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14 Immunosuppression with Cyclophosphamide Significantly Prolongs High Level Expression of IDUA Mediated by the Sleeping Beauty Transposon System Molecular Therapy Volume 19, Supplement 1, May 2011 Co[.]
DNA VECTOROLOGY expression was silenced as with the standard plasmid vector These results suggested that spacer DNA regardless of its source >1kb caused the minicircle constructs to be silenced even in the absence of plasmid BB To further establish this idea, 500bp of plasmid BB was used as spacer to make new constructs These constructs also showed persistent expression suggesting the size of the non-transcribed insert and not the source of the spacer DNA was the critical factor for DNA silencing To better understand the mechanism of plasmid silencing, we attempted to determine if leaky transcriptional termination across the spacer sequences was an important parameter for silencing To this, we added a robust transcriptional terminator between the poly A site and DNA spacer Even with an additional terminator, only spacers of > 1kb silenced the transgene Our data suggest that the distance between the poly A site and promoter region influences DNA silencing These results have important implications for designing DNA plasmid vectors for gene therapy 12 Development of a High-Throughput Quantitative Method for Assessing Plasmid Nuclear Import Holly M Reynolds,1 David A Dean.1 Pediatrics, University of Rochester, Rochester, NY One area of interest in gene therapy is designing strategies that can allow DNA to be delivered only to a specific cell type or tissue Historically, these strategies have included: application of the agent directly to desired tissue, exploitation of receptor-ligand interactions, and using cell-specific promoters to drive transcription Our lab has developed a novel and robust method for cell-specific delivery that allows for nuclear import in non-dividing cells by a sequence specific process that requires the cytoplasmic binding of transcription factors, these factors provide transport across the nuclear membrane Namely, this exploits the fact that promoter sequences bind transciption factors and in some cases, when the Nuclear Localization Signal is oriented in the correct way, allow for taxiing across the nuclear membrane To date, we have discovered DNA sequences that provide cell-specific import in smooth muscle cells, osteoblasts, endothelial cells and alveolar type epithelial cells Identifying such plasmid sequences, however, is a tedious and time-consuming task, requiring cytoplasmic microinjection of individual plasmids containing potential sequences into hundreds to thousands of individual cells and determining nuclear localization of the plasmids by in situ hybridization and/ or fluorescence microscopy We have developed a simpler, higherthroughput assay that allows us to screen a large number of potential sequences for cell-specific nuclear entry in a large panel of cell types Using a 96-well electroporator with optimized field strengths, we can induce cell uptake of pools of fluorescently-labeled plasmids containing various DNA sequences After incubation, these cells are fixed and run, in a 96-well format, on an AMNIS Imagestream with a nuclear stain This instrument is essentially a flow cytometer that images each cell in up to fluorescent channels with a 40X or 60X objective as it moves through the stream The instrument is capable of collecting data from up to 5000 cells per minute and images can then be analyzed with software to assess and quantify subcellular localization of the plasmids Using this approach, we have shown that plasmids carrying the ubiquitously active SV40 DNA nuclear targeting sequence localize to the nuclei of multiple cell types while isogenic plasmids lacking this sequence remain in the cytoplasm unless incubation times are greatly increased to allow for cell division (and concomitant breakdown of the nuclear envelope) This approach should greatly enhance our ability to screen DNA sequence libraries in a wide variety of cell types to identify nuclear localizing DNA sequences for cell-specific gene therapy 13 Robust Long-Term Gene Expression in Mouse Liver Via the Novel hAT Transposon TcBuster Lauren E Woodard,1 Aparna Kaja,1 Robert H Hice,2 Peter W Atkinson,2 Nancy L Craig,3 Matthew H Wilson.1 Department of Medicine, Division of Nephrology, Baylor College of Medicine, Houston, TX; 2Department of Entomology & Institute for Integrative Genome Biology, University of California, Riverside, Riverside, CA; 3Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD TcBuster is a recently discovered mobile DNA element from the hAT superfamily Colony count assays in HEK 293 cells using varying amounts of transposon and transposase indicate that maximal transposition occurred using 500 ng of pTCBNeo and 100 ng of pCMV-TCB Out of 48 TcBuster integration sites sequenced from these cells, 62.5% were in genes and 4.2% were in exons, suggesting a preference for transcriptionally active areas Comparing different transposons by colony count assay in HEK 293 cells, the transposition of TcBuster was approximately equivalent to hAT family member Tol2 and about half that of piggyBac, depending on the ratio of transposon to transposase plasmids When an equal amount of transposase and transposon plasmids were used, all three systems were approximately equal However, when an excess of transposase (200 ng of transposon and 800 ng of transposase plasmids) or an excess of transposon (900 ng of transposon and 100 ng of transposase plasmids) were provided, piggyBac outperformed both hAT systems, indicating the significant flexibility of the piggyBac system As for the hAT systems, when the amount of transposase plasmid was higher, the Tol2 system was superior to TcBuster, whereas when the amount of transposase plasmid was low, the TcBuster system was superior to Tol2 After transfection with a construct carrying an HA-tagged version of the TcBuster transposase, staining revealed localization to unique intranuclear rodlets We hypothesize that the rodlets may serve a protective role, limiting free transposase and thus DNA damage In support of this hypothesis, as the amount of pCMV-HA-TCB is increased beyond 100 ng, the number of rodlets per nuclei increased exponentially as the number of colonies formed decreased To compare TcBuster with piggyBac for integration into mammalian chromosomes in vivo, adult female FVB mice were given hydrodynamic injections containing luciferase transposon plasmids with or without the plasmid encoding the transposase Mice were imaged using the IVIS system to determine the levels of liver-specific luciferase expression at timepoints out to 24 weeks At the later timepoints, the normalized luciferase values for groups given either TcBuster or piggyBac transposase were both statistically significantly higher than groups given transposon alone (p