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The epidermal growth factor receptor (EGFR) is upregulated in majority of head and neck squamous cell carcinoma (HNSCC) tumors. In preclinical models inhibition of EGFR using several strategies results in tumor growth inhibition. However, clinical trials using agents that inhibit EGFR activation and signaling demonstrated limited antitumor efficacy. Another approach involving downmodulation of EGFR protein levels using antisense DNA sequences has been reported to be highly effective in inhibiting HNSCC growth in preclinical models. However, DNA based agents are prone to nuclease degradation necessitating intratumoral delivery of the agent. To facilitate systemic delivery of EGFR antisense oligonucleotides, we modified the DNA backbone to a pseudo-peptide moiety containing guanidinium groups called guanidinium-peptide nucleic acids (GPNA). This novel class of molecules binds the target DNA or RNA in a highly sequence specific manner, is resistant to nulceases and proteases, has a strong affinity for complementary DNA and RNA sequences. Experimental procedures: We designed a GPNA antisense oligomer complementary to 16 bases on the EGFR mRNA (EGFRAS GPNA). Transfection efficiency of the GPNA oligomer was determined using GFP expressing vectors and confocal imaging. Localization of the EGFRAS GPNA within the endoplasmic reticulum (ER) was determined using an ER specific dye from Molecular Probes. Immunoblotting and RT-PCR determinations were carried out on EGFR levels in HNSCC cells treated with EGFRAS GPNA. In vitro antitumor effects were assessed using the Cell Titre Glo assay. Antitumor effects of EGFRAS GPNA (5 mg/kg daily injections) were examined on local intratumoral and systemic intraperitoneal delivery in HNSCC subcutaneous tumor bearing athymic mice. In order to compare the antitumor efficacy of the EGFRAS GPNA to EGFR inhibitors currently in clinical use, we treated HNSCC xenograft tumor bearing mice with either EGFRAS GPNA (5 mg/kg body weight daily), the scrambled control GPNA (5 mg/kg body weight daily), cetuximab (0.8 mg/mouse thrice a week) or erlotinib (90 mg/kg daily via oral gavage). Results: Our data demonstrate that EGFRAS GPNA can be successfully introduced into HNSCC cells in the presence of serum containing medium with almost 100 % transfection efficiency. Further, EGFRAS GPNA treatment results in anti-tumor effects in vitro by down modulating EGFR mRNA levels. Our data demonstrate that EGFRAS GPNA has similar antitumor effects on HNSCC xenografts when delivered systemically via intraperitoneal injections or locally via intratumoral injections. A scrambled control GPNA had no antitumor effects in vivo demonstrating that EGFRAS GPNA specifically inhibited HNSCC growth. Our data demonstrate that EGFRAS GPNA has comparable antitumor effects to FDA approved EGFR inhibitors. Further, EGFRAS GPNA downmodulated EGFR mRNA levels in the HNSCC xenografts. Conclusions: EGFRAS GPNA has high cellular uptake and specifically downmodulates EGFR levels in HNSCC cells. When delivered systemically, EGFRAS GPNA has antitumor efficacy comparable to that of erlotinib and cetuximab. A novel strategy to target EGFR when systemically administered has immense potential to improve current therapeutic approaches for head and neck cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C175.