Publications by authors named "Wendell A Lim"

109 Publications

T cell circuits that sense antigen density with an ultrasensitive threshold.

Science 2021 Feb 25. Epub 2021 Feb 25.

Cell Design Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA.

Overexpressed tumor associated antigens (e.g., HER2 and epidermal growth factor receptor) are attractive targets for therapeutic T cells, but toxic "off-tumor" cross-reaction with normal tissues expressing low levels of target antigen can occur with Chimeric Antigen Receptor (CAR) T cells. Inspired by natural ultrasensitive response circuits, we engineered a two-step positive feedback circuit that allows T cells to discriminate targets based on a sigmoidal antigen density threshold. In this circuit, a low affinity synthetic Notch receptor for HER2 controls the expression of a high affinity CAR for HER2. Increasing HER2 density thus has cooperative effects on T cells-it both increases CAR expression and activation-leading to a sigmoidal response. T cells with this circuit show sharp discrimination between target cells expressing normal amounts of HER2 and cancer cells expressing 100-fold more HER2, both in vitro and in vivo.
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http://dx.doi.org/10.1126/science.abc1855DOI Listing
February 2021

T cells selectively filter oscillatory signals on the minutes timescale.

Proc Natl Acad Sci U S A 2021 Mar;118(9)

HHMI, University of California, San Francisco, CA 94158;

T cells experience complex temporal patterns of stimulus via receptor-ligand-binding interactions with surrounding cells. From these temporal patterns, T cells are able to pick out antigenic signals while establishing self-tolerance. Although features such as duration of antigen binding have been examined, our understanding of how T cells interpret signals with different frequencies or temporal stimulation patterns is relatively unexplored. We engineered T cells to respond to light as a stimulus by building an optogenetically controlled chimeric antigen receptor (optoCAR). We discovered that T cells respond to minute-scale oscillations of activation signal by stimulating optoCAR T cells with tunable pulse trains of light. Systematically scanning signal oscillation period from 1 to 150 min revealed that expression of CD69, a T cell activation marker, reached a local minimum at a period of ∼25 min (corresponding to 5 to 15 min pulse widths). A combination of inhibitors and genetic knockouts suggest that this frequency filtering mechanism lies downstream of the Erk signaling branch of the T cell response network and may involve a negative feedback loop that diminishes Erk activity. The timescale of CD69 filtering corresponds with the duration of T cell encounters with self-peptide-presenting APCs observed via intravital imaging in mice, indicating a potential functional role for temporal filtering in vivo. This study illustrates that the T cell signaling machinery is tuned to temporally filter and interpret time-variant input signals in discriminatory ways.
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http://dx.doi.org/10.1073/pnas.2019285118DOI Listing
March 2021

DNA scaffolds enable efficient and tunable functionalization of biomaterials for immune cell modulation.

Nat Nanotechnol 2021 Feb 14;16(2):214-223. Epub 2020 Dec 14.

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA.

Biomaterials can improve the safety and presentation of therapeutic agents for effective immunotherapy, and a high level of control over surface functionalization is essential for immune cell modulation. Here, we developed biocompatible immune cell-engaging particles (ICEp) that use synthetic short DNA as scaffolds for efficient and tunable protein loading. To improve the safety of chimeric antigen receptor (CAR) T cell therapies, micrometre-sized ICEp were injected intratumorally to present a priming signal for systemically administered AND-gate CAR-T cells. Locally retained ICEp presenting a high density of priming antigens activated CAR T cells, driving local tumour clearance while sparing uninjected tumours in immunodeficient mice. The ratiometric control of costimulatory ligands (anti-CD3 and anti-CD28 antibodies) and the surface presentation of a cytokine (IL-2) on ICEp were shown to substantially impact human primary T cell activation phenotypes. This modular and versatile biomaterial functionalization platform can provide new opportunities for immunotherapies.
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http://dx.doi.org/10.1038/s41565-020-00813-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878327PMC
February 2021

Precise T cell recognition programs designed by transcriptionally linking multiple receptors.

Science 2020 11;370(6520):1099-1104

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.

Living cells often identify their correct partner or target cells by integrating information from multiple receptors, achieving levels of recognition that are difficult to obtain with individual molecular interactions. In this study, we engineered a diverse library of multireceptor cell-cell recognition circuits by using synthetic Notch receptors to transcriptionally interconnect multiple molecular recognition events. These synthetic circuits allow engineered T cells to integrate extra- and intracellular antigen recognition, are robust to heterogeneity, and achieve precise recognition by integrating up to three different antigens with positive or negative logic. A three-antigen AND gate composed of three sequentially linked receptors shows selectivity in vivo, clearing three-antigen tumors while ignoring related two-antigen tumors. Daisy-chaining multiple molecular recognition events together in synthetic circuits provides a powerful way to engineer cellular-level recognition.
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http://dx.doi.org/10.1126/science.abc6270DOI Listing
November 2020

Engineering cytokines and cytokine circuits.

Science 2020 11;370(6520):1034-1035

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA.

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http://dx.doi.org/10.1126/science.abb5607DOI Listing
November 2020

Engineering synthetic morphogen systems that can program multicellular patterning.

Science 2020 10;370(6514):327-331

Cell Design Institute, Department of Cellular and Molecular Pharmacology, and Howard Hughes Medical Institute, University of California San Francisco, San Francisco, CA 94158, USA.

In metazoan tissues, cells decide their fates by sensing positional information provided by specialized morphogen proteins. To explore what features are sufficient for positional encoding, we asked whether arbitrary molecules (e.g., green fluorescent protein or mCherry) could be converted into synthetic morphogens. Synthetic morphogens expressed from a localized source formed a gradient when trapped by surface-anchoring proteins, and they could be sensed by synthetic receptors. Despite their simplicity, these morphogen systems yielded patterns reminiscent of those observed in vivo. Gradients could be reshaped by altering anchor density or by providing a source of competing inhibitor. Gradient interpretation could be altered by adding feedback loops or morphogen cascades to receiver cell response circuits. Orthogonal cell-cell communication systems provide insight into morphogen evolution and a platform for engineering tissues.
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http://dx.doi.org/10.1126/science.abc0033DOI Listing
October 2020

Discriminatory Power of Combinatorial Antigen Recognition in Cancer T Cell Therapies.

Cell Syst 2020 Sep 10;11(3):215-228.e5. Epub 2020 Sep 10.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Cell Design Institute and Center for Synthetic Immunology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Precise discrimination of tumor from normal tissues remains a major roadblock for therapeutic efficacy of chimeric antigen receptor (CAR) T cells. Here, we perform a comprehensive in silico screen to identify multi-antigen signatures that improve tumor discrimination by CAR T cells engineered to integrate multiple antigen inputs via Boolean logic, e.g., AND and NOT. We screen >2.5 million dual antigens and ∼60 million triple antigens across 33 tumor types and 34 normal tissues. We find that dual antigens significantly outperform the best single clinically investigated CAR targets and confirm key predictions experimentally. Further, we identify antigen triplets that are predicted to show close to ideal tumor-versus-normal tissue discrimination for several tumor types. This work demonstrates the potential of 2- to 3-antigen Boolean logic gates for improving tumor discrimination by CAR T cell therapies. Our predictions are available on an interactive web server resource (antigen.princeton.edu).
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http://dx.doi.org/10.1016/j.cels.2020.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814417PMC
September 2020

The Design Principles of Biochemical Timers: Circuits that Discriminate between Transient and Sustained Stimulation.

Cell Syst 2019 09 11;9(3):297-308.e2. Epub 2019 Sep 11.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 600 16th Street, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Cell Design Initiative, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Many cellular responses for which timing is critical display temporal filtering-the ability to suppress response until stimulated for longer than a given minimal time. To identify biochemical circuits capable of kinetic filtering, we comprehensively searched the space of three-node enzymatic networks. We define a metric of "temporal ultrasensitivity," the steepness of activation as a function of stimulus duration. We identified five classes of core network motifs capable of temporal filtering, each with distinct functional properties such as rejecting high-frequency noise, committing to response (bistability), and distinguishing between long stimuli. Combinations of the two most robust motifs, double inhibition (DI) and positive feedback with AND logic (PF), underlie several natural timer circuits involved in processes such as cell cycle transitions, T cell activation, and departure from the pluripotent state. The biochemical network motifs described in this study form a basis for understanding common ways cells make dynamic decisions.
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http://dx.doi.org/10.1016/j.cels.2019.07.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763348PMC
September 2019

High-throughput multicolor optogenetics in microwell plates.

Nat Protoc 2019 07 24;14(7):2205-2228. Epub 2019 Jun 24.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA.

Optogenetic probes can be powerful tools for dissecting complexity in cell biology, but there is a lack of instrumentation to exploit their potential for automated, high-information-content experiments. This protocol describes the construction and use of the optoPlate-96, a platform for high-throughput three-color optogenetics experiments that allows simultaneous manipulation of common red- and blue-light-sensitive optogenetic probes. The optoPlate-96 enables illumination of individual wells in 96-well microwell plates or in groups of wells in 384-well plates. Its design ensures that there will be no cross-illumination between microwells in 96-well plates, and an active cooling system minimizes sample heating during light-intensive experiments. This protocol details the steps to assemble, test, and use the optoPlate-96. The device can be fully assembled without specialized equipment beyond a 3D printer and a laser cutter, starting from open-source design files and commercially available components. We then describe how to perform a typical optogenetics experiment using the optoPlate-96 to stimulate adherent mammalian cells. Although optoPlate-96 experiments are compatible with any plate-based readout, we describe analysis using quantitative single-cell immunofluorescence. This workflow thus allows complex optogenetics experiments (independent control of stimulation colors, intensity, dynamics, and time points) with high-dimensional outputs at single-cell resolution. Starting from 3D-printed and laser-cut components, assembly and testing of the optoPlate-96 can be accomplished in 3-4 h, at a cost of ~$600. A full optoPlate-96 experiment with immunofluorescence analysis can be performed within ~24 h, but this estimate is variable depending on the cell type and experimental parameters.
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http://dx.doi.org/10.1038/s41596-019-0178-yDOI Listing
July 2019

Engineering cell-cell communication networks: programming multicellular behaviors.

Curr Opin Chem Biol 2019 10 28;52:31-38. Epub 2019 May 28.

Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, and Center for Systems and Synthetic Biology, University of California San Francisco, San Francisco, CA 94158, USA. Electronic address:

Cell-cell communication governs the biological behaviors of multicellular populations such as developmental and immunological systems. Thanks to intense genetic analytical studies, the molecular components of cell-cell communication pathways have been well identified. We also have been developing synthetic biology tools to control cellular sensing and response systems that enable engineering of new cell-cell communication with design-based regulatory features. Recently, using these molecular backgrounds, synthetic cellular networks have been built and tested to understand the basic principles of multicellular biological behaviors. These approaches will provide new capabilities to control and program desired biological behaviors with engineered cell-cell communication to apply them toward cell-based therapeutics.
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http://dx.doi.org/10.1016/j.cbpa.2019.04.020DOI Listing
October 2019

Programming self-organizing multicellular structures with synthetic cell-cell signaling.

Science 2018 07 31;361(6398):156-162. Epub 2018 May 31.

Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, and Center for Systems and Synthetic Biology, University of California, San Francisco, CA 94158, USA.

A common theme in the self-organization of multicellular tissues is the use of cell-cell signaling networks to induce morphological changes. We used the modular synNotch juxtacrine signaling platform to engineer artificial genetic programs in which specific cell-cell contacts induced changes in cadherin cell adhesion. Despite their simplicity, these minimal intercellular programs were sufficient to yield assemblies with hallmarks of natural developmental systems: robust self-organization into multidomain structures, well-choreographed sequential assembly, cell type divergence, symmetry breaking, and the capacity for regeneration upon injury. The ability of these networks to drive complex structure formation illustrates the power of interlinking cell signaling with cell sorting: Signal-induced spatial reorganization alters the local signals received by each cell, resulting in iterative cycles of cell fate branching. These results provide insights into the evolution of multicellularity and demonstrate the potential to engineer customized self-organizing tissues or materials.
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http://dx.doi.org/10.1126/science.aat0271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492944PMC
July 2018

Building a Stable Relationship: Ensuring Homeostasis among Cell Types within a Tissue.

Cell 2018 02;172(4):638-640

Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, Center for Systems and Synthetic Biology, University of California, San Francisco, CA 94158, USA. Electronic address:

Many processes controlling cell growth and death are well characterized for individual cell lineages, but how ensembles of different cell types in a tissue regulate collective size and composition remains unclear. In this issue of Cell, Zhou et al. employ experiments and theory to uncover design principles of tissue homeostasis arising from cross-talk between fibroblasts and macrophages.
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http://dx.doi.org/10.1016/j.cell.2018.01.024DOI Listing
February 2018

Design of Tunable Oscillatory Dynamics in a Synthetic NF-κB Signaling Circuit.

Cell Syst 2017 11 25;5(5):460-470.e5. Epub 2017 Oct 25.

Center for Quantitative Biology and Peking-Tsinghua Joint Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China; The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing 100871, China. Electronic address:

Although oscillatory circuits are prevalent in transcriptional regulation, it is unclear how a circuit's structure and the specific parameters that describe its components determine the shape of its oscillations. Here, we engineer a minimal, inducible human nuclear factor κB (NF-κB)-based system that is composed of NF-κB (RelA) and degradable inhibitor of NF-κB (IκBα), into the yeast, Saccharomyces cerevisiae. We define an oscillation's waveform quantitatively as a function of signal amplitude, rest time, rise time, and decay time; by systematically tuning RelA concentration, the strength of negative feedback, and the degradation rate of IκBα, we demonstrate that peak shape and frequency of oscillations can be controlled in vivo and predicted mathematically. In addition, we show that nested negative feedback loops can be employed to specifically tune the frequency of oscillations while leaving their peak shape unchanged. In total, this work establishes design principles that enable function-guided design of oscillatory signaling controllers in diverse synthetic biology applications.
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http://dx.doi.org/10.1016/j.cels.2017.09.016DOI Listing
November 2017

Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control.

Mol Cell 2017 Sep 17;67(5):757-769.e5. Epub 2017 Aug 17.

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. Electronic address:

Cell signaling networks coordinate specific patterns of protein expression in response to external cues, yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway and monitoring a target gene's transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit; repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded by both dynamic filters and logic gates to shape target gene responses in a context-specific manner.
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http://dx.doi.org/10.1016/j.molcel.2017.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591080PMC
September 2017

Synthetic Immunology: Hacking Immune Cells to Expand Their Therapeutic Capabilities.

Annu Rev Immunol 2017 04;35:229-253

Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158; email:

The ability of immune cells to survey tissues and sense pathologic insults and deviations makes them a unique platform for interfacing with the body and disease. With the rapid advancement of synthetic biology, we can now engineer and equip immune cells with new sensors and controllable therapeutic response programs to sense and treat diseases that our natural immune system cannot normally handle. Here we review the current state of engineered immune cell therapeutics and their unique capabilities compared to small molecules and biologics. We then discuss how engineered immune cells are being designed to combat cancer, focusing on how new synthetic biology tools are providing potential ways to overcome the major roadblocks for treatment. Finally, we give a long-term vision for the use of synthetic biology to engineer immune cells as a general sensor-response platform to precisely detect disease, to remodel disease microenvironments, and to treat a potentially wide range of challenging diseases.
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http://dx.doi.org/10.1146/annurev-immunol-051116-052302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555230PMC
April 2017

CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells.

Sci Rep 2017 04 7;7(1):737. Epub 2017 Apr 7.

Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, 94158, USA.

Immunotherapies with chimeric antigen receptor (CAR) T cells and checkpoint inhibitors (including antibodies that antagonize programmed cell death protein 1 [PD-1]) have both opened new avenues for cancer treatment, but the clinical potential of combined disruption of inhibitory checkpoints and CAR T cell therapy remains incompletely explored. Here we show that programmed death ligand 1 (PD-L1) expression on tumor cells can render human CAR T cells (anti-CD19 4-1BBζ) hypo-functional, resulting in impaired tumor clearance in a sub-cutaneous xenograft model. To overcome this suppressed anti-tumor response, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing and lentiviral transduction to generate PD-1 deficient anti-CD19 CAR T cells. Pdcd1 (PD-1) disruption augmented CAR T cell mediated killing of tumor cells in vitro and enhanced clearance of PD-L1+ tumor xenografts in vivo. This study demonstrates improved therapeutic efficacy of Cas9-edited CAR T cells and highlights the potential of precision genome engineering to enhance next-generation cell therapies.
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http://dx.doi.org/10.1038/s41598-017-00462-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428439PMC
April 2017

The Principles of Engineering Immune Cells to Treat Cancer.

Cell 2017 02;168(4):724-740

Center for Cellular Immunotherapies, the Department of Pathology and Laboratory Medicine at the Perelman School of Medicine, and the Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Chimeric antigen receptor (CAR) T cells have proven that engineered immune cells can serve as a powerful new class of cancer therapeutics. Clinical experience has helped to define the major challenges that must be met to make engineered T cells a reliable, safe, and effective platform that can be deployed against a broad range of tumors. The emergence of synthetic biology approaches for cellular engineering is providing us with a broadly expanded set of tools for programming immune cells. We discuss how these tools could be used to design the next generation of smart T cell precision therapeutics.
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http://dx.doi.org/10.1016/j.cell.2017.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553442PMC
February 2017

Interrogating cellular perception and decision making with optogenetic tools.

J Cell Biol 2017 Jan 21;216(1):25-28. Epub 2016 Dec 21.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158

Optogenetics promises to deepen our understanding of how cells perceive and respond to complex and dynamic signals and how this perception regulates normal and abnormal function. In this study, we present our vision for how these nascent tools may transform our view of fundamental cell biological processes.
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http://dx.doi.org/10.1083/jcb.201612094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223619PMC
January 2017

Engineering Therapeutic T Cells: From Synthetic Biology to Clinical Trials.

Annu Rev Pathol 2017 Jan 5;12:305-330. Epub 2016 Dec 5.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco 94158-2517; email:

Engineered T cells are currently in clinical trials to treat patients with cancer, solid organ transplants, and autoimmune diseases. However, the field is still in its infancy. The design, and manufacturing, of T cell therapies is not standardized and is performed mostly in academic settings by competing groups. Reliable methods to define dose and pharmacokinetics of T cell therapies need to be developed. As of mid-2016, there are no US Food and Drug Administration (FDA)-approved T cell therapeutics on the market, and FDA regulations are only slowly adapting to the new technologies. Further development of engineered T cell therapies requires advances in immunology, synthetic biology, manufacturing processes, and government regulation. In this review, we outline some of these challenges and discuss the contributions that pathologists can make to this emerging field.
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http://dx.doi.org/10.1146/annurev-pathol-052016-100304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557092PMC
January 2017

Engineering dynamical control of cell fate switching using synthetic phospho-regulons.

Proc Natl Acad Sci U S A 2016 11 7;113(47):13528-13533. Epub 2016 Nov 7.

Howard Hughes Medical Institute, San Francisco, CA 94158;

Many cells can sense and respond to time-varying stimuli, selectively triggering changes in cell fate only in response to inputs of a particular duration or frequency. A common motif in dynamically controlled cells is a dual-timescale regulatory network: although long-term fate decisions are ultimately controlled by a slow-timescale switch (e.g., gene expression), input signals are first processed by a fast-timescale signaling layer, which is hypothesized to filter what dynamic information is efficiently relayed downstream. Directly testing the design principles of how dual-timescale circuits control dynamic sensing, however, has been challenging, because most synthetic biology methods have focused solely on rewiring transcriptional circuits, which operate at a single slow timescale. Here, we report the development of a modular approach for flexibly engineering phosphorylation circuits using designed phospho-regulon motifs. By then linking rapid phospho-feedback with slower downstream transcription-based bistable switches, we can construct synthetic dual-timescale circuits in yeast in which the triggering dynamics and the end-state properties of the ON state can be selectively tuned. These phospho-regulon tools thus open up the possibility to engineer cells with customized dynamical control.
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http://dx.doi.org/10.1073/pnas.1610973113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127309PMC
November 2016

Complex transcriptional modulation with orthogonal and inducible dCas9 regulators.

Nat Methods 2016 Dec 24;13(12):1043-1049. Epub 2016 Oct 24.

Department of Bioengineering, Stanford University, Stanford, California, USA.

The ability to dynamically manipulate the transcriptome is important for studying how gene networks direct cellular functions and how network perturbations cause disease. Nuclease-dead CRISPR-dCas9 transcriptional regulators, while offering an approach for controlling individual gene expression, remain incapable of dynamically coordinating complex transcriptional events. Here, we describe a flexible dCas9-based platform for chemical-inducible complex gene regulation. From a screen of chemical- and light-inducible dimerization systems, we identified two potent chemical inducers that mediate efficient gene activation and repression in mammalian cells. We combined these inducers with orthogonal dCas9 regulators to independently control expression of different genes within the same cell. Using this platform, we further devised AND, OR, NAND, and NOR dCas9 logic operators and a diametric regulator that activates gene expression with one inducer and represses with another. This work provides a robust CRISPR-dCas9-based platform for enacting complex transcription programs that is suitable for large-scale transcriptome engineering.
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http://dx.doi.org/10.1038/nmeth.4042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436902PMC
December 2016

Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors.

Cell 2016 Oct 29;167(2):419-432.e16. Epub 2016 Sep 29.

Department of Cellular & Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA; UCSF Center for Systems and Synthetic Biology, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, San Francisco, CA 94158, USA. Electronic address:

Redirecting T cells to attack cancer using engineered chimeric receptors provides powerful new therapeutic capabilities. However, the effectiveness of therapeutic T cells is constrained by the endogenous T cell response: certain facets of natural response programs can be toxic, whereas other responses, such as the ability to overcome tumor immunosuppression, are absent. Thus, the efficacy and safety of therapeutic cells could be improved if we could custom sculpt immune cell responses. Synthetic Notch (synNotch) receptors induce transcriptional activation in response to recognition of user-specified antigens. We show that synNotch receptors can be used to sculpt custom response programs in primary T cells: they can drive a la carte cytokine secretion profiles, biased T cell differentiation, and local delivery of non-native therapeutic payloads, such as antibodies, in response to antigen. SynNotch T cells can thus be used as a general platform to recognize and remodel local microenvironments associated with diverse diseases.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072533PMC
http://dx.doi.org/10.1016/j.cell.2016.09.011DOI Listing
October 2016

Modular engineering of cellular signaling proteins and networks.

Curr Opin Struct Biol 2016 08 15;39:106-114. Epub 2016 Jul 15.

Howard Hughes Medical Institute, United States; Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, 94158, United States. Electronic address:

Living cells respond to their environment using networks of signaling molecules that act as sensors, information processors, and actuators. These signaling systems are highly modular at both the molecular and network scales, and much evidence suggests that evolution has harnessed this modularity to rewire and generate new physiological behaviors. Conversely, we are now finding that, following nature's example, signaling modules can be recombined to form synthetic tools for monitoring, interrogating, and controlling the behavior of cells. Here we highlight recent progress in the modular design of synthetic receptors, optogenetic switches, and phospho-regulated proteins and circuits, and discuss the expanding role of combinatorial design in the engineering of cellular signaling proteins and networks.
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http://dx.doi.org/10.1016/j.sbi.2016.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127285PMC
August 2016

CRISPR/Cas9 for Human Genome Engineering and Disease Research.

Annu Rev Genomics Hum Genet 2016 08 23;17:131-54. Epub 2016 May 23.

Department of Bioengineering, Stanford University, Stanford, California 94305; email: ,

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, a versatile RNA-guided DNA targeting platform, has been revolutionizing our ability to modify, manipulate, and visualize the human genome, which greatly advances both biological research and therapeutics development. Here, we review the current development of CRISPR/Cas9 technologies for gene editing, transcription regulation, genome imaging, and epigenetic modification. We discuss the broad application of this system to the study of functional genomics, especially genome-wide genetic screening, and to therapeutics development, including establishing disease models, correcting defective genetic mutations, and treating diseases.
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http://dx.doi.org/10.1146/annurev-genom-083115-022258DOI Listing
August 2016

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function.

Elife 2016 04 28;5. Epub 2016 Apr 28.

Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, United States.

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo.
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http://dx.doi.org/10.7554/eLife.13450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4880442PMC
April 2016

Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits.

Cell 2016 Feb 28;164(4):770-9. Epub 2016 Jan 28.

Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, San Francisco, CA 94158, USA. Electronic address:

T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen "bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2016.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4752902PMC
February 2016

Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors.

Cell 2016 Feb 28;164(4):780-91. Epub 2016 Jan 28.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute. Electronic address:

The Notch protein is one of the most mechanistically direct transmembrane receptors-the intracellular domain contains a transcriptional regulator that is released from the membrane when engagement of the cognate extracellular ligand induces intramembrane proteolysis. We find that chimeric forms of Notch, in which both the extracellular sensor module and the intracellular transcriptional module are replaced with heterologous protein domains, can serve as a general platform for generating novel cell-cell contact signaling pathways. Synthetic Notch (synNotch) pathways can drive user-defined functional responses in diverse mammalian cell types. Because individual synNotch pathways do not share common signaling intermediates, the pathways are functionally orthogonal. Thus, multiple synNotch receptors can be used in the same cell to achieve combinatorial integration of environmental cues, including Boolean response programs, multi-cellular signaling cascades, and self-organized cellular patterns. SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues.
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http://dx.doi.org/10.1016/j.cell.2016.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4752866PMC
February 2016

Mapping the functional versatility and fragility of Ras GTPase signaling circuits through in vitro network reconstitution.

Elife 2016 Jan 14;5. Epub 2016 Jan 14.

Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.

The Ras-superfamily GTPases are central controllers of cell proliferation and morphology. Ras signaling is mediated by a system of interacting molecules: upstream enzymes (GEF/GAP) regulate Ras's ability to recruit multiple competing downstream effectors. We developed a multiplexed, multi-turnover assay for measuring the dynamic signaling behavior of in vitro reconstituted H-Ras signaling systems. By including both upstream regulators and downstream effectors, we can systematically map how different network configurations shape the dynamic system response. The concentration and identity of both upstream and downstream signaling components strongly impacted the timing, duration, shape, and amplitude of effector outputs. The distorted output of oncogenic alleles of Ras was highly dependent on the balance of positive (GAP) and negative (GEF) regulators in the system. We found that different effectors interpreted the same inputs with distinct output dynamics, enabling a Ras system to encode multiple unique temporal outputs in response to a single input. We also found that different Ras-to-GEF positive feedback mechanisms could reshape output dynamics in distinct ways, such as signal amplification or overshoot minimization. Mapping of the space of output behaviors accessible to Ras provides a design manual for programming Ras circuits, and reveals how these systems are readily adapted to produce an array of dynamic signaling behaviors. Nonetheless, this versatility comes with a trade-off of fragility, as there exist numerous paths to altered signaling behaviors that could cause disease.
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http://dx.doi.org/10.7554/eLife.12435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775219PMC
January 2016

Expanding the CRISPR imaging toolset with Staphylococcus aureus Cas9 for simultaneous imaging of multiple genomic loci.

Nucleic Acids Res 2016 05 5;44(8):e75. Epub 2016 Jan 5.

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94143, USA Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143, USA

In order to elucidate the functional organization of the genome, it is vital to directly visualize the interactions between genomic elements in living cells. For this purpose, we engineered the Cas9 protein from Staphylococcus aureus (SaCas9) for the imaging of endogenous genomic loci, which showed a similar robustness and efficiency as previously reported for Streptococcus pyogenes Cas9 (SpCas9). Imaging readouts allowed us to characterize the DNA-binding activity of SaCas9 and to optimize its sgRNA scaffold. Combining SaCas9 and SpCas9, we demonstrated two-color CRISPR imaging with the capability to resolve genomic loci spaced by <300 kb. Combinatorial color-mixing further enabled us to code multiple genomic elements in the same cell. Our results highlight the potential of combining SpCas9 and SaCas9 for multiplexed CRISPR-Cas9 applications, such as imaging and genome engineering.
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http://dx.doi.org/10.1093/nar/gkv1533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856973PMC
May 2016