We developed a non-viral vector, a combination of HIV-1 Tat peptide modified with histidine and cysteine (mTat) and polyethylenimine, jetPEI (PEI), displaying the high efficiency of plasmid DNA transfection with little toxicity. Since the highest efficiency of INTERFERin (INT), a cationic amphiphilic lipid-based reagent, for small interfering RNA (siRNA) transfection among six commercial reagents was shown, we hypothesized that combining mTat/PEI with INT would improve transfection efficiency of siRNA delivery. To elucidate the efficacy of the hybrid vector for siRNA silencing, β-actin expression was measured after siRNA β-actin was transfected with mTat/PEI/INT or other vectors in HSC-3 human oral squamous carcinoma cells. mTat/PEI/INT/siRNA produced significant improvement in transfection efficiency with little cytotoxicity compared to other vectors and achieved ≈ 100% knockdown of β-actin expression compared to non-treated cells. The electric charge of mTat/PEI/INT/siRNA was significantly higher than INT/siRNA. The particle size of mTat/PEI/INT/siRNA was significantly smaller than INT/siRNA. Filipin III and β-cyclodextrin, an inhibitor of caveolae-mediated endocytosis, significantly inhibited mTat/PEI/INT/siRNA transfection, while chlorpromazine, an inhibitor of clathrin-mediated endocytosis, did not inhibit mTat/PEI/INT/siRNA transfection. Furthermore, the transfection efficiency of mTat/PEI/INT at 4 °C was significantly lower than 37 °C. These findings demonstrated the feasibility of using mTat/PEI/INT as a potentially attractive non-viral vector for siRNA delivery.

Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR₂) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR₂. We report here a role for cathepsin S in PAR₂-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR₂ in Na_v1.8-positive neurons (Par₂Na_v1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR₂ on neurons.

Oral cancer patients suffer pain at the site of the cancer. Calcitonin gene related polypeptide (CGRP), a neuropeptide expressed by a subset of primary afferent neurons, promotes oral cancer growth. CGRP also mediates trigeminal pain (migraine) and neurogenic inflammation. The contribution of CGRP to oral cancer pain is investigated in the present study. The findings demonstrate that CGRP-immunoreactive (-ir) neurons and neurites innervate orthotopic oral cancer xenograft tumors in mice. Cancer increases anterograde transport of CGRP in axons innervating the tumor, supporting neurogenic secretion as the source of CGRP in the oral cancer microenvironment. CGRP antagonism reverses oral cancer nociception in preclinical oral cancer pain models. Single-cell RNA-sequencing is used to identify cell types in the cancer microenvironment expressing the CGRP receptor components, receptor activity modifying protein 1 Ramp1 and calcitonin receptor like receptor (CLR, encoded by Calcrl). Ramp1 and Calcrl transcripts are detected in cells expressing marker genes for Schwann cells, endothelial cells, fibroblasts and immune cells. Ramp1 and Calcrl transcripts are more frequently detected in cells expressing fibroblast and immune cell markers. This work identifies CGRP as mediator of oral cancer pain and suggests the antagonism of CGRP to alleviate oral cancer pain.

Developmental endothelial locus-1 (DEL-1) has traditionally been characterized within the scientific community as having anti-inflammatory properties with potential inhibitory effects on osteoclast formation. Our investigation challenges this paradigm by examining Del-1 expression in RAW264.7 cells and bone marrow-derived macrophages (BMMs) during osteoclastogenesis, as well as its functional impact on osteoclast development and activity. Our experimental findings revealed that Del-1 mRNA levels were markedly elevated in cells stimulated by the receptor activator of the nuclear factor κB ligand compared to unstimulated precursors. When cultured with varying concentrations of recombinant DEL-1, osteoclast differentiation increased in a dose-dependent manner. Furthermore, BMMs isolated from ovariectomized mice exhibited significantly higher Del-1 mRNA expression than those from control animals. To confirm DEL-1’s role, we employed RNA interference techniques, demonstrating that DEL-1 silencing in RAW264.7 cells substantially reduced osteoclast formation. These results suggest that DEL-1 plays a previously unrecognized role in promoting osteoclastogenesis and may contribute to bone metabolism imbalances in conditions like osteoporosis, highlighting its complex role in skeletal homeostasis and its potential as a therapeutic target.

Gene therapy has emerged as a promising therapeutic approach across various oral diseases. This review examines current applications and future prospects of gene therapy in dentistry, focusing on five key areas: oral cancer, cancer-related pain, xerostomia (dry mouth), dental caries, and periodontal disease. Recent advances in viral and non-viral vectors have enabled more efficient gene delivery systems, with particular success in cancer pain management through µ-opioid receptor gene transfer and xerostomia treatment using aquaporin-1 gene therapy. For periodontal applications, gene therapy strategies include both immunomodulation and tissue regeneration approaches using growth factors like platelet-derived growth factor and bone morphogenetic proteins. While significant progress has been made, particularly in treating radiation-induced xerostomia and oral cancer pain, challenges remain in vector optimization and delivery methods. Clinical trials, predominantly in Phase I, indicate both the potential and current limitations of gene therapy in oral healthcare. This review synthesizes current evidence and outlines future directions for gene therapy applications in oral medicine and dentistry.