Genetic Engineering of the Kidney to Permanently Silence MHC Transcripts During ex vivo Organ Perfusion

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.

Fit-For-All iPSC-Derived Cell Therapies and Their Evaluation in Humanized Mice With NK Cell Immunity

“…Researchers have access to several molecular tools to disrupt β2M expression, and the choice depends on the outcome needed. Post-transcriptional manipulation by RNA interference (RNAi), either using small interfering RNA (siRNA) or short hairpin RNA (shRNA), stably downregulated or silenced the expression of HLA proteins in B-lymphocyte cell lines ( 58 ) in conjunction with human ESCs ( 59 ), iPSCs ( 60 ), primary human hepatocytes ( 61 ), endothelial cells ( 62 , 63 ) and in a perfusion system at the whole organ level in rat kidney ( 64 ). Silencing with RNAi proved to be stable even after the iPSCs were differentiated to megakaryocytes and even platelets ( 60 ).…”

Generation of vascular chimerism within donor organs

“…Consequently, it has been suggested that direct targeting of the graft vasculature may reduce alloimmunity and prolong graft survival 10 . Previous efforts to modify the extent of endothelial activation were made in the form of immunocloaking the vasculature using a temporary barrier 11 and silencing MHC antigen expression 12 – 14 .…”

Treatment of transplant kidneys during machine perfusion

“…Experimental evidence for the delivery of MSCs [13–15], gene therapies [28], nanoparticles [30,31] and other IRI targeted [33–39] therapies during NMP is increasing and the results have been positive. However, none of the approaches have been tested in clinical practice.…”

“…delivered lentiviral vectors directly to the organ. This delivered encoding shRNA targeting beta2 microglobulin and the class II transactivator, in addition to a sequence for a secreted nanoluciferase [28]. After kidney transplantation and 6 weeks follow up, bioluminescence was detected in the plasma and urine of recipients of engineered kidneys, indicating stable gene expression.…”

“…The transcript levels of beta 2 microglobulin and the class II transactivator were decreased by 70% in kidneys expressing specific shRNAs. In addition, there was an alteration in cytokine secretion trending towards an immunoregulatory profile, and the vector was restricted to the graft as detected by biodistribution assays [28].…”