Cationic Polymers and Polymeric Micelles as Plasmid DNA and CRISPR-Cas9 Ribonucleoprotein Delivery Vehicles

Abstract

Millions of people are currently suffering from genetic diseases and disorders worldwide and the traditional protein-based treatments are both expensive and require repetitive injections to maintain long-term effects. Gene therapy, as an alternative, holds great potential by direct delivery of genetic materials such as nucleic acids and genome editing machineries into human body to achieve long-term therapeutic protein expression, malfunctioning gene silencing, and native gene alternation. As a crucial step towards gene therapy, the delivery of genetic materials remains a major challenge, and affordable, efficient, and well-defined delivery vehicles are urgently needed. Synthetic polymers have been explored as plasmid DNA delivery vehicles for decades owing to their low production cost, chemical flexibility, low immunotoxicity, and the ability to encapsulate biomacromolecules. However, the precise control of polymeric vehicle properties by structure-tuning is a general challenge. Hence, the fundamental understanding of polymeric vehicle’s structure-property relationships is of great importance. In addition, polymers as well-documented nucleic acid delivery vehicles, are largely underexplored for their ability to encapsulate, stabilize and deliver RNA-protein conjugates, such as CRISPR/Cas9 ribonucleoprotein, a recently emerged versatile genome editing tool, presumably due to the inherent structural differences between long, semiflexible DNA and globular RNA-protein payload. Herein, several classes of polymeric delivery vehicles were synthesized and investigated, namely, cationic linear polymers, block copolymers, and polymeric micelles. Initially, a systematic comparison of linear polymers and micelles were performed, and vehicle architecture were shown to largely affect DNA complexation ability, complex physical properties, and biological performance. Later on, polymeric micelles were explored as well-defined CRISPR/Cas9 ribonucleoprotein delivery vehicles, and the solvent condition was found to be a key factor that affect particle complexation and gene-editing efficiency. Finally, polymers with liver-targeting ability and cellular membrane-penetration property have been developed and studied for their gene delivery efficiency and cytotoxicity.