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Sleeping beauty transposon

José Eduardo Vargas, Leonardo Chicaybam, Renato Tetelbom Stein, Amilcar Tanuri, Andrés Delgado-Cañedo, Martin H Bonamino
Gene therapy protocols require robust and long-term gene expression. For two decades, retrovirus family vectors have offered several attractive properties as stable gene-delivery vehicles. These vectors represent a technology with widespread use in basic biology and translational studies that require persistent gene expression for treatment of several monogenic diseases. Immunogenicity and insertional mutagenesis represent the main obstacles to a wider clinical use of these vectors. Efficient and safe non-viral vectors are emerging as a promising alternative and facilitate clinical gene therapy studies...
October 12, 2016: Journal of Translational Medicine
Suneel A Narayanavari, Shreevathsa S Chilkunda, Zoltán Ivics, Zsuzsanna Izsvák
Sleeping Beauty (SB) is the first synthetic DNA transposon that was shown to be active in a wide variety of species. Here, we review studies from the last two decades addressing both basic biology and applications of this transposon. We discuss how host-transposon interaction modulates transposition at different steps of the transposition reaction. We also discuss how the transposon was translated for gene delivery and gene discovery purposes. We critically review the system in clinical, pre-clinical and non-clinical settings as a non-viral gene delivery tool in comparison with viral technologies...
October 4, 2016: Critical Reviews in Biochemistry and Molecular Biology
Fabienne Cocchiarella, Maria Carmela Latella, Valentina Basile, Francesca Miselli, Melanie Galla, Carol Imbriano, Alessandra Recchia
The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raises increasing interest for gene therapy application, including genome modification and, more recently, induced pluripotent stem cells (iPS) reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by an integrating retroviral vector (iRV), has been circumvented by the transient delivery of SB100X using retroviral mRNA transfer. In this study, we developed an alternative, safe, and efficient transposase delivery system based on a tetracycline-ON regulated expression cassette and the rtTA2(S)-M2 transactivator gene transiently delivered by integration-defective lentiviral vectors (IDLVs)...
2016: Molecular Therapy. Methods & Clinical Development
R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, M Hudecek
Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation. Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs)...
August 5, 2016: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
Karen M Mann, Justin Y Newberg, Michael A Black, Devin J Jones, Felipe Amaya-Manzanares, Liliana Guzman-Rojas, Takahiro Kodama, Jerrold M Ward, Alistair G Rust, Louise van der Weyden, Christopher Chin Kuan Yew, Jill L Waters, Marco L Leung, Keith Rogers, Susan M Rogers, Leslie A McNoe, Luxmanan Selvanesan, Nicholas Navin, Nancy A Jenkins, Neal G Copeland, Michael B Mann
A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis...
September 2016: Nature Biotechnology
Gregory M Vercellotti, Ping Zhang, Julia Nguyen, Fuad Abdulla, Chunsheng Chen, Phong Nguyen, Carlos Nowotny, Clifford J Steer, Ann Smith, John D Belcher
Sickle cell disease (SCD) patients have low serum hemopexin (Hpx) levels due to chronic hemolysis. We hypothesize that in SCD mice, hepatic overexpression of hemopexin will scavenge the proximal mediator of vascular activation, heme, and will inhibit inflammation and microvascular stasis. To examine the protective role of Hpx in SCD, we transplanted bone marrow from NY1DD SCD mice into Hpx(-/-) or Hpx(+/+) C57BL/6 mice. Dorsal skin fold chambers were implanted in week 13 post-transplant and microvascular stasis (% non-flowing venules) evaluated in response to heme infusion...
July 19, 2016: Molecular Medicine
Philip Boehme, Wenli Zhang, Manish Solanki, Eric Ehrke-Schulz, Anja Ehrhardt
For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells...
2016: Molecular Therapy. Nucleic Acids
Huanhuan Joyce Chen, Zhubo Wei, Jian Sun, Asmita Bhattacharya, David J Savage, Rita Serda, Yuri Mackeyev, Steven A Curley, Pengcheng Bu, Lihua Wang, Shuibing Chen, Leona Cohen-Gould, Emina Huang, Xiling Shen, Steven M Lipkin, Neal G Copeland, Nancy A Jenkins, Michael L Shuler
Refined cancer models are needed to bridge the gaps between cell line, animal and clinical research. Here we describe the engineering of an organotypic colon cancer model by recellularization of a native human matrix that contains cell-populated mucosa and an intact muscularis mucosa layer. This ex vivo system recapitulates the pathophysiological progression from APC-mutant neoplasia to submucosal invasive tumor. We used it to perform a Sleeping Beauty transposon mutagenesis screen to identify genes that cooperate with mutant APC in driving invasive neoplasia...
August 2016: Nature Biotechnology
Helga Eyjolfsdottir, Maria Eriksdotter, Bengt Linderoth, Göran Lind, Bengt Juliusson, Philip Kusk, Ove Almkvist, Niels Andreasen, Kaj Blennow, Daniel Ferreira, Eric Westman, Inger Nennesmo, Azadeh Karami, Taher Darreh-Shori, Ahmadul Kadir, Agneta Nordberg, Erik Sundström, Lars-Olof Wahlund, Anders Wall, Maria Wiberg, Bengt Winblad, Åke Seiger, Lars Wahlberg, Per Almqvist
BACKGROUND: Targeted delivery of nerve growth factor (NGF) has emerged as a potential therapy for Alzheimer's disease (AD) due to its regenerative effects on basal forebrain cholinergic neurons. This hypothesis has been tested in patients with AD using encapsulated cell biodelivery of NGF (NGF-ECB) in a first-in-human study. We report our results from a third-dose cohort of patients receiving second-generation NGF-ECB implants with improved NGF secretion. METHODS: Four patients with mild to moderate AD were recruited to participate in an open-label, phase Ib dose escalation study with a 6-month duration...
2016: Alzheimer's Research & Therapy
Azra Fatima, Dina Ivanyuk, Stefan Herms, Stefanie Heilmann-Heimbach, Orla O'Shea, Charlotte Chapman, Zsuszanna Izsvák, Martin Farr, Jürgen Hescheler, Tomo Šarić
We report here the generation of human iPS cell line UKKi009-A from dermal fibroblasts of a patient carrying heterozygous mutation c.3035-3045delTCCCTCGATGC, p.Leu1012Pro (fs*55) in KCNH2 gene leading to long QT syndrome type 2 (LQT2). We used the Sleeping Beauty transposon-based plasmids expressing OSKM along with microRNAs 307/367 to reprogram the fibroblasts. The iPS cells possess pluripotent stem cell characteristics and differentiate to cell lineages of all three germ layers. This cell line can serve as a source for in vitro modeling of LQT2...
March 2016: Stem Cell Research
Rajagopal N Aravalli, Chang W Park, Clifford J Steer
The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17...
August 26, 2016: Biochemical and Biophysical Research Communications
Soo-Young Yum, Song-Jeon Lee, Hyun-Min Kim, Woo-Jae Choi, Ji-Hyun Park, Won-Wu Lee, Hee-Soo Kim, Hyeong-Jong Kim, Seong-Hun Bae, Je-Hyeong Lee, Joo-Yeong Moon, Ji-Hyun Lee, Choong-Il Lee, Bong-Jun Son, Sang-Hoon Song, Su-Min Ji, Seong-Jin Kim, Goo Jang
Here, we efficiently generated transgenic cattle using two transposon systems (Sleeping Beauty and Piggybac) and their genomes were analyzed by next-generation sequencing (NGS). Blastocysts derived from microinjection of DNA transposons were selected and transferred into recipient cows. Nine transgenic cattle have been generated and grown-up to date without any health issues except two. Some of them expressed strong fluorescence and the transgene in the oocytes from a superovulating one were detected by PCR and sequencing...
2016: Scientific Reports
Chiara F Magnani, Nice Turazzi, Fabrizio Benedicenti, Andrea Calabria, Erika Tenderini, Sarah Tettamanti, Greta M P Giordano Attianese, Laurence J N Cooper, Alessandro Aiuti, Eugenio Montini, Andrea Biondi, Ettore Biagi
Chimeric antigen receptor (CAR)-modified T-cell adoptive immunotherapy is a remarkable therapeutic option proven effective in the treatment of hematological malignancies. In order to optimize cell manufacturing, we sought to develop a novel clinical-grade protocol to obtain CAR-modified cytokine-induced killer cells (CIKs) using the Sleeping Beauty (SB) transposon system. Administration of irradiated PBMCs overcame cell death of stimulating cells induced by non-viral transfection, enabling robust gene transfer together with efficient T-cell expansion...
June 13, 2016: Oncotarget
Takahiro Kodama, Justin Y Newberg, Michiko Kodama, Roberto Rangel, Kosuke Yoshihara, Jean C Tien, Pamela H Parsons, Hao Wu, Milton J Finegold, Neal G Copeland, Nancy A Jenkins
Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs)...
June 14, 2016: Proceedings of the National Academy of Sciences of the United States of America
Amer M Najjar, Pallavi R Manuri, Simon Olivares, Leo Flores, Tiejuan Mi, Helen Huls, Elizabeth J Shpall, Richard E Champlin, Nashaat Turkman, Vincenzo Paolillo, Jason Roszik, Brian Rabinovich, Dean A Lee, Mian Alauddin, Juri Gelovani, Laurence J N Cooper
PURPOSE: We have incorporated a positron emission tomography (PET) functionality in T cells expressing a CD19-specific chimeric antigen receptor (CAR) to non-invasively monitor the adoptively transferred cells. PROCEDURES: We engineered T cells to express CD19-specific CAR, firefly luciferase (ffLuc), and herpes simplex virus type-1 thymidine kinase (TK) using the non-viral-based Sleeping Beauty (SB) transposon/transposase system adapted for human application. Electroporated primary T cells were propagated on CD19(+) artificial antigen-presenting cells...
May 31, 2016: Molecular Imaging and Biology: MIB: the Official Publication of the Academy of Molecular Imaging
Z Bősze, P Major, I Baczkó, K E Odening, L Bodrogi, L Hiripi, A Varró
Since the creation of the first transgenic rabbit thirty years ago, pronuclear microinjection remained the single applied method and resulted in numerous important rabbit models of human diseases, including cardiac deficiencies, albeit with low efficiency. For additive transgenesis a novel transposon mediated method, e.g., the Sleeping Beauty transgenesis, increased the efficiency, and its application to create cardiac disease models is expected in the near future. The targeted genome engineering nuclease family, e...
July 2016: Progress in Biophysics and Molecular Biology
Engin Gürlevik, Bettina Fleischmann-Mundt, Jennifer Brooks, Ihsan Ekin Demir, Katja Steiger, Silvia Ribback, Tetyana Yevsa, Norman Woller, Arnold Kloos, Dmitrij Ostroumov, Nina Armbrecht, Michael P Manns, Frank Dombrowski, Michael Saborowski, Moritz Kleine, Thomas C Wirth, Helmut Oettle, Güralp O Ceyhan, Irene Esposito, Diego F Calvisi, Stefan Kubicka, Florian Kühnel
BACKGROUND & AIMS: Even after potentially curative R0 resection, patients with pancreatic ductal adenocarcinoma (PDAC) have a poor prognosis owing to high rates of local recurrence and metastasis to distant organs. However, we have no suitable transgenic animal models for surgical interventions. METHODS: To induce formation of pancreatic tumor foci, we electroporated oncogenic plasmids into pancreata of LSL-KrasG12D × p53fl/fl mice; mutant Kras was expressed in p53fl/fl mice using a sleeping beauty transposon...
August 2016: Gastroenterology
Takahiro Kodama, Emilie A Bard-Chapeau, Justin Y Newberg, Michiko Kodama, Roberto Rangel, Kosuke Yoshihara, Jerrold M Ward, Nancy A Jenkins, Neal G Copeland
BACKGROUND & AIMS: High-throughput sequencing technologies have identified thousands of infrequently mutated genes in hepatocellular carcinomas (HCCs). However, high intratumor and intertumor heterogeneity, combined with large numbers of passenger mutations, have made it difficult to identify driver mutations that contribute to the development of HCC. We combined transposon mutagenesis with a high-throughput screen of a small-hairpin RNA (shRNA) library to identify genes and pathways that contribute to HCC development...
August 2016: Gastroenterology
Orsolya I Hoffmann, Andrea Kerekes, Nandor Lipták, Laszlo Hiripi, Szilard Bodo, Gabor Szaloki, Sabine Klein, Zoltan Ivics, Wilfried A Kues, Zsuzsanna Bosze
The Sleeping Beauty transposon system was established as a robust and efficient method for germline transgenesis in different mammalian species. The generation of transgenic mice, rats, rabbits and swine carrying an identical Venus reporter construct delivered by transposon-mediated gene transfer enables comparative studies of gene expression in these lines of mammalian models. Whereas comparable expression patterns of the Venus reporter were found in somatic tissues, preliminary studies suggested that a striking difference in reporter expression may exist in mature spermatozoa of these species...
2016: PloS One
Junyan Tao, Emily Xu, Yifei Zhao, Sucha Singh, Xiaolei Li, Gabrielle Couchy, Xin Chen, Jessica Zucman-Rossi, Maria Chikina, Satdarshan P S Monga
: Hepatocellular cancer (HCC) remains a significant therapeutic challenge due to its poorly understood molecular basis. In the current study, we investigated two independent cohorts of 249 and 194 HCC cases for any combinatorial molecular aberrations. Specifically we assessed for simultaneous HMET expression or hMet activation and catenin β1 gene (CTNNB1) mutations to address any concomitant Met and Wnt signaling. To investigate cooperation in tumorigenesis, we coexpressed hMet and β-catenin point mutants (S33Y or S45Y) in hepatocytes using sleeping beauty transposon/transposase and hydrodynamic tail vein injection and characterized tumors for growth, signaling, gene signatures, and similarity to human HCC...
November 2016: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
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