title: Stable gene transfer to human CD34(+) hematopoietic cells using the Sleeping Beauty transposon.
abstract: OBJECTIVE
  Methods of gene transfer to hematopoietic stem cells that result in stable integration may provide treatments for many inherited and acquired blood diseases. It has been demonstrated previously that a gene delivery system based on the Sleeping Beauty (SB) transposon can be derived where a plasmid transiently expressing the SB transposase can mediate the stable chromosomal integration of a codelivered second plasmid containing a gene expression unit flanked by the inverted repeats derived from the transposon.
  
  
  METHODS
  Plasmid DNA containing the elements required for SB transposition was delivered to hematopoietic cells via electroporation. Integrated transgene (enhanced green fluorescent protein [eGFP]) expression was assessed in vitro and in vivo.
  
  
  RESULTS
  In the K562 human hematopoietic cell line, we observed stable expression of eGFP in >60% of cells for over 2 months after electroporation of the two plasmids; in contrast, in control cells either not treated with transposase or exposed to a defective mutant transposase, the level of gene expression had fallen to near background (<0.1%) by 2 weeks. In purified human cord blood CD34(+) progenitor cells, the transposase led to stable gene transfer at levels up to 6% for over 4 weeks, but gene transfer to more primitive nonobese diabetic/severe combined immunodeficient repopulating cells or CD34(+)/CD38(-) in long-term culture was low and electroporation of the cells with plasmid DNA caused significant cell death.
  
  
  CONCLUSIONS
  The long-term stable expression highlights the potential of this transposase-based gene delivery method for ameliorating diseases affecting the hematopoietic system, although further improvements in gene transfer efficacy are needed.
authors.affiliation: Division of Research Immunology/Bone Marrow Transplant, The Saban Research Institute of Childrens Hospital Los Angeles, Departments of Pediatrics, Los Angeles, CA 90027, USA.
authors.forename: Roger P
authors.initials: RP
authors.lastname: Hollis
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Nightingale
authors.forename: Xiuli
authors.initials: X
authors.lastname: Wang
authors.forename: Karen A
authors.initials: KA
authors.lastname: Pepper
authors.forename: Xiao-Jin
authors.initials: XJ
authors.lastname: Yu
authors.forename: Lora
authors.initials: L
authors.lastname: Barsky
authors.forename: Gay M
authors.initials: GM
authors.lastname: Crooks
authors.forename: Donald B
authors.initials: DB
authors.lastname: Kohn
mesh_terms: D018952:Antigens, CD34; D004251:DNA Transposable Elements / Q000235:genetics; D018274:Electroporation / Q000379:methods; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D016254:Mutagenesis, Insertional / Q000379:methods; D013997:Time Factors; D019895:Transposases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D018952:Antigens, CD34; D004251:DNA Transposable Elements; D019895:Transposases