Systematic Improvement of Quantification and Formulation of Synthetic Protein Nanoparticles for Gene Delivery

Abstract

Gene therapy is a promising field for the generation of new and more effective therapies, but its application is stymied by the lack of safe and efficient delivery vehicles. Furthermore, gene delivery system optimization is hindered by inadequate methods for payload quantification within nanoparticles. The work presented in this thesis establishes methods to formulate and quantify plasmid DNA (pDNA) loading in protein carriers that drive maximal gene expression towards two aims: Aim 1: Engineering and characterization of plasmid DNA payloads in synthetic protein nanoparticles and Aim 2: Structure function relationships related to protein nanoparticle composition and reporter gene expression. The pDNA loading of synthetic proteins nanoparticles (SPNPs) has been fully characterized, and a method for probing the effect of composition on biological outputs has been established. With clinical translation as the ultimate goal, the fundamental discoveries presented herein enable the further development and in vivo application of SPNP vectors. Here, the characterization and formulation of SPNPs are studied and optimized for their effect on reporter gene expression in a model cell line. The efficiency of SPNP production is important to systems with precious or difficult to manufacture payloads. This thesis also examines processing methods for SPNPs, and the mass loss and repeatability of the current centrifugal filtration method is compared to a new syringe filtration method. In Chapter 2, we developed a strategy for fluorescent plasmid backbone labeling (PBL) that enables the characterization of SPNPs loaded with pDNA with lower measurement bias than the conventional methods tested. This allowed us to determine the loading percentage as well as model the maximum loading of pDNA in SPNPs. We next combined pDNA and particle size quantifications to determine the distribution of pDNA within a SPNP population; this allowed us to examine SPNP loading with a granularity not widely reported in literature. An in-depth protocol for the PBL strategy is presented in Chapter 3 to assist future researchers in adapting the PBL strategy for various systems and nucleotides. In Chapter 4, the structure function relationship between SPNP composition and reporter gene expression was investigated by varying three key parameters: polyethylenimine (PEI) amount, SV40 content, and pDNA loading percentage. A rational design strategy was developed to interrogate the effect of changing PEI, SV40 and pDNA on reporter gene expression (GFP %). We found that PEI content positively correlated with GFP % (correlation coefficient = 0.91), while no other SPNP component (SV40) or biological output (viability, uptake) did. The work presented here establishes strategies for quantifying and formulating SPNPs in vitro for eventual application in organismal studies and human health. This thesis presents a platform for quantification and design of gene delivery vectors that can be extended to various nucleotides, genes, and target applications.