Developing Endosomolytic Polymer Porous Silicon Nanocomposites for Delivery of Diverse Gene Therapies

Abstract

With recent advances in biomedical research, a focus has been placed on the engineering and optimization of diverse genetic therapies, which can alleviate diseased states that are otherwise insurmountable. Among the various classes and methods, RNA interference (RNAi) and CRISPR/Cas9 mediated gene therapy appear promising and offer possibilities for temporarily silencing genes through, and removal/replacement of malfunctioning genetic code, respectively. At present, specific and efficient delivery of these types of genetic therapies are the two main hurdles preventing their realization as practical therapeutic intervention at the bedside. In this body of work I will present to you a pH-responsive, porous silicon based nanoparticle (PSNP) polymer composites system that is capable of loading, delivering, and potentiating efficient knock-down/knock-out using two different classes of genetic therapies. The first class uses pH responsive polymer coated PSNPs to deliver a non-ionic, hydrophobic peptide nucleic acid (PNA) therapy that targets microRNA 122, a small non-coding RNA which in some diseased states of the liver, is overexpressed and impairs liver homeostasis via and RNAi meditated mechanism. The large internal surface are of PSNPs and its anionic surface allows for high loading efficiency of the PNA and electrostatic deposition of our lab’s gold standard polymer PEG-DB, Respectively. Secondly, we will demonstrate that with simple surface modifications, we can tune the PSNPs to delivery anionic Cas9 ribonucleoprotein (Cas9 RNP) targeted to lox-p sites in the genomic loci of cells and animals. Efficient delivery and genomic modification is characterized by a fluorescence shift from orange to green in the cells and can be cytometrically and visually quantified, while the animal model is a fluorescent “turn-on” method. Additionally, we will show that we can target endogenous MMP13 loci to induce gene knockout via frameshift generation by non-homologous end joining DNA repair mechanisms.