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. 2013 Jun 28;8(6):e68201.
doi: 10.1371/journal.pone.0068201. Print 2013.

Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer

Anne-Christine Field  1 , Conrad Vink, Richard Gabriel, Roua Al-Subki, Manfred Schmidt, Nicholas Goulden, Hans Stauss, Adrian Thrasher, Emma Morris, Waseem Qasim
Affiliations

Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer

Anne-Christine Field et al. PLoS One. .

Abstract

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 αβ 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. WT1-TCR vector constructs.
A. Schematic representation of the pSIN second-generation lentiviral (Lv) vector encoding the codon-optimized, murinized hybrid HLA-A0201–restricted pWT126-specific additional cysteines modified TCR α- and β-chain genes separated by a self-cleaving porcine teschovirus 2A sequence (P2A). LTR indicates long terminal repeat; m, murine SFFV, spleen-forming focus virus; and WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. B. Schematic representation of the plasmid Sleeping Beauty (SB) vector consisting of a transgene expression cassette, encoding the codon-optimized, murinized hybrid HLA-A0201–restricted pWT126-specific additional cysteines modified TCR α- and β-chain genes separated by a self-cleaving porcine teschovirus 2A sequence (P2A), flanked by inverted repeats; and the hyperactive SB transposase SB100X. CMV indicates cytomegalovirus promoter; IL, left inverted repeat; IR, right inverted repeat; m, murine; SFFV, spleen-forming focus virus.
Figure 2
Figure 2. WT1-TCR expression and function in human primary T cells.
A. WT1-TCR cell surface expression. Percentages of Vβ2.1 positive cells in CD8+ T cells for each HLA-A2+ donor are shown after WT1-TCR lentiviral (Lv) or Sleeping Beauty (SB) vector gene transfers. CD3/CD28 microbeads-activated PBMCs were transduced at a multiplicity of infection of 20 or nucleofected with 5 µgs transposon and transposase plasmids. *p<0.05. B. Antigen-specific effector function. Mean percentages of IFNγ positive cells in CD8+ T cells from HLA-A2+ donors assayed by MACS Cytokine Secretion Assay of human primary CD8+ T cells, after WT1-TCR lentiviral (Lv) or Sleeping Beauty (SB) vector gene transfer, restimulated with either the specific WT1 peptide, anti-CD3 Ab (positive control) or irrelevant peptide (negative control), and autologous PBMCs (top). Representative dot plots of IFNγ secretion are also shown after WT1-TCR Lv (bottom left) or SB (bottom right) gene transfer.
Figure 3
Figure 3. Antigen-specific responses of human primary T cells.
A. Murine Cβ/human Vβ2.1 expression and WT1 tetramer binding are shown in representative human primary lymphocyte dot plots, after SFFV-driven WT1-TCR lentiviral vector (Lv) or Sleeping Beauty (SB) gene transfer and in unmodified controls. CD3/CD28 microbeads-activated PBMCs were transduced at a multiplicity of infection of 20 or nucleofected with 5 µgs transposon and transposase plasmids and expanded with T2 cells coated with specific peptide pWT126 and irradiated autologous PBMCs as feeder cells. B. In vitro cytotoxicity of donor T cells against specific peptide-loaded T2 target cells, Lv versus SB. Percentages of cytolytic activity of T cells, after WT1-TCR lentiviral (Lv) or Sleeping Beauty (SB) vector gene transfers, was tested in a 51Cr release CTL assay. CD3/CD28 microbeads-activated PBMCs were transduced at a multiplicity of infection of 20 or nucleofected with 5 µgs transposon and transposase plasmids and expanded with the specific WT1 peptide and autologous irradiated PBMCs. Both WT1-TCR-modified donor T cells recognized and killed their specific targets (2 way ANOVA, p<0.001 at all E:T ratios compared to GFP-modified or unmodified cells).
Figure 4
Figure 4. In vivo cytotoxicity of murine primary T cells.
In vivo cytotoxicity of modified mouse splenocytes against CFSE-labelled peptide-loaded mouse target B cells. A2Kb Tg mice, 2 days after intravenous injection of syngeneic effector cells modified with Sleeping Beauty (SB) WT1-TCR vector system, were again intravenously injected with a 1∶1 mix of relevant: irrelevant peptide-loaded A2Kb Tg target B cells, differentially labelled with CFSE (specific WT126 peptide, 1.5 µM CFSE, and irrelevant WT235 peptide for WT1-TCR, 0.15 µM CFSE). Eighteen hours later, splenocytes of injected animals were harvested and analysed by FACS to identify CFSE-labelled cells. Control A2Kb Tg mice were injected with only CFSE-labelled peptide-loaded target B cells or with unmodified splenocytes as effectors. Antigen-specific cytotoxicity was calculated as [1- (number of relevant peptide-loaded targets in experimental mice/number of irrelevant peptide-loaded targets in experimental mice)/(number of relevant peptide-loaded targets in control mice/number of irrelevant peptide-loaded targets in control mice )] ×100.
Figure 5
Figure 5. Integration profiles in human primary T cells by LM-PCR.
CD3/CD28 microbeads-activated PBMCs were transduced at a multiplicity of infection of 20 (Lv) or nucleofected with both 5 µgs transposon and transposase plasmids (SB). Both vectors encoded the identical WT1-TCR transgene. Genomic DNA was extracted and lentivirus-chromosome or transposon-chromosome junctions were recovered by ligation-mediated PCR and sequenced. A. Genome-wide mapping of vector integrations at the chromosome level. Sequences were mapped to the University of California at Santa Cruz (UCSC) human genome by BLAT search and integration sites were depicted relative to chromosomes using the UCSC Genome Graphs tool. Lv in Blue, SB in Red. B. Frequency of integration sites of vectors within RefSeq genes. 1,000 random integration sites were generated by bioinformatics, as already described . C. Proximity of integration sites to transcription start sites (TSS). RefSeq genes containing integration sites were divided by length into 4 equally sized regions and 1 upstream region (0–5 kb), and the proportion of integration sites within each region was counted. To allow statistical comparison of integration preferences with average genomic content, 1,000 random chromosomal sites were generated by multiplying the total length of the genome by a random number between 0 and 1 and converting this value to a chromosomal coordinate. Vector integration frequencies are expressed relative to the proportion of random sites within each region.
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References

    1. Jena B, Dotti G, Cooper LJ (2010) Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor. Blood 116: 1035–1044 blood-2010-01-043737 [pii];10.1182/blood-2010-01-043737 [doi] - DOI - PMC - PubMed
    1. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, et al. (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314: 126–129. - PMC - PubMed
    1. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, Le Deist F, Wulffraat N, et al. (2003) A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 348: 255–256. - PubMed
    1. Ott MG, Schmidt M, Schwarzwaelder K, Stein S, Siler U, et al. (2006) Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 12: 401–409. - PubMed
    1. Scholler J, Brady TL, Binder-Scholl G, Hwang WT, Plesa G, et al. (2012) Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci Transl Med 4: 132ra53 4/132/132ra53 [pii];10.1126/scitranslmed.3003761 [doi] - DOI - PMC - PubMed

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