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

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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.