific tissue.

[0111] 7.1.3.1 Methods for detecting mRNA levels in recombinant minipigs In certain embodiments, human or humanized CD47 and SIRPA mRNA from the recombinant minipigs described in Sections 7.1.1 and 7.1.2 are detected by the techniques described herein. In some embodiments, porcine CD47 and SIRPA mRNA from the recombinant minipigs described in Sections 7.1.1 and 7.1.2 are undetectable using the techniques described herein.

[0112] Several methods of detecting or quantifying mRNA levels are known in the art. Exemplary methods include, but are not limited to, Northern blots, ribonuclease protection assays, PCR-based methods (e.g., quantitative PCR), RNA sequencing, Fluidigm® analysis, and the like. Human or humanized CD47 and SIRPA mRNA sequences can be used to prepare probes that are at least partially complementary to specific fragments of the human mRNA sequence, but not to their porcine counterparts. Similarly, porcine CD47 and SIRPA mRNA sequences can be used to prepare probes that are at least partially complementary to specific fragments of the porcine mRNA sequence, but not to their human counterparts. The probes can then be used to detect the presence of human or humanized and porcine CD47 and SIRPA mRNA in a sample using any suitable assay, such as PCR-based methods, Northern blotting, dipstick assay, TaqMan™ assay, and the like.

[0113] In other embodiments, a nucleic acid assay can be prepared to examine human or humanized CD47 and SIRPA expression in a biological sample. The assay typically includes a solid support and at least one nucleic acid contacted to the support. The nucleic acid corresponds to at least a portion of an mRNA that is unique to human CD47 and SIRPA but not present in the porcine counterpart. Similarly, the nucleic acid corresponds to at least a portion of an mRNA that is unique to porcine CD47 and SIRPA but not present in the human counterpart. The assay can also have a means for detecting changes in the expression of the mRNA in the sample. The assay method can vary depending on the type of mRNA information desired. Exemplary methods include, but are not limited to, Northern blots and PCR-based methods (e.g., qRT-PCR). Methods such as qRT-PCR can also accurately quantify the amount of the mRNA in a sample.

[0114] A typical mRNA assay may include the steps of (1) obtaining a surface-bound probe of interest, (2) hybridizing a population of mRNA to the surface-bound probe under conditions sufficient to provide specific binding, (3) washing after hybridization to remove nucleic acids that are not specifically bound to the surface-bound probe, and (4) detecting the hybridized mRNA. The reagents used in each of these steps and the conditions for their use may vary depending on the particular application.

[0115] Other methods, such as PCR-based methods, can also be used to detect the expression of human or humanized CD47 and SIRPA. An example of a PCR method can be found in U.S. Patent No. 6,927,024, which is incorporated herein by reference in its entirety. An example of a RT-PCR method can be found in U.S. Patent No. 7,122,799, which is incorporated herein by reference in its entirety. A method of fluorescent in situ PCR is described in U.S. Patent No. 7,186,507, which is incorporated herein by reference in its entirety.

[0116] In some embodiments, quantitative reverse transcription PCR (qRT-PCR) can be used to both detect and quantify RNA targets (Bustin et al., Clin. Sci. 2005, 109:365-379). In some embodiments, qRT-PCR-based assays can be useful in measuring mRNA levels during cell-based assays. Examples of qRT-PCR-based methods can be found, for example, in U.S. Patent No. 7,101,663, which is incorporated herein by reference in its entirety.

[0117] In contrast to conventional reverse transcription PCR and agarose gel analysis, qRT-PCR gives quantitative results. An additional advantage of qRT-PCR is its relative ease and convenience of use. Instruments for qRT-PCR, such as Applied Biosystems 7500, are commercially available, as are reagents, such as TaqMan® Sequence Detection Chemistry. For example, TaqMan® Gene Expression Assays can be used according to the manufacturer's instructions. These kits are pre-formulated gene expression assays for rapid, reliable detection and quantification of human, mouse, and rat mRNA transcripts. An exemplary qRT-PCR program is, for example, 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, followed by 60°C for 1 minute.

[0118] 7.1.3.2 Methods for detecting polypeptide or protein levels in recombinant minipigs In one aspect, provided herein are methods of detecting human or humanized CD47 and SIRPα polypeptides or proteins produced in a recombinant minipig, such as the recombinant minipig described in Sections 7.1.1 and 7.1.2. In some embodiments provided herein, porcine CD47 and SIRPα polypeptides or proteins are undetectable in the recombinant minipig described in 7.1.1 and 7.1.2 described herein.

[0119] Various protein detection and quantification methods can be used to measure the levels of human or humanized CD47 and SIRPα. Any suitable protein quantification method can be used. In some embodiments, antibody-based methods are used. Exemplary methods that can be used include, but are not limited to, immunoblotting (Western blot), ELISA, immunohistochemistry, immunofluorescence, flow cytometry, cytometric bead array, mass spectrometry, and the like. Several types of ELISA are commonly used, including direct ELISA, indirect ELISA, and sandwich ELISA.

[0120] 7.1.4 Other genetic modifications The recombinant minipigs provided herein may be further modified in addition to CD47 and SIRPA. Such further modifications include, for example, knockout of alpha-1,3-galactosyltransferase and modification of cytokine receptors. In some embodiments, the minipigs provided herein do not express alpha-1,3-galactosyltransferase. In some embodiments, the minipigs provided herein further express human CD55, human CD46, human CD59, IL-3R, or any combination thereof. See, for example, Nomura et al. (2020), Xenotransplantation. 2020;27:e12549, U.S. Patent No. 9,883,939, and U.S. Patent No. 9,980,471 B2.

[0121] With reference to the transplantation methods in Section 7.3, such further genetic modifications may be used in connection with minipigs that are donors of organ transplants, and such further modifications may also be used in connection with minipigs that are donors of hematopoietic stem cells (e.g., in the case of bone marrow transplants).

[0122] 7.2 Cells, tissues and organs derived from engineered minipigs Cells, tissues, organs or body fluids from the transgenic donor minipigs described in section 7.1 may be used in methods of transplantation (e.g., xenotransplantation).

[0123] In some embodiments, cells of the transgenic donor mini pig described in section 7.1 may be used in methods of transplantation (e.g., xenotransplantation). In some particular embodiments, red blood cells from the transgenic donor mini pig are used. In some particular embodiments, granulocytes from the transgenic donor mini pig are used. In some particular embodiments, agranulocytes from the transgenic donor mini pig are used. In some particular embodiments, platelets from the transgenic donor mini pig are used. In some particular embodiments, neurons from the transgenic donor mini pig are used. In some particular embodiments, glial cells from the transgenic donor mini pig are used. In some particular embodiments, muscle cells from the transgenic donor mini pig are used. In some particular embodiments, chondrocytes from the transgenic donor mini pig are used. In some particular embodiments, bone cells from the transgenic donor mini pig are used. In some particular embodiments, skin cells from the transgenic donor mini pig are used. In some particular embodiments, endothelial cells from the transgenic donor mini pig are used. In some particular embodiments, epithelial cells from the transgenic donor mini pig are used. In some specific embodiments, adipocytes from a transgenic donor mini pig are used. In some specific embodiments, sperm from a transgenic donor mini pig are used. In some specific embodiments, eggs from a transgenic donor mini pig are used.

[0124] In some embodiments, tissues of the transgenic donor mini pig described in Section 7.1 may be used in the method of transplantation (e.g., xenotransplantation). In some specific embodiments, connective tissue from the transgenic donor mini pig is used. In some specific embodiments, epithelial tissue from the transgenic donor mini pig is used. In some specific embodiments, muscle tissue from the transgenic donor mini pig is used. In some specific embodiments, neural tissue from the transgenic donor mini pig is used.

[0125] In some embodiments, organs from transgenic donor minipigs described in section 7.1 may be used in methods of transplantation (e.g., xenotransplantation). In some particular embodiments, a skeleton from a transgenic donor minipigs is used. In some particular embodiments, a joint from a transgenic donor minipigs is used. In some particular embodiments, a ligament from a transgenic donor minipigs is used. In some particular embodiments, a tendon from a transgenic donor minipigs is used. In some particular embodiments, a salivary gland from a transgenic donor minipigs is used. In some particular embodiments, an esophagus from a transgenic donor minipigs is used. In some particular embodiments, a trachea from a transgenic donor minipigs is used. In some particular embodiments, a stomach from a transgenic donor minipigs is used. In some particular embodiments, a small intestine from a transgenic donor minipigs is used. In some particular embodiments, a large intestine from a transgenic donor minipigs is used. In some particular embodiments, a liver from a transgenic donor minipigs is used. In some particular embodiments, a gallbladder from a transgenic donor minipigs is used. In some particular embodiments, a mesentery from a transgenic donor minipig is used. In some particular embodiments, a pancreas from a transgenic donor minipig is used. In some particular embodiments, a lung from a transgenic donor minipig is used. In some particular embodiments, a heart from a transgenic donor minipig is used. In some particular embodiments, a pancreatic islet from a transgenic donor minipig is used. In some particular embodiments, a kidney from a transgenic donor minipig is used. In some particular embodiments, a bladder from a transgenic donor minipig is used. In some particular embodiments, a urethra from a transgenic donor minipig is used.In some particular embodiments, a uterus from a transgenic donor minipig is used. In some particular embodiments, a pituitary gland from a transgenic donor minipig is used. In some particular embodiments, a pineal gland from a transgenic donor minipig is used. In some particular embodiments, a thyroid gland from a transgenic donor minipig is used. In some particular embodiments, a parathyroid gland from a transgenic donor minipig is used. In some particular embodiments, a skin from a transgenic donor minipig is used. In some particular embodiments, an adrenal gland from a transgenic donor minipig is used. In some particular embodiments, an artery from a transgenic donor minipig is used. In some particular embodiments, a vein from a transgenic donor minipig is used. In some particular embodiments, a capillary from a transgenic donor minipig is used. In some particular embodiments, a lymphatic vessel from a transgenic donor minipig is used. In some particular embodiments, a lymph node from a transgenic donor minipig is used. In some particular embodiments, a bone marrow from a transgenic donor minipig is used. In some particular embodiments, a thymus from a transgenic donor minipig is used. In some specific embodiments, a spleen from a transgenic donor mini pig is used. In some specific embodiments, a cornea from a transgenic donor mini pig is used. In some specific embodiments, a retina from a transgenic donor mini pig is used. In some specific embodiments, an iris from a transgenic donor mini pig is used.

[0126] 7.3 Porting method Provided herein is a method of transplanting a graft from a first donor, e.g., a recombinant minipig as described in Section 7.1, with or without bone marrow from a second donor, e.g., a recombinant minipig as described in Section 7.1. In particular, methods of harvesting cells, tissues, and organs from transgenic donor minipig are described in Section 7.3.1. Preparation of the graft, the process of the graft, and post-transplant procedures are described in Section 7.3.2. Recipient patient groups are described in Section 7.3.3. Xenograft tolerance can be improved by the methods described in Section 7.3.4. Transplant outcomes can be measured as described in Section 7.3.7.

[0127] 7.3.1 Graft harvesting method In some embodiments, the methods provided herein include harvesting a graft from a recombinant minipig as described in Section 7.1.

[0128] Surgical procedures for harvesting grafts from minipigs are well known in the art, see, for example, Tena et al. (2017), Transplantation 101:316-21, and Nomura et al. (2020), Xenotransplantation 2020;27:e12549, the contents of which are incorporated herein by reference in their entirety.

[0129] In some embodiments, during the transition period after the graft is removed from the donor animal but before it is transplanted into the recipient, the graft is stored in a specific container to maintain the viability of the graft. In some specific embodiments, the container allows for perfusion of the graft at warm and / or cold temperatures to extend the useful life of the graft. In some specific embodiments, the graft is stored in the container with a suitable fluid sample. In some specific embodiments, the graft is stored in the container with a sufficient oxygen supply. U.S. Patent No. 6,673,594, incorporated herein by reference in its entirety.

[0130] 7.3.2 Graft transplantation method In some aspects, the method provided herein for transplanting a graft derived from a recombinant minipig into a recipient comprises (a) harvesting the graft from the recombinant minipig as described in Section 7.1, and (b) transplanting the graft into the primate. In some embodiments, the recipient is a primate. In some specific embodiments, the recipient is a human. In some embodiments, the graft is a cell, tissue, or organ, and the organ can be a heart, kidney, pancreatic islet, liver, pancreas, lung, intestine, skin, small intestine, trachea, cornea, or a combination thereof.

[0131] In some aspects, the transplantation methods provided herein include (a) harvesting bone marrow from a recombinant minipig as described in Section 7.1, (b) transplanting the bone marrow into a recipient, (c) harvesting a graft for the same recipient from another recombinant minipig as described in Section 7.1, and (d) transplanting the graft into the same recipient.

[0132] In some embodiments, the hematopoietic stem cells and the donor cells, tissues, and organs are derived from the same donor animal. In other embodiments, the hematopoietic stem cells and the donor cells, tissues, and organs are taken from two different but genetically matched donor animals. As used herein, "genetically matched" may refer to the homology between genes, e.g., MHC genes. In some embodiments, the genetically matched donor animals are fully matched with respect to MHC. In some embodiments, the hematopoietic stem cells and the donor cells, tissues, and organs are derived from two different animals of the same highly inbred group.

[0133] In certain embodiments, the step of transplanting donor cells, tissues, and organs from a mini pig is performed at least 7 days after the step of transplanting hematopoietic stem cells (e.g., bone marrow) from another mini pig. In certain embodiments, the step of transplanting donor cells, tissues, and organs from a mini pig is performed at least 14 days after the step of transplanting hematopoietic stem cells (e.g., bone marrow) from another mini pig. In certain embodiments, the step of transplanting donor cells, tissues, and organs from a mini pig is performed at least 21 days after the step of transplanting hematopoietic stem cells (e.g., bone marrow) from another mini pig. In certain embodiments, the step of transplanting donor cells, tissues, and organs from a mini pig is performed at least 28 days after the step of transplanting hematopoietic stem cells (e.g., bone marrow) from another mini pig. In certain embodiments, the step of transplanting donor cells, tissues, and organs from a mini pig is performed at least 35 days after the step of transplanting hematopoietic stem cells (e.g., bone marrow) from another mini pig. In certain embodiments, the step of transplanting donor cells, tissues, and organs from a mini pig is performed at least 42 days after the step of transplanting hematopoietic stem cells (e.g., bone marrow) from another mini pig. In certain embodiments, the step of transplanting donor cells, tissues, and organs from a mini pig is performed at least 49 days after the step of transplanting hematopoietic stem cells (e.g., bone marrow) from another mini pig. In certain embodiments, the step of transplanting donor cells, tissues, and organs from a mini pig is performed at least 56 days after the step of transplanting hematopoietic stem cells (e.g., bone marrow) from another mini pig. The present disclosure includes methods and techniques described in Watanabe et al., Xenotransplantation, 2020, 27:e12552 and Nomura et al., Xenotransplantation, 2020, 27:e12549 for transgenic expression of human CD47 in donor cells.

[0134] The hematopoietic stem cells can be any cell type. In certain embodiments, the cells are hematopoietic stem cells, lymphocytes, or bone marrow cells. In some embodiments, a mixed population of hematopoietic cells is transplanted from a first donor animal (e.g., a miniature pig) to a recipient. In certain embodiments, the porcine hematopoietic stem cells are obtained from bone marrow, peripheral blood, umbilical cord blood, fetal liver, or embryonic stem cells. The hematopoietic stem cells can be transplanted by any suitable method known in the art, for example, by the method described in Section 7.3.4.3 below. In some embodiments, the hematopoietic stem cells are transplanted into the recipient by, for example, intraosseous bone marrow transplantation as described in Watanabe et al. (2019), Xenotransplantation. 2019; 00: e12552.

[0135] 7.3.3 Transplant recipients In preferred embodiments, the patient (e.g., recipient of one or more donor grafts) treated according to the methods described herein is a human patient. As used herein, the terms "subject" and "patient" are used interchangeably and include any human or non-human mammal. Non-limiting examples include members of human, equine, porcine, bovine, rat, murine, canine, and feline species. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human. In certain embodiments, the subject is a human adult. In some embodiments, the subject is a human child. In certain embodiments, the subject is a human and receives one or more donor grafts from a porcine donor. In other specific embodiments, the subject is a non-human primate (e.g., a baboon, a cynomolgus monkey, or a rhesus monkey) and receives one or more grafts from a porcine donor.

[0136] In some embodiments, the patient treated according to the methods described herein requires a kidney transplant. The patient may require a kidney transplant due to kidney failure or rejection of the donor kidney. Renal failure may have several causes, including but not limited to high blood pressure (hypertension), physical injury, diabetes, kidney disease (polycystic kidney disease, glomerular disease) and autoimmune disorders, such as lupus. Renal failure may be acute or chronic. Renal failure may also be diagnosed by clinical tests, such as glomerular filtration rate, blood urea nitrogen, and serum creatinine, by imaging tests (ultrasound, computed tomography) or kidney biopsy. In some embodiments, the patient treated according to the methods described herein has stage 1, 2, 3, 4, or 5 kidney disease.

[0137] In some embodiments, the patient treated according to the methods described herein requires a heart transplant. The patient may require a heart transplant due to heart failure or coronary artery disease. Heart failure or coronary artery disease may have several causes, including but not limited to dilated cardiomyopathy, restrictive myopathy, hypertrophic cardiomyopathy, valvular heart disease, congenital heart disease, and ventricular arrhythmias. End-stage heart failure or severe coronary artery disease may be acute or chronic. Heart failure and coronary artery disease may be diagnosed by clinical tests, such as blood tests, chest x-rays, electrocardiograms (ECG), echocardiograms, stress tests, cardiac computed tomography (CT) scans, magnetic resonance imaging (MRI), coronary angiograms, myocardial biopsies, cardiac catheterization, and angiograms.

[0138] In some embodiments, the patient treated according to the methods described herein requires a pancreatic islet transplant. The patient may require a pancreatic islet transplant due to a lack of insulin production capacity, which may have several causes, including but not limited to type 1 diabetes. The patient may be diagnosed with laboratory tests, such as glycosylated hemoglobin (A1C) tests, random blood glucose tests, and fasting blood glucose tests.

[0139] In some embodiments, the patient treated according to the methods described herein requires liver transplantation. The patient may require liver transplantation due to liver failure or liver cancer. Liver failure or liver cancer may have several causes, including but not limited to cirrhosis, hepatitis B infection, hepatitis C infection, alcoholic liver disease, non-alcoholic fatty liver disease, genetic diseases that affect the liver, such as but not limited to hemochromatosis and Wilson's disease, primary biliary cirrhosis, primary sclerosing cholangitis, and biliary atresia. Liver failure may be acute or chronic. Liver failure may be diagnosed by laboratory tests, such as blood tests, ultrasound, CT scan, MRI, and liver biopsy.

[0140] In some embodiments, the patient treated according to the methods described herein requires a pancreas transplant. The patient may require a pancreas transplant due to lack of insulin production capacity, which may have several causes, including but not limited to type 1 diabetes. The patient may be diagnosed with laboratory tests, such as glycosylated hemoglobin (A1C) test, random blood glucose test, and fasting blood glucose test.

[0141] In some embodiments, the patient treated according to the methods described herein requires a lung transplant. The patient may require a lung transplant due to lung failure or lung cancer. The lung failure or lung cancer may have several causes, including but not limited to emphysema, pulmonary embolism, pulmonary fibrosis, pulmonary hypertension, and chronic obstructive pulmonary disease (COPD), including cystic fibrosis. The lung failure may be acute or chronic. The lung failure may be diagnosed by clinical tests, such as physical examination, pulse oximetry, and arterial blood gas test.

[0142] In some embodiments, the patient treated according to the methods described herein requires an intestinal transplant. The patient may require an intestinal transplant due to intestinal failure. Intestinal failure may have several causes, including but not limited to, short bowel syndrome (SBS), chronic intestinal pseudo-obstruction (CIPO), intraperitoneal non-metastatic tumor, ischemia, Crohn's disease, trauma, motility disorder, intestinal volvulus, necrotizing enterocolitis, gastroschisis, omphalocele, intestinal obstruction, microvillus inclusion disease, intractable infantile diarrhea, autoimmune enteritis, and intestinal polyposis. Intestinal failure may be acute or chronic. Intestinal failure may be diagnosed by laboratory tests, such as abdominal computed tomography (CT) scan, abdominal X-ray, barium enema / follicle gastrointestinal tract radiography, blood tests, colonoscopy, sigmoidoscopy, gastric emptying test, gastroduodenal manometry, gastric accommodation scintigraphy, upper endoscopy, wireless capsule gastrointestinal monitoring system.

[0143] In some embodiments, the patient treated according to the methods described herein is in need of a skin graft. Patients who may require a skin graft may be due to a number of causes, including, but not limited to, skin infections, deep burns, large open wounds, bed sores, skin ulcers, or skin cancer.

[0144] In some embodiments, the patient treated according to the methods described herein requires a tracheal transplant. The patient may require a tracheal transplant due to airway damage. Airway damage may have several causes, including, but not limited to, tuberculosis, mucoepidermoid carcinoma, adenoid cystic carcinoma, bronchomalacia, tracheoesophageal fistula, and tracheotomy.

[0145] In some embodiments, the patient treated according to the methods described herein requires a corneal transplant. The patient may require a corneal transplant due to severe vision impairment. Severe vision impairment may have several causes, including but not limited to eye infection, eye inflammation, corneal thinning, degenerative vision diseases such as Fuchs' dystrophy, keratoconus, corneal perforation, corneal scarring, and bullous keratopathy. Severe vision impairment may be acute or chronic. Severe vision impairment may be diagnosed by clinical examination, such as comprehensive eye examination and corneal topography.

[0146] In some embodiments, the patient treated according to the methods described herein is in need of vascular tissue transplantation. The patient may be in need of vascular tissue transplantation due to poorly functioning, diseased or missing blood vessels. The type of vascular tissue provided may be, but is not limited to, the saphenous vein and femoral vessels from the lower limbs, and the aortoiliac artery from the abdomen. The reasons for receiving vascular tissue transplantation may be several, including, but not limited to, peripheral vascular disease, chronic dialysis treatment, severe thrombosis, and abdominal aortic aneurysm.

[0147] 7.3.4 Methods for improving xenograft tolerance Additional treatment can be used before, at the same time, or after the transplantation method described herein.Additional treatment is generally aimed at improving the tolerance of xenograft in recipient, but other treatments are contemplated.Therefore, the transplantation method provided herein can include one or more additional treatments, such as treatments that inhibit T cells, block complement, or otherwise downregulate the immune response of the recipient to the transplant.

[0148] In some embodiments, the recipient is thymectomized and / or splenectomized.

[0149] In some embodiments, the recipient is irradiated, e.g., total body irradiation. In certain embodiments, the recipient is irradiated with 5-10 Gy or 10-15 Gy. In some embodiments, thymic irradiation may be used. In some embodiments, the recipient is administered a low dose of radiation (e.g., sublethal total body irradiation of 100 rad to 400 rad). Local thymic irradiation may be used.

[0150] The blood of a subject undergoing transplantation according to the methods described herein may contain antibodies that target the xenograft. Such antibodies may be eliminated by organ perfusion and / or transplantation of tolerance-inducing bone marrow. Natural antibodies may be absorbed from the recipient's blood by hemoperfusion of the donor species' liver. Similarly, antibody-producing cells may be present in the recipient. Such antibody-producing cells may be eliminated, for example, by irradiation or drug treatment. In certain embodiments, grafts, cells, tissues, or organs used for transplantation may be genetically modified so that they are not recognized by antibodies present in the host (e.g., the cells are a-1,3-galactosyltransferase deficient), as described in Section 7.1.4.

[0151] In some embodiments, donor stromal tissue is administered, hi certain embodiments, the donor stromal tissue is obtained from fetal liver, thymus, and / or fetal spleen and transplanted into the recipient, for example, within the kidney capsule.

[0152] 7.3.4.1 Immunosuppressive therapy In some embodiments, the patient undergoing xenotransplantation according to the methods described herein undergoes immunosuppressive therapy. The immunosuppressive therapy can be any FDA-approved therapy that has been shown to reduce transplant rejection and / or improve xenotransplantation outcomes. Non-limiting examples of immunosuppressive therapy include calcineurin inhibitors (e.g., tacrolimus or cyclosporine), antiproliferative agents (e.g., antimetabolites such as mycophenolic acid, 6-mercaptopurine or its prodrug azathioprine), inhibitors of mammalian target of rapamycin (mTOR) (e.g., sirolimus, rapamycin), steroids (e.g., prednisone), cell cycle inhibitors (azathioprine or mycophenolate mofetil), lymphodepleting agents (e.g., antithymocyte globulin or antibodies, e.g., alemtuzumab, siplizumab or basiliximab), and costimulatory blockers (e.g., belatacept). See, e.g., Chung et al. (2020)., Ann Transl Med. Mar;8(6):409, van der Mark et al. (2020), Eur Respir Rev;29:190132, and Benvenuto et al. (2018), J Thorac Dis 10:3141-3155.

[0153] Immunosuppressive therapy may be administered as induction therapy (perioperatively or immediately after surgery), maintenance doses, or for acute rejection. Induction therapy typically includes basiliximab, antithymocyte globulin, or alemtuzumab. Immunosuppressive therapy may also be administered as maintenance therapy, which is often required to continue for the recipient's lifetime. Maintenance immunosuppressive therapy typically includes calcineurin inhibitors (tacrolimus or cyclosporine), antiproliferative agents (mycophenolic acid or azathioprine), and corticosteroids. Immunosuppressive therapy for acute rejection typically includes thymoglobulin or mycophenolic acid. See, for example, Chung et al. (2020), Ann Transl Med. Mar; 8: 409 and Benvenuto et al., (2018) J Thorac Dis 10: 3141-3155.

[0154] Non-limiting examples of immunosuppressants include: (1) antimetabolites, such as purine synthesis inhibitors (inosine monophosphate dehydrogenase (IMPDH) inhibitors, such as azathioprine, mycophenolic acid, and mycophenolate mofetil), pyrimidine synthesis inhibitors (e.g., leflunomide and teriflunomide), and folate metabolic antagonists (e.g., methotrexate); (2) calcineurin inhibitors, such as tacrolimus, cyclosporine A, pimecrolimus, and voclosporin; and (3) TNF-alpha. inhibitors such as thalidomide and lenalidomide, (4) IL-1 receptor antagonists such as anakinra, (5) inhibitors of the mammalian target of rapamycin (mTOR) such as rapamycin (sirolimus), deforolimus, everolimus, temsirolimus, zotarolimus, and biolimus A9, (6) corticosteroids such as prednisone, and (7) antibodies against any one of a number of cellular or serum targets, including antilymphocyte globulin and antithymocyte globulin.

[0155] Non-limiting exemplary cellular targets and their respective inhibitory compounds include complement component 5 (e.g., eculizumab), tumor necrosis factor (TNF) (e.g., infliximab, adalimumab, certolizumab pegol, afelimomab, and golimumab), IL-5 (e.g., mepolizumab), IgE (e.g., omalizumab), BAYX (e.g., nerelimomab), interferon (e.g., faralimomab), IL-6 (e.g., elsilimomab), IL-12 and IL-13 (e.g., lebrikizumab and ustekinumab), CD3 (e.g., muromonab-CD3, otelixizumab, teplizumab, visilizumab), CD4 (e.g., clenoliximab, keliximab, and zanolimumab), CDI la (e.g., efalizumab), CD18 (e.g., erlizumab), CD20 (e.g., afutuzumab, ocrelizumab, pascolizumab), CD23 (e.g., lumiliximab), CD40 (e.g., teneliximab, toralizumab), CD62L / L-selectin (e.g., acelizumab), CD80 (e.g., galiximab), CD147 / basigin (e.g., gavilimomab), CD154 (e.g., ruprix These include, but are not limited to, BlyS (e.g., belimumab), CTLA-4 (e.g., ipilimumab, tremelimumab), CAT (e.g., bertilimumab, lerdelimumab, metelimu- mab), integrins (e.g., natalizumab), IL-6 receptor (e.g., tocilizumab), LFA-1 (e.g., odulimomab), and IL-2 receptor / CD25 (e.g., basiliximab, daclizumab, inolimomab).

[0156] In some embodiments, the transplantation methods provided herein include inducing tolerance in the recipient, for example, by inducing mixed chimerism. "Mixed chimerism" is generally understood to describe a state in which the lymphohematopoietic system of a recipient of allogeneic hematopoietic stem cells contains a mixture of host and donor cells. This state is usually achieved by either bone marrow or mobilized peripheral blood stem cell transplantation. Mixed chimerism can be transient or stable. See, for example, Sachs et al. (2014), Cold Spring Harb Perspect Med 2014;4:a015529, U.S. Patent No. 6,296,846, and U.S. Patent No. 6,306,651. Mixed chimerism can also be achieved by simultaneous transplantation of thymus tissue from a donor animal. See, for example, International Patent Application Publication No. WO2020 / 061272.

[0157] 7.3.4.2 Vascularized thymus transplantation In some embodiments, the patient treated according to the methods described herein receives a vascularized thymus transplant. See, for example, International Patent Application Publication No. PCT WO2020 / 061272. Thymus tissue can be prepared for transplantation by embedding under the autologous kidney capsule for revascularization. The vascularized thymus transplant can be, for example, a "thymic kidney", i.e., a kidney prepared by transplanting thymus tissue from a donor under the donor's own kidney capsule. See, for example, Yamada et. al., Transplantation 68(11):1684-1692(1999), Yamada et al., J Immunol 164:3079-3086(2000) and Yamada et al., Transplantation 76(3):530-536(2003). The vascularized thymus transplant can also be a vascularized thymus lobe transplanted separately from the kidney. See, e.g., LaMattina et al., Transplantation 73(5):826-831(200) and Kamano et al., Proc Natl Acad Sci USA 101(11):3827-3832(2004).

[0158] 7.3.4.3 Hematopoietic stem cell transplantation Stem cell engraftment and hematopoiesis across interspecies barriers can be enhanced by providing a hematopoietic stromal environment from the donor species. The stromal matrix provides species-specific factors, such as hematopoietic growth factors, adhesion molecules, and their ligands, required for the interaction of hematopoietic stem cells with the stromal environment.

[0159] Since the liver is the primary hematopoietic site in the fetus, fetal liver can also serve as an alternative to bone marrow as a source of hematopoietic stem cells. As an alternative or adjunct to transplantation, fetal liver cells can be administered in a fluid suspension. The thymus is the primary site of T cell maturation. Each organ contains an organ-specific stromal matrix that can support the differentiation of the respective undifferentiated stem cells transplanted into the host. Thymic stromal tissue can be irradiated prior to transplantation.

[0160] Porcine hematopoietic chimerism can lead to donor-specific non-reactivity in mixed lymphocyte reaction, lack of anti-donor IgG antibody production, and acceptance of the donor graft. Thus, mixed chimerism can induce tolerance in highly different xenogeneic combinations and may have clinical potential to prevent xenograft rejection. See, for example, Griesemer et al., Immunol.Rev.2014;258(1):241-258; Sachs et al.(2014), Cold Spring Harb Perspect Med 2014;4:a015529. To induce mixed chimerism, the recipient can be injected with donor bone marrow cells (BMC), or another source of hematopoietic stem cells, such as fetal liver suspension. The hematopoietic stem cells can be harvested from any source, such as bone marrow or peripheral blood stem cells. See, for example, Sachs et al. (2014), Cold Spring Harb Perspect Med 2014;4:a015529. The donor's BMCs home to the appropriate site in the recipient, grow and proliferate adjacent to the remaining host cells, forming a chimeric lymphohematopoietic population. This process exposes newly formed B cells (and the antibodies they produce) to donor antigens, resulting in the recognition of the graft as self. Tolerance to the donor is also observed at the T cell level in animals in which engraftment of hematopoietic stem cells, e.g., bone marrow cells, has been achieved. Transplantation of thymic tissue (e.g., vascularized thymus or thymic kidney) can induce T cell tolerance by generating a T cell repertoire that is unresponsive to the xenograft. The use of xenogeneic donors offers the possibility of using bone marrow cells and organs derived from the same animal, or from a genetically matched animal. In the case of bone marrow transplantation, the recipient may be administered a low dose of radiation. Optionally, the recipient may be treated with an agent that depletes complement, such as cobra venom factor (eg, on day -1).

[0161] 7.3.5 How to assess transplantation In some embodiments, xenotransplantation using a graft produced in accordance with the present disclosure reduces the administration of immunosuppressive therapy to the recipient following transplantation of the donor cells, tissues, and / or organs, as compared to the current standard of care. In some embodiments, xenotransplantation using a graft produced in accordance with the present disclosure reduces the incidence of short-term rejection of the donor cells, tissues, and / or organs, as compared to the current standard of care. In some embodiments, xenotransplantation using a graft produced in accordance with the present disclosure reduces the incidence of long-term rejection of the donor cells, tissues, and / or organs, as compared to the current standard of care. In some embodiments, xenotransplantation using a graft produced in accordance with the present disclosure extends the survival of the donor cells, tissues, and / or organs, as compared to the current standard of care. In some embodiments, xenotransplantation using a graft produced in accordance with the present disclosure improves the function of the donor cells, tissues, and / or organs in the recipient receiving the donor cells, tissues, and / or organs, as compared to the current standard of care. In some embodiments, xenotransplantation using a graft produced according to the present disclosure improves the corresponding disease targeted for mediated by the graft in the recipient of the donor's cells, tissues, and / or organs, as indicated by the corresponding biomarker(s), compared to the current standard of care. In some embodiments, xenotransplantation using a graft produced according to the present disclosure normalizes the function of other non-targeted organ systems in the recipient of the donor's cells, tissues, and / or organs. In some embodiments, xenotransplantation using a graft produced according to the present disclosure increases the survival rate of the recipient of the donor's cells, tissues, and / or organs compared to the current standard of care. In some embodiments, xenotransplantation using a graft produced according to the present disclosure increases the quality of life of the recipient of the donor's cells, tissues, and / or organs compared to the current standard of care.

[0162] In certain embodiments, xenotransplantation using a transplant produced according to the present disclosure reduces the amount of immunosuppressant administered to the recipient as compared to the amount of immunosuppressant typically administered to a comparable transplant recipient (e.g., a person of the same sex and age, height, and / or weight). In one embodiment, the methods provided herein reduce the amount of immunosuppressant administered to the recipient by 10%. In one embodiment, the methods provided herein reduce the amount of immunosuppressant administered to the recipient by 10-20%. In one embodiment, the methods provided herein reduce the amount of immunosuppressant administered to the recipient by 20-30%. In one embodiment, the methods provided herein reduce the amount of immunosuppressant administered to the recipient by 30-40%. In one embodiment, the methods provided herein reduce the amount of immunosuppressant administered to the recipient by 40-50%. In one embodiment, the methods provided herein reduce the amount of immunosuppressant administered to the recipient by 50-60%. In one embodiment, the methods provided herein reduce the amount of immunosuppressant administered to the recipient by 60-70%. In one embodiment, the methods provided herein reduce the amount of immunosuppressant administered to the recipient by 70-80%. In one embodiment, the methods provided herein reduce the amount of immunosuppressant administered to the recipient by 80-90%. In one embodiment, the methods provided herein reduce the amount of immunosuppressant administered to the recipient by more than 90%.

[0163] In some embodiments, xenotransplantation using a transplant produced according to the present disclosure reduces the frequency of administration of immunosuppressive therapy to the recipient as compared to that typically administered to a comparable recipient (e.g., a person of the same sex and age, height, and / or weight). In one embodiment, the methods provided herein reduce the frequency of immunosuppressive agents administered to the recipient by 10%. In one embodiment, the methods provided herein reduce the frequency of immunosuppressive agents administered to the recipient by 10-20%. In one embodiment, the methods provided herein reduce the frequency of immunosuppressive agents administered to the recipient by 20-30%. In one embodiment, the methods provided herein reduce the frequency of immunosuppressive agents administered to the recipient by 30-40%. In one embodiment, the methods provided herein reduce the frequency of immunosuppressive agents administered to the recipient by 40-50%. In one embodiment, the methods provided herein reduce the frequency of immunosuppressive agents administered to the recipient by 50-60%. In one embodiment, the methods provided herein reduce the frequency of immunosuppressants administered to the recipient by 60-70%. In one embodiment, the methods provided herein reduce the frequency of immunosuppressants administered to the recipient by 70-80%. In one embodiment, the methods provided herein reduce the frequency of immunosuppressants administered to the recipient by 80-90%. In one embodiment, the methods provided herein reduce the frequency of immunosuppressants administered to the recipient by more than 90%.

[0164] In some embodiments, xenotransplantation using a transplant produced according to the present disclosure reduces the duration of immunosuppressive therapy administered to the recipient compared to that typically administered to a comparable recipient (e.g., a person of the same sex and age, height, and / or weight). In one embodiment, the methods provided herein reduce the duration of immunosuppressive therapy administered to the recipient by 10%. In one embodiment, the methods provided herein reduce the duration of immunosuppressive therapy administered to the recipient by 10-20%. In one embodiment, the methods provided herein reduce the duration of immunosuppressive therapy administered to the recipient by 20-30%. In one embodiment, the methods provided herein reduce the duration of immunosuppressive therapy administered to the recipient by 30-40%. In one embodiment, the methods provided herein reduce the duration of immunosuppressive therapy administered to the recipient by 40-50%. In one embodiment, the methods provided herein reduce the duration of immunosuppressive therapy administered to the recipient by 50-60%. In one embodiment, the method provided herein reduces the treatment duration of the immunosuppressive therapy administered to the recipient by 60-70%. In one embodiment, the method provided herein reduces the treatment duration of the immunosuppressive therapy administered to the recipient by 70-80%. In one embodiment, the method provided herein reduces the treatment duration of the immunosuppressive therapy administered to the recipient by 80-90%. In one embodiment, the method provided herein reduces the treatment duration of the immunosuppressive therapy administered to the recipient by more than 90%.

[0165] In some embodiments, xenotransplantation using transplants produced according to the present disclosure reduces the incidence of short-term rejection of donor cells, tissues, and / or organs as described in Section 7.1 compared to those derived from recombinant minipigs in which only CD47 has been engineered (i.e., the porcine CD47 gene has been deleted or inactivated and a human CD47 gene has been inserted). In other embodiments, the transplantation methods described herein reduce the incidence of long-term rejection of donor cells, tissues, and / or organs as described in Section 7.1 compared to those derived from recombinant minipigs in which only CD47 has been engineered (i.e., the porcine CD47 gene has been deleted or inactivated and a human CD47 gene has been inserted).

[0166] In some embodiments, xenotransplantation using grafts produced in accordance with the present disclosure reduces the incidence of short-term rejection of the donor cells, tissues, and / or organs (e.g., donor cells, tissues, and / or organs described in Section 7.1) compared to allogeneic donor cells, tissues, and / or organs. In other embodiments, xenotransplantation using grafts produced in accordance with the present disclosure reduces the incidence of long-term rejection of the donor cells, tissues, and / or organs (e.g., donor cells, tissues, and / or organs described in Section 7.1) compared to allogeneic donor cells, tissues, and / or organs.

[0167] In some embodiments, xenotransplantation using transplants produced according to the present disclosure extends the survival of donor cells, tissues, and / or organs obtained from a recombinant minipig described herein, as compared to donor cells, tissues, and / or organs obtained from a recombinant minipig in which only CD47 has been engineered (i.e., the porcine CD47 gene has been deleted or inactivated and the human CD47 gene has been inserted). In one embodiment, the method provided herein extends the survival of the donor cells, tissues, and / or organs by less than 10%. In one embodiment, the method provided herein extends the survival of the donor cells, tissues, and / or organs by less than 10-25%. In one embodiment, the method provided herein extends the survival of the donor cells, tissues, and / or organs by 25-50%. In one embodiment, the method provided herein extends the survival of the donor cells, tissues, and / or organs by 50-75%. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 75-100%. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 100-200%. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 200-300%. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by more than 300%. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 1-5 years. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 5-10 years. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 10-15 years. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 15-20 years. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 20-25 years. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 25-30 years.

[0168] In other embodiments, xenotransplantation using transplants produced according to the present disclosure extends the survival of donor cells, tissues, and / or organs derived from the recombinant minipigs described in Section 7.1, as compared to allogeneic donor cells, tissues, and / or organs. In one embodiment, the methods provided herein extend the survival of the donor cells, tissues, and / or organs by less than 10%. In one embodiment, the methods provided herein extend the survival of the donor cells, tissues, and / or organs by less than 10-25%. In one embodiment, the methods provided herein extend the survival of the donor cells, tissues, and / or organs by 25-50%. In one embodiment, the methods provided herein extend the survival of the donor cells, tissues, and / or organs by 50-75%. In one embodiment, the methods provided herein extend the survival of the donor cells, tissues, and / or organs by 75-100%. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 100-200%. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 200-300%. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by more than 300%. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 1-5 years. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 5-10 years. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 10-15 years. In one embodiment, the methods provided herein extend the survival of the donor's cells, tissues, and / or organs by 15-20 years. In one embodiment, the methods provided herein extend the survival of the donor cells, tissues, and / or organs by 20-25 years.In one embodiment, the methods provided herein extend the survival of the donor cells, tissues, and / or organs by 25-30 years.

[0169] In some embodiments, xenotransplantation using a transplant produced according to the present disclosure allows the donor's cells, tissues, and / or organs to survive in the recipient for at least 6 months. In other embodiments, the methods provided herein allow the donor's cells, tissues, and / or organs to survive in the recipient for at least 1 year. In other embodiments, the methods provided herein allow the donor's cells, tissues, and / or organs to survive in the recipient for at least 5 years. In other embodiments, the methods provided herein allow the donor's cells, tissues, and / or organs to survive in the recipient for at least 10 years. In other embodiments, the methods provided herein allow the donor's cells, tissues, and / or organs to survive in the recipient for at least 15 years. In other embodiments, the methods provided herein allow the donor's cells, tissues, and / or organs to survive in the recipient for at least 20 years.

[0170] In some embodiments, xenotransplantation using transplants produced according to the present disclosure results in improved function of the donor's cells, tissues, and / or organs after transplantation compared to those derived from recombinant minipigs in which only CD47 has been engineered (i.e., the porcine CD47 gene has been deleted or inactivated and the human CD47 gene has been inserted). In other embodiments, the methods provided herein result in improved function of the donor's cells, tissues, and / or organs after transplantation compared to cells, tissues, and / or organs from an allogeneic donor.

[0171] In some embodiments, xenotransplantation using transplants produced according to the present disclosure improves the corresponding disease targeted by the transplant in recipients receiving the donor cells, tissues, and / or organs, as indicated by the corresponding biomarker(s), compared to those derived from recombinant minipigs in which only CD47 has been engineered (i.e., the porcine CD47 gene has been deleted or inactivated and the human CD47 gene has been inserted). In other embodiments, the methods provided herein improve the corresponding disease targeted by the transplant in recipients receiving the donor cells, tissues, and / or organs, as indicated by the corresponding biomarker(s), compared to the allogeneic donor cells, tissues, and / or organs.

[0172] In some embodiments, xenotransplantation using transplants produced according to the present disclosure results in normal function of other non-targeted organ systems in recipients receiving donor cells, tissues, and / or organs derived from the recombinant minipigs described in Section 7.1 compared to recipients receiving donor cells, tissues, and / or organs derived from recombinant minipigs in which only CD47 has been engineered (i.e., the porcine CD47 gene has been deleted or inactivated and the human CD47 gene has been inserted). In other embodiments, the methods provided herein result in normal function of other non-targeted organ systems in recipients receiving donor cells, tissues, and / or organs derived from the recombinant minipigs described in Section 7.1 compared to recipients receiving allogeneic donor cells, tissues, and / or organs.

[0173] In some embodiments, xenotransplantation using transplants produced according to the present disclosure extends survival of recipients receiving donor cells, tissues, and / or organs derived from a recombinant minipig described herein (e.g., a recombinant minipig described in Section 7.1) as compared to transplants derived from recombinant minipigs in which only CD47 has been engineered (i.e., the porcine CD47 gene has been deleted or inactivated and the human CD47 gene has been inserted). In one embodiment, the methods provided herein extend survival of the recipient by less than 10%. In one embodiment, the methods provided herein extend survival of the recipient by less than 10-25%. In one embodiment, the methods provided herein extend survival of the recipient by 25-50%. In one embodiment, the methods provided herein extend survival of the recipient by 50-75%. In one embodiment, the methods provided herein extend survival of the recipient by 75-100%. In one embodiment, the methods provided herein extend survival of the recipient by 100-200%. In one embodiment, the methods provided herein extend the survival of the recipient by 200-300%. In one embodiment, the methods provided herein extend the survival of the recipient by more than 300%. In one embodiment, the methods provided herein extend the survival of the recipient by 1-5 years. In one embodiment, the methods provided herein extend the survival of the recipient by 5-10 years. In one embodiment, the methods provided herein extend the survival of the recipient by 10-15 years. In one embodiment, the methods provided herein extend the survival of the recipient by 15-20 years. In one embodiment, the methods provided herein extend the survival of the recipient by 20-25 years. In one embodiment, the methods provided herein extend the survival of the recipient by 25-30 years.

[0174] In some embodiments, xenotransplantation using transplants produced according to the present disclosure extends survival of a recipient receiving donor cells, tissues, and / or organs derived from a recombinant minipig described herein (e.g., a recombinant minipig described in Section 7.1) compared to allogeneic donor cells, tissues, and / or organs. In one embodiment, the methods provided herein extend survival of the recipient by less than 10%. In one embodiment, the methods provided herein extend survival of the recipient by less than 10-25%. In one embodiment, the methods provided herein extend survival of the recipient by 25-50%. In one embodiment, the methods provided herein extend survival of the recipient by 50-75%. In one embodiment, the methods provided herein extend survival of the recipient by 75-100%. In one embodiment, the methods provided herein extend survival of the recipient by 100-200%. In one embodiment, the methods provided herein extend survival of the recipient by 200-300%. In one embodiment, the methods provided herein extend the survival of the recipient by more than 300%. In one embodiment, the methods provided herein extend the survival of the recipient by 1-5 years. In one embodiment, the methods provided herein extend the survival of the recipient by 5-10 years. In one embodiment, the methods provided herein extend the survival of the recipient by 10-15 years. In one embodiment, the methods provided herein extend the survival of the recipient by 15-20 years. In one embodiment, the methods provided herein extend the survival of the recipient by 20-25 years. In one embodiment, the methods provided herein extend the survival of the recipient by 25-30 years.

[0175] In some embodiments, xenotransplantation using transplants produced according to the present disclosure enhances the quality of life of recipients receiving donor cells, tissues, and / or organs derived from the recombinant minipigs described in Section 7.1, as compared to recipients receiving donor cells, tissues, and / or organs derived from recombinant minipigs in which only CD47 has been engineered (i.e., the porcine CD47 gene has been deleted or inactivated and the human CD47 gene has been inserted). In other embodiments, the methods provided herein enhance the quality of life of recipients receiving donor cells, tissues, and / or organs derived from the recombinant minipigs described in Section 7.1, as compared to recipients receiving allogeneic donor cells, tissues, and / or organs.

[0176] In certain embodiments, xenotransplantation into baboons using donor cells, tissues, and / or organs derived from recombinant minipigs produced according to the present disclosure is advantageous compared to xenotransplantation into baboons using donor cells, tissues, and / or organs derived from recombinant minipigs in which only CD47 has been engineered (i.e., the porcine CD47 gene has been deleted or inactivated and the human CD47 gene has been inserted). For example, in some embodiments, xenotransplantation into a baboon using donor cells, tissues, and / or organs derived from a recombinant mini-pig produced according to the present disclosure results in reduced administration of immunosuppressive therapy, reduced incidence of short-term rejection of the donor cells, tissues, and / or organs, reduced incidence of long-term rejection of the donor cells, tissues, and / or organs, extended survival, better function of the donor cells, tissues, and / or organs, normal function of other non-targeted organ systems, extended survival of the baboon, a higher quality of life for the baboon, or any combination of the above, compared to xenotransplantation into a baboon using donor cells, tissues, and / or organs derived from a recombinant mini-pig in which only CD47 has been engineered.

[0177] In another specific embodiment, xenotransplantation into humans using donor cells, tissues, and / or organs derived from recombinant minipigs produced according to the present disclosure is advantageous compared to transplantation using non-engineered donor cells, tissues, and / or organs. For example, in some embodiments, xenotransplantation into humans using donor cells, tissues, and / or organs derived from recombinant minipigs produced according to the present disclosure results in reduced administration of immunosuppressive therapy, reduced incidence of short-term rejection of the donor cells, tissues, and / or organs, reduced incidence of long-term rejection of the donor cells, tissues, and / or organs, extended survival, better function of the donor cells, tissues, and / or organs, amelioration of corresponding disease as indicated by corresponding biomarker(s), normal function of other non-targeted organ systems, extended survival of the human recipient, higher quality of life of the human recipient, or any combination of the above, compared to transplantation using non-engineered donor cells, tissues, and / or organs.

[0178] Proteinuria is characterized by elevated levels of protein in the urine and can be a symptom of decreased renal function and potentially renal failure. In yet other specific embodiments, when kidneys derived from the recombinant minipigs described in Section 7.1 are transplanted into a recipient, no proteinuria is observed. In yet other specific embodiments, when kidneys derived from the recombinant minipigs described in Section 7.1 are transplanted into a recipient, the severity of proteinuria is reduced.

[0179] In some embodiments, the severity of the proteinuria is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95%. In some embodiments, patients treated according to the methods provided herein do not experience proteinuria, defined as the excretion of more than 150 mg of protein in the urine per day. In some embodiments, patients treated according to the methods provided herein may experience transient proteinuria that resolves within 1, 2, 3, 3-7, 7-10, 10-14 days, or 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8 weeks, or 1, 2, 3, 4, 5, 6 months after transplantation.

[0180] In some embodiments, recipients treated with the methods described herein who develop proteinuria have a total protein concentration in the urine of less than about 60 mg per day, less than about 80 mg per day, less than about 100 mg per day, less than about 120 mg per day, less than about 140 mg per day, less than about 160 mg per day, less than about 200 mg per day, less than about 220 mg per day, less than about 240 mg per day, less than about 260 mg per day, less than about 280 mg per day, less than about 300 mg per day, less than about 320 mg per day, less than about 340 mg per day, less than about 360 mg per day, less than about 380 mg per day, or less than about 400 mg per day.

[0181] In some embodiments, recipients treated with the methods described herein who develop proteinuria have albumin concentrations in the urine of less than about 5 mg per day, less than about 10 mg per day, less than about 20 mg per day, less than about 30 mg per day, less than about 40 mg per day, less than about 50 mg per day, less than about 60 mg per day, less than about 70 mg per day, less than about 80 mg per day, less than about 90 mg per day, or less than about 100 mg per day.

[0182] In some embodiments, patients treated according to the methods described herein have a protein to creatinine ratio in a 24 hour urine sample that is less than about 0.2, less than about 0.4, less than about 0.6, less than about 0.8, or less than about 1. In some embodiments, patients treated according to the methods described herein have an albumin to creatinine ratio in a 24 hour urine sample that is less than about 0.02, less than about 0.04, less than about 0.06, less than about 0.08, or less than about 0.1.

[0183] In some embodiments, the risk of developing proteinuria in a recipient treated with the methods described herein is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% compared to the risk in a recipient of a kidney from a donor in which only endogenous porcine CD47 has been replaced with a human homolog. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] EXAMPLES

[0184] 8. Working Example Below are descriptions of exemplary protocols that can be used to demonstrate the advantages of the methods and compositions described herein. These protocols are not intended to limit the scope of what the inventors regard as their disclosure, nor are they intended to represent all of the experiments that have been performed or may be performed.

[0185] 8.1 Example 1 Recombinant minipigs are generated by replacing endogenous porcine CD47 and SIRPA with human homologs. Briefly, fetal fibroblasts are collected from α-1,3-galactosyltransferase knockout minipigs. The cells are transfected with (i) a vector carrying the CDS sequence of human CD47 flanked by two homologous DNA arms of the porcine CD47 sequence required for homologous recombination, (ii) a vector carrying the CDS sequence of human SIRPA flanked by two homologous DNA arms of the porcine SIRPA sequence required for homologous recombination, (iii) a vector carrying Cas9, (iv) a vector carrying sgRNA targeting mouse CD47 gene, and (v) a vector carrying sgRNA targeting mouse SIRPA gene. Prior to this transfection, the vectors are verified by sequence analysis. After transfection, the cells are cultured in a medium for recovery. After recovery, the cells are split into single cells and cultured for a few more days to form colonies. Positive colonies are selected by PCR and sequence analysis to confirm the successful replacement of endogenous porcine CD47 and SIRPA with their human homologs. Minipig oocytes are collected for somatic cell nuclear transfer. Denuded oocytes are enucleated and cells from the above positive colonies are used as donor cells to be injected into the perivitelline space of the oocytes. DC pulses are used for cell fusion. The reconstructed embryos are surgically transferred into the oviducts of surrogate pigs, which give birth to recombinant minipigs for xenotransplantation. Sequence analysis is performed to confirm that endogenous porcine CD47 and SIRPA are indeed replaced by their human homologs in the resulting recombinant minipigs.

[0186] Kidneys are harvested from the above recombinant minipigs in which endogenous porcine CD47 and SIRPA have been replaced with human homologs and transplanted into recipient baboons along with bone marrow from the same minipigs. As a comparison, kidneys are harvested from recombinant minipigs in which only endogenous porcine CD47 has been replaced with human homologs and transplanted into another group of recipient baboons along with bone marrow from the same minipigs. The following parameters are measured and compared between the two groups: (i) proteinuria, assessed by measuring urinary protein concentration after transplantation, (ii) survival time without evidence of rejection of the transplanted kidney, (iii) inflammatory response, including generalized edema and serum cytokine levels (e.g., IL-6), along with a comprehensive physical examination of the recipient baboons' other organ systems, (iv) life span of the recipient baboons, and (v) postmortem analysis of any possible atrophy and abnormalities of the transplanted kidney.

[0187] 8.2 Example 2 - Humanized Porcine CD47 The following example demonstrates that porcine CD47 can be humanized by replacing a portion of porcine CD47 with a portion of human CD47. Specifically, porcine CD47 was humanized by replacing porcine exon 2 with human exon 2, allowing expression of humanized CD47 from the native porcine CD47 promoter in transgenic pigs under the appropriate lineage and temporal expression pattern.

[0188] Briefly, a homologous recombination vector, 47X2R, was developed to effect replacement of exon 2. The 47X2R vector consisted of the last 1000 bp of porcine CD47 intron 1, followed by exon 2 of human CD47, followed by the first 1020 bp of porcine intron 2 (SEQ ID NO: 6). Silent nucleotide substitutions in human CD47 exon 2 (G to A at position 1095 of the vector and ACGC instead of CAGA beginning at position 1314) were made to prevent CRISPR / Cas9 cleavage at potential CRISPR guide sites within the vector.

[0189] Three potential RNA guide pairs near the junction of intron 1 and exon 2 were identified for use as an RNP complex in combination with Cas9 D10A nickase to introduce a double-stranded break into the porcine CD47 gene. The guide pair sequences tested, which refer to SEQ ID NO: 7, were (1) 47US-F1 (nt 43-63) and 47US-R1 (complement of nt 6-25), (2) 47US-F3 (nt 138-157) and 47US-R3 (complement of nt 95-114), and (3) 47US-F3 and 47US-R4 (complement of nt 113-132).

[0190] RNP complexes composed of the above guide pairs and Cas9 D10A were nucleofected into porcine fetal fibroblasts, and the efficiency of loss of CD47 expression due to simultaneous loss of function at both CD47 alleles was assessed using FACS analysis of transfected cells stained with anti-CD47 monoclonal antibody CC2C6. Both guide pairs 47US-F3 / 47US-R3 and 47US-F3 / 47US-R4 resulted in a high percentage of CD47 null cells (Figure 1C and Figure 1D, Table 2).

[0191] [Table 2]

[0192] Next, homologous replacement of CD47 exon 2 was performed using vector 47X2R and CRISPR / Cas9 D10A. Guide pair 47US-F3 / 47US-R3 was used as an exemplary set of guide pairs. Specifically, the vector and RNP complex were nucleofected into fibroblasts derived from miniature pig fetuses transgenic for human CD55 and CD59. Transfected cells were sorted based on binding to anti-CD47 monoclonal antibody B6H12, which binds to human CD47 but not to porcine CD47. Analysis of the sorted population demonstrated that a very high percentage of this population expressed humanized CD47. It was further demonstrated that a very high percentage of this population was null for porcine CD47 (presumably due to mutations introduced by Cas9 cleavage of the untargeted CD47 allele) by staining with the anti-CD47 monoclonal antibody CC26, which binds both human and porcine CD47 when blocked with the human-specific monoclonal B6H12 (Figure 2). Loss of porcine CD47 function is the desired outcome of this modification.

[0193] In summary, this example demonstrates that porcine CD47 can be humanized by introducing a portion of human CD47 (eg, exon 2 of human CD47) into the homologous region of porcine CD47.

[0194] 8.3 Example 3 - Humanized Porcine SIRPA The humanized porcine CD47 cells are used as donor cells in somatic cell nuclear transfer to generate humanized CD47 fetuses. In this process, the nucleus of a somatic cell is transferred into an enucleated oocyte (e.g., a metaphase II oocyte), and the complex is then activated. The reconstructed embryo is then cultured and transferred into a recipient synchronized with pregnancy. This population is then subjected to genomic, RNA (e.g., RT-PCR), and / or protein analysis, e.g., to confirm the expected structure of the transgenic locus, RNA, and protein expression, and to determine whether porcine CD47 has been altered in this process.

[0195] This CD47 fetus is the starting material for humanization of the SIRPA gene using methods similar to those described in Example 2.

[0196] 9. Equivalents Although the present disclosure has been described in detail with reference to specific embodiments thereof, it will be understood that functionally equivalent variations are within the scope of the present disclosure. Indeed, various modifications of the present disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

[0197] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

1. A recombinant miniature pig, (a)(i) a deleted or functionally inactivated endogenous gene encoding porcine CD47, wherein expression of the endogenous gene encoding porcine CD47 is regulated by a porcine CD47 regulatory element; and (ii) a transgene encoding human CD47 inserted into the pig genome, wherein expression of the human CD47-encoding transgene is regulated by the pig CD47 regulatory element; and (b)(i) a deleted or functionally inactivated endogenous gene encoding porcine SIRPα, wherein expression of the endogenous gene encoding porcine SIRPα is regulated by a porcine SIRPA regulatory element; and (ii) a transgene encoding a human SIRPα inserted into the pig genome, wherein expression of the human SIRPα-encoding transgene is regulated by the pig SIRPA regulatory element; The recombinant minipig comprising:

2. The recombinant minipig according to claim 1, wherein the minipig is an alpha-1,3 galactosyltransferase-deficient minipig.

3. The recombinant minipig of claim 2, wherein the alpha-1,3 galactosyltransferase-deficient minipig is a major histocompatibility complex (MHC) inbred minipig.

4. The recombinant minipig of claim 1, wherein the human CD47 comprises SEQ ID NO: 1, 2, or 3.

5. The recombinant minipig of claim 1 , wherein the human SIRPA comprises SEQ ID NO: 4 or 5.

6. 2. The recombinant minipig of claim 1, wherein the transgenes encoding human SIRPα and human CD47 are inserted into the genome of the minipig by homologous recombination.

7. 2. The recombinant minipig of claim 1, wherein the transgenes encoding human SIRPα and human CD47 are inserted into the pig genome by non-homologous end joining.

8. 2. The recombinant minipig of claim 1, wherein the transgenes encoding human SIRPα and human CD47 are inserted into the pig genome by recombinase-mediated cassette exchange.

9. 2. The recombinant minipig of claim 1, wherein the transgenes encoding human SIRPα and human CD47 are inserted into the pig genome by site-specific nucleases.

10. 10. The recombinant minipig of claim 9, wherein the site-specific nuclease is selected from the group consisting of zinc finger, ZFN dimer, ZF nickase, transcription activator-like effector nuclease (TALEN), and CRISPR / Cas9.

11. 2. The recombinant minipig of claim 1, wherein the expression of the transgene encoding human CD47 is regulated by the porcine CD47 regulatory element of NCBI gene ID: 397042.

12. 2. The recombinant minipig of claim 1, wherein expression of the transgene encoding human SIRPα is regulated by the porcine SIRPA regulatory element of NCBI gene ID: 494566.

13. 2. The recombinant minipig of claim 1, wherein the expression of human CD47 protein is substantially similar to the expression pattern of endogenous porcine CD47 as determined by immunohistochemistry.

14. 2. The recombinant minipig of claim 1, wherein the expression of human SIRPα protein is substantially similar to the expression pattern of endogenous porcine SIRPα as determined by immunohistochemistry.

15. A recombinant miniature pig, (a) a humanized CD47 gene, and / or (b) Humanized SIRPA gene The recombinant minipig comprising:

16. The recombinant minipig of claim 15, wherein the humanized CD47 gene comprises a porcine CD47 gene comprising exon 2 of human CD47.

17. A cell derived from the recombinant miniature pig according to any one of claims 1 to 16.

18. An oocyte derived from the recombinant miniature pig according to any one of claims 1 to 16.

19. Sperm derived from the recombinant miniature pig according to any one of claims 1 to 16.

20. A tissue derived from the recombinant miniature pig according to any one of claims 1 to 16.

21. An organ derived from the recombinant miniature pig according to any one of claims 1 to 16.

22. 17. The recombinant miniature pig according to any one of claims 1 to 16, for use in a method for transplanting a graft derived from a first recombinant miniature pig into a primate, said method comprising: (a) harvesting a graft from the first recombinant minipig according to any one of claims 1 to 16; and (b) implanting the graft into the primate. The recombinant minipig according to any one of claims 1 to 16, comprising:

23. The recombinant minipig of claim 22, wherein the primate is a human.

24. 23. The recombinant minipig of claim 22, wherein the graft comprises a cell, tissue, or organ.

25. 25. The recombinant minipig of claim 24, wherein the organ is selected from the group consisting of heart, kidney, pancreatic islet, liver, pancreas, lung, intestine, skin, trachea, and cornea, or a combination thereof.

26. A recombinant mini-pig as described in claim 22, wherein the method further comprises harvesting bone marrow from a second recombinant mini-pig as described in any one of claims 1 to 16, and transplanting the bone marrow into the same primate.

27. 27. The recombinant minipig of claim 26, wherein the bone marrow is transplanted at least 28 days prior to transplantation from the first recombinant pig.

28. 27. The recombinant minipig of claim 26, wherein the first recombinant minipig and the second recombinant minipig are the same recombinant pig.

29. 27. The recombinant minipig of claim 26, wherein the first recombinant minipig and the second recombinant minipig are derived from a highly inbred herd of minipigs.

30. 27. The recombinant minipig of claim 26, wherein the first recombinant minipig and the second recombinant minipig are genetically compatible minipigs.

31. 27. The recombinant minipig of claim 26, wherein the first recombinant minipig and the second recombinant minipig are MHC-matched.

32. 23. The recombinant minipig of claim 22, wherein a graft derived from the first recombinant minipig survives within the recipient for at least 6 months, 1 year, 5 years, 10 years, 15 years, or 20 years.

33. 23. The recombinant minipig of claim 22, wherein a graft derived from the first recombinant minipig functions within the recipient for at least 6 months, 1 year, 5 years, 10 years, 15 years, or 20 years.

34. 23. The recombinant minipig of claim 22, wherein the recipient requires a 90%, 80%, 70%, 60%, or 50% reduction in immunosuppressive therapy.

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