Manufacturing innovation to drive down cell therapy costs.

Chimeric antigen receptor T cells (CAR-T) have demonstrated their potential to revolutionize cancer treatment. However, manufacturing remains a challenge. Multiple manufacturing innovations [e.g., vector and gene engineering, process improvements, hardware innovation, digital innovation, and point-of-care (POC) manufacturing] have the potential to help realize the full potential of CAR-T therapies.

Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria.

We report here the behavior of naturally occurring and rationally engineered preQ1 riboswitches and their application to inducible gene regulation in mycobacteria. Because mycobacteria lack preQ1 biosynthetic genes, we hypothesized that preQ1 could be used as an exogenous nonmetabolite ligand to control riboswitches in mycobacteria. Selected naturally occurring preQ1 riboswitches were assayed and successfully drove preQ1-dependent repression of a green fluorescent protein reporter in Mycobacterium smegmatis Using structure-based design, we engineered three preQ1 riboswitches from Thermoanaerobacter tencongensis, Bacillus subtilis, and Lactobacillus rhamnosus toward achieving higher response ratios and increased repression. Assuming a steady-state model, variants of the T. tencongensis riboswitch most closely followed the predicted trends. Unexpectedly, the preQ1 dose response was best described by a model with a second, independent preQ1 binding site. This behavior was general to the preQ1 riboswitch family, since the wild type and rationally designed mutants of riboswitches from all three bacteria behaved analogously. Across all variants, the response ratios, which describe expression in the absence versus the presence of preQ1, ranged from <2 to ∼10, but repression in all cases was incomplete up to 1 mM preQ1. By reducing the transcript expression level, we obtained a preQ1 riboswitch variant appropriate for inducible knockdown applications. We further showed that the preQ1 response is reversible, is titratable, and can be used to control protein expression in mycobacteria within infected macrophages. By engineering naturally occurring preQ1 riboswitches, we have not only extended the tools available for inducible gene regulation in mycobacteria but also uncovered new behavior of these riboswitches.IMPORTANCE Riboswitches are elements found in noncoding regions of mRNA that regulate gene expression, typically in response to an endogenous metabolite. Riboswitches have emerged as important tools for inducible gene expression in diverse organisms. We noted that mycobacteria lack the biosynthesis genes for preQ1, a ligand for riboswitches from diverse bacteria. Predicting that preQ1 is not present in mycobacteria, we showed that it controls optimized riboswitches appropriate for gene knockdown applications. Further, the riboswitch response is subject to a second independent preQ1 binding event that has not been previously documented. By engineering naturally occurring riboswitches, we have uncovered a new behavior, with implications for riboswitch function in its native context, and extended the tools available for inducible gene regulation in mycobacteria.

A riboswitch-based inducible gene expression system for mycobacteria.

Research on the human pathogen Mycobacterium tuberculosis (Mtb) would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.

From SELEX to cell dual selections for synthetic riboswitches.

Synthetic riboswitches have emerged as useful tools for controlling gene expression to reprogram cellular behavior. However, advancing beyond proof-of-principle experiments requires the ability to quickly generate new synthetic riboswitches from RNA libraries. In this chapter, we provide a step-by-step overview of the process of obtaining synthetic riboswitches for use in Escherichia coli, starting from a randomized RNA library.

Synthetic riboswitches that induce gene expression in diverse bacterial species.

We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-negative and Gram-positive bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.

Engineering bacteria to recognize and follow small molecules.

The ability to recognize and react to specific environmental cues allows bacteria to localize to environments favorable to their survival and growth. Synthetic biologists have begun to exploit the chemosensory pathways that control cell motility to reprogram how bacteria move in response to novel signals. Reprograming is often accomplished by designing novel protein or RNA parts that respond to specific small molecules not normally recognized by the natural chemosensory pathways. Additionally, cell motility and localization can be coupled to bacterial quorum sensing, potentially allowing consortia of cells to perform complex tasks.

Emerging applications of riboswitches in chemical biology.

Living systems use RNA sequences known as riboswitches to detect the concentrations of small-molecule metabolites within cells and to regulate the expression of genes that produce these metabolites. Like their natural counterparts, synthetic riboswitches also regulate gene expression in response to small molecules. Because synthetic riboswitches can be engineered to respond to nonendogenous small molecules, they are powerful tools for chemical and synthetic biologists interested in understanding and reprogramming cellular behavior. In this review, we present an overview of natural riboswitches, highlight recent studies toward developing synthetic riboswitches and provide an overview of emerging applications of these RNA switches in chemical biology.

High-throughput screens to discover synthetic riboswitches.

Synthetic riboswitches constructed from RNA aptamers provide a means to control bacterial gene expression using exogenous ligands. A common theme among riboswitches that function at the translational level is that the RNA aptamer interacts with the ribosome-binding site (RBS) of a gene via an intervening sequence known as an expression platform. Structural rearrangements of the expression platform convert ligand binding into a change in gene expression. While methods for selecting RNA aptamers that bind ligands are well established, few general methods have been reported for converting these aptamers into synthetic riboswitches with desirable properties. We have developed two such methods that not only provide the throughput of genetic selections, but also feature the quantitative nature of genetic screens. One method, based on cell motility, is operationally simple and requires only standard consumables; while the other, based on fluorescence-activated cell sorting (FACS), is particularly adept at identifying synthetic riboswitches that are highly dynamic and display very low levels of background expression in the absence of the ligand. Here we present detailed procedures for screening libraries for riboswitches using the two methods.

Riboswitches in unexpected places--a synthetic riboswitch in a protein coding region.

In natural and engineered systems, cis-RNA regulatory elements such as riboswitches are typically found within untranslated regions rather than within the protein coding sequences of genes. However, RNA sequences with important regulatory roles can exist within translated regions. Here, we present a synthetic riboswitch that is encoded within the translated region of a gene and represses Escherichia coli gene expression greater than 25-fold in the presence of a small-molecule ligand. The ability to encode riboswitches within translated regions as well as untranslated regions provides additional opportunities for creating new genetic control elements. Furthermore, evidence that a riboswitch can function in the translated region of a gene suggests that future efforts to identify natural riboswitches should consider this possibility.

Guiding bacteria with small molecules and RNA.

Chemotactic bacteria navigate their chemical environment by coupling sophisticated information processing capabilities to molecular motors that propel the cells forward. The ability to reprogram bacteria to follow entirely new chemical signals would create powerful new opportunities in bioremediation, bionanotechnology, and synthetic biology. However, the complexities of bacterial signaling and limitations of current protein engineering methods combine to make reprogramming bacteria to follow novel molecules a difficult task. Here we show that by using a synthetic riboswitch rather than an engineered protein to recognize a ligand, E. coli can be guided toward and precisely localized to a completely new chemical signal.

A genetic toolbox for creating reversible Ca2+-sensitive materials.

A major goal of polymer science is to develop "smart" materials that sense specific chemical signals in complex environments and respond with predictable changes in their mechanical properties. Here, we describe a genetic toolbox of natural and engineered protein modules that can be rationally combined in manifold ways to create reversible self-assembling materials that vary in their composition, architecture, and mechanical properties. Using this toolbox, we produced several materials that reversibly self-assemble in the presence of Ca2+ and characterized these materials using particle-tracking microrheology. The properties of these materials could be predicted from the dilute solution behavior of their component modules, suggesting that this toolbox may be generally useful for creating new stimuli-sensitive materials.