2000 Fiscal Year Final Research Report Summary
PRELIMINARY EVALUATION OF THE TREATMENT OF CONGENITAL THROMBOTIC DISORDERS BY DNA-RNA OLIGOMUCLEOTIDE
Project/Area Number |
11671009
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Research Category |
Grant-in-Aid for Scientific Research (C)
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Allocation Type | Single-year Grants |
Section | 一般 |
Research Field |
Hematology
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Research Institution | KYOTO PREFECTURAL UNIVERSITY OF MEDICINE |
Principal Investigator |
TSUJI Hajime KYOTO PREFECTURAL UNIVERSITY OF MEDICINE, BLOOD TRANSFUSION MEDICINE, ASSISTANT PROFESSOR, 医学部, 助教授 (90188532)
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Project Period (FY) |
1999 – 2000
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Keywords | OLIGONECLEOTIDE / THROMBOSIS / GENE / ANTITHROMBIN |
Research Abstract |
Antithrombin (AT) is the major plasma inhibitor of thrombin, and is a member of the serine proteinase inhibitor (serpin) superfamily. It is mainly synthesized by the hepatocytes, and its gene (13.5 kb) has been assigned to chromosome 1q (23.1-23.9), consisting of 7 exons and 6 introns. Inherited AT deficiency is a well-recognized risk factor for the development of venous thromboembolism, which has now been shown to be caused by a spectrum of genetic defects. In order to fundamentally cure the disease, targeted correction of the disease related mutation by introducing homologous recombination is considered to be the most effective strategy for gene therapy. A novel and unique method of site-directed mutagenesis using chimeric RNA/DNA oligonucleotide (RDO) was introduced by Kmiec et al in 1996, which was later proven to be effective in introducing mutagenesis in the factor IX in vivo by Steer et al in 1998, suggesting its possibility as a novel tool to treat congenital hemophilic and thr
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ombogenic disease arising from a point mutation. The aim of this study is to confirm the effectiveness of this novel method to the introduction of targeted nucleotide exchange in human AT gene to produce a model of AT deficiency. A chimeric 2'-O-methylated-RNA/DNA oligonucleotide containing sequences complementary to 25 bases of the human AT gene was constructed as a duplex containing either 5390C to T, 5393A to T or 7431C to G substitution to introduce missense mutation. As a targeting cell, human hepatoma cell line HuH-7 was used, within which the expression of AT mRNA was confirmed by RT-PCR.Transfection of the oligonucleotide was performed with SuperFect Transfection Reagent^<TM> (SFTR ; Qiagen Co., Germany). The transfection efficiency was determined by nuclear and cellular up-take of FITC-labeled all-DNA oligonucleotide confirmed with confocal microscopy. Successful mutagenesis were screened and confirmed by PCR-RFLP and direct-sequencing using SEQ4x4 personal sequencing system (Amersham Pharmacia Biotech, UK), respectively. As a result, transfection efficiency with SFTR was between 60 and 80% and it was possible to introduce all three types of mutagenesis into human AT gene in HuH-7 cells in vitro. Although it is necessary to further elucidate its effectiveness in correcting inherited AT gene mutation in vivo, the present results suggest that the RDO-gene-therapy could be an alternative treatment for patients with congenital AT deficiency. Less
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