Purpose: Cell migration and proliferation are still an open issues concerning atherogenesis and restenosis. The exploitation of ribozyme for the inactivation of genes involved in the phenotype changes responsible for the pathological state, could be a promising and innovative technique. The Purpose of this study was to set up a method for the delivery and the validation of a ribozyme in Vascular Smooth Muscle Cells (VSM cells). Methods: A minimal ribozyme was tailored on PDGFR-b mRNA target sequence by an "in silico" approach and synthesized with an oligonucleotide synthesizer apparatus. The delivery was accomplished with a new polyplexed vehicle. The complex vehicle-ribozyme was administered to porcine VSM cells in culture in the presence of serum. Cells were stimulated by PDGF-BB after 24 hours and their migration activity monitored according to an "in vitro wound model". Results: A differential migratory activity was observed: cells stimulated with PDGF-BB, invaded extensively the scratched surface, whereas cells pre-treated with ribozyme (before growth factor stimulation) displayed a considerably reduction of this repair activity. A computational measure of repair demonstrated that the ribozyme reduced the cell migration of about 90%. Conclusions: These results are very encouraging since the activity of ribozyme was demonstrated in a biological model. Moreover, the vehicle preserved the gene-drug from nucleases and it was highly effective also in the presence of serum, condition thatmimic the effective environment of vascular tissue. Such pilot experiments open actual perspectives for "in vivo" local gene knock-down treatments suggesting therapeutic strategies based on multiple ribozymes targeting different crucial pathological genes.
Ribozymes as specific therapeutic agents against smooth muscle cell migration and proliferation: a new perspective in gene therapy
CECCHETTINI, ANTONELLA;TEDESCHI, LORENA;
2010-01-01
Abstract
Purpose: Cell migration and proliferation are still an open issues concerning atherogenesis and restenosis. The exploitation of ribozyme for the inactivation of genes involved in the phenotype changes responsible for the pathological state, could be a promising and innovative technique. The Purpose of this study was to set up a method for the delivery and the validation of a ribozyme in Vascular Smooth Muscle Cells (VSM cells). Methods: A minimal ribozyme was tailored on PDGFR-b mRNA target sequence by an "in silico" approach and synthesized with an oligonucleotide synthesizer apparatus. The delivery was accomplished with a new polyplexed vehicle. The complex vehicle-ribozyme was administered to porcine VSM cells in culture in the presence of serum. Cells were stimulated by PDGF-BB after 24 hours and their migration activity monitored according to an "in vitro wound model". Results: A differential migratory activity was observed: cells stimulated with PDGF-BB, invaded extensively the scratched surface, whereas cells pre-treated with ribozyme (before growth factor stimulation) displayed a considerably reduction of this repair activity. A computational measure of repair demonstrated that the ribozyme reduced the cell migration of about 90%. Conclusions: These results are very encouraging since the activity of ribozyme was demonstrated in a biological model. Moreover, the vehicle preserved the gene-drug from nucleases and it was highly effective also in the presence of serum, condition thatmimic the effective environment of vascular tissue. Such pilot experiments open actual perspectives for "in vivo" local gene knock-down treatments suggesting therapeutic strategies based on multiple ribozymes targeting different crucial pathological genes.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
