The piggyBac transposon system is naturally active, originally derived from the 
cabbage looper moth. 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 
insect, mammalian, and human cells6 including primary human T cells. Recently, a 
hyperactive piggyBac transposase was generated improving gene transfer 
efficiency. Human T lymphocytes are of clinical interest for adoptive 
immunotherapy of cancer. Of note, the first clinical trial involving transposon 
modification of human T cells using the Sleeping beauty transposon system has 
been approved. 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 gene. Analysis of genomic 
integration sites revealed a lack of preference for integration into or near 
known proto-oncogenes. 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 model. We have also used 
piggyBac to generate human T cells resistant to rapamycin, which should be 
useful in cancer therapies where rapamycin is utilized. 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 antigens, we have also used 
piggyBac to add an inducible safety switch in order to eliminate gene modified 
cells if needed. The large cargo capacity of piggyBac has also enabled gene 
transfer of a large rapamycin resistant mTOR molecule (15 kb). Therefore, we 
present a non-viral methodology for stable gene-modification of primary human T 
cells for a wide variety of purposes.