Exosomes are cell-derived nanovesicles (50–150 nm), which mediate intercellular communication, and are candidate therapeutic agents. However, inefficiency of exosomal message transfer, such as mRNA, and lack of methods to create designer exosomes have hampered their development into therapeutic interventions. Here, we report a set of EXOsomal transfer into cells (EXOtic) devices that enable efficient, customizable production of designer exosomes in engineered mammalian cells. These genetically encoded devices in exosome producer cells enhance exosome production, specific mRNA packaging, and delivery of the mRNA into the cytosol of target cells, enabling efficient cell-to-cell communication without the need to concentrate exosomes. Further, engineered producer cells implanted in living mice could consistently deliver cargo mRNA to the brain. Therapeutic catalase mRNA delivery by designer exosomes attenuated neurotoxicity and neuroinflammation in in vitro and in vivo models of Parkinson’s disease, indicating the potential usefulness of the EXOtic devices for RNA delivery-based therapeutic applications.

Synthetic biology has advanced the design of standardized control devices that program cellular functions and metabolic activities in living organisms. Rational interconnection of these synthetic switches resulted in increasingly complex designer networks that execute input-triggered genetic instructions with precision, robustness and computational logic reminiscent of electronic circuits. Using trigger-controlled transcription factors, which independently control gene expression, and RNA-binding proteins that inhibit the translation of transcripts harbouring specific RNA target motifs, we have designed a set of synthetic transcription–translation control devices that could be rewired in a plug-and-play manner. Here we show that these combinatorial circuits integrated a two-molecule input and performed digital computations with NOT, AND, NAND and N-IMPLY expression logic in single mammalian cells. Functional interconnection of two N-IMPLY variants resulted in bitwise intracellular XOR operations, and a combinatorial arrangement of three logic gates enabled independent cells to perform programmable half-subtractor and half-adder calculations. Individual mammalian cells capable of executing basic molecular arithmetic functions isolated or coordinated to metabolic activities in a predictable, precise and robust manner may provide new treatment strategies and bio-electronic interfaces in future gene-based and cell-based therapies.

The possibility to externally control gene expression is of fundamental importance in both basic and applied life sciences. Although there are some techniques available to regulate expression in mammalian cells, they rely on the presence of ligand-sensing transcription factors, making it necessary to generate cell lines or organisms that stably express these regulatory factors. In recent years, mechanisms relying on direct RNA-ligand interactions for controlling gene expression have been both discovered in nature and engineered artificially. Among the latter, RNA switches relying on catalytically active RNA have been described. In principle, ligand-dependent triggering of mRNA self-cleavage should be a universal mechanism in order to control gene expression in a variety of organisms. Nevertheless, no examples of such aptazymes acting as RNA-based switches have been reported so far in mammalian cells. Here we present the theophylline-induced activation of an mRNA-based hammerhead ribozyme, resulting in an off-switch of gene expression. Starting from an artificial aptazyme switch reported to function in bacteria, we identified and optimized important parameters such as artificial start codons and the communicating sequence connecting ribozyme and aptamer, resulting in an RNA switch that allows for controlling transgenic expression in mammalian cells without the need to express a corresponding ligand-sensing transcription factor.

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Synthetic biology concerns the engineering of man-made living biomachines from standardized components that can perform predefined functions in a (self-)controlled manner. Different research strategies and interdisciplinary efforts are pursued to implement engineering principles to biology. The "top-down" strategy exploits nature's incredible diversity of existing, natural parts to construct synthetic compositions of genetic, metabolic, or signaling networks with predictable and controllable properties. This mainly application-driven approach results in living factories that produce drugs, biofuels, biomaterials, and fine chemicals, and results in living pills that are based on engineered cells with the capacity to autonomously detect and treat disease states in vivo. In contrast, the "bottom-up" strategy seeks to be independent of existing living systems by designing biological systems from scratch and synthesizing artificial biological entities not found in nature. This more knowledge-driven approach investigates the reconstruction of minimal biological systems that are capable of performing basic biological phenomena, such as self-organization, self-replication, and self-sustainability. Moreover, the syntheses of artificial biological units, such as synthetic nucleotides or amino acids, and their implementation into polymers inside living cells currently set the boundaries between natural and artificial biological systems. In particular, the in vitro design, synthesis, and transfer of complete genomes into host cells point to the future of synthetic biology: the creation of designer cells with tailored desirable properties for biomedicine and biotechnology.

RNAs are ideal for the design of gene switches that can monitor and program cellular behavior because of their high modularity and predictable structure-function relationship. We have assembled an expression platform with an embedded modular ribozyme scaffold that correlates self-cleavage activity of designer ribozymes with transgene translation in bacteria and mammalian cells. A design approach devised to screen ribozyme libraries in bacteria and validate variants with functional tertiary stem-loop structures in mammalian cells resulted in a designer ribozyme with a protein-binding nutR-boxB stem II and a selected matching stem I. In a mammalian expression context, this designer ribozyme exhibited dose-dependent translation control by the N-peptide, had rapid induction kinetics and could be combined with classic small molecule-responsive transcription control modalities to construct complex, programmable genetic circuits.

All metabolic activities operate within a narrow pH range that is controlled by the CO2-bicarbonate buffering system. We hypothesized that pH could serve as surrogate signal to monitor and respond to the physiological state. By functionally rewiring the human proton-activated cell-surface receptor TDAG8 to chimeric promoters, we created a synthetic signaling cascade that precisely monitors extracellular pH within the physiological range. The synthetic pH sensor could be adjusted by organic acids as well as gaseous CO2 that shifts the CO2-bicarbonate balance toward hydrogen ions. This enabled the design of gas-programmable logic gates, provided remote control of cellular behavior inside microfluidic devices, and allowed for CO2-triggered production of biopharmaceuticals in standard bioreactors. When implanting cells containing the synthetic pH sensor linked to production of insulin into type 1 diabetic mice developing diabetic ketoacidosis, the prosthetic network automatically scored acidic pH and coordinated an insulin expression response that corrected ketoacidosis.

Synthetic biologists have advanced the design of trigger-inducible gene switches and their assembly into input-programmable circuits that enable engineered human cells to perform arithmetic calculations reminiscent of electronic circuits. By designing a versatile plug-and-play molecular-computation platform, we have engineered nine different cell populations with genetic programs, each of which encodes a defined computational instruction. When assembled into 3D cultures, these engineered cell consortia execute programmable multicellular full-adder logics in response to three trigger compounds.