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.

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The ability to engineer synthetic circuits and devices in mammalian cells has enabled a multitude of exciting applications in industrial biotechnology and medical therapy. In spite of the recent availability of powerful genome engineering tools such as CRISPR-Cas9, the process of designing and implementing functioning genetic circuits remains painstakingly slow and fraught with inexplicable failures. The unexpected divergence between intended and actual function of synthetic circuits can be attributed to several factors, most notably the contextual background in which these circuits operate. In particular, t he dependence of synthetic circuits on cellular resources which are limited leads to unintended dynamic couplings between the various exogenous components of the circuit, as well as with endogenous components of the host cell. The consumption of these resources by synthetic circuits thus exerts a burden on the host cell that reduces its capacity to support additional circuits, potentially resulting in counter-intuitive functional behaviors and detrimental effects on host physiology. In spite of its critical importance, gene expression burden in mammalian cells remains largely unstudied. Here, we comprehensively investigate the impact of host resource limitations on synthetic constructs in mammalian cells. We show that effects of both transcriptional and translational resource limitations can be observed and that each can lead to the coupling of independent, co-expressed synthetic genes, which in turn imposes trade-offs in their expression and diminishes performance. We next explore the role of post-transcriptional regulators, such as microRNAs (miRNAs) and RNA binding proteins (RBPs) and show that they can redistribute resources in a way that limits burden-induced coupling effects. To quantify and predict the influence of burden on gene expression in engineered cells, we describe a modelling framework that allows to incorporate the effect of limited resources into classical models of gene expression. Based on this framework, we identify network topologies that mitigate burden and then implement these topologies using endogenous and synthetic miRNA-based circuits that buffer the expression of genes of interest from fluctuations in cellular resources. Among other regulators, microRNAs can conveniently tailor synthetic device regulation in different cell lines and tissues, as well as during dynamic changes of cellular states and downstream information processing, or in pathological conditions. This study thus establishes a foundation for context-aware predictions of in vivo synthetic circuit performance and paves the way towards a more rational synthetic construct design in mammalian cells.

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.

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Resource competition can be the cause of unintended coupling between co-expressed genetic constructs. Here we report 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 co-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 take resource demand into consideration when designing mammalian constructs to achieve robust and optimised gene expression.

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.

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