Citotoksičnost plazmidne DNA, vnešene z elektrogenskim prenosom

Polak Schwery, Tjaša (author), Čemažar, Maja (mentor), Kamenšek, Urška (comentor)

Gensko zdravljenje temelji na uporabi nukleinskih kislin (DNA ali RNA), ki jih vnašamo v celice s terapevtskim namenom. Cilj terapevtskih pristopov je selektivno uničiti tumorske celice, brez vpliva na okoliške zdrave celice in tkivo. Zelo pomembno je celostno zdravljenje bolezni. Zaželeno je, da zdravljenje sproži posebno imunogeno obliko celične smrti, ki nato sproži imunski odziv proti rakavim celicam. Metoda, ki veliko obeta, je prenos genov z električnim tokom, kar imenujemo genski elektroprenos. Med aplikacijami je ena zanimivejših imunostimulacija. Plazmidno DNA z zapisom za različne imunostimulatorne molekule vnesemo v celice tumorjev ali normalnih tkiv. Tako postanejo tkiva proizvajalci teh molekul, ki lahko delujejo lokalno ali pa se izločajo v krvni obtok. Na ta način vplivamo na celice, ki so neposredno izpostavljene zdravljenju, in na oddaljene nezdravljene zasevke. Namen naše naloge je bil raziskati uspešnost vnosa plazmidne DNA z metodo elektroporacije v mišje melanomske celice B16.F10 in določiti citotoksičnost te metode. Plazmidno DNA smo uspešno vnesli v melanomske celice in dokazali citotoksičen učinek vnesenih plazmidov. Citotoksičen učinek DNA lahko izkoristimo predvsem v protokolih vakcinacije proti tumorjem, kot dopolnilno zdravljenje. Raziskali smo tudi, katero vrsto celične smrti povzročijo vneseni plazmidi. Ugotovili smo, da plazmidi, vneseni z metodo elektroporacije, vplivajo na umiranje celic v fazi pozne apoptoze oziroma nekroze. Na ta način pride do sproščanja antigenov iz melanomskih celic, kar in vivo spodbudi imunski odziv.

• Language: Slovenian
• Keywords: elektrogenski prenos, melanomske celice, plazmidi, pVAX1, pControl, pEGFP N1, kaspaza-1, kaspaza-3
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [T. Polak Schwery]
• Year: 2023
• PID: 20.500.12556/RUL-150748
• UDC: 616-006(043.2)
• COBISS.SI-ID: 169193475
• Publication date in RUL: 22.09.2023

Secondary language

• Language: English
• Title: CYTOTOXICITY OF PLASMID DNA DELIVERED BY GENE ELECTROTRANSFER

Abstract

Gene therapy is based on the use of nucleic acids (DNA or RNA) that are introduced into cells for therapeutic purposes. The aim of therapeutic approaches is to selectively destroy tumour cells without affecting surrounding healthy cells and tissue. The integrated treatment of the disease is very important. It is desirable that the treatment induces a specific immunogenic form of cell death, which then triggers an immune response against cancer cells. A method that shows great promise is gene transfer by means of an electric current, which is called electrogenic transfer. One of the more interesting applications is immunostimulation. Plasmid DNA with a transcript for various immunostimulatory molecules is introduced into tumour cells or normal tissues. The tissues thus become producers of these molecules, which can act locally or be secreted into the bloodstream. In this way, cells directly exposed to the treatment and distant untreated tumours are affected. The aim of our work was to investigate the transfection efficiency and cytotoxicity of plasmid DNA delivery by electroporation into murine melanoma cells B16.F10. We successfully delivered plasmid DNA into melanoma cells and demonstrated the cytotoxic effect of the delivered plasmids. The cytotoxic effect of DNA can be exploited mainly in vaccination protocols against tumours, as an adjunctive treatment. We have also investigated which type of cell death is induced by the introduced plasmids. We found that plasmids introduced by electroporation induce cell death. They are responsible for the formation of late apoptotic and necrotic cells. This leads to the release of antigens from melanoma cells, which stimulates an immune response in vivo.

• Keywords: gene electrotransfer, melanoma cells, plasmids, pVAX1, pControl, pEGFP N1, caspaza-1, caspaza-3