Molecular Desighn of DNA-Encapsulating Nano-Particle Capable of Nuclear Targeting

在哺乳动物细胞中的基因转移和表达将成为未来先进医学应用所需的技术。病毒载体有其固有的机制，可以跨越细胞膜和核膜两大屏障传递遗传物质，因此在当前的医学应用中受到青睐。然而，在许多临床应用中需要更安全的非病毒载体。非病毒载体的性能已通过加强跨细胞膜运输得到改善。然而，目前还没有一种技术能够实现大的DNA分子跨核膜的运输。在这个项目中，我研究了允许包裹纳米粒子的DNA有效核运输的条件。为此，我采用噬菌体Lambda作为模型。基于SV40 T抗原的多种具有核转运活性的肽（核转运信号，NLS）通过与两种主要衣壳蛋白之一的D蛋白融合在噬菌体颗粒头部表面显示。所有这些NLS肽辅助D核运输的蛋白质,但只有少数可以协助运输纳米大小的粒子。我以噬菌体颗粒通过显微注射进入细胞质的核转运为指标，成功地优化了信号，使高达5%的颗粒可以通过核孔复合物（NPC）主动转运到细胞核中。另一方面，我也发现nls在细胞质中介导的颗粒破坏，可能是通过蛋白酶体，限制了核运输的效率。这是第一个证据，证明在核转运研究中最常用的来自SV40 T抗原的NLS肽不一定适合大颗粒的转运。

Gene transfer and expression in mammalian cells should become a technology required in advanced medical application in future. Virus vectors are favored in current medical application as they have their intrinsic machinery to deriver the genetic materials across two major barriers, the cell membrane and the nuclear membrane. However, safer non-viral vectors are desired in many clinical applications. Performance of non-viral vectors has been improved through enhancing the transport across the cell membrane. However, no technology realizing the transport of large DNA molecules across the nuclear membrane has been established yet. In this project, I investigate the conditions allowing the efficient nuclear transport of the DNA encapsulating nano-sized particles. For this purpose, I employ bacteriophage Lambda as a model. Various peptides with nuclear transport activity(nuclear transport signal, NLS) based on T antigen of SV40 were displayed on the surface of the head of the phage particle by fusing them to D protein, one of two major capsid proteins. All of these NLS peptide assisted the nuclear transport of the D proteins, but only a few could assist the transport of nano sized particles. By using the nuclear transport of the phage particles injecting into the cytoplasm by microinjection as an Index, I succeeded to optimize the signals so that up to 5% of the particles could be transported into the nucleus actively through the nuclear pore complex(NPC). On the other hand, I also found that the NLS-mediated particle disruption in the cytoplasm, possibly through the proteasome, limited the efficiency of the nuclear transport. This is the first evidence that the NLS peptide derived from SV40 T antigen, used most popularly in the research of nuclear transport, may not necessarily be suited for transport of large particles.