title: β-globin matrix attachment region improves stable genomic expression of the Sleeping Beauty transposon.
abstract: The liver is an attractive target for gene therapy due to its extensive capability for protein production and the numerous diseases resulting from a loss of gene function it normally provides. The Sleeping Beauty Transposon (SB-Tn)(1) system is a non-viral vector capable of delivering and mediating therapeutic transgene(s) insertion into the host genome for long-term expression. A current challenge for this system is the low efficiency of integration of the transgene. In this study we use a human hepatoma cell line (HuH-7) and primary human blood outgrowth endothelial cells (BOECs) to test vectors containing DNA elements to enhance transposition without integrating themselves. We employed the human β-globin matrix attachment region (MAR) and the Simian virus 40 (SV40) nuclear translocation signal to increase the percent of HuH-7 cells persistently expressing a GFP::Zeo reporter construct by ∼50% for each element; while combining both did not show an additive effect. Interestingly, both elements together displayed an additive effect on the number of insertion sites, and in BOECs the SV40 alone appeared to have an inhibitory effect on transposition. In long-term cultures the loss of plasmid DNA, transposase expression and mapping of insertion sites demonstrated bona fide transposition without episomal expression. These results show that addition of the β-globin MAR and potentially other elements to the backbone of SB-Tn system can enhance transposition and expression of therapeutic transgenes. These findings may have a significant influence on the use of SB transgene delivery to liver for the treatment of a wide variety of disorders.
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, 55455.
authors.forename: Lucas
authors.initials: L
authors.lastname: Sjeklocha
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, 55455.
authors.forename: Yixin
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, 55455.
authors.forename: Meghan C
authors.initials: MC
authors.lastname: Daly
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, 55455.|Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota, 55455.
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Steer
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, 55455.
authors.forename: Betsy T
authors.initials: BT
authors.lastname: Kren
mesh_terms: D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D042783:Endothelial Cells / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D024243:HMGB1 Protein / Q000378:metabolism; D006801:Humans; D045170:Matrix Attachment Regions; D010957:Plasmids; D061251:Primary Cell Culture; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D019076:Transgenes; D019895:Transposases / Q000235:genetics / Q000378:metabolism; D055544:beta-Globins / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements; D024243:HMGB1 Protein; D011994:Recombinant Proteins; D055544:beta-Globins; D049452:Green Fluorescent Proteins; D019895:Transposases; C494471:sleeping beauty transposase, human