Unexpectedly High Copy Number of Random Integration but Low Frequency of Persistent Expression of the Sleeping Beauty Transposase After Trans Delivery in Primary Human T Cells

In this study, the authors demonstrated that the Sleeping Beauty (SB) transposon system can mediate stable expression of both reporter and therapeutic genes in human primary T cells and that trans delivery is at least 3-fold more efficient than cis delivery. One concern about trans delivery is the potential for integration of the transposase-encoding sequence into the cell genome with the possibility of continued expression, transposon remobilisation, and insertional mutagenesis. To address this concern, human peripheral blood lymphocytes were nucleofected with transposase plasmid and a DsRed transposon. Eighty-eight stable DsRed+ T cell clones were generated and found to be negative for the transposase-encoding sequence by PCR analysis of genomic DNA. Genomic PCR was possible for transposase in 5 of 15 bulk T cell populations that were similarly transfected and selected for transgene expression where copy numbers were unexpectedly high (0.007-0.047 per cell) by quantitative PCR. Transposase-positive bulk T cells lacked transposase plasmid demonstrated by Hirt (episomal) extracted DNA and showed no detectable transposase by Southern hybridisation, Western blot, and quantitative RT-PCR analyses. Cytogenetic and array comparative genomic hybridisation analyses of the only identified transposase-pos. clone showed no chromosomal abnormality or tumour formation in nude mice although transposon remobilisation was detected. The data suggests that SB delivery via plasmid in T cells should be carried out with caution because of unexpectedly high copy numbers of randomly integrated SB transposase. The Sleeping Beauty (SB) transposon system is a non-viral method that can be used for integration and persistent transgene expression in a variety of somatic cell types. Here, Huang et al. report on the safety of these SB vectors; specifically, they examine the frequency of random integration by SB in human primary T cells. They also look at whether an SB+ T-cell clone triggers numeric or structural chromosome alterations and tumour growth in nude mice.

Authors: Huang, Xin; Haley, Kari; Wong, Marianna; Guo, Hongfeng; Lu, Changming; Wilber, Andrew; Zhou, Xianzheng. ;Full Source: Human Gene Therapy 2010, 21(11), 1577-1590 (USA). ;