Vaults as Nonviral Nucleic Acid Delivery Vehicles

描述（由申请人提供）：基因治疗需要经济实惠、可扩展的技术，将感兴趣的基因特异性递送至靶细胞的核区室，而不触发免疫反应。尽管病毒和非病毒载体被设计用来将DNA传递到细胞中，但人们对非病毒载体的兴趣日益增长，因为它们有望克服操纵病毒所固有的许多限制，包括其复杂性、难以适应大规模生产以及易于触发免疫反应。该提案中描述的非病毒方法，即天然存在的纳米级结构的工程，是独特的且很大程度上是前所未有的。然而，考虑到在成功实现基因治疗之前必须克服的重大挑战，有必要采取大胆的新方法。穹窿颗粒是一种高度保守、丰富且普遍存在的纳米胶囊，具有巨大的内部容量，可以封装和保护核酸等不稳定的生物化合物。尽管该颗粒的质量为 13 MDa，几乎是核糖体大小的四倍，但该颗粒仅由三种蛋白质的多个副本和一种或多种小型非翻译 RNA 组成。这些蛋白质之一，MVP，是颗粒的主要结构蛋白质。当在昆虫细胞中表达时，96 个 MVP 副本可以组装成基本的穹窿胶囊。 MVP cDNA 的分子操作允许将特定的肽和蛋白质包装到穹窿腔中。该提案概述了一种操纵穹窿纳米胶囊的策略，使其可用于特异性包装核酸。此外，还描述了一种用细胞靶向信号修饰颗粒的方法，这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.