Developing peptide technologies to combat cancer and monogenetic disorders

Abstract

Epithelial cells form the secretory glands and line and cover nearly all organs and body surfaces, respectively. Epithelial cell dysfunction manifests in the vast majority of cancers as well as in common monogenetic disorders, such as cystic fibrosis (CF). Therapeutic strategies to treat such diseases are available, including targeted intracellular protein degradation and gene therapy; however, their targeted use in treating epithelial cell dysfunction is not currently possible. Recently a cell-penetrating peptide (CPP) named Xentry was discovered which has the potential to deliver therapeutics exclusively into adherent cells, and therefore into epithelial cells. Here it is shown that Xentry can be used to deliver proteolysis targeting chimeras (PROTACs) into cultured epithelial breast cancer cell lines to degrade the HER2 and MUC1 oncoproteins which have been associated with resistance to the breast cancer drug tamoxifen. PROTAC-mediated MUC1 degradation was associated with modest reductions in cancer cell viability, though there was only weak synergy with the tamoxifen metabolite 4-hydroxytamoxifen (4-OHT). Here it is also shown that Xentry appended with protamine, a nucleic acid-binding motif, can deliver mRNA and plasmid DNA into cultured epithelial cell lines. Moreover, mRNAs encoding proteins lost in CF and hereditary diffuse gastric cancer (HDGC), namely the cystic fibrosis transmembrane conductance regulator and E-cadherin, were delivered into cell lines derived from respective disease-specific tissues. Further, the addition of cell type-specific homing motifs to Xentry showed some ability to enhance the targeting of Xentry uptake. Notably, the co-application of lysosomotropic agents chloroquine or hydroxychloroquine, or nucleic acid-sensing toll-like receptor (TLR) antagonist E6446 during nucleic acid delivery improved gene expression levels approximately ten-fold, with protein expression observed in ~2-10% of cells. Together these results provide evidence that Xentry has the potential to target the delivery of therapeutics into epithelial cells, and it is anticipated that the present study may provide a starting point for further preclinical investigations. Finally, the ability of E6446 to enhance nucleic acid transfection here signifies E6446 as a novel agent for general use in pre-clinical studies in this context. Further, the findings suggest that E6446 may be uniquely suited to enhancing gene therapy delivery in general, and that this notion should be explored further.