Nonviral gene delivery with the Sleeping Beauty transposon system

Effective gene therapy requires robust delivery of the desired genes into the relevant target cells, long-term gene expression and minimal risks of secondary effects. Non-viral gene transfer approaches typically result in only short-lived transgene expression in primary cells, due to the lack of nuclear maintenance of the vector over several rounds of cell division. The development of efficient and safe non-viral vectors equipped with an integrating feature would thus greatly facilitate clinical gene therapy studies. The latest generation transposon technology based on the Sleeping Beauty (SB) transposon may potentially overcome some of these limitations. SB was recently shown to provide efficient stable gene transfer after non-viral gene delivery into therapeutically relevant primary cell types, including human hematopoietic progenitors, mesenchymal stem cells, muscle stem/progenitor cells (myoblasts), iPSCs and T cells. These cells are relevant targets for stem cell biology and for regenerative medicine and gene- and cell-based therapies of complex genetic diseases. Moreover, the first-in-man clinical trial has recently been launched to use redirected T cells engineered with SB for gene therapy of B cell lymphoma. We discuss aspects of cellular delivery of the SB transposon system, transgene expression provided by integrated transposon vectors, target site selection of the transposon vectors and potential risks associated with random genomic insertion

Sleeping Beauty hits them all: transposon-mediated saturation mutagenesis in the mouse germline

The Sleeping Beauty (SB) transposon emerged as a useful tool for applications such as germline and somatic cell insertional mutagenesis and now shows its usefulness again by facilitating saturating germline mutagenesis in mice

Our Administrative System of Criminal Justice

To commemorate our founding in 1914, the Board of Editors has selected six influential pieces published by the Law Review over the past 100 years and will republish one piece in each issue. The fourth piece selected by the Board is Our Administrative System of Criminal Justice, an article written by Gerard E. Lynch that is among the most cited works in the Law Review’s history. This article illustrates how the practice of plea bargaining blurs the boundaries between adversarial and inquisitorial criminal justice systems. Judge Lynch now sits on the Second Circuit having eventually succeeded the late Judge Joseph M. McLaughlin, who also is honored in the pages of this book for the permanent mark he left on Fordham Law School and the Law Review. We think it is fitting that the Law Review feature two of the many contributions that judges of the Second Circuit have made to legal education and scholarship in this issue

Human endogenous retrovirus K Rec forms a regulatory loop with MITF that opposes the progression of melanoma to an invasive stage

In the human genome, HERV-K(HML2) is the most recently endogenized retrovirus (ERV). While HERV-K(HML2) transcription is observed in healthy tissues, various cancers showed the upregulation of retroviral derived endogenized accessory products (e.g., envelope (Env), Np9 and Rec). Still, it is not clear whether the different HERV-K-derived genes contribute to a disease, or they are mere by-products. Here, we focus on the potential role of Rec in melanoma. Our in vitro model and high throughput data mining, including single-cell transcriptome analyses of patent’s material, reveal that Rec expression marks the proliferative (still controllable) stage of melanoma, and is involved in maintaining a delicate balance between cell proliferation and invasion. Thus, similar to melanocyte-inducing transcription factor (MITF), Rec is a sensitive marker of melanoma progression. Our Rec-knockdown in vitro system can faithfully model a subpopulation (MITF malignancy) of melanoma cells in human patients. Like Env, Rec modulates an endothelial-mesenchymal transition (EMT)-like process of cancer progression; however, they seem to affect the phenotype switch inversely. Rec inhibits the transition to the invasive state by altering the expression level of some key determinants of the EMT-like process, including MITF that directly binds the LTR5 _Hs of HERV-K. The Hominoid-specific HERV-K products might explain certain species-specific features of melanoma progression, and pinpoint to the limitation of using animal models in melanoma studies

Critical role for Piccolo in synaptic vesicle retrieval

Loss of function of the presynaptic active zone protein Piccolo has recently been linked to a devastating disease causing brain atrophy. Here, we address how Piccolo inactivation adversely affects synaptic function and thus may contributes to neuronal loss. Our analysis shows that Piccolo is critical for the activity dependent recycling and maintenance of synaptic vesicles (SVs). Specifically, we find that boutons lacking Piccolo have deficits in the Rab5/EEA1 dependent formation of early endosomes and thus the recycling of SVs. Mechanistically, impaired Rab5 function was caused by the reduced synaptic recruitment of Pra1, known to interact selectively with the zinc fingers of Piccolo. Importantly, over-expression of GTPase deficient Rab5 or the Znf1 domain of Piccolo restores the size and recycling of SV pools. These data provide a molecular link between the active zone and endosome sorting at synapses providing hints to how Piccolo contributes to both developmental and psychiatric disorders

Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery

The Sleeping Beauty (SB) transposase and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy. The potential cytotoxicity of transposases requires careful assessment, considering that residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and resulting uncontrollable transposition. Comparing retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, we found that conventional SB transposase, SB100X and a newly developed codon-optimized SB100Xo may trigger premitotic arrest and apoptosis. Cell stress induced by continued SB overexpression was self-limiting due to the induction of cell death, which occurred even in the absence of a co-transfected transposable element. The cytotoxic effects of SB transposase were strictly dose dependent and heralded by induction of p53 and c-Jun. Inactivating mutations in SB’s catalytic domain could not abrogate cytotoxicity, suggesting a mechanism independent of DNA cleavage activity. An improved approach of retrovirus particle-mediated mRNA transfer allowed transient and dose-controlled expression of SB100X, supported efficient transposition and prevented cytotoxicity. Transposase-mediated gene transfer can thus be tuned to maintain high efficiency in the absence of overt cell damage

Retargeting transposon insertions by the adeno-associated virus Rep protein

The Sleeping Beauty (SB), piggyBac (PB) and Tol2 transposons are promising instruments for genome engineering. Integration site profiling of SB, PB and Tol2 in human cells showed that PB and Tol2 insertions were enriched in genes, whereas SB insertions were randomly distributed. We aimed to introduce a bias into the target site selection properties of the transposon systems by taking advantage of the locus-specific integration system of adeno-associated virus (AAV). The AAV Rep protein binds to Rep recognition sequences (RRSs) in the human genome, and mediates viral integration into nearby sites. A series of fusion constructs consisting of the N-terminal DNA-binding domain of Rep and the transposases or the N57 domain of SB were generated. A plasmid-based transposition assay showed that Rep/SB yielded a 15-fold enrichment of transposition at a particular site near a targeted RRS. Genome-wide insertion site analysis indicated that an approach based on interactions between the SB transposase and Rep/N57 enriched transgene insertions at RRSs. We also provide evidence of biased insertion of the PB and Tol2 transposons. This study provides a comparative insight into target site selection properties of transposons, as well as proof-of-principle for targeted chromosomal transposition by composite protein-protein and protein-DNA interactions

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

The phylogenetically distinct early human embryo

The phylogenetic singularity of the human embryo remains unresolved as cell types of the human blastocyst have resisted classification. Combining clustering of single cellular transcriptomes and dynamically expressed genes we resolve the cell types. This unveils the missing inner cell mass (ICM) and reveals classical step-wise development. Conversely, numerous features render our blastocyst phylogenetically distinct: unlike mice, our epiblast is self-renewing and we have blastocyst non-committed cells (NCCs), part of an apoptosis-mediated quality control/purging process. At the transcriptome-level all primate embryos are distinct as the pluripotent cell types are uniquely fast evolving. A substantial fraction of gene expression gain and loss events between human and new-world monkeys involve endogenous retrovirus H (ERVH). Human pluripotent cells are unique in which (H)ERVH's are active, the extent to which these modulate neighbour gene expression and their ability to suppress mutagenic transposable elements. Current naive cultures are heterogeneous and both developmentally and phylogenetically "confused"

Passport, a native Tc1 transposon from flatfish, is functionally active in vertebrate cells

The Tc1/mariner family of DNA transposons is widespread across fungal, plant and animal kingdoms, and thought to contribute to the evolution of their host genomes. To date, an active Tc1 transposon has not been identified within the native genome of a vertebrate. We demonstrate that Passport, a native transposon isolated from a fish (Pleuronectes platessa), is active in a variety of vertebrate cells. In transposition assays, we found that the Passport transposon system improved stable cellular transgenesis by 40-fold, has an apparent preference for insertion into genes, and is subject to overproduction inhibition like other Tc1 elements. Passport represents the first vertebrate Tc1 element described as both natively intact and functionally active, and given its restricted phylogenetic distribution, may be contemporaneously active. The Passport transposon system thus complements the available genetic tools for the manipulation of vertebrate genomes, and may provide a unique system for studying the infiltration of vertebrate genomes by Tc1 elements