(D-155) TRPV1-based Sonogenetics for Immunotherapy

Introduction:: In recent years, sonogenetics has emerged as a promising alternative to optogenetics, utilizing ultrasound as an external modality to induce genetic outcomes in engineered cells, particularly in the field of neuromodulation. The heat-sensitive TRPV1 channel serves as a valuable component in sonogenetic systems, enabling focused ultrasound-induced heat sensing. Previous research has demonstrated the potential of introducing TRPV1 into neurons to modulate neural activity and locomotor behavior. Sonogenetics based on the TRPV1 channel can become a powerful tool for broad applications if we can efficiently transduce the TRPV1 mediated calcium influx into user-defined transcriptional activities. In this study, we integrated our CaDox system, a genetic transducer designed as a calcium and doxycycline dependent AND logic circuit, to redirect the heat-induced calcium signaling into therapeutic outcomes. To test the feasibility of this sonogenetic system in CAR T therapy, we engineered HEK293T cells to express a truncated CD19 antigen upon pulsed heat shock, which can be recognized by anti-CD19 CAR T cells. The results revealed significant killing in the group activated by heating and doxycycline incubation. This innovative approach enables precise, spatiotemporal control of gene expression and opens up new opportunities in immunotherapy.

Materials and Methods:: HEK293T cells were purchased from the ATCC and cultured in DMEM supplemented with 10% HI-FBS and 1% penicillin/streptomycin. Primary T cells were cultured in X-Vivo 15 (Lonza) supplemented with 5% HI-FBS, 50µM β-Mercaptoehanol, and 1% recombinant human IL-2. For plasmid construction, Gibson assembly method was used. Inducible genetic circuits were transduced to cells via lentiviral infection and further sorted with a fluorescence marker. For the gene inducibility assay, Dual-Glo (Promega) was used to measure inducible firefly luciferase and constitutive ranilla luciferase, and for the killing assay, CellTiter-Glo 2.0 cell viability assay (Promega) was used.

Results, Conclusions, and Discussions::
Our initial goal was to assess if TRPV1 could be activated by heat. We transiently transfected HEK293T cells with TRPV1, R-Geco1 (a calcium sensor), and NFAT-EGFP to monitor calcium influx and NFAT translocation. Upon heating to 43°C, we observed increased intracellular calcium in TRPV1-positive cells and subsequent NFAT translocation, confirming TRPV1 activation upon heat exposure (Fig 1B). Next, we investigated whether TRPV1-mediated calcium influx and NFAT translocation would be sufficient to drive the CaDox circuit. We treated HEK293T cells stably expressing the CaDox system under various conditions, including doxycycline (100 nM), ionomycin (1mg/mL), and heating (43°C), with and without TRPV1. As expected, the doxycycline- and the ionomycin-treated group showed strong induction regardless of TRPV1 (Fig. 1C). Importantly, in TRPV1-expressing cells, robust gene activation was observed with doxycycline and heat treatment, indicating that TRPV1-mediated calcium signaling effectively activates the CaDox circuit. To assess the potential of this system for immunotherapy applications, we engineered HEK293T cells to express the CaDox system with the tCD19 antigen as an inducible marker and treated the cells with doxycycline and/or heat. Consistent with previous results, we found significant tCD19 expression only with the combined doxycycline and heating treatment (Fig. 1D). Lastly, we co-cultured the activated cells with anti-CD19 CAR T cells and observed that the heat- and doxycycline-induced antigen presentation was sufficient to mediate CAR-mediated killing (Fig. 1E). In conclusion, our study demonstrates that TRPV1-mediated calcium signaling, in combination with the CaDox system, effectively drives user-defined gene expression in response to heat and doxycycline treatment. This innovative approach holds significant potential for immunotherapy applications, as exemplified by the successful activation of anti-CD19 CAR T cells, and paves the way for further exploration of sonogenetics in diverse therapeutic contexts.




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