Targeted Subcellular Delivery of Oligonucleotides and Proteins

描述（由申请人提供）：寡核苷酸和蛋白质的靶向亚细胞递送。 由于分子/细胞生物学和基因组学领域的惊人进展，基于核酸的治疗剂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在以治疗有效的模式和量将这些大分子化合物递送至其细胞和亚细胞靶位点中伴随的更大困难。该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。ON的强大应用之一是它们作为各种受体的配体的用途，特别是免疫细胞的受体。考虑到疫苗对病毒感染和肿瘤的预防性和治疗性使用的巨大重要性，将探索ON在实现和定制疫苗接种的免疫结果方面的强大能力;该提议集中于可以将ON和蛋白质抗原放入所需细胞类型的适当亚细胞区室的有效递送系统。为了测试所提出的递送系统的递送能力，我们将首先检查以下假设：与免疫刺激序列ON（ISS-ON）共包封在携带抗原的递送系统内的内体透化的溶血素O（LLO）可以将显著程度的抗原递送至胞质溶胶（即，抗原呈递的MHCI途径）以及MHCII区室，同时增强ISS-ON向内吞囊泡中的同源受体的递送。与对照免疫相比，携带抗原和ISS-ON的LLO-脂质体将用于测试免疫刺激和免疫调节作用。LLO的作用，除了其通过内体溶解增强胞质递送的能力之外，还将使用细胞生物学和免疫学方法研究和检查内体成熟的修饰和减缓其向溶酶体的进展。递送的表征和优化将使用抗原呈递细胞的原代培养物在体外进行，并且使用小鼠模型在体内进行。然后将使用病毒核蛋白测试工作递送载体用于抗病毒疫苗。本项目产生的最佳疫苗递送载体将是一种非病毒/非细菌递送载体，类似于能够侵入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，这是诱导稳健的Th 1型细胞介导免疫的理想载体。本项目研制的疫苗靶向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.