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Development of Antisense Therapy for Hematological Mailgnancies Targeted Their Rearranged Genes.

针对血液恶性肿瘤的重排基因开发反义疗法。

基本信息

批准号：
11670983
负责人：
MAEKAWA Taira
金额：
$ 2.3万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670983/
关键词：
antisense apoptosis erythroleukemia 白血病分子標的治療

项目摘要

In this research project, to overcome the non-antisense effects possessed by antisense oligodeoxynucleotides (AS ODN) with phosphorothioate (PS) backbone that are conventionally used in experiments, we have developed a new analogue with chimeric backbone. The ODNs were 18 nucleotides in length, and the last three nucleotides at both ends had their internucleotidic PS linkages. The central portion of ODNs has phosphodiester linkages. These chimeric ODNs with partial PS-modification, in which end-capping was used to prevent nuclease hydrolysis, were used because of their significantly-decreased propensity for nonspecific and nonantisense effects sometimes observed in studies using uniformly PS-modified ODNs. Using these chimeric AS ODNs, we have clarified followings concerning the mechanism of suppression of the proliferation of human hematopoietic and leukemic cells by AS ODN for specific and rearranged genes.(1) C-myc AS ODN could inhibit the proliferation of human leukemia cells HL6O, … More and the abrupt up-regulation of number of leukemia cells in G1/S boundary. c-myc AS ODN also inhibited the proliferation of synovial cells from patients with rheumatoid arthritits and induced the apoptosis through Fas/Fas ligand system.(2) We have established the synthesis and purification methods of AS ODN with chimeric backbone structure of phosphorothioate and phosphodiester linkages. The first three linkages of both 5'- and 3'-end are synthesized by phosphorothioate chemistry. We have shown these chimeric analogues were resistant to the degradation by nuclease.(3) The above chimeric analogue have much less non-specific effects or aptamer effects that are often encountered when AS ODNs with all-phosphorothioate linkages are used.(4) These chimeric analogues showed specific antisense effects when used in combination with DOTAP (N-[1-(2, 3-Dioleoxyloxy) propyl]-N, N, N-trimethyl- ammoniummethylsulfate) as a drug delivery system into cells.(5) We have shown that AS ODNs with chimeric backbones against Bcl-2, c-myc, erythropoietin receptor, and stromal-derived factor 1 effectively work as functional antisense molecules, and we have found the novel target antisense sequence to reduce the Bcl-2 expression.(6) Also, using chimeric AS ODNs against c-mpl, we have shown that megakaryocytic progenitors produce thrombopoietin by themselves. Less

在本研究项目中，为了克服实验中常用的具有硫代磷酸酯（PS）骨架的反义寡核苷酸（AS ODN）所具有的非反义效应，我们开发了一种新的具有嵌合骨架的类似物。ODNs长18个核苷酸，两端的最后3个核苷酸具有其核苷酸间的PS键。ODN的中心部分具有磷酸二酯键。这些嵌合ODN与部分PS修饰，其中封端用于防止核酸酶水解，被使用，因为它们显着降低的倾向，有时在使用均匀PS修饰的ODN的研究中观察到的非特异性和非反义作用。利用这些嵌合AS ODN，我们阐明了以下关于特异性和重排基因的AS ODN抑制人造血细胞和白血病细胞增殖的机制。(1)C-myc AS ODN能抑制人白血病细胞HL 60的增殖， ...更多信息 G1/S期白血病细胞数量的突然增加。c-myc AS ODN还能抑制类风湿关节炎滑膜细胞增殖，并通过Fas/Fas配体系统诱导滑膜细胞凋亡。(2)我们建立了硫代磷酸酯和磷酸二酯键嵌合骨架结构的AS ODN的合成和纯化方法。5 '-和3'-末端的前三个键通过硫代磷酸酯化学合成。我们已经证明这些嵌合类似物对核酸酶的降解具有抗性。(3)上述嵌合类似物具有少得多的非特异性效应或适体效应，而这些非特异性效应或适体效应在使用具有全硫代磷酸酯键的AS ODN时经常遇到。(4)当与DOTAP（N-[1-（2，3-二油氧基）丙基]-N，N，N-三甲基-硫酸铵）组合作为药物递送系统进入细胞时，这些嵌合类似物显示出特异性反义作用。(5)我们已经证明，AS ODNs与嵌合骨干Bcl-2，c-myc，促红细胞生成素受体，基质衍生因子1有效地工作作为功能性反义分子，我们已经发现了新的目标反义序列，以减少Bcl-2的表达。(6)此外，使用嵌合AS ODN对c-mpl，我们已经表明，巨核细胞祖细胞产生血小板生成素本身。少