A versatile plasmid architecture for mammalian synthetic biology (VAMSyB)

Haellman, Viktor; Strittmatter, Tobias; Bertschi, Adrian; Stücheli, Pascal; Fussenegger, Martin

doi:10.1016/j.ymben.2021.04.003

Cited by 5 publications

(5 citation statements)

References 40 publications

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“…This technique exploits high pressure, volume, and plasmid concentrations to facilitate the uptake of DNA by hepatocytes, with very low or no vector delivery to other organs, such as the heart, spleen, or kidneys. [ 28 , 32 , 33 ] To increase the plasmid delivery efficiency, we first combined all SWEET components into a single vector (pSG151), [ 34 ] which was successfully validated in vitro by measuring xylose‐inducible SEAP production (Figure S5 , Supporting Information), and we then determined the optimal amount of plasmid to be delivered in vivo (Figure S6 , Supporting Information). SWEET‐engineered mice that received xylose by o.g.…”

Section: Resultsmentioning

confidence: 99%

Regulation of Transgene Expression by the Natural Sweetener Xylose

et al. 2022

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Next‐generation gene and engineered‐cell therapies benefit from incorporating synthetic gene networks that can precisely regulate the therapeutic output in response to externally administered signal inputs that are safe, readily bioavailable and pleasant to take. To enable such therapeutic control, a mammalian gene switch is designed to be responsive to the natural sweetener xylose and its functionality is assessed in mouse studies. The gene switch consists of the bacterial transcription regulator XylR fused to a mammalian transactivator, which binds to an optimized promoter in the presence of xylose, thereby allowing dose‐dependent transgene expression. The sensitivity of SWEET (sweetener‐inducible expression of transgene) is improved by coexpressing a xylose transporter. Mice implanted with encapsulated SWEET‐engineered cells show increased blood levels of cargo protein when taking xylose‐sweetened water or coffee, or highly concentrated apple extract, while they do not respond to intake of a usual amount of carrots, which contain xylose. In a proof‐of‐concept therapeutic application study, type‐1 diabetic mice engineered with insulin‐expressing SWEET show lowered glycemia and increased insulin levels when administered this fairly diabetic‐compliant sweetener, compared to untreated mice. A SWEET‐based therapy appears to have the potential to integrate seamlessly into patients’ life‐style and food habits in the move toward personalized medicine.

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Section: Resultsmentioning

confidence: 99%

Regulation of Transgene Expression by the Natural Sweetener Xylose

et al. 2022

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“…The TetO 7 -SEAP construct was cloned on a Tier-3 vector flanked by Sleeping Beauty transposase recognition sites 46 and also encoded constitutive expression of the puromycin resistance gene. It was transfected into HEK293T cells capitalizing on the Sleeping Beauty transposon protocol 47 .…”

Section: Methodsmentioning

confidence: 99%

Combinatorial protein dimerization enables precise multi-input synthetic computations

et al. 2023

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Bacterial transcription factors (TFs) with helix-turn-helix (HTH) DNA-binding domains have been widely explored to build orthogonal transcriptional regulation systems in mammalian cells. Here we capitalize on the modular structure of these proteins to build a framework for multi-input logic gates relying on serial combinations of inducible protein–protein interactions. We found that for some TFs, their HTH domain alone is sufficient for DNA binding. By fusing the HTH domain to TFs, we established dimerization dependent rather than DNA-binding-dependent activation. This enabled us to convert gene switches from OFF-type into more widely applicable ON-type systems and to create mammalian gene switches responsive to new inducers. By combining both OFF and ON modes of action, we built a compact, high-performance bandpass filter. Furthermore, we were able to show cytosolic and extracellular dimerization. Cascading up to five pairwise fusion proteins yielded robust multi-input AND logic gates. Combinations of different pairwise fusion proteins afforded a variety of 4-input 1-output AND and OR logic gate configurations.

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“…HEK-293T cells were transfected with a hyperactive Sleeping Beauty (SB) transposase (pTS395) expression vector ( 29 ) in a 1:5 (weight/weight) ratio with pAna366 and pAna368 vectors, containing SB recognition sites and encoding a puromycin resistance marker and a blue fluorescent protein. The medium was exchanged 12 h after transfection and cells were incubated for 48 h before the addition of selection medium containing 4 μg/mL puromycin.…”

Section: Methodsmentioning

confidence: 99%

Evolution of molecular switches for regulation of transgene expression by clinically licensed gluconate

Teixeira

Xue

Huang

et al. 2023

Nucleic Acids Research

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Synthetic biology holds great promise to improve the safety and efficacy of future gene and engineered cell therapies by providing new means of endogenous or exogenous control of the embedded therapeutic programs. Here, we focused on gluconate as a clinically licensed small-molecule inducer and engineered gluconate-sensitive molecular switches to regulate transgene expression in human cell cultures and in mice. Several switch designs were assembled based on the gluconate-responsive transcriptional repressor GntR from Escherichia coli. Initially we assembled OFF- and ON-type switches by rewiring the native gluconate-dependent binding of GntR to target DNA sequences in mammalian cells. Then, we utilized the ability of GntR to dimerize in the presence of gluconate to activate gene expression from a split transcriptional activator. By means of random mutagenesis of GntR combined with phenotypic screening, we identified variants that significantly enhanced the functionality of the genetic devices, enabling the construction of robust two-input logic gates. We also demonstrated the potential utility of the synthetic switch in two in vivo settings, one employing implantation of alginate-encapsulated engineered cells and the other involving modification of host cells by DNA delivery. Then, as proof-of-concept, the gluconate-actuated genetic switch was connected to insulin secretion, and the components encoding gluconate-induced insulin production were introduced into type-1 diabetic mice as naked DNA via hydrodynamic tail vein injection. Normoglycemia was restored, thereby showcasing the suitability of oral gluconate to regulate in situ production of a therapeutic protein.

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A versatile plasmid architecture for mammalian synthetic biology (VAMSyB)

Cited by 5 publications

References 40 publications

Regulation of Transgene Expression by the Natural Sweetener Xylose

Regulation of Transgene Expression by the Natural Sweetener Xylose

Combinatorial protein dimerization enables precise multi-input synthetic computations

Evolution of molecular switches for regulation of transgene expression by clinically licensed gluconate

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