Mammalian designer cells: Engineering principles and biomedical applications.

Biotechnology Journal

Biotechnol. J. 2015, 10, 1005–1018

DOI 10.1002/biot.201400642

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Review

Mammalian designer cells: Engineering principles and biomedical applications Mingqi Xie1 and Martin Fussenegger1,2 1 Department 2 Faculty

of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland of Life Science, University of Basel, Basel, Switzerland

Biotechnology is a widely interdisciplinary field focusing on the use of living cells or organisms to solve established problems in medicine, food production and agriculture. Synthetic biology, the science of engineering complex biological systems that do not exist in nature, continues to provide the biotechnology industry with tools, technologies and intellectual property leading to improved cellular performance. One key aspect of synthetic biology is the engineering of deliberately reprogrammed designer cells whose behavior can be controlled over time and space. This review discusses the most commonly used techniques to engineer mammalian designer cells; while control elements acting on the transcriptional and translational levels of target gene expression determine the kinetic and dynamic profiles, coupling them to a variety of extracellular stimuli permits their remote control with user-defined trigger signals. Designer mammalian cells with novel or improved biological functions not only directly improve the production efficiency during biopharmaceutical manufacturing but also open the door for cell-based treatment strategies in molecular and translational medicine. In the future, the rational combination of multiple sets of designer cells could permit the construction and regulation of higher-order systems with increased complexity, thereby enabling the molecular reprogramming of tissues, organisms or even populations with highest precision.

Received 24 FEB 2015 Revised 02 APR 2015 Accepted 08 MAY 2015

Keywords: Biocomputer · Cell therapy · Gene regulation · Mammalian production · Trigger-inducible designer cell

1 Introduction Much of what we recognize as life results from the remarkable capacity of living cells to continuously measure, process and store environmental information with

Correspondence: Prof. Martin Fussenegger, Mattenstrasse 26, CH-4058 Basel, Switzerland E-mail: [email protected] Abbreviations: BCI, brain-computer interface; cAMP, cyclic adenosine monophosphate; CAR, chimeric antigen receptor; cGMP, cyclic guanosine monophosphate; CRISPR,clustered regulatory interspaced short palindromic repeat; DAG, diacylglycerol; DSB, double strand break; dsDNA, double stranded DNA; DTA, diphtheria toxin α-chain; E2F4, activation domain of the human E2F transcription factor 4; FDA, US Food and Drug Administration; GFP, green fluorescent protein; GPCR, G-protein coupled receptor; gRNA, guide RNA; HDR, homology directed repair; HHR, hammerhead ribozyme; HSV-TK, thymidine kinase from herpes simplex virus; IP3,inositol

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high precision [1–3]. Additionally, the production capacity and accuracy of biological macromolecules from a living cell remains unrivaled by any type of man-made machines or factories [4, 5]. Biotechnology has always profited from this natural resource. Pioneering work

trisphosphate; KRAB, human kox1-derived Krueppel-associated box protein; lacZ, gene in the lac operon of Escherichia coli encoding β-galactosidase; loxP, locus of crossover (x) in P1; miRNA, micro RNA; NHEJ, nonhomologous end-joining; NIR, near-infrared (light); NO, nitric oxide; Pol III, RNA polymerase III; RBP, RNA-binding protein; RISC, RNA-induced silencing complex; scFv, single chain variable fragment; shRNA, small hairpin RNA; siRNA, small interfering RNA; STING, stimulator of interferon genes; TALE, Xanthomonas-derived transcription activator-like effectors; TCR, T-cell receptor; TNF-α, tumor necrosis factor α; UREX, uric acid responsive transgene expression; UTR, un-translated region of an mRNA molecule; VP16, herpes simplex derived virion protein 16; VP64, four tandem-repeats of a minimal VP16 motif; ZF, zinc-finger containing factors.

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Biotechnol. J. 2015, 10, 1005–1018


involved biotransformation reactions using unique organisms to convert biomass and other feedstocks into desired products such as fuels, food and antibiotics [4, 6]. The recombinant DNA revolution in the mid-1970s further allowed biotechnologists to use engineered microbial cells for the production of heterologous products, thereby convincingly outperforming chemical factories in terms of catalytic activity, product specificity and economic requirements for price, temperature and space [4, 5, 7–10]. Today, industrial demands for biotechnological products have dramatically shifted from small-molecule chemicals and biofuels towards complex therapeutic proteins such as antibodies, cytokines, growth factors or peptide hormones [7, 10–13]. The biological activity of these proteins often depends on complex structures resulting from unique posttranslational modifications such as glycosylation, truncation and secretion – processes that are available in mammalian host cells at a high standard [6, 10, 14]. Therefore, despite the drastically higher maintenance costs than bacterial or fungal production systems, investment in and insight into mammalian cell technologies will be indispensable for the generation of present and future biologics [11]. Synthetic biology, a research field that interprets biology as an engineering discipline, has undergone dramatic growth throughout the past decade and is poised to revolutionize biotechnology and medicine [4, 15, 16]. Research in synthetic biology continuously produces novel autonomous biological systems such as artificial cellular units that can be precisely controlled and re-used in an interdisciplinary and problem-oriented manner [10, 13, 14, 16]. The core components of such synthetic biological systems are designer cells, i.e. artificial cellular units designed by humans to behave in a predictable way [3]. By considering cells as robust biological computers that reliably translate an external signal into protein production, the engineering of designer cells is based on creating independent control elements to enable triggerinducible target gene activities [17–20]. In this review, we discuss various gene regulatory tools that can be used or interconnected to enable precise programming of mammalian designer cells. Section 2 illustrates a multitude of control elements that act at the transcriptional, translational and genomic levels of mammalian gene expression. Judicious combinations of those control elements enable the creation of gene expression profiles with temporal and dynamic precision. Section 3 shows recent strategies that enable remote control of designer cells with user-defined trigger signals differing in their spatial distance to the transcription machinery. For instance, synthetic optogenetic [21–23] and cybernetic control elements [24] represent the farthest accession points from which gene expression of a mammalian cell can be controlled by monochromatic light or human mind, respectively. The rational assembly of gene control

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tools discussed in this work enables efficient, reliable and predictable engineering of designer mammalian cells with novel and improved functions that are tailored to solve important problems in biotechnology and medicine [8, 9, 16, 18, 25, 26].

2 Control of mammalian gene expression in the intracellular compartment 2.1 Gene regulation at the transcriptional level Gene regulation at the transcriptional level is commonly achieved with transcription factors, nuclear proteins that bind to a specific DNA-sequence within a target promoter region, whereupon gene transcription is either induced or repressed [27]. The first building blocks in mammalian synthetic biology were synthetic transregulators engineered on the basis of prokaryotic transcriptional repressors that are under allosteric control of their cognate ligands [14]. Most of these prokaryotic transcription factors (pTFs) are homodimeric proteins belonging to the TetR, GntR and MarR families of transcriptional repressors, and consist of a C-terminal ligand binding domain (LBD) and an N-terminal DNA-binding domain (DBD) [28–30]. In the absence of a trigger molecule, the DBD of a transcription factor binds to a cognate DNA operator sequence with high specificity and affinity [18, 27]. However, binding of a pTF-specific ligand to the LBD triggers a change in the protein conformation, thereby rendering the DBD incompatible with DNA-binding [14, 28]. The simple binary character of this protein-DNA interaction has been widely used in synthetic biology research to engineer tight, robust and orthogonal transgene switches in mammalian cells [30–32].

2.1.1 Synthetic transactivators, transsilencers and transrepressors Synthetic mammalian transactivation systems are generic constructs in which a pTF is fused to a transcriptional activator domain (such as VP16, p65 or E2F4) and activates transcription upon binding to minimal eukaryotic promoters engineered to contain pTF-specific operator sequences [20, 33–35]. Likewise, fusion of the same transcription factors to transcriptional repressor domains (such as KRAB) forms synthetic mammalian transsilencers that repress target gene transcription when bound to cognate operator sites placed upstream or downstream of a mammalian constitutive promoter, by steric means and/or by chromatin-based epigenetic silencing [3, 20, 36]. Whereas a mammalian transactivation system can be used to build an off-type switch for gene expression in mammalian cells (i.e. the presence of a ligand shuts off gene transcription), the ligand-dependent binding of a transsilencer or a transrepressor (i.e. a pTF without any fusion domain) to its cognate promoter


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Figure 1. Synthetic binary gene switches. (A) Generic construction of trigger-inducible transregulator systems. Binding of a pTF-specific ligand abolishes the pTF’s ability to bind to its cognate DNA sequence (OTF, red). Depending on the effector domain fused to pTF (AD, activation domain [green]; RD, repression domain [black]), this interaction leads to a ligand-triggered off/on-switch in mammalian cells. (B) Synthetic transregulators with programmable DNA-specificity. The DNA-binding domain (DBD) is composed of a ZF-, TALE-, or dCas9-protein and can be engineered to bind almost any DNA sequence of choice (purple). Depending on the effector domain (ED) fused to the DBD, targeted genes in either episomal vectors or host chromosomal regions can be activated or silenced. (C) Synthetic RNA switch regulating translational initiation. Incorporation of RBP-binding sites into the 5’-UTR of the target gene mRNA allows the initiation of translation to be regulated by RBP. Protein expression occurs only in the absence of active RBP in the cytoplasm. (D) Synthetic riboswitches optimized for controlling translation in mammalian cells. Ribozyme structures (RS, blue) engineered into the 3’-UTR of target mRNAs form a catalytically active secondary structure leading to mRNA cleavage and inhibition of translation. The presence of a trigger peptide abolishes the catalytic structure of the ribozyme and restores translation-compatible mRNA molecules. Note: UGA represents a generic stop codon. (E) Translational inhibition by RNA interference. Short regulatory RNA molecules (shRNA, siRNA or miRNA) bind and knock down a target mRNA molecule that contains a complementary nucleotide sequence after formation of a catalytic complex with host endonucleases (RISC). (F) Use of site-specific recombinases to regulate gene expression. Capitalizing on programmable recombination events (excision, inversion), the activity of a recombinase can be directed to either reconstitute or disrupt a functional transcription unit. In the case of the Cre recombinase, if the target-site pair is placed in parallel orientations, the flanked DNA sequence is excised. If the target sites have opposing orientations, the flanked DNA segment is inverted. Abbreviations: goi, gene of interest; pTF, prokaryotic transcription factor; Pconst., constitutive promoter; Pmin., minimal promoter; POI, protein of interest.


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generates an on-type switch following a classical derepression mechanism [6] (Fig. 1A). Because bacteria are often exposed to a variety of harsh environmental stimuli and hence require robust transcriptional response systems for survival, they have evolved a large number of transcriptional regulators (pTFs) that respond to diverse signals [30]. Therefore, using a generic approach to engineer synthetic transactivators, transrepressors and transsilencers, it became possible to build a variety of binary on/off-gene switches in mammalian cells, using different pTF-specific trigger molecules (Fig. 1A). A key advantage of converting bacterial transcription factors into synthetic transregulators is their orthogonality to the eukaryotic genome and to each other: the DNA-binding domain of a pTF remains specific to its cognate DNA sequence [31]. Furthermore, the functional scope of prokaryotic transcription factors can be systematically expanded by designing novel characteristics such as reversed allosteric control [37–40], tailored sequence specificity [41] or ligand affinity [42, 43]. Today, the availability of a huge portfolio of triggerinducible synthetic transgene switches has led to many advances in medical and scientific research [20, 32] (see [3] for a comprehensive list). First, analogous to the personal computer, in which digital computing results from the precise combination of binary 0/1 units, the rational assembly of orthogonal gene switches allows the engineering of apparently digital features at a biological level [2, 3, 44, 45]. For example, the combination of different synthetic gene switches has enabled the creation of complex gene expression profiles in a single cell, reminiscent of toggle switches [46], logic gates [47], band-pass filters [48], memory devices [49, 50] and changeover relays [20]. Recently, a unique gene switch has been engineered that for the first time broke the binary restriction of transcriptional regulators. Based on the ability of the Comamonas testosteroni-derived repressor CbaR to accept multiple small-molecule ligands that either abolish or enhance CbaR’s DNA binding conformation, Xie et al. engineered a dual-input mammalian gene switch that is under the antagonistic control of the FDA-approved food additives benzoate and vanillate [51]. Benzoate activates gene expression from the KRAB-CbaR transsilencer-regulated promoter, and the addition of vanillate would terminate this transcriptional event at any user-defined point in time. Such a gene regulation profile might be of great interest for industrial production processes in which a particular batch-reaction can be flexibly initiated, held and resumed with biologically inert and nontoxic trigger compounds. Second, synthetic transgene switches can further be applied as powerful drug-screening tools. For example, Weber et al. engineered a synthetic mammalian gene switch based on the EthR repressor, an orphan Mycobacterium tuberculosis-derived transcription factor that represses genes whose expression would confer vulnera-

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bility of the bacterium to the prodrug ethionamide [52]. Operation of this gene switch as a cell-based screening platform in mammalian cells has led to the identification of a FDA-approved strawberry flavor 2-phenylethylbutyrate as a ligand to trigger the release of EthR from its cognate promoter [53]. Applying 2-phenylethyl-butyrate to a pathogenic strain of M. tuberculosis dramatically increased this bacterium’s susceptibility to ethionamide. This work highlights the medical impact of mammalian synthetic biology, as many pharmaceutical companies have been motivated to adopt similar adjuvant treatment strategies to restore the efficacy of antibiotics in multidrug-resistant bacteria [54]. Third, a synthetic transregulator system can be engineered into a compact self-sufficient prosthetic network if its trigger compound represents a systemic disease marker [55, 56]. For example, the UREX system engineered by Kemmer et al. relied on the synthetic transsilencer KRABHucR’s ability to derepress transgene expression upon binding to uric acid at concentrations that were typical in diseases such as tumor lysis syndrome and gout [56, 57]. By judiciously choosing a secretion-engineered mammalian urate oxidase (smUOX) as the system’s output transgene, UREX-transgenic mammalian cells were able to restore blood urate homeostasis in a self-sufficient manner when implanted into a mouse model of acute hyperuricemia, in which smUOX converted excessive uric acid into the renally secretable compound allantoin [57]. In a new generation of transcriptional regulators, the modularized blueprint of designing synthetic transregulators is retained, but pTF is substituted with a ZF-, TALEor dCas9 domain because these proteins can be engineered to bind a DNA sequence of choice with customizable affinity [27, 32, 58] (Fig. 1B). In general, synthetic transcriptional regulators containing ZF-, TALE- or dCas9-derived DNA-binding domains perform similarly to their counterparts consisting of prokaryotic transcription factors [18]. However, the lack of a trigger-inducible control element to regulate their instant activity often requires their expression from an upstream pTF-regulated promoter [30]. Nonetheless, the programmability of the cognate DNA sequence enables these transcriptional regulators to regulate the expression of both transgenes and endogenous genes, whereas pTF-derived transregulators remain restricted to episome-derived transgene control [59]. Indeed, fusion of a nonspecific nuclease to a site-specific ZF/TALE-domain can recruit the enzyme onto a chromosomal region of interest, where it can modify the genetic code of the host cell via a mechanism discussed in section 2.3.2.

2.1.2 Split transcription factors The modularity of synthetic transregulators is further underscored by the finding that the DNA-binding domain and the effector domain do not always require physical



Biotechnol. J. 2015, 10, 1005–1018



Figure 2. Synthetic gene control devices with biocomputational capacity. (A) Synthetic two-hybrid devices. In a split transcription factor, the DNA-binding domain and the effector domain are expressed from different promoters (P1, P2) as separate proteins, each fused to a dimerization partner (DimA, DimB) of a protein complex. Only simultaneous expression of both split transcription factor components or inducible protein dimerization can reconstitute the transregulatory activity of the target promoter. (B) Synthetic three-input and logic-gate. Individual TALE-fragments (TALEN, TALEC) are incapable of activating gene expression from a cognate TALE-promoter. By integrating TALEN and TALEC into a modular protein-splicing machinery, full TALE can be reconstituted when IntA, IntB and IntC form a catalytically active structure for intein excision. (C) Genetic DPDT-changeover relay. Synthetic multipartite transactivators (pTFA-pTFB-pTFC-VP16) can be targeted to different cognate promoters with multiple pTF-specific input signals. DPDT relay switches are electromagnetically operated mechanical switches that control one or several electric circuits by a fully isolated low-power signal. Asymmetries in the binding affinity of a bipartite transactivator’s individual pTF-moieties lead to the activation of only one cognate promoter (Promoter A, circuit 1) at low protein concentrations. Trigger-inducible promoter switching allows the reversible activation of another cognate promoter (Promoter B, circuit 2). (D) Synthetic control device regulating alternative RNA splicing. Protein-binding RNA-motifs placed next to regulatory sequences (red) in the primary transcript regulate the splicing pattern of a gene. In the absence of protein binding, isoform A is translated. Binding of nuclear proteins to key intronic regions promotes alternative RNA splicing events such as exon exclusion, resulting in the generation and translation of isoform B.

binding to drive transgene expression. Capitalizing on the red-light-induced dimerization of the proteins PhyB and PIF6, Müller et al. constructed a trigger-inducible split transcription factor consisting of two separate fusion proteins: PIF6 was fused to the tetracycline repressor TetR, and PhyB was fused to VP16 [60]. Red-light-induced PIF6/PhyB-dimerization enabled the final reconstitution of the functional synthetic transactivator TetR-VP16, thereby activating gene expression from a TetR-specific target promoter [13]. This type of two-hybrid technology (Fig. 2A) is an excellent platform for high-throughput screening studies of putative intracellular protein-protein interactions or synchronized transcriptional activities [2]. For example, Nissim et al. engineered a dual-promoter integrator in which each of the two mutually independent


promoters drives expression of one split component of the transcription factor [61]. By placing these split transcription factor components under the control of endogenous cancer-related constitutive promoters, this dual-promoter integrator could be used to gain insight into the development of malignancy in mammalian cells [61]. Similarly, Lienert et al. used a split-intein proteinsplicing strategy of TALE fragments to build synthetic AND logic gates in mammalian cells [59]. The TALE fragments expressed from each individual promoter were incapable of activating transcription on their own. Only if both fragments were present simultaneously was an active TALE protein reconstituted via a protein splicing mechanism [62], which in turn activates transcription from cognate promoters. This design strategy has also

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been expanded to engineer a three-input AND logic gate, thereby highlighting the biocomputational capacity of transcription regulatory devices (Fig. 2B) [59].

2.1.3 Multipartite transcription factors Instead of separating the protein domains of transcriptional regulators, Folcher et al. created a novel type of synthetic transregulator by daisy-chaining multiple prokaryotic transcription factors to produce synthetic multipartite transactivators (pTFA-pTFB-VP16, pTFA-pTFB-pTFCVP16) whose targeting to different cognate promoters is determined by multiple pTF-specific input signals [20]. Asymmetries in the binding affinity of the individual pTFmoieties to their respective promoters resulted in gene expression characteristics reminiscent of double-pole double-throw (DPDT) relay switches, a unique control topology that requires the composite transactivator to move from one synthetic promoter to the next one in a trigger-inducible but reversible manner, thereby enabling the sequential activation of different sets of transgenes for a preset duration (Fig. 2C).

63]. Recently, Ausländer et al. unraveled the design principles of riboswitches (trigger-inducible ribozymes) in mammalian cells by systematically expanding and generalizing a conserved HHR architecture [63]. In the absence of trigger compounds, a designer riboswitch engineered into the 3’-UTR of a target mRNA transcript forms a catalytically active secondary structure that leads to continuous cleavage of the mRNA molecule and prevents translation. However, the presence of a trigger peptide abolishes the self-cleavage ability of this ribozyme, thereby restoring stabilized mRNA molecules for ribosome recognition and initiation of translation (Fig. 1D). Furthermore, the authors identified not only universal sequence-structure correlations that are critical for correct switching performances but also flexible regions within the ribozyme molecule that are tolerant to further modification and engineering. It has also been shown that all riboswitches designed from this scheme were fully compatible with synthetic transcription-based gene circuits; this work may serve as a blueprint for the rational design of triggerinduced RNA switches to control transgene translation in mammalian cells.

2.2 Gene regulation at the translational level 2.2.3 Control of alternative RNA splicing In recent years, RNA-centered switches have gained increased attention owing to their small size, modularity, sequence specificity and predictable structure-function relationship [2, 17, 63]. Compared with transcriptional regulation systems, RNA-based systems are relatively fast acting and hence represent invaluable tools to finetune time-dependent gene expression dynamics [10, 59, 63].

2.2.1 Translational inhibition The simplest way to control translational initiation adopts a steric hindrance mechanism similar to the inhibition of transcription by synthetic transrepressors. RBPs, such as the archaeal ribosomal protein L7Ae, bind to cognate RNA motifs with high affinity and specificity [64]. Incorporation of such RNA motifs into the 5’-UTR of the mRNA transcript of a target gene allows translation initiation to be regulated by the activity of L7Ae [65]. In the absence of L7Ae, translation of the target mRNA occurs normally to induce constitutive protein expression. However, trigger-induced transcription of L7Ae interrupts the translation event by the binding of the expressed protein to the target mRNA, thus blocking the ribosomal machinery (Fig. 1C).

2.2.2 Riboswitches Ribozymes are small catalytic RNA structures with sequence-specific RNA cleavage activity [66]. In mammalian cells, the ribozyme most widely used to regulate gene expression is the HHR, a self-cleaving ribozyme that degrades the RNA molecule it is residing in, thus irreversibly inhibiting the translation of targeted mRNAs [18,

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In eukaryotes, a unique post-transcriptional regulatory mechanism enables the generation of different protein isoforms from a same primary mRNA transcript. Depending on which RNA segments are marked as exons or introns by regulatory proteins, particular segments on the primary RNA construct can be selectively skipped and recombined to generate different mRNA molecules prior to nuclear export and translation initiation [67]. In engineering synthetic gene control elements to regulate the splicing pattern of a gene, protein-binding RNA-motifs placed next to regulatory sequences in the primary transcript permit trigger-inducible exon exclusion [68] (Fig. 2D). For example, Culler et al. engineered a highly sophisticated sensor-effector control device that linked endogenous disease markers to programmed cell death [13, 69]. An RNA aptamer designed to recognize the p50 and p65 subunits of the endogenous transcription factor NF-κB was inserted into key intronic locations in the primary transcript of an apoptotic gene HSV-TK [69]. In the absence of p50 and p65, a nonapoptotic isoform of HSV-TK is translated. However, elevated extracellular levels of inflammatory cytokines such as TNF-α activate the NF-κB pathway in most mammalian cells, leading to p50 and p65 translocation into the nucleus, where they bind to the aptamer and eventually induce cell death by promoting the generation of the pro-apoptotic isoform of HSV-TK.

2.2.4 RNA interference RNA interference (RNAi) is a pathway found in many eukaryotic cells, and it plays a vital role in the immune



Biotechnol. J. 2015, 10, 1005–1018


response to viruses or other foreign genetic material [70]. Different types of short regulatory RNA molecules (shRNA, siRNA or miRNA) that consist of approximately 20 nucleotides can bind and knock down a target mRNA molecule containing a complementary nucleotide sequence. Depending on the degree of sequence complementarity with the short RNAs, the targeted mRNA can undergo either cleavage or translational repression [71] (Fig. 1E). However, the success of RNAi-based gene switches is often limited by the difficulty of engineering and expressing the short regulatory RNA fragments in mammalian cells [58]. To facilitate the use of this technique, Leisner et al. recently reported a comprehensive protocol for miRNA design, construction and expression [72]. Deans et al. convincingly described how RNA-targeted systems can achieve unprecedented control capacity when coupled to transcriptional regulation [73]. By combining the repressing contributions of a synthetic transrepressor’s steric hindrance with the shRNA-induced degradation of a target mRNA, the authors engineered a trigger-induced gene switch termed LTRi (LacI-TetRRNAi) that allowed for tight, tunable and reversible control over transgene expression [35]. The tightness of this gene switch is of special note as cells fully survived the off-state when the gene switch was regulating the expression of DTA, a protein that is so toxic that one single molecule would be sufficient to kill the cell [73]. Additionally, miRNAs are excellent selective cues for tissue and disease-state identification [74]. For example, Xie et al. have engineered a synthetic multi-input gene circuit that permitted the specific recognition and destruction of cancer cells [13, 56, 75]. This device was capable of calculating the oncogenic state of a human cell by continuously aligning its current level of endogenous miRNA markers with a predefined cancer-specific signature, which required the expression level of one set of miRNAs to be high and the expression level of another set to be simultaneously low. Complementary target sequences of the endogenous miRNAs were placed in the 3’-UTR of each transcript, coding for both the apoptotic effector protein hBax and the LTRi components that tightly regulate hBax expression [18]. The mRNA transcript responsible for hBax expression was engineered such that the absence of only one set of miRNA markers protected hBax from RNAi-mediated degradation. Meanwhile, the mRNA transcripts encoding the LTRi components were targeted for RNA interference by another set of miRNA markers. Consequently, the predefined high/low signature of a cancer cell is matched when the LTRi-encoding mRNA transcripts were degraded and the hBax transcript was protected, leading to self-managed apoptosis in cancer cells [75].



2.3 Gene regulation through DNA sequence editing Although control devices combining transcriptional and translational regulatory elements already enable gene expression regulation with highest levels in robustness [76], tightness [73] and biocomputational capacity [3, 65, 75], they are generally restricted to the processing of a fixed set of genetic raw material. In contrast, control elements that can tune the temporal availability or even existence of a particular DNA segment allow for a more drastic intervention and further augment the control capacity of gene expression in mammalian cells [77].

2.3.1 DNA inversion, deletion and insertion with recombinases Recombinases are enzymes that catalyze directionally sensitive DNA exchange reactions between short (30–40 bp) target site sequences, leading to either gene excision/insertion or fragment inversion [78]. Excellently reviewed in [79, 80], site-specific recombinases are commonly used in genetic engineering to modify a gene fragment in the DNA of living cells, resulting either in reconstitution of a functional transcription unit or in nonsense mutants that are incompatible with gene expression [18, 78] (Fig. 1F). The most commonly used tool for DNA manipulation is the P1 bacteriophage-derived Cre, an enzyme that mediates site-specific recombination reactions between a pair of direction-sensitive DNA sequences named loxP [81]. If the loxP pair is placed in the same orientation, the reaction produces excision of the DNA segment flanked by the loxP sequences [80]. In contrast, if the loxP elements have opposing orientations, the recombination results in inversion of the DNA segment [80] (Fig. 1F). Cre’s unique mechanism of “writing” particular information on target DNA sequences renders site-specific recombinase systems invaluable tools for the design of control elements that store and process genetic memory in single cells [77, 79, 82]. Indeed, incorporation of DNAinverting switches into mammalian gene-control devices has enabled the engineering of novel features, such as time-delayed output production or irreversible cellular genotypes [74]. In a classical transcriptional regulation setup, the transcription rate of a target gene is defined by the synthetic transregulator’s innate characteristics, such as its own transcription rate, the affinity for its cognate promoter or its protein stability. However, Lapique et al. used the DNA-inverting switch to engineer a synthetic time-delay control element that could flexibly tune the kinetic parameters of a transcriptional regulation device [77]. The coding region of the output sequence was initially placed in the transcriptionally inactive antisense orientation, thus permitting the transcription of this gene only after expression of the Cre recombinase, which even-

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Figure 3. Synthetic control devices that generate genetic memory. (A) Synthetic time-delay element based on Cre-mediated DNA-inversion. The time-consuming reconstitution of a transcription unit enables a modification of a synthetic transregulator’s fixed kinetic parameters. (B) Synthetic lineage tracing device used for genetic labeling. In stem cells, asymmetric cell division produces an identical daughter cell and a nonidentical progeny cell. In the stem cell, a cell-type specific promoter (PSp.) drives transcription of GFP and Cre-activated lacZ transcription. In the differentiated progeny cells, the absence of the stem cell-specific promoter leads to termination of GFP expression, while expression of lacZ remains active due to an inherited memory of Cre-recombination in the progenitor cell. The resulting blue/green pattern of the tissue allows for accurate interpretation of the underlying differentiation mechanism. (C) Gene targeting. When directed to a target genomic locus, endonucleases induce DSBs on a mammalian chromosome. Co-delivery of a donor plasmid bearing locus-specific homology arms with the target site manipulates the host cell’s HDR mechanism to integrate a donor sequence into the DSB-region, resulting in targeted nucleotide alterations in the host genome. (i) Induction of DSBs with chimeric nucleases ZFN (ZF fused to FokI nuclease) or TALEN (TALE fused to FokI nuclease). (ii) Induction of DSBs with the CRISPR-derived Cas9/gRNA complex. Programmable nucleotide-binding elements are shown in yellow.

tually restores the sense orientation by Cre-mediated inversion [77] (Fig. 3A). Similarly, such a trigger-induced irreversible gene switch is an excellent platform for cell fate studies that require long-term storage of spatiotemporal influences as inheritable memory. For example, a synthetic gene circuit consisting of a GFP-marked Cre-recombinase expressed from a cell-type specific promoter and a reporter construct producing Cre-activated lacZ expression has enabled the construction of a powerful lineage-tracing device for studying cell proliferation in regenerative tissues [83, 84] (Fig. 3B). Stable integration of this device into the mouse genome has led to the identification and verification of multipotent stem cells in the crypt base of the small intestine [85, 86]. More specifically, the promoter that drives GFP and Cre expression was known to be active only in certain stem cells that have the unique characteristics of continuous self-renewal and differentiation, resulting in expression of both GFP and LacZ in these cells. Consequently, the finding that the surrounding tissue exclu-

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sively expressed LacZ was an undeniable indication that these cells were indeed differentiated from the same progenitor, but the quiescence of the stem cell-specific promoter in progeny cells led to the termination of GFP expression (Fig. 3B).

2.3.2 Gene targeting with nucleases Currently, genome editing in mammalian cells is best achieved with programmable nucleases that can be engineered to target specific genomic loci with high precision and efficiency [87]. As briefly introduced in section 2.1.1, fusion of a ZF or TALE protein to a nuclease such as the restriction endonuclease FokI results in a chimeric enzyme consisting of a DNA-binding domain with programmable sequence-specificity and a nonspecific DNA cleavage domain [18, 58]. These chimeric nucleases (ZFN/TALEN) induce DSBs at desired genomic loci, which in turn trigger the endogenous DNA repair machinery of living cells [88] (Fig. 3C). Co-delivery of a donor plasmid bearing locus-specific homology arms with





the target site prompts a mammalian cell to initiate a highly accurate repair mechanism based on homologous recombination [89, 90]. Before entering mitosis, cells naturally maintain their genomic integrity by consulting the highly homologous template provided by the sister chromatid to repair DSBs in a copy-and-paste manner [90]. Therefore, a manipulation technique based on HDR would enable error-free nucleotide alterations through targeted integration of exogenously provided DNA sequences into a genomic locus of interest [88] (Fig. 3C). Currently, ZFN/TALEN-mediated gene targeting has been widely adopted to generate animal models for genetically linked diseases [58]. Furthermore, TALEN-based gene targeting has become the technology of choice to stably integrate synthetic transgene control circuits into the mammalian genome [88]. An alternative class of nucleases that has currently emerged for effective gene targeting is derived from the bacterial CRISPR system [18]. The key component of the CRISPR system is the Cas9 protein, a RNA-guided effector nuclease capable of cleaving sequence-specific dsDNA [87, 91]. The Cas9 protein forms a complex with short RNA molecules that can translocate to target DNA sites, where the binding of a 20-nt spacer region in the RNA strand to a complementary target DNA sequence triggers the endonuclease activity of Cas9 to generate a blunt DSB [32, 88, 89, 91, 92]. Recent experiments have shown that all RNA moieties required for binding, guiding and inducing the Cas9 protein can be engineered as a single chimeric transcript called gRNA, which can be delivered into a mammalian cell either by ectopic expression from a human Pol III promoter or by direct transfection of an in vitro-generated construct [32, 89, 91, 92]. Consequently, expression of the Cas9/gRNA complex in mammalian cells was sufficient to generate DSBs at almost arbitrarily targeted genomic sites that are complementary to an engineered gRNA sequence [18, 89]. Analogous to the ZFN/TALEN-mediated gene targeting approach, the targeted sequence can be modified through manipulation of endogenous DSB-repair mechanisms [87, 89, 92] (Fig. 3C). Optimization studies of the programmable sequence specificity of the CRISPR/Cas9 system for the regulation of genes in a write-protected manner included genetic mutation of Cas9 to generate a catalytically inactive form, called dCas9 or Cas9m [18, 27, 93, 94]. In such a CRISPRi technique, the endonuclease activity of Cas9 is abolished while still allowing the targeting of dCas9 to any dsDNA sequence by co-delivering an appropriate gRNA [30, 91]. When recruited to target DNA sites, dCas9 itself was shown to be sufficient for repressing both synthetic and endogenous genes through steric blocking of transcription initiation [94]. However, fusion of dCas9 to various effector domains such as activators, repressors or recombinases enables these synthetic regulator proteins to function analogously to synthetic transregulators [58] (Fig. 1B). For example, fusing the KRAB domain to dCas9


significantly improved the repressor performance [18]. Additionally, co-expression of a dCas9-VP64 fusion protein with various gRNA molecules enabled the selective upregulation of endogenous target genes in multiple mammalian cell types [32].

3 Control of mammalian gene expression from the cell surface A variety of biological phenomena in mammalian cells, including metabolism, cell division, differentiation and disease progression, are typified by a prolonged cellular response to a transient environmental signal [50]. Such responses are mediated by intracellular signaling pathways, which are the cell’s major interface with the environment [95]. They consist of a complex network of chemicals, enzymes and protein scaffolds that collectively transduce and amplify an extracellular signal into the nucleus or towards other specialized compartments in the cytoplasm, and a cellular response can be elicited through gene transcription or posttranscriptional modifications [95, 96]. The principles and examples introduced in this section elucidate general design strategies to allow systematic rewiring of different extracellular signals to the transcription machinery. Unlike classical transregulators in which the trigger compound monitoring a designer cell is generally restricted to small hydrophobic nucleus-permeable molecules, cell membrane receptors accept a much wider variety of signal molecules with a minimal residence time at the cell surface [97] (Fig. 4). Coupling these triggers to synthetic gene-control devices that act on the intracellular compartment would greatly expand the scope of spatial control points and allow the convenient and demand-driven remote control of cellular and genetic behavior.

3.1 Targeting signaling pathways with second messengers In mammalian cells, binding of a cell-surface receptor to its cognate ligand often induces a transient shift in the concentrations of various second messengers in the cytoplasm, such as cyclic nucleotides (cAMP, cGMP), cyclic di-nucleotides (c-di-GMP, c-di-AMP), Ca2+, IP3, DAG or NO [98]. Many cellular events that are initiated by second messengers result in transcription of certain genes by activating endogenous promoters. Careful examination of this dependency has led to the discovery of a set of DNA binding sites that have been considered second messenger-specific response elements [99–103]. Isolation and combination of these response elements into synthetic minimal promoters allowed for the engineering of cognate promoters that are under tight regulation of these second messengers (Fig. 4).

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Figure 4. Self-sufficient prosthetic networks. After implantation of mammalian designer cells into a patient, a particular disease marker in the host’s blood triggers the expression of a secreted therapeutic protein. Self-sufficiency is achieved when the secreted drug reduces the concentration of the disease marker into a range that is no longer pathologic and at the same time insufficient to keep activating the designer cell. (Left) Signal reception at the transcriptional level. A nuclear-permeable disease metabolite triggers transgene expression from a cognate transsilencer-regulated promoter. (Right) Signal reception at the cell surface. Binding of a cell-surface receptor to its cognate ligand often induces a concentration shift of various second messengers (SMs) in the cytoplasm. This activity can be scored with synthetic promoters that contain SM-specific response elements.

3.1.1 Rewiring ion channel activity In an approach combining synthetic biology with nanotechnology, Stanley et al. coated a modified temperaturesensitive TRPV1 channel with iron oxide nanoparticles to engineer a transgene expression device in mammalian cells in a system capable of delivering protein therapeutics in animals [104, 105]. Briefly, the iron oxide particles coating ectopically expressed TRPV1 were able to absorb and store thermal energy when the cell was exposed to radio waves of low and medium frequency. When the local temperature of the cell membrane exceeded a threshold value, TRPV1 was activated, allowing a transient influx of calcium ions. These elevated levels of intracellular calcium induced transgene expression regulated by a Ca2+responsive promoter.

3.1.2 Rewiring the human immune system Capitalizing on recent findings that the human antimicrobial STING pathway is activated by bacteria-derived c-di-GMP [106], Folcher et al. linked a Rhodobacter sphaeroides NIR-light-activated c-di-GMP-producing enzyme with a human interferon promoter to engineer a

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mammalian transgene expression device that is controlled by NIR illumination [24]. To adapt this device for facilitated remote control in biomedical applications, the authors also engineered an electroencephalographybased BCI device capable of converting human brain waves of different intensities into an accordingly strong NIR-signal on a self-powered LED lamp. Therefore, when mice received designer cells embedded inside biocompatible LED chamber implants, different levels of transgene expression could be triggered by reciprocal mental states of a human investigator such as concentration or meditation. By merging synthetic biology with cybernetics, to allow brain waves to remotely control cellular behavior in a wireless manner, this work represents an impressive milestone in boosting future gene delivery methods to adopt noninvasive and discrete trigger signals.

3.1.3 Rewiring GPCR activity GPCRs are responsive to a plethora of stimuli, both endogenous (biogenic amines, cations, lipids, peptides and glycoproteins) and exogenous (therapeutic drugs,





photons, tastants and odorants) [107]. To control gene expression in mammalian designer cells with GPCR agonists, cells can be engineered to ectopically express a receptor that is activated by a desired trigger compound [13]. Depending on the selected Gα-protein, a variety of second messenger responsive promoters can be used to capture this signaling with a transcriptional message [98] (Fig. 4). For example, Ausländer et al. linked the Gαs-mediated signaling pathway of a proton-responsive GPCR with a cAMP-responsive promoter (PCRE) to engineer a designer mammalian cell whose transcriptional activity could be regulated by its extracellular pH or CO2 content [108]. When implanting these designer cells into mice that suffer from severe diabetic ketoacidosis, the abnormally acidic blood in the animals was able to trigger PCRE-driven insulin expression in a self-sufficient manner (Fig. 4). Analogously, by coupling a histamine-responsive GPCR to the PCRE-driven transcription of a quantitative reporter protein, Ausländer et al. also engineered a cellbased allergy profiler capable of scoring the allergen-triggered release of histamine in human whole-blood samples [109]. Remarkably, not only did the scores of this allergy profiler fully correlate with results obtained from standard diagnostic tools such as the skin-prick test but the all-in vitro format of this mammalian designer cell also did not require a patient’s direct exposure to potential allergens. Therefore, this device might qualify as an excellent candidate for the diagnosis of infants or sensitive patients suffering from skin disorders [109].

Martin Fussenegger is Professor of Biotechnology and Bioengineering at the Department of Biosystems Science and Engineering (D-BSSE) of ETH Zurich in Basel as well as at the University of Basel. He graduated with Werner Arber at the Biocenter of the University of Basel (1992), obtained his Ph.D. in Medical Microbiology (1994) at the Max Planck Institute of Biology (Tübingen) and continued his postdoctoral studies on host-pathogen interactions at the Max Planck Institute of Infection Biology (Berlin, 1995). Subsequently, he joined the ETH Institute of Biotechnology (1996), where he received his habilitation in 2000, and became Swiss National Science Foundation Professor of Molecular Biotechnology in 2002, prior to being awarded a Chair in Biotechnology and Bioengineering at the ETH Institute for Chemical and Bioengineering in 2004. On a presidential mission, he moved to Basel in 2008 to build up the D-BSSE of the ETH Zurich. Martin Fussenegger is a fellow of the American Institute for Medical and Biological Engineering (AIMBE) and a member of the Swiss Academy of Engineering Sciences.

Mingqi Xie is a Ph.D. student in Prof. Fussenegger’s group at the ETH Zurich, Basel, Switzerland. Current research topics involve the engineering of self-sufficient therapeutic gene circuits for diabetes treatment and the

4 Conclusions and perspectives

construction of orthogonal triggerinducible gene switches.

Synthetic biology is increasingly providing valuable information for applications in the clinic, in the biotechnology industry and in basic molecular research [16]. Instead of building cells from scratch [110], the engineering approach of synthetic biologists consisting of rational reassembly of well-characterized components from different kingdoms of life provides new insight into the control logic and overall behavior of cellular systems and enables the design of cells with new and useful function [1, 2, 18]. In this respect, synthetic designer cells can also be regarded as “computerized living cells” because the engineered cellular behavior directly results from precise computations between high-fidelity biomolecules that were brought together by engineers with the mindsets of computer programmers [1, 2, 4, 10]. Designer cell engineering enormously facilitates research that requires in-depth understanding of singlecell behavior. In an attempt to challenge current issues in pharmaceutical immunology, T lymphocytes have been reprogrammed ex vivo to express synthetic receptors called CARs, i.e. modular transmembrane fusion proteins of a TCR’s cytoplasmic signaling domains with an extra-


cellular antigen-targeting element, which is most often a scFv-antibody [12, 18]. When adoptively transferred into the patient, these CAR-expressing designer cells were able to migrate towards cancer cells and kill those cells displaying scFv-specific antigens on their surfaces. Although this field remains in its infancy, clinical trials have already shown significant antitumor activities in multiple types of cancer [111]. With synthetic biology-based circuits becoming increasingly complex and multilayered, the next step in mammalian synthetic biology might challenge the move from engineering single designer cells to synthetic higher-order systems that function in a truly multicellular fashion [3, 5, 13, 16]. Indeed, similar to the rational reassembly of gene control elements to engineered designer cells, synthetic intercellular communication devices can also be engineered by the judicious interconnection of wellunderstood single designer cells [13]. In the future, rational combination of multiple sets of designer cells might

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allow the construction and regulation of higher-order systems with increased complexity, thereby enabling the reprogramming of tissues, organisms or even populations with high precision.

We thank Marc Folcher and Lina Schukur for critical comments on the manuscript. Work in the M.F. lab is supported by a European Research Council (ERC) advanced grant (ProNet – No. 321381) and, in part, by the National Centre of Competence in Research (NCCR) Molecular Systems Engineering. The authors declare no financial or commercial conflict of interest.

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ISSN 1860-6768 · BJIOAM 10 (7) 927–1090 (2015) · Vol. 10 · July 2015

Systems & Synthetic Biology · Nanobiotech · Medicine

7/2015 CHO cells Systems biology Genome engineering

Cover illustration

Mammalian Production Systems www.biotechnology-journal.com

Special issue: Mammalian Production Systems. This special issue is edited by Nicole Borth and Lars Nielsen. It focuses on the use of CHO cells and their improvement to produce recombinant proteins. The articles cover the application of CRISPR technology, systems biology and modeling approaches as well as bioinformatics methods. The cover visualizes the highly interconnected metabolic pathway map of CHO cells based on a genome-scale metabolic reconstruction (blue: reactions, red: molecular species). Image by Michael Hanscho.

Biotechnology Journal – list of articles published in the July 2015 issue. Editorial: On the cusp of rational CHO cell engineering Lars Nielsen and Nicole Borth

http://dx.doi.org/10.1002/biot.201500375

Review Industrial production of clotting factors: Challenges of expression, and choice of host cells Sampath R. Kumar

Review CHOgenome.org 2.0: Genome resources and website updates


Benjamin G. Kremkow, Jong Youn Baik, Madolyn L. MacDonald and Kelvin H. Lee

Review Mammalian designer cells: Engineering principles and biomedical applications


Mingqi Xie and Martin Fussenegger

Review Optimizing eukaryotic cell hosts for protein production through systems biotechnology and genome-scale modeling Jahir M. Gutierrez and Nathan E. Lewis

http://dx.doi.org/10.1002/biot.201400647 Review Towards next generation CHO cell biology: Bioinformatics methods for RNA-Seq-based expression profiling Craig Monger,Paul S. Kelly, Clair Gallagher, Martin Clynes, Niall Barron and Colin Clarke

http://dx.doi.org/10.1002/biot.201500107 Review Epigenetic regulatory elements: Recent advances in understanding their mode of action and use for recombinant protein production in mammalian cells Niamh Harraghy, David Calabrese, Igor Fisch, Pierre-Alain Girod, Valérie LeFourn, Alexandre Regamey and Nicolas Mermod

http://dx.doi.org/10.1002/biot.201400649 Review CRISPR/Cas9-mediated genome engineering of CHO cell factories: Application and perspectives

http://dx.doi.org/10.1002/biot.201400642 Research Article NF-κB, CRE and YY1 elements are key functional regulators of CMV promoter-driven transient gene expression in CHO cells Adam J. Brown, Bernie Sweeney, David O. Mainwaring, David C. James

http://dx.doi.org/10.1002/biot.201400744 Research Article Re-programming CHO cell metabolism using miR-23 tips t he balance towards a highly productive phenotype Paul S. Kelly, Laura Breen, Clair Gallagher, Shane Kelly, Michael Henry, Nga T. Lao, Paula Meleady, Donal O’Gorman, Martin Clynes and Niall Barron

http://dx.doi.org/10.1002/biot.201500101 Research Article Chemical manipulation of the mTORC1 pathway in industrially relevant CHOK1 cells enhances production of therapeutic proteins Nazanin Dadehbeigi and Alan J. Dickson


Jae Seong Lee, Lise Marie Grav, Nathan E. Lewis and Helene Faustrup Kildegaard




Research Article Low glucose depletes glycan precursors, reduces site occupancy and galactosylation of a monoclonal antibody in CHO cell culture

Research Article Deep sequencing reveals different compositions of mRNA transcribed from the F8 gene in a panel of FVIII-producing CHO cell lines

Carina Villacrés, Venkata S. Tayi, Erika Lattová, Hélène Perreault and Michael Butler

Christian S. Kaas, Gert Bolt, Jens J. Hansen, Mikael R. Andersen and Claus Kristensen



Research Article Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin Jong Youn Baik, Hussain Dahodwala, Eziafa Oduah, Lee Talman, Trent R. Gemmill, Leyla Gasimli, Payel Datta, Bo Yang, Guoyun Li, Fuming Zhang, Lingyun Li, Robert J. Linhardt, Andrew M. Campbell, Stephen F. Gorfien and Susan T. Sharfstein




Engineering Stem Cells for Biomedical Applications.

The community FabLab platform: applications and implications in biomedical engineering.

Photoreconfigurable polymers for biomedical applications: chemistry and macromolecular engineering.

Assessing asymmetry using reflection and rotoinversion in biomedical engineering applications.

Core-Crosslinked Polymeric Micelles: Principles, Preparation, Biomedical Applications and Clinical Translation.

Cerenkov luminescence imaging: physics principles and potential applications in biomedical sciences.

Mouse genome engineering using designer nucleases.

Microdosimetry: Principles and applications.

Hormesis: principles and applications.

Synthesis and optimization of chitosan nanoparticles: Potential applications in nanomedicine and biomedical engineering.

Special section guest editorial: Optical coherence tomography and interferometry: advanced engineering and biomedical applications.

Engineering ultrasmall water-soluble gold and silver nanoclusters for biomedical applications.

Preparation and evaluation of cerium oxide-bovine hydroxyapatite composites for biomedical engineering applications.

Superlattice valley engineering for designer topological insulators.

Biomedical Applications of Dental and Oral-Derived Stem Cells.

Aptamers: Biomedical Interest and Applications.

Bottom-Up Engineering of Well-Defined 3D Microtissues Using Microplatforms and Biomedical Applications.

Engineering of microscale three-dimensional pancreatic islet models in vitro and their biomedical applications.

Enzymes: principles and biotechnological applications.

S-layers: principles and applications.

Nanofiber: Synthesis and biomedical applications.

Biomedical applications of nisin.

Biomedical applications of collagens.

STARR-seq - principles and applications.