Ribozymes as a Tool to Inhibit Artery
Smooth Muscle Cell Proliferation Following Percutaneous Transluminal Angioplasty.
G. Grassi1, A. Kuhn1,
J.J. Schnorr1, H. Koehn1,
M. Grassi3, G. Bauriedel2,
P. Di Nardo4, R. Kandolf1.
1Department of Molecular Pathology, University
of Tuebingen, Germany, 2Department
of Cardiology, University of Bonn, Germany; 3Department
of Chemical Engineering, University of Trieste, Italy; 4Department
of Molecular Cardiology, University of Tor Vergata, Roma, Italy.
Dietary fats play a relevant role in the development of atherosclerosis
which very often results in the partial occlusion of the vessel lumen.
In this case a possible therapeutical approach is represented by percutaneous
transluminal angioplasty (PTA). However, in about 30 to 50 % of the cases
PTA is followed by artery re-occlusion mostly due to vascular smooth muscle
cell proliferation (VSMC). Therefore, we based our therapeutical approach
on the inhibition VSMC growth by hammerhead ribozymes. These are short
RNA molecules capable of specifically cleave a phosphodiester bond within
a RNA molecule. As ribozyme targets we have chosen the mRNAs of E2F1 and
cyclin E genes which play a key role in the transition from G1 to S phase
of the cell cycle. Ribozyme accessible cleavage sites were mapped on the
two full length mRNAs by the RNase H digestion technique. Based on these
results, ribozymes directed against the two targets were generated with
8 nucleotides per binding arm and tested in vitro under single turnover
conditions. Cleavage reactions were performed under physiological conditions
of temperature and pH. The most active ribozymes had kreact/Km ratios between
50 and 80x104 M-1min-1
which are among the best values so far described for ribozymes targeted
against long mRNAs. In addition we investigated the effect on ribozyme
cleavage efficiency of the variation of the binding arm lengths. As a result
of these experiments we found that the optimal binding arm length was in
the range of 8-12 nucleotides. The in vivo effectiveness of the
most active ribozymes will be tested in human smooth muscle cells grown
either in the presence or in the absence of the specific active ribozyme.
The possible ribozyme antisense effect will be tested using inactive ribozymes
bearing a mutation in the active core which abolishes the catalytic activity
but not the possibility to bind to the target. Cell cycle progression will
be evaluated by facs analysis. In conclusion our data represent the first
example of in vitro highly active ribozymes targeted against cyclin
E and E2F1 mRNAs. In addition, preliminary in vivo studies indicate
that our ribozymes are active also in vivo.