In this study, we compared the genomic integration efficiencies and transposition site preferences of Sleeping Beauty (SB or SB11), Tol2, and piggyBac (PB) transposon systems in primary T cells derived from peripheral blood lymphocytes (PBL) and umbilical cord blood (UCB). We found that PB demonstrated the highest efficiency of stable gene transfer in PBL-derived T cells, whereas SB11 and Tol2 mediated intermediate and lowest efficiencies, respectively. Southern hybridization analysis demonstrated that PB generated the highest number of integrants when compared to SB and Tol2 in both PBL and UCB T cells. Tol2 and PB appeared more likely to promote clonal expansion than SB, which may be in part due to the dysregulated expression of cancer-related genes near the insertion sites. Genome-wide integration analysis demonstrated that SB, Tol2, and PB integrations occurred in all the chromosomes without preference. Additionally, Tol2 and PB integration sites were mainly localized near transcriptional start sites (TSSs), CpG islands and DNaseI hypersensitive sites, whereas SB integrations were randomly distributed. These results suggest that SB may be a preferential choice of the delivery vector in T cells due to its random integration site preference and relatively high efficiency, and support continuing development of SB-mediated T-cell phase I trials.
Bibliographical noteFunding Information:
We thank Sonja Nodland (University of Minnesota Masonic Cancer Center) for major editing, R. Scott McIvor, David Largaespada, and Perry Hackett (University of Minnesota Center for Genome Engineering, Minneapolis, MN) for critical review of the manuscript. We also thank John E. Wagner for providing cord blood, Malcolm J. Fraser (University of Notre Dame, Notre Dame, IN) for providing piggyBac vectors, Andrew C. Wilber (Southern Illinois University, Springfield, IL) for providing Sleeping Beauty transposon recoverable vector, Kevin A.T. Silverstein (Bioinformatics Core Facility at Masonic Cancer Center, University of Minnesota) for the independent evaluation on the data, and Linan Ma (Biostatistics and Informatics Core at the Masonic Cancer Center, University of Minnesota) for initial statistical analysis. This work was supported by grants from the Children's Cancer Research Fund in Minneapolis, Alliance for Cancer Gene Therapy, the Gabrielle's Angel (formerly G&P) Foundation for Cancer Research, the Sidney Kimmel Foundation for Cancer Research Kimmel Scholar Program, the University of Minnesota Translational Research Grant, the University Minnesota Medical School Dean's Commitment (X.Z.), and Leukemia Research Fund. E.M. was a recipient of American Society of Hematology Research Trainee Award. P.B. was a recipient of the Undergraduate Research Opportunity Program Award and the Multicultural Summer Research Opportunities Award. This project was funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E (X.W.). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. Contribution statement: X.H. designed and performed the research, analyzed the data, and wrote the paper; H.G., S.T., E.M., and P.B. performed the research. Y.-C.J. performed DNA sequencing and analyzed the data; Q.C. performed the statistical analyses; Z.J.T. and Y.C.K. performed integration site analysis; S.C.E. provided mini Tol2 construct and discussed the research; X.W. preformed integration bioinformatic analyses and wrote the paper; S.M.W. analyzed the data and wrote the paper; X.Z. oversaw the research and wrote the paper. The authors declare no competitive financial interests.