The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5-6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications.

To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole-body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain-specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposonrelated genetic abnormality, and p53 was not overexpressed. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures. STEM CELLS 2007;25:371-379

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The Sleeping Beauty (SB) transposon is an emerging tool for transgenesis, gene discovery, and therapeutic gene delivery in mammals. Here we studied 1,336 SB insertions in primary and cultured mammalian cells in order to better understand its target site preferences. We report that, although widely distributed, SB integration recurrently targets certain genomic regions and shows a small but significant bias toward genes and their upstream regulatory sequences. Compared to those of most integrating viruses, however, the regional preferences associated with SB-mediated integration were much less pronounced and were not significantly influenced by transcriptional activity. Insertions were also distinctly nonrandom with respect to intergenic sequences, including a strong bias toward microsatellite repeats, which are predominantly enriched in noncoding DNA. Although we detected a consensus sequence consistent with a twofold dyad symmetry at the target site, the most widely used sites did not match this consensus. In conjunction with an observed SB integration preference for bent DNA, these results suggest that physical properties may be the major determining factor in SB target site selection. These findings provide basic insights into the transposition process and reveal important distinctions between transposon-and virus-based integrating vectors.Approximately half of the mammalian genome is derived from ancient transposable elements. Although the two general types of transposable elements, (DNA) transposons and retrotransposons, are often regarded as "selfish DNA parasites" or "junk DNA," their frequent movement in and out of host cell chromosomes has played a significant role in genome diversification and evolution. Members of the Tc1/mariner family of DNA transposons are extremely widespread in nature (44) and can function independently of species-specific host factors (29,56). Although the vast majority of elements present in vertebrate genomes are nonfunctional (14, 32), an active Tc1-like element called Sleeping Beauty (SB) was recently reconstructed from ancient transposon fossils found within fish genomes (20).SB elements transpose by a cut-and-paste mechanism that requires the sequence-specific binding of the SB transposase to the transposon ends (25). This transposition process involves the precise excision and reintegration of the transposon from one DNA site to another site, which invariably contains a TA dinucleotide that is duplicated upon insertion. The transfer of DNA strands at the insertion site is mediated by the transposase catalytic core domain, which contains a conserved DDE motif shared by a large group of recombinase proteins, including the V(D)J recombinase and retrovirus integrases (44). SB is capable of efficient transposition in a variety of cell types (24), including human, mouse, and fish cells, and is an emerging tool for genetic research on vertebrates, with potential applications for transgenesis (22), functional genomics (1, 5-9, 11, 12, 16, 18, 19, 21, 33), and human gene therapy...

HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly.

Adeno-associated virus serotype 8 (AAV8) is currently emerging as a powerful gene transfer vector, owing to its capability to efficiently transduce many different tissues in vivo. While this is believed to be in part due to its ability to uncoat more readily than other AAV serotypes such as AAV2, understanding all the processes behind AAV8 transduction is important for its application and optimal use in human gene therapy. Here, we provide the first report of a cellular receptor for AAV8, the 37/67-kDa laminin receptor (LamR). We document binding of LamR to AAV8 capsid proteins and intact virions in vitro and demonstrate its contribution to AAV8 transduction of cultured cells and mouse liver in vivo. We also show that LamR plays a role in transduction by three other closely related serotypes (AAV2, -3, and -9). Sequence and deletion analysis allowed us to map LamR binding to two protein subdomains predicted to be exposed on the AAV capsid exterior. Use of LamR, which is constitutively expressed in many clinically relevant tissues and is overexpressed in numerous cancers, provides a molecular explanation for AAV8's broad tissue tropism. Along with its robust transduction efficiency, our findings support the continued development of AAV8-based vectors for clinical applications in humans, especially for tumor gene therapy.Adeno-associated virus (AAV) is an increasingly popular gene transfer vector with a number of inherent advantages over other vectors, including a lack of pathogenicity and the ability to mediate long-term gene expression in a variety of tissues in vivo. Of particular benefit is the feasibility to pseudotype recombinant AAV genomes (typically derived from the AAV serotype 2 [AAV2] prototype) with capsids from any of the over 100 identified naturally occurring human or nonhuman viral isolates (4,8,9,11,25) or with synthetic "designer" shells engineered via capsid DNA shuffling or mutagenesis (18). This results in vector particles with distinct properties, including unique tissue biodistribution and transduction profiles, and thus significantly contributes to the versatility of the AAV vector system. Thus far, AAV2 has been the primary serotype tested in clinical trials, but many alternative serotypes that offer specific advantages for certain diseases are currently in preclinical development. Among these, perhaps the most interesting candidate is AAV8. We and others recently documented unusually robust and sustained transgene expression from AAV8 vectors in numerous tissues, including the liver, heart, and skeletal muscle (9,20,36). In fact, AAV8 resulted in up to 20-foldhigher liver transduction in mice than AAV2 did, despite an 83% amino acid similarity of the two viruses (20). We also showed that the higher rate of AAV8 capsid uncoating compared to AAV2 may be responsible for the increased transduction efficiency (31), allowing AAV8 to be transduced in almost 100% of hepatocytes (20). A second advantage of AAV8 is that since it is a primate virus (isolated from rhesus monkeys), vectors derived th...

The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules.DOI: http://dx.doi.org/10.7554/eLife.04114.001

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