Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment.

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.

Pharmacologically triggered hydrogel for scheduling hepatitis B vaccine administration.

The simplification of current vaccine administration regimes is of crucial interest in order to further sustain and expand the high impact of vaccines for public health. Most vaccines including the vaccine against hepatitis B need several doses to achieve protective immunization. In order to reduce the amount of repetitive injections, depot-based approaches represent a promising strategy. We present the application of novobiocin-sensitive biohybrid hydrogels as a depot for the pharmacologically controlled release of a vaccine against hepatitis B. Upon subcutaneous implantation of the vaccine depot into mice, we were able to release the vaccine by the oral administration of the stimulus molecule novobiocin resulting in successful immunization of the mice. This material-based vaccination regime holds high promises to replace classical vaccine injections conducted by medical personnel by the simple oral uptake of the stimulus thereby solving a major obstacle in increasing hepatitis B vaccination coverage.

Synthetic two-way communication between mammalian cells.

The design of synthetic biology-inspired control devices enabling entire mammalian cells to receive, process and transfer metabolic information and so communicate with each other via synthetic multichannel networks may provide new insight into the organization of multicellular organisms and future clinical interventions. Here we describe communication networks that orchestrate behavior in individual mammalian cells in response to cell-to-cell metabolic signals. We engineered sender, processor and receiver cells that interact with each other in ways that resemble natural intercellular communication networks such as multistep information processing cascades, feed-forward-based signaling loops, and two-way communication. The engineered two-way communication devices mimicking natural control systems in the development of vertebrate extremities and vasculature was used to program temporal permeability in vascular endothelial cell layers. These synthetic multicellular communication systems may inspire future therapies or tissue engineering strategies.

The food additive vanillic acid controls transgene expression in mammalian cells and mice.

Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin.

Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida-mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies.

Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch.

Applications of conditional gene expression, whether for therapeutic or basic research purposes, are increasingly requiring mammalian gene control systems that exhibit far tighter control properties. While numerous approaches have been used to improve the widely used Tet-regulatory system, many applications, particularly with respect to the engineering of synthetic gene networks, will require a broader range of tightly performing gene control systems. Here, a generically applicable approach is described that utilizes intronically encoded siRNA on the relevant transregulator construct, and siRNA sequence-specific tags on the reporter construct, to minimize basal gene activity in the off-state of a range of common gene control systems. To demonstrate tight control of residual expression the approach was successfully used to conditionally express the toxic proteins RipDD and Linamarase. The intronic siRNA concept was also extended to create a new generation of compact, single-vector, autoinducible siRNA vectors. Finally, using improved regulation systems a mammalian epigenetic toggle switch was engineered that exhibited superior in vitro and in vivo induction characteristics in mice compared to the equivalent non-intronic system.

A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1.

We describe the design and detailed characterization of 6-hydroxy-nicotine (6HNic)-adjustable transgene expression (NICE) systems engineered for lentiviral transduction and in vivo modulation of angiogenic responses. Arthrobacter nicotinovorans pAO1 encodes a unique catabolic machinery on its plasmid pAO1, which enables this Gram-positive soil bacterium to use the tobacco alkaloid nicotine as the exclusive carbon source. The 6HNic-responsive repressor-operator (HdnoR-O(NIC)) interaction, controlling 6HNic oxidase production in A.nicotinovorans pAO1, was engineered for generic 6HNic-adjustable transgene expression in mammalian cells. HdnoR fused to different transactivation domains retained its O(NIC)-binding capacity in mammalian cells and reversibly adjusted transgene transcription from chimeric O(NIC)-containing promoters (P(NIC); O(NIC) fused to a minimal eukaryotic promoter [P(min)]) in a 6HNic-responsive manner. The combination of transactivators containing various transactivation domains with promoters differing in the number of operator modules as well as in their relative inter-O(NIC) and/or O(NIC)-P(min) spacing revealed steric constraints influencing overall NICE regulation performance in mammalian cells. Mice implanted with microencapsulated cells engineered for NICE-controlled expression of the human glycoprotein secreted placental alkaline phosphatase (SEAP) showed high SEAP serum levels in the absence of regulating 6HNic. 6HNic was unable to modulate SEAP expression, suggesting that this nicotine derivative exhibits control-incompatible pharmacokinetics in mice. However, chicken embryos transduced with HIV-1-derived self-inactivating lentiviral particles transgenic for NICE-adjustable expression of the human vascular endothelial growth factor 121 (VEGF121) showed graded 6HNic response following administration of different 6HNic concentrations. Owing to the clinically inert and highly water-soluble compound 6HNic, NICE-adjustable transgene control systems may become a welcome alternative to available drug-responsive homologs in basic research, therapeutic cell engineering and biopharmaceutical manufacturing.

Engineered Streptomyces quorum-sensing components enable inducible siRNA-mediated translation control in mammalian cells and adjustable transcription control in mice.

BACKGROUND: Recent advances in functional genomics, gene therapy, tissue engineering, drug discovery and biopharmaceuticals production have been fostered by precise small-molecule-mediated fine-tuning of desired transgenes. METHODS: Capitalizing on well-evolved quorum-sensing regulatory networks in Streptomyces coelicolor we have designed a mammalian regulation system inducible by the non-toxic butyrolactone SCB1. Fusion of the S. coelicolor SCB1 quorum-sensing receptor ScbR to the human Kox-1-derived transsilencing domain reconstituted a mammalian transsilencer (SCS) able to repress transcription from SCS-specific operator-containing promoters in a reverse SCB1-adjustable manner. RESULTS: This quorum-sensing-derived mammalian transgene control system (Q-ON) enabled precise SCB1-specific fine-tuning of (i) desired transgene transcription in a variety of mammalian/human cell lines and human primary cells, (ii) small interfering RNA-mediated posttranscriptional knockdown (siRNA) in mammalian cells, and (iii) dosing of a human glycoprotein in mice. CONCLUSIONS: As exemplified by Q-ON technology, bacterial quorum-sensing regulons may represent a near-infinite source for the design of mammalian gene control systems compatible with molecular interventions relevant to future gene therapy and tissue engineering scenarios.

Streptomyces-derived quorum-sensing systems engineered for adjustable transgene expression in mammalian cells and mice.

Prokaryotic transcriptional regulatory elements have been adopted for controlled expression of cloned genes in mammalian cells and animals, the cornerstone for gene-function correlations, drug discovery, biopharmaceutical manufacturing as well as advanced gene therapy and tissue engineering. Many prokaryotes have evolved specific molecular communication systems known as quorum-sensing to coordinate population-wide responses to physiological and/or physicochemical signals. A generic bacterial quorum-sensing system is based on a diffusible signal molecule that prevents binding of a repressor to corresponding operator sites thus resulting in derepression of a target regulon. In Streptomyces, a family of butyrolactones and their corresponding receptor proteins, serve as quorum-sensing systems that control morphological development and antibiotic biosynthesis. Fusion of the Streptomyces coelicolor quorum-sensing receptor (ScbR) to a eukaryotic transactivation domain (VP16) created a mammalian transactivator (SCA) which binds and adjusts transcription from chimeric promoters containing an SCA-specific operator module (P(SPA)). Expression of erythropoietin or the human secreted alkaline phosphatase (SEAP) by this quorum-sensor-regulated gene expression system (QuoRex) could be fine-tuned by non-toxic butyrolactones in a variety of mammalian cells including human primary and mouse embryonic stem cells. Following intraperitoneal implantation of microencapsulated Chinese hamster ovary cells transgenic for QuoRex-controlled SEAP expression into mice, the serum levels of this model glycoprotein could be adjusted to desired concentrations using different butyrolactone dosing regimes.