Optimization of Polymeric Micelles as a Delivery System for CRISPR/Cas9 Ribonucleoprotein

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immune system in bacteria that has been harnessed to perform precise genetic modifications in mammalian cells. Among CRISPR-associated (Cas) proteins, Cas9 has emerged as a transformative tool in gene editing due to its simplicity, high specificity, and versatility compared to the other Cas proteins and gene-editing technologies. Despite its potential to cure genetic disorders, the clinical application of Cas9 has been hindered by challenges such as immunogenicity, off-target effects, and low delivery efficiency. To address these issues, this dissertation explores using polymeric nanoparticle systems to encapsulate and protect Cas9 and facilitate targeted delivery while minimizing toxicity and enhancing efficiency. In this dissertation, I evaluate the ability of different block-copolymers to create polymeric micelles as a delivery platform for Cas9. We first assessed the cationic polymer, poly-L-lysine, but found the highly positive charge created difficulties in micelle synthesis, leading me to change my approach. To improve my results, I designed a poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA) polymeric micelle system capable of encapsulating and delivering Cas9 ribonucleoprotein (RNP). The micelles formed at an average diameter of 52.24 ± 1.12 nm with a ζ-potential of -16.4 ± 7.61 mV. To improve the nanocarrier, I introduced TAT, a cell-penetrating peptide, to the surface of our nanoparticle. I discovered that increasing amounts of TAT both increase the micelles' cellular uptake and extend protein release to near zero order. Finally, Cas9 RNP-loaded PEG-b-PLA micelles did lead to some gene knockout when incubated with a HEK293-GFP-expressing cell line. The results presented in this dissertation demonstrate that PEG-b-PLA micelles are capable of effectively delivering Cas9 RNP to a target gene, inducing knockout, and providing a promising nanocarrier system for potential therapeutic applications for genetic disorders. In the future, this technology will be tested as a therapeutic for GM1 gangliosidosis. In these efforts, we have explored the blood-brain barrier (BBB) bypassing capability of polymeric nanoparticles when bound to Apolipoprotein E. Combined, these two thrusts of my research could lead to paradigm-shifting approaches to cure childhood neurodegenerative conditions through gene knockout and eventual gene modification.