| Co-Investigator(Kenkyū-buntansha) |
MORIGUCHI Atsushi Osaka University Graduate School of Medicine Assistant Professor, 医学系研究科, 助手 (10273666)
MATSUMOTO Kunio Osaka University Graduate School of Medicine Associate Professor, 医学系研究科, 助教授 (90201780)
KANEDA Yasufumi Osaka University Graduate School of Medicine Professor, 医学系研究科, 教授 (10177537)
MORISHITA Ryuichi Osaka University Graduate School of Medicine Associate Professor, 医学系研究科, 助教授 (40291439)
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| Budget Amount *help |
¥5,600,000 (Direct Cost: ¥5,600,000)
Fiscal Year 2000: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
In this study we developed an efficient gene transfer method into the kidney and several strategies for gene therapy to renal diseases. We first reported the successful gene transfer into the rat kidney by using HVJ- liposome method (BBRC 1992). We have demonstrated potential of gene therapy to renal disease, especially glomerulonephritis, by combination of decoy strategy and conventional HVJ-liposome method (JASN 1995, Exp Nephrol 1997). Results drawn from this study are shown as below. Crescentic glomerulonephritis was induced in primed Wistar rats by injection of sheep anti-glomerular basement membrane serum. Thirty minutes after injection, rats were anaesthetised and the left kidney perfused with NF-κB decoy ODN or scrambled ODN control mixed with a virus-liposome complex, and then killed 7 days later. Animals given the scrambled control ODN developed severe glomerulonephritis by day 7 ; with heavy proteinuria, glomerular crescents and interstitial lesions, marked leukocytic infilt
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ration and up-regulated renal expression of cytokines (IL-1 and TNF-α) and adhesion molecules (ICAM-1). In contrast, NF-κB decoy ODN treatment substantially inhibited the disease with a 50% reduction in proteinuria, a 3-fold reduction in histologic damage, a 50% reduction in leukocytic infiltration and a 50-80% reduction in the renal expression of cytokines and leukocyte adhesion molecules. As the other strategy we focused on transcription factor E2F.E2F decoy oligonucleotides treatment specifically inhibited mRNA expression of PCNA and cdk2 kinase in kidneys injured with anti-Thy 1. This was associated with a significant decrease in number of glomerular cells in S phase, and attenuation of glomerular injury assessed histologically. These evidences suggest that intrarenal delivery of E2F decoy oligonucleotides prevents the induction of cell cycle regulatory gene expression and proliferation. Finally, in this study we have developed renal glomerulus-specific gene transfer using HVJ-liposome with anti-Thy 1 antibody, OX-7. By systemic delivery of FITC-labeled ODN by HVJ-liposome coupled with OX-7 we observed fluorescence in renal glomeruli from 2 hours after transfection. To examine the efficacy of this delivery system, NF-κB or scrambled decoy ODN was given by HVJ-liposomes coupled with OX-7 into crescent glomerulonephritis, anti-GBM model. Animals given the scrambled decoy ODN developed severe glomerulonephritis by day 7 with heavy proteinuria, glomerular crescents, marked leukocyte infiltration and up-regulated renal expression of IL-1β, INF-α and ICAM-1. In contrast, NF-κB decoy ODN treatment substantially inhibited the disease with a reduction in proteinuria, histologic damage, leukocyte infiltration and the renal expression of inflammatory cytokines. In conclusion, this study has demonstrated that systemic delivery of HVJ-liposome coupled with OX-7 resulted in efficient ODN transfer in rat glomeruli. NF-κB, but not scrambled decoy ODN given systemically by HVJ-liposome complexed with OX-7 showed clear therapeutic potential for glomerulonephritis. Less
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