Wider application of small-for-size partial liver grafting by gene transfer using teromerase reverse transcriptase (TERT).

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1. Development of rat liver transplantation model using small-for-size graft:Rat liver transplantation model using small-for-size graft (30% of whole liver volume) was established, in which 100% 3-day mortality was assured.2. Development of gene-transfer technique:(1) Gene transfer using electroporation (EP) technique:An EP-mediated teromerase reverse transcriptase (TERT) gene transfer into human hepatocyte primary cultrure was carried out. A 10% of transfection effectivcess was achieved. However, gene transfer of TERT did not extend the survival of the cells. A direct damege to hepatocytes by EP technique was suggested.(2) Gene transfer into cold-preserved rat liver:Ex vivo EP-mediated or hydrodynamic gene transfer into a cold-preserved (UW solution, 4℃) rat liver graft was evaluated using rat liver transplantation model. The luciferase gene expression in the graft after liver transplantation was found to be increased. However, significant graft damage due to a gene transfer was observed.Therefore, development of an alternative strategy using nano-carrier was indicated.(3) In vivo IL-12 gene transfer using EP technique under immunosuppression:Using mice model carrying hepatoma cells (MH134), in vivo IL-12 gene transfer using EP technique was carried out under immunosuppression by FK506. Intratumoral administration of mIL-12 vector elevated intratumoral IL-12 and IFN-γ on the days 7 and 14 of transfer and significantly inhibited not only the growth of HCC, but also the angiogenesis in the tumor through expression of antiangiogenetic chemokaine IP-10.(4) Gene transfer using hydrodynamic method:As an alternative to EP technique, gene transfer using hydrodynamic method was established. Expression of inciferase gene peaked at 4 hour after injection. The mechanism of the method has not been elucidated so far, thus further investigation was indicated.