Ligand-enhanced polymeric nanoparticles for targeted RNA-based gene therapy

Nucleic acids and their analogs have the capacity to augment and regulate gene expression in order to beget unparalleled levels of therapeutic specificity and potency for the treatment of chronic diseases such as cancer. The success of gene therapy is intimately aligned with the efficacy of associated delivery systems. In this dissertation, I develop a poly(lactic-co-glycolic acid) (PLGA) nanoparticle delivery system that can induce various forms of RNA-based gene regulation, including siRNA-mediated knockdown of gene expression, relief of microRNA-induced downregulation of tumor suppressor genes, and controlled alternative splicing of cancer oncogenes. PLGA nanoparticles (loaded with 142.7 +/- 23.8 pmol siRNA/mg nanoparticle) that were coated with molecules that impart the delivery enhancements of cell-specific targeting (i.e. folate) and improved cellular uptake (i.e. the cell-penetrating peptide, penetratin) were effective at achieving siRNA-induced gene knockdown in cultured cells and in a subcutaneous tumor model (60% relative to control tumors). To the best of my knowledge, this work presents the first nanoscale delivery systems to employ a combination of these exact ligands for gene therapy---importantly, the ligands were attached using a PEGylated phospholipid (DSPE-PEG) tethering strategy that allowed for a high density (∼200-2000 molecules per nanoparticle) of functional ligand display. My results suggest that when attached in tandem both folate and penetratin improved the delivery capabilities of nanoparticles via two modes: by facilitating cellular uptake and by enhancing the avidity between nanoparticles and target cells. This multifunctional nanoparticle platform was also utilized to deliver antisense nucleic acids that block microRNA activity and alter splicing. Toward this end, I found that chemically-enhanced charge-neutral nucleic acid analogs (specifically, morpholinos and peptide nucleic acids) could be encapsulated into PLGA nanoparticles (∼500 molecules per nanoparticle) without the experimental electrostatic manipulations that typically confound nucleic acid delivery. The anti-cancer efficacy of this nanoparticulate system was evaluated in cell culture and mouse tumor models. Both forms of nanoparticle-mediated gene therapy reduced cell viability---in part, through augmentation of apoptosis. In the treatment of epithelial cancer cells, altering the splicing pattern of Mcl-1 to produce its pro-apoptotic isoform had in an IC50 of ∼ 360nM, and blocking the activity of the microRNA, miR-155, had an IC50 of ∼80nM. Inhibition of miR-155 was further investigated in vivo. Certain microRNAs have been shown to have oncogenic functions, e.g. mir-155; further, some cancers are dependent on the expression of these microRNA oncogenes. I investigated the ability of nanoparticles that deliver microRNA-blocking peptide nucleic acids to inhibit miR-155 in a lymphoma tumor model of miR-155-addiction. Attenuation of this critical microRNA target produced anti-tumor effects (after ∼1 week, tumors showed a 5-fold decrease in growth after local delivery and a 2-fold decrease after systemic administration) that correlated with suppression of miR-155 levels, which confirmed that this nanoparticle delivery system is a promising therapeutic platform. This work is germane to the fields of drug delivery and cancer gene therapy, as it presents a nanoscale technology that has been tailored to effectively and safely achieve controlled expression of cancer-associated genes by evoking multiple methods of gene regulation.