Biodegradable Polymer-Based Nanoparticle for mRNA Gene Therapy Delivery to the Brain

The five-year survival rate of high-grade brain cancer patients remain very low at 5% despite many years of research. The lack of effective treatments for brain tumors are mostly contributed to the complicated structure of brain tissue that renders drug delivery a challenging task. Chapter 1 provides an overview on the current status of brain tumor treatments and the challenges associated with them. Many efforts have been put into the design of various type of drug delivery systems, including viral, liposomal, and polymeric materials. However, many limitations such as immunogenicity, instability, and cytotoxicity still exist. Recently, our group has published a formulation for PEGylated poly(β-amino ester) or PBAE-based nanoparticles that exhibited widespread gene delivery in brain tumors. Here, we would like to adapt the formulation and investigate its ability in compacting mRNA, as mRNA possesses several advantages over DNA, including cytosolic delivery and the lack of danger for insertional mutagenesis. Several structural differences exist between mRNA and DNA, and therefore an optimization of particle formulation is necessary. Chapter 2 discusses the optimization process of formulating PEG-PBAE-based mRNA particles, which involves the addition of tripolyphosphate (TPP). I demonstrated that the addition of TPP at a mRNA to TPP mass ratio of 1:2 led to great improvements in the polydispersity index, surface charge, and stability of the nanoparticles. With the optimized particle formulation, I conducted in vitro and ex vivo experiments to compare the transfection efficiency of the PEG-PBAE mRNA particles and the PEG-PBAE DNA particles. Two types of reporter genes were used: enhanced green fluorescent protein (EGFP) and luciferase (Fluc), and two types of cell lines were tested: 9L and GL261. The experiments generated consistent results, showing that the mRNA-carrying particles had higher cell-uptake and transgene expression compared to the DNA-carrying particles. Multiple particles tracking ex vivo experiments confirmed that both types of particles exhibited similar diffusion in brain tissue, which was expected due to their similarity in size and charge. The PEG-PBAE TPP mRNA particles showed promising data in vitro that provide a strong foundation for future in vivo experiments, including distribution and transfection in brain tumors and survival studies with mRNA gene therapy. I expect the mRNA particles to demonstrate two main advantages: 1) higher cell uptake leads to higher efficacy and 2) higher transfection efficiency of mRNA particles allows for lower dosages to be administered and thereby minimizing potential cytotoxicity