Efficient ex vivo inhibition of perforin and Fas ligand expression by chimeric tRNA-hammerhead ribozymes.
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.
Diabetes Research Institute, University of Miami School of Medicine, FL 33136, USA.
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Ricordi
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Inverardi
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Podack
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Pastori
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D007150:Immunohistochemistry; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000235:genetics; D054353:Perforin; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D052899:Pore Forming Cytotoxic Proteins; D017433:Protein Structure, Secondary; D012313:RNA / Q000032:analysis / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*; D014162:Transfection; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D016337:RNA, Catalytic; D011993:Recombinant Fusion Proteins; D019014:fas Receptor; D054353:Perforin; D012313:RNA; D012343:RNA, Transfer