ANTISENSE OLIGONUCLEOTIDE APPROACHES TO BCL-2

BCL-2 的反义寡核苷酸方法

• 批准号: 3203973
• 负责人: JOHN C REED
• 金额: $ 14.5万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-23 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3203973
• 关键词: antisense nucleic acid; autologous transplantation; bone marrow transplantation; cytotoxicity; flow cytometry; genetically modified animals; high performance liquid chromatography; human tissue; immunofluorescence technique; laboratory mouse; leukemia; lymphoma; molecular cloning; neoplasm /cancer immunotherapy; neoplastic cell; nucleic acid hybridization; oligonucleotides; oncogenes; oncoproteins; polymerase chain reaction; reagent /indicator

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.

当前的癌症治疗方法缺乏特异性。 鉴于癌基因在许多人的发病机理中的重要性 肿瘤，这些基因及其编码蛋白似乎是明显的目标 特定治疗摄政症的攻击。 反义寡脱氧核苷酸 （ODN）已用作研究工具的特定消融 培养细胞中的癌基因功能。 最近，我们两者都应用了 正常磷酸二酯（PO）和抗核酸酶耐药磷酸盐剂（PS） Bcl2（B细胞淋巴瘤/白血病-2）癌基因的反义ODN和 已经实现了序列特异性抑制Bcl2蛋白的产生和 淋巴瘤和白血病细胞系的生长。 重要的是，因为bcl2是 在癌基因中，其独特的能力不仅可以调节细胞 增长但也生存，这些BCL2反义ODN也可以 序列特异性杀死培养中的淋巴瘤和白血病细胞。 现在，我们建议将BCL2反义ODN的研究扩展到 为治疗性优化这些试剂的最终目标 自体之前的体内清除骨髓 移植。 首先，我们将进一步优化我们的BCL2反义ODN 关于序列和长度，使用完整的未修饰的po-odn 培养的细胞和体内翻译系统。 然后，尝试 将磷酸胸酯ODN的核酸酶抗性与 快速吸收未修饰的磷酸化酯ODN，我们将准备杂交 仅在其末端含有硫修饰的ODN，将 这些部分的硫磺修饰和完整的合成版本 添加5'或3'-胆固醇的PS-ODN。 杂种和均匀的ODN （有和没有胆固醇）然后将 细胞摄取，血清核酸酶敏感性，序列特异性和 人淋巴瘤和白血病细胞系中反义作用的机制。 最后，我们将尝试定义反义的优化条件 ODN介导的表达BCL2的恶性细胞的选择性根除 来自正常的骨髓。