Synthetic mammalian signaling circuits for robust cell population control.

In multicellular organisms, cells actively sense and control their own population density. Synthetic mammalian quorum-sensing circuits could provide insight into principles of population control and extend cell therapies. However, a key challenge is reducing their inherent sensitivity to "cheater" mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal mammalian cell-cell communication and quorum sensing. We designed a paradoxical population control circuit, termed "Paradaux," in which auxin stimulates and inhibits net cell growth at different concentrations. This circuit limited population size over extended timescales of up to 42 days of continuous culture. By contrast, when operating in a non-paradoxical regime, population control became more susceptible to mutational escape. These results establish auxin as a versatile "private" communication system and demonstrate that paradoxical circuit architectures can provide robust population control.

Organs-on-a-chip models for biological research.

We explore the utility of bioengineered human tissues-individually or connected into physiological units-for biological research. While much smaller and simpler than their native counterparts, these tissues are complex enough to approximate distinct tissue phenotypes: molecular, structural, and functional. Unlike organoids, which form spontaneously and recapitulate development, "organs-on-a-chip" are engineered to display some specific functions of whole organs. Looking back, we discuss the key developments of this emerging technology. Thinking forward, we focus on the challenges faced to fully establish, validate, and utilize the fidelity of these models for biological research.

The Emerging Landscape of Immune Cell Therapies.

Cell therapies present an entirely new paradigm in drug development. Within this class, immune cell therapies are among the most advanced, having already demonstrated definitive evidence of clinical benefits in cancer and infectious disease. Numerous features distinguish these "living therapies" from traditional medicines, including their ability to expand and contract in proportion to need and to mediate therapeutic benefits for months or years following a single application. Continued advances in fundamental immunology, genetic engineering, gene editing, and synthetic biology exponentially expand opportunities to enhance the sophistication of immune cell therapies, increasing potency and safety and broadening their potential for treatment of disease. This perspective will summarize the current status of immune cell therapies for cancer, infectious disease, and autoimmunity, and discuss advances in cellular engineering to overcome barriers to progress.

The Principles of Engineering Immune Cells to Treat Cancer.

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.

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

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.

A Receptor for All Occasions.

Cells communicate with their environment, in part, through cell surface receptors. Engineering receptors that both sense arbitrary inputs and provide outputs orthogonal to endogenous signaling pathways has been a challenge. Now, Lim and colleagues report a system based on synthetic Notch receptors that allows independent control of both inputs and outputs in diverse cell types.

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

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.

Designing cell function: assembly of synthetic gene circuits for cell biology applications.

Synthetic biology is the discipline of engineering application-driven biological functionalities that were not evolved by nature. Early breakthroughs of cell engineering, which were based on ectopic (over)expression of single sets of transgenes, have already had a revolutionary impact on the biotechnology industry, regenerative medicine and blood transfusion therapies. Now, we require larger-scale, rationally assembled genetic circuits engineered to programme and control various human cell functions with high spatiotemporal precision in order to solve more complex problems in applied life sciences, biomedicine and environmental sciences. This will open new possibilities for employing synthetic biology to advance personalized medicine by converting cells into living therapeutics to combat hitherto intractable diseases.

Expanding chemistry through in vitro and in vivo biocatalysis.

Living systems contain a vast network of metabolic reactions, providing a wealth of enzymes and cells as potential biocatalysts for chemical processes. The properties of protein and cell biocatalysts-high selectivity, the ability to control reaction sequence and operation in environmentally benign conditions-offer approaches to produce molecules at high efficiency while lowering the cost and environmental impact of industrial chemistry. Furthermore, biocatalysis offers the opportunity to generate chemical structures and functions that may be inaccessible to chemical synthesis. Here we consider developments in enzymes, biosynthetic pathways and cellular engineering that enable their use in catalysis for new chemistry and beyond.

CellNet--where your cells are standing.

The manufacturing of clinically relevant cells is a widely used strategy in regenerative medicine. Cahan et al. develop a network biology platform named CellNet to accurately assess the fidelity of such cells and spot aberrant regulatory networks, and Morris et al. apply this platform to improve cell manufacturing.

Dissecting engineered cell types and enhancing cell fate conversion via CellNet.

Engineering clinically relevant cells in vitro holds promise for regenerative medicine, but most protocols fail to faithfully recapitulate target cell properties. To address this, we developed CellNet, a network biology platform that determines whether engineered cells are equivalent to their target tissues, diagnoses aberrant gene regulatory networks, and prioritizes candidate transcriptional regulators to enhance engineered conversions. Using CellNet, we improved B cell to macrophage conversion, transcriptionally and functionally, by knocking down predicted B cell regulators. Analyzing conversion of fibroblasts to induced hepatocytes (iHeps), CellNet revealed an unexpected intestinal program regulated by the master regulator Cdx2. We observed long-term functional engraftment of mouse colon by iHeps, thereby establishing their broader potential as endoderm progenitors and demonstrating direct conversion of fibroblasts into intestinal epithelium. Our studies illustrate how CellNet can be employed to improve direct conversion and to uncover unappreciated properties of engineered cells.

CellNet: network biology applied to stem cell engineering.

Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering.

Programming multicellular assembly with synthetic cell adhesion molecules.

Cell adhesion molecules are ubiquitous in multicellular organisms, specifying precise cell-cell interactions in processes as diverse as tissue development, immune cell trafficking and the wiring of the nervous system1-4. Here we show that a wide array of synthetic cell adhesion molecules can be generated by combining orthogonal extracellular interactions with intracellular domains from native adhesion molecules, such as cadherins and integrins. The resulting molecules yield customized cell-cell interactions with adhesion properties that are similar to native interactions. The identity of the intracellular domain of the synthetic cell adhesion molecules specifies interface morphology and mechanics, whereas diverse homotypic or heterotypic extracellular interaction domains independently specify the connectivity between cells. This toolkit of orthogonal adhesion molecules enables the rationally programmed assembly of multicellular architectures, as well as systematic remodelling of native tissues. The modularity of synthetic cell adhesion molecules provides fundamental insights into how distinct classes of cell-cell interfaces may have evolved. Overall, these tools offer powerful abilities for cell and tissue engineering and for systematically studying multicellular organization.

Co-opting signalling molecules enables logic-gated control of CAR T cells.

Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity3-5; however, a truly safe and effective logic-gated CAR has remained elusive6. Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity7 and fibrosis8. In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering.

Multiple Input Sensing and Signal Integration Using a Split Cas12a System.

The ability to integrate biological signals and execute a functional response when appropriate is critical for sophisticated cell engineering using synthetic biology. Although the CRISPR-Cas system has been harnessed for synthetic manipulation of the genome, it has not been fully utilized for complex environmental signal sensing, integration, and actuation. Here, we develop a split dCas12a platform and show that it allows for the construction of multi-input, multi-output logic circuits in mammalian cells. The system is highly programmable and can generate expandable AND gates with two, three, and four inputs. It can also incorporate NOT logic by using anti-CRISPR proteins as an OFF switch. By coupling the split dCas12a design to multiple tumor-relevant promoters, we provide a proof of concept that the system can implement logic gating to specifically detect breast cancer cells and execute therapeutic immunomodulatory responses.

Bioengineering methods for analysis of cells in vitro.

Efforts in the interdisciplinary field of bioengineering have led to innovative methods for investigating the complexities of cell responses in vitro. These approaches have emphasized the reduction of complex multicomponent cellular microenvironments into distinct individual signals as a means to both (a) better construct mimics of in vivo microenvironments and (b) better deconstruct microenvironments to study them. Microtechnology tools, together with advances in biomaterials, have been fundamental to this progress by enabling the tightly controlled presentation of environmental cues and the improved systematic analysis of cellular perturbations. In this review, we describe bioengineering approaches for controlling and measuring cell-environmental interactions in vitro, including strategies for high-throughput analysis. We also describe the mechanistic insights gained by the use of these novel tools, with associated applications ranging from fundamental biological studies, in vitro modeling of in vivo processes, and cell-based therapies.

Global View of Domain-Specific O-Linked Mannose Glycosylation in Glycoengineered Cells.

Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved posttranslational modification that fulfills important biological roles during embryonic development. Three nonredundant enzyme families, POMT1/POMT2, TMTC1-4, and TMEM260, selectively coordinate the initiation of protein O-Man glycosylation on distinct classes of transmembrane proteins, including α-dystroglycan, cadherins, and plexin receptors. However, a systematic investigation of their substrate specificities is lacking, in part due to the ubiquitous expression of O-Man glycosyltransferases in cells, which precludes analysis of pathway-specific O-Man glycosylation on a proteome-wide scale. Here, we apply a targeted workflow for membrane glycoproteomics across five human cell lines to extensively map O-Man substrates and genetically deconstruct O-Man initiation by individual and combinatorial knockout of O-Man glycosyltransferase genes. We established a human cell library for the analysis of substrate specificities of individual O-Man initiation pathways by quantitative glycoproteomics. Our results identify 180 O-Man glycoproteins, demonstrate new protein targets for the POMT1/POMT2 pathway, and show that TMTC1-4 and TMEM260 pathways widely target distinct Ig-like protein domains of plasma membrane proteins involved in cell-cell and cell-extracellular matrix interactions. The identification of O-Man on Ig-like folds adds further knowledge on the emerging concept of domain-specific O-Man glycosylation which opens for functional studies of O-Man-glycosylated adhesion molecules and receptors.

Nanoinjection: A Platform for Innovation in Ex Vivo Cell Engineering.

ConspectusIn human cells, intracellular access and therapeutic cargo transport, including gene-editing tools (e.g., CRISPR-Cas9 and transposons), nucleic acids (e.g., DNA, mRNA, and siRNA), peptides, and proteins (e.g., enzymes and antibodies), are tightly constrained to ensure healthy cell function and behavior. This principle is exemplified in the delivery mechanisms of chimeric antigen receptor (CAR)-T cells for ex-vivo immunotherapy. In particular, the clinical success of CAR-T cells has established a new standard of care by curing previously incurable blood cancers. The approach involves the delivery, typically via the use of electroporation (EP) and lentivirus, of therapeutic CAR genes into a patient's own T cells, which are then engineered to express CARs that target and combat their blood cancer. But the key difficulty lies in genetically manipulating these cells without causing irreversible damage or loss of function─all the while minimizing complexities of manufacturing, safety concerns, and costs, and ensuring the efficacy of the final CAR-T cell product.Nanoinjection─the process of intracellular delivery using nanoneedles (NNs)─is an emerging physical delivery route that efficiently negotiates the plasma membrane of many cell types, including primary human T cells. It occurs with minimal perturbation, invasiveness, and toxicity, with high efficiency and throughput at high spatial and temporal resolutions. Nanoinjection promises greatly improved delivery of a broad range of therapeutic cargos with little or no damage to those cargos. A nanoinjection platform allows these cargos to function in the intracellular space as desired. The adaptability of nanoinjection platforms is now bringing major advantages in immunomodulation, mechanotransduction, sampling of cell states (nanobiopsy), controlled intracellular interrogation, and the primary focus of this account─intracellular delivery and its applications in ex vivo cell engineering.Mechanical nanoinjection typically exerts direct mechanical force on the cell membrane, offering a straightforward route to improve membrane perturbation by the NNs and subsequent transport of genetic cargo into targeted cell type (adherent or suspension cells). By contrast, electroactive nanoinjection is controlled by coupling NNs with an electric field─a new route for activating electroporation (EP) at the nanoscale─allowing a dramatic reduction of the applied voltage to a cell and so minimizing post-EP damage to cells and cargo, and overcoming many of the limitations of conventional bulk EP. Nanoinjection transcends mere technique; it is an approach to cell engineering ex vivo, offering the potential to endow cells with new, powerful features such as generating chimeric antigen receptor (CAR)-T cells for future CAR-T cell technologies.We first discuss the manufacturing of NN devices (Section 2), then delve into nanoinjection-mediated cell engineering (Section 3), nanoinjection mechanisms and interfacing methodologies (Section 4), and emerging applications in using nanoinjection to create functional CAR-T cells (Section 5).

Novel extragenic genomic safe harbors for precise therapeutic T-cell engineering.

Cell therapies that rely on engineered immune cells can be enhanced by achieving uniform and controlled transgene expression in order to maximize T-cell function and achieve predictable patient responses. Although they are effective, current genetic engineering strategies that use γ-retroviral, lentiviral, and transposon-based vectors to integrate transgenes, unavoidably produce variegated transgene expression in addition to posing a risk of insertional mutagenesis. In the setting of chimeric antigen receptor (CAR) therapy, inconsistent and random CAR expression may result in tonic signaling, T-cell exhaustion, and variable T-cell persistence. Here, we report and validate an algorithm for the identification of extragenic genomic safe harbors (GSH) that can be efficiently targeted for DNA integration and can support sustained and predictable CAR expression in human peripheral blood T cells. The algorithm is based on 7 criteria established to minimize genotoxicity by directing transgene integration away from functionally important genomic elements, maximize efficient CRISPR/Cas9-mediated targeting, and avert transgene silencing over time. T cells engineered to express a CD19 CAR at GSH6, which meets all 7 criteria, are curative at low cell dose in a mouse model of acute lymphoblastic leukemia, matching the potency of CAR T cells engineered at the TRAC locus and effectively resisting tumor rechallenge 100 days after their infusion. The identification of functional extragenic GSHs thus expands the human genome available for therapeutic precision engineering.