magistrsko delo
Timotej Sotošek (Avtor), Duško Lainšček (Mentor), Gregor Gunčar (Član komisije za zagovor), Vera Župunski (Član komisije za zagovor), Marko Dolinar (Komentor)

Povzetek

Nov preboj zdravljenja raka se je zgodil znotraj imunoterapije, ko so bile celice CAR-T predstavljene kot od komponent MHC neodvisna adaptivna T-celična terapija za zdravljenje B-celičnih neoplazij. Priprava celic CAR-T običajno poteka z zunanjo spremembo pacientovih avtolognih limfocitov T z virusnimi vektorji, pri čemer je integracija dostavljenega transgena v genom naključna. Posledice tega so lahko insercijske mutageneze, kar povzroča stranske učinke terapije, med drugim aktivacijo onkogenov ter poslabšanje celičnega produkta. Rešitev slednjih nevšečnosti leži v uporabi sistema CRISPR/Cas, ki omogoča tarčno vstavitev terapevtskega transgena v genom. Namen magistrskega dela je priprava kodirajočega zaporedja za transgen CD19 CAR ter drugih ključnih komponent za delovanje izboljšanega sistema CRISPR/Cas, CCExo. Omenjene komponente smo nato z nevirusnim vnosom (elektroporacija, lipidni nanodelci) dostavili v človeške celice CD3(+) z namenom razvoja celic CAR-T in vitro. V eksperimentalnem delu smo uspešno pripravili komponente CCExo v obliki mRNA in gRNA TRAC in vitro ter te skupaj s transgenom za humani CD19 CAR v obliki DNA vnesli v človeške celice CD3(+) z elektroporacijo. Uspešnost priprave celic CAR T smo dokazali s citometrijo, njihovo funkcionalnost pa z spremljanjem tarčne citotoksičnosti in citokinskega profila. Nadalje smo komponente za generacijo celic CD19 CAR-T zapakirali v lipidne nanodelce (LNP). Te smo za učinkovitejšo dostavo v celice T konjugirali s protitelesi proti humanemu površinskemu označevalcu celic T, CD5. Po uspešnem dokazu dostave komponent CCExo in transgena z LNP s prej naštetimi metodami smo poskuse nadaljevali in vivo. V humanizirane miši smo injicirali prej omenjene LNP z namenom tvorbe celic CD19 CAR-T v živem organizmu brez potrebe predhodne zunanje modifikacije celic T. Po enem tednu od injiciacije LNP smo iz miši izolirali kri in vranice ter s prej omenjenimi metodami dokazali, da smo uspešno pripravili celice CAR-T in vivo. S poskusi smo pokazali, da je sistem CCExo v kombinaciji z LNP bolj učinkovit pri pripravi celic CAR-T in vitro ter in vivo kot klasični sistem CRISPR/Cas9.

Ključne besede

CCExo;LNP;CAR-T in vivo;CRISPR/Cas9;tarčna integracija;magistrska dela;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [T. Sotošek]
UDK: 577.27:616-006.6(043.2)
COBISS: 254626563 Povezava se bo odprla v novem oknu
Št. ogledov: 127
Št. prenosov: 35
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Use of lipid nanoparticles to deliver an enhanced CRISPR/Cas system for targeted integration of the CD19-CAR transgene into T lymphocytes for cancer immunotherapy
Sekundarni povzetek: A new breakthrough in cancer treatment occurred in immunotherapy when CAR-T cells were introduced as an MHC-independent adaptive T-cell therapy for the treatment of B cell neoplasia. CAR-T cell preparation is usually carried out by exogenous modification of the patient's autologous T lymphocytes with viral vectors, whereby the integration of the delivered transgene into the genome is random. The consequences of this can be insertional mutagenesis, which causes side effects of the therapy, including the activation of oncogenes and the deterioration of the cellular product. The solution to the latter inconvenience lies in the use of the CRISPR/Cas system, which has the ability to target genomic insertion of a therapeutic transgene. The purpose of the master's thesis is to prepare the coding sequence for the CD19 CAR transgene and other key components for the operation of the improved CRISPR/Cas system, CCExo. The aforementioned components were then delivered into human CD3(+) cells by non-viral delivery (electroporation, lipid nanoparticles) with the aim of developing CAR-T cells in vitro. In the experimental part, we successfully prepared CCExo components in the form of mRNA and gRNA TRAC in vitro, and electroporated them together with the human CD19 CAR transgene in the form of dsDNA into human CD3(+) cells. The success of the preparation of CAR-T cells was demonstrated by cytometry, and their functionality by monitoring target cytotoxicity and cytokine profile. Furthermore, we packaged the components for the generation of CD19 CAR-T cells into lipid nanoparticles (LNPs), which were conjugated with antibodies against the human T cell surface marker, CD5, for more efficient delivery to T cells. After successfully demonstrating the delivery of CCExo components and the transgene with LNPs using the previously listed methods, we additionally performed in vivo experiments. We injected previously mentioned LNPs into humanized mice with the aim of generating CD19 CAR-T cells in vivo without the need of exogenous modification of T cells. After one week of LNP injection, blood and spleens were isolated from the mice and, using the aforementioned methods, we demonstrated that we had successfully prepared CAR-T cells in vivo. We demonstrated through experiments that the CCExo system in combination with LNP is more efficient in preparing CAR-T cells in vitro and in vivo than the conventional CRISPR/Cas9 system.
Sekundarne ključne besede: CCExo;LNP;CAR-T in vivo;CRISPR/Cas9;cancer immunotherapy;targeted integration;Rak (bolezen);Imunoterapija;Lipidi;Nanodelci;Univerzitetna in visokošolska dela;
Vrsta dela (COBISS): Magistrsko delo/naloga
Študijski program: 1000377
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Strani: 1 spletni vir (1 datoteka PDF (97 str.))
ID: 27382880