Transposon-modified antigen-specific T lymphocytes for sustained therapeutic protein delivery in vivo
Matthew H. Wilson
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. Vaccination stimulates long-term T-cell engraftment, persistence, and transgene expression enabling detection of modified cells up to 300 days after adoptive transfer. We demonstrate adoptive transfer of antigen-specific T cells expressing erythropoietin (EPO) elevating the hematocrit in mice for more than 20 weeks. We extend our observations to human T cells demonstrating inducible EPO production from Epstein–Barr virus (EBV) antigen-specific T lymphocytes. Our results reveal antigen-specific T lymphocytes to be an effective delivery platform for therapeutic molecules such as EPO in vivo, with important implications for other diseases that require peptide therapy.We engineered a series of piggyBac vectors for genetic modification of T cells to enable tracking of lymphocytes, quantitation of their persistence in vivo, and to express both murine and human EPO (Fig. 1). We first genome-modified murine CD8+ lymphocytes with the pT-effluc-thy1.1 transposon, confirmed luciferase expression from transferred cells by bioluminescent imaging, and observed thy1.1 expression by flow cytometry. We routinely observed that ~35% of the cells were transgene positive after 24 h of in culture (Fig. 2a).The plasmid vectors pT-effluc-thy1.131, pCMV-m7pB32, and pCMV-SB100X32 were described previously. The pT-EF1α-mEPO was synthesized by Cyagen Biosciences (Santa Clara, CA). The pTSB-CAG-OVA vector to encode a kozak consensus sequence followed by the nine amino-acid peptide sequence corresponding to chicken ovalbumin 257–264 and a stop codon (5′-GATCGCCACCATGAGTATAATCAACTTTGAAAAACTGTAACCGG-3′) by replacing the piggyBac terminal repeats (TRs) in pT-CAGLuc33 with the Sleeping Beauty TRs and swapping out the luciferase cDNA for the OVA peptide. The vector pT-Tight-hEPO was generated by blunt cloning the Tet-ON element from pT-Tet-ON (Clontech, Mountain View, CA) into the multiple cloning site (MCS) of the zeo-pT-MCS vector31 to make zeo-pT-MCS-Tet-ON. The TRE-hEPO element was cloned into the XhoI site of zeo-pT-MCS-Tet-ON generating TRE-hEPO-MCS-Tet-ON. We then synthesized the 250 bp core insulator sequence from the chicken B-globin 5′HS4 element with flanking AscI and AgeI sites (Genescript, NJ). This was then cloned into the MCS of the zeo-pT-TRE-hEPO-MCS-Tet-ON vector to generate pT-Tight-hEPO. All plasmid vectors were confirmed by DNA sequencing.This work was supported in part by National Institutes of Health grant DK093660 to M.H.W., Department of Veterans Affairs grant BX002190 to M.H.W., and the Vanderbilt O’Brien Kidney Center (NIH DK114809) and the Vanderbilt Institute for Clinical and Translational Research (NIH UL1TR002243). S.S. was supported in part by the HHMI Med into Grad Training Grant through the Baylor College of Medicine Translational Biology and Molecular Medicine Program. R.T.O. was supported by DK007569. Some core services were performed through Vanderbilt University Medical Center’s Digestive Disease Research Center supported by NIH grant P30DK058404.The authors declare no competing interests.Electronic supplementary materialSupplementary Information accompanies this paper at 10.1038/s41467-018-03787-8.Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.