Novel chimeric p16 and p27 molecules with increased antiproliferative activity for vascular disease gene therapy

We describe the construction and characterization of a series of novel cycl in-dependent kinase inhibitors with increased antiproliferative activity fo r use in the genetic treatment of hyperproliferative cell disorders, such a s angioplasty-induced restenosis. These inhibitors were generated through t he fusion of truncated versions of the p27 gene to the full-length p16 gene . Biochemical-ly, the p27-p16 chimeric molecules were of comparable potency to the parental p27 in inhibiting the activities of several cyclin-depende nt kinases in vitro. Replication-deficient adenoviruses encoding the parent al p16, p27 genes, or their derivatives were created to assess the potency of the novel cyclin-dependent kinase inhibitor chimeric molecules to inhibi t vascular smooth muscle cell proliferation, which is the seminal event in the restenosis process. One of the p27-p16 chimeric molecules, W9, was obse rved to be the most potent inhibitor of human primary smooth muscle and end othelial cell proliferation when compared to the p16, p27, p27 derivatives or several alternative p27-p16 chimeric molecules. Overexpression of the W9 chimeric molecule in human coronary artery smooth muscle cells induced hum an coronary artery smooth muscle cell growth arrest in G(1) but did not ind uce cell apoptosis. Recombinant adenoviral vectors that express this W9 chi meric cyclin-dependent kinase inhibitor molecule constitute a novel potent antiproliferative agent for the treatment of restenosis.