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NAKED DNA: METHODS NAKED DNA: METHODS 21 Molecular Evidence for Sleeping BeautyMediated Transposition and Long-Term Expression In Vivo Paul R Score,1 Joel L Frandsen,1 Jennifer Jeske,1 Perry B Hackett,1,2 David A Largaespada,1 R Scott McIvor.1 Gene Therapy Program, Institue of Human Genetics, Dept of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 2Discovery Genomics Inc., Minneapolis, MN Long-term expression is most effectively achieved through stable integration of expression constructs into genomic DNA, preventing degradation and ensuring transfer to future progeny The Sleeping Beauty Transposon system (SB) offers a non-viral means to achieve long-term gene expression by mediating efficient integration of genecarrying transposons Using a hydrodynamic injection technique to deliver DNA to the livers of mice, several groups have shown that the Sleeping Beauty system can be used to achieve long-term in vivo expression However, in the experiments it is often difficult to distinguish expression from episomal, integrated, or transposed elements To better understand SB’s efficiency at mediating in vivo transposition, we have designed a system to silence expression from non-transposed elements and thus reduce or remove expression from episomal or randomly integrated plasmids A transposon plasmid pT2F/Cage (a transposon carrying a murine erythropoietin (epo) gene transcriptionally regulated by a CMV enhancer / b-actin promoter combination) was engineered to contain loxP sites positioned to interrupt expression upon Cre-mediated recombination Upon transposition these sites become segregated, thus protecting the expression construct from Cre-mediated recombination and subsequent silencing Mx1Cre inducible mice were injected with pT2F/Cage with or without transposase-encoding plasmid At to weeks post-injection, in the absence of transposase, Cre induction reduced the expression of epo to about 1% of that seen in the group that included the transposase plasmid, which maintained epo levels near that of the uninduced groups These results indicate a substantial level of DNA-mediated expression not associated with transposition, but which can be quantitatively distinguished from transposition by its sensitivity to Cre recombinase They also provide additional evidence for the effectiveness of the Sleeping Beauty transposon system as an in vivo DNA-mediated gene transfer strategy for achieving long-term expression We anticipate that this adaptation of the LoxP-Cre recombinase system will be extremely useful for the study of in vivo transposition as a gene therapy strategy, whether mediated by Sleeping Beauty or by other transposon systems P.B Hackett, D.A Largaespada, and R.S McIvor have a financial interest in Discovery Genomics Inc 22 Development and Analyses of Hyperactive Forms of the Sleeping Beauty Transposase Stephen R Yant,1 Jacob G Mikkelsen,1 Jason Hoyt,1 Hui Xu,1 Mark A Kay.1 Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA, United States Previously, we have shown that nonviral and adenovirus-based vectors encoding the Sleeping Beauty (SB) transposase/transposon system support the stable integration of therapeutic genes in as many as 6% of mouse hepatocytes in vivo Genomic integration occurs via a highly ordered process called DNA transposition and is triggered by the physical association of transposase monomers that are bound to transposon end sequences In this report, we have attempted to improve the overall efficiency of SB-mediated transposition in mammalian cells by altering the affinity of transposase subunits to itself and/or to the protein binding sites Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy located within the transposon terminal repeats Through site-directed mutagenesis, we have produced a total of ninety-six different transposase mutants, each containing a single amino acid substitution within the N-terminal DNA binding and subunit multimerization domains of the SB transposase gene These mutants were cloned into plasmids to permit high-level mammalian expression and then tested for their ability to support transposition of a neomycinmarked transposon following co-transfection and G418-resistant growth selection in HeLa cells Results indicate that twenty-one of these transposase mutants were ~1.5-to-4-fold more active than wild-type transposase, whereas nineteen other mutants were completely defective for transposition These results suggest that many of these residues play an important role in Sleeping Beauty’s biological activity Currently, we are using a yeast-based reporter system to investigate the relative DNA binding affinities of these mutants in an attempt to achieve a better understanding of the molecular mechanism(s) of hyperactivity In order to test whether the combination of individual hyperactive mutations could be used to further improve the level of transposition in host cells, we have analyzed a small subclass of transposase ‘double’ mutants using our HeLa cell transposition assay Interestingly, some combinations of these hyperactive mutations had an additive effect on transposase activity while other combinations produced transposase proteins that were less active relative to each of the single mutants These results suggest that efficient SB-mediated transposition may require a delicate balance between assembly of a stable transposase/DNA complex and rapid dissociation of this complex during re-insertion of excised elements Although additional changes to the transposase gene are ongoing, we have already obtained two SB mutants which are 5-to-8 times more active than the wild-type transposase In addition, we have recently generated a modified transposon vector that exhibits a two-fold improvement in transposition in HeLa cells compared to the wild-type vector We are presently combining all of these improvements and are studying the activity of this new hyperactive system in vivo following hydrodynamic-based delivery to mouse liver In summary, our research demonstrates the potential for transposase evolution and suggests that dramatic improvements in the integration frequencies of transposon-based vectors will be readily obtainable These improvements should greatly extend the utility of this integrating vector system for both basic research and human gene therapy applications 23 Cleavage/Excision of Plasmid DNA In Vivo Leads to Increased Maintenance and Persistence of Transgenes Expression in Mouse Efren Riu,1 Zan Huang,1 Mark A Kay.1 Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA Persistence of transgene expression is a major limitation for non viral mediated gene therapy approaches We have recently established that bacterial DNA sequences can variably affect transcription of transgenes in vivo To establish if covalent linkage of the bacterial DNA to the transgene was critical for transcriptional repression, we analyzed whether cleavage/excision of bacterial plasmid DNA sequences in vivo influenced the long-term transgene expression in mouse liver To this, the human alpha-1-antitrypsin (hAAT) and human clotting factor IX (hFIX) reporter genes were flanked by two I-SCeI meganuclease recognition sites, and co-injected together with a plasmid encoding the I-SCeI cDNA or a control plasmid DNA into mouse liver Two weeks after DNA administration, mice injected with the reporter gene alone or with the irrelevant control plasmid showed very low serum levels of hAAT and hFIX, which remained low throughout the length of the experiment However, animals that expressed I-SCeI had a 5-10 fold increase in serum hAAT and hFIX that persisted for at least months (length of S9 NAKED DNA: METHODS study) In addition, expression of I-SCeI resulted in cleavage and excision of hAAT and hFIX expression cassettes from plasmid backbone, as established by Southern blot analyses This suggested that covalent linkage of bacterial DNA markedly diminished longterm transgene expression in vivo Therefore, to increase the persistence of transgenes in vivo and, thus, optimize non viral gene therapy approaches, the excision of the transgenes from plasmid backbone and the removal of bacterial DNA should be considered 24 New Insight into Cellular Uptake of Nucleic Acids: A Role for the Nucleic AcidConducting Channel Edgar Leal-Pinto,1 Avelino Teixeira,1 Scott C Henderson,2 Mary E Hawkins,3 Paul E Klotman,1 Basil Hanss.1 Nephrology/Medicine, Mt Sinai School of Medicine, New York, NY, United States; 2Molecular, Cell, and Developmental Biology, Mt Sinai School of Medicine, New York, NY, United States; Pediatric Branch, National Cancer Institute, NIH, Bethesda, MD, United States Much of the published literature suggests that receptor-mediated endocytosis is the primary mechanism of cellular uptake of oligodeoxynucleic acids (ODN) In many of these studies ODNs are labeled with a large, hydrophobic fluorophore such as FAM (a derivative of fluorescein) We have hypothesized that the presence of a large hydrophobic molecule on the ODN would change the kinetics, if not the mechanism, of uptake To test this hypothesis we developed the methodology to examine ODN uptake in cultured cells using a new structural analog of 2-deoxyguanosine, 3-Methyl8-(2-deoxy-β-D-ribofuranosyl) isoxanthopterin (3MI) 3MI is highly fluorescent and is very closely related in structure to guanosine As such, it does not introduce significant structural or steric changes to an ODN For these studies, ODN was labeled with 3MI or FAM (FAM-ODN) and comparisons of uptake kinetics in LLC-PK1 cells (a renal epithelial cell line derived from pig kidney) were made using multi-photon laser scanning microscopy The rate of 3MI-ODN uptake is significantly (p