Abstract The incidence of human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) is rising and while treatment options are available, they remain limited primarily to surgical excision, radiation therapy, and chemotherapy which often result in long term morbidity. RNA interference (RNAi) appears to be a promising therapeutic tool for the treatment of many diseases, including HPV+ related cancers, through targeting viral oncogenes, E6 and E7, with small interfering RNAs (siRNA). The expression of E6 and E7 viral oncoproteins inhibits the p53 and pRb tumor suppressors, respectively, resulting in uncontrolled cellular proliferation. Knockdown of E6 and E7 in vitro has been shown to induce apoptosis in both HPV+ cervical cell carcinoma and HPV+ head and neck squamous cell carcinoma cell lines, indicating that E6 and E7 are viable therapeutic targets for RNAi therapy. However, the therapeutic application of RNAi requires a delivery platform that can overcome numerous challenges typically associated with this form of therapy. Peptide carriers show great promise as siRNA carriers based on the diversity of their physiochemical properties and functions. Recently, we demonstrated that a novel peptide carrier we designed, termed RD3AD, enhanced the intracellular delivery and availability of therapeutic, chemically synthesized siRNAs in oral cancer cells, in vitro, to levels that were ~4x that of Lipofectamine 3000, which some would consider to be the “gold standard” positive control for in vitro delivery of nucleic acids. It therefore stands to reason that RD3AD could be an effective delivery vehicle for siRNAs designed to target the E6 and E7 viral oncogenes (siE6 and siE7) in HNSCC cells. RD3AD-siRNA complexes were also observed to localize to cellular projections identified as filopodia. These observations were indicative of patterns previously reported for cellular uptake of viruses, bacteria, activated receptors, lipo/polyplexes, and exosomes, which utilized filopodia to undergo retrograde transport toward the cell, resulting in significant enhancements of uptake. It is therefore possible, given the observations with RD3AD, that this peptide is exploiting similar mechanisms. Thus, our overarching hypothesis is that RD3AD-siE6/7 (siE6 or siE7) complexes enter HPV+ oral cancer cells after binding to cellular filopodia and silence viral oncogenic E6 or E7 mRNA, in vitro. We will test our hypothesis through the following specific aims. Aim 1: Identify the RD3AD receptor on filopodia and exploit this receptor for cell specific targeting. Aim 2: Determine the mechanism(s) of RD3AD-siRNA complex uptake. In aggregate, this proposal will establish a molecular therapeutic option for HPV+ HNSCCs, while describing a novel mechanism of cell-specific delivery and uptake by a peptide nanocarrier. The results from this study will contribute significantly to a solution for a critical unmet clinical need and will be particularly impactful on ...