DEVELOPMENT OF GENE THERAPY FOR CARDIOVASCULAR DISEASES

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Adenoviral vectors for gene transfer to cardiovascular systems were investigated.The role of domestic institute was to establish the in vitro and in vivo model of cardiovascular diseases. In vitro system includes cardiac myocytes from adult rats or new born rats, human umbilical venous endothelial cells, and vascular smooth muscle cells. All were successfully prepared in primary culture for gene transfer target cells. In vivo system includes myocardial infarction model by coronary ligation, mouse heart transplant model, viras induced myocarditis and cardiomyopathy model, and have established. Further establishment of long QT syndrome model are on going.For the candidate genes, cytokines, growth factors, ion channels, transcription factors are prepared. To test the functions of these factors, molecular and biochemical analysis including northern blottings, western blottings, gel shift assays, and others are performed in various myocaridal disease models. Adenovirus vectors including mod … More ified form with polylysin or other chemicals were constructed and tested in the oversea collaborators. Adenovirus containing reporter genes, beta-galactosidase were used as a reference. Target cells were 293 cells, COS cells or CHO cells. Control viruses works fine and recombinant adenovirus containing cDNAs of interest were tested in these cells and in mice. Tissue plasminogen activator, tissue plasminogen activator inhibitor, endothelial nitric oxide synthase, preproendothelin are tested as candidate genes for in vivo gene transfers. Polylysin conjugated adenovirus were also tested in cultured cells to transfer reporter genes. In all cell types listed above are prone to successful transfer by modified adenovirus. Considering that this system has vertually no limit in cDNA size while recombinant adenovirus limits its insert size less than 7kb, larger genes which could not be integrated in recombinant virus may have opportunity of gene transfer. Also, adenovirus is inactivated with during conjugation with polylysin, the risk of biaohazard is minimized in this system. Gene transfer with conventional non viral vectors are on going for growth factors, cytokines, and ion channels including thier native forms and mutant forms.In conclusion, this collaborative research was fruitful and successful. Modified and nonmodified adenovirus gene transefer for cardiovascular system was successfully performed for establishing disease models, identifying candidate genes, constructing vectors, and testing gene transfers. Further applications of more genes in in vivo system are to be continued. Less