No abstract

The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3 0 and 5 0 arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 Â 10 8 CAR19 T cells per one electroporation of 1 Â 10 7 peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, earlyphase CAR T cell products.

The paper deals with simulating the circumstances of switching surge and trigger current at switching-in of the T402 autotransformer recently installed in Križovany, with the tests conducted by means of ATP (Alternative Transient Program) simulation software for simulating transient electromagnetic phenomena. The target has been to determine temporal curves for the most critical surge instances against earth and between the phases and the curves for trigger current with and without surge limiters connected, and to determine by means of simulations the size and statistical frequency of switching surges and based on a controlled switching simulation, to describe the elimination of transient phenomena during the process and in reference to the simulations and to operating manual for the PSD02 Siemens control unit, suggest the required configuration settings for the unit. K e y w o r d s: switching surge, trigger current, substation, transformer, T402 transformer, EMTP -ATP, Križovany, surge current