Efficient ex vivo inhibition of perforin and Fas ligand expression by chimeric tRNA-hammerhead ribozymes

Zhimei Du, Camillo Ricordi, Luca A Inverardi, Eckhard Podack, Ricardo Pastori

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1551-1560
Number of pages10
JournalHuman Gene Therapy
Volume9
Issue number11
StatePublished - Jul 20 1998

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Perforin
Fas Ligand Protein
Transfer RNA
Catalytic RNA
Graft vs Host Disease
RNA
RNA Polymerase III
Homologous Transplantation
Papilloma
Bone Marrow Transplantation
Rodentia
Cytoplasm
Plasmids
hammerhead ribozyme
Viruses
Polymerase Chain Reaction
Research
Proteins

ASJC Scopus subject areas

  • Genetics

Cite this

Efficient ex vivo inhibition of perforin and Fas ligand expression by chimeric tRNA-hammerhead ribozymes. / Du, Zhimei; Ricordi, Camillo; Inverardi, Luca A; Podack, Eckhard; Pastori, Ricardo.

In: Human Gene Therapy, Vol. 9, No. 11, 20.07.1998, p. 1551-1560.

Research output: Contribution to journalArticle

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