Polymeric Nanoparticles for Delivery of Ribonucleic Acids: Overcoming the Endosomal Barrier and Going Beyond the Liver

核糖核酸（RNA）被包装成纳米颗粒（NPs）或包含在分子偶联物中以传递到细胞中，可以敲低基因表达，阐明基因功能，表达蛋白质，上调基因和编辑基因组，并准备彻底改变医学领域。广泛的研究已经发表了NP递送系统，其中RNA被包装与阳离子聚合物或阳离子脂质。尽管在该领域取得了重大进展，但基于rna的系统面临的主要挑战仍然是交付。传递系统通过囊泡（内体）内化到细胞中，囊泡中的包裹构成了主要的细胞内屏障和当前研究的中心问题。此外，静脉给药后的RNA递送目前几乎完全针对肝脏。申请人在壳聚糖的生产和表征方面具有成熟的专业知识，壳聚糖是一种由氨基葡萄糖组成的带正电的生物聚合物，来源于甲壳类动物的外壳。壳聚糖具有生物相容性，在生物医学领域有着广泛的应用。申请人用RNA形成了壳聚糖的NPs，并表明这些NPs在体外有效，并且在静脉给药后特异性地在肾脏中积累。虽然这些结果是有希望的，但这些纳米系统的逃离核内体的能力、有效性和静脉注射后的生物分布必须进一步研究和改进。本研究计划的长期目标是开发一种新型的壳聚糖基系统家族，该系统具有增强的逃避核内体的能力，以改善RNA的递送，并在静脉给药后生物分布到肝脏以外的器官。另一个长期目标是进一步了解细胞内化和内体逃逸的基本机制。本提案旨在通过合成大量化学修饰的壳聚糖库来扩展我们之前的工作，这些壳聚糖将与促进内体释放的部分结合使用，以产生全新的递送系统。这些纳米系统将以其物理化学和结构特性以及细胞内化，细胞内运输，功效和生物分布为特征。本项目前5年招收高分子化学、胶体科学、细胞/分子生物学背景的博士研究生4名，本科生1名/年。这项工作将为高效的壳聚糖基RNA传递系统提供设计原则、结构-活性关系和细胞内生物学功能机制，并将创建一个知识库，使这些纳米系统在基因表达、基因敲除和体内应用方面的适用性得到极大的扩展。该研究项目的成果将对基因治疗、纳米技术和细胞生物学等领域具有重要意义。

Ribonucleic acid (RNA) packaged into nanoparticles (NPs) or included in molecular conjugates for delivery to cells can knock-down gene expression, elucidate gene function, express proteins, upregulate genes and edit the genome and is poised to revolutionize the field of medicine. Extensive research has been published for NP delivery systems where RNA is packaged with cationic polymers or cationic lipids. Despite significant progress achieved in the field, delivery is still the main challenge that faces RNA-based systems. Delivery systems are internalized into cells in vesicles (endosome) and entrapment in these vesicles constitutes a major intracellular barrier and a central current research problem. Additionally, delivery of RNA following intravenous administration is currently almost exclusively targeted to the liver. The applicant has an established expertise in the production and characterization of chitosan, a positively charged biopolymer composed of glucosamine that is derived from crustacean shells. Chitosan is biocompatible and is used extensively in biomedical applications. The applicant has formed NPs of chitosan with RNA and showed that these NPs are efficient in vitro and specifically accumulate in kidneys following their intravenous administration. Although these results are promising, the ability to escape the endosome, the efficacy and the biodistribution following intravenous injection of these nanosystems must be further examined and improved. A long-term objective of this research program is to develop a novel family of chitosan-based systems with increased ability to escape the endosome for improved delivery of RNA and that biodistribute to organs other than liver following intravenous administration. Another long-term objective is to further our understanding of cellular internalization and endosomal escape fundamental mechanisms. This proposal aims to extend our previous work by synthesizing a large library of chemically modified chitosans that will be used in combination with moieties facilitating endosomal release to produce entirely new delivery systems. These nanosystems will be characterized for their physicochemical and structural properties and their cellular internalization, intracellular trafficking, efficacy and biodistribution. For the first 5 years of this research program, 4 PhD students with background in polymer chemistry, colloidal science, and cell/molecular biology and 1 undergraduate student/year will be recruited. This work will provide design principles, structure-activity relationships and identify intracellular mechanisms of biological function for efficient chitosan-based RNA delivery systems and will create a knowledge base that will allow for a tremendous expansion in the applicability of these nanosystems for gene expression, gene knockdown and in vivo applications. The outcomes of this research program will be significant for the fields of gene therapy, nanotechnology and cell biology.