Development of mRNA vectors for the expression of molecular constructs in target cells

Razvoj široko uporabnih gensko spremenjenih imunskih celic je usmerjen v zdravljenje malignih bolezni, med katere spadajo tudi B-celični raki. V ta namen se predvsem limfocite T, ki spadajo med celice, ki jih je težje transformirati, modificira, da izražajo himerni antigenski receptor ali CAR. Ena od metod, ki kaže uspeh pri transformacijah teh celic, je elektrogenski transfer z elektroporacijo, pri čemer do sedaj uporabljene vektorje, kot sta pDNA in retrovirusni vektorji, zamenjuje mRNA, ki je manj toksična. V magistrskem delu smo skušali doseči spreminjanje imunskih celic s prenosom mRNA z elektroporacijo. Najprej smo oba plazmida (s CAR in GFP) pripravili za prepis s PCR in tako linearizirali in pomnožili želen odsek. S komercialnim kompletom smo DNA in vitro prepisali v mRNA in jo v procesu opremili z ustreznimi konstrukti za stabilnost (5\u27kapa in 3\u27 poli(A) rep) ter jo očistili. Izbrane celice smo elektroporirali z mikroporatorjem Neon Transfection System v kombinaciji dveh različnih pogojev in pri petih različnih količinah mRNA. Ugotovili smo, da je z elektroporacijo mRNA v celicah Jurkat možno izraziti izbrane molekulske konstrukte, vendar nismo dosegli pričakovane ravni uspešnosti izražanja. Sklepamo, da je v splošnem za doseg visoke uspešnosti transformacije in preživetja elektroporiranih celic pred eksperimentom nujna dodatna optimizacija elektroporacije kljub uporabi pogojev, navedenih v literaturi. Potrebno bi bilo optimizirati pripravo in kakovost mRNA vektorjev.The development of widely used genetically modified immune cells is aimed at the treatment of malignant diseases including B cell cancers. For these purposes, T lymphocytes, which are among hard-to-transfect cells, are modified to express a chimeric antigen receptor or CAR. One of the methods showing success in transfecting these cells is electrogenic transfer by electroporation, whereby the vectors used so far, such as pDNA and retroviral vectors, are replaced by mRNA, which is less toxic. In the master\u27s thesis, we tried to achieve the modification of immune cells with mRNA transfer by electroporation. Firstly, both plasmids (with CAR and GFP) were prepared for transcription by PCR to linearize and amplify the desired fragment. Using a commercial kit, DNA was transcribed in vitro and equipped with appropriate stability constructs (5\u27cap and 3\u27poly(A) tail) and purified. Selected cells were electroporated with the Neon Transfection System microporator in a combination of two different conditions and five different mRNA quantities. We found that it is possible to express selected molecular constructs by electroporation of mRNA in Jurkat cells, but we did not achieve the expected level of expression success. We conclude that in in general, to achieve high transformation efficiency and survival of electroporated cells before the experiment, additional optimization of electroporation is necessary, despite the use of conditions listed in the literature. The preparation and quality of mRNA vectors should be optimized