Targeted Subcellular Delivery of Oligonucleotides and Proteins

描述(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步，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演变为临床现实。然而，这些目标的成功实现依赖于克服随之而来的更大的困难，即以有效的治疗方式和数量将这些大分子化合物输送到它们的细胞和亚细胞靶点。这项建议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。ONS的一个强大的应用是用作各种受体的配体，特别是免疫细胞的受体。考虑到预防和治疗使用疫苗预防和治疗病毒感染和肿瘤的巨大重要性，将探讨ONS在实现和定制疫苗免疫结果方面的强大能力；这项提议的重点是一种有效的递送系统，该系统可以将ONS和蛋白质抗原放入所需细胞类型的适当的亚细胞间隔中。为了测试建议的递送系统的递送能力，我们将首先检验这样的假设，即携带抗原的递送系统内的内体通透性李斯特溶素O(LLO)与免疫刺激序列ONS(ISS-ONS)共包裹可以将大量抗原递送到胞浆(即MHCI抗原递呈途径)以及MHCII隔室，同时增强ISS-ONS递送到内吞泡内的同源受体的递送。携带抗原和ISS的LLO脂质体将被用来测试免疫刺激和调节效果，并与对照免疫进行比较。LLO的作用，除了它通过内溶促进胞浆递送的能力外，在修改内体成熟和减缓其向溶酶体的进展方面的作用将被用细胞生物学和免疫学的方法来研究和检验。传递的特征和优化将在体外进行，使用抗原呈递细胞的原代培养，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工作传递载体进行测试。该项目产生的最佳疫苗输送载体将是一种类似细菌颗粒的非病毒/非细菌输送载体，能够入侵细胞，并携带免疫监测机制(S)识别的免疫刺激/调制信号，这是诱导强大的Th1型细胞免疫的理想选择。本项目研制的强效疫苗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.