A new approach to gene therapy using Sleeping Beauty to genetically modify clinical-grade T cells to target CD19

Singh H Huls H Kebriaei P Cooper LJ
Source: Immunol Rev
Publication Date: (2014)
Issue: 257(1): 181-190
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b

Quiescent T cells (typically 1-2 × 107 mononuclear cells per cuvette) from PB and UCB are electroporated in the presence of DNA plasmids coding for second-generation CD19-specific CAR [designated CD19RCD28 (34), that co-activates T cells via chimeric CD3-? and CD28] and SB11 transposase (transposon to transposase ratio of 3:1, 15:5 µg per cuvette) using the human T-cell kit and Nucleofector II electroporation device. Excellent Nucleofector reference with good graphs and figures


The advent of efficient approaches to the genetic modification of T cells has provided investigators with clinically appealing methods to improve the potency of tumor-specific clinical grade T cells. For example, gene therapy has been successfully used to enforce expression of chimeric antigen receptors (CARs) that provide T cells with ability to directly recognize tumor-associated antigens without the need for presentation by human leukocyte antigen. Gene transfer of CARs can be undertaken using viral-based and non-viral approaches. We have advanced DNA vectors derived from the Sleeping Beauty (SB) system to avoid the expense and manufacturing difficulty associated with transducing T cells with recombinant viral vectors. After electroporation, the transposon/transposase improves the efficiency of integration of plasmids used to express CAR and other transgenes in T cells. The SB system combined with artificial antigen-presenting cells (aAPC) can selectively propagate and thus retrieve CAR(+) T cells suitable for human application. This review describes the translation of the SB system and aAPC for use in clinical trials and highlights how a nimble and cost-effective approach to developing genetically modified T cells can be used to implement clinical trials infusing next-generation T cells with improved therapeutic potential.