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

Muldoon, Joseph J.; Donahue, Patrick S.; Dolberg, Taylor B; Leonard, Joshua N.

doi:10.1016/j.cobme.2017.10.004

Cited by 8 publications

(3 citation statements)

References 52 publications

(57 reference statements)

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“…Engineered cell-based therapies are a promising strategy for the targeted treatment of many diseases ( 1–4 ). Central to this approach is the use of genetically encoded sense-and-response programs, which cause the cell to enact a therapeutic function upon detection of specified cues.…”

Section: Introductionmentioning

confidence: 99%

Elucidation and refinement of synthetic receptor mechanisms

et al. 2020

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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 (FRET)-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.

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Section: Introductionmentioning

confidence: 99%

Elucidation and refinement of synthetic receptor mechanisms

et al. 2020

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“…Cell‐based therapeutics use genetically engineered cells as vehicles of delivery for biologics in the treatment or prevention of diseases [ 1 , 2 ]. In these therapies, the stable integration of transgenes, most commonly through lentiviral or retroviral methods, is critical for consistent clinical responses in patients [ 3 ].…”

Section: Introductionmentioning

confidence: 99%

Design of a synthesis‐friendly hypoxia‐responsive promoter for cell‐based therapeutics

Lam

Truong

2021

Engineering in Life Sciences

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Towards the goal of making ‘smart’ cell therapies, one that recognizes disease conditions (e.g. hypoxia) and then produces mitigating biologics, it is important to develop suitable promoters. Currently, hypoxia responsive promoters are composed of strongly repeated sequences containing hypoxia response elements upstream of a minimal core promoter. Unfortunately, such repeated sequences have inherent genomic instability that may compromise the long‐term consistency of cell‐based therapeutics. Thus, we designed a synthesis‐friendly hypoxia‐inducible promoter (named SFHp) that has GC content between 25% and 75% and no repeats greater than 9 base pairs. In HEK293 cells stably integrated with genes regulated by synthetic SFHp, we demonstrated inducible reporter expression with fluorescent proteins, cell morphology rewiring with our previously engineered RhoA protein and intercellular cell signalling with secreted cytokines. These experiments exemplify the potential usage of SFHp in cell‐based therapeutics with integrated genetic circuits that inducibly respond to the disease microenvironment.

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“…Engineered cell-based therapies are a promising strategy for the targeted treatment of many diseases (1)(2)(3)(4). Central to this approach is the use of genetically encoded sense-and-response programs, which cause the cell to enact a therapeutic function upon detection of specified cues.…”

Section: Introductionmentioning

confidence: 99%

Elucidation and refinement of synthetic receptor mechanisms

Edelstein¹,

Donahue

Muldoon³

et al. 2020

Preprint

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Synthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors confer unique capabilities for detecting environmental ligands and transducing signals to control downstream gene expression events such as therapeutic protein production. For many applications, it would be useful to understand how receptor design choices impart desirable performance metrics and trade-offs. Towards this goal, we employed the existing modular extracellular sensor architecture (MESA) and systematically characterized biosensors with previously unexamined protein domain choices. A key finding that might extend to other receptor systems is that choice of transmembrane domain (TMD) is highly consequential. To provide mechanistic insights, we adopted and employed a FRET-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, and we apply this understanding to rationally tune receptors. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.

show abstract

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

Cited by 8 publications

References 52 publications

Elucidation and refinement of synthetic receptor mechanisms

Elucidation and refinement of synthetic receptor mechanisms

Design of a synthesis‐friendly hypoxia‐responsive promoter for cell‐based therapeutics

Elucidation and refinement of synthetic receptor mechanisms

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