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Objective: To evaluate the clinically relevant anti-CD40 antibody iscalimab for baseline immunosuppression in a preclinical pig-to-rhesus renal xenograft model., Summary Background Data: CD40/CD40L co-stimulation blockade-based immunosuppression has been more successful than calcineurin-based protocols in prolonging xenograft survival in preclinical models., Methods: GGTA1 knockout/CD55 transgenic pig kidneys were transplanted into rhesus monkeys (n = 6) receiving an iscalimab-based immunosuppressive regimen., Results: Two grafts were lost early (22 and 26 days) because of ectatic donor ureters with otherwise normal histology. The other recipients survived 171, 315, 422, and 439 days with good renal function throughout the posttransplant course. None of the recipients experienced serious infectious morbidity., Conclusions: It may be reasonable to evaluate an iscalimab-based immunosuppressive regimen in clinical renal xenotransplantation., (© 2024 The Author(s). Xenotransplantation published by Wiley Periodicals LLC.)

Attack of donor tissues by pre-formed anti-pig antibodies is well known to cause graft failure in xenotransplantation. Genetic engineering of porcine donors to eliminate targets of these pre-formed antibodies coupled with advances in immunosuppressive medicines have now made it possible to achieve extended survival in the pre-clinical pig-to-non-human primate model. Despite these improvements, antibodies remain a risk over the lifetime of the transplant, and many patients continue to have pre-formed donor-specific antibodies even to highly engineered pigs. While therapeutics exist that can help mitigate the detrimental effects of antibodies, they act broadly potentially dampening beneficial immunity. Identifying additional xenoantigens may enable more targeted approaches, such as gene editing, to overcome these challenges by further eliminating antibody targets on donor tissue. Because we have found that classical class I swine leukocyte antigens are targets of human antibodies, we now examine whether related pig proteins may also be targeted by human antibodies. We show here that non-classical class I swine leukocyte proteins (SLA-6, -7, -8) can be expressed at the surface of mammalian cells and act as antibody targets., (© 2024 The Author(s). Xenotransplantation published by John Wiley & Sons Ltd.)

Prolonged survival in preclinical renal xenotransplantation demonstrates that early antibody mediated rejection (AMR) can be overcome. It is now critical to evaluate and understand the pathobiology of late graft failure and devise new means to improve post xenograft outcomes. In renal allotransplantation the most common cause of late renal graft failure is transplant glomerulopathy-largely due to anti-donor MHC antibodies, particularly anti-HLA DQ antibodies. We evaluated the pig renal xenograft pathology of four long-surviving (>300 days) rhesus monkeys. We also evaluated the terminal serum for the presence of anti-SLA class I and specifically anti-SLA DQ antibodies. All four recipients had transplant glomerulopathy and expressed anti-SLA DQ antibodies. In one recipient tested for anti-SLA I antibodies, the recipient had antibodies specifically reacting with two of three SLA I alleles tested. These results suggest that similar to allotransplantation, anti-MHC antibodies, particularly anti-SLA DQ, may be a barrier to improved long-term xenograft outcomes., (© 2024 The Authors. Xenotransplantation published by John Wiley & Sons Ltd.)

Pig liver xenotransplantation is limited by a thrombocytopenic coagulopathy that occurs immediately following graft reperfusion. In vitro and ex vivo studies from our lab suggested that the thrombocytopenia may be the result of a species incompatibility in platelet glycosylation. Realization that platelet α-granules contain antibodies caused us to reevaluate whether the thrombocytopenia in liver xenotransplantation could occur because IgM and IgG from inside platelet α-granules bound to pig liver sinusoidal endothelial cells (LSECs). Our in vitro analysis of IgM and IgG from inside α-granules showed that platelets do carry xenoreactive antibodies that can bind to known xenoantigens. This study suggests that thrombocytopenia occurring following liver xenotransplantation could occur because of xenoreactive antibodies tethering human platelets to the pig LSEC enabling the platelet to be phagocytosed. These results suggest genetic engineering strategies aimed at reducing xenoantigens on the surface of pig LSEC will be effective in eliminating the thrombocytopenia that limits survival in liver xenotransplantation., (© 2023 The Authors. Xenotransplantation published by John Wiley & Sons Ltd.)

Organ supply remains inadequate to meet the needs of many patients who could benefit from allotransplantation. Xenotransplantation, the use of animals as organ donors, provides an opportunity to alleviate this challenge. Pigs are widely accepted as the ideal organ donor, but humans and nonhuman primates have strong humoral immune responses to porcine tissue. Although carbohydrate xenoantigens have been studied intensively, the primate Ab response also targets class I and class II swine leukocyte Ags (SLAs). Human Abs that recognize HLAs can cross-react with SLA molecules because epitopes can be shared across species. However, ∼15% of people may also exhibit Abs toward class II SLAs despite lacking Abs that also recognize class II HLAs. Here, we extend these studies to better understand human Ab responses toward class I SLAs. When tested against a panel of 18 unique class I SLA proteins, 14 of 52 sera samples collected from patients in need of an organ transplant contained Abs that bound class I SLAs. Class I SLA-reactive sera may contain IgM only, IgG, only, or IgM and IgG capable of recognizing the pig proteins. The presence of class I HLA-reactive Abs was not essential to generating anti-class I SLA Ig. Last, anti-class I SLA reactivity varied by serum; some recognized a single SLA allele, whereas others recognized multiple class I SLA proteins., (Copyright © 2023 The Authors.)

Genetically engineered pigs with multiple gene deletions and insertions are predicted to extend porcine to human xenograft survival. Several genes have been successfully knocked out and inserted, yet more have failed to produce viable animals for unexplained reasons. The effects of gene editing on cellular homeostasis may be the cause of reduced embryo fitness, failed pregnancies, or poor piglet viability. The elements of cellular dysfunction such as endoplasmic reticulum stress and oxidative stress induced by gene editing may additively affect the quality of genetically engineered cells to be used for cloning. Evaluating the impact of each gene edit on cellular fitness for cloning will allow researchers to maintain the cellular homeostasis of engineered cells that were validated as candidates for cloning and the production of porcine organ donors.

A safe, efficacious, and clinically applicable immunosuppressive regimen is necessary for islet xenotransplantation to become a viable treatment option for diabetes. We performed intraportal transplants of wild-type adult porcine islets in 25 streptozotocin-diabetic cynomolgus monkeys. Islet engraftment was good in 21, partial in 3, and poor in 1 recipient. Median xenograft survival was 25 days with rapamycin and CTLA4Ig immunosuppression. Adding basiliximab induction and maintenance tacrolimus to the base regimen significantly extended median graft survival to 147 days (p < .0001), with three animals maintaining insulin-free xenograft survival for 265, 282, and 288 days. We demonstrate that this regimen suppresses non-Gal anti-pig antibody responses, circulating effector memory T cell expansion, effector function, and infiltration of the graft. However, a chronic systemic inflammatory state manifested in the majority of recipients with long-term graft survival indicated by increased neutrophil to lymphocyte ratio, IL-6, MCP-1, CD40, and CRP expression. This suggests that this immunosuppression regimen fails to regulate innate immunity and resulting inflammation is significantly associated with increased incidence and severity of adverse events making this regimen unacceptable for translation. Additional studies are needed to optimize a maintenance regimen for regulating the innate inflammatory response., (© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1 - /HLAG1 + pigs to explore their use as organ and cell donors that may extend xenograft survival and function in both preclinical nonhuman primate (NHP) models and future clinical trials. In the present study, HLA-G1 was expressed from the porcine ROSA26 locus by homology directed repair (HDR) mediated knock-in (KI) with simultaneous deletion of α-1-3-galactotransferase gene (GGTA1; GTKO) using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) gene-editing system. GTKO/HLAG1 + pigs showing immune inhibitory functions were generated through somatic cell nuclear transfer (SCNT). The presence of HLA-G1 at the ROSA26 locus and the deletion of GGTA1 were confirmed by next generation sequencing (NGS) and Sanger's sequencing. Fibroblasts from piglets, biopsies from transplantable organs, and islets were positive for HLA-G1 expression by confocal microscopy, flow cytometry, or q-PCR. The expression of cell surface HLA-G1 molecule associated with endogenous β2-microglobulin (β2m) was confirmed by staining genetically engineered cells with fluorescently labeled recombinant ILT2 protein. Fibroblasts obtained from GTKO/HLAG1 + pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4 + and CD8 + T cells, B cells and natural killer (NK) cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1 + genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes. The findings presented here suggest that the HLA-G1 + transgene can be stably expressed from the ROSA26 locus of non-fetal maternal tissue at the cell surface. By providing an immunomodulatory signal, expression of HLA-G1 + may extend survival of porcine pancreatic islet and organ xenografts., Competing Interests: BH has an equity interest in and serves as paid executive officer and director of Diabetes Free, Inc., an organization that may commercially benefit from the results of this research. This interest has been reviewed and managed by the University of Minnesota in accordance with its conflict of interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rao, Hosny, Kumbha, Naqvi, Singh, Swanson, Levy, Matson, Steinhoff, Forneris, Walters, Hering and Burlak.)

Objective: Pig-to-primate renal xenotransplantation is plagued by early antibody-mediated graft loss which precludes clinical application of renal xenotransplantation. We evaluated whether temporary complement inhibition with anti-C5 antibody Tesidolumab could minimize the impact of early antibody-mediated rejection in rhesus monkeys receiving pig kidneys receiving costimulatory blockade-based immunosuppression., Methods: Double (Gal and Sda) and triple xenoantigen (Gal, Sda, and SLA I) pigs were created using CRISPR/Cas. Kidneys from DKO and TKO pigs were transplanted into rhesus monkeys that had the least reactive crossmatches. Recipients received anti-C5 antibody weekly for 70 days, and T cell depletion, anti-CD154, mycophenolic acid, and steroids as baseline immunosuppression (n = 7). Control recipients did not receive anti-C5 therapy (n = 10)., Results: Temporary anti-C5 therapy reduced early graft loss secondary to antibody-mediated rejection and improved graft survival (P < 0.01). Deleting class I MHC (SLA I) in donor pigs did not ameliorate early antibody-mediated rejection (table). Anti-C5 therapy did not allow for the use of tacrolimus instead of anti-CD154 (table), prolonging survival to a maximum of 62 days., Conclusion: Inhibition of the C5 complement subunit prolongs renal xenotransplant survival in a pig to non-human primate model., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Understanding the anti-carbohydrate antibody response toward epitopes expressed on porcine cells, tissues, and organs is critical to advancing xenotransplantation toward clinical application. In this study, we determined IgM and IgG antibody specificities and relative concentrations in five cynomolgus monkeys at baseline and at intervals following intraportal xenotransplantation of adult porcine islets. This study utilized a carbohydrate antigen microarray that comprised more than 400 glycoconjugates, including historically reported α-Gal and non-α-Gal carbohydrate antigens with various modifications. The elicited anti-carbohydrate antibody responses were predominantly IgM compared to IgG in 4 out of 5 monkeys. Patterns of elicited antibody responses greater than 1.5 difference (log2 base units; 2.8-fold on a linear scale) from pre-serum to post-serum sampling specific for carbohydrate antigens were heterogeneous and recipient-specific. Increases in the elicited antibody response to α-Gal, Sda, GM2 antigens, or Lexis X antigen were found in individual monkeys. The novel carbohydrate structures Galβ1-4GlcNAcβ1-3Galβ1 and N-linked glycans with Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ structure were common targets of elicited IgM antibodies. These results provide important insights into the carbohydrate epitopes that elicit antibodies following pig-to-monkey islet xenotransplantation and reveal possible targets for gene editing., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: BJH has an equity interest in and serves as an executive officer of Diabetes-Free, Inc, an organization that may commercially benefit from the results of this research. This interest has been reviewed and managed by the University of Minnesota in accordance with its Conflict of Interest policies, and does not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction: Macrophages contribute to xenograft rejection by direct cytotoxicity and by amplifying T cell-mediated immune responses. It has been shown that transgenic expression of hCD47 protects porcine cells from human macrophages by restoring the CD47-SIRPα self-recognition signal. It has also been reported that the long 3' untranslated region (3'UTR) of the hCD47 gene, which is missing from constructs previously used to make hCD47 transgenic pigs, is critical for efficient cell surface expression in human cells. The aim of this study was to investigate the impact of a modified form of the 3'UTR on the expression, localization, and function of hCD47 in transfected porcine cells., Methods: hCD47 constructs with and without the modified 3'UTR were knocked into the GGTA1 locus in porcine fetal fibroblasts using CRISPR. Flow cytometry of the transfected cells was used to analyze hCD47 localization. Endoplasmic reticulum (ER), mitochondrial, and oxidative stress were examined by gene expression analysis and confocal microscopy. Phagocytosis of transfected cells by human macrophages was measured by flow cytometry, and stimulation of human/non-human (NHP) primate lymphocytes by the cells was examined using a PBMCs proliferation assay., Results: Cells transfected with the construct lacking the 3'UTR (hCD47(3'UTR-)) exhibited predominantly intracellular expression of hCD47, and showed evidence of ER stress, dysregulated mitochondrial biogenesis, oxidative stress, and autophagy. Inclusion of the 3'UTR (hCD47(3'UTR+)) decreased intracellular expression of hCD47 by 36% and increased cell surface expression by 53%. This was associated with a significant reduction in cellular stress markers and a higher level of protection from phagocytosis by human macrophages. Furthermore, hCD47(3'UTR+) porcine cells stimulated significantly less proliferation of human/NHP T cells than hCD47(3'UTR-) cells., Conclusion: Our results suggest the potential benefits of using hCD47 constructs containing the 3'UTR to generate genetically engineered hCD47-expressing donor pigs., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Genetically engineered porcine donors are a potential solution for the shortage of human organs for transplantation. Incompatibilities between humans and porcine donors are largely due to carbohydrate xenoantigens on the surface of porcine cells, provoking an immune response which leads to xenograft rejection., Materials and Methods: Multiplex genetic knockout of GGTA1, β4GalNT2, and CMAH is predicted to increase the rate of xenograft survival, as described previously for GGTA1. In this study, the clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 system was used to target genes relevant to xenotransplantation, and a method for highly efficient editing of multiple genes in primary porcine fibroblasts was described., Results: Editing efficiencies greater than 85% were achieved for knockout of GGTA1, β4GalNT2, and CMAH., Conclusion: The high-efficiency protocol presented here reduces scale and cost while accelerating the production of genetically engineered primary porcine fibroblast cells for in vitro studies and the production of animal models.

Objectives: Islet transplantation is an emerging treatment option for type 1 diabetes but its application is limited by the shortage of human pancreas donors. Characterization of the N- and O-glycan surface antigens that vary between human and genetically engineered porcine islet donors could shed light on targets of antibody mediated rejection., Methods: N- and O-glycans were isolated from human and adult porcine islets and analyzed using matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS/MS)., Results: A total of 57 porcine and 34 human N-glycans and 21 porcine and 14 human O-glycans were detected from cultured islets. Twenty-eight of which were detected only from porcine islets, which include novel xenoantigens such as high-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues. Porcine islets have terminal N-glycolylneuraminic acid (NeuGc) residue in bi-antennary N-glycans and sialyl-Tn O-glycans. No galactose-α-1,3-galactose (α-Gal) or Sda epitope were detected on any of the islets., Conclusions: These results provide important insights into the potential antigenic differences of N- and O-glycan profiles between human and porcine islets. Glycan differences may identify novel gene targets for genetic engineering to generate superior porcine islet donors., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: BJH has an equity interest in and serves as an executive officer of Diabetes-Free, Inc, an organization that may commercially benefit from the results of this research. This interest has been reviewed and managed by the University of Minnesota in accordance with its Conflict of Interest policies, and does not alter our adherence to PLOS ONE policies on sharing data and materials.

Progress has been made in overcoming antibody-mediated rejection of porcine xenografts by deleting pig genes that produce unique carbohydrate epitopes. Pigs deficient in galactose α-1,3 galactose (gene modified: GGTA1) and neu5Gc (gene modified: CMAH) have reduced levels of human antibody binding. Previously we identified α-fucose as a glycan that was expressed in high levels on cells of GGTA1/CMAH KO pigs. To validate the α-fucose phenotype observed previously we compared lectin affinity toward human and pig serum glycoproteins by dot blot analysis and confocal microscopy. Human anti-fucose antibody isolated by affinity chromatography was tested for specificity to L-fucose by custom macroarray. The affinity and cytotoxicity of the isolated human anti-fucose antibody toward human and GGTA1/CMAH KO pig PBMCs was determined by flow cytometry. Dot blot and confocal analysis support out previous findings that α-fucose is more highly expressed in pigs than humans. Pig kidney glomeruli and tubules contain abundant α-fucose and may represent focal sites for anti-α-fucose antibody binding. The Isolated human anti-fucose IgA, IgG and IgM bound to GGTA1/CMAH KO pig PBMC and were cytotoxic. Interestingly, the isolated human IgG cross reacted with the methyl pentose, L-rhamnose. Human anti-fucose antibody bound and was cytotoxic to GGTA1/CMAH KO pig peripheral blood monocytes. We have shown that α-fucose is an abundant target for cytotoxic human antibody in the organs of genetically modified pigs important to xenotransplantation., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pigs are especially useful large animal models, however, limited availability of commercially available antibodies for immunoblotting presents a significant obstacle facing preclinical xenotransplantation research. Major histocompatibility complex class I (MHC-I) molecule expression enhancement by nucleotide-binding oligomerization domain (NOD)-like receptor family with a caspase recruitment domain (CARD) containing caspase 5 (NLRC5) is fundamental to understanding porcine xenoantigen presentation. Swine Leukocyte Antigens (SLAs) are the porcine MHC homologs for human leukocyte antigens. SLA-I is a known xenoantigen that causes T cell activation. NLRC5, SLA-I, and B2M are all targets of immune modulation in genetically engineered pigs in xenotransplantation research with the goal to reduce SLA-I expression. In the present study, the human anti-NLRC5 (ab105411), anti-NLRC5 (ab117624), anti-NLRC5 N-terminal (ab178767), anti-HLA E (ab203082), anti-HLA E (ab135826), anti-HLA E (ab2216) and anti-β 2 M (ab75853) antibodies were examined using immunoblots for porcine cross-reactivity. The anti-human antibodies ab117624, ab105411, ab178767, ab2216, and ab75853 cross reacted with cognate proteins in porcine cell lysates. Antibody reagents from this study will allow for validation of NLRC5, B2M, MHC-I expression in future research studies. In addition, following the methodology described in this study for other xenotransplantation targets may provide an alternative to custom antibody development., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Engineering of α-Galactosyltransferase gene-knockout pigs circumvented hyperacute rejection of pig organs after xenotransplantation in non-human primates. Overcoming this hurdle revealed the importance of non-α-Gal carbohydrate antigens in the immunobiology of acute humoral xenograft rejection., Methods: This study analyzed serum from seven naïve cynomolgus monkeys (blood type O/B/AB = 3/2/2) for the intensity of natural IgM and IgG signals using carbohydrate antigen microarray, which included historically reported α-Gal and non-α-Gal carbohydrate antigens with various modifications., Results: The median (range) of IgM and IgG signals were 12.71 (7.23-16.38) and 9.05 (7.23-15.90), respectively. The highest IgM and IgG signals with narrowest distribution were from mono- and disaccharides, followed by modified structures. Natural anti-α-Gal antibody signals were medium to high in IgM (11.2-15.9) and medium in IgG (8.5-11.6) spectra, and was highest with Lac core structure (Galα1-3Galβ1-4Glc, iGb3) and lowest with LacNAc core structure (Galα1-3Galβ1-4GlcNAc). Similar signal intensities (up to 15.8 in IgM and up to 11.8 in IgG) were observed for historically detected natural non-α-Gal antigens, which included Tn antigen, T antigen, GM2 glycolipid, and Sd a antigen. The hierarchical clustering analysis revealed the presence of clusters of anti-A antibodies and was capable of distinguishing between the blood group B and AB non-human primates., Conclusions: The results presented here provide the most comprehensive evaluation of natural antibodies present in cynomolgus monkeys., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Xenotransplantation of porcine islets has emerged in recent decades as a potential treatment for type 1 diabetes (T1D). Current methods of detection, indicative of successful engraftment, occur downstream of actual islet death. Epigenetic biomarkers can be detected in circulating cell-free DNA (cfDNA) to provide an earlier indication of graft dysfunction., Aims: The present study identified a biomarker of islet death using differential methylation of the insulin gene, INS, originating from β-cells in porcine islets., Materials & Methods: Pyrosequencing primers specific for porcine INS were designed to quantify hypomethylation along 12 cysteine-guanine dinucleotide (CpG) sites, including three sites in the cyclic adenosine monophosphate (cAMP) response element (CRE) binding protein 2 (CRE2) binding region of the 5' untranslated region (UTR) and nine sites within intron 2., Results: PCR amplification of bisulfite-converted DNA combined with pyrosequencing data support the conclusion that hypomethylated porcine INS is specific to islet origin., Conclusion: Moreover, the results of this study indicate a highly specific epigenetic biomarker, capable of detecting a single islet, supporting the measurement of cfDNA as a biomarker for transplanted islet death. Defining the epigenetic characteristics of porcine-derived islets within cfDNA will be crucial to develop a better understanding of graft survival immunology for transplantation., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Handmade cloning is a zona-free nuclear transfer approach and an economical, efficient, and simple micromanipulation-free alternative to dolly based traditional cloning (TC). In this study, based on handmade cloning with minor modifications, an optimized bi-oocyte fusion (BOF) cloning method was established to produce GGTA1 KO porcine embryos using the CRISPR/Cas9 gene editing system. The GGTA1 gene is responsible for the generation of Gal epitopes on the surface of porcine cells, triggering hyperacute immune rejection in preclinical porcine-to-human xenotransplantation. The purpose of the present study is to establish an efficient protocol for activation of porcine oocyte cytoplast-fibroblast fused constructs developed to GGTA1 KO blastocysts by the zona-free bi-oocyte fusion cloning method. High percentages of cleavage (90 ± 2.6%) and blastocyst rates (39 ± 4.0%) were achieved upon treatment with demecolcine-assisted oocyte enucleation followed by 6 V alternating current for proper alignment and single-step fusion technique using a single direct current pulse of 1.0 kV/cm for 9 μs duration, compared to the double-step fusion method with combined chemical activation using thimerosal and dithiothreitol. Overall blastocyst rate was higher for oocyte enucleation by demecolcine (0.4 μg/ml) and 45 min incubation (42 ± 1.5%) compared to without demecolcine incubation followed by complete chemical thimerosal/dithiothreitol activation (33 ± 1.1%). The blastocyst rate (39 ± 1.0%) was found to be significantly higher 1 h post-electrofusion, compared to at 0 and 4 h (28 ± 1.5 and 6 ± 1.5%, respectively). Blastocyst development rates for GGTA1 knockout embryos (38 ± 1.76%) were comparable to those obtained with wild-type embryos (41.1 ± 0.67%). In conclusion, we achieved high overall efficiency in production of GGTA1 KO blastocysts by modified HMC protocol., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The ever-increasing disparity between the lack of organ donors and patients on the transplant waiting list is increasing worldwide. For the past several decades xenotransplantation has led the way to correct this deficit and remains clearly the only feasible option to provide a means to meet the demand for patients in need of an organ transplant. Xenotransplantation's ability to provide a specifically designed unlimited supply of organs, suited to treat the various needs for transplant organs and cells, has recently been championed by successful pre-clinical trials that have run long-term in non-human primate studies. In this review we show how these improvements have come about due to long-term dedicated research and recent advances in biomedical engineering technology, such as genome editing tools including zinc finger nucleases, TALEN, and CRISPER/Cas9 which have paved the way for significant breakthroughs in improving xenograft outcomes through genetic modifications to the donor source pig. Other novel approaches include the development of decellularized porcine tissue, such as corneas which can now be transplanted into patients with the minimal need for immunosuppression or other side effects. Further genetic variants of the porcine genome are also now being optimized to abrogate rejection. The emergence of new modalities such as; mesenchymal stem cells, donor thymic vascularization, in vivo bioreactors, chemokine and cytokine therapies have come to show improvements in xenograft outcomes. Furthermore, new studies confirm the safety status of using porcine xenografts, verifying that with current technologies and approaches, the issue of PERV transmission is a moot point. These breakthroughs and technological advancements push the reality of xenotransplantation one step closer to the clinic., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: BJH has an equity interest in and serves as an executive officer of Diabetes-Free, Inc, an organization that may commercially benefit from the results of this research. This interest has been reviewed and managed by the University of Minnesota in accordance with its Conflict of Interest policies, and does not alter our adherence to PLOS ONE policies on sharing data and materials.

Immune tolerance to allografts has been pursued for decades as an important goal in transplantation. Administration of apoptotic donor splenocytes effectively induces antigen-specific tolerance to allografts in murine studies. Here we show that two peritransplant infusions of apoptotic donor leukocytes under short-term immunotherapy with antagonistic anti-CD40 antibody 2C10R4, rapamycin, soluble tumor necrosis factor receptor and anti-interleukin 6 receptor antibody induce long-term (≥1 year) tolerance to islet allografts in 5 of 5 nonsensitized, MHC class I-disparate, and one MHC class II DRB allele-matched rhesus macaques. Tolerance in our preclinical model is associated with a regulatory network, involving antigen-specific Tr1 cells exhibiting a distinct transcriptome and indirect specificity for matched MHC class II and mismatched class I peptides. Apoptotic donor leukocyte infusions warrant continued investigation as a cellular, nonchimeric and translatable method for inducing antigen-specific tolerance in transplantation.