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

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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