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.
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