A synthetic angiotensin II/ACE2-based hormone shunt controlling experimental hypertension

Domain: neuroscience
Published: 2026-04-11
Editorial outcome: major_revision

Abstract
Arterial hypertension is the leading preventable cause of various life-threatening medical conditions and comorbidities that affect over 1.3 billion adults, causing over 7.5 million annual deaths worldwide. Capitalizing on a synthetic biology-inspired engineering approach, we design a fully human, antihypertensive gene circuit called ARCH (autonomous regulator of chronic hypertension), which precisely monitors and efficiently controls angiotensin-dependent, renin-angiotensin system (RAS)-driven hypertension, in which angiotensin II (Ang-II) is persistently elevated, in a reversible, self-sufficient, and closed-loop manner. We utilize the ectopically expressed native type-1 angiotensin receptor (AT1R) to monitor the blood level of the hypertension biomarker Ang-II. We then rewire the AT1R activation during hypertension to a synthetic promoter to coordinate the expression of clinically licensed, secretion-engineered, soluble therapeutic angiotensin-converting enzyme 2 (stACE2), which degrades Ang-II and restores normotension. Implantation of microencapsulated ARCH-transgenic human cells into male hypertensive mice restores and maintains normal blood pressure.

Introduction
Arterial hypertension is a multifactorial medical condition involving dysfunction of the renin-angiotensin system (RAS), which fine-tunes blood pressure, fluid and electrolyte balance, and systemic vascular resistance 1, 2. Lifelong treatment is required to prevent a range of life-threatening complications 3 such as acute aortic syndromes 4, acute myocardial infarction 5, and stroke 5, as well as a variety of comorbidities including metabolic syndrome 6, blindness 7, diabetes 8, and dementia 9. Arterial hypertension currently affects over 30% of the global population of individuals above the age of 50 years, having dramatically increased since the mid-2000s 10, and is the major cause of death and disability throughout the world 2. Since persistent high blood pressure develops slowly over time, often without obvious symptoms, arterial hypertension remains undiagnosed in almost 40% of the population, developing into a "silent killer" 11.

Current treatment options are limited to lifelong daily use of antihypertensive prescription drugs 12 including calcium channel blockers (e.g., nifedipine), angiotensin II receptor blockers (e.g., valsartan), diuretics (e.g., chlorthalidone), angiotensin-converting enzyme (ACE) inhibitors (e.g., enalapril), and beta blockers (e.g., atenolol). Unfortunately, each class of drugs has severe contraindications, dosing challenges, and multiple side effects, including asthma, miscarriage, and diabetes 13, and a single drug is often not sufficient to restore normal blood pressure. Thus, despite the availability of a wide range of licensed antihypertensive drugs that generate over 25 billion USD in revenue annually 14, over 80% of patients do not have their blood pressure under good control 2 and medication adherence remains below 50% 15.

Consequently, new antihypertensive treatment strategies are urgently needed. This is particularly challenging, since blood-pressure homeostasis is a complex process intricately fine-tuned by the RAS 1. When blood flow is reduced or \u03b21-adrenergic receptors are stimulated, the proprotein convertase furin in juxtaglomerular cells of the kidney activates endogenous systemic prorenin, releasing renin into the circulation 16. In the bloodstream, renin converts liver-released angiotensinogen into the decapeptide angiotensin I, which is cleaved by ACE on the surface of vascular endothelial cells to generate the octapeptide angiotensin II (Ang-II) 17. Ang-II activates type-1 angiotensin receptor (AT1R), which mediates a potent vasoconstrictive effect, resulting in an increase of blood pressure 18. Ang-II also triggers the delivery of aldosterone, which increases the blood pressure by promoting reabsorption of sodium and release of potassium 19.

Blood pressure homeostasis is maintained by the action of angiotensin-converting enzyme 2 (ACE2) 18. ACE2 is present in membrane-bound form (mACE2) on many cells in the intestines, kidney and heart until it is converted to soluble ACE2 (sACE2) by the enzyme ADAM17 17 20. sACE2 indirectly lowers the blood pressure by catalyzing the hydrolysis of Ang-II to angiotensin 1-7, a vasodilator that directly decreases blood pressure by binding to MasR receptors 21. mAECE2 and sACE2 have recently attracted attention as drug targets and decoy therapeutics for emergency Covid-19 treatment since mACE2 interacts with the spike protein of many coronaviruses, including SARS-CoV-2, and serves as their cellular entry point 22, 23.

Despite the development of many medications that interfere with the vasoconstrictive or vasorelaxant axes of the RAS to maintain blood pressure within the optimal range 24, dosage and timing must be carefully controlled, and poor patient compliance can have severe consequences 25. Indeed, dosing and medication adherence remain major concerns in the long-term management of many chronic disorders 15, 26. One approach to overcome these issues is to develop cell-based therapies using implanted cells that dynamically rewire biomarker sensing to secretion of protein therapeutics in a closed-loop manner. Indeed, such devices have the potential to be foundation stones of future personalized medicine 27-30. Specifically, genetic closed-loop systems in which critical blood biomarker concentrations dynamically manage the release of biopharmaceuticals that attenuate the medical condition and decrease the biomarker load would be able to autonomously restore and maintain a healthy homeostatic state. Closed-loop systems have already been successfully validated for the treatment of experimental arthritis 31, 32, obesity 33, and diabetes 34-36.

While Ang-II is not a universal biomarker of hypertension and Ang-II-independent forms exist 37, 38, ARCH is designed to target angiotensin-dependent hypertension, in which pathological Ang-II elevation can persist over extended periods across diverse etiologies (e.g., genetic predisposition, lifestyle, obesity, trauma) 39. Moreover, the well-known inter-individual variability in Ang-II dynamics motivated a closed-loop precision-therapy design, in which the feedback controller autonomously self-tunes therapeutic output to patient-specific needs. Likewise, owing to its central role in vasorelaxant counter-regulation via enzymatic cleavage of Ang-II, soluble ACE2 (sACE2) is an attractive antihypertensive protein therapeutic whose clinical safety and dosing have been evaluated in humans, including studies in patients with COVID-19 40. Notably, elevated Ang-II has also been implicated in pathophysiological processes beyond hypertension, including cardiovascular remodeling 41, atherogenesis 42, renal fibrosis 43, insulin resistance 44, and tumor angiogenesis 45, underscoring the broader relevance of Ang-II homeostasis. Here, we describe a fully human, antihypertensive gene circuit called ARCH (autonomous regulator of chronic hypertension), which automatically restores and maintains normal blood pressure levels in mice suffering from angiotensin-dependent hypertension through modulation of Ang-II homeostasis by dynamically rewiring ectopic AT1R-mediated Ang-II sensing to a chimeric promoter driving the expression of secretion-engineered, stabilized ACE2 (stACE2).

Results
Design and validation of a human antihypertensive sensor-effector shunt. Binding of Ang-II to the G-protein-coupled receptor (GPCR) AT1R serves as the central control unit in the vasoconstrictive axis of the RAS 18. Therefore, Ang-II is a key biomarker and AT1R is a native biosensor of arterial hypertension 39. Upon binding of Ang-II, AT1R interacts with endogenous G-proteins (G\u03b1s, G\u03b1q/11, G\u03b112/13) to activate the cyclic adenosine monophosphate (cAMP), Ca2+, and Rho GTPase-dependent intracellular signaling cascades 46. These in turn can be rerouted to a synthetic promoter (PCSN, CRE-SRE-NFAT-PCSNmin) driving therapeutic interventions such as the expression of a desired protein therapeutic (Fig. 1a). To endow human cells with Ang-II sensor capacity, we ectopically expressed human AT1R in human embryonic kidney cells (HEK-293T) using a constitutive mammalian expression vector (pGU52, PCAG-hAT1R-pA). hAT1R is exclusively localized in the plasma membrane of transfected HEK-293T cells, and binding of Ang-II triggers the cAMP signaling pathway and activates synthetic promoters such as PCSN (Supplementary Fig. 1). Indeed, upon co-transfecting the constitutive AT1R expression vector (pGU52, PCAG-hAT1R-pA) and a reporter expression vector encoding PCSN-driven stabilized secretion-engineered luciferase (pGU23, PCSN-SS-nLuc-Fc-pA), Ang-II-inducible reporter gene expression was observed in a variety of rodent (BHK-21, NIH/3T3), canine (MDCK), primate (COS-7), and human (HEK-293T, hMSC, HepG2) cells, indicating that AT1R-mediated Ang-II sensing is functional in a wide variety of mammalian cell types and species, which is crucial for translational potential (Fig. 1b). Since the Ang-II sensor performed most robustly in HEK-293T cells, we used this human cell line for all subsequent experiments to establish an all-human ARCH circuit. The comparatively strong response in HEK-293T cells likely reflects cell-type-specific factors, including molecular context and transfection efficiency 47. Notably, HEK-293T cells express the Simian Virus 40 (SV40) large T-antigen, which enhances episomal plasmid replication and supports high levels of transient transgene expression, making this cell line particularly useful for preclinical gene circuit validation 48-52. To determine the optimal Ang-II triggered AT1R-mediated signaling route, we designed a variety of isogenic vectors containing synthetic promoters with specific operator sites or combinations thereof, such as the nuclear factor of activated T cells (NFAT; pMX57, PNFAT3-SEAP-pA), cAMP response element (CRE; pCK53, PCRE-SEAP-pA), sterol response element (SRE; pGU232, PSRE6-SEAP-pA), CRE combined with SRE and NFAT (CSN; pYL1, PCSN-SEAP-pA), and nuclear factor kappa light chain enhancer of activated B cells (NF\u03baB; pKR32, PNF\u03baB-SEAP-pA) to express reporter genes in an Ang-II-inducible manner (Fig. 1c). Since the broad-spectrum daisy-chaining the CRE, SRE, and NFAT operator modules showed best-in-class Ang-II-triggered AT1R-mediated activation, it was chosen to drive effector gene expression in follow-up experiments (Fig. 1c).

Characterization and optimization of the antihypertensive synthetic human sensor-effector shunt. To assess the specificity of AT1R-mediated cAMP signaling and the downstream PCSN response, HEK-293T cells co-transfected with pGU52 (PCAG-hAT1R-pA) and pGU23 (PCSN-SS-nLuc-Fc-pA) were stimulated with a panel of angiotensin analogs spanning both the classical and the counter-regulatory arms of the RAS 53 (Fig. 2a). AT1R was fully activated by Ang-II, and to a lesser extent by the Ang-II degradation product Ang-III, whereas the other angiotensin analogs tested did not trigger receptor signaling (Fig. 2b). Aiming to further boost the sensitivity of the sensor-effector shunt, we co-transfected varying amounts of receptor (pGU52) and reporter (pGU23) plasmids. The relative ratio of the system components indeed impacted on the overall sensitivity, and optimal performance was observed at a 2:3 receptor-reporter ratio (Fig. 2c, d). The sensor-effector shunt was fully reversible over consecutive cycles of Ang-II-mediated ON and OFF switching (Fig. 2e).

Although AT1R taps into endogenous cAMP signaling to address the synthetic PCSN promoter, this signaling pathway was insulated from interference by endogenous GPCRs employing the same signaling cascade. Indeed, neither somatostatin, norepinephrine, carbachol, niacin nor the synthetic pain-killing opioid DADLE was able to activate cAMP signaling and PCSN when AT1R was ectopically overexpressed, despite the presence of the respective cognate GPCRs (SSTR5, ADRA1B, CHRM3, GPR109A, and OPRD1) 54 (Fig. 2f). This observation is in line with previous reports suggesting that ectopic expression of GPCRs titrates the associated G proteins away from endogenous GPCRs, thereby insulating the rewired synthetic promoter from undesired interference 55. Transient activation of endogenous signaling cascades by heterologous GPCRs, other cell-surface receptors or channels does not have any detectable impact on host cells 31, 36, 55-65.

Since the AT1R-triggered reporter gene expression is probably insufficient to capture the subnanomolar Ang-II levels reached during arterial hypertension, we designed a fully human plug-in feed-forward gene circuit to amplify the Ang-II input and boost the overall output of the device (Supplementary Fig. 2). The booster plug-in consists of a genetic adapter encoding a PCSN-driven human sterol regulatory element transcription factor 1 (hSREBF1) 66 (pGU234, PCSN-hSREBF1E-pA) that can amplify expression of any transgene and boost the release of desired biopharmaceuticals (Supplementary Fig. 2). However, since we found that we could achieve comparable sensor-effector performance with monoclonal cell lines containing the basic AT1R-based input device, we chose to retain the simpler, more compact basic design as it would be preferable for future cell-based therapies.

Design and validation of the antihypertensive effector device. Soluble angiotensin-converting enzyme 2 (sACE2), which degrades Ang-II, is a central effector of the vasorelaxant axis of the RAS 67 and was clinically evaluated in Covid-19 studies 40, 68, 69. sACE2 should therefore be an excellent antihypertensive effector protein. Since sACE2 is generated by ADAM17-mediated conversion of its membrane-bound form mACE2 70, we considered direct Ang-II-triggered AT1R-mediated PCSN-driven expression of a secretion-engineered, stabilized, therapeutic ACE2 variant (stACE2) to establish the autonomous regulator of chronic hypertension (ARCH). We profiled different variants of stACE2 for Ang-II degradation in a cell-based assay using pcDNA3-sACE2(WT)-Fc(IgA1) (immunoglobulin A (IgA) fusion, PhCMV-stACE2 (IgA1)-pA), pcDNA3-sACE2(WT)-Fc(IgG1) (immunoglobulin G (IgG) fusion, PhCMV-stACE2 (IgG1)-pA), as well as pcDNA3-sACE2-WT(732) (wild-type sACE2, PhCMV-sACE2-pA) (Fig. 2g). Since the IgG1-tagged secretion-engineered stACE2-derived proteinase domain combined the most compact 844-amino-acid format with the maximum expression level and optimal angiotensin cleavage activity, we chose it as the therapeutic effector for ARCH. We further validated the enzymatic activity of the therapeutic effector (stACE2 fusion protein) using a fluorometric assay (Supplementary Fig. 3). We additionally confirmed that the recombinant fusion protein is inhibited by MLN4760, a competitive ACE2 inhibitor. It should be noted that beyond catalytic blockade, MLN4760 binding has been reported to induce conformational changes in soluble ACE2 that can modulate non-catalytic interactions and, depending on the assay format, could affect antibody-based detection in a context-dependent manner71-73.

Design of ARCH-transgenic monoclonal human cells (HEKARCH). Transient co-transfection of ARCH components did not provide sufficient sensitivity to detect Ang-II within the physiologically relevant range. This limitation is consistent with the variability inherent to transient expression, where differences of plasmid copy number and the absence of defined genomic integration can alter transcriptional control parameters, including ligand sensitivity, basal activity (leakiness), inducibility, and absolute expression levels 50, 74. This issue is well recognized across mammalian synthetic gene circuits, where transient co-transfection can introduce substantial cell-to-cell variability 49-52. We therefore generated stably transgenic HEK-293T cell lines with constitutive expression of human AT1R (pGU52, PCAG-hAT1R-pA) and PCSN-driven expression of secretion-engineered stACE2 (pGU139, PCSN-stACE2-IgG1-P2A-SEAP-pA) (Fig. 3a, b). To exclude genetic drift or transgene silencing, we evaluated circuit performance across multiple passages and observed stable function over passage numbers aligned with established industrial benchmarks for HEK-293T-derived production cell lines 75, 76 (Fig. 3c). Indeed, Ang-II-triggered dose-response profiles indicated that the polyclonal cell line (EC50 = 5.5 \u00d7 10-10 M) showed a 250-fold higher Ang-II sensitivity than transiently transfected cell cultures (EC50 = 1.4 \u00d7 10-7 M), driving half-maximal effective concentrations (EC50) into the clinically relevant subnanomolar range (Fig. 3d). Selecting the stable monoclonal cell line HEKARCH from the polyclonal cell population further boosted ARCH performance (EC50 = 4.5 \u00d7 10-10 M), affording over 300-fold higher Ang-II sensitivity compared to the basic transient configuration and driving ARCH\u2019s sensitivity well into the clinically relevant sensitivity range (Fig. 3d). Upon stimulation with clinically relevant doses of Ang-II, HEKARCH produced and secreted stACE2 (Fig. 3e). To quantify the temporal dynamics of stACE2 activity, we monitored circuit output over time and observed a progressive depletion of Ang-II consistent with enzymatic degradation (Fig. 3f).

Interactions of ARCH with canonical hypertension medications. To profile possible interactions between HEKARCH and medications targeting its components, we exposed HEKARCH cells to a variety of antihypertensive medications at physiologically relevant, therapeutic levels (5 \u03bcM). HEKARCH-triggered stACE2 release could be blocked by clinically licensed antihypertensive drugs such as valsartan (an AT1R antagonist), which interferes with ARCH\u2019s Ang-II detection and signaling. Other blood pressure therapeutics with targets unrelated to ARCH did not interfere with the activity of this antihypertensive hormone shunt within the therapeutic concentration ranges, suggesting that ARCH should be compatible with combination regimes without direct molecular cross-reactivity that would compromise its function (Fig. 4a). Valsartan and ARCH-mediated stACE2 exhibited comparable efficacy in a kinetic assay (Fig. 4b).

ARCH-based control of experimental hypertension. To validate ARCH\u2019s potential to sense Ang-II levels associated with arterial hypertension and produce therapeutic doses of stACE2 to restore and maintain normal blood pressure in an automatic, self-sufficient, and closed-loop manner, we microencapsulated 5 \u00d7 106 HEKARCH into coherent beads of clinically licensed alginate and implanted them subcutaneously in hypertensive mice in an Ang-II infusion model 77-79 (Fig. 5a). ARCH was able to detect and correct hypertension (>140 mmHg systolic), restore normal blood pressure (<120 mmHg systolic) within 24 hours after implantation on day 4, and maintain the systolic pressure within 100 to 120 mmHg for the remainder of the study (Fig. 5b). The diastolic pressure and the mean arterial pressure (MAP) profiles also supported the therapeutic potential of ARCH (Supplementary Fig. 4). In contrast, a single high-dose treatment of hypertensive mice with the clinically licensed antihypertensive drug valsartan only gradually reduced blood pressure over 72 hours, while other hypertension medications failed to reverse the high blood pressure after a single administration, suggesting that ARCH compares favorably with established antihypertensive drugs for the treatment of experimental hypertension (Fig. 5c, Supplementary Fig. 5). No hypotensive excursions were seen either in hypertensive mice receiving the ARCH-based cell therapy or in wild-type animals implanted with HEKARCH as evidenced by blood pressure profiling and behavioral monitoring (Fig. 5d). Blood level analyses confirmed that only ARCH-treated hypertensive mice showed increased stACE2 in their bloodstream (Fig. 5e), at levels that correlated with those of the co-cistronically expressed reporter protein SEAP (Fig. 5f). Overall, these results represent a proof-of-concept for cell-based therapy to achieve ARCH-mediated closed-loop control of arterial hypertension. In order to verify the host compatibility of the therapeutic strategy, the cell-laden capsules were harvested at two weeks post-implantation together with the surrounding peritoneal lavage. Upon flow-cytometric analysis of the peritoneal lavage and subsequent leukocyte profiling, no abnormality was detected in host immune cell composition 80 (Supplementary Figs. 6, 7).

Closed-loop control of obesity-associated RAS dysregulation. Elevated Ang-II has been implicated in diverse pathophysiological processes 41-45. Because obesity is associated with RAS activation, partly driven by adipose tissue as an endocrine organ that upregulates local angiotensin signaling 81, we used an obesity model to test ARCH functionality under conditions of disease-relevant RAS dysregulation. In high-fat diet-fed mice, both plasma and renal Ang-II levels were elevated, and decreased following ARCH implantation (Figs. 6a, b, Supplementary Fig. 8). Intraperitoneal bolus administration of an ACE2 inhibitor transiently blunted the Ang-II lowering, indicating that stACE2-dependent enzymatic degradation is the primary therapeutic mechanism. Reduced plasma Ang-II levels correlated with increased circulating soluble ACE2 (Fig. 6c), whereas renal ACE2 levels remained unchanged (Fig. 6d). Plasma Ang-(1-7) showed a transient increase in the treatment group but this was not sustained (Fig. 6e), and renal Ang-(1-7) remained unchanged (Fig. 6f). Finally, consistent with reduced AT1R signaling, the circulating and renal renin concentrations increased in line with compensatory renin release from juxtaglomerular cells (Figs. 6g, h).

ARCH responsiveness to human serum. To probe translational relevance, we cultured HEKARCH cells in sera from hypertensive individuals. Participant spanned elevated blood pressure (>120 mmHg), stage 1 hypertension (>130 mmHg), and stage 2 hypertension (>140 mmHg) based on systolic values (Fig. 7a) and exhibited isolated systolic hypertension, with diastolic blood pressures in the normal range (<80 mmHg) (Fig. 7b). To enrich for RAS activity, sera were selected from individuals not receiving ACE inhibitors and in the top quintile for renin and angiotensinogen (Figs. 7c, d). Consistent with this stratification, sera containing higher levels of Ang-II activated the AT1R/reporter axis and stACE2 expression in HEKARCH (Figs. 7e, f).

Methods
Ethics Statement. This study complies with all relevant ethical regulations and all experiments comply with the European Community Council (2010/63/EU), approved by the French Republic (project No. DR2018-40v5 and APAFIS #16753) and Swiss animal welfare legislation (art. 18, 141, 30), approved by the Veterinary Office of Canton Basel-Stadt (GenoMet; national No.: 35863, cantonal No.: 3200).

Plasmid design. The cloning strategies and oligonucleotide sequences used in this study can be found in Supplementary Data 1 in the supplementary information. Briefly, the gene expression vectors used in this study were constructed through digestion with high-fidelity restriction endonucleases (New England Biolabs, Massachusetts, USA) followed by ligation with T4 DNA ligase (New England Biolabs, cat. No. M0202S) with or without PCR amplification using custom oligonucleotides (Merck Life Science, UK) and Q5\u00ae High-Fidelity DNA polymerase (New England Biolabs, cat. No. M0491S). Quick calf intestinal alkaline phosphatase (CIP, New England Biolabs, cat. No. M0525S) was used to prevent self-ligation of empty backbones. Cloned vectors were transformed into XL10-Gold ultracompetent cells (New England Biolabs, cat. No. C3040H), subsequently purified using a miniprep kit (Zymo Research, Irvine, California, USA, cat. No. D4054), and validated via Sanger sequencing (Microsynth AG, Switzerland).

Cell culture and transfection. Human embryonic kidney (HEK-293T, ATCC: CRL-3216), COS-7 (ATCC: CRL-1651), MDCK (ATCC: CCL-34), BHK-21 (ATCC: CCL-10), NIH/3T3 (ATCC: CRL-1658), and HepG2 (ATCC: HB-8065) cells were cultured in full medium consisting of Dulbecco\u2019s modified Eagle\u2019s medium (DMEM, high glucose, GlutaMAXTM Supplement, GibcoTM, cat. No. 10566016) supplemented with 10% fetal bovine serum (FBS, heat-inactivated at 56\u00b0C for 30 min, Sigma-Aldrich, cat. No. F7524) and 1% penicillin-streptomycin (GibcoTM, cat. No. 15070063, final concentration 50 U\u00b7mL\u22121 penicillin and 50 \u03bcg\u00b7mL\u22121 streptomycin) at 37\u00b0C in a humidified atmosphere containing 5% CO2. Immortalized human mesenchymal stem cells (hMSC-TERT) were a gift from Moustapha Kassem (University Hospital of Aarhus and University Hospital of Odense, Denmark) and were treated in the same way. Cells were trypsinized at 90-95% confluency with 0.05% trypsin-EDTA (Life Technologies, California, USA; cat. No. 25300-054) for 2 minutes at 37\u00b0C, then the trypsin was diluted 1:5 in full medium, and the mixture was centrifuged at 250 \u00d7 g for 1 minute. The supernatant was discarded and the cell pellet was resuspended in full medium. The number of cells in suspension was determined using a CellDrop brightfield cell counter (DeNovix, Delaware, USA). For passaging, cells were seeded in culture dishes in the appropriate volume of culture medium at 15,000 cell\u00b7cm-1 surface area. For fast-forward transfection for in vitro experiments, cells were seeded in 96-well plates at 30,000 cells\u00b7100 \u03bcL-1 per well and cells in each well were immediately transfected with 150 ng total plasmid DNA:600 ng polyethyleneimine (PEI; Polysciences Inc.) mixture in 50 \u03bcL FBS-free and antibiotic-free medium for 16 hours.

Monoclonal cell line generation. For HEKARCH stable cell generation, HEK-293T cells were seeded in 6-well plates at 600,000 cells \u00b7 2 mL-1 per well and cells in each well were immediately transfected with 2,500 ng plasmid DNA (500 ng pGU52 + 2,000 ng pGU139):250 ng Sleeping Beauty transposase vector (pSB100X):12,000 ng PEI mixture in 1 mL FBS-free and antibiotic-free medium for 16 hours. Stable cell selection was carried out for 10 days with blasticidin (Invitrogen, 5 \u03bcg\u00b7mL-1) and puromycin (Invitrogen, 2.5 \u03bcg\u00b7mL-1) Next, the dual antibiotic-resistant polyclonal population underwent FACS-mediated single cell sorting for dual Ypet and mRuby fluorescence. Of the 96 clones picked, 25 surviving clones were screened for Ang-II sensitivity and protein expression. Following clonal expansion, the best-performing clone (HEKARCH) was used for further experiments.

Inducers. Ang-II (cat. No. 05-23-0101), Ang-III (cat. No. 10385), Ang-IV (cat. No. A0230), Ang-(1-7) (cat. No. A9202), alamandine (cat. No. SML1374); nifedipine (cat. No. N7634), valsartan (cat. No. SML0142), chlorthalidone (cat. No. SML0591), enalapril (cat. No. E6888), atenolol (cat. No. A7655), norepinephrine (cat. No. A7257), [D-Ala2, D-Leu5]enkephalin (DADLE, cat. No. E7131), carbachol (cat. No. C4382), somatostatin (cat. No. S9129), and niacin (cat. No. N4126) were purchased from Sigma-Aldrich. Ang-(1-5) (cat. No. HY-P1839A) was purchased from MedChemExpress. Ang-A (cat. No. P001208) and Ang-(1-9) (cat. No. P001207) were purchased from Aapptec. MLN-4760 (cat. No. 3345) was purchased from Tocris. Stock solutions were prepared in either Dulbecco\u2019s phosphate-buffered saline (DPBS, ThermoFisher Scientific, cat. No. J67670.K2) whenever possible or dimethyl sulfoxide (DMSO, Sigma-Aldrich, cat. No. D8418) if necessary. Stock solutions were aliquoted to avoid multiple freeze-thaw cycles and diluted to the required working concentrations before each experiment.

Fluorescence microscopy. For fluorescence imaging, cells expressing fluorescent fusion proteins were seeded in black 96-well plates with a transparent flat bottom (Greiner Bio-One, cat. No. 655096). YFP fluorescence was acquired with 520 nm laser excitation 560 nm emission using a Nikon WF3 + X-light SD confocal microscope.

Flow cytometry. Anti-mouse CD16/32 (93, 101302, 1:200 dilution), anti-mouse CD4 (RM4-5, 100527, 1:500 dilution), anti-mouse CD8\u03b2 (YTS256.7.7, 126617 and 126633, 1:500 dilution), anti-mouse F4/80 (BM8, 123107, 1:500 dilution), anti-mouse CD25 (PC61, 102011, 1:500 dilution), anti-mouse/human CD44 (IM7, 103006, 1:500 dilution), anti-mouse CX3CR1 (SA011F11, 149009, 1:500 dilution), anti-mouse PD-1 (29F.1A12, 135205, 1:500 dilution), anti-mouse CD11c (N418, 117308, 1:500 dilution), and anti-mouse CD80 (16-10A1, 104734, 1:500 dilution) antibodies were purchased from BioLegend. For surface marker staining, cells harvested from peritoneal lavage were collected in U-bottom 96-well plates, blocked with anti-mouse CD16/32 antibody, and incubated with the indicated antibodies at 4\u00b0C for 20 min, followed by live/dead staining with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, D9542, final concentration: 0.1 \u03bcg ml-1. Cells were washed and resuspended in FACS buffer containing 0.2% w/v bovine serum albumin (BSA, Sigma-Aldrich, A3059) and 2 mM EDTA (Invitrogen, AM9260G) in DPBS for flow-cytometric analyses. Flow cytometry was performed using a CytoFLEX S Flow Cytometer with CytExpert Software v2.1 (Beckman Coulter) at the Single Cell Facility (SCF) of D-BSSE. Data analyses were performed using FlowJo 10.9 (Tree Star). Gate margins were determined by isotype controls and fluorescence-minus-one controls.

Cell encapsulation. Subcutaneous implants for in vivo experiments were produced by encapsulating HEKARCH as well as native HEK-293T in alginate-poly-(l-lysine)-alginate (400 \u00b5m beads; 200 cells/capsule) using an Inotech Encapsulator Research Unit IE-50R (Buchi Labortechnik AG) according to the manufacturer\u2019s protocol, using a 200 \u00b5m nozzle with a vibration frequency of 1024 Hz and 1200 V for bead dispersion, with a 20 mL syringe operated at a flow rate of 500 units. The cell-laden capsules were kept in full culture medium overnight at 37\u00b0C following the encapsulation procedure. Prior to subcutaneous administration, the capsules were washed three times with serum- and antibiotic-free medium.

Reporter assays. For in vitro secreted embryonic alkaline phosphatase (SEAP) analysis, 20 \u00b5L cell supernatant from cells expressing SEAP was diluted in 80 \u00b5L water and heat-inactivated at 65\u00b0C for 35 minutes with continuous shaking at 300 rpm. The assay mixture containing 80 \u00b5L buffer (20 mM homoarginine, 1 mM MgCl2, 21% (v/v) diethanolamine) and 20 \u00b5L substrate (20 mM pNPP, Acros Organics BVBA) was added immediately prior to absorbance measurement at 405 nm using a Tecan M1000 plate reader (TECAN AG, Switzerland). For SEAP analysis of in vivo plasma samples, a luminescence reporter gene assay kit (Abcam, cat. No. ab133077) was used according to the manufacturer\u2019s protocol. For in vitro nanoluciferase analysis, the Nano-Glo Luciferase Assay System (Promega, Wisconsin, USA, cat. No. N1110) was used according to the manufacturer\u2019s protocol.

RAS measurements. (i) Cell-based stACE2 assay. Two transfections were carried out in parallel. For the first transfection, 50 ng of vectors encoding for constitutive expression of different variants of the therapeutic stACE2 (pcDNA3-sACE2(WT)-Fc(IgA1) (immunoglobulin A (IgA) fusion, PhCMV-stACE2 (IgA1)-pA), pcDNA3-sACE2(WT)-Fc(IgG1) (immunoglobulin G (IgG) fusion, PhCMV-stACE2 (IgA1)-pA), as well as pcDNA3-sACE2-WT(732) (wild-type sACE2, PhCMV-sACE2-pA)) was transfected into native HEK-293T with 100 ng pcDNA3.1 (+) filler plasmid. A constitutive eGFP expression vector (pFOX12, PhCMV-eGFP-pA) was used as a control. For the second transfection, plasmids encoding for CAG-driven AT1R expression (pGU52, 10 ng) and CSN-driven nLuc reporter expression (pGU23, 20 ng) were transfected in native HEK-293T cells with 120 ng pcDNA3.1 (+) filler plasmid. The next day, the medium in the second plate was replaced with a mixture of 75 \u00b5L supernatant from the first plate and 75 \u00b5L of 2 \u00d7 10-6 M Ang-II. The mixture was incubated at 37\u00b0C for 5 minutes prior to this. Luminescence was quantified 24 hours after this medium change. (ii) stACE2 quantification. A human ACE2 ELISA Kit (ThermoFisher Scientific, cat. no. EH489RB) was used according to the manufacturer\u2019s protocols to quantify stACE2 for both in vitro cell culture supernatants and in vivo plasma samples. Cell culture supernatants were diluted with assay diluent 1:1 and plasma samples were diluted with assay diluent 1:2. (iii) Assay of stACE2-mediated Ang-II degradation kinetics. HEKARCH cells were stimulated with Ang-II (1 \u00d7 10-6 M) to trigger stACE2 secretion, and culture supernatants were collected at 1, 2, 4, 6, 8, 24 h post-stimulation. In parallel, native HEK-293T cells were transiently transfected with plasmids encoding PCAG-driven AT1R (pGU52, 10 ng) and PCSN-driven nLuc reporter (pGU23, 20 ng), supplemented with 120 ng pcDNA3.1 (+) filler plasmid. For the bioassay, transfected HEK-293T cells were induced with 75 \u00b5L HEKARCH supernatant at each time point and 75 \u00b5L of 2 \u00d7 10-8 M Ang-II. The mixture was incubated at 37\u00b0C for 5 minutes prior to this. Luminescence was quantified 24 hours after this medium change. (iv) Ang-II quantification. HEKARCH cells were transiently transfected with a PCSN-driven NanoLuc reporter plasmid (pGU23; 120 ng per well). After transfection, cells were incubated for 24 h with in vitro samples (cell-culture supernatants) or in vivo samples (plasma or serum). NanoLuc luminescence was measured and Ang-II concentrations were determined by interpolation from an Ang-II standard curve assayed in parallel. For each sample matrix (supernatant, plasma, serum), the corresponding matrix-only control (no Ang-II) was included and signals were background-corrected. (v) Ang-(1\u20137) quantification. Ang-(1\u20137) concentrations in in vivo samples were quantified using an Ang-(1\u20137) ELISA kit (Abbexa; cat. no. abx251960). (vi) Renin quantification. Renin levels in in vivo samples were quantified using a mouse renin 1 ELISA kit (RayBiotech; cat. no. ELM-Renin1-1). (vii) ACE2 activity assay. ACE2 enzymatic activity was measured using a fluorometric ACE2 activity assay kit (Abcam; cat. no. ab273297).

Animal experiments. Experiments involving the Ang-II-dependent hypertension model were performed according to the directive of the European Community Council (2010/63/EU), approved by the French Republic (project No. DR2018-40v5 and APAFIS #16753), and carried out by Ghislaine Charpin-El Hamri (license No. 69266309) at the Institut Universitaire de Technologie of the Universit\u00e9 Claude Bernard Lyon 1, F-696226, Villeurbanne Cedex, France. Male C57BL/6J mice, aged 12 weeks, weighing 24-26 g were purchased from Janvier Labs (53940 Le Genest-Saint-Isle, France) and acclimatized for one week (n = 4). The standard hypertensive mouse model was generated on day 2 through cranial dorsal subcutaneous implantation of micro-osmotic pumps (ALZET, Model 1002, DURECT Corporation, California, USA) loaded with 100 \u00b5L aqueous solution of 2.88 mM Ang-II (Calbiochem, cat. no. 05-23-0101, Germany) to achieve 500 ng\u00b7kg-1\u00b7min-1 infusion via osmosis. Therapeutic cell capsules were injected subcutaneously (1 mL, 5 x 106 cells per mouse) on day 4 in the caudal region. Blood pressure measurements were taken every day in the morning using the non-invasive BP-2000 tail-cuff machine (Bioseb, France). Mice were trained and acclimatized to tail-cuff plethysmography. During measurements, animals were maintained at 37\u00b0C using the instrument\u2019s temperature controller and restrained using appropriately sized holders according to the manufacturer\u2019s guidelines to ensure reliable recordings. On day 10, blood samples were harvested for further analysis. For drug injections, the maximal human doses were converted to corresponding mouse doses taking account of both weight and surface area as described 109. For leukocyte profiling, encapsulated cells were injected intraperitoneally (1 mL, 5 x 106 cells per mouse) and recovered together with the surrounding intraperitoneal lavage at two weeks post-injection. For RAS profiling, obesity-model experiments were conducted according to the Swiss animal welfare legislation (art. 18, 141, 30), were approved by the Veterinary Office of Canton Basel-Stadt (GenoMet; national No.: 35863, cantonal No.: 3200), and were carried out by Yu-Qing Xie at the ETH Zurich (D-BSSE, CH-4056, Basel, Switzerland). Male B6.V-Lepob/JRj (ob/ob) mice (4 weeks old) were obtained from Janvier Labs (France), acclimatized for one week (n = 4), and fed a high fat diet (SAFE-Lab, Augy, France; cat. No. U8956) unless otherwise indicated. Therapeutic cell capsules were administered intraperitoneally (1 mL, 5 x 106 cells per mouse), and blood was sampled twice weekly for subsequent analyses. All mice were continuously monitored (appearance, breathing, eyes, behavior, posture, locomotion, seizure, and weight) throughout the study to ensure their well-being and accurate data collection. All mice were housed in the joint animal facility of Biozentrum University of Basel & D-BSSE ETHZ and were kept in individually ventilated cages, at 19\u201323 \u00b0C, with 45\u201365% humidity and with a 12-h dark/light cycle.

Human samples. All participants provided written informed consent and the study was approved by the Ethics Commission Northwest- and Central Switzerland (IRB00003565). Serum was obtained from hypertensive patients with high angiotensinogen and renin levels (top quintile for each), who were not receiving ACE-inhibitor therapy. Angiotensinogen and renin were measured with a SOMAscan v4 (SomaLogic, Colorado, USA).

Statistical analysis. The unpaired, two\u2010tailed Welch's t-test was applied with a 95% confidence interval with a minimum of three replicates per sample using GraphPad Prism 8 (v 9.2.0, GraphPad Software Inc.) and Microsoft Excel (v 16.51, Microsoft Inc.). Error bars represent mean \u00b1 SD for in vitro measurements and mean \u00b1 SEM for in vivo measurements (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). For the in vivo time course experiments, a linear mixed model (m) was used in R to describe the average responses for each group on each day; m = lmer(bp~ht*treat*day+(1|mouse),data = d), where lmer() is the linear mixed-effects regression function, in this case modeling blood pressure (bp) looking at two-by-two groups; hypertensive (ht) versus non-hypertensive (nonht) and treatment (treat) versus control (ctrl). A minimum of 3 replicates were used for each condition for in vitro experiments. For in vivo experiments, a minimum of 4 were used. In accordance with European legislation (Directive 2010/63/EC) and the corresponding national regulations governing animal experimentation in the EU, the UK, and Switzerland, animal experiments are conducted under the principles of replacement, reduction, and refinement (3R), with the aim of using the minimum number of animals required to achieve the scientific objective. No data were excluded from the analyses. All in vitro data were successfully reproduced at least twice with proper controls to ensure that no external factors (cell viability, transfection efficiency, etc.) affect data accuracy. For in vivo experiments, mice allocation into different groups were randomized. Investigators were blinded to group allocation during data collection and analysis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Unal et al. report a fully human closed-loop gene circuit (ARCH) for autonomous blood pressure regulation. The authors\nrewire AT1R into a biosensor that detects angiotensin II (AT-II) and couple it to the expression of a secretion-engineered\nsoluble ACE2 (stACE2). They demonstrate restoration and maintenance of normal blood pressure following implantation of\nmicroencapsulated ARCH-transgenic human cells, providing convincing proof-of-concept evidence. Overall, this study\nrepresents a significant advance in the application of synthetic biology to chronic disease therapy and offers a promising\napproach that warrants further investigation. Before publication, I have some minor concerns as follows:\n1. Lines 244-249, the AT-II sensor was functional across multiple mammalian cell types, with the strongest response\nobserved in HEK-293T cells. Could the authors speculate on which factors might underlie these differences, such as\nreceptor expression levels or cell-type-specific signaling efficiency?\n2. Lines 314-321, the stable monoclonal HEKARCH cell line showed ~300-fold higher AT-II sensitivity compared to\ntransiently transfected cells. Could the authors explain the potential factors underlying this enhanced sensitivity? For\nexample, is this improvement due to increased copy number, higher expression stability, or other cell-intrinsic mechanisms?\nClarifying these factors could provide valuable guidance for improving the sensitivity of similar systems in future studies.\n3. Lines 294-304: sACE2 is described as a licensed ad hoc drug for the treatment of Covid-19. Given that hypertension is a\nchronic condition requiring long-term treatment, could the authors discuss the potential risks associated with prolonged\nACE2 protein secretion? Although ACE2 expression and secretion are controlled by the AT-II sensor, it would be important\nfor the authors to comment on what levels or duration of AT-II sensor-responsive ACE2 expression could be considered\nsafe.\n4. Could the authors provide information on the response kinetics of the system? Specifically, how rapidly can the AT-II\nsensor–controlled ACE2 expression respond to changes in AT-II levels, and how quickly does this translate into measurable\nblood pressure reduction?\n5. If possible, could the authors verify whether the system is capable of responding to blood samples from hypertensive\npatients? Demonstrating responsiveness in clinically relevant samples would further support the translational potential and\ntherapeutic relevance of the system.\n6. This study demonstrates synthetic gene circuits with potential for treating chronic diseases. To contextualize these\nfindings and highlight their potential applications, the authors may refer to representative previous works, including synthetic\ngene circuits for treating chronic diseases via inhalation (PMID: 38321056) and for diabetes therapy in large animals (PMID:\n31645456, 34608325).\n7. The black bars obscure the distribution of individual data points; please consider using a different color to improve\nvisibility. Regarding Fig.1a, the current schematic is overly simplified. It would be helpful to provide a more detailed\nillustration of the signaling pathway and the operational process of the system.", "reviewer_2": "In this study, Unal et al. designed a cell-based therapy for hypertension called ARCH (autonomous regulator of chronic\nhypertension), which utilized human cells co-transfected with type-1 angiotensin receptor (AT1R) and a chimeric promoter\n(PCSN, CRE-SRE-NFAT-Pmin) driven soluble therapeutic ACE2 (stACE2). Increased blood Angiotensin-II (Ang-II) levels\nunder hypertensive conditions would activate AT1R, which then induce the expression of stACE2 through the chimeric\npromoter. The authors first tested this system in cultured cells and then tested its therapeutic potential in the Ang-II infusion\nmodel of hypertension in mice. The same research group has designed similar closed-loop systems for the treatment of\nexperimental arthritis, obesity, and diabetes, but its application in hypertension is new. However, there is a major concern of\nutilizing Ang-II levels as the activation switch, as blood Ang-II levels in patients with hypertension vary. Although Ang-II\nlevels are usually elevated in patients with severe essential hypertension, renal hypertension, and malignant hypertension,\nits levels are usually normal in benign and uncomplicated essential hypertension. Moreover, blood Ang-II levels can be\naltered by drugs including many anti-hypertensive medications. Thus, the utility of such a system in hypertensive patients is\nquestionable.\nIn addition, the following concerns are noted about the proposed cell-based therapy:\n1. Plasma Ang-1-7 levels should be measured in the in vivo validation experiment.\n2. The Ang-II infusion model is an acute, not chronic, model of experimental hypertension.\n3. The anti-hypertensive drugs used in Figure 4a affect Ang-II levels in vivo, thus this cell-based (HEK 293) assay cannot\nrule out the interactions of ARCH with canonical hypertension medications.\n4. The tai cuff method is less reliable than radiotelemetry, especially for diastolic blood pressure measurement.\n5. What behavioral monitoring was conducted in these mice (line 347)?\n6. The authors stated that “the cell-containing capsules were harvested at two weeks post-implantation together with the\nsurrounding peritoneal lavage (line 352-253),” but this is not consistent with how the cells were implanted, line 214-215\n“Therapeutic cell capsules were injected subcutaneously (1 mL, ~5 million cells per mouse) on day 4 in the caudal region.”\n7. Leukocyte activation markers should be included in the leukocyte profiling (Supplementary Figure 5).\n8. A two-tailed t-test is more appropriate than the one-tailed t-test used in this study. What statistical methods were used\nwhen more than two groups were compared?\n9. Please list the number of mice used in each group in the in vivo experiment.", "reviewer_3": "Unal et al showed a proof-of-concept therapy based on a synthetic biology approach to treat hypertension. The technology\ninvolved the development of a fully human, closed-loop gene circuit named ARCH (Autonomous Regulator of Chronic\nHypertension), that leverages the angiotensin II type 1 receptor (AT1R) and a stabilized, secretion-engineered angiotensin-\nconverting enzyme 2 (stACE2), to autonomously sense and regulate blood pressure. This is a highly innovative approach,\nand experiments clearly showed possible application.\n1. It is not clear why the authors included an NFAT response element in the synthetic promoter, because, as depicted in\nFigure 1C, PNFAT did not respond to Ang II.\n2. In supplementary figure 2, the authors showed a basic (panel a), and a booster (panel b) sensor design. It is not clear\nwhether the experiments performed in the manuscript were done using the basic or the booster design.\n3. In figure 2b, angiotensin A, angiotensin-(1-9,) and angiotensin-(1-5) should also be tested.\n4. In figure 2f, the drugs were added in the presence of Ang II? Because in the figure appears as - AT-II + Drug\n5. Only protein levels of stACE2 were measured. Because the enzymatic activity of ACE2 is important for its biological effect,\nstACE2 activity should also be measured.\n6. How stable is the HEKARCH (Stable, monoclonal) cell line? Because synthetic gene circuits may undergo genetic drift or\nsilencing over time.\n7. The in vivo experiments were performed using an Ang II-infused mouse model. Because hypertension is multifactorial and\nnot solely Ang II dependent, a different hypertension model should be tested, for instance, on spontaneously hypertensive\nrats.\n8. In the in vivo experiment, a control verifying that the decrease in blood pressure is due to stACE2should be included, i.e.,\nby using an inhibitor of stACE2.\n9. In the animals, are the levels of Ang II decreased and the levels of Ang-(1-7) increased?\n10. Produces this approach also a reduction of cardiac remodeling, vascular remodeling?", "reviewer_4": "The study approach is unique and novel model to use ARCH-transgenic human cells and elevate stACE2 to treat AT-II\nhypertension. In this approach, it is expected that stACE2 will break down (reduce) hypertensive AT-II and increase anti-\nhypertensive Ang1-7 peptide hormones. However, to validate these mechanisms, some measurements, as follows, are\nimportant.\n1. Plasma as well as tissue, particularly kidney levels of AT-II and Ang (1-7) will tell whether the levels of these peptide\nhormones really changed and contributed to lowering the BP. Please measure the levels of these peptides.\n2. ACE2 activity, not just the expression, is critical to correlate with the changes in AT-II and Ang(1-7).\n3. Blocking/reducing AT1R is known to increase the renin activity, which has independently profibrotic activity via pro-\nrenin receptors. So please measure renin activity in the plasma and the kidney.\n4. AT-II model of hypertension is good in animal models to understand the mechanism. For translational purposes, a salt-\nsensitive model of hypertension could be more helpful."}, "author_response": "Response to the reviewers’ comments\nReviewer 1\nUnal et al. report a fully human closed-loop gene circuit (ARCH) for autonomous blood\npressure regulation. The authors rewire AT1R into a biosensor that detects angiotensin II (AT-\nII) and couple it to the expression of a secretion-engineered soluble ACE2 (stACE2). They\ndemonstrate restoration and maintenance of normal blood pressure following implantation of\nmicroencapsulated ARCH-transgenic human cells, providing convincing proof-of-concept\nevidence. Overall, this study represents a significant advance in the application of synthetic\nbiology to chronic disease therapy and offers a promising approach that warrants further\ninvestigation. Before publication, I have some minor concerns as follows:\nWe thank the reviewer for their positive overall assessment that ARCH provides\nconvincing proof-of-concept and represents a significant advance toward chronic disease\ntherapy. We have addressed all minor concerns, most notably with an expanded discussion of\nlong-term safety under feedback control, additional kinetic characterization, and a\ndemonstration of responsiveness to sera from hypertensive patients, as detailed below.\n1. Lines 244-249, the AT-II sensor was functional across multiple mammalian cell types, with\nthe strongest response observed in HEK-293T cells. Could the authors speculate on which\nfactors might underlie these differences, such as receptor expression levels or cell-type-specific\nsignaling efficiency?\nThe observed differences in Ang-II sensor performance across cell types likely reflect a\ncombination of (i) differences in effective AT R/reporter expression levels (driven by\n1\nvariations in cell-type-dependent transfection efficiency and plasmid expression capacity) and\n(ii) differences in cell-intrinsic coupling efficiency of AT R to the downstream\n1\ncAMP/PKA/CREB cascade that activates the P promoter. HEK-293T cells are widely used\nCSN\nfor preclinical circuit validation because they typically support robust plasmid-mediated\nexpression and show wide dynamic ranges in transient reporter assays (Yang, et al., Nature\nCommunications, 2021, PMID: 34226556; Nissim, et al., Cell, 2017, PMID: 29056342; De\nCarluccio, et al., Nature Communications, 2024, PMID: 38632224). More broadly, variable\nresponse strengths across cell lines are commonly attributable to differences in transfection\nefficiency and endogenous cellular components that modulate signaling performance (Wang,\net al., Nature Biomedical Engineering, 2018, PMID: 31015627; Liu, et al., Cell, 2018, PMID:\n29937224; Wu, et al., Nature Communications, 2020, PMID: 32709899; Mahameed et al.,\nNature Biomedical Engineering, 2025, PMID: 39953326). We have now incorporated this\nrationale into the revised Results text to clarify why HEK-293T provided the most robust\nresponse.\n2. Lines 314-321, the stable monoclonal HEKARCH cell line showed ~300-fold higher AT-II\nsensitivity compared to transiently transfected cells. Could the authors explain the potential\nfactors underlying this enhanced sensitivity? For example, is this improvement due to increased\ncopy number, higher expression stability, or other cell-intrinsic mechanisms? Clarifying these\nfactors could provide valuable guidance for improving the sensitivity of similar systems in\nfuture studies.\nWe thank the reviewer for raising this important point. The marked gain in Ang-II\nsensitivity in the stable monoclonal HEK line likely reflects reduced expression\nARCH\n1\nheterogeneity and improved stoichiometric balance relative to transient transfection. In\ntransiently transfected cultures, and likewise in polyclonal stable populations, effective copy\nnumber and expression levels vary substantially across cells, and polyclonal integration further\nintroduces site-dependent expression differences due to random genomic insertion. By\ncontrast, monoclonal stable lines originate from single cells and can be screened top-down for\nfavorable circuit properties (high inducibility, low basal activity, and optimal expression\nbalance), which is a standard strategy in circuit engineering and cell-line development (Huang,\net al., Adv. Sci., 2024, PMID: 38526196; Unal, et al., Cell Metab., 2025, PMID: 40972574).\nThe above factors are well known to affect transcriptional control parameters, including\napparent ligand sensitivity, leakiness, inducibility, and absolute output, as observed in our and\nothers’ mammalian gene-circuit work (Mahameed et al., Nat Biomed Eng, 2025, PMID:\n39953326; Chen et al., Nat Chem Biol, PMID: 34782743). We have now clarified this in the\nrevised Results section (lines 357–361).\n3. Lines 294-304: sACE2 is described as a licensed ad hoc drug for the treatment of Covid-19.\nGiven that hypertension is a chronic condition requiring long-term treatment, could the authors\ndiscuss the potential risks associated with prolonged ACE2 protein secretion? Although ACE2\nexpression and secretion are controlled by the AT-II sensor, it would be important for the\nauthors to comment on what levels or duration of AT-II sensor-responsive ACE2 expression\ncould be considered safe.\nWe agree with the reviewer that long-term safety is a central consideration when selecting a\ntherapeutic output for a gene circuit. First, although ACE2 is predominantly membrane-bound,\nit is physiologically shed and measurable in the circulation as soluble ACE2 in healthy\nindividuals (Batlle et al., Cell, 2022, PMID: 35623327). Second, recombinant soluble ACE2\nhas been clinically evaluated in humans, including in COVID-19 studies, at doses that are\napproximately three orders of magnitude higher than the maximal output achieved when\nARCH is fully activated; the ARCH-level exposure is only 3-4 times greater than endogenous\ncirculating concentrations (Zoufaly et al., Lancet Respir Med, 2020, PMID: 33131609; Kawabe\net al., Front Med, 2022, PMID: 35155493). Importantly, ARCH was intentionally engineered\nas a feedback-controlled closed-loop system to minimize unnecessary long-term exposure:\nwhen Ang-II levels are in the normotensive range, the circuit remains effectively quiescent and\ntransgene output is at background (Fig. 5e, f; blue bars). We have now made this feedback-\nbased safety rationale more explicit in the Discussion (lines 508-510).\n4. Could the authors provide information on the response kinetics of the system? Specifically,\nhow rapidly can the AT-II sensor–controlled ACE2 expression respond to changes in AT-II\nlevels, and how quickly does this translate into measurable blood pressure reduction?\nWe thank the reviewer for highlighting the importance of response kinetics. ARCH is\na transcriptionally regulated circuit and therefore operates on transcription, translation, and\nsecretion timescales. Following Ang-II stimulation, secreted transgene output becomes\ndetectable in the culture medium within 4 hours (Fig. 4b). This comment prompted us to add a\ndedicated kinetic analysis of Ang-II depletion mediated by secreted stACE2, which directly\nquantifies the time course of Ang-II modulation (new Fig. 3f). Nevertheless, it is important to\nnote that ARCH is intended for angiotensin-dependent chronic hypertension, where\ndysregulation persists for prolonged periods and requires long-term management rather than\nminute-scale control (Li et al., Mol Ther Nucleic Acids, 2023, PMID: 37449045). We have\nclarified this intended use case and the relevance of the observed kinetics in the revised text.\n2\n5. If possible, could the authors verify whether the system is capable of responding to blood\nsamples from hypertensive patients? Demonstrating responsiveness in clinically relevant\nsamples would further support the translational potential and therapeutic relevance of the\nsystem.\nWe thank the reviewer for this excellent suggestion. We obtained serum samples from\nhypertensive patients at University Hospital Basel, selecting individuals with high RAS\nactivity, defined as the top 20% for renin and angiotensinogen, and not receiving ACE\ninhibitors. Using these samples, we now show that HEK is activated by patients’ sera and\nARCH\ninduces stACE2 output in samples with elevated Ang-II (new Fig. 7).\n6. This study demonstrates synthetic gene circuits with potential for treating chronic diseases.\nTo contextualize these findings and highlight their potential applications, the authors may refer\nto representative previous works, including synthetic gene circuits for treating chronic diseases\nvia inhalation (PMID: 38321056) and for diabetes therapy in large animals (PMID: 31645456,\n34608325).\nAs suggested, we have incorporated the cited studies into the Discussion to better\ncontextualize ARCH within the broader landscape of synthetic gene circuits for long-term\nmanagement of chronic diseases and to support its translational potential.\n7. The black bars obscure the distribution of individual data points; please consider using a\ndifferent color to improve visibility. Regarding Fig.1a, the current schematic is overly\nsimplified. It would be helpful to provide a more detailed illustration of the signaling pathway\nand the operational process of the system.\nThank you. We have improved the visibility of the control data points throughout the\nrelevant plots and also revised the circuit schematic to provide a more informative\nrepresentation of ARCH (revised Fig. 1a).\nReviewer 2\nIn this study, Unal et al. designed a cell-based therapy for hypertension called ARCH\n(autonomous regulator of chronic hypertension), which utilized human cells co-transfected\nwith type-1 angiotensin receptor (AT1R) and a chimeric promoter (PCSN, CRE-SRE-NFAT-\nPmin) driven soluble therapeutic ACE2 (stACE2). Increased blood Angiotensin-II (Ang-II)\nlevels under hypertensive conditions would activate AT1R, which then induce the expression\nof stACE2 through the chimeric promoter. The authors first tested this system in cultured cells\nand then tested its therapeutic potential in the Ang-II infusion model of hypertension in mice.\nThe same research group has designed similar closed-loop systems for the treatment of\nexperimental arthritis, obesity, and diabetes, but its application in hypertension is new.\nHowever, there is a major concern of utilizing Ang-II levels as the activation switch, as blood\nAng-II levels in patients with hypertension vary. Although Ang-II levels are usually elevated\nin patients with severe essential hypertension, renal hypertension, and malignant hypertension,\nits levels are usually normal in benign and uncomplicated essential hypertension. Moreover,\nblood Ang-II levels can be altered by drugs including many anti-hypertensive medications.\nThus, the utility of such a system in hypertensive patients is questionable.\nmechanistic heterogeneity of hypertension. We agree that Ang-II is not a universal biomarker\nacross all patient subtypes. Accordingly, we have clarified in the manuscript that ARCH is\npositioned for angiotensin-dependent, RAS-driven hypertension in which Ang-II is persistently\nelevated. We further emphasize that the well-established inter-individual variability in Ang-II\ndynamics motivated a closed-loop design for ARCH, which enables autonomous adjustment\nof stACE2 output in response to patient-specific biomarker levels and therapeutic demand. This\nclosed-loop design also means that ARCH would be compatible with most combination\nregimens.\nIn addition, the following concerns are noted about the proposed cell-based therapy:\n1. Plasma Ang-1-7 levels should be measured in the in vivo validation experiment.\nWe agree that a more comprehensive analysis of the angiotensin axis is important to\nsubstantiate autonomous, feedback-controlled regulation in vivo. In response, we introduced\nan additional in vivo setting of RAS dysregulation using a high-fat diet obesity model, in which\nadipose tissue RAS activation increases Ang-II production and release (Yasue et al., Am J\nHypertens, 2010, PMID: 20057360). We then quantified key components of the axis in both\nplasma and kidney, including Ang-II, Ang-(1–7), soluble ACE2, and renin, to directly link\nARCH activation and stACE2 output with modulation of the endogenous RAS. These data\ndemonstrate elevation of plasma and renal Ang-II in obese mice and its attenuation following\nARCH implantation, accompanied by increased circulating soluble ACE2, a transient plasma\nAng-(1–7) increase, and compensatory renin elevation (new Fig. 6).\n2. The Ang-II infusion model is an acute, not chronic, model of experimental hypertension.\nThe Ang-II infusion paradigm can be implemented as either an acute or a chronic\nmodel, depending on the dosing regimen and exposure time. Acute designs typically use bolus\nadministration or short infusions of the order of minutes to hours, whereas chronic hypertension\nmodels commonly employ continuous Ang-II delivery via osmotic minipumps for 1–4 weeks\n(or longer). Our in vivo experiments used this established chronic infusion format, which has\nbeen widely applied to model sustained Ang-II-driven hypertension (Crowley et al., PNAS,\n2006, PMID: 17090678; Guzik et al., J Exp Med, 2007, PMID: 17875676; Dikalova et al., Circ\nRes, 2010, PMID: 20448215).\n3. The anti-hypertensive drugs used in Figure 4a affect Ang-II levels in vivo, thus this cell-\nbased (HEK 293) assay cannot rule out the interactions of ARCH with canonical hypertension\nmedications.\nWe agree with the reviewer that many antihypertensive drugs modulate Ang-II levels\nin vivo and would therefore influence ARCH indirectly by changing the input signal to the\nfeedback loop. For example, ACE inhibitors reduce Ang-II formation. However, the purpose\nof Fig. 4a was to address a different question, namely whether commonly used\nantihypertensives with targets unrelated to ARCH directly interfere with circuit function\nthrough molecular cross-reactivity within their therapeutic concentration ranges. In this assay,\nthese agents did not measurably perturb ARCH activity, supporting the compatibility of ARCH\nwith combination regimens. As expected, AT R blockers constitute an exception because they\n1\nprevent Ang-II sensing and therefore shut down the circuit. We have clarified this distinction\nand rationale in the revised text (lines 385-387).\n4\n4. The tail cuff method is less reliable than radiotelemetry, especially for diastolic blood\npressure measurement.\nWe agree that radiotelemetry provides unmatched temporal resolution for blood\npressure monitoring. We considered telemetry, but selected tail-cuff plethysmography because\nit was fit for purpose for the primary question addressed here, namely detecting sustained\ndifferences in systolic blood pressure between groups over time. ARCH operates through\ntranscription, translation, and secretion, and its functional response therefore occurs on the\norder of hours, rather than capturing circadian or beat-to-beat dynamics (new Fig. 3f; Chen et\nal., Nat Med, 2015, PMID: 26259032; Li et al., Nat Commun, 2022, PMID: 35668093). A\nparticular consideration was that tail-cuff measurement is non-invasive and is recommended\nfor assessing substantial group differences and longitudinal changes in systolic blood pressure,\nconsistent with American Heart Association guidance (Kurtz et al., Hypertension, 2005, PMID:\n15611363). Given that animals already underwent major procedures in this study, we aimed to\nminimize additional surgical burden and stress associated with telemetry implantation, which\ncan itself affect cardiovascular readouts (Cantalupo et al., Nat Med, 2015, PMID: 26301690;\nSantisteban et al., Nat Neurosci, 2024, PMID: 38049579; Harrison et al., Hypertension, 2024,\nPMID: 37990918). We also implemented standard quality-control measures for tail-cuff\nreliability, including the use of a BP-2000 system with temperature control and appropriately\nsized restrainers, and one week of pre-training during acclimatization. These methodological\ndetails have now been added to the Methods section (lines 237–240).\n5. What behavioral monitoring was conducted in these mice (line 347)?\nAnimal welfare monitoring was performed in accordance with the Swiss Animal\nWelfare Act using a protocol-specific, standardized score sheet for each animal, incorporating\nboth physical and behavioral assessments. The monitored parameters included appearance,\nrespiration, eyes, behavior, posture, locomotion, seizure activity, and body weight, each\nrecorded as part of a welfare score to guide intervention if needed. We have now included these\nmonitoring criteria in the Methods section (lines 252–253).\n6. The authors stated that “the cell-containing capsules were harvested at two weeks post-\nimplantation together with the surrounding peritoneal lavage (line 352-253),” but this is not\nconsistent with how the cells were implanted, line 214-215 “Therapeutic cell capsules were\ninjected subcutaneously (1 mL, ~5 million cells per mouse) on day 4 in the caudal region.”\nWe thank the reviewer for their careful reading of the text. The statement regarding\nintraperitoneal recovery referred to the leukocyte profiling experiment, which was performed\nas a separate host-compatibility study using intraperitoneal implantation and subsequent\nperitoneal lavage collection. This was inadvertently conflated with the subcutaneous\nimplantation procedure described for the blood-pressure study in the Methods. We have now\ncorrected and clarified the Methods section accordingly (lines 242–244) to clearly distinguish\nthe two in vivo protocols.\n7. Leukocyte activation markers should be included in the leukocyte profiling (Supplementary\nFigure 5).\nWe appreciate this suggestion. We repeated the leukocyte profiling experiment and\nexpanded the panel to include commonly used leukocyte activation markers (new Fig. S6).\nimplanted animals compared with non-implanted controls. Thus, there was no evidence of\novert immune activation under the conditions tested.\n8. A two-tailed t-test is more appropriate than the one-tailed t-test used in this study. What\nstatistical methods were used when more than two groups were compared?\nWe thank the reviewer for pointing this out. We have now consistently applied two-\ntailed statistical testing throughout the study. In vitro comparisons involved two groups and\nwere analyzed using two-tailed t-tests. For the in vivo systolic blood pressure time-course data,\nwe used a linear mixed model in R to account for repeated measurements within animals and\nto estimate group-wise mean responses over time. Full details are provided in the Methods.\n9. Please list the number of mice used in each group in the in vivo experiment.\nThe group sizes were already specified in the Fig. 5 legend; we have now additionally\nincluded the number of animals per group in the Methods to ensure this information is\nimmediately accessible and consistently reported throughout the manuscript.\nReviewer 3\nUnal et al showed a proof-of-concept therapy based on a synthetic biology approach to treat\nhypertension. The technology involved the development of a fully human, closed-loop gene\ncircuit named ARCH (Autonomous Regulator of Chronic Hypertension), that leverages the\nangiotensin II type 1 receptor (AT1R) and a stabilized, secretion-engineered angiotensin-\nconverting enzyme 2 (stACE2), to autonomously sense and regulate blood pressure. This is a\nhighly innovative approach, and experiments clearly showed possible application.\nWe thank the reviewer for their positive overall assessment and for recognizing the\ninnovation and potential applicability of ARCH as a fully human, closed-loop gene circuit for\nautonomous blood-pressure regulation. In response to the comments, we have further\nstrengthened the manuscript with additional in vivo mechanistic validation of RAS modulation,\nincluding expanded angiotensin-axis profiling and pharmacological inhibition of ACE2\nactivity, and we have also provided further support for translational relevance by testing\nresponsiveness to sera from hypertensive patients. We address each point in detail below.\n1. It is not clear why the authors included an NFAT response element in the synthetic promoter,\nbecause, as depicted in Figure 1C, PNFAT did not respond to Ang II.\nWe selected the synthetic P promoter because it integrates three repeats of CRE,\nCSN\nSRE, and NFAT operator modules and provides robust, low-leakage, highly inducible\ntranscriptional control across multiple signaling pathways. P was previously engineered and\nCSN\ncharacterized as a broadly responsive promoter architecture for mammalian gene circuits (Liu\net al., Nucleic Acids Res, 2018, PMID: 30219861) and has shown superior performance in\ndiverse cascade settings (Mahameed et al., Nat Commun, 2022, PMID: 36446786; Stefanov et\nal., Small, 2022, PMID: 36084222). Consistent with these reports, P also provided strong\nCSN\ninducible control downstream of AT1R-mediated cAMP signaling in the ARCH context.\n6\n2. In supplementary figure 2, the authors showed a basic (panel a), and a booster (panel b)\nsensor design. It is not clear whether the experiments performed in the manuscript were done\nusing the basic or the booster design.\nAll experiments reported in this manuscript were performed using the core ARCH\nconfiguration without the booster module, as stated in the Results (lines 336–338). We included\nthe booster plug-in only as an optional design extension to illustrate a modular topology that\ncould be readily integrated to increase gain if required in future applications, but it is not\nnecessary for the conclusions or in vivo performance demonstrated here.\n3. In figure 2b, angiotensin A, angiotensin-(1-9,) and angiotensin-(1-5) should also be tested.\nWe thank the reviewer for this suggestion. We have now expanded the specificity panel\nto include Ang-A, Ang-(1–9), and Ang-(1–5), thereby providing a more comprehensive\nassessment of angiotensin analog responsiveness (revised Fig. 2a, b).\n4. In figure 2f, the drugs were added in the presence of Ang II? Because in the figure appears\nas - AT-II + Drug\nIn Fig. 2f, the indicated drugs were applied instead of Ang-II (that is, Ang-II was\nomitted) to test whether they could directly activate the AT R/P reporter axis. We have\nrevised the figure legend to state this explicitly.\n5. Only protein levels of stACE2 were measured. Because the enzymatic activity of ACE2 is\nimportant for its biological effect, stACE2 activity should also be measured.\nWe agree that more comprehensive angiotensin-axis profiling would strengthen the\nmechanistic basis for autonomous, feedback-controlled regulation. Accordingly, in a new in\nvivo experiment we quantified circulating stACE2 together with Ang-II and Ang-(1–7) (new\nFig. 6). stACE2 levels increased in the treatment group in parallel with attenuation of Ang-II,\nconsistent with enzymatic Ang-II cleavage in vivo. This mechanism aligns well with the in\nvitro time-resolved Ang-II depletion kinetics we now include (new Fig. 3f) and also with\nestablished ACE2 biology within the vasoprotective RAS axis (Paz Ocaranza et al., Nat Rev\nCardiol, 2020, PMID: 31427727).\n6. How stable is the HEKARCH (Stable, monoclonal) cell line? Because synthetic gene circuits\nmay undergo genetic drift or silencing over time.\nWe agree this is an important issue. We have now assessed HEK circuit\nARCH\nperformance across multiple passage numbers (P10, P20, and P30) and observed stable\ninducible function across this range (new Fig. 3c), supporting the robustness of the engineered\nmonoclonal line. This passage window is consistent with commonly used industrial\nqualification ranges for HEK-293T-derived production cell lines, which have frequently been\nvalidated over 20–40 passages without evidence of genetic drift or functional decline (Grieger\net al., Mol Ther, 2015, PMID: 26437810).\n7. The in vivo experiments were performed using an Ang II-infused mouse model. Because\nhypertension is multifactorial and not solely Ang II dependent, a different hypertension model\nshould be tested, for instance, on spontaneously hypertensive rats.\nhave clarified in the manuscript that ARCH is positioned for angiotensin-dependent, RAS-\ndriven hypertension. As the reviewer kindly suggested, we introduced an additional disease-\nrelevant model of obesity-associated RAS dysregulation, in which adipose RAS activation\nincreases Ang-II production (Yasue et al., Am J Hypertens, 2010, PMID: 20057360). In this\nmodel, ARCH implantation reduced elevated plasma and renal Ang-II, and the results directly\nsupport the expected Ang-II-dependent mechanism (new Fig. 6). In addition, we performed a\nnegative-control experiment in an Ang-II-independent hypertension setting using the\nspontaneously hypertensive BPH/2 strain, which has been reported to exhibit neurogenic,\nRAS-independent hypertension (Jackson et al., Front Physiol, 2019, PMID: 31681017;\nJackson et al., Hypertens Res, 2020, PMID: 32427944). As expected for a circuit that acts\nthrough the Ang-II/AT R axis, ARCH had no apparent effect on the blood pressure profile in\nchange the interpretation of the mechanistic data provided in new Fig. 6, we did not include it\nin the revised manuscript. We would, however, be happy to incorporate this dataset into the\nSupplementary Information if the editor or reviewers consider it useful.\nFigure R1 | ARCH in BPH/2 mice. ARCH is designed to regulate Ang-II-dependent\nhypertension via the Ang-II/AT R axis and therefore is not expected to affect neurogenic, RAS-\n1\nindependent hypertension. Consistent with this specificity, ARCH implantation did not\nsignificantly change systolic blood pressure in the spontaneously hypertensive BPH/2 strain.\nStatistical analysis was performed by two-way ANOVA. Data are shown as mean ± SEM; n =\n4; ns, not significant.\n8. In the in vivo experiment, a control verifying that the decrease in blood pressure is due to\nstACE2should be included, i.e., by using an inhibitor of stACE2.\nWe thank the reviewer for this excellent suggestion. We have now added an in vivo\npharmacological control using the ACE2 inhibitor MLN-4760. In the new experimental group,\nmice received ARCH implants and two bolus doses of MLN-4760. Inhibition of ACE2 activity\ntransiently abolished the ARCH-mediated reduction in Ang-II, providing clear evidence that\nAng-II attenuation is driven by stACE2 enzymatic activity (new Fig. 6).\n9. In the animals, are the levels of Ang II decreased and the levels of Ang-(1-7) increased?\nWe have now performed longitudinal profiling of key RAS components, including\ncirculating stACE2, Ang-II, and Ang-(1–7) (new Fig. 6). In the treatment group, increased\nstACE2 coincided with sustained attenuation of Ang-II and an early increase in Ang-(1–7),\n8\nconsistent with ACE2-mediated conversion of Ang-II within the vasoprotective RAS axis (Paz\nOcaranza et al., Nat Rev Cardiol, 2020, PMID: 31427727). The Ang-(1–7) difference was\ntransient, which is consistent with downstream metabolism of Ang-(1–7) into peptides such as\nAng-(1–5) and alamandine. As ARCH acts upstream by reducing Ang-II, it is not expected to\ndirectly modulate these downstream processing steps (revised Fig. 2a).\n10. Produces this approach also a reduction of cardiac remodeling, vascular remodeling?\nWe thank the reviewer for raising this point. Elevated Ang-II has been implicated not\nonly in hypertension but also in additional pathophysiological processes, including\ncardiovascular remodeling, atherogenesis, renal fibrosis, insulin resistance, and tumor\nangiogenesis (Domenighetti et al., Hypertension, 2005, PMID: 15998712; Lovren et al., Am J\nPhysiol Heart Circ Physiol, 2008, PMID: 18660448; Mezzano et al., Hypertension, 2001,\nPMID: 11566946; Underwood, Curr Hypertens Rep, 2013, PMID: 23242734; Egami et al., J\nClin Invest, 2003, PMID: 12840060). Accordingly, sustained Ang-II attenuation through\nARCH could, in principle, confer broader vasoprotective effects beyond blood-pressure\ncontrol, including potential mitigation of Ang-II-driven cardiovascular remodeling. This would\nbe an interesting direction for future work.\nReviewer 4\nThe study approach is unique and novel model to use ARCH-transgenic human cells and\nelevate stACE2 to treat AT-II hypertension. In this approach, it is expected that stACE2 will\nbreak down (reduce) hypertensive AT-II and increase anti-hypertensive Ang1-7 peptide\nhormones. However, to validate these mechanisms, some measurements, as follows, are\nimportant.\nWe thank the reviewer for the constructive assessment and helpful suggestions. We\nhave addressed the key points by strengthening in vivo mechanistic validation of RAS\nmodulation, including expanded angiotensin-axis profiling and introduction of an ACE2-\ninhibitor control, and by clarifying the experimental design and interpretation throughout the\nmanuscript. We respond to each point in detail below.\n1. Plasma as well as tissue, particularly kidney levels of AT-II and Ang (1-7) will tell whether\nthe levels of these peptide hormones really changed and contributed to lowering the BP. Please\nmeasure the levels of these peptides.\nWe agree that broader profiling of the angiotensin axis is important to substantiate\nautonomous, feedback-controlled regulation in vivo. In response, we introduced an additional\ndisease-relevant model of RAS dysregulation using high-fat diet–induced obesity, in which\nadipose RAS activation increases Ang-II production (Yasue et al., Am J Hypertens, 2010,\nPMID: 20057360). We then quantified key RAS components in both plasma and kidney,\nincluding Ang-II, Ang-(1–7), soluble ACE2 (as a readout of stACE2 output), and renin. These\nmeasurements directly link ARCH activation to attenuation of elevated Ang-II, accompanied\nby an early Ang-(1–7) increase and compensatory renin elevation (new Fig. 6).\n2. ACE2 activity, not just the expression, is critical to correlate with the changes in AT-II and\nAng(1-7).\ninhibitor MLN-4760. In the new experimental group, mice received ARCH implants and two\n two\n\ntwo bolus doses of MLN-4760. ACE2 inhibition transiently abrogated the ARCH-mediated\nreduction in Ang-II, providing evidence that Ang-II attenuation is driven by stACE2 enzymatic\nactivity (new Fig. 6).\n3. Blocking/reducing AT1R is known to increase the renin activity, which has independently\nprofibrotic activity via pro-renin receptors. So please measure renin activity in the plasma and\nthe kidney.\nIndeed, suppression of AT R signaling is known to trigger compensatory renin release\n1\nfrom juxtaglomerular cells, leading to increased circulating renin levels (Belyea et al., Clin Sci,\n2023, PMID: 36503993; Nagalakshmi et al., Acta Physiol, 2023, PMID: 37309075). Consistent\nwith this physiology, ARCH implantation increased renin concentrations in both plasma and\nkidney relative to disease controls (new Fig. 6g, h). We agree that elevated renin could, in\nprinciple, engage (pro)renin receptor–linked, Ang-II-independent signaling pathways,\nincluding profibrotic responses. However, Ang-II itself is the major driver of fibrosis via AT R-\n1\ndependent induction of profibrotic mediators, and sustained Ang-II attenuation is a central\ntherapeutic strategy for preventing end-organ damage in RAS-driven disease (Brenner et al., N\nEngl J Med, 2001, PMID: 11565518; Lewis et al., N Engl J Med, 2001, PMID: 11565517). We\nhave clarified this compensatory renin response and its interpretation in the revised text and\nadded an explicit statement to the Discussion highlighting this consideration for future long-\nterm evaluation (lines 494–497).\n4. AT-II model of hypertension is good in animal models to understand the mechanism. For\ntranslational purposes, a salt-sensitive model of hypertension could be more helpful.\nWe thank the reviewer for this suggestion. We agree that salt-sensitive hypertension is\nclinically important. However, high salt intake has been reported to lower circulating Ang-II\nwhile promoting Ang-II hypersensitivity (Crestani et al., Vascul Pharmacol, 2014, PMID:\n24321189). Since ARCH is an Ang-II-sensing, Ang-II–degrading closed-loop circuit designed\nto respond to elevated Ang-II, we consider a salt-sensitive model would not be suitable as a\nprimary model to test ARCH. We have clarified this in the manuscript to avoid potential\nmisunderstanding. Instead, we selected an obesity-associated RAS dysregulation model, in\nwhich Ang-II elevation is a prominent feature, to further evaluate ARCH in a disease-relevant\ntranslational context (new Fig. 6).\n10"}, {"round": 2, "reviewer_comments": {"reviewer_1": "They have addressed all my concerns, so I have no further questions.", "reviewer_2": "The authors did a good job addressing the previous critiques. However, I have the following remaining concerns:\n1. Figure 1a shows four pathways downstream of AT1R, but only one pathway is shown in Figure 3a. This is confusing\nsince the broad-spectrum PCSN with combined CRE, SRE, and NFAT operator modules was selected for the ARCH\nsystem.\n2. Figure 6: In the obesity model, please include regular diet control. What are the methods for Ang-II and Ang-1-7 peptide\nmeasurement? What are the Ang-II levels in pg/mL? Are the values consistent with published literature?\n3. The plasma Ang-II levels in Figures 6 and 7 are very different. Please explain.\n4. The sample size (4 animals per group) is low for the in vivo experiments.\n5. Lines 387-388 “Valsartan and ARCH exhibited comparable efficacy in a kinetic assay.”\nShould it be Valsartan and stACE2 exhibited comparable efficacy in a kinetic assay?", "reviewer_3": "Unal et al performed most of the suggested changes. However, some concerns still remained:\n1. In Figure 3f, Ang II levels were measured as a readout of stACE2 activity. However, a decrease in Ang II levels could also\nbe produced by aminopeptidase II, dipeptidyl aminopeptidase, neprilysin, etc. All these enzymes are present in HEK293\ncells. Therefore, I recommend a direct measure of stACE2 enzymatic activity.\n2. On page 8, line 390, it should be clearly stated that the hypertension model is an Ang-II-infused mouse model.\n3. Incorporate the fig R1 as supplementary data in the manuscript.\n4. The quantification methods for Ang II, Ang-(1-7), and renin are not described in methods.\n5. Arrows of Figure 6 (a, c, e, g) are not described in the respective legend. In the same figure, why does the addition of an\ninhibitor of ACE2 reduce the amount of ACE2? ACE2 inhibitors should not reduce plasma ACE2 levels; instead, they should\nonly inhibit its enzymatic activity, thus preventing the conversion of Ang II to Ang-(1-7).", "reviewer_4": "The manuscript is adequately revised with additional data as asked in the original review. However, this reviewer could not\nlocate the methods used to quantify angiotensin II and angiotensin (1-7) peptides in mice plasma and kidney samples.\nMethod for renin measurement is mentioned under human samples; if the same method is used in animals' samples, please\nstate so."}, "author_response": "Reviewer #1 (Remarks to the Author):\nThey have addressed all my concerns, so I have no further questions.\nThank you.\nReviewer #2 (Remarks to the Author):\nThe authors did a good job addressing the previous critiques. However, I have the following\nremaining concerns:\nThank you. We addressed the remaining concerns below.\n1. Figure 1a shows four pathways downstream of AT1R, but only one pathway is shown in\nFigure 3a. This is confusing since the broad-spectrum PCSN with combined CRE, SRE, and\nNFAT operator modules was selected for the ARCH system.\nTo enhance clarity, we retained the detailed AT R downstream signaling map in Fig. 1a as a\n1\nconceptual overview. Fig. 3a serves a different purpose and was intentionally streamlined to\nhighlight the ARCH circuit architecture and the broad-spectrum P promoter (integrating\nCSN\nCRE, SRE, and NFAT operator modules), rather than re-drawing the full upstream signaling\nnetwork. The Fig. 3 legend now explicitly states that P integrates these signaling inputs,\nCSN\nand the label “Rewired signalling” is used in the schematic.\n2. Figure 6: In the obesity model, please include regular diet control. What are the methods for\nAng-II and Ang-1-7 peptide measurement? What are the Ang-II levels in pg/mL? Are the\nvalues consistent with published literature?\nThank you for your feedback. We performed a regular-diet control experiment in the obesity\nmodel and found that normal chow (NC)-fed mice did not show elevated plasma Ang II levels.\nAs these animals did not exhibit the obesity-associated increase in Ang II that the ARCH circuit\nis designed to counteract, the control dataset was not shown in the previous version. In response\nto the reviewer’s request, we now provide these data as new Supplementary Fig. 8.\nAng II was quantified using our cell-based HEK reporter assay, whereas Ang-(1–7) and\nARCH\nrenin were quantified using corresponding commercial ELISA kits. These measurement\nprocedures are now described in the Methods section (lines 224–234). In addition, as requested,\nAng II is now reported in absolute units in the revised Figs. 6a-b, 7e. Across groups, plasma\nAng II values were in the range of approximately 20–200 pg/mL, which is within the operating\nrange of our reporter assay and is consistent with published reports using comparable\nexperimental settings and quantification approaches (Yiannikouris, Hypertension, 2013,\nPMID: 23108647; Visniauskas, AJP-Heart, 2023, PMID: 36930656). We also note that\nabsolute circulating RAS peptide concentrations can vary substantially across studies, often\nover orders of magnitude, depending on sample handling, extraction, and assay platform (e.g.,\nimmunoassay vs. LC–MS/MS) as well as cohort characteristics (Gurley et al., JCI, 2006,\nPMID: 16878172; Gomolak et al., Front Physiol, 2014, PMID: 25400581; Santos et al., Life\nSci, 2014, PMID: 25219880). Accordingly, we emphasize relative differences within the same\nexperimental and analytical framework as the most directly interpretable readout.\n3. The plasma Ang-II levels in Figures 6 and 7 are very different. Please explain.\ngiven experiment and panel. Accordingly, RLU values from different figures should not be\ncompared quantitatively because they depend on background signal, instrument settings, data\nprocessing, and independent experimental runs. In Fig. 7e, the RLU axis was additionally\nrescaled to facilitate visualization alongside the clinical RFU readouts. To avoid any ambiguity,\nand in line with point 2 above, we now provide plasma Ang II levels in absolute units in the\nrevised Figs. 6a-b, 7e.\n4. The sample size (4 animals per group) is low for the in vivo experiments.\nIn accordance with European legislation (Directive 2010/63/EC) and the corresponding\nnational regulations governing animal experimentation in the EU, the UK, and Switzerland,\nanimal experiments are conducted under the principles of replacement, reduction, and\nrefinement (3R), with the aim of using the minimum number of animals required to achieve\nthe scientific objective. For each animal license, group sizes are predefined and justified during\nthe approval process based on power calculations that incorporate the expected effect size and\nvariance. Accordingly, group sizes in the range of 3 ≤ n ≤ 6 are widely used and accepted for\nmouse in vivo studies in high-impact research, including in vivo assessments of synthetic\nbiology and protein-based interventions. Representative examples include Vicent et al.,\nScience, 2023, PMID: 37824640; Roybal et al., Cell, 2016, PMID: 26830879; Reddy et al.,\nScience, 2024, PMID: 39636990; Cohen et al., Science Translational Medicine, 2026, PMID:\n41637528; Franko et al., Nature Communications, 2025, PMID: 41198646; Xue et al.,\nAdvanced Science, 2025, PMID: 40899507; Lin et al., Nature Nanotechnology, 2025, PMID:\n40325210; Huang et al., NAR, 2025, PMID: 40114374; Mahameed et al., Nature Biomedical\nEngineering, 2025, PMID: 39953326. Importantly, the observed effects were consistent across\nanimals within each group, and statistical analyses were performed as described in the\nMethods. Given the prospective ethical approval with predefined group sizes and the\nconsistency of the observed effects, we believe the current cohorts were sufficient to address\nthe study objectives.\n5. Lines 387-388 “Valsartan and ARCH exhibited comparable efficacy in a kinetic assay.”\nShould it be Valsartan and stACE2 exhibited comparable efficacy in a kinetic assay?\nThe sentence has been reworded for clarity to explicitly refer to the ARCH-mediated stACE2\neffector, thereby avoiding any ambiguity between the device (ARCH) and its therapeutic output\n(stACE2) (lines 403-404).\nReviewer #3 (Remarks to the Author):\nUnal et al performed most of the suggested changes. However, some concerns still remained:\nThank you. We addressed the remaining concerns below.\n1. In Figure 3f, Ang II levels were measured as a readout of stACE2 activity. However, a\ndecrease in Ang II levels could also be produced by aminopeptidase II, dipeptidyl\naminopeptidase, neprilysin, etc. All these enzymes are present in HEK293 cells. Therefore, I\nrecommend a direct measure of stACE2 enzymatic activity.\nbecause additional peptidases present in HEK293 cells can also contribute to Ang II turnover.\nIn the previous version, we therefore relied on the combined evidence of stACE2 production\ntogether with Ang II depletion and the consequent increase in Ang-(1–7). In response to your\nrecommendation, we now also provide a direct measurement of enzymatic activity for the\nrecombinantly produced stACE2 fusion protein using a fluorometric ACE2 activity assay (new\nSupplementary Fig. 3).\n2. On page 8, line 390, it should be clearly stated that the hypertension model is an Ang-II-\ninfused mouse model.\nThe text has been revised to explicitly state that the hypertension model is an Ang II-infused\nmouse model (lines 406-410).\n3. Incorporate the fig R1 as supplementary data in the manuscript.\nIn the previous revision, we provided Fig. R1 in the rebuttal as a negative-control dataset in an\nAng II-independent hypertension setting (spontaneously hypertensive BPH/2 strain), which has\nbeen reported to exhibit neurogenic, RAS-independent hypertension (Jackson et al., Front\nPhysiol, 2019, PMID: 31681017; Jackson et al., Hypertens Res, 2020, PMID: 32427944). As\nexpected for a circuit designed to act specifically through the Ang II/AT R axis, ARCH had no\n1\nmeasurable effect on the blood-pressure profile in this model. This outcome is therefore a\npredictable null result that follows directly from the defined input dependency of the system\nand does not add any mechanistic insight. Given the substantial expansion of the dataset across\ntwo rounds of revision (new Figs. 3f, 6a-h, 7a-f, and Supplementary Figs. 3a-b, 7, and 8),\nwe prefer to keep the Supplementary Information focused on experiments that directly support\nthe ARCH mechanism and therapeutic conclusions, and have therefore not incorporated Fig.\nR1, as we believe it would simply increase manuscript volume without strengthening any\nconclusion.\n4. The quantification methods for Ang II, Ang-(1-7), and renin are not described in methods.\nThank you for pointing this out. Ang II was quantified using our cell-based HEK reporter\nARCH\nassay, whereas Ang-(1–7) and renin were quantified using corresponding commercial ELISA\nkits. These quantification procedures have now been added to the Methods section (lines 224–\n234).\n5. Arrows of Figure 6 (a, c, e, g) are not described in the respective legend. In the same figure,\nwhy does the addition of an inhibitor of ACE2 reduce the amount of ACE2? ACE2 inhibitors\nshould not reduce plasma ACE2 levels; instead, they should only inhibit its enzymatic activity,\nthus preventing the conversion of Ang II to Ang-(1-7).\nThe arrows in Fig. 6a,c,e,g indicate the timing of key experimental steps. To improve\ninterpretability, we have expanded the description and now annotate this information directly\nin the respective panels in addition to the figure legend (revised Fig. 6a,c,e,g).\nRegarding the ACE2 inhibitor, MLN4760 is a competitive ACE2 inhibitor that blocks catalytic\nactivity. In addition, MLN4760 binding has been reported to induce conformational changes\nin soluble ACE2 that can influence non-catalytic interactions and, depending on the assay\nformat, may affect antibody-based detection of soluble ACE2 (Towler et al., J Biol Chem,\n3\n2004, PMID: 14754895; Nami et al., J Hypertens, 2021, PMID: 34188005; Trozzi et al., Org\nBiomol Chem, 2022, PMID: 35420112). We therefore infer that the reduced signal observed in\nthe presence of MLN4760 reflects altered detectability/assay readout rather than a true\nreduction in ACE2 abundance. This clarification has been added to the main text (lines 364-\n369), supported by new Supplementary Fig. 3, and the y-axis label has been adjusted\naccordingly (revised Fig. 6c).\nReviewer #4 (Remarks to the Author):\nThe manuscript is adequately revised with additional data as asked in the original review.\nHowever, this reviewer could not locate the methods used to quantify angiotensin II and\nangiotensin (1-7) peptides in mice plasma and kidney samples. Method for renin measurement\nis mentioned under human samples; if the same method is used in animals' samples, please\nstate so.\nThank you for pointing this out. Ang II was quantified using our cell-based HEK reporter\nARCH\nassay, whereas Ang-(1–7) and renin were quantified using corresponding commercial ELISA\nkits for mouse samples. The renin measurement in animals was performed using the same\nELISA-based method described for human samples. ACE2 activity was measured using a\nfluorometric ACE2 activity assay. These quantification procedures have now been explicitly\nadded to the Methods section (lines 224-234).\n4"}, {"round": 3, "reviewer_comments": {"reviewer_2": "I have no additional comments.", "reviewer_3": "The manuscript is adequately revised with corrections as requested.", "reviewer_4": "Authors have clarified my concerns and questions and revised the manuscript adequately."}, "author_response": "Reviewer #1 (Remarks to the Author): (Previous round)\nThey have addressed all my concerns, so I have no further questions.\nThank you.\nThank you.\nThank you.\nThank you."}]

Decision letter