Achieving immunological tolerance using genetic engineering to silence human leukocyte antigen molecules within a donated organ may be an effective way to reduce the risk of T cell–mediated rejection. T cell–mediated rejection is caused by host T cells recognizing nonself-variants of major histocompatibility complex (MHC) class I and II molecules expressed on endothelial cells of a transplanted organ.1 It is characterized by lymphocytic infiltration of the tubules, interstitium or arterial intima. small interfering RNAs (siRNAs) silence genes by disrupting mRNA and reducing protein translation.2 They can be designed to target MHC class I and MHC class II molecules. siRNAs were established in 1998, but difficulties with their delivery, stability, and efficiency have impeded their translation into clinical practice.2 The stability can be compromised in the extracellular environment by nucleases or enzymes that degrade the genetic material. Chemical modifications can be made at the phosphate backbone, ribose, or base of the siRNA to enhance stability and efficiency.2 The conjugation of siRNA with cholesterol is 1 of the most effective chemical modifications described.3 There is growing interest and application of ex vivo normothermic machine perfusion (NMP) in kidney transplantation as an alternative method of preservation to hypothermia. It is recognized that NMP provides an ideal setting for the delivery of therapeutics to an isolated organ.4 This limits any potential systemic exposure to the recipient. Typically, a red blood-based solution is used to perfuse an organ at near or normal body temperature.5 The restoration of cellular function allows the active synthesis of agents administered during perfusion. Lin et al6 described the application of siRNA-Cholesterols (siRNA-Chol) targeting β2 microglobulin and Class II major histocompatibility transactivator (CIITA). β2 microglobulin is a component of MHC class I molecules presented on all nucleated cells and is encoded by the B2M gene. CIITA is transcription factor responsible for MHC class II expression. The siRNAs (siRNA B2-Chol and siCIITA-Chol) were delivered to rodent kidneys during NMP at 37°C with an autologous whole blood-based solution over a period of 3 h.6 Following delivery, kidneys were then orthotopically transplanted into mismatched recipient animals replicating an acute rejection model. The transplant kidneys were compared with a static cold storage and NMP negative control kidneys (150 nM siNC-Chol). None of the rats survived in the static cold storage control group, whereas survival was extended to 10 d in the treated and 8 d in the negative control NMP kidneys. This suggests that even alone NMP may have some benefit on reducing acute rejection, perhaps through the depletion of passenger leukocytes from the donated kidney during NMP. Kidney function was improved in treated kidneys with lower serum creatinine levels on days 6 and 7 posttransplant compared with the negative control. Histological evaluation showed better preservation of renal structure, and reduced tubulitis, glomerulopathy and peripheral tubular capillary vasculitis. On day 3 posttransplant, treated kidneys showed reduced expression of B2M and CIIT compared with the negative controls. This was confirmed at a level of protein expression and by flow cytometry showing reduced presence of MHC class I and II. However, by day 7, there was no significant difference between the treated and negative control kidneys and re-expression of MHC was evident. Treated kidneys also showed reduced presence of T and B cells, downregulation of proinflammatory cytokines and upregulation of anti-inflammatory cytokines, and a reduction in IgG and IgM antibodies by day 7 posttransplant. The siRNA-Chol demonstrated a good safety profile with no enhancement of injury markers, alteration of morphology, and intact tissue integrity. Several kidneys were treated with siNC-Chol-Cy5fluorescence during NMP and animals recovered until day 3 posttransplant to validate and confirm the direct targeting of siRNA-Chol in the kidney. Other studies have shown a more sustained downregulation of MHC molecules with the application of RNAs combined with lentivirus vectors. Yuzefovych et al administered a lentiviral vector encoding short hairpin (shRNAs) targeting β2 microglobulin and CIITA to the kidney during subnormothermic ex vivo perfusion.7 Transcript levels of β2 microglobulin and the CIIT were decreased by 70% after 6 wk posttransplantation. There were alterations in cytokine levels but no evidence of cytotoxicity. In the lung, Figueiredo et al demonstrated the permanent downregulation of swine leukocyte antigen during ex vivo lung perfusion by lentiviral transduction of short hairpin RNAs again targeting mRNAs encoding β2 microglobulin and CIITA.8 This reduced the incidence of rejection in a porcine allogeneic lung transplant model. Recipients of treated lungs also had lower donor-specific antibodies and lower levels of proinflammatory cytokines post-transplant. Remarkably several of the animals remained alive without immunosuppression during the 2-y monitoring period. It could be argued that what Lin et al has shown is not novel and not as effective as these previous studies. Nonetheless, in the kidney, this study is the first to use an acute rejection transplant model to assess the effects of the siRNA-Chol. The study provides commanding evidence of the efficiency of the siRNA-Chol to prevent acute T cell–mediated rejection. The application of ‘naked’ RNAs offer less off-target effects compared with the use of viral vectors. Viral vectors carry a high risk of immunogenicity and mutagenesis in immunocompromised recipients. There is still a long road to translation and more sustained strategies need to be developed. The authors suggested that their siRNA therapy could enable immunosuppression to be reduced in the initial post-transplant phase to minimize any side effects or complications, which is plausible. Future work will need to investigate strategies to extend gene suppression and test the effect in more clinically relevant models. In conclusion, the delivery of siRNAs using machine perfusion technology offers a promising strategy to reduce organ immunogenicity by targeting MHC class I and class II expression.
Sarah A Hosgood (Thu,) studied this question.