Generation of CAR-T Cells with Sleeping Beauty Transposon Gene Transfer

Human T lymphocytes that transgenically express a chimeric antigen receptor (CAR)Chimeric antigen receptor (CAR) have proven efficacy and safety in gene- and cell-based immunotherapy of certain hematological cancers. Appropriate gene vectors and methods of genetic engineeringGenetic engineering are required for therapeutic cell products to be biologically potent and their manufacturing to be economically viable. Transposon-based gene transferGene transfer satisfies these needs, and is currently being evaluated in clinical trials. In this protocol we describe the basic Sleeping Beauty (SB) transposon Transposons vector components required for stable gene integration in human cells, with special emphasis on minicircle Minicircle (MC) DNADNA vectors and the use of synthetic mRNA. We provide a protocol for functional validation of the vector components in cultured human cell lines on the basis of fluorescent reporter gene expression. Finally, we provide a protocol for CAR-T cellCAR T-cell engineering and describe assays that address transgene Transgenes expression, biological potency and genomic vector copy numbersVector copy numbers in polyclonal cell populations. Because transposonsTransposons allow virus-free gene transferGene transfer with naked nucleic acids, the protocol can be adopted by any laboratory equipped with biological safety level S1 facilities.