NON-VIRAL GENE THERAPY VECTORS--EFFICIENT NUCLEAR IMPORT

尽管非病毒载体被认为具有高的潜在性， 未来大规模的临床基因治疗应用，它们的用途是 目前受到产量的限制，产量是几个数量级 小于主要的病毒载体系统。多种原因，在 取决于具体配方的可变比例， 导致非病毒载体的总产量低。 在所有 案例中，一个因素降低产量一至三个数量级 是DNA从细胞质到其 最终目的地，核心。 在成熟生物体的大多数细胞中， 在有丝分裂后， 膜的 虽然已经尝试改善核 通过连接到DNA合成肽上使载体易位，或 含有核定位信号（NLS）的蛋白质，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，当很少的副本的载体 将存在于每个细胞中。 该项目的目的是探索一种 新的原则，这有希望更有效的核 易位 由于蛋白质输入途径是基于一条链 要输入的蛋白质与转运因子的相互作用， 这个项目的中心思想是把载体DNA放在入口处， 这条链的点，在那里它必须与大量的竞争， 天然蛋白质等待进口，但在链的末端， 就在穿过核孔的最后一步之前。 在 生物化学术语，这一概念转化为使用靶向部分 不是NLS，而是转运因子karyopherins β，在正常情况下， 在核孔处对接复合物。 三个已知的 将研究人类核转运蛋白β的作用能力， 作为核靶向部分。 每一个最小的碎片 将测定保留完全易位能力的核转运蛋白， 用于改进的输送系统。我们预计，这一做法将 确保DNA快速、高效地转移到 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.