Reducing the immunogenicity of the heart by genetic engineering during ex vivo heart perfusion

Heart transplantation (HTx) is associated with major hurdles related to the limited number of organs available for transplantation, the risk of rejection due to genetic discrepancies and the immunosuppression (IS) burden. Previously, we showed that the silencing of swine leucocyte antigen (SLA) class I and class II expression in pulmonary grafts enabled graft survival after allogeneic lung transplantation even in the absence of IS. In this study, we aimed at evaluating the feasibility to decrease the immunogenicity of the heart.

Lentiviral vectors encoding for shRNAs targeting β2-microglobulin sh(β2m) and class II transactivator (shCIITA) were delivered to the heart during two hours of normothermic ex-vivo heart perfusion (EVHP). Hearts perfused with lentiviral vectors encoding for non‐specific shRNA (shNS) or non-transduced (non-TD) hearts served as controls. After perfusion different cardiac cell types wer isolated, culture in vitro and evaluated for the expression of the reporter gene in luminescence assays and the SLA expression by real-time PCR and flow cytometry analyses.

Transduction efficiency was extremely high in all types of cells in the heart tissue including endothelial cells, fibroblasts and cardiomyocytes. Compared to shNS transduced hearts, silenced hearts showed up to 95% decreased SLA class I and up to 65% SLA class II-DQ expression, including the vascular endothelium as the major interface between donor and recipient. Histological analyses, perfusate troponin T (non-TD: 0.4±0.1 vs. TD: 0.2±0.1 ng/ml) and LDH levels (Abs A₄₉₀-A₆₉₀: non-TD: 0.961±0.1 vs. TD: 0.860±0.1) indicated no additional cell injury or tissue damage caused by lentiviral vector transduction in comparison to non-TD hearts after normothermic EVHP. Additionally, pro-inflammatory cytokine secretion signatures did not significantly differ between shβ2m/shCIITA and shNS transduced hearts.

This study clearly shows the potential of heart genetic engineering and its use in creating immunological graft invisibility. It is very likely that this technology will open up a completely new direction in overcoming organ rejection.

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