OLIGODEOXYNECLEOTIDE INHIBITION OF CELL PROLIFERATION

这项工作的目标是测试新的、基因特异性的dna疗法。 用于治疗活化人实体瘤和白血病 C-myc癌基因在两种动物模型中的表达。中心假设是，有些人 主干和终端改造的组合将产生最大 效力和特异度。反义或反义DNA对小鼠的抗肿瘤作用 在细胞培养中观察到了癌基因和病毒基因。 实验室和其他，以剂量依赖和序列特定的方式。 初步结果表明，反义DNA甲基膦 抑制c-myc转基因小鼠B细胞c-myc的表达 正常反义DNA抑制肿瘤形成的实验研究 C-Ha-ras基因转化裸鼠NIH3T3细胞。在这 建议的正常、硫代磷和甲基膦酸类DNA，带有和 不需要5‘-二甲氧基三苯基和/或3’-胆固醇基衍生物即可 加速细胞摄取和缓慢排泄，将被合成。 已知三个敏感的c-myc信使核糖核酸靶点：5‘Pl转录 起始位点[5‘-DGCA CAG CTC GGG GGT]，第一内含子/第二外显子 连接[5‘-dGGC TGC TGG AGC GGG]和起始密码子环 [5‘-DAAC GTT Gag GGG CAT]；上游已知两个敏感的DNA靶标 Pl：推测的反平行三链位点[5‘-dTGG GTG GGG TGG GTG]， 和重叠的H-DNA位点[5‘-dACC CTC ccc ACC CTC]。 置乱、感觉和野生类型的控件也将被合成，如 以及较短的低聚物。然后将对不同的衍生品进行测试 在c-myc转基因中抑制肿瘤生长的相对能力 小鼠--B细胞淋巴瘤动物模型及C57BL/Balb/c裸鼠 裸鼠皮下移植人早幼粒细胞HL-60细胞。 来自处理动物和对照动物的转化细胞将被分析 C-myc p65的表达、增殖和分化，以及 肿瘤的生长将被测量。序列特异性、长度依赖性、 将确定剂量依赖关系和抑制时间过程 每一个衍生品。将研究三种给药方案：每日 经腹膜腔内推注，经皮下持续输注 泵，每日通过静脉注射脂质体。阿司匹林的药代动力学 低聚物将在血清、尿液和粪便中进行测量。在发生以下情况时 反义或反义基因可显著降低肿瘤的大小和生长速度 抗基因治疗，来自治疗肿瘤的细胞将被测试 以确定治疗后的肿瘤细胞是否具有致瘤性 由于DNA治疗，进行了终末分化。这个 C-myc表达与MHC-I类抗原的相互关系将是 免疫监测与细胞毒性T淋巴细胞的相关研究 敏感度。这些结果应该允许候选人的收敛 基因特异性DNA的衍生、长度、 以及适合于临床前试验的序列。

The goal of this work is to test novel, gene-specific DNA therapeutics for treatment of human solid tumors and leukemias induced by activated c-myc oncogene in two animal models. The central hypothesis is that some combination of backbone and terminal modification will yield maximum potency and specificity. Antisense or antigene DNA efficacy against oncogenes and viral genes has been observed in cell culture in this laboratory and others, in a dose dependent and sequence specific manner. Preliminary results indicate that antisense DNA methylphosphonates inhibit c-myc expression in B-cells of c-myc transgenic mice, and that normal antisense DNA inhibits tumorigenesis by pretreated c-Ha-ras-transformed NIH3T3 cells in athymic nude mice. In this proposal, normal, phosphorothioate, and methylphosphonate DNAs, with and without 5' dimethoxytrityl and/or 3' cholesteryl derivatization to accelerate cellular uptake and slow excretion, will be synthesized. Three sensitive c-myc mRNA targets are known: the 5' Pl transcription start site [5'-dGCA CAG CTC GGG GGT], the first intron/second exon junction [5'-dGGC TGC TGG AGC GGG], and the initiation codon loop [5'-dAAC GTT GAG GGG CAT]; two sensitive DNA targets are known upstream of Pl: the putative antiparallel triplex site [5'-dTGG GTG GGG TGG GTG], and the overlapping putative H-DNA site [5'-dACC CTC CCC ACC CTC]. Scrambled, sense, and wild type controls will also be synthesized, as well as shorter oligomers. The different derivatives will then be tested for their relative ability to inhibit tumor growth in c-myc-transgenic mice, an animal model for B-cell lymphoma, and in C57BL/Balb/c athymic nude mice implanted subcutaneously with human promyelocytic HL-60 cells. Transformed cells from treated and control animals will be analyzed for c-myc p65 expression, proliferation, and differentiation, and rate of tumor growth will be measured. Sequence specificity, length dependence, dose dependence, and time course of inhibition will be determined for each derivative. Three regimens of administration will be studied: daily via intraperitoneal bolus, continuous infusion via subcutaneous Alza pump, and daily via intravenous liposomes. Pharmacokinetics of the oligomers will be measured in serum, urine, and feces. In the event that tumors are significantly reduced in size and growth rate by antisense or antigene treatment, cells from treated tumors will be tested for tumorigenicity in order to determine whether the treated tumor cells underwent terminal differentiation as a result of DNA therapy. The interrelationship of c-myc expression with MHC class I antigens will be studied relative to immune surveillance and cytotoxic T lymphocyte sensitivity. These results should allow convergence of the candidate gene-specific DNAs to an optimum combination of derivatization, length, and sequence which will be suitable for preclinical trials.