Mammalian Synthetic Biology

Synthetic Transcription Factors

Engineered biological circuits provide insights into the underlying biology of living cells and offer potential solutions to a range of medical and industrial challenges. A prerequisite for efficient engineering of sophisticated circuits is a library of regulatory devices that can be connected to create new and predictable behaviors. Complex and sophisticated phenotypes in eukaryotic cells manifest from layered regulatory networks and specific expression programs involving the regulated transcription of many genes. Eukaryotic transcriptional factors (TFs) can integrate multiple signals and perform complex, combinatorial functions on promoters to modulate gene expression patterns. Rewiring endogenous transcriptional networks by synthetic TFs is a powerful strategy for interrogating cellular functions and controlling cellular phenotypes. Our goal is to establish a robust pipeline for the production and screening of synthetic transcription factors (sTFs), incorporating the DNA binding domains of TAL effectors (TALEs) and the CRISPR/Cas system. The major goal will be to scale up production and screening to enable isolation in an unbiased manner of sTFs that can bind to endogenous mammalian cis-regulatory elements and subsequently activate or repress associated mammalian genes. sTFs will provide valuable tools that could be exploited in many SB applications including biosensing, drug screening, the in vitro production of mammalian cell types for disease modeling or directly as therapeutic agents (e.g. gene therapy).

Stable gene insertion

Systems for targeted gene or synthetic circuit integration into reliable safe harbors or landing pads are needed for the stable, predictable and controlled levels of transgene expression that are essential for effective synthetic biology approaches in mammalian systems. The current technology relies on the transgenes integrating randomly into the host genome resulting in highly variable protein expression between different transfectants and integration into gene-coding regions that can disrupt normal cell processes or areas of instability and transgene silencing.

Site-specific recombinases have been used as tools to generate recombinant mammalian cells. The most commonly used systems Cre/loxP and Flp/FRT regenerate the original substrates after recombination and are efficient at deletion by excising DNA located between directly repeated recombination sites. This innate reversibility is, however, problematic for stable DNA integration. Recently the serine integrases e.g. ϕC31 or ϕBT1have been investigated for use in mammalian cells because they can catalyse highly efficient irreversible recombination. Further, accessory proteins exist, Recombination Directionality Factors (RDFs), that can reverse the serine integrase recombination reaction and so provide a novel versatile system allowing efficient DNA integration, cassette deletion or exchange.

Memory and computation

Memory and computation are central enablers of many envisioned applications of synthetic biological engineering, including programming and tracking cell fate, diagnostics, and bio-manufacturing platforms, and are a major goal in mammalian SB. Much of the synthetic biological computing power takes the form of combinatorial logic circuits that combine a number of inputs using logic operations to provide an output. Inputs and outputs are typically binary: for example, presence or absence of a chemical input, transcription on or off. These devices have no memory of past events: they simply process and take action on current inputs. In contrast, regulatory circuits with memory can respond differentially depending on their past history. This "sequential logic" is necessary for any temporally evolving program and could be used to specify a series of gene expression patterns.

Synthetic circuits with memory have been implemented using networks of transcriptional promoters and regulators. Memory can be maintained for generations but is dependent on gene expression, so information is lost when the inducer input is removed or protein synthesis ceases.

In both yeast and mammalian cells, we are developing an alternative approach using site-specific recombination for construction of Boolean logic gates and synthetic counting circuits with the ability to memorize information by implementing changes in DNA sequence.

Engineered Cell lines for Drug Testing

There is a great need to replace in vivo animal testing with cell-line based in vitro toxicological and efficacy assays. Growing evidence suggests that drugs interact with diverse molecular targets mediating both therapeutic and toxic effects. These complex interactions cannot be predicted from chemical structures alone and many toxicity assays rely on a simplistic cell death phenotype that does not give any mechanistic insight into the mode of toxicity. Traditionally, screening for drug safety starts at a late phase of lead optimization. Any liability discovered then can easily block development pipelines and cause high late attrition rates with resultant escalating costs. However, recent advances have the potential to provide the pharmaceutical industry with platforms to produce simple, fast and cost effective in vitro screening assays, applicable to the early phases of drug discovery and that rapidly highlight potential problems making drug development more cost effective.

Cells are filled with natural biosensors – molecular systems that measure the state of the cell and respond by regulating host processes. Systems biology is helping to unravel regulatory networks and crosstalk between these networks. We are using that understanding to design and build synthetic signalling networks to reroute information flow and to engineer sensing and output circuits that perform logic computations to deliver mechanistic information on the cell state, e.g. disease states, and responses to perturbations, e.g. drug treatments.