OLIGODEOXYNUCLEOTIDE INHIBITION OF CELL PROLIFERATION

这项工作的目标是测试新的基因特异性DNA疗法 用于治疗激活的人实体瘤和白血病 c-myc癌基因在两种动物模型中的表达。 核心假设是， 主链和末端修饰的组合将产生最大的 效力和特异性。 反义或反基因DNA对 癌基因和病毒基因已经在细胞培养中观察到， 实验室和其他，以剂量依赖性和序列特异性方式。 初步结果表明，反义DNA甲基膦酸 抑制c-myc转基因小鼠B细胞中c-myc表达， 正常反义DNA通过预处理抑制肿瘤发生 c-Ha-ras转化的NIH 3 T3细胞在无胸腺裸鼠中。 在这 建议，正常，硫代磷酸酯，甲基膦酸酯DNA，与和 没有5'二甲氧基三苯甲基和/或3'胆固醇基衍生化， 加速细胞吸收和缓慢排泄，将被合成。 已知三种敏感的c-myc mRNA靶点： 起始位点[5 '-dGCA CAG CTC GGG GGT]，第一内含子/第二外显子 接头[5 '-dGGC TGC TGG AGC GGG]和起始密码子环 [5 '-dAAC GTT GAG GGG CAT];已知上游有两个敏感的DNA靶标 P1的：推定的反平行三链体位点[5 '-dTGG GTG GGG TGG GTG]， 和重叠的推定H-DNA位点[5 '-dACC CTC CCC ACC CTC]。 也将合成加扰、有义和野生型对照，如 以及更短的低聚物。 然后将测试不同的衍生品 因为它们在c-myc转基因小鼠中抑制肿瘤生长的相对能力 小鼠，B细胞淋巴瘤的动物模型，以及C57 BL/Balb/c无胸腺 皮下植入人早幼粒细胞HL-60细胞的裸鼠。 将分析来自处理动物和对照动物的转化细胞， c-myc p65表达、增殖和分化，以及 测量肿瘤生长。 序列特异性，长度依赖性， 剂量依赖性和抑制的时程将被确定， 每个衍生物。 将研究三种给药方案：每日一次 通过腹膜内推注，通过皮下Alza持续输注 泵和每日通过静脉注射脂质体。 药代动力学 在血清、尿液和粪便中测量低聚物。 的情况下 肿瘤的大小和生长速度显著降低， 在抗基因治疗中，将测试来自经治疗肿瘤的细胞的 致瘤性，以确定治疗的肿瘤细胞是否 由于DNA治疗而经历了终末分化。 的 c-myc表达与MHC I类抗原的相互关系将是 与免疫监视和细胞毒性T淋巴细胞相关的研究 灵敏度 这些结果应该允许候选人的收敛 基因特异性DNA的衍生化，长度， 以及将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.