| Project/Area Number |
12470246
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General surgery
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| Research Institution | Saitama Medical School |
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Principal Investigator |
KYO Shunei Saitama Medical School, Dep. Cardio-thoracic Surgery, Professor, 医学部, 教授 (30153232)
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| Co-Investigator(Kenkyū-buntansha) |
TANABE Hiroaki Saitama Medical School, Dep. Cardio-thoracic Surgery, Assistant Professor, 医学部, 講師 (80281301)
KOUMOTO Osami Saitama Medical School, Dep. Cardio-thoracic Surgery, Assistant Professor, 医学部, 講師 (00261967)
GOJO Satoshi Saitama Medical School, Cardiovascular Surgery, Assistant Professor, 医学部, 講師 (90316745)
MAZDA Osamu Kyoto Prefectural Univ. of Med., Dep. Microbiol., Associate Professor, 大学院・医学研究科, 助教授 (00271164)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥14,500,000 (Direct Cost: ¥14,500,000)
Fiscal Year 2001: ¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 2000: ¥9,100,000 (Direct Cost: ¥9,100,000)
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| Keywords | Gene Therapy / Heart failure / LVSA / Naked DNA / Electroporation / Gene gun / Adrenergic receptor / siRNA |
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| Research Abstract |
Recently, molecular mechanisms of heart diseases have been investigated and pathogenesis as well as pathophysiology is understood at molecular levels. Genetic regulation of critical genes may greatly contribute in the therapeutic intervention of cardiac disorders, particularly those that are resistant for the conventional therapies. In this concern, we planned to establish systems feasible for controlling genes of interest in vivo in the myocardium. As a gain-of-function approach, we tried nonviral delivery method to transfer reporter or therapeutic genes into the beating heart. Beta2-adrenergic receptor (bAR) gene was introduced into an EBV-based plasmid vector, which we have previously shown to exert extremely powerful expression in various cells and tissues, and the resultant plasmid was injected into the myocardium of cardiomyopathic hamsters as naked DNA. The treated animals showed significant elevation in the FS and cardiac output. Systemic administration of isoprotelenol further
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augmented the cardiac contraction, suggesting that the catecholamine response of the failing heart was elevated by the genetic transfer. The hear rate was not significantly increased. RT-PCR analyses demonstrated that the expression of the transgene was strictly restricted in the injected region of the heart. In another approach we also succeeded in delivering naked EBV-plasmid vector into the rat myocardium through the particle-mediated transfer system. More recently we also transferred plasmid vector-based transfer into beating heart of a larger animal, i.e., dogs, showing that significant expression of marker genes can be obtained.
We also performed loss-of-function strategies to control genetic expression in vivo. As a first trial, we transferred the short interfering RNA (siRNA) to regulate exogenous or endogenous gene function in skeletal muscle cells by means of the electroporation in vivo. The siRNA transfer quite effectively suppressed expression of luciferase marker gene that had been transduced simultaneosly with the siRNA. In case transgenic mice carring the EGFP marker gene was tested, transduction of siRNA specific for GFP resulted in the significant reduction in the GFP fluorescence.
Collectively, we succeeded in establishing strategies to transduce or regulate gene in vivo using non-viral or naked DNA systems. These procedures may become quite useful in the gene therapy as well as the therapeutic molecular targeting for severe cardiac disorders. The system may also be applicable to functional genomics investigations for the heart. Less
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