Introduction

In recent years, significant progress has been made in engineering cells to respond in a programmable, user-defined manner. Synthetic biology1 approaches have enabled engineered cells to exhibit complex computer-like behavior2,3,4, endowing them with the ability to conduct diverse, non-native tasks5,6,7. Control over engineered biological systems, reliant on specific inputs, can be achieved at the transcriptional8,9,10, translational11,12 or post-translational level13,14,15,16,17, with transcriptionally controlled systems being the most widely adopted due to their robustness18,19. Many synthetic transcriptional switches are based on bacterial transcription factors responsive to small-molecule inputs, fused to viral or mammalian transactivation domains to drive transgene expression in mammalian cells9,10,20. Although these have been adopted to control expression of various therapeutic genes in mouse studies, they often require high concentrations of the input molecules, making them unsuitable for long-term translational applications. Alternative approaches to engineer sense-response systems leverage mammalian cell membrane receptors21, channels22, or intracellular sensing components23. These components are less immunogenic and are more likely to detect physiological or disease-relevant input concentrations. The signal transmission of these receptors often depends on native cell signaling cascades, which culminate in the activation of transgene expression from a synthetic promoter. Taking advantage of a vast repertoire of these mammalian sensors, cells have been engineered to sense microbe-derived formyl peptides24, bile acids25, menthol21, fatty acids26, inflammatory cytokines27,28, and to activate therapeutic gene expression in mouse models of liver injury25, chronic pain29, and psoriasis27. Both closed- and open-loop controlled biological systems offer promising approaches for precise regulation of cell-based therapies in response to physiologically relevant cues.

Many organisms, including humans, exhibit natural oscillations that affect a range of biological processes. These rhythmic fluctuations, known as circadian rhythms, are governed by an autonomous, intrinsic timekeeping system called the circadian clock. This clock operates in a roughly 24-h cycle and integrates external environmental changes and internal physiological cues to enable robust adaptation to the surrounding environment30,31. Environmental cues, termed zeitgebers, entrain the circadian rhythm by either advancing or delaying the circadian clock, thus ensuring synchronization with the solar day. The primary circadian clock, located in the suprachiasmatic nucleus of the hypothalamus coordinates various brain areas and peripheral tissues throughout the body via neural and hormonal signals32. It regulates the circadian secretion of diffusible endocrine signals, such as thyrotropin (TSH)33, melatonin (MTN)34, and cortisol35, which exhibit an oscillatory pattern over the course of the day. We hypothesized that such endogenous rhythmic signals could be repurposed to power the expression of therapeutic genes in engineered cells, enabling circadian regulation of cell therapies. As cortisol levels are highly susceptible to stress and external perturbations36,37, we focused on TSH and MTN as more reliable candidates for circadian input signals.

To implement this strategy, we design a gene switch using human receptors responsive to these circadian hormones and their associated signaling cascades. After screening various candidates, we identify the melatonin receptor 1A (MTNR1A), which relies on the native cAMP signaling cascade for signal transmission, as the most promising component for constructing a circadian gene switch. Fine-tuning MTNR1A expression and optimizing the reporter construct yielded a system with minimal leakiness and a high dynamic range, responsive specifically to melatonin levels characteristic of the nocturnal phase, while remaining inactive during daytime levels. The switch exhibits tunable, robust, and reversible kinetics of transgene expression. To illustrate the therapeutic potential of this circadian rhythm sense-response system, we connect the melatonin-sensing module to a glucagon-like peptide-1 (GLP-1) expression actuator module. GLP-1 is a clinically approved treatment for type-2 diabetes and obesity. Engineered cells implanted in mice detect and respond to both physiological and experimentally manipulated melatonin, producing GLP-1 at night or in response to exogenous melatonin. In type-2 diabetic mice, implantation of these engineered cells successfully restores normoglycemia through melatonin-induced GLP-1 release. This work presents a proof-of-concept for harnessing endogenous hormonal rhythms to drive therapeutic gene expression in engineered cells. Such circadian gene switches hold promise for a wide range of applications, including drug discovery, chronobiology research and precise regulation of next-generation cell and gene therapies.

Results

Design of mammalian circadian rhythm sense-response systems

To build a circadian rhythm-controlled transcription system, we focused on two circadian biomarkers that display pulsatile levels in the bloodstream, TSH and melatonin, which are both present at low levels during the day and peak during the night (Fig. 1a). An ideal circadian sense-response system would exhibit minimal background transgene expression under day-time hormone levels, while elevated night-time levels would trigger receptor activation, and robust transgene expression (Fig. 1b). We screened candidate human G protein-coupled receptors (GPCRs) that could serve as biosensors for these inputs and translate a circadian signal to transcription activation of desired transgenes. Specifically, we tested the TSH receptor (TSHR) and the melatonin receptors MTNR1A and MTNR1B. All candidate systems were evaluated in transiently transfected human embryonic kidney (HEK293T) cells using human placental secreted embryonic alkaline phosphatase (SEAP)38 as a reporter protein, expressed under the control of a synthetic promoter containing cAMP response elements (CRE) to monitor activation of the cAMP signaling pathway. TSH stimulation led to SEAP expression via activation of constitutively expressed TSHR (Fig. 1c); however, high basal leakiness in the absence of TSH resulted in poor inducibility. In contrast, of the two melatonin receptors tested, MTNR1A showed strong performance with low background activity and robust SEAP induction upon melatonin stimulation (Fig. 1d). MTNR1B, on the other hand, failed to elicit melatonin-dependent SEAP expression (Fig. 1d). Based on these results, the melatonin-inducible system using MTNR1A was identified as the most promising approach for circadian rhythm-driven transgene expression and was selected for further optimization and characterization.

Fig. 1: Design of mammalian circadian rhythm sense-response systems.

a Oscillatory pattern of circadian biomarkers TSH and melatonin in vivo, showing distinct peaks during the night. b Schematics of a cell-based system responsive to circadian biomarker. Circadian rhythm receptor ectopically expressed on the cell surface monitors circadian biomarker levels. Elevated concentration triggers the receptor and activates a signaling pathway that subsequently leads to transgene expression activation and secretion of protein of interest (POI) from the cells. c Transgene expression activation by GPCR-based TSHR receptor. Cells were transfected with TSHR (PSV40-TSHR-pA, pSG14) and a reporter construct containing a PcAMP-driven SEAP (pVH421) expression cassette. SEAP secretion levels were determined 24 h after induction with TSH (30 pM). d Transgene expression activation by GPCR-based melatonin receptors: MTNR1A and MTNR1B. HEK293T cells were co-transfected with a combination of melatonin receptor MTNR1A (PmPGK-MTNR1A-pA, pNF111) or MTNR1B (PCMV-MTNR1B-pA, pNF82) and a reporter construct containing PcAMP-driven SEAP expression cassette (pNF421). After induction with 100 nM melatonin (MTN) for 24 h, SEAP secretion levels in the cell supernatant were determined. In (c, d), data are shown as mean ± SD of n = 3 biological replicates. Individual data are shown as filled circles with fold induction indicated above the bars. In (c, d), statistical significance was calculated with a two-tailed unpaired t-test. Source data are provided as a Source Data file.

Tuning the melatonin-responsive transcriptional switch

Given that several pathways have been implicated in melatonin signaling39,40, we assessed whether MTNR1A activation by melatonin could induce transcription from a range of reporter promoters responsive to calcium signaling (NFAT), mitogen-activated protein kinase (MAPK/ERK), JAK/STAT, NF-κB, TGF-β/Smad and AhR. Using transient co-transfection assays, we observed that melatonin-inducible transgene expression was restricted to constructs containing CRE sites (Fig. 2a), confirming that in our system, MTNR1A primarily signals through the cAMP pathway. To increase the performance of the circadian rhythm-responsive gene expression system, we systematically screened combinations of constitutive promoters driving MTNR1A expression (CMV, SV40, mPGK, or EF1α) with different synthetic CRE-containing promoters controlling SEAP expression, in order to identify configurations offering both high inducibility and strong transgene expression capacity. We evaluated three reporter plasmids, pCK53 (Fig. 2b), pSP16 (Fig. 2c), and pVH421 (Fig. 2d), which differ in the spacer sequences between CRE sites and their downstream minimal promoters (Supplementary Table 1). Head-to-head comparisons identified the combination of the mPGK promoter (PmPGK) driving MTNR1A and the pVH421 reporter as the most effective, yielding the highest SEAP expression and robust fold induction in response to melatonin (Fig. 2e, f). This optimized configuration was used in all further experiments.

Fig. 2: Optimization of melatonin sense-response system.

a MTNR1A signaling orthogonality. MTNR1A was co-transfected with reporters for the indicated signaling pathways, followed by MTN induction for 24 h before determining SEAP expression. b–d Effects of various constitutive promoters driving MTNR1A expression and PcAMP promoter variants on the system’s inducibility and SEAP expression capacity. MTNR1A was placed under the control of four constitutive promoters with different strengths, namely PSV40, PCMV, PmPGK, and PEF1α. After combinatorial co-transfection with PcAMP promoter variants (pCK53, pSP16, and pVH421; see Supplementary Table 1 for details) driving SEAP expression, cells were treated with melatonin (100 nM) and SEAP was profiled 24 h thereafter. e, f Heat-maps illustrating the performance of promoter and reporter combinations presented as maximal transgene expression capacity (e) and fold-inductions, calculated as a ratio of melatonin-induced to un-induced SEAP expression levels (f) (n = 3 biological replicates). In (a–d), data are shown as mean ± SD of n = 3 biological replicates, with individual data shown as filled circles. Statistical significance was calculated with a two-tailed unpaired t-test. Source data are provided as a Source Data file.

Characterization of the melatonin-responsive system

To assess the generalizability of the system, we tested its functionality across multiple mammalian cell lines, including Chinese hamster ovary cells (CHO), widely used in biopharmaceutical manufacturing, and human mesenchymal stem cells (hMSC), a common cell chassis in many cell therapy trials7. Broad responsiveness to melatonin was observed across all tested lines, although to varying extents, likely reflecting differences in transfection efficiency (Fig. 3a). In humans, plasma melatonin levels typically oscillate from low pM levels during the day phase41 to nighttime peaks of up to 700 pM42. We therefore evaluated whether our transcriptional switch exhibits appropriate sensitivity to the varying melatonin levels to differentiate between circadian phases. Dose-response assays showed that SEAP expression was significantly induced at melatonin concentrations as low as 100 pM (Fig. 3b), aligning with night-time physiological levels.

Fig. 3: Characterization of circadian rhythm sense-response system.

a Melatonin-inducible transgene expression across various mammalian cell lines transfected with MTNR1A (pNF111) and PcAMP driven SEAP reporter (pVH421). SEAP activity was determined 24 h after MTN (100 nM) treatment. b Sensitivity and tunability of the circadian rhythm sense-response system in HEK293T cells co-transfected with pNF111 and pVH421. SEAP expression was profiled 24 h after addition of indicated melatonin levels. c Sensitivity and adjustability of the circadian rhythm sense-response system by clinically approved drugs. Cells co-transfected with pNF111 and pVH421 were induced with MTNR1A agonists at the indicated concentrations for 24 h before determining SEAP expression levels. d Melatonin-induced secretion kinetics of SEAP and nLuc from HEK293T cells transfected with pNF111 and either SEAP (pVH421) or nLuc (pNF392) reporter plasmids. Cells were induced with 10 nM MTN and SEAP or nLuc expression levels were determined at indicated time points after induction. e Melatonin-dependent SEAP secretion from a monoclonal cell line (C16) with a genome integrated MTNR1A and PcAMP driven SEAP reporter construct. SEAP activity was profiled 24 h after induction with indicated MTN concentrations. f SEAP secretion dynamics from clone C16. Cells were treated with specified MTN concentrations and SEAP levels were determined at indicated time points. g Reversibility of melatonin-induced SEAP expression. Cells were cultivated for 96 h, with media exchanged every 24 h, alternating between standard or melatonin (10 nM)-supplemented medium. SEAP levels were determined at indicated time points. h Melatonin-dependent GLP-1 secretion from a monoclonal cell line (C33) with a genome integrated MTNR1A (pNF396) and PcAMP driven GLP-1 construct (pNF395). GLP-1 levels were determined by ELISA 48 h after induction with the indicated MTN concentrations. In (a–d, f, g), data are shown as mean ± SD of n = 3 biological replicates and in (e) as mean of n = 2 biological replicates. In (h), data are shown as mean ± SD of n = 4 biological replicates. Where no bar is shown, the SD is smaller than the symbol. In (a, c, d, h), individual data are shown as filled circles. Statistical significance was calculated with a two-tailed unpaired t-test in (a, d) or one-way ANOVA in (c, f, h). Source data are provided as a Source Data file.

To explore the potential for open-loop control, we tested the system’s responsiveness to four clinically approved MTNR1A agonists, namely piromelatine, tasimelteon, ramelteon, and agomelatine43 (Fig. 3c). These compounds all triggered dose-dependent SEAP expression, offering a pharmacologically tunable method for controlling transgene output. Similar sensitivities were observed across all the agonists, except for piromelatine, which exhibited a lower dynamic range. Importantly, these agonists feature extended half-lives compared to melatonin, making them more suitable for sustained in vivo applications. Next, we characterized the expression kinetics of the system by tracking the levels of SEAP and nanoluciferase (nLuc) over time, following melatonin stimulation (Fig. 3d). A significant increase in reporter expression was observed at 6 h post-induction in comparison to non-stimulated controls.

For long-term experiments, we established cell populations with genomically integrated switch components using a Sleeping Beauty transposase-based system44. Single cell clones were isolated via FACS, and several high-performing clones were identified based on transgene expression and fold induction (Supplementary Fig. 1a, b). One of the top performing clones showed up to 40-fold induction and exhibited dose-dependent SEAP expression upon melatonin treatment (Fig. 3e). We further profiled its secretion dynamics across a range of melatonin concentrations over time (Fig. 3f). Additionally, the system could be toggled ON or OFF by alternately culturing the cells in melatonin-containing or melatonin-free medium, demonstrating reversible transgene expression (Fig. 3g).

Next, to showcase the therapeutic ability of the circadian rhythm sense-response system, we focused on obesity, a major health burden in the 21st century45,46, and selected GLP-147 as the effector molecule due to its well-established clinical efficacy47. We connected the melatonin-sensing module to an actuator encoding GLP-1 and nLuc, separated by a self-cleaving peptide, allowing simultaneous regulation and streamlined screening. A stable cell population with genomically integrated switch components was generated, and high-performing clones were identified based on nLuc expression and fold induction (Supplementary Fig. 2a). Secondary screening of the top five candidates confirmed dose-dependent production of the transgene products (Supplementary Fig. 2b), with clone 33 (C33) identified as the top performer; this clone was selected for all further experiments. This optimized cell line, named HEKGLP-1, exhibited melatonin-dependent GLP-1 production, with an induction level of above 100 pM (Fig. 3h)—aligning with physiological night-time melatonin levels.

In vivo validation of melatonin-induced GLP-1 secretion

To evaluate the therapeutic potential of the melatonin-responsive system in vivo, HEKGLP-1 cells were encapsulated in coherent semipermeable alginate-poly-L-lysine-alginate microbeads (Fig. 4a), a clinically available cell delivery strategy for treatment of diabetes48. In vitro, melatonin treatment of encapsulated cells led to robust GLP-1 secretion compared to untreated controls, confirming effective induction within the capsule matrix (Fig. 4b). Encapsulated HEKGLP-1 cells were then implanted into melatonin-deficient C57BL/6J mice (Supplementary Fig. 3). Following oral administration of exogenous melatonin, GLP-1 secretion was induced in vivo (Fig. 4c), with similar activation kinetics to the reporter proteins in vitro (Fig. 3b). To evaluate the potential to synchronize therapeutic transgene expression with endogenous melatonin oscillations, we implanted encapsulated HEKGLP-1 into C3H/HeJ mice49, which produce melatonin endogenously (Supplementary Fig. 3). Circulating GLP-1 levels were significantly higher in C3H/HeJ mice compared to C57BL/6J mice, indicating activation of HEKGLP-1 by endogenous melatonin and robust therapeutic gene expression (Fig. 4d, left). These results confirmed the existence of strain-specific differences in melatonin production. As expected, melatonin levels increased under light deprivation, which stimulates pineal synthesis50, and were suppressed under constant illumination (Fig. 4d, right), as determined by a custom-designed reporter assay (Supplementary Fig. 4). It is important to note, however, that mouse and human circadian physiology and melatonin profiles differ significantly51,52. Further studies will be required to determine precisely how therapeutic transgene expression can be coupled to the human circadian rhythm.

Fig. 4: Validation of the melatonin-induced therapeutic transgene product expression in mice.

a Schematics of HEKGLP-1 cells, microencapsulated in microbeads and implanted into mice. The synthetic gene network continuously tracks the phase of the diurnal rhythm by sensing environmental melatonin levels and programs cells to produce GLP-1 when melatonin levels are high. The microscopy image shows cells inside alginate-poly-L-lysine-alginate beads (scale bar: 200 µm). b GLP-1 secretion from encapsulated HEKGLP-1 cells cultivated without or with melatonin (1 nM) profiled in supernatants 48 h after induction. Numbers show fold-changes between GLP-1 levels of melatonin-treated and GLP-1 levels of DMSO-treated (control) samples. c Activation kinetics of melatonin-dependent target gene expression in vivo. One day after implantation of 5 × 106 microencapsulated HEKGLP-1 cells into C57BL/6J mice, animals were given a single oral dose of 710 µg/kg melatonin, then GLP-1 levels in the bloodstream were monitored over 24 h. Mice receiving ddH2O were used as negative controls. d GLP-1 activation in response to endogenous melatonin levels (left) and light-dependent manipulation of endogenous melatonin production (right) in mice. Male C3H/HeJ or C57BL/6J mice were housed in the dark or exposed to constant illumination (1 mW/cm2, 24 h) before receiving intraperitoneal implants of microencapsulated HEKGLP-1 (5 × 106 cells per mouse). GLP-1 levels (right) and corresponding blood melatonin levels (left) were quantified 24 h after implantation. e Melatonin-dependent transgene expression in vivo. One day after implantation of microencapsulated HEKGLP-1 cells into C57BL/6J mice, animals received various doses of melatonin and GLP-1 levels in the bloodstream were quantified after 24 h. f, g Therapeutic efficacy of melatonin-dependent GLP-1 production. db/db mice received intraperitoneal implants of microencapsulated HEKGLP-1 (5 × 106 cells per mouse) and indicated MTN oral dose. GLP-1 and fasting glycemia levels were recorded after 24 h. C57BL/6J mice (WT) were used as healthy controls. Data in (b) are shown as mean ± SD (n = 4 biological replicates) and in (c–g) as mean ± SEM (n = 5 mice per group). Therapeutically active GLP-1 concentrations in the bloodstream of db/db mice are indicated by a dashed line ( > 50 pM; efficacy threshold). Statistical significance was calculated with a two-tailed unpaired t-test in (**b–**d) or one-way ANOVA in (e, f, g). Source data are provided as a Source Data file.

To explore pharmacologically controlled activation, we leveraged the melatonin-deficient background of C57BL/6J mice49, where basal GLP-1 expression remained below the ~50 pM threshold required for therapeutic efficacy53. This low baseline effectively locked the system in an OFF state (Fig. 4d, left). Upon oral melatonin administration, GLP-1 expression increased in a dose-dependent manner (Fig. 4e), demonstrating controllability of the gene switch with this clinically approved over-the-counter drug typically taken at night. Finally, we evaluated therapeutic efficacy in type-2 diabetic db/db mice. Implantation of encapsulated HEKGLP-1 cells followed by titrated oral melatonin dosing led to graded increases in circulating GLP-1 levels (Fig. 4f), ultimately restoring normoglycemia once therapeutically effective thresholds were reached (Fig. 4g). This suggests the feasibility of developing personalized nighttime medications where therapeutic protein secretion is precisely coupled to endogenous or exogenous sleep-promoting hormones in the bloodstream.

Discussion

The circadian rhythm sense-and-response system developed in this study expands the synthetic biology toolbox by enriching the repertoire of receptor-based gene-switches54 available to augment human cells with new functionalities operating in a predictable, user-defined manner. To minimize immunogenicity and maximize translational potential, we focused on human-based sensors to build the gene switch. Among the receptors tested for their ability to sense molecules with oscillatory patterns in vivo, MTNR1A conferred the most robust transgene activation in response to elevated melatonin. While previous works report decreased cAMP levels upon MTNR1A activation39, our results suggest that HEK293T cells overexpressing MTNR1A can elevate cAMP levels in response to melatonin, leading to CREB activation and induction of cAMP-dependent promoters. We confirmed that only promoters containing CRE were responsive to melatonin, while synthetic promoters driven by alternative signaling pathways were inactive.

We systematically characterized different combinations of constitutive promoters driving MTNR1A expression and synthetic promoters driving transgene expression, achieving either tight regulation of transgene expression or a balance of high expression and low leakiness, thereby making it possible to tailor system performance for specific applications. Importantly, the sensitivity of the developed system matches physiologically relevant melatonin concentrations41, so that the system is switched OFF at daytime melatonin levels, while the increase of melatonin during the night-phase of the diurnal rhythm triggers robust transgene expression, effectively distinguishing between phases of the circadian rhythm cycle. However, it should be noted that endogenous melatonin levels decline with age41, and this may cause insufficient therapeutic transgene activation in vivo, potentially limiting translational applications in older populations. Nevertheless, in such cases, readily available melatonin supplements55 could be used to provide sufficient levels to activate transgene expression. Alternatively, we showed that clinically approved melatonin receptor drug agonists43, such as Valdoxan® or Hetlioz®, with improved pharmacodynamic and pharmacokinetic properties, can trigger transgene expression, and thus could also enable exogenous control of the therapeutic output.

The present system with GLP-1 as a therapeutic output may have translational potential for circadian rhythm-regulated once-daily therapy for obesity47 or type-2 diabetes treatment56. The engineered cells enabled melatonin-dependent GLP-1 expression, with robust GLP-1 production at melatonin levels that occur in the night phase of the diurnal rhythm. Further, when implanted in vivo into C3H/HeJ mice—a standard mouse model with an intact biosynthesis pathway for endogenous melatonin49,52—the cells could distinguish different melatonin concentrations produced by circadian manipulations, producing corresponding GLP-1 output levels. In contrast, upon implantation into melatonin-deficient C57BL/6J mice, we showed how externally applied melatonin enabled high and dose-dependent activation of GLP-1 release into the circulation. Furthermore, this strategy enabled restoration of normoglycemia in type-2 diabetic db/db mice. In providing a cell-therapy approach where therapeutic protein secretion is precisely coupled to circadian biomarkers, this work may pave the way to the development of personalized night-time medication regimens for patients suffering from sleep disorder-associated metabolic diseases. Current treatment regimens using GLP-1-derived medications, such as semaglutide57, are typically administered as once-weekly injections, which can be inconvenient for long-term use from the patient’s perspective and may cause undesirable side effects58,59 due to high systemic doses. A promising alternative is gene therapy delivering constitutively expressed GLP-1 via viral vectors such as adeno-associated virus60. However, this approach lacks control over therapeutic protein levels once expression is established in the patient. Our system addresses this limitation by leveraging endogenous melatonin oscillations for basal control, while also permitting external modulation of transgene expression. MTNR1A agonists can be used to enhance GLP-1 production, while antagonists61 could allow suppression in the case of adverse effects. This dual-mode regulation promises precise therapeutic dosing, minimizes side effects, and holds the potential for improved clinical outcomes.

We believe this system provides a foundation for gene- and cell-based therapeutic applications that require once-daily therapeutic dosing. Further, the actuator is modular and compatible with a wide range of transgenes beyond GLP-1, enabling easy substitution with alternative therapeutic proteins—such as monoclonal antibodies, erythropoietin62, or other biologics to address diverse disease contexts. This would be particularly valuable for chronic conditions requiring steady-state therapeutics, while still allowing dose adjustment in the form of a pill. Alternatively, as a gene-based therapy, the genetic components of the circadian sense-response system could be delivered to cells exposed to higher melatonin levels, such as those close to the pineal gland or cerebrospinal fluid, where melatonin levels can be up to 20 times higher than in the general circulation63.

The platform could also serve as a cell-based biosensor for studying circadian regulation in vivo. Engineered cells responsive to melatonin fluctuations could be used to investigate how specific biological or environmental conditions influence melatonin dynamics. A luminescence-based output would enable non-invasive, real-time monitoring in living organisms.

Due to its high sensitivity and robust performance, the system has strong potential for high-throughput screening of candidate small molecules or biologics for the ability to act as MTNR1A agonists, providing a high-performance, cell-based tool that should greatly facilitate the drug discovery process43. Its characteristics make it well-suited for identifying therapeutic candidates while providing deeper insights into physiological function—accelerating the discovery of new therapeutics targeting circadian regulation, sleep disorders, and related physiological processes. Finally, our system complements recent chronogenetic approaches that use core clock gene promoters64, contributing to the advancement of the field of chrono-pharmacology65. By leveraging the temporal dynamics of hormonal rhythms, this platform opens up avenues for circadian-informed therapeutic interventions.

Methods

Ethical statement

This study was carried out in full compliance with all relevant ethical regulations and animal welfare legislation in China. The experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Westlake University, conducted in accordance with the Animal Care Guidelines of the Ministry of Science and Technology of the People’s