20.500.12556/RUL-124827 Priprava molekulskih konstruktov himernih antigenskih receptorjev za prenos v limfocite T Preparation of chimeric antigen receptors’ molecular constructs for their transfer into T lymphocytes Adoptivna celična terapija z limfociti z izraženim himernim antigenskim receptorjem (CAR T) je od njene odobritve za zdravljenje akutne limfoblastne levkemije (ALL) in difuznega velikoceličnega B-limfoma (DLBCL) predmet številnih raziskav, ki so usmerjene v možnosti razširitve uporabnosti CAR T še na zdravljenje drugih rakavih bolezni. V okviru magistrske naloge smo želeli transpozonski plazmid, ki nosi zaporedje za himerni antigenski receptor, z metodami molekulskega kloniranja preurediti tako, da himerni antigenski receptor ne bi prepoznaval površinskih molekul CD19, pač pa molekule CD20. Tako bi limfociti T, v katere bi ta novi plazmid elektroporirali, s svojim citotoksičnim delovanjem še vedno prepoznavali predvsem maligne celice pri DLCBL in ALL, vendar pa bi bila njihova učinkovitost zaznave, zaradi večje gostote molekul CD20 na teh celicah, bolj učinkovita. Pri našem delu smo uporabljali moderno metodo sestavljanje po Gibsonu (ang. Gibson assembly). Ugotovili smo, da sta bila plazmida, ki smo ju izbrali zaradi visokih deležev citozina in gvanina v zaporedjih, ključnih za kloniranje in posledično tvorbo lasnic, neprimerna. Na osnovi naših izsledkov predlagamo alternativne pristope k tovrstnem kloniranju. Adoptive cell therapy with lymphoctes expressing chimeric antigen receptor (CAR) has been a subject of great interest since the approval of CAR-T therapy for treating acute lymphoblast leukaemia (ALL) and diffuse large B cell lymphoma (DLBCL), especially due to its potential of expansion to treat other malignancies. The intention of this masters thesis was to rearrange a plasmid, bearing a sequence for a chimeric antigen receptor with methods of molecular cloning in such a way, that the receptor would stop recognising CD19 antigen, but would instead be directed to CD20 molecules. After a succesful rearrangement, the lymphocites with an electroporated plasmid bearing such a sequence would still be mostly recognising ALL and DLCBL cells, but their efficiency of recognition would be much greater due to a higher density of CD20 molecules expressed on the surface of these malignant cells. For our work we used a modern technique of molecular cloning, the Gibson assembly. We concluded that the plasmids used for cloning were unsuitable for our work due to a large percentange of cytosine and guanine nucleotides present in their key sequences. Based on our conclusions we suggest alternative solutions for cloning. Himerni antigenski receptor sestavljanje po Gibsonu molekulsko kloniranje variabilni fragment enojne verige protitelesa scFvCD20 transpozon Chimeric antigen receptor Gibson assemby molecular cloning single chain variable fragment scFvCD20 transposon true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2021-02-21 07:45:03 2021-02-21 07:45:15 2022-09-04 04:01:43 0000-00-00 00:00:00 2021 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 79881 1648.pdf 1648.pdf 1 5D8DB4DB930BD7D76966443B99EA325A bc3df5a250b4555648eada5632a6785b7aee368d95e7099662af0d73e606a75b a23fcdd8-a1bb-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=140445 Fakulteta za farmacijo 0 0 0