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1Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, 2Department of Medicine, Division of Nephrology, Baylor College of Medicine, 3Department of Immunology and Pathology, Shinshu University School of Medicine, 4Center for Cell and Gene Therapy, Baylor College of Medicine, 5Department of Pediatrics, Baylor College of Medicine, 6Program in Cell and Molecular Biology, Baylor College of Medicine, 7Department of Molecular Virology and Microbiology, Baylor College of Medicine, 8Michael E. DeBakey VA Medical Center
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We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.
Saha, S., Nakazawa, Y., Huye, L. E., Doherty, J. E., Galvan, D. L., Rooney, C. M., et al. piggyBac Transposon System Modification of Primary Human T Cells. J. Vis. Exp. (69), e4235, doi:10.3791/4235 (2012).
The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.
1. Isolation of PBMCs from Human Blood
2. Coating Plates with anti-CD28 and anti-CD3 Antibody for Stimulating T cells
3. Nucleofection of Unstimulated T Cells
4. Nonspecific Stimulation of T Cells
7. Analysis of Gene Expression
Day 8 (Optional)
8. Expansion of T cells
A schematic demonstrating the steps in genetically modifying human T lymphocytes with a reporter gene (eGFP) is shown in Figure 1. These plasmids are available upon request from the authors. A schemtic demonstrating the steps in genetically modified human T lymphocytes with a reporter gene (eGFP) is showin in Figure 2. It is necessary to activate T cells in order to get them to divide, expand, and propagate in culture. Modified human T cells were then cultured and analyzed using flow cytometry for gene expression on Day 1 and Day 7. Shown are results from one donor in Figure 3. Cells were stained with allophycocyanin (APC)-conjugated anti-CD8, analyzed for eGFP (the transgene) and APC fluorescence by a FACSCalibur equipped with the filter set for 4 fluorescence signals using Cell Quest software (Becton Dickinson). We have previously observed gene modification of both CD4 and CD8 positive T cells and herein demonstrate CD8 positive cells as an example7. Although we analyzed for single transgene expression herein, piggyBac has also been used for multi-gene (or multiplexed) gene transfer in human cells17. The decrease in eGFP expression between day 1 and day 7 is likely due to the fact that not all transfected cells undergo stable integration of transposon DNA.
Figure 1. Schematic of plasmids used for piggyBac mediated gene modification of human T cells. CMV, cytomegalovirus promoter; intron, SV40 intron for mRNA stabilization; piggyBac, transposase cDNA; SV40 pA, polyadenylation site; pUC, origin of replication; b-lactamase, ampicillin resistance gene; PB3'IR, piggyBac 3' inverted repeat; PB5'IR, piggyBac 5' inverted repeat; ZeoR, zeocin resistance gene; R6K Ori, origin of replication; IVS, intervening sequence; eGFP, flourescent reporter gene. Note: antibiotics were used for bacterial selection and growth but were not used in T cell cultures. Click here to view larger figure.
Figure 2. Schematic describing the modification of primary human T cells using the piggyBac transposon system.
Figure 3. Stable transgene expression in T cells modified with the piggyBac transposon system. Left panels: eGFP expression on Day 1. Right panels: eGFP expression on day 7. Click here to view larger figure.
The method described herein enables stable transgene modification of primary human T lymphocytes. We have previously tested the use of the piggyBac transposon system to modify T cells to express a reporter gene (for more than 4 weeks), a non-immunogenic suicide gene, a chimeric antigen receptor for adoptive immunotherapy (for more than 100 days), and to engineer resistance to immunosuppressive medications7,13-15. Non-viral modification of T cells for adoptive immunotherapy and other applications should be much less expensive and therefore more widely utilized than retroviral transduction. The use of new hyperactive piggyBac elements should increase the feasibility of manufacturing of stable transgene modified human T cells9. Although not described herein, one can achieve necessary numbers of stably transfected T cells for potential clinical application. Using a combination of piggyBac-mediated gene transfer and aK562 (artificial antigen presenting) feeder cells, an initial yield of about 2 x 106 stably transfected T cells can be expanded by 4 to 5 logs to over 1010 transduced T cells in 4 to 5 weeks and to 1012 in 6 to 7 weeks7. Analysis of piggyBac integration sites in human T cells showed no bias towards proto-oncogenes, however, it did show a predilection for integrating into highly expressed genes in activated T cells when using the nucleofection technology outlined above13. PiggyBac represents a promising methodology for stable genetic modification of human T cells for a wide variety of applications.
No conflicts of interest declared.
SS is supported in part by the HHMI Med into Grad Training Grant through the TBMM Program. MHW is supported in part by a career development award from the Department of Veterans Affairs and the generous support of Dr. and Mrs. Harold M. Selzman. This work was also supported in part by NIH lymphoma SPORE grant P50CA126752 and NIH R01 DK093660.
|Advanced RPMI 1,640||LifeTechnologies||12633020|
|Hyclone Fetal Bovine Serum||Fisher Scientific||SH3008803|
|Human IL-15 Recombinant Protein||eBioscience||14-8159|
|EndoFree Plasmid Maxi Kit||Qiagen||12362|
|Human T Cell Nucleofector Kit||Lonza||VPA-1002|
|Anti-Human CD28||BD Pharmingen||555725|
|24 Well Tissue Culture Treated Plate||BD Falcon||353047|
|24 Well Non Tissue Culture Treated Plate||BD Falcon||351147|
|Complete T cell media composition
1x Advanced RPMI 1,640
5% Heat Inactivated Fetal Bovine Serum
2 mM GlutamaxIM-I
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