The evolution of synthetic receptor systems.

Receptors enable cells to detect, process and respond to information about their environments. Over the past two decades, synthetic biologists have repurposed physical parts and concepts from natural receptors to engineer synthetic receptors. These technologies implement customized sense-and-respond programs that link a cell's interaction with extracellular and intracellular cues to user-defined responses. When combined with tools for information processing, these advances enable programming of sophisticated customized functions. In recent years, the library of synthetic receptors and their capabilities has substantially evolved-a term we employ here to mean systematic improvement and expansion. Here, we survey the existing mammalian synthetic biology toolkit of protein-based receptors and signal-processing components, highlighting efforts to evolve and integrate some of the foundational synthetic receptor systems. We then propose a generalized strategy for engineering and improving receptor systems to meet defined functional objectives called a 'metric-enabled approach for synthetic receptor engineering' (MEASRE).

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.

Elucidating Design Principles for Engineering Cell-Derived Vesicles to Inhibit SARS-CoV-2 Infection.

The ability of pathogens to develop drug resistance is a global health challenge. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody (mAb) therapies and vaccine-induced sera. Decoy nanoparticles-cell-mimicking particles that bind and inhibit virions-are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, quantitative understanding as to how design features impact performance of these therapeutics is lacking. To address this gap, this study presents a systematic, comparative evaluation of various biologically derived nanoscale vesicles, which may be particularly well suited to sustained or repeated administration in the clinic due to low toxicity, and investigates their potential to inhibit multiple classes of model SARS-CoV-2 virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus-decoy binding affinities. In addition, these cell-mimicking vesicles effectively inhibit model SARS-CoV-2 variants that evade mAbs and recombinant protein-based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS-CoV-2 and other viral infections.

Nanofountain Probe Electroporation Enables Versatile Single-Cell Intracellular Delivery and Investigation of Postpulse Electropore Dynamics.

Introducing exogenous molecules into cells with high efficiency and dosage control is a crucial step in basic research as well as clinical applications. Here, the capability of the nanofountain probe electroporation (NFP-E) system to deliver proteins and plasmids in a variety of continuous and primary cell types with appropriate dosage control is reported. It is shown that the NFP-E can achieve fine control over the relative expression of two cotransfected plasmids. Finally, the dynamics of electropore closure after the pulsing ends with the NFP-E is investigated. Localized electroporation has recently been utilized to demonstrate the converse process of delivery (sampling), in which a small volume of the cytosol is retrieved during electroporation without causing cell lysis. Single-cell temporal sampling confers the benefit of monitoring the same cell over time and can provide valuable insights into the mechanisms underlying processes such as stem cell differentiation and disease progression. NFP-E parameters that maximize the membrane resealing time, which is essential for increasing the sampled volume and in meeting the challenge of monitoring low copy number biomarkers, are identified. Its application in CRISPR/Cas9 gene editing, stem cell reprogramming, and single-cell sampling studies is envisioned.

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.

Therapeutic applications of extracellular vesicles: clinical promise and open questions.

This review provides an updated perspective on rapidly proliferating efforts to harness extracellular vesicles (EVs) for therapeutic applications. We summarize current knowledge, emerging strategies, and open questions pertaining to clinical potential and translation. Potentially useful EVs comprise diverse products of various cell types and species. EV components may also be combined with liposomes and nanoparticles to facilitate manufacturing as well as product safety and evaluation. Potential therapeutic cargoes include RNA, proteins, and drugs. Strategic issues considered herein include choice of therapeutic agent, means of loading cargoes into EVs, promotion of EV stability, tissue targeting, and functional delivery of cargo to recipient cells. Some applications may harness natural EV properties, such as immune modulation, regeneration promotion, and pathogen suppression. These properties can be enhanced or customized to enable a wide range of therapeutic applications, including vaccination, improvement of pregnancy outcome, and treatment of autoimmune disease, cancer, and tissue injury.

Stabilization of exosome-targeting peptides via engineered glycosylation.

Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics.

Contributions of unique intracellular domains to switchlike biosensing by Toll-like receptor 4.

Toll-like receptors (TLRs) mediate immune recognition of both microbial infections and tissue damage. Aberrant TLR signaling promotes disease; thus, understanding the regulation of TLR signaling is of medical relevance. Although downstream mediators of TLR signaling have been identified, the detailed mechanism by which ligand binding-mediated dimerization induces downstream signaling remains poorly understood. Here, we investigate this question for TLR4, which mediates responsiveness to bacterial LPS and drives inflammatory disease. TLR4 exhibits structural and functional features that are unique among TLRs, including responsiveness to a wide variety of ligands. However, the connection between these structural features and the regulation of signaling is not clear. Here, we investigated how the unique intracellular structures of TLR4 contribute to receptor signaling. Key conclusions include the following. 1) The unique intracellular linker of TLR4 is important for achieving LPS-inducible signaling via Toll/IL-1 receptor (TIR) domain-containing adapter-inducing interferon-ß (TRIF) but less so for signaling via myeloid differentiation primary response 88 (MyD88). 2) Membrane-bound TLR4 TIR domains were sufficient to induce signaling. However, introducing long, flexible intracellular linkers neither induced constitutive signaling nor ablated LPS-inducible signaling. Thus, the initiation of TLR4 signaling is regulated by a mechanism that does not require tight geometric constraints. Together, these observations necessitate refining the model of TLR4 signal initiation. We hypothesize that TLR4 may interact with an inhibitory partner in the absence of ligand, via both TIR and extracellular domains of TLR4. In this speculative model, ligand binding induces dissociation of the inhibitory partner, triggering spontaneous, switchlike TIR domain homodimerization to initiate downstream signaling.

Adding energy minimization strategy to peptide-design algorithm enables better search for RNA-binding peptides: Redesigned λ N peptide binds boxB RNA.

Our previously developed peptide-design algorithm was improved by adding an energy minimization strategy which allows the amino acid sidechains to move in a broad configuration space during sequence evolution. In this work, the new algorithm was used to generate a library of 21-mer peptides which could substitute for λ N peptide in binding to boxB RNA. Six potential peptides were obtained from the algorithm, all of which exhibited good binding capability with boxB RNA. Atomistic molecular dynamics simulations were then conducted to examine the ability of the λ N peptide and three best evolved peptides, viz. Pept01, Pept26, and Pept28, to bind to boxB RNA. Simulation results demonstrated that our evolved peptides are better at binding to boxB RNA than the λ N peptide. Sequence searches using the old (without energy minimization strategy) and new (with energy minimization strategy) algorithms confirm that the new algorithm is more effective at finding good RNA-binding peptides than the old algorithm. © 2016 Wiley Periodicals, Inc.

Spatial and functional heterogeneities shape collective behavior of tumor-immune networks.

Tumor growth involves a dynamic interplay between cancer cells and host cells, which collectively form a tumor microenvironmental network that either suppresses or promotes tumor growth under different conditions. The transition from tumor suppression to tumor promotion is mediated by a tumor-induced shift in the local immune state, and despite the clinical challenge this shift poses, little is known about how such dysfunctional immune states are initiated. Clinical and experimental observations have indicated that differences in both the composition and spatial distribution of different cell types and/or signaling molecules within the tumor microenvironment can strongly impact tumor pathogenesis and ultimately patient prognosis. How such "functional" and "spatial" heterogeneities confer such effects, however, is not known. To investigate these phenomena at a level currently inaccessible by direct observation, we developed a computational model of a nascent metastatic tumor capturing salient features of known tumor-immune interactions that faithfully recapitulates key features of existing experimental observations. Surprisingly, over a wide range of model formulations, we observed that heterogeneity in both spatial organization and cell phenotype drove the emergence of immunosuppressive network states. We determined that this observation is general and robust to parameter choice by developing a systems-level sensitivity analysis technique, and we extended this analysis to generate other parameter-independent, experimentally testable hypotheses. Lastly, we leveraged this model as an in silico test bed to evaluate potential strategies for engineering cell-based therapies to overcome tumor associated immune dysfunction and thereby identified modes of immune modulation predicted to be most effective. Collectively, this work establishes a new integrated framework for investigating and modulating tumor-immune networks and provides insights into how such interactions may shape early stages of tumor formation.

Systems hematology: an introduction.

Hematologists have traditionally studied blood and its components by simplifying it into its components and functions. A variety of new techniques have generated large and complex datasets. Coupled to an appreciation of blood as a dynamic system, a new approach in systems hematology is needed. Systems hematology embraces the multi-scale complexity with a combination of mathematical, engineering, and computational tools for constructing and validating models of biological phenomena. The validity of mathematical modeling in hematopoiesis was established early by the pioneering work of Till and McCulloch. This volume seeks to introduce to the various scientists and physicians to the multi-faceted field of hematology by highlighting recent works in systems biology. Deterministic, stochastic, statistical, and network-based models have been used to better understand a range of topics in hematopoiesis, including blood cell production, the periodicity of cyclical neutropenia, stem cell production in response to cytokine administration, and the emergence of drug resistance. Future advances require technological improvements in computing power, imaging, and proteomics as well as greater collaboration between experimentalists and modelers. Altogether, systems hematology will improve our understanding of normal and abnormal hematopoiesis, better define stem cells and their daughter cells, and potentially lead to more effective therapies.

Engineered cell-based therapies: a vanguard of design-driven medicine.

Engineered cell-based therapies are uniquely capable of performing sophisticated therapeutic functions in vivo, and this strategy is yielding promising clinical benefits for treating cancer. In this review, we discuss key opportunities and challenges for engineering customized cellular functions using cell-based therapy for cancer as a representative case study. We examine the historical development of chimeric antigen receptor (CAR) therapies as an illustration of the engineering design cycle. We also consider the potential roles that the complementary disciplines of systems biology and synthetic biology may play in realizing safe and effective treatments for a broad range of patients and diseases. In particular, we discuss how systems biology may facilitate both fundamental research and clinical translation, and we describe how the emerging field of synthetic biology is providing novel modalities for building customized cellular functions to overcome existing clinical barriers. Together, these approaches provide a powerful set of conceptual and experimental tools for transforming information into understanding, and for translating understanding into novel therapeutics to establish a new framework for design-driven medicine.

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.

Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions.

Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.

HaloTag display enables quantitative single-particle characterisation and functionalisation of engineered extracellular vesicles.

Extracellular vesicles (EVs) play key roles in diverse biological processes, transport biomolecules between cells and have been engineered for therapeutic applications. A useful EV bioengineering strategy is to express engineered proteins on the EV surface to confer targeting, bioactivity and other properties. Measuring how incorporation varies across a population of EVs is important for characterising such materials and understanding their function, yet it remains challenging to quantitatively characterise the absolute number of engineered proteins incorporated at single-EV resolution. To address these needs, we developed a HaloTag-based characterisation platform in which dyes or other synthetic species can be covalently and stoichiometrically attached to engineered proteins on the EV surface. To evaluate this system, we employed several orthogonal quantification methods, including flow cytometry and fluorescence microscopy, and found that HaloTag-mediated quantification is generally robust across EV analysis methods. We compared HaloTag-labelling to antibody-labelling of EVs using single vesicle flow cytometry, enabling us to measure the substantial degree to which antibody labelling can underestimate proteins present on an EV. Finally, we demonstrate the use of HaloTag to compare between protein designs for EV bioengineering. Overall, the HaloTag system is a useful EV characterisation tool which complements and expands existing methods.

TLR3-specific double-stranded RNA oligonucleotide adjuvants induce dendritic cell cross-presentation, CTL responses, and antiviral protection.

Maturation of dendritic cells (DC) to competent APC is essential for the generation of acquired immunity and is a major function of adjuvants. dsRNA, a molecular signature of viral infection, drives DC maturation by activating TLR3, but the size of dsRNA required to activate DC and the expression patterns of TLR3 protein in DC subsets have not been established. In this article, we show that cross-priming CD8α(+) and CD103(+) DC subsets express much greater levels of TLR3 than other DC. In resting DC, TLR3 is located in early endosomes and other intracellular compartments but migrates to LAMP1(+) endosomes on stimulation with a TLR3 ligand. Using homogeneous dsRNA oligonucleotides (ONs) ranging in length from 25 to 540 bp, we observed that a minimum length of ∼90 bp was sufficient to induce CD86, IL-12p40, IFN-ß, TNF-α, and IL-6 expression, and to mature DC into APC that cross-presented exogenous Ags to CD8(+) T cells. TLR3 was essential for activation of DC by dsRNA ONs, and the potency of activation increased with dsRNA length and varied between DC subsets. In vivo, dsRNA ONs, in a size-dependent manner, served as adjuvants for the generation of Ag-specific CTL and for inducing protection against lethal challenge with influenza virus when given with influenza nucleoprotein as an immunogen. These results provide the basis for the development of TLR3-specific adjuvants capable of inducing immune responses tailored for viral pathogens.

Teaching systematic, reproducible model development using synthetic biology.

We present an educational unit to teach computational modeling, a vital part of chemical engineering curricula, through the lens of synthetic biology. Lectures, code, and homework questions provide conceptual and practical introductions to each computational method involved in the model development process, along with perspectives on how methods can be iterated upon to arrive at a final model. Ultimately, this content can be applied broadly to address questions in synthetic biology and classical chemical engineering.

Comparative Evaluation of Synthetic Cytokines for Enhancing Production and Performance of NK92 Cell-Based Therapies.

Off-the shelf immune cell therapies are potentially curative and may offer cost and manufacturing advantages over autologous products, but further development is needed. The NK92 cell line has a natural killer-like phenotype, has efficacy in cancer clinical trials, and is safe after irradiation. However, NK92 cells lose activity post-injection, limiting efficacy. This may be addressed by engineering NK92 cells to express stimulatory factors, and comparative analysis is needed. Thus, we systematically explored the expression of synthetic cytokines for enhancing NK92 cell production and performance. All synthetic cytokines evaluated (membrane-bound IL2 and IL15, and engineered versions of Neoleukin-2/15, IL15, IL12, and decoy resistant IL18) enhanced NK92 cell cytotoxicity. Engineered cells were preferentially expanded by expressing membrane-bound but not soluble synthetic cytokines, without compromising the radiosensitivity required for safety. Some membrane-bound cytokines conferred cell-contact independent paracrine activity, partly attributable to extracellular vesicles. Finally, we characterized interactions within consortia of differently engineered NK92 cells.