Gene Switching Research Articles

Inhibition of γ/β Globin Gene Switching in CD 34+ Derived Erythroid Cells by BCL11A RNA Silencing.

The induction of fetal haemoglobin (Hb F), due to the sustained clinical effects, is one of the most promising methods for the treatment of β hemoglobinopathies, such as thalassemia major and sickle cell disease (SCD). Inhibition of γ-globin gene silencing, possibly is a suitable strategy to induce HbF expression in these patients. In this study, the possibility of increasing HbF in the CD34+ derived erythroid cells was investigated by BCL11A inhibition using specific small-interfering RNAs (siRNAs). Human peripheral blood-derived hematopoietic stem cells were isolated and differentiated to erythroid cells. Erythroid maturation was investigated using cell morphology parameters and flow cytometry analysis of CD235a expression On day 20, siRNA complementary to BCL11A was transfected to differentiating cells via electroporation. BCL11A expression was evaluated through real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbant assay (ELISA). β actin was used as the reference gene to confirm the relative expression level of BCL11A gene mRNA. 48 hours after transfection, BCL11A siRNA significantly reduced BCL11A mRNA levels and consequently led to 2.0 fold decrease in corresponding protein. On the 28th day, haemoglobin electrophoresis results showed that Hb F levels in transfected erythroid cells increased 3.3-fold when compared with non transfected cells. In this study we showed that BCL11A inhibition in erythroid cells could increase fetal hemoglobin, and this strategy can be the basis for designing a γ globin expressing cellular system to increase Hb F in patients with thalassemia and SCD.

Epigenetic Modulators as Potential Multi-targeted Drugs Against Hedgehog Pathway for Treatment of Cancer.

The Sonic hedgehog signalling is known to play a crucial role in regulating embryonic development, cancer stem cell maintenance and tissue patterning. Dysregulated hedgehog signalling has been reported to affect tumorigenesis and drug response in various human malignancies. Epigenetic therapy relying on DNA methyltransferase and Histone deacetylase inhibitors are being proposed as potential drug candidates considering their efficiency in preventing development of cancer progenitor cells, killing drug resistant cells and also dictating "on/off" switch of tumor suppressor genes and oncogenes. In this docking approach, epigenetic modulators were virtually screened for their efficiency in inhibiting key regulators of SHH pathway viz., sonic hedgehog, Smoothened and Gli using polypharmacological approach. The control drugs and epigenetic modulators were docked with PDB protein structures using AutoDock vina and further checked for their drug-likeness properties. Further molecular dynamics simulation using VMD and NAMD, and MMP/GBSA energy calculation were employed for verifying the stability and entropy of the ligand-receptor complex. EPZ-6438 and GSK 343 (EZH2 inhibitors), CHR 3996 and Mocetinostat (HDAC inhibitors), GSK 126 (HKMT inhibitor) and UNC 1215 (L3MBTL3 antagonist) exhibited multiple-targeted approach in modulating HH signalling. This is the first study to report these epigenetic drugs as potential multi-targeted hedgehog pathway inhibitors. Thus, epigenetic polypharmacology approach can be explored as a better alternative to challenges of acute long term toxicity and drug resistance occurring due to traditional single targeted chemotherapy in the future.

Transcriptomics of the grape berry shrivel ripening disorder

Key messageThe lower expression at veraison of several ripening master regulators “switch genes” can play a central role in the induction of the berry shrivel ripening physiological disorder in grapevine.Berry shrivel (BS) is a ripening physiological disorder affecting grape berry with visible symptoms appearing after veraison. Berry shrivel leads to shrinking berries with a reduced weight and a lower content of sugars and anthocyanins. In this study, for the first time a transcriptomic analysis coupled with selected metabolites quantification was undertaken to understand the metabolic modifications induced by the disorder. Different stages of berry development were considered including pre- and symptomatic berries. No metabolic alterations in the berry transcriptome and in the metabolite content was observed in pre-symptomatic and pre-veraison samples. Interestingly, at veraison, with still not visible symptoms appearing on the berry, a subset of genes, called switch genes previously suggested as master regulators of the ripening onset in grape berries, were strongly lower expressed in BS. Later during the ripening phase and with visible symptoms of the disorder, more than 3000 genes were differentially expressed. The genes up-regulated were related to hormone biosynthesis, response to stress and the phenylpropanoid pathway, while the genes down-regulated during ripening belonged mainly to the flavonoid pathway, and the sugar metabolism. In agreement, BS berries showed lower content of sugars and anthocyanins from the onset of veraison onward, while the amount of acids was not significantly affected. In conclusion, these results highlight a pivotal role of the switch genes in grapevine ripening, as well as their possible contribution to induce the ripening disorder berry shrivel, although it remains unclear whether this is part of the cause or consequences of the BS disorder.

Distinct genome-wide alternative polyadenylation during the response to silicon availability in the marine diatom Thalassiosira pseudonana.

The post-transcriptional regulation involved in the responses of diatoms to silicon is poorly understood. Using a poly(A)-tag sequencing (PAT-seq) technique that interrogates only the junctions of 3'-untranslated region (UTR) and the poly(A) tails at the transcriptome level, a comprehensive comparison of alternative polyadenylation (APA) was performed to understand the role of post-transcriptional regulation in various silicon-related cellular responses for the marine diatom Thalassiosira pseudonana. In total, 23701 poly(A) clusters and 6894 APA genes, treated with silicon starvation and replenishment, were identified at nine time points. Significant APA was found in numerous genes (e.g. five cingulin genes) closely associated with the silicon-starvation response, girdle bands and valve synthesis, suggesting that many genes participated in the responses to silicon availability and biosilica formation through changes in transcript isoforms. The poly(A) site usage profiles were distinct during various stages of silicon biomineralization responses. Moreover, a correlation between APA and expression levels of APA switching genes was also discovered. This is an interesting study that presents a genome-wide profile of transcript ends in diatoms, which is distinct from that of higher plants, animals and other microalgae. This work provides an important resource to understand a different aspect of cell-wall synthesis.

A CRISPR/Cas9-based central processing unit to program complex logic computation in human cells

Controlling gene expression with sophisticated logic gates has been and remains one of the central aims of synthetic biology. However, conventional implementations of biocomputers use central processing units (CPUs) assembled from multiple protein-based gene switches, limiting the programming flexibility and complexity that can be achieved within single cells. Here, we introduce a CRISPR/Cas9-based core processor that enables different sets of user-defined guide RNA inputs to program a single transcriptional regulator (dCas9-KRAB) to perform a wide range of bitwise computations, from simple Boolean logic gates to arithmetic operations such as the half adder. Furthermore, we built a dual-core CPU combining two orthogonal core processors in a single cell. In principle, human cells integrating multiple orthogonal CRISPR/Cas9-based core processors could offer enormous computational capacity.

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.

BRAFV600E-mutant cancers display a variety of networks by SWIM analysis: prediction of vemurafenib clinical response.

Several studies have shown that different tumour types sharing a driver gene mutation do not respond uniformly to the same targeted agent. Our aim was to use an unbiased network-based approach to investigate this fundamental issue using BRAFV600E mutant tumours and the BRAF inhibitor vemurafenib. We applied SWIM, a software able to identify putative regulatory (switch) genes involved in drastic changes to the cell phenotype, to gene expression profiles of different BRAFV600E mutant cancers and their normal counterparts in order to identify the switch genes that could potentially explain the heterogeneity of these tumours' responses to vemurafenib. We identified lung adenocarcinoma as the tumour with the highest number of switch genes (298) compared to its normal counterpart. By looking for switch genes encoding for kinases with homology sequences similar to known vemurafenib targets, we found that thyroid cancer and lung adenocarcinoma have a similar number of putative targetable switch gene kinases (5 and 6, respectively) whereas colorectal cancer has just one. We are persuaded that our network analysis may aid in the comprehension of molecular mechanisms underlying the different responses to vemurafenib in BRAFV600E mutant tumours.

GmZPR3d Interacts with GmHD-ZIP III Proteins and Regulates Soybean Root and Nodule Vascular Development.

Fabaceans produce two major classes of symbiotic nodules: the indeterminate type characterized by a persistent meristem, and the determinate type that lacks a persistent meristem. The class III homeodomain leucine zipper (HD-ZIP III) transcription factor family influence development of multiple lateral organs and meristem maintenance, but their role in determinate nodule development is not known. HD-ZIP III protein activity is post-translationally regulated by members of the small leucine zipper protein (ZPR) family in arabidopsis. We characterized the ZPR gene family in soybean and evaluated their ability to interact with two key members of GmHD-ZIP III family through yeast two-hybrid assays. GmZPR3d displayed the strongest interaction with GmHD-ZIP III-2 among the different pairs evaluated. GmHD-ZIP III-1, -2, and GmZPR3d showed overlapping expression patterns in the root stele and in nodule parenchyma tissues. Over-expression of GmZPR3d resulted in ectopic root secondary xylem formation, and enhanced expression of vessel-specific master switch genes in soybean. The nodules in ZPR3d over-expressing roots were larger in size, had a relatively larger central zone and displayed increased nodule vascular branching. The results from this study point to a key role for GmZPR3d in soybean root and nodule development.

Finite-time adaptive stability of gene regulatory networks

This paper is concerned with the finite-time adaptive stability of genetic regulatory networks(GRNs). Traditionally, the finite-time stability criterion is often established based on the prior information on gene regulatory parameters. However, it is difficult to acquire the above prior information for GRNs. To overcome the above difficulty, we design controllers in non-switching and switching gene regulatory networks with adaptive technique, respectively. Then, we derive a sufficient criterion for system to achieve finite-time stability under the action of an adaptive controller and give an estimate of the steady-state time. Furthermore, we can also shorten the setting time required for the system to achieve finite-time stability under the designed adaptive controller. Finally, two numerical examples are given to illustrate the effectiveness of the proposed design method.

Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology: Enzymatic Cascades and Directed Evolution.

ConspectusArtificial metalloenzymes (ArMs) result from anchoring a metal-containing moiety within a macromolecular scaffold (protein or oligonucleotide). The resulting hybrid catalyst combines attractive features of both homogeneous catalysts and enzymes. This strategy includes the possibility of optimizing the reaction by both chemical (catalyst design) and genetic means leading to achievement of a novel degree of (enantio)selectivity, broadening of the substrate scope, or increased activity, among others. In the past 20 years, the Ward group has exploited, among others, the biotin–(strept)avidin technology to localize a catalytic moiety within a well-defined protein environment. Streptavidin has proven versatile for the implementation of ArMs as it offers the following features: (i) it is an extremely robust protein scaffold, amenable to extensive genetic manipulation and mishandling, (ii) it can be expressed in E. coli to very high titers (up to >8 g·L–1 in fed-batch cultures), and (iii) the cavity surrounding the biotinylated cofactor is commensurate with the size of a typical metal-catalyzed transition state. Relying on a chemogenetic optimization strategy, varying the orientation and the nature of the biotinylated cofactor within genetically engineered streptavidin, 12 reactions have been reported by the Ward group thus far. Recent efforts within our group have focused on extending the ArM technology to create complex systems for integration into biological cascade reactions and in vivo.With the long-term goal of complementing in vivo natural enzymes with ArMs, we summarize herein three complementary research lines: (i) With the aim of mimicking complex cross-regulation mechanisms prevalent in metabolism, we have engineered enzyme cascades, including cross-regulated reactions, that rely on ArMs. These efforts highlight the remarkable (bio)compatibility and complementarity of ArMs with natural enzymes. (ii) Additionally, multiple-turnover catalysis in the cytoplasm of aerobic organisms was achieved with ArMs that are compatible with a glutathione-rich environment. This feat is demonstrated in HEK-293T cells that are engineered with a gene switch that is upregulated by an ArM equipped with a cell-penetrating module. (iii) Finally, ArMs offer the fascinating prospect of “endowing organometallic chemistry with a genetic memory.” With this goal in mind, we have identified E. coli’s periplasmic space and surface display to compartmentalize an ArM, while maintaining the critical phenotype–genotype linkage. This strategy offers a straightforward means to optimize by directed evolution the catalytic performance of ArMs. Five reactions have been optimized following these compartmentalization strategies: ruthenium-catalyzed olefin metathesis, ruthenium-catalyzed deallylation, iridium-catalyzed transfer hydrogenation, dirhodium-catalyzed cyclopropanation and carbene insertion in C–H bonds. Importantly, >100 turnovers were achieved with ArMs in E. coli whole cells, highlighting the multiple turnover catalytic nature of these systems.

Applications of high-throughput sequencing to analyze and engineer ribozymes.

A large number of catalytic RNAs, or ribozymes, have been identified in the genomes of various organisms and viruses. Ribozymes are involved in biological processes such as regulation of gene expression and viral replication, but biological roles of many ribozymes still remain unknown. Ribozymes have also inspired researchers to engineer synthetic ribozymes that function as sensors or gene switches. To gain deeper understanding of the sequence-function relationship of ribozymes and to efficiently engineer synthetic ribozymes, a large number of ribozyme variants need to be examined which was limited to hundreds of sequences by Sanger sequencing. The advent of high-throughput sequencing technologies, however, has allowed us to sequence millions of ribozyme sequences at low cost. This review focuses on the recent applications of high-throughput sequencing to both characterize and engineer ribozymes, to highlight how the large-scale sequence data can advance ribozyme research and engineering.

Timed GDNF gene therapy using an immune-evasive gene switch promotes long distance axon regeneration.

Neurosurgical repair in patients with proximal nerve lesions results in unsatisfactory recovery of function. Gene therapy for neurotrophic factors is a powerful strategy to promote axon regeneration. Glial cell line-derived neurotrophic factor (GDNF) gene therapy promotes motor neuron survival and axon outgrowth; however, uncontrolled delivery of GDNF results in axon entrapment. We report that time-restricted GDNF expression (1 month) using an immune-evasive doxycycline-inducible gene switch attenuated local axon entrapment in avulsed reimplanted ventral spinal roots, was sufficient to promote long-term motor neuron survival (24 weeks) and facilitated the recovery of compound muscle action potentials by 8 weeks. These improvements were associated with an increase in long-distance regeneration of motor axons. In contrast, persistent GDNF expression impaired axon regeneration by inducing axon entrapment. These findings demonstrate that timed expression can resolve the deleterious effect of uncontrolled growth factor delivery and shows that inducible growth factor gene therapy can be employed to enhance the efficacy of axon regeneration after neurosurgical repair of a proximal nerve lesion in rats. This preclinical study is an important step in the ongoing development of a neurotrophic factor gene therapy for patients with severe proximal nerve lesions.

Evolution of Disease Defense Genes and Their Regulators in Plants.

Biotic stresses do damage to the growth and development of plants, and yield losses for some crops. Confronted with microbial infections, plants have evolved multiple defense mechanisms, which play important roles in the never-ending molecular arms race of plant–pathogen interactions. The complicated defense systems include pathogen-associated molecular patterns (PAMP) triggered immunity (PTI), effector triggered immunity (ETI), and the exosome-mediated cross-kingdom RNA interference (CKRI) system. Furthermore, plants have evolved a classical regulation system mediated by miRNAs to regulate these defense genes. Most of the genes/small RNAs or their regulators that involve in the defense pathways can have very rapid evolutionary rates in the longitudinal and horizontal co-evolution with pathogens. According to these internal defense mechanisms, some strategies such as molecular switch for the disease resistance genes, host-induced gene silencing (HIGS), and the new generation of RNA-based fungicides, have been developed to control multiple plant diseases. These broadly applicable new strategies by transgene or spraying ds/sRNA may lead to reduced application of pesticides and improved crop yield.

Enrichment of Cxcl12 promoter with TET2: A possible link between promoter demethylation and enhanced gene expression in the absence of PARP-1

Previously, we described the link between C-X-C motif chemokine 12 (Cxcl12) gene induction and DNA hypomethylation in the absence of poly(ADP-ribose) polymerase 1 (PARP-1). We have now firmly established that demethylation is the primary cause of gene induction on the basis of Cxcl12 gene upregulation upon treatment with the demethylating agent 5-azacytidine (5-aza). Since the demethylation state of Cxcl12 is favored by PARP-1 absence, we investigated the presence of ten-eleven translocation (TET) proteins on the Cxcl12 promoter in order to corroborate the relationship between the demethylation process and increased gene expression that occurs in the absence of PARP-1. Analysis was performed on the promoter region within CpG islands of Cxcl12 from control mouse embryonic fibroblasts (NIH3T3) and PARP-1 knock-out mouse embryonic fibroblasts (PARP1-/-). The lack of PARP-1 increased the abundance of TET2 on the Cxcl12 promoter, suggesting that TET-mediated demethylation provoked by the absence of PARP-1 could account for the observed increased expression of this chemokine. Deciphering the regulation of DNA (de)methylation factors that control Cxcl12 expression may provide an additional therapeutic approach in pharmacological interventions where gene switching on or off based on targeted stimulation or inhibition is necessary. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173020]

Origin and Pathogenesis of B Cell Lymphomas.

Immunoglobulin (IG) gene remodeling by V(D)J recombination plays a central role in the generation of normal B cells, and somatic hypermutation and class switching of IG genes are key processes during antigen-driven B cell differentiation. However, errors of these processes are involved in the development of B cell lymphomas. IG locus-associated translocations of proto-oncogenes are a hallmark of many B cell malignancies. Additional transforming events include inactivating mutations in various tumor suppressor genes and also latent infection of B cells with viruses, such as Epstein-Barr virus. Many B cell lymphomas require B cell antigen receptor expression, and in several instances, chronic antigenic stimulation plays a role in lymphoma development and/or sustaining tumor growth. Often, survival and proliferation signals provided by other cells in the microenvironment are a further critical factor in lymphoma development and pathophysiology. Many B cell malignancies derive from germinal center B cells, most likely because of the high proliferation rate of these cells and the high activity of mutagenic processes.

Photoelectrochemical biosensor for hydroxymethylated DNA detection and T4-β-glucosyltransferase activity assay based on WS2 nanosheets and carbon dots

5-Hydroxymethylcytosine (5hmC) plays an important role in switching genes on and off in mammals, and it is implicated in both embryonic development and cancer progression. Herein, a novel photoelectrochemical (PEC) biosensor was developed for 5hmC detection based on WS2 nanosheets as the photoactive material and boronic acid functionalized carbon dots (B-CDs) for signal amplification unit. This biosensor can also be used for T4-β-glucosyltransferase (β-GT) activity assessment. Firstly, WS2 nanosheets and gold nanoparticles (AuNPs) were immobilized on an ITO electrode surface. Then probe DNA was immobilized on this electrode surface via Au-S bond. Afterwards, the complementary DNA containing 5hmC was then captured on the modified electrode surface by hybridization. Subsequently, β-GT transferred glucose from uridine diphosphoglucose to the hydroxyl groups of the 5hmC residues. After glycosylation, B-CDs could further be immobilized on the modified electrode surface resulting in a strong photocurrent. The PEC biosensor afforded high selectivity, excellent sensitivity and good reproducibility, with detection limits of 0.0034 nM and 0.028 unit/mL for 5hmC and β-GT, respectively. Results demonstrate that the photoelectrochemical strategy introduced here based on WS2 nanosheets and B-CDs offers a versatile platform for hydroxymethylated DNA detection, β-GT activity assessment and β-GT inhibitor screening.

The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of HBG Promoter DNA Methylation

Pharmacological or genetic induction of fetal hemoglobin (HbF, α2γ2) in adult red blood cells is a proven strategy to ameliorate the clinical symptoms of sickle cell disease (SCD) and β-thalassemia. Therefore, efforts are underway to better understand mechanisms that mediate the perinatal switch from HbF to adult hemoglobin (HbA, α2β2). We performed a CRISPR-Cas9/guide (g) RNA screen to identify novel proteins that regulate HbF production in HUDEP-2 cells, a human erythroid line that normally expresses HbA. We identified UHRF1 (ubiquitin-like with PHD and RING finger domains 1) as a repressor of HbF production. UHRF1 binds hemi-methylated DNA and recruit DNA methyltransferase 1 (DNMT1) to ensure faithful maintenance of DNA methylation during DNA replication. Numerous UHRF1-interacting proteins, including DNMT1, EHMT1/2 and HDAC2 are associated with γ-globin repression. We used CRISPR/Cas9 and RNA interference to validate UHRF1 as a HbF regulator. Compared to non-targeting gRNA UHRF1 disruption using Cas9 + 2 separate gRNAs increased the γ-globin/γ+β-globin RNA ratio from 1.9 to 25.8/27.1% (P<0.01), increased the fraction of HbF immunostaining cells (“F-cells”) from 7.5 to 25.1/35.4% and increased HbF protein from 2.10 to 16.3/15.0% (P<0.01) in HUDEP-2 cells. Compared to a control luciferase shRNA, 2 different UHRF1 shRNAs increased theγ-globin/γ+β-globin RNA ratio from 9.68% to 21.59/28.93% (P<0.01), increased the F-cell fraction from 37.9 to 49.8/55.6% and increased HbF protein from 9.1 to 16.18/18.5% (P<0.05) in erythroid cells derived from normal adult peripheral blood CD34+ cells. UHRF1 deficiency did not alter erythroid maturation or expression of key transcription factor genes that regulate HbF expression in HUDEP-2 or CD34+ cells (BCL11A, ZBTB7A, MYB and KLF1). UHRF1 mutant proteins defective in recognizing H3K9me2 (FW237/238AA), binding to hemi-methylated DNA (R491A) or ubiquitination of H3K23 to enhance DNMT1 recruitment (C741A), were unable to repress HBG1/HBG2. These mutations have the most profound effects on maintaining DNA methylation, indicating that UHRF1 represses HBG1/HBG2 in HUDEP-2 cells through this mechanism.UHRF1 knockout induced genome-wide demethylation including 6 CpG sites located at positions -162, -53, -50, +6, +17, +50 positions relative to the γ-globin (HBG1 and HBG2) transcription start site. Demethylation of these sites is thought to be required for γ-globin de-repression. However, forced demethylation of these cytosines in HUDEP-2 cells using specific gRNAs + dead (d) Cas9-TET1 was not sufficient to activate γ-globin expression when UHRF1 was present. Additionally, dCas9-DNMT3a-mediated methylation of the HBG promoter CpG residues in UHRF1 knockdown HUDEP-2 cells did not inhibit γ-globin expression in UHRF1 knockout HUDEP-2 cells. Based on these studies, we conclude that: 1) UHRF1 regulates γ-globin transcription; 2) demethylation of CpG sites at the HBG gene promoters is neither necessary or sufficient for γ-globin induction; 3) UHRF1 regulates γ-to-β globin gene switching either by methylating DNA regions other than those present around the HBG promoter or through non-canonical activities. Distinguishing these mechanisms will elucidate further our understanding of globin gene switching and could identify new pathways for pharmacological induction of HbF. DisclosuresNo relevant conflicts of interest to declare.

Generation of regulable EGFRvIII targeted chimeric antigen receptor T cells for adoptive cell therapy of glioblastoma

Adoptive immunotherapy using chimeric antigen receptors-modified T cells (CAR-T) is a promising approach for cancer treatment. However, CARs currently applied in the clinics cannot be effectively regulated and the safety of CAR-T cell therapies remains a major concern. To improve the safety of CAR-T cells, we designed a synthetic splitting CAR (ssCAR) that can regulate T cell functions exogenously. Epidermal growth factor receptor variant III (EGFRvIII) was used as a molecular target for ssCAR. Our results indicate that both EGFRvIII and small molecule are needed for the activation of the ssCAR-T cells. AP21967 dose-dependently increased the expression of T cell activation, production of cytokines and extent of cell lysis. In conclusion, the gene switch designed in this study allows for temporal and spatial control over engineered T cells in a dose-and time-dependent manner by AP21967. Our work demonstrates the feasibility and improved safety profile of this novel treatment approach.

SWIM tool application to expression data of glioblastoma stem-like cell lines, corresponding primary tumors and conventional glioma cell lines

BackgroundIt is well-known that glioblastoma contains self-renewing, stem-like subpopulation with the ability to sustain tumor growth. These cells – called cancer stem-like cells – share certain phenotypic characteristics with untransformed stem cells and are resistant to many conventional cancer therapies, which might explain the limitations in curing human malignancies. Thus, the identification of genes controlling the differentiation of these stem-like cells is becoming a successful therapeutic strategy, owing to the promise of novel targets for treating malignancies.MethodsRecently, we developed SWIM, a software able to unveil a small pool of genes – called switch genes – critically associated with drastic changes in cell phenotype. Here, we applied SWIM to the expression profiling of glioblastoma stem-like cells and conventional glioma cell lines, in order to identify switch genes related to stem-like phenotype.ResultsSWIM identifies 171 switch genes that are all down-regulated in glioblastoma stem-like cells. This list encompasses genes like CAV1, COL5A1, COL6A3, FLNB, HMMR, ITGA3, ITGA5, MET, SDC1, THBS1, and VEGFC, involved in “ECM-receptor interaction“ and “focal adhesion” pathways. The inhibition of switch genes highly correlates with the activation of genes related to neural development and differentiation, such as the 4-core OLIG2, POU3F2, SALL2, SOX2, whose induction has been shown to be sufficient to reprogram differentiated glioblastoma into stem-like cells. Among switch genes, the transcription factor FOSL1 appears as the brightest star since: it is down-regulated in stem-like cells; it highly negatively correlates with the 4-core genes that are all up-regulated in stem-like cells; the promoter regions of the 4-core genes harbor a consensus binding motif for FOSL1.ConclusionsWe suggest that the inhibition of switch genes in stem-like cells could induce the deregulation of cell communication pathways, contributing to neoplastic progression and tumor invasiveness. Conversely, their activation could restore the physiological equilibrium between cell adhesion and migration, hampering the progression of cancer. Moreover, we posit FOSL1 as promising candidate to orchestrate the differentiation of cancer stem-like cells by repressing the 4-core genes’ expression, which severely halts cancer growth and might affect the therapeutic outcome. We suggest FOSL1 as novel putative therapeutic and prognostic biomarker, worthy of further investigation.

A synthetic free fatty acid-regulated transgene switch in mammalian cells and mice.

Trigger-inducible transgene expression systems are utilized in biopharmaceutical manufacturing and also to enable controlled release of therapeutic agents in vivo. We considered that free fatty acids (FFAs), which are dietary components, signaling molecules and important biomarkers, would be attractive candidates as triggers for novel transgene switches with many potential applications, e.g. in future gene- and cell-based therapies. To develop such a switch, we rewired the signal pathway of human G-protein coupled receptor 40 to a chimeric promoter triggering gene expression through an increase of intracellular calcium concentration. This synthetic gene switch is responsive to physiologically relevant FFA concentrations in different mammalian cell types grown in culture or in a bioreactor, or implanted into mice. Animal recipients of microencapsulated sensor cells containing this switch exhibited significant transgene induction following consumption of dietary fat (such as Swiss cheese) or under hyperlipidaemic conditions, including obesity, diabetes and lipodystrophy.