magistrsko delo
Mojca Skrbinek (Author), Anže Smole (Reviewer), Duško Lainšček (Mentor)

Abstract

Trenutne metode proizvodnje celic CAR-T z virusno transdukcijo imajo omejitve zaradi ne-tarčne integracije terapevtskih transgenov v genom in morebitne insercijske mutageneze. V magistrskem delu smo se osredotočili na ne-virusno genetsko modifikacijo celic CAR-T s tehnologijo CRISPR/Cas9 in njegovo izboljšano različico. Glavni cilj je izboljšati učinkovitost in varnost z naprednim sistemom preurejanja genoma, CCExo, ki omogoča tarčno vstavljanje zapisa za receptor CAR v genom limfocitov T. Oblikovali smo različno dolge HDRt z zapisom za transgen CD19-CAR in RCC za povečanje učinkovitosti HDR. Transgen smo vstavljali v dva specifična lokusa, TRAC in TRBC1, ki sta del T-celičnega receptorja. Rezultati kažejo, da je sistem CRISPR/Cas9 učinkovit pri vstavljanju transgena ne glede na dolžino homolognih zaporedij in prisotnost RCC. CCExo je pokazal povečano učinkovitost tarčnega funkcionalnega vstavljanja transgena CD19-CAR v genom limfocitov T v primerjavi s klasičnim sistemom CRISPR/Cas9, zlasti pri krajših HDRt ob prisotnosti RCC. TRBC1 tarčna integracija transgena je bila dodatno povečana pri elektroporaciji z ribonukleoproteini v primerjavi s plazmidno elektroporacijo. Uporaba tehnologije CRISPR/Cas9 in njegove izboljšane različice CCExo se izkazuje kot obetavna pot za izboljšanje priprave terapevtskih celic ter terapije s celicami CAR-T in razširitev njene uporabe pri zdravljenju raka.

Keywords

CAR-T;CRISPR/Cas9;imunoterapija raka;preurejanje genoma;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [M. Skrbinek]
UDC: 602.68:606:616-006.6(043.2)
COBISS: 164252419 Link will open in a new window
Views: 342
Downloads: 0
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Other data

Secondary language: English
Secondary title: Targeted integration of CD19-CAR transgenic constructs into the T cell genome using an enhanced CRISPR/Cas9 system
Secondary abstract: Current methods of producing CAR-T cells with viral transduction have limitations due to non-target integration of therapeutic transgenes into the genome and potential insertional mutagenesis. In this master's thesis, we focused on non-viral genetic modification of CAR-T cells with CRISPR/Cas9 technology and its enhanced version. The main goal is to improve efficiency and safety with an advanced genome editing system, CCExo, which allows targeted insertion of the CAR receptor into the T lymphocyte genome. We designed HDRts of different lengths with a genetic sequence for the CD19-CAR transgene and RCC to increase HDR efficiency. We inserted the transgene into two specific loci, TRAC and TRBC1, which are part of the T-cell receptor. The results show that the CRISPR/Cas9 system is effective in inserting the transgene regardless of the length of homologous sequences and the presence of RCC. Compared to the classic CRISPR/Cas9 system, CCExo showed increased efficiency of targeted functional insertion of the CD19-CAR transgene into the T lymphocyte genome, especially with shorter HDRts in the presence of RCC. TRBC1 target transgene integration was further increased with electroporation with ribonucleoproteins compared to plasmid electroporation. The use of CRISPR/Cas9 technology and its improved version CCExo is proving to be a promising path for improving the preparation of therapeutic cells and CAR-T cell therapy and expanding its use in cancer treatment.
Secondary keywords: cancer imunotherapy;genome editing;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 2025-09-07
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij biotehnologije
Pages: 1 spletni vir (1 datoteka PDF (XV, 72 str., [20] str. pril.))
ID: 19917029