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

在本研究项目中，为了克服实验中常规使用的带有硫代磷酸(PS)骨架的反义寡核苷酸(AS ODN)所具有的非反义效应，我们开发了一种带有嵌合骨架的新的类似物。ODN全长18个核苷酸，最后3个核苷酸在两端都有核苷酸间的PS连接。ODN的中心部分具有磷酸二酯键。这些带有部分PS修饰的嵌合ODN，其中末端封端用于防止核酸酶水解，因为它们显著降低了在使用均匀PS修饰的ODN的研究中有时观察到的非特异性和非反义效应的倾向。利用这些嵌合的寡核苷酸，我们阐明了针对特定基因和重排基因的反义寡核苷酸抑制人造血细胞和白血病细胞增殖的机制：(1)C-myc反义寡核苷酸可以抑制人白血病细胞HL60、…的增殖。G1/S交界处白血病细胞数量骤增。C-myc反义寡核苷酸还可通过Fas/Fas配体系统抑制类风湿关节炎患者滑膜细胞的增殖并诱导细胞凋亡。(2)建立了具有硫代磷酸和磷酸二酯嵌合骨架结构的反义寡核苷酸的合成和纯化方法。用硫代化学法合成了5‘端和3’端的前三个键。这些嵌合类似物与DOTAP(N-[1-(2，3-二油氧氧基)丙基]-N，N，N-三甲基-甲基硫酸铵)作为药物输送系统进入细胞时，显示出特异性的反义作用。(5)我们已经证明，作为具有嵌合骨架的ODN，抗Bc-2，c-myc，EPO受体，而基质衍生因子1有效地作为功能反义分子，我们发现了新的靶向反义序列来降低Bcl2的表达。(6)利用嵌合的c-MPL反义寡核苷酸，我们还证明了巨核系祖细胞可以自己产生血小板生成素。较少

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