Building Synthetic Biosensors Using Red Blood Cell Proteins.

As the use of engineered cell therapies expands from pioneering efforts in cancer immunotherapy to other applications, an attractive but less explored approach is the use of engineered red blood cells (RBCs). Compared to other cells, RBCs have a very long circulation time and reside in the blood compartment, so they could be ideally suited for applications as sentinel cells that enable in situ sensing and diagnostics. However, we largely lack tools for converting RBCs into biosensors. A unique challenge is that RBCs remodel their membranes during maturation, shedding many membrane components, suggesting that an RBC-specific approach may be needed. Toward addressing this need, here we develop a biosensing architecture built on RBC membrane proteins that are retained through erythropoiesis. This biosensor employs a mechanism in which extracellular ligand binding is transduced into intracellular reconstitution of a split output protein (including either a fluorophore or an enzyme). By comparatively evaluating a range of biosensor architectures, linker types, scaffold choices, and output signals, we identify biosensor designs and design features that confer substantial ligand-induced signal in vitro. Finally, we demonstrate that erythroid precursor cells engineered with our RBC-protein biosensors function in vivo. This study establishes a foundation for developing RBC-based biosensors that could ultimately address unmet needs including noninvasive monitoring of physiological signals for a range of diagnostic applications.

Building synthetic biosensors using red blood cell proteins.

As the use of engineered cell therapies expands from pioneering efforts in cancer immunotherapy to other applications, an attractive but less explored approach is the use of engineered red blood cells (RBCs). Compared to other cells, RBCs have a very long circulation time and reside in the blood compartment, so they could be ideally suited for applications as sentinel cells that enable in situ sensing and diagnostics. However, we largely lack tools for converting RBCs into biosensors. A unique challenge is that RBCs remodel their membranes during maturation, shedding many membrane components, suggesting that an RBC-specific approach may be needed. Towards addressing this need, here we develop a biosensing architecture built on RBC membrane proteins that are retained through erythropoiesis. This biosensor employs a mechanism in which extracellular ligand binding is transduced into intracellular reconstitution of a split output protein (including either a fluorophore or an enzyme). By comparatively evaluating a range of biosensor architectures, linker types, scaffold choices, and output signals, we identify biosensor designs and design features that confer substantial ligand-induced signal in vitro. Finally, we demonstrate that erythroid precursor cells engineered with our RBC protein biosensors function in vivo. This study establishes a foundation for developing RBC-based biosensors that could ultimately address unmet needs including non-invasive monitoring of physiological signals for a range of diagnostic applications.

RNA Sequence and Structure Determinants of Pol III Transcriptional Termination in Human Cells.

The precise mechanism of transcription termination of the eukaryotic RNA polymerase III (Pol III) has been a subject of considerable debate. Although previous studies have clearly shown that multiple uracils at the end of RNA transcripts are required for Pol III termination, the effects of upstream RNA secondary structure in the nascent transcript on transcriptional termination is still unclear. To address this, we developed an in cellulo Pol III transcription termination assay using the recently developed Tornado-Corn RNA aptamer system to create a Pol III-transcribed RNA that produces a detectable fluorescent signal when transcribed in human cells. To study the effects of RNA sequence and structure on Pol III termination, we systematically varied the sequence context upstream of the aptamer and identified sequence characteristics that enhance or diminish termination. For transcription from Pol III type 3 promoters, we found that only poly-U tracts longer than the average length found in the human genome efficiently terminate Pol III transcription without RNA secondary structure elements. We observed that RNA secondary structure elements placed in proximity to shorter poly-U tracts induced termination, and RNA secondary structure by itself was not sufficient to induce termination. For Pol III type 2 promoters, we found that the shorter poly-U tract lengths of 4 uracils were sufficient to induce termination. These findings demonstrate a key role for sequence and structural elements within Pol III-transcribed nascent RNA for efficient transcription termination, and demonstrate a generalizable assay for characterizing Pol III transcription in human cells.

Computation-guided optimization of split protein systems.

Splitting bioactive proteins into conditionally reconstituting fragments is a powerful strategy for building tools to study and control biological systems. However, split proteins often exhibit a high propensity to reconstitute, even without the conditional trigger, limiting their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific or often ineffective. Here, we report a computational design strategy grounded in fundamental protein biophysics to guide experimental evaluation of a sparse set of mutants to identify an optimal functional window. We hypothesized that testing a limited set of mutants would direct subsequent mutagenesis efforts by predicting desirable mutant combinations from a vast mutational landscape. This strategy varies the degree of interfacial destabilization while preserving stability and catalytic activity. We validate our method by solving two distinct split protein design challenges, generating both design and mechanistic insights. This new technology will streamline the generation and use of split protein systems for diverse applications.

Elucidation and refinement of synthetic receptor mechanisms.

Synthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors enable cell-based therapies to perform complex tasks such as regulating therapeutic gene expression in response to sensing physiological cues. Although multiple synthetic receptor systems now exist, many aspects of receptor performance are poorly understood. In general, it would be useful to understand how receptor design choices influence performance characteristics. In this study, we examined the modular extracellular sensor architecture (MESA) and systematically evaluated previously unexamined design choices, yielding substantially improved receptors. A key finding that might extend to other receptor systems is that the choice of transmembrane domain (TMD) is important for generating high-performing receptors. To provide mechanistic insights, we adopted and employed a Förster resonance energy transfer-based assay to elucidate how TMDs affect receptor complex formation and connected these observations to functional performance. To build further insight into these phenomena, we developed a library of new MESA receptors that sense an expanded set of ligands. Based upon these explorations, we conclude that TMDs affect signaling primarily by modulating intracellular domain geometry. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.

Building with intent: technologies and principles for engineering mammalian cell-based therapies to sense and respond.

The engineering of cells as programmable devices has enabled therapeutic strategies that could not otherwise be achieved. Such strategies include recapitulating and enhancing native cellular functions and composing novel functions. These novel functions may be composed using both natural and engineered biological components, with the latter exemplified by the development of synthetic receptor and signal transduction systems. Recent advances in implementing these approaches include the treatment of cancer, where the most clinical progress has been made to date, and the treatment of diabetes. Principles for engineering cell-based therapies that are safe and effective are increasingly needed and beginning to emerge, and will be essential in the development of this new class of therapeutics.

Rewiring human cellular input-output using modular extracellular sensors.

Engineered cell-based therapies comprise a promising emerging strategy for treating diverse diseases. Realizing this promise requires new tools for engineering cells to sense and respond to soluble extracellular factors, which provide information about both physiological state and the local environment. Here, we report such a biosensor engineering strategy, leveraging a self-contained receptor-signal transduction system termed modular extracellular sensor architecture (MESA). We developed MESA receptors that enable cells to sense vascular endothelial growth factor (VEGF) and, in response, secrete interleukin 2 (IL-2). By implementing these receptors in human T cells, we created a customized function not observed in nature-an immune cell that responds to a normally immunosuppressive cue (VEGF) by producing an immunostimulatory factor (IL-2). Because this platform utilizes modular, engineerable domains for ligand binding (antibodies) and output (programmable transcription factors based upon Cas9), this approach may be readily extended to novel inputs and outputs. This generalizable approach for rewiring cellular functions could enable both translational applications and fundamental biological research.