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Evaluation of long-term transgene expression in piggyBac-modified human T lymphocytes.

ABSTRACT: The piggyBac transposon system is a promising nonviral method to genetically modify T cells for immunotherapeutic applications. To evaluate the regulation and stability of transgene expression in human T cells modified with piggyBac-transposons, peripheral blood mononuclear cells were nucleofected with transposase and an enhanced green fluorescence protein (eGFP)-expressing transposon. Single-cell clones that were subsequently stimulated and expanded exhibited homogenous eGFP expression for >26 weeks in culture. CD3 stimulation of the T-cell receptor together with CD28-mediated costimulation resulted in an approximate 10-fold transient increase in eGFP expression, but immunomodulatory cytokines, including interferon-?, interleukin-12, interleukin-4, and transforming growth factor-?, did not alter transgene expression in actively dividing, activated, or resting T cells. Epigenetic modification with 5-azacytidine or trichostatin-A increased transgene expression indicating that piggyBac-mediated transgene expression could be modulated by methylation or histone acetylation, respectively. We performed transposon copy number analysis of populations of stably transfected T cells, comparing transposon plasmids of 5.6 and 3.5 kb. The smaller vector achieved an average of 22 transposon copies per cell, whereas the larger vector achieved 1.6 copies/cell, implying that transposon copy number can be engineered to be low or high depending on the vector used. Our results provide important insight into the ability of piggyBac to achieve stable genetic modification of T cells for immunotherapy applications and how transgene expression might be regulated by TCR activation, cytokines, and epigenetic mechanisms.

SUBMITTER: Nakazawa Y

PROVIDER: S-EPMC3521868 | biostudies-other | 2013 Jan

REPOSITORIES: biostudies-other

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Evaluation of long-term transgene expression in piggyBac-modified human T lymphocytes.

Nakazawa Yozo Y Saha Sunandan S Galvan Daniel L DL Huye Leslie L Rollins Lisa L Rooney Cliona M CM Wilson Matthew H MH

Journal of immunotherapy (Hagerstown, Md. : 1997) 20130101 1

The piggyBac transposon system is a promising nonviral method to genetically modify T cells for immunotherapeutic applications. To evaluate the regulation and stability of transgene expression in human T cells modified with piggyBac-transposons, peripheral blood mononuclear cells were nucleofected with transposase and an enhanced green fluorescence protein (eGFP)-expressing transposon. Single-cell clones that were subsequently stimulated and expanded exhibited homogenous eGFP expression for >26 ...[more]

PMID: 23211626

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Project description:A major obstacle in the treatment of human immunodeficiency virus type 1 (HIV-1) is a sub-population of latently infected CD4(+) T lymphocytes. The cellular and viral mechanisms regulating HIV-1 latency are not completely understood, and a promising technique for probing the regulation of HIV-1 latency is single-cell time-lapse microscopy. Unfortunately, CD4(+) T lymphocytes rapidly migrate on substrates and spontaneously detach, making them exceedingly difficult to track, hampering single-cell level studies. To overcome these problems, we built microdevices with a three-level architecture. The devices contain arrays of finger-like microchannels to "corral" T-lymphocyte migration, round wells that are accessible to pipetting, and microwells connecting the microchannels with the round wells. T lymphocytes that are loaded into a well first settle into the microwells and then to microchannels by gravity. Within the microchannels, T lymphocytes are in favorable culture conditions because they are in physical contact with each other, under no mechanical stress, and fed from a large reservoir of fresh medium. Most importantly, T lymphocytes in the microchannels are not exposed to any flow and their random migration is restricted to a nearly one-dimensional region, greatly facilitating long-term tracking of multiple cells in time-lapse microscopy. The devices have up to nine separate round wells, making it possible to test up to nine different cell lines or medium conditions in a single experiment. Activated primary CD4(+) T lymphocytes, resting primary CD4(+) T lymphocytes, and THP-1 monocytic leukemia cells loaded into the devices maintained viability over multiple days. The devices were used to track the fluorescence level of individual primary CD4(+) T lymphocytes expressing green fluorescent protein (GFP) for up to 60 hours (h) and to quantify single-cell gene-expression kinetics of four different HIV-1 variants. The kinetics of GFP expression from the lentiviruses in the primary CD4(+) T lymphocytes agree with previous measurements of these lentiviral vectors in the immortalized Jurkat T lymphocyte cell line. The proposed devices offer a simple, robust approach to long-term single-cell studies of environmentally sensitive primary lymphocytes.

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Evaluation of long-term transgene expression in piggyBac-modified human T lymphocytes.

Publications

Evaluation of long-term transgene expression in piggyBac-modified human T lymphocytes.

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