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

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

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

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

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

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"

Mutant screen reveals the Piccolo's control over depression and brain-gonad crosstalk

Successful sexual reproduction involves a highly complex, genetically encoded interplay between animal physiology and behavior. Here we developed a screen to identify genes essential for rat reproduction based on an unbiased methodology involving mutagenesis via the Sleeping Beauty transposon. As expected, our screen identified genes where reproductive failure was connected to gametogenesis (Btrc, Pan3, Spaca6, Ube2k) and embryogenesis (Alk3, Exoc6b, Slc1a3, Tmx4, Zmynd8). In addition, our screen identified Atg13 (longevity) Dlg1 and Pclo (neuronal disorders), previously not associated with reproduction. Dominant Pclo traits caused epileptiform activity and affected genes supporting GABAergic synaptic transmission (Gabra6, Gabrg3), and animals exhibited a compromised crosstalk between the brain and gonads via disturbed GnRH signaling. Recessive Pclo traits disrupted conspecific recognition required for courtship/mating and were mapped to allelic markers for major depressive disorder (Grm5, Htr2a, Sorcs3, Negr1, Drd2). Thus, Pclo-deficiency in rats link neural networks controlling sexual motivation to Pclo variants that have been associated with human neurological disorders

Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34 + cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is 3c20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34 + cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34 + cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4\u20138 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol. Ivics and collegues refined the Sleeping Beauty transposon system for gene transfer in human hematopoietic stem and progenitor cells by vectorizing the transposon components as minicircle DNA and synthetic mRNA. The advanced vector system enables efficient and safe non-viral engineering of hematopoietic cells that can be transplanted into immunodeficient mice

Recapitulating early human development with 8C-like cells

In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An in vitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development