20.500.12556/RUL-138993 Načrtovanje inhibitornih domen za uravnavanje celične signalizacije CAR-T Design of inhibitory domains for CAR-T cell regulation Celična terapija s celicami CAR-T je povzročila izredno učinkovite ter v nekaterih primerih trajne klinične rezultate pri zdravljenju raka. Vseeno pa pri terapiji s celicami CAR-T obstajajo omejitve, vključno s smrtno nevarnim sindromom sproščanja citokinov in nevrotoksičnostjo. Za lajšanje simptomov neželenih učinkov se večinoma uporabljajo zdravilne učinkovine, a je za učinkovito in varno terapijo bolj smiselno uvesti mehanizme, ki omogočajo uravnavanje neželenih učinkov na ravni celične signalizacije CAR-T. V okviru magistrskega dela smo za uravnavanje celične signalizacije CAR-T s pristopi sintezne biologije načrtovali šest različnih inhibitornih domen, ki temeljijo na naravnih mehanizmih signalizacije celic T. Inhibitorne domene ter receptor CAR smo povezali s kemijsko inducibilnimi dimerizacijskimi domenami, tako da je signalizacija celic CAR-T inhibirana le ob dodatku eksogene majhne molekule. Za indukcijo heterodimerizacije smo uporabili danoprevir. Izražanje receptorjev CAR in funkcionalnost inhibitornih domen smo preučevali na celični liniji Jurkat s pretočno citometrijo in testom ELISA. Celice Jurkat smo uspešno elektroporirali s pripravljenimi genskimi konstrukti in potrdili izražanje CAR na površini celic. Vsi konstrukti so v odsotnosti danoprevirja sprožili celično aktivacijo. Ob dodatku danoprevirja, ki v nizkih koncentracijah ni citotoksičen, je popolno inhibicijo dosegel le konstrukt z inhibitorno domeno na osnovi fosfataze CD45. Pri inhibitornih domenah na osnovi ZAP-70 je prišlo do delne inhibicije, pri inhibitornih domenah na osnovi PD1 pa inhibicije ni bilo. CAR-T cell therapy has resulted in extremely effective and sustained clinical results in the treatment of cancer. However, there are limitations to CAR-T therapy, including life-threatening cytokine release syndrome and neurotoxicity. To alleviate the symptoms of side effects active substances are mostly used, but for effective and safe therapy it would be prefereable to introduce mechanisms that allow the regulation of side effects at the CAR-T cell signaling level. In our master's thesis, we designed six different inhibitory domains based on native T cell signaling mechanisms to regulate CAR-T cell signaling. Inhibitory domains and CAR were fused to chemically inducible dimerization domains, so that only with the addition of small exogenous molecule CAR-T signaling is inhibited. A small molecule danoprevir was used to induce heterodimerization. The expression of constructs and the functionality of inhibitory domains were studied on Jurkat cells by flow cytometry and ELISA. Jurkat cells were successfully electroporated with prepared constructs and their expression on the cell surface was confirmed. All constructs induced cell activation in the absence of danoprevir. With the addition of danoprevir, which is not cytotoxic at low concentrations, only construct with the CD45-based inhibitory domain achieved complete inhibition. Partial inhibition occurred withZAP-70 inhibitory domains and no inhibition with PD1 inhibitory domain. CAR-T celična terapija imunoterapija raka danoprevir ZAP-70 CD45 PD1 CAR-T cell therapy cancer imunotherapy danoprevir ZAP-70 CD45 PD1 true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2022-08-27 07:16:11 2022-08-27 07:16:19 2023-01-10 15:32:24 0000-00-00 00:00:00 2022 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2023-08-24 208030 119511299 23782.pdf 23782.pdf 1 F59C341F999C85DACFB32DF856EBDAFC aab727d89e69eef94a140e81e2586e0cf44c2f7e27526fb5aa4eecf3c54e492d 5c08fbf5-25c7-11ed-92af-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=159497 Biotehniška fakulteta 0 0 0