Locked nucleic acid modified DNA enzymes targeting early growth response-1 inhibit human vascular smooth muscle cell growth

摘要

Smooth muscle cell (SMC) proliferation and migration are key processes that occur in the pathogenesis of atherosclerosis and post-angioplasty restenosis. In the present study, we designed locked nucleic acid (LNA)-modified DNAzymes targeting a specific region spanning the translational start site of human EGR-1, an immediate-early gene, wherein two of the nucleotides in each of the 9+9 hybridizing arms of the DNAzyme were substituted with LNA monomers. In vitro cleavage experiments revealed that the LNA- modified DNAzyme (LzF4) cleaved a 32P-labelled 388 nt EGR-1 transcript with greater efficacy than its native unmodified phosphodiester counterpart, DzF. The scrambled versions of these molecules, LzF4SCR and DzFSCR, did not display any ability to cleave the transcript. Western blot analysis revealed that both active molecules abrogated serum-inducible EGR-1 protein expression in primary human aortic SMCs and inhibited serum-inducible SMC proliferation in a dose-dependent and non-toxic manner. SMC proliferation was inhibited by >50% with LzF4 at concentrations as low as 20 nM, whereas inhibition by DzF at this concentration was not evident. Finally, LzF4 and DzF inhibited SMC regrowth from the wound edge after mechanical injury in vitro. In contrast, neither DzFSCR nor LzF4SCR had any influence on EGR-1 protein expression, SMC proliferation or regrowth. These findings provide the first functional demonstration of LNA-modified DNAzyme efficacy in a biological setting of any kind. These studies also demonstrate that LNA modification increases DNAzyme potency without necessarily compromising specificity.