WELL-DEFINED MULTIFUNCTIONAL POLYMERIC NANOCARRIERS FOR EFFECTIVE GENE DELIVERY

描述（由申请人提供）：尽管在基因治疗中非病毒载体比病毒载体有一些优势，但很少有临床试验研究非病毒系统。非病毒基因方法表现不佳的一个主要原因是它们的转染效率低，特别是在体内。对细胞运输过程中主要限制因素的广泛调查提供了一系列工具，有望在成功使用非病毒系统时绕过细胞外和细胞内障碍。高分子载体在设计和功能化方面表现出极大的灵活性。可生物降解或不可降解的聚阳离子通常用作DNA浓缩载体，修饰一些官能团有助于靶向和运输到细胞核。然而，目前的方法并没有真正定义好，因为修饰的聚阳离子是静电的，并且随机地与阴离子基因络合。载体制备的定义不明确，功能很少，在适当的地方暴露的官能团不足，是导致效率低的主要原因。本应用中提出的一种新方法是适应从注射部位到细胞核的基因运输的所有功能（尽可能多），包括在血流中屏蔽带正电荷的表面，细胞靶向配体，内溶剂，从复合体中控制DNA释放，核孔扩张和核输入。所有这些功能将被纳入纳米级组装通过一层一层的方法，导致三层基因载体；地核，地幔和地壳层。更重要的是，纳米结构有望以明确的方式将功能基团暴露在细胞内区室所需的位置，每一层在发挥其应有的作用后将被剥离，留下最小的成分用于整合基因的核输入。这种独特的组装方法确保了未来翻译研究的药物基因配方的高潜力。长期目标将在四个具体目标上实现：前三个目标分别为核心层、地幔层和壳层。每一层将为目标细胞定制；增殖和非增殖细胞，内体pH值和配体受体。第四个具体目标将在体内测试和证明所提出的概念。

DESCRIPTION (provided by applicant): Despite several advantages of non-viral vectors over viral vectors in gene therapy, few clinical trials have investigated non-viral systems. A major reason for the suboptimal performance of non-viral gene approaches is their low transfection efficiency especially in vivo. Extensive investigations into the major limiting factors during the cellular trafficking processes have provided spectra of tools anticipated to circumvent the extracellular and intracellular obstacles in successful use of non-viral systems. Polymeric vectors demonstrate great flexibility in their design and functionalization. Biodegradable or non-degradable polycations are often used as DNA condensing vectors and modifying a few functional groups helps targeting and trafficking into the nucleus. However, current approaches are not truly well-defined because the modified polycations are electrostatically and randomly complexed with anionic genes. A poorly defined vector preparation with a few functionalities and inadequate exposure of the functional groups at the right places are primarily responsible for low efficiency. A novel approach proposed in this application is to accommodate all functionalities (as many as needed) for gene trafficking from an injection site to the nucleus, which include shielding the positively charged surface in the blood stream, cell targeting ligands, endosomolytic agents, controlled release of DNA from polyplex, nuclear pore dilation and nuclear import. All these functionalities will be incorporated into nanoscaled assemblies constructed by a layer-by-layer method, resulting in triple layered gene vectors; the core, mantle, and the shell layers. More importantly the nanoconstruct is expected to expose the functional groups at the place required in the intracellular compartments in a well defined manner and each layer will be peeled-off after serving its due role, leaving minimal components for nuclear import of the incorporated gene. This unique assembly approach assures high potential for pharmaceutical gene formulations for future translational studies. The long term goal will be accomplished in four specific aims: first three aims for core layer, mantle layer and shell layer respectively. Each layer will be customized for target cells; proliferating and non-proliferating cells, endosomal pH, and ligand receptor. The fourth specific aim will test and prove the proposed concept in vivo. PUBLIC HEALTH RELEVANCE: Despite several advantages of non-viral vectors over viral owns in gene therapy, particularly in reproducible production of pharmaceutical formulations, most clinical trials for gene therapy employed viral systems. This application is for development of polymeric gene carriers based on a new design concept, which is anticipated to present a high potential for a translational study, for acceptable transfection efficiency in vivo and have minimal toxicity. The novel and unique design principle will accommodate all functionalities required for effective gene transfection and these functionalities will be exposed in the appropriate intracellular compartments where needed the most in a well-defined fashion. The nano-sized gene carrier will be constructed from pharmaceutically-friendly polymers.