Stief, Tanja (2022): Functional replacement of the T-cell receptor in primary human T cells for the treatment of refractory adenovirus infections. Dissertation, LMU München: Faculty of Biology |
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Abstract
Adenoviral (AdV) infections can cause life-threatening infections, especially in paediatric patients, following haematopoietic stem cell transplantations due to transient lacking T-cell immunity. AdV hexon-protein derived peptide LTDLGQNLLY (LTDL)-specific T cells have proven to induce cross-reactive protection, thus being highly attractive targets for adoptive T-cell transfer (ACT). The aim of this project was to redirect primary human T cells by replacing endogenous T-cell receptors (TCRs) with a LTDL-specific TCR using CRISPR/Cas9 technology. Simultaneous knock out (KO) of the complete endogenous TCR will prevent harmful TCR mispairing and alloreactivity. Stable and highly efficient genetic KO of the endogenous TCR in primary human T cells was confirmed on protein as well as on genetic level. TCR-KO T cells are phenotypically very similar to unedited cells but fail to produce IFNγ upon stimulation, thereby functionally demonstrating TCR disruption. Whole-genome sequencing of CRISPR/Cas9-edited cells revealed no significant increase of mutations, although analysis of predicted gRNA-dependent off-target sites revealed two putative off-target mutations for the gRNA targeting TCR β chain. Functional analysis of two novel LTDL-specific TCRs revealed strong, target-specific effector functions for both of them. CRISPR/Cas9-mediated knock in (KI) using non-viral delivery of one protective LTDL-specific TCR rescued IFNγ production upon LTDL-specific stimulation and demonstrated LTDL-specific cytotoxic capacity, cytokine secretion and proliferation. The targeted and in-frame integration into the endogenous TCR α chain was combined with an additional KO of the β chain to prevent TCR mispairing and enables expression under the control of the endogenous promoter. In conclusion, redirecting primary human T cells by replacing endogenous TCRs with a LTDL-specific TCR using CRISPR/Cas9 is feasible, resulting in target-specific cytotoxic T cells with strong effector functions. The combined TCR KO/KI procedure could outcompete transduction based conventional TCR editing strategies and therefore presents a safe and powerful tool for the treatment of refractory viral infections in the immunocompromised host.
Item Type: | Theses (Dissertation, LMU Munich) |
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Keywords: | TCR replacement, CRISPR/Cas9, Adenovirus |
Subjects: | 500 Natural sciences and mathematics 500 Natural sciences and mathematics > 570 Life sciences |
Faculties: | Faculty of Biology |
Language: | English |
Date of oral examination: | 4. April 2022 |
1. Referee: | Leonhardt, Heinrich |
MD5 Checksum of the PDF-file: | 5be89080b70c7b9c029a6e1047b0cd6e |
Signature of the printed copy: | 0001/UMC 28799 |
ID Code: | 29963 |
Deposited On: | 08. Jun 2022 08:08 |
Last Modified: | 08. Jun 2022 08:08 |