DNA-RNA chimeric hammerhead ribozyme to transforming growth factor-beta(1)mRNA inhibits the exaggerated growth of vascular smooth muscle cells from spontaneously hypertensive rats

Objective: The purpose of this study was to develop DNA-RNA chimeric hammer head ribozyme against transforming growth factor-beta(1) (TGF-beta(1)) mRNA as a gene therapy agent for arterial proliferative diseases. Methods: a 38 -base hammerhead ribozyme against rat TGF-beta(1) mRNA, to produce cleavage at the GUC sequence at nucleotide 825 according to the secondary structure of rat TGF-beta(1) mRNA was designed. To enhance its stability, we synthes ized a DNA-RNA chimeric ribozyme with two phosphorothioate linkages at the 3'-terminal. We also synthesized a mismatch ribozyme with single base chang e in the catalytic loop region as a control. These ribozymes were delivered into rat vascular smooth muscle cells (VSMC) from spontaneously hypertensi ve rats (SHR) and normotensive Wistar-Kyoto (WKY) rats by lipofectin-mediat ed transfection, and their biological effects were investigated. Results: A ccording to in vitro cleavage studies, the synthetic ribozyme can cleave th e synthetic substrate RNA into two RNA fragments. Chimeric ribozyme signifi cantly inhibited DNA synthesis in VSMC from SHR but not in cells from WKY r ats. Mismatch ribozyme showed only a little effect on growth of VSMC from S HR. Chimeric ribozyme significantly inhibited proliferation of VSMC from SH R; in contrast, the proliferation of-VSMC from WKY rats was significantly i ncreased by this chimeric ribozyme. Mismatch ribozyme did not affect prolif eration of VSMC from either rat strain. Chimeric hammerhead ribozyme to rat TGF-beta(1) dose-dependently inhibited TGF-beta(1) mRNA expression detecte d by reverse transcription and polymerase chain reaction analysis in VSMC f rom both rat strains. Chimeric hammerhead ribozyme to rat TGF-beta(1) also dose-dependently inhibited TGF-P, protein production detected by Western bl ot analysis. Conclusions: The present results demonstrated that our designe d DNA-RNA chimeric hammerhead ribozyme to TGF-beta(1) mRNA might be a usefu l gene therapy agent for hypertensive vascular diseases. (C) 2000 Elsevier Science B.V. All rights reserved.