CD4(+)Foxp3(+) regulatory T cells (Tregs) play an essential role in suppressing transplant rejection, but their role within the graft and heterogeneity in tolerance are poorly understood. Here, we compared phenotypic and transcriptomic characteristics of Treg populations within lymphoid organs and grafts in an islet xenotransplant model of tolerance. We showed Tregs were essential for tolerance induction and maintenance. Tregs demonstrated heterogeneity within the graft and lymphoid organs of tolerant mice. A subpopulation of CD127(hi) Tregs with memory features were found in lymphoid organs, presented in high proportions within long -surviving islet grafts, and had a transcriptomic and phenotypic profile similar to tissue Tregs. Importantly, these memory -like CD127(hi) Tregs were better able to prevent rejection by effector T cells, after adoptive transfer into secondary Rag(-/-) hosts, than naive Tregs or unselected Tregs from tolerant mice. Administration of IL -7 to the CD127(hi)Treg subset was associated with a strong activation of phosphorylation of STAT5. We proposed that memory -like CD127(hi) Tregs developed within the draining lymph node and underwent further genetic reprogramming within the graft toward a phenotype that had shared characteristics with other tissue or tumor Tregs. These findings suggested that engineering Tregs with these characteristics either in vivo or for adoptive transfer could enhance transplant tolerance.

Background: Porcine neonatal islet cell clusters (NICC) are widely accepted as a potential means for treatment of type 1 diabetes amid significant issues of low donor organ availability for allotransplantation and the poor yield of islet cells from donor pancreata. Several studies have shown NICC xenotransplantation reverses hyperglycaemia in large animals and non-human primates; however, these are varied in results and methodology. By developing an optimised method for pancreas retrieval and NICC culture, we hope to develop a universal protocol for NICC xenotransplantation to be used for clinical translation. Aim: To optimise NICC culture and evaluate the short-term efficacy (100 days) for their use in pig-to-baboon xenotransplantation. Methods: A retrospective audit of 9 NICC preparations from which NICC were transplanted into diabetic baboons was reviewed to determine which factors yielded the greatest NICC yield and provided good function. NICC were retrieved from piglets ranging from 3-6 days of age, digested with 25 mg/ml collagenase, and filtered through a 500um sieve. Tissue was plated in Petri dishes in Hams F10 medium (containing additives) for 6 days in a 37°C, 5% CO2 incubator. Full media changes were performed on days one and three. 10 ml of Ham’s F10 medium with additives was added on day 5 before a final count and transplant on day 6. The 9 NICC preps were transplanted into 9 baboons, each of which received 5 doses of anti-CD2 at 5 mg/kg from day -3 to day 21 along with fortnightly anti-CD154 at 20-30 mg/kg and belatacept at 20 mg/kg. With daily maintenance immunosuppression of tacrolimus at 5 mg/kg or sirolimus at 2 mg/kg. Prior to immunosuppression, blood was collected to establish baseline immunological parameters which were assessed by flow cytometry. Results: Over the 9 NICC isolations, the most common age for pancreatectomy of the piglets was 3 days (4 preps), followed by 4 (2 preps), 5 (2 preps) and 6 days (1 prep). The difference in age, however, did not significantly alter the yield of islet equivalents (IEQ), a mean of 7,060 1,696IEQ per gram of pancreas (p-gmP) was produced for all preps combined, however, day 3 pancreatectomy demonstrated higher IEQ/ p-gmP with a mean of 7,794 1,802 IEQ/ p-gmP. In 8 of the 9 recipients, there was detectable porcine C peptide (PcPep) produced within 1 month, with an average PcPep of 97pmol/L, and all baboons achieved normoglycaemia with functional IVGTTs over the duration of the treatment protocol. In all recipients, the immunosuppressive therapy was effective in suppressing total white cell count including all key immune cells (B cells, T cells, monocytes and granulocytes) with all grafts surviving for over 100 days. Discussion/Conclusion: The current NICC isolation technique allowed optimisation to produce the greatest IEQ yield with the resulting transplants achieving normoglycemia in less than 1 month and providing normoglycaemia and PcPep production for up-to 12 months. NH&MRC Project Grant Support.

BACKGROUND Pig islet xenotransplantation is a potential treatment for type 1 diabetes. We have shown that maintenance immunosuppression is required to protect genetically modified (GM) porcine islet xenografts from T cell-mediated rejection in baboons. Local expression of a depleting anti-CD2 monoclonal antibody (mAb) by the xenograft may provide an alternative solution. We have previously reported the generation of GGTA1 knock-in transgenic pigs expressing the chimeric anti-CD2 mAb diliximab under an MHC class I promoter (MHCIP). In this study, we generated GGTA1 knock-in pigs in which MHCIP was replaced by the β-cell-specific porcine insulin promoter (PIP), and compared the pattern of diliximab expression in the two lines. METHODS A PIP-diliximab knock-in construct was prepared and validated by transfection of NIT-1 mouse insulinoma cells. The construct was knocked into GGTA1 in wild type (WT) porcine fetal fibroblasts using CRISPR, and knock-in cells were used to generate pigs by somatic cell nuclear transfer (SCNT). Expression of the transgene in MHCIP-diliximab and PIP-diliximab knock-in pigs was characterised at the mRNA and protein levels using RT-qPCR, flow cytometry, ELISA and immunohistochemistry. Islets from MHCIP-diliximab and control GGTA1 KO neonatal pigs were transplanted under the kidney capsule of streptozotocin-diabetic SCID mice. RESULTS NIT-1 cells stably transfected with the PIP-diliximab knock-in construct secreted diliximab into the culture supernatant, confirming correct expression and processing of the mAb in β cells. PIP-diliximab knock-in pigs showed a precise integration of the transgene within GGTA1. Diliximab mRNA was detected in all tissues tested (spleen, kidney, heart, liver, lung, pancreas) in MHCIP-diliximab pigs, but was not detectable in PIP-diliximab pigs. Likewise, diliximab was present in the serum of MHCIP-diliximab pigs, at a mean concentration of 1.8 μg/mL, but was not detected in PIP-diliximab pig serum. An immunohistochemical survey revealed staining for diliximab in all organs of MHCIP-diliximab pigs but not of PIP-diliximab pigs. Whole genome sequencing (WGS) of a PIP-diliximab pig identified a missense mutation in the coding region for the dixilimab light chain. This mutation was also found to be present in the fibroblast knock-in clone used to generate the PIP-diliximab pigs. Islet xenografts from neonatal MHCIP-diliximab pigs restored normoglycemia in diabetic immunodeficient mice, indicating no overt effect of the transgene on islet function, and demonstrated expression of diliximab in situ. CONCLUSION Diliximab was widely expressed in MHCIP-diliximab pigs, including in islets, consistent with the endogenous expression pattern of MHC class I. Further investigation is required to determine whether the level of expression in islets from the MHCIP-diliximab pigs is sufficient to prevent T cell-mediated islet xenograft rejection. The unexpected absence of diliximab expression in the islets of PIP-diliximab pigs was probably due to a mutation in the transgene arising during the generation of the knock-in cells used for SCNT.

Background: Our preclinical model demonstrates long-term restoration of blood glucose control in diabetic immunosuppressed baboons transplanted with GTKO-hCD55-hCD59 porcine neonatal islet cell clusters (NICCs). However, T cell-mediated rejection inevitably occurs following withdrawal of maintenance immunosuppression. We hypothesise local expression of an anti-T-cell monoclonal antibody by the xenografts may prevent rejection. We previously reported generation of two transgenic (Tg) pig lines, using CRISPR to integrate a transgene for diliximab, a chimeric anti-human CD2 monoclonal antibody, into GGTA1. Transgene expression was directed by either a mouse MHC class I promoter (MHCIP) for ubiquitous tissue expression, or the pig insulin promoter (PIP) for islet-specific expression. Aim: To compare diliximab expression in the Tg pig lines and to examine local expression in a pig-to-mouse islet xenograft model. Methods: Diliximab expression was tested by ELISA (serum), RT-qPCR and immunohistochemistry (tissues). MHCIP-diliximab and control GTKO NICCs were transplanted under the kidney capsule of streptozotocin-diabetic SCID mice. NICC xenograft maturation and function were assessed by blood sugar levels (BSL) and intraperitoneal glucose tolerance tests. Results: Diliximab mRNA and protein were detected in spleen, kidney, heart, liver, lung, and pancreas of MHCIP-diliximab pigs, and in serum at a mean concentration of 1.8 μg/ml. Tissue expression ranged from strong in spleen to patchy in pancreas, with weak expression in islets. MHCIP-diliximab NICC xenografts restored normoglycemia in diabetic immunodeficient mice, indicating no overt effect of the transgene on islet function, and demonstrated weak expression of diliximab in situ on day 85. Diliximab was not detected in serum or non-pancreatic tissues of PIP-diliximab pigs, but unexpectedly was also absent in islets. Discussion/Conclusion: Diliximab was expressed in all tested organs of MHCIP-diliximab pigs including the pancreas. Islet cell function appeared to be unaffected by the transgene, and expression by islet xenografts in mice was detected at the transplant site. Despite weak basal expression in MHCIP-diliximab islets, we predict that in the transplant setting, pro-inflammatory cytokines produced by activated T cells recruited to the site will induce the MHCIP to upregulate diliximab expression; whether this will be sufficient to prevent T-cell mediated islet xenograft rejection awaits further investigation. Surprisingly, the PIP-diliximab pigs failed to express diliximab in islets, despite in vitro validation of the transgene construct and confirmation of correct integration into GGTA1. Sequencing analysis is underway to assess the integrity of the transgene in the genome of the PIP-diliximab pigs. National Health & Medical Research Council of Australia (Project Grants #1061868 and #1156889) and the Juvenile Diabetes Research Foundation (Project 3-SRA-2017-366-S-B).

Introduction: CD4+Foxp3+regulatory T-cells (Tregs) are essential for islet xenotransplant tolerance. We identified a novel memory-like CD127+/highTreg subset in the spleen of tolerant mice following CTLA4-Fc/MR-1 induction and demonstrated their potent suppressive capacity in an adaptive-transfer mouse model. Our aims were to: (1) Further characterise CD127+/high Tregs. (2) Investigate transcriptional profile of CD4+Foxp3+Treg and non-Foxp3 CD4+ subsets in transplant tolerance. Methods:DEpletion of REGulatory T-cells (DEREG) mice carrying a GFP transgene under a Foxp3 promoter were used as recipients in a porcine NICC xenotransplant model. Cell-subsets were selected with BD Influx Cell Sorter based on expression of CD4, GFP, and CD127 or CD45, CD4 and GFP. Bulk RNA-Seq and flow-cytometry profiled the transcriptomes and phenotypes of Treg subsets (CD127highTreg, CD127-/lowTreg, and all Treg) from the spleen (sp), draining lymph node (DLN) and grafts of tolerant-mice (day-100) compared to non-transplant mice. Quantitative PCR (qPCR) was used to assess expression of Il10/Tgfb1/Ifnγ/Il2/Il7//Il18//Il33/Ctla4 in spleen, ALN, DLN and graft cells of transplant mice (CTLA4-Fc/MR-1-treatment), rejection mice (no treatment) at day-8/100 and non-transplant mice. Imaging mass cytometry (17 antibodies) was used to evaluate the graft-infiltrating immune cells in tolerant-(day-8, 20, 100) and rejection-(day-8, 20) groups. Results: A high proportion of CD127highTregs was observed in tolerant-grafts compared to tolerant-spleens [25.6±3.1% vs 14.8±0.4%]. Ebi3(IL-35), Il-10 and Blimp-1 expression were upregulated in splenic CD127highTregs of day-100 transplant-mice compared to naïve-Tregs. We identified 1740 differentially expressed genes (DEGs)(FDR<0.05) from 15 pairwise-comparisons that distinguished between CD45+CD4-, Foxp3-CD4+T-cells, and Treg subsets with: similar expression patterns between naive and tolerant CD45+CD4- cells; minor differences in Foxp3-CD4+T cells; and notable differences across Treg subsets where transcriptional profile was not uniform. Between different Treg subsets, 9 paired cross-comparisons identified 427 DEGs. In tolerant mice, both sp- and DLN-CD127+Tregs showed an effector/memory Treg profile. Compared to naïve-Treg or CD127-/lowTreg subsets, graft-Treg and CD127highTregs displayed upregulated DEGs including Il7r, Kctd12, Cxcr6, Ctla2a, Anxa1, H2-Ab, Klrk1, Klrg1, Ccl5, Id2, Ccr2, Adam8, Il18r1, Il1rl that have been reported in multiple tissue/tumour Treg subsets with memory features. Additionally, measured by qPCR, increased Il7 expression and decreased Il2 expression in day-100 vs day-8 tolerant-graft suggest activated Tregs may preferentially utilise IL-7 or IL-35 over IL-2 (more broadly acting). Conclusion: Memory-like CD127+/highTregs are critical for maintaining tolerance and may share a transcriptional trajectory with other tissue/tumour Tregs. National Health and Medical Research Council (NHMRC; Grants: GNT1013185 & GNT1125456). JDRF/Australian Research Council (Grant: 4-SRA-2016-265-M-B). Diabetes Australia (Grant: Y16G-HUMI). Westmead Scientific Platforms, Westmead Institute for Medical Research. Department of Animal Care at Westmead Hospital. Cancer Institute New South Wales. Ian Potter Foundation. Ramaciotti Facility for Human Systems Biology. Sydney Cytometry, University of Sydney.

Xenotransplantation has seen recent global interest peak as a result of several clinical xenotransplants being performed in decedents and a live cardiac recipient. However, underpinning these latest transplants have been decades of invested scientific research programs that have been developing the ideal donor source animals to avoid the overwhelming hyperacute xenograft rejection seen using nongenetically modified animal organs, tissues, and cells. However, this also needs to be undertaken along with the development of safe and efficacious xenotransplantation technologies, immunosuppression, monitoring, disease screening, patient selection, societal education, and acceptance. Paralleling the advent of such extraordinary transplants have been several decades of establishment of world xenotransplantation authorities such as the International Xenotransplantation Association, and the development of guidance documents and regulations for the assessment of these cutting-edge technologies. Similar to all new technologies there remain outdated concerns and fears of the theoretical potential for transmission of xenozoonosis, ethical concerns, and outdated or appropriately educated societal concerns and religious views of the benefits or risks and issues for xenotransplantation use of organs, tissues, or cells from animals to human beings. Here, we discuss the development of xenotransplantation and the intricate balance in managing the various challenges with which we are faced: in the absolute benefits of xenotransplantation and the dichotomy in balancing the pros and cons of xenotransplantation with social, religious, ethical, scientific, and medical opinions. Ultimately, the benefits are to those patients suffering from the many and various diseases that drive the need for xenotransplantation. The hope is that it will be implemented as soon as possible to help the many millions of patients who can truly benefit.

Background: With the recent progress of xenotransplantation moving to the clinic there is renewed enthusiasm in the field of xenotransplantation, but the need for strong immunosuppressive and anti-inflammatory agents remains. The effects and safety of these agents need to be reviewed in detail to allow progress to the clinic. This project aimed to evaluate the short-term (100 days) effects of immunosuppression on haematological and immunological parameters following neonatal porcine islet cell cluster (NICC) xenotransplantation in a diabetic non-human primate model. Methods: Four baboons received 5 doses of anti-CD2 at 5mg/kg from day -3 to day 21 along with fortnightly anti-CD154 administration at 20-30mg/kg and belatacept administration at20mg/kg. This was followed by daily maintenance immunosuppression of tacrolimus at 5mg/kg or sirolimus at 2mg/kg. Prior to immunosuppression administration, blood was collected to establish baseline haematological and immunological parameters. Full haematological parameters were assayed and immunological parameters including B cells, T cells, monocytes, and granulocytes were assessed by flow cytometry. Results: The immunosuppressive therapy was highly effective in suppressing total white cell counts in all recipients. By day 100, B and T cells were depleted by 50-60% compared to baseline. In the 14-day gap between treatment with anti-CD154+belatacept, the immune cells (particularly B and T cells) recovered but were suppressed at each subsequent time point as compared to previous levels. There was a gradual reversal of the CD4:CD8 ratio, with a reduction of CD4+ cells and an increase in CD8+ cells over the 100 days. Tregs, a key component of achieving tolerance, gradually increased from day 30-60. Other hematological parameters such as mean corpuscular hemoglobin concentration (MCHC) and red cell count (RCC) remained unaffected by immunosuppression. Conclusion: This novel combination of immunosuppressive agents is effective and safe in baboons receiving neonatal porcine islet cell cluster xenotransplantation.

Background: We found previously that depletion of CD4+Foxp3+Tregs at early time (within 20 days) and later time (80 days) of transplantation abrogated pig-islet-xenograft tolerance in mice induced by short-term CTLA4-Fc/MR-1 treatment. We also identified memory-like CD127+/highCD4+GFP+/Foxp3+Tregs (CD127+/highTreg) in spleen of tolerant mice following CTLA4-Fc/MR-1 induction and demonstrated their potent suppressive capacity in an adaptive-transfer model. Aims: 1) Further characterise tissue CD127+/high Tregs. 2) Investigate transcriptional profile of CD4+Foxp3+Treg and non-Foxp3 CD4+ subsets in transplant tolerance. Methods: We used DEpletion of REGulatory T cells (DEREG) mice, which carry the enhanced GFP transgene under Foxp3 promoter as recipients of NICC transplantation tolerance model. Cell-subsets were selected with FACS/Cell Sorter based on positive or negative expressions of CD4, GFP, and CD127 or CD45, CD4 and GFP. mRNA expression of Il-10, Tgf-β, Blimp-1, Ebi3 (reflecting IL-35) of CD127+/high Tregs was assessed using TaqMan® Gene Expression Assay. Bulk RNA-Seq revealed the transcriptional profiles of CD127+/highTreg, CD127-/low Treg, CD4+GFP-Foxp3+ Treg, non-Foxp3 CD4+, and CD45+CD4- subsets from spleens (sp), graft draining-lymphocytes (DLN/dln), or grafts in mice with 100-day tolerant-graft induced by CTLA4-Fc/MR-1 blockade or naïve DEREG-mice. Results: RT-PCR showed Ebi3, Il-10, Blimp-1 significantly increased in splenic CD127+/high Tregs compared to naïve-CD4+GFP-Foxp3+Tregs or non-Foxp3 CD4+T cells. The proportion of CD127+/high Tregs was higher in tolerant grafts (25.6±3.1%) than tolerant spleens (14.8±0.4%). 15 pairwise-comparisons identified 1740 differentially expressed genes (DEGs) (FDR<0.05) that clearly distinguished between CD45+CD4-, Foxp3-CD4+T, and Treg subsets; with no striking differences seen for CD45+CD4- cells (spleen) and mild differences in Foxp3-CD4+T cells (spleen) between naive and tolerant-groups; and diverse differences within Treg subsets. Next, 9 paired cross-comparisons between different Treg subsets identified 427 DEGs and showed large difference between graft-Treg and Treg subsets of spleen or DLN; moderate differences between spTreg and dlnTreg subsets; and minor differences within the three Treg subsets of spleen or DLN. Further, compared to naïve-Treg or CD127-/low Treg subsets, graft-Tregs shared many upregulated-DEGs across dlnCD127+/high Treg, and/or spCD127+/high Treg including Il7r, Kctd12, Cxcr6, Ctla2a, Anxa1, H2-Ab1 (an MHC-II gene), Klrk1, Klrg1, Ccl5, Id2, Ccr2, Adam8, Il18r1, Il1rl that have been reported in multiple tissue/tumour Treg subsets with memory features and high suppressive functions in both mice and/or humans. Conclusion: Tissue-Tregs (CD127+/high Tregs) developed in graft, spleen and DLN of transplant-tolerant mice share a transcriptional trajectory with other tissue/tumour Tregs.

XenotransplantationVolume 28, Issue 2 e12674 LITERATURE UPDATE Xenotransplantation literature update, November/December 2020 Wayne J. Hawthorne, The Centre for Transplant & Renal Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia The Department of Surgery, Westmead Hospital, University of Sydney, Westmead, NSW, AustraliaSearch for more papers by this authorAdwin Thomas, The Centre for Transplant & Renal Research, The Westmead Institute for Medical Research, Westmead, NSW, AustraliaSearch for more papers by this authorChristopher Burlak, Corresponding Author cburlak@umn.edu Department of Surgery, Schulze Diabetes Institute, University of Minnesota Medical School, Minneapolis, MN, USA Correspondence Christopher Burlak, Department of Surgery, Schulze Diabetes Institute, University of Minnesota Medical School, Minneapolis, MN, USA. Email: cburlak@umn.eduSearch for more papers by this author Wayne J. Hawthorne, The Centre for Transplant & Renal Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia The Department of Surgery, Westmead Hospital, University of Sydney, Westmead, NSW, AustraliaSearch for more papers by this authorAdwin Thomas, The Centre for Transplant & Renal Research, The Westmead Institute for Medical Research, Westmead, NSW, AustraliaSearch for more papers by this authorChristopher Burlak, Corresponding Author cburlak@umn.edu Department of Surgery, Schulze Diabetes Institute, University of Minnesota Medical School, Minneapolis, MN, USA Correspondence Christopher Burlak, Department of Surgery, Schulze Diabetes Institute, University of Minnesota Medical School, Minneapolis, MN, USA. Email: cburlak@umn.eduSearch for more papers by this author First published: 21 March 2021 https://doi.org/10.1111/xen.12674Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Volume28, Issue2March/April 2021e12674 RelatedInformation

XenotransplantationVolume 28, Issue 5 e12710 LITERATURE UPDATE Updateon xenotransplantation for May/June 2021 Wayne J. Hawthorne, Corresponding Author wayne.hawthorne@sydney.edu.au Centre for Transplant & Renal Research, Westmead Institute for Medical Research, Westmead, New South Wales, Australia Department of Surgery, Westmead Clinical School, Westmead Hospital, University of Sydney, Westmead, New South Wales, Australia Correspondence Wayne J Hawthorne, Department of Surgery, Westmead Clinical School, Westmead Hospital, University of Sydney, Westmead NSW 2145, Australia. Email: wayne.hawthorne@sydney.edu.au Christopher Burlak, Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA. Email: cburlak@umn.eduSearch for more papers by this authorErin Fuller, Centre for Transplant & Renal Research, Westmead Institute for Medical Research, Westmead, New South Wales, AustraliaSearch for more papers by this authorAdwin Thomas, Centre for Transplant & Renal Research, Westmead Institute for Medical Research, Westmead, New South Wales, AustraliaSearch for more papers by this authorJoseph Sushil Rao, Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, Minnesota, USA Solid Organ Transplantation, Department of Surgery, University of Minnesota, Minneapolis, Minnesota, USASearch for more papers by this authorChristopher Burlak, Corresponding Author cburlak@umn.edu Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, Minnesota, USA Correspondence Wayne J Hawthorne, Department of Surgery, Westmead Clinical School, Westmead Hospital, University of Sydney, Westmead NSW 2145, Australia. Email: wayne.hawthorne@sydney.edu.au Christopher Burlak, Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA. Email: cburlak@umn.eduSearch for more papers by this author Wayne J. Hawthorne, Corresponding Author wayne.hawthorne@sydney.edu.au Centre for Transplant & Renal Research, Westmead Institute for Medical Research, Westmead, New South Wales, Australia Department of Surgery, Westmead Clinical School, Westmead Hospital, University of Sydney, Westmead, New South Wales, Australia Correspondence Wayne J Hawthorne, Department of Surgery, Westmead Clinical School, Westmead Hospital, University of Sydney, Westmead NSW 2145, Australia. Email: wayne.hawthorne@sydney.edu.au Christopher Burlak, Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA. Email: cburlak@umn.eduSearch for more papers by this authorErin Fuller, Centre for Transplant & Renal Research, Westmead Institute for Medical Research, Westmead, New South Wales, AustraliaSearch for more papers by this authorAdwin Thomas, Centre for Transplant & Renal Research, Westmead Institute for Medical Research, Westmead, New South Wales, AustraliaSearch for more papers by this authorJoseph Sushil Rao, Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, Minnesota, USA Solid Organ Transplantation, Department of Surgery, University of Minnesota, Minneapolis, Minnesota, USASearch for more papers by this authorChristopher Burlak, Corresponding Author cburlak@umn.edu Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, Minnesota, USA Correspondence Wayne J Hawthorne, Department of Surgery, Westmead Clinical School, Westmead Hospital, University of Sydney, Westmead NSW 2145, Australia. Email: wayne.hawthorne@sydney.edu.au Christopher Burlak, Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA. Email: cburlak@umn.eduSearch for more papers by this author First published: 07 October 2021 https://doi.org/10.1111/xen.12710Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Volume28, Issue5September/October 2021e12710 RelatedInformation

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.