OLIGONUCLEOTIDE INHIBITION OF CELL PROLIFERATION

这项工作的目标是开发新的、针对基因的dna疗法。 用于治疗C-myc癌基因活化诱发的实体瘤 E(MU)-myc转基因小鼠，B细胞淋巴瘤动物模型。这个 中心假设是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 在细胞中观察到了对癌基因和病毒基因的疗效 在本实验室和其他实验室的培养和动物中，呈剂量依赖关系 和特定于序列的方式。迄今为止的进展表明，反义 DNA抑制E(MU)-myc转基因小鼠B细胞c-myc的表达 预防性反义DNA治疗抑制小鼠先天性肿瘤的发生 这条线。已知三个敏感的c-myc mRNA靶点：5‘P1 转录起始点[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)及其起始 密码子环[5‘-DCAC GTT Gag GGG CAT]；已知两个敏感的DNA靶标 PI上游：推测的反平行三链位点[5‘-dTGG GTG GGG TGG GTG]，以及可能的重叠H-DNA位点[5‘-dACC CTC CCC ACC CTC]。在这项建议中，立体正则ALL-R甲基膦和ALL-S 硫代磷酸脱氧核糖核酸将被合成。立体规则齐聚物 会更有威力，因为它们的底座比 外消旋低聚物。置乱、感觉和野生类型的控件也将 合成的，以及较短的齐聚物。在项目后期，嵌合体 含有2-3个中心磷酸二酯残基和10-13个外围立体规则 甲基膦或2‘-O-甲基膦残留量 合成的。不同的衍生品将由微型 渗透泵，并测试其相对抑制肿瘤的能力 E(MU)-myc转基因小鼠的生长，均为肿瘤前预防 起病，并在肿瘤发病后治疗。肿瘤的发病率和 将测量进展情况，并将治疗和转化的细胞 将对对照动物进行c-myc mRNA和myc p65蛋白的分析。 表达、增殖和分化特征。剂量 相关性、序列特异性、长度相关性和时间进程 将确定每个主干衍生物的抑制作用。 低聚物的药代动力学将在血清、尿液和 粪便。在抗c-myc治疗不能清除肿瘤的情况下，活检样本 将进行筛选以确定随后激活的癌基因，这些癌基因 然后会受到新的反义DNA的攻击。反义细胞- 将对退化的肿瘤进行致瘤性测试，以确定 经处理的肿瘤细胞是否经历了终末分化 DNA治疗的结果，或构成残留病。这些结果 应该允许反义DNA收敛到最佳组合 派生、长度和序列将适用于 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.