OLIGONUCLEOTIDE INHIBITION OF CELL PROLIFERATION

这项工作的目标是开发新的，基因特异性的DNA疗法 用于治疗由活化的C-myc癌基因诱导的实体瘤 E（mu）-myc转基因小鼠，B细胞淋巴瘤的动物模型。的 核心假设是DNA骨架修饰的某种组合 将产生最大的效力和特异性。反义或反基因DNA 已经在细胞中观察到抗癌基因和病毒基因功效 在本实验室和其他实验室的动物中， 和序列特异性方式。迄今为止的进展表明， DNA抑制E（mu）-myc转基因小鼠B细胞中的c-myc表达，并且 预防性反义DNA治疗抑制先天性肿瘤发生 这条线。已知三种敏感的c-myc mRNA靶点： 转录起始位点[5'-dGCA CAG CTC GGG GGT]，第一个 内含子/第二外显子连接（5'-dGGC TGC TGG AGC GGG），以及起始于内含子/第二外显子连接的内含子/第二外显子连接（5'-dGGC TGC TGG AGC GGG）。 密码子环[5'-dCAC GTT GAG GGG CAT];已知两种敏感的DNA靶标 Pi上游：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 反恐委员会]。在该建议中，立体规则的全R甲基膦酸酯和全S 将合成硫代磷酸酯DNA。有规立构低聚物 会更有效，因为它们的碱基配对比 外消旋低聚物。加扰、感测和野生型对照也将被 合成的，以及较短的低聚物。在这个项目的后期， 具有2 - 3个中心磷酸二酯残基和10 - 13个外围有规立构 甲基膦酸酯或2'-O-甲基甲基膦酸酯残基将被 合成了不同的衍生物将通过微- 渗透泵，并测试其抑制肿瘤的相对能力 在E（mu）-myc转基因小鼠中的生长， 发病，以及肿瘤发病后的治疗。肿瘤发病率和 将测量进展，并将来自经处理的和 对照动物将分析c-myc mRNA和MYC p65蛋白 表达、增殖和分化特征。剂量 依赖性、序列特异性、长度依赖性和时间过程。 将测定每种骨架衍生物的抑制作用。 低聚物的药代动力学将在血清、尿液和血浆中测量。 粪便当抗c-myc治疗不能消融肿瘤时， 将被筛选，以确定随后激活的癌基因， 会被新的反义DNA攻击反义- 将测试消退的肿瘤的致瘤性，以确定 治疗的肿瘤细胞是否经历了终末分化， DNA治疗的结果，或构成残留疾病。这些结果 应该允许反义DNA收敛到以下的最佳组合： 衍生化，长度和序列，这将适合于 转化为临床试验。

The goal of this work is to develop novel, gene-specific DNA therapeutics for treatment of solid tumors induced by activated C-myc oncogene in E(mu)-myc transgenic mice, an animal model for B-cell lymphoma. The central hypothesis is that some combination of DNA backbone modifications will yield maximum potency and specificity. Antisense or antigene DNA efficacy against oncogenes and viral genes has been observed in cell culture and in animals in this laboratory and others, in a dose dependent and sequence specific manner. Progress to date indicates that antisense DNA inhibits c-myc expression in B-cells of E(mu)-myc transgenic mice, and prophylactic antisense DNA therapy inhibits congenital tumorigenesis in this line. Three sensitive c-myc mRNA targets are known: the 5' P1 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'-dCAC GTT GAG GGG CAT]; two sensitive DNA targets are known upstream of Pi: the putative antiparallel triplex site [5'-dTGG GTG GGG TGG GTG], and the overlapping putative H-DNA site [5'-dACC CTC CCC ACC CTC]. In this proposal, stereoregular all-R methylphosphonate and all-S phosphorothioate DNAs will be synthesized. The stereoregular oligomers will be more potent because they basepair much more tightly than the racemic oligomers. Scrambled, sense, and wild type controls will also be synthesized, as well as shorter oligomers. Later in the project, chimeras with 2-3 central phosphodiester residues and 10-13 outlying stereoregular methylphosphonate or 2'-O-methyl methylphosphonate residues will be synthesized. The different derivatives will be administered by micro- osmotic pumps, and tested for their relative ability to inhibit tumor growth in E(mu)-myc transgenic mice, both prophylactically before tumor onset, and therapeutically after tumor onset. The rates of tumor onset and progression will be measured, and transformed cells from treated and control animals will be analyzed for c-myc mRNA and MYC p65 protein expression, proliferation, and differentiation characteristics. Dose dependence, sequence specificity, length dependence, and time course of inhibition will be determined for each backbone derivative. Pharmacokinetics of the oligomers will be measured in serum, urine, and feces. Where anti-c-myc therapy fails to ablate tumors, biopsy samples will be screened to identify the subsequent activated oncogenes, which will then be attacked with new antisense DNAs. Cells from antisense- regressed tumors will be tested for tumorigenicity in order to determine whether the treated tumor cells underwent terminal differentiation as a result of DNA therapy, or constitute residual disease. These results should allow convergence of antisense DNAs to an optimum combination of derivatization, length, and sequence which will be suitable for translation to clinical trials.