A Ratiometric Gene-Switch System for miRNA Sensing and Gene Regulation.

microRNAs (miRNAs) are a class of non-coding, small RNAs that play an important role in diverse biological processes and diseases. By regulating the expression of eukaryotic genes post-transcriptionally in a sequence-specific manner, miRNAs are widely used to design synthetic RNA switches. However, most of the RNA switches are often dependent on the corresponding ligand molecules, whose specificity and concentration would affect the efficiency of synthetic RNA circuits. Here, a fused transcriptional repressor Gal4BD-Rluc based gene-switch system Gal-miR for miRNA visualization and gene regulation is described. By placing a luciferase downstream gene under the control of endogenous miRNA machinery, the Gal-miR system makes the conversion of miRNA-mediated gene silencing into a ratiometric bioluminescent signal, which quantitatively reflected miRNA-206 activity during myogenic differentiation. Moreover, it demonstrates that this gene-switch system can effectively inhibit breast cancer cell viability, migration and invasion under the control of specific miRNAs by replacing the downstream gene with melittin functional gene. The study proposes a powerful modular genetic design for achieving precise control of transgene expression in a miRNA responsive way, as well as visualizing the dynamics of miRNA activity.

Reprogramming a Doxycycline-Inducible Gene Switch System for Bacteria-Mediated Cancer Therapy.

PURPOSE: Attenuated Salmonella typhimurium is a potential biotherapeutic antitumor agent because it can colonize tumors and inhibit their growth. The present study aimed to develop a doxycycline (Doxy)-inducible gene switch system in attenuated S. typhimurium and assess its therapeutic efficacy in various tumor-bearing mice models. PROCEDURES: A Doxy-inducible gene switch system comprising two plasmids was engineered to trigger the expression of cargo genes (Rluc8 and clyA). Attenuated S. typhimurium carrying Rluc8 were injected intravenously into BALB/c mice bearing CT26 tumors, and bioluminescence images were captured at specified intervals post-administration of doxycycline. The tumor-suppressive effects of bacteria carrying clyA were evaluated in BALB/c mice bearing CT26 tumors and in C57BL/6 mice bearing MC38 tumors. RESULTS: Expression of the fimE gene, induced only in the presence of Doxy, triggered a unidirectional switch of the POXB20 promoter to induce expression of the cargo genes. The switch event was maintained over a long period of bacterial culture. After intravenous injection of transformed Salmonella into mice bearing CT26 tumors, the bacteria transformed with the Doxy-inducible gene switch system for Rluc8 targeted only tumor tissues and expressed the payloads 2 days after Doxy treatment. Notably, bacteria carrying the Doxy-inducible gene switch system for clyA effectively suppressed tumor growth and prolonged survival, even after just one Doxy induction. CONCLUSIONS: These results suggest that attenuated S. typhimurium carrying this novel gene switch system elicited significant therapeutic effects through a single induction triggering and were a potential biotherapeutic agent for tumor therapy.

Effect of the switch status of Helicobacter pylori outer inflammatory protein A on gastric diseases.

Helicobacter pylori OipA (Outer Inflammatory Protein A) is an outer membrane protein that takes role in the adherence and colonization to the stomach. oipA gene expression is regulated by the slipped-strand mispairing mechanism through a hypermutable CT dinucleotide repeat motif in the 5΄ region. Alterations in the CT number repeats cause frame-shift mutations to result in phase variation of oipA expression. While a functional "On" status has been recognized as a risk factor for peptic ulcer diseases and gastric cancer in many studies, some controversial findings still exist. To this end, this study compiled the sequence data of oipA from 10 different studies between 2000-2019 and 50 oipA DNA sequences from our own research that examined the relationship between the phase On/Off status of oipA and gastric diseases based on CT repeat number. Overall, we have reached 536 oipA DNA sequences from patients. This large collection of oipA sequences first clarified the absolute conservation of the peptide-pentamer of FWLHA for phase ''On'' status, suggesting this pentamer as a superior marker for the determination of oipA status than counting the number of CT repeats. Combining the sequence and patient data, we have re-analyzed the association between the ''On'' status of oipA and gastric diseases. Our results showed a strong association between oipA ''On'' status and gastric cancer supporting previous findings. We also investigated the AlphaFold2 computed structure of OipA that adopts a beta-barrel fold closely resembling to the autotransporter family of H. pylori. Altogether, this study confirms a strong association between oipA ''On'' statuses and severe gastrointestinal diseases like cancer and provides useful insights into the FWLHA pentamer as an indicator of "On" status of oipA putative autotransporter function rather than CT repeats number.

Remote control of transgene expression using noninvasive near-infrared irradiation.

This study investigated whether noninvasive near-infrared (NIR) energy could be transduced into heat in deep-seated organs in which adenovirus type-5 vectors tend to accumulate, thereby activating heat shock protein (HSP) promoter-mediated transgene expression, without local administration of photothermal agents. NIR irradiation of the subdiaphragmatic and left dorsocranial part of the abdominal cavity of adult immunocompetent C3H/HeNRj mice with an 808-nm laser effectively increased the temperature of the irradiated regions of the liver and spleen, respectively, resulting in the accumulation of the heat-inducible HSP70 protein. Spatial control of transgene expression was achieved in the NIR-irradiated regions of the mice administered an adenoviral vector carrying a firefly luciferase (fLuc) coding sequence controlled by a human HSP70B promoter, as assessed by bioluminescence and immunohistochemistry analyses. Levels of reporter gene expression were modulated by controlling NIR power density. Spatial control of transgene expression through NIR-focused activation of the HSP70B promoter, as well as temporal regulation by administering rapamycin was achieved in the spleens of mice inoculated with an adenoviral vector encoding a rapamycin-dependent transactivator driven by the HSP70B promoter and an adenoviral vector carrying a fLuc coding sequence controlled by the rapamycin-activated transactivator. Mice that were administered rapamycin and exposed to NIR light expressed fLuc activity in the splenic region, whereas no activity was detected in mice that were only administered rapamycin or vehicle or only NIR-irradiated. Thus, in the absence of any exogenously supplied photothermal material, remote control of heat-induced transgene expression can be achieved in the liver and spleen by means of noninvasive NIR irradiation.

Facile Expansion of the Variety of Orthogonal Ligand/Aptamer Pairs for Artificial Riboswitches.

An RNA aptamer that induces suitable conformational changes upon binding to a user-defined ligand allows us to artificially construct a riboswitch, a ligand-dependent and cis-acting gene regulatory RNA. Although such an aptamer can be obtained through in vitro selection, it is still challenging to rationally expand the variety of orthogonal ligand/aptamer (ligand/riboswitch) pairs. To achieve this in a facile, selection-free way, we herein focused on a specific type of ligand, 6-nt nanosized DNA (nDNA) and its aptamer that was previously selected to construct a eukaryotic artificial riboswitch. Specifically, we merely mutated one or more possible Watson-Crick base pairs in the nDNA/aptamer (nDNA/riboswitch) interactions into another base pair or pairs. Using two sets that each had 16 comprehensive mutations, we obtained three groups of several orthogonal nDNA/riboswitch pairs. These pairs could be used to create complex gene circuits, including multiple simultaneous and/or multistep cascading regulations in synthetic biology.

Application of the FLP/LoxP-FRT recombination system to switch the eGFP expression in a model prokaryote.

In prokaryotes, few studies have applied the flippase (FLP)/P1-flippase recombination target (LoxP-FRT) recombination system to switch gene expression. This study developed a new method for switching gene expression by constructing an FLP/LoxP-FRT site-specific recombination system in Escherichia coli. To this end, we placed the Nos terminator flanked by a pair of LoxP-FRT in front of enhanced green fluorescent protein (eGFP). The Nos terminator was used to block the expression of the eGFP. When a plasmid expressing FLP was available, deletion of the Nos terminator would allow expression of eGFP. The regulatory effect was demonstrated by eGFP expression. The efficiency of the gene switch was calculated as high as 89.67%. The results showed that the FLP/LoxP-FRT recombinase system could be used as a gene switch to regulate gene expression in prokaryotes. This new method for switching gene expression could simplify the gene function analysis in E. coli and other prokaryotes, as well as eukaryotes.

Enhancement of nanoparticle-mediated double suicide gene expression driven by 'E9-hTERT promoter' switch in dedifferentiated thyroid cancer cells.

Differentiated thyroid cancer (DTC), such as papillary thyroid cancer, has a good prognosis after routine treatment. However, in the course of treatment, 5% to 20% of cases may dedifferentiate and can be transformed into dedifferentiated DTC (deDTC) or anaplastic thyroid cancer, leading to treatment failure. To date, several drugs have been used effectively for dedifferentiated thyroid cancer, whereas gene therapy may be a potential method. Literature reported that double suicide genes driven by human telomerase reverse transcriptase promoter (hTERTp) can specifically express in cancer cells and kill them. However, the weak activity of hTERTp limits its further research. To overcome this weakness, we constructed a novel chitosan nanocarrier containing double suicide genes driven by a 'gene switch' (a cascade of radiation enhancer E9 and a hTERTp). The vector was labeled with iodine-131 (131I). On one hand, E9 can significantly enhance the activity of hTERTp under the weak radiation of 131I, thereby increasing the expression of double suicide genes in deDTC cells. On the other hand, 131I also plays a certain killing role when it enters host cells. The proposed nanocarrier has good specificity for deDTC cells and thus deserves further study.

Optimized pharmacological control over the AAV-Gene-Switch vector for regulable gene therapy.

Gene therapy in its current design is an irreversible process. It cannot be stopped in case of unwanted side effects, nor can expression levels of therapeutics be adjusted to individual patient's needs. Thus, the Gene-Switch (GS) system for pharmacologically regulable neurotrophic factor expression was established for treatment of parkinsonian patients. Mifepristone, the synthetic steroid used to control transgene expression of the GS vector, is an approved clinical drug. However, pharmacokinetics and -dynamics of mifepristone vary considerably between different experimental animal species and depend on age and gender. In humans, but not in any other species, mifepristone binds to a high-affinity plasma carrier protein. We now demonstrate that the formulation of mifepristone can have robust impact on its ability to activate the GS system. Furthermore, we show that a pharmacological booster, ritonavir (Rtv), robustly enhances the pharmacological effect of mifepristone, and allows it to overcome gender- and species-specific pharmacokinetic and -dynamic issues. Most importantly, we demonstrate that the GS vector can be efficiently controlled by mifepristone in the presence of its human plasma carrier protein, α1-acid glycoprotein, in a "humanized" rat model. Thus, we have substantially improved the applicability of the GS vector toward therapeutic use in patients.

Intracellular Unnatural Catalysis Enabled by an Artificial Metalloenzyme.

Artificial metalloenzymes, constructed by incorporating a synthetic catalyst into the internal spaces of a protein scaffold, can perform noncanonical chemical transformations that are not possible using natural enzymes. The addition of cell-permeable modules to artificial metalloenzymes allows for noncanonical catalysis to be implemented as a function of mammalian cells. In this chapter, we describe a protocol for controlling cellular function through a cascade consisting of an artificial metalloenzyme and a gene-circuit engineered via synthetic biology.

Rational design and optimization of synthetic gene switches for controlling cell-fate decisions in pluripotent stem cells.

Advances in synthetic biology have enabled robust control of cell behavior by using tunable genetic circuits to regulate gene expression in a ligand-dependent manner. Such circuits can be used to direct the differentiation of pluripotent stem cells (PSCs) towards desired cell types, but rational design of synthetic gene circuits in PSCs is challenging due to the variable intracellular environment. Here, we provide a framework for implementing synthetic gene switches in PSCs based on combinations of tunable transcriptional, structural, and posttranslational elements that can be engineered as required, using the vanillic acid-controlled transcriptional activator (VanA) as a model system. We further show that the VanA system can be multiplexed with the well-established reverse tetracycline-controlled transcriptional activator (rtTA) system to enable independent control of the expression of different transcription factors in human induced PSCs in order to enhance lineage specification towards early pancreatic progenitors. This work represents a first step towards standardizing the design and construction of synthetic gene switches for building robust gene-regulatory networks to guide stem cell differentiation towards a desired cell fate.

A Blue Light-Inducible CRISPR-Cas9 System for Inhibiting Progression of Melanoma Cells.

Melanoma is an aggressive skin tumor that shows a high mortality rate and level of metastasis. BRAF gene mutation (BRAF V600E) is directly related to the occurrence of melanoma. In this study, a light-inducible gene expression system was designed to control the Cas9 transcription, which could then cleave the BRAF V600E. To prove the potential utility of this system in melanoma, the physiological function of melanoma cells was tested. It illustrated that the light-induced CRISPR-Cas9 system could inhibit the progression of G361 and A375 cells. Thus, this system may provide a novel therapeutic strategy of melanoma intervention.

Generation of mouse iPS cells using an inducible expression of transgenes via the cumate gene-switch.

We applied an inducible gene expression system that utilizes the p-cmt operon, the cumate gene-switch, to generate mouse induced pluripotent stem (iPS) cells. Mouse embryonic fibroblast (MEF) E6E7-MEF cells were transfected with a single cumate gene-switch vector enabling concomitant expression of Oct4, Sox2, c-Myc, Klf4, and Gfp. Then, the cells were cultured with cumate, a monoterpene. An increase in colonies positive for alkaline phosphatase activity was observed dose-dependently with cumate. In the absence of cumate, the expression of GFP, a marker for transgene expression, was undetectable in tightly aggregated iPS cell-like colonies with endogenous expression of NANOG and OCT4. From primary MEFs using the cumate gene-switch, we also isolated iPS cells expressing endogenous NANOG, OCT4, SOX2, KLF4, and SSEA1 with hypo-methylated genomic promoter regions of endogenous Nanog and Oct4. In embryoid bodies with the progression of differentiation, expression of markers for all three germ layers was detected, and contracting cardiomyocytes were observed. Overall, we suggest that the cumate gene-switch is applicable for the generation of mouse iPS cells. The cumate gene-switch in combination with other inducible systems, such as the tet system, may provide useful approaches for analyzing the roles of transgenes underlying the establishment of iPS cells.

CROX (Cluster Regulation of RUNX) as a Potential Novel Therapeutic Approach.

Comprehensive inhibition of RUNX1, RUNX2, and RUNX3 led to marked cell suppression compared with inhibition of RUNX1 alone, clarifying that the RUNX family members are important for proliferation and maintenance of diverse cancers, and "cluster regulation of RUNX (CROX)" is a very effective strategy to suppress cancer cells. Recent studies reported by us and other groups suggested that wild-type RUNX1 is needed for survival and proliferation of certain types of leukemia, lung cancer, gastric cancer, etc. and for their one of metastatic target sites such as born marrow endothelial niche, suggesting that RUNX1 often functions oncogenic manners in cancer cells. In this review, we describe the significance and paradoxical requirement of RUNX1 tumor suppressor in leukemia and even solid cancers based on recent our findings such as "genetic compensation of RUNX family transcription factors (the compensation mechanism for the total level of RUNX family protein expression)", "RUNX1 inhibition-induced inhibitory effects on leukemia cells and on solid cancers through p53 activation", and "autonomous feedback loop of RUNX1-p53-CBFB in acute myeloid leukemia cells". Taken together, these findings identify a crucial role for the RUNX cluster in the maintenance and progression of cancers and suggest that modulation of the RUNX cluster using the pyrrole-imidazole polyamide gene-switch technology is a potential novel therapeutic approach to control cancers.

Orthogonal Protein-Responsive mRNA Switches for Mammalian Synthetic Biology.

The lack of available genetic modules is a fundamental issue in mammalian synthetic biology. Especially, the variety of genetic parts for translational control are limited. Here we report a new set of synthetic mRNA-based translational switches by engineering RNA-binding proteins (RBPs) and RBP-binding RNA motifs (aptamers) that perform strong translational repression. We redesigned the RNA motifs with RNA scaffolds and improved the efficiency of the repression to target RBPs. Using new and previously reported mRNA switches, we demonstrated that the orthogonality of translational regulation was ensured among five different RBP-responsive switches. Moreover, the new switches functioned not only with plasmid introduction, but also with RNA-only delivery, which provides a transient and safer regulation of expression. The translational regulators using RNA-protein interactions provide an alternative strategy to construct complex genetic circuits for future cell engineering and therapeutics.

[Development and application of optogenetic tools].

Dynamic variations of the cell microenvironment can affect cell differentiation, cell signaling pathways, individual growth, and disease. Optogenetics combines gene-encoded protein expression with optical controlling, and offers a novel, reversible, non-invasive and spatiotemporal-specific research tool to dynamically or reversibly regulate cell signaling pathways, subcellular localization and gene expression. This review summarizes the types of optogenetic components and the involved cellular signaling pathways, and explores the application and future prospects of the light-controlled cell signaling pathways.

Reducing recombinant protein expression during CHO pool selection enhances frequency of high-producing cells.

Chinese hamster ovary (CHO) cells are the most widely used mammalian host for industrial-scale production of monoclonal antibodies (mAbs) and other protein biologics. Isolation of rare high-producing CHO cell lines from heterogeneous populations of stable transfectants is a daunting task and delays the process of manufacturing of novel biologics. A variety of factors that contribute to the low frequency of high-producing clones have been described; however, the impact of metabolic burden and other stresses (eg. ER stress) associated with sustained high-level expression of recombinant protein (r-protein) during selection of stable transfectants has not been fully appreciated. CHO cell line development has not traditionally received much optimization in this area because the vast majority of platforms use constitutive expression systems to produce biologics. Previously, we developed a cell line (CHOBRI/rcTA) containing a robust inducible expression system, based on the cumate gene switch, that allows r-protein expression to be down-regulated during selection. Using this switch, we generated inducible CHOBRI/rcTA pools expressing an Fc-fusion protein within two weeks of transfection with volumetric productivity of up to 1.1 g/L at 17 days post-induction in a fed-batch culture process. Herein, we show that the ability to regulate r-protein expression during pool generation confers a substantial advantage for selecting high-producing stable clones. Reducing expression levels ("off-state") during pool selection dramatically enhances high-producer frequency compared to a pool in which expression was maintained at a high level during selection ("on-state", mimicking a constitutive expression system). Overexpression of the r-protein during the pool selection process negatively affects pool recovery and is associated with subtle but significant increases in BiP expression and cell death compared to pool selection in the "off-state". Our data shows that the cumate gene switch is a valuable platform for stable clone generation and supports the wider application of inducible systems for scalable production of biologics in CHO cells.

Immunization by Replication-Competent Controlled Herpesvirus Vectors.

Replication-competent controlled virus vectors were derived from the virulent herpes simplex virus 1 (HSV-1) wild-type strain 17syn+ by placing one or two replication-essential genes under the stringent control of a gene switch that is coactivated by heat and an antiprogestin. Upon activation of the gene switch, the vectors replicate in infected cells with an efficacy that approaches that of the wild-type virus from which they were derived. Essentially no replication occurs in the absence of activation. When administered to mice, localized application of a transient heat treatment in the presence of systemic antiprogestin results in efficient but limited virus replication at the site of administration. The immunogenicity of these viral vectors was tested in a mouse footpad lethal challenge model. Unactivated viral vectors-which may be regarded as equivalents of inactivated vaccines-induced detectable protection against lethality caused by wild-type virus challenge. Single activation of the viral vectors at the site of administration (rear footpads) greatly enhanced protective immune responses, and a second immunization resulted in complete protection. Once activated, vectors also induced far better neutralizing antibody and HSV-1-specific cellular immune responses than unactivated vectors. To find out whether the immunogenicity of a heterologous antigen was also enhanced in the context of efficient transient vector replication, a virus vector constitutively expressing an equine influenza virus hemagglutinin was constructed. Immunization of mice with this recombinant induced detectable antibody-mediated neutralization of equine influenza virus, as well as a hemagglutinin-specific cellular immune response. Single activation of viral replication resulted in a severalfold enhancement of these immune responses.IMPORTANCE We hypothesized that vigorous replication of a pathogen may be critical for eliciting the most potent and balanced immune response against it. Hence, attenuation/inactivation (as in conventional vaccines) should be avoided. Instead, the necessary safety should be provided by placing replication of the pathogen under stringent control and by activating time-limited replication of the pathogen strictly in an administration region in which pathology cannot develop. Immunization will then occur in the context of highly efficient pathogen replication and uncompromised safety. We found that localized activation in mice of efficient but limited replication of a replication-competent controlled herpesvirus vector resulted in a greatly enhanced immune response to the virus or an expressed heterologous antigen. This finding supports the above-mentioned hypothesis and suggests that the vectors may be promising novel agents worth exploring for the prevention/mitigation of infectious diseases for which efficient vaccination is lacking, in particular in immunocompromised patients.

Generation of glucose-sensitive insulin-secreting beta-like cells from human embryonic stem cells by incorporating a synthetic lineage-control network.

We previously reported novel technology to differentiate induced pluripotent stem cells (IPSCs) into glucose-sensitive insulin-secreting beta-like cells by engineering a synthetic lineage-control network regulated by the licensed food additive vanillic acid. This genetic network was able to program intricate expression dynamics of the key transcription factors Ngn3 (neurogenin 3, OFF-ON-OFF), Pdx1 (pancreatic and duodenal homeobox 1, ON-OFF-ON) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A, OFF-ON) to guide the differentiation of IPSC-derived pancreatic progenitor cells to beta-like cells. In the present study, we show for the first time that this network can also program the expression dynamics of Ngn3, Pdx1 and MafA in human embryonic stem cell (hESC)-derived pancreatic progenitor cells and drive differentiation of these cells into glucose-sensitive insulin-secreting beta-like cells. Therefore, synthetic lineage-control networks appear to be a robust methodology for differentiating pluripotent stem cells into somatic cell types for basic research and regenerative medicine.

External Noise and External Signal Induced Transition of Gene Switch and Coherence Resonance in the Genetic Regulatory System.

The transition of gene switch induced by external noises (multiplicative external noise and additive external noise) and external signals is investigated in the genetic regulatory system. Results show that the state-to-state transition of gene switch as well as resonant behaviors, such as the explicit coherence resonance (ECR), implicit coherence resonance (ICR) and control parameter coherence biresonance (CPCBR), can appear when noises are injected into the genetic regulatory system. The ECR is increased with the increase of the control parameter value when starting from the supercritical Hopf bifurcation parameter point, and there exists a critical control parameter value for the occurrence of ECR. However, the ICR is decreased as the control parameter value is increased when starting from the subcritical Hopf bifurcation point. In particular, the coherence of ECR is higher and more sensitive to noise than that of ICR. When an external signal is introduced into the system, the enhancement or suppression of the CPCBR and the number of peaks strongly depend on the frequency and amplitude of the external signal. Furthermore, the gene regulation system can selectively enhance or decrease the noise-induced oscillation signals at preferred frequency and amplitude of an external signal.

An Efficient Light-Inducible P53 Expression System for Inhibiting Proliferation of Bladder Cancer Cell.

Optogenetic gene expression systems enable spatial-temporal modulation of gene transcription and cell behavior. Although applications in biomedicine are emerging, the utility of optogenetic gene switches remains elusive in cancer research due to the relative low gene activation efficiency. Here, we present an optimized CRISPR-Cas9-based light-inducible gene expression device that controls gene transcription in a dose-dependent manner. To prove the potential utility of this device, P53 was tested as a functional target in the bladder cancer cell models. It was illustrated that the light-induced P53 inhibited proliferation of 5637 and UMUC-3 cell effectively. The "light-on" gene expression system may demonstrate a novel therapeutic strategy for bladder cancer intervention.