The use of exogenous nucleic acids has great potential for therapeutic applications in numerous diseases,
including cancer. However, the delivery of nucleic acids is challenging, as they face numerous biological barriers,
and thus require sophisticated delivery platform technologies. Despite the development of many promising non-
viral carrier technologies, such as lipids, polymers, and peptides, achieving a clearer understanding of the
mechanisms they employ to facilitate cell entry of nucleic acid-based drug cargos is, in-part, critical to advancing
their clinical translation. As such, in our quest to further the development of a cell-penetrating peptide (CPP)
carrier design for siRNA-based human oral and oropharyngeal cancer (OOC) therapy, we recently reported that
a particular peptide variant, RD3AD, which exhibited enhanced siRNA uptake and gene silencing compared to
the parent CPP, was found to direct siRNAs to specific cell-surface protrusions, identified as filopodia.
Intriguingly, filopodia are highly dynamic, elongated, and thin cellular processes that have been reported to
facilitate the highly efficient cellular entry of viruses, bacteria, exosomes, and other biological
macromolecules/complexes. Moreover, because filopodia function as sensory antennae in probing the cellular
environment, which is important for cell motility through complex 3D microenvironments, they contain cell-surface
receptors, such as integrins, that can interact with and interpret extracellular cues. Interestingly, integrins have
been reported to assemble along filopodia in similar localization patterns as RD3AD-siRNA complexes, thus
implying that the RD3AD peptide carrier potentially targets integrins on filopodia as a means of cell entry. With
filopodia having also been implicated in driving cancer cell migration and invasion, with their densities and lengths
correlated with cancer progression and increased invasiveness, and increased levels of integrins also being
associated with OOC and metastasis, the goal of the current proposal is to elucidate the mechanisms underlying
filopodia-mediated intracellular uptake of the RD3AD-siRNA complex, as well as determine its ability to target
and deliver siRNAs to migrating/invading cancer cells. To accomplish this goal, we will use biochemical and
molecular biology approaches to: (1) uncover the mechanisms of filopodia-directed cell entry of the RD3AD-
siRNA complex in oral cancer cells; and (2) demonstrate the ability of RD3AD to target and deliver complexed
siRNAs to migrating/invading cancer cells and 3D tumor spheroids in vitro, as well as malignant tumors in vivo.
The outcomes of the proposed high-risk, high-reward research are expected to uncover the mechanisms
governing the intracellular delivery of the RD3AD-siRNA complex via its associations with filopodia and/or
integrins, as well as its propensity to target migrating/invading cancer cells. The significance being that the
knowledge gained will aid in future and more informed development of a CPP-based molecular targeted nucleic
acid therapy for human OOC intervention.
