Abstract
The U6 expression system was explored for efficient expression of a ribozyme against the human proto-oncogene c-neu. A hammerhead ribozyme (neuRz) and the control mutant ribozyme (MRz) were targeted to cleave c-neu mRNA at the tyrosine kinase domain. In vitro cleavage showed that neuRz was very active while MRz was not. Near-maximal target cleavage observed at a low ribozyme:target ratio (0.1) suggests that neuRz has good activity and turnover capability under physiological conditions, i.e., <5 mM MgCI2 and 37°C. Chimeric U6 ribozyme was expressed at about 5 × 106 copies/cell at 48 h in the ovarian carcinoma cell line SKOV-3.ip1. Partial down-regulation of c-neu mRNA and protein was observed in a dose-dependent manner in cells transiently transfected with U6neuRz- but not with MRz-containing plasmid. Sorted transient transfectants demonstrated dramatic growth inhibition with the neuRz-expressing cells. Our results demonstrate that the U6 expression system is very efficient and suitable for the expression of a hammerhead ribozyme. Moreover, nonviral delivery of the neuRz-expressing plasmid resulted in specific down-regulation of c-neu and, subsequently, growth inhibition of ovarian cancer cells overexpressing c-neu.
Original language | English (US) |
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Pages (from-to) | 169-177 |
Number of pages | 9 |
Journal | Molecular Therapy |
Volume | 3 |
Issue number | 2 |
DOIs | |
State | Published - 2001 |
Externally published | Yes |
Keywords
- Gene therapy
- HER-2/neu ribozyme
- Ovarian cancer growth inhibition
- U6 RNA
ASJC Scopus subject areas
- Molecular Medicine
- Molecular Biology
- Genetics
- Pharmacology
- Drug Discovery