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Efficient non-viral gene delivery into human hematopoietic stem cells by minicircle Sleeping Beauty transposon vectors

Authors

  • M. Holstein
  • C. Mesa-Nuñez
  • C. Miskey
  • E. Almarza
  • V. Poletti
  • M. Schmeer
  • E. Grueso
  • J.C. Ordóñez Flores
  • D. Kobelt
  • W. Walther
  • M.K. Aneja
  • J, Geiger
  • H.B. Bonig
  • Z. Izsvák
  • M. Schleef
  • C. Rudolph
  • F. Mavilio
  • J.A. Bueren
  • G. Guenechea
  • Z. Ivics

Journal

  • Molecular Therapy

Citation

  • Mol Ther 26 (4): 1137-1153

Abstract

  • The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4-8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.


DOI

doi:10.1016/j.ymthe.2018.01.012