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An electrogenetic interface to program mammalian gene expression by direct current

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An electrogenetic interface to program mammalian gene expression by direct current Empty An electrogenetic interface to program mammalian gene expression by direct current

Post by Chromium6 Mon Sep 25, 2023 12:26 am

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https://www.nature.com/articles/s42255-023-00850-7

   Article
   Open Access
   Published: 31 July 2023

An electrogenetic interface to program mammalian gene expression by direct current

   Jinbo Huang, Shuai Xue, Peter Buchmann, Ana Palma Teixeira & Martin Fussenegger

Nature Metabolism volume 5, pages 1395–1407 (2023)Cite this article

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Abstract

Wearable electronic devices are playing a rapidly expanding role in the acquisition of individuals’ health data for personalized medical interventions; however, wearables cannot yet directly program gene-based therapies because of the lack of a direct electrogenetic interface. Here we provide the missing link by developing an electrogenetic interface that we call direct current (DC)-actuated regulation technology (DART), which enables electrode-mediated, time- and voltage-dependent transgene expression in human cells using DC from batteries. DART utilizes a DC supply to generate non-toxic levels of reactive oxygen species that act via a biosensor to reversibly fine-tune synthetic promoters. In a proof-of-concept study in a type 1 diabetic male mouse model, a once-daily transdermal stimulation of subcutaneously implanted microencapsulated engineered human cells by energized acupuncture needles (4.5 V DC for 10 s) stimulated insulin release and restored normoglycemia. We believe this technology will enable wearable electronic devices to directly program metabolic interventions.

Main

Interconnected smart electronic devices are increasingly dominating our daily lives and shaping our health awareness1; however, electronic and biological systems function in radically different ways and are largely incompatible due to the lack of a functional communication interface. While biological systems are analog, programmed by genetics, updated slowly by evolution and controlled by ions flowing through insulated membranes, electronic systems are digital, programmed by readily updatable software and controlled by electrons flowing through insulated wires. Electrogenetic interfaces that would enable electronic devices to control gene expression remain the missing link in the path to full compatibility and interoperability of the electronic and genetic worlds2.

Synthetic biology has taken up this challenge by assembling simple analog gene switches into complex gene circuits that can program cellular behavior with the logic-processing functionality of electronic circuits such as oscillators3, timers4, memories5, band-pass filters6 and relay switches7 as well as analog-to-digital converters8, half-adders9 and even full-adders10. The utility of many of these gene circuits has been demonstrated in the experimental control of diverse medical conditions, including cancer3, bacterial infections11, chronic pain12 and diabetes13. Gene circuits typically incorporate trigger-inducible gene switches that are controlled by small-molecular compounds such as antibiotics14, vitamins15, food additives16, cosmetics17 or volatile fragrances8. As differences in bioavailability, pleiotropic side effects and pharmacodynamics may jeopardize the overall regulatory performance of such triggers in a mammalian host, attention has increasingly turned to non-molecular traceless physical cues such as electromagnetic waves, including light18,19, magnetic fields20, radio waves21 and heat22; however, physically triggered gene switches may require high energy input21, may involve unphysiological chemical or inorganic cofactors with side effects19, poor bioavailability23 or short half-lives24, may suffer from illumination-based cytotoxicity25 and may be confounded by any fever-associated medical condition22.

Thus, there is a need for a device to permit direct battery-powered, cofactor-free, time- and voltage-dependent electrical fine-tuning of mammalian gene expression to set the stage for wearable-based electro-controlled gene expression with the potential to connect medical interventions to an internet of the body or the internet of things. Pioneering attempts to design electro-inducible gene expression in bacteria26,27,28,29,30 and mammalian cells31,32,33 proved promising in cell cultures, but were either incompatible with in vivo applications due to the cytotoxicity, limited bioavailability and poor clinical compatibility of electrosensitive redox compounds26,31 or required high-voltage alternating current controlled by complex bioelectronic implants with limited longevity32. Such devices are not suitable for use in battery-powered wearables to program therapeutic transgene expression in implanted cells32.

In humans, reactive oxygen species (ROS) are produced by electron-transfer reactions during respiratory processes in the mitochondria and peroxisomes, during mitochondrial cytochrome P450 activity in steroidogenic tissues and by NADPH oxidase in immune cells during immune responses34. The Kelch-like ECH-associated protein 1 (KEAP1) is an important tumor and metastasis suppressor that also acts as a native ROS biosensor35. Under quiescent conditions, KEAP1 sequesters and primes the nuclear factor erythroid 2 p45-related factor 2 (NRF2) for proteasomal destruction35. In the presence of elevated ROS, KEAP1 releases NRF2, which translocates to the nucleus to coordinate antioxidant and anti-inflammatory responses by binding to antioxidant-response elements (AREs)35.

Inspired by the fact that electrodes delivering DC at low voltage can rapidly generate free electrons and radical species that lead to mediator-free production of ROS at low, non-cytotoxic levels36,37,38, we set out to design the missing link for DC-powered electrogenetic target gene modulation in human cells, which we refer to as DC-actuated regulation technology (DART). DC-based generation of hydrogen peroxide was recently applied to establish an electrogenetic system using engineered bacterial cells growing at the surface of an electrode, which was able to activate transgene expression upon electrical stimulation30. Here we designed an electrogenetic interface consisting of genetic components that render human cells responsive to DC-triggered electrostimulation and enable exclusive, DC-adjustable transgene expression. We initially found that the ROS levels produced in human embryonic kidney cells (HEK293) by exposure to 4.5 V DC for 10 s were not sufficient to substantially activate KEAP1/NRF2; however, the cells could be hypersensitized to electroinduced ROS by ectopic expression of native KEAP1 and NRF2. Then, rewiring of NRF2 to synthetic ARE-containing promoters enabled direct and cofactor-free DC-powered fine-tuning of the expression of therapeutic transgenes such as the insulin gene. For a proof of concept, we implemented DART-based remote control of insulin expression in type1 diabetic mice. Stimulation of subcutaneously implanted engineered cells with World Health Organization (WHO)-approved and US Food and Drug Administration (FDA)-licensed acupuncture needle electrodes at 4.5 V DC for 10 s once per day triggered the production of sufficient insulin to attenuate postprandial glycemic excursions and restore normoglycemia.

Results

Design and characterization of DC-controlled gene expression

To sensitize native human cells for electrostimulated ROS-mediated transgene expression control, we co-transfected human embryonic kidney cells (HEK293) with constitutive KEAP1 (pJH1004, PhCMV-KEAP1-pA) and NRF2 (pJH1003, PhCMV-NRF2-pA) expression vectors as well as the reporter construct pJH1005 (PDART-SEAP-pA; PDART, OARE-PhCMVmin) encoding the human model glycoprotein SEAP (human placental secreted alkaline phosphatase) under the control of a synthetic NRF2-dependent promoter containing an ARE operator site (Supplementary Table 1). Protons and chlorine ions are generated in the cell culture medium at the electrodes36,37 (Fig. 1a) and lead to the production of ROS at levels38 that can trigger the release of NRF2 from KEAP1, resulting in NRF2-mediated expression of a gene of interest from the NRF2-specific synthetic PDART promoter (Fig. 1b).

Fig. 1: Design of the direct-current-activated transgene expression switch in mammalian cells.
figure 1

a, Schematic illustration of the stimulation setup for monolayer cultures. Each well of a 24-well plate has two platinum wires that function as anode and cathode, placed 0.6 cm apart submerged in the culture medium. When electric current is applied, bubbles form around the electrodes, with production of chlorine gas at the anode and hydrogen gas at the cathode. b, Schematic representation of the electrogenetic circuit based on the NRF2/KEAP1 antioxidative response. Upon electrical stimulation, the formation of ROS is sensed by constitutively expressed NRF2 and KEAP1 complexes localized in the cytoplasm, which triggers the translocation of NRF2 to the nucleus, where it activates expression of the gene of interest by binding to ARE sites in the upstream synthetic promoter. Under non-stimulating conditions, NRF2 is continuously targeted to the 26S proteasome for degradation. c, SEAP produced by transiently transfected HEK293 cells (KEAP1, pJH1004; NRF2, pJH1003; PDART-SEAP, pJH1005) upon stimulation by DC with 10 V for 15 s (DC10V) and 5 V for 20 s (DC5V). d, SEAP produced by cells transfected with only ARE reporter (PDART-SEAP, pJH1005) or together with KEAP1 (pJH1004) and NRF2 variants (wild-type NRF2, pJH1003; NRF2-VP64, pJH1175) and reporter (pJH1005). Cells were stimulated with DC5V for 20 s. e, SEAP produced by cells co-transfected with KEAP1 (pJH1004), NRF2 fused to tetracycline-dependent transactivator TetR-VP64 (NRF2-TetR-VP64, pJH1181) and the cognate reporter (PTRE-SEAP-pA, pMF111). The cells were stimulated with DC5V for 20 s. f, SEAP produced by cells co-transfected with reporter constructs containing one (DART1), two (DART2), three (DART3) and four (DART4) ARE repeats in the promoter region and stimulated with DC5V for 20 s. Data are mean ± s.d., n = 4. P values were calculated between stimulated and non-stimulated controls.

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For electrostimulation, the engineered cells were cultivated in standard 24-well plates containing customized lids that serve to immerse 0.5-mm platinum electrodes in the culture medium at a separation distance of 6 mm (Fig. 1a, Supplementary Fig. 1a,b and Supplementary Video 1). We observed significantly increased SEAP expression upon electrostimulation with DC at 10 V for 15 s or 5 V for 20 s (Fig. 1c). Alternating current (AC) pulse programs delivering similar energy to these DC programs (for instance, 1-ms pulses of 5 V at 1 Hz during 2.78 h) resulted in a less than two-fold increase in SEAP expression and significantly decreased cell viability (Supplementary Figs. 1c–f and 2). Indeed, to reach comparable SEAP expression levels, AC stimulation needs to deliver more energy and run for longer time periods (6 h for 1-ms pulses (Supplementary Fig. 2c); or over 1 h for 10-ms pulses (Supplementary Fig. 2e)), again with a negative impact on cell viability (Supplementary Figs. 1c–f and 2). Therefore, hereafter we focused on DC as our sole energy source. Notably, DC stimulation programs below 5 V and 20 s had no impact on cell viability (Supplementary Figs. 2 and 3a), medium composition (Supplementary Fig. 3b–e and Supplementary Tables 2–5), growth kinetics or overall protein production capacity of stimulated cells (Supplementary Fig. 4), indicating that DC-mediated electrostimulation can indeed be specifically rewired to DART promoters to fine-tune transgene expression in human cells. Electrostimulated HEK293 cells exclusively transfected with pJH1005 showed substantially less SEAP induction, indicating that concomitant coexpression of KEAP1 and NRF2 increases the sensitivity of the human cells to electrostimulated ROS production (Fig. 1d). We also tested whether NRF2 fused to the strong transactivation domain VP64, which is based on four tandem repeats of the Herpes simplex virus early transcriptional activator VP16 (pJH1175, PhCMV-NRF2-VP64-pA), could further increase the electrostimulated response; however, this modification increased basal as well as electrostimulated SEAP expression, resulting in a substantially lower overall induction fold (3.7×) compared to native NRF2 (7.2×) (Fig. 1d). Likewise, we observed lower fold induction (3.9×) of NRF2 fused to the tetracycline-dependent transactivator TetR-VP64 (pJH1181, PhCMV-NRF2-TetR-VP64-pA) activating SEAP expression from a promoter containing tetracycline-response elements (TRE) (pMF111, PTRE-SEAP-pA; PTRE, OTetR-PhCMVmin) (Fig. 1e). Further analyses with PDART variants containing different ARE tandem repeats revealed that a single ARE repeat provided low basal expression but also the lowest maximum expression level, while more tandem repeats achieved substantially higher maximum expression levels at the expense of higher basal expres​sion(Fig. 1f). Therefore, the priority for either lowest leakiness or highest expression level will determine the choice of PDART variants, as is also the case for other transcription-control modalities39.

A fluorescence-based assay showed that intracellular ROS levels were increased over two-fold at 1 h after DC electrostimulation at 5 V for 20 s and returned to non-stimulated levels within 6 h, suggesting that electrostimulated SEAP expression was indeed mediated by ROS36,37,38 (Extended Data Fig. 1a). Notably, cells treated with the ROS scavenger N-acetyl-L-cysteine (NAC)40 failed to increase SEAP expression upon electrostimulation (Extended Data Fig. 1b,c). In contrast, cells treated with inhibitors of different ROS-generating enzymes, such as GKT136901, AEBSF, ML171 and VAS2870 (ref. 41), could still respond to electrostimulation by increasing SEAP expres​sion(Extended Data Fig. 1b,c), supporting the view that the ROS generation results from the DC power stimulation rather than from endogenous sources. Furthermore, although hydrogen peroxide (H2O2), oltipraz, diquat dibromide42 and aspirin43 can induce ROS surges in cells, the DART system showed no SEAP response to these chemicals (Extended Data Fig. 2), suggesting that DART is not responsive to the free radicals generated by these chemicals (for instance, hydroxyl radicals (•OH) from H2O2 treatment44). Conversely, DC electrostimulation generates free electrons that can be received by different molecules to form a wider range of radicals and some of them can trigger the DART system by activating transgene expression from the synthetic PDART promoter. We also analyzed the resulting current, the ROS levels, SEAP response and impact on cell viability when the applied DC voltages were measured in a three-electrode setup incorporating an Ag/AgCl reference electrode, thereby demonstrating the tunability of the DART system in vitro (Extended Data Fig. 3). Next-generation sequencing revealed that DC electrostimulation at 5 V for 10 s had negligible impact at the transcriptome level, but 5 V for 25 s had a slight effect, with a small set of genes mainly associated with antioxidant response being differentially expressed compared to non-stimulated cells (Extended Data Fig. 4 and Supplementary List). These results indicate that stimulation times <25 s do not interfere with DART control and all follow-up experiments were conducted accordingly.
Characterization and validation of DART

Profiling transgene expression levels following voltage-dependent DC electrostimulation for 15 s revealed similar expression levels between 5 and 12.5 V, whereas higher voltages generated higher ROS levels that decreased cell viability (Fig. 2a and Supplementary Fig. 5a). With the DC power set to 5 V, the expression level of the target gene could be precisely adjusted (Fig. 2b and Supplementary Fig. 5b,c) and distinct induction profiles were maintained over longer periods of time (Fig. 2c), during which correlating timelapse fluorescence microscopical analysis of enhanced green fluorescent protein showed no substantial leakiness of the DART system (Supplementary Fig. 5d–f and Supplementary Videos 2 and 3). Nevertheless, exposure times beyond 30 s significantly decreased both cell viability and SEAP expres​sion(Supplementary Fig. 5g,h), likely as a result of gas bubbles and pH changes occurring at the electrodes37 (Supplementary Fig. 6a–e). The induction kinetics revealed significant levels of electrostimulated protein production in the culture supernatant within 4 h (Fig. 2d), which is consistent with the behavior of professional secretory cells32. Additionally, DC-powered transgene expression control was fully reversible, showing similar induction and repression profiles over several cycles of ON-to-OFF and OFF-to-ON switching (Fig. 2e).

Fig. 2: In vitro characterization of the DART system.
figure 2

a,b, SEAP levels 24 h after electrical stimulation in culture supernatants of HEK293 cells co-transfected with the DART constructs (pJH1003, pJH1004 and pJH1005). Stimulation for 15 s at the indicated voltages (a), or with 5 V DC for the indicated time periods (b). c, SEAP production kinetics over 72 h by engineered cells exposed to 5 V DC for the indicated periods of time. The induction factors were calculated between stimulated and non-stimulated group at 25 s. d, SEAP produced within 6 h by DART-engineered cells after exposure to 5 V DC for 20 s. The induction factors were calculated between the indicated groups. e, Reversibility of the DART switch. Engineered cells were alternately cultured for 24 h cycles in medium without electrostimulation (OFF) or treated with 5 V DC for 20 s (ON) and SEAP production was measured in the culture supernatants. Every 24 h, the culture medium was exchanged and the cell density was re-adjusted. f, SEAP produced by different mammalian cell lines transiently transfected with the DART constructs and stimulated with 5 V DC for 20 s. Non-stimulated cultures were used as controls. All data are mean ± s.d.; n = 4. P values were calculated between stimulated and non-induced control. Statistical designations with different colors refer to the corresponding data points with the same color (c).

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To further assess the versatility of the DART system, we transiently transfected a set of mammalian cell lines (Fig. 2f). Despite variations in transfection efficiency11 and ROS sensitivity, resulting in different fold inductions and maximum expression levels, DART functionality was validated in all the tested cell lines, including human mesenchymal stem cell-derived cell line (hMSC-TERT), suggesting that DART will be compatible with a wide range of applications (Fig. 2f and Supplementary Fig. 6f). Taking into account the fold induction, basal and maximum expression levels, we selected HEK293 and hMSC-TERT cells for use in follow-up experiments (Fig. 2f).
DART stimulation by off-the-shelf consumer batteries

To check the suitability of widely available DC power sources, as well as compatibility with portable and wearable electronic devices, we next examined electrostimulation of gene expression using standard off-the-shelf DC supplies such as alkaline batteries, button cells, mobile chargers and portable power banks. We first tested one, two and three 1.5 V AA and AAA batteries that provide 1.5, 3 and 4.5 V when connected in series, respectively. Although voltages <3 V required longer induction times of 2–60 min to produce sufficient ROS to trigger significant SEAP levels, stimulation with three AA or three AAA batteries for 10 s was sufficient to induce SEAP expression, which peaked at 25 s of stimulation (Fig. 3a–c and Extended Data Fig. 5a–c), showing induction profiles comparable to those generated by a 5 V power supply.

Fig. 3: SEAP expression by DART-engineered cells stimulated for the indicated time periods with various DC supplies.
figure 3

a–c, One (a), two (b) and three (c) Duracell or Energizer alkaline 1.5 V AA batteries. d, Two Duracell or Energizer lithium button cells (CR2032) provide about 6 V. e, One Duracell or Energizer 9 V block alkaline battery. f, One A220/504 A Exell battery. SEAP levels were measured 24 h after stimulation. All data are mean ± s.d.; n = 4. P values were calculated between stimulated and non-induced control.

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The 3 V CR2032 lithium button cell battery, which is widely used to drive wearable devices, triggered SEAP expression in up to 30 min, whereas a 6 V in-series setup of two CR2032s programmed peak SEAP expression levels in <20 s (Fig. 3d and Extended Data Fig. 5d). Furthermore, a 6 V in-series setup powered by four AA or four AAA batteries produced protein expression profiles comparable to those generated by the 2 × CR2032 configuration, corroborating the voltage-dependence of the electrogenetic gene switch independently of the type of power source (Extended Data Fig. 5e,f). This was further confirmed by using a mobile phone charger and a power bank that typically provide a 5 V output (Extended Data Fig. 5g,h).

We also tested higher-voltage batteries, including 9 V block, 12 V 23 A alkaline and 15 V Exell A220 504 A batteries in single (Fig. 3e,f and Extended Data Fig. 5i) or in-series tandem (Extended Data Fig. 5j–l) configurations. Measurements of SEAP expression confirmed an inverse correlation between voltage (1.5–30 V) and peak expression level times (5 s to 60 min) across all DC power sources (Fig. 3e,f and Extended Data Fig. 5i–l). Most notably, none of the tested battery configurations substantially impacted cell viability during the indicated stimulation times (Supplementary Figs. 7 and Cool, confirming that the DC-powered electrogenetic interface is robust, safe and tunable across a wide range of voltages, battery types and stimulation times.
Battery-powered insulin expression for the treatment of T1D

As a challenging in vivo proof-of-principle, we chose to treat experimental type1 diabetes (T1D), because diabetes is a chronic disease that shows dramatically increasing prevalence globally and requires dynamically demanding management45. For DC-sensitive control of insulin production and release, we established stable HEK293 and hMSC-TERT cell lines engineered for constitutive expression of KEAP1 (ITR-PhCMV-KEAP1-P2A-BlastR-pA-ITR, pJH1054), NRF2 (ITR-PhCMV-NRF2-pA:PRPBSA-ECFP-P2A-PuroR-pA-ITR, pJH1101) and NRF2-dependent expression of insulin (ITR-PDART4-SEAP-P2A-mINS-pA:PmPGK-ZeoR-pA-ITR, pJH1169, PDART4, OARE4-PhCMVmin) (Extended Data Fig. 6a). To maximize the dynamic range of insulin expression, we used the synthetic promoter variant PDART4, which contains four tandem ARE operator sites (Fig. 1f). Several monoclonal cell lines were profiled for DC-controlled insulin expression and the best-in-class hMSC-TERT cell clone DCINS (Extended Data Fig. 6b,c), which showed improved electrostimulated insulin fold induction and release compared to transiently transfected isogenic cell populations (Extended Data Fig. 6c), was selected for treatment of experimental T1D. We confirmed by qPCR and western blotting that the DCINS cell line had increased levels of KEAP1 and NRF2 transcripts and proteins when compared to the parental cell line (Extended Data Fig. 6d–g). Stimulation of DCINS cells with 4.5 V DC from three AA batteries for exposure times between 5 s and 25 s could precisely regulate SEAP (Fig. 4a) and insulin (Fig. 4b) expression levels and distinct induction profiles were maintained over longer periods of time (Fig. 4c). Examination of the induction kinetics revealed that electrostimulated protein production reached a significant level in the culture supernatant within 3 h (Fig. 4d), which is faster than in the case of transiently transfected cells (Fig. 2d). Furthermore, the DCINS cells exhibited excellent reversibility of SEAP (Fig. 4e) and insulin (Fig. 4f) expression in response to ON-OFF-ON or OFF-ON-OFF stimulation patterns at 24-h intervals.
Fig. 4: In vitro characterization of the monoclonal cell line.
figure 4

a,b, SEAP (a) and insulin (b) levels in culture supernatants of the monoclonal DCINS cells stably containing the DART system 24 h after electrical stimulation. The voltage to stimulate the cells was provided by three AA batteries (4.5 V DC) for the indicated periods of time. c, SEAP production kinetics during 72 h after exposure of DCINS cells to 4.5 V DC for the indicated time periods. The induction factors were calculated between non-stimulated and stimulated group at 25 s. d, SEAP produced during the first 2, 3 and 6 h by DCINS cells non-stimulated or stimulated with 4.5 V DC for 20 s. The induction factors were calculated between the indicated groups. e,f, Reversibility of DCINS cells expressing SEAP (e) and insulin (f). DCINS cells were alternately cultured for 24-h cycles in medium treated with 5 V DC for 20 s (ON) or without electrostimulation (OFF). Culture supernatant samples were collected every 12 h for analysis of SEAP and insulin production. The culture medium was exchanged and the cell density was re-adjusted every 24 h. All data are means ± s.d.; n = 4. The P value indicates the significance of differences in the mean values; indicated group versus the non-stimulated group (a,b,d). The statistical designations with different colors refer to the corresponding data points with the same color (c).

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As DC-electrostimulated DART-controlled insulin expression does not require any complex control electronics, we used a triple AA battery pack providing 4.5 V DC, wired via a simple manual ON/OFF power switch to two customized platinized acupuncture needles (Supplementary Fig. 9a–i and Supplementary Table 6) located 6 mm apart at the implantation site to stimulate DCINS subcutaneously implanted on the back of type1 diabetic mice (Fig. 5a,b). Before implantation, we confirmed that the DCINS cells microencapsulated in clinically licensed semi-permeable alginate showed precise time-dependent insulin release when stimulated with 4.5 V DC (Supplementary Fig. 9j). To confirm that insulin is produced and secreted in response to direct electrostimulation of implanted cells, we used a negative control group with similar dorsal implantation, but stimulated with acupuncture needles placed 3 cm away from the implant site. In the treated group, a single 4.5-V electrostimulation for 10 s per day attenuated fasting glycemia within 2 d and normoglycemia was restored over the whole treatment period of 4 weeks (Fig. 5c). In agreement with these results, glycated hemoglobin (HbA1c) levels were attenuated in electrostimulated T1D mice over 5 weeks of treatment, reaching similar levels to those of wild-type mice (Extended Data Fig. 7a). In contrast, when stimulating T1D mice with two electrically conductive sticky patches attached to the implantation site instead of the platinized acupuncture needles, the animals remained as hyperglycemic as non-stimulated T1D mice, indicating that this stimulation method cannot trigger insulin production from implanted DCINS cells (Extended Data Fig. 7b,c). We also assessed how mice responded to the acupuncture needle stimulation when pretreated with NAC (ROS scavenger) or inhibitors of ROS-generating enzymes. In line with the in vitro results, only NAC abrogated the effect of electrostimulation, with mice showing blood glucose and insulin levels similar to those of non-stimulated mice (Extended Data Fig. 7d,e). Macroscopic assessment of the implantation site 4 weeks after transplantation showed no obvious differences between stimulated and non-stimulated mice or compared to non-transplanted wild-type mice (Supplementary Fig. 10a–c). To confirm that stimulation has no impact on surrounding tissues, we analyzed tissues adjacent to the electrode site of stimulated and non-stimulated mice according to ISO 10993-6 (ref. 46). We observed no material or electrostimulation-related cytotoxicity and no notable local-immune response around the implantation site (Supplementary Fig. 10d–g and Supplementary Table 7). There was no apparent histopathological difference between the electrode-containing non-stimulated and electrode-containing electrically stimulated groups (Supplementary Fig. 10d–g and Supplementary Table 7). Profiling of inflammatory mediators in serum of non-stimulated and electrostimulated groups also showed no significant differences (Extended Data Fig. 8a–c). Also, the short electrostimulation had no apparent effect on blood pH (Extended Data Fig. 8d). Glucose tolerance tests (GTTs) revealed that electrostimulated insulin production and release not only restored glucose homeostasis, but also attenuated postprandial glycemic excursion compared to negative control groups without any implant, or with non-stimulated implants, or with implants but distant electrostimulation (Fig. 5d and Extended Data Fig. 8e). Glycemic control was completely reversible when the electrostimulation status was switched from OFF to ON or from ON to OFF at 3-d intervals (Fig. 5e). Electrostimulated animals had significantly higher blood insulin levels than the control groups, confirming that DC-powered induction of insulin production and secretion indeed restored glucose homeostasis in type 1 diabetic mice (Fig. 5f). Furthermore, we profiled the blood glucose levels several times a day, during 24 h after electrostimulation with DC 4.5 V for 10 s and did not observe any hypo- or hyper-glycemic episodes (Fig. 5g). In agreement with this finding, the time-course analysis of other biomarkers of insulin deficiency, namely insulin, ketones, triglycerides and glucagon, revealed no significant differences between electrostimulated T1D mice and wild-type animals, whereas those biomarkers were significantly lower (insulin) and higher (ketones, triglycerides and glucagon) in non-stimulated T1D mice, respectively (Extended Data Fig. 9).
Fig. 5: Adaptation and validation of the DART system to treat type1 diabetic mice.
figure 5

a, Scheme showing how encapsulated DART-engineered cells implanted in the back of mice are stimulated. b, Picture showing the customized acupuncture needles connected to the alkaline batteries. c, Fasting glycemia was recorded before implantation (day 0) and for four consecutive weeks after implantation in three groups of T1D mice with DCINS cell implants, namely non-stimulated mice, mice stimulated for 10 s at the implantation site (stimulated) and mice with cell implants stimulated for 10 s with electrodes placed on their back 3 cm away from the implant site (distantly stimulated). Wild-type mice and T1D mice without implants were also used as controls. d, Intraperitoneal GTT was performed on mice 3 d after implantation of microencapsulated cells and after fasting for 8 h. e, Reversibility of DART-mediated glycemic control. Microencapsulated cells were percutaneously electrostimulated at the implantation site while reversing the ON-OFF/OFF-ON stimulation every third day, using 4.5 V DC for 10 s as ON. f, Blood insulin levels of non-stimulated and electrostimulated animals were profiled 1, 2, 3 and 4 weeks after implantation. Stimulated and distantly stimulated (3 cm away from the implant site) groups were treated with 4.5 V DC for 10 s per day (c,f). g, Blood glucose excursions on day 4 after implantation. Blood samples were collected at several time points for analysis of glucose in wild-type mice and T1D mice non-stimulated and stimulated with DC 4.5 V for 10 s. Time zero corresponded to midnight and electrostimulation was performed at 6:00. All data are mean ± s.d.; n = 5; the values were normalized to the wild-type group. The experiment was performed once in g. P values indicates the significance of differences in the mean values; stimulated group versus non-stimulated group (blue) or wild-type group (black) (c,d); stimulated group versus non-stimulated group (g); two groups versus each other (e); and stimulated group versus the indicated group (f).

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Tunability of DART in vivo

To assess the tunability of the DART system in vivo, we stimulated T1D mice during different time periods (between 5 to 15 s) using two to five AA batteries and one 9 V block battery as power sources, providing 3, 4.5, 6, 7.5 and 9 V of DC, respectively. We observed voltage-dependent as well as time-dependent blood glucose (Fig. 6a,b) and blood insulin (Fig. 6c,d) tunability as well as concomitant fine-tuning of other biomarkers of insulin deficiency, ketones, triglycerides and glucagon (Extended Data Fig. 10a–f), showing that metabolic homeostasis could be restored by applying 4.5 V electrostimulation for 10 s or more (Fig. 6b,d) and confirming the in vitro tunability of DART-transgenic cells (Figs. 2b,c and 4a–c, Extended Data Figs. 3g,h and 7j and Supplementary Fig. 5c).
Fig. 6: Evaluation of the tunability of DART system in T1D mice.
figure 6

a–d, Blood glucose (a,b) and insulin (c,d) levels of T1D mice with DCINS cell implants electrostimulated with different voltages (3, 4.5, 6, 7.5 and 9 V) for 5 s (a,c) and for different periods of time (0, 5, 10 and 15 s) at 4.5 V (b,d). The voltages were applied with two to five AA batteries and one 9 V block battery, respectively. T1D and wild-type mice without any treatment were used as controls. The blood samples were taken after 6 h of fasting. The corresponding blood profiles of biomarkers of insulin deficiency, ketones, triglycerides and glucagon are shown in Extended Data Fig. 10a–f, Time-series blood glucose (e) and insulin (f) monitoring on day 4 after implantation in T1D mice non-stimulated or stimulated once, twice, three or four times per day with DC 4.5 V for 10 s according to the scheme in Extended Data Fig. 10g. The corresponding blood profiles of biomarkers of insulin deficiency, ketones, triglycerides and glucagon are shown in Extended Data Fig. 10h–j. All data are mean ± s.d.; n = 5; the experiment was performed once and values were normalized to the wild-type group. The P value indicates the significance of differences in the mean values. Statistical designations with different colors refer to the corresponding data points with the same color (e,f).

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Finally, we assessed how the implanted DART system responds to up to four electrostimulations per day, to mimic the need for multiple daily insulin injections in some patients with T1D. To mimic the pattern in humans, we scheduled repeated fasting–feeding cycles for the mice throughout the day (Extended Data Fig. 10g). In all electrostimulated groups, the glucose and insulin levels fluctuated around the wild-type levels and all showed significant differences from the non-stimulated group at each time point (Fig. 6e,f). To make a comprehensive assessment of the diabetic status of all groups, we took blood samples every 3 h in the daytime and tested for ketone bodies, triglycerides and glucagon. The results indicated that all the electrostimulation schedules, from one to four per day, significantly improved the glycemic state of diabetic mice (Extended Data Fig. 10h–j).
Discussion

DART provides a reversible and tunable electrogenetic interface operated by simple, readily available low-voltage DC sources, such as batteries that are widely used to power portable or wearable electronic devices18,47. Notably, DART requires very little power and overall energy to control target gene expression. As a single electrical stimulation using two electrodes 6 mm apart for only 10 s is sufficient to trigger production and release of sufficient daily insulin, we estimate the electrical DC power required for a daily insulin shot to be around 0.06 W, which would enable a simple 4.5 V triple AA battery pack to operate for more than 5 years, while providing a daily therapeutic dose. In principle, operating times could be further optimized by decreasing the resistance by reducing the electrode separation, or as we have shown, by using higher-voltage and higher-capacity battery packs to achieve even shorter induction times. Furthermore, control of DART requires only a simple manual electrical ON/OFF switch. Also, as DART can directly stimulate engineered cells that are microencapsulated in US FDA-approved alginate microcontainers that are clinically licensed and validated for human islet transplantation48 via WHO-approved and US FDA-licensed acupuncture needles49, the remote control is simple and does not require the use of complex, failure-prone bioelectronic implants, which are particularly challenging to operate in a tissue environment32. Indeed, our first electrogenetic cell implant device relied on the sensitization of the cells to electrical fields by coexpression of two ion channels linked to endogenous signaling cascades and encapsulation of the cells in a complex wireless-powered bioelectronic implant, activated by AC at high voltage with extended stimulation times32. In contrast, the DART technology capitalizes on intracellular ROS sensors that sensitize alginate-encapsulated cells to direct battery-powered low-voltage DC stimulation within seconds via two simple acupuncture needles and without the need to use or implant any electronics. In fact, electrostimulation by acupuncture needles is already standard practice in traditional Chinese medicine for the treatment of inflammation, chronic pain, muscle spasm and neurological disorders50,51, representing a therapeutic modality that is approved by the WHO49 and practiced on a worldwide basis. As the DART system does not need hours at higher voltages but only seconds at lower voltages to actuate transgene expression, it has higher energy efficiency and safety. Thus, we believe rapid, electronics-free direct battery-powered low-voltage DC control of therapeutic transgenes in human cells is a leap forward, representing the missing link that will enable wearables to control genes in the not-so-distant future.

On the molecular side of the electrogenetic interface, DART taps into the ubiquitous KEAP1/NRF2-mediated sensing of ROS. ROS production is a part of the cellular respiratory process34,52, but we found that intrinsic levels of ROS production and the KEAP1/NRF2-mediated ROS management system do not interfere with DART and do not substantially induce DART-specific promoters. Instead, human cells need to be sensitized to electro-inducible ROS production by ectopic expression of native KEAP1 and NRF2, which reroute ROS sensing to synthetic PDART promoters driving biopharmaceutical production. As with other synthetic transcription-control modalities, activation of endogenous genes cannot be ruled out completely7; however, comparative deep-sequencing analysis suggested that any such effect is minimal. Also, mass spectroscopic analysis of the culture medium showed no substantial difference between non-stimulated and electrostimulated cell cultures, indicating that DART activation does not cause sufficient electrolysis in the culture medium to perturb cellular systems. Furthermore, DART is exclusively composed of endogenous components, involving simple ectopic expression of native endogenous factors KEAP1 and NRF1 and target promoters assembled by fusing native tandem ARE elements to a minimal promotor box, which should minimize risks that can be associated with cell engineering, such as neoplastic transformation.

Qualitatively, electrostimulated ROS production is triggered by the creation of potentially hazardous chlorine ions and chlorine gas as well as protons and hydrogen gas at the anode and cathode, respectively; however, we found that electrostimulation had no negative impact either on the viability or the transcriptome of the cells or on the medium composition, presumably due to the low voltage and short induction times of only a few seconds. Indeed, a single daily electrostimulation of implanted engineered cells at 4.5 V for 10 s triggered production and release of sufficient insulin to restore normoglycemia in experimental T1D, exhibiting comparable efficacy to long-acting insulin therapies that can maintain fairly stable blood-sugar levels for 24 h18,22,32. DART control also provided sufficient insulin to rapidly attenuate postprandial glycemic excursion, as shown by GTTs. In addition, DART control was reversible and also finely tunable by varying the voltage and/or electroinduction period. Notably, we also evaluated several biomarkers of insulin deficiency in animals treated with the DART system using different batteries, different induction periods and different frequencies. The improvements of glucose and insulin levels confirm that DART is an efficient system. Furthermore, the improvement of ketone levels in diabetic mice would also reduce the risk of diabetic ketoacidosis.

While we chose DART-controlled insulin production for proof-of-concept validation, it should be straightforward to link DART control to the in situ production and dosing of a wide range of biopharmaceuticals. We believe simple electrogenetic interfaces such as DART that functionally interconnect analog biological systems with digital electronic devices hold great promise for a variety of future gene- and cell-based therapies, including closed-loop genetic interventions, real-time dosing and global telemetric monitoring by medical staff or algorithms.

Chromium6

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