TCR and CAR Engineering of Primary Human T Cells.

The efficient expression of T-cell receptors (TCRs) or chimeric antigen receptors (CARs) in primary human T cells is crucial for preclinical testing of receptor properties for adoptive T-cell therapies. Multiple streams of technological platforms have been developed in the recent decades to genetically modify primary T cells including nonviral platforms such as transposon-based systems (PiggyBac, Sleeping Beauty), TALENs, or CRISPR-Cas9). The production of CAR- or TCR-encoding retroviral vectors, however, is still the most commonly used technique both in preclinical as well as in clinical settings.In this chapter we describe a comprehensive 12-day protocol for (a) generating high-titered gamma-retroviral vector particles containing the transgene of interest (e.g., TCR , CAR ), (b) the isolation, activation and rapid expansion of primary T cells and (c) the stable genetic engineering of these T cells with the transgene for subsequent characterization.

Efficient Non-Viral T-Cell Engineering by Sleeping Beauty Minicircles Diminishing DNA Toxicity and miRNAs Silencing the Endogenous T-Cell Receptors.

Transposon-based vectors have entered clinical trials as an alternative to viral vectors for genetic engineering of T cells. However, transposon vectors require DNA transfection into T cells, which were found to cause adverse effects. T-cell viability was decreased in a dose-dependent manner, and DNA-transfected T cells showed a delayed response upon T-cell receptor (TCR) stimulation with regard to blast formation, proliferation, and surface expression of CD25 and CD28. Gene expression analysis demonstrated a DNA-dependent induction of a type I interferon response and interferon-ß upregulation. By combining Sleeping Beauty transposon minicircle vectors with SB100X transposase-encoding RNA, it was possible to reduce the amount of total DNA required, and stable expression of therapeutic TCRs was achieved in >50% of human T cells without enrichment. The TCR-engineered T cells mediated effective tumor cell killing and cytokine secretion upon antigen-specific stimulation. Additionally, the Sleeping Beauty transposon system was further improved by miRNAs silencing the endogenous TCR chains. These miRNAs increased the surface expression of the transgenic TCR, diminished mispairing with endogenous TCR chains, and enhanced antigen-specific T-cell functionality. This approach facilitates the rapid non-viral generation of highly functional, engineered T cells for immunotherapy.