Transposon-modified antigen-specific T lymphocytes for sustained therapeutic protein delivery in vivo.

A cell therapy platform permitting long-term delivery of peptide hormones in vivo would be a significant advance for patients with hormonal deficiencies. Here we report the utility of antigen-specific T lymphocytes as a regulatable peptide delivery platform for in vivo therapy. piggyBac transposon modification of murine cells with luciferase allows us to visualize T cells after adoptive transfer.

Regulated Expansion and Survival of Chimeric Antigen Receptor-Modified T Cells Using Small Molecule-Dependent Inducible MyD88/CD40.

Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4 and CD8 T cells following pathogen challenge or in graft-versus-host disease (GvHD) settings, suggesting that these costimulatory pathways may be co-opted to improve CAR-T cell persistence and function. Here, we present a novel strategy to activate TLR and CD40 signaling in human T cells using inducible MyD88/CD40 (iMC), which can be triggered in vivo via the synthetic dimerizing ligand, rimiducid, to provide potent costimulation to CAR-modified T cells.

Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells.

Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns.

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

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.

piggyBac transposon system modification of primary human T cells.

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.