Targeted Subcellular Delivery of Oligonucleotides and Proteins

描述（由申请人提供）：靶向亚细胞递送寡核苷酸和蛋白质。由于分子/细胞生物学和基因组学领域的惊人进展，基于核酸的治疗药物和其他大分子正在努力从概念上令人满意的实验方法发展到临床现实。然而，这些目标的成功实现依赖于克服伴随而来的更大困难，以治疗有效的方式和数量将这些大分子化合物递送到它们的细胞和亚细胞靶点。本提案的主要目标是表征和开发有效的递送策略和递送载体，以实现基于寡核苷酸（ON）和蛋白质的药物的治疗目标。网络的强大应用之一是它们作为各种受体的配体，特别是免疫细胞的配体。考虑到针对病毒感染和肿瘤的疫苗的预防和治疗用途的巨大重要性，将探索网络在启用和调整疫苗接种的免疫结果方面的强大能力；这一建议的重点是一种有效的递送系统，可以将蛋白抗原和蛋白抗原放入所需细胞类型的适当亚细胞区室。为了测试所提出的递送系统的递送能力，我们将首先检验一个假设，即在携带抗原的递送系统内，内核体通透性李斯特菌溶素O （LLO）与免疫刺激序列蛋白（ISS-ONs）共包被，可以将大量抗原递送到细胞质（即抗原呈递的MHCI途径）以及MHCII室，同时增强ISS-ONs向内吞囊泡中同源受体的递送。携带抗原和iss - on的低密度脂蛋白脂质体将用于测试免疫刺激和调节作用，并与对照免疫接种进行比较。LLO的作用，除了其通过内溶酶增强细胞质递送的能力，在内溶体成熟的修饰和减缓其向溶酶体的进展中，将使用细胞生物学和免疫学方法进行研究和检查。表征和优化递送将在体外进行，使用抗原提呈细胞的原代培养，并在体内使用小鼠模型。然后将使用抗病毒疫苗的病毒核蛋白对有效的递送载体进行测试。该项目得出的最佳疫苗递送载体将是一种非病毒/非细菌递送载体，类似于能够入侵细胞并携带免疫监视机制识别的免疫刺激/调节信号的细菌颗粒，这是诱导强大的th1型细胞介导免疫的理想载体。本项目研制的强效疫苗制剂靶向递送载体对人类公共卫生和国家生物防御具有重要意义。

DESCRIPTION (provided by applicant): Targeted Subcellular Delivery of Oligonucleotides and Proteins. Nucleic acid-based therapeuticals and other macromolecules, as a result of astounding advances in the fields of molecular/cellular biology and genomics, are striving to evolve from conceptually-satisfying experimental approaches to a clinical reality. However, successful implementation of these goals relies on overcoming the accompanying greater difficulties in delivering these large molecular compounds to their cellular and subcellular target sites in therapeutically effective modes and quantities. The major goal of this proposal is to characterize and develop efficient delivery strategies and delivery vectors for the realization of therapeutic goals of oligonucleotide (ON)- and protein-based drugs. One of the powerful applications of ONs is their usage as ligands for a variety of receptors, especially those of the immune cells. Considering the enormous importance of prophylactic and therapeutic usage of vaccines against viral infections and tumors, the powerful capacity of ONs in enabling and tailoring the immunologic outcome of vaccination will be explored; this proposal is focused on an efficient delivery system that can put ONs and protein antigens into the appropriate subcellular compartments of desired cell types. To test the delivery capabilities of the proposed delivery systems, we will first examine the hypothesis that endosome-permeabilizing listeriolysin O (LLO) co-encapsulated with immunostimulating sequence ONs (ISS-ONs) inside antigen-carrying delivery systems can deliver a significant extent of antigen to the cytosol (i.e., MHCI pathway of antigen presentation) as well as MHCII compartments while enhancing the delivery of ISS-ONs to the cognate receptors in the endocytic vesicles. The antigen- and ISS-ON-carrying LLO-liposomes will be used to test the immuno-stimulating and -modulating effects in comparison with control immunizations. The role of LLO, in addition to its ability to enhance cytosolic delivery via endosomolysis, in the modification of endosome maturation and slowing down its progression into lysosomes will be investigated and examined using cell biological and immunological methods. The characterization and optimization of delivery will be done in in vitro, using primary cultures of antigen-presenting cells, and in vivo using mouse models. The working delivery carrier will be then tested using viral nucleoproteins for anti-viral vaccines. The optimal vaccine delivery vehicle resulting from this project will be a non-viral/ non-bacterial delivery vector resembling a bacterial particle capable of invading cells and carrying immuno-stimulating/modulation signals recognized by immune surveillance mechanism(s), which is ideal for inducing robust, Th1-type, cell-mediated immunity. Targeted delivery carrier for robust vaccine formulations developed in this project will have great significances in human public health and nation's biodefense.