MIRELLA: a mathematical model explains the effect of microRNA-mediated synthetic genes regulation on intracellular resource allocation.

Competition for intracellular resources, also known as gene expression burden, induces coupling between independently co-expressed genes, a detrimental effect on predictability and reliability of gene circuits in mammalian cells. We recently showed that microRNA (miRNA)-mediated target downregulation correlates with the upregulation of a co-expressed gene, and by exploiting miRNAs-based incoherent-feed-forward loops (iFFLs) we stabilise a gene of interest against burden. Considering these findings, we speculate that miRNA-mediated gene downregulation causes cellular resource redistribution. Despite the extensive use of miRNA in synthetic circuits regulation, this indirect effect was never reported before. Here we developed a synthetic genetic system that embeds miRNA regulation, and a mathematical model, MIRELLA, to unravel the miRNA (MI) RolE on intracellular resource aLLocAtion. We report that the link between miRNA-gene downregulation and independent genes upregulation is a result of the concerted action of ribosome redistribution and 'queueing-effect' on the RNA degradation pathway. Taken together, our results provide for the first time insights into the hidden regulatory interaction of miRNA-based synthetic networks, potentially relevant also in endogenous gene regulation. Our observations allow to define rules for complexity- and context-aware design of genetic circuits, in which transgenes co-expression can be modulated by tuning resource availability via number and location of miRNA target sites.

Three-dimensionally Patterned Scaffolds Modulate the Biointerface at the Nanoscale.

The main aim of cell instructive materials is to guide in a controlled way cellular behavior by fine-tuning cell-material crosstalk. In the last decades, several efforts have been spent in elucidating the relations between material cues and cellular fate at the nanoscale and in the development of novel strategies for gaining a superior control over cellular function modulation. In this context, a particular attention has been recently paid to the role played by cellular membrane rearrangement in triggering specific molecular pathways linked to the regulation of different cellular functions. Here, we characterize the effect of linear microtopographies upon cellular behavior in three-dimensional (3D) environments, with particular focus on the relations linking cytoskeleton structuration to membrane rearrangement and internalization tuning. The performed analysis shown that, by altering the cellular adhesion processes at the micro- and nanoscale, it is possible to alter the membrane physical state and cellular internalization capability. More specifically, our findings pointed out that an increased cytoskeletal structuration influences the formation of nanoinvagination membrane process at the cell-material interface and the expression of clathrin and caveolin, two of the main proteins involved in the endocytosis regulation. Moreover, we proved that such topographies enhance the engulfment of inert polystyrene nanoparticles attached on 3D patterned surfaces. Our results could give new guidelines for the design of innovative and more efficient 3D cell culture systems usable for diagnostic, therapeutic, and tissue engineering purposes.

Engineered Protease-Responsive RNA-Binding Proteins (RBPs) to Expand the Toolbox of Synthetic Circuits in Mammalian Cells.

Genetically encoded sensor-actuator circuits aim at reprogramming cellular functions and are inspired by intracellular networks: from the input signal (sensor) to the desired output response (actuator). In the last years, circuits with posttranscriptional regulation of gene expression have aroused great interest for their potential in the biomedical space. Posttranscriptional modulation can be achieved with ribozymes, riboswitches (simple regulatory elements based on RNA secondary structures), noncoding RNAs, and RNA-binding proteins (RBPs). RBPs are proteins that recognize specific motifs on the mRNA target inducing mRNA decay or translation inhibition. The use of RBPs deriving from different species in mammalian cells has allowed to create sophisticated and multilayered regulatory networks, addressing the previous limitation of regulatory orthogonal parts that can be assembled in synthetic devices. In this chapter, we describe the engineering and tests of protease-responsive RNA-binding proteins (L7Ae and MS2-cNOT7) to expand the toolbox of synthetic circuits in mammalian cells.

Transcriptional gene network inference from a massive dataset elucidates transcriptome organization and gene function.

We collected a massive and heterogeneous dataset of 20 255 gene expression profiles (GEPs) from a variety of human samples and experimental conditions, as well as 8895 GEPs from mouse samples. We developed a mutual information (MI) reverse-engineering approach to quantify the extent to which the mRNA levels of two genes are related to each other across the dataset. The resulting networks consist of 4 817 629 connections among 20 255 transcripts in human and 14 461 095 connections among 45 101 transcripts in mouse, with a inter-species conservation of 12%. The inferred connections were compared against known interactions to assess their biological significance. We experimentally validated a subset of not previously described protein-protein interactions. We discovered co-expressed modules within the networks, consisting of genes strongly connected to each other, which carry out specific biological functions, and tend to be in physical proximity at the chromatin level in the nucleus. We show that the network can be used to predict the biological function and subcellular localization of a protein, and to elucidate the function of a disease gene. We experimentally verified that granulin precursor (GRN) gene, whose mutations cause frontotemporal lobar degeneration, is involved in lysosome function. We have developed an online tool to explore the human and mouse gene networks.

Resource-aware construct design in mammalian cells.

Resource competition can be the cause of unintended coupling between co-expressed genetic constructs. Here we report the quantification of the resource load imposed by different mammalian genetic components and identify construct designs with increased performance and reduced resource footprint. We use these to generate improved synthetic circuits and optimise the co-expression of transfected cassettes, shedding light on how this can be useful for bioproduction and biotherapeutic applications. This work provides the scientific community with a framework to consider resource demand when designing mammalian constructs to achieve robust and optimised gene expression.

Biodiversity: the overlooked source of human health.

Biodiversity is the measure of the variation of lifeforms in a given ecological system. Biodiversity provides ecosystems with the robustness, stability, and resilience that sustains them. This is ultimately essential for our survival because we depend on the services that natural ecosystems provide (food, fresh water, air, climate, and medicine). Despite this, human activity is driving an unprecedented rate of biodiversity decline, which may jeopardize the life-support systems of the planet if no urgent action is taken. In this article we show why biodiversity is essential for human health. We raise our case and focus on the biomedicine services that are enabled by biodiversity, and we present known and novel approaches to promote biodiversity conservation.

Construction and modelling of an inducible positive feedback loop stably integrated in a mammalian cell-line.

Understanding the relationship between topology and dynamics of transcriptional regulatory networks in mammalian cells is essential to elucidate the biology of complex regulatory and signaling pathways. Here, we characterised, via a synthetic biology approach, a transcriptional positive feedback loop (PFL) by generating a clonal population of mammalian cells (CHO) carrying a stable integration of the construct. The PFL network consists of the Tetracycline-controlled transactivator (tTA), whose expression is regulated by a tTA responsive promoter (CMV-TET), thus giving rise to a positive feedback. The same CMV-TET promoter drives also the expression of a destabilised yellow fluorescent protein (d2EYFP), thus the dynamic behaviour can be followed by time-lapse microscopy. The PFL network was compared to an engineered version of the network lacking the positive feedback loop (NOPFL), by expressing the tTA mRNA from a constitutive promoter. Doxycycline was used to repress tTA activation (switch off), and the resulting changes in fluorescence intensity for both the PFL and NOPFL networks were followed for up to 43 h. We observed a striking difference in the dynamics of the PFL and NOPFL networks. Using non-linear dynamical models, able to recapitulate experimental observations, we demonstrated a link between network topology and network dynamics. Namely, transcriptional positive autoregulation can significantly slow down the "switch off" times, as compared to the non-autoregulated system. Doxycycline concentration can modulate the response times of the PFL, whereas the NOPFL always switches off with the same dynamics. Moreover, the PFL can exhibit bistability for a range of Doxycycline concentrations. Since the PFL motif is often found in naturally occurring transcriptional and signaling pathways, we believe our work can be instrumental to characterise their behaviour.

gDesigner: computational design of synthetic gRNAs for Cas12a-based transcriptional repression in mammalian cells.

Synthetic networks require complex intertwined genetic regulation often relying on transcriptional activation or repression of target genes. CRISPRi-based transcription factors facilitate the programmable modulation of endogenous or synthetic promoter activity and the process can be optimised by using software to select appropriate gRNAs and limit non-specific gene modulation. Here, we develop a computational software pipeline, gDesigner, that enables the automated selection of orthogonal gRNAs with minimized off-target effects and promoter crosstalk. We next engineered a Lachnospiraceae bacterium Cas12a (dLbCas12a)-based repression system that downregulates target gene expression by means of steric hindrance of the cognate promoter. Finally, we generated a library of orthogonal synthetic dCas12a-repressed promoters and experimentally demonstrated it in HEK293FT, U2OS and H1299 cells lines. Our system expands the toolkit of mammalian synthetic promoters with a new complementary and orthogonal CRISPRi-based system, ultimately enabling the design of synthetic promoter libraries for multiplex gene perturbation that facilitate the understanding of complex cellular phenotypes.

Engineering Protein-Based Parts for Genetic Devices in Mammalian Cells.

Synthetic biology has been advancing cellular and molecular biology studies through the design of synthetic circuits capable to examine diverse endogenously or exogenously driven regulatory pathways. While early genetic devices were engineered to be insulated from intracellular crosstalk, more recently the need of achieving dynamic control of cellular behavior has led to the development of smart interfaces that connect signal information (sensor) to desired output activation (actuator). Sensor-actuator circuits can respond to diverse inputs, including small molecules, exogenous and endogenous mRNA, noncoding RNA (i.e., miRNA), and proteins to regulate downstream events, transcriptionally, posttranscriptionally, and translationally. These devices require attentive engineering to either create complex chimeric proteins or modify protein structures to be amenable to the specific circuits' architecture and/or purpose.In this chapter, we describe how to implement two different protein-based devices in mammalian cells: (1) a modular platform that sense and respond to disease-associated proteins and (2) a protein-based system that allows simultaneous regulation of RNA translation and protein activity, via RNA-protein and newly engineered protein-protein interactions.

Mitigating losses: how scientific organisations can help address the impact of the COVID-19 pandemic on early-career researchers.

Scientific collaborations among nations to address common problems and to build international partnerships as part of science diplomacy is a well-established notion. The international flow of people and ideas has played an important role in the advancement of the 'Sciences' and the current pandemic scenario has drawn attention towards the genuine need for a stronger role of science diplomacy, science advice and science communication. In dealing with the COVID-19 pandemic, visible interactions across science, policy, science communication to the public and diplomacy worldwide have promptly emerged. These interactions have benefited primarily the disciplines of knowledge that are directly informing the pandemic response, while other scientific fields have been relegated. The effects of the COVID-19 pandemic on scientists of all disciplines and from all world regions are discussed here, with a focus on early-career researchers (ECRs), as a vulnerable population in the research system. Young academies and ECR-driven organisations could suggest ECR-powered solutions and actions that could have the potential to mitigate these effects on ECRs working on disciplines not related to the pandemic response. In relation with governments and other scientific organisations, they can have an impact on strengthening and creating fairer scientific systems for ECRs at the national, regional, and global level.

Correction: Mitigating losses: how scientific organisations can help address the impact of the COVID-19 pandemic on early-career researchers.

[This corrects the article DOI: 10.1057/s41599-021-00944-1.].

Engineering Intracellular Protein Sensors in Mammalian Cells.

Proteins can function as biomarkers of pathological conditions, such as neurodegenerative diseases, infections or metabolic syndromes. Engineering cells to sense and respond to these biomarkers may help the understanding of molecular mechanisms underlying pathologies, as well as to develop new cell-based therapies. While several systems that detect extracellular proteins have been developed, a modular framework that can be easily re-engineered to sense different intracellular proteins was missing. Here, we describe a protocol to implement a modular genetic platform that senses intracellular proteins and activates a specific cellular response. The device operates on intracellular antibodies or small peptides to sense with high specificity the protein of interest, triggering the transcriptional activation of output genes, through a TEV protease (TEVp)-based actuation module. TEVp is a viral protease that selectively cleaves short cognate peptides and is widely used in biotechnology and synthetic biology for its high orthogonality to the cleavage site. Specifically, we engineered devices that recognize and respond to protein-biomarkers of viral infections and genetic diseases, including mutated huntingtin, NS3 serine-protease, Tat and Nef proteins to detect Huntington's disease, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections, respectively. Importantly, the system can be hand tailored for the desired input-output functional outcome, such as fluorescent readouts for biosensors, stimulation of antigen presentation for immune response, or initiation of apoptosis to eliminate unhealthy cells.

Precision Tools in Immuno-Oncology: Synthetic Gene Circuits for Cancer Immunotherapy.

Engineered mammalian cells for medical purposes are becoming a clinically relevant reality thanks to advances in synthetic biology that allow enhanced reliability and safety of cell-based therapies. However, their application is still hampered by challenges including time-consuming design-and-test cycle iterations and costs. For example, in the field of cancer immunotherapy, CAR-T cells targeting CD19 have already been clinically approved to treat several types of leukemia, but their use in the context of solid tumors is still quite inefficient, with additional issues related to the adequate quality control for clinical use. These limitations can be overtaken by innovative bioengineering approaches currently in development. Here we present an overview of recent synthetic biology strategies for mammalian cell therapies, with a special focus on the genetic engineering improvements on CAR-T cells, discussing scenarios for the next generation of genetic circuits for cancer immunotherapy.

Characterization and mitigation of gene expression burden in mammalian cells.

Despite recent advances in circuit engineering, the design of genetic networks in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here, we demonstrate that transiently expressed genes in mammalian cells compete for limited transcriptional and translational resources. This competition results in the coupling of otherwise independent exogenous and endogenous genes, creating a divergence between intended and actual function. Guided by a resource-aware mathematical model, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the use of endogenous miRNAs as elementary components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells.

Synthetic biology has the potential to bring forth advanced genetic devices for applications in healthcare and biotechnology. However, accurately predicting the behavior of engineered genetic devices remains difficult due to lack of modularity, wherein a device's output does not depend only on its intended inputs but also on its context. One contributor to lack of modularity is loading of transcriptional and translational resources, which can induce coupling among otherwise independently-regulated genes. Here, we quantify the effects of resource loading in engineered mammalian genetic systems and develop an endoribonuclease-based feedforward controller that can adapt the expression level of a gene of interest to significant resource loading in mammalian cells. Near-perfect adaptation to resource loads is facilitated by high production and catalytic rates of the endoribonuclease. Our design is portable across cell lines and enables predictable tuning of controller function. Ultimately, our controller is a general-purpose device for predictable, robust, and context-independent control of gene expression.

Cell-Free Protein Synthesis as a Prototyping Platform for Mammalian Synthetic Biology.

The field of mammalian synthetic biology is expanding quickly, and technologies for engineering large synthetic gene circuits are increasingly accessible. However, for mammalian cell engineering, traditional tissue culture methods are slow and cumbersome, and are not suited for high-throughput characterization measurements. Here we have utilized mammalian cell-free protein synthesis (CFPS) assays using HeLa cell extracts and liquid handling automation as an alternative to tissue culture and flow cytometry-based measurements. Our CFPS assays take a few hours, and we have established optimized protocols for small-volume reactions using automated acoustic liquid handling technology. As a proof-of-concept, we characterized diverse types of genetic regulation in CFPS, including T7 constitutive promoter variants, internal ribosomal entry sites (IRES) constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression. Our data shows simple regulatory elements for use in mammalian cells can be quickly prototyped in a CFPS model system.

Protein-based parts and devices that respond to intracellular and extracellular signals in mammalian cells.

Synthetic biology aims to rewire cellular activities and functionality by implementing genetic circuits with high biocomputing capabilities. Recent efforts led to the development of smart sensing interfaces which integrate multiple inputs to activate desired outputs in a highly specific and sensitive manner. In this review, we highlight protein-based interfaces that sense intracellular or extracellular cues providing information about dynamic environmental changes and cellular state. We will also discuss different mechanisms of regulation of gene expression connected to the sensors to develop diagnostic and therapeutic devices. We conclude discussing challenges and opportunities for biomedical applications of synthetic mammalian protein-based devices.

Engineered Cell-Based Therapeutics: Synthetic Biology Meets Immunology.

Synthetic Biology has enabled new approaches to several medical applications including the development of immunotherapies based on bioengineered cells, and most notably the engineering of T-cells with tumor-targeting receptors, the Chimeric Antigen Receptor (CAR)-T cells. CAR-T-cells have successfully treated blood tumors such as large B-cell lymphoma and promise a new scenario of therapeutic interventions also for solid tumors. Learning the lesson from CAR-T cells, we can foster the reprogramming of T lymphocytes with enhanced survival and functional activity in depressing tumor microenvironment, or to challenge diseases such as infections, autoimmune and chronic inflammatory disorders. This review will focus on the most updated bioengineering approaches to increase control, and safety of T-cell activity and to immunomodulate the extracellular microenvironment to augment immune responses. We will also discuss on applications beyond cancer treatment with implications toward the understanding and cure of a broader range of diseases by means of mammalian cells engineering.

Membrane Poration Mechanisms at the Cell-Nanostructure Interface.

3D vertical nanostructures have become one of the most significant methods for interfacing cells and the nanoscale and for accessing significant intracellular functionalities such as membrane potential. As this intracellular access can be induced by means of diverse cellular membrane poration mechanisms, it is important to investigate in detail the cell condition after membrane rupture for assessing the real effects of the poration techniques on the biological environment. Indeed, differences of the membrane dynamics and reshaping have not been observed yet when the membrane-nanostructure system is locally perturbed by, for instance, diverse membrane breakage events. In this work, new insights are provided into the membrane dynamics in case of two different poration approaches, optoacoustic- and electro-poration, both mediated by the same 3D nanostructures. The experimental results offer a detailed overview on the different poration processes in terms of electrical recordings and membrane conformation.

Engineering protein-protein devices for multilayered regulation of mRNA translation using orthogonal proteases in mammalian cells.

The development of RNA-encoded regulatory circuits relying on RNA-binding proteins (RBPs) has enhanced the applicability and prospects of post-transcriptional synthetic network for reprogramming cellular functions. However, the construction of RNA-encoded multilayer networks is still limited by the availability of composable and orthogonal regulatory devices. Here, we report on control of mRNA translation with newly engineered RBPs regulated by viral proteases in mammalian cells. By combining post-transcriptional and post-translational control, we expand the operational landscape of RNA-encoded genetic circuits with a set of regulatory devices including: i) RBP-protease, ii) protease-RBP, iii) protease-protease, iv) protein sensor protease-RBP, and v) miRNA-protease/RBP interactions. The rational design of protease-regulated proteins provides a diverse toolbox for synthetic circuit regulation that enhances multi-input information processing-actuation of cellular responses. Our approach enables design of artificial circuits that can reprogram cellular function with potential benefits as research tools and for future in vivo therapeutics and biotechnological applications.