UNASSIGNED: CAR T cells have generated great excitement due to their remarkable clinical response rates in selected hematologic malignancies. However, these engineered immune cells are living drugs which are hard to control after administration.
UNASSIGNED: We discuss small molecule-regulated switch systems which can potentially be used to control CAR T cell function within the patient, as well as the most important obstacles in the CAR T cell field, which might be overcome with those switch systems.
UNASSIGNED: There is an urgent need to develop advanced switch systems. Once available, we expect that they will open up new avenues for future CAR T cell generations.

[This corrects the article DOI: 10.3389/fmed.2022.1052318.].

Gene therapy would greatly benefit from a method to regulate therapeutic gene expression temporally. Riboswitches are small RNA elements that have been studied for their potential use in turning transgene expression on or off by ligand binding. We compared several tetracycline and toyocamycin-inducible ON-riboswitches for a drug responsive transgene expression. The tetracycline-dependent K19 riboswitch showed the best control and we successfully applied it to different transgenes. The induction of gene expression was 6- to 10-fold, dose-dependent, reversible, and occurred within hours after the addition of a clinically relevant tetracycline dose, using either plasmid or adeno-associated virus (AAV) vectors. To enhance the switching capacity, we further optimized the gene cassette to control the expression of a potential therapeutic gene for cardiovascular diseases, VEGF-B. Using two or three riboswitches simultaneously reduced leakiness and improved the dynamic range, and a linker sequence between the riboswitches improved their functionality. The riboswitch function was promoter-independent, but a post-transcriptional WPRE element in the expression cassette reduced its functionality. The optimized construct was a dual riboswitch at the 3\' end of the transgene with a 100 bp linker sequence. Our study reveals significant differences in the function of riboswitches and provides important aspects on optimizing expression cassette designs. The findings will benefit further research and development of riboswitches.

Immune cell-based therapies can induce potent antitumor effects but are also often associated with severe toxicities. We previously developed a PD-1-based small molecule-regulated reversible T cell activation switch to control the activity of cellular immunotherapy products. This chemically regulated and SH2-delivered-inhibitory tail (CRASH-IT) switch relies on the noncovalent interaction of switch SH2 domains with phosphorylated ITAM motifs in either chimeric antigen receptors or T cell receptors. After this interaction, the immunoreceptor tyrosine-based inhibition motif/switch motif (ITIM/ITSM) containing PD-1 domain present in the CRASH-IT switch induces robust inhibition of T cell signaling, and CRASH-IT-mediated suppression of T cell activity can be reversed by small molecule-induced switch proteolysis. With the aim to develop improved second-generation switch systems, we here analyze the possibility space of both the immune cell receptor docking and inhibitory signaling domains that allow control over T cell activity. Importantly, these analyses demonstrate that the inhibitory domains that most potently suppress antigen receptor signaling in primary human T cells are not derived from inhibitory receptors, such as PD-1 and BTLA, that are endogenously expressed in T cells, but include ITIM/ITSM containing inhibitory domains derived from receptors present in myeloid cells. In addition, we demonstrate that physical proximity of the inhibitory domain to the antigen receptor is crucial to efficiently suppress T cell activation, as only switch designs that employ SH2 domains directly interacting with ITAM motifs in antigen receptors efficiently and reversibly inhibit T cell functionality. These data demonstrate the flexible and interchangeable nature of immune cell signaling domains, and inform the design of a synthetic proximity-based switch system with a superior dynamic range.