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Engineering of synthetic gene circuits for (re‐)balancing physiological processes in chronic diseases

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Synthetic biology is a promising multidisciplinary field that brings together experts in scientific disciplines from cell biology to engineering with the goal of constructing elements that do not occur in nature for use in various applications, such as the development of novel approaches to improving healthcare management. Current disease treatment strategies are typically based on the diagnosis of phenotypic changes, which are often the result of an accumulation of endogenous metabolic defects in the human body. These defects occur when the tight regulation of physiological processes is disturbed by genetic alterations, protein function losses, or environmental changes. Such disturbances can result in the development of serious disorders that are often associated with aberrant physiological levels of certain biomolecules (e.g., metabolites, cytokines, and growth factors), which may lead to specific pathogenic states. However, these aberrant levels can also serve as biomarkers for the precise detection and specification of disease types. Clinical interventions are often conducted during the late stages of disease pathogenesis because of a lack of early detection of these physiological disturbances, which results in disease treatment rather than prevention. Therefore, advanced therapeutic tools must be developed to link therapeutic intervention to early diagnosis. Recent advances in the field of synthetic biology have enabled the design of complex gene circuits that can be linked to a host's metabolism to autonomously detect disease‐specific biomarkers and then reprogrammed to produce and release therapeutic substances in a self‐sufficient and automatic fashion, thereby restoring the physiological balance of the host and preventing the progression of the disease. This concept offers a unique opportunity to design treatment protocols that could replace conventional strategies, especially for diseases with complex and recurrent dynamics, such as chronic diseases. WIREs Syst Biol Med 2016, 8:402–422. doi: 10.1002/wsbm.1345 This article is categorized under: Translational, Genomic, and Systems Medicine > Diagnostic Methods Translational, Genomic, and Systems Medicine > Therapeutic Methods Translational, Genomic, and Systems Medicine > Translational Medicine
Ligand‐controlled transcription factor (TF)‐based gene circuits. (a) Synthetic gene circuit to restore uric acid homeostasis. Increased levels of uric acid in the bloodstream are detected by the sensor component (mUTS), a synthetic TF that consists of the uric acid‐responsive HucR fused to the transsilencer domain KRAB (KRAB‐HucR). In the absence of uric acid, mUTS represses the expression of the output by binding to hucO8. In the presence of uric acid, mUTS is released from hucO8 to allow for the expression of smUOX, which is released into the bloodstream to convert uric acid into the more soluble and renally secretable allantoin. (b) A synthetic thyroid hormone (TH)‐responsive gene circuit for the treatment of Graves’ disease. TH is detected by the synthetic TH‐sensing receptor (TSR), which is a fusion protein that contains the yeast GAL4 DNA‐binding domain (GAL4DBD) that constitutively binds its cognate operator site (upstream activating sequence, UAS) and the ligand‐(thyroid‐) binding domain of the human TH receptor (TRαLBD). In the absence of TH, the TSR is associated with a co‐repressor complex, which leads to inhibited expression of the output. In the presence of TH, TSR is associated with a co‐activator complex to induce the expression of the output effector thyroid‐stimulating hormone receptor antagonist (TSHAntag), which is released into the circulation to restore TH homeostasis in mice. (c) Synthetic gene circuit for the treatment of obesity. A lipid‐responsive sensor (LSR) was constructed by fusing the ligand‐binding domain of the human PPARα (PPARαLBD) to the phloretin‐responsive repressor (TtgR). (i) In the absence of fatty acids, PPARα‐TtgR bound to TtgR‐specific operator sites (OTtgR) inhibits gene expression by associating with a co‐repressor complex. (ii) In the presence of fatty acids, PPARα‐TtgR associates with a co‐activator complex to induce the expression of the appetite‐suppressing peptide hormone (pramlintide). (iii) Independent of the presence or absence of fatty acids, the addition of phloretin leads to the dissociation of PPARα‐TtgR from DNA, thereby inhibiting the expression of pramlintide.
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Disease targeting with cell‐based therapies. (a) T cells are isolated from patients and then genetically modified with chimeric antigen receptors capable of binding tumor‐associated antigens on targeted tumor cells. After a period of in vitro expansion of the engineered T cells, the cells are reinfused into the same patient, where they are expected to bind to their target antigens on tumor cells, become active, and induce tumor‐killing processes. (b) Synthetic gene circuits can be designed to respond to disease‐specific input signals through input‐adjusted expression and the release of therapeutic outputs. These circuits can be uploaded into mammalian cells that are encapsulated inside semipermeable hydrogels, which protect the engineered cells from destruction by the immune system but allow for the diffusion of nutrients and therapeutic molecules. These microcapsules are intraperitoneally injected into animals to validate the therapeutic potency of the constructed synthetic gene networks.
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Rewired intracellular signaling cascades. (a) Synthetic dopamine‐triggered signaling cascade to attenuate high blood pressure. The human dopamine‐responsive G protein‐coupled receptor (dopamine receptor 1, DRD1) is activated by extracellular dopamine to trigger the endogenous cAMP‐dependent pathway, thereby resulting in the activation of cAMP‐responsive element‐binding protein 1 (CREB1). Activated CREB1 binds to specific operator‐site cAMP‐response elements (CRE) to drive the expression of the antihypertensive arterial natriuretic peptide (ANP). (b) A synthetic pH‐controlled signaling cascade to restore glucose homeostasis. The G protein‐coupled receptor TDAG8 is activated by extracellular proton levels to trigger the pH‐adjusted activation of the intracellular cAMP‐dependent signaling cascade, which is rewired to the expression of secretion‐engineered insulin (furin‐cleavable proinsulin I) in response to pH changes resulting from diabetic ketoacidosis. (c) Rewired NFkB‐triggered signaling cascade for disease‐targeted cell death. Extracellular tumor necrosis factor (TNF) binds and activates its receptor TNFR to trigger an intracellular signaling cascade that results in the translocation of NFkB into the nucleus to regulate the expression of the output transgene. A RNA‐based device is composed of specific aptamers designed to recognize nuclear NFkB proteins. These aptamers are localized at key intronic regions near an alternatively spliced exon that contains a stop codon, which was part of a three‐exon, two‐intron minigene fused to the suicide gene (herpes simplex virus‐thymidine kinase, HSV‐TK). The binding of TNF to TNFR induces the NFkB signaling pathway, which in turn regulates the exclusion of the alternatively spliced exon containing the stop codon, thereby linking activated intracellular proteins to reprogrammed cell death. (d) Synthetic cytokine converter for the treatment of the autoimmune disease psoriasis. A synthetic gene network that consists of sequentially interconnected signaling cascades quantifies the level of proinflammatory psoriasis‐associated cytokines TNF and interleukin 22 (IL22), processes their level with AND gate logic, and coordinates the adjusted production of the anti‐inflammatory cytokines interleukin 4 (IL4) and interleukin 10 (IL10). TNF binds its cognate receptor (TNFR) to trigger endogenous NFkB‐mediated expression of human IL22 receptor α subunit (IL22RA). In the presence of IL22, IL22RA heterodimerizes with endogenous human IL10 receptor β subunit (IL10RB) on mammalian HEK‐293T cells to trigger an intracellular JAK/STAT signaling cascade that results in the translocation of STAT3 into the nucleus, which activates STAT3‐responsive promoters and drives the expression of IL4 and IL10. Expressed therapeutic IL4 and IL10 are released into the blood circulation to reduce the ongoing inflammation and restore the skin's morphology.
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Translational, Genomic, and Systems Medicine > Diagnostic Methods
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