Gene therapy: can we prevent/modulate apoptosis in EC?

Abstract

Abstract Purpose Regardless of the inciting cause, CEC loss is a common denominator of corneal graft failure. CEC loss during storage results in significant loss of suitable tissue for grafting, CEC loss after transplantation is a major cause of graft failure. The purpose of this study is to investigate the role of apoptosis in CEC in order to prevent CEC loss during storage. Methods Gene transfer of Lenti‐Bcl‐xL or –p35 was accomplished in human donor corneas, primary cultured CEC and an immortalized CEC line and compared to untreated controls. Cell death (apoptosis) was induced by Actinomycin or Etoposide (external vs. internal apoptotic pathway, respectively). In addition, CEC loss during preservation was studied both during Optisol GS (4C) and organ culture storage (37C, Biochrome Medium I). Both storage media were diluted with PBS to promote cell loss. CEC were enumerated, apoptosis was detected by TUNEL staining and confocal microscopy. Results The percentage of TUNEL‐positive CEC provoked by the apoptotic inducers was significantly reduced relative to controls. Transfected corneas preserved an almost intact endothelial monolayer while controls nearly entirely lost vital CEC. During long‐term storage experiments at 4C and at 37C, CEC counts in corneas expressing anti‐apoptotic genes remained significantly higher compared to the controls. Conclusion Protection of CEC by anti‐apoptotic genes appears to be an effective method to reduce CEC loss during storage. The application of this technique could increase the amount of high quality grafts in eye banking and further reduce graft failure following corneal transplantation, and is be of specific interest as to precut corneas and DSAEK procedures.