MODULATION OF CELL GROWTH BY ANTISENSE AND ANTIGENE REAGENTS

该项目的重点有三个方面：（1）确定吸收的特点， 未修饰和修饰的寡核苷酸的细胞内加工;（2） 在几种体外模型中利用反义和反基因技术 用于识别细胞增殖/病毒中关键事件的系统 复制;（3）研究反义和反义基因的功效 作为基因表达的体内调节剂的试剂。 (1)我们已经将未修饰的寡核苷酸的摄取表征为能量- 依赖性内吞过程，由至少一个细胞表面介导， 结合蛋白 我们设计了一种新的技术来研究寡核苷酸的摄取， 细胞内定位以及与蛋白质和核酸的结合 acids. 这种非侵入性技术将允许亚细胞定位 随着时间的推移，一个内在的寡核苷酸。 (2)我们已经证实，c-myc抑制对正常和 恶性淋巴样细胞和一些伯基特淋巴瘤细胞可以 特别是在体外用新的c-myc反义物抑制生长。 我们 已经证实N-myc抑制导致生长减少， 继发于神经外胚层来源的分化状态的改变 细胞系 我们已经证明，TGF和自分泌的中断， 袢对上皮细胞和间充质细胞具有膀胱抑制作用。 (3)我们已经证明，连续皮下灌注的一个 oligo可以在体内显著抑制其靶基因的表达， 复制了用DNA或RNA观察到的其他体外现象 反义 该模型允许体内测试反义寡核苷酸， 有效性和毒性。 我们已经证明了连续 灌注鞘内模型，以研究反义寡核苷酸在 更相关的临床前体内模型系统。

The focus of this project is three-fold: (1) to characterize uptake and intracellular processing of unmodified and modified oligonucleotides; (2) to utilize antisense and antigene technology in several in vitro model systems to identify critical events in cell proliferation/viral replication; and (3) to study the efficacy of antisense and antigene reagents as in vivo modulators of gene expression. (1) We have characterized the uptake of unmodified oligos as an enery- dependent, endocytic process, mediated by at least one cell surface- binding protein. We have devised a novel technique to study oligo 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 that 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 reduction in growth secondary to alteration in differentiative status of neuroectoderm-derived cell lines. We have demonstrated that interruption of TGF and autocrine loops is cystostatic for epithelial and mesenchymal cells. (3) We have demonstrated that continuous subcutaneous perfusion of an oligo can significantly arrest in vivo its targeted gene expression and reproduces other in vitro phenomena observed with either DNA or RNA antisense. This model permits in vivo testing of antisense oligos for efficacy and toxicity. We have demonstrated the feasibility of continuous perfusion intrathecal model to study the clinical efficacy of antisense in a more relevant pre-clinical in vivo model system.