No abstract

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 ex vivo 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.

In contrast to the whole liver, primary hepatocytes are highly immunogenic. Thus, alternative strategies of immunomodulation after hepatocyte transplantation are of special interest. Silencing of HLA class I expression is expected to reduce the strength of allogeneic immune responses and to improve graft survival. In this study, primary human hepatocytes (PHH) were isolated using a two‐step‐collagenase perfusion‐technique and co‐cultured with allogeneic lymphocytes in terms of a mixed lymphocyte hepatocyte culture. Expression of HLA class I on PHH was silenced using lentiviral vectors encoding for β2‐microglobulin‐specific short hairpin RNA (shβ2m) or non‐specific shRNA (shNS) as control. The delivery of shβ2m into PHH caused a decrease by up to 96% in β2m transcript levels and a down‐regulation of HLA class I cell surface expression on PHH by up to 57%. Proliferative T cell alloresponses against HLA‐silenced PHH were significantly lower than those observed form fully HLA‐expressing PHH. In addition, significantly lower secretion of pro‐inflammatory cytokines was observed. Levels of albumin, urea and aspartate‐aminotransferase did not differ in supernatants of cultured PHH. In conclusion, silencing HLA class I expression on PHH might represent a promising approach for immunomodulation in the transplant setting without compromising metabolic function of silenced hepatocytes.

Diabetes mellitus is characterized by chronic hyperglycaemia caused by abnormalities in insulin secretion, action or both. Eighty to 90% of the cases of diabetes in children and adolescents are suffering from type 1 diabetes resulting from complete autoimmune destruction of pancreatic β-cells through cellular immune responses. [1][2][3] Human allogeneic pancreas transplantation represents a successful treatment of type 1 diabetes.Nevertheless, because of its complexity and the risks associated, such as formation of blood clots, resurgence of pancreatitis and undesired side-effects from the immunosuppressive treatments, it is not considered as routine therapy. 4,5 Human allogeneic transplantation of encapsulated islets has

Low haemocompatibility of left ventricular assist devices (LVAD) surfaces necessitates anticoagulative therapy. Endothelial cell (EC) seeding can support haemocompatibility, however, the availability of autologous ECs is limited. In contrast, allogeneic ECs are readily available in sufficient quantity, but HLA disparities induce harmful immune responses causing EC loss. In this study, we investigated the feasibility of using allogeneic low immunogenic ECs to endothelialize LVAD sintered inflow cannulas (SIC). To reduce the immunogenicity of ECs, we applied an inducible lentiviral vector to deliver short-hairpins RNA to silence HLA class I expression. HLA class I expression on ECs was conditionally silenced by up to 70%. Sufficient and comparable endothelialization rates were achieved with HLA-expressing or HLA-silenced ECs. Cell proliferation was not impaired by cell-to-Sintered Inflow Cannulas (SIC) contact or by silencing HLA expression. The levels of endothelial phenotypic and thrombogenic markers or cytokine secretion profiles remained unaffected. HLA-silenced ECs-coated SIC exhibited reduced thrombogenicity. In contrast to native ECs, HLA-silenced ECs showed lower cell lysis rates when exposed to allogeneic T cells or specific anti-HLA antibodies. Allogeneic HLA-silenced ECs could potentially become a valuable source for LVAD endothelialization to reduce immunogenicity and correspondingly the need for anticoagulative therapy which can entail severe side effects.