Genetic and Chemically Programmed Nanoparticles for Prodrug Therapy

描述(由申请人提供)：病毒样颗粒(VLP)代表了用于疾病靶向的最具特征和最容易定制的纳米结构。它们是X射线结晶学常规分析的最大组件，也是拥有基因组的最小实体，因此它们在化学和生物学世界之间占据了独特的位置。我们最近的工作集中在直径28 nm的二十面体颗粒上，该颗粒来自噬菌体Q？的外壳蛋白。这种结构在大肠杆菌中高产率和高纯度地重组生产，对热、可变的pH、有机溶剂和条件非常稳定 化学修饰。它也不被哺乳动物细胞上的特异性受体识别，因此可以通过在衣壳的外表面和内室安装官能团来赋予其所需的特性。我们已经开发出在Q？VLP的外部对多肽进行遗传编码的方法，同时在内部包装多个活性酶的副本。外部基团用于与选定的受体结合，以靶向特定类型的细胞，或展示用于相同目的的抗体。内部的酶可以将良性的前药转化为细胞毒剂，催化效率高，同时防止蛋白酶攻击和变性。我们将进一步开发和提高生产这些颗粒的技术，目标是更精确地控制它们的组成，使用更多功能组件。我们还将把它们应用于体外和体内前药激活的概念验证测试，以确定这种治疗方法的范围和局限性。对于它们最终的临床应用，其后果是 还必须确定抗颗粒免疫反应的大小。因此，我们将首次对免疫前对这种纳米颗粒的循环寿命和生物分布的影响进行定量评估，并根据这些目标测试降低其免疫原性和调理作用的复杂方法。这些努力的结果将是蛋白质纳米颗粒设计和生产方面的新能力，对使用靶向VLP包装酶的前药物治疗优点的令人兴奋的临床前评估，以及对纳米颗粒靶向的基础理解和避免调理和免疫途径的重大贡献。

DESCRIPTION (provided by applicant): Virus-like particles (VLPs) represent the best-characterized and most easily tailored nanostructures for the targeting of disease. They are the largest assemblies to be routinely analyzed by x-ray crystallography, and the smallest entities to possess a genome, and thus they occupy a unique place between the worlds of chemistry and biology. Our recent efforts have focused on the 28 nm diameter icosahedral particle derived from the coat protein of bacteriophage Q¿. This structure, produced recombinantly in E. coli in high yield and purity, is very stable toward heat, variable pH, organic solvents, and conditions of chemical modification. It is also recognized by no specific receptor on mammalian cells, and therefore may be imbued with desired properties by installing functional groups on the outside surface and inside compartment of the capsid. We have developed methods to genetically encode polypeptides on the exterior of Q¿ VLPs while at the same time packaging multiple copies of active enzymes on the inside. The outside groups serve to bind to chosen receptors for the targeting of specific cell types or to display antibodies for the same purpose. The enzymes on the inside can convert benign prodrugs to cytotoxic agents with high catalytic efficiency, while being protected from protease attack and denaturation. We will further develop and enhance the technology to produce these particles, with the goal of more precisely controlling their composition and using more functional components. We will also apply them to proof-of-concept tests of prodrug activation both in vitro and in vivo to determine the scope and limitations of this therapeutic approach. For their eventual clinical application, the consequences of the anti-particle immune response must also be determined. We will therefore conduct the first quantitative assessment of the effects of pre-immunization on the circulation lifetime and biodistribution of this type of nanoparticle, and test sophisticated methods to diminish their immunogenicity and opsonization in the context of these goals. The result of these efforts will be new capabilities in protein nanoparticle design and production, an exciting preclinical evaluation of the merits of prodrug therapy using targeted VLP-packaged enzymes, and a major contribution to the fundamental understanding of nanoparticle targeting and avoidance of opsonization and immunological pathways.