Precision off-the-shelf natural killer cell therapies for oncology with logic-gated gene circuits.

Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis (5-year survival rate of 30.5% in the United States). Designing cell therapies to target AML is challenging because no single tumor-associated antigen (TAA) is highly expressed on all cancer subpopulations. Furthermore, TAAs are also expressed on healthy cells, leading to toxicity risk. To address these targeting challenges, we engineer natural killer (NK) cells with a multi-input gene circuit consisting of chimeric antigen receptors (CARs) controlled by OR and NOT logic gates. The OR gate kills a range of AML cells from leukemic stem cells to blasts using a bivalent CAR targeting FLT3 and/or CD33. The NOT gate protects healthy hematopoietic stem cells (HSCs) using an inhibitory CAR targeting endomucin, a protective antigen unique to healthy HSCs. NK cells with the combined OR-NOT gene circuit kill multiple AML subtypes and protect primary HSCs, and the circuit also works in vivo.

Cas9-assisted biological containment of a genetically engineered human commensal bacterium and genetic elements.

Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Engineered biosensing bacteria outfitted with such circuits can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiring thyA via horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacterium Bacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine.

Building smart CAR T cell therapies: The path to overcome current challenges.

Successful implementation of adoptive cell therapy (ACT) of cancer requires comprehensively addressing biological and practical challenges. This approach has been largely overlooked, resulting in a gap between the potential of ACT and its actual effectiveness. We summarize the most promising technical strategies in creating an "ideal" ACT product, focusing on chimeric antigen receptor (CAR)-engineered cells. Since many requirements for effective ACT are common to most cancers, what we outline here might have a broader impact.

A systems-level mass spectrometry-based technique for accurate and sensitive quantification of the RNA cap epitranscriptome.

Chemical modifications of transcripts with a 5' cap occur in all organisms and function in many aspects of RNA metabolism. To facilitate analysis of RNA caps, we developed a systems-level mass spectrometry-based technique, CapQuant, for accurate and sensitive quantification of the cap epitranscriptome. The protocol includes the addition of stable isotope-labeled cap nucleotides (CNs) to RNA, enzymatic hydrolysis of endogenous RNA to release CNs, and off-line enrichment of CNs by ion-pairing high-pressure liquid chromatography, followed by a 17 min chromatography-coupled tandem quadrupole mass spectrometry run for the identification and quantification of individual CNs. The total time required for the protocol can be up to 7 d. In this approach, 26 CNs can be quantified in eukaryotic poly(A)-tailed RNA, bacterial total RNA and viral RNA. This protocol can be modified to analyze other types of RNA and RNA from in vitro sources. CapQuant stands out from other methods in terms of superior specificity, sensitivity and accuracy, and it is not limited to individual caps nor does it require radiolabeling. Thanks to its unique capability of accurately and sensitively quantifying RNA caps on a systems level, CapQuant can reveal both the RNA cap landscape and the transcription start site distribution of capped RNA in a broad range of settings.

BioAutoMATED: An end-to-end automated machine learning tool for explanation and design of biological sequences.

The design choices underlying machine-learning (ML) models present important barriers to entry for many biologists who aim to incorporate ML in their research. Automated machine-learning (AutoML) algorithms can address many challenges that come with applying ML to the life sciences. However, these algorithms are rarely used in systems and synthetic biology studies because they typically do not explicitly handle biological sequences (e.g., nucleotide, amino acid, or glycan sequences) and cannot be easily compared with other AutoML algorithms. Here, we present BioAutoMATED, an AutoML platform for biological sequence analysis that integrates multiple AutoML methods into a unified framework. Users are automatically provided with relevant techniques for analyzing, interpreting, and designing biological sequences. BioAutoMATED predicts gene regulation, peptide-drug interactions, and glycan annotation, and designs optimized synthetic biology components, revealing salient sequence characteristics. By automating sequence modeling, BioAutoMATED allows life scientists to incorporate ML more readily into their work.

Optimization of Host Cell-Compatible, Antimicrobial Peptides Effective against Biofilms and Clinical Isolates of Drug-Resistant Bacteria.

Here, we describe the continued synthetic molecular evolution of a lineage of host-compatible antimicrobial peptides (AMP) intended for the treatment of wounds infected with drug-resistant, biofilm-forming bacteria. The peptides tested are variants of an evolved AMP called d-amino acid CONsensus with Glycine Absent (d-CONGA), which has excellent antimicrobial activities in vitro and in vivo. In this newest generation of rational d-CONGA variants, we tested multiple sequence-structure-function hypotheses that had not been tested in previous generations. Many of the peptide variants have lower antibacterial activity against Gram-positive or Gram-negative pathogens, especially variants that have altered hydrophobicity, secondary structure potential, or spatial distribution of charged and hydrophobic residues. Thus, d-CONGA is generally well tuned for antimicrobial activity. However, we identified a variant, d-CONGA-Q7, with a polar glutamine inserted into the middle of the sequence, that has higher activity against both planktonic and biofilm-forming bacteria as well as lower cytotoxicity against human fibroblasts. Against clinical isolates of Klebsiella pneumoniae, innate resistance to d-CONGA was surprisingly common despite a lack of inducible resistance in Pseudomonas aeruginosa reported previously. Yet, these same isolates were susceptible to d-CONGA-Q7. d-CONGA-Q7 is much less vulnerable to AMP resistance in Gram-negative bacteria than its predecessor. Consistent with the spirit of synthetic molecular evolution, d-CONGA-Q7 achieved a critical gain-of-function and has a significantly better activity profile.

Development of membrane-active peptide therapeutics in oncology.

Membrane-active peptides play an essential role in many living organisms and their immune systems and counter many infectious diseases. Many have dual or multiple mechanisms and can synergize with other molecules, like peptides, proteins, and small molecules. Although membrane-active peptides have been intensively studied in the past decades and more than 3500 sequences have been identified, only a few received approvals from the US Food and Drug Administration. In this review, we investigated all the peptide therapeutics that have entered the market or were subjected to preclinical and clinical studies to understand how they succeeded. With technological advancement (e.g., chemical modifications and pharmaceutical formulations) and a better understanding of the mechanism of action and the potential targets, we found at least five membrane-active peptide drugs that have entered preclinical/clinical phases and show promising results for cancer treatment. We summarized our findings in this review and provided insights into membrane-active anticancer peptide therapeutics.

Full-colour Jabuticaba-like nanostructures via the multiplex and orthogonal self-assembly of protein-conjugated quantum dots with engineered biofilms.

The integration of inorganic components with bacterial biofilms is of great significance for expanding the functionality of artificial biological materials. However, so far, the complexities and functionalities of biofilm-based scaffolds assembled via metal-peptide coordination chemistries remain limited. Here, we present a platform for the multiplexed and specific coupling of recombinant protein-functionalized fluorescent red-green-blue (RGB) quantum dots (QDs) with engineered biofilms to form Jabuticaba-like nanostructures. Full-color living Jabuticaba-like nanostructures have been achieved through the interaction of extracellular peptides that are fabricated by biofilms with the proteins that modify the surface of the RGB QDs through orthogonal SpyTag/SpyCatcher, IsopeptagN/PilinN, and IsopeptagC/PilinC pairs. We envision that living cell populations will enable the multiplexable, scalable and bottom-up assembly of versatile materials that integrate both abiotic and biotic components into multifunctional systems.

Ultra-lightweight living structural material for enhanced stiffness and environmental sensing.

Natural materials such as bone, wood, and bamboo can inspire the fabrication of stiff, lightweight structural materials. Biofilms are one of the most dominant forms of life in nature. However, little is known about their physical properties as a structural material. Here we report an Escherichia coli biofilm having a Young's modulus close to 10 â€‹GPa with ultra-low density, indicating a high-performance structural material. The mechanical and structural characterization of the biofilm and its components illuminates its adaptable bottom-up design, consisting of lightweight microscale cells covered by a dense network of amyloid nanofibrils on the surface. We engineered E. coli such that 1) carbon nanotubes assembled on the biofilm, enhancing its stiffness to over 30 â€‹GPa, or that 2) the biofilm sensitively detected heavy metal as an example of an environmental toxin. These demonstrations offer new opportunities for developing responsive living structural materials to serve many real-world applications.

Enhancing nutritional niche and host defenses by modifying the gut microbiome.

The gut microbiome is essential for processing complex food compounds and synthesizing nutrients that the host cannot digest or produce, respectively. New model systems are needed to study how the metabolic capacity provided by the gut microbiome impacts the nutritional status of the host, and to explore possibilities for altering host metabolic capacity via the microbiome. Here, we colonized the nematode Caenorhabditis elegans gut with cellulolytic bacteria that enabled C. elegans to utilize cellulose, an otherwise indigestible substrate, as a carbon source. Cellulolytic bacteria as a community component in the worm gut can also support additional bacterial species with specialized roles, which we demonstrate by using Lactobacillus plantarum to protect C. elegans against Salmonella enterica infection. This work shows that engineered microbiome communities can be used to endow host organisms with novel functions, such as the ability to utilize alternate nutrient sources or to better fight pathogenic bacteria.

A synthetic transcription platform for programmable gene expression in mammalian cells.

Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. Here we create a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized libraries of guide RNAs, binding motifs of synthetic operators, transcriptional activators, and additional genetic regulatory elements express mammalian genes in a highly predictable and tunable manner. We demonstrate the programmable control of reporter genes episomally and chromosomally, with up to 25-fold more activity than seen with the EF1α promoter, in multiple cell types. We use these circuits to program the secretion of human monoclonal antibodies and to control T-cell effector function marked by interferon-γ production. Antibody titers and interferon-γ concentrations significantly correlate with synthetic promoter strengths, providing a platform for programming gene expression and cellular function in diverse applications.

An N-capping asparagine-lysine-proline (NKP) motif contributes to a hybrid flexible/stable multifunctional peptide scaffold.

Structural diversity drives multiple biological activities and mechanisms of action in linear peptides. Here we describe an unusual N-capping asparagine-lysine-proline (NKP) motif that confers a hybrid multifunctional scaffold to a computationally designed peptide (PaDBS1R7). PaDBS1R7 has a shorter α-helix segment than other computationally designed peptides of similar sequence but with key residue substitutions. Although this motif acts as an α-helix breaker in PaDBS1R7, the Asn5 presents exclusive N-capping effects, forming a belt to establish hydrogen bonds for an amphipathic α-helix stabilization. The combination of these different structural profiles was described as a coil/N-cap/α-helix scaffold, which was also observed in diverse computational peptide mutants. Biological studies revealed that all peptides displayed antibacterial activities. However, only PaDBS1R7 displayed anticancer properties, eradicated Pseudomonas aeruginosa biofilms, decreased bacterial counts by 100-1000-fold in vivo, reduced lipopolysaccharide-induced macrophages stress, and stimulated fibroblast migration for wound healing. This study extends our understanding of an N-capping NKP motif to engineering hybrid multifunctional peptide drug candidates with potent anti-infective and immunomodulatory properties.

A conserved Bacteroidetes antigen induces anti-inflammatory intestinal T lymphocytes.

The microbiome contributes to the development and maturation of the immune system. In response to commensal bacteria, intestinal CD4+ T lymphocytes differentiate into functional subtypes with regulatory or effector functions. The development of small intestine intraepithelial lymphocytes that coexpress CD4 and CD8αα homodimers (CD4IELs) depends on the microbiota. However, the identity of the microbial antigens recognized by CD4+ T cells that can differentiate into CD4IELs remains unknown. We identified ß-hexosaminidase, a conserved enzyme across commensals of the Bacteroidetes phylum, as a driver of CD4IEL differentiation. In a mouse model of colitis, ß-hexosaminidase-specific lymphocytes protected against intestinal inflammation. Thus, T cells of a single specificity can recognize a variety of abundant commensals and elicit a regulatory immune response at the intestinal mucosa.

Engineering the human gut commensal Bacteroides thetaiotaomicron with synthetic biology.

The role of the microbiome in health and disease is attracting the attention of researchers seeking to engineer microorganisms for diagnostic and therapeutic applications. Recent progress in synthetic biology may enable the dissection of host-microbiota interactions. Sophisticated genetic circuits that can sense, compute, memorize, and respond to signals have been developed for the stable commensal bacterium Bacteroides thetaiotaomicron, dominant in the human gut. In this review, we highlight recent advances in expanding the genetic toolkit for B. thetaiotaomicron and foresee several applications of this species for microbiome engineering. We provide our perspective on the challenges and future opportunities for the engineering of human gut-associated bacteria as living therapeutic agents.

Synthetic molecular evolution of antimicrobial peptides.

As we learn more about how peptide structure and activity are related, we anticipate that antimicrobial peptides will be engineered to have strong potency and distinct functions and that synthetic peptides will have new biomedical applications, such as treatments for emerging infectious diseases. As a result of the enormous number of possible amino acid sequences and the low-throughput nature of antimicrobial peptide assays, computational tools for peptide design and optimization are needed for direct experimentation toward obtaining functional sequences. Recent developments in computational tools have improved peptide design, saving labor, reagents, costs, and time. At the same time, improvements in peptide synthesis and experimental platforms continue to reduce the cost and increase the throughput of peptide-drug screening. In this review, we discuss the current methods of peptide design and engineering, including in silico methods and peptide synthesis and screening, and highlight areas of potential improvement.

Engineered Living Hydrogels.

Living biological systems, ranging from single cells to whole organisms, can sense, process information, and actuate in response to changing environmental conditions. Inspired by living biological systems, engineered living cells and nonliving matrices are brought together, which gives rise to the technology of engineered living materials. By designing the functionalities of living cells and the structures of nonliving matrices, engineered living materials can be created to detect variability in the surrounding environment and to adjust their functions accordingly, thereby enabling applications in health monitoring, disease treatment, and environmental remediation. Hydrogels, a class of soft, wet, and biocompatible materials, have been widely used as matrices for engineered living cells, leading to the nascent field of engineered living hydrogels. Here, the interactions between hydrogel matrices and engineered living cells are described, focusing on how hydrogels influence cell behaviors and how cells affect hydrogel properties. The interactions between engineered living hydrogels and their environments, and how these interactions enable versatile applications, are also discussed. Finally, current challenges facing the field of engineered living hydrogels for their applications in clinical and environmental settings are highlighted.

Sense-and-Respond Payload Delivery Using a Novel Antigen-Inducible Promoter Improves Suboptimal CAR-T Activation.

Chimeric antigen receptor (CAR)-T cell therapies demonstrate the clinical potential of lymphocytes engineered with synthetic properties. However, CAR-T cells are ineffective in most solid tumors, partly due to inadequate activation of the infused lymphocytes at the site of malignancy. To selectively enhance antitumor efficacy without exacerbating off-target toxicities, CAR-T cells can be engineered to preferentially deliver immunostimulatory payloads in tumors. Here, we report a novel antigen-inducible promoter for conditional payload expression in primary human T cells. In therapeutic T cell models, the novel NR4A-based promoter induced higher reporter gene expression than the conventional NFAT-based promoter under weakly immunogenic conditions, where payload expression is most needed. Minimal activity was detected from the inducible promoters in the absence of antigen and after withdrawal of stimulation. As a functional proof-of-concept, we used the NR4A-based promoter to express cytokines in an antimesothelin CAR-T model with suboptimal stimulation and observed improved proliferation compared to T cells engineered with the conventional NFAT promoter or CAR alone. Our system achieves CAR-directed payload expression under weakly immunogenic conditions and could enable the next generation of cell therapies with enhanced antitumor efficacy.

Predicting Membrane-Active Peptide Dynamics in Fluidic Lipid Membranes.

Understanding the interactions between peptides and lipid membranes could not only accelerate the development of antimicrobial peptides as treatments for infections but also be applied to finding targeted therapies for cancer and other diseases. However, designing biophysical experiments to study molecular interactions between flexible peptides and fluidic lipid membranes has been an ongoing challenge. Recently, with hardware advances, algorithm improvements, and more accurate parameterizations (i.e., force fields), all-atom molecular dynamics (MD) simulations have been used as a "computational microscope" to investigate the molecular interactions and mechanisms of membrane-active peptides in cell membranes (Chen et al., Curr Opin Struct Biol 61:160-166, 2020; Ulmschneider and Ulmschneider, Acc Chem Res 51(5):1106-1116, 2018; Dror et al., Annu Rev Biophys 41:429-452, 2012). In this chapter, we describe how to utilize MD simulations to predict and study peptide dynamics and how to validate the simulations by circular dichroism, intrinsic fluorescent probe, membrane leakage assay, electrical impedance, and isothermal titration calorimetry. Experimentally validated MD simulations open a new route towards peptide design starting from sequence and structure and leading to desirable functions.

A warm-start digital CRISPR/Cas-based method for the quantitative detection of nucleic acids.

Nucleic acids-based molecular diagnostic tools incorporating the CRISPR/Cas system are being developed as rapid and sensitive methods for pathogen detection. However, most CRISPR/Cas-based diagnostics lack quantitative detection ability. Here, we report Warm-Start RApid DIgital Crispr Approach (WS-RADICA) for the rapid, sensitive, and quantitative detection of nucleic acids. WS-RADICA detected as little as 1 copy/µl SARS-CoV-2 RNA in 40 min (qualitative detection) or 60 min (quantitative detection). WS-RADICA can be easily adapted to various digital devices: two digital chips were evaluated for both DNA and RNA quantification, with linear dynamic ranges of 0.8-12777 copies/µL for DNA and 1.2-18391 copies/µL for RNA (both R2 values > 0.99). Moreover, WS-RADICA had lower detection limit and higher inhibitor tolerance than a bulk RT-LAMP-Cas12b reaction and similar performance to RT-qPCR and RT-dPCR. To prove its performance on nucleic acids derived from live virus, WS-RADICA was also validated to detect and quantify human adenovirus and herpes simplex virus. Given its speed, sensitivity, quantification capability, and inhibitor tolerance, WS-RADICA shows great promise for a variety of applications requiring nucleic acid quantification.

High-Throughput CRISPR Screens To Dissect Macrophage-Shigella Interactions.

Shigellosis causes most diarrheal deaths worldwide, particularly affecting children. Shigella invades and replicates in the epithelium of the large intestine, eliciting inflammation and tissue destruction. To understand how Shigella rewires macrophages prior to epithelium invasion, we performed genome-wide and focused secondary CRISPR knockout and CRISPR interference (CRISPRi) screens in Shigella flexneri-infected human monocytic THP-1 cells. Knockdown of the Toll-like receptor 1/2 signaling pathway significantly reduced proinflammatory cytokine and chemokine production, enhanced host cell survival, and controlled intracellular pathogen growth. Knockdown of the enzymatic component of the mitochondrial pyruvate dehydrogenase complex enhanced THP-1 cell survival. Small-molecule inhibitors blocking key components of these pathways had similar effects; these were validated with human monocyte-derived macrophages, which closely mimic the in vivo physiological state of macrophages postinfection. High-throughput CRISPR screens can elucidate how S. flexneri triggers inflammation and redirects host pyruvate catabolism for energy acquisition before killing macrophages, pointing to new shigellosis therapies. IMPORTANCE Treatment for shigellosis is becoming increasingly difficult as resistance to antibiotics becomes more prevalent. One way to prevent this significant public health problem from developing into a full-blown crisis is to approach shigellosis intervention from the point of view of the host. So far, little is known about the specific biological pathways that might be modulated in macrophages, sentinel cells of the innate immune system, to strengthen the response to Shigella infection. In this work, we conducted CRISPR screens to comprehensively decipher the complexity of macrophage-Shigella interactions and to discover new potential therapeutic interventions against Shigella flexneri infection. Our work highlights systematic genetic perturbation strategies to provide direct causal evidence showing how intracellular pathogens manipulate innate immune cells.