Transposable elements can be considered as natural, nonviral gene-delivery vehicles and are valuable and widely used tools for germ-line transgenesis and insertional mutagenesis in invertebrate ...systems such as flies and worms. Such tools were not available for genome manipulations in vertebrates until recently, when an active element was resurrected from transposon fossils found in fish genomes. This element, the Sleeping Beauty transposon, shows efficient transposition in cells of a wide range of vertebrates, including humans. Sleeping Beauty transposition is a cut-and-paste process, during which the element “jumps” from one DNA molecule to another. Transposon integration into chromosomes provides the basis for long-term, or possibly permanent, transgene expression in transgenic cells and organisms. Thus, the reconstruction of the Sleeping Beauty element generated considerable interest in developing efficient and safe vectors for vertebrate transgenesis as well as for human gene therapy. In this review we summarize our current knowledge of Sleeping Beauty biology and describe the strengths and current limitations of transposon technology for gene therapeutic applications.
Transposable elements (TEs) are major components of eukaryotic genomes. However, the extent of their impact on genome evolution, function, and disease remain a matter of intense interrogation. The ...rise of genomics and large-scale functional assays has shed new light on the multi-faceted activities of TEs and implies that they should no longer be marginalized. Here, we introduce the fundamental properties of TEs and their complex interactions with their cellular environment, which are crucial to understanding their impact and manifold consequences for organismal biology. While we draw examples primarily from mammalian systems, the core concepts outlined here are relevant to a broad range of organisms.
Totipotent cells have more robust developmental potency than any other cell types, giving rise to both embryonic and extraembryonic tissues. Stable totipotent cell cultures and deciphering the ...principles of totipotency regulation would be invaluable to understand cell plasticity and lineage segregation in early development. Our approach of remodeling the pericentromeric heterochromatin and re-establishing the totipotency-specific broad H3K4me3 domains promotes the pluri-to-totipotency transition. Our protocol establishes a closer match of mouse 2-cell (2C) embryos than any other 2C-like cells. These totipotent-like stem cells (TLSCs) are stable in culture and possess unique molecular features of the mouse 2C embryo. Functionally, TLSCs are competent for germline transmission and give rise to both embryonic and extraembryonic lineages at high frequency. Therefore, TLSCs represent a highly valuable cell type for studies of totipotency and embryology.
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•DOT1L inhibition collapses chromocenters to induce an alternative 2C-like state•KDM5B inhibition promotes remodeling of broad H3K4me3 to stabilize totipotency•TLSCs derived from early embryos and ESCs molecularly resemble mouse 2C embryos•TLSCs have embryonic and extraembryonic developmental potency and can form blastoids
By using a chemical cocktail to remodel the pericentromeric heterochromatin and re-establish a totipotent-specific H3K4me3 landscape, Wang and colleagues establish a totipotent-like stem cell line that faithfully recapitulates mouse two-cell embryos.
Cell senescence suppresses tumors by arresting cells at risk of becoming malignant. However, this process in turn can affect the microenvironment, leading to acquisition of a senescence-associated ...secretory phenotype (SASP) that renders senescent cells proinflammatory and results in tumor progression. But how is SASP controlled? In this issue of the JCI, Attig and Pape et al. describe the role of chimeric calbindin 1 (CALB1) transcripts, which are driven by an upstream human endogenous retrovirus subfamily H (HERVH) element. The authors propose that in lung squamous cell carcinoma (LUSC), HERVH-driven isoforms of calbindin (HERVH-CALB1) counteract SASP. As an alternative promoter, HERVH drove calbindin isoforms that prevented cancer cell senescence and associated inflammation, which was associated with better patient survival. We comment on the similarities between HERVH-CALB1-related cellular fitness in cancer and early embryogenesis and discuss the potential benefits of HERVH-driven chimeric transcripts.
Molecular medicine has entered a high-tech age that provides curative treatments of complex genetic diseases through genetically engineered cellular medicinal products. Their clinical implementation ...requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective and economically viable manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are prevalent in ongoing pre-clinical and translational research. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here we review several recent refinements of the system, including the development of optimized transposons and hyperactive SB variants, the vectorization of transposase and transposon as mRNA and DNA minicircles (MCs) to enhance performance and facilitate vector production, as well as a detailed understanding of SB's genomic integration and biosafety features. This review also provides a perspective on the regulatory framework for clinical trials of gene delivery with SB, and illustrates the path to successful clinical implementation by using, as examples, gene therapy for age-related macular degeneration (AMD) and the engineering of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy.
Although translational selection to favour codons that match the most abundant tRNAs is not readily observed in humans, there is nonetheless selection in humans on synonymous mutations. We ...hypothesize that much of this synonymous site selection can be explained in terms of protection against unwanted RNAs - spurious transcripts, mis-spliced forms or RNAs derived from transposable elements or viruses. We propose not only that selection on synonymous sites functions to reduce the rate of creation of unwanted transcripts (for example, through selection on exonic splice enhancers and cryptic splice sites) but also that high-GC content (but low-CpG content), together with intron presence and position, is both particular to functional native mRNAs and used to recognize transcripts as native. In support of this hypothesis, transcription, nuclear export, liquid phase condensation and RNA degradation have all recently been shown to promote GC-rich transcripts and suppress AU/CpG-rich ones. With such 'traps' being set against AU/CpG-rich transcripts, the codon usage of native genes has, in turn, evolved to avoid such suppression. That parallel filters against AU/CpG-rich transcripts also affect the endosomal import of RNAs further supports the unwanted transcript hypothesis of synonymous site selection and explains the similar design rules that have enabled the successful use of transgenes and RNA vaccines.
Members of the
Tc1/mariner superfamily of transposons isolated from fish appear to be transpositionally inactive due to the accumulation of mutations. Molecular phylogenetic data were used to ...construct a synthetic transposon,
Sleeping Beauty, which could be identical or equivalent to an ancient element that dispersed in fish genomes in part by horizontal transmission between species. A consensus sequence of a transposase gene of the salmonid subfamily of elements was engineered by eliminating the inactivating mutations.
Sleeping Beauty transposase binds to the inverted repeats of salmonid transposons in a substrate-specific manner, and it mediates precise cut-and-paste transposition in fish as well as in mouse and human cells.
Sleeping Beauty is an active DNA-transposon system from vertebrates for genetic transformation and insertional mutagenesis.
Transposable elements can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of class II transposable elements ...(DNA transposons) can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest (be it a fluorescent marker, a small hairpin (sh)RNA expression cassette, a mutagenic gene trap or a therapeutic gene construct) cloned between the inverted repeat sequences of a transposon-based vector can be used for stable genomic insertion in a regulated and highly efficient manner. This methodological paradigm opened up a number of avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture, the production of germline transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species, and therapy of genetic disorders in humans. Sleeping Beauty (SB) was the first transposon shown to be capable of gene transfer in vertebrate cells, and recent results confirm that SB supports a full spectrum of genetic engineering including transgenesis, insertional mutagenesis, and therapeutic somatic gene transfer both ex vivo and in vivo. The first clinical application of the SB system will help to validate both the safety and efficacy of this approach. In this review, we describe the major transposon systems currently available (with special emphasis on SB), discuss the various parameters and considerations pertinent to their experimental use, and highlight the state of the art in transposon technology in diverse genetic applications.
The Sleeping Beauty (SB) transposon is a promising technology platform for gene transfer in vertebrates; however, its efficiency of gene insertion can be a bottleneck in primary cell types. A ...large-scale genetic screen in mammalian cells yielded a hyperactive transposase (SB100X) with approximately 100-fold enhancement in efficiency when compared to the first-generation transposase. SB100X supported 35-50% stable gene transfer in human CD34(+) cells enriched in hematopoietic stem or progenitor cells. Transplantation of gene-marked CD34(+) cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution. In addition, SB100X supported sustained (>1 year) expression of physiological levels of factor IX upon transposition in the mouse liver in vivo. Finally, SB100X reproducibly resulted in 45% stable transgenesis frequencies by pronuclear microinjection into mouse zygotes. The newly developed transposase yields unprecedented stable gene transfer efficiencies following nonviral gene delivery that compare favorably to stable transduction efficiencies with integrating viral vectors and is expected to facilitate widespread applications in functional genomics and gene therapy.
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