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. 2017 Feb 22;25(2):443–455. doi: 10.1016/j.ymthe.2016.11.008

A Synthetic-Biology-Inspired Therapeutic Strategy for Targeting and Treating Hepatogenous Diabetes

Shuai Xue 1, Jianli Yin 1, Jiawei Shao 1, Yuanhuan Yu 1, Linfeng Yang 1, Yidan Wang 1, Mingqi Xie 1,2, Martin Fussenegger 2, Haifeng Ye 1,∗
PMCID: PMC5368401  PMID: 28153094

Abstract

Hepatogenous diabetes is a complex disease that is typified by the simultaneous presence of type 2 diabetes and many forms of liver disease. The chief pathogenic determinant in this pathophysiological network is insulin resistance (IR), an asymptomatic disease state in which impaired insulin signaling in target tissues initiates a variety of organ dysfunctions. However, pharmacotherapies targeting IR remain limited and are generally inapplicable for liver disease patients. Oleanolic acid (OA) is a plant-derived triterpenoid that is frequently used in Chinese medicine as a safe but slow-acting treatment in many liver disorders. Here, we utilized the congruent pharmacological activities of OA and glucagon-like-peptide 1 (GLP-1) in relieving IR and improving liver and pancreas functions and used a synthetic-biology-inspired design principle to engineer a therapeutic gene circuit that enables a concerted action of both drugs. In particular, OA-triggered short human GLP-1 (shGLP-1) expression in hepatogenous diabetic mice rapidly and simultaneously attenuated many disease-specific metabolic failures, whereas OA or shGLP-1 monotherapy failed to achieve corresponding therapeutic effects. Collectively, this work shows that rationally engineered synthetic gene circuits are capable of treating multifactorial diseases in a synergistic manner by multiplexing the targeting efficacies of single therapeutics.

Keywords: synthetic biology, synthetic gene circuit, prosthetic gene network, biomedical engineering, gene and cell-based therapy, oleanolic acid, hepatogenouse diabetes, glucagon-like peptide-1

Graphical Abstract

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Ye and colleagues describe a promising and realistic therapeutic strategy for the clinical application of synthetic-biology-based and oleanolic-acid-controlled gene circuits for treating hepatogenous diabetes, the currently frequently discussed disease profile characterized by the simultaneous presence of diabetes mellitus, chronic liver diseases, and insulin resistance.

Introduction

Hepatogenous diabetes, which is the complex and bi-directional relationship linking type 2 diabetes mellitus and many forms of chronic liver disease, has recently been the focus of intense new interest.1, 2, 3 Type 2 diabetes mellitus is typified by persistent hyperglycemia resulting from an impaired hepatic insulin-mediated glucose uptake, insulin resistance (IR), and the progressive exhaustion of pancreatic β cells;4, 5 however, many early stages of liver disorders, such as non-alcoholic fatty liver disease, fail to trigger symptoms or significant discomfort until eventual routine laboratory assessments reveal a profile of metabolic impairments that is indicative of the simultaneous presence of diabetes and liver failure.2, 6 As a result, most type 2 diabetes mellitus patients suffer from a large number of liver disorders, including elevated liver enzymes, fatty liver disease, dyslipidemia, cirrhosis, and an increased susceptibility to hepatocellular carcinoma and hepatitis C virus infection.7, 8, 9 Therefore, the development of novel diagnostic, prognostic and therapeutic tools that act early in attenuating key metabolic impairments of hepatogenous diabetes is urgently needed in biomedical research.10, 11, 12

In recent years, increasing numbers of opinions in the fields of gastroenterology and endocrinology have specified IR (a state preceding the development of overt diabetes in which increased circulating free fatty acids impair insulin signaling in target tissues, resulting in excessive levels of blood insulin that are insufficient for achieving glycemic control2, 4) as a chief pathogenic determinant of hepatogenous diabetes and therefore an ideal target for therapeutic intervention.8, 9, 13, 14, 15 However, pharmacotherapies can be dangerous for liver disease patients, because these drugs may induce fatal metabolic burdens on the liver during their hepatic first pass. Consequently, most preferred treatment options for hepatogenous diabetes include hepatoprotective medications that target type 2 diabetes.2, 7 The subcutaneous injection of glucagon-like-peptide 1 (GLP-1) receptor agonists,16 which are analogs of the GLP-1 peptide hormone that is naturally released from the intestine after meal ingestion to (1) stimulate postprandial insulin release from pancreatic β cells, (2) improve hepatic insulin sensitivity, (3) slow gastric emptying, and (4) inhibit glucagon secretion,17, 18, 19, 20 has evolved as the treatment option of choice for most type 2 diabetes cases and shows optimal therapeutic efficacy for parallel liver disease symptoms.6, 21, 22

To reduce the metabolic burden on the liver caused by many drug delivery routes,7 several studies have sought to stimulate endogenous GLP-1 secretion by activating signaling pathways regulated by the G-protein-coupled receptor GPBAR1.23, 24 GPBAR1, also known as TGR5, is activated by a variety of amphipathic metabolites, including bile acids, to stimulate the release of GLP-1 from intestinal cells via a cyclic AMP (cAMP)-dependent pathway.25 However, this phenomenon could only be achieved in transgenically modified mice with dramatically increased GPBAR1 expression levels; in wild-type animals, both natural and synthetic GPBAR1 agonists failed to activate endogenous GLP-1 expression into a bioactive range with observable therapeutic activity.22 Therefore, a biomedical approach that enables GPBAR1-mediated GLP-1 production but does not necessitate host genetic interventions to achieve therapeutic efficacy would be highly attractive for targeting and treating essential symptoms of hepatogenous diabetes.

In this work, we capitalized on the recent discovery of oleanolic acid (OA) as a strong GPBAR1 agonist and engineered a synthetic-biology-inspired OA-triggered gene expression device in human embryonic kidney cells, which have previously been shown to have optimal production capacities for antidiabetic proteins.26, 27, 28 OA (3β-hydroxyolean-12-en-28-oic acid) is a pentacyclic triterpenoid compound that is widely distributed in the plant kingdom as a major constituent of olive leaves, apples, and red beets. Currently, its hepatoprotective, anti-inflammatory, and antidiabetic functions as active ingredients in complementary and alternative medicine (CAM) are also gaining clinical acceptance.29, 30, 31 When implanting immunoprotective microcapsules containing OA-regulated short human GLP-1 (shGLP-1)-expressing cells into a mouse model of hepatogenous diabetes, only the combined action of OA administration with shGLP-1 secretion led to the rapid and simultaneous attenuation of many disease-specific metabolic failures such as glucose intolerance, IR, hyperglycemia, dyslipidemia, and excessive liver enzyme levels (Figure 1). Furthermore, a doxycycline (Dox)-induced regulatory element integrated into the circuit enabled the rapid and on-demand termination of transcription, which provided an increased control capacity for unforeseeable scenarios in vivo. Collectively, this therapeutic gene circuit assembled through synthetic-biology-based design principles compresses the most promising drug targets for treating hepatogenous diabetes into a compact, effective, and accessible delivery system. In particular, we believe that this concept of a combined therapy in which a non-toxic phytochemical compound coordinates the expression of an antidiabetic protein at optimal bioavailable concentrations will maximally exploit the therapeutic potential of biological systems, which should foster the development of synthetic cell-based treatment strategies for clinical applications in molecular and translational medicine.32, 33, 34, 35, 36

Figure 1.

Figure 1

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An Abstract Diagram Showing the Synthetic-Biology-Inspired Therapeutic Strategy to Combat Hepatogenous Diabetes

When hepatogenous diabetic mice implanted with immunoprotective microcapsules containing oleanolic acid (OA)-controlled shGLP-1-expressing mammalian cells and orally administrated with OA (an extract from olives), the combined action of OA administration with shGLP-1 expression led to a rapid and simultaneous attenuation of many metabolic failures such as hyperglycemia, dyslipidemia (high triacylglycerol [TG] levels), and excessive liver enzyme levels (such as aspartate transaminase [AST] and alanine aminotransferase [ALT]).

Results

Design of an OA-Inducible Dox-Repressible Gene Circuit

In human cells, OA binds to the G-protein-coupled receptor GPBAR1 (G-protein-coupled bile acid receptor) to trigger an intracellular surge of the second messenger cAMP via a Gαs-protein-mediated signal transduction cascade.11, 25 The binding of cAMP molecules to the regulatory subunits of protein kinase A (PKA) triggers the translocation of the catalytic subunits of PKA into the nucleus, where they phosphorylate a variety of protein substrates at their serine or threonine residues.37 To couple a synthetic transcription unit to this OA-triggered PKA-dependent signaling pathway, we engineered a hybrid transcription factor TetR-CREB1 (pXS13; PhCMV-TetR-CREB1-pA) consisting of a TetR DNA-binding domain and a PKA-substrate CREB1 (Figure 2). In the absence of OA, constitutively expressed TetR-CREB1 binds to a TetR-specific inducible promoter PhCMV*-1 (PhCMV*-1, tetO7-PhCMVmin) and is incapable of activating transgene expression. However, the OA-triggered nuclear accumulation of PKA phosphorylates CREB1, rendering it a strong transactivator and triggering transgene expression from PhCMV*-1-driven vectors. This transactivation module can be further regulated by another trigger compound, Dox, the presence of which in the cell would induce a dissociation of TetR-CREB1 from PhCMV*-1 and lead to the immediate termination of transgene expression independent of the phosphorylation status of CREB1. Therefore, TetR-CREB1 functions as a synthetic dual-input transcriptional regulator that enables OA-inducible Dox-repressible transgene expression in human cells (Figure 2).

Figure 2.

Figure 2

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Design of a Synthetic OA-Inducible Dox-Repressible Gene Expression Device

The binding of OA to ectopically expressed human G-protein-coupled bile acid receptor (GPBAR1) triggers the Gαs-mediated activation of adenylate cyclase (AC), which converts ATP to cyclic AMP (cAMP). cAMP binds to the regulatory subunits of protein kinase A (PKA), the catalytic subunits of which translocate into the nucleus, where they phosphorylate the cAMP-responsive binding protein 1 (CREB1). Phosphorylated TetR-CREB1 fusion proteins trans-activate minimal inducible promoters engineered to contain TetR-specific operator sites (PhCMV*-1; tetO7-PhCMVmin). The presence of doxycycline (Dox) renders the TetR-domain incapable of binding tetO7, resulting in the immediate termination of transgene expression. PConst, constitutive promoter; pA, polyadenylation signal.

Construction of an Optimal Expression Platform for OA-Inducible Dox-Repressible Transgene Expression

An optimal OA-inducible Dox-repressible gene switch should enable (1) low basal transgene expression levels in the absence of OA, (2) high transgene induction ratios in the presence of OA, and (3) complete transcriptional inactivity in the presence of Dox. Therefore, we transfected human cells with TetR-CREB1 (pXS13; PhCMV-TetR-CREB1-pA), pMF111 (PhCMV*-1-SEAP-pA) (a reporter vector containing PhCMV*-1 driving human placental secreted alkaline phosphatase [SEAP]), and various GPBAR1-expression vectors that differ in their constitutive promoters (pXS16, MCS-GPBAR1-pA; pXS20, PNFAT-GPBAR1-pA; pXS21, PARE-GPBAR1-pA; pXS22, PCRE-GPBAR1-pA; pXS23, PCRE-tight-GPBAR1-pA; pXS24, PhCMV*-1-GPBAR1-pA; pXS25, PhCMV-GPBAR1-pA; pXS26, PSV40-GPBAR1-pA; pXS43, PNF-κB-GPBAR1-pA) at various plasmid ratios and found that the combination of pXS25/pXS13/pMF111 showed optimal OA-inducible Dox-repressible SEAP regulation (Figure S1). Therefore, this combination was used in all following experiments. The confocal imaging of fluorescently tagged GPBAR1 (pXS44, PhCMV-GPBAR1-EGFP-pA) confirmed the efficient expression and membrane localization of the receptor upon transfection (Figure S2), whereas OA treatment of pXS25-transfected human cells triggered a rapid (Figure S3) and PKA-dependent (Figure S4) intracellular cAMP surge. Importantly, neither the OA treatment nor plasmid transfection affected the viability or metabolic integrity of human cells (Figure S5), thereby excluding falsifying perturbations from the experimental conditions. Additionally, OA did not activate other Gαs-protein-dependent membrane receptors that involve cAMP-dependent PKA signaling, thereby confirming exclusive signal rewiring (Figure S6), and this specificity to GPBAR1 was further corroborated by the finding that this circuit was insensitive to physiological levels of a representative composition of human serum bile acids (chenodeoxycholic acid [CDCA], 31%; deoxycholic acid [DCA], 31%; cholic acid [CA], 35%; lithocholic acid [LCA], 1%; and ursodeoxycholic acid [UDCA], 2%38, 39) (Figure S7).

Characterization of the OA-Inducible Dox-Repressible Gene Circuit in Vitro and in Mice

An assessment of the pXS25/pXS13/pMF111-encoded OA-inducible Dox-repressible gene circuit in various mammalian cell lines revealed optimal expression performances in various derivatives of human embryonic kidney cells and mesenchymal stem cells, suggesting that this system is applicable to a variety of human cell types (Figure 3A). SEAP expression could be dose-dependently activated not only by chemically synthesized 3β-hydroxyolean-12-en-28-oic acid pure compounds (Figure 3B) but also by solubilized OA tablets that are commonly available as over-the-counter (OTC) drugs (Figure 3C). Individual time-course experiments of enhanced yellow fluorescent protein (EYFP) or SEAP expression revealed tight (Figure 3D), rapid (Figures 3D and 3E), and reversible (Figure S8) induction kinetics resulting from a precise, robust, and tunable OA-triggered remote control. The Dox-triggered termination module further expanded the control capacity over the transcription unit in which OA-activated expression was dose-dependently reversed by Dox administration (Figure 3F); initially repressed SEAP expression states could also be released by the removal of Dox at any user-defined point in time (Figure 3G). For the long-term study of transgene expression kinetics in mammalian cells, the synthetic OA-controlled gene circuit was further integrated into HEK293 cells using the Sleeping Beauty transposon system.40, 41, 42 SEAP expression could be dose-dependently activated in the stable HEKGPBAR1-SEAP cells for 6 days (Figure 4A), and OA-activated expression could be dose-dependently reversed by Dox (Figure 4B). The fluorescent micrographs further confirmed the tight and time-dependent transgene profiles (Figure 4C).

Figure 3.

Figure 3

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Characterization of the Synthetic OA-Inducible Dox-Repressible Gene Expression Device in Mammalian Cells

(A) OA-inducible Dox-repressible SEAP expression in various mammalian cell lines. HeLa, CHO-K1, HEK293, Hana3A, and hMSC-TERT cells were co-transfected with pXS25 (PhCMV-GPBAR1-pA), pXS13 (PhCMV-TetR-CREB1-pA) and pMF111 (PhCMV*-1-SEAP-pA) in a 1:20:20 ratio (w/w) and cultured in cell culture medium containing OA (20 μM), a combination of Dox (5 ng/mL) and OA (20 μM), or no additional supplement (control). SEAP levels in the culture supernatants were profiled after 72 hr. Data represent mean ± SD; n = 3 independent experiments. (B and C) Dose-dependent OA-inducible SEAP expression. HEK293 cells were co-transfected with pXS25, pXS13, and pMF111 in a 1:20:20 ratio (w/w) and cultured in cell culture medium containing various OA concentrations. SEAP levels in the culture supernatants were profiled every 24 hr. Data represent mean ± SD; n = 3 independent experiments. (D) Fluorescence micrographs profiling EYFP expression in HEK293 cells cotransfected with pXS25, pXS13, and pHY74 (PhCMV*-1-EYFP-pA) in a 1:20:20 ratio (w/w) and cultivated for 72 hr in the presence or absence of 20 μM OA. (E) SEAP expression kinetics of pXS25/pXS13/pMF111-cotransfected HEK293 cells cultivated for various periods of time in the presence of 20 μM OA. Data represent mean ± SD; n = 3 independent experiments. (F) Dose-dependent Dox-repressible SEAP expression. pXS25/pXS13/pMF111-transgenic HEK293 cells were cultivated in the presence of 20 μM OA and various concentrations of Dox. SEAP levels in the culture supernatants were profiled every 24 hr. Data represent mean ± SD; n = 3 independent experiments. (G) Dox-mediated SEAP derepression. pXS25/pXS13/pMF111-transgenic HEK293 cells were cultivated in the presence of OA (20 μM) and Dox (5 ng/mL) for various lengths of time prior to exchanging the cell culture medium to fresh Dox-free DMEM supplemented with 20 μM OA. SEAP levels in the culture supernatants were quantified accordingly. The various colored arrows correspond to the removal action of Dox at different time points. Data represent mean ± SD; n = 3 independent experiments.

Figure 4.

Figure 4

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Long-Term Characterization of the Synthetic OA-Inducible Dox-Repressible Gene Expression Device in HEKGBAR1-SEAP Cells

(A) Dose-dependent OA-inducible SEAP expression. pXS101/pXS103/pXS102-transgenic HEKGPBAR1-SEAP cells were cultured in culture medium containing various OA concentrations. SEAP levels in the culture supernatants were profiled every 2 days. Data represent mean ± SD; n = 3 independent experiments. (B) Dose-dependent Dox-repressible SEAP expression. pXS101/pXS103/pXS102-transgenic HEKGPBAR1-SEAP cells were cultivated in the presence of 20 μM OA and various concentrations of Dox. SEAP levels in the culture supernatants were profiled every 2 days. (C) Fluorescence micrographs profiling EGFP expression in HEKGPBAR1-SEAP cells and cultivated for 6 days in the presence or absence of 20 μM OA.

To validate the OA-inducible Dox-repressible gene circuit in vivo, we microencapsulated pXS25/pXS13/pMF111-transgenic human cells into coherent, semi-permeable, and immunoprotective alginate-poly-(L-lysine)-alginate beads (Figure S9), which are attractive because of their potential for vascularization to support appropriate oxygen supply to the encapsulated cells (Figure 5A).43, 44, 45 This clinically validated implant technology enables the free diffusion of metabolites, nutrients, and proteins of lower molecular weight (<72 kDa)46, 47, 48, 49, 50 across the biocompatible capsule membrane while shielding their cellular content from physical contact with the host’s immune system (Figure 5A).51, 52 Notably, SEAP levels in the bloodstream of mice were correlated with increased doses of OA administered via either the injection of PBS-buffered 3β-hydroxyolean-12-en-28-oic acid pure compounds (Figure 5B) or the oral uptake of solubilized OA tablets at clinically approved dosages (Figure 5C). Dox-triggered SEAP repression was also completely achieved in vivo even in the presence of a half-maximal activating OA dosage, thereby enabling tight and precise remote control over implant activities during the clinical investigations (Figure 5D).

Figure 5.

Figure 5

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OA-Triggered SEAP Expression in Wild-Type Mice

(A) Principle of the microcapsule implants technology. Mammalian cells engineered to carry custom-designed genetic circuits are microencapsulated into alginate-poly-(L-lysine)-alginate beads that embed into the capillary structures of animal tissues upon implantation. The pore size of the capsules can be tuned so that oxygen, nutrients, and growth factors freely diffuse across the membrane, while larger compounds such as antibodies or immune cells are shielded from the cellular content. In the case of microencapsulated pXS25/pXS13/pMF111-transgenic HEK293 cells, OA or Dox administered to mice via either injection or oral uptake reaches the implant through the systemic circulation to remotely control capsule activities. (B and C) Dose-dependent OA-inducible SEAP expression. Male C57BL/6J mice were intraperitoneally implanted with 2 × 106 microencapsulated pXS25/pXS13/pMF111- transgenic HEK293 cells (200 cells/capsule) and received three daily doses of (B) injected OA solution or (C) orally administered tablets. SEAP levels in the bloodstream of mice were quantified at 48 hr after implantation. Data are expressed as mean ± SEM; statistics via two-tailed t test; n = 8 mice. ***p < 0.001 versus control. (D) Dose-dependent Dox-repressible SEAP expression. Male C57BL/6J mice were intraperitoneally implanted with 2 × 106 microencapsulated pXS25/pXS13/pMF111-transgenic HEK293 cells (200 cells/capsule) and received orally administered OA tablets (3 × 10 mg/kg/day) and daily injections of Dox. SEAP levels in the bloodstream of the mice were quantified at 48 hr after implantation. Data are shown as mean ± SEM; statistics via two-tailed t test; n = 8 mice. **p < 0.01 and ***p < 0.001 versus control.

Attenuation of Hepatogenous Diabetes Symptoms in db/db Mice

Hepatogenous diabetes patients are typically characterized by the simultaneous presence of a variety of metabolic impairments such as IR, excessive liver enzyme activities, hyperglycemia, glucose intolerance, and dyslipidemia.1, 2, 7, 10 Pharmacotherapies capable of targeting all of those symptoms are rare, and excessive drug consumption is problematic for patients with liver dysfunction.53, 54 Currently, the insulin-sensitizing hormone GLP-1, which is increasingly preferred in type 2 diabetes therapies,16, 55 has also received wide acceptance in treating liver disorders. In an attempt to design a customized therapy against hepatogenous diabetes, we capitalized on the congruent pharmacological activities of OA and GLP-1 targeting IR and relieving the metabolic burden on the liver and pancreas and thus engineered a shGLP-1 expression unit under the control of the OA-inducible Dox-repressible promoter architecture (Figure S10A). The expression of absolute (Figure S10B) and bioactive amounts of shGLP-1 (Figure S10C) was dose- and time-dependently triggered by solubilized OA tablets in vitro and in mice (Figures S11A and S11B), highlighting the modularity and interoperability of engineering synthetic gene circuits.

To create a mouse model for hepatogenous diabetes, we used db/db mice in which mutations in the leptin receptor gene successively cause obesity, IR, glucose intolerance, hyperglycemia, type 2 diabetes, and liver steatosis.10 The male db/db mice used in this study showed not only persistent hyperglycemia (26.7 ± 1.22 mM) upon reception but also excessive aspartate transaminase (AST) (Figure S12A) and alanine aminotransferase (ALT) levels (Figure S12B), thereby confirming the simultaneous presence of type 2 diabetes and liver disease symptoms and validating the eligibility of this hepatogenous diabetes disease model (Table S1). When db/db mice were implanted with the microencapsulated transgenic HEK293 cells containing OA-inducible Dox-repressible shGLP-1 expression device, OA triggered an accumulation of shGLP-1 levels in the bloodstream within 48 hr (Figure 6A) that was sufficient for substantial restoration of the animals’ glycemic control (Figure 6B) and insulin sensitivity (Figure 6C). Importantly, control experiments confirmed that this increase in serum shGLP-1 exclusively resulted from the TetR-CREB1-regulated promoter (pHY73, PhCMV*-1-shGLP1-pA); in wild-type mice, neither OA nor other GPBAR1-agonists such as LCA (a bile acid derivative with high GPBAR1 affinity) or CCl4 (carbon tetrachloride, a hepatotoxin that causes acute surges of serum bile acids) could stimulate the secretion of endogenous GLP-1 through GPBAR1-mediated pathways22, 23, 24, 25 (Figure S13). Furthermore, OA-triggered shGLP-1 expression also rapidly attenuated the most critical hepatogenous diabetes symptoms, such as IR (Figure 6D), excessive liver enzyme activity (Figures 6E–6G), and dyslipidemia (Figure 6H), whereas a monotherapy with commercial OA tablets or with only shGLP-1 failed to elicit significant changes in this investigated disease profile (Figures S14 and S15). These results suggest that combined therapy as designed with this synthetic gene circuit is capable of rapidly reversing complex and multifactorial diseases, such as hepatogenous diabetes, before various symptoms aggravate or induce more severe disorders.

Figure 6.

Figure 6

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OA-Triggered shGLP-1 Expression in a Mouse Model of Hepatogenous Diabetes

(A) OA-triggered shGLP-1 expression. Male db/db mice were intraperitoneally implanted with 2 × 106 microencapsulated pXS25/pXS13/pHY73 (PhCMV*-1-shGLP1-pA)-transgenic HEK293 cells (200 cells/capsule) and received orally administered OA tablets (3 × 100 mg/kg/day). Active GLP-1 levels in the bloodstream of the mice were quantified 48 hr after implantation. (B and C) Intraperitoneal (B) glucose and (C) insulin tolerance test of db/db mice. 48 hr after implantation, the same groups of mice as in (A) received an intraperitoneal injection of (B) aqueous 1.5 g/kg D-glucose or (C) 1 U/kg recombinant human insulin, and the glycemic profile of each animal was tracked every 30 min. (D–H) Effect of OA-triggered shGLP-1 expression on homeostasis model assessment for insulin resistance (HOMA-IR) (D), AST (E), ALT (F), the AST/ALT ratio (G), and triacylglycerol levels (H) in db/db mice scored at 48 hr after implantation (same mice as in A). All mouse data are shown as mean ± SEM, and the analysis was performed with a two-tailed t test (n = 8 mice). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control.

In order to evaluate the long-term therapeutic efficacy of the OA-inducible Dox-repressible shGLP-1 expression device in db/db mice, the synthetic OA-inducible circuit was uploaded into the Sleeping Beauty (SB) transposon system,40, 41, 42 which allowed the stable introduction of the synthetic OA-inducible circuit into mammalian cells. When db/db mice were thrice implanted with pXS101/pXS103/pXS107-transgenic HEKGPBAR1-shGLP-1 cells every 5 days, OA triggered a long-term stable accumulation of shGLP-1 levels in the bloodstream within 15 days (Figure 7A) that was sufficient for significant restoration of the animals’ glycemic control (Figure 7B) and insulin sensitivity (Figure 7C). Moreover, OA-triggered shGLP-1 expression further attenuated hepatogenous diabetes symptoms, such as IR (Figure 7D), excessive liver enzyme activities (Figures 7E–7G), and dyslipidemia (Figure 7H), in 15 days. The mice that received treatment also showed significant reduction of food (Figure 7I) and water (Figure 7J) intake and decreased body weight changes (Figure 7K). After 15 days of treatment, HbA1c levels were significantly decreased (Figure 7L). Liver histological analysis further revealed a significant reduction of fatty accumulation in hepatocytes of treated mice (Figures 7M and 7N). Collectively, these results demonstrate that the long-term efficacy of the synthetic-biology-inspired design strategy to engineer a therapeutic gene circuit enables a synergistic action of OA and shGLP-1 that improves liver function and relieves diabetes (Table S1).

Figure 7.

Figure 7

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Long-Term Therapeutic Effect of OA-Triggered shGLP-1 Expression in a Mouse Model of Hepatogenous Diabetes

(A) OA-triggered shGLP-1 expression. Male db/db mice were intraperitoneally implanted with 2 × 106 microencapsulated pXS101/pXS103/pXS107-transgenic HEKGPBAR1-shGLP-1 cells (200 cells/capsule) on days 0, 5, and 10 and received orally administered OA tablets (3 × 100 mg/kg/day). Active GLP-1 levels in the bloodstream of the mice were scored every 3 days after implantation. (B and C) Intraperitoneal glucose (B) and insulin (C) tolerance test of db/db mice. 15 days after implantation, the same groups of mice as in (A) received an intraperitoneal injection of 1.5 g/kg D-glucose (B) or 1 U/kg recombinant human insulin (C), and the glycemic profile of each animal was tracked every 30 min. (D–J) Effect of OA-triggered shGLP-1 expression on HOMA-IR (D), AST (E), ALT (F), the AST/ALT ratio (G), triacylglycerol levels (H), and food (I) and water (J) intake in db/db mice scored on day 15 after implantation (same mice as in A). (K) Body weights of the same treated db/db mice were profiled every 3 days as well. (L) Change in hemoglobin A1c (HbA1c) levels by OA administration over 15 days. (M and N) H&E-staining-based histological analysis of liver sections. 15 days after implantation, mice were sacrificed for liver histological analysis. Representative of liver micrographs from control mice treated with vehicle (M) and mice treated with OA (N). The arrows represent fatty metamorphosis of the liver. Scale bar, 500 μm. Mouse data (A–L) are shown as the mean ± SEM, and the analysis was performed with a two-tailed t test (n = 6 mice). *p < 0.05, **p < 0.01, and ***p < 0.001 versus control.

Discussion

In recent years, the use of botanical materials or phytochemicals, either as a potential source of new drugs or as pharmacologically active ingredients in CAM formulations, has received broad acceptance by the general public.56, 57 As a natural triterpenoid widely found in several dietary and medicinal plants (e.g., Olea europaea, Viscum album L., or Aralia chinensis L.), OA has been used in Chinese medicine for the treatment of liver disorders for over 20 years, and commercial OA tablets are available as OTC drugs in Chinese pharmacies for the treatment of acute and chronic hepatitis infections.30, 58 Although they are slow-acting, herbal medicines and medical nutrition therapies are recommended for liver disease patients over pharmacotherapies that utilize chemically synthesized drugs, because the former elicit milder metabolic burdens on a defective liver during the unavoidable hepatic first pass of xenobiotics.7, 53, 59

Synthetic biology, which is the science of reassembling characterized, optimized, and cataloged biological building blocks to engineer designer cells to solve predetermined problems in biotechnology and medicine,32 continually leads to novel therapeutic strategies for treating complex multifactorial diseases.36, 60, 61, 62 Implants carrying therapeutic designer cells can be engineered for the inducible production of a therapeutic protein with a customizable trigger signal, such as disease markers,26, 63, 64, 65 light,28 food additives,66, 67 or cosmetics,68 thereby synchronizing the activity of the drugs with a patient’s internal health state or lifestyle. In this work, we engineered a synthetic therapeutic designer cell that compensates for the intrinsically slow-acting character of OA-based monotherapies. OA and GLP-1 share similar pharmacological activities in attenuating IR, liver dysfunction, and glucose intolerance,21, 30 and a synthetic OA-triggered shGLP-1-expressing device implanted into mice with hepatogenous diabetes enabled a dramatic amplification of the therapeutic effect at the same OA dosage. Only the combined action of OA administration triggering in vivo shGLP-1 secretion was able to attenuate many disease-specific metabolic failures of hepatogenous diabetes over 15 days (Figure 7); the administration of OA or shGLP-1 alone failed to achieve rapid therapeutic effects. Therefore, in this therapeutic strategy, OA not only acts as an chemical inducer but also has additional therapeutic benefits and might be a preferred inducer compared to previous inducible systems (such as benzonic acid,66 paraben,67 and vanillic acid68).

Collectively, the therapeutic designer cell engineered in this work integrates all potential advantages of this novel strategy to treat a multifactorial disease such as hepatogenous diabetes. First, both OA and GLP-1 target IR, enabling the treatment of an essential pathogenic determinant in the progression from acute metabolic abnormalities to chronic liver disease and type 2 diabetes at the earliest possible stage.2 Second, the implantation of microencapsulated human designer cells is a clinically validated and immunoprotective therapeutic setup in which the host and graft communicate solely via secretory metabolites that diffuse across a semi-permeable biocompatible membrane.44, 49, 52 Third, a Dox-triggered safety switch engineered into the designer cell architecture enables the precise and on-demand termination of gene circuit function during any unforeseeable scenarios in clinical applications. In the not-too-distant future, the optimization of this design architecture into a technical platform for (pre)-clinical studies in humans69, 70, 71 would greatly foster the biomedical impact of synthetic biology.

Materials and Methods

Study Design

The aim of this study was to demonstrate a novel therapeutic strategy of targeting and rapidly reversing hepatogenous diabetes (a disease profile characterized by simultaneous symptoms of chronic liver diseases and type 2 diabetes mellitus). For in vivo experiments, 12-week-old male db/db mice (BKS.Cg-Dock7m +/+ Leprdb/J, derived from C57BL/6J mice, Charles River Laboratory) were chosen as a disease model for hepatogenous diabetes as blood analysis confirmed pathologic levels of glucose, insulin, aspartate transaminase, and alanine aminotransferase in the serum of untreated animals. Therapeutic implants were engineered by microencapsulating OA-inducible GLP-1-expressing human embryonic kidney cells into biocompatible, semi-permeable, and immunoprotective alginate-poly-(L-lysine)-alginate beads. Therapeutic efficacy was assessed at 48 hr after implantation by comparing the animals’ glucose tolerance, insulin sensitivity, homeostatic model assessment (HOMA) IR values, AST/ALT ratio, and serum triglyceride levels in the presence or absence of orally administered OA. The combined action of OA and GLP-1 was confirmed by control experiments showing that oral administration of OA was unable to elicit corresponding therapeutic effects in mice that did not harbor circuit-transgenic implants. In all experiments, mice were randomly assigned to the individual groups of six to eight mice, and the experimenters were blinded to the analysis of all samples. The choice of sample size was based on published reports of quantifying diabetic symptoms in mice.72 Comparisons among groups were performed using Student’s t test, and the results were expressed as the means ± SEM. All mice were sacrificed after termination of experiments.

Plasmid Construction

Comprehensive design and construction details for all expression vectors are listed in Table S2. The assembly of some plasmids was performed by Gibson Assembly73 with a Seamless Assembly Cloning Kit (Obio Technology, catalog number BACR(C)20144001) according to the manufacturer’s instructions. All new constructs were confirmed using Sanger sequencing (GENEWIZ).

Cell Culture and Transfections

Human embryonic kidney cells (HEK293, ATCC: CRL-11268), human cervical adenocarcinoma cells (HeLa, ATCC: CCL-2), HEK293-derived Hana3A cells engineered for the stable expression of Gαoλϕ and chaperones RTP1/RTP2/REEP1,74 telomerase-immortalized human mesenchymal stem cells (hMSC-TERT75), and Chinese hamster ovary cells (CHO-K1, ATCC: CCL-61) were cultured in DMEM (Gibco, catalog number 31600-083, lot number 1237526) supplemented with 10% (v/v) fetal bovine serum (FBS; Biological Industries, catalog number 04-001-1C, lot number 1413865) and 1% (v/v) penicillin/streptomycin solution (Biowest, catalog number L0022-100, lot number S10252L0022). All cell types were cultured at 37°C in a humidified atmosphere containing 5% CO2.

All cell lines were transfected with an optimized polyethyleneimine (PEI)-based protocol.67 For cell culture experiments, 5 × 104 cells seeded per well of a 24-well plate 18 hr prior to transfection were incubated for 6 hr with 100 μL of a 3:1 PEI/DNA mixture (w/w) (polyethyleneimine, molecular weight 40,000, stock solution 1 mg/mL in ddH2O; Polysciences, catalog number 24765, lot number 663735) containing 0.3 μg total DNA in serum- and antibiotic-free DMEM. For mouse experiments, 6.5 × 106 HEK293 cells were seeded into a 150-mm cell culture dish 18 hr prior to transfection and incubated for 6 hr with 1,500 μL of a 3:1 PEI/DNA mixture (w/w) containing 30 μg total DNA in serum- and antibiotic-free DMEM. Cell concentrations and viability were profiled with a Cellometer cell counter (Nexcelom Bioscience).

Generation of Stable Cell Lines

The polyclonal HEKGPBAR1-SEAP population, transgenic for OA-inducible and Dox-repressible SEAP expression, was constructed by co-transfecting 5 × 104 HEK293 cells with 100 ng pXS101 (ITR-PhCMV-CREB1-TetR-2A-mCherry-2A-puromycin-pA-ITR), 100 ng pXS102 (ITR-PhCMV*-1-SEAP-2A-EGFP-2A-puromycin-pA-ITR), 10 ng pXS103 (ITR-PhCMV-GPBAR1-pA-ITR), and 20 ng Sleeping Beauty transposase expression vector pCMV-T7-SB100 (PhCMV-SB100X-pA).42 After selection with 1 μg/mL puromycin for 2 weeks, the surviving population HEKGPBAR1-SEAP was stimulated with 20 μM OA for 48 hr and then sorted by fluorescence-activated cell sorting (FACS) into different subpopulations according to different red- and green-fluorescence intensities. The subpopulation with the top 5% mCherry and EGFP intensity, HEKGPBAR1-SEAP, showed the highest sensitivity to OA and was used for following studies.

The polyclonal HEKGPBAR1-shGLP-1 population, transgenic for OA-inducible shGLP-1 expression, was constructed by co-transfecting 5 × 104 HEK293 cells with 100 ng pXS101, 100 ng pXS107 (ITR-PhCMV*-1-shGLP-1-2A-EGFP-2A-puromycin- pA-ITR), 10 ng pXS103 and 20 ng Sleeping Beauty transposase expression vector pCMV-T7-SB100. After selection with 1 μg/mL puromycin for 2 weeks, the surviving population HEKGBAR1-shGLP-1 was stimulated by 20 μM OA for 48 hr and then FACS-sorted into different subpopulations according to different red- and green-fluorescence intensities. The subpopulation with top 5% mCherry and EGFP intensity HEKGPBAR1-shGLP-1 showed highest sensitivity to OA and was used for following studies.

Chemicals and Drugs

OA (catalog number O5504), poly-L-lysine hydrobromide (catalog number P7890), methylthiazolyldiphenyl-tetrazolium bromide (MTT; catalog number M2128), and human recombinant insulin (catalog number 91077C) were purchased from Sigma-Aldrich. CDCA (catalog number C104902), CA (catalog number C103692), LCA (catalog number U107242, lot number 40112061), UDCA (catalog number U110695), and CCL4 (catalog number C112043) were purchased from Aladdin. L-homoarginine hydrochloride (catalog number A602842), DCA (catalog number A100613), Dox (catalog number DB0889), and D-glucose (catalog number G0188) were purchased from Sangon Biotech. p-NPP disodium hexahydrate (catalog number 0364-25G) was purchased from AMRESCO. Sodium alginate (catalog number 11059993) was purchased from BÜCHI Labortechnik AG. OA tablets (Jiuzhitang; state medical permit number H20003499; 20 mg OA per tablet; other excipients: starch, talcum powder, magnesium stearate, lauryl sodium sulfate, and carboxymethyl starch sodium) were purchased at local pharmacies. PKA inhibitor H89 was ordered from Selleck Chemicals (catalog number S1582).

Analytical Assays

ALT

ALT levels in mouse serum were quantified using an ALT assay kit (BioSino & Biotechnology Science, catalog number 0010) and read at 520 nm with an Olympus autobiochemical analyzer (Olympus AU680).

AST

AST levels in mouse serum were quantified using an AST assay kit (BioSino & Biotechnology Science, catalog number 0020) and read at 450 nm with an Olympus autobiochemical analyzer (Olympus AU680).

Triglycerides

Triglyceride (TG) levels in mouse serum were quantified using a TG assay kit (BioSino & Biotechnology Science, catalog number 20090) and read at 540 nm with an Olympus autobiochemical analyzer (Olympus AU680).

Hemoglobin A1c

Hemoglobin A1c (HbA1c) levels were measured using a Bio-Rad D10 Automated HbA1c Analyzer (Bio-Rad).

cAMP assay

Intracellular cAMP levels were determined using an ELISA according to the manufacturer’s protocol (R&D Systems, catalog number SKGE002B, lot number 316451).

Confocal Microscopy

EGFP expression was visualized using a laser scanning confocal microscope (LSCM; Leica TCS SP5) equipped with a Leica HCX PL APO 63× oil-immersion objective (numerical aperture 1.4) and Leica Application Suite software (version 2.0.2).

Fluorescence Microscopy

EYFP expression was visualized using an Olympus microscope (Olympus IX71, TH4-200) equipped with an Olympus digital camera (Olympus DP71), a 10× objective, a 488-nm/509-nm (B/G/R) excitation/emission filter set, and Image-Pro Express C software (version ipp6.0).

Glycemia

Blood glucose levels of the mice were measured with a commercial glucometer (Contour Next; Bayer HealthCare; detection range: 0.5–35 mM).

MTT Assay

In brief, 1 × 104 HEK293 cells were seeded per well of a 96-well plate and cultured for 72 hr in the presence of various concentrations of OA; 10 μL MTT (5 mg/mL in PBS) was then added to each well. 4 hr after incubation at 37°C in a humidified atmosphere containing 5% CO2, 150 μL DMSO was added to each well. After complete solubilization of the purple formazan crystals, the plate was read with a microplate reader (BioTek Instruments) at 490 nm.

GLP-1 ELISA

shGLP-1 levels in culture supernatants and active GLP-1 levels in mouse serum were quantified with the High-Sensitivity GLP-1 Active ELISA Kit (Merck Millipore, catalog number EGLP-35K, lot number 2639195).

Insulin ELISA

Insulin levels in mouse serum were quantified with the Mouse Insulin ELISA Kit (Mercodia AB, Sylveniusgatan 8A, SE-754 50, catalog number 10-1247-01, lot number 24243).

SEAP Expression

The expression of human placental SEAP in cell culture supernatants was quantified using a p-nitrophenylphosphate-based light absorbance time course.76 Briefly, 120 μL substrate solution (100 μL 2× SEAP assay buffer containing 20 mM homoarginine, 1 mM MgCl2, and 21% diethanolamine [pH 9.8], and 20 μL substrate solution containing 120 mM p-nitrophenylphosphate) was added to 80 μL heat-inactivated (65°C, 30 min) cell culture supernatant, and the light absorbance was recorded at 405 nm (37°C) for 30 min using a Synergy H1 hybrid multi-mode microplate reader (BioTek Instruments) using Gen5 software (version: 2.04). SEAP levels in mouse serum were quantified using a chemiluminescence-based assay (Roche Diagnostics, catalog number 11779842001, lot number 10514400).66

Animal Experiments

Microcapsule Implants

Intraperitoneal implants were produced by encapsulating transgenic HEK293 cells into coherent alginate-poly-(L-lysine)-alginate beads (400 μm; 200 cells/capsule) using a B-395 Pro encapsulator (BÜCHI Labortechnik) set to the following parameters: a 200-μm nozzle with a vibration frequency of 1,300 Hz, a 25-mL syringe operated at a flow rate of 450 U, and 1.10-kV voltage for bead dispersion.20 12-week-old male wild-type C57BL/6J mice (East China Normal University Laboratory Animal Center) or 12-week-old male db/db mice (BKS.Cg-Dock7m +/+ Leprdb/J, derived from C57BL/6J mice, Charles River Laboratory) were intraperitoneally injected with 800 μL DMEM containing 2 × 106 microencapsulated human cells (200 transgenic HEK293 cells/capsule). Control compounds (OA and Dox) were prepared in PBS (200 μL/mouse) and administered orally or via injection at 1 hr (at the earliest) after implantation. Blood samples were collected via centrifugation (10 min, 5,000 rpm) of clotted blood (37°C, 0.5 hr and then 4°C, 2 hr) at 48 hr after implantation.

Glucose Tolerance Test

At 48 hr after implantation, fasted mice (16 hr) received an intraperitoneal injection of aqueous 1.5 g/kg D-glucose, and the glycemic profile of each animal was tracked every 30 min.

Insulin Tolerance Test

At 48 hr after implantation, fasted mice (4 hr) received an intraperitoneal injection of 1 U/kg recombinant human insulin, and the glycemic profile of each animal was tracked every 30 min.

HOMA-IR

The approximation equation for insulin resistance index was calculated according to the formula: HOMA-IR = [fasting glucose (mmol/L) × fasting insulin (mU/L)]/22.5.77

Histology

Mouse livers from biopsies were immersed in PBS for 10 min, fixed in 10% neutral buffered formaldehyde (NBE) solution for 24 hr and then embedded in paraffin wax using a paraffin-embedding machine (Leica EG1150H). Paraffin-embedded liver tissue was cut at 5 μm thickness on a Leica Microtomes (Leica RM2235). Sections were stained with H&E. Images were obtained using a Leica microscope (Leica DM4000 B LED) equipped with a Leica digital camera (Leica DFC310 FX) and analyzed with software (LAS V4.2).

Ethics

All experiments involving animals were performed according to the protocol approved by the East China Normal University (ECNU) Animal Care and Use Committee and in direct accordance with the Ministry of Science and Technology of the People’s Republic of China on Animal Care guidelines. The protocol was approved by the ECNU Animal Care and Use Committee (protocol ID m20140301).

Statistical Analyses

All in vitro data represent the mean ± SD of three independent experiments (n = 3). For animal experiments, each treatment group consisted of eight mice (n = 6 or 8). Comparisons between groups were performed using Student’s t test, and the results are expressed as mean ± SEM. Differences were considered statistically significant at p < 0.05. Prism 5 software (version 5.01; GraphPad Software) was used for statistical analysis.

Author Contributions

H.Y., S.X., M.X., and M.F. designed the project, analyzed the results, and wrote the manuscript. H.Y., S.X., J.Y., J.S., L.Y., Y.Y., and Y.W. performed the experimental work.

Conflicts of Interest

The authors declare no competing financial interests.

Acknowledgments

We thank Prof. Z.R. Wu for providing GLP-1 receptor expression plasmid (pGLP1R). All data pertaining to this study are in the paper. All genetic sequences for constructs generated in this study are available with a material transfer agreement (contact H.Y.).

This work was supported by grants from the National Key Research and Development Program of China, Stem Cell and Translational Research (2016YFA0100300), the National Natural Science Foundation of China (31470834, 31522017, and 31670869), the Science and Technology Commission of Shanghai Municipality (15QA1401500 and 14JC1401700), and the Thousand Youth Talents Plan (H.Y.).

Footnotes

Supplemental Information includes fifteen figures and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.ymthe.2016.11.008.

Supplemental Information

Document S1. Figures S1–S15 and Tables S1 and S2
mmc1.pdf (1.7MB, pdf)
Document S2. Article plus Supplemental Information
mmc2.pdf (4.4MB, pdf)

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Associated Data

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Supplementary Materials

Document S1. Figures S1–S15 and Tables S1 and S2
mmc1.pdf (1.7MB, pdf)
Document S2. Article plus Supplemental Information
mmc2.pdf (4.4MB, pdf)

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