Silencing HLA Class I Expression in Human Corneas to Decrease the Risk of Graft Rejection after Keratoplasty

Abstract

The high variability of the human histocompatibility leukocyte antigen (HLA) complex, and of the minor histocompatibility antigens significantly contributes to elicit an immune response after allogeneic cornea transplantation, and may lead to the development of acute or chronic rejection. In addition, often inevitable second cornea transplantation is always associated with an increased risk of graft rejection. This project aims to permanently reduce HLA expression on cornea transplants to decrease the risk of graft rejection after keratoplasty. This method represents a new therapeutic concept and may contribute to prevent graft rejection. For this purpose, lentiviral vectors encoding short hairpin RNAs (shRNA) targeting β2-microglobulin were used to induce the downregulation of HLA class I expression. Levels of β2-microglobulin mRNA or cell surface HLA class I expression were determined by Real-Time PCR and flow cytometry, respectively. Cell viability tests using 7-Aminoactinomycin (7-AAD) and Propidium iodide (PI) staining were performed after transduction. Light and fluorescence microscopic analysis were carried out to evaluate the integrity of the corneal endothelium. A mean of 92%±7% of the total cornea cells were transduced. In comparison to the non-engineered cornea, the delivery of the β2-microglobulin-specific shRNA caused a reduction by up to 95% in β2-microglobulin transcript levels as detected at day 5 and day 15 after cornea transduction. Furthermore, the reduction of β2-microglobulin levels induced a decrease in the cell surface expression of HLA class I proteins by up to 90%. Microscopic analysis of the cornea endothelium showed that this cell layer was not affected by the transduction procedure or the silencing effect. The integrity of the HLA class I silenced endothelium and the absence of a positive signal upon 7-AAD/PI staining indicated the absence of off target effects that might impair the endothelial function or viability. This data demonstrates the feasibility of silencing HLA class I expression in the original 3D tissue-structure by RNAi-mediated nucleic acid targeting. As in vivo data in an allogeneic rat model showed a superior survival of RT1-silenced cells, silencing HLA expression has the potential to overcome alloimmunization and tissue rejection caused by HLA mismatches in allogeneic keratoplasty.