Efficient Ex Vivo Inhibition of Perforin and Fas Ligand Expression by Chimeric tRNA–Hammerhead Ribozymes

Abstract

Graft-versus-host disease (GVHD) is a feared complication of allogeneic bone marrow transplantation. Research in rodent models has linked perforin and Fas ligand (FasL), two components of independent lytic pathways, with the induction of GVHD. In this study we characterized two hammerhead ribozymes that cleave their target perforin and Fas ligand RNAs with high efficiency in CTLL-2 cells. The perforin and Fas ligand ribozymes were expressed from a tRNA-directed RNA polymerase III promoter that was inserted in an episomal multicopy plasmid derived from papilloma virus. Chimeric anti-perforin and anti-FasL tRNA–ribozymes had sequences engineered in order to have specific secondary structure effects. These sequence modifications allow the formation of a 5′ → 3′ stem structure and also place the ribozyme in a flexible bulge region that keeps the ribozyme separated from the tRNA domain. Northern and RT in situ PCR analyses showed high levels of transcription and efficient transportation to the cytoplasm. The expression of perforin and FasL in CTLL-2 cells was significantly reduced as assessed by RNA and protein analyses.

Overview summary

We have expressed ex vivo tRNA–hammerhead ribozymes against two components of cytotoxic T cell, perforin and Fas ligand, that previous research had linked to induction of graft-versus-host disease following allogeneic bone marrow transplantation. The chimeric tRNA hammerhead molecules were engineered to form a 5′ → 3′ structure and to place the ribozyme in a flexible region of the molecule separated from the tRNA domain. We found that both ribozymes efficiently inhibited the expression of perforin and Fas ligand in the cytolytic CTLL-2 cell line.