Secondary abstract: |
CAR-T cell therapy is a form of cancer immunotherapy that has proven itself as an effective cure for treating various haematological malignancies. Generally, viral transduction is used for the preparation of therapeutic cells, yet due to its numerous disadvantages various non-viral methods for CAR-T cell preparation are being investigated, which would be safer, cheaper, and more effective. Hence, as part of the master’s thesis, we prepared an improved version of the CRISPR/Cas9 technology, named CCExo-streptavidin, which enables efficient integration of genes at the target site in the cell genome and thus the preparation of therapeutic cells. The components of the CCExo-streptavidin system were designed and prepared so that they enabled insertion of the second generation anti-CD19 CAR receptor coding region into the cell genome in place of the TRAC gene, which was knocked out. The effectiveness of the CCExo streptavidin system was demonstrated in vivo on the CD4+ T lymphocyte cell line Jurkat and on human primary CD3+ T lymphocytes. It was shown as a higher expression of the CAR receptor on the surface of the treated cells, a higher concentration of IL-2 in the co-culture of T lymphocytes and target cancer cells Raji-fLUC, and a higher cytotoxic activity of the therapeutic CD8+ cells compared to the classic CRISPR/Cas9 system. Further optimization of the CCExo-streptavidin system enabled us to determine the optimal concentration of the components for electroporation of Jurkat cells, yet the optimization can be further improved and carried out additionally for the electroporation of human primary T lymphocytes. Lastly, we aspired to insert the CAR transgene also in the TRBC1 locus, but with little success. |