Hepatitis C virus RNA functionally sequesters miR-122.

Hepatitis C virus (HCV) uniquely requires the liver-specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during HCV infection showed robust AGO binding on the HCV 5'UTR at known and predicted miR-122 sites. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 "sponge" effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV-induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV.

Engineered bacterial swarm patterns as spatial records of environmental inputs.

A diverse array of bacteria species naturally self-organize into durable macroscale patterns on solid surfaces via swarming motility-a highly coordinated and rapid movement of bacteria powered by flagella. Engineering swarming is an untapped opportunity to increase the scale and robustness of coordinated synthetic microbial systems. Here we engineer Proteus mirabilis, which natively forms centimeter-scale bullseye swarm patterns, to 'write' external inputs into visible spatial records. Specifically, we engineer tunable expression of swarming-related genes that modify pattern features, and we develop quantitative approaches to decoding. Next, we develop a dual-input system that modulates two swarming-related genes simultaneously, and we separately show that growing colonies can record dynamic environmental changes. We decode the resulting multicondition patterns with deep classification and segmentation models. Finally, we engineer a strain that records the presence of aqueous copper. This work creates an approach for building macroscale bacterial recorders, expanding the framework for engineering emergent microbial behaviors.

The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. METHODS: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting-purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. RESULTS: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor ß was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB-IL34/CCL8 signaling that promotes macrophage chemotaxis. CONCLUSIONS: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.

Synchronized cycles of bacterial lysis for in vivo delivery.

The widespread view of bacteria as strictly pathogenic has given way to an appreciation of the prevalence of some beneficial microbes within the human body. It is perhaps inevitable that some bacteria would evolve to preferentially grow in environments that harbor disease and thus provide a natural platform for the development of engineered therapies. Such therapies could benefit from bacteria that are programmed to limit bacterial growth while continually producing and releasing cytotoxic agents in situ. Here we engineer a clinically relevant bacterium to lyse synchronously ata threshold population density and to release genetically encoded cargo. Following quorum lysis, a small number of surviving bacteria reseed the growing population, thus leading to pulsatile delivery cycles. We used microfluidic devices to characterize the engineered lysis strain and we demonstrate its potential as a drug delivery platform via co-culture with human cancer cells in vitro. Asa proof of principle, we tracked the bacterial population dynamics in ectopic syngeneic colorectal tumours in mice via a luminescent reporter. The lysis strain exhibits pulsatile population dynamics in vivo, with mean bacterial luminescence that remained two orders of magnitude lower than an unmodified strain. Finally, guided by previous findings that certain bacteria can enhance the efficacy of standard therapies, we orally administered the lysis strain alone or in combination with a clinical chemotherapeutic to a syngeneic mouse transplantation model of hepatic colorectal metastases. We found that the combination of both circuit-engineered bacteria and chemotherapy leads to a notable reduction of tumour activity along with a marked survival benefit over either therapy alone.Our approach establishes a methodology for leveraging the tools of synthetic biology to exploit the natural propensity for certain bacteria to colonize disease sites.

Rapid screening of engineered microbial therapies in a 3D multicellular model.

Synthetic biology is transforming therapeutic paradigms by engineering living cells and microbes to intelligently sense and respond to diseases including inflammation, infections, metabolic disorders, and cancer. However, the ability to rapidly engineer new therapies far outpaces the throughput of animal-based testing regimes, creating a major bottleneck for clinical translation. In vitro approaches to address this challenge have been limited in scalability and broad applicability. Here, we present a bacteria-in-spheroid coculture (BSCC) platform that simultaneously tests host species, therapeutic payloads, and synthetic gene circuits of engineered bacteria within multicellular spheroids over a timescale of weeks. Long-term monitoring of bacterial dynamics and disease progression enables quantitative comparison of critical therapeutic parameters such as efficacy and biocontainment. Specifically, we screen Salmonella typhimurium strains expressing and delivering a library of antitumor therapeutic molecules via several synthetic gene circuits. We identify candidates exhibiting significant tumor reduction and demonstrate high similarity in their efficacies, using a syngeneic mouse model. Last, we show that our platform can be expanded to dynamically profile diverse microbial species including Listeria monocytogenes, Proteus mirabilis, and Escherichia coli in various host cell types. This high-throughput framework may serve to accelerate synthetic biology for clinical applications and for understanding the host-microbe interactions in disease sites.

A sensing array of radically coupled genetic 'biopixels'.

Although there has been considerable progress in the development of engineering principles for synthetic biology, a substantial challenge is the construction of robust circuits in a noisy cellular environment. Such an environment leads to considerable intercellular variability in circuit behaviour, which can hinder functionality at the colony level. Here we engineer the synchronization of thousands of oscillating colony 'biopixels' over centimetre-length scales through the use of synergistic intercellular coupling involving quorum sensing within a colony and gas-phase redox signalling between colonies. We use this platform to construct a liquid crystal display (LCD)-like macroscopic clock that can be used to sense arsenic via modulation of the oscillatory period. Given the repertoire of sensing capabilities of bacteria such as Escherichia coli, the ability to coordinate their behaviour over large length scales sets the stage for the construction of low cost genetic biosensors that are capable of detecting heavy metals and pathogens in the field.

A synchronized quorum of genetic clocks.

The engineering of genetic circuits with predictive functionality in living cells represents a defining focus of the expanding field of synthetic biology. This focus was elegantly set in motion a decade ago with the design and construction of a genetic toggle switch and an oscillator, with subsequent highlights that have included circuits capable of pattern generation, noise shaping, edge detection and event counting. Here we describe an engineered gene network with global intercellular coupling that is capable of generating synchronized oscillations in a growing population of cells. Using microfluidic devices tailored for cellular populations at differing length scales, we investigate the collective synchronization properties along with spatiotemporal waves occurring at millimetre scales. We use computational modelling to describe quantitatively the observed dependence of the period and amplitude of the bulk oscillations on the flow rate. The synchronized genetic clock sets the stage for the use of microbes in the creation of a macroscopic biosensor with an oscillatory output. Furthermore, it provides a specific model system for the generation of a mechanistic description of emergent coordinated behaviour at the colony level.

Engineering Probiotic E. coli Nissle 1917 for Release of Therapeutic Nanobodies.

Bioengineered probiotics enable new opportunities to improve cancer treatment strategies due to their tumor-colonizing capabilities. Here, we will describe the development of a probiotic E. coli Nissle 1917 platform encoding a synchronized lysis mechanism for the localized and sustained release of blocking nanobodies against immune checkpoint molecules like programmed cell death protein-ligand 1 and cytotoxic T lymphocyte-associated protein-4. Specifically, we will detail the experimental protocols needed to (1) encode and validate binding of recombinantly produced checkpoint blockade nanobodies, (2) evaluate the therapeutic efficacy and safety of the probiotic platform in syngeneic tumor-bearing mice, and (3) analyze the immunophenotype of the tumor microenvironment.

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells.

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Bacterial therapies at the interface of synthetic biology and nanomedicine.

Bacteria are emerging as living drugs to treat a broad range of disease indications. However, the inherent advantages of these replicating and immunostimulatory therapies also carry the potential for toxicity. Advances in synthetic biology and the integration of nanomedicine can address this challenge through the engineering of controllable systems that regulate spatial and temporal activation for improved safety and efficacy. Here, we review recent progress in nanobiotechnology-driven engineering of bacteria-based therapies, highlighting limitations and opportunities that will facilitate clinical translation.

"Tumor-selective treatment of metastatic pancreatic cancer with an engineered, probiotic living drug".

Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges for effective treatment, with systemic chemotherapy often proving inadequate due to poor drug delivery and the tumor's immunosuppressive microenvironment. Engineered bacteria present a novel approach to target PDAC, leveraging their ability to colonize tumors and deliver therapeutic payloads. Here, we engineered probiotic Escherichia coli Nissle 1917 (EcN) to produce the pore-forming Theta toxin (Nis-Theta) and evaluated its efficacy in a preclinical model of PDAC. Probiotic administration resulted in selective colonization of tumor tissue, leading to improved overall survival compared to standard chemotherapy. Moreover, this strain exhibited cytotoxic effects on both primary and distant tumor lesions while sparing normal tissues. Importantly, treatment also modulated the tumor microenvironment by increasing anti-tumor immune cell populations and reducing immunosuppressive markers. These findings demonstrate the potential of engineered probiotic bacteria as a safe and effective therapeutic approach for PDAC, offering promise for improved patient outcomes.

Engineering tumor-colonizing E. coli Nissle 1917 for detection and treatment of colorectal neoplasia.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment. Here, first, we demonstrate selective colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition and orthotopic models of CRC. We next undertake an interventional, double-blind, dual-centre, prospective clinical trial, in which CRC patients take either placebo or EcN for two weeks prior to resection of neoplastic and adjacent normal colorectal tissue (ACTRN12619000210178). We detect enrichment of EcN in tumor samples over normal tissue from probiotic-treated patients (primary outcome of the trial). Next, we develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate. Oral delivery of this strain results in increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. To assess therapeutic potential, we engineer EcN to locally release a cytokine, GM-CSF, and blocking nanobodies against PD-L1 and CTLA-4 at the neoplastic site, and demonstrate that oral delivery of this strain reduces adenoma burden by ~50%. Together, these results support the use of EcN as an orally-deliverable platform to detect disease and treat CRC through the production of screening and therapeutic molecules.

Engineered Bacteria for Short-Chain-Fatty-Acid-Repressed Expression of Biotherapeutic Molecules.

Short-chain fatty acids (SCFA) such as propionate and butyrate are critical metabolites produced by the gut microbiota. Microbiome dysbiosis resulting in altered SCFA profiles is associated with certain diseases, including inflammatory bowel diseases (IBD), characterized by a reduction in butyrate concentration and active intestinal inflammation. There is an increasing interest in the use of engineered bacteria as diagnostic and therapeutic tools for gut diseases. In this study, we developed genetic circuits capable of sensing SCFA concentrations to build biosensors that express a response protein (superfolder green fluorescent protein [sfGFP]) in amounts inversely proportional to the SCFA concentration. We also built biotherapeutics expressing the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) using the same logic. The propionate biotherapeutic expressed larger amounts of mouse GM-CSF in the absence of propionate. The butyrate biotherapeutics presented the expected behavior only at the beginning of the kinetics and an accelerated response in the absence of butyrate. Overall, these genetic systems may function as complementary diagnostic tools for measuring SCFAs and as delivery vehicles for biotherapeutic molecules. IMPORTANCE Short-chain fatty acids are key molecules produced by the gut microbiome. Their concentrations are altered in certain diseases. Here, we created molecular biosensors that quantify the absence of propionate and butyrate, using logic "NOT" gates and bacterial promoters. Finally, we show that these genetic systems could be useful for the delivery of therapeutic molecules in the gut, in the absence of these acids.

Engineered bacteria launch and control an oncolytic virus.

The ability of bacteria and viruses to selectively replicate in tumors has led to synthetic engineering of new microbial therapies. Here we design a cooperative strategy whereby S. typhimurium bacteria transcribe and deliver the Senecavirus A RNA genome inside host cells, launching a potent oncolytic viral infection. Then, we engineer the virus to require a bacterially delivered protease in order to achieve virion maturation, demonstrating bacterial control over the virus. This work extends bacterially delivered therapeutics to viral genomes, and the governing of a viral population through engineered microbial interactions. One-Sentence Summary: Bacteria are engineered to act as a synthetic "capsid" delivering Senecavirus A genome and controlling its spread.

Chemokines expressed by engineered bacteria recruit and orchestrate antitumor immunity.

Tumors use multiple mechanisms to actively exclude immune cells involved in antitumor immunity. Strategies to overcome these exclusion signals remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor-localized delivery of therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16K42A) offer therapeutic benefit in multiple mouse tumor models, an effect mediated via recruitment of CD8+ T cells. Furthermore, we target the presentation of tumor-derived antigens by dendritic cells, using a second engineered bacterial strain expressing CCL20. This led to type 1 conventional dendritic cell recruitment and synergized with hCXCL16K42A-induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to recruit and activate innate and adaptive antitumor immune responses, offering a new cancer immunotherapy strategy.

Probiotic-guided CAR-T cells for solid tumor targeting.

A major challenge facing tumor-antigen targeting therapies such as chimeric antigen receptor (CAR)-T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. By contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be engineered as antigen-independent platforms for therapeutic delivery. To bridge these approaches, we developed a platform of probiotic-guided CAR-T cells (ProCARs), in which tumor-colonizing probiotics release synthetic targets that label tumor tissue for CAR-mediated lysis in situ. This system demonstrated CAR-T cell activation and antigen-agnostic cell lysis that was safe and effective in multiple xenograft and syngeneic models of human and mouse cancers. We further engineered multifunctional probiotics that co-release chemokines to enhance CAR-T cell recruitment and therapeutic response.

Colorectal cancer detection and treatment with engineered probiotics.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate the phenomenon of selective, long-term colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition. We show that, after oral administration, adenomas can be monitored over time by recovering EcN from stool. We also demonstrate specific colonization of EcN to solitary neoplastic lesions in an orthotopic murine model of CRC. We then exploit this neoplasia-homing property of EcN to develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate, and demonstrate that oral delivery of this strain results in significantly increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. We also assess EcN engineered to locally release immunotherapeutics at the neoplastic site. Oral delivery to mice bearing adenomas, reduced adenoma burden by ∻50%, with notable differences in the spatial distribution of T cell populations within diseased and healthy intestinal tissue, suggesting local induction of robust anti-tumor immunity. Together, these results support the use of EcN as an orally-delivered platform to detect disease and treat CRC through its production of screening and therapeutic molecules.

Queueing up for enzymatic processing: correlated signaling through coupled degradation.

High-throughput technologies have led to the generation of complex wiring diagrams as a post-sequencing paradigm for depicting the interactions between vast and diverse cellular species. While these diagrams are useful for analyzing biological systems on a large scale, a detailed understanding of the molecular mechanisms that underlie the observed network connections is critical for the further development of systems and synthetic biology. Here, we use queueing theory to investigate how 'waiting lines' can lead to correlations between protein 'customers' that are coupled solely through a downstream set of enzymatic 'servers'. Using the E. coli ClpXP degradation machine as a model processing system, we observe significant cross-talk between two networks that are indirectly coupled through a common set of processors. We further illustrate the implications of enzymatic queueing using a synthetic biology application, in which two independent synthetic networks demonstrate synchronized behavior when common ClpXP machinery is overburdened. Our results demonstrate that such post-translational processes can lead to dynamic connections in cellular networks and may provide a mechanistic understanding of existing but currently inexplicable links.

A rapid screening platform to coculture bacteria within tumor spheroids.

The prevalence of tumor-colonizing bacteria along with advances in synthetic biology are leading to a new generation of living microbial cancer therapies. Because many bacterial systems can be engineered to recombinantly produce therapeutics within tumors, simple and high-throughput experimental platforms are needed to screen the large collections of bacteria candidates and characterize their interactions with cancer cells. Here, we describe a protocol to selectively grow bacteria within the core of tumor spheroids, allowing for their continuous and parallel profiling in physiologically relevant conditions. Specifically, tumor spheroids are incubated with bacteria in a 96-well low-adhesion plate followed by a series of washing steps and an antibiotic selection protocol to confine bacterial growth within the hypoxic and necrotic core of tumor spheroids. This bacteria spheroid coculture (BSCC) system is stable for over 2 weeks, does not require specialized equipment and is compatible with time-lapse microscopy, commercial staining assays and histology that uniquely enable analysis of growth kinetics, viability and spatial distribution of both cellular populations, respectively. We show that the procedure is applicable to multiple tumor cell types and bacterial species by varying protocol parameters and is validated by using animal models. The BSCC platform will allow the study of bacteria-tumor interactions in a continuous manner and facilitate the rapid development of engineered microbial therapies.