Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy.

Despite its success in several clinical trials, cancer immunotherapy remains limited by the rarity of targetable tumor-specific antigens, tumor-mediated immune suppression, and toxicity triggered by systemic delivery of potent immunomodulators. Here, we present a proof-of-concept immunomodulatory gene circuit platform that enables tumor-specific expression of immunostimulators, which could potentially overcome these limitations. Our design comprised de novo synthetic cancer-specific promoters and, to enhance specificity, an RNA-based AND gate that generates combinatorial immunomodulatory outputs only when both promoters are mutually active. These outputs included an immunogenic cell-surface protein, a cytokine, a chemokine, and a checkpoint inhibitor antibody. The circuits triggered selective T cell-mediated killing of cancer cells, but not of normal cells, in vitro. In in vivo efficacy assays, lentiviral circuit delivery mediated significant tumor reduction and prolonged mouse survival. Our design could be adapted to drive additional immunomodulators, sense other cancers, and potentially treat other diseases that require precise immunological programming.

Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells.

RNA-based regulation and CRISPR/Cas transcription factors (CRISPR-TFs) have the potential to be integrated for the tunable modulation of gene networks. A major limitation of this methodology is that guide RNAs (gRNAs) for CRISPR-TFs can only be expressed from RNA polymerase III promoters in human cells, limiting their use for conditional gene regulation. We present new strategies that enable expression of functional gRNAs from RNA polymerase II promoters and multiplexed production of proteins and gRNAs from a single transcript in human cells. We use multiple RNA regulatory strategies, including RNA-triple-helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based RNA processing. Using these tools, we efficiently modulate endogenous promoters and implement tunable synthetic circuits, including multistage cascades and RNA-dependent networks that can be rewired with Csy4 to achieve complex behaviors. This toolkit can be used for programming scalable gene circuits and perturbing endogenous networks for biology, therapeutic, and synthetic biology applications.

Cellular heterogeneity mediates inherent sensitivity-specificity tradeoff in cancer targeting by synthetic circuits.

Synthetic gene circuits are emerging as a versatile means to target cancer with enhanced specificity by combinatorial integration of multiple expression markers. Such circuits must also be tuned to be highly sensitive because escape of even a few cells might be detrimental. However, the error rates of decision-making circuits in light of cellular variability in gene expression have so far remained unexplored. Here, we measure the single-cell response function of a tunable logic AND gate acting on two promoters in heterogeneous cell populations. Our analysis reveals an inherent tradeoff between specificity and sensitivity that is controlled by the AND gate amplification gain and activation threshold. We implement a tumor-mimicking cell-culture model of cancer cells emerging in a background of normal ones, and show that molecular parameters of the synthetic circuits control specificity and sensitivity in a killing assay. This suggests that, beyond the inherent tradeoff, synthetic circuits operating in a heterogeneous environment could be optimized to efficiently target malignant state with minimal loss of specificity.

Contact guidance and collective migration in the advancing epithelial monolayer.

At the edge of a confluent cell layer, cell-free empty space is a cue that can drive directed collective cellular migration. Similarly, contact guidance is also a robust mechanical cue that can drive cell migration. However, it is unclear which of the two effects is stronger, and how each mechanism affects collective migration. To address this question, here we explore the trajectories of cells migrating collectively on a substrate containing micropatterned grooves (10-20 µm in periodicity, 2 µm in height) compared with unpatterned control substrates. Compared with unpatterned controls, the micropatterned substrates attenuated path variance by close to 70% and augmented migration coordination by more than 30%. Together, these results show that contact guidance can play an appreciable role in collective cellular migration. Also, our result can provide insights into tissue repair and regeneration with the remodeling of the connective tissue matrix.

Genetic Modification of Tumor-Infiltrating Lymphocytes, Peripheral T Cells, and T-Cell Model Cell Lines.

Genetic modification of tumor-infiltrating lymphocytes (TILs) or circulating T cells has become an important avenue in cancer therapy. Here we describe a comprehensive method for establishing and expanding TIL cultures and genetically modifying them with a gene of interest (GOI) via retroviral transduction or mRNA transfection. The method includes all the important steps starting with TIL extraction from tumors through to the maintenance of the genetically modified TILs. The protocol includes instructions for retroviral transduction and mRNA transfection of circulating T cells or T-cell lines. The GOIs most commonly introduced into the target cells are chimeric antigen receptors (CARs); genetic adjuvants, such as membrane-bound interleukins; and antitumor T-cell receptors (TCRs).

A tunable dual-promoter integrator for targeting of cancer cells.

Precise discrimination between similar cellular states is essential for autonomous decision-making scenarios, such as in vivo targeting of diseased cells. Discrimination could be achieved by delivering an effector gene expressed under a highly active context-specific promoter. Yet, a single-promoter approach has linear response and offers limited control of specificity and efficacy. Here, we constructed a dual-promoter integrator, which expresses an effector gene only when the combined activity of two internal input promoters is high. A tunable response provides flexibility in choosing promoter inputs and effector gene output. Experiments using one premalignant and four cancer cell lines, over a wide range of promoter activities, revealed a digital-like response of input amplification following a sharp activation threshold. The response function is cell dependent with its overall magnitude increasing with degree of malignancy. The tunable digital-like response provides robustness, acts to remove input noise minimizing false-positive identification of cell states, and improves targeting precision and efficacy.

The impact of gender on the clinical presentation, management, and surgical outcomes of patients with native-joint septic arthritis.

RATIONALE, AIMS AND OBJECTIVES: Approximately 20 000 cases of septic arthritis (SA) occur in the U.S. yearly. We examined whether gender-related differences exist in the presentation, management, and outcomes of patients with native joint septic arthritis (NJSA). METHODS: This was a retrospective study of medical files of patients aged 18 years and older admitted between 1998 and 2015 to a single tertiary care hospital and diagnosed with NJSA. All study subjects had positive synovial fluid or blood cultures and each was managed surgically. Patients' charts were reviewed for demographics, comorbidities, clinical presentations, microbiology profiles, management, and outcomes. Cases of osteomyelitis, septic bursitis, prosthetic joint, and culture-negative SA were excluded. RESULTS: Of 324 NJSA patients, those who were female (n = 130; 40.1%) were significantly older at presentation than males (mean age: 63.6 vs 58.3; P = .006). Prior joint pathology was more common amongst females, including osteoarthritis (20.8% vs 12.9%; P = .04) and rheumatoid arthritis (10% vs 3.6%; P = .03). Female patients had a higher frequency of hip involvement (17.7% vs 10.8%; P = .05). No differences were observed in clinical presentations, culture results, medical management, or outcomes between genders. CONCLUSIONS: Compared to men, women with NJSA presented at an older age and had more prior joint pathology and a higher frequency of hip involvement. These differences, however, had no significant impact on the clinical presentation, medical management, or outcomes of NJSA.

Synthetic Circuit-Driven Expression of Heterologous Enzymes for Disease Detection.

The integration of nanotechnology and synthetic biology could lay the framework for new classes of engineered biosensors that produce amplified readouts of disease states. As a proof-of-concept demonstration of this vision, here we present an engineered gene circuit that, in response to cancer-associated transcriptional deregulation, expresses heterologous enzyme biomarkers whose activity can be measured by nanoparticle sensors that generate amplified detection readouts. Specifically, we designed an AND-gate gene circuit that integrates the activity of two ovarian cancer-specific synthetic promoters to drive the expression of a heterologous protein output, secreted Tobacco Etch Virus (TEV) protease, exclusively from within tumor cells. Nanoparticle probes were engineered to carry a TEV-specific peptide substrate in order to measure the activity of the circuit-generated enzyme to yield amplified detection signals measurable in the urine or blood. We applied our integrated sense-and-respond system in a mouse model of disseminated ovarian cancer, where we demonstrated measurement of circuit-specific TEV protease activity both in vivo using exogenously administered nanoparticle sensors and ex vivo using quenched fluorescent probes. We envision that this work will lay the foundation for how synthetic biology and nanotechnology can be meaningfully integrated to achieve next-generation engineered biosensors.

Modeling SARS-CoV-2 Infection in Mice Using Lentiviral hACE2 Vectors Infers Two Modes of Immune Responses to SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a severe global pandemic. Mice models are essential to investigate infection pathology, antiviral drugs, and vaccine development. However, wild-type mice lack the human angiotensin-converting enzyme 2 (hACE2) that mediates SARS-CoV-2 entry into human cells and consequently are not susceptible to SARS-CoV-2 infection. hACE2 transgenic mice could provide an efficient COVID-19 model, but are not always readily available, and practically restricted to specific strains. Therefore, there is a dearth of additional mouse models for SARS-CoV-2 infection. We applied lentiviral vectors to generate hACE2 expression in interferon receptor knock-out (IFNAR1-/-) mice. Lenti-hACE2 transduction supported SARS-CoV-2 replication in vivo, simulating mild acute lung disease. Gene expression analysis revealed two modes of immune responses to SARS-CoV-2 infection: one in response to the exposure of mouse lungs to SARS-CoV-2 particles in the absence of productive viral replication, and the second in response to productive SARS-CoV-2 infection. Our results infer that immune response to immunogenic elements on incoming virus or in productively infected cells stimulate diverse immune effectors, even in absence of type I IFN signaling. Our findings should contribute to a better understanding of the immune response triggered by SARS-CoV-2 and to further elucidate COVID-19.

A high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS).

Cell state-specific promoters constitute essential tools for basic research and biotechnology because they activate gene expression only under certain biological conditions. Synthetic Promoters with Enhanced Cell-State Specificity (SPECS) can be superior to native ones, but the design of such promoters is challenging and frequently requires gene regulation or transcriptome knowledge that is not readily available. Here, to overcome this challenge, we use a next-generation sequencing approach combined with machine learning to screen a synthetic promoter library with 6107 designs for high-performance SPECS for potentially any cell state. We demonstrate the identification of multiple SPECS that exhibit distinct spatiotemporal activity during the programmed differentiation of induced pluripotent stem cells (iPSCs), as well as SPECS for breast cancer and glioblastoma stem-like cells. We anticipate that this approach could be used to create SPECS for gene therapies that are activated in specific cell states, as well as to study natural transcriptional regulatory networks.

An autonomous system for identifying and governing a cell's state in yeast.

We present an approach for an autonomous system that detects a particular state of interest in a living cell and can govern cell fate accordingly. Cell states could be better identified by the expression pattern of several genes than of a single one. Therefore, autonomous identification can be achieved by a system that measures the expression of these several genes and integrates their activities into a single output. We have constructed a system that diagnoses a unique state in yeast, in which two independent pathways, methionine anabolism and galactose catabolism, are active. Our design is based on modifications of the yeast two-hybrid system. We show that cells could autonomously report on their state, identify the state of interest, and inhibit their growth accordingly. The system's sensitivity is adjustable to detect states with limited dynamic range of inputs. The system's output depends only on the activity of input pathways, not on their identity; hence it is straightforward to diagnose any pair of inputs. A simple model is presented that accounts for the data and provides predictive power. We propose that such systems could handle real-life states-of-interest such as identification of aberrant versus normal growth.

The onset of p53-dependent apoptosis plays a role in terminal differentiation of human normoblasts.

The p53 tumor suppressor gene was found to play a role in the differentiation of several tissue types. We report here that p53-dependent apoptosis plays a role in the final stages of physiological differentiation of normoblasts, resulting in nuclear condensation and expulsion without cell death. Blood samples of healthy newborns, cord blood as well as bone marrow, were analysed for apoptosis by TUNEL and p53 expression by immunostaining. While some samples exhibited simultaneously several distinct patterns of apoptosis, such as perinuclear, diffused nuclear or nuclear apoptotic bodies, others presented a single defined pattern. Overexpression of p53 protein was detected in normoblasts exhibiting either perinuclear or diffused nuclear p53, corresponding to the nuclear apoptotic pattern in the same sample. Similar results were also evident with colonies cultivated for 12-14 days in culture. Differentiated erythroid colonies exhibited overexpression of p53 and positive TUNEL staining only in the normoblasts. We further examined the state of caspase 3/7 and observed a decrease of this activated enzyme during erythroid differentiation in culture. This study suggests a novel role for apoptosis in normoblast differentiation where nuclear degradation occurs with a delay in the actual cell death. A pivotal role for the p53-dependent apoptosis in the erythroid lineage development is implied. However, this apoptotic process is not fully executed because of the exhaustion in caspase 3/7 and thus cells are diverted towards final stages of differentiation.