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

核糖核酸(RNA)被包装成纳米粒子(NPs)或包含在分子偶联物中用于传递到细胞中，可以下调基因表达、阐明基因功能、表达蛋白质、上调基因和编辑基因组，并有望给医学领域带来革命性的变化。已经发表了大量关于NP递送系统的研究，其中RNA与阳离子聚合物或阳离子脂类包装在一起。尽管在这一领域取得了重大进展，但交付仍然是基于RNA的系统面临的主要挑战。给药系统被内化到囊泡(内体)中的细胞中，而被困在这些囊泡中构成了一个主要的细胞内屏障，也是当前研究的一个中心问题。此外，静脉给药后的RNA输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拥有成熟的专业知识，壳聚糖是一种由从甲壳动物贝壳中提取的氨基葡萄糖组成的带正电的生物聚合物。壳聚糖具有良好的生物相容性，在生物医学领域有着广泛的应用。申请人已经与RNA形成了壳聚糖的NPs，并表明这些NPs在体外是有效的，并且在静脉给药后特异性地积累在肾脏中。尽管这些结果很有希望，但静脉注射这些纳米系统后逃逸内体的能力、有效性和生物分布仍需进一步检查和改进。*该研究计划的长期目标是开发一种新的基于壳聚糖的系统家族，该系统具有更强的逃逸内体的能力，从而改善RNA的输送，并在静脉给药后生物分布到肝脏以外的器官。另一个长期目标是加深我们对细胞内化和内体逃逸基本机制的理解。这项提议旨在通过合成一个化学修饰的壳聚糖大文库来扩展我们之前的工作，该文库将与促进内体释放的部分结合使用，以产生全新的给药系统。这些纳米系统将根据它们的物理化学性质和结构特性以及它们的细胞内化、细胞内运输、功效和生物分布来表征。在这项研究计划的前5年，将招募4名具有聚合物化学、胶体科学和细胞/分子生物学背景的博士生和1名本科生/年。*这项工作将提供有效的壳聚糖RNA递送系统的设计原则、结构-活性关系和识别生物功能的细胞内机制，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。这项研究计划的结果将对基因治疗、纳米技术和细胞生物学领域具有重要意义。

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.**