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.

Day 0
1. Isolation of PBMCs from Human Blood
<ol>
	<li>Collect 20 ml of fresh human blood using venipuncture into Na-heparin vacutainer tubes.</li>
	<li>Mix blood and Advanced RPMI 1,640 in 1:1 (v/v) ratio.</li>
	<li>Add 20 ml lymphoprep medium to a 50 ml centrifuge tube (25 &deg;C). Slowly layer 25-30 ml of blood-RPMI 1,640 mix on top of the lymphoprep.</li>
	<li>Centrifuge at 400 x g for 40 min without brakes.</li>
	<li>Collect both distinct and fuzzy layers using a disposable pipette into 10...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac+transposon-based+inducible+gene+expression+in+vivo+after+somatic+cell+gene+transfer.">PiggyBac transposon-based inducible gene expression in vivo after somatic cell gene transfer.</a> Mol. Ther. 17 (12), 2115 (2009).</li><li>Nakanishi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac+transposon-mediated+long-term+gene+expression+in+mice.">piggyBac transposon-mediated long-term gene expression in mice.</a> Mol. Ther. 18 (4), 707 (2010).</li><li>Wilson, M. H., Coates, C. J., George, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac+Transposon-mediated+Gene+Transfer+in+Human+Cells.">PiggyBac Transposon-mediated Gene Transfer in Human Cells.</a> Mol. Ther. 15 (1), 139 (2007).</li><li>Nakazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+PiggyBac+transposon+system+for+the+sustained+genetic+modification+of+human+T+lymphocytes.">Optimization of the PiggyBac transposon system for the sustained genetic modification of human T lymphocytes.</a> J. Immunother. 32 (8), 826 (2009).</li><li>Raja Manuri, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac+transposon/transposase+system+to+generate+CD19-specific+T+cells+for+treatment+of+B-lineage+malignancies.">piggyBac transposon/transposase system to generate CD19-specific T cells for treatment of B-lineage malignancies.</a> Hum. Gene Ther. 21 (4), 427 (2010).</li><li>Doherty, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperactive+piggyBac+gene+transfer+in+human+cells+and+in+vivo.">Hyperactive piggyBac gene transfer in human cells and in vivo.</a> Hum. 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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 shoul...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Lympholyte</td>
 <td>Cedarlane</td>
 <td>CL5015</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Advanced RPMI 1,640</td>
 <td>LifeTechnologies</td>
 <td>12633020</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hyclone Fetal Bovine Serum</td>
 <td>Fisher Scientific</td>
 <td>SH3008803</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GlutaMAX-I Supplement</td>
 <td>LifeTechnologies</td>
 <td>35050-061</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Human IL-15 Recombinant Protein</td>
 <td>eBioscience</td>
 <td>14-8159</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EndoFree Plasmid Maxi Kit</td>
 <td>Qiagen</td>
 <td>12362</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Amaxa Nucleofector</td>
 <td>Lonza</td>
 <td>AAD-1001S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Human T Cell Nucleofector Kit</td>
 <td>Lonza</td>
 <td>VPA-1002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CD8-APC</td>
 <td>Southern Biotech</td>
 <td>9536-11</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-Human CD3</td>
 <td>eBioscience</td>
 <td>16-0037-81</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-Human CD28</td>
 <td>BD Pharmingen</td>
 <td>555725</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>24 Well Tissue Culture Treated Plate</td>
 <td>BD Falcon</td>
 <td>353047</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>24 Well Non Tissue Culture Treated Plate</td>
 <td>BD Falcon</td>
 <td>351147</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Complete T cell media composition 
 1x Advanced RPMI 1,640 
 5% Heat Inactivated Fetal Bovine Serum 
 2 mM GlutamaxIM-I</td>
 </tr>
 </tbody>
</table>

piggybac transposon system modification of primary human t cells

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.

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 cyt...

Watch this Scientific Journal Video about piggyBac Transposon System Modification of Primary Human T Cells at JoVE.com

piggyBac Transposon System Modification of Primary Human T Cells

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.

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

piggybac-transposon-system-modification-of-primary-human-t-cells

Research

JoVE Journal

Immunology and Infection

17.1K visualizações.  Baylor College of Medicine. Descreve-se um método para modificar geneticamente células T primárias humanas com um transgene, utilizando o não-viral PiggyBac Sistema de transposon. As células T modificado para utilizar o PiggyBac Transposon sistema mostra a expressão do transgene estável.