Mechanogenetics for the remote and noninvasive control of cancer immunotherapy

Abstract

While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and noninvasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) that is activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK293T cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered the Jurkat T-cell line and primary T cells (peripheral blood mononuclear cells) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells. This approach is modular and can be extended for remote-controlled activation of different cell types with high spatiotemporal precision for therapeutic applications.

Keywords: cancer immunotherapy; mechanogenetics; remote control; synthetic biology; ultrasound.

Fig. 1.

Design of synthetic genetic circuits remotely activatable by ultrasound. (A) Schematic drawing of ultrasound-induced cell activation and gene expression. Microbubbles can be coupled to the surface of a cell, where mechanosensitive Piezo1 channels are expressed. Upon exposure to ultrasound waves, the mechanical stimulation can activate the Piezo1 ion channels. The subsequent calcium entry triggers the downstream pathways, including calcineurin activation, NFAT dephosphorylation, and translocation into the nucleus. The nucleus-translocated NFAT can bind to upstream response elements to initiate gene expression through one-stage or two-stage genetic transducing modules. (B) Diagram of an integrated system of ultrasound stimulation and FRET imaging. The CCD camera captures fluorescent images of FRET biosensors with two emission filters controlled by a filter changer (480DF40 for CFP and 535DF35 for YFP). The ultrasonic transducer was driven by a function generator and a power amplifier. (C) The time courses of the normalized FRET/ECFP (enhanced cyan fluorescent protein) ratio (mean ± SEM) of a D3cpv calcium biosensor in HEK293T cells before and after 5 s of ultrasound stimulation under different conditions with or without the presence of the three components (Piezo1, microbubbles, and Ca²⁺) in the medium: with all three components (black, n = 32), without Piezo1 (orange, n = 37), without microbubbles (green, n = 10), without Ca²⁺ (brown, n = 14), or without both Piezo1 and microbubbles (blue, n = 11). (D) Representative FRET/ECFP ratio images of the D3cpv calcium biosensor in HEK293T cells expressed with (Top) or without (Bottom) Piezo1 before (Left) and after (Right) ultrasound stimulation. (Scale bar: 20 μm.)

Fig. 2.

Characterization of GTMs remotely activatable by ultrasound. (A) The design of ReCoM GTMs. Calcium-responding elements consisting of SRE, CRE, and NFAT RE were followed by target genes of interest (GOI) controlled by minimal promoter (a, P_min) or minimal CMV promoter (b, P_CMVmin). For two-stage constructs, LexA-VPR was designed as the product of stage 1 gene cassette, which can bind to the LexA-responding element and activate stage 2 target genes (c, d, e, and f). (B) Ultrasound stimulation induced gene products of different GTMs. Microbubble-coupled HEK293T cells transfected with Piezo1 and GTMs showed a significant increase in luciferase gene expression upon ultrasound stimulation (n = 3). (C) Lonomycin stimulation induces gene expression. HEK293T cells transfected with Piezo1 and genetic cassettes showed a significant increase in luciferase gene expression 16 h after ionomycin stimulation (n = 3). (D) Fluorescence microscopy images of HEK293T cells transfected with Piezo1 and gene activation cassette a (mNeonGreen as the reporter) without (Left) and 16 h after (Right) ultrasound stimulation (for 10 min). (Scale bar: 40 μm.) Error bars indicate SEM, *P < 0.05, **P < 0.01 from two-tailed Student t test.

Fig. 3.

Remote-controlled activation of Jurkat T cells with ReCoM. (A) Jurkat cells were transfected with inducible ReCoM-CAR. After ultrasound stimulation, they were mixed with antigen CD19-expressing target tumor cells and evaluated for their activation level (CD69 expression). (B) The representative FRET/ECFP ratio images of the D3cpv calcium biosensor in Jurkat cells before (Left) and after (Right) ultrasound stimulation. (Scale bar: 10 μm.) (C) The FRET/ECFP ratio percentage change of D3cpv calcium FRET biosensor averaged among multiple Jurkat cells before and 10 s after ultrasound stimulation (n = 15). (D) Normalized expression of anti–CD19-CAR in Jurkat cells 16 h after ultrasound stimulation (n = 3). (E) Representative histograms of T-cell activation in Jurkat cells by quantifying the expression of the cell-surface protein marker CD69. Jurkat and Toledo mixtures were stained with Alexa647-conjugated anti-CD69 antibody and analyzed by flow cytometry. (F) The bar graphs represent CD69 up-regulation (normalized percentage of CD69-positive cells) in ultrasound-induced Jurkat cells upon Toledo cell engagement (n = 8). Error bars indicate SEM, *P < 0.05, ***P < 0.001 from two-tailed Student t test.

Fig. 4.

Remote-controlled activation of PBMCs with ReCoM. (A) PBMCs were transfected with inducible ReCoM-CAR. After ultrasound stimulation, they were mixed with antigen CD19-expressing target tumor cells and evaluated for killing efficiency by functional assays. (B) The Fluo-4 calcium indicator intensity change in PBMCs before and after 5 s of ultrasound stimulation (n = 4). (C) Expression of ReCoM-CAR in PBMCs 16 h after ultrasound stimulation (n = 5). (D) Cytotoxicity of target Nalm6 tumor cells caused by CAR expression upon ultrasound stimulation in PBMC cells transfected with ReCoM-CAR GTMs, measured by luciferase-based killing assay (n = 4). Cytotoxicity of plain PBMCs upon ultrasound stimulation was also measured (n = 3). Error bars indicate SEM, *P < 0.05, **P < 0.01, ***P < 0.001 from two-tailed Student t test.