Emerging mammalian gene switches for controlling implantable cell therapies.

Engineered cell-based therapies have emerged as a new paradigm in modern medicine, with several engineered T cell therapies currently approved to treat blood cancers and many more in clinical development. Tremendous progress in synthetic biology over the past two decades has allowed us to program cells with sophisticated sense-and-response modules that can effectively control therapeutic functions. In this review, we highlight recent advances in mammalian synthetic gene switches, focusing on devices designed for therapeutic applications. Although many gene switches responding to endogenous or exogenous molecular signals have been developed, the focus is shifting towards achieving remote-controlled production of therapeutic effectors by stimulating implanted engineered cells with traceless physical signals, such as light, electrical signals, magnetic fields, heat or ultrasound.

A Wnt-mediated phenotype switch along the epithelial-mesenchymal axis defines resistance and invasion downstream of ionising radiation in oral squamous cell carcinoma.

BACKGROUND: Dynamic transitions of tumour cells along the epithelial-mesenchymal axis are important in tumorigenesis, metastasis and therapy resistance. METHODS: In this study, we have used cell lines, 3D spheroids and tumour samples in a variety of cell biological and transcriptome analyses to highlight the cellular and molecular dynamics of OSCC response to ionising radiation. RESULTS: Our study demonstrates a prominent hybrid epithelial-mesenchymal state in oral squamous cell carcinoma cells and tumour samples. We have further identified a key role for levels of E-cadherin in stratifying the hybrid cells to compartments with varying levels of radiation response and radiation-induced epithelial-mesenchymal transition. The response to radiation further entailed the generation of a new cell population with low expression levels of E-cadherin, and positive for Vimentin (ECADLow/Neg-VIMPos), a phenotypic signature that showed an enhanced capacity for radiation resistance and invasion. At the molecular level, transcriptome analysis of spheroids in response to radiation showed an initial burst of misregulation within the first 30 min that further declined, although still highlighting key alterations in gene signatures. Among others, pathway analysis showed an over-representation for the Wnt signalling pathway that was further confirmed to be functionally involved in the generation of ECADLow/Neg-VIMPos population, acting upstream of radiation resistance and tumour cell invasion. CONCLUSION: This study highlights the functional significance and complexity of tumour cell remodelling in response to ionising radiation with links to resistance and invasive capacity. An area of less focus in conventional radiotherapy, with the potential to improve treatment outcomes and relapse-free survival.

Switching Protein Localization by Site-Directed RNA Editing under Control of Light.

Site directed RNA editing is an engineered tool for the posttranscriptional manipulation of RNA and proteins. Here, we demonstrate the inclusion of additional N- and C-terminal protein domains in an RNA editing-dependent manner to switch between protein isoforms in mammalian cell culture. By inclusion of localization signals, a switch of the subcellular protein localization was achieved. This included the shift from the cytoplasm to the outer-membrane, which typically is inaccessible at the protein-level. Furthermore, the strategy allows to implement photocaging to achieve spatiotemporal control of isoform switching. The strategy does not require substantial genetic engineering, and might well complement current optogenetic and optochemical approaches.

Use of Hydroxyapatite Doping to Enhance Responsiveness of Heat-Inducible Gene Switches to Focused Ultrasound.

Recently, we demonstrated that ultrasound-based hyperthermia can activate cells containing a heat-activated and ligand-inducible gene switch in a spatio-temporally controlled manner. These engineered cells can be incorporated into hydrogel scaffolds (e.g., fibrin) for in vivo implantation, where ultrasound can be used to non-invasively pattern transgene expression. Due to their high water content, the acoustic attenuation of fibrin scaffolds is low. Thus, long ultrasound exposures and high acoustic intensities are needed to generate sufficient hyperthermia for gene activation. Here, we demonstrate that the attenuation of fibrin scaffolds and the resulting hyperthermia achievable with ultrasound can be increased significantly by doping the fibrin with hydroxyapatite (HA) nanopowder. The attenuation of a 1% (w/v) fibrin scaffold with 5% (w/v) HA was similar to soft tissue. Transgene activation of cells harboring the gene switch occurred at lower acoustic intensities and shorter exposures when the cells were encapsulated in HA-doped fibrin scaffolds versus undoped scaffolds. Inclusion of HA in the fibrin scaffold did not affect the viability of the encapsulated cells.

Expression switching of three genes encoding light-independent protochlorophyllide oxidoreductase in Chlorella protothecoides.

Three chloroplast genes, chlL, chlN and chlB, encoding the light-independent protochlorophyllide oxidoreductase (LIPOR) in Chlorella protothecoides CS-41 growing either photoautotrophically, mixotrophically or heterotrophically, were all transcribed constitutively independent of illumination and presence of glucose. Steady-state amounts of all three transcripts in the light-grown cells were, however, approximately two- to three-fold greater than those in the dark-grown cells. In addition, Western blotting demonstrated that approximately the same amount of protein was present in cultures grown mixtrophically or heterotrophically both containing glucose. However, much less protein was in photoautotrophic cells. These results suggest that LIPOR activity depends on post-transcriptional and post-translational regulation. Moreover, the fact that LIPOR accumulates in the light-grown cells indicates that LIPOR, which was thought to work only in darkness, may partially account for the protochlorophyllide photoreduction in light. Therefore, LIPOR switches on both in light and in darkness.

Analysis of DNA repair pathways of Schizosaccharomyces pombe by means of swi-rad double mutants.

In Schizosaccharomyces pombe 11 different switching genes (swi1 to swi10 and rad22) are known which are involved in mating-type (MT) switching. Mutations in swi5, swi9, swi10 and rad22 also cause an increased radiation sensitivity. We tested whether the survival of these mutants after UV irradiation is influenced by caffeine. We included rad1 and rad13 mutants in our experiments which do not affect MT switching. Several double and triple mutants were constructed. We were able to assign the switching genes to different repair pathways: swi9 and swi10 are involved in excision repair, rad22 has a function in recombination repair, while swi5 appears to be involved in a hitherto unknown pathway. This 'swi5 pathway' is stimulated (!) by caffeine. Previously it was found that the swi5 mutation also reduces meiotic recombination. As to rad genes, we found a few inconsistencies with previous reports in the literature.