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优于病毒载体，但很少有临床试验研究非病毒系统。非病毒基因方法的次优性能的主要原因是它们的低转染效率，特别是在体内。对细胞运输过程中的主要限制因素的广泛研究提供了预期在成功使用非病毒系统中规避细胞外和细胞内障碍的工具谱。聚合物载体在其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缩合载体，并且修饰一些官能团有助于靶向和运输到细胞核中。然而，目前的方法并没有真正明确定义，因为改性的聚阳离子是静电和随机复合阴离子基因。具有少量功能的定义不明确的载体制剂和在正确位置处的官能团暴露不足是效率低的主要原因。本申请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（根据需要），其包括屏蔽血流中的带正电荷的表面、细胞靶向配体、内体溶解剂、DNA从多聚物的受控释放、核孔扩张和核输入。所有这些功能都将被整合到通过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，地幔层和外壳层。更重要的是，预期纳米构建体以明确定义的方式在细胞内区室中所需的位置处暴露官能团，并且每一层在发挥其应有的作用后将被剥离，留下最少的组分用于掺入基因的核输入。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。长期目标将分四个具体目标来实现：前三个目标分别是核层、幔层和壳层。每层将针对靶细胞、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。 公共卫生相关性：尽管在基因治疗中，特别是在药物制剂的可重复生产中，非病毒载体优于病毒载体，但大多数基因治疗的临床试验采用病毒系统。本申请是基于新的设计概念开发聚合物基因载体，预期其具有用于翻译研究的高潜力，用于可接受的体内转染效率并且具有最小的毒性。新颖且独特的设计原理将适应有效基因转染所需的所有功能，并且这些功能将以明确定义的方式暴露在最需要的适当细胞内区室中。纳米级基因载体将由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.