Vaults as Nonviral Nucleic Acid Delivery Vehicles

描述（由申请人提供）：基因治疗需要负担得起的、可扩展的技术，用于在不引发免疫反应的情况下将感兴趣的基因特异性递送到靶细胞的核室。尽管病毒和非病毒载体已被设计用于将DNA传递到细胞，但人们对非病毒载体的兴趣越来越大，因为它们有望克服操纵病毒所固有的许多限制，包括它们的复杂性、难以适应大规模生产和易于引发免疫反应。在这个提议中描述的非病毒方法，一个自然发生的纳米级结构的工程，是独特的，很大程度上是前所未有的。然而，考虑到在基因治疗取得成功之前必须克服的重大挑战，大胆的新方法是必要的。拱顶粒子是一种高度保守、丰富且普遍存在的纳米胶囊，具有较大的内部容量，可以包封和保护核酸等不稳定的生物化合物。尽管该颗粒的13mda质量使其大小几乎是核糖体的四倍，但该颗粒仅由三种蛋白质和一个或多个小的未翻译rna的多个副本组成。其中一种蛋白质，MVP，是粒子的主要结构蛋白。当在昆虫细胞中表达时，MVP的96个拷贝可以组装成基本的拱顶囊。MVP cDNA的分子操作允许将特定肽和蛋白质包装到拱顶管腔中。这一建议概述了一种策略来操纵拱顶纳米胶囊，使其可以用于特异性包装核酸。此外，本文还描述了一种用细胞靶向信号修饰颗粒的方法，该方法将允许这些结构特异性靶向正常或肿瘤细胞。

DESCRIPTION (provided by applicant): Gene therapy requires affordable, scaleable technology for the specific delivery of a gene of interest to the nuclear compartment of a target cell without triggering an immune response. Although viral and nonviral vectors have been designed to deliver DNA to cells, interest in nonviral vectors has grown from the promise that they can overcome numerous limitations inherent in manipulating viruses including their complexity, difficulty to adapt to large-scale production and ease of triggering an immune response. The nonviral approach described in this proposal, the engineering of a naturally-occurring nano scale structure, is unique and largely unprecedented. However, given the significant challenges that must be overcome before successful gene therapy can be achieved, a bold new approach is warranted. The vault particle is a highly conserved, abundant and ubiquitous nanocapsule with a large internal capacity to encapsulate and protect labile biological compounds such as nucleic acids. Although the 13 MDa mass of the particle makes it nearly four times the size of the ribosome, the particle is composed of multiple copies of just three proteins and one or more small untranslated RNAs. One of these proteins, MVP, is the major structural protein of the particle. When expressed in insect cells, 96 copies of MVP can assemble into the basic vault capsule. Molecular manipulation of the MVP cDNA allows packaging of specific peptides and proteins into the vault lumen. This proposal outlines a strategy to manipulate the vault nanocapsule so that it can be used to specifically package nucleic acids. In addition, an approach is delineated to modify the particle with cell targeting signals that will allow the specific targeting of these structures to normal or neoplastic cells.