Current approaches to cancer treatment suffer from a lack of specificity. Given the importance of oncogenes in the pathogenesis of many human tumors, these genes and their encoded proteins seem obvious targets for attack by specific therapeutic regents. Antisense oligodeoxynucleotides (ODNs) have been used as research tools for the specific ablation of oncogene function in cultured cells. Recently, we have applied both normal phosphodiester (PO) and nuclease-resistant phosphorothioate (PS) antisense ODNs to the BCL2 (B cell lymphoma/leukemia-2) oncogene, and have achieved sequence-specific inhibition of BCL2 protein production and growth of lymphoma and leukemia cell lines. Importantly, because BCL2 is unique among oncogenes in its capacity to regulate not only cellular growth but also survival, these BCL2 antisense ODNs can also sequence-specifically kill lymphoma and leukemia cells in culture. We now propose to extend our studies of BCL2 antisense ODNs towards an ultimate goal of optimizing these reagents for the therapeutic application of ex vivo purging of bone marrow prior to autologous transplantation. First, we will further optimize our BCL2 antisense ODNs with regards to sequence and length, using unmodified PO-ODNs in intact cultured cells and in in vivo translation systems. Then, in an attempt to combine the nuclease-resistance of phosphorothioate ODNs with the rapid uptake of unmodified phosphodiester ODNs, we will prepare hybrid ODNs that contain sulfur modifications only at their ends and will synthesize versions of these partially sulfur-modified and complete PS-ODNs with 5' or 3'-cholesterol additions. Hybrid and uniform ODNs (with and without cholesterol) will then be contrasted with regards to cellular uptake, serum-nuclease sensitivity, sequence-specificity, and mechanisms of antisense action in human lymphoma and leukemia cell lines. Finally, we will attempt to define optimized conditions for the antisense ODN-mediated selective eradication of BCL2-expressing malignant cells from normal bone marrow.
