Emerging biomedical applications of synthetic biology

Abstract

Synthetic biology aims to create functional devices, systems and organisms with novel and useful functions on the basis of catalogued and standardized biological building blocks. Although they were initially constructed to elucidate the dynamics of simple processes, designed devices now contribute to the understanding of disease mechanisms, provide novel diagnostic tools, enable economic production of therapeutics and allow the design of novel strategies for the treatment of cancer, immune diseases and metabolic disorders, such as diabetes and gout, as well as a range of infectious diseases. In this Review, we cover the impact and potential of synthetic biology for biomedical applications.

Figure 1. Mammalian gene expression control strategies.

a | Repression-based expression control. A repressor protein binds to its operator and thus prevents activation of the promoter and expression of the gene of interest. In response to an inducer, the repressor dissociates from the operator, the promoter is derepressed, and the gene of interest is expressed. b | Activation-based expression control. A minimal promoter (P_min) is activated when a chimeric transcription factor that is constructed by fusing a repressor protein to a transcription activation domain binds to its operator. In the presence of an inducer, the repressor protein–transcription-activation domain complex dissociates from its operator, P_min is no longer activated, and transcription of the gene of interest is prevented. c | mRNA transcript-based expression control. A self-cleaving ribozyme is fused to a small-molecule-binding aptamer and introduced into the 3′ untranslated region (UTR) of a gene of interest. In the absence of the inducer, the ribozyme undergoes self-cleavage, thereby eliminating the poly(A) tail (pA) from the open reading frame and preventing translation. However, in the presence of the inducer, the aptamer undergoes a conformational change, which inactivates the ribozyme and allows translation to occur.

Figure 2. Synthetic biology for understanding and preventing disease.

a | A female-specific dominant-lethal gene network for mosquito control. Mosquitoes were engineered to express an intron-containing variant of the tetracycline (TET) transactivator (tTA) under the control of a flight-muscle-specific promoter (P_FM). In male mosquitoes, the intron is not spliced out, which prevents correct tTA translation. In female progeny, however, functional tTA translation is restored by sex-specific mRNA splicing. This results in the activation of the tTA-responsive promoter P_TET and the expression of a toxic gene that triggers a flightless phenotype. If mosquitoes are raised in the presence of tetracycline (TET), tTA is prevented from activating P_TET, which results in a normal phenotype. However, following their release into the TET-free environment, engineered males mate with wild-type females. This transmits the female-specific dominant flightless phenotype and should eventually result in the reduction or extinction of the wild-type population. b | Propagation of a selfish gene converting a heterozygous into a homozygous host. The homing endonuclease I-SceI is expressed and cleaves its cognate restriction site (RS) on the homologous chromosome. Following end resection and repair, the I-SceI expression cassette is inserted into the second chromosome. pA, poly(A) tail.

Figure 3. Mammalian-cell-based drug discovery.

a | Identification of antibiotics. In Chinese hamster ovary (CHO-K1) cells, the streptogramin-responsive repressor (PIP) was expressed by a constitutive promoter (P_const). PIP binds to its multimeric operator (PIR3) and represses expression of the reporter gene secreted alkaline phosphatase (SEAP). Exposing this screening cell line to a small molecule library only resulted in SEAP production for compounds that were streptogramin-like, cell-permeable and non-toxic (indicated by the brown star). b | Discovery of small molecules that are able to overcome antibiotic resistance. The Mycobacterium tuberculosis antibiotic resistance regulator (EthR) was fused to the herpes-simplex-derived transcriptional activator (VP16) and expressed in human embryonic kidney cells (HEK293-T) under the control of a constitutive promoter (P_const). When EthR–VP16 binds to its cognate operator (O_EthR), the minimal promoter (P_min) is activated, which results in expression of the reporter gene SEAP. A screen is performed to identify a cell-permeable, non-toxic molecule (indicated by the yellow star) that prevents EthR binding to O_EthR, stopping SEAP expression. c | Overcoming resistance to ethionamide in M. tuberculosis. In M. tuberculosis, EthR represses transcription of both the Baeyer–Villiger monooxygenase (EthA) and itself in a negative feedback loop. When 2-phenylethylbutyrate (indicated by the pink star) is added, it prevents EthR binding its target promoter (labelled 'P' in the figure). This derepresses EthA production, thereby turning ethionamide into a cytotoxic compound that kills the mycobacterium. pA, poly(A) tail.

Figure 4. Drug delivery.

Interactive biohybrid material based on the interaction of a repressor protein with its cognate DNA operator motif. Homodimeric tetracycline repressor (TetR) is converted into a single-chain repressor (scTetR) by connecting two TetR subunits through a flexible peptide linker, and it is tagged with six histidines (scTetR–His₆). This molecule is coupled to a polymer and is mixed with a polyacrylamide that has copies of a tetracycline operator (tetO) attached to it. scTetR binds to tetO so crosslinks are formed, making a hydrogel. When tetracycline is added, scTetR releases tetO, and the gel is dissolved. This can be used to release another molecule that was attached to the polymer — in this case, the cytokine interleukin 4 (IL-4).

Figure 5. Bacterial and viral cancer therapy.

a | Population-density-dependent invasion of cancer cells. After intravenous injection, Escherichia coli accumulates in cancer tissue, where it reaches high population densities. E. coli is engineered to link the quorum-sensing receptor LuxR to an autoinducer 1 (AI-1)-inducible promoter (P_lux). P_lux is also used to drive luxI and the invasin gene inv. LuxI produces AI-1, generating a positive feedback loop that coordinates invasion throughout the population. b | Acetylsalicylic acid (Aspirin)-triggered killing of cancer cells after invasion of Salmonella spp. Salmonella spp. naturally invade cancer cells after intravenous injection. Salmonella spp. were engineered with a Pseudomonas putida-derived signal-amplifying two-level cascade in which NahR controls salicylate promoter (P_sal)-driven xylS2 expression and XylS2 then triggers a XylS2-dependent promoter (P_m)-driven expression of the cytosine deaminase (labelled CD in the figure). Salicylate induces both NahR-based P_sal and XylS2-mediated P_m activation. Mammalian cells are resistant to 5-fluorocytosine because they lack cytosine deaminase, which converts 5-fluorocytosine into the toxic cancer therapeutic 5-fluorouracil. c | Invasive bacteria suppress oncogene expression. E. coli is engineered to constitutively co-express a catenin β-1-specific short hairpin RNA (shRNA), Listeria monocytogenes listeriolysin (LLO*) and inv under control of the bacteriophage T7 promoter (P_T7). They invade cancer cells (using the Inv protein), escape from the phagosome (using LLO*) and knock down the catenin β-1 oncogene (using shRNA). d | Therapeutic protein transduction. Lentiviral particles are produced using an integrase-negative helper vector (designated 'helper' in the figure) and a constitutive expression vector encoding the protein of interest (designated 'protein' in the figure) fused to viral protein R (VPR) and a protease cleavage site (PC). This can be delivered to any target cell in the absence of viral nucleic acids and proteins. An example application is described in the main text. pA, poly(A) tail. P_EF1α, elongation factor 1 alpha (EF1α) promoter.

Figure 6. Synthetic genetic cancer classifiers.

a | A transformation-sensing cancer kill switch can consist of a two-input, transformation-sensing device with 'AND' logic. The device constantly monitors the transformation state of a cell and produces a kill signal when two malignancy markers occur. Two independent malignancy-sensitive promoters drive expression of two chimeric proteins (DocS–VP16 and Gal4_BD–Coh2). When they are simultaneously expressed, both proteins dimerize to form a synthetic transcription factor that binds Gal4 operator sites (O_Gal4), induces downstream minimal promoters (P_min) and triggers expression of the herpes simplex virus type 1 thymidine kinase (TK1). In the presence of ganciclovir, the system is cytotoxic. b | A microRNA (miRNA)-based cancer classifier that discriminates cancer cells from non-transformed cells by scoring high and low expression profiles of a set of cancer-specific miRNAs. The classifier consists of high and low miRNA sensors that exclusively promote output gene expression if the specific input miRNAs are expressed at high or low levels, respectively. In the high miRNA sensor, high-target miRNA concentrations prevent translation of mRNAs encoding the reverse tetracycline-dependent transactivator (rtTA) and the repressor of the lactose operon (LacI). This results in derepression of transcription of the output gene (labelled 'Output' in the figure). In the low miRNA sensor, the output-gene-encoding mRNA is only translated when low-target miRNA concentrations are present. c | By combining different high and low miRNA sensors, the classifier can be customized to sense predetermined profiles of high and low miRNA levels, such as the ones that are typically produced by cancer cells and respond with expression of the apoptosis-inducing human BCL2-associated X protein (BAX). pA, poly(A) tail.

Figure 7. Advanced therapeutic and prosthetic networks.

a | Light-triggered transcription control of blood glucose homeostasis. The synthetic phototransduction cascade consists of rewired melanopsin and nuclear factor of activated T cells (NFAT) control circuits. Photo-isomerization of the 11-cis-retinal chromophore (R) by blue light (~480 nm) activates melanopsin. This sequentially turns on Gaq-type G protein (GAQ), phospholipase C (PLC) and phosphokinase C (PKC) and triggers Ca²⁺ ion influx via transient receptor potential channels (TRPCs) and possibly also from the endoplasmic reticulum. This Ca²⁺ ion surge activates calmodulin (CaM) to calcineurin (CaN), which dephosphorylates NFAT. NFAT then translocates into the nucleus, where it binds to specific promoters (P_NFAT) and coordinates transgene transcription. When linked to the glucagon-like peptide (GLP1), this mechanism allowed light-controlled blood glucose homeostasis to be achieved in a mouse model of type 2 diabetes. b | Prosthetic network for the treatment of tumour lysis syndrome and gout. Implanted sensor–effector cells are used to monitor serum urate levels constantly: they import urate via a transgenic human uric acid transporter (URAT1). Urate prevents binding of the uric acid-sensitive transsilencer (KRAB–HucR, which is the uricase regulator linked to a KRAB domain) to its operator (hucO₈). This operator controls expression of secretion-engineered urate oxidase (smUOX), so smUOX is expressed when urate concentration reaches pathological levels. smUOX mediates conversion of urate into allantoin. Expression of smUOX stops when urate concentration reaches oxidative-stress-protective urate levels. pA, poly(A) tail. Part a is modified, with permission, from Ref. © (2011) American Academy for the Advancement of Science.