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，将感兴趣的基因特定地输送到目标细胞的核间室，而不会引发免疫反应。尽管病毒和非病毒载体被设计成将DNA运送到细胞，但人们对非病毒载体的兴趣源于这样的承诺，即它们可以克服操纵病毒所固有的众多限制，包括它们的复杂性、难以适应大规模生产以及容易引发免疫反应。这项提案中描述的非病毒式方法，即设计一种自然产生的纳米级结构，是独一无二的，在很大程度上是前所未有的。然而，考虑到在实现成功的基因治疗之前必须克服的重大挑战，一种大胆的新方法是必要的。拱顶粒子是一种高度保守、丰富和普遍存在的纳米胶囊，具有很大的内部容量，可以包裹和保护核酸等不稳定的生物化合物。尽管该颗粒的13mda质量使其几乎是核糖体大小的四倍，但该颗粒只由三种蛋白质和一个或多个未翻译的小RNA的多个副本组成。其中一种蛋白质MVP是颗粒的主要结构蛋白。当在昆虫细胞中表达时，96个拷贝的MVP可以组装成基本的拱顶胶囊。通过对MVP基因进行分子操作，可以将特定的多肽和蛋白质包装到血管腔中。这项提案概述了一种操纵拱顶纳米胶囊的策略，以便它可以被用来专门包装核酸。此外，描述了一种用细胞靶向信号修饰颗粒的方法，该信号将允许将这些结构特异性地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.