Expression of transgenes in peripheral blood mononuclear cells and their characterisation

Adoptivna celična terapija, ki temelji na uporabi gensko modificiranih limfocitov-T reprogramiranih za prepoznavo določenih tumor-specifičnih antigenov se je izkazala za uspešno metodo zdravljenja nekaterih vrst rakavih obolenj. Uspešnost je bila dokazana predvsem pri nekaterih terapijah B-celičnih levkemij, kot je naprimer akutna limfoblastna levkemija ter nekaterih ne-hodgkinovih limfomov. Pri tem so bili uporabljeni s himernim antigenskim receptorjem spremenjeni limfociti T (CAR-T) usmerjeni proti označevalcu CD19, značilnemu za limfocite B. V okviru magistrske naloge smo skušali s pomočjo elektrogenskega prenosa v humane enojedrne celice periferne krvi (PBMC) vstaviti zapis za anti-CD19 CAR. Določili smo optimalne pogoje za nespecifično aktivacijo in razmnoževanje gensko spremenjenih PBMC. Z uporabo plazmida z zapisom za GFP smo optimizirali pogoje elektroporacije. Celice smo okarakterizirali s pretočno citometrijo in fluorescenčno mikroskopijo. Optimalni pogoji transfekcije za sočasni vnos plazmidov z zapisom za CAR in encim transpozazo, ki je omogočila trajno spremembo genoma limfocitov T so bili pri enem pulzu napetosti 2250V in trajanju 20 ms. Prisotnost CAR smo dokazali neposredno s pretočno citometrijo in posredno z funkcijskim ELISA testom, kjer smo dokazovali prisotnost IL-2.Adoptive cell therapy based on the use of genetically modified T cells, programmed to identify specific tumor-specific antigens, has been proven to be a successful method of treating certain types of cancer. The efficacy has been proven primarily in the treatment of B-cell leukemias, such as, for example, acute lymphoblastic leukemia and some types of non-hodgkin`s lymphoma. In this case, the anti-CD19 CAR-T cells targeted against the B- lymphocyte antigen CD19 were used. In this thesis we attempted to insert anti-CD19 CAR molecular construct into human peripherial mononuclear blood cells by electroporation . First, we determined the optimum concentrations of reagents for non-specific activation and amplification of PBMC. Optimization of the electroporation conditions was carried out using the eGFP plasmid. The cells were characterized by flow cytometry and by fluorescence microscopy. Optimal transfection conditions for the simultaneous introduction of plasmids with CAR and the transposase enzyme, which allowed a permanent change in the genome of T lymphocytes, were at a pulse of 2250V and a duration of 20 ms. The presence of CAR was demonstrated in a direct manner with flow cytometry and in a indirect manner using the functional ELISA test for the IL-2 detection