An engineered epigenetic transgene switch in mammalian cells.

In multicellular systems cell identity is imprinted by epigenetic regulation circuits, which determine the global transcriptome of adult cells in a cell phenotype-specific manner. By combining two repressors, which control each other's expression, we have developed a mammalian epigenetic circuitry able to switch between two stable transgene expression states after transient administration of two alternate drugs. Engineered Chinese hamster ovary cells (CHO-K1) showed toggle switch-specific expression profiles of a human glycoprotein in culture, as well as after microencapsulation and implantation into mice. Switch dynamics and expression stability could be predicted with mathematical models. Epigenetic transgene control through toggle switches is an important tool for engineering artificial gene networks in mammalian cells.

Macrolide-based transgene control in mammalian cells and mice.

Heterologous mammalian gene regulation systems for adjustable expression of multiple transgenes are necessary for advanced human gene therapy and tissue engineering, and for sophisticated in vivo gene-function analyses, drug discovery, and biopharmaceutical manufacturing. The antibiotic-dependent interaction between the repressor (E) and operator (ETR) derived from an Escherichia coli erythromycin-resistance regulon was used to design repressible (E(OFF)) and inducible (E(ON)) mammalian gene regulation systems (E.REX) responsive to clinically licensed macrolide antibiotics (erythromycin, clarithromycin, and roxithromycin). The E(OFF) system consists of a chimeric erythromycin-dependent transactivator (ET), constructed by fusing the prokaryotic repressor E to a eukaryotic transactivation domain that binds and activates transcription from ETR-containing synthetic eukaryotic promoters (P(ETR)). Addition of macrolide antibiotic results in repression of transgene expression. The E(ON) system is based on E binding to artificial ETR-derived operators cloned adjacent to constitutive promoters, resulting in repression of transgene expression. In the presence of macrolides, gene expression is induced. Control of transgene expression in primary cells, cell lines, and microencapsulated human cells transplanted into mice was demonstrated using the E.REX (E(OFF) and E(ON)) systems. The macrolide-responsive E.REX technology was functionally compatible with the streptogramin (PIP-regulated and tetracycline (TET-regulated expression systems, and therefore may be combined for multiregulated multigene therapeutic interventions in mammalian cells and tissues.

Dual-regulated expression of C/EBP-alpha and BMP-2 enables differential differentiation of C2C12 cells into adipocytes and osteoblasts.

CCAAT/enhancer-binding proteins (C/EBPs) as well as bone morphogenic proteins (BMPs) play essential roles in mammalian cell differentiation in shaping adipogenic and osteoblastic lineages in particular. Recent evidence suggested that adipocytes and osteoblasts share a common mesenchymal precursor cell phenotype. Yet, the molecular details underlying the decision of adipocyte versus osteoblast differentiation as well as the involvement of C/EBPs and BMPs remains elusive. We have engineered C2C12 cells for dual-regulated expression of human C/EBP-alpha and BMP-2 to enable independent transcription control of both differentiation factors using clinically licensed antibiotics of the streptogramin (pristinamycin) and tetracycline (tetracycline) classes. Differential as well as coordinated expression of C/EBP-alpha and BMP-2 revealed that (i) C/EBP-alpha may differentiate C2C12 myoblasts into adipocytes as well as osteoblasts, (ii) BMP-2 prevents myotube differentiation, (iii) is incompetent in differentiating C2C12 into osteoblasts and (iv) even decreases C/EBP-alpha's osteoblast-specific differentiation potential but (v) cooperates with C/EBP-alpha on adipocyte differentiation, (vi) osteoblast formation occurs at low C/EBP-alpha levels while adipocyte-specific differentiation requires maximum C/EBP-alpha expression and that (vii) BMP-2 may bias the C/EBP-alpha-mediated adipocyte versus osteoblast differentiation switch towards fat cell formation. Dual-regulated expression technology enabled precise insight into combinatorial effects of two key differentiation factors involved in adipocyte/osteoblast lineage control which could be implemented in rational reprogramming of multipotent cells into desired cell phenotypes tailored for gene therapy and tissue engineering.

A genetic time-delay circuitry in mammalian cells.

Gene expression circuitries with time-delayed expression profiles regulate key events, such as oscillating systems, noise elimination, and coordinated multi-step processes, in all organisms from bacteria to mammalian cells. We present the rational synthesis of a genetic circuit displaying time-delayed expression in silico and in mammalian cells. The network is based on a time-delay circuit, where the tetracycline-responsive transactivator (tTA) induces expression of the pristinamycin-responsive repressor PIP-KRAB, which silences expression of the terminal human placental secreted alkaline phosphatase (SEAP). While the addition of pristinamycin I inactivates PIP-KRAB and results in the immediate resumption of SEAP expression, addition of tetracycline abolishes PIP-KRAB synthesis. Consequently, SEAP production remains repressed until the PIP-KRAB buffer in the cell is eliminated. We characterized in silico and in vivo the time-delayed expression properties and analyzed the impact of the size and stability of the PIP-KRAB buffer on fine-tuning of the response kinetics. This tunable time-delay circuitry represents a biologic building block for emulating a fundamental circuit topology in integrated artificial synthetic gene networks for the design of tailor-made cell types and organisms.

Hysteresis in a synthetic mammalian gene network.

Bistable and hysteretic switches, enabling cells to adopt multiple internal expression states in response to a single external input signal, have a pivotal impact on biological systems, ranging from cell-fate decisions to cell-cycle control. We have designed a synthetic hysteretic mammalian transcription network. A positive feedback loop, consisting of a transgene and transactivator (TA) cotranscribed by TA's cognate promoter, is repressed by constitutive expression of a macrolide-dependent transcriptional silencer, whose activity is modulated by the macrolide antibiotic erythromycin. The antibiotic concentration, at which a quasi-discontinuous switch of transgene expression occurs, depends on the history of the synthetic transcription circuitry. If the network components are imbalanced, a graded rather than a quasi-discontinuous signal integration takes place. These findings are consistent with a mathematical model. Synthetic gene networks, which are able to emulate natural gene expression behavior, may foster progress in future gene therapy and tissue engineering initiatives.

Semi-synthetic mammalian gene regulatory networks.

In recent years gene network engineers have celebrated spectacular success: Genetic devices such as epigenetic toggle switches and oscillating networks have been engineered and pioneered a new ever-increasing scientific community known as synthetic biology. While synthetic biology was until recently restricted to network assembly and testing in prokaryotes, decisive advances have been achieved in eukaryotic systems based on current availability of different human-compatible transgene control technologies. Most prominent examples include the epigenetic gene network enabling metastable fully inheritable transgene expression states in mice, artificial regulatory cascades managing multi-level expression control and Boolean-type BioLogic gates supporting near-digital expression readout. The majority of transgene control networks available to date are fully synthetic and integrate artificial extracellular signals in a desired host metabolism-independent manner. Yet, in order to develop their full anticipated therapeutic potential, synthetic transgene control circuits need to be well interconnected with the host cell's regulatory networks in order to enable physiologic control of prosthetic molecular expression units. We have designed three semi-synthetic transcription control networks able to integrate physiologic oxygen levels and artificial antibiotic signals to produce expression readout with NOT IF or NOR-type Boolean logic or discrete multi-level control of several intracellular and secreted model product proteins. Subtle differences in the regulation performance of the endogenous oxygen-sensing system in CHO-K1 and human HT-1080 switched the semi-synthetic network's readout from a classic four-level (high, medium, low, basal) regulatory cascade to a network enabling six discrete transgene expression levels. These findings are in excellent correspondence with a mathematical model. Prosthetic networks, precisely embedded in host regulatory networks and co-fine-tuned by physiologic as well as pharmacologic input signals, will foster future advances in gene therapy and tissue engineering.

Artificial regulatory networks and cascades for discrete multilevel transgene control in mammalian cells.

Prototype drug-adjustable heterologous transcription control systems designed for gene therapy applications typically show sigmoid dose-response characteristics and enable fine-tuning of therapeutic transgenes only within a narrow inducer concentration range of a few nanograms. However, the design of clinical dosing regimes which achieve tissue-specific concentrations with nanogram precision is yet a "mission impossible." Therefore, most of today's transcription control systems operate as ON/OFF switches and not in a true adjustable mode. The availability of robust transcription control configurations which lock expression of a single therapeutic transgene at desired levels in response to fixed clinical doses of different inducers rather than minute concentration changes of a single inducer would be highly desirable. Based on in silico predictions, we have constructed a variety of mammalian artificial regulatory networks by interconnecting the tetracycline- (TET(OFF)), streptogramin- (PIP(OFF)), and macrolide- (E(OFF)) repressible gene regulation systems as linear (auto)regulatory cascades. These networks enable multilevel expression control of several transgenes in response to different antibiotics or allow titration of a single transgene to four discrete expression levels by clinical dosing of a single antibiotic: 1) high expression in the absence of any antibiotic (+++), 2) medium level expression following addition of tetracycline (++), 3) low level expression in response to the macrolide erythromycin (+), and 4) complete repression by streptogramins such as pristinamycin (-). The first-generation artificial regulatory networks exemplify modular interconnections of different heterologous gene regulations systems to achieve multigene expression, fine-tuning, or to design novel control networks with unprecedented transgene regulation properties. Such higher-level transcription control modalities will lead the way towards composite artificial regulatory networks able to effect complex therapeutic interventions in future gene therapy and tissue engineering scenarios.

BioLogic gates enable logical transcription control in mammalian cells.

The architecture of gene regulatory networks is reminiscent of electronic circuits. Modular building blocks that respond in a logical way to one or several inputs are connected to perform a variety of complex tasks. Gene circuit engineers have pioneered the construction of artificial gene regulatory networks with the intention to pave the way for the construction of therapeutic gene circuits for next-generation gene therapy approaches. However, due to the lack of a critical amount of eukaryotic cell-compatible gene regulation systems, the field has so far been limited to prokaryotes. Recent development of several mammalian cell-compatible expression control systems laid the foundations for the assembly of transcription control modules that can respond to several inputs. Herein, three approaches to evoke combinatorial transcription control have been followed: (i) construction of artificial promoters with up to three operator sites for regulatory proteins, and (ii) parallel and (iii) serial linking of two gene regulation systems. We have combined tetracycline-, streptogramin-, macrolide-, and butyrolactone transcription control systems to engineer BioLogic gates of the NOT IF-, AND-, NOT IF IF-, NAND-, OR-, NOR-, and INVERTER-type in mammalian cells, which are able to respond to up to three different small molecule inputs. BioLogic gates enable logical transcriptional control in mammalian cells and, in combination with modern transduction technologies, could serve as versatile tools for regulated gene expression and as building blocks for complex artificial gene regulatory networks for applications in gene therapy, tissue engineering, and biotechnology.

Novel promoter/transactivator configurations for macrolide- and streptogramin-responsive transgene expression in mammalian cells.

BACKGROUND: The recently developed heterologous macrolide- (E.REX system) and streptogramin- (PIP system) responsive gene regulation systems show significant differences in their regulation performance in diverse cell lines. METHODS: In order to provide optimal regulation modalities for a wide variety of mammalian cell lines, we have performed a detailed analysis of E.REX and PIP systems modified in (i) the transactivation domains of the antibiotic-dependent transactivators, (ii) the type of minimal promoter used, and (iii) the spacing between the operator module and the minimal promoter. RESULTS: These novel E.REX and PIP regulation components showed not only dramatically improved regulation performance in some cell types, but also enabled their use in cell lines which had previously been inaccessible to regulated transgene expression. CONCLUSIONS: Due to their modular set-up the novel E.REX and PIP regulation systems presented here are most versatile and ready for future upgrades using different cell-specific key regulation components.

Versatile macrolide-responsive mammalian expression vectors for multiregulated multigene metabolic engineering.

The novel macrolide-inducible and -repressible mammalian gene regulation systems (E.REX) have been cloned into a variety of sophisticated expression configurations including (1) multi-purpose expression vectors, (2) pTRIDENT-based artificial operons, (3) dual-regulated expression strategies for independent control of two different transgenes, (4) autoregulated vectors for one-step installation of adjustable multigene expression, and (5) oncoretroviral and lentiviral plasmids for transduction of macrolide-, streptogramin- and tetracycline-dependent transactivators and production of cell lines supporting independent control of three different transgenes. This vector portfolio represents a construction kit-like toolbox for efficient installation of adjustable gene expression responsive to clinically licensed antibiotics and enables the design of multiregulated multigene metabolic engineering strategies required for biopharmaceutical manufacturing, gene therapy, and tissue engineering.

Synergies of microtissue design, viral transduction and adjustable transgene expression for regenerative medicine.

In the past decade, regenerative medicine has evolved as an interdisciplinary field, integrating expertise from the medical, life- and material-science communities. Recent advances in tissue engineering, gene therapy, gene-function analysis, animal-free drug testing, drug discovery, biopharmaceutical manufacturing and cell-phenotype engineering have capitalized on a core technology portfolio including artificial microtissue design, viral transduction and precise transcription dosing of therapeutic or phenotype-modulating transgenes. We provide a detailed overview on recent progress in these core technologies and comment on their synergistic impact on current and future human therapies.