Sleeping Beauty Research Articles

PDTM-20. THE HISTONE MUTATION H3.3-G34R ENCOUNTERED IN PEDIATRIC HIGH GRADE GLIOMA MODIFIES THE TUMOR IMMUNE MICROENVIRONMENT RENDERING IT MORE PERMISSIVE FOR IMMUNE MEDIATED THERAPIES

Abstract Pediatric high grade gliomas (pHGGs) have a median survival (MS) of 9–15 months and are the most common malignant brain tumors in children. No significant improvement in the MS of these patients has been registered in decades. Thus, a representative in vivo model to study these tumors is critical. Recurrent mutations in genes encoding histones H3.3 and H3.1 have been described, which are distinctive of pHGGs. To stablish a mouse model for pHGG harboring the mutation H3.3-G34R, which co-occurs with inactivating mutations in ATRX and TP53, we used the Sleeping Beauty transposase system (H3.3-G34R group). The MS of mice harboring H3.3-G34R mutation was significantly longer than the MS of the WT-H3.3 control group (p< 0.0001). We performed RNA-Sequencing of these tumors which yielded 233 differentially expressed genes between these two groups (p< 0.05). Gene set enrichment analysis of the upregulated genes in H3.3-G34R group showed significant GOs related to immune response activation and response to IFNγ. Thus, we next studied the tumor microenvironment (TME) to determine the distinctive immune cell populations, at the phenotypic and functional level. We observed that the H3.3-G34R TME exhibited less frequency of Gr-1+/CD11b+ cells (myeloid-derived suppressor cells: MDSC). MDSC are immature myeloid cells that impair T cell functions and the adaptive immune response. Moreover, the H3.3-G34R TME had increased numbers of total macrophages (p< 0.05), a higher proportion of M1 anti-tumor macrophages, and higher levels of CD8+ T cells (p< 0.01). Also, the H3.3-G34R renders glioma cells more sensitive to IFNγ regulation as it induced a higher pSTAT1/STAT1 ratio in H3.3-G34R cells. These results shed light on the interaction between the immune system and the H3.3-G34R glioma cells and how this could be harnessed to develop immune mediated therapies specific for pHGG harboring the H3.3-G34R mutation.

Direct Inference, Reichenbach's Principle, and the Sleeping Beauty Problem

AbstractA group of philosophers led by the late John Pollock has applied a method of reasoning about probability, known as direct inference and governed by a constraint known as Reichenbach's principle, to argue in support of ‘thirdism’ concerning the Sleeping Beauty Problem. A subsequent debate has ensued about whether their argument constitutes a legitimate application of direct inference. Here I defend the argument against two extant objections charging illegitimacy. One objection can be overcome via a natural and plausible definition, given here, of the binary relation ‘logically stronger than’ between two properties that can obtain even when the respective properties differ from one another in ‘arity’; given this definition, the Pollock group's argument conforms to Reichenbach's principle. Another objection prompts a certain refinement of Reichenbach's principle that is independently well-motivated. My defense of the Pollock group's argument has epistemological import beyond the Sleeping Beauty problem, because it both widens and sharpens the applicability of direct inference as a method for inferring single-case epistemic probabilities on the basis of general information of a probabilistic or statistical nature.

Generation of CAR+ T Lymphocytes Using the Sleeping Beauty Transposon System.

Adoptive immunotherapy of cancer using T cells expressing chimeric antigen receptors (CARs) is now an approved treatment for non-Hodgkin lymphoma (NHL) and B cell acute lymphoblastic leukemia (B-ALL), inducing high response rates in patients. The infusion products are generated by using retro- or lentiviral transduction to induce CAR expression in T cells followed by an in vitro expansion protocol. However, use of viral vectors is cumbersome and is associated with increased costs due to the required high titers, replication-competent retrovirus (RCR) detection and production/use in a biosafety level 2 culture rooms, and additional quality control tests. Nonviral methods, like the Sleeping Beauty transposon system, can stably integrate in the genome of target cells and can be delivered using straightforward methods like electroporation. This chapter describes a protocol for T cell genetic modification using Sleeping Beauty transposon system and electroporation with the Lonza Nucleofector II device for the stable expression of CAR molecules in T lymphocytes.

Preclinical Evaluation of a Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System in Choroidal Neovascularization

Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats, and transfected primary cells (transfected RPE [tRPE] and transfected IPE [tIPE] cells) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinases 2 and 9 (MMP-2/9), and microglial/macrophage markers were measured. Injection with tRPE/IPE cells significantly reduced the leakage area at 7 and 14 days and the CNV area at 7 days. There was a significant increase in PEDF and the PEDF/VEGF ratio with tRPE cells and a reduction in the MMP-2 activity. Our data demonstrated that ex vivo non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.

A highly soluble Sleeping Beauty transposase improves control of gene insertion.

The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk for genome instability, and by the inability to use transposase protein directly. Here, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T-cells, which exhibit potent anti-tumor activity in vitro and in xenograft mice. We found that hsSB spontaneously penetrates cells, enabling modification of iPSCs and generation of CAR-T cells without the use of transfection reagents. Titration of hsSB to modulate genomic integration frequency achieved as few as two integrations per genome.

HEROINES VICTIMIZED BY FEMALE VIOLENCE IN FAIRY TALES: JORINDA, RAPUNZEL, SLEEPING BEAUTY, CINDERELLA AND SNOW WHITE

Despite the term ‘violence’ associates the force which strong and powerful people use against the weak in order to have their ways, there lies a deeper truth beneath the surface. According to Erich Fromm, it is possible to say that violence actually stems from impotency or different kinds of weakness and feeling of deficiency. Hence, the origin of violence is psychological. From this point of view, it can be clearly noticed that violence has begun to lose its male character and gains a new and female attribution when examined under the light of some recent researches regarding myths, fairy tales and some religious narratives as the indicators of behavioural patterns in time immemorial. In these narratives, women can also apply violence, even to women. Especially in some famous fairy tales like Jorinda and Joringel, Rapunzel, Sleeping Beauty, Cinderella and Snow White, the female violence against female figures becomes strikingly obvious. So, in this study we have turned our perspective to the feminine world in these fairy tales and have tried to analyse the female violence victimizing the heroines by using some theories of analytical psychology and psychoanalysis.

The occurrence of 'Sleeping Beauty' publications in medical research: Their scientific impact and technological relevance.

We investigate publications in medical research that have gone unnoticed for a number of years after being published and then suddenly become cited to a significant degree. Such publications are called Sleeping Beauties (SBs). This study focuses on SBs that are cited in patents. We find that the increasing trend of the relative number of SBs comes to an end around 1998. However, still a constant fraction of publications becomes an SB. Many SBs become highly cited publications, they even belong to the top-10 to 20% most cited publications in their field. We measured the scaling of the number of SBs in relation to the sleeping period length, during-sleep citation-intensity, and with awakening citation-intensity. We determined the Grand Sleeping Beauty Equation for these medical SBs which shows that the probability of awakening after a period of deep sleep is becoming rapidly smaller for longer sleeping periods and that the probability for higher awakening intensities decreases extremely rapidly. The exponents of the scaling functions show a time-dependent behavior which suggests a decreasing occurrence of SBs with longer sleeping periods. We demonstrate that the fraction of SBs cited by patents before scientific awakening exponentially increases. This finding shows that the technological time lag is becoming shorter than the sleeping time. Inventor-author self-citations may result in shorter technological time lags, but this effect is small. Finally, we discuss characteristics of an SBs that became one of the highest cited medical papers ever.

Advanced Establishment of Stable Recombinant Human Suspension Cell Lines Using Genotype-Phenotype Coupling Transposon Vectors.

Stable mammalian, namely human, suspension cell lines play a pivotal role in red biotechnology production scenarios for the generation of state-of-the-art biologics. However, selection of genetically modified and highly productive cell populations-prior to the establishment of clonal lines-is often challenging. To overcome this limitation, we first describe an optimized transient transfection protocol using the inexpensive reagent polyethylenimine (PEI) and human 293F cells. Transposon donor vectors derived from Sleeping Beauty encompassing a cassette with the reporter gene encoding for the green fluorescent protein (GFP) coupled with an internal ribosome entry site (IRES) to the expression of puromycin-resistance are employed to readily detect transfected cells. Upon stable transfection in the presence and absence of transposase expression, respectively,and subsequent antibiotic selection, GFP expression using flow cytometry analysis, cell viability, and cell density can be examined over a range of up to 3 weeks. Owing to the integration of high vectorcopy numbers into the target cell genome, transposase-mediated transposition of transposon donor vectors is instrumental in the faster establishment of recombinant cell population as compared to the classical stable transfection of plasmid DNA.

High-level protein production in erythroid cells derived from in vivo transduced hematopoietic stem cells

AbstractWe developed an in vivo hematopoietic stem cell (HSC) transduction approach that involves HSC mobilization from the bone marrow into the peripheral bloodstream and the IV injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. HDAd5/35++ vectors target human CD46, a receptor that is abundantly expressed on primitive HSCs. Transgene integration is achieved by a hyperactive Sleeping Beauty transposase (SB100x) and transgene marking in peripheral blood cells can be increased by in vivo selection. Here we directed transgene expression to HSC-derived erythroid cells using β-globin regulatory elements. We hypothesized that the abundance and systemic distribution of erythroid cells can be harnessed for high-level production of therapeutic proteins. We first demonstrated that our approach allowed for sustained, erythroid-lineage specific GFP expression and accumulation of GFP protein in erythrocytes. Furthermore, after in vivo HSC transduction/selection in hCD46-transgenic mice, we demonstrated stable supraphysiological plasma concentrations of a bioengineered human factor VIII, termed ET3. High-level ET3 production in erythroid cells did not affect erythropoiesis. A phenotypic correction of bleeding was observed after in vivo HSC transduction of hCD46+/+/F8−/− hemophilia A mice despite high plasma anti-ET3 antibody titers. This suggests that ET3 levels were high enough to provide sufficient noninhibited ET3 systemically and/or locally (in blood clots) to control bleeding. In addition to its relevance for hemophilia A gene therapy, our approach has implications for the therapy of other inherited or acquired diseases that require high levels of therapeutic proteins in the blood circulation.

Bir Peri Masalı Örneği: Ludwig Tieck’in Elfler Adlı Masalı Üzerine Bir İnceleme

İlk kez 14. yüzyılda görülmeye başlayan peri masalları; içerisinde gizemi, büyüyü ve olağanüstüyü barındıran bir yazın anlayışına karşılık gelir. Gizem, insanoğlunda merak duygusunu tetikleyen ve bu bağlamda ilgi uyandıran başlıca olgulardan biridir. Peri masalı türü, içinde yaşanılan dünyanın gerçekliğe dayalı döngüsü içerisinde olağanüstüyü deneyimlemeye, gerçek yaşam düzleminin içerisine yerleştirilen bu olağanüstü deneyimlerden anlamlar çıkarmaya ve bu anlamlar doğrultusunda gizemin sır perdesini aralamaya hizmet eder. Türün görülmeye başlanmasından kısa bir süre sonra çok sayıda eserler verilmesinin ve bu denli ilgi görmesinin asıl sebebi de budur. İnsanoğlunun gizem karşısındaki ilgisi, Charles Perrault, Grimm Kardeşler ve Hans Christian Andersen gibi peri masalı yazarlarının popülarite kazanmasını sağlar. Her üç yazarın peri masalı (fairy tales) türündeki eserlerinin birçoğu (Kırmızı Başlıklı Kız, Uyuyan Güzel, Külkedisi, Pamuk Prenses v.b) ilk kez yayımlandığı yıldan günümüze değin hemen her çocuğun hayatında önemli ölçüde yer alır. Çocuklukta başlayan bu serüven hayat boyu sürer ve peri masalları artık yetişkinliğe erişmiş olan okurun ilgisini çekmeye devam eder. Çalışmada, peri masalı türü ekseninde Alman Romantik Dönemi yazarı Ludwig Tieck’in Elfler adlı masalı ele alınacak ve masal, peri masalı türünde sıklıkla yer alan öğeler ve karakterler bağlamında incelenecektir. Ayrıca çalışmada, kuramcıların peri masalı hakkındaki verileri esas alınacak ve masal, metin odaklı yöntem kullanılarak çözümlenecektir.

A Phase II Study Using the Administration of Autologous T-Cells Engineered Using the Sleeping Beauty Transposon/Transposase System to Express T-Cell Receptors Reactive Against Mutated Neoantigens in Patients With Metastatic Cancer

A Phase II Study Using the Administration of Autologous T-Cells Engineered Using the Sleeping Beauty Transposon/Transposase System to Express T-Cell Receptors Reactive Against Mutated Neoantigens in Patients With Metastatic Cancer

In vivo CRISPR screening in CD8 T cells with AAV-Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma.

Identification of T cell targets to improve immunotherapies is of prime interest. To facilitate large-scale CRISPR screens directly in T cells in vivo, here, we developed a hybrid genetic screening system with adeno associated virus (AAV) and the Sleeping Beauty (SB) transposon, where the transposon is nested in the viral vector. The approach enables efficient gene editing in primary murine T cells and genomic integration of the sgRNA cassette for screen readout. We performed focused in vivo AAV-SB-CRISPR screens in CD8+ T cells in mouse models of glioblastoma (GBM) and identified membrane protein targets. Adoptive transfer of CD8+ T cells with Pdia3, Mgat5, Emp1, or Lag3 gene editing enhance the survival of GBM-bearing mice in both syngeneic and TCR-transgenic models. Transcriptome profiling, single cell sequencing, cytokine assays, and T cell signaling analysis showed that Pdia3 editing in T cells enhances effector functions. Engineered PDIA3 mutant EGFRvIII chimeric antigen T cells are more potent in antigen-specific killing of human GBM cells.

Translation, Illustration, Transcreation. From the Fairy Tales of Charles Perrault to Classic Fairy Tales Retold

The article proposes that the fairy-tale tradition unfolds as a collective, dynamic and transformative process of transcreation across languages, contexts and media for different purposes and publics. It notably draws attention to the role of translators, illustrators and publishers whose collaborations have produced new versions of the familiar stories in print form, which have in turn inspired highly innovative projects for children and adults reflecting personal takes on the stories. As a case study, I trace the editorial history of Angela Carter’s translation of The Fairy Tales of Charles Perrault (1977) illustrated by Martin Ware, which paved the way for Carter’s own collection of ‘ stories about fairy stories ’, The Bloody Chamber and Other Stories (1979), in contrapuntal fashion. The translation was reissued with child-friendly illustrations by Michael Foreman in Sleeping Beauty and Other Favourite Fairy Tales (1982), also published by Victor Gollancz, which later fed into Classic Fairy Tales Retold and Illustrated by Michael Foreman (2005)1.

Engraftment of mesothelin chimeric antigen receptor using a hybrid Sleeping Beauty/minicircle vector into NK-92MI cells for treatment of pancreatic cancer

BackgroundWe have previously demonstrated in vitro cytotoxicity of mesothelin-chimeric antigen receptor autologous T cells against pancreatic cancer cells using lentiviral vectors, but these vectors pose safety concerns. Here, we incorporated Sleeping Beauty and minicircle design enhancements into interleukin-2–secreting natural NK-92MI cells to eliminate both bacterial and viral components and address inhibition by the tumor microenvironment. MethodsParental (conventional deoxyribonucleic acid)-mesothelin-chimeric antigen receptor and minicircle-mesothelin-chimeric antigen receptor vectors were electroporated into NK-92MI cells and engraftment was visualized by immunofluorescence analysis with protein-L staining. Interferon-γ and granzyme B secretion were measured by enzyme-linked immunosorbent assay from cocultures of parental-mesothelin-chimeric antigen receptors and minicircle-mesothelin-chimeric antigen receptors with human pancreatic cancer cells, and cytotoxicity of chimeric antigen receptor NK-92MI cells was tested against three pancreatic cancer cell lines. ResultsCloning of mesothelin-chimeric antigen receptor Sleeping Beauty into a minicircle vector removed its bacterial backbone and reduced its size with improved electroporation efficiency. Chimeric antigen receptor engraftment, Interferon-γ and granzyme B secretion, and specific lysis against all three pancreatic cancer lines were significantly increased with minicircle-mesothelin-chimeric antigen receptor versus parental-mesothelin-chimeric antigen receptor NK-92MI cells. ConclusionWe provide proof of concept that allogeneic mesothelin-chimeric antigen receptor NK-92MI cells with hybrid Sleeping Beauty and minicircle technologies provide increased engraftment and cytotoxicity in vitro with potential safety benefits when translated to the clinical arena.

A transition stage co-citation criterion for identifying the awakeners of sleeping beauty publications

The role played by citing papers in triggering the recognition of sleeping beauty (SB) papers may be concealed by the traditional co-citation criteria for screening awakeners for two reasons: the increasing number of citations received by the SB and/or the decay of co-citations between the citing paper and the SB after the SB’s awakening. Here, we used the co-citation frequency of the SB and the citing paper for the shortest period immediately following the publication of the citing paper during which the SB received at least a specified number of citations to identify which of the citing papers published before or shortly after the SB’s awakening served as the ‘awakener’. This proposed transition stage co-citation criterion can disclose the important role of awakeners in awakening SBs, which might be neglected by the traditional co-citation criterion. Examples show that this method can identify awakener(s) that the traditional co-citation criteria fail to identify for some SBs, especially for those that do not have an awakener according to the traditional criteria. Another advantage of the proposed method is that once determined, the results for identifying awakeners by the proposed method do not vary with time. A modification of the transition stage co-citation criterion to flexibly treat SBs with fewer citations is also provided.

La restitution de l’« effet-monde » dans le récit

Abstract This article aims to study the recreation of the “world-effect” in the translation of a narrative text. Using a corpus made up of three Chinese translations of Charles Perrault’s Sleeping Beauty published in different periods, we find that in the translation of children’s literature, the translator may face various difficulties (ex. the sociohistorical constraints, editorial policy…). Thus, he often tends to adopt different strategies (Chinesizing, exotisizing, infantilizing) which would inevitably lead to different effects for the target readership.

CRISPR-Cas9–mediated gene knockout in intestinal tumor organoids provides functional validation for colorectal cancer driver genes

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide Sleeping Beauty transposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations (Apc and Kras mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that Acvr1b, Acvr2a, and Arid2 could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53 in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-β (TGF-β) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets.

Sleeping Beauty Mouse Models of Cancer: Microenvironmental Influences on Cancer Genetics.

The Sleeping Beauty (SB) transposon insertional mutagenesis system offers a streamlined approach to identify genetic drivers of cancer. With a relatively random insertion profile, SB is uniquely positioned for conducting unbiased forward genetic screens. Indeed, SB mouse models of cancer have revealed insights into the genetics of tumorigenesis. In this review, we highlight experiments that have exploited the SB system to interrogate the genetics of cancer in distinct biological contexts. We also propose experimental designs that could further our understanding of the relationship between tumor microenvironment and tumor progression.

Dispelling the Charm of Fairy tales in Elfriede Jelinek’s Miniature Plays

The article analyzes Elfriede Jelinek’s Snow White and Sleeping Beauty as reinterpretations of classic fairytales. Weronika Kosecka’s conception of the postmodern fairytale is referred to so as to illuminate Jelinek’s engagement with the issues of gender and the body in the texts under discussion.

HBx-K130M/V131I Promotes Liver Cancer in Transgenic Mice via AKT/FOXO1 Signaling Pathway and Arachidonic Acid Metabolism.

Chronic hepatitis B viral (HBV) infection remains a high underlying cause for hepatocellular carcinoma (HCC) worldwide, while the genetic mechanisms behind this remain unclear. This study elucidated the mechanisms contributing to tumor development induced by the HBV X (HBx) gene of predominantly Asian genotype B HBV and its common HBx variants. To compare the potential tumorigenic effects of K130M/V131I (Mut) and wild-type (WT) HBx on HCC, the Sleeping Beauty (SB) transposon system was used to deliver HBx Mut and WT into the livers of fumarylacetoacetate hydrolase (Fah)-deficient mice and in the context of transformation related protein 53 (Trp53) deficiency. From our results, HBx Mut had a stronger tumorigenic effect than its WT variant. Also, inflammation, necrosis, and fibrosis were evident in HBx experimental animals. Reduction of forkhead box O1 (FOXO1) with increased phosphorylation of upstream serine/threonine kinase (AKT) was detected under HBx Mut overexpression. Thus, it is proposed that HBx Mut enhances disease progression by reducing FOXO1 via phosphorylation of AKT. At the metabolomic level, HBx altered the expression of genes that participated in arachidonic acid (AA) metabolism, as a result of inflammation via accumulation of proinflammatory factors such as prostaglandins and leukotriene in liver. Taken together, the increased rate of HCC observed in chronic hepatitis B patients with K130M/V131I-mutated X protein, may be due to changes in AA metabolism and AKT/FOXO1 signaling. IMPLICATIONS: Our findings suggested that HBx-K130M/V131I-mutant variant promoted HCC progression by activating AKT/FOXO1 pathway and inducing stronger inflammation in liver via AA metabolism.