<p>The success of gene medicine ultimately depends on the efficient intracellular delivery and sustained expression of nucleic acid therapeutics, yet nonviral gene delivery performed with cationic polymer carriers has been chronically hindered by the slow release of nucleic acid payloads at their targets, as well as the transient nature of exogenous transgene expression. Polymer-nucleic acid nanocomplexes made with passive gene carriers using traditional bulk methods have proven inadequate for most translational applications. The objective of this work is to improve nonviral gene delivery through the selection, formulation, and application of improved nanoparticles. </p><p> After screening a number of number of cationic polymer delivery systems ranging from natural to synthetic, high molecular weight to low, binary and ternary, we identified a bioreducible linear poly(amido amine) able to give sustained, robust expression of both DNA and RNA through serial dosing. We next turned our attention to the process of nanocomplex assembly. Traditional assembly via bulk mixing is poorly controlled, and the poor quality of these nanocomplexes is a significant impediment to both the establishment of robust structure-function relationships and the advancement of nonviral gene delivery. So, we developed an emulsion-based microfluidic nanomanufacturing platform to better control the self-assembly process, and thus the physical properties of nanocomplexes. Confined mixing within picoliter droplets generates self-assembled nanocomplexes that are more uniform and more effective. This microfluidic nanomanufacturing approach possesses broad utility in the production of polymer-nucleic acid nanocomplexes; we demonstrated that its benefits extend to multiple gene carriers, a range of nucleic acid payloads, and translationally relevant cell types. Then, we applied the improved nanomanufactured particles to begin to address an unmet clinical need, namely the lack of a safe and ethical source of cells to treat neurodegenerative diseases. Nonviral cellular reprogramming strategies eliminate the integration of viral DNA sequences and represent a potentially safer alternative to viral transdifferentiation methods to generate therapeutic cells. Using nanomanufactured polymer-nucleic acid nanocomplexes, we improved the efficiency of the nonviral cellular reprogramming of fibroblasts directly to functional induced neuronal cells. </p><p> Nonviral gene therapy will continue to demand more sophisticated delivery systems to continue to progress. Microfluidic nanomanufacturing represents a reproducible and scalable platform to synthesize more uniform and effective nanocomplexes that not only improves their functional performance, but may also help establish clearer structure-function relationships that will inform future gene carrier design. Complementing the innovative chemical and biological approaches to create multifunctional nanoparticles, this study indicates that microfluidic nanomanufacturing can serve as a parallel physical strategy to both optimize the properties of polymer-nucleic acid nanocomplexes and improve their performance in applications with important clinical implications.</p> / Dissertation