Oral cancer causes more prevalent and severe pain than any other cancer. Oral cancer patients suffer from severe, chronic, mechanically-induced pain; talking and eating are profoundly disrupted. Opioids are plagued with side effects and ineffective as tolerance develops; there is no satisfactory treatment for oral cancer pain. Our long-term goal is to develop cancer-targeting non-viral gene therapy to disrupt nociceptive signaling in the cancer microenvironment with minimal off-target effects and translate our approach to patients. We created two non-viral vectors with excellent transfection efficiency and no cytotoxicity: a cell-permeable peptide com- bined with a cationic lipid for DNA; and, a lipopolymer for RNA. Non-viral transfection with OPRM1 (μ-opioid receptor gene, MOR) DNA led to re-expression of MOR and partial attenuation of nociception (i.e., pain) in cancer mouse models. Protease-activated receptor-2 (PAR2) is elevated in the primary afferent neurons that innervate the cancer and drive pain. Knockdown of F2rl1 (PAR2 gene) partially attenuated nociception. While both approaches partially reduce cancer pain, our goal is elimination. Complete analgesia poses a challenge; there are multiple and redundant pain pathways, and genomic heterogeneity in oral cancer produces variations in the pathways. As a strategy to obstruct these multiple and varied pathways, we posit that a combination of OPRM1 re-expression and F2RL1 downregulation in the cancer could eliminate pain. The central hypothesis is that a non-viral vector approach to gain OPRM1 re-expression and F2RL1 downregulation within the oral can- cer will eliminate cancer pain and fully restore function. The rationale for this project is that demonstration of therapeutic efficacy of co-delivery of OPRM1 DNA and F2RL1 RNAi or CRISPR/Cas9 will yield a scientifically rigorous framework for development of non-opioid therapies that can be translated to patients. The central hy- pothesis will be tested in two specific aims: 1) Determine the feasibility and efficacy of ex vivo transfection (i.e., transfection of oral cancer cells with dual genes prior to generation of a xenograft mouse model); and, in vivo transfection (i.e., direct inoculation of genes into the oral cancer in the chemical carcinogen mouse model) with OPRM1 DNA and/or F2RL1 RNAi or CRISPR/Cas9 with the non-viral vectors to eliminate cancer-induced pain in mouse models; 2) Measure pain in oral cancer patients, analyze OPRM1 and F2RL1 expression in their can- cers relative to matched normal tissue, and analyze the correlation between dysregulation of these genes and patients’ pain. The results will give us data regarding the proportion of oral cancer patients who might benefit from co-delivery of DNA and RNA and set the stage for a clinical trial. The research proposed in this applica- tion is innovative because co-delivery of DNA and RNA into a cancer with non-viral vectors for the manage- ment of pain has not been done. The proposed research is significant because it is expected to prove strong scientific justification for the continued development and future clinical trials of novel dual gene therapy. Ulti- mately, such knowledge might lead to development of novel non-opioid therapies for cancer pain.

Project Narrative The proposed research is relevant to public health because it focuses on developing a novel and alternative strategy using non-viral dual gene (DNA and RNA) delivery methodology to treat oral cancer pain. Once such strategies have been developed, there is the potential for a significant advance in anti-pain therapy for oral cancer patients suffering severe debilitating pain. Thus, the proposed research is relevant to the part of the NIH’s mission that pertains to reducing illness and disability.