Smart-watch-programmed green-light-operated percutaneous control of therapeutic transgenes

Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.


Supplementary Figure 2 | Microscopy analysis of the Glow Control components.
HEK293T cells were transfected with either (a) pMMZ351 (PSV40-Myr-TtCBD-GFP-pA) or (b) pMMZ429 (PSV40-TtCBD-TetR-VPR-mCherry-pA) and induced with green light (545 nm; 12 h/day, 15 sec ON and 45 sec OFF and 88 µW/cm 2 ) for 48 h. Control groups were kept in dark. Cells were fixed with 4% PFA and stained with DAPI. Scale bars, 10 µm. A representative image of three replicates from each group is shown.

Supplementary Figure 3 | Optimization of plasmid ratio in Glow Control system. The Glow
Control system plasmids (pMMZ269; PSV40-Myr-TtCBD-pA, and transactivator pMMZ272; PSV40-TtCBD-TetR-VP16-pA) were co-transfected with a constant amount of the PTET reporter (pTS1017; PTetO7-hCMVmin-SEAP-pA). Different ratios of Myr-TtCBD (constant amount in b and reducing in a and c) to transactivator (constant amount in a and increasing in b and c) were applied to optimize SEAP expression in the Glow Control system. Numbers below are amounts of DNA (ng/well) used for co-transfection in HEK293T cells seeded in 96-well plates. Transfected cells were illuminated with green light (545 nm; 15 sec ON/45 sec OFF and 88 µW/cm 2 for 12 h/day) and SEAP expression was measured 48 h after first illumination. Control cells were kept in the dark. Bars represent the mean ± s.d. (n = 3), and numbers above the bars indicate fold changes of reporter (SEAP) expression compared to the corresponding dark control. Source data are provided as a Source Data file.

Supplementary Figure 5 | Comparison of N-terminal or C-terminal tagging of TtCBD in improved version of Glow Control. a, Schematic representation of plasma membrane-anchored
TtCBD tagged C-terminally (C-term) or N-terminally (N-term) with negatively supercharged GFP. b, HEK293T cells were co-transfected with pMMZ272 (PSV40-TtCBD-TetR-VP16-pA), pTS1017 (TetO7-PhCMVmin-SEAP-pA) and either pMMZ284 (PSV40-Myr-TtCBD-nGFP-pA) or pMMZ410 (PSV40-Myr-nGFP-TtCBD-pA). Cells were illuminated with green light (545 nm; 15 sec ON/45 sec OFF and 88 µW/cm 2 for 12 h/day) and SEAP expression was measured 48h after first illumination. Control cells kept in dark. Bars represent the mean ± s.d. (n = 3), and numbers above the bars indicate fold changes of reporter (SEAP) expression compared to the corresponding dark control. Source data are provided as a Source Data file.  Figure 3c. HEK293T cells were illuminated with pulsed green light (ON, indicated by green box) or kept in the dark (OFF, indicated by grey box) and SEAP levels were profiled every 12 h for 48 h. After 24 h, the culture medium was changed and the cell density was adjusted to 5 x10 4 cells/well of a 96-well plate. The pattern of light/dark cycles is indicated above each plot. The values are mean ± s.d., n = 3 biological replicates. b, In vitro reversibility of Glow Control in a continuous culture. Transfected HEK293T cells were illuminated with pulsed green light (ON; green box) or were kept in the dark (OFF; dark box) and 10 µl of supernatant (replaced by the same volume of fresh complete media) was taken at each indicated time point for SEAP measurement. The pattern of light/dark cycles is indicated above each plot. The values are mean ± s.d., n = 6 biological replicates. c, TtCBD-TA half-life measurement. HEK293T cells were transfected with TtCBD-TA (pMMZ516; PCMV-HA-TtCBD-TetR-VP16-pA) or a degron-tagged version of TtCBD-TA (MMZ518; PCMV-Ubk-HA-TtCBD-TetR-VP16-pA) and were cultured in presence of the protein synthesis inhibitor cycloheximide (CHX, 25 μg/mL). Protein half-life was measured by western blotting after the indicated times of treatment. A representative image of three replicates is shown. and pTS1017 were microencapsulated and subcutaneously implanted in RjOrl:SWISS (CD-1) mice. The mice were illuminated with green light (545 nm, 12 h/day, 15 sec ON/45 sec OFF, 300 µW/cm 2 ) from LEDs mounted on the ceiling of the cage. b, Quantification of SEAP levels in blood stream of light-shower-treated mice for three days. Values are mean ± SEM (n = 5), and statistical significance between the Light and Dark groups at the indicated time points were calculated using a two-tailed, paired Student's t-test. *, p<0.05; ***, p<0.001. c, Mice were illuminated with a programmable, smart-watch-mimicking LED patch (see Supplementary Figure 9) (545 nm, e.g., 3 h, constant or pulsed for 12 h/day, 15 sec ON/45 sec OFF, 150 µW/cm 2 ), which was fixed with sutures to the shaved back of the mice. Source data are provided as a Source Data file. The LED case is a 3D printed plastic frame containing a printed circuit board (PCB) to harvest the energy from a magnetic field to drive two LEDs. a, A top view of the plastic case carrying the PCB, from inside (top) and outside (down). b, Top view of the PCB harboring green LEDs from the front (top) and back (down). c, The plastic case equipped with the PCB in an open (top) or closed and finalized (down) format. d-f, A functional smart-watchmimicking LED patch; d, Dimensions, e, LED patch dimensions, with a coin for reference, and f, weight of the complete LED patch. g, The LED patch is stitched face down onto the mouse skin. A magnetic field generator provides wireless energy transmission to power the LEDs. The field generator is also connected to Arduino TM , allowing control of light intensity and illumination time. h, working LED patch on a mouse in the light and in the dark.

Supplementary Figure 14 | Characterization of insulin level upon green light activation of implanted Glow Control cells in vivo.
Effect of hGLP1 expression on insulin levels in T2D mice after illumination with or without green light for 12 days (related to Fig. 4h). Bars represent the mean ± s.d. (n = 6). Statistical significance between Lpr db/db mice of the indicated groups on day 12 were calculated using a one-way ANOVA. ns, not significant, ***, p<0.001; ****, p<0.0001. Source data are provided as a Source Data file.

Supplementary Figure 15 | Functionality of the encapsulated Glow Control cells. a, Glow
Control-hGLP1 cells encapsulated in alginate-poly(L-lysine)-alginate microcapsules were recovered from the dorsal subcutaneous space of sacrificed Lpr db/db mice 12 days after transplantation (left side). Encapsulated cells were washed with DMEM and imaged with a bright-field microscope (right side). Scale bar is 200 µm. A representative image of three replicates is shown. b, Viability of microencapsulated Glow Control-hGLP1 cells. 200 recovered capsules from illuminated and un-illuminated Lpr db/db mice 12 days after transplantation, together with capsules that had been kept in an incubator for 12 days, were subjected to MTT assay (n = 3), c, In vitro quantitative evaluation of Glow Control-hGLP1-LH2 before encapsulation, after encapsulation and after recovery on the 12th day after transplantation. Cells were either illuminated with pulsed green light (545 nm, 12 h/day, 15 sec ON/45 sec OFF, and 88 µW/cm 2 cells) for 48 h or kept in the dark. Bars represent the mean ± s.d. (n = 3 independent experiments). d, In vitro reversibility of Glow Control-hGLP1 cells (related to Figure 4j) in the presence (ON) or absence (OFF) of pulsed green light (545 nm, 12 h/day, 30 sec ON/30 sec OFF, 150 µW/cm 2 ). Values are mean ± s.d. for n = 3 cell culture experiments. Statistical significance was calculated using a two-way ANOVA. ***, p<0.001; ****, p<0.0001. Source data are provided as a Source Data file.