EFFICIENT EX-VIVO INHIBITION OF PERFORIN AND FAS LIGAND EXPRESSION BYCHIMERIC TRANSFER-RNA-HAMMERHEAD RIBOZYMES

Graft-versus-host disease (GVHD) is a feared complication of allogenei c bone marrow transplantation. Research in rodent models has linked pe rforin and Fas ligand (FasL), two components of independent lytic path ways, with the induction of GVHD, In this study we characterized two h ammerhead ribozymes that cleave their target perforin and Fas ligand R NAs with high efficiency in CTLL-2 cells. The perforin and Fas ligand ribozymes were expressed from a tRNA-directed RNA polymerase III promo ter that was inserted in an episomal multicopy plasmid derived from pa pilloma virus. Chimeric anti-perforin and anti-Fast tRNA-ribozymes had sequences engineered in order to have specific secondary structure ef fects. 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 effici ent transportation to the cytoplasm, The expression of perforin and Ea st in CTLL-2 cells was significantly reduced as assessed by RNA and pr otein analyses.