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Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates

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 ∼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.

Transposon-mediated genome manipulation in vertebrates

Transposable elements are DNA segments with the unique ability to move about in the genome. This inherent feature can be exploited to harness these elements as gene vectors for genome manipulation. Transposon-based genetic strategies have been established in vertebrate species over the last decade, and current progress in this field suggests that transposable elements will serve as indispensable tools. In particular, transposons can be applied as vectors for somatic and germline transgenesis, and as insertional mutagens in both loss-of-function and gain-of-function forward mutagenesis screens. In addition, transposons will gain importance in future cell-based clinical applications, including nonviral gene transfer into stem cells and the rapidly developing field of induced pluripotent stem cells. Here we provide an overview of transposon-based methods used in vertebrate model organisms with an emphasis on the mouse system and highlight the most important considerations concerning genetic applications of the transposon systems.

Comparative analysis of transposable element vector systems in human cells

Transposon-based gene vectors have become indispensable tools in vertebrate genetics for applications ranging from insertional mutagenesis and transgenesis in model species to gene therapy in humans. The transposon toolkit is expanding, but a careful, side-by-side characterization of the diverse transposon systems has been lacking. Here we compared the Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposons with respect to overall activity, overproduction inhibition (OPI), target site selection, transgene copy number as well as long-term expression in human cells. SB was the most efficient system under conditions where the availability of the transposon DNA is limiting the transposition reaction including hard-to-transfect hematopoietic stem/progenitor cells (HSCs), and the most sensitive to OPI, underpinning the need for careful optimization of the transposon components. SB and PB were about equally active, and both more efficient than Tol2, under nonrestrictive conditions. All three systems provided long-term transgene expression in human cells with minimal signs of silencing. Indeed, mapping of Tol2 insertion sites revealed significant underrepresentation within chromosomal regions with H3K27me3 histone marks typically associated with transcriptionally repressed heterochromatin. SB, Tol2, and PB constitute complementary research tools for gene transfer in mammalian cells with important implications for fundamental and translational research.

Stable gene transfer and expression in cord blood-derived CD34+ hematopoietic stem and progenitor cells by a hyperactive Sleeping Beauty transposon system

Here we report stable gene transfer in cord blood-derived CD34(+) hematopoietic stem cells using a hyperactive nonviral Sleeping Beauty (SB) transposase (SB100X). In colony-forming assays, SB100X mediated the highest efficiency (24%) of stable Discosoma sp red fluorescent protein (DsRed) reporter gene transfer in committed hematopoietic progenitors compared with both the early-generation hyperactive SB11 transposase and the piggyBac transposon system (1.23% and 3.8%, respectively). In vitro differentiation assays further demonstrated that SB100X-transfected CD34(+) cells can develop into DsRed(+) CD4(+)CD8(+) T (3.17%-21.84%; median, 7.97%), CD19(+) B (3.83%-18.66%; median, 7.84%), CD56(+)CD3(-) NK (3.53%-79.98%; median, 7.88%), and CD33(+) myeloid (7.59%-15.63%; median, 9.48%) cells. SB100X-transfected CD34(+) cells achieved approximately 46% engraftment in NOD-scid IL2gammac(null) (NOG) mice. Twelve weeks after transplantation, 0.57% to 28.96% (median, 2.79%) and 0.49% to 34.50% (median, 5.59%) of total human CD45(+) cells in the bone marrow and spleen expressed DsRed, including CD19(+) B, CD14(+) monocytoid, and CD33(+) myeloid cell lineages. Integration site analysis revealed SB transposon sequences in the human chromosomes of in vitro differentiated T, B, NK, and myeloid cells, as well as in human CD45(+) cells isolated from bone marrow and spleen of transplanted NOG mice. Our results support the continuing development of SB-based gene transfer into human hematopoietic stem cells as a modality for gene therapy.

Germline transgenic pigs by Sleeping Beauty transposition in porcine zygotes and targeted integration in the pig genome.

Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X) transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes, resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases.

The Ancient mariner Sails Again: Transposition of the Human Hsmar1 Element by a Reconstructed Transposase and Activities of the SETMAR Protein on Transposon Ends

ABSTRACT Hsmar1, one of the two subfamilies of mariner transposons in humans, is an ancient element that entered the primate genome lineage ∼50 million years ago. Although Hsmar1 elements are inactive due to mutational damage, one particular copy of the transposase gene has apparently been under selection. This transposase coding region is part of the SETMAR gene, in which a histone methylatransferase SET domain is fused to an Hsmar1 transposase domain. A phylogenetic approach was taken to reconstruct the ancestral Hsmar1 transposase gene, which we named Hsmar1-Ra. The Hsmar1-Ra transposase efficiently mobilizes Hsmar1 transposons by a cut-and-paste mechanism in human cells and zebra fish embryos. Hsmar1-Ra can also mobilize short inverted-repeat transposable elements (MITEs) related to Hsmar1 (MiHsmar1), thereby establishing a functional relationship between an Hsmar1 transposase source and these MITEs. MiHsmar1 excision is 2 orders of magnitude more efficient than that of long elements, thus providing an explanation for their high copy numbers. We show that the SETMAR protein binds and introduces single-strand nicks into Hsmar1 inverted-repeat sequences in vitro. Pathway choices for DNA break repair were found to be characteristically different in response to transposon cleavage mediated by Hsmar1-Ra and SETMAR in vivo. Whereas nonhomologous end joining plays a dominant role in repairing excision sites generated by the Hsmar1-Ra transposase, DNA repair following cleavage by SETMAR predominantly follows a homology-dependent pathway. The novel transposon system can be a useful tool for genome manipulations in vertebrates and for investigations into the transpositional dynamics and the contributions of these elements to primate genome evolution.

Hybrid Lentivirus-transposon Vectors With a Random Integration Profile in Human Cells

Gene delivery by human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) is efficient, but genomic integration of the viral DNA is strongly biased toward transcriptionally active loci resulting in an increased risk of insertional mutagenesis in gene therapy protocols. Nonviral Sleeping Beauty (SB) transposon vectors have a significantly safer insertion profile, but efficient delivery into relevant cell/tissue types is a limitation. In an attempt to combine the favorable features of the two vector systems we established a novel hybrid vector technology based on SB transposase-mediated insertion of lentiviral DNA circles generated during transduction of target cells with integrase (IN)-defective LVs (IDLVs). By construction of a lentivirus-transposon hybrid vector allowing transposition exclusively from circular viral DNA substrates, we demonstrate that SB transposase added in trans directs efficient transposon mobilization from DNA circles in vector-transduced cells. Both transfected plasmid DNA and transduced IDLVs can serve as the source of active transposase. Most important, we demonstrate that the SB transposase overrides the natural lentiviral integration pathway and directs vector integration less frequently toward transcriptional units, resulting in a random genomic integration profile. The novel hybrid vector system combines the attractive features of efficient gene delivery by viral transduction and a safer genomic integration profile by DNA transposition.

The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor

Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on the measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen-presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with an SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells.

Applying a “Double-Feature” Promoter to Identify Cardiomyocytes Differentiated from Human Embryonic Stem Cells Following Transposon-Based Gene Delivery†‡

Human embryonic stem (HuES) cells represent a new potential tool for cell‐therapy and gene‐therapy applications. However, these approaches require the development of efficient, stable gene delivery, and proper progenitor cell and tissue separation methods. In HuES cell lines, we have generated stable, enhanced green fluorescent protein (EGFP)‐expressing clones using a transposon‐based (Sleeping Beauty) system. This method yielded high percentage of transgene integration and expression. Similarly to a lentiviral expression system, both the undifferentiated state and the differentiation pattern of the HuES cells were preserved. By using the CAG promoter, in contrast to several other constitutive promoter sequences (such as CMV, elongation factor 1α, or phosphoglycerate kinase), an exceptionally high EGFP expression was observed in differentiated cardiomyocytes. This phenomenon was independent of the transgene sequence, methods of gene delivery, copy number, and the integration sites. This “double‐feature” promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifically labeling differentiated cardiomyocytes, was assessed by transcriptional profiling. We found a positive correlation between CAG promoter‐driven EGFP transcription and expression of cardiomyocyte‐specific genes. Our experiments indicate an efficient applicability of transposon‐based gene delivery into HuES cells and provide a novel approach to identify differentiated tissues by exploiting a nontypical behavior of a constitutively active promoter, thereby avoiding invasive drug selection methods. Stem Cells 2009;27:1077–1087

Technology transfer from worms and flies to vertebrates: transposition-based genome manipulations and their future perspectives

To meet the increasing demand of linking sequence information to gene function in vertebrate models, genetic modifications must be introduced and their effects analyzed in an easy, controlled, and scalable manner. In the mouse, only about 10% (estimate) of all genes have been knocked out, despite continuous methodologic improvement and extensive effort. Moreover, a large proportion of inactivated genes exhibit no obvious phenotypic alterations. Thus, in order to facilitate analysis of gene function, new genetic tools and strategies are currently under development in these model organisms. Loss of function and gain of function mutagenesis screens based on transposable elements have numerous advantages because they can be applied in vivo and are therefore phenotype driven, and molecular analysis of the mutations is straightforward. At present, laboratory harnessing of transposable elements is more extensive in invertebrate models, mostly because of their earlier discovery in these organisms. Transposons have already been found to facilitate functional genetics research greatly in lower metazoan models, and have been applied most comprehensively in Drosophila. However, transposon based genetic strategies were recently established in vertebrates, and current progress in this field indicates that transposable elements will indeed serve as indispensable tools in the genetic toolkit for vertebrate models. In this review we provide an overview of transposon based genetic modification techniques used in higher and lower metazoan model organisms, and we highlight some of the important general considerations concerning genetic applications of transposon systems.