MODULATION OF CELL GROWTH BY ANTISENSE AND ANTIGENE REAGENTS

"这个项目的重点有三个方面：（1） 表征未修饰的和 修饰的寡核苷酸;（2）利用反义和反基因 技术在几个体外模型系统，以确定关键 细胞增殖/病毒复制的要素;以及（3）研究 反义和反基因试剂作为体内 基因表达(1)我们已经描述了 修饰的寡核苷酸作为能量依赖性的内吞过程， 由至少一种细胞表面结合蛋白介导。我们有 设计了一种新的技术来研究olig摄取，细胞内 定位以及与蛋白质和核酸的结合。这 非侵入性技术将允许随着时间的推移进行亚细胞定位 一个内在的寡头。(2)我们已经证实c-myc 抑制作用对正常和恶性淋巴样细胞具有细胞抑制作用， 一些伯基特淋巴瘤细胞可以特异性地生长停滞， 体外与一种新的c-myc反义。我们已经证实N-myc 抑制导致生长减少，其次是 神经外胚层来源的细胞系的分化状态。(3)我们 已经证明c-myc反义特别有效 针对几种实体瘤，包括人类和大鼠胶质瘤， 胚细胞瘤在实体瘤中，c-myc反义寡核苷酸具有， 除了其反义作用之外， 非反义介导的，对细胞附着的影响 细胞外基质(4)我们已经鉴定出能够 特异性抑制bcr-abl酪氨酸激酶和其它酪氨酸 激酶通过与蛋白质的直接相互作用。(5)我们一直 能够显著延长动物存活时间 在注射用c-myc反义核酸处理的肿瘤细胞后。 同时，这种反义治疗对正常的 骨髓细胞这些最后的发现导致了 使用反义核酸进行I期临床试验所需的过程 （c-myc靶向）和抗蛋白（bcr-abl靶向） 寡核苷酸作为骨髓净化剂。最近，我们 已经研究了体内递送合成的 靶向VEGF和FGF的核酶用于治疗胶质母细胞瘤。 我们使用的是大鼠中枢神经系统模型， 通过渗透泵颅内给药。初步数据显示， 抗VEGF和抗FGF核酶的组合显著地 如果同时开始给药，则动物存活率降低 作为肿瘤细胞接种（使用人胶质母细胞瘤，U87）。 正在进行的研究将确定核酶在 脑组织和肿瘤，以及这些核酶的体外有效性 肿瘤细胞VEGF和FGF mRNA的表达。的额外 动物研究，我们已经证明了显着的抗肿瘤活性 独特的CpG寡核苷酸小鼠单次接种 该寡核苷酸在肿瘤接种前或接种后都能够 阻止肿瘤生长或导致肿瘤停滞/消退， 超过70%的动物。作用机制似乎 涉及宿主免疫系统，但NK细胞似乎不 必需的. "

"The focus of this project is three-fold: (1) to characterize uptake and intra- cellular processing of unmodified and modified oligonucleotides; (2) to utilize antisense and antigene technology in several in vitro model systems to identify critical elements in cell proliferation/viral replication; and (3) to study the efficacy of antisense and antigene reagents as in vivo modulators of gene express- ion. (1) We have characterized the uptake of modified oligos as an energy-depend- ent, endocytic process, mediated by at least one cell surface-binding protein. We have devised a novel technique to study olig uptake, intracellular localization, and association with protein and nucleic acids. This non-invasive technique will permit subcellular localization over time of an internalized oligo. (2) We have confirmed that c-myc inhibition is cytostatic for normal and malignant lymphoid cells and some Burkitt lymphoma cells can be specifically growth-arrested in vitro with a novel c-myc antisense. We have confirmed that N-myc inhibition leads to re- duction in growth secondary to alteration in differentiative status of neuroecto- derm-derived cell lines. (3) We have demonstrated that c-myc antisense is particularly effective against several solid tumors, including human and rat glio- blastoma. In solid tumors, the c-myc antisense oligonucleotide has, in addition to its antisense effects, a sequence-specific, non-antisense mediated, effect on cellular attachment to extracellular matrix. (4) We have identified sequences capable of specifically inhibiting bcr-abl tyrosine kinase and other tyrosine kinases via direct interaction with the proteins. (5) We have been able to demonstrate significant prolongation of animal survival following injection of tumor cells treated with antisense to c-myc. At the same time such antisense treatment has no effect on normal bone marrow cells. These last findings have led to initiation of the process required to undertake a Phase I clinical trial using antisense (c-myc-targeted) and anti-protein (bcr-abl-targeted) oligonucleotides as bone marrow purging agents. Most recently, we have been investigating the efficacy of in vivo delivery of synthetic ribozymes targeting VEGF and FGF for treatment of glioblastoma. We are using a rat CNS model in which ribozymes are delivered intracranially via osmotic pump. Preliminary data suggest that a combination of anti-VEGF and anti-FGF ribozymes significantly prolongs animal survival if administration is begun at the same time as tumor cell inoculation (using a human glioblastoma, U87). Ongoing studies will determine the distribution of ribozymes in brain tissue and tumor, and in vitro effectiveness of these ribozymes toward VEGF and FGF mRNA in the tumor cells. In additional animal studies, we have demonstrated significant anti-tumor activity of a unique CpG oligonucleotide. A single inoculation of mice with this oligonucleotide either pre- or post-tumor inoculation is able to prevent tumor growth, or cause tumor stasis/regression, in greater than 70 percent of animals. The mechanism of action appears to involve the host immune system, but NK cells do not appear to be required."