Porcine SLA class ii knockout products and methods

Abstract

The present disclosure relates to transgenic animals with nuclear genomes comprising knockouts of both alleles of an SLA-DQ gene and / or an SLA-DR gene. The disclosure provides transgenic pigs, transplant products from the transgenic pigs and xenotransplantation methods utilizing the transplant products for treatment of transplant rejection, especially in humans.

Description

PCT / US25 / 52258 23 October 2025 (23.10.2025)Cross-Reference to Related Applications

[0001] This application claims priority to Provisional Application No. 63 / 711,228, filed October 24, 2024, and to Provisional Application No. 63 / 769,605, filed March 10, 2025, which are incorporated herein by reference in their entirety.Statement of Government Support

[0002] This invention was made with government support under U01 AI126322 awarded by the National Institutes of Health. The government has certain rights in the invention.Field

[0003] The present disclosure relates to transgenic animals with nuclear genomes comprising knockouts of both alleles of an SLA-DQ gene and / or an SLA-DR gene. The disclosure provides transgenic pigs, transplant products from the transgenic pigs and xenotransplantation methods utilizing the transplant products for treatment of transplant rejection, especially in humans.Background

[0004] The porcine major histocompatibility complex (MHC) is known as the swine leukocyte antigens (SLA) and has a very similar structure to the human leukocyte antigen (HLA). The SLA complex is located on porcine chromosome 7 (SSC7) and consists of three gene clusters, the SLA class I, III, and II. SLA class II, which displays strong homology to HLA class II, are alphabetically designated after the HLA class II genes as SLA-DR, DQ, DM and DO.

[0005] Late failure of renal xenografts in preclinical animal models is characterized by transplant glomerulopathy with thrombotic microangiopathy that is likely due to chronic antibody mediated rejection (AMR) (1, 2). In allotransplantation transplant glomerulopathy is associated with the presence of ant-HLA class II antibodies, either pre-transplant or de novo post-transplant (3, 4). Anti-HLA-DQ antibodies are significantly more prevalent in allotransplantation and are associated with worse long-term outcomes with more significant transplant glomerulopathy (5, 6). The likelihood of developing de novo anti-HLA-DQ is directly correlated with the degree of amino acid (AA) mismatch between donor and recipient, and the tacrolimus levels necessary to prevent the formation of anti-HLA-DR and -DQ antibodies can be predicted based upon the degree of AA mismatch as well (7-12).PCT / US25 / 52258 23 October 2025 (23.10.2025)Significant attention in allotransplantation is being directed at HLA-DQ matching and trying to balance organ availability with long term outcome for recipients.

[0006] In rhesus monkeys, long-term surviving recipients of pig renal xenografts (>300 days) have donor specific anti-SLA-DQ antibodies that are likely triggers for the development of transplant glomerulopathy and thrombotic microangiopathy that characterizes their graft failure (13). In waitlisted patients, we showed that a significant proportion of anti-HLA-DR and / or anti-HLA-DQ antibodies will cross react with SLA-DQ and SLA-DR on the pig donor endothelium, posing a barrier to xenotransplantation for these patients. We also found anti-SLA-DQ and SLA-DR antibodies in some patients with no HLA sensitization (14). SLA-DQ is expressed on the vascular endothelium of pig kidneys (15). It is contemplated herein that these findings suggest that antibodies directed against SLA-DQ and / or SLA-DR may pose a significant barrier to long-term success of renal xenotransplantation.

[0007] There remains a need in the art for products and methods for xenotransplantation.Summary

[0008] In this disclosure, we evaluated sera from naive and HLA class II sensitized patients for the presence of anti-SLA-DQ antibodies against seven different SLA molecules. Next, we generated SLA-DQ KO pigs and SLA-DR KO pigs on the GGTA1 / B4GALNT2 KO background. We used lymphocytes from these pigs and patient serum to evaluate the impact of deleting SLA-DQ or SLA-DR. Our findings have implications for clinical xenotransplantation, and the SLA-DQ KO and / or SLA-DR KO could be a potential engineering solution to this present barrier to an ideal crossmatch.

[0009] As mentioned above, the most common cause of late graft failure in renal allotransplantation is chronic antibody mediated rejection caused by donor specific antibodies against class II HLA, particularly HLA DQ. In preclinical renal xenotransplantation, graft failure 1 -month post-transplant is characterized by glomerulopathy and IgG staining in the glomerulus. Rhesus renal xenograft recipients with late graft failure also have anti-SLA-DQ antibodies present in their serum suggesting that, like allotransplantation, late xenograft failure may be driven by anti-donor MHC class II antibodies, particularly SLA-DQ and / or SLA-DR. Some patients have anti-SLA-DQ and SLA-DR antibodies, but the magnitude of this problem is unclear.

[0010] We evaluated patient sera for the presence of anti-SLA-DQ antibodies in engineered immortalized cells, to determine patients’ reactivity towards seven different SLA-DQ molecules. Next, we created GGTA1 / B4GALNT2 / SLA-DQ KO pigs so that we could evaluate the impact of SLA-DQ on the level of anti-pig antibodies by performingPCT / US25 / 52258 23 October 2025 (23.10.2025)crossmatches with serum from naive and HLA class II sensitized patients and SLA-DQ KO PBMCs. We also created GGTA1 / B4GALNT2 / SLA-DR KO pigs to evaluate the impact of SLA-DR.

[0011] Naive and HLA class II sensitized patients had some anti-SLA-DQ IgM and IgG that were pan specific rather than SLA-DQ allele specific. Crossmatching patient sera with PBMCs from the SLA-DQ KO pigs revealed that many patients had anti-SLA-DQ antibody. Eliminating SLA-DQ reduced human IgM and IgG binding to primary pig cells

[0012] SLA-DQ and SLA-DR are xenoantigens for most patients. SLA-DQ KO pigs and SLA-DR KO pigs are contemplated herein to help address this problem.

[0013] The disclosure thus provides a transgenic pig comprising in its nuclear genome a knockout of both alleles of an SLA-DQ gene, an SLA-DR gene or both an SLA-DQ gene and SLA-DR gene.

[0014] Transgenic pigs provided herein are engineered to ultimately prevent pairing and cell surface expression of SLA proteins in the transgenic pig, either by eliminating DQ proteins or eliminating DR proteins, or eliminating both. Depending on the genotype of the pig, knockout of both alleles of at least one of the DQa, DQp, DRa and DRp genes is contemplated. As one example, for a pig where the DRa and p proteins pair, and the goal to prevent expression of DR the pig is engineered to knockout both alleles of one or both of DRa and DRp genes. As another example, for a pig where the DRa proteins pair, and the goal to prevent expression of DR the pig is engineered to knockout both alleles of the DRa gene.

[0015] Thus, the disclosure contemplates a variety of SLA gene knockouts. The following are non-limiting examples. The knockout may be both alleles of an SLA-DQa gene. The knockout may be both alleles of an SLA-DQp gene. The knockout may be both alleles of an SLA-DRa gene. The knockout may be both alleles of an SLA-DRp gene. The knockout may be both alleles of an SLA-DQp gene and both alleles of an SLA-DRa gene. The knockout may be both alleles of an SLA-DQa gene and both alleles of an SLA-DRp gene.

[0016] The disclosure provides a porcine transplant product isolated from a transgenic pig provided herein, wherein the transplant product is an organ, tissue, transfusion product or cell. The porcine transplant product can be a porcine organ, tissue, transfusion product or cell is skin, heart, liver, kidneys, lung, pancreas, thyroid, small bowel, whole blood or a blood component.

[0017] The disclosure provides a method of preparing a transplant product for xenotransplantation into a human, the method comprising isolating the transplant productPCT / US25 / 52258 23 October 2025 (23.10.2025)from a transgenic pig comprising in its nuclear genome a knockout of both alleles of an SLA-DQ gene, an SLA-DR gene or both an SLA-DQ gene and SLA-DR gene. The porcine transplant product can be an organ, tissue, transfusion product or cell. The porcine transplant can be skin, heart, liver, kidneys, lung, pancreas, thyroid, small bowel, whole blood or a blood component.

[0018] The disclosure provides a method of increasing the duration of the period between when a human subject is identified as a subject in need of a human transplant product and when the human transplant occurs, comprising administering a porcine transplant product to the human subject in a therapeutically effective manner, wherein the porcine transplant product is isolated from a transgenic pig comprising in its nuclear genome a knockout of both alleles of the SLA-DQ gene, the SLA-DR gene or both the SLA-DQ gene and SLA-DR gene. The porcine transplant product can be an organ, tissue, transfusion product or cell. The porcine transplant can be skin, heart, liver, kidneys, lung, pancreas, thyroid, small bowel, whole blood or a blood component.

[0019] The disclosure provides a method of improving a transplant rejection-related symptom in a human subject comprising administering a porcine transplant product to a subject in need thereof, wherein the porcine transplant product is isolated from a transgenic pig comprising in its nuclear genome a knockout of both alleles of the SLA-DQ gene, the SLA-DR gene or both the SLA-DQ gene and SLA-DR gene, and wherein a transplant rejection-related symptom is improved as compared to when porcine transplant product from a wild-type pig is transplanted into a human subject. The transplant rejection-related symptom can be a hyperacute rejection (HAR) symptom. The transplant rejection-related symptom can be a chronic antibody-mediated rejection (AMR) symptom. The porcine transplant product can be an organ, tissue, transfusion product or cell. The porcine transplant can be skin, heart, liver, kidneys, lung, pancreas, thyroid, small bowel, whole blood or a blood component.

[0020] The disclosure provides a transgenic pig or method as set out in any Summary paragraph above, wherein the gene is the SLA-DQ gene. The disclosure provides a transgenic pig or method as set out in any summary paragraph above, wherein the gene is the SLA-DR gene. The disclosure provides a transgenic pig or method as set out in any Summary paragraph above, wherein both the SLA-DQ gene and SLA-DR gene alleles are knocked out.

[0021] The disclosure provides a transgenic pig or method as set out in any Summary paragraph above, wherein the transgenic pig further comprises in its nuclear gene a knockout of both allelles of one or more of the a(1,3)-galactosyltransferase (GGTA1) gene,PCT / US25 / 52258 23 October 2025 (23.10.2025)cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene and (31,4 N-acetylgalactosaminyltransferase (p4GalNT2) gene. The transgenic pig or methodcan be a transgenic pig comprising in its nuclear gene a knockout of both alleles of both the GGTA1 gene and the p4GalNT2 gene. The transgenic pig or method can be a transgenic pig comprising in its nuclear gene a knockout of both alleles of each of the GGTA1 gene, CMAH gene and the p4GalNT2 gene.Brief Description of the Drawings

[0022] The patent or application file contains at least one drawing executed in color.Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] Figure 1 shows binding of human IgM and IgG to various SLA-DQ proteins. Eight variants of an immortal cell line (eC1 R) which express seven individual SLA-DQ proteins or an empty vector as a negative control. Expression levels of each SLA-DQ protein in these cells were revealed by flow cytometry of the cells after incubation with a monoclonal antibody specific for SLA-DQ (A black outline histogram). Negative controls were cells evaluated without the SLA-DQ monoclonal antibody (gray histograms) and the empty vector cell line incubated with and without the SLA-DQ monoclonal antibody (top row SLA-DQa and -DQb as “none”). IgM (B) and IgG (C) binding to each of these cell lines was examined for 40 human sera. 22 of the sera lacked class II HLA antibodies (white boxes), and 18 sera contained IgG specific for class II HLA proteins (gray boxes). Median fluorescence intensity (MFI) was plotted as a measure of immunoglobulin binding. In panels B (IgM) and C (IgG), open circles represent values obtained with SLA-DQ positive cells. Red circles represent MFI values observed when examining each serum against the SLA deficient cell line. Pigs 022-1, 022-4 and WT pig (D).

[0024] Figure 2 shows SLA-DQ disruption. (A) The SLA-DR and SLA-DQ alleles found in the haplotypes of the pigs in this disclosure are shown. Also noted are the pairings of class II SLA a / b chains which can potentially generate cell surface proteins where at least one chain comes from either DQa or DQp proteins. (B) The guide RNAs used to disrupt SLA-DQ cell surface expression targeted exons 2 and 3 in both SLA DQp loci. The (*) notes which exons and protein domains were targeted by the CRISPR tool. Horizontal arrows indicate the PCR primer binding sites, in exons 1 and 8, used when amplifying cDNA from SLA-DQB1 transcripts to evaluate gene modifications post CRISPR treatment. (C) The contour plots show the expression of SLA-DR (y-axis) and SLA-DQ (x-axis) without IFN-g stimulation of the fibroblasts (left plot). IFN-g treatment drove expression of SLA-DR and SLA-DQ inPCT / US25 / 52258 23 October 2025 (23.10.2025)unedited cells (middle plot). Note there are very few SLA-DR single positive cells in the unedited cells (quadrant marked by an * in the middle plot). Editing generated a population of cells which express SLA-DR but not SLA-DQ (right plot, population marked by **) after IFN-g treatment.

[0025] Figure 3 shows phenotypic analyses of SLA-DQ knockout piglets. Pig PBMC were incubated with fluorescently conjugated antibodies for CD21, SLA-DR, and SLA-DQ and analyzed by flow cytometry. PBMC from animals containing functional SLA-DQ genes and from genetically matched animals having SLA-DQ gene disruptions were compared. The percentage of CD21 -positive cells are shown. Representative contour plots and histograms from an SLA-DQ negative (A) and an SLA-DQ positive pig (panel B) are shown. MFI of the negative controls are noted in black numbers and histograms while the SLA-DQ and -DR expression is noted in gray numbers and histograms. (C) MFI of SLA-DR (black bars) and SLA-DQ (gray bars) expression on CD21 -positive PBMC are shown for three SLA-DQ knockout pigs and two SLA-DQ expressing pigs. Numbers beneath the graph indicate the gene insertions or deletions present at each SLA-DQ allele. ND indicates that the precise deletion could not be determined. (D) Confocal microscopy was used to determine SLA-DQ and SLA-DR expression in kidney, liver, heart, spleen and pancreas from wild type and SLA-DQ knockout animals. Expression of CD31 (heart, liver, kidney), CD21 (spleen) and, insulin (pancreas) was also evaluated in the tissues.

[0026] Figure 4 shows human antibody binding to PBMC from SLA-DQ knockout pigs. 17 sera were collected from patients lacking anti-HLA antibodies, and another 17 sera were collected from patients known to have anti-class II HLA antibodies. SLA-DQ knockout pig PBMC and SLA-DQ positive pig PBMC were incubated with these sera and the binding of IgG and IgM determined using flow cytometry. The effect of SLA-DQ knockout was evaluated by calculating a binding of ratio where the IgG (or IgM) MFI on the DQ negative PBMC was divided by the IgG (or IgM) MFI on the DQ positive PBMC. Ratios < 1 indicate less binding to SLA-DQ knockouts than to SLA-DQ positive cells, (panel A) IgG and IgM binding ratios for serum devoid of HLA reactive IgG. (B) IgG and IgM binding ratios for serum containing HLA reactive IgG.

[0027] Figure 5 shows the approach to obtaining SLA-DRa knockout cells. The DRa gene map with introns and exons are shown as are the CRISPR Cas9 guide RNAs used to disrupt the SLA-DRa gene. The gRNA sites are in exon 2 of the gene which encodes the alpha 1 domain of the mature protein.

[0028] Figure 6 shows plots of SLA-DR and SLA-DQ expression in fibroblasts pre- and post-editing. After treating the fibroblasts (cells used to make cloned pigs) with thePCT / US25 / 52258 23 October 2025 (23.10.2025)gRNA / Cas9 described in Figure 1, the fibroblasts were evaluated for loss of SLA-DR protein expression. This was achieved by treating the cells with interferon gamma (IFNy) for 1 to 4 days to induce SLA-DR and SLA-DQ cell surface expression. Cells lacking SLA-DR expression but still expressing SLA-DQ were sorted to obtain a population of SLA-DR negative / SLA-DQ positive cells. These cells are noted in the box in the lower right of each plot. The plot on the left is IFNy treated, parent fibroblasts that had not been CRISPR treated. The plot on the right is those same fibroblasts after editing with the CRISPR described in Figure 1. A small but clear population of SLA-DR negative, SLA-DQ positive cells can be seen.

[0029] Figure 7 shows plots of SLA-DR and SLA-DQ expression in cloned (transgenic) piglet fibroblasts. Edited fibroblasts from Figure 6 were used in SCNT and embryo transfer to create cloned pigs. Skin fibroblasts from cloned piglets were treated with I FNg to induce SLA-DR and SLA-DQ gene expression. Fibroblasts from unedited pigs were included for comparison. The graph in the lower left (WT no IFNg) were unedited fibroblasts showing that in the absence of IFNy, no SLA-DQ is expressed and very little SLA-DR. The upper left graph (WT + IFNy) shows that interferon-gamma treatment is needed to induce SLA-DR and SLA-DQ to high levels. The remaining graphs show the piglet number and the result of treating fibroblasts from each animal with IFNy and staining for SLA-DR and SLA-DQ expression. The cells from the edited animals lack SLA-DR expression but continue to express high levels of SLA-DQ when induced with IFNy.

[0030] Figure 8 shows editing of SLA-DR alleles. PCR was used to amplify the portions of the open reading frames of both SLA-DR alleles (SLA-DRa*02:01:01 and SLA-DRa*04:01). The PCR products were subjected to Sanger sequencing. This showed alleles had either had many bases deleted, or the PCR product was not detected indicating gene disruptions that prevented stable transcripts from being produced. Primers used to create these PCR fragments are noted in Figure 5.

[0031] Figure 9 shows graphs of SLA-DR and SLA-DQ expression in cells from cloned pig kidney, liver and heart. Kidney, liver, and heart were collected from a cloned pig and stained for CD31 (green), and anti-SLA-DR (Red top graph, DR-KO) or anti-SLA-DQ (Red bottom graph, DR-KO). Cells that had not their class II SLA genes edited were used as positive controls to demonstrate SLA-DR and SLA-DQ expression typically occur in each tissue.

[0032] Figure 10 shows GGTA1 / B4GALNT2 / SLA DR KO kidneys were transplanted into rhesus monkeys (A). Immunosuppression was given as described (B).PCT / US25 / 52258 23 October 2025 (23.10.2025)Detailed Description

[0033] As used herein, “subject,” “individual,” or “patient” are used interchangeably and refer to any member of the phylum Chordata, including, without limitation, humans and other primates, including non-human primates, such as rhesus macaques, chimpanzees, and other monkey and ape species; farm animals, such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rabbits, mice, rats, and guinea pigs; birds, including domestic, wild, and game birds, such as chickens, turkeys, and other gallinaceous birds, ducks, and geese; and the like. The term does not denote a particular age or gender. Thus, the term includes adult, young, and newborn individuals as well as males and females.

[0034] As used herein, the terms “wild-type,” “naturally occurring,” “endogenous” and “unmodified” are used herein to mean the typical (or most common) form, appearance, phenotype, or strain existing in nature; for example, the typical form of cells, organisms, polynucleotides, proteins, macromolecular complexes, genes, RNAs, DNAs, or genomes as they occur in, and can be isolated from, a source in nature. The wild-type form, appearance, phenotype, or strain serve as the original parent before an intentional modification. Thus, edited, mutant, variant, engineered, recombinant, and modified forms are not wild-type forms.

[0035] The term "transgenic mammal" refers to a transgenic mammal wherein a given gene has been edited (i.e., mutated, deleted or disrupted). It is to be emphasized that the term is to be intended to include all progeny generations. Thus, the founder animal and all F1, F2, F3 and so on progeny thereof are included, regardless of whether progeny were generated by somatic cell nuclear transfer (SCNT) from the founder animal or a progeny animal or by traditional reproductive methods. By "single transgenic" is meant a transgenic mammal wherein one gene has been edited. By "double transgenic" is meant a transgenic mammal wherein two genes have been edited. By "triple transgenic" is meant a transgenic mammal wherein three genes have been edited. By "quadruple transgenic" is meant a transgenic mammal wherein four genes have been edited. When editing of a gene results in a lack of a functional gene product in a cell (or in a transgenic animal) it is referred to as a “knockout” (KO).

[0036] In principle, transgenic animals may have one or both copies of the gene of interest edited. In the case where only one copy or allele of the nucleic acid of interest is edited, the animal is termed a "heterozygous transgenic animal". The term "null" mutation encompasses both instances in which the two copies of a nucleotide sequence of interest are disrupted differently but for which the disruptions overlap such that some geneticPCT / US25 / 52258 23 October 2025 (23.10.2025)material has been removed from both alleles, and instances in which both alleles of the gene of interest share the same disruption. Disruptions of genes of interest may occur in at least one cell of the transgenic animal, at least a plurality of the animal's cells, at least half the animal's cells, at least a majority of animal's cells, at least a supermajority of the animal's cells, at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the animal's cells.

[0037] The term "chimera", "mosaic" or "chimeric transgenic mammal" refers to a transgenic mammal with a knockout in some of its genome-containing cells. A chimera has at least one cell with an unaltered gene sequence, at least several cells with an unaltered gene sequence or a plurality of cells with an unaltered sequence.

[0038] The term "heterozygote" or "heterozygotic transgenic mammal" refers to a transgenic mammal with an edit of one of a chromosome pair (one allele) in all of its genome containing cells.

[0039] The term "homozygote" or "homozygotic transgeneic mammal" refers to a transgenic mammal with an edit of both members of a chromosome pair (both alleles) in all of its genome containing cells. A "homozygous alteration" refers to an alteration on both members of a chromosome pair.

[0040] A "non-human mammal" of the application includes mammals such as rodents, sheep, dogs, ovine such as sheep, bovine such as beef cattle and milk cows, and swine such as pigs and hogs. Although the application provides an illustrative non-human animal (pigs), other animals can similarly be genetically modified.

[0041] An “edit” is a detectable change in the genetic material in the animal that is transmitted to the animal's progeny. An edit is a change in one or more deoxyribonucleotides, such as an insertion, deletion or substitution.

[0042] By "pig" is intended any pig known to the art including, but not limited to, a wild pig, domestic pig, mini pigs, a Sus scrofa pig, a Sus scrofa domesticus pig, as well as in-bred pigs. Without limitation the pig can be selected from the group comprising Landrace, Yorkshire, Hampshire, Duroc, Chinese Meishan, Chester White, Berkshire Goettingen, Landrace / York / Chester White, Yucatan, Barna Xiang Zhu, Wuzhishan, Xi Shuang Banna and Pietrain pigs. Porcine organs, tissues, cells or transfusion products are organs, tissues, cells or transfusion products from a pig.

[0043] The alpha 1,3 galactosyltransferase (aGal, GGTA, GGT 1, GT, aGT, GGTA1, GGTA-1) gene encodes an enzyme (GT, aGal, a1,3 galactosyltransferase). Ensemble transcript ENSSSCG00000005518 includes the porcine GGTA1 nucleotide sequence.Functional a1,3 galactosyltransferase catalyzes formation of galactose-a1,3-galactosePCT / US25 / 52258 23 October 2025 (23.10.2025)(aGal, Gal, Gal, gall,3gal, gall-3ga1) residues on glycoproteins. The galactose-a1,3-galactose (aGal) residue is an antigenic epitope or antigen recognized by the human immunological system. One CRISPR target sequence in the gene is in exon 3 of the gene, near the start codon.

[0044] The pig CMAH gene (Ensembl transcript ENSSSCT00000075719.2) encodes the enzyme Cytidine monophospho-N-acetylneuraminic acid hydroxylase which adds a hydroxyl group to the sialic acid variant Neu5Ac to create Neu5Gc. Humans lack a functional CMAH gene; therefore, Neu5Gc is an antigen to humans. Inactivating the pig gene via genetic engineering eliminates Neu5Gc on pig cells reducing the xenoantigen burden. One CRISPR target occurs at the junction of the third intron and fourth exon which encompasses the sequence (5’-GAGTAAGGTACGTGATCTGT-3’).

[0045] The pi,4 N-acetylgalactosaminyltransferase gene encodes the pi,4 N-acetylgalactosaminyltransferase 2 glycosyltransferase (B4GalNT2). Functional B4GalNT2 produces Sda-like glycans (Dall'Olio et al (2014) Biochemica Biophysica Acta 1840:443-453 and Blanchard et al (1983) JBC 258:7691-7685). The Ensembl database ENSSSCG00000030269 entry includes the B4GalNT2 cDNA and amino acid sequences. One CRISPR target region in the gene is found in exon 2 and is (5’-CTGTATCGAGGAACACGCTT-3’).

[0046] Knockouts of the pig GGTA1, CMAH and B4GalNT2 genes are described in Martens, Gregory R., et al., Transplantation 101.4 (2017): e86-e92.

[0047] SLA-DR and SLA-DQ proteins are each comprised of alpha and beta chains. All of these gene loci (SLA-DR alpha, SLA-DR beta, SLA-DQ alpha, and SLA-DQ beta) exhibit polymorphisms. The SLA genes and alleles specific to the Examples herein are found in the NCBI database (DRa*02:01:02, Accession: KJ776450.1; DRa*04:01, Accession:KJ776449.1; DRb1*04:03, Accession: KJ776455.1; DRb1*10:01, Accession: KJ776456.1; DQa1*02:04, Accession: KJ776452.1; DQa1*01:01, Accession: KJ776451.1; DQb1*03:03, Accession: KJ776453.1; DQb1*06:01, Accession: KJ776454.1). Additional allelic variants found at each of these loci can be found in the IPD MHC-2.0 database which accumulates and verifies novel SLA gene sequences [Maccari G, Robinson J, Ballingall K, Guethlein LA, Grimholt U, Kaufman J, Ho OS, De Groot NG, Flicek P, Bontrop RE, Hammond JA and Marsh SGE IPD-MHC 2.0: an improved inter-species database for the study of the major histocompatibility complex. Nucleic Acids Res. (2017), 45: D860-D864)]. It is known in the art that different individuals have different permutations of binding pairs of SLA-DR and SLA-DQ proteins. In some individuals, SLA-DQ alpha chains form binding pairs with SLA-DQ beta chains. In some individuals, SLA-DR alpha chains form binding pairs with SLA-DR betaPCT / US25 / 52258 23 October 2025 (23.10.2025)chains. In some individuals, SLA-DQ alpha chains form binding pairs with SLA-DR beta chains. In some individuals, SLA-DR alpha chains form binding pairs with SLA-DQ beta chains. SLA proteins assembled in binding pairs are transported to the cell surface, while SLA proteins that do not form binding pairs are degraded in cells. Thus, generally, knockouts are contemplated herein that prevent formation of SLA binding pairs.

[0048] The phrases "edited gene" and “engineered gene” are intended to encompass insertion, interruption, or deletion of a nucleotide sequence of interest wherein the edited gene either encodes a polypeptide having an altered amino acid sequence that differs from the amino acid sequence of the endogenous polypeptide, encodes a polypeptide having fewer amino acid residues than the endogenous amino acid sequence or does not encode a polypeptide although the wild-type nucleotide sequence of interest encodes a polypeptide.

[0049] The present disclosure provides transgenic animals lacking expression of functional SLA-DQ and / or SLA-DR genes. The animal can be any mammal suitable for xenotransplantation. In a specific embodiment, the animal is a pig. “SLA-DQ KO”, for example, refers to cells or animals that lack expression of functional SLA-DQ. “SLA-DR KO”, for example, refers to cells or animals that lack expression of functional SLA-DR.

[0050] Expression may be analyzed by any means known in the art including, but not limited to, RT-PCR, Western blots, Northern blots, microarray analysis, immunoprecipitation, radiological assays, polypeptide purification, spectrophotometric analysis, Coomassie staining of acrylamide gels, ELISAs, 2-D gel electrophoresis, in situ hybridization, chemiluminescence, silver staining, enzymatic assays, ponceau S staining, multiplex RT-PCR, immunohistochemical assays, radioimmunoassay, colorimetric assays, immunoradiometric assays, positron emission tomography, fluorometric assays, fluorescence activated cell sorter staining of permeablized cells, radioimunnosorbent assays, real-time PCR, hybridization assays, sandwich immunoassays, flow cytometry, SAGE, differential amplification or electronic analysis. Expression may be analyzed directly or indirectly. Indirect expression analysis may include but is not limited to, analyzing levels of a product catalyzed by an enzyme to evaluate expression of the enzyme. See for example, Ausubel et al, eds (2013) Current Protocols in Molecular Biology, Wiley-lnterscience, New York, N. Y. and Coligan et al (2013) Protocols in Protein Science, Wiley-lnterscience New York, NY. Gene expression assays for porcine ASGR1 are commercially available (Applied BiosystemsTM, Carlsbad CA).

[0051] A “transplant product” encompasses organs, tissue, cells and / or transfusion products from an animal for use as xenografts. A transplant product from transgenic (e.g., knockout) pigs can be isolated from a prenatal, neonatal, immature or fully mature animal.PCT / US25 / 52258 23 October 2025 (23.10.2025)The transplant product may be used as temporary or permanent organ replacement for a human subject in need of an organ transplant.

[0052] Any porcine organ from a transgenic pig provided herein can be used including, but not limited to, the brain, heart, lungs, eye, stomach, pancreas, kidneys, liver, intestines, uterus, bladder, skin, hair, nails, ears, glands, nose, mouth, lips, spleen, gums, teeth, tongue, salivary glands, tonsils, pharynx, esophagus, large intestine, small intestine, small bowel, rectum, anus, thyroid gland, thymus gland, bones, cartilage, tendons, ligaments, suprarenal capsule, skeletal muscles, smooth muscles, blood vessels, blood, spinal cord, trachea, ureters, urethra, hypothalamus, pituitary, pylorus, adrenal glands, ovaries, oviducts, uterus, vagina, mammary glands, testes, seminal vesicles, penis, lymph, lymph nodes and lymph vessels.

[0053] Any porcine tissue from a transgenic pig provided herein can be used including, but not limited to, epithelium, connective tissue, blood, bone, cartilage, muscle, nerve, adenoid, adipose, areolar, brown adipose, cancellous muscle, cartilaginous, cavernous, chondroid, chromaffin, dartoic, elastic, epithelial, fatty, fibrohyaline, fibrous, Gamgee, gelatinous, granulation, gut-associated lymphoid, skeletal muscle, Haller's vascular, indifferent, interstitial, investing, islet, lymphatic, lymphoid, mesenchymal, mesonephric, multilocular adipose, thymus tissue, mucous connective, myeloid, nasion soft, nephrogenic, nodal, osteoid, osseus, osteogenic, bone marrow, retiform, periapical, reticular, smooth muscle, hard hemopoietic and subcutaneous tissue, devitalized animal tissues including heart valves, skin, and tendons, and vital porcine skin.

[0054] Any cells from a transgenic pig provided herein can be used including, but not limited to, epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, Islet of Langerhans cells, pancreatic insulin secreting cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, aortic endothelial cells, microvascular endothelial cells, fibroblasts, liver stellate cells, aortic smooth muscle cells, cardiac myocytes, neurons, Kupferr cells, smooth muscle cells, Schwann cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, thymus cells, parathyroid cells, parotid cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, heart cells, pacemaker cells, spleen cells, antigen presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testicular cells, germ cells, egg cells, leydig cells, peritubularPCT / US25 / 52258 23 October 2025 (23.10.2025)cells, sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, dopaminergic cells, squamous epithelial cells, osteocytes, osteoblasts, osteoclasts, bone marrow, embryonic stem cells, fibroblasts and fetal fibroblasts.

[0055] Nonviable derivatives from a transgenic pig provided herein can be used including, but not limited to, tissues stripped of viable cells by enzymatic or chemical treatment these tissue derivatives can be further processed through crosslinking or other chemical treatments prior to use in transplantation. The nonviable derivatives include extracellular matrix derived from a variety of tissues, including skin, bone, urinary, bladder or organ submucosal tissues. In addition, tendons, joints, and bones stripped of viable tissue to including but not limited to heart valves and other nonviable tissues as medical devices are provided. Serum or medium suitable for cell culture and isolated from a transgenic pig of the disclosure is provided. Components of porcine transgenic organs, tissues or cells are also provided. Components may also be modified through any means known in the art including but not limited to crosslinking and aldehyde crosslinking. Components may vary depending on the larger organ or tissue from which the component is obtained. Skin components may include, but are not limited to, stripped skin, collagen, epithelial cells, fibroblasts and dermis. Bone components may include but are not limited to collagen and extracellular matrix. Heart components may include but are not limited to valves and valve tissue.

[0056] As used herein, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. An isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype.

[0057] As used herein, the term “propagate” means to grow or alter the phenotype of a cell or population of cells. The term “grow” or “expand” refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, support cells, or any chemical or biological compound necessary for obtaining the desired number of cells or cell type. In one embodiment, the growing / expansion of cells results in the regeneration of tissue.PCT / US25 / 52258 23 October 2025 (23.10.2025)

[0058] As used herein, the term “culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation or division of cells.

[0059] As used herein, the “lineage” of a cell defines the heredity of the cell, i.e. its predecessors and progeny. The lineage of a cell places the cell within a hereditary scheme of development and differentiation.

[0060] As used herein, the term “substantially homogeneous” describes a population of cells in which more than about 50%, or alternatively more than about 60%, or alternatively more than 70%, or alternatively more than 75%, or alternatively more than 80%, or alternatively more than 85%, or alternatively more than 90%, or alternatively more than 95%, or alternatively more than 99% of the cells are of the same or similar phenotype. Phenotype can be determined by a pre-selected cell surface marker or other marker.

[0061] As used herein, the term “purified population” of cells of interest refers to the cell population that has been isolated away from substantially all other cells that exist in their native environment, but also when the proportion of the cells of interest in a mixture of cells is greater than would be found in their native environment. For example, a purified population of cells represents an enriched population of the cells of interest, even if other cells and cell types are also present in the enriched population. In some embodiments, a purified population of cells represents at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% of a mixed population of cells, with the proviso that the cells of interest comprise a greater percentage of the total cell population in the “purified” population than they did in the population prior to the purification.

[0062] As used herein, the term “population of cells” can refer to a collection of more than one cell that is identical (clonal) or non-identical in phenotype and / or genotype.

[0063] As used herein, “cell culture” refers to cells grown under controlled condition(s). A primary cell culture is a culture of cells, tissues, or organs, taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is often measured by the amount of time required for the cells to double in number,PCT / US25 / 52258 23 October 2025 (23.10.2025)otherwise known as the doubling time. As used herein, the term “cell colony” or “colony” refers to a grouping of closely associated cells formed as a result of cell growth.

[0064] Methods for treatment provided herein

[0065] As used herein, the terms “treatment,” “treating,” and the like, refer to administering a product or carrying out a procedure, for the purposes of obtaining an effect. The effect is prophylactic in terms of completely or partially preventing disease or symptom thereof in a subject at risk and / or is therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease in a subject. “Treatment,” as used herein, includes treatment of a disease or disorder in a mammal (in particular transplant rejection), particularly in a human, and includes: (a) prophylactic treatment, that is preventing the disease or a symptom of a disease from occurring in a subject which is predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that are associated with or caused by a primary disease), when used in conjunction with prophylactic methods, the term treatment means that, after treatment, a smaller number of subjects (on average) develop the undesired disease or disorder or progress in severity of symptoms; (b) therapeutic treatment inhibiting the disease, i.e., arresting its development; and (c) therapeutic treatment relieving the disease, i.e., causing regression of the disease. Treating refers to any clinical indicia of success in the treatment or amelioration or prevention, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of an examination by a physician. Accordingly, the term “treating” includes the administration of the transplant products provided herein to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with transplant rejection. The term “therapeutic effect” refers to the reduction, elimination, or prevention of transplant rejection, symptoms of transplant rejection, or side effects of transplant rejection in the subject.

[0066] As used herein, the term “delivery” refers to routes, approaches, formulations, technologies, and systems for transporting a transplant product to the body as needed to safely achieve its desired therapeutic effect. The route of delivery can be any suitable route, including but not limited to surgical, intravascular, intravenous, intraarterial, intramuscular, cutaneous, subcutaneous, percutaneous, intradermal, and intraepidermal routes.

[0067] As used herein, the term “effective amount” refers to a concentration or amount of transplant product, that is effective for producing an intended therapeutic result for the treatment of transplant rejection in a patient in need thereof. It will be appreciated that thePCT / US25 / 52258 23 October 2025 (23.10.2025)effective amount of product to be administered will vary depending on the specifics of the subject to be treated, including but not limited to size or total volume / surface area to be treated, as well as proximity of the site of administration to the location of the region to be treated, among other factors familiar to the medicinal biologist and / or treating physician.

[0068] As used herein, the terms “effective period (or time)” and “effective conditions” refer to a period of time or other controllable conditions (e.g., temperature, humidity for in vitro methods), necessary or preferred for transplant product to achieve its intended result.

[0069] An “effective response” in accordance with the present disclosure is achieved when the subject experiences partial or total alleviation or reduction of signs or symptoms of illness, retarding progression, cure, prolongation of survival, or other objective responses. The expected progression-free survival times can be measured in months to years, depending on prognostic factors including the number of relapses, stage of disease, and other factors. Prolonging survival includes without limitation times of at least 1 month (mo.), about at least 2 mos., about at least 3 mos., about at least 4 mos., about at least 6 mos., about at least 1 year, about at least 2 years, about at least 3 years, etc. Overall survival is also measured, for example, in months to years. Alternatively, an effective response can be that a subject’s symptoms remain static.

[0070] As used herein, the term “control” or “control group” refers to an alternative subject or sample used in an experiment for comparison purpose. A control can be “positive” or “negative”.

[0071] As used herein, the term “concurrently” refers to simultaneous ( / .e., in conjunction) administration.

[0072] As used herein, the term “sequentially” refers to separate ( / .e., at different times) administration. In one embodiment, the administration is staggered such that two or more pharmaceutically active ingredients are delivered separately at different times.

[0073] As used herein, the terms “autologous transfer”, “autologous transplantation”, “autograft” and the like refer to treatments wherein the transplant product originates from the subject. The terms “allogeneic transfer”, “allogeneic transplantation”, “allograft” and the like refer to treatments wherein the transplant is of the same species as the subject but is not derived from the subject. A cell transfer in which the organ has been histocompatibly matched with a recipient is sometimes referred to as a syngeneic transfer.

[0074] "Xenotransplantation" encompasses any procedure that involves the transplantation, implantation or infusion of cells, tissues, organs and / or transfusion product into a recipient subject from a different species. Xenotransplantation in which the recipient isPCT / US25 / 52258 23 October 2025 (23.10.2025)a human is particularly envisioned. A xenotransplant include, but is not limited to, a vascularized xenotransplant, partially vascularized xenotransplant, unvascularized xenotransplant, xenodressings, xenobandages, xenostructures and xenotransfusions.

[0075] “Transplant rejection” occurs when transplanted tissue, organs, cells or material are not accepted by the recipient's body. In transplant rejection, the recipient's immune system attacks the transplanted material. Multiple types of transplant rejection exist and may occur separately or together. Rejection processes included but are not limited to hyperacute rejection (HAR), acute humoral xenograft rejection reaction (AHXR), thrombocytopenia, acute humoral rejection, hyperacute vascular rejection, antibody mediated rejection and graft versus host disease. By "hyperacute rejection" we mean rejection of the transplanted material or tissue occurring or beginning within the first 24 hours post-transplant involving one or more mechanisms of rejection. Rejection encompasses but is not limited to "hyperacute rejection", "humoral rejection", "acute humoral rejection", "cellular rejection" and "antibody mediated rejection". The acute humoral xenograft reaction (AHXR) is characterized by a spectrum of pathologies including, but not limited to, acute antibody mediated rejection occurring within days of transplant, the development of thrombotic microangiopathy (TMA), microvascular angiopathy, pre-formed non-Gal IgM and IgG binding, complement activation, microvascular thrombosis and consumptive thrombocytopenia within the first few weeks post-transplant.

[0076] The disclosure provides a method of improving a rejection related symptom in a patient comprising transplanting a transplant product provided herein, wherein one or more rejection related symptoms is improved as compared to when tissue from a wild-type pig is transplanted into a human. Rejection-related symptoms include but are not limited to hyperacute rejection related symptoms and acute humoral xenograft reaction related symptoms. Rejection related symptoms may include, but are not limited to, thrombotic microangiopathy (TMA), microvascular angiopathy, pre-formed non-Gal IgM and IgG binding, complement activation, agglutination, fibrosis, microvascular thrombosis, consumptive thrombocytopenia, consumptive coagulopathy, profound thrombocytopenia, refractory coagulopathy, graft interstitial hemorrhage, mottling, cyanosis, edema, thrombosis, necrosis, fibrin thrombi formation, systemic disseminated intravascular coagulation, IgM deposition in glomerular capillaries, IgG deposition in glomerular capillaries, elevated creatinine levels, elevated BUN levels, T cell infiltrate, infiltrating eosinophils, infiltrating plasma cells, infiltrating neutrophils, arteritis, antibody binding to endothelium, altered expression of IGOS, CTLA-4, BTLA, PD-1, LAG-3, or TIM-3, and systemic inflammation.

[0077] "Hyperacute rejection related symptom" is intended to encompass any symptom known to the field as related to or caused by hyperacute rejection. It is recognized thatPCT / US25 / 52258 23 October 2025 (23.10.2025)hyperacute rejection related symptoms may vary depending upon the type of organ, tissue or cell that was transplanted. Hyperacute rejection related symptoms may include, but are not limited to, thrombotic occlusion, hemorrhage of the graft vasculature, neutrophil influx, ischemia, mottling, cyanosis, edema, organ failure, reduced organ function, necrosis, glomerular capillary thrombosis, lack of function, hemolysis, fever, clotting, decreased bile production, asthenia, hypotension, oliguria, coagulopathy, elevated serum aminotransferase levels, elevated alkaline phosphatase levels, jaundice, lethargy, acidosis and hyperbilirubenemia and thrombocytopenia.

[0078] Thrombocytopenia is a quantity of platelets below the normal range of 140,000 to 440,000 / pl. Thrombocytopenia related symptoms include, but are not limited to, internal hemorrhage, intracranial bleeding, hematuria, hematemesis, bleeding gums, abdominal distension, melena, prolonged menstruation, epistaxis, ecchymosis, petechiae or purpura. Uptake of human platelets by pig livers contributes to the development of thrombocytopenia in xenograft recipients.

[0079] Platelets, also known as thrombocytes, are enucleate fragments of megakaryocytes involved in blood coagulation, hemostasis and blood thrombus formation. Human platelets are routinely isolated through a variety of methods including, but not limited to, platelet apheresis, plateletpheresis and ultracentrifugation.

[0080] The phrase "platelet uptake" is intended to encompass the incorporation of a platelet into a liver or liver cell. While not being limited by mechanism, such uptake may occur through a phagocytic process. Platelet uptake may be monitored by any platelet uptake monitoring assay known in the art. Platelet uptake monitoring assays include, but are not limited to immunological methods, western blots, immunoblotting, microscopy, confocal microscopy, transmission electron microscopy and phagosome isolation. It is recognized that the appropriate platelet uptake monitoring assay may depend upon the type of label used. Platelet uptake may be measured as a percentage of total platelets absorbed, percentage of total platelets not absorbed, a ratio of absorbed to unabsorbed platelets, percentage of cells absorbing at least one platelet, percentage of cells not absorbing a platelet, or number of platelets absorbed per cell. It is recognized that platelet uptake by more than one cell type may contribute to the total platelet uptake of the liver. Total platelet uptake by an animal liver may include platelet uptake by liver sinusoidal endothelial cells, platelet uptake by Kuppffer cells, platelet uptake by LSECs and Kupffer cells and platelet uptake by additional cell types. It is recognized that platelet uptake by different cell types may contribute similar or disparate fractions of the total platelet uptake by a liver. Thus an alteration, inhibition, reduction, decrease, or lowering of platelet uptake by a liver comprises an alteration, inhibition, reduction, decrease, or lowering of platelet uptake by one or more liver cell types.PCT / US25 / 52258 23 October 2025 (23.10.2025)

[0081] While not being limited by mechanism, platelet uptake may occur through phagocytosis by LSEC and Kupffer cells. Phagocytosis is characterized by the formation of an endosome which by the fusion of lysosomes containing degradative enzymes becomes a phagosome.

[0082] Any method of evaluating, assessing, analyzing, measuring, quantifying, or determining a rejection related symptom known in the art may be used with the claimed compositions and methods. Methods of analyzing a rejection related symptom may include, but are not limited to, laboratory assessments including CBC with platelet count, coagulation studies, liver function tests, flow cytometry, immunohistochemistry, standard diagnostic criteria, immunological methods, western blots, immunoblotting, microscopy, confocal microscopy, transmission electron microscopy, IgG binding assays, IgM binding assays, expression assays, creatinine assays and phagosome isolation.

[0083] A "skin related product" encompasses products isolated from skin and products intended for use with skin. Skin related products isolated from skin or other tissues may be modified before use with skin. Skin related products include but are not limited to replacement dressings, burn coverings, dermal products, replacement dermis, dermal fibroblasts, collagen, chondroitin, connective tissue, keratinocytes, cell-free xenodermis, cell-free pig dermis, composite skin substitutes and epidermis and temporary wound coverings. See for example Matou-Kovd et al (1994) Ann Med Burn Club 7:143, herein incorporated by reference in its entirety.

[0084] The attachment period of a skin related product is the time between application of the skin related product to a human subject and natural separation of the skin related product from the human subject. When a human subject's skin wound has sealed, a skin related product may be removed by natural separation or mechanical separation. However natural separation of a skin related product from a human subject may occur prematurely. Premature natural separation occurs before separation is desired by a medical practitioner. By way of example and not limitation, premature natural separation may occur before the wound has been sealed. Premature natural separation may also be termed "sloughing", "shedding", or "flaking". Clinical management of premature natural separation may include reapplication of a skin related product, dressing application, bandage application, administering antibiotic, and administering fluids. A skin wound may be sealed by any means known in the art including but not limited to by growth of the subject's skin and by skin grafting. Reduced premature separation encompasses a decreased, lower, less frequent, diminished, smaller amount of natural separation of a skin related product before separation is desired by a medical practitioner. The reduced premature separation may relate to a lower number of complete, a lower number of partial premature separation events, andPCT / US25 / 52258 23 October 2025 (23.10.2025)involvement of a smaller portion of the skin related product in a partial premature separation event than compared to a skin related product obtained from a wild-type pig. A skin related product of the instant application may also exhibit an increased, lengthened, improved, extended, or expanded attachment period. Use of a skin related product of the instant application may increase the duration of the attachment period.

[0085] A skin wound encompasses any injury to the integument including but not limited to an open wound, burn, laceration, ulcer, leg ulcer, foot ulcer, melanoma removal, cancer removal, plastic surgery, and bite.

[0086] By "surgically attaching" is intended joining, combining, uniting, attaching, fastening, connecting, joining or associating through any surgical method known in the art.

[0087] The disclosure provides transfusion products, i.e., non-human material suitable for xenotransfusions. Transfusion products include, but are not limited to, blood, whole blood, plasma, serum, red blood cells, platelets, and white bloods cells. Such transfusion products may be isolated, enriched or purified. Methods of isolating, enriching or purifying transfusion products suitable for transfusion are known in the art.

[0088] Other terminology and disclosure

[0089] As used herein, the term “comprising” or “comprises” means that the compositions and methods include the steps and / or elements recited, but do not exclude other steps or elements. When used herein, the term "comprising" can be substituted with the term "containing" or "including" or "having." When used herein, the term "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the subject matter of a claim. “Consisting of” shall mean excluding more than trace elements of other ingredients and / or substantial method steps.

[0090] As used herein, “may,” “may comprise,” “may be,” “can,” “can comprise,” “can be” and “is contemplated herein” all indicate something envisaged by the inventors that is functional and available as part of the subject matter provided.

[0091] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any element, e.g., any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0092] When a range of values is provided herein, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value inPCT / US25 / 52258 23 October 2025 (23.10.2025)that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0093] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes the concrete number, e.g., about 10 includes 10.

[0094] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure.

[0095] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. This disclosure is intended to provide support for all such combinations.

[0096] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials for the purpose for which the publications are cited. If any material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0097] AbbreviationsAA- amino acidAMR - antibody mediated rejectionB4GalNt2 - Beta-1,4-N-acetyl-galactosaminyltransferase 2CMAH - cytidine monophosphate-N-acetylneuraminic acid hydroxylaseCPF1- Cas12CIITA- Class II TransactivatorEGM-2MV - microvascular endothelial cell growth medium-2FBS - fetal bovine serumFITC - fluorescein isothiocyanateGFP - green fluorescence proteinGGTA1- glycoprotein, alpha-galactosyltransferase 1HEPES - 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHLA - human leukocyte antigenKO- knockoutIFN-y- interferon gammaMEM-a - minimum essential medium-alphaPCT / US25 / 52258 23 October 2025 (23.10.2025)NCSU-23 - North Carolina State University-23PBMC- peripheral blood mononuclear cellsPBS - phosphate-buffered salinePSI - pounds per square inchRT-PCR - reverse transcriptase polymerase chain reactionsgRNA - single guide ribonucleic acidSCNT - somatic cell nuclear transferSLA -swine leukocyte antigenExamples

[0098] While the following example describes specific embodiments, variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be placed on the invention.Example 1Methods

[0099] Human and Animal Research Oversight

[0100] Human serum samples were collected by venipuncture under a protocol approved by the University of Miami Institutional Review Board. All animal work was performed under IACUC approved protocols at the University of Miami.

[0101] Development of e-C1R Cells Expressing SLA-DQ Proteins

[0102] DNA constructs, synthesized to contain the open reading frames of SLA-DQa alleles and SLA-DQb alleles connected by an internal ribosome entry sequence, were cloned into the mammalian expression vector pREP4 (Invitrogen). Alpha and beta chain pairs are shown in Figure 1 A. Relevant SLA open reading frame cDNA sequences were obtained from the IMGT MHC database (16). These constructs were electroporated into the engineered human B-lymphoblastoid C1 R cell line (eC1 R), which was subsequently maintained with hygromycin at 200 ug / ml (Invitrogen) in RPMI plus 10% FBS. The e-C1 R lack multiple surface molecules (HLA class I and class II, Fc-receptor, and IgG), which cause background binding of antibodies in human sera (17). SLA-DQ expression was validated using anti-class ll-DQ (Bio-Rad, CA) and a secondary Ab-AF-647-conjugated Fragment Donkey Anti-Mouse IgG (JacksonlmmunoResearch, PA).

[0103] Evaluating Antibody Binding to SLA-DQ proteins in eC1R Cells

[0104] To assess the levels of SLA-DQ reactive IgG and IgM in patient sera, a flow cytometric crossmatch assay was performed as described previously (17). Briefly, patient sera in 1:4 dilution were tested against each eC1 R cell line expressing individual SLA-DQ proteins. Alexa Fluor 488 goat anti-human IgG (catalog no. 109-546-097, Jackson ImmunoResearchPCT / US25 / 52258 23 October 2025 (23.10.2025)Laboratories Inc.) or Alexa Fluor 488 goat anti-human IgM (catalog no. 109-546-129, Jackson ImmunoResearch Laboratories Inc.) were used as the secondary Abs. Flow cytometric data were acquired using a BD FACSLyric flow cytometer. A gating strategy using forward scatter (FSC) and side scatter (SSC), followed by comparing FSC-height versus FSC-area to identify singlet cells, was used to define the population of cells being analyzed. Flow cytometry files were analyzed in FlowJo version 10 (BD Biosciences, Ashland, OR).

[0105] Cell Culture of Cells Used to Create SLA-DQ Knockout Pigs

[0106] Fetal fibroblasts from a cloned GGTA1 / b4GalNt2 KO pig with known class II SLA DR and DQ alleles were used in this study (1, 18). Fetal fibroblasts cultured in stem cell media (FFSCs) were resuspended and cultured in MEM-a (Invitrogen, Carlsbad, CA) plus EGM-MV (Lonza, Basel, Switzerland) supplemented with 10% FBS (HyClone, Logan, UT), 10% horse serum (Invitrogen), 12mM HEPES (Sigma-Aldrich, St. Louis, MO) and 1% penicillin / streptomycin (Life Technologies, Grand Island, NY) and cultured in collagen-l-coated plates (Becton Dickinson, Bedford, MA) at 38.5°C, 5%CO2 and 10%O2.

[0107] Generation of CRISPR Tools to Inactivate SLA-DQb Genes.

[0108] Plasmid pSpCas9(BB)-2A-GFP (Addgene # 48138, donated by Feng Zhang) was used to clone the designed annealed oligos. Custom oligos were selected using the web tool CRISPOR (crisDor.tefor.net) and purchased from IDT (Integrated DNA Technologies, Morrisville, NC). Insertion of annealed oligos into Bbsl site was performed using previously published protocols (19). gRNAs were designed to target exons 2 and 3 of the beta chain for SLA-DQ gene (Alleles SLA-DQb1*03.03 and *06.01.

[0109] Transfection and Recovery of CRISPR Targeted Cells

[0110] Fetal fibroblast cells in early passage (passage 3), were expanded to about 1 x106cells and transfected with the two plasmids encoding the SLA-DQB gRNA, Cas9 and green fluorescent protein (GFP). Cells, resuspended in R buffer and 1.5μg of plasmid DNA, were transfected using the Neon® Transfection System (Invitrogen, Life Technologies, CA). 100μL Neon tips were used to deliver two 20 millisecond pulses of 1400V. Transfected cells were seeded into collagen l-coated plates 6-well plate (Becton Dickinson, MA) with MEM-a / EGM-MV (Invitrogen, CA / Lonza, Switzerland) media supplemented with 10% FBS (HyClone, UT), 10% horse serum (Invitrogen, CA), 12 mM HEPES (Sigma-Aldrich, MO) without antibiotics. Media was removed on the next day, and fresh media containing 1% penicillin / streptomycin (Life Technologies, NY) was added. Cells were cultured at 38.5 -C, 5% CO2 and 10% O2.

[0111] After 3 days of transfection, GFP positive cells were sorted and expanded to a population of about 5x106cells. Cells were induced with recombinant porcine IFN-gamma (IFN-g) for 60h prior to testing for SLA-DR and SLA-DQ expression. Cells were confirmed to be SLA Class ll-DQ negative and Class ll-DR positive by incubation with anti SLA-DQ and anti-SLA-DR antibodies respectively. This was achieved by staining with anti-class ll-DQPCT / US25 / 52258 23 October 2025 (23.10.2025)(Bio-Rad, CA; clone K274.3G8) for 30 min and rinsing with PBS twice before staining for another 30 min with the conjugated secondary antibody - Donkey Anti-Mouse IgG Alexa Fluor 647(Jackson ImmunoResearch, PA). Cells were rinsed again twice with PBS and blocked with Normal Mouse Serum (EMD Millipore Corporation, CA) for 10 min. The final staining was performed with a directly labeled anti-class II DR-FITC (Bio-Rad, CA; clone 2E9 / 13). All incubations were done on ice in the dark.

[0112] Cells that did not express SLA-DQ were sorted using a Beckman Coulter MoFlo Astrios EQ cell sorter equipped with 5 lasers and Summit 6.2 software (Beckman Coulter Inc., USA). The cell sorter was operated with a 100pm nozzle, at 25 psi, and an event rate of approximately 10,000 events per second. This was performed at the Flow Cytometry Core Facility in the Diabetes Research Institute at the Miami Miller School of Medicine.

[0113] Verification of SLA-DQ Inactivation by DNA Sequencing

[0114] Genomic DNA was isolated from pig cells using the QIAmp DNA mini kit (Qiagen). RNA samples were isolated using RNeasy Plus mini kit (Qiagen) following the manufacturer’s protocol. RNA quality and quantity were affirmed by Agilent bioanalyzer analysis (Core Facility of the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine). RNA samples were reverse transcribed using a OneStep RT-PCR kit (Qiagen). The PCR primer sequences used to amplify SLA-DQb transcripts amplify the entire open reading frame (ORF start: 5’-ATGTCTGGGATGGTGGCTCT-3+; ORF stop: 5’-CCAACCCCAAAGTATCTTCAGGA-3’). PCR products were purified and ligated into the pCR4-TOPO TA vector (Invitrogen). Transformed bacteria were plated into Luria-Bertani agar containing 50pg / mL kanamycin for clone selection. Plasmids were isolated using the QIAprep Spin Miniprep kit (Qiagen). Nucleotide sequences were performed by the Sanger method using custom sequencing service (Azenta, South Plainfield, NJ).

[0115] Somatic cell nuclear transfer (SCNT)

[0116] SCNT was performed as described previously (20) using in vitro matured oocytes (DeSoto Biosciences Inc., St. Seymour, TN, USA). Cumulus cells were removed from the oocytes by pipetting in 0.1% hyaluronidase. Only oocytes with normal morphology and a visible polar body were selected for SCNT. Oocytes were incubated in manipulation media (Ca2+-free NCSU-23 with 5% FBS) containing 5 pg / ml bisbenzimide and 7.5 pg / ml cytochalasin B for 15 min. Oocytes were enucleated by removing the first polar body plus metaphase II plate, and one cell was injected into the perivitelline space of each enucleated oocyte. Couples were fused and activated simultaneously by two DC pulses of 180 V for 50 ps (BTX cell electroporator, Harvard Apparatus, Hollison, MA, USA) in 280 mM mannitol, 0.1 mM CaCl2, and 0.05 mM MgCl2. Activated embryos were placed back in NCSU-23 medium with 0.4% bovine serum albumin (BSA) and cultured at 38.5°C, 5% CO2 in a humidifiedPCT / US25 / 52258 23 October 2025 (23.10.2025)atmosphere for <1 h, before being transferred into the recipient. Recipients were synchronized occidental gilts on their first day of estrus.

[0117] Pig PBMCs Isolation for Use in Flow Phenotyping and Crossmatchina Assays

[0118] Peripheral blood mononuclear cells (PBMCs) were isolated from pig whole blood collected in Acid citrate dextrose (ACD) using Ficoll-Paque Plus (Cytiva) separation.Anticoagulated pig blood was diluted 1:1 with phosphate-buffered saline (PBS) Ca2+ / Mg2+free and layered onto Ficoll-Paque. Samples were centrifuged at 400g for 30 minutes and the PBMC layer was transferred to a new tube where red blood cells (RBC) lysis was performed using DI water for 15-30 seconds. Viable cells were counted using trypan blue (Thermo Fisher).

[0119] Flow Cytometric Analysis of SLA-DR and SLA-DQ on Peripheral B Lymphocytes

[0120] To evaluate SLA-DR expression, pig PBMC were incubated simultaneously with directly conjugated CD21 -PE (MA1 -19753, Invitrogen) and directly conjugated anti-SLA-DR-FITC (29E / 13, Biorad) antibodies. To evaluate SLA-DQ expression, PBMCs were incubated with unconjugated anti-SLA-DQ (Bio-Rad, CA; clone K274.3G8) and then with secondary antibody -Donkey Anti-Mouse IgG Alexa Fluor 647 (JacksonlmmunoResearch, PA). Next, cells were blocked with Normal Mouse Serum (EMD Millipore Corporation, CA) and then incubated with the anti-CD21 antibody. Cells were fixed with Fixation Buffer (Biolegend).

[0121] Flow cytometric analyses gated on lymphocytes by FSC (forward scatter) and SSC (side scatter) area followed by gating on singlet cells using FSC area vs FSC height. CD21 positive cells (B-lymphocytes) were gated and analyzed for SLA-DR or SLA-DQ expression.

[0122] Microscopic Analysis of Class II SLA Expression in Pic Tissues

[0123] The tissue expression of SLA-DR (2E9 / 13; Biorad), -DQ (K274.3G8; Biorad), CD21 (MA1 -19753; Invi trogen), CD31 (#AF3387; R& D systems), or insulin (ab151742;Abeam) was evaluated using an IX81 / FV1000 confocal microscope. Tissue sections (8pm) were fixed in 4% paraformaldehyde then blocked with 0.5% bovine serum albumin (BSA) in PBS followed by labeling with anti-pig CD31 (#AF3387; R& D systems) followed by secondary antibodies, donkey anti-mouse IgG Alexa Fluor 488, donkey anti-rat IgG Alexa Fluor 647 (Jackson Immunoresearch Laboratories). Confocal microscopy images were captured using the settings of the negative control for that experiment. Images were uniformly cropped or adjusted in Powerpoint (Microsoft).

[0124] Human Serum Absorption with Pic Red Blood Cells

[0125] To remove non-SLA xenoreactive antibodies in human serum, an equal volume of pig DKO SLA-DQ deficient RBCs was incubated with each human serum for 1 hour at room temperature. Depleted serum was recovered by centrifugation of the RBC / serum mixture at 20,000 g for 1 minute.PCT / US25 / 52258 23 October 2025 (23.10.2025)

[0126] Human Anti-Pig PBMC Flow Cytometric Crossmatchina Assay

[0127] The following steps were all performed at room temperature. Ficoll-Paque isolated pig PBMCs 0.2 x 106were incubated with 25% heat-inactivated and RBC absorbed human serum for 30 minutes and then washed twice with Minimum Essential Medium Eagle (MEM) (SigmaAldrich). PBMCs were incubated with donkey anti-human IgG Alexa Fluor 488 (cat# 709-546-098) or goat anti-human IgM Alexa Fluor 488 (cat# 109-546-129) (1 / 200) (Jackson ImmunoResearch) for 30 minutes. PBMCs were washed twice and fixed with Fixation Buffer (Biolegend) for 15 min. Samples were acquired on a FACS Lyric flow cytometer (BD Biosciences) and analysis was performed using FlowJo software (v.10.9.0, TreeStar).Results

[0128] We developed seven cell lines, each expressing a unique SLA-DQ protein, to add to our understanding of SLA-DQ as a humoral xenoantigen. The SLA-DQ proteins studied and the cell surface abundance of each are shown in Figure 1 A. Next, we incubated these cells with 40 human sera (Figure 1 B and C); 18 sera were from patients that produced class II HLA specific IgG, and 22 naive sera did not contain IgG specific for class II HLA proteins. The levels of IgG and IgM that bound the cell surface were examined using flow cytometry. In 39 of 40 sera, the empty vector background control (red symbols in Figure 1 B) yielded the lowest IgM median-fluorescence intensity (MFI) of all cell lines tested, with the sole exception being serum 22. For IgG binding, the empty vector control generated the lowest MFI in 30 of 40 sera tested (red symbols in Figure 1 C; samples 1, 3, 4, 9, 10, 11, 17, 18, 20, 25, 27, 33, 34, 35, 36, 37, 38, 6, 7, 13, 14, 15,16, 21, 22, 23, 30, 31, 39, 40). These data reveal a broad humoral reactivity of human antibodies towards SLA-DQ that occurs without requiring HLA-specific antibodies to be present. Therefore, we wanted to determine if eliminating SLA-DQ from pigs could reduce patients’ antibody binding to cells from those animals. This necessitated the creation of pigs devoid of SLA-DQ proteins.

[0129] When designing SLA-DQ knockout animals, we relied on our prior biosynthetic analyses of the class II SLA proteins specific to our pig model. The SLA haplotypes present in these animals produce SLA-DQ beta chains capable of cross pairing with SLA-DR alpha chains (reference 14, and Figure 2A). Therefore, we inactivated both alleles of the SLA-DQ beta chains to prevent expression of cell surface SLA-DQ proteins and SLA-DRa / DQb hybrids. SLA-DQb targets of the CRISPR tools are shown in Figure 2B. IFN-γ stimulation of pig fibroblasts was required in vitro to induce expression of SLA-DR and SLA-DQ (Figure 2C, middle panel). When those cells were edited with SLA-DQ-specific gRNA, we observed a population of fibroblasts expressing only SLA-DR (Figure 2C, right panel). These edited cells were isolated by FACS and used to produce genetically engineered animals lackingPCT / US25 / 52258 23 October 2025 (23.10.2025)SLA-DQ. The Table below provides a summary of the cloning success rates and the resultant SLA-DQ gene modifications.PCT / US25 / 52258 23 October 2025 (23.10.2025)Table: Cloning ResultsPCT / US25 / 52258 23 October 2025 (23.10.2025)

[0130] In pigs, B-cells (CD21+PBMCs) possess functional class II SLA genes expressing abundant SLA-DQ and SLA-DR. Notably, when B-cells, from the peripheral blood and spleen of SLA-DQb KO pigs were examined, we observed that these cells do not make cell surface SLA-DQ proteins while expressing SLA-DR (Figures 3A, B, and C for peripheral blood; Figure 3D, spleen). Moreover, SLA-DQ proteins are also absent on the solid organs from these pigs including kidney, liver, heart, pancreas and spleen (Figure 3D). As observed in the B cells, the tissues continue to express SLA-DR. Finally, to determine if reducing human antibody binding to pig cells could be achieved by eliminating SLA-DQ on pig primary cells, we incubated PBMC from these animals with human sera collected from transplant patients. Apart from the SLA-DQ genes, all animals contain identical genetic backgrounds to minimize variations in other potential antigens.17 samples were tested from people lacking HLA IgG (Figure 4, panel A) and another 17 samples were tested from people who had circulating HLA IgG (Figure 4, panel B). Eliminating SLA-DQ on pig PBMC reduced the binding of human IgG and IgM when compared to PBMC from SLA-DQ expressing animals. For human sera lacking HLA IgG, 14 of 17 samples showed lower IgG and 12 of 17 and had less IgM binding SLA-DQ deficient PBMC. Similarly, in the 17 patients with antibodies specific to class II HLA, 12 had reduced IgG and 11 had lower IgM binding to PBMC isolated from SLA-DQ knockout pigs than PBMC originating from SLA-DQ expressing animals.Discussion

[0131] Late graft failure (>100 days) in preclinical renal xenotransplantation is characterized by glomerulopathy, proteinuria, and the presence of donor specific anti-SLA-DQ antibodies in recipient serum (13,21). This resembles what occurs in late allograft failure where glomerulopathy and donor specific anti-class II HLA antibodies, especially HLA-DQ, are routinely found in recipient sera (12). Increasing numbers of AA mismatches between donor and recipient HLA-DR and -DQ make late AMR more likely to occur (9-12). Since the degree of class II MHC mismatch between pig and human will be greater than that which occurs in allotransplantation we evaluated recipients for anti-SLA-DQ antibodies prior to receiving a transplant. Our eC1 r SLA-DQ transfectants suggest many patients will have anti-SLA-DQ antibodies prior to receiving a xenograft. Anti-SLA-DQ reactivity was generalized to the different SLA-DQ alleles we evaluated, making a “magic” SLA-DQ that avoids DSA unlikely. There were patients with no HLA sensitization who had anti-SLA-DQ antibodies, consistent with our earlier results (14).

[0132] To further study the role of SLA-DQ as a xenoantigen, we describe the creation of 4 healthy (5 total) SLA-DQ KO pigs on the GGTA1 / p4GalNt2 KO background. At the time of publication, the animals have been alive and healthy for 10 months. One pig had intestinalPCT / US25 / 52258 23 October 2025 (23.10.2025)volvulus and was sacrificed at 1-week of age. All cell types tested from these animals exhibit total loss of cell surface SLA-DQ proteins. Work with H-2 and HLA showed that it is possible to delete class II a or p chains and still have class II expression because of the interlocus hybrid pairing of SLA-DR and SLA-DQ chains (22,23). Our prior work showed that while SLA-DQc' and SLA-DR13 failed to create functional molecules, SLA-DRa and SLA-DQp formed hybrid proteins detectable at the cell surface (14). Consequently, we chose to disrupt SLA-DQb genes to block synthesis of normal and hybrid SLA-DQ proteins in pigs.

[0133] Evaluation of naive and allosensitized patient serum with the SLA-DQ KO pig PBMC yielded unexpected results, since at least half of the patients had some anti-SLA-DQ antibodies regardless of allosensitization status. In allotransplantation, the presence of antiFI LA-DQ antibodies pre-transplant is associated with reduced long term graft survival and development of glomerulopathy. Given the results of our crossmatching it seems likely that SLA-DQ KO may be helpful for some patients with regards to reducing the antibody barrier to long-term graft success. Evaluation of the SLA-DQ KO in a preclinical renal xenograft model will be critical to determine the impact of SLA-DQ on long term graft survival.

[0134] Though Figure 1 indicates most patients contain IgM and IgG that recognize SLA-DQ, we noted in Figure 4 that eliminating SLA-DQ expression on pig cells did not provide universal benefit. Some samples exhibited no improvement or improvement for only IgG or IgM, and other measurements increased on the SLA-DQ deficient PBMC. To better understand these observations, the assay used in Figure 4 will need to be modified in the future. In this first iteration, we chose to simply mix pig PBMC with human serum followed by detection reagents for IgG or IgM. Lymphocytes, the bulk of PBMC, typically contain about 4% B-cells which all express class II proteins. CD8 positive lymphocytes also make class II SLA proteins, but CD4 cells do not (24). Therefore, many cells in our assay only contribute background noise rather than SLA-specific signal, likely reducing the assay sensitivity. In addition, we have noted that many patients contain antibodies to SLA-DR as well (14). It is possible that some of the sera used here contain antibodies which recognize the SLA-DR expressed in our SLA-DQ knockouts (Figure 3). Differences in SLA-DR expression levels by the SLA-DQ KO PBMC and the unedited comparator PBMC, may explain the increased binding observed in some samples. Nevertheless, of the 34 total patients analyzed, 18 showed reduced IgG and IgM binding to the SLA-DQ knockout pig PBMC.

[0135] As in allotransplantation, crossmatching may be used to avoid patients with antibodies that recognize SLA-DR. We chose to modify one gene at a time in the donor pig to simplify the analysis of each change. An alternative approach is to create pigs lacking both SLA-DQ and SLA-DR genes. This has been achieved by others who have created animals with an inactive Class II transactivator (CIITA) gene. CIITA is a transcription factorPCT / US25 / 52258 23 October 2025 (23.10.2025)that drives expression of SLA-DR and SLA-DQ in pigs (25-27). Loss of class II expression altered the relative abundance of CD4+ versus CD8+ T-cells; otherwise, those animals appeared healthy. Though not analyzed here, we expect SLA-DQ knockouts to have less of an impact on the pig immune system than CIITA inactivation. In addition, we prefer to disrupt the SLA structural genes rather than transcription factors which drive their expression. We aim to avoid any possibility of SLA gene activity that may occur for reasons that we do not yet understand. In the homologous human system, CIITA also drives transcription of class II HLA genes (28). Notably, class II HLA gene expression has been detected in the absence of functional CIITA (29).

[0136] Concerns regarding deleting any part of the MHC of a donor pig include: (i) the susceptibility to infectious pathogens and long-term health of the pig, (ii) the ability of the DQ knockout pig organ to participate in a protective immune response mounted by a human xenograft recipient exposed to various pathogens. Deletion of any class II MHC antigens is directly related to the CD4+ T cell response which is an important part of the viral immune response via promotion of CD8+ T cell responses as well as humoral immunity, a significant consideration given the concerns for viral zoonotic risks associated with xenotransplantation. There are other examples of class II deletions that occur naturally in mammals that suggest that the immune system will not necessarily be compromised by the deletion of SLA-DQ. All species of cats have deleted their entire set of DQ alleles in the feline leukocyte antigen complex (30, 31). The cheetah had been considered a model of disease susceptibility because of low genetic diversity at the genes of the MHC complex, but even with the low numbers of expressed MHC DRp alleles and no DQ alleles, the immunocompetence of free-ranging cheetahs in Namibia was not compromised (32). In addition, there are strains of naturally occurring mice that lack the H-2 l / E antigens (HLA-DR equivalent) that are healthy without apparent effects regarding disease susceptibility (33). Thus far, the SLA-DQ KO pigs have shown no compromise with regards to immunocompetence, consistent with the findings in other species with limited class II allele expression.

[0137] The SLA-DQ KO pigs should be useful for patients with anti-HLA-DQ antibodies that cross react with SLA-DQ, as well as those patients with anti-SLA-DQ antibodies who are not sensitized. The absence of the SLA-DQ in the pig renal xenograft should improve the cross match for those with anti-SLA-DQ antibodies that if allowed to react in renal xenograft containing SLA-DQ would result in accelerated transplant glomerulopathy and graft loss. These SLA-DQ KO pigs will enable the testing of the hypothesis that de novo anti-SLA-DQ antibodies are critical to the pathogenesis of transplant glomerulopathy and proteinuria seen in late renal xenotransplantation in preclinical models.PCT / US25 / 52258 23 October 2025 (23.10.2025)

[0138] These pigs will also be useful in the evaluation of SLA-DQ and its role in T cell mediated xenograft rejection in preclinical as well as in vitro models. Previous work has shown that renal xenograft survival was more dependent upon CD4+T cells and that the in vitro proliferative response of rhesus CD4+T cells was significantly reduced in cells devoid of SLA class I. These pigs will allow more detailed dissection of the mechanism of T cell rejection in preclinical as well as clinical models. It is also possible that some version of SLA class II deletion in donor pigs could replace the need for T cell depletion as part of the induction immunosuppression regimen that has resulted in improved survival in preclinical models (21). These SLA KO pigs will enable studies that begin to address these questions.

[0139] In summary, we have shown that many patients both naive and HLA class II sensitized have anti-SLA-DQ antibodies. The antibodies recognize multiple SLA-DQ and are not specific to a single allele. We have produced healthy pigs devoid of SLA-DQ on the GGTA1 / p4GalNt2 KO background with no expression of SLA-DQ in transplantable organs. These pigs should be valuable for delineating the role of SLA-DQ in the development of transplant glomerulopathy in late xenograft failure. These pigs are also contemplated to offer a viable approach to transplanting human recipients who have anti-SLA-DQ antibodies.Example 2

[0140] A parallel approach to that described in Example 1 was used to obtain SLA-DRa knockout cells. In the pigs of the Examples herein, SLA-DRa can pair with SLA-DQ, but SLA-DQa does not pair with SLA-DR to form functional proteins. Therefore, to generate pigs without any cell surface SLA-DR, we disrupted SLA-DRa protein expression.

[0141] Figure 5 shows CRISPR Cas9 guide RNAs used to disrupt the SLA-DRa gene. The gRNA sites are in exon 2 of the gene which encodes the alpha 1 domain of the mature protein.

[0142] After treating fibroblasts (the cells used to make cloned pigs) with the gRNA / Cas9 shown in Figure 5, the fibroblasts were evaluated for loss of SLA-DRa expression. This was achieved by treating the cells with interferon gamma (I FNy) for 1 to 4 days to induce SLA-DR and SLA-DQ cell surface expression. Cells lacking SLA-DR expression but still expressing SLA-DQ were sorted to obtain a population of SLA-DR negative / SLA-DQ positive cells. Figure 6 shows these cells in the box in the lower right of each plot. The plot on the left is IFNy-treated parent fibroblasts that had not been CRISPR treated. The plot on the right is those same fibroblasts after CRISPR / Cas9 editing.PCT / US25 / 52258 23 October 2025 (23.10.2025)

[0143] Edited fibroblasts from Figure 6 were used in SCNT and embryo transfer to create cloned (transgenic) pigs. Skin fibroblasts were edited from cloned piglets and treated with IFNy to induce SLA-DR and SLA-DQ gene expression. Fibroblasts from unedited pigs were included for comparison. The Figure 7 graph in the lower left (WT no IFNy) were unedited cells showing that in the absence of IFNy, no SLA-DQ is expressed and very little SLA-DR. The Figure 7 upper left graph (WT + IFNy) shows that interferon-gamma treatment is needed to induce SLA-DR and SLA-DQ to high levels. The remaining Figure 7 graphs show the piglet number and the result of treating fibroblasts from each animal with IFNy and staining for SLA-DR and SLA-DQ expression. The cells from the edited animals lack SLA-DR expression but continue to express high levels of SLA-DQ when induced with IFNy.

[0144] PCR was used to amplify the open reading frames of both SLA-DR alleles (SLA-DRa*02:01:01 and SLA-DRa*04:01). The PCR products were subjected to Sanger sequencing. Figure 8 shows alleles had either had many bases deleted, or the PCR product was not detected indicating gene disruptions that prevented stable transcripts from being produced.

[0145] Kidney, liver, and heart were collected from a cloned pig and stained for CD31 (Figure 9 green), and anti-SLA-DR (Figure 9 Red top graph, DR-KO) or anti-SLA-DQ (Figure 9 Red bottom graph, DR-KO). Cells that had not their class II SLA genes edited were used as positive controls to demonstrate SLA-DR and SLA-DQ expression typically occur in each tissue.

[0146] Thus, use of transplant products from SLA-DQ and / or SLA-DR KO pigs is contemplated herein to offer a viable approach for transplanting human recipients who have anti-SLA-DQ and / or anti-SLA-DR antibodies.Example 3

[0147] SLA DR KO pigs on the GGTA1 / B4GALNT2 KO background were born healthy and devoid of infectious proclivities. The pigs grew normally and are now more than 1-year old. The pigs intended for preclinical experiments described below and in Figure 10 were devoid of any zoonotic viruses except for PERV C as the pigs were PERV C+ from the outset. The present disclosure contemplates TKO SLA DR KO clinical pigs are PERV C negative. Four rhesus monkeys received GGTA1 / B4GALNT2 / SLA DR KO kidneys as follows.

[0148] Preclinical Experiment SeriesPCT / US25 / 52258 23 October 2025 (23.10.2025)

[0149] Case 1 (Vienna): A GGTA1 / B4GALNT2 / SLA DR KO kidney was transplanted into a rhesus monkey using an anti-CD154 based immunosuppression regimen.

[0150] The immunosuppressive protocol used T cell depletion, B cell depletion, and transient anti-complement therapy as induction immunosuppression. The T cell depletion rids the recipient of memory T cells that cause costimulation blockade-resistant xenograft rejection. The anti-complement therapy prevents early IgM mediated graft loss. The baseline immunosuppression in these transplants consisted of anti-CD154 (5c8), mycophenolic acid, and steroids. Unlike other xenograft efforts in the art which utilize CD40 / CD154 based costimulation blockade maintenance immunosuppression, no tacrolimus or rapamycin was added to the protocol.

[0151] The Vienna recipient was screened using standard PBMC flow cytometric crossmatches with GGTA1 / B4GALNT2 KO, GGTA1 / B4GALNT2 / SLA DR KO, and GGTA1 / B4GALNT2 / SLA DQ KO PBMCs. The recipient rejected the GGTA1 / B4GALNT2 / SLA DR KO kidney at 1-week post-transplant, and explant pathology showed thrombotic microangiopathy and cellular infiltration. Immunofluorescent staining showed diffuse IgG staining throughout which is unusual for xenograft rejection at this time point. A post-transplant SLA assay showed that the recipient was in fact sensitized to the donor SLA, having anti-IgG antibodies against both SLA DQ alleles with MFIs of X and Y on flow cytometric crossmatch. This is contemplated herein to be the cause of the early graft loss in the Vienna recipient.

[0152] Case 2 (Wellington): A GGTA1 / B4GALNT2 / SLA DR KO kidney was transplanted into a rhesus monkey using the anti-CD154 based immunosuppression regimen. This recipient was also screened using standard PBMC flow cytometric crossmatches with GGTA1 / B4GALNT2 KO, GGTA1 / B4GALNT2 / SLA DR KO, and GGTA1 / B4GALNT2 / -SLA DQ KO PBMCs. This recipient did well until post-transplant day 126 when it rejected the kidney. Explant pathology of this kidney showed glomerulopathy with evidence of membranous nephropathy and linear IgG glomerular deposits. There was an abundance of glomerular IgM staining which is unusual for rejection more than 1 -month post-xenotransplant. Once again, a post-transplant SLA assay showed that the recipient was in fact sensitized to the donor SLA, having anti-IgM antibodies against donor SLA-1*12:01 allele with MFIs >4000 on flow cytometric crossmatch pre-transplant. Usually the anti-pig IgM (glycan and SLA) decreases during the first month under the cover of complement inhibition with anti-C5, but in this recipient the IgM levels never dipped below MFIs >4000 on flow crossmatch testing.PCT / US25 / 52258 23 October 2025 (23.10.2025)

[0153] This pre-clinical experiment series presented an important opportunity for screening recipients with post-transplant SLA crossmatches to determine whether immunosuppression can be withdrawn or reduced. It is contemplated herein that the Wellington graft loss would have been avoided by continuing the anti-C5 antibody past day 75 until the IgM levels decreased.

[0154] The Vienna and Wellington cases show that careful pre-transplant screening of potential recipients with advanced histocompatibility testing for both anti-glycan and anti-SLA antibodies can eliminate choosing recipients for xenotransplant that have no opportunity to benefit long term.

[0155] Cases 3 & 4 (Helsinki & Oxford): These two recipients received GGTA1 / B4GALNT2 / SLA DR KO pig kidney transplants using the same protocols and both achived the experimental endpoint (180 days) with good renal function, normal hemoglobin, albumin, and total protein.

[0156] Specific measurements

[0157] Renal function: Recipients 2, 3, and 4 maintained serum creatinine levels at 1.2 mg / dL or less for the duration of their graft survival. Recipient 2 had a late sharp increase in serum creatinine on post-transplant days 124-126 coinciding with graft losing AMR.

[0158] Proteinuria and albuminuria: While not measured directly, serum total protein, albumin, and globulin remained in the normal range until very late in the course when rejection was occurring in Wellington (day 126 rejector). In the two long surviving recipients, each of the levels remained in the normal range throughout, indicating that significant proteinuria was very unlikely.

[0159] Hemoglobin maintenance: All recipients were able to maintain Hgb levels above 8 mg / dL throughout their post-transplant course and none required exogenous erythropoietin (EPO) infusion to augment Hgb levels.

[0160] Platelet counts: Platelet counts remained above 100 X103 / uL, levels consistent with normal coagulation function.

[0161] Infectious issues: No infectious issues were found in these four GGTA1 / B4GALNT2 / SLA DR KO kidney transplants. CMV is endemic in rhesus macaques and is often reactivated in the face of intense immunosuppression, requiring anti-viral therapy. Specifically, there was no reactivation of rhCMV in any of the recipients at any timepoint. The lack of infectious problems suggests that the immunosuppression used to achieve prolonged survival is indicated for clinical use. In addition, the deletion of SLA DR in the donor kidney does not appear to increase the risk for post-transplant infections.PCT / US25 / 52258 23 October 2025 (23.10.2025)

[0162] Pre-clinical experiment series keys

[0163] Immunosuppression required strong depletional induction with T cell and B cell depleting agents, temporary complement inhibition, CD40 / CD154 costimulation blockade, mycophenolic acid and steroids.

[0164] Pigs whose genetic makeup eliminated significant xenoantigens minimized the number of anti-pig DSA present in recipient serum. To accomplish this, slightly different pigs must be used in the pre-clinical and clinical scenario. The pre-clinical scenario utilizes a GGTA1 / B4GALNT2 KO background, while the clinical applications require the addition of a CMAH KO to accommodate the human-specific Neu5Gc antigen present in the donor pig.Documents Mentioned1. Adams, A. B., S. C. Kim, G. R. Martens, J. M. Ladowski, J. L. Estrada, L. M. Reyes, C. Breeden, A. Stephenson, D. E. Eckhoff, M. Tector, and A. J. Tector. 2018. Xenoantigen Deletion and Chemical Immunosuppression Can Prolong Renal Xenograft Survival. Ann Surg 268: 564-573.2. Tector, A. J., A. B. Adams, and M. Tector. 2023. Current Status of Renal Xenotransplantation and Next Steps. Kidney3604: 278-284.3. Issa, N., F. G. Cosio, J. M. Gloor, S. Sethi, P. G. Dean, S. B. Moore, S. DeGoey, and M. D. Stegall. 2008. Transplant glomerulopathy: risk and prognosis related to anti-human leukocyte antigen class II antibody levels. Transplantation 86: 681-685.4. Cosio, F. G., J. M. Gloor, S. Sethi, and M. D. Stegall. 2008. Transplant glomerulopathy. Am J Transplant 8: 492-496.5. Tambur, A. R., V. Kosmoliaptsis, F. H. J. Claas, R. B. Mannon, P. Nickerson, and M. Naesens. 2021. Significance of HLA-DQ in kidney transplantation: time to reevaluate human leukocyte antigen-matching priorities to improve transplant outcomes? An expert review and recommendations. Kidney Int 100: 1012-1022.6. Sapir-Pichhadze, R., K. Tinckam, K. Quach, A. G. Logan, A. Laupacis, R. John, J.Beyene, and S. J. Kim. 2015. HLA-DR and -DQ eplet mismatches and transplant glomerulopathy: a nested case-control study. Am J Transplant 15: 137-148.7. Davis, S., C. Wiebe, K. Campbell, C. Anobile, M. Aubrey, E. Stites, M. Graklals, E.Pomklret, P. Nickerson, and J. E. Cooper. 2021. Adequate tacrolimus exposure modulates the impact of HLA class II molecular mismatch: a validation study in an American cohort. Am J Transplant 21: 322-328.PCT / US25 / 52258 23 October 2025 (23.10.2025)8. Wiebe, C., V. Kosmoliaptsis, D. Pochinco, I. W. Gibson, J. Ho, P. E. Birk, A. Goldberg, M. Karpinski, J. Shaw, D. N. Rush, and P. W. Nickerson. 2019. HLA-DR / DQ molecular mismatch: A prognostic biomarker for primary alloimmunity. Am J Transplant 19: 1708-1719.9. Wiebe, C., and P. W. Nickerson. 2020. More precise donor-recipient matching: the role of eplet matching. Curr Opin Nephrol Hypertens 29: 630-635.10. Wiebe, C., and P. W. Nickerson. 2020. Molecular Mismatch-the Renaissance of HLA in Kidney Transplantation. J Am Soc Nephrol 31: 1922-1925.11. Wiebe, C., D. N. Rush, T. E. Nevins, P. E. Birk, T. Blydt-Hansen, I. W. Gibson, A.Goldberg, J. Ho, M. Karpinski, D. Pochinco, A. Sharma, L. Storsley, A. J. Matas, and P. W. Nickerson. 2017. Class II Eplet Mismatch Modulates Tacrolimus Trough Levels Required to Prevent Donor-Specific Antibody Development. J Am Soc Nephrol 28: 3353-3362.12. Senev, A., M. Coemans, E. Lerut, V. Van Sandt, J. Kerkhouls, L. Daniels, M. V.Driessche, V. Compernolle, B. Sprangers, E. Van Loon, J. Callemeyn, F. Claas, A. R.Tambur, G. Verbeke, D. Kuypers, M. P. Emonds, and M. Naesens. 2020. Eplet Mismatch Load and De Novo Occurrence of Donor-Specific Anti-H LA Antibodies, Rejection, and Graft Failure after Kidney Transplantation: An Observational Cohort Study. J Am Soc Nephrol 31: 2193-2204.13. Ladowski JM, Tector M, Martens G, Wang ZY, Burlak C, Reyes L, Estrada J, Adams A, Tector AJ. Late graft failure of pig-to-rhesus renal xenografts has features of glomerulopathy and recipients have anti-swine leukocyte antigen class I and class II antibodies.Xenotransplantation. 2024 May;31(3):e12862.14. Ladowski, J. M., G. R. Martens, L. M. Reyes, Z. Y. Wang, D. E. Eckhoff, V. Hauptfeld-Dolejsek, M. 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Claims

ClaimsWe claim:

1. A transgenic pig comprising in its nuclear genome a knockout of both alleles of an SLA-DQ gene, an SLA-DR gene or both an SLA-DQ gene and SLA-DR gene.

2. A porcine transplant product isolated from the transgenic pig of claim 1, wherein the transplant product is an organ, tissue, transfusion product or cell.

3. The porcine transplant product of claim 2, wherein said porcine organ, tissue, transfusion product or cell is skin, heart, liver, kidneys, lung, pancreas, thyroid, small bowel, whole blood or a blood component.

4. A method of preparing a transplant product for xenotransplantation into a human, the method comprising isolating the transplant product from a transgenic pig comprising in its nuclear genome a knockout of both alleles of an SLA-DQ gene, ane SLA-DR gene or both an SLA-DQ gene and SLA-DR gene.

5. The method of claim 4, wherein the porcine transplant product is an organ, tissue, transfusion product or cell.

6. The method of claim 5, wherein the porcine organ, tissue, transfusion product or cell is skin, heart, liver, kidneys, lung, pancreas, thyroid, small bowel, whole blood or a blood component.

7. A method of increasing the duration of the period between when a human subject is identified as a subject in need of a human transplant product and when the human transplant occurs, comprising administering a porcine transplant product to the human subject in a therapeutically effective manner,wherein the porcine transplant product is isolated from a transgenic pig comprising in its nuclear genome a knockout of both alleles of an SLA-DQ gene, an SLA-DR gene or both an SLA-DQ gene and SLA-DR gene.

8. The method of claim 7, wherein the porcine transplant product is an organ, tissue, transfusion product or cell.

9. The method of claim 8, wherein the porcine transplant product is skin, heart, liver, kidneys, lung, pancreas, thyroid, small bowel, whole blood or a blood component.

10. A method of improving a transplant rejection-related symptom in a human subject comprising administering a porcine transplant product to a subject in need thereof, wherein the porcine transplant product is isolated from a transgenic pig comprising in its nuclear genome a knockout of both alleles of an SLA-DQ gene, an SLA-DR gene or both an SLA-DQ gene and SLA-DR gene, andwherein a transplant rejection-related symptom is improved as compared to when porcine transplant product from a wild-type pig is transplanted into a human subject.

11. The method of claim 10, wherein the transplant rejection-related symptom is a hyperacute rejection (HAR) symptom.

12. The method of claim 10, wherein the transplant rejection-related symptom is a chronic antibody-mediated rejection (AMR) symptom.

13. The method of any of claims 11-13, wherein the porcine transplant product is an organ, tissue, transfusion product or cell.

14. The method of claim 13, wherein the porcine transplant product is skin, heart, liver, kidneys, lung, pancreas, thyroid, small bowel, whole blood or a blood component.

15. The transgenic pig or method of any preceding claim, wherein the gene is an SLA-DQ gene.

16. The transgenic pig or method of any preceding claim, wherein the gene is an SLA-DR gene.

17. The transgenic pig or method of any preceding claim, wherein both alleles of both an SLA-DQ gene and SLA-DR gene are knocked out.

18. The transgenic pig or method of any preceding claim, wherein the transgenic pig further comprises in its nuclear genome a knockout of both alleles of one or more of the a(1,3)-galactosyltransferase (GGTA1) gene, cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene and 01,4 N-acetylgalactosaminyltransferase (04GalNT2) gene.

19. The method of claim 18, wherein the transgenic pig comprises in its nuclear gene a knockout of both alleles of both the GGTA1 gene and the p4GalNT2 gene.

20. The method of claim 18, wherein the transgenic pig comprises in its nuclear gene a knockout of both alleles of each of the GGTA1 gene, CMAH gene and the p4GalNT2 gene.

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