WELL-DEFINED MULTIFUNCTIONAL POLYMERIC NANOCARRIERS FOR EFFECTIVE GENE DELIVERY

描述(申请人提供)：尽管非病毒载体在基因治疗中比病毒载体有几个优点，但很少有临床试验研究非病毒系统。非病毒基因方法性能不佳的一个主要原因是它们的低转染率，特别是在体内。对细胞贩运过程中的主要限制因素的广泛调查提供了一系列工具，预计可绕过成功使用非病毒系统的细胞外和细胞内障碍。聚合物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常被用作DNA浓缩载体，对少数官能团进行修饰有助于靶向和转运到细胞核。然而，目前的方法并不是真正明确的，因为修饰的聚阳离子是静电和随机络合的阴离子基因。低效率的主要原因是载体制备的定义不明确，功能性很少，官能团在正确的位置暴露不足。在这一应用中提出的一种新方法是适应从注射部位到核的基因转移的所有功能(根据需要)，包括屏蔽血流中带正电的表面、细胞靶向配体、内溶剂、从复合体控制DNA释放、核孔扩张和核输入。所有这些功能都将被整合到由逐层方法构建的纳米级组件中，从而产生三层基因载体：核心层、外套层和壳层。更重要的是，纳米结构有望以明确的方式暴露细胞内隔间中所需位置的官能团，每一层都将在发挥其应有作用后被剥离，留下最少的组件用于整合基因的核进口。这种独特的组装方法确保了药物基因制剂在未来翻译研究中的高潜力。长期目标将通过四个具体目标来实现：第一个三个目标分别是核心层、地幔层和壳层。每一层都将针对靶细胞、增殖和非增殖细胞、内体pH和配体受体进行定制。第四个具体目标将在体内测试和证明所提出的概念。 与公共卫生相关：尽管非病毒载体在基因治疗方面比病毒载体有几个优势，特别是在可重复生产药物制剂方面，但大多数基因治疗的临床试验都使用病毒系统。这一应用是基于一种新的设计概念开发的聚合物基因载体，预计这将为翻译研究提供高潜力，在体内获得可接受的转染效率，并具有最小的毒性。新颖而独特的设计原则将适应有效的基因转染所需的所有功能，这些功能将以明确的方式暴露在最需要的适当的细胞内隔间中。这种纳米大小的基因载体将由对药物友好的聚合物构建。

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.