Targeting nucleic acids for pancreatic cancer: disease modeling and therapy

Abstract

Pancreatic cancer is responsible for nearly 40,000 deaths in the U.S. annually, with a dismal 5-year survival rate below 7%. The poor therapeutic outcomes reflect a paucity of new approaches targeting the genomic underpinnings of pancreatic ductal adenocarcinomas (PDAC, the vast majority of pancreatic cancers) as well as our inability to overcome the desmoplastic stromal barrier characteristic of PDAC. RNA interference through siRNA holds promise in targeting key mutations driving PDAC, such as oncogenic KRAS; however, a nucleic acid delivery vehicle that homes to PDAC and breaches the stroma does not yet exist. Noting that the novel cyclic peptide iRGD mediates tumor targeting and penetration through interactions with [alpha][upsilon][beta]3/5 integrins and neuropilin-1, we hypothesized that "tandem" peptides combining a cell-penetrating peptide and iRGD can complex with siRNA to form tumor-penetrating nanocomplexes (TPNs) effective in delivering siRNA to PDAC. Such a nanoscale carrier could provide a practical means of bridging our understanding of PDAC as a genetic disease to the clinic. Furthermore, the modular aspect of these self-assembled particles permits them to accommodate alternate cargoes or targeting domains, and we have proposed that tandem peptide complexes could be extended to applications outside of RNA interference, particularly for the delivery of components for CRISPR/Cas9-mediated gene editing. This delivery system could be applied to generate improved animal models of pancreatic cancer. In this work, we first designed, characterized, and optimized iRGD-based TPNs for RNAi in pancreatic cancer cells in vitro, showing robust knockdown of single and multiple targets. In order to stabilize these nanoparticles for systemic administration, we then devised and compared diverse, non-covalent materials for formulating TPNs with polyethylene glycol (PEG). The best material in this capacity, a peptide-PEG conjugate, reduced accumulation of TPNs in off-target organs and improved circulation kinetics, while preserving functional knockdown capacity. Incorporating this approach with the iRGD tandem peptides, we studied the translational potential of PEGylated iRGD TPNs to deliver siRNA to various models of pancreatic cancer and have begun therapeutic testing with siRNA targeting KRAS. Finally, we have adapted the tandem peptide platform to mediate delivery of CRISPR/Cas9 components. Particle configurations to deliver guide RNA alone, guide RNA with a DNA template for homology-directed repair, and guide RNA with Cas9 protein have all shown efficacy in gene editing in vitro, important steps toward creating sporadic mutations to model PDAC. Thus, we have established a versatile approach to the delivery of nucleic acids for studying and treating pancreatic cancer.