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.

It has been previously shown that integrase-defective HIV-1-based gene vectors can serve, with moderate efficiency, as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) transposase. Here, we describe the enhanced gene transfer properties of a HIV-1/SB hybrid vector that allows efficient DNA transposition, facilitated by the hyperactive SB100X transposase, from vector DNA intermediates in primary human cells. Potent transposase-dependent integration of genetic cargo carried by the hybrid HIV-1/SB vector (up to 160-fold above background) is reported in human cell lines as well as in primary human fibroblasts and keratinocytes. The efficiency of transgene integration in context of the newly developed hybrid vector is comparable with that of conventional lentiviral vectors (LVs). Integration profiles of integrating HIV-1-derived vectors and SB transposons mobilized from LVs are investigated by deep sequencing of a large number of integration sites. A significant bias of lentiviral integrations in genes is reported, confirming that biological properties of the viral integration machinery facilitate preferred insertion into actively transcribed genomic regions. In sharp contrast, lentiviral insertions catalyzed by the SB100X transposase are far more random with respect to genes. Based on these properties, HIV-1/SB vectors may become valuable tools for genetic engineering and therapeutic gene transfer.

Up to 13% of women may experience symptoms of depression during pregnancy or in the postpartum period. Depression during pregnancy has been associated with an increased risk of adverse neurodevelopmental outcomes in the child and epigenetic mechanisms could be one of the biological pathways to explain this association. In 844 mother–child pairs from the Avon Longitudinal Study of Parents and Children, we carried out an epigenome-wide association study (EWAS) to investigate associations between prospectively collected data on maternal depression ascertained by the Edinburgh Postnatal Depression Scale in pregnancy and DNA methylation in the cord blood of newborn offspring. In individual site analysis, we identified two CpG sites associated with maternal depression in the middle part of pregnancy. In our regional analysis, we identified 39 differentially methylated regions (DMRs). Seven DMRs were associated with depression at any time point during pregnancy, 7 associated with depression in mid-pregnancy, 23 were associated with depression in late pregnancy, and 2 DMRs were associated with depression throughout pregnancy. Several of these map to genes associated with psychiatric disease and brain development. We attempted replication in The Generation R Study and could not replicate our results. Although our findings in ALSPAC suggest that maternal depression could be associated with cord blood DNA methylation the results should be viewed as preliminary and hypothesis generating until further replicated in a larger sample.

DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.