Razvoj DNA cepiva proti metastazam melanoma z vdihavanjem na podlagi analize značilnosti transgene ekspresije gole pDNA in trojni kompleks v mišjem pljučnem tkivu

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Melanoma is the deadliest form of skin cancer. After reaching metastasis, survival rates drop, therefore early diagnosis and a certain lifestyle greatly reduce the risk.[1] Potential causes for melanoma are: family history, personal characteristic ( pale skin or bright eye color ), sun exsposure and atypical (asymmetric) mole.[2]

DNA vaccines

Protein antigens are encoded into DNA, during DNA vaccine production. Antigen-specific humoral and cell-mediated immunity are induced. A great number of melanoma antignes have been repoted, which is why DNA vaccines have potential in prevention and treatment of melanoma. DNA vaccines are produced because they are versatile, inexpensive, easy to manufacture, easy to store and safe to handle. Pulmonary administration route (inhalation) is used in this experiment, because the lungs contain highly responsive immune system.[3]

Materials and methods

Model pDNA (pCMV-Luc, pEGFP-C1, or pZsGreen) was used to evaluate transfection efficacy after inhalation in mice. Gene expression of naked pDNA and gene expression of the ternary complexes were compared. 1) Pulmonary administration of naked pCMV-Luc or the ternary complex containing the pCMV-Luc was performed to monitore gene expression. 6, 24 and 48 hours after administration liver, kidneys, spleen, heart and lungs were observed. Luciferase activity was measured and was indicated as relative light units (RLUs) per gram of tissue. 2)Naked pEGFP-C1 and ternary complexes containing pEGFP-C1 were administrated intrapulmonary to visualize the accumulation and gene expression of the complexes. 24 hours after the administration GFP expression levels in the lungs were observed using fluorescent microscopy. 3) Naked pZsGreen1-N1 and the ternary complex containing pZsGreen1-N1 were used too as a model pDNA. In addition DiD and Dil in 5% glusoce were administrated too. This step was done by multicolor deep imaging system using the tissue clearing reagent.[3]

Naked pDNA, ternary complexes containing pUb-M or ternanry complexes containing pEGFP-C1 were administrated three times biweekly. Luciferase activity and tumor volume was monitored, becuse it was belived that it will prevent melanoma metastasis. Th1 Cytokine (IFN-gama, TNF-alpha, and IL-6 ) secretion was monitored too.[3]


Luciferase activity in the lungs 6 and 24 h after administration of naked pCMV-Luc and the ternary complex was significantly higher than that in other organs. After the administration with naked pEGFP-C1 weak fluorescence was noticed. On the other hand, after the administration with the ternary complex containing pEGFP-C1 strong fluorescence was noticed. ZsGreen1 expression was noticed in alveolar macrophage and/or alveolar epithelial type II cells-rich sites in the lungs. All three, pEGFP-C1 complex, naked pUb-M and the Pub-M complex inhibited the lung metastasis, according to reduced luciferase activity and reduced tumor growth. The most effective of all was the pUb-M complex, and the least effective was pEGFP-C1 complex. Splenic cells immunized by the Pub-M complex secreted the largest amounts of IFN-gama, TNF-alpha, and IL-6.[3]


Pulmonary administration is shown to be an effective route for the delivery of DNA vaccines. Both, naked pDNA and the ternary complex of pDNA showed high gene expression in alveolar macrophage and/or alveolar epithelial type II cells-rich sites in lungs. Since the ternary complex showed stronger effect, it can be concluded that it can be efficiently taken up by alveolar macrophages than naked pDNA. The pUb-M complex is the most effective in the melanoma metastasis inhibition, as same as in the secretion of Th1 cytokines (IFN-gama and TNF-alpha).[3]


1.Davis LE, Shalin SC, Tackett AJ. Current state of melanoma diagnosis and treatment. Cancer Biol Ther. 2019;20(11):1366-1379. doi:10.1080/15384047.2019.1640032

2.Heistein JB, Acharya U. Malignant Melanoma. [Updated 2020 Dec 5]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-.

3.Kodama, Yukinobu and Nakashima, Mikiro and Nagahara, Tadayuki and Oyama et. al, Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues, Pharmaceutics, 12, 2020.

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