Transgenic swine, methods of making and uses thereof, and methods of making human immune system mice

Abstract

To provide improved porcine thymus tissue for heterotransplantation.SOLUTION: There are provided a transgenic swine, comprising one or more nucleotide sequences encoding one or more HLA I polypeptides and / or one or more HLA II polypeptides inserted into one or more native SLA loci of the swine genome, and thymus tissue derived from the transgenic swine.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] Cross - reference to other applications This application is a related application to U.S. Patent Application No. 62 / 924,228 filed on October 22, 2019, and U.S. Patent Application No. 62 / 925,859 filed on October 25, 2019, and claims priority based thereon. The entire contents of the above two patent applications are incorporated herein by reference in their entirety.

[0002] Explanation of government rights The present invention was made with government support under grant number AI045897 awarded by the National Institutes of Health. The federal government has certain rights in the present invention.

[0003] The present disclosure provides a transgenic pig comprising one or more nucleotide sequences encoding one or more HLA I polypeptides and / or one or more HLA II polypeptides inserted into one or more natural SLA loci of the pig genome, a method for producing the same, and a method for using the same.

[0004] The present disclosure also provides a method for producing a human immune system mouse, which is an improved method.

Background Art

[0005] Human immune system (HIS) mice have great potential for the study of human autoimmune diseases, transplantation, and infectious diseases. The major tissue required to form a stable human immune system in immunodeficient mice is human fetal thymus tissue, which creates a highly functional and diverse human T cell repertoire. Postnatal human thymus tissue lacks the ability to proliferate T cells even when transplanted under the mouse kidney capsule (if it had its original proliferative ability, the thymus tissue could generate numerous human T cells and become larger than the kidney). In immunodeficient mice, human T cells develop to some extent in the natural thymus of the mouse, but the thymus function is abnormal and defective, and only a very small number of human T cells are generated, and normal thymic education necessary for proper immune tolerance induction does not occur. Therefore, human fetal thymus tissue is considered optimal for the HIS mouse model. However, human fetal tissue cannot be used for research. Therefore, another tissue source is needed.

[0006] Fetal porcine thymus tissue can be an alternative. Fetal porcine (SW) thymus (THY) tissue also has the same proliferative characteristics as human (HU) fetal THY tissue when transplanted into immunodeficient mice, and highly supports the stable proliferation of human thymocytes and the peripheral immune reconstitution of human CD34+ cells. However, since HLA molecules are not present in SW thymic epithelial cells (TEC), negative selection of conventional T cells and positive selection of regulatory T cells that recognize HLA-restricted antigens (TRA) produced by TEC are limited. Also, positive selection of human T cells that can recognize foreign antigens in relation to the HLA of an individual is limited. Therefore, when using fetal porcine thymus tissue to produce HIS mice, improvement is needed. Furthermore, improvement is also needed when using porcine thymus tissue for other applications (such as xenotransplantation to humans).

[0007] This specification describes a method for producing human immune system mice improved using fetal porcine thymus tissue. This specification also describes transgenic pigs.

Summary of the Invention

[0008] This specification provides transgenic pigs, methods for producing such pigs, and uses of such pigs.

[0009] In one embodiment, the transgenic pig comprises one or more nucleotide sequences encoding one or more HLA I polypeptides and / or one or more HLA II polypeptides inserted into one or more natural SLA loci of the pig genome.

[0010] In some embodiments, the human HLA is selected from the group consisting of HLA I polypeptides and HLA II polypeptides. In some embodiments, the human HLA I is selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the HLA I polypeptide is HLA-A2.

[0011] In some embodiments, the HLA II polypeptide is selected from the group consisting of HLA-DP, HLA-DM, HLA-DO, HLA-DQ, and HLA-DR. In some embodiments, the HLA II polypeptide is HLA-DQ8 or SLA-Dra. In some embodiments, the HLA-DQ8 polypeptide targets the natural SLA-DQα locus with a bicistronic vector encoding HLA-DQ8 (HLA-DQAl:03:01:01 and HLA-DQB1:03:02:01).

[0012] In some embodiments, the natural SLA locus is SLA-1, SLA-2, or SLA-3. In some embodiments, the SLA locus is the SLA-DQα or SLA-DR locus. In some embodiments, the nucleic acid is inserted or integrated after the natural SLA promoter. In some embodiments, the nucleic acid encoding the HLA polypeptide is inserted or integrated at the intron 1 / exon 2 junction of the natural SLA locus.

[0013] In some embodiments, the nucleic acid encoding the HLA polypeptide is inserted or integrated into the native SLA locus using a target vector. In some embodiments, the vector is bicistronic. In some embodiments, the vector is promoterless.

[0014] In some embodiments, the vector further comprises a high-efficiency IRES element.

[0015] In some embodiments, the vector further comprises a polyadenylation site. In some embodiments, the polyadenylation site is rabbit β-globin.

[0016] Also provided herein are methods for producing the transgenic pigs and uses thereof (including, but not limited to, xenotransplantation into human subjects).

[0017] Provided herein is a method for producing a human immune system mouse, which is an improved method.

[0018] In some embodiments, the method includes removing the thymus of the mouse and introducing porcine fetal thymus tissue and human CD34+ cells into the mouse. In some embodiments, the human CD34+ cells are derived from umbilical cord blood.

[0019] In some embodiments, the method includes removing the thymus of the mouse and introducing porcine fetal thymus tissue of a transgenic pig as described herein into the mouse.

[0020] For purposes of specifically illustrating the present invention, specific embodiments of the present invention are shown in the drawings. However, the present invention is not limited to the detailed configurations and means of the embodiments shown in the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0022] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is then translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include mRNA splicing in eukaryotic cells.

[0023] As used herein, the term "isolated" refers to a molecule, biological agent, or cell material that is substantially free of other substances.

[0024] As used herein, the term "functional" may also be used in connection with modifying a molecule, biological agent, or cell material for the purpose of achieving a particular specialized effect.

[0025] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used synonymously with any length of polymeric nucleotides, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivative nucleotide bases.

[0026] The terms "protein", "peptide", and "polypeptide" are synonymous with each other and, in the broadest sense, refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another aspect, the subunits may be linked by other bonds, such as esters, ethers, etc. It is essential that one protein or peptide contains at least two amino acids, but there is no limit to the maximum number of amino acids that a protein sequence or peptide sequence can contain. The term "amino acid" herein refers to either natural and / or non-natural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0027] As used herein, "targeting", "targets" (third person singular), or "targeted" means partial cleavage or non-cleavage of the covalent backbone of a polynucleotide. In one embodiment, an inactivated Cas protein (or dCas) targets a nucleotide sequence after formation of a DNA-binding complex with a guide RNA. Since the nuclease activity of dCas is completely or partially inactivated, dCas binds to the sequence without cleaving or fully cleaving the sequence. In some embodiments, targeting of a gene sequence or its promoter by dCas inhibits or interferes with the transcription and / or expression of the polynucleotide or gene.

[0028] The term "Cas9" means a CRISPR-associated endonuclease, as the name indicates. Non-limiting examples of Cas9 described herein include, for example, Cas9 of UniProtKB G3ECR1 (CAS9_STRTR) or Cas9 of Staphylococcus aureus, as well as nuclease-inactive (dead) Cas9, their orthologs and biological equivalents. Orthologs include Cas9 from Streptococcus pyogenes ("spCas9"), Streptococcus thermophilus, Legionella pneumophila, Neisseria lactamica, Neisseria meningitidis, or Francisella novicida; and Cpf1 (exhibiting a cleavage function similar to Cas9) from various bacterial species (including Acidaminococcus spp. and Francisella novicida U112), but are not limited thereto.

[0029] The term "CRISPR" in this specification refers to a sequence-specific gene manipulation technique based on the clustered regularly interspaced short palindromic repeats pathway. CRISPR can be used for the purpose of gene editing and / or gene regulation, as well as simply targeting a protein to a specific genomic location. Gene editing refers to a technique in genetic engineering that changes the nucleotide sequence of a target polynucleotide by introducing deletions, insertions, or base substitutions into the polynucleotide sequence. Gene regulation means increasing or decreasing the production of a specific gene product (such as a protein or RNA).

[0030] The term "gRNA" or "guide RNA" in this specification refers to a guide RNA sequence used to target specific genes for the purpose of modification using CRISPR technology. Techniques for designing gRNA and donor therapeutic polynucleotides with respect to target specificity are well known in the art. For example, Doench et al., 2014, Nature biotechnology 32(12):1262-7, Mohr et al., 2016, FEBS Journal 3232-38, and Graham et al., 2015, Genome Biol. 16:260. gRNA is a fusion polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA); or a polynucleotide comprising, consisting essentially of, or further consisting of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In some aspects, gRNA is a synthetic composition (Kelley et al., 2016, J of Biotechnology 233:74-83). Biological equivalents of the gRNA described herein include, but are not limited to, polynucleotides or targeting molecules that can direct Cas9 or its equivalents to a specific nucleotide sequence (such as a specific region of the cell's genome).

[0031] The term "embryo" refers to the early stage of development of a multicellular organism. Generally, in organisms that reproduce sexually, embryogenesis means a part of the life cycle that begins immediately after fertilization and continues through the formation of body structures (such as tissues and organs). Each embryo begins development as a single zygote, which is a single cell resulting from the fusion of gametes (i.e., fertilization of a female egg cell by a male sperm cell). In the first stage of embryogenesis, the single-celled zygote undergoes rapid multiple rounds of cell division (called cleavage) to form a blastula.

[0032] As used herein, "transgenic" and grammatical equivalents, if any, include the genome of a donor animal that has been modified to introduce a non-native gene from a different species at a site that is not a homologous or endogenous site in the donor animal genome, such that (if present) the homologous and endogenous gene remains intact in whole or in part. As used herein, "transgene," "transgenic," and grammatical equivalents do not include reprogrammed genomes, knockouts, or other modifications as described herein.

[0033] As used herein, "immune tolerance" means inhibition or reduction of the ability of a transplant recipient to initiate an immune response, e.g., against donor antigens (or otherwise, e.g., as would be expected to respond when a non-self MHC antigen enters the recipient). Immune tolerance can include humoral responses, cellular responses, or both humoral and cellular responses. The concept of immune tolerance includes both complete immune tolerance and partial immune tolerance. In other words, immune tolerance as used herein includes any degree of inhibition of the ability of a transplant recipient to initiate an immune response, e.g., an immune response against donor antigens.

[0034] As used herein, "hematopoietic stem cell" refers to a cell that can develop into mature myeloid and / or lymphoid cells. Preferably, the hematopoietic stem cell is capable of long-term repopulation of the myeloid lineage and / or the lymphoid lineage. Stem cells derived from the umbilical cord blood of a recipient or donor can be used in the methods of the present disclosure.

[0035] As used herein, "minipig" refers to a minipig that is fully or partially inbred.

[0036] As used herein, "transplant" refers to a body part, organ, tissue, cell, or portion thereof.

[0037] Abbreviations SW-pig HU-human TEC - thymic epithelial cells TMC - thymic mesenchymal cells WBC - white blood cells DP - double - positive cells (CD4+ and CD8+) SP - single - positive cells (either CD4+ or CD8+) Treg - regulatory T cells LN - lymph nodes TRA - tissue - specific self - antigen HSC - human hematopoietic cells NSG - NOD scid common gamma chain knockout SCNT - somatic cell nuclear transfer

[0038] The present disclosure provides a transgenic pig comprising a nucleotide sequence encoding an HLA I or HLA II polypeptide inserted into the SLA locus of the pig genome, a method of producing such a transgenic pig, and a method of using such a transgenic pig.

[0039] The present disclosure also provides a human immune system (HIS) mouse produced using the thymus of a transgenic pig fetus, a human - immunized mouse produced using the thymus and umbilical cord blood - derived CD34+ cells of a pig fetus, and a method of producing such HIS mice.

[0040] Transgenic pig Previously, the inventors have shown that stable proliferation of human thymocytes occurs in porcine thymic grafts (Nikolic et al., 1999; Shimizu et al., 2008; Kalscheuer et al., 2014). However, compared to human fetal thymus, peripheral human T cells generated in pigs show a slight decrease in HLA-restricted immune function and homeostasis as well as immune tolerance to tissue-specific self-antigens. Adding transgenic HLA molecules to porcine thymic tissue may overcome most of these limitations. Accordingly, this specification discloses multiple lines of transgenic pigs expressing common HLA alleles instead of some porcine leukocyte antigen (SLA, corresponding to HLA in pigs) molecules. These transgenic pigs can be used as a source of thymic tissue and as donor tissue for multiple purposes including creating HIS mice. Transgene expression of common HLA molecules improves positive selection of HLA-restricted human T cells and generation of functional regulatory T (Treg) cells that efficiently interact with human antigen-presenting cells (APCs) in the periphery, and also improves negative selection of human TRA-reactive T cells, reducing the risk of autoimmunity.

[0041] In baboons receiving porcine thymus-kidney grafts, evidence of de novo recipient (baboon) thymocyte proliferation in the porcine thymic graft, appearance of recent thymic emigrants in the periphery, and donor-specific non-responsiveness in Elispot assays and MLR assays, as well as reduction of non-Gal natural antibodies has been shown. The latter may reflect absorption by the porcine kidney, but only minimal IgM binding was detected in these xenografts, no complement binding, and no significant lesions were observed. Thus, the results obtained with this model demonstrate the potential of thymus-kidney composite xenografts to induce immune tolerance in primates.

[0042] Limitations in generating a human T cell repertoire in xenogeneic pig thymus include selective recognition of microbial antigens on porcine MHC, which is useful in protecting the graft but does not optimize protection against microbial pathogens infecting the host, and the inability to achieve negative selection of conventional T cells and positive selection of Tregs that recognize human tissue-specific self-antigens (TRA). In fact, studies in humanized mice have shown that when human T cells develop in pigs rather than in human thymic grafts, the response to peptides presented by human APCs after immunization is reduced.

[0043] One approach to overcoming this limitation is the creation of a "hybrid thymus" by injecting recipient thymic epithelial cells obtained from thymectomy samples or generated from stem cells into porcine thymic tissue. Hybrid thymuses have been created from postnatal thymic donors, and these hybrid thymuses promote immunotolerance of human T cells to human TRA.

[0044] Porcine thymic grafts have been shown to support the development of a normal and diverse mouse or human T cell repertoire, and these T cells are specifically tolerant to the xenogeneic porcine donor. However, recognition of foreign antigens presented by recipient HLA molecules in the periphery is only suboptimal. Thus, it is possible that immune function has not reached an optimal level. As already shown in International Application No. PCT / US2019 / 0051865, which is an application by the same applicant, this can be overcome by providing recipient TECs to porcine-human hybrid thymic grafts. The reason is that these TECs are involved in positive selection, and as a result, T cells that can more easily recognize foreign antigens presented by recipient HLA molecules in the periphery are obtained. With respect to porcine thymic grafts, the survival, homeostasis, and function of T cells that do not encounter "positive selection" ligands in the periphery are only suboptimal. When thymocytes have low-affinity T cell receptors that recognize MHC / peptide complexes, the positive selection ligand is the complex present on the surface of TECs that rescue thymocytes from programmed cell death. By providing recipient TECs to porcine-human hybrid thymus, positive selection of T cells that detect the same ligand on recipient cells in the periphery becomes possible, and normal viability, homeostasis, and function are conferred. By using hybrid thymus in this way instead of simple porcine thymus, it becomes possible to improve the function and autoimmunity tolerance of the human T cell repertoire generated in porcine thymus, and the development of immune tolerance to pigs is also possible. In short, it is possible to improve the function and autoimmunity of the human T cell repertoire generated in porcine thymus by using transgenic porcine thymus. Therefore, the transgenic pigs of the present disclosure can also be used as a source of donor thymic tissue.

[0045] The Sachs miniature pig colony was established from two founder animals by Dr. David Sachs in the 1970s. The MHC (porcine leukocyte antigen, SLA) of these animals was serologically determined by Dr. Sachs, and three SLA homozygous partial inbred lines are maintained along with multiple SLA-region recombinants. These pigs can serve as an animal source for the transgenic pigs disclosed herein (U.S. Patent Nos. 6,469,229 (Sachs), 7,141,716 (Sachs); the disclosures of which are incorporated herein by reference in their entireties). The production of such pigs by the methods described herein and / or the use of such pigs and their progeny after production can be used in the practice of the present disclosure, and the uses include, but are not limited to, the use of organs, tissues, and / or cells derived from such pigs.

[0046] In some embodiments, cells derived from such pigs are used as a starting material. In some embodiments, the cells are fibroblasts. In some embodiments, the cells are derived from GTA1 null, SLA haplotype h homozygous Sachs miniature pigs (SLA-1*02:01, SLA-1*07:01, SLA-2*02:01, SLA-3 null, SLA-DRA*01:01:02, SLA-DRB*02:01, SLA-DQA*02:02:01, SLA-DQB*04:01:01). Due to the nature of these animals being partial inbred lines, the progeny will have a high degree of genetic similarity.

[0047] In some embodiments, cells that have already been modified by the insertion or integration of a nucleic acid sequence encoding an HLA polypeptide into the native SLA locus are used as a starting material.

[0048] In humans, the major histocompatibility complex (MHC) molecules are the human leukocyte antigen ( H uman L eukocyte AIt is represented by HLA, which is an acronym for (ntigens), and these are encoded in the HLA region located on chromosome 6p21.3. The HLA segment is divided into three regions (from centromere to telomere); namely, class II, class III, and class I. These cell surface proteins are involved in the regulation of the immune system in humans. The HLA genes are highly polymorphic, which means that this gene has many different alleles, thereby enabling fine-tuning of the adaptive immune system. As a result of the historical discovery as a factor in organ transplantation, the proteins encoded by certain genes are also known as antigens. Different classes have different functions.

[0049] The HLAs corresponding to MHC class I (A, B, and C) are all in the HLA class 1 group and present peptides derived from within the cell. Usually, these specific peptides are small polymers and are about 9 amino acids in length. The foreign antigens presented by MHC class I attract killer T cells that destroy the cell (also called CD8-positive or cytotoxic T cells). The MHC class 1 protein associates with β2-microglobulin, which, unlike the HLA protein, is encoded by a gene on chromosome 15.

[0050] The HLAs corresponding to MHC class II (DP, DM, DO, DQ, and DR) present antigens derived from outside the cell to T lymphocytes. These specific antigens stimulate the proliferation of T helper cells (also sometimes called CD4-positive T cells), which in turn then stimulate antibody-producing B cells to promote antibody production against that specific antigen. Self-antigens are suppressed by regulatory T cells. The genes that are affected are known to encode four different regulatory factors that control the transcription of MHC class II genes.

[0051] The HLA corresponding to MHC class III encodes components of the complement system.

[0052] In addition to the genes encoding the six major antigen-presenting proteins, there are numerous genes, many of which are involved in immune functions and are present in the HLA complex.

[0053] The diversity of HLA in the human population is a facet of disease defense, and as a result, the probability that two unrelated individuals have identical HLA molecules at all loci is extremely low. Historically, the identification of HLA genes was based on the high likelihood of successful organ transplantation between individuals with similar HLA.

[0054] Each human cell expresses six MHC class I alleles (one HLA-A, one HLA-B, and one HLA-C allele from each parent) and six to eight MHC class II alleles (one HLA-DP and HLA-DQ from each parent, and one or two HLA-DR, and combinations thereof). The diversity of MHC in the human population is high, with at least 350 alleles for the HLA-A gene, at least 620 alleles for HLA-B, at least 400 alleles for DR, and at least 90 alleles for DQ. In humans, MHC class II molecules are encoded by three different loci, HLA-DR, -DQ, and -DP, which show approximately 70% similarity to each other. Polymorphism is a prominent feature of MHC class II genes. This genetic diversity becomes a problem in xenotransplantation, where the recipient's immune response, which determines the success of engraftment and post-transplant survival, is the most important factor.

[0055] In some embodiments, the disclosure includes modifying a pig by inserting or integrating a nucleic acid encoding one or more human HLA polypeptides into one or more native SLA loci of the pig.

[0056] In some embodiments, the human HLA is selected from the group consisting of HLA I polypeptide and HLA II polypeptide. In some embodiments, the human HLA I is selected from the group consisting of HLA-A, HLA-A2, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the HLA I polypeptide is HLA-A2. In some embodiments, the HLA II polypeptide is selected from the group consisting of HLA-DP, HLA-DM, HLA-DO, HLA-DQ, and HLA-DR. In some embodiments, the HLA II polypeptide is HLA-DQ8.

[0057] In some embodiments, the human HLA is a known HLA polypeptide. Such HLA sequences are available, for example, from the IPD-IMGT / HLA database (available from ebi.ac.uk / ipd / imgt / hla / ) and the International ImMunoGeneTics Information System RTM (available from imgt.org). For example, HLA-A1, B8, DR17 are the most common HLA haplotypes in Caucasians, with a frequency of 5%. Therefore, the methods of the present disclosure can be implemented by using the known HLA sequence information in combination with the methods described herein.

[0058] In some embodiments, the nucleic acid encoding the human HLA polypeptide is derived from a specific human individual. In some embodiments, a transgenic pig is created using a nucleic acid encoding a human HLA polypeptide derived from a specific human individual and thymic tissue or other cells, and the tissue or organ of the transgenic pig is introduced into the same specific human individual. In these embodiments, the human leukocyte antigen (HLA) gene derived from the specific human individual who receives the xenotransplant from the transgenic pig is identified and sequenced. It will be understood that the identification and sequencing of specific HLA alleles can be performed by methods known in the art.

[0059] It may also be possible to introduce a known human HLA sequence or an HLA sequence that has been identified and sequenced, derived from a specific human individual, under the control of the SLA promoter of the vector (for example, those having 90%, 95%, 98%, 99%, or 100% sequence homology to the HLA sequence).

[0060] In some embodiments, the nucleic acid encoding the HLA polypeptide can be optimized and modified to have the HLA polypeptide sequence or to mimic the HLA alleles of the recipient mammal.

[0061] In some embodiments, the HLA polypeptide is fused to another protein. In some embodiments, the protein is human beta-2 microglobulin (B2M). In some embodiments, HLA-A2 is fused to B2M. Introducing HLA-A2 and human B2m as a fusion protein will ensure that the heterotypic interaction between HLA-A2 and porcine B2m does not interfere with HLA-A2 surface expression.

[0062] In some embodiments, the native SLA locus is SLA I. In some embodiments, the native SLA locus is SLA-1 or SLA-2. In some embodiments, the SLA locus is the SLA-DQα locus. In some embodiments, the nucleic acid is inserted or integrated behind the native SLA promoter. In some embodiments, the nucleic acid encoding the HLA polypeptide is inserted or integrated at the intron 1 / exon 2 junction of the native SLA locus.

[0063] In some embodiments, a nucleic acid encoding the HLA polypeptide is inserted or integrated into the native SLA locus using a target vector. In some embodiments, the vector is bicistronic. In some embodiments, the vector is promoterless. Using a promoterless vector design ensures that cells expressing the human B2m / HLA-A2 fusion target the DQA gene at a very high rate.

[0064] In some embodiments, the vector further comprises a high-efficiency IRES element.

[0065] In some embodiments, the vector further comprises a polyadenylation site. In some embodiments, the polyadenylation site is rabbit β-globin.

[0066] Methods of modifying the SLA locus by integrating or inserting a nucleic acid encoding an HLA polypeptide include the use of site-specific nucleases described below.

[0067] Thus, provided herein are methods of creating transgenic pigs. In one aspect, the HLA genes of a particular human individual recipient are sequenced and used to construct a target vector for introduction into porcine cells. In another aspect, known human HLA genotypes in the WHO database may be used to construct a target vector for introduction into porcine cells. A target vector as described herein is constructed using a nucleic acid encoding the HLA polypeptide. A CRISPR-Cas9 plasmid can be prepared. In porcine cells, a CRISPR cleavage site of the SLA / MHC locus is identified, and one or more gRNA sequences targeting the designed cleavage site are cloned into one or more CRISPR-Cas9 plasmids. The CRISPR-Cas9 plasmid is then introduced into porcine cells together with the target vector.

[0068] Once the modification is complete, the cells are screened for the desired modification using methods known in the art. Cells having the desired modification can be used as donor cells for somatic cell nuclear transfer (SCNT) for the purpose of nuclear transfer / embryo transfer and the production of transgenic pig fetuses and piglets, which can also be carried out by methods known in the art.

[0069] At approximately 40 weeks, transgenic pig fetuses are recovered. These fetuses are analyzed for the expression and proper integration of the desired HLA gene. Fetuses found to have proper integration are further utilized as a source of cell lines for SCNT cloning to produce more fetuses and piglets. At approximately 56 - 70 weeks, fetuses are recovered for thymus isolation.

[0070] These fetuses are also used to produce transgenic founder boars.

[0071] The thymic tissue of transgenic pig fetuses has many uses, including but not limited to the production of modified human immune system (HIS) mice as described below.

[0072] Cells, tissues, and / or organs (including thymic tissue) from transgenic pig fetuses can be used for xenotransplantation and can also be used to normalize or restore reduced thymic and reconstituted T cell function in a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0073] Cells, tissues, and organs derived from transgenic pigs for the purpose of xenotransplantation will have reduced rejection compared to cells, tissues, and organs derived from wild - type pigs.

[0074] Further included in the present disclosure is a method of xenotransplantation in a recipient mammal that is a first biological species, the method including transplanting thymic tissue into the recipient mammal, wherein the thymic tissue is derived from the transgenic pig described herein.

[0075] The present disclosure also provides a method of restoring or inducing immunocompetence in a recipient mammal that is a first biological species, the method including introducing thymic tissue into the recipient mammal, wherein the thymic tissue is derived from the transgenic pig described herein.

[0076] The present disclosure also provides a method of restoring or promoting thymus-dependent ability in a recipient mammal that is a first biological species, in which T cell progenitor cells develop into functional T cells, the method including introducing thymic tissue into the recipient mammal that is a first biological species, wherein the thymic tissue is derived from the transgenic pig described herein.

[0077] In one embodiment, the recipient mammal has essentially no thymic function prior to the introduction of the thymic tissue. In another embodiment, the thymus of the recipient mammal is removed prior to the introduction of the thymic tissue. In yet another embodiment, the recipient mammal has an immunodeficiency.

[0078] The second biological species may be a pig (such as a transgenic pig).

[0079] The first biological species may be a primate (such as a non-human primate or a human).

[0080] In one embodiment, the recipient mammal is a human, and the donor mammal is the transgenic pig described herein. In some embodiments, the recipient human is a source of nucleic acid encoding an HLA polypeptide that is introduced into a pig to create the transgenic pig. In some embodiments, the nucleic acid encoding the HLA polypeptide is known in the art.

[0081] In one embodiment, the thymic tissue is transplanted into a recipient mammal. For example, the thymic tissue may be transplanted as a thymic lobe in which angiogenesis is mainly occurring, or as a thymus-kidney composite graft. The thymic tissue may be transplanted into the muscle of the recipient. The thymic tissue may be transplanted only into the quadriceps femoris of the recipient, or may be transplanted into another transplantation site (for example, renal capsule and greater omentum) in addition to the quadriceps femoris.

[0082] CRISPR / Cas and other endonucleases Any suitable nuclease may be used in this method for producing a transgenic pig. A nuclease is an enzyme that hydrolyzes nucleic acids. Nucleases can also be classified into endonucleases or exonucleases. Endonucleases belong to a group of enzymes that catalyze the hydrolysis of the bonds between nucleic acids inside a DNA molecule or an RNA molecule. Exonucleases belong to a group of enzymes that catalyze the reaction of hydrolyzing a single nucleotide from the end of a DNA strand or an RNA strand. Nucleases may also be classified based on whether they specifically digest DNA or RNA. A nuclease that specifically catalyzes the hydrolysis of DNA is sometimes called deoxyribonuclease or DNase, while a nuclease that specifically catalyzes the hydrolysis of RNA is sometimes called ribonuclease or RNase. There are nucleases specific for either single-stranded or double-stranded nucleic acid sequences. There are also enzymes having the properties of both exonucleases and endonucleases. Furthermore, there are enzymes that can digest both DNA sequences and RNA sequences.

[0083] Non-limiting examples of endonucleases include zinc finger nucleases (ZFNs), ZFN dimers, zinc finger nickases, transcription activator-like effector nucleases (TALENs), or RNA-guided DNA endonucleases (e.g., CRISPR / Cas). Meganucleases are endonucleases characterized by the ability to recognize and cleave long DNA sequences (12 base pairs or more). In this method of introducing double-strand breaks in the host genome, any meganuclease can be used if suitable, and such meganucleases include endonucleases of the LAGLIDADG and PI-Sce families.

[0084] In one aspect of the present disclosure, an RNA-guided endonuclease is provided. The RNA-guided endonuclease also includes at least one nuclease domain and at least one domain that interacts with a guide RNA. The RNA-guided endonuclease is guided to a specific nucleic acid sequence (or target site) by the guide RNA. The guide RNA interacts with the RNA-guided endonuclease as well as the target site, and if the RNA-guided endonuclease is guided to the target site, a double-strand break can be introduced into the target site nucleic acid sequence. Since the guide RNA confers specificity to target cleavage, the endonuclease of the RNA-guided endonuclease is general and can be used with different guide RNAs to cleave different target nucleic acid sequences.

[0085] As an example of an RNA-guided sequence-specific nuclease system that can be used with the methods and compositions described herein, the CRISPR system (Wiedenheft 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) can be mentioned. In the CRISPR (clustered regularly interspaced short palindromic repeats) system, sequence-specific cleavage of target DNA is utilized with RNA-guided DNA binding cleavage. The guide RNA / Cas combination confers site specificity to the nuclease. Single-stranded guide RNA (sgRNA) contains about 20 nucleotides, which is complementary to the target genomic DNA sequence located upstream of the genomic PAM (protospacer adjacent motif) site (e.g., NGG) and the RNA constant scaffold region. The Cas (CRISPR-associated) protein binds to the sgRNA and the target DNA to which the sgRNA binds, and introduces a double-strand break at a specific site upstream of the PAM site. Cas9 has two independent nuclease domains homologous to the HNH endonuclease and the RuvC endonuclease, and by introducing a mutation into either of these two domains, the Cas9 protein can be converted into a nickase that introduces a single-strand break (Cong et al., 2013 Science 339:819-823). It is particularly contemplated that the methods and compositions of the present disclosure can be used with single-stranded inducible or double-stranded inducible Cas9, and also with other RNA-guided DNA nucleases (such as other bacterial Cas9-like systems). The sequence-specific nucleases of the methods and compositions described herein can be engineered from any organism, can be chimeric, or can be isolated. The nuclease can be introduced into cells in the form of DNA, mRNA, and protein.

[0086] Those skilled in the art will understand that it is possible to generate gRNAs with respect to target specificity targeting specific genes, optionally targeting genes associated with a disease, disorder, or pathological condition. Thus, when used in combination with Cas9, the guide RNA enhances the target specificity of the CRISPR / Cas9 system. In further aspects such as the selection of a promoter, it is also possible to provide additional mechanisms for achieving target specificity (e.g., promoter selection for a polynucleotide encoding a guide RNA that promotes expression in a specific organ or tissue). Thus, the selection of a suitable gRNA for a specific disease, disorder, or pathological condition is contemplated herein. In one embodiment, the gRNA hybridizes to a gene or allele containing a single nucleotide polymorphism (SNP).

[0087] Non-limiting examples of suitable CRISPR / Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966.

[0088] In one embodiment, the RNA-guided endonuclease is derived from a type II CRISPR / Cas system. In certain embodiments, the RNA-guided endonuclease is derived from a Cas9 protein. The Cas9 protein may be derived from the following bacteria: Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.) Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp.) Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina.

[0089] In some embodiments, the nucleotide sequence encoding a Cas (e.g., Cas9) nuclease is modified to alter its protein activity. In some embodiments, the Cas (e.g., Cas9) nuclease is a catalytically inactive Cas (e.g., Cas9) (or a catalytically inactivated / deficient Cas9 or dCas9). In one embodiment, the dCas (e.g., dCas9) is a Cas protein (e.g., Cas9) lacking endonuclease activity due to point mutations present in one or both of the endonuclease catalytic sites (RuvC and HNH) of wild-type Cas (e.g., Cas9). For example, dCas9 contains mutations in catalytically active residues (D10 and H840) and thus has no nuclease activity. In some cases, the dCas has reduced cleavage ability with respect to both the complementary and non-complementary strands of the target DNA. In some cases, the dCas9 has two types of mutations, D10A and H840A, in the amino acid sequence of Streptococcus pyogenes Cas9. In some embodiments, when the catalytic activity of dCas9 is reduced or absent (e.g., when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 mutations, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A), the Cas protein can still specifically bind to the target DNA because it is still induced by the DNA targeting sequence of the target polynucleotide (e.g., gRNA) as long as it retains the ability to interact with the Cas binding sequence of the target polynucleotide (e.g., gRNA).

[0090] Inactivation of the Cas endonuclease activity results in a catalytically inactivated Cas (dCas, e.g., dCas9). Since dCas can bind to DNA but cannot cleave it, it inhibits the transcription of target genes by creating a physical barrier to the action of transcription factors. This CRISPR effect works reversibly at the transcriptional level. This strategy is called CRISPR interference or CRISPRi. In CRISPR interference (CRISPRi), a dCas fusion protein (e.g., dCas fused to another protein or a portion thereof) can also be used in the methods of the present disclosure. In some embodiments, dCas is fused to a (transcription) repressor domain or a transcriptional silencer. Non-limiting examples of transcriptional repression domains include the Kruppel-associated box (KRAB) domain, the ERF repressor domain (ERD), the mSin3A interaction domain (SID) domain, a concatamer of SID (e.g., SID4X), or homologs thereof. A non-limiting example of a transcriptional silencer is heterochromatin protein 1 (HP1). CRISPRi may be modified by fusing Cas (e.g., dCas) to the Kruppel-associated box repression domain (KRAB) that enhances the inhibitory effect of Cas. Gilbert et al., 2013. Cell 154(2):442-51.

[0091] The second-generation CRISPRi strongly suppresses via the PUF-KRAB repressor. PUF proteins (so named due to their resemblance to Drosophila Pumilio and C. elegans fem-3 binding factor) are known to be involved in the regulation of mRNA stability and translation. These proteins contain a unique RNA-binding domain known as the PUF domain. RNA-binding PUF domains (such as the domain of the human Pumilio 1 protein, also referred to herein as PUM) contain eight repeated sequences that bind to consecutive bases in an antiparallel manner (each repeat is referred to as a PUF motif or PUF repeat), and each repeat recognizes a single base respectively; that is, PUF repeats R1-R8 recognize nucleotides N8-N1 respectively. For example, PUM consists of eight tandem repeats, and each repeat is composed of 34 amino acids that fold into a high-density domain consisting of an alpha helix. PUF and its derivatives or functional variants are programmable RNA-binding domains that can be utilized in the present methods and systems, and can be used as part of a PUF domain fusion that directs an effector domain to a specific PUF-binding sequence on a target polynucleotide (e.g., gRNA).

[0092] The present method may utilize CRISPR deletion (CRISPRd). CRISPRd takes advantage of the tendency of DNA repair strategies to direct towards non-homologous end joining (NHEJ) by default, and does not require a donor template for the repair of cleaved strands. Instead, Cas first introduces a double-strand break (DSB) into the gene containing the mutation, and then non-homologous end joining occurs; insertions and / or deletions (INDELs) are introduced, damaging the sequence, thereby inhibiting gene expression or preventing proper protein folding. This strategy can be particularly applicable to dominant pathologies where it is possible to fully restore the phenotype to wild type by disrupting the mutant dominant allele and retaining the intact wild-type allele.

[0093] In certain embodiments, the Cas enzyme may be a catalytically defective Cas (e.g., Cas9) or dCas, or a Cas nickase or nickase.

[0094] The Cas enzyme (e.g., Cas9) may be modified to function as a nickase (so named because it introduces a “nick” into DNA by inducing a single-strand break instead of a double-strand break). As used herein, the term “Cas nickase” or “nickase” means a Cas protein that can cleave only one strand of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas nickase may be any of the nickases disclosed in U.S. Patent No. 10,167,457 (the contents of this reference are hereby incorporated by reference in their entirety). In one embodiment, the Cas (e.g., Cas9) nickase has an active HNH nuclease domain and can cleave the non-target strand of DNA (i.e., the strand to which the gRNA is bound). In one embodiment, the Cas (e.g., Cas9) nickase has an inactive RuvC nuclease domain and cannot cleave the target strand of DNA (i.e., the strand that is the subject of base editing). In some embodiments, the Cas nickase cleaves the target strand of the double-stranded nucleic acid molecule, which means that the Cas nickase cleaves the strand that base pairs (is complementary to) the gRNA (e.g., sgRNA) bound to the Cas. In some embodiments, the Cas nickase cleaves the non-target strand of the double-stranded nucleic acid molecule that is not the subject of base editing, which means that the Cas nickase cleaves the strand that does not base pair with the gRNA (e.g., sgRNA) bound to the Cas. Other suitable Cas9 nickases will be apparent to those skilled in the art based on the present disclosure and the knowledge in the art; and they are included within the scope of the present invention.

[0095] In CRISPR activation (CRISPRa), dCas may be fused to an activation domain (such as VP64 or VPR). Such dCas fusion proteins may be used with the constructs described herein for gene activation. In some embodiments, dCas is fused to an epigenetic regulatory domain (such as a histone demethylase domain or a histone acetyltransferase domain). In some embodiments, dCas is fused to LSD1 or p300, or a portion thereof. In some embodiments, the dCas fusion is used for CRISPR-based epigenetic regulation. In some embodiments, dCas or Cas is fused to the Fokl nuclease domain. In some embodiments, Cas or dCas fused to the Fokl nuclease domain is utilized for genome editing. In some embodiments, Cas or dCas is fused to a fluorescent protein (e.g., GFP, RFP, mCherry, etc.). In some embodiments, the Cas / dCas protein fused to a fluorescent protein is used for labeling and / or visualizing genomic loci or identifying cells that express the Cas endonuclease. Generally, CRISPR / Cas proteins include at least one RNA recognition domain and / or RNA binding domain. The RNA recognition domain and / or RNA binding domain interacts with the guide RNA. CRISPR / Cas proteins can also include a nuclease domain (i.e., a DNase domain or an RNase domain), a DNA binding domain, a helicase domain, an RNAse domain, a protein-protein interaction domain, a dimerization domain, and other domains.

[0096] In addition to the CRISPR-Cas systems that have been characterized in detail, a novel CRISPR enzyme called Cpf1 (Cas protein 1 of subtype PreFran) can also be utilized in the present methods and systems (Zetsche et al., 2015, Cell). Cpf1 is a single RNA-guided endonuclease lacking tracrRNA and utilizes a T-rich protospacer adjacent motif. The authors demonstrated that Cpf1 has properties different from those of Cas9 and mediates potent DNA interference. Thus, in one embodiment of the present invention, the CRISPR-Cpf1 system can be utilized to cleave a desired region within a target gene.

[0097] In a further embodiment, the nuclease is a transcription activator-like effector nuclease (TALEN). TALENs contain a TAL effector domain that binds to a specific nucleotide sequence and an endonuclease domain that catalyzes double-strand break at the target site (International Application No. WO2011072246; Miller et al., 2011 Nat. Biotechnol. 29:143-148; Cermak et al., 2011 Nucleic Acid Res. 39:e82). Since sequence-specific endonucleases can inherently be modular, DNA binding specificity can be obtained by arranging one or more modules. Bibikova et al., 2001 Mol. Cell. Biol. 21:289-297; Boch et al., 2009 Science 326:1509-1512.

[0098] ZFNs can comprise two or more (e.g., 2-8, 3-6, 6-8 or more) array-specific DNA binding domains (e.g., zinc finger domains) fused to an effector endonuclease domain (e.g., the FokI endonuclease). Porteus et al., 2005 Nat. Biotechnol. 23:967-973; Kim et al., 2007 Proceedings of the National Academy of Sciences of USA, 93:1156-1160; U.S. Patent No. 6,824,978; International Publication Nos. WO1995 / 09233 and WO1994018313.

[0099] In one embodiment, the nuclease is a site-specific nuclease selected from the group consisting of omega, zinc finger, TALEN, and CRISPR / Cas, or selected from that group.

[0100] The sequence-specific endonucleases of the methods and compositions described herein can be engineered from any organism, can be chimeric, or can be isolated. Endonucleases can be engineered, for example, by mutagenesis, to recognize a particular DNA sequence. Seligman et al., 2002 Nucleic Acids Research 30:3870-3879. Combinatorial assembly is a method capable of associating or fusing protein subunits from different enzymes. Arnould et al., 2006 Journal of Molecular Biology 355:443-458. In certain embodiments, by combining these two approaches (mutagenesis and combinatorial assembly), an endonuclease engineered to recognize a desired DNA sequence can be created.

[0101] The sequence-specific nuclease can be introduced into cells in the form of a protein or in the form of a nucleic acid (such as mRNA or cDNA) encoding the sequence-specific nuclease. The nucleic acid can be introduced as part of a larger construct (such as a plasmid or viral vector) or directly, for example, by electroporation, lipid vesicles, viral transporters, microinjection, and gene gun. Similarly, the construct containing one or more transgenes can be introduced by a method suitable for cell introduction of the nucleic acid.

[0102] The guide RNA used in the method of the present disclosure can be designed to bind the Cas-gRNA complex to a predetermined cleavage site in the genome by induction of the guide RNA. In one embodiment, the cleavage site may be selected to excise a fragment or sequence containing the region of the frameshift mutation. In a further embodiment, the cleavage site may be selected to excise a fragment or sequence containing the supernumerary chromosome.

[0103] Regarding Cas family enzymes (such as Cas9) that bind well to DNA, the target sequence in genomic DNA can be complementary to the gRNA sequence, and a correct protospacer adjacent motif or "PAM" sequence may be located immediately downstream thereof. "Complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either of the conventional Watson-Crick type or other non-conventional type. The percentage of complementarity is the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence. Perfect complementarity is not necessarily required as long as there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex. The target sequence can include any polynucleotide (such as a DNA or RNA polynucleotide). For the Cas9 protein, mismatches located distal to the PAM are acceptable. Depending on the bacterial species from which Cas9 is derived, the PAM sequence varies. The most widely used CRISPR system is derived from Streptococcus pyogenes, and its PAM sequence is NGG located immediately at the 3' end of the sgRNA recognition sequence. Examples of PAM sequences of CRISPR systems derived from exemplary bacterial species include Streptococcus pyogenes (NGG), Neisseria meningitidis (NNNNGATT), Thermophilus (NNAGAA), and Treponema denticola (NAAAAC).

[0104] The gRNA used in the present disclosure has a length of about 5 to 100 nucleotides, or a length greater than that (the length being, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides or more). In one embodiment, the gRNA has a length of about 15 to about 30 nucleotides (the length being, for example, about 15 to 29, 15 to 26, 15 to 25; 16 to 30, 16 to 29, 16 to 26, 16 to 25, or about 18 to 30, 18 to 29, 18 to 26, or 18 to 25 nucleotides).

[0105] Many computational tools have been developed to assist in the design of gRNA (see Prykhozhij et al., 2015 PLoS ONE 10(3); Zhu et al., 2014 PLoS ONE 9(9); Xia et al., 2014 Bioinformatics. Jan 21(2014)); Heigwer et al., 2014 Nat Methods 11(2):122 - 123). Methods and tools for guide RNA design are described in detail in Zhu 2015 Frontiers in Biology 10(4):289 - 296, which is incorporated herein by reference. There are also publicly available software tools that can be used to assist in the design of gRNA (http: / / www.genscript.com / gRNA - design - tool.html).

[0106] Human Immune System (HIS) mouse The availability of NOD-scid common gamma chain-deficient (NSG) mice, which lack mouse T cells, B cells, and NK cells and are severely immunodeficient, has greatly increased the possibility of creating human immune system (HIS) mice. One of the major requirements for creating HIS mice with optimal immune function is the availability of human thymic tissue. Human fetal thymic tissue aids in the stable proliferation of human thymocytes derived from injected fetal or adult CD34+ cells; the injected cells maintain a stable supply of T cell progenitors to the thymus, and in the bone marrow, B cells, DCs, and monocytes are generated, and these generated cells are distributed peripherally and function as antigen-presenting cells (APCs) to T cells developing within the human fetal thymic graft (Lan et al., 2004; Lan et al., 2006; Melkus et al., 2006). T cells generated de novo in the human thymic graft are tolerant to the murine host, presumably as a result of elimination by detectable mouse APCs in the graft (Kalscheuer et al., 1999). The natural mouse thymus can generate human T cells at low levels, but due to the abnormal structure of the mouse thymus, normal negative selection does not occur (Khosravi Maharlooei et al., 2019). This, combined with slow peripheral T cell reconstitution and the resulting highly lymphopenia-induced proliferation (LIP), causes severe autoimmune syndromes that can be prevented by removing the natural mouse thymus (Khosravi Maharlooei et al., 2019). In contrast, transplantation of human fetal thymic tissue into HIS mice that receive CD34+ hematopoietic stem cell / progenitor cells (HSPCs) results in a human thymus with a normal structure containing easily distinguishable cortex, medulla, and Hassall's corpuscles. In this human thymus, relatively rapid reconstitution of naive human T cells occurs peripherally, resulting in a significant reduction in LIP and less autoimmunity compared to that seen in T cells developing in the natural NSG mouse thymus.

[0107] Considering the availability and issues in the use of human fetal tissues, it would be desirable to find other sources of thymic tissue that can function similarly to those derived from human fetuses. The inventors have previously shown that stable proliferation of human thymocytes occurs in porcine thymic grafts transplanted into immunodeficient mice receiving human HSPCs (Nikolic et al., 1999; Shimizu et al., 2008; Kalscheuer et al., 2014). The use of porcine fetal thymic tissue provides an alternative to human fetal thymic tissue for generating normal and functional human T cells (including Tregs) with a broad TCR repertoire. However, as suggested by the responses to immunization and as shown by the finding that positive selection of thymocytes expressing HLA-restricted transgenic TCR20 is defective in porcine thymus (Figures 6 and 8), the selection of human T cells that mediate optimal HLA-restricted immune functions peripherally seems to be limited because there are no HLA molecules on porcine thymic epithelial cells (TECs). Furthermore, the positive selection ability of HLA-restricted Tregs that recognize human tissue-specific self-antigens (TRA) produced by TECs, and the negative selection of effector T cells that recognize these TRA / HLA complexes also seem to be limited in porcine thymus. Compared to human fetal thymus, peripheral human T cells generated in pigs show slight impairment in HLA-restricted immune functions and homeostasis as well as immune tolerance to tissue-specific self-antigens (Kalscheuer et al., 2012). Most of these limitations could be overcome by conferring transgenic HLA molecules on porcine thymic tissue.

[0108] Two improved methods for obtaining HIS mice that do not rely on the use of human fetal tissues are presented herein.

[0109] In one embodiment, a HIS mouse is generated by introducing porcine fetal thymic tissue and human CD34+ cells into a mouse. In some embodiments, the human CD34+ cells are derived from umbilical cord blood. In some embodiments, the human CD34+ cells are derived from adult tissue. In some embodiments, the adult tissue is bone marrow. In some embodiments, the CD34+ cells are derived from mobilized peripheral blood hematopoietic stem cells.

[0110] In a further embodiment, a HIS mouse is generated by introducing fetal thymic tissue derived from the transgenic pigs described herein.

[0111] In some embodiments, as recently reported (Khosravi Maharlooei et al., 2019), the thymus of the mouse is removed prior to thymic tissue introduction. In some embodiments, the mouse is also irradiated. In some embodiments, the mouse is a NOD scid common gamma chain knockout (NSG) mouse.

[0112] Porcine fetal thymus can be transplanted under the renal capsule of the mouse. When injecting human umbilical cord blood-derived CD34+ cells into the mouse, the cells can be injected before, after, or simultaneously with thymic transplantation.

[0113] The HIS mouse model can be widely applied in research areas where T cells play an important role. Such areas include, but are not limited to, the following:

[0114] · HIV infection and other infections. This model has been used to demonstrate that porcine thymus confers resistance to HIV infection compared to human fetal thymic tissue (Hongo et al., 2007).

[0115] ·Biology of Tregs (including development in the thymus, transport and homeostasis in peripheral tissues). When Tregs are generated in porcine thymus, their development and function are good, but there are subtle differences in phenotypes due to different peripheral homeostasis, which is shown by the use of this model to be potentially improved by conferring HLA molecules on thymus tissue. Furthermore, this model enables the determination of the distribution, viability and activity of ex vivo-expanded Tregs after injection (e.g., the activity to suppress graft rejection), so this model will be useful for the study of Treg therapy.

[0116] ·Transplantation immunity. HIS mice created with human fetal thymus tissue or porcine fetal thymus tissue and human fetal or adult CD34+ cells have been shown to be able to reject human and porcine skin and islet allografts and xenografts (Lan et al., 2004; Shimizu et al., 2008; Zhao et al., 1997; Zhao et al., 1998). On the other hand, those created with porcine fetal thymus tissue specifically tolerate skin grafts sharing the SLA of the thymus donor (Kalscheuer et al., 2014). The mice created by the methods described herein can also be used to reject allogeneic human skin grafts. The above data suggest that this model will be beneficial for preclinical studies to investigate transplantation immunity and approaches to induce immune tolerance to allografts and xenografts. Also, approaches using mixed chimeras and porcine thymus-thymus transplantation for xenograft immune tolerance, which are currently under investigation, will be optimized by this model.

[0117] ·Autoimmunity. Transduction of CD34+ cells with TCRs that recognize islet autoantigens will facilitate studies using this model on how the development of autoreactive T cells in the thymus and immune tolerance to autoantigens in both the thymus and periphery are controlled. This well-defined model with a highly reproducible thymic HLA genotype allows easy study of additional TCRs specific for autoantigens.

[0118] ·Infectious diseases such as COVID-19. There is a great need for models containing the human immune system to investigate the effects of the human immune system on the pathology of COVID-19. The unavailability of human fetal tissues has been a major challenge for such research. By using HLA-transgenic fetal pig thymus tissues as an alternative to human fetal thymus, this problem could be overcome.

[0119] By using transgenic pigs in the production of HIS mice, a better model than HIS mice produced using human fetal thymus can be obtained. The reason is that the MHC (SLA) and HLA transgenes underlying each donor are the same, and the pigs are fairly inbred as a whole. One of the difficulties in using human fetal tissues is that the HLA and the overall genetic background vary from donor to donor, which becomes a variable that hinders the reproducibility of HIS mouse research.

[0120] Examples The present invention will be more fully understood from the details of the experiments described below. However, it will be readily understood by those skilled in the art that the specific methods and results discussed therein are merely illustrative of the invention as more particularly described in the claims that follow the examples.

[0121] Example 1: Genetic modification of pigs using CRISPR-assisted homologous recombination Two types of pig genetic modifications were created for the purpose of specifically showing that gene modification in pigs is possible by CRISPR-assisted homologous recombination when combined with an appropriate selection strategy for cells in which accurate targeting has been achieved.

[0122] In the first modification, the coding sequences of four human genes were introduced into the GGTA1 locus of the Sachs minipig using CRISPR-assisted homologous recombination (Figure 1). In this case, targeted integration into the GGTA1 locus was a "safe harbor" with respect to transgene expression because this genomic region is not subject to tight temporal or lineage-dependent transcriptional repression. The 2A self-splicing element was used to express the four transgenes in two groups from the ubiquitous CAG promoter. Clonal selection of cells in which accurate targeting had been achieved was straightforward in this case, which means that transgene expression could be used as a positive marker and cells heterozygous for the null GGTA1 allele were transformed with the vector (loss of GGTA1 expression). Population-based rapid cell selection yielded an efficient somatic cell nuclear transfer (SCNT) donor population for the production of cloned fetuses and piglets.

[0123] Next, the second modification was introduced into the fibroblasts obtained from the cloned fetuses with the first modification, which was rather complex. In this case, sequences encoding both chains of the human IL-3 receptor under the control of the native IL-3 receptor alpha-chain promoter needed to be introduced to achieve appropriate lineage and temporal specificity of human IL3R expression. The major obstacle to the selection of target-achieved cells in this case was the lack of IL3R expression in the fibroblasts required for SCNT cloning. Furthermore, the disruptive loss of endogenous ILR3 expression due to targeted integration of indels was expected to be highly deleterious, if not lethal, so gene modification was limited to only one allele of the native ILRa locus. From the perspective of cloning, it was desirable to obtain a non-clonal donor cell population in which cells with accurate targeting were sufficiently enriched while minimizing population doubling as much as possible.

[0124] The strategy and results of this experiment are shown in Figure 2.

[0125] Since a highly enriched SCNT donor cell population with minimal doubling was required, a vector without a selection marker promoter was used. Since fibroblasts do not express IL3Ra, we decided to investigate whether the biased expression of neighboring genes (SLC25A6, mitochondrial nucleotide transporter) could be used as a marker for proper target achievement. By tagging the SLC25A6 transcription unit using a GFP coding sequence linked via a 2A self-cleaving peptide, a highly reliable selection strategy is provided, but it was unclear whether such complex modifications (replacing a genomic sequence of more than 15 kbp with a vector sequence of more than 7 kbp) could be achieved with sufficiently efficient donor cell selection.

[0126] CRISPR guide RNAs predicted to cleave one allele of the IL3Ra gene in already modified fetal cells were selected and tested with the exemplified vector. In preliminary transformation, it was revealed that when paired guide RNAs were used in combination with "nickase"-type Cas9, a population containing fairly discrete high-GFP and low-GFP subpopulations was obtained. Flow analysis of a population obtained with one such combination is shown in Figure 2B. PCR analysis showed that cells in the sorted high-GFP subpopulation included cells with proper integration at both ends of the vector (Figure 2C). When cells from this population were used for SCNT at approximately 24 doublings (well before the average clonal senescence at 32 doublings), 8 live fetuses were obtained from 3 nulliparous embryo recipient pigs. Genome analysis and RT-PCR analysis revealed that all 8 fetuses had the intended genetic modification (Figures 2D and 2E). Another pregnancy using this donor cell population was continued until birth, and live offspring expressing the transgene were obtained.

[0127] Collectively, the above modifications demonstrated that multi-cistronic target modifications can be sequentially introduced into pigs using a non-clonal donor cell selection strategy for rapidly generating pigs with multiple genetic modifications.

[0128] Example 2: HLA-A2 Transduction: Generation of d40 Transgenic Pig Fetuses and Genotype / Phenotype Evaluation Starting Material Fibroblasts from GGTA1 null, SLA haplotype h homozygous Sachs minipigs (SLA-l*02:01, SLA-2*02:01, SLA-3 null, SLA-DRA*01:01:02, SLA-DRB*02:01, SLA-DQA*02:02:01, SLA-DQB*04:01:01) were used as starting material for genetic modification. In previous transgenic projects, cells of this strain were successfully cloned, and a large breeding colony was maintained by CCTI for xenotransplantation research. This will facilitate the expansion of HLA gene transfer research to supply thymic tissue to the research community. Due to the characteristic that this animal is partially inbred, the offspring will have a very high genetic similarity.

[0129] Overall Strategy All transgenic modifications are made by targeted insertion behind the native SLA promoter. This ensures the appropriate lineage and temporal expression pattern. Potential problems associated with inappropriate HLA expression in the placenta during development are also avoided by this. Both chains of the transgenic molecule are introduced simultaneously. Sequential modification is used at the fetal stage to rapidly generate first HLA-A2 transgenic thymic material and then HLA-A2 / HLA-DQ8 transgenic thymic material.

[0130] To introduce both HLA modifications, a promoterless gene targeting vector is used, which minimizes the number of cell divisions before use in somatic cell nuclear transfer (SCNT) and allows selection of a highly enriched population of non-cloned cells for properly targeted cells. This is similar to the promoter targeting modification approach used in Example 1 with the IL3 receptor chain, but the process of vector design is considerably simplified because both class I and class II molecules are expressed normally or inducibly in the fibroblasts required for SCNT cloning.

[0131] Generation of d40 Cloned Transgenic Fetuses The coding sequence of HLA-A2 is introduced downstream of either the SLA-1 promoter or the SLA-2 class I promoter. These loci are alternative to each other with respect to the intended modification, and the choice between them is determined based on sequencing of intron 1 of both and evaluation of optimal CRISPR guide RNA sites.

[0132] HLA-A2 is expressed as a fusion of human beta2-microglobulin (B2M) and the HLA-A2 alpha chain. There have been previous reports in mice regarding transgenic expression of such fusions (Kotsiou et al., 2011; Pascolo et al., 1997), and by using it for the purposes herein, it is ensured that the heterotypic interaction between HLA-A2 and porcine B2m does not interfere with surface expression of HLA-A2.

[0133] CRISPR / Cas9-assisted homologous recombination is used for targeting the fusion cassette. Targeting of HLA-A2 is limited to one allele of the SLA I gene; the other allele may be in a null state; although mutation of the second allele is not thought to affect porcine immunity, HLA-A2 expression may increase due to reduced expression of the endogenous class I alpha chain.

[0134] Vector Construction for HLA-A2 Integration Regarding the target vector for HLA-A2 integration, a schematic diagram is shown in Figure 3. The human B2M-HLA-A2 cassette is introduced at the intron 1 / exon 2 junction by homologous recombination between the homologous arms of the vector, which have the same sequence as that of the natural gene (white and blue segments). Mature human B2M is introduced at this site, which is accompanied by the signal peptide provided by exon 1; this signal peptide ends 1 bp from the splice site, so a fusion protein is made without causing a change in the B2M protein sequence. A paired CRISPR guide RNA is selected for an appropriate sequence site near the end of intron 1 and the beginning of exon 2 and inserted into a plasmid expressing Cas9 nickase activity.

[0135] Selection of Modified Fibroblasts for SCNT The target plasmid and the CRISPR / Cas9 guide plasmid are introduced into fibroblasts by nucleofection and subjected to the first selection 3 to 5 days later. The selection is by flow sorting of cells stained with an HLA-A2 specific antibody (clone BB7.2, Biolegend). A preliminary single-sort analysis is performed with the selected guide pair to determine the pair with the highest targeting efficiency based on HLA-A2 expression. For SCNT donor cell selection, two similar selections are used to maximize the enrichment of expressing cells. This population is then subjected to genomic analysis and RT-PCR analysis to confirm whether the transgenic locus has the expected structure and whether there is expression at the RNA level, and to determine whether the second SLA locus has changed in this process.

[0136] Generation and Characterization of d40 Transgenic Fetuses Select SCNT donor cells are used for nuclear transfer / embryo transfer, and the resulting fetuses are recovered at approximately 40 days of gestation. In any case in the porcine engineering project, a two-step cloning process is used. By recovering at 40 days of gestation, confirmation of gene structure and, in many cases, confirmation of transgene expression can be performed at the clone level before determining the line for further cloning of the clone. Furthermore, cells that have undergone minimal culturing from early fetuses tend to have a significantly higher cloning efficiency than those after expansion culture in an in vitro selection process. Finally, this enables "renewal" of the line regarding the in vitro survival period essential for additive genetic modification (for example, sequential introduction of HLADQ8).

[0137] For the characterization of HLA-A2 transgenic fetuses, genomic PCR is used to confirm the structure of the expected integration site, RT-PCR is used to confirm proper RNA expression, and flow cytometry analysis is used to confirm surface expression.

[0138] Example 3: HLA-A2 / HLA-DQ8 transduction: Generation of d40 transgenic porcine fetuses and genotyping / phenotyping evaluation Using the same overall strategy and targeted expression with a promoter-free vector targeting the natural promoter, transgenic pigs (HLA-A2 / HLA-DQ8) are produced by performing cell selection based on HLA-DQ8 expression described in Example 2. In contrast to SLA class I, SLA class II is not normally expressed on fibroblasts. To determine whether induction of class II expression by interferon gamma, as seen in human and mouse fibroblasts, is possible in porcine fetal fibroblasts, primary fetal fibroblasts were exposed to porcine IFN-γ (80 ng / ml), and then porcine DR and DQ pan-allele surface expression was observed by flow cytometry. After 6 days of treatment with IFN-γ, it was revealed that surface expression of both DR and DQ was strongly induced in almost all cells (Figure 4); most cells highly expressed both after 3 days of induction. Importantly, such treatment did not seem to have any effect on the morphology or proliferation of these cells. Therefore, inducible expression of class II is a viable option for transgenic HLA-DQ8 expression by the natural class II promoter in cells required for SCNT cloning.

[0139] Proper class II expression depends on the function of accessory molecules (including CD74; also HLA-DM in humans). From the expression of HLA-DQ8 in transgenic mice, pigs are also likely to have all the appropriate activities regarding HLA-DQ8 expression (Cheng et al., 1996). Mouse studies have suggested that expression of endogenous MHC-II molecules can limit exogenous MHC-II expression, probably by competition. HLA-DQ8 expression targets the natural SLA-DQA locus. This targeting event itself abolishes the function of one SLA-DQA allele. Due to the nature of CRISPR-mediated modification, loss-of-function related to indels occurs similarly in most cells to non-target alleles.

[0140] Vector construction A schematic diagram of the targeting vector for HLA-DQ8 integration is shown in Figure 5.

[0141] For HLA-A2 transfection, both the alpha and beta chains are introduced in a single transfection step. For DQ8, the coding sequences of the two chains are ligated to the highly efficient IRES element that has been well used in other bicistronic expression vectors. Since the functional impact of the amino acid addition to the HLA-DQ alpha chain is unknown, in this case, it is preferable that the ligation to the IRES is a ligation to a self-splicing element. Similar to the addition in HLA-A2, exon 1 of the native locus is used to confer a leader sequence in HLA-DQ8 as well, resulting in the addition of a single amino acid at the N-terminus.

[0142] Selection of Modified Fibroblasts for SCNT The HLA-A2 transgenic d40 fetal cells created as described in Example 2 serve as the starting material for the modified introduction of HLA-DQ8. Preliminary SCNT donor cell transformation is carried out by the method described in Example 2. Numerous anti-pan haplotype human DQ antibodies are commercially available. First, IFN-g-induced porcine fibroblasts are screened to obtain candidate cells that do not bind to porcine DQ dimers for the purpose of discrimination. Next, a second screening is performed on these candidates, which is carried out using IFN-g-induced porcine fibroblasts individually transformed with the expression constructs of HLA-DQA*03:01 and HLA-DQB1*03:02 for the purpose of removing antibodies that recognize interspecies dimers. The selection of candidate cells that meet these criteria is carried out by the method described in Example 2. Similar to Example 2, this flow sorted population is also subjected to genomic analysis and RT-PCR analysis to confirm the expected structure and RNA expression of the transgenic locus.

[0143] Generation and Characterization of d40 Transgenic Fetuses Genomic analysis and RNA analysis are carried out in the same manner as in the case of HLA-A2 modification described in Example 2.

[0144] Example 4: Preparation of d56-70 Thymic Tissue Expressing HLA-A2 and HLA-A2 / HLA-DQ8 Early fetal cell lines created by the methods described in Examples 2 and 3 and confirmed by genotype and phenotype were transported to a facility equipped with laboratories for cell culture, oocyte maturation, and embryo reconstruction, and also to a surgical facility for embryo transfer and the delivery of fetuses and piglets. SCNT cloning was performed to obtain fetuses on days 56 - 70. After genotyping and phenotyping of the fetal conformation, thymus isolation was performed by methods known in the art.

[0145] Example 5: Reproduction of HLA - A2 / HLA - DQ8 Transgenic Founder Boar For SCNT to produce founder boars, d40 fetal cells that have been prepared as described in Examples 2 and 3 and confirmed for genotype / phenotype are utilized. Transgenic piglets are raised until they reach the transferable age (8 - 16 weeks) and then transferred to a state - of - the - art livestock agricultural facility for large - scale animal breeding, rearing, and further livestock handling.

[0146] Example 6: Importance of HLA Sharing between Thymus and Peripheral APCs for Human T - Cell Homeostasis in HIS Mice Method Six - to - eight - week - old female NOD scid common gamma chain knockout (NSG) mice purchased from Jackson Laboratories were thymectomized by a previously reported method (Khosravi Maharlooei et al., 2019). Two weeks later, these mice were subjected to sublethal total body irradiation (1 Gy), and then 1 - mm 3 fragments of porcine fetal thymus tissue or human thymus tissue were surgically transplanted under the renal capsule.

[0147] Next, mixed chimeric donor HIS mice were created by transplanting two types of allogeneic CD34+ cells that do not share HLA (#1 and #2) and the autologous fetal thymus of donor #1 into thymectomized NSG mice. Two groups of adoptive recipient (AR) mice were created by injecting CD34+ cell #1 or #2 into thymectomized NSG mice (athymic). Twenty weeks after transplantation, the T cells of the mixed chimeras were intravenously injected into AR1 and AR2 mice. See Figure 6A.

[0148] Results On the 10th day after adoptive immunotransfer, in AR1 mice where the APC was HLA-autologous to the donor thymus with T cell selection, the proportion of proliferating (Ki67+) T cells was significantly higher than that in AR2 mice containing only allogeneic HLA. See Figure 6B.

[0149] These studies indicate that thymic HLA is required on peripheral APCs to maximize lymphopenia-inducing properties that support the proliferation of peripheral human T cells, emphasizing the importance of studies to endow porcine thymus with human thymic epithelial cells or HLA molecules to achieve normal immune homeostasis.

[0150] Example 7: Comparison of Human Immunoreconstitution in HIS Mice Methods Humanized mice were generated by transplanting porcine fetal thymus under the renal capsule of thymectomized and irradiated NOD scid common γ-chain knockout (NSG) mice by the method described in Example 6.

[0151] Next, these mice were injected with human umbilical cord blood-derived CD34+ cells. Two batches of humanized mice were generated using the same porcine fetal thymus and different umbilical cord blood CD34+ cells. CD34+ cells were isolated using a human CD34 MicroBead Kit (Miltenyi Biotech). To prevent rejection reactions caused by existing human thymocytes from the graft, i.e., rejection reactions against porcine thymus tissue and / or allogeneic human umbilical cord blood CD34+ cells injected, residual T cells in the CD34+ cell inoculum were depleted, and residual thymocytes released from human fetal thymus tissue were depleted. For this purpose, anti-CD2 mAb LoCD2b (400 μg / mouse) was intraperitoneally injected once a week for two weeks (days 0, 7, and 14).

[0152] The reconstitution of humanized mice generated in different experiments using human fetal thymus tissue and autologous fetal liver-derived CD34+ cells was included for comparison.

[0153] Peripheral blood collection from the mice was started at week 4, and the human CD3 cell concentration in the blood was measured.

[0154] Flow cytometric analysis of peripheral blood was performed at week 15 to examine the number of cell populations; the cell populations were as follows: CD4 and CD8 T cells, naive and memory CD4 and CD8 T cells, regulatory T cells (Tregs) and follicular helper T cells (Tfhs); T cell, B cell, and myeloid cell populations including B cell subsets, monocytes, and dendritic cells (DCs) (including classical DCs (cDC1 and cDC2) and plasmacytoid DCs (pDCs)).

[0155] Results As shown in Figure 7A, based on human cells in the peripheral blood of the mice, human T cell reconstitution in the two batches of mice generated with porcine fetal thymus and human CD34+ cells was comparable to that generated with human fetal thymus.

[0156] As shown in Fig. 7B, it was found by high-low detection that naive T cells were present at a high percentage (%) in the CD4 and CD8 subsets. CD4+CD25 high CD127 low The generation of regulatory T cells was also demonstrated.

[0157] Example 8: Continuous monitoring and analysis of HIS mice The mice prepared by the method described in Example 7 are further monitored as follows.

[0158] Plasma immunoglobulin (IgM and IgG) levels are tracked and compared by ELISA every 4 weeks after transplantation.

[0159] When it is expected that the reconstitution of human cells in HIS mice is completed at 14 to 16 weeks after transplantation, half of the animals in each group are euthanized and the whole group is compared for tissue size, tissue structure, cellularity, and cell populations in peripheral blood, lymph nodes, spleen, and thymus. The flow cytometry panel for examining immune cell populations is shown in Table 1. Histological tests are performed using small pieces of each lymphoid tissue (including spleen, lymph nodes, and thymus), and the structures of these tissues are compared. For all HIS mice, serum immunoglobulin (IgM and IgG) levels are measured by ELISA. Furthermore, in vitro assays of proliferation, cytokine production, and cytotoxicity in response to pan-TCR stimulation (anti-CD3 / CD28 beads), alloantigen stimulation, heterologous antigen stimulation, and tetanus toxoid neoantigen stimulation are used to compare the functions of human T cells in the periphery of each group of mice. Proliferation is determined by CFSE intracellular dye dilution. Cytokine production, including IL-2 and IFN-γ, is assayed by intracellular staining. For alloantigen and heterologous antigen stimulation, allogeneic human PBMC and third-party porcine PBMC are used as stimulators. Isolated splenic T cells from HIS mice are labeled with CFSE and co-cultured for 6 days at a 1:1 ratio with irradiated stimulators. CFSE dilution of human CD4 and CD8 T cells is determined by flow cytometry. For tetanus toxoid neoantigen stimulation, DCs are generated using cord blood-derived or fetal liver-derived CD34+ cells used for HIS mouse generation. To differentiate into dendritic cells, CD34+ cells are cultured for 13 days in the presence of human cytokines (including stem cell factor, GM-CSF, and IL-4). CD34-derived DCs are pulsed with tetanus toxoid neoantigen and then matured with TNF-α and PGE2, and then co-cultured with CFSE-labeled isolated splenic T cells for 7 days. Proliferating T cells are determined by flow cytometry. Monocytes are stimulated with LPS, and TNF-α, IL-6, and IL-10 production in the supernatant is determined by ELISA.

[0160] Monitor the remaining HIS mice until week 30 at most to examine the persistence of the reconstruction of each lineage and also examine the onset of graft-versus-host disease / autoimmune diseases. Collect blood from the mice every 4 weeks to determine human cell engraftment. Evaluate the mice for graft-versus-host disease / autoimmunity starting from week 20 after transplantation, and evaluate them twice a week until week 30 using the following evaluation system. Conduct all analyses in the same manner as described above.

[0161] Evaluation system: Weight loss (%): <10%, 0; <10 - 15%, 1; <15 - 20%, 2; >20%, 3 Posture: Normal, 0; Slight kyphosis at rest, 1; Moderate kyphosis, can walk around normally, 2; Severe kyphosis, reduced movement and walking, 3 Coat condition: Normal, 0; Slight coat roughness, 1; Moderate coat roughness, 2; Severe coat roughness, soiling by porphyrin on the head or forelimbs, 3 Activity: Normal, 0; Slight to moderate reduction, 1; Active only during feeding or drinking or stimulation, 2; Difficulty getting up, unable to move when stimulated, 3

[0162] Animals with any signs of GVHD (score of 2 or more) are monitored daily and weighed every other day. Animals with a total score of 6 or more are monitored daily and weighed daily. Animals with a total score of 9 or more, or animals with a score of 3 in any one category, are euthanized.

[0163] In these tests, human reconstitution after transplantation of fetal porcine thymus and cord blood-derived CD34+ cells is compared with human reconstitution after transplantation of human fetal thymus and fetal CD34+ cells. The results indicate that human reconstitution in HIS mice generated with fetal porcine thymus and cord blood-derived CD34+ cells is similar to human reconstitution in HIS mice generated with human fetal thymus and fetal CD34+ cells. Once human cell reconstitution in peripheral blood is confirmed (about 4 months after transplantation), the test is initiated, and the in vivo immune function of these mice is examined by determining thymic selection of a transgenic human T cell receptor (TCR) with specific constraints and rejection of human allogeneic skin grafts as follows.

[0164]

Table 1

[0165] Example 9: Comparison of Selection of HLA-A2 Restricted TCR in HIS Mice In thymectomized NSG mice, the selection of HLA-A2 restricted TCR when reconstituted from cord blood CD34+ cells is compared between fetal thymus tissue defined by SLA and HLA-A2+ human fetal thymus tissue. When lentiviral transduction is used for human CD34+ cells in HU / HU mice, it has been revealed that positive selection of the human HLA-A2 restricted TCR MARTI occurs in HLA-A2+ human thymus but not in SLAkm porcine thymus (Figure 8). This test showed that this TCR is also not positively selected in homozygous SLAhh fetal porcine thymus, because this is the porcine SLA used for HLA transgene introduction in the transgenic pigs of Examples 2 and 3.

[0166] Using the method outlined in Example 7, three groups of mice are generated using porcine fetal thymus (SLAhh) or human fetal thymus and MART-1-TCR-transduced fetal liver-derived or cord blood-derived CD34+ cells (Table 2). For transduction of CD34+ cells, RetroNectin-coated plates are used and human fetal liver-derived or cord blood CD34+ cells are pre-stimulated by incubating for 3 hours each in Stemline II medium containing 10 μg / mL protamine sulfate and 60 ng / mL, 150 ng / mL, and 300 ng / mL of recombinant human IL-3, Flt3 ligand, and stem cell factor. After transduction of the cells overnight at a multiplicity of infection of 30, the cells are harvested and prepared for intratibial injection. A small number of transduced CD34+ cells are cultured for 4 days in stem cell medium without protamine sulfate, and then the transduction efficiency is evaluated by flow cytometry. For optimal homeostasis of human T cells selected by HLA-A2, HLA-A2+ fetal liver CD34+ cells or cord blood CD34+ cells are used for generation of HIS mice. For HLA typing, after isolation of CD34+ cells from CD34-negative fetal liver or cord blood cells, DNA is extracted using the DNeasy Blood & Tissue Kit (Qiagen). HLA typing at the allele level by the Sanger method is performed to determine the HLA type of the tissue. During the tissue typing assay, human fetal CD34+ cells and cord blood CD34+ cells are frozen.

[0167] Fourteen to sixteen weeks after transplantation, after human cell reconstitution is complete in HIS mice, the mice are euthanized and analyzed. The percentage and absolute number of MART-1+ thymocytes in the double-negative (CDla+)(including CD7+ early thymocytes), double-positive, CD4 single-positive, and CD8 single-positive subsets are examined with selection markers (CD69, PD1, CCR7). Positive selection deficiency of HLA class I-restricted TCR MART1 is observed in porcine fetal thymus.

[0168] To identify transgenic T cells, fluorescent dye-labeled MART1 tetramers are used, while GFP is utilized as a marker representing the origin of gene-introduced HSPC. By GFP+ and GFP− thymocytes at each stage of thymic development, an internally consistent comparison regarding the selection levels of transgenic T cells and non-transgenic T cells in each mouse individual can be obtained. These tests conducted during the production of transgenic pigs (Examples 3 and 4) provide a baseline that serves as a criterion when evaluating the effect of the HLA-A2 transgene in porcine fetal thymus on HLA-A2-restricted human T cell selection in porcine thymus. The detailed panel is shown in Table 3 below. The analysis is performed by Aurora spectral flow cytometry.

[0169]

Table 2

[0170]

Table 3

[0171] For HLA typing, after isolating CD34+ cells from CD34-negative fetal liver or umbilical cord blood cells, DNA is extracted using the DNeasy Blood & Tissue Kit (Qiagen). HLA typing at the allele level is performed by the Sanger method to determine the HLA type of the tissue. During the tissue typing test, human fetal CD34+ cells and umbilical cord blood CD34+ cells are frozen.

[0172] At 14 - 16 weeks after transplantation, after human cell reconstitution is completed in HIS mice, the mice are euthanized for analysis. The percentage and absolute number of clone 5+ thymocytes in double-negative (CD1a+)(including CD7+ early thymocytes), CD69+ and CD69- double-positive, CD4 single-positive and CD8 single-positive subsets are examined with negative selection markers (PD1, CCR7). In HLA-DQ8+ thymus, to detect the Treg lineage differentiation of thymocytes with this TCR, the analysis also includes Treg markers (CD25 and CD127). The detailed panel is shown in Table 5 below. The analysis is performed by Aurora spectral flow cytometry.

[0173] Since the insulin peptide recognized by this TCR is expected to be produced by medullary thymic epithelial cells (mTEC), the positive selection of this TCR depends on HLA-DQ8 expression by thymic epithelium. Therefore, defective positive selection of HLA class II-restricted TCR clone 5 is observed in fetal pig thymus.

[0174] However, cross-reactive determinants may exist and the factor may be produced in SLAhh pig thymus where positive selection of this TCR is possible. In this case, it is determined whether negative selection of thymocytes with this TCR occurs in the reconstituted pig thymus containing HLA-DQ8+ CD34+ cells.

[0175] Preliminary data in HLA-DQ8+ human thymus suggests that HLA-DQ8 on CD34 cell-derived APCs is required for negative selection of this TCR (see Figure 8). This may still occur in porcine thymus containing human HLA-DQ8+ APCs. The reason is that the insulin B (9-23) peptide is identical in porcine and human insulin molecules, and human APCs in porcine thymus grafts capture and present it. To identify transgenic T cells, a fluorescent dye-labeled clone 5Vβ-specific mAb (Vβ21.3) is used, while GFP is utilized as a marker representing the origin of gene-introduced HSPC. GFP+ and GFP− thymocytes at each stage of thymic development provide an internally consistent comparison regarding the selection levels of Tg and non-Tg T cells in each mouse individual. These tests performed during the generation of transgenic pigs (Examples 3 and 4) provide a baseline criterion for evaluating the effect of the HLA-DQ8 transgene in porcine fetal thymus on HLA DQ8-restricted human T cell selection in porcine thymus.

[0176]

Table 4

[0177]

Table 5

[0178] Example 11: Comparison of Allogeneic Human Skin Graft Rejection in HIS Mice To examine the human immune system function in HIS mice created with different thymuses and CD34+ cells, their allogeneic skin graft rejection capabilities were compared. For this purpose, HIS mice were generated by transplanting porcine fetal or human thymus and CD34+ cells derived from CB or fetal liver (Table 6) by the method outlined in Example 7.

[0179] At 14 to 16 weeks after transplantation, a stratified (2.3 mm) skin sample obtained from an allogeneic human donor is transplanted onto the outer chest wall. The skin grafts are evaluated daily from day 7 for 4 weeks and then examined at least once every 3 days. When the survival status of the graft is less than 10%, the graft is defined as rejected. HIS mice generated with two types of thymus and CD34+ cells can all reject allogeneic skin grafts.

[0180]

Table 6

[0181] Example 12: Comparison between non-transgenic pig thymus and HLA-A2 transgenic pig thymus for human cell reconstitution As shown in Example 7, HIS mice generated with porcine fetal thymus and cord blood-derived CD34+ cells have minor functional defects in T cells (such as reduced HLA-restricted antigen responses and selection of TCR-transduced T cells in the thymus, etc.) compared to HIS mice generated with fetal thymus and autologous fetal liver-derived CD34+ cells. The main reason is as follows: In porcine thymus, porcine leukocyte antigen (SLA) molecules rather than HLA molecules mediate the positive selection of thymocytes, and only a very small subset of these selected T cells show sufficient cross-reactivity to human HLA and recognize peptide antigens presented by HLA of DCs derived from the CD34 cell donor. This model is optimized using transgenic (Tg) porcine fetal thymus expressing common HLA molecules (including HLA-A2 and HLA-DQ8).

[0182] Using the HLA-A2 transgenic porcine fetal thymus of Example 3, immune reconstitution and immune function are compared between HIS mice generated with non-transgenic fetal thymus and HIS mice generated with HLA-A2 transgenic porcine fetal thymus.

[0183] Using thymectomized NSG mice, two types of HIS mice are generated using transgenic and non-transgenic porcine fetal thymus and CB CD34+ cells by the methods outlined in Example 7 and as described in Table 7.

[0184] After generating these HIS mice, the mice are monitored as follows.

[0185] Human immune cell reconstitution is monitored and the two types of HIS mice are compared by measuring the peripheral blood concentrations of the repopulation rate and T cell, B cell, and myeloid cell populations (CD4 and CD8 T cells, naive and memory CD4 and CD8 T cells, regulatory T cells (Tregs) and follicular helper T cells (Tfh); B cell subsets, monocytes, and DCs (including classical DCs (cDC1 and cDC2) and plasmacytoid DCs (pDCs)). Every 4 weeks after transplantation, peripheral blood is collected from the HIS mice and red blood cells are lysed with ACK buffer. Flow cytometry analysis of the peripheral blood is performed to determine the percentage (%) and absolute number of each population. The absolute number of each population is calculated by counting the beads. The percentage (%) of mice achieving reconstitution in each group of HIS mice is also examined. The panel used to examine the immune cell populations is shown in Table 1.

[0186] In three types of HIS mice, plasma immunoglobulin (IgM and IgG) levels are monitored and compared by ELISA every 4 weeks after transplantation.

[0187] When it is expected that the reconstitution of human cells in HIS mice is completed at 14 - 16 weeks after transplantation, half of the animals in each group are euthanized and the tissue size, tissue structure, cellularity, and cell populations in peripheral blood, lymph nodes, spleen, and thymus are compared. The flow cytometry panel for testing immune cell populations is the same as that in Table 1. Histological tests are performed using small pieces of each lymphoid tissue (including spleen, lymph nodes, and thymus) to compare the structures of these tissues. For all HIS mice, serum immunoglobulin (IgM and IgG) levels are measured by ELISA. Furthermore, in vitro assays of proliferation, cytokine production, and cytotoxicity in response to pan-TCR stimulation (anti-CD3 / CD28 beads), alloantigen stimulation, heterologous antigen stimulation, and tetanus toxoid neoantigen stimulation are used to compare the functions of human T cells in the periphery of each group of mice. Proliferation is determined by CFSE intracellular dye dilution. Cytokine production including IL-2 and IFN-γ is assayed by intracellular staining. For alloantigen and heterologous antigen stimulation, allogeneic human PBMC and third-party porcine PBMC are used as stimulators. Isolated splenic T cells from HIS mice are labeled with CFSE and co-cultured for 6 days at a 1:1 ratio with irradiated stimulators. CFSE dilution of human CD4 and CD8 T cells is determined by flow cytometry. For tetanus toxoid neoantigen stimulation, DCs are generated using CB CD34+ cells used for generating HIS mice. To differentiate into dendritic cells, CD34+ cells are cultured for 13 days in the presence of human cytokines (including stem cell factor, GM-CSF, and IL-4). CD34-derived DCs are pulsed with tetanus toxoid neoantigen and then matured with TNF-α and PGE2, and then co-cultured with CFSE-labeled isolated splenic T cells for 7 days. Proliferating T cells are determined by flow cytometry. Monocytes are stimulated with LPS, and TNF-α, IL-6, and IL-10 production in the supernatant is determined by ELISA.

[0188] Monitor the remaining HIS mice up to the 30th week at the longest, examine the persistence of the reconstitution of each lineage, and also examine the onset of graft-versus-host disease / autoimmune diseases. Blood samples of the mice are taken every 4 weeks to determine human cell engraftment. The evaluation of the mice for graft-versus-host disease starts from the 20th week after transplantation and is carried out twice a week until the 30th week using the evaluation system described in Example 6. Any analysis performed at this time point is the same as the analysis at 14 - 16 weeks.

[0189] Similar bone marrow reconstitution is observed among these groups. In recipients of HLA transgenic pig thymus, immune reconstitution and immune function can be promoted.

[0190]

Table 7

[0191] Example 13: Compare the immune tolerance of human T cells generated in HLA-A2 transgenic pig fetal thymus to HLA-A2 molecules In HIS generated in HLA-A2 transgenic pig fetuses thymus, a major feature of human T cells that develop is expected to be immune tolerance to HLA-A2. The reason is that HLA-A2 reactive T cells are removed by negative selection by thymic epithelial cells expressing HLA-A2, and / or HLA-A2 reactive T cells are suppressed by Tregs selected by TECs expressing HLA-A2. For this purpose, the immune tolerance of T cells generated in HLA-A2-Tg to human Tg HLA molecules is compared with that of non-Tg pig fetal thymus. To remove the negative selection of HLA-A2 reactive T cells by CD34+ cell-derived APCs, HIS mice were generated using CD34+ cells derived from HLA-A2-CB. The generated HIS mouse groups are shown in Table 8. At 14 - 16 weeks after transplantation, splenic T cells and mature thymic T cells were isolated and immune tolerance to HLA-A2 (expected to be observed only in recipients of HLA-A2-Tg pig fetal thymus) was tested in vitro using donor pig-derived DCs. DCs were prepared from porcine fetal liver leukocytes, recovered at the time of fetal thymus recovery, and frozen until use. Fetal liver leukocytes were cultured for 13 days in the presence of porcine stem cell factor, GM-CSF, and IL-4 to differentiate into DCs. To determine the effect of HLA-A2 transgenic expression on Treg suppression of HLA-A2 responses, these tests included Treg depletion.

[0192]

Table 8

[0193] Example 14: Comparison of HIS mice prepared as controls and HIS mice prepared with HLA-A2-Tg pig fetal thymus for the selection of HLA-A2 restricted TCR Regarding the selection of HLA-A2-restricted TCR and MART1, compare HIS mice generated in non-Tg controls with HIS mice generated in HLA-A2-Tg pig fetal thymus. After injecting MART-1-transduced HLA-A2+ CB CD34+ cells into sub-lethally irradiated thymectomized NSG mice, transplant non-Tg control or HLA-A2-Tg pig fetal thymus (Table 9).

[0194]

Table 9

[0195] At 14 - 16 weeks after transplantation, after human cell reconstitution is completed in HIS mice, the mice are euthanized for analysis. Examine the percentage (%) and absolute numbers of MART-1+ thymocytes in the double-negative (CDla+)(including CD7+ early thymocytes), double-positive, CD4 single-positive, and CD8 single-positive subsets with other negative selection markers (CD69, PD1, CCR7). Enhanced positive selection of HLA class I-restricted TCR MART1 is expected in HLA-A2+ Tg pig fetal thymus. To identify transgenic T cells, use fluorescent dye-labeled MART1 tetramers, while GFP is used as a marker representing the origin of gene-introduced HSPC. GFP+ and GFP- thymocytes at each stage of thymic development provide an internally consistent comparison regarding the selection levels of Tg and non-Tg T cells in each mouse individual. The detailed panel is shown in Table 4 below. The analysis is performed by Aurora spectral flow cytometry.

[0196] Under the hypothesis that HLA-A2 enhances positive selection in porcine thymus, thereby resulting in a greater number of MART1+ T cells being transported to the periphery, enumerate MART1+ and negative CD8+ T cells in the periphery (blood, spleen, lymph nodes) of each mouse. Label MART1+ cells with the cell proliferation dye eFluor450, incubate the cells with autologous DCs and MART1 peptides added in a stepwise amount, measure proliferation, and examine the function of peripheral MART1+ cells by measuring other markers for the activation of GFP+ T cells.

[0197] Example 15: Comparison of allogeneic skin graft rejection by HIS mice generated with HLA-A2-Tg porcine fetal thymus To examine the immune system function in HIS mice generated with HLA-A2-Tg thymus and CD34+ cells, compare the allogeneic skin graft rejection ability of HIS mice. For this purpose, generate HIS mice by transplanting HLA-A2-Tg or non-Tg control porcine fetal thymus and CB CD34+ cells into sub-lethally irradiated thymectomized NSG mice (Table 10). At 14 - 16 weeks after transplantation, transplant a split-thickness (2.3 mm) skin sample from an allogeneic human donor onto the chest wall. Skin grafts are evaluated daily from day 7 for 4 weeks and then examined at least once every 3 days. Define that a graft is rejected when its survival state is less than 10%. When thymic and peripheral human APCs share HLA molecules, peripheral T cell function is more stable, and a more rapid graft rejection reaction occurs in HLA-A2-Tg recipients than in recipients of control porcine thymic grafts.

[0198]

Table 10

[0199] Example 16: Comparison of non-Tg porcine thymus and HLA-A2 / DQ8-Tg porcine thymus for human cell reconstitution When HLA-A2 / DQ8-Tg porcine fetal thymuses are available, the immune reconstitution and immune function in HIS mice generated using non-Tg porcine fetal thymuses are compared with those of HIS mice generated using HLA-A2 / DQ8-Tg porcine fetal thymuses. Using thymectomized NSG mice, two types of HIS mice are generated using HLA-A2-Tg and HLA-A2 / DQ8-Tg porcine fetal thymuses and CD34+ cells derived from HLA-DQ8+ CB by the method as described in Table 11. For the optimal homeostasis of human T cells selected by HLA-DQ8, the presence of HLA-DQ8+ APCs in the periphery is required, and thus CD34+ cells derived from HLA-DQ8+CB are used for the generation of HIS mice. To optimize immune function by retaining both class I and class II HLA alleles shared by the thymus and peripheral APCs, HLA-A2+DQ8+ CD34+ cells are used.

[0200]

Table 11

[0201] After the generation of these HIS mice, the mice are monitored as follows.

[0202] For human immune cell reconstitution, the repopulation rate and the peripheral blood concentrations of T cell, B cell, and myeloid cell populations (CD4 and CD8 T cells, naive and memory CD4 and CD8 T cells, regulatory T cells (Tregs) and follicular helper T cells (Tfh); B cell subsets, monocytes, and DCs (including classical DCs (cDC1 and cDC2) and plasmacytoid DCs (pDCs)) are measured to monitor and compare the two types of HIS mice. Every 4 weeks after transplantation, peripheral blood is collected from the HIS mice and the red blood cells are lysed with ACK buffer. Flow cytometry analysis of the peripheral blood is performed to determine the percentage (%) and absolute number of each population. The absolute number of each population is calculated by counting the beads. The percentage (%) of mice achieving reconstitution in each group of HIS mice is also examined. The panel used to examine the immune cell populations is shown in Table 2.

[0203] In three types of HIS mice, serum immunoglobulin (IgM and IgG) levels are monitored and compared by ELISA every four weeks after transplantation.

[0204] When it is expected that the reconstitution of human cells in HIS mice is completed at 14 - 16 weeks after transplantation, half of the animals in each group are euthanized and the tissue size, tissue structure, cellularity, and cell populations in peripheral blood, lymph nodes, spleen, and thymus are compared. The flow cytometry panel for testing immune cell populations is the same as that in Table 2. Histological tests are performed using small pieces of each lymphoid tissue (including spleen, lymph nodes, and thymus) to compare the structures of these tissues. Serum immunoglobulin (IgM and IgG) levels are measured by ELISA for all HIS mice. Furthermore, in vitro assays of proliferation, cytokine production, and cytotoxicity in response to pan-TCR stimulation (anti-CD3 / CD28 beads), alloantigen stimulation, heterologous antigen stimulation, and tetanus toxoid neoantigen stimulation are used to compare the functions of human T cells in the periphery of each group of mice. Proliferation is determined by CFSE intracellular dye dilution. Cytokine production, including IL-2 and IFN-γ, is assayed by intracellular staining.

[0205] For allogeneic antigen and heterologous antigen stimulation, allogeneic human PBMCs and third-party porcine PBMCs are used as stimulators. Isolated splenic T cells from HIS mice are labeled with CFSE, irradiated, and co-cultured with the irradiated stimulators at a 1:1 ratio for 6 days. The CFSE dilution of human CD4 and CD8 T cells is determined by flow cytometry. For tetanus toxoid neoantigen stimulation, DCs are generated using CB CD34+ cells used for the generation of HIS mice. To differentiate into dendritic cells, CD34+ cells are cultured for 13 days in the presence of human cytokines (including stem cell factor, GM-CSF, and IL-4). CD34-derived DCs are pulsed with tetanus toxoid neoantigen, then matured with TNF-α and PGE2, and then co-cultured with CFSE-labeled isolated splenic T cells for 7 days. The determination of proliferating T cells is performed by flow cytometry. Monocytes are stimulated with LPS, and the production of TNF-α, IL-6, and IL-10 in the supernatant is determined by ELISA.

[0206] The remaining HIS mice are monitored until week 30 at the latest to examine the persistence of the reconstitution of each lineage and also to examine the onset of graft-versus-host disease / autoimmune diseases. Blood samples are taken from the mice every 4 weeks to determine human cell engraftment. The evaluation of the mice for graft-versus-host disease is started from week 20 after transplantation and is performed twice a week until week 30 using the evaluation system described in Example 8. Any analysis performed at this time is the same as the analysis at weeks 14 - 16.

[0207] Example 17: Comparison of T cells generated in HLA-A2 / DQ8-Tg porcine fetal thymus and T cells generated in HLA-A2-Tg porcine fetal thymus with respect to immune tolerance to HLA-DQ8 Regarding immune tolerance to the human Tg HLA-DQ8 molecule, compare T cells generated in HLA-A2 / DQ8-Tg with T cells generated in non-Tg pig fetal thymus. To remove negative selection of HLA-DQ8-reactive T cells by CD34+ cell-derived APCs, HIS mice were generated using HLA-DQ8-CB CD34+ cells. The HIS mouse groups created for this task are shown in Table 12. At 14 - 16 weeks after transplantation, spleen T cells and mature thymus T cells were isolated and immune tolerance to HLA-DQ8 (expected to be observed only in recipients of HLA-A2 / DQ8-Tg pig fetal thymus) was tested in vitro using donor pig-derived DCs. DCs were prepared from pig fetal liver leukocytes, recovered at the time of fetal thymus recovery, and frozen until use. Fetal liver leukocytes were cultured for 13 days in the presence of porcine stem cell factor, GM-CSF, and IL-4 to differentiate into DCs. Since the presence of HLA DQ8 on TECs is thought to enable positive selection of Tregs with these specificities, these tests include Treg depletion.

[0208]

Table 12

[0209] Example 18: For the selection of HLA-DQ8-restricted TCRs, compare HIS mice created as controls with HIS mice created using HLA-A2 / DQ8-Tg pig fetal thymus For the selection of HLA-DQ8-restricted TCR (clone 5), compare HIS mice created with non-Tg controls with HIS mice created using HLA-A2 / DQ8-Tg pig fetal thymus. Sublethally irradiated thymectomized NSG mice were injected with clone 5 transduced CB CD34+ cells, and then non-Tg controls or HLA-A2 / DQ8-Tg pig fetal thymus were transplanted (Table 13).

[0210]

Table 13

[0211] Fourteen to sixteen weeks after transplantation, when human cell reconstitution is complete in HIS mice, the HIS mice are euthanized for analysis. The percentage (%) and absolute numbers of clone 5+ thymocytes in double-negative (CDla+)(including CD7+ early thymocytes), double-positive CD69+ and CD69-, CD4 single-positive and CD8 single-positive subsets are examined with negative selection markers (PD1, CCR7). In HLA-DQ8+ thymus, Treg markers (CD25 and CD127) are also evaluated to detect the Treg lineage differentiation of thymocytes with this TCR. The detailed panel is shown in Table 5 below. The analysis is performed by Aurora spectral flow cytometry. Since the insulin peptide recognized by this TCR is expected to be produced by medullary TEC (mTEC), the negative selection of this TCR is expected to depend on HLA-DQ8 expression by thymic epithelium. Enhanced positive selection of HLA class Il-restricted TCR clone 5 is expected to be seen in HLA-A2 / DQ8-Tg fetal porcine thymus compared to non-Tg porcine thymus. Preliminary data in HLA-DQ8+ human thymus suggest that for the negative selection of this TCR, in addition to expression on TEC, HLA-DQ8 is also required on CD34 cell-derived APCs (see Figure 9). Therefore, it may also be possible to test the negative selection of clone 5+ T cells by using HLA-DQ8+ CB CD34+ cells for the generation of HIS mice. To identify transgenic T cells, a fluorescent dye-labeled clone 5 Vβ-specific mAb (Vβ21.3) is used, while GFP is used as a marker representing the origin of gene-transduced HSPC. GFP+ and GFP- thymocytes at each stage of thymic development provide internally consistent comparisons regarding the selection levels of Tg and non-Tg T cells in each mouse individual.

[0212] Example 19: Comparing allogeneic skin graft rejection by HIS mice generated from HLA-A2 / DQ8 Tg fetal porcine thymus To examine the immune system function in HLA-A2 / DQ8-Tg thymus and HIS mice generated with CD34+ cells, the allogeneic skin graft rejection ability of HIS mice was compared. For this purpose, HIS mice were created by transplanting HLA-A2 / DQ8-Tg or non-Tg control porcine fetal thymus and CB CD34+ cells (Table 10). At 14-16 weeks after transplantation, an allogeneic human donor split-thickness (2.3 mm) skin sample was transplanted onto the outer chest wall. Skin grafts were evaluated daily from day 7 for 4 weeks and then examined at least once every 3 days. When the survival status of the graft was less than 10%, the graft was defined as rejected.

[0213]

Table 14

Claims

1. 1. A method for producing a human immune system (HIS) mouse, comprising: (a) performing a thymectomy on a mouse; (b) injecting the mouse (i) fetal porcine thymus tissue; and (ii) human CD34+ cells; and A method comprising:

2. The method described in claim 1, wherein the human CD34+ cells are fetal cells.

3. The method described in claim 1, wherein the human CD34+ cells are adult cells.

4. The method described in claim 1, wherein the human CD34+ cells are derived from umbilical cord blood.

5. The method of claim 1, wherein the fetal pig thymus tissue comprises one or more nucleotide sequences encoding one or more HLA I polypeptides.

6. The method of claim 1, wherein the fetal pig thymus tissue comprises one or more nucleotide sequences encoding one or more HLA II polypeptides.

7. The method of claim 1, wherein the fetal pig thymus tissue comprises one or more nucleotide sequences encoding one or more HLA I polypeptides and one or more HLA II polypeptides.

8. A method described in any one of claims 5 to 7, wherein the one or more nucleotide sequences are inserted into a natural SLA locus in the pig genome.

9. The method of claim 8, wherein the one or more nucleotide sequences are inserted downstream of a native SLA I promoter.

10. 1. A method for producing a human immune system (HIS) mouse, comprising: (a) performing a thymectomy on a mouse; (b) introducing fetal pig thymus tissue into said mouse; Including, the porcine fetal thymus tissue comprises one or more nucleotide sequences encoding one or more HLA I polypeptides and / or one or more HLA II polypeptides inserted into one or more naturally occurring SLA loci of the porcine genome; method.

11. The method of claim 10, wherein the one or more nucleotide sequences encode an HLA I polypeptide and are inserted into a naturally occurring SLA I locus.

12. The method of claim 11, wherein the SLA I locus is selected from the group consisting of SLA-1 and SLA-2.

13. The method of claim 11, wherein the HLA I polypeptide comprises HLA-A2 fused to human beta-2 microglobulin (B2M).

14. The method of claim 11, wherein the one or more nucleotide sequences are inserted downstream of a native SLA I promoter.

15. The method of any one of claims 11 to 13, wherein the one or more nucleotide sequences are inserted at the intron 1 / exon 2 junction of the SLA I locus.

16. The method of claim 11, wherein the one or more nucleotide sequences further encode an HLA II polypeptide and are inserted into a natural SLA-DQα locus.

17. The method of claim 10, wherein the one or more nucleotide sequences encode an HLA II polypeptide and are inserted into a naturally occurring SLA-DQα locus.

18. The method of claim 16 or 17, wherein the HLA II polypeptide comprises an HLA-DQ8 polypeptide.

19. The method of claim 18, wherein the nucleotides encoding the HLA-DQ8 polypeptide are targeted to the natural SLA-DQα locus by a bicistronic vector encoding HLA-DQ8 (HLA-DQA1:03:01:01 and HLA-DQB1:03:02:01).

20. The method described in claim 19, wherein the bicistronic vector further contains a high-efficiency IRES element.

21. The method of any one of claims 11 to 20, wherein one or more nucleotide sequences encoding the HLA II polypeptide are inserted downstream of a native SLA DQα promoter.

22. The method of any one of claims 11 to 20, wherein one or more nucleotide sequences encoding the HLA II polypeptide are inserted at the intron 1 / exon 2 junction of the SLA DQα locus.

23. The method of claim 10, wherein the HLA I polypeptide is selected from the group consisting of HLA-A, HLA-A2, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, and the HLA II polypeptide is selected from the group consisting of HLA-DP, HLA-DM, HLA-DO, HLA-DQ, and HLA-DR.

24. A method described in any one of claims 10 to 23, further comprising introducing human CD34+ cells into the mouse.

25. The method described in claim 24, wherein the human CD34+ cells are fetal cells.

26. The method described in claim 24, wherein the human CD34+ cells are adult cells.

27. ​​The method described in claim 24, wherein the human CD34+ cells are derived from umbilical cord blood.

Images