piggyBac Transposon System Modification of Primary Human T Cells

Summary

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.

Abstract

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth^1,2. 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 insect^1,2, mammalian^3-5, and human cells6 including primary human T cells^7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency^9,10.

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.

Protocol

Day 0

1. Isolation of PBMCs from Human Blood

1. Collect 20 ml of fresh human blood using venipuncture into Na-heparin vacutainer tubes.
2. Mix blood and Advanced RPMI 1,640 in 1:1 (v/v) ratio.
3. Add 20 ml lymphoprep medium to a 50 ml centrifuge tube (25 °C). Slowly layer 25-30 ml of blood-RPMI 1,640 mix on top of the lymphoprep.
4. Centrifuge at 400 x g for 40 min without brakes.
5. Collect both distinct and fuzzy layers using a disposable pipette into 10 ml of 1x PBS (25 °C) and bring the volume up to 50 ml with 1x PBS.
6. Centrifuge at 450 x g for 10 min.
7. Aspirate the supernatant....

Results

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.......

Discussion

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 medications^7,13-15. Non-viral modification of T cells for adoptive immunotherapy and other applications shoul.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lympholyte	Cedarlane	CL5015
Advanced RPMI 1,640	LifeTechnologies	12633020
Hyclone Fetal Bovine Serum	Fisher Scientific	SH3008803
GlutaMAX-I Supplement	LifeTechnologies	35050-061
Human IL-15 Recombinant Protein	eBioscience	14-8159
EndoFree Plasmid Maxi Kit	Qiagen	12362
Amaxa Nucleofector	Lonza	AAD-1001S
Human T Cell Nucleofector Kit	Lonza	VPA-1002
CD8-APC	Southern Biotech	9536-11
Anti-Human CD3	eBioscience	16-0037-81
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