Genomic Analysis of Sleeping Beauty Transposon Integration in Human Somatic Cells

The Sleeping Beauty (SB) transposon is a non-viral integrating vector system with proven efficacy for gene transfer and functional genomics. However, integration efficiency is negatively affected by the length of the transposon. To optimize the SB transposon machinery, the inverted repeats and the transposase gene underwent several modifications, resulting in the generation of the hyperactive SB100X transposase and of the high-capacity \u2018\u2018sandwich\u2019\u2019 (SA) transposon. In this study, we report a side-by-side comparison of the SA and the widely used T2 arrangement of transposon vectors carrying increasing DNA cargoes, up to 18 kb. Clonal analysis of SA integrants in human epithelial cells and in immortalized keratinocytes demonstrates stability and integrity of the transposon independently from the cargo size and copy number-dependent expression of the cargo cassette. A genome-wide analysis of unambiguously mapped SA integrations in keratinocytes showed an almost random distribution, with an overrepresentation in repetitive elements (satellite, LINE and small RNAs) compared to a library representing insertions of the first-generation transposon vector and to gammaretroviral and lentiviral libraries. The SA transposon/SB100X integrating system therefore shows important features as a system for delivering large gene constructs for gene therapy application

One-step Multiplex Transgenesis via Sleeping Beauty Transposition in Cattle

Genetically modified cattle are important for developing new biomedical models and for an improved understanding of the pathophysiology of zoonotic diseases. However, genome editing and genetic engineering based on somatic cell nuclear transfer suffer from a low overall efficiency. Here, we established a highly efficient one-step multiplex gene transfer system into the bovine genome.Fil: Garrels, Wiebke. Institut für Nutztiergenetik; AlemaniaFil: Talluri, Thirumala R.. Institut für Nutztiergenetik; AlemaniaFil: Apfelbaum, Ronja. Institut für Nutztiergenetik; AlemaniaFil: Carratalá, Yanet P.. Institut für Nutztiergenetik; AlemaniaFil: Bosch, Pablo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Pötzsch, Kerstin. Paul Ehrlich Institute; AlemaniaFil: Grueso, Esther. Paul Ehrlich Institute; AlemaniaFil: Ivics, Zoltan. Paul Ehrlich Institute; AlemaniaFil: Kues, Wilfred. Institut für Nutztiergenetik; Alemani

Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs

We present the first study of the 3D kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the early host response in a large lung volume using a combination of tissue imaging and transcriptomics. This approach allowed us to make a number of important findings:Spatially restricted antiviral response is shown, including the formation of monocytic macrophage clusters and upregulation of the major histocompatibility complex II in infected epithelial cells.The monocyte-derived macrophages are linked to SARS-CoV-2 clearance, and the appearance of these cells is associated with post-infection endothelial damage; thus, we shed light on the role of these cells in infected tissue.An early onset of tissue repair occurring simultaneously with inflammatory and necrotizing processes provides the basis for longer-term alterations in the lungs

Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells

The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the non-viral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4 and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed putative iPS cells showed long term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5 and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the three germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies towards the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model

Latest Advances for the Sleeping Beauty Transposon System: 23 Years of Insomnia but Prettier than Ever: Refinement and Recent Innovations of the Sleeping Beauty Transposon System Enabling Novel, Nonviral Genetic Engineering Applications

The Sleeping Beauty transposon system is a nonviral DNA transfer tool capable of efficiently mediating transposition-based, stable integration of DNA sequences of choice into eukaryotic genomes. Continuous refinements of the system, including the emergence of hyperactive transposase mutants and novel approaches in vectorology, greatly improve upon transposition efficiency rivaling viral-vector-based methods for stable gene insertion. Current developments, such as reversible transgenesis and proof-of-concept RNA-guided transposition, further expand on possible applications in the future. In addition, innate advantages such as lack of preferential integration into genes reduce insertional mutagenesis-related safety concerns while comparably low manufacturing costs enable widespread implementation. Accordingly, the system is recognized as a powerful and versatile tool for genetic engineering and is playing a central role in an ever-expanding number of gene and cell therapy clinical trials with the potential to become a key technology to meet the growing demand for advanced therapy medicinal products

Jumping Ahead with Sleeping Beauty: Mechanistic Insights into Cut-and-Paste Transposition

Sleeping Beauty (SB) is a transposon system that has been widely used as a genetic engineering tool. Central to the development of any transposon as a research tool is the ability to integrate a foreign piece of DNA into the cellular genome. Driven by the need for efficient transposon-based gene vector systems, extensive studies have largely elucidated the molecular actors and actions taking place during SB transposition. Close transposon relatives and other recombination enzymes, including retroviral integrases, have served as useful models to infer functional information relevant to SB. Recently obtained structural data on the SB transposase enable a direct insight into the workings of this enzyme. These efforts cumulatively allowed the development of novel variants of SB that offer advanced possibilities for genetic engineering due to their hyperactivity, integration deficiency, or targeting capacity. However, many aspects of the process of transposition remain poorly understood and require further investigation. We anticipate that continued investigations into the structure–function relationships of SB transposition will enable the development of new generations of transposition-based vector systems, thereby facilitating the use of SB in preclinical studies and clinical trials

Immuntherapien zur Behandlung der chronischen Hepatitis-B-Virusinfektion – eine Übersicht unter besonderer Berücksichtigung von CAR-T-Zellen

ZusammenfassungDerzeit leiden weltweit mehr als 250 Mio. Menschen an einer chronischen Infektion mit Hepatitis-B-Virus (CHB). Eine chronische Infektion geht mit einem erhöhten Risiko der Entwicklung einer Leberfibrose/-zirrhose und der Entwicklung eines hepatozellulären Karzinoms einher. Derzeit versterben jährlich ca. 0,8–1 Mio. Menschen an den Folgen einer chronischen Infektion. Eine Schwierigkeit bei der Therapie der CHB besteht darin, dass das virale Genom in Form eines Minichroms sehr lange Zeit persistieren kann bzw. dass virale Sequenzen in das Wirtsgenom inserieren können. Chronische Infektionen sind häufig durch funktionale Defekte der zellulären Immunantwort, insbesondere der T‑Zell-Antwort charakterisiert, was einer Eliminierung HBV-infizierter Zellen entgegensteht.Immuntherapien zur Heilung der CHB zielen daher darauf ab, die antivirale Funktion der zellulären Immunantwort wiederherzustellen. Im Rahmen dieser Übersicht sollen verschiedene aktuelle Ansätze zur Immuntherapie der CHB beschrieben werden, insbesondere gentechnisch veränderte autologe T‑Zellen als mögliches Werkzeug zur Therapie der CHB. Weiterhin werden die Modulation von Checkpointinhibitoren der Immunantwort, metabolische T‑Zelltherapien und die therapeutische Impfung zur Stimulation der T‑Zellantwort als immuntherapeutische Strategien zur Therapie der chronischen HBV-Infektion zusammenfassend dargestellt.</jats:p

Immuntherapien zur Behandlung der chronischen Hepatitis-B-Virusinfektion – eine Übersicht unter besonderer Berücksichtigung von CAR-T-Zellen

More than 250 million people worldwide suffer from chronic infection with hepatitis B virus (CHB). Chronic infection is associated with an increased risk of developing liver fibrosis/cirrhosis and hepatocellular carcinoma. Approximately 0.8-1 million people die annually as a result of CHB. One difficulty in the treatment of CHB is that the viral genome can persist for a very long time in the form of a minichromosome, and viral sequences can insert themselves into the host genome. Chronic infections are often characterized by functional defects of the cellular immune response, especially the T‑cell response, which prevents the elimination of HBV-infected cells.Immunotherapies aiming to cure CHB therefore aim to restore the antiviral function of the cellular immune response. In this review, various current approaches to immunotherapy of CHB are described, in particular the use of genetically modified autologous T cells as a possible tool for therapy. Furthermore, the modulation of checkpoint inhibitors of the immune response, metabolic T cell therapies, and therapeutic vaccination to stimulate the T‑cell response are summarized as immunotherapeutic strategies for treating CHB