Characterisation of transgenic pigs expressing a human T cell-depleting anti-CD2 monoclonal antibody.

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 ß-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 ß 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.

Xenotransplantation literature update, November/December 2019.

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.

Intragraft memory-like CD127hiCD4+Foxp3+ Tregs maintain transplant tolerance.

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 CD127hi 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 CD127hi 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 CD127hi Treg subset was associated with a strong activation of phosphorylation of STAT5. We proposed that memory-like CD127hi 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.

Ethics and Theoretical Issues in Kidney Xenotransplantation.

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.