Comparison of Lentiviral and Sleeping Beauty Mediated alpha beta T Cell Receptor Gene Transfer

Anne-Christine Field, Conrad Vink, Richard Gabriel, Roua Al-Subki, Manfred Schmidt, Nicholas Goulden, Hans Stauss, Adrian Thrasher, Emma Morris, Waseem Qasim
Source: PLoS ONE
Publication Date: (2013)
Issue: 8(6): e68201
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
T cell, mouse - C57BL/6
Species: mouse
Tissue Origin: blood
Culture Media:
Nucleofector® I/II/2b

Human T cells: PBMCs were activated using magnetic beads conjugated to antibodies to CD3 and CD28 (Invitrogen, Stockholm, Sweden), at a ratio of 1:1 for 48 hours in X-VIVO 10 medium supplemented with human AB serum and 100 U/ml IL2 [22]. The cells were Lentiviral infection at MOI of 20 Nucleofection: DNA supercoiled plasmids (5 mg each) encoding the SB transposon and the SB transposase, using an Amaxa Nucleofector II device and Human T Cell Nucleofector Kit and protocol for Stimulated Human T cells (Lonza). After 48– 72 hours after expression of TCR transgenes was analysed by flow cytometry. Mouse T cells: Substrate: SB transposon and the SB transposase DNA plasmids Kit: Mouse T Cell Kit Program: according to OP Not possible to transefct via lentivirus Summary (Lonza): Transfer of recombinant T cell receptors, e.g. chimeric antigen receptors (CARs) into T cells is used to redirect T cell immunity against certain tumor or viral antigens. In this publication, the authors have compared lentiviral transduction versus Nucleofection™ of an enhanced Sleeping Beauty (SB) transposition system, to deliver a T cell receptor against Wilms’ Tumor antigen 1, a tumor antigen overexpressed by malignant hematological and certain non-hematological tumors. They could show that SB-mediated transfer of antigen-specific T cell receptor into T cells using Nucleofection™ achieves results comparable to lentivirus while providing certain advantages: As a non-viral method it offers a more flexible and less costly pathway for rapid evaluation of different receptor configurations and may allow for exploration of therapeutic applications in early phase clinical studies.


Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and ab T cell receptors, the current platforms are costly, time consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm’s tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies.