ANTISENSE OLIGONUCLEOTIDE APPROACHES TO BCL-2

目前的癌症治疗方法缺乏特异性。 鉴于癌基因在许多人类肿瘤发病机制中的重要性， 肿瘤，这些基因及其编码的蛋白质似乎是明显的目标 被特定的治疗试剂攻击 反义寡核苷酸 （ODN）已被用作特异性消融的研究工具， 癌基因在培养细胞中的功能。 最近，我们将两者都应用于 正常磷酸二酯（PO）和抗核酸酶硫代磷酸酯（PS） BCL 2（B细胞淋巴瘤/白血病-2）癌基因的反义ODN，和 实现了BCL 2蛋白产生的序列特异性抑制， 淋巴瘤和白血病细胞系的生长。 重要的是，因为BCL 2是 在癌基因中是独一无二的，它不仅能够调节细胞内的 生长以及存活，这些BCL 2反义ODN还可以 序列特异性地杀死培养中的淋巴瘤和白血病细胞。 我们现在建议将BCL 2反义ODN的研究扩展到 优化这些试剂用于治疗的最终目标 在自体移植之前应用离体净化骨髓 移植 首先，我们将进一步优化我们的BCL 2反义ODNs 关于序列和长度，使用未修饰的PO-ODNs， 培养细胞和体内翻译系统。 然后，为了 将硫代磷酸ODN的核酸酶抗性与 快速摄取未修饰的磷酸二酯ODNs，我们将制备杂交 ODN仅在其末端含有硫修饰， 合成这些部分硫改性和完全硫改性 具有5'或3'-胆固醇添加的PS-ODN。 混合ODN和均匀ODN (with不含胆固醇），然后将与 细胞摄取，血清核酸酶敏感性，序列特异性，和 在人类淋巴瘤和白血病细胞系中的反义作用机制。 最后，我们将尝试定义反义寡核苷酸的优化条件。 ODN介导的BCL 2表达恶性细胞的选择性根除 从正常的骨髓中提取

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.