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Organ gene therapy represents a promising tool to correct diseases or improve graft survival after transplantation. Polymorphic variation of the major histocompatibility complex (MHC) antigens remains a major obstacle to long-term graft survival after transplantation. Previously, we demonstrated that MHC-silenced cells are protected against allogeneic immune responses. We also showed the feasibility to silence MHC in the lung. Here, we aimed at the genetic engineering of the kidney toward permanent silencing of MHC antigens in a rat model. We constructed a sub-normothermic perfusion system to deliver lentiviral vectors encoding shRNAs targeting β2-microglobulin and the class II transactivator to the kidney. In addition, the vector contained the sequence for a secreted nanoluciferase. After kidney transplantation (ktx), we detected bioluminescence in the plasma and urine of recipients of an engineered kidney during the 6 weeks of post-transplant monitoring, indicating a stable transgene expression. Remarkably, transcript levels of β2-microglobulin and the class II transactivator were decreased by 70% in kidneys expressing specific shRNAs. Kidney genetic modification did not cause additional cell death compared to control kidneys after machine perfusion. Nevertheless, cytokine secretion signatures were altered during perfusion with lentiviral vectors as revealed by an increase in the secretion of IL-10, MIP-1α, MIP-2, IP-10, and EGF and a decrease in the levels of IL-12, IL-17, MCP-1, and IFN-γ. Biodistribution assays indicate that the localization of the vector was restricted to the graft. This study shows the potential to generate immunologically invisible kidneys showing great promise to support graft survival after transplantation and may contribute to reduce the burden of immunosuppression.

Xenotransplantation offers a promising alternative to circumvent the lack of donated human organs available for transplantation. Different attempts to improve the survival of xenografts led to the generation of transgenic pigs expressing various combinations of human protective genes or knocked out for specific antigens. Currently, testing the efficiency of porcine organs carrying different genetic modifications in preventing xenogeneic immune responses completely relies on in vitro assays, humanized mouse models, or non-human primate transplantation models. However, these tests are often associated with major concerns due to reproducibility and generation of insufficient data as well as they raise ethical, logistical, and economic issues. In this study, we investigated the feasibility of specifically assessing the strength of human T-cell responses towards the kidneys of wild-type (WT) or transgenic pigs overexpressing human programmed death-1 ligand 1 (hPD-L1) during ex vivo kidney perfusion (EVKP). Human T cells were shown to adhere to the endothelium and transmigrate into WT and hPD-L1 kidneys. However, transcript levels of TNF-a and IFN-y as well as cytotoxic molecules such as granzyme B and perforin secreted by human T cells were significantly decreased in the tissue of hPD-L1 kidneys in comparison to WT kidneys. These results were confirmed via in vitro assays using renal endothelial cells (ECs) isolated from WT and hPD-L1 transgenic pigs. Both CD4 + and CD8 + T cells showed significantly lower proliferation rates after exposure to hPD-L1 porcine renal ECs in comparison to WT ECs. In addition, the secretion of pro-inflammatory cytokines was significantly reduced in cultures using hPD-L1 ECs in comparison to WT ECs. Remarkably, hPD-L1 EC survival was significantly increased in cytotoxic assays. This study demonstrates the feasibility of evaluating the human response of specific immune subsets such as human T cells towards the whole xenograft during EVKP. This may represent a robust strategy to assess the potency of different genetic modifications to prevent xenogeneic immune responses and thereby predict the risk of immune rejection of new genetically engineered xenografts.

Heart transplantation is associated with major hurdles, including the limited number of available organs for transplantation, the risk of rejection due to genetic discrepancies, and the burden of immunosuppression. In this study, we demonstrated the feasibility of permanent genetic engineering of the heart during ex vivo perfusion. Lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin (shβ2m) and class II transactivator (shCIITA) were delivered to the graft during two hours of normothermic EVHP. Highly efficient genetic engineering was indicated by stable reporter gene expression in endothelial cells and cardiomyocytes. Remarkably, swine leucocyte antigen (SLA) class I and SLA class II expression levels were decreased by 66% and 76%, respectively, in the vascular endothelium. Evaluation of lactate, troponin T, and LDH levels in the perfusate and histological analysis showed no additional cell injury or tissue damage caused by lentiviral vectors. Moreover, cytokine secretion profiles (IL-6, IL-8, and TNF-α) of non-transduced and lentiviral vector-transduced hearts were comparable. This study demonstrated the ex vivo generation of genetically engineered hearts without compromising tissue integrity. Downregulation of SLA expression may contribute to reduce the immunogenicity of the heart and support graft survival after allogeneic or xenogeneic transplantation.

Colorectal cancer (CRC), a severe complication of inflammatory bowel diseases, is a common type of cancer and accounts for high mortality. CRC can be modeled in mice by application of the tumor promoter, azoxymethane (AOM), in combination with dextran sodium sulfate (DSS), which are able to induce colitis-like manifestations. Active colitis correlates with high mucosal concentrations of histamine, which, together with the histamine receptor subtype 4 (H4R), provide a pro-inflammatory function in a mouse colitis model. Here, we analyzed whether H4R is involved in the pathogenesis of AOM/DSS-induced CRC in mice. As compared to wild type (WT) mice, AOM/DSS-treated mice lacking H4R expression (TM) demonstrate ameliorated signs of CRC, i.e., significantly reduced loss of body weight, stiffer stool consistency, and less severe perianal bleeding. Importantly, numbers and diameters of tumors and the degree of colonic inflammation are dramatically reduced in TM mice as compared to WT mice. This is concomitant with a reduced colonic inflammatory response involving expression of cyclooxygenase 2 and the production of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2. We conclude that H4R is involved in the tumorigenesis of chemically-induced CRC in mice via cyclooxygenase 2 expression and, probably, CXCL1 and CXCL2 as effector molecules.

Colorectal cancer (CRC), a severe complication of inflammatory bowel diseases, is a common type of cancer and accounts for high mortality. CRC can be modeled in mice by application of the tumor promoter, azoxymethane (AOM), in combination with dextran sodium sulfate (DSS), which are able to induce colitis-like manifestations. Active colitis correlates with high mucosal concentrations of histamine, which, together with the histamine receptor subtype 4 (H R), provide a pro-inflammatory function in a mouse colitis model. Here, we analyzed whether H R is involved in the pathogenesis of AOM/DSS-induced CRC in mice. As compared to wild type (WT) mice, AOM/DSS-treated mice lacking H R expression (TM) demonstrate ameliorated signs of CRC, i.e., significantly reduced loss of body weight, stiffer stool consistency, and less severe perianal bleeding. Importantly, numbers and diameters of tumors and the degree of colonic inflammation are dramatically reduced in TM mice as compared to WT mice. This is concomitant with a reduced colonic inflammatory response involving expression of cyclooxygenase 2 and the production of C-X-C motif chemokine ligand 1 (CXCL1) and CXCL2. We conclude that H R is involved in the tumorigenesis of chemically-induced CRC in mice via cyclooxygenase 2 expression and, probably, CXCL1 and CXCL2 as effector molecules.

Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis‐antagonizing transcription factor (AATF)/Che‐1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA , BAX and BAK promoter regions to repress p53‐driven expression of these pro‐apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF‐depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho‐mimicking AATF point mutant results in marked adriamycin resistance in vivo . Nuclear AATF enrichment appears to be selected for in p53‐proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53‐proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53‐driven apoptosis and commend this pathway as a target for DNA damage‐sensitizing therapeutic regimens. DNA damage activates the tumour suppressor p53, resulting in either cell‐cycle arrest or apoptosis. One mechanism that selectively represses apoptosis‐promoting genes involves phosphorylation of the transcription factor AATF/Che‐1 by the p38‐activated kinase MK2.