Sleeping beauty system to redirect T-cell specificity for human applications

J Immunother. 2013 Feb;36(2):112-23. doi: 10.1097/CJI.0b013e3182811ce9.


The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electrotransfer of CD19-specific SB DNA plasmids in peripheral blood mononuclear cells and propagation on CD19 artificial antigen presenting cells was used to numerically expand CD3 T cells expressing CAR. By day 28 of coculture, >90% of expanded CD3 T cells expressed CAR. CAR T cells specifically killed CD19 target cells and consisted of subsets expressing biomarkers consistent with central memory, effector memory, and effector phenotypes. CAR T cells contracted numerically in the absence of the CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCR Vα and TCR Vβ repertoire. Quantitative fluorescence in situ hybridization revealed that CAR T cells preserved the telomere length. Quantitative polymerase chain reaction and fluorescence in situ hybridization showed CAR transposon integrated on average once per T-cell genome. CAR T cells in peripheral blood can be detected by quantitative polymerase chain reaction at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the nonviral SB system for the investigational treatment of CD19 B-cell malignancies (currently under 3 INDs: 14193, 14577, and 14739).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antigens, CD19 / genetics
  • Antigens, CD19 / immunology*
  • Antigens, CD19 / metabolism
  • CD3 Complex / metabolism
  • Cell Line, Tumor
  • Cells, Cultured
  • Electroporation
  • Gene Transfer Techniques
  • Glioblastoma / immunology
  • Glioblastoma / therapy
  • Humans
  • Immunotherapy, Adoptive*
  • Mice
  • Receptors, Antigen, T-Cell / immunology
  • Recombinant Fusion Proteins / biosynthesis
  • T-Lymphocytes / transplantation*
  • Transposases / genetics*


  • Antigens, CD19
  • CD3 Complex
  • Receptors, Antigen, T-Cell
  • Recombinant Fusion Proteins
  • Transposases
  • sleeping beauty transposase, human