Shaping the future US bioeconomy through safety, security, sustainability, and social responsibility.

Biomanufacturing practitioners and researchers describe the norms that should govern the growing, global field, to include safety, security, sustainability, and social responsibility. These '4S Principles' should be broadly adopted so that the future of the field may provide the greatest benefits to society.

Comparing research and development, launch, and scale up timelines of 18 vaccines: lessons learnt from COVID-19 and implications for other infectious diseases.

Over the next decade, millions of deaths could be prevented by increasing access to vaccines in low-income and middle-income countries (LMICs). The COVID-19 pandemic has demonstrated that the research and development (R&D), launch and scale up timelines of vaccines can be drastically shortened. This study compares such timelines for eighteen vaccines and identifies lessons and implications for accelerating the R&D, launch and scale up process for other vaccine candidates. To replicate the rapid R&D process of the COVID-19 vaccines, future vaccine R&D should capitalise on public-private knowledge sharing partnerships to promote technology innovation, establish regional clinical trial centres and data sharing networks to optimise clinical trial efficiency, and create a funding mechanism to support research into novel vaccine platforms that may prove valuable to quickly developing vaccine candidates in future global health emergencies. To accelerate the launch timeline, future efforts to bring safe and efficacious vaccines to market should include LMICs in the decision-making processes of global procurement and delivery alliances to optimise launch in these countries, strengthen the WHO prequalification and Emergency Use Listing programs to ensure LMICs have a robust and transparent regulatory system to rely on, and invest in LMIC regulatory and manufacturing capacity to ensure these countries are vaccine self-sufficient. Lastly, efforts to accelerate scale up of vaccines should include the creation of regional pooled procurement mechanisms between LMICs to increase purchasing power among these countries and an open line of clear communication with the public regarding pertinent vaccine information to combat misinformation and vaccine hesitancy.

Multidimensional Characterization of Parts Enhances Modeling Accuracy in Genetic Circuits.

Mathematical models can aid the design of genetic circuits, but may yield inaccurate results if individual parts are not modeled at the appropriate resolution. To illustrate the importance of this concept, we study transcriptional cascades consisting of two inducible synthetic transcription factors connected in series. Despite the simplicity of this design, we find that accurate prediction of circuit behavior requires mapping the dose responses of each circuit component along the dimensions of both its expression level and its inducer concentration. Using this multidimensional characterization, we were able to computationally explore the behavior of 16 different circuit designs. We experimentally verified a subset of these predictions and found substantial agreement. This method of biological part characterization enables the use of models to identify (un)desired circuit behaviors prior to experimental implementation, thus shortening the design-build-test cycle for more complex circuits.

De novo design of protein logic gates.

The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo-designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the TIM3 gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions.

Publisher Correction: Modular and tunable biological feedback control using a de novo protein switch.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells.

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods for the assembly of mammalian DNA circuits are laborious, slow, and expensive. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible, and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be assembled into transcriptional units and further weaved into complex circuits. We showcase the capabilities of the MTK by using it to generate single-integration landing pads, create and deliver libraries of protein variants and sgRNAs, and iterate through dCas9-based prototype circuits. As a biological proof of concept, we demonstrate how the MTK can speed the generation of noninfectious viral circuits to enable rapid testing of pharmacological inhibitors of emerging viruses that pose a major threat to human health.

Modular and tunable biological feedback control using a de novo protein switch.

De novo-designed proteins1-3 hold great promise as building blocks for synthetic circuits, and can complement the use of engineered variants of natural proteins4-7. One such designer protein-degronLOCKR, which is based on 'latching orthogonal cage-key proteins' (LOCKR) technology8-is a switch that degrades a protein of interest in vivo upon induction by a genetically encoded small peptide. Here we leverage the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signalling pathways and synthetic gene circuits. We first generate synthetic negative and positive feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signalling molecules, illustrating the ease with which this strategy can be used to rewire complex endogenous pathways. We next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit9, to quantify the feedback capabilities and operational range of the feedback control circuit. The designed nature of degronLOCKR proteins enables simple and rational modifications to tune feedback behaviour in both the synthetic circuit and the mating pathway. The ability to engineer feedback control into living cells represents an important milestone in achieving the full potential of synthetic biology10,11,12. More broadly, this work demonstrates the large and untapped potential of de novo design of proteins for generating tools that implement complex synthetic functionalities in cells for biotechnological and therapeutic applications.

De novo design of bioactive protein switches.

Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix 'cage' with a single interface that can interact either intramolecularly with a terminal 'latch' helix or intermolecularly with a peptide 'key'. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage-key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering.