NON-VIRAL GENE THERAPY VECTORS--EFFICIENT NUCLEAR IMPORT

尽管非病毒载体被认为具有很高的潜力 未来大规模的临床基因治疗应用，它们的用途是 目前受限于它们的收益率，这是几个数量级的 比主要的病毒载体系统要小。多重原因，在 根据具体配方的不同，可变比例为 对非病毒载体的总体低产量负责。总而言之， 案例，一个使产量减少一到三个数量级的因素 是DNA从细胞质到其自身的低效转位 最终目的地，原子核。在成熟生物体的大多数细胞中， 它们是有丝分裂后的，转位被一个完整的核阻止 薄膜。尽管已经做出了改善核电的尝试 通过附着到DNA合成肽或 含有核定位信号的蛋白质(NLSS)，改进 都是次要的。预计未来的情况会更糟。 暂时优化的情况下，当向量的副本非常少时 将出现在每个细胞中。这个项目的目的是探索一种 新原则，它为更高效的核能提供了希望 易位。因为蛋白质输入途径是基于一系列 要进口的蛋白质与运输因子的相互作用 这个项目的中心思想是将载体DNA放在入口处 在这个链条上，它必须与广大的 天然蛋白质等待进口，但在链条的末端， 就在穿过核孔的最后一步之前。在……里面 生物化学术语这个概念翻译成用于靶向部分 不是NLS，而是核粘附素β转运因子，在正常情况下 情况使复合体停靠在核孔处。这三个人都知道 人类核粘附素β将被研究它们的作用能力 作为核靶向部分。每个片段的最小片段 核粘附素保留完整的易位能力将被确定， 用于改进的输送系统。我们预计，这种做法将 确保DNA快速高效地跨 核膜。

Although non-viral vectors are believed to hold high potential for future large scale clinical gene therapy applications, their use is currently limited by their yield, which is several orders of magnitude smaller than for the major viral vector systems. Multiple causes, in variable proportions depending on the specific formulation, are responsible for the overall low yield of non-viral vectors. In all cases, a factor reducing the yield by one to three orders of magnitude is the inefficient translocation of the DNA from the cytoplasm to its final destination, the nucleus. In most cells of the mature organism, which are postmitotic, the translocation is blocked by an intact nuclear membrane. Although attempts have been made to improve the nuclear translocation of the vectors by attaching to DNA synthetic peptides or proteins containing Nuclear Localization Signals (NLSs), improvements have been minor. The situation is expected to be even worse for future tentatively optimized situations, when very few copies of the vector will be present in each cell. The aim of this project is to explore a new principle, which holds the promise for much more efficient nuclear translocation. As the protein import pathway is based on a chain of interactions of the proteins to be imported with transport factors, the central idea of this project is to place the vector DNA not at the entry point of this chain, where it has to compete with the vast number of native proteins waiting to be imported, but at the end of the chain, right before the final step of crossing the nuclear pore. In biochemical terms this concept translates into using as targeting moiety not an NLS, but the transport factors karyopherins beta, which in normal situations dock the complexes at the nuclear pores. All three known human karyopherins beta will be investigated for their ability to act as nuclear targeting moieties. The smallest fragment of each karyopherin retaining full translocation capacity will be determined, for use in improved delivery systems. We expect that this approach will assure rapid and highly efficient translocation of the DNA across the nuclear membrane.