Improving non-viral gene delivery: Polymer carriers for spatial and temporal control of nucleic acid release.

摘要

Gene therapy has garnered significant interest over the past few decades as an innovative approach for regulating protein expression towards the treatment of (i) genetic disorders including cystic fibrosis, severe combined immunodeficiency (SCID), and muscular dystrophy, (ii) acquired/infectious diseases such as Hepatitis and HIV/AIDS, and (iii) multiple varieties of cancer. Non-viral gene delivery approaches have received particular attention due their low toxicity, high nucleic acid storage capacity, and tailorability as compared to their viral counterparts. However, the efficiency and clinical realization of non-viral delivery vehicles has been limited by an incomplete understanding of their assembly, subcellular trafficking, and intracellular release mechanisms. Thus, the goal of this work was to develop strategies to understand and direct gene association and release using non-viral carriers, with the aim of improving current delivery platforms. Specific milestones of these studies include (1) the identification of the structural and functional consequences of fluorescent labeling of polymeric carriers for intracellular investigations, (2) the development of materials with tunable gene association and release capacity, as well as (3) the identification of novel non-viral approaches to address the functional requirements of gene transfer while promoting versatile preparation methods and facile cell-responsive delivery.;A systematic investigation of the impact of fluorescent modification of an established polymer carrier, polyethylenimine, indicated that the incorporation of hydrophobic labeling moieties weakened gene association and promoted increased heterogeneity in polyplex structure. Additionally, cellular investigations identified extracellular aggregation and reduced polyplex uptake as a result of this routinely employed structural modification. In the design of controllable methods for promoting gene association and release, a polymeric delivery platform with light-responsive groups along the cationic segment of the polymer backbone was designed to provide controlled nucleic acid assembly and a user-defined method for intracellular release. Additionally, light-mediated polyplex destabilization demonstrated significant utility in siRNA delivery as observed through efficient siRNA release and enhanced protein silencing compared to a commercial Lipofectamine lipoplex. The demonstrated benefits of this stimuli-responsive delivery method motivated a cell-responsive approach to control gene association and release. A novel polymer-peptide conjugate with functional capabilities for controlled release, cell targeting, and endosomal escape was developed to address the numerous functional requirements in the gene delivery pathway.;Ultimately, the clinical realization of gene therapies will rely on the development of methods to elucidate and control gene association and release along the delivery pathway. This will require the development of delivery vehicles whose physical and biochemical properties enable them to appropriately navigate the intra- and extracellular space. In total, this dissertation provides new evidence of the role of structural modifications (including fluorescent probes, targeting ligands and stimuli-responsive groups) for promoting efficient gene association and subsequent delivery and contributes to the growing body of literature to improve our fundamental understanding of the processing of non-viral carriers.