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Emerging biomedical applications of synthetic biology

Emerging biomedical applications of synthetic biology REVIEWS Emerging biomedical applications of synthetic biology 1,2 3,4 Wilfried Weber and Martin Fussenegger 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. By applying engineering principles to biology, sy- n protein expression dynamics with high precision and Aptamer thetic biology has become the science of reassembling predictable logic in response to external cues or physi - An oligonucleic acid that binds to a specific target, such catalogued and standardized biological components in ological pathways. Because most control components as a chemical compound, a systematic and rational manner to create and eng- i function in different bacterial and eukaryotic species a protein or a nucleic acid. 36,47–49 neer functional biological designer devices, systems after minor refinements , gene switches and net- Aptamers were found to and organisms with predictable, useful and novel fun -c work blueprints that were pioneered in bacteria or yeast control riboswitches in natural tions. Synthetic biology is able to use an inventory of are often fully functional in mammalian cells. Examples systems, but they can also be selected in vitro. biomolecular parts compiled over 50 years of molecular of synthetic networks with similar components and cir - 1–4 biological and functional genomic research , as well as cuit topology in bacterial and mammalian cells include: 5,6 42,43 15,20,23,45 technology that has made it possible to analyse, syn- regulatory cascades , epigenetic toggle switches , 7–9 10 11 12,13 22,50,51 30,52 thesize , assemble , modify and transfer genetic hysteretic circuits , molecular timing devices , components into living organisms. synt hetic eco-sensing systems, synthetic quorum- 46,53,54 Although it has recently become possible to reco- n sensing systems, synthetic hormone systems , band- 21,30,55 struct a living organism after transfer of a synthetic pass filters and different types of oscillators that genome that has been assembled from chemically sy-n program rhythmic transgene expression with a tun- Faculty of Biology, University 13,14 16,26–29,56,57 thesized nucleic acid pieces , the rational design of able frequency and amplitude . Most of these of Freiburg, Schänzlestrasse 1, state-of-the-art biological circuits with predicable fun -c first-generation synthetic circuits operated in isolation D‑79104 Freiburg, Germany. tions remains challenging and is apparently limited to a without any interface with the metabolism of the host BIOSS Centre for Biological 15–31 Signalling Studies, University handful genes . cell, and they were used to program specific cellular of Freiburg, Hebelstrasse 25, 58–62 Synthetic circuitry is composed of basic heterologous functions using heterologous external input signals . D‑79104 Freiburg, Germany. control components that fine-tune transgene expression Key challenges that often occur in the biomedical Department of Biosystems in response to specific exogenous cues or endogenous setting are the need for drug–target specificity, precise Science and Engineering, ETH Zurich, Mattenstrasse metabolites . These gene switches include trigger- drug-dosing regimes, minimizing side effects, shorten- 33–36 26, CH‑4058 Basel, inducible protein–DNA or aptamer–transcript inter- ing the diagnosis-to-treatment timelines and avoiding Switzerland. 37–39 actions , which control transcription and translation drug-resistance of pathogens. As synthetic biology ena - Faculty of Science, University in response to heterologous and endogenous input sig - bles the engineering of complex, high-precision control of Basel, Mattenstrasse 26, nals( FIG. 1). The standardized design of gene switches has devices that couple sensing and delivery mechanisms, CH‑4058 Basel, Switzerland. 40,41 Correspondence to M.F.  improved functional compatibiliy t and has enabled this emerging science may be able to offer tools that are e‑mail: the construction of higher-order networks — includ - suited to meeting current biomedical challenges in new fussenegger@bsse.ethz.ch 42,43 ing multi-trigger inputs and sequential control , ways. A decade after the pioneering synthetic networks doi:10.1038/nrg3094 20,23,44,45 16,19,28,46 18,20 mutual control and feedback control of were reported , the first successful therapeutic appli - Published online 29 November 2011 circuit components — that are able to provide complex cations in animal models of prominent human diseases NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 21 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS C +PFWEGT Epigenetic toggle switches 4 4 Genetic circuits that can be switched between two stable expression states (for example, 4 4 an ‘on’ and an ‘off’ state) by a transient stimulus. In the 2 1 )GPG R# 2 1 )GPG R# absence of a switching stimulus, the expression state is locked and inherited across cell generations. Hysteretic circuits D +PFWEGT Genetic circuits whose 4 4 response dynamics depend # # on a combination of past and present states. 4 4 1 2 )GPG R# 1 2 )GPG R# OKP OKP Eco-sensing The capacity of a species to sense and score its surrounding ecosystem, for example, to identify the type and population density of neighbouring species. Quorum sensing %NGCXCIG A small-molecule-based E #RVCOGT chemical language by which R# bacteria communicate within 9KVJQWV and across populations (the )GPG 4KDQ\[OG KPFWEGT ‘quorum’). Production and response to quorum-sensing molecules is correlated with 9KVJ )GPG R# population density. KPFWEGT Band-pass filters These are devices that are CPUNCVKQP selectively induced within a specific concentration range )GPG R# of the input signal. At lower or higher trigger levels, the band-pass filter shuts down output signals. )GPGQH 2 T 1 )GPG R# N # CEVKXCVKQP RT QVGKP KPVGT GUV FQOCKP Figure 1 | Mammalian gene expression control strategies. a | Repression-based expression control. A repressor 0CVWT G4G XKGYU ^)GPGVKEU 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 ) is activated when a chimeric min 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 is no longer activated, and transcription of the gene of interest is prevented. c | mRNA min 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. 63–65 are starting to emerge . This Review focuses on the Understanding disease mechanisms recent progress in synthetic biology towards improv - Pathogen mechanisms. The availability of affordable ing human health, including diagnostic applications, technology for synthesizing and assembling sequences 68 69 design of novel preventive care strategies, progress for proteins or for viral and bacterial genomes with in drug discovery, design and delivery, and develo -p increased speed has dramatically improved our under - ment of novel treatment strategies, such as prosthetic standing of host–pathogen interactions and of disease networks. Synthetic biology holds the promise of pro - mechanisms. The synthetic biology principle of ‘analysis viding unique opportunities for major advances in by synthesis’ provides mechanistic insight by combin- the improvement of human health in the twenty-first ing rapid synthesis, assembly, shuffling and mutation 4,66,67 century . of individual genetic components with straightforward 22 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved RQN[ # VCK VQT TC 1RG QOQVG 2T UUQT TG 4GR CPUETKRVKQP 6T 6T REVIEWS functional analysis. For example, the genome of the Disease prevention H1N1 virus that was responsible for the 1918 Spanish Vaccines. High-throughput and high-precision assem- influenza pandemic was synthesized using sequence bly and engineering of entire genomes from well- information from genomic pieces that were extracted defined genetic components using synthetic biology from permafrost-conserved tissue samples. Functional principles has provided new opportunities for the analysis of the reconstructed virus provided new insight design of attenuated pathogens for use as vaccines. For into the key virulence factors of the pathogen: namely, a example, primates immunized with virus-like particles haemagglutinin variant that induces membrane fusion that were produced by selective expression of particular without trypsin activation and a modified polymerase chikungunya virus (CHIKV) structural proteins were that enhances viral replication . The same study also protected against viraemia after a high-dose challenge; revealed that a combination of eight genes was respon - even immunodeficient mice that were treated with sible for the exceptional virulence of the Spanish infl -u monkey-derived antibodies survived subsequent lethal 68,70 81 enza strain . This finding may help to identify the doses of CHIKV . DNA synthesis and assembly has also 71–73 pandemic potential of future virus variants . played an essential part in pioneering a safe live vaccine Synthesis and analysis of chimeric viruses have also against the poliovirus . The poliovirus was attenuated made a substantial contribution to the understanding of by systematic genome-scale changes of adjacent pairs of coronavirus zoonoses that were responsible for the severe codons from over- to underrepresented codon sets in acute respiratory syndrome (SARS) pandemic of 2002 viral capsid genes (for example, GCC|GAA is strongly and 2003. The characterization of the history of the SARS under-represented compared with GCA|GAG, although coronavirus, especially its switch in tropism, was pa- r both encode Ala–Glu). These changes reduced tran-s ticularly challenging, as its direct ancestors could not be lation and impaired the replication competence and propagated in laboratory models. However, after a 30 kb infectivity of the virus. This attenuated poliovirus pro - SARS-like bat coronavirus was designed to contain the vides protective immunization in mice and offers a high receptor-binding spike protein of its human homologue , safety standard given the low probability that all 631 the synthesized chimeric virus was able to replicate in culture individual changes will revert and thus reconstitute and infect mice . These in vivo studies revealed infection- infectious wild-type viruses. The genome-engineering enhancing mutations in the spike protein and established approach used here could represent a general strategy this surface protein as a key factor that is responsible for for designing live vaccines against infectious diseases. tropism switches in coronavirus zoonoses . Reconstruction Other promising vaccination concepts include using of pathogens by DNA synthesis can also be used for the antigen-producing immunostimulatory liposomes as 75,76 83 production of diagnostic high-density antigen arrays , genetically programmable synthetic vaccines and the such as those used to profile post-Lyme-disease syn - production of heat-stable oral algae-based vaccines to 76 84,85 drome or the humoural immune responses to hepatitis C protect against Staphylococcus aureus infections . and the human immunodeficiency virus (HIV) . Vector control. Suppression of insect vector populations Immune systems. Synthetic biology has recently pro - using transgenic viral strains that harbour conditional vided new insight into disorders that are related to dominant-lethal synthetic circuitry may control the deficiencies of the immune system, which is known transmission of malaria parasites and dengue viruses for its particularly complex control circuits and cel -lu and could eventually control the spread of untreatable 86–88 lar interaction networks. For example, dysfunction of diseases . Mosquitoes that are transgenic for a tet- B-lymphocyte activation underlies several physiological racycline-dependent transactivator (tTA) that is exclu - disorders . Functional reconstitution and analysis of the sively expressed in the female’s indirect flight muscle human B cell antigen receptor (BCR) signalling cascade can only be propagated in the presence of tetracycline, in insect cells revealed that BCRs are not activated by which represses the transcription of this gene. However, antigen-specific crosslinking, as presented in textbooks, the absence of tetracycline leads to the development of 87,88 but instead have an autoinhibitory oligomeric confor - a female-specific flightless phenotype . Putting the mation on resting B lymphocytes that shifts to an active eggs of this transgenic mosquito into the ecosystem Immunostimulatory liposomes dissociated form when antigens bind . This triggers the results in male-only releases; female mosquitoes remain Liposomes that are decorated signalling cascade, which results in antibody production grounded and cannot feed, mate or take blood meals, with antigenic peptides and the onset of a humoural immune response. which effectively represents a lethal phenotype. Males or proteins that elicit an Also, construction of a representation of the complete do not transmit the disease, but they disseminate the immune response. human peptidome engineered for display on the surface synthetic circuit across the resident wild-type mosquito 80 87,88 Gene drive system of T7 phages enabled Church and colleagues to discover population (FIG. 2a). These are molecular devices new autoantigens. They used patient-derived autoanti- Similarly, a synthetic homing endonuclease-based that promote the spreading of bodies to enrich autoantigenic peptides displayed on gene drive system could be used to spread genetic modi - a specific gene throughout a the phages; they could then identify the antigens by fication, such as malaria resistance, from engineered target population by taking advantage of a mechanism high-throughput sequence analysis . Knowledge of the mosquitoes to the field population. Homing endonu - that multiplies the specific antigens that are involved in autoimmune processes is cleases typically produce a single sequence-specific gene in the host genome. important for understanding disease aetiology, develo- p double-strand break in the host genome that is repaired Gene drive systems produce ing accurate diagnostic tests and designing drugs that by homologous recombination using the homing endo - non-Mendelian patterns of inheritance. neutralize autoreactive immune cells. nuclease gene (HEG) as a template. Consequently, NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 23 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS 'PIKPGGT GF JQOQ\[IQWU 'PIKPGGT GF 6'6 &GCVJ JGVGT Q\[IQWU 9KNFV[RG s6'6 &GCVJ × s6'6 +PVT QP V6 # R# 2 +PVT QP V6 # R# (/ 6'6 V6 # R# 2 6 QZKEIGPG R# (NKIJVNGUU 6'6 9KNFV[RGKPUGEV *GVGT Q\[IQWURT QIGP[ *QOQ\[IQWURT QIGP[ + 5EG + + 5EG + + 5EG + 4GRCKT CPUIGPG + 5EG + QRWNCVKQPKPXCUKQP 2 QHVT + 5EG + %NGCXCIG 'PIKPGGT GFKPUGEV GPFT GUGEVKQP )GPG VKQPPWODGT Figure 2 | Synthetic biology for understanding and preventing disease. a | A female-specific dominant-lethal 0CVWT G4G XKGYU ^)GPGVKEU 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 ). In male FM 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 and the expression of a toxic gene that triggers a flightless phenotype. If mosquitoes TET are raised in the presence of tetracycline (TET), tTA is prevented from activating P , which results in a normal TET 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. 24 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved TC REVIEWS the selfish HEG is copied to the broken chromosome so prevents EthA-mediated conversion of the last- in a gene conversion process referred to as ‘homing’. line-defence antibiotic ethionamide into a pathogen- Expressing the HEG I-SceI under the control of a male killing metabolite. The chemical 2 -phenyethylbutyrate, germline promoter enabled efficient homing in transhet - best known for its strawberry flavour, was the first com- erozygous males and rapid genetic drive, which led to pound found that specifically inactivated EthR and so HEG invasion in caged mosquito populations (FIG. 2b). triggered ethA expression and re-established the sen - By engineering the sequence specificity of other HEGs sitivity of Mycobacterium tuberculosis to ethionamide (for example, I-AniI or I-CreI), the gene drive concept (FIG. 3d). Further work revealed other EthR-inactivating could, in principle, be used to knock in or knock out ethionamide booster compounds; these have also been gene functions that target the mosquito’s ability to serve successfully tested in a mouse model of human tuber - 89 98 as a disease vector . culosis . Restoring drug sensitivity by pharmacological Field tests of release of insects carrying dominant inhibition of master resistance regulators may be widely lethals (RIDL) technology using first-generation tTA- applicable . transgenic mosquitoes have already been conducted in A further example of the use of synthetic circuitry Grand Cayman. First, a small-scale release confirmed for drug discovery is provided by mammalian cells that transgenic males could survive, mate with wild that are conditionally arrested in the G1 phase of the females and produce transgenic larvae, and then the cell cycle by circuitry controlling the expression o f full field trial showed an 80% reduction in the num - the cycline-dependent kinase inhibitor p27. These cells bers of wild mosquitoes about 11 weeks after release. reproducibly formed a mixture of isogenic subpopul- a As the study site was not isolated and the surrounding tions of proliferation-inhibited cells and proliferating areas contained high densities of wild-type mosqui - cells that had spontaneously escaped the synthetic cell toes, scoring the actual suppression efficiency remains cycle block . These cells could be used as a cell-based challenging . cancer model and could be used to screen for anticancer compounds that selectively eliminate proliferating cells 100,101 Drug development while leaving arrested ones intact . Drug discovery. Synthetic mammalian transcription cir - cuits consisting of a chimeric small-molecule-responsive Drug production and drug delivery. The synthetic path- transcription factor and a cognate synthetic promoter ways that are created by assembling enzymatic cascades were originally designed for future gene-based thera - or networks in bacteria, yeast and plants have been pies, and the aim was to adjust therapeutic transgene instrumental for the large-scale economic production of expression in mammalian cells in response to a pha -r high-value drug and drug precursor compounds, as well 34,47,49,91 macologically active substance . As most chimeric as for the biosynthesis of new secondary metabolites transcription factors are derived from repressors that with novel therapeutic activities. Examples include com - 102,103 104–106 manage drug resistance in bacteria (for example, resist - plex polyketides , halogenated alkaloids and the ance to antibiotics ) and are promiscuous for structur- precursors of the anti-malaria drug artemisinin (which ally related compounds, mammalian cells containingi s produced by the company Amyris, for example) such circuitry could also be used in ‘reverse mode’, and of the anti-cancer compound taxol . For produc- as integrated screening devices for the class-specific tion of these compounds, it was necessary to overcome 33,93 discovery of new drug candidates (for example, new several challenges, including the functional expression antibiotics ) (FIG. 3a). When mammalian cells that are of complex biosynthetic enzymes (such as cytochrome transgenic for the screening circuit are exposed to a P450 monooxygenases ) and the overall orchestra - compound library, they detect and modulate reporter tion of the multistep pathway to avoid accumulation of gene expression in the presence of a non-toxic, cell- (toxic) intermediate products and to ensure metabolic permeable and bioavailable molecule that has a class-channelling . specific core structure and corresponding drug activity Small-molecule-responsive protein–protein and (for example, antibiotic activity) (FIG.  3b). Using the protein–DNA interactions that are used to pioneer 36,49,110 same screening setup, compounds have been detected gene switches in mammalian cells have also been that lock the transcription factor onto the DNA, which successfully re-engineered in the design of trigger- 111–115 may block induction of antibiotic resistance in patho - inducible biohybrid materials for drug delivery . gens and render them drug-sensitive (for example, see Using synthetic protein–polyacrylamide and DNA– Bioversys). Using such compounds alongside the spe - polyacrylamide monomers, hydrogels can be produced Polyketides cific antibiotic may offer novel anti-infective treatment that dissolve when specific ligands are supplied (FIG. 4). These constitute a group opportunities and a new life cycle for established an-ti Biopharmaceuticals (for example, vascular endothelial of secondary metabolites biotics (FIG. 3c). Other trigger-inducible transcription growth factor (VEGF)) supplied during gel formation produced through linear control systems can be used in this manner as well, such are loaded into the hydrogel and can be released in a decarboxylative condensation as those that are responsive to streptogramin , tetracy- dose-dependent manner after subcutaneous implanta - of acetyl-CoA with several 91 95 malonyl-CoA-derived extender cline or macrolide antibiotics , anti-diabetes drugs or tion into mice and oral administration of the trigger units to a polyketide chain. 96,97 115 immunosuppressive lactones . compound . It is thought that any trigger-inducible Many pharmacologically active One example of the efficacy of a transcription circuit protein–protein and protein–DNA interactions could compounds, such as antibiotics system involves the bacterial transcriptional repres - be used to produce drug-sensing and drug-releasing and anti-cancer drugs, belong 114,116,117 to the polyketide class. sor EthR. EthR represses transcription of ethA and hydrogels . NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 25 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS %JGOKECNNKDT CT[ 6 QZKE 0QPRGTOGCDNG C %*1- 0QGȭGEV 2 2 2+2 R# 2+2 R# EQPUV EQPUV 2+2 2+2 2+2 2+2 2 2+4 5'#2 R# 2 2+4 5'#2 R# EQPUV EQPUV D *'-6 2 2 GVJ4 82 R# GVJ4 82 R# EQPUV EQPUV 82 82 1 2 5'#2 R# 1 2 5'#2 R# 'VJ4 OKP 'VJ4 OKP E /VWDGT EWNQUKU 2JGP[NGVJ[NDWV[T CVG 2 2 2 2 GVJ# GVJ4 GVJ# GVJ4 'VJ# 'VJKQPCOKFG 'VJKQPCOKFG 0CVWT G4G XKGYU ^)GPGVKEU 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 ). When const EthR–VP16 binds to its cognate operator (O ), the minimal promoter (P ) is activated, which results in expression of EthR min 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 , stopping SEAP expression. c | Overcoming resistance to ethionamide EthR 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. 26 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 REVIEWS Novel treatments for infections constitutively express DspB: an enzyme that hydrolyses SOS DNA repair Breaking bacterial resistance by designer phages. β-1,6-N-acetyl-d-glucosamine, which is an adhesin Genetically encoded repair program protecting against Biofilms are surface-associated bacterial communi - that is required for biofilm formation and integrity in DNA damage. In prokaryotes, ties that are encased in a hydrated extracellular po- ly Staphylococcus spp. and Escherichia coli clinical iso - the repair program is meric substance (EPS) matrix that is composed of lates. The initial infection of a bacterial biofilm with coordinated by LexA and RecA. polysaccharides, proteins, nucleic acids and lipids. this bacteriophage (known as T7 ) results in rapid DspB They are crucial to the pathogenesis of many clinically multiplication of the phage and expression of DspB. Persister cells Dormant individual cells within important bacteria and exhibit resistance both to the Following lysis, T7 and DspB are released into the DspB a bacterial population that immune system and to antimicrobial treatments, mak - biofilm, which leads to re-infection and degradation o f show a high tolerance to 118,119 ing them difficult to eradicate . Collins and co-l β-1,6-N-acetyl-d-glucosamine. During the process antimicrobials. leagues successfully engineered bacteriophage T7 to of T7 infection, bacterial biofilm cell counts are DspB reduced by 99.997% — over two orders of magnitude greater than when a non-enzymatic phage is used . In a follow-up study, bacteriophage M13 was eng-i neered to express LexA3, which suppresses the SOS DNA repair system that bacteria require to counteract 121–123 VGV1 antibiotic-induced oxidative stress . Infection by VGV1 +. VGV1 this designer phage sensitizes E. coli to quinolone anti- biotics. Use of this phage increases the survival of mice that are infected with E. coli, decreases the survival of antibiotic-resistant bacteria, persister cells and biofilm +. cells and reduces the number of antibiotic-resistant bac - VGV1 VG V1 teria that arise from an antibiotic-treated population. It also acts as a strong adjuvant for other bactericidal antibiotics . The designer phage platform can be used to produce other antibiotic adjuvants . Although it was once abandoned after the intro - duction of antibiotics, phage therapy is currently being revisited in several clinical trials around the world as the 6 GVT CE[ENKPG prevalence of multidrug-resistant pathogens is dramat -i cally increasing. Although phage therapy may face clini - cal challenges associated with development of bacterial phage resistance, phage neutralization by the immune +. system and pharmacokinetics, the field will certainly receive an impetus from designer phages . Engineered probiotic bacteria decrease pathogen VGV1 virulence. Bacteria can communicate with each other using a chemical language known as quorum sensing. VGV1 Individual bacteria produce and secrete signalling m- ol VGV1 ecules (called autoinducers) that are common to mu - l tiple species or are species-specific. These molecules VGV1 accumulate as the population grows and can bind to receptors that coordinate colony-wide gene expression or manipulate the behaviour of other bacterial popu - VGV1 lations. For example, Vibrio cholerae produces cholera +. autoinducer 1 (CAI-1) and autoinducer 2 (AI-2), which trigger repression of key virulence factors. Feeding 2 QN[OGT infant mice with a probiotic E. coli that naturally pro - duces AI-2 and has been engineered to constitutively UE6 GV4s*KU synthesize CAI-1 significantly increased the animals’ survival rate after ingesting V. cholerae . This suggests that such an approach could be an economic strategy to 0CVWT G4G XKGYU ^)GPGVKEU Figure 4 | Drug delivery. Interactive biohybrid material based on the interaction of prevent infectious diseases. Unlike antibiotics, quorum- a repressor protein with its cognate DNA operator motif. Homodimeric tetracycline sensing-based interventions do not kill pathogens but repressor (TetR) is converted into a single-chain repressor (scTetR) by connecting two reprogram their behaviour; this strategy may be free of TetR subunits through a flexible peptide linker, and it is tagged with six histidines selection pressure and therefore may be less prone to (scTetR–His ). This molecule is coupled to a polymer and is mixed with a polyacrylamide develop resistance. In another study,commensal bacteria that has copies of a tetracycline operator (tetO) attached to it. scTetR binds to tetO so were equipped with synthetic circuitry to stimulate crosslinks are formed, making a hydrogel. When tetracycline is added, scTetR releases glucose-dependent insulin production in intestinal 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). epithelial cells. NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 27 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS Cancer therapies Figure 5 | Bacterial and viral cancer therapy. a | Population-density-dependent invasion of cancer cells. Despite decades of progress in cancer therapy, a major After intravenous injection, Escherichia coli accumulates in challenge remains: how to specifically target and sele - c cancer tissue, where it reaches high population densities. tively kill neoplastic cells that develop within native and E. coli is engineered to link the quorum-sensing receptor implanted tissue and relocate within the organism to LuxR to an autoinducer 1 (AI-1)-inducible promoter (P ). lux form metastasis. Therefore, therapeutic strategies that P is also used to drive luxI and the invasin gene inv. LuxI lux are designed to eliminate cancer cells must be extremely produces AI-1, generating a positive feedback loop that precise to exclusively target diseased tissue while le- av coordinates invasion throughout the population. ing normal tissue intact. Although native cytotoxicity b | Acetylsalicylic acid (Aspirin)-triggered killing of cancer or the constitutive expression of anticancer compounds cells after invasion of Salmonella spp. Salmonella spp. have demonstrated some potential in animal studies and naturally invade cancer cells after intravenous injection. Salmonella spp. were engineered with a Pseudomonas human clinical trials , trigger-inducible drug expres - putida-derived signal-amplifying two-level cascade in sion circuits delivered by tumour-invasive bacteria or which NahR controls salicylate promoter (P )-driven xylS2 sal tumour-transducing viral particles may improve can - expression and XylS2 then triggers a XylS2-dependent cer therapy. Synthetic biologists have recently designed promoter (P )-driven expression of the cytosine deaminase a few anti-cancer devices that provide precise timing, (labelled CD in the figure). Salicylate induces both location and dosing of drug production by external cues NahR-based P and XylS2-mediated P activation. sal m and could provide greater intra-tumoural effects while Mammalian cells are resistant to 5-fluorocytosine minimizing systemic toxicity. because they lack cytosine deaminase, which converts 5-fluorocytosine into the toxic cancer therapeutic Bacterial synthetic devices. After intravenous injection or 5-fluorouracil. c | Invasive bacteria suppress oncogene expression. E. coli is engineered to constitutively oral administration, many bacterial species (for example , co-express a catenin β-1-specific short hairpin RNA E.  coli and Salmonella spp.) naturally sense and self- (shRNA), Listeria monocytogenes listeriolysin (LLO*) and inv propel towards tumours. These bacteria have also been under control of the bacteriophage T7 promoter (P ). They T7 engineered to selectively invade and proliferate in tumour invade cancer cells (using the Inv protein), escape from the tissues and to produce cytotoxic compounds as well as phagosome (using LLO*) and knock down the catenin β-1 reporter proteins for non-invasive follow-up monitoring oncogene (using shRNA). d | Therapeutic protein of tumour regression . These bacteria express flagella transduction. Lentiviral particles are produced using an to penetrate tissue and chemotactic receptors to promote integrase-negative helper vector (designated ‘helper’ in migration towards aspartate produced by viable cancer the figure) and a constitutive expression vector encoding cells, ribose released by necrotic tissue or hypoxic regions the protein of interest (designated ‘protein’ in the figure) fused to viral protein R (VPR) and a protease cleavage site generated by the hyper-metabolic activities of neoplastic (PC). This can be delivered to any target cell in the absence cells. After they have reached the tumour site, the bac - of viral nucleic acids and proteins. An example application teria then either proliferate in the extracellular space or is described in the main text. pA, poly(A) tail. P , EF1a invade the tumour cells. In either situation, selective cyt-o elongation factor 1 alpha (EF1a ) promoter. toxicity was engineered by expressing toxins, cytokines, tumour antigens, pro-apoptotic factors or prodrug- converting enzymes . Non-invasive E. coli has success- fully been programmed to invade cultured tumour cells in control expression of drug components following sys - a hypoxia-responsive or population-density-dependen t temic administration of the trigger molecule in mice manner. The corresponding circuitries consist of the in tumour cells that have been invaded by Salmonella anaerobically induced formate dehydrogenase promoter spp. . The device is based on a circuit that is derived driving the Yersinia pseudotuberculosis invasin gene (inv), from Pseudomonas putida, which controls expression of which mediates invasion using specific integrin receptors cytosine deaminase in a salicylate-inducible manner . that are typically expressed on tumour cells. Population- Mammalian cells normally lack cytosine deaminase, density-dependent invasion requires an engineered quo - which means that they are resistant to 5-fluorocytosine rum-sensing circuit that triggers inv expression after the because this enzyme is needed to convert 5-fluoro- bacterial population has reached a threshold size a t cytosine into the cytotoxic molecule 5 -fluorouracil. Pharmacokinetics the tumour site. This circuitry consists of quorum-sens- Tumour-bearing mice were injected with attenuated The action of drugs in the body ing receptor LuxR that co-induces luxI (which encodes Salmonella enterica engineered with the P.  putida- over a period of time. It covers the enzyme producing the quorum-sensing messenger derived circuit and then treated with 5-fluorocytosine. absorption of the drug as well as its distribution, tissue autoinducer 1 (AI-1)), and inv. AI-1-triggered, LuxR- The mice showed significant tumour regression when localization, biotransformation mediated expression of luxI represents a positive feedback fed with acetylsalicylic acid (Aspirin) , which is rapidly and excretion. loop that amplifies inv expression and AI-1 production; converted to salicylate after intake by the anima(FIG.  l 5b). this coordinates and broadcasts the invasion order across RNAi is a potent and highly conserved mechanism Commensal bacteria the entire population (FIG. 5a). for the targeted knockdown of mRNA translation by Commensal bacteria live in close contact with the host. Tumour-invading bacteria have also been engi - small RNAs. Non-pathogenic E. coli was engineered to In this special type of neered for trigger-inducible drug expression after express a short RNA hairpin that triggers RNAi against symbiosis, one partner is entering tumour cells. In addition tol -arabinose- catenin β-1, which is a colon-cancer-specific onco- benefited, whereas the 131 133 and γ-irradiation-induced drug expression, a synthetic gene . These bacteria, which were also engineered to other is neither benefited nor harmed. salicylate-triggered expression device has been used to express proteins to mediate cellular invasion and escape 28 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS from the phagosome, were administered orally or intra - C %CPEGTEGNN venously and significantly reduced catenin - β 1 levels in 'EQNK the intestinal epithelium and in human colon cancer xenografts in mice (FIG. 5c). Combining various bacte- rial anti-cancer treatment strategies may increase safety, 2 2 NWZ4 NWZ+ KPX NWZ NWZ +PX specificity and efficiency in future clinical trials. .WZ+ Viral synthetic devices. Viruses have also been success- fully engineered to transduce specific cells by expressing epitopes that are recognized by particular cell-surface receptors and to express prodrug convertases and #+ cytokines for use in cancer therapy . Most of these onc- olytic viruses carry coding viral nucleic acids, which may cause side effects owing to recombination with the host %CPEGTEGNN chromosome or proviral elements that are already in the host cell. Recently, synthetic viral particles have been designed that lack coding nucleic acids and that excl -u 5CNOQPGNNC sively package therapeutic proteins, which can be released in a dose-dependent manner . For example, viral particles carrying linamarase from Manihot esculenta PCJ4 2 2 Z[N5 2 %& were injected into human breast cancer xenografts in PCJ4 UCN O mice that had been treated with the non-toxic natural ȯWQT QWT CEKN product linamarin; these viruses triggered efficient %& tumour regression owing to the cyanide produced by linamarase-mediated conversion of linamarin (FIG. 5d). ȯWQT QE[VQUKPG Similarly, protein-carrying viral nanoparticles have been used to deliver site-specific DNA recombinases, such as FLP, to precisely integrate or excise genetic components 5CNKE[NCVG on the host chromosome . They might also be used to deliver native or chimeric transcription factors that could transiently control the expression of target genes %CPEGTEGNN that are involved in therapeutic interventions, lineage control or induction of pluripotency . A transformation sensor for cancer therapy. Gene ther- UJ40# .. 1 KPX +PX apy advances for cancer include virus-mediated delivery of cytotoxic effector genes controlled by cancer-specific .. 1 138,139 promoters or delivery of chimeric adaptor proteins to link tyrosine kinase signalling to the apoptosis-induc - GV ing caspase machinery . Most promoters and control O40# circuits that coordinate simple reactions such as these are inherently noisy and only allow linear responses, which means limited control of specificity and efficacy. However, using two internal input signals can improve fidelity, mediate sharp response profiles and ensure robust biochemical processes . Using decision-making circuits as blueprints, Nissim and Bar-Ziv designed a tunable dual promoter integrator (DPI) to target cancer .GPVKXKT CN RCTVKENGU cells precisely. The DPI consists of two native promo -t ers that are concurrently activated by two independent transcription factors. Each cancer-sensing promoter produces a different fusion protein in proportion to its 2T QVGKP activity, and these two proteins assemble together as a chimeric transcription factor. This transcription factor then activates a synthetic promoter that controls expres- XRTsRE  RT QVGKP '( α sion of the herpes simplex virus type 1 thymidine kinase (TK1), which is cytotoxic in the presence of nucleotide JGNRGT '( α analogues, such as ganciclovir (FIG. 6a). The DPI could be optimized for a specific cancer cell type by using different 2T QFWEGTEGNN 4GEGKXGTEGNN combinations of input promoters and effector genes, as well as by modulating the assembly efficiency and half life 0CVWT G4G XKGYU ^)GPGVKEU NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 29 © 2011 Macmillan Publishers Limited. All rights reserved R# R# TI 6C :[N5 0CJ4 .WZ4 REVIEWS C +PRWV +PRWV 1WVRWV $GPKIP $GPKIP 0QPG $GPKIP /CNKIPCPV 0QPG /CNKIPCPV $GPKIP 0QPG /CNKIPCPV /CNKIPCPV %GNNFGCVJ FQE5 X2 R# /CNKIPCPE[ &QE5 &QE5 %QJ %QJ /CNKIPCPE[ )CN $& )CN $& 1 2 6- R# )CN OKP ICN EQJ R# $& 6- )CPEKENQXKT D *KIJOK40#UGPUQT OK40# .QYOK40#UGPUQT OK40# 2 TV6 # OK40# R# %/8 VC GV 2 OK40# R# 1WVRWV OK40# R# NCE+ 2 64' VC GV VC GV NCE1 1WVRWV R# %#) %GNNURGEKȮEOK40# OK40# OK40# OK40# OK40# OK40# OK40# GZRT GUUKQPRCVVGTP JKIJ JKIJ NQY NQY NQY NQY %GNNENCUUKȮGTEKT EWKV *KIJUGPUQT *KIJUGPUQT .QYUGPUQT .QYUGPUQT .QYUGPUQT .QYUGPUQT 1WVRWV $#: 5[UVGOT GURQPUG Figure 6 | Synthetic genetic cancer classifiers. a | A transformation-sensing cancer kill switch can consist of a 0CVWT G4G XKGYU ^)GPGVKEU 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 –Coh2). When they are simultaneously BD expressed, both proteins dimerize to form a synthetic transcription factor that binds Gal4 operator sites (O ), Gal4 induces downstream minimal promoters (P ) and triggers expression of the herpes simplex virus type 1 thymidine min 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. 30 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved TI TI TI REVIEWS of the chimeric transactivator components. So far, a set of the protein to the aptamers controls splicing of the mini - three promoters have been characterized in detail, but the gene; when the central exon is spliced out, the transgene DPI design may accommodate other suitable promoters. is expressed at high levels, and when it remains unspliced, The recently developed ‘cell-type classifier’ is concep - the transgene is expressed at low levels. Such a device was tually similar to the DPI, as it can also be programmed configured to sense subunits of nuclear factor kappa B to destroy cells that express a specific set of neoplastic (NFκB) or β-catenin (which are neoplastic markers) and markers . The cell-type classifier combines transcri- p to express the herpes simplex virus thymidine kinase. tion and translation control components in a single Thymidine kinase renders cells susceptible to ganciclo - scalable synthetic circuit that senses expression levels vir, so this device only operated as a cancer kill switch in of a set of (currently up to six) endogenous microRNAs the presence of the cancer markers and gangcicolvir . (miRNAs); it triggers an apoptosis-inducing response The modular configuration of the RNA sensor–actuator only if those levels match a preset profile. The cell-typede vice allows it to be tailored to different intracellular classifier combines sensor modules for the detection of proteins and even to multi-protein input using specific highly and lowly expressed miRNA (FIG.  s 6b). For clinical intronic aptamers. Also, responsiveness and performance implementation, both the DPI and the cell-type classifier can be tuned by placing the aptamers at different loc- a must either be delivered to the cancer tissue, or they must tions within the introns. The availability of compact RNA provide a fail-safe mechanism that constantly eliminates sensor–actuators that are easy to design and to alter and transforming cells from engineered tissue implants. that control transgene expression in response to intracel - lular levels of key proteins may also improve the ability to Other emerging tools for biomedicine link metabolic disease states with gene-based therapeutic Novel treatment strategies will require new technologies interventions. to sense and control disease. Synthetic biologists have designed new devices that could sense key physiological Optogenetic devices in blood glucose homeostasis. Light activities and have found new ways to dose therapeutic is becoming increasingly popular as a traceless, molecule- interventions precisely in response to external physical free input signal for triggering transgene expression in cues. Such synthetic devices could have wide-ranging living systems. Bacteria have been engineered to record 149–151 biomedical applications. projected images with gigapixel resolution and to adjust transgene expression in response to multi- RNA controllers of cell proliferation. Thus far, synthetic chromatic input , and now genetic light switches have control devices that are designed to interface with host also been designed to control gene expression and metabolism and to reprogram cellular behaviour have shape of mammalian cells . largely been limited to heterologous transcription fac - Devices that convert light pulses into transcription tors. RNA controllers may represent an alternative. They may foster novel therapeutic opportunities in future are straightforward to design and can be integrated into gene- and cell-based therapies and may improve the ma- n a single expression unit containing sensors (aptamers), ufacturing of difficult-to-produce protein pharmaceut- i gene-regulatory components (ribozymes) and effector cals, such as cancer therapeutics. An illustrative example 39,143,144 transgenes . The inherent modularity and com - is light-controlled expression of the glucagon-like p-ep patibility of RNA-based control components enables tide 1 (GLP1), which is a promising drug candidate for them to be independently optimized or exchanged. the treatment of type 2 diabetes (FIG. 7a). An optoge- For example, an RNA control device consisting of a netic device that enables light-triggered gene expres - drug-responsive aptamer linked to a ribozyme in the sion in human cells was designed. This involves ectopic 3′ untranslated region (UTR) of a cytokine expression expression of melanopsin in human embryonic kidney unit enabled trigger-inducible inactivation of ribozyme- cells and functional rewiring of signalling downstream mediated transcript cleavage and full transgene expr- es of melanopsin; the cascade integrates blue-light-pulse- sion in the presence of the input signal . This synthetic triggered photoreception and produces a reversible and RNA control device was applied to control proliferation sustained intensity-dependent transcription response. Melanopsin of engineered primary human T cells and enabled ext- er When placed in hollow fibre containers and implanted A vitamin-A-dependent, G-protein-coupled receptor nal control of the expansion of transgenic T cells that into mice, transgene expression in the engineered light- that is expressed in are implanted into mice. Synthetic RNA control devices sensitive cells could be controlled remotely by an o -pti intrinsically photosensitive could provide the advance that is necessary to enable cal fibre . Illuminating mice that carried subcutaneous retinal ganglion cells T cell therapy ; by contrast, state-of-the-art, trigger- implants of microencapsulated photo-responsive cells Prosthetic networks inducible expansion of engineered T cells using chimeric also enabled transdermal control of transgene expr-es Networks that replace existing antigen receptors has only led to moderate proliferation sion and of corresponding protein levels in the blood of cellular functionality that 146,147 and poor survival of T cells in clinical trials . treated animals. This system was able to attenuate glycae - is ill-driven or out of order. Another use for synthetic RNA is the design of pro- mic excursions and to control glucose homeostasis in a They represent molecular grammable sensor–actuator devices that convert levels mouse model of human type 2 diabetes . prostheses for non-functional cellular activity; they differ of an intracellular protein into a discrete high or low from other synthetic networks 148 transgene-expression state . The RNA devices consist Prosthetic networks. Prosthetic networks are synthetic that add useful functionality of a three-exon, two-intron minigene followed by the sensor–effector devices that act as molecular prostheses. but do not replace transgene. The introns contain protein-sensing aptamers, When engineered into cells and functionally connected non-functional cellular networks. and the central exon includes a stop codon. Binding of to host metabolism, they sense, monitor and score NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 31 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS Figure 7 | Advanced therapeutic and prosthetic networks. a | Light-triggered %C transcription control of blood glucose homeostasis. The synthetic phototransduction $NWG cascade consists of rewired melanopsin and nuclear factor of activated T cells (NFAT) NKIJV 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 642% /GNCPQRUKP 2+ (GAQ), phospholipase C (PLC) and phosphokinase C (PKC) and triggers Ca ion influx via transient receptor potential channels (TRPCs) and possibly also from the 2+ endoplasmic reticulum. This Ca ion surge activates calmodulin (CaM) to calcineurin )#3 (CaN), which dephosphorylates NFAT. NFAT then translocates into the nucleus, where it binds to specific promoters (P ) and coordinates transgene transcription. When NFAT 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. 2.% 2-% b | Prosthetic network for the treatment of tumour lysis syndrome and gout. %C/ 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 (hucO ). This operator %C0 controls expression of secretion-engineered urate oxidase (smUOX), so smUOX is 0( expressed when urate concentration reaches pathological levels. smUOX mediates %[VQRNCUO 'PFQRNCUOKE T GVKEWNWO conversion of urate into allantoin. Expression of smUOX stops when urate 0WENGWU concentration reaches oxidative-stress-protective urate levels. pA, poly(A) tail. Part a is modified, with permission, from REF. 65 © (2011) American Academy for the 0( Advancement of Science. ).2 R# 0( $NQQFXGUUGN 1* 7T CVG #NNCPVQKP *1 0* 0 1* 74# 6 1* 5GPUQTsGȭGEVQTEGNN *1 0* 0 1* CVG 7T 2 -4#$ JWE4 R# 2 JWE1 R# UO71 : 58 58 6 KOG 0CVWT G4G XKGYU ^)GPGVKEU disease-relevant metabolites, process off-level concen - concentrations and restores urate homeostasis by con- trations and coordinate adjusted diagnostic, preventive trolled expression of a urate oxidase — which reduces or therapeutic responses in a seamless, automatic and excessive urate concentration while preserving levels self-sufficient manner. that are suitable for radical scavenging — could repr -e An example of the use of a prosthetic network is sent a treatment strategy for hyperuricaemic disorders . the sensing of metabolites to improve control of urate In brief, human cells that contain such a prosthetic net - homeostasis (FIG.  7b). Moderate levels of uric acid, work have recently been designed by combining: the uric which scavenges radicals, are deemed to be beneficial. acid sensor HucR , which manages oxidative stress pro- However, a transient surge in uric acid that is released by tection in Deinococcus radiodurans; the human uric acid dying cells during cancer therapy leads to tumour lysis transporter URAT1 (also known as SLC22A12), which syndrome, and chronic hyperuricaemia can result in increases the intracellular uric acid levels and thus the gout. Humans are particularly sensitive to imbalances of sensitivity of the prosthetic circuit; and a secretion- urate homeostasis because they lack uricolytic activity. A engineered urate oxidase (smUOX) that is clinicall y prosthetic network that constantly monitors blood urate licensed for the treatment of the tumour lysis syndrome . 32 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved #6 #6 #6 2JQURJCVG REVIEWS 94,98 The prosthetic uric-acid-responsive expression network may imminently develop into shorter drug discovery (UREX) was able to sense uric acid concentrations pre - and drug development timelines, increased precision 112,114 cisely and to activate secretion of smUOX when the uric of drug delivery and production of new and more 104–109 acid concentration was at pathologic levels. Secretion affordable medicines . Ultimately, sophisticated thera - of smUOX is stopped as soon as the uric acid concen- peutic sensor–effector devices that can sense disturbances, tration has returned to the homeostasis level. This was seek out pathological conditions and restore function are impressively demonstrated when UREX-transgenic cells on the roadmap. Such therapeutic networks that connect were implanted into urate-oxidase-deficient mice, which diagnostic input with therapeutic output may provide develop acute hyperuricaemia with symptoms that are all-in-one diagnostic, preventive and therapeutic sol-u similar to human gout. UREX was able to degrade urate tions in future gene- and cell-based therapies. Matching and restore urate homeostasis in the blood, resulting in diagnostic outcome with high-end therapies has recently the dissolution of uric acid crystal deposits in the kidney become a focus of the pharmaceutical industry, which of treated animals . Its straightforward design may allow has declared personalized medicine as the treatment UREX to serve as a blueprint for the assembly of other strategy of the future. Tools that will have a tremendous prosthetic networks that sense metabolic disturbances impact in future biomedical applications include: using and circulating pathologic metabolites. light-activated triggers to bring about a precise therapeu - tic response in cells , programming bacteria to seek and 128,132,133 An artificial insemination device. Artificial insemi- destroy cancer cells and using synthetic circuitry 64,65 nation is standard practice to facilitate both human to keep crucial metabolites at homeostatic levels , to reproduction and livestock breeding. Because of broad manage disease-controlled expansion or to eliminate 31,148,159 variations in oestrus expression and ovulation timing, specific cell populations . Recent work has shown coordinating sperm delivery with female oestrus is still a that this is, in principle, possible and that some devices major challenge. Ovulation is triggered at a specific time are working as expected and are producing a therapeutic 64,65 when the pituitary gland releases luteinizing hormone, impact in animal models of human diseases . Implants which binds to the luteinizing hormone receptor (LHR) consisting of engineered microencapsulated cells repr -e and coordinates the release of the oocyte. By integra- t sent a way of introducing prosthetic networks with a pre- ing synthetic signalling cascades with advanced biom-a defined function instead of directly targeting the host cells terials, Kemmer and colleagues designed an artificial with the genetic material. Although implants containing insemination device that coordinates sperm delivery with cells with engineered prosthetic networks are certainly the oestrus control. The artificial insemination device con - most promising way forward, they will limit biomedical sists of cellulose sulphate capsules containing bull sperma pplications to extracellular disease metabolites that can 63,156,157 and sensor cells . The sensor cells are engineered to be therapeutically addressed through the vascular system. express LHR constitutively so that when it is activated, it However, there is still a long way to go until synthetic- triggers expression of cellulase that can be secreted. After biology-based biomedical devices will be a clinical reality. implantation into the cow’s uterus, the sensor cell line Placing therapeutic circuits in specific cells of a patient constantly monitors the animal’s luteinizing hormone and making sure that there will be no interference with levels, and the oestrus-triggered surge in luteinizing ho - r human metabolism are the most important challenges. mone levels leads to the production of secreted cellulase, Th erefore, clinical use of synthetic-biology-based devices which degrades the implanted capsule and results in the and therapeutic scenarios will face the same scientific, timely delivery of the sperm and successful conception. ethical and legal issues as any gene- and cell-based th - er Fine tuning of the designer cascade could enable its use apy, but they may offer more complex control dynamics in other species, including in humans. and are therefore expected to have a higher therapeutic impact. 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NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 35 © 2011 Macmillan Publishers Limited. All rights reserved http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Reviews. Genetics Pubmed Central

Emerging biomedical applications of synthetic biology

Nature Reviews. Genetics , Volume 13 (1) – Nov 29, 2011

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Pubmed Central
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© Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2011
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1471-0056
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1471-0064
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10.1038/nrg3094
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Abstract

REVIEWS Emerging biomedical applications of synthetic biology 1,2 3,4 Wilfried Weber and Martin Fussenegger 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. By applying engineering principles to biology, sy- n protein expression dynamics with high precision and Aptamer thetic biology has become the science of reassembling predictable logic in response to external cues or physi - An oligonucleic acid that binds to a specific target, such catalogued and standardized biological components in ological pathways. Because most control components as a chemical compound, a systematic and rational manner to create and eng- i function in different bacterial and eukaryotic species a protein or a nucleic acid. 36,47–49 neer functional biological designer devices, systems after minor refinements , gene switches and net- Aptamers were found to and organisms with predictable, useful and novel fun -c work blueprints that were pioneered in bacteria or yeast control riboswitches in natural tions. Synthetic biology is able to use an inventory of are often fully functional in mammalian cells. Examples systems, but they can also be selected in vitro. biomolecular parts compiled over 50 years of molecular of synthetic networks with similar components and cir - 1–4 biological and functional genomic research , as well as cuit topology in bacterial and mammalian cells include: 5,6 42,43 15,20,23,45 technology that has made it possible to analyse, syn- regulatory cascades , epigenetic toggle switches , 7–9 10 11 12,13 22,50,51 30,52 thesize , assemble , modify and transfer genetic hysteretic circuits , molecular timing devices , components into living organisms. synt hetic eco-sensing systems, synthetic quorum- 46,53,54 Although it has recently become possible to reco- n sensing systems, synthetic hormone systems , band- 21,30,55 struct a living organism after transfer of a synthetic pass filters and different types of oscillators that genome that has been assembled from chemically sy-n program rhythmic transgene expression with a tun- Faculty of Biology, University 13,14 16,26–29,56,57 thesized nucleic acid pieces , the rational design of able frequency and amplitude . Most of these of Freiburg, Schänzlestrasse 1, state-of-the-art biological circuits with predicable fun -c first-generation synthetic circuits operated in isolation D‑79104 Freiburg, Germany. tions remains challenging and is apparently limited to a without any interface with the metabolism of the host BIOSS Centre for Biological 15–31 Signalling Studies, University handful genes . cell, and they were used to program specific cellular of Freiburg, Hebelstrasse 25, 58–62 Synthetic circuitry is composed of basic heterologous functions using heterologous external input signals . D‑79104 Freiburg, Germany. control components that fine-tune transgene expression Key challenges that often occur in the biomedical Department of Biosystems in response to specific exogenous cues or endogenous setting are the need for drug–target specificity, precise Science and Engineering, ETH Zurich, Mattenstrasse metabolites . These gene switches include trigger- drug-dosing regimes, minimizing side effects, shorten- 33–36 26, CH‑4058 Basel, inducible protein–DNA or aptamer–transcript inter- ing the diagnosis-to-treatment timelines and avoiding Switzerland. 37–39 actions , which control transcription and translation drug-resistance of pathogens. As synthetic biology ena - Faculty of Science, University in response to heterologous and endogenous input sig - bles the engineering of complex, high-precision control of Basel, Mattenstrasse 26, nals( FIG. 1). The standardized design of gene switches has devices that couple sensing and delivery mechanisms, CH‑4058 Basel, Switzerland. 40,41 Correspondence to M.F.  improved functional compatibiliy t and has enabled this emerging science may be able to offer tools that are e‑mail: the construction of higher-order networks — includ - suited to meeting current biomedical challenges in new fussenegger@bsse.ethz.ch 42,43 ing multi-trigger inputs and sequential control , ways. A decade after the pioneering synthetic networks doi:10.1038/nrg3094 20,23,44,45 16,19,28,46 18,20 mutual control and feedback control of were reported , the first successful therapeutic appli - Published online 29 November 2011 circuit components — that are able to provide complex cations in animal models of prominent human diseases NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 21 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS C +PFWEGT Epigenetic toggle switches 4 4 Genetic circuits that can be switched between two stable expression states (for example, 4 4 an ‘on’ and an ‘off’ state) by a transient stimulus. In the 2 1 )GPG R# 2 1 )GPG R# absence of a switching stimulus, the expression state is locked and inherited across cell generations. Hysteretic circuits D +PFWEGT Genetic circuits whose 4 4 response dynamics depend # # on a combination of past and present states. 4 4 1 2 )GPG R# 1 2 )GPG R# OKP OKP Eco-sensing The capacity of a species to sense and score its surrounding ecosystem, for example, to identify the type and population density of neighbouring species. Quorum sensing %NGCXCIG A small-molecule-based E #RVCOGT chemical language by which R# bacteria communicate within 9KVJQWV and across populations (the )GPG 4KDQ\[OG KPFWEGT ‘quorum’). Production and response to quorum-sensing molecules is correlated with 9KVJ )GPG R# population density. KPFWEGT Band-pass filters These are devices that are CPUNCVKQP selectively induced within a specific concentration range )GPG R# of the input signal. At lower or higher trigger levels, the band-pass filter shuts down output signals. )GPGQH 2 T 1 )GPG R# N # CEVKXCVKQP RT QVGKP KPVGT GUV FQOCKP Figure 1 | Mammalian gene expression control strategies. a | Repression-based expression control. A repressor 0CVWT G4G XKGYU ^)GPGVKEU 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 ) is activated when a chimeric min 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 is no longer activated, and transcription of the gene of interest is prevented. c | mRNA min 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. 63–65 are starting to emerge . This Review focuses on the Understanding disease mechanisms recent progress in synthetic biology towards improv - Pathogen mechanisms. The availability of affordable ing human health, including diagnostic applications, technology for synthesizing and assembling sequences 68 69 design of novel preventive care strategies, progress for proteins or for viral and bacterial genomes with in drug discovery, design and delivery, and develo -p increased speed has dramatically improved our under - ment of novel treatment strategies, such as prosthetic standing of host–pathogen interactions and of disease networks. Synthetic biology holds the promise of pro - mechanisms. The synthetic biology principle of ‘analysis viding unique opportunities for major advances in by synthesis’ provides mechanistic insight by combin- the improvement of human health in the twenty-first ing rapid synthesis, assembly, shuffling and mutation 4,66,67 century . of individual genetic components with straightforward 22 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved RQN[ # VCK VQT TC 1RG QOQVG 2T UUQT TG 4GR CPUETKRVKQP 6T 6T REVIEWS functional analysis. For example, the genome of the Disease prevention H1N1 virus that was responsible for the 1918 Spanish Vaccines. High-throughput and high-precision assem- influenza pandemic was synthesized using sequence bly and engineering of entire genomes from well- information from genomic pieces that were extracted defined genetic components using synthetic biology from permafrost-conserved tissue samples. Functional principles has provided new opportunities for the analysis of the reconstructed virus provided new insight design of attenuated pathogens for use as vaccines. For into the key virulence factors of the pathogen: namely, a example, primates immunized with virus-like particles haemagglutinin variant that induces membrane fusion that were produced by selective expression of particular without trypsin activation and a modified polymerase chikungunya virus (CHIKV) structural proteins were that enhances viral replication . The same study also protected against viraemia after a high-dose challenge; revealed that a combination of eight genes was respon - even immunodeficient mice that were treated with sible for the exceptional virulence of the Spanish infl -u monkey-derived antibodies survived subsequent lethal 68,70 81 enza strain . This finding may help to identify the doses of CHIKV . DNA synthesis and assembly has also 71–73 pandemic potential of future virus variants . played an essential part in pioneering a safe live vaccine Synthesis and analysis of chimeric viruses have also against the poliovirus . The poliovirus was attenuated made a substantial contribution to the understanding of by systematic genome-scale changes of adjacent pairs of coronavirus zoonoses that were responsible for the severe codons from over- to underrepresented codon sets in acute respiratory syndrome (SARS) pandemic of 2002 viral capsid genes (for example, GCC|GAA is strongly and 2003. The characterization of the history of the SARS under-represented compared with GCA|GAG, although coronavirus, especially its switch in tropism, was pa- r both encode Ala–Glu). These changes reduced tran-s ticularly challenging, as its direct ancestors could not be lation and impaired the replication competence and propagated in laboratory models. However, after a 30 kb infectivity of the virus. This attenuated poliovirus pro - SARS-like bat coronavirus was designed to contain the vides protective immunization in mice and offers a high receptor-binding spike protein of its human homologue , safety standard given the low probability that all 631 the synthesized chimeric virus was able to replicate in culture individual changes will revert and thus reconstitute and infect mice . These in vivo studies revealed infection- infectious wild-type viruses. The genome-engineering enhancing mutations in the spike protein and established approach used here could represent a general strategy this surface protein as a key factor that is responsible for for designing live vaccines against infectious diseases. tropism switches in coronavirus zoonoses . Reconstruction Other promising vaccination concepts include using of pathogens by DNA synthesis can also be used for the antigen-producing immunostimulatory liposomes as 75,76 83 production of diagnostic high-density antigen arrays , genetically programmable synthetic vaccines and the such as those used to profile post-Lyme-disease syn - production of heat-stable oral algae-based vaccines to 76 84,85 drome or the humoural immune responses to hepatitis C protect against Staphylococcus aureus infections . and the human immunodeficiency virus (HIV) . Vector control. Suppression of insect vector populations Immune systems. Synthetic biology has recently pro - using transgenic viral strains that harbour conditional vided new insight into disorders that are related to dominant-lethal synthetic circuitry may control the deficiencies of the immune system, which is known transmission of malaria parasites and dengue viruses for its particularly complex control circuits and cel -lu and could eventually control the spread of untreatable 86–88 lar interaction networks. For example, dysfunction of diseases . Mosquitoes that are transgenic for a tet- B-lymphocyte activation underlies several physiological racycline-dependent transactivator (tTA) that is exclu - disorders . Functional reconstitution and analysis of the sively expressed in the female’s indirect flight muscle human B cell antigen receptor (BCR) signalling cascade can only be propagated in the presence of tetracycline, in insect cells revealed that BCRs are not activated by which represses the transcription of this gene. However, antigen-specific crosslinking, as presented in textbooks, the absence of tetracycline leads to the development of 87,88 but instead have an autoinhibitory oligomeric confor - a female-specific flightless phenotype . Putting the mation on resting B lymphocytes that shifts to an active eggs of this transgenic mosquito into the ecosystem Immunostimulatory liposomes dissociated form when antigens bind . This triggers the results in male-only releases; female mosquitoes remain Liposomes that are decorated signalling cascade, which results in antibody production grounded and cannot feed, mate or take blood meals, with antigenic peptides and the onset of a humoural immune response. which effectively represents a lethal phenotype. Males or proteins that elicit an Also, construction of a representation of the complete do not transmit the disease, but they disseminate the immune response. human peptidome engineered for display on the surface synthetic circuit across the resident wild-type mosquito 80 87,88 Gene drive system of T7 phages enabled Church and colleagues to discover population (FIG. 2a). These are molecular devices new autoantigens. They used patient-derived autoanti- Similarly, a synthetic homing endonuclease-based that promote the spreading of bodies to enrich autoantigenic peptides displayed on gene drive system could be used to spread genetic modi - a specific gene throughout a the phages; they could then identify the antigens by fication, such as malaria resistance, from engineered target population by taking advantage of a mechanism high-throughput sequence analysis . Knowledge of the mosquitoes to the field population. Homing endonu - that multiplies the specific antigens that are involved in autoimmune processes is cleases typically produce a single sequence-specific gene in the host genome. important for understanding disease aetiology, develo- p double-strand break in the host genome that is repaired Gene drive systems produce ing accurate diagnostic tests and designing drugs that by homologous recombination using the homing endo - non-Mendelian patterns of inheritance. neutralize autoreactive immune cells. nuclease gene (HEG) as a template. Consequently, NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 23 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS 'PIKPGGT GF JQOQ\[IQWU 'PIKPGGT GF 6'6 &GCVJ JGVGT Q\[IQWU 9KNFV[RG s6'6 &GCVJ × s6'6 +PVT QP V6 # R# 2 +PVT QP V6 # R# (/ 6'6 V6 # R# 2 6 QZKEIGPG R# (NKIJVNGUU 6'6 9KNFV[RGKPUGEV *GVGT Q\[IQWURT QIGP[ *QOQ\[IQWURT QIGP[ + 5EG + + 5EG + + 5EG + 4GRCKT CPUIGPG + 5EG + QRWNCVKQPKPXCUKQP 2 QHVT + 5EG + %NGCXCIG 'PIKPGGT GFKPUGEV GPFT GUGEVKQP )GPG VKQPPWODGT Figure 2 | Synthetic biology for understanding and preventing disease. a | A female-specific dominant-lethal 0CVWT G4G XKGYU ^)GPGVKEU 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 ). In male FM 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 and the expression of a toxic gene that triggers a flightless phenotype. If mosquitoes TET are raised in the presence of tetracycline (TET), tTA is prevented from activating P , which results in a normal TET 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. 24 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved TC REVIEWS the selfish HEG is copied to the broken chromosome so prevents EthA-mediated conversion of the last- in a gene conversion process referred to as ‘homing’. line-defence antibiotic ethionamide into a pathogen- Expressing the HEG I-SceI under the control of a male killing metabolite. The chemical 2 -phenyethylbutyrate, germline promoter enabled efficient homing in transhet - best known for its strawberry flavour, was the first com- erozygous males and rapid genetic drive, which led to pound found that specifically inactivated EthR and so HEG invasion in caged mosquito populations (FIG. 2b). triggered ethA expression and re-established the sen - By engineering the sequence specificity of other HEGs sitivity of Mycobacterium tuberculosis to ethionamide (for example, I-AniI or I-CreI), the gene drive concept (FIG. 3d). Further work revealed other EthR-inactivating could, in principle, be used to knock in or knock out ethionamide booster compounds; these have also been gene functions that target the mosquito’s ability to serve successfully tested in a mouse model of human tuber - 89 98 as a disease vector . culosis . Restoring drug sensitivity by pharmacological Field tests of release of insects carrying dominant inhibition of master resistance regulators may be widely lethals (RIDL) technology using first-generation tTA- applicable . transgenic mosquitoes have already been conducted in A further example of the use of synthetic circuitry Grand Cayman. First, a small-scale release confirmed for drug discovery is provided by mammalian cells that transgenic males could survive, mate with wild that are conditionally arrested in the G1 phase of the females and produce transgenic larvae, and then the cell cycle by circuitry controlling the expression o f full field trial showed an 80% reduction in the num - the cycline-dependent kinase inhibitor p27. These cells bers of wild mosquitoes about 11 weeks after release. reproducibly formed a mixture of isogenic subpopul- a As the study site was not isolated and the surrounding tions of proliferation-inhibited cells and proliferating areas contained high densities of wild-type mosqui - cells that had spontaneously escaped the synthetic cell toes, scoring the actual suppression efficiency remains cycle block . These cells could be used as a cell-based challenging . cancer model and could be used to screen for anticancer compounds that selectively eliminate proliferating cells 100,101 Drug development while leaving arrested ones intact . Drug discovery. Synthetic mammalian transcription cir - cuits consisting of a chimeric small-molecule-responsive Drug production and drug delivery. The synthetic path- transcription factor and a cognate synthetic promoter ways that are created by assembling enzymatic cascades were originally designed for future gene-based thera - or networks in bacteria, yeast and plants have been pies, and the aim was to adjust therapeutic transgene instrumental for the large-scale economic production of expression in mammalian cells in response to a pha -r high-value drug and drug precursor compounds, as well 34,47,49,91 macologically active substance . As most chimeric as for the biosynthesis of new secondary metabolites transcription factors are derived from repressors that with novel therapeutic activities. Examples include com - 102,103 104–106 manage drug resistance in bacteria (for example, resist - plex polyketides , halogenated alkaloids and the ance to antibiotics ) and are promiscuous for structur- precursors of the anti-malaria drug artemisinin (which ally related compounds, mammalian cells containingi s produced by the company Amyris, for example) such circuitry could also be used in ‘reverse mode’, and of the anti-cancer compound taxol . For produc- as integrated screening devices for the class-specific tion of these compounds, it was necessary to overcome 33,93 discovery of new drug candidates (for example, new several challenges, including the functional expression antibiotics ) (FIG. 3a). When mammalian cells that are of complex biosynthetic enzymes (such as cytochrome transgenic for the screening circuit are exposed to a P450 monooxygenases ) and the overall orchestra - compound library, they detect and modulate reporter tion of the multistep pathway to avoid accumulation of gene expression in the presence of a non-toxic, cell- (toxic) intermediate products and to ensure metabolic permeable and bioavailable molecule that has a class-channelling . specific core structure and corresponding drug activity Small-molecule-responsive protein–protein and (for example, antibiotic activity) (FIG.  3b). Using the protein–DNA interactions that are used to pioneer 36,49,110 same screening setup, compounds have been detected gene switches in mammalian cells have also been that lock the transcription factor onto the DNA, which successfully re-engineered in the design of trigger- 111–115 may block induction of antibiotic resistance in patho - inducible biohybrid materials for drug delivery . gens and render them drug-sensitive (for example, see Using synthetic protein–polyacrylamide and DNA– Bioversys). Using such compounds alongside the spe - polyacrylamide monomers, hydrogels can be produced Polyketides cific antibiotic may offer novel anti-infective treatment that dissolve when specific ligands are supplied (FIG. 4). These constitute a group opportunities and a new life cycle for established an-ti Biopharmaceuticals (for example, vascular endothelial of secondary metabolites biotics (FIG. 3c). Other trigger-inducible transcription growth factor (VEGF)) supplied during gel formation produced through linear control systems can be used in this manner as well, such are loaded into the hydrogel and can be released in a decarboxylative condensation as those that are responsive to streptogramin , tetracy- dose-dependent manner after subcutaneous implanta - of acetyl-CoA with several 91 95 malonyl-CoA-derived extender cline or macrolide antibiotics , anti-diabetes drugs or tion into mice and oral administration of the trigger units to a polyketide chain. 96,97 115 immunosuppressive lactones . compound . It is thought that any trigger-inducible Many pharmacologically active One example of the efficacy of a transcription circuit protein–protein and protein–DNA interactions could compounds, such as antibiotics system involves the bacterial transcriptional repres - be used to produce drug-sensing and drug-releasing and anti-cancer drugs, belong 114,116,117 to the polyketide class. sor EthR. EthR represses transcription of ethA and hydrogels . NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 25 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS %JGOKECNNKDT CT[ 6 QZKE 0QPRGTOGCDNG C %*1- 0QGȭGEV 2 2 2+2 R# 2+2 R# EQPUV EQPUV 2+2 2+2 2+2 2+2 2 2+4 5'#2 R# 2 2+4 5'#2 R# EQPUV EQPUV D *'-6 2 2 GVJ4 82 R# GVJ4 82 R# EQPUV EQPUV 82 82 1 2 5'#2 R# 1 2 5'#2 R# 'VJ4 OKP 'VJ4 OKP E /VWDGT EWNQUKU 2JGP[NGVJ[NDWV[T CVG 2 2 2 2 GVJ# GVJ4 GVJ# GVJ4 'VJ# 'VJKQPCOKFG 'VJKQPCOKFG 0CVWT G4G XKGYU ^)GPGVKEU 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 ). When const EthR–VP16 binds to its cognate operator (O ), the minimal promoter (P ) is activated, which results in expression of EthR min 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 , stopping SEAP expression. c | Overcoming resistance to ethionamide EthR 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. 26 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 'VJ4 REVIEWS Novel treatments for infections constitutively express DspB: an enzyme that hydrolyses SOS DNA repair Breaking bacterial resistance by designer phages. β-1,6-N-acetyl-d-glucosamine, which is an adhesin Genetically encoded repair program protecting against Biofilms are surface-associated bacterial communi - that is required for biofilm formation and integrity in DNA damage. In prokaryotes, ties that are encased in a hydrated extracellular po- ly Staphylococcus spp. and Escherichia coli clinical iso - the repair program is meric substance (EPS) matrix that is composed of lates. The initial infection of a bacterial biofilm with coordinated by LexA and RecA. polysaccharides, proteins, nucleic acids and lipids. this bacteriophage (known as T7 ) results in rapid DspB They are crucial to the pathogenesis of many clinically multiplication of the phage and expression of DspB. Persister cells Dormant individual cells within important bacteria and exhibit resistance both to the Following lysis, T7 and DspB are released into the DspB a bacterial population that immune system and to antimicrobial treatments, mak - biofilm, which leads to re-infection and degradation o f show a high tolerance to 118,119 ing them difficult to eradicate . Collins and co-l β-1,6-N-acetyl-d-glucosamine. During the process antimicrobials. leagues successfully engineered bacteriophage T7 to of T7 infection, bacterial biofilm cell counts are DspB reduced by 99.997% — over two orders of magnitude greater than when a non-enzymatic phage is used . In a follow-up study, bacteriophage M13 was eng-i neered to express LexA3, which suppresses the SOS DNA repair system that bacteria require to counteract 121–123 VGV1 antibiotic-induced oxidative stress . Infection by VGV1 +. VGV1 this designer phage sensitizes E. coli to quinolone anti- biotics. Use of this phage increases the survival of mice that are infected with E. coli, decreases the survival of antibiotic-resistant bacteria, persister cells and biofilm +. cells and reduces the number of antibiotic-resistant bac - VGV1 VG V1 teria that arise from an antibiotic-treated population. It also acts as a strong adjuvant for other bactericidal antibiotics . The designer phage platform can be used to produce other antibiotic adjuvants . Although it was once abandoned after the intro - duction of antibiotics, phage therapy is currently being revisited in several clinical trials around the world as the 6 GVT CE[ENKPG prevalence of multidrug-resistant pathogens is dramat -i cally increasing. Although phage therapy may face clini - cal challenges associated with development of bacterial phage resistance, phage neutralization by the immune +. system and pharmacokinetics, the field will certainly receive an impetus from designer phages . Engineered probiotic bacteria decrease pathogen VGV1 virulence. Bacteria can communicate with each other using a chemical language known as quorum sensing. VGV1 Individual bacteria produce and secrete signalling m- ol VGV1 ecules (called autoinducers) that are common to mu - l tiple species or are species-specific. These molecules VGV1 accumulate as the population grows and can bind to receptors that coordinate colony-wide gene expression or manipulate the behaviour of other bacterial popu - VGV1 lations. For example, Vibrio cholerae produces cholera +. autoinducer 1 (CAI-1) and autoinducer 2 (AI-2), which trigger repression of key virulence factors. Feeding 2 QN[OGT infant mice with a probiotic E. coli that naturally pro - duces AI-2 and has been engineered to constitutively UE6 GV4s*KU synthesize CAI-1 significantly increased the animals’ survival rate after ingesting V. cholerae . This suggests that such an approach could be an economic strategy to 0CVWT G4G XKGYU ^)GPGVKEU Figure 4 | Drug delivery. Interactive biohybrid material based on the interaction of prevent infectious diseases. Unlike antibiotics, quorum- a repressor protein with its cognate DNA operator motif. Homodimeric tetracycline sensing-based interventions do not kill pathogens but repressor (TetR) is converted into a single-chain repressor (scTetR) by connecting two reprogram their behaviour; this strategy may be free of TetR subunits through a flexible peptide linker, and it is tagged with six histidines selection pressure and therefore may be less prone to (scTetR–His ). This molecule is coupled to a polymer and is mixed with a polyacrylamide develop resistance. In another study,commensal bacteria that has copies of a tetracycline operator (tetO) attached to it. scTetR binds to tetO so were equipped with synthetic circuitry to stimulate crosslinks are formed, making a hydrogel. When tetracycline is added, scTetR releases glucose-dependent insulin production in intestinal 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). epithelial cells. NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 27 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS Cancer therapies Figure 5 | Bacterial and viral cancer therapy. a | Population-density-dependent invasion of cancer cells. Despite decades of progress in cancer therapy, a major After intravenous injection, Escherichia coli accumulates in challenge remains: how to specifically target and sele - c cancer tissue, where it reaches high population densities. tively kill neoplastic cells that develop within native and E. coli is engineered to link the quorum-sensing receptor implanted tissue and relocate within the organism to LuxR to an autoinducer 1 (AI-1)-inducible promoter (P ). lux form metastasis. Therefore, therapeutic strategies that P is also used to drive luxI and the invasin gene inv. LuxI lux are designed to eliminate cancer cells must be extremely produces AI-1, generating a positive feedback loop that precise to exclusively target diseased tissue while le- av coordinates invasion throughout the population. ing normal tissue intact. Although native cytotoxicity b | Acetylsalicylic acid (Aspirin)-triggered killing of cancer or the constitutive expression of anticancer compounds cells after invasion of Salmonella spp. Salmonella spp. have demonstrated some potential in animal studies and naturally invade cancer cells after intravenous injection. Salmonella spp. were engineered with a Pseudomonas human clinical trials , trigger-inducible drug expres - putida-derived signal-amplifying two-level cascade in sion circuits delivered by tumour-invasive bacteria or which NahR controls salicylate promoter (P )-driven xylS2 sal tumour-transducing viral particles may improve can - expression and XylS2 then triggers a XylS2-dependent cer therapy. Synthetic biologists have recently designed promoter (P )-driven expression of the cytosine deaminase a few anti-cancer devices that provide precise timing, (labelled CD in the figure). Salicylate induces both location and dosing of drug production by external cues NahR-based P and XylS2-mediated P activation. sal m and could provide greater intra-tumoural effects while Mammalian cells are resistant to 5-fluorocytosine minimizing systemic toxicity. because they lack cytosine deaminase, which converts 5-fluorocytosine into the toxic cancer therapeutic Bacterial synthetic devices. After intravenous injection or 5-fluorouracil. c | Invasive bacteria suppress oncogene expression. E. coli is engineered to constitutively oral administration, many bacterial species (for example , co-express a catenin β-1-specific short hairpin RNA E.  coli and Salmonella spp.) naturally sense and self- (shRNA), Listeria monocytogenes listeriolysin (LLO*) and inv propel towards tumours. These bacteria have also been under control of the bacteriophage T7 promoter (P ). They T7 engineered to selectively invade and proliferate in tumour invade cancer cells (using the Inv protein), escape from the tissues and to produce cytotoxic compounds as well as phagosome (using LLO*) and knock down the catenin β-1 reporter proteins for non-invasive follow-up monitoring oncogene (using shRNA). d | Therapeutic protein of tumour regression . These bacteria express flagella transduction. Lentiviral particles are produced using an to penetrate tissue and chemotactic receptors to promote integrase-negative helper vector (designated ‘helper’ in migration towards aspartate produced by viable cancer the figure) and a constitutive expression vector encoding cells, ribose released by necrotic tissue or hypoxic regions the protein of interest (designated ‘protein’ in the figure) fused to viral protein R (VPR) and a protease cleavage site generated by the hyper-metabolic activities of neoplastic (PC). This can be delivered to any target cell in the absence cells. After they have reached the tumour site, the bac - of viral nucleic acids and proteins. An example application teria then either proliferate in the extracellular space or is described in the main text. pA, poly(A) tail. P , EF1a invade the tumour cells. In either situation, selective cyt-o elongation factor 1 alpha (EF1a ) promoter. toxicity was engineered by expressing toxins, cytokines, tumour antigens, pro-apoptotic factors or prodrug- converting enzymes . Non-invasive E. coli has success- fully been programmed to invade cultured tumour cells in control expression of drug components following sys - a hypoxia-responsive or population-density-dependen t temic administration of the trigger molecule in mice manner. The corresponding circuitries consist of the in tumour cells that have been invaded by Salmonella anaerobically induced formate dehydrogenase promoter spp. . The device is based on a circuit that is derived driving the Yersinia pseudotuberculosis invasin gene (inv), from Pseudomonas putida, which controls expression of which mediates invasion using specific integrin receptors cytosine deaminase in a salicylate-inducible manner . that are typically expressed on tumour cells. Population- Mammalian cells normally lack cytosine deaminase, density-dependent invasion requires an engineered quo - which means that they are resistant to 5-fluorocytosine rum-sensing circuit that triggers inv expression after the because this enzyme is needed to convert 5-fluoro- bacterial population has reached a threshold size a t cytosine into the cytotoxic molecule 5 -fluorouracil. Pharmacokinetics the tumour site. This circuitry consists of quorum-sens- Tumour-bearing mice were injected with attenuated The action of drugs in the body ing receptor LuxR that co-induces luxI (which encodes Salmonella enterica engineered with the P.  putida- over a period of time. It covers the enzyme producing the quorum-sensing messenger derived circuit and then treated with 5-fluorocytosine. absorption of the drug as well as its distribution, tissue autoinducer 1 (AI-1)), and inv. AI-1-triggered, LuxR- The mice showed significant tumour regression when localization, biotransformation mediated expression of luxI represents a positive feedback fed with acetylsalicylic acid (Aspirin) , which is rapidly and excretion. loop that amplifies inv expression and AI-1 production; converted to salicylate after intake by the anima(FIG.  l 5b). this coordinates and broadcasts the invasion order across RNAi is a potent and highly conserved mechanism Commensal bacteria the entire population (FIG. 5a). for the targeted knockdown of mRNA translation by Commensal bacteria live in close contact with the host. Tumour-invading bacteria have also been engi - small RNAs. Non-pathogenic E. coli was engineered to In this special type of neered for trigger-inducible drug expression after express a short RNA hairpin that triggers RNAi against symbiosis, one partner is entering tumour cells. In addition tol -arabinose- catenin β-1, which is a colon-cancer-specific onco- benefited, whereas the 131 133 and γ-irradiation-induced drug expression, a synthetic gene . These bacteria, which were also engineered to other is neither benefited nor harmed. salicylate-triggered expression device has been used to express proteins to mediate cellular invasion and escape 28 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS from the phagosome, were administered orally or intra - C %CPEGTEGNN venously and significantly reduced catenin - β 1 levels in 'EQNK the intestinal epithelium and in human colon cancer xenografts in mice (FIG. 5c). Combining various bacte- rial anti-cancer treatment strategies may increase safety, 2 2 NWZ4 NWZ+ KPX NWZ NWZ +PX specificity and efficiency in future clinical trials. .WZ+ Viral synthetic devices. Viruses have also been success- fully engineered to transduce specific cells by expressing epitopes that are recognized by particular cell-surface receptors and to express prodrug convertases and #+ cytokines for use in cancer therapy . Most of these onc- olytic viruses carry coding viral nucleic acids, which may cause side effects owing to recombination with the host %CPEGTEGNN chromosome or proviral elements that are already in the host cell. Recently, synthetic viral particles have been designed that lack coding nucleic acids and that excl -u 5CNOQPGNNC sively package therapeutic proteins, which can be released in a dose-dependent manner . For example, viral particles carrying linamarase from Manihot esculenta PCJ4 2 2 Z[N5 2 %& were injected into human breast cancer xenografts in PCJ4 UCN O mice that had been treated with the non-toxic natural ȯWQT QWT CEKN product linamarin; these viruses triggered efficient %& tumour regression owing to the cyanide produced by linamarase-mediated conversion of linamarin (FIG. 5d). ȯWQT QE[VQUKPG Similarly, protein-carrying viral nanoparticles have been used to deliver site-specific DNA recombinases, such as FLP, to precisely integrate or excise genetic components 5CNKE[NCVG on the host chromosome . They might also be used to deliver native or chimeric transcription factors that could transiently control the expression of target genes %CPEGTEGNN that are involved in therapeutic interventions, lineage control or induction of pluripotency . A transformation sensor for cancer therapy. Gene ther- UJ40# .. 1 KPX +PX apy advances for cancer include virus-mediated delivery of cytotoxic effector genes controlled by cancer-specific .. 1 138,139 promoters or delivery of chimeric adaptor proteins to link tyrosine kinase signalling to the apoptosis-induc - GV ing caspase machinery . Most promoters and control O40# circuits that coordinate simple reactions such as these are inherently noisy and only allow linear responses, which means limited control of specificity and efficacy. However, using two internal input signals can improve fidelity, mediate sharp response profiles and ensure robust biochemical processes . Using decision-making circuits as blueprints, Nissim and Bar-Ziv designed a tunable dual promoter integrator (DPI) to target cancer .GPVKXKT CN RCTVKENGU cells precisely. The DPI consists of two native promo -t ers that are concurrently activated by two independent transcription factors. Each cancer-sensing promoter produces a different fusion protein in proportion to its 2T QVGKP activity, and these two proteins assemble together as a chimeric transcription factor. This transcription factor then activates a synthetic promoter that controls expres- XRTsRE  RT QVGKP '( α sion of the herpes simplex virus type 1 thymidine kinase (TK1), which is cytotoxic in the presence of nucleotide JGNRGT '( α analogues, such as ganciclovir (FIG. 6a). The DPI could be optimized for a specific cancer cell type by using different 2T QFWEGTEGNN 4GEGKXGTEGNN combinations of input promoters and effector genes, as well as by modulating the assembly efficiency and half life 0CVWT G4G XKGYU ^)GPGVKEU NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 29 © 2011 Macmillan Publishers Limited. All rights reserved R# R# TI 6C :[N5 0CJ4 .WZ4 REVIEWS C +PRWV +PRWV 1WVRWV $GPKIP $GPKIP 0QPG $GPKIP /CNKIPCPV 0QPG /CNKIPCPV $GPKIP 0QPG /CNKIPCPV /CNKIPCPV %GNNFGCVJ FQE5 X2 R# /CNKIPCPE[ &QE5 &QE5 %QJ %QJ /CNKIPCPE[ )CN $& )CN $& 1 2 6- R# )CN OKP ICN EQJ R# $& 6- )CPEKENQXKT D *KIJOK40#UGPUQT OK40# .QYOK40#UGPUQT OK40# 2 TV6 # OK40# R# %/8 VC GV 2 OK40# R# 1WVRWV OK40# R# NCE+ 2 64' VC GV VC GV NCE1 1WVRWV R# %#) %GNNURGEKȮEOK40# OK40# OK40# OK40# OK40# OK40# OK40# GZRT GUUKQPRCVVGTP JKIJ JKIJ NQY NQY NQY NQY %GNNENCUUKȮGTEKT EWKV *KIJUGPUQT *KIJUGPUQT .QYUGPUQT .QYUGPUQT .QYUGPUQT .QYUGPUQT 1WVRWV $#: 5[UVGOT GURQPUG Figure 6 | Synthetic genetic cancer classifiers. a | A transformation-sensing cancer kill switch can consist of a 0CVWT G4G XKGYU ^)GPGVKEU 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 –Coh2). When they are simultaneously BD expressed, both proteins dimerize to form a synthetic transcription factor that binds Gal4 operator sites (O ), Gal4 induces downstream minimal promoters (P ) and triggers expression of the herpes simplex virus type 1 thymidine min 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. 30 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved TI TI TI REVIEWS of the chimeric transactivator components. So far, a set of the protein to the aptamers controls splicing of the mini - three promoters have been characterized in detail, but the gene; when the central exon is spliced out, the transgene DPI design may accommodate other suitable promoters. is expressed at high levels, and when it remains unspliced, The recently developed ‘cell-type classifier’ is concep - the transgene is expressed at low levels. Such a device was tually similar to the DPI, as it can also be programmed configured to sense subunits of nuclear factor kappa B to destroy cells that express a specific set of neoplastic (NFκB) or β-catenin (which are neoplastic markers) and markers . The cell-type classifier combines transcri- p to express the herpes simplex virus thymidine kinase. tion and translation control components in a single Thymidine kinase renders cells susceptible to ganciclo - scalable synthetic circuit that senses expression levels vir, so this device only operated as a cancer kill switch in of a set of (currently up to six) endogenous microRNAs the presence of the cancer markers and gangcicolvir . (miRNAs); it triggers an apoptosis-inducing response The modular configuration of the RNA sensor–actuator only if those levels match a preset profile. The cell-typede vice allows it to be tailored to different intracellular classifier combines sensor modules for the detection of proteins and even to multi-protein input using specific highly and lowly expressed miRNA (FIG.  s 6b). For clinical intronic aptamers. Also, responsiveness and performance implementation, both the DPI and the cell-type classifier can be tuned by placing the aptamers at different loc- a must either be delivered to the cancer tissue, or they must tions within the introns. The availability of compact RNA provide a fail-safe mechanism that constantly eliminates sensor–actuators that are easy to design and to alter and transforming cells from engineered tissue implants. that control transgene expression in response to intracel - lular levels of key proteins may also improve the ability to Other emerging tools for biomedicine link metabolic disease states with gene-based therapeutic Novel treatment strategies will require new technologies interventions. to sense and control disease. Synthetic biologists have designed new devices that could sense key physiological Optogenetic devices in blood glucose homeostasis. Light activities and have found new ways to dose therapeutic is becoming increasingly popular as a traceless, molecule- interventions precisely in response to external physical free input signal for triggering transgene expression in cues. Such synthetic devices could have wide-ranging living systems. Bacteria have been engineered to record 149–151 biomedical applications. projected images with gigapixel resolution and to adjust transgene expression in response to multi- RNA controllers of cell proliferation. Thus far, synthetic chromatic input , and now genetic light switches have control devices that are designed to interface with host also been designed to control gene expression and metabolism and to reprogram cellular behaviour have shape of mammalian cells . largely been limited to heterologous transcription fac - Devices that convert light pulses into transcription tors. RNA controllers may represent an alternative. They may foster novel therapeutic opportunities in future are straightforward to design and can be integrated into gene- and cell-based therapies and may improve the ma- n a single expression unit containing sensors (aptamers), ufacturing of difficult-to-produce protein pharmaceut- i gene-regulatory components (ribozymes) and effector cals, such as cancer therapeutics. An illustrative example 39,143,144 transgenes . The inherent modularity and com - is light-controlled expression of the glucagon-like p-ep patibility of RNA-based control components enables tide 1 (GLP1), which is a promising drug candidate for them to be independently optimized or exchanged. the treatment of type 2 diabetes (FIG. 7a). An optoge- For example, an RNA control device consisting of a netic device that enables light-triggered gene expres - drug-responsive aptamer linked to a ribozyme in the sion in human cells was designed. This involves ectopic 3′ untranslated region (UTR) of a cytokine expression expression of melanopsin in human embryonic kidney unit enabled trigger-inducible inactivation of ribozyme- cells and functional rewiring of signalling downstream mediated transcript cleavage and full transgene expr- es of melanopsin; the cascade integrates blue-light-pulse- sion in the presence of the input signal . This synthetic triggered photoreception and produces a reversible and RNA control device was applied to control proliferation sustained intensity-dependent transcription response. Melanopsin of engineered primary human T cells and enabled ext- er When placed in hollow fibre containers and implanted A vitamin-A-dependent, G-protein-coupled receptor nal control of the expansion of transgenic T cells that into mice, transgene expression in the engineered light- that is expressed in are implanted into mice. Synthetic RNA control devices sensitive cells could be controlled remotely by an o -pti intrinsically photosensitive could provide the advance that is necessary to enable cal fibre . Illuminating mice that carried subcutaneous retinal ganglion cells T cell therapy ; by contrast, state-of-the-art, trigger- implants of microencapsulated photo-responsive cells Prosthetic networks inducible expansion of engineered T cells using chimeric also enabled transdermal control of transgene expr-es Networks that replace existing antigen receptors has only led to moderate proliferation sion and of corresponding protein levels in the blood of cellular functionality that 146,147 and poor survival of T cells in clinical trials . treated animals. This system was able to attenuate glycae - is ill-driven or out of order. Another use for synthetic RNA is the design of pro- mic excursions and to control glucose homeostasis in a They represent molecular grammable sensor–actuator devices that convert levels mouse model of human type 2 diabetes . prostheses for non-functional cellular activity; they differ of an intracellular protein into a discrete high or low from other synthetic networks 148 transgene-expression state . The RNA devices consist Prosthetic networks. Prosthetic networks are synthetic that add useful functionality of a three-exon, two-intron minigene followed by the sensor–effector devices that act as molecular prostheses. but do not replace transgene. The introns contain protein-sensing aptamers, When engineered into cells and functionally connected non-functional cellular networks. and the central exon includes a stop codon. Binding of to host metabolism, they sense, monitor and score NATUR E R EVIEWS | GENETICS VOLUME 13 | JANUARY 2012 | 31 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS Figure 7 | Advanced therapeutic and prosthetic networks. a | Light-triggered %C transcription control of blood glucose homeostasis. The synthetic phototransduction $NWG cascade consists of rewired melanopsin and nuclear factor of activated T cells (NFAT) NKIJV 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 642% /GNCPQRUKP 2+ (GAQ), phospholipase C (PLC) and phosphokinase C (PKC) and triggers Ca ion influx via transient receptor potential channels (TRPCs) and possibly also from the 2+ endoplasmic reticulum. This Ca ion surge activates calmodulin (CaM) to calcineurin )#3 (CaN), which dephosphorylates NFAT. NFAT then translocates into the nucleus, where it binds to specific promoters (P ) and coordinates transgene transcription. When NFAT 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. 2.% 2-% b | Prosthetic network for the treatment of tumour lysis syndrome and gout. %C/ 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 (hucO ). This operator %C0 controls expression of secretion-engineered urate oxidase (smUOX), so smUOX is 0( expressed when urate concentration reaches pathological levels. smUOX mediates %[VQRNCUO 'PFQRNCUOKE T GVKEWNWO conversion of urate into allantoin. Expression of smUOX stops when urate 0WENGWU concentration reaches oxidative-stress-protective urate levels. pA, poly(A) tail. Part a is modified, with permission, from REF. 65 © (2011) American Academy for the 0( Advancement of Science. ).2 R# 0( $NQQFXGUUGN 1* 7T CVG #NNCPVQKP *1 0* 0 1* 74# 6 1* 5GPUQTsGȭGEVQTEGNN *1 0* 0 1* CVG 7T 2 -4#$ JWE4 R# 2 JWE1 R# UO71 : 58 58 6 KOG 0CVWT G4G XKGYU ^)GPGVKEU disease-relevant metabolites, process off-level concen - concentrations and restores urate homeostasis by con- trations and coordinate adjusted diagnostic, preventive trolled expression of a urate oxidase — which reduces or therapeutic responses in a seamless, automatic and excessive urate concentration while preserving levels self-sufficient manner. that are suitable for radical scavenging — could repr -e An example of the use of a prosthetic network is sent a treatment strategy for hyperuricaemic disorders . the sensing of metabolites to improve control of urate In brief, human cells that contain such a prosthetic net - homeostasis (FIG.  7b). Moderate levels of uric acid, work have recently been designed by combining: the uric which scavenges radicals, are deemed to be beneficial. acid sensor HucR , which manages oxidative stress pro- However, a transient surge in uric acid that is released by tection in Deinococcus radiodurans; the human uric acid dying cells during cancer therapy leads to tumour lysis transporter URAT1 (also known as SLC22A12), which syndrome, and chronic hyperuricaemia can result in increases the intracellular uric acid levels and thus the gout. Humans are particularly sensitive to imbalances of sensitivity of the prosthetic circuit; and a secretion- urate homeostasis because they lack uricolytic activity. A engineered urate oxidase (smUOX) that is clinicall y prosthetic network that constantly monitors blood urate licensed for the treatment of the tumour lysis syndrome . 32 | JANUARY 2012 | VOLUME 13 w w w.nature.com/reviews/genetics © 2011 Macmillan Publishers Limited. All rights reserved #6 #6 #6 2JQURJCVG REVIEWS 94,98 The prosthetic uric-acid-responsive expression network may imminently develop into shorter drug discovery (UREX) was able to sense uric acid concentrations pre - and drug development timelines, increased precision 112,114 cisely and to activate secretion of smUOX when the uric of drug delivery and production of new and more 104–109 acid concentration was at pathologic levels. Secretion affordable medicines . Ultimately, sophisticated thera - of smUOX is stopped as soon as the uric acid concen- peutic sensor–effector devices that can sense disturbances, tration has returned to the homeostasis level. This was seek out pathological conditions and restore function are impressively demonstrated when UREX-transgenic cells on the roadmap. Such therapeutic networks that connect were implanted into urate-oxidase-deficient mice, which diagnostic input with therapeutic output may provide develop acute hyperuricaemia with symptoms that are all-in-one diagnostic, preventive and therapeutic sol-u similar to human gout. UREX was able to degrade urate tions in future gene- and cell-based therapies. Matching and restore urate homeostasis in the blood, resulting in diagnostic outcome with high-end therapies has recently the dissolution of uric acid crystal deposits in the kidney become a focus of the pharmaceutical industry, which of treated animals . Its straightforward design may allow has declared personalized medicine as the treatment UREX to serve as a blueprint for the assembly of other strategy of the future. Tools that will have a tremendous prosthetic networks that sense metabolic disturbances impact in future biomedical applications include: using and circulating pathologic metabolites. light-activated triggers to bring about a precise therapeu - tic response in cells , programming bacteria to seek and 128,132,133 An artificial insemination device. Artificial insemi- destroy cancer cells and using synthetic circuitry 64,65 nation is standard practice to facilitate both human to keep crucial metabolites at homeostatic levels , to reproduction and livestock breeding. Because of broad manage disease-controlled expansion or to eliminate 31,148,159 variations in oestrus expression and ovulation timing, specific cell populations . Recent work has shown coordinating sperm delivery with female oestrus is still a that this is, in principle, possible and that some devices major challenge. Ovulation is triggered at a specific time are working as expected and are producing a therapeutic 64,65 when the pituitary gland releases luteinizing hormone, impact in animal models of human diseases . Implants which binds to the luteinizing hormone receptor (LHR) consisting of engineered microencapsulated cells repr -e and coordinates the release of the oocyte. By integra- t sent a way of introducing prosthetic networks with a pre- ing synthetic signalling cascades with advanced biom-a defined function instead of directly targeting the host cells terials, Kemmer and colleagues designed an artificial with the genetic material. Although implants containing insemination device that coordinates sperm delivery with cells with engineered prosthetic networks are certainly the oestrus control. The artificial insemination device con - most promising way forward, they will limit biomedical sists of cellulose sulphate capsules containing bull sperma pplications to extracellular disease metabolites that can 63,156,157 and sensor cells . The sensor cells are engineered to be therapeutically addressed through the vascular system. express LHR constitutively so that when it is activated, it However, there is still a long way to go until synthetic- triggers expression of cellulase that can be secreted. After biology-based biomedical devices will be a clinical reality. implantation into the cow’s uterus, the sensor cell line Placing therapeutic circuits in specific cells of a patient constantly monitors the animal’s luteinizing hormone and making sure that there will be no interference with levels, and the oestrus-triggered surge in luteinizing ho - r human metabolism are the most important challenges. mone levels leads to the production of secreted cellulase, Th erefore, clinical use of synthetic-biology-based devices which degrades the implanted capsule and results in the and therapeutic scenarios will face the same scientific, timely delivery of the sperm and successful conception. ethical and legal issues as any gene- and cell-based th - er Fine tuning of the designer cascade could enable its use apy, but they may offer more complex control dynamics in other species, including in humans. and are therefore expected to have a higher therapeutic impact. 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