Adoptive cell therapy with lymphoctes expressing chimeric antigen receptor (CAR) has been a subject of great interest since the approval of CAR-T therapy for treating acute lymphoblast leukaemia (ALL) and diffuse large B cell lymphoma (DLBCL), especially due to its potential of expansion to treat other malignancies. The intention of this masters thesis was to rearrange a plasmid, bearing a sequence for a chimeric antigen receptor with methods of molecular cloning in such a way, that the receptor would stop recognising CD19 antigen, but would instead be directed to CD20 molecules. After a succesful rearrangement, the lymphocites with an electroporated plasmid bearing such a sequence would still be mostly recognising ALL and DLCBL cells, but their efficiency of recognition would be much greater due to a higher density of CD20 molecules expressed on the surface of these malignant cells. For our work we used a modern technique of molecular cloning, the Gibson assembly. We concluded that the plasmids used for cloning were unsuitable for our work due to a large percentange of cytosine and guanine nucleotides present in their key sequences. Based on our conclusions we suggest alternative solutions for cloning.
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