Vaccination with a piggyBac plasmid with transgene integration potential leads to sustained antigen expression and CD8+ T cell responses

Abstract

DNA vaccination with plasmid has conventionally involved vectors designed for transient expression of antigens in injected tissues. Next generation plasmids are being developed for site-directed integration of transgenes into safe sites in host genomes and may provide an innovative approach for stable and sustained expression of antigens for vaccination. The goal of this study was to evaluate in vivo antigen expression and the generation of cell mediated immunity in mice injected with a non-integrating plasmid compared to a plasmid with integrating potential. Hyperactive piggyBac transposase-based integrating vectors (pmhyGENIE-3) contained a transgene encoding either eGFP (pmhyGENIE-3-eGFP) or luciferase (pmhyGENIE-3-GL3), and were compared to transposase-deficient plasmids with the same transgene and DNA backbone. Both non-integrating and integrating plasmids were equivalent at day 1 for protein expression at the site of injection. While protein expression from the non-integrating plasmid was lost by day 14, the pmhyGENIE-3 was found to exhibit sustained protein expression up to 28 days post-injection. Vaccination with pmhyGENIE-3-eGFP resulted in a robust CD8+ T cell response that was threefold higher than that of non-integrating plasmid vaccinations. Additionally we observed in splenocyte restimulation experiments that only the vaccination with pmhyGENIE-3-eGFP was characterized by IFNγ producing CD8+ T cells. Overall, these findings suggest that plasmids designed to direct integration of transgenes into the host genome are a promising approach for designing DNA vaccines. Robust cell mediated CD8+ T cell responses generated using integrating plasmids may provide effective, sustained protection against intracellular pathogens or tumor antigens.

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

“…To assess the response to vaccination we adoptively transferred pT-effluc-thy1.1 transposon-modified CD8+ cells, and tracked engraftment by in vivo bioluminescence imaging. Transposon vaccines have been shown to produce sustained antigen expression and improved antigen-specific T-cell responses in vivo 17 . We chose the Sleeping Beauty system for our vaccine, to avoid inducing an immune response to the piggyBac transposase, which was used for T-cell modification to enable long-term transgene expression.…”

Calpain‐2 inhibitor treatment preferentially reduces tumor progression for human colon cancer cells expressing highest levels of this enzyme

“…Cells were cultured in RPMI media with 10% fetal bovine serum and 1% antibiotic–antimycotic solution (all from Gibco/Thermo Fisher Scientific). For in vivo experiments, HCC2998, HCT116, and HCT15 stable cell lines expressing luciferase were generated using a piggyBac plasmid as described previously . The calpain‐2 inhibitor, zLLY‐CH2F, was purchased from EMD Millipore (Life Science) and used at 20 μ g/mL final concentration in cell culture experiments and 0.75 mg/kg in mice as described previously .…”

Vy-PER: eliminating false positive detection of virus integration events in next generation sequencing data

“…Two primary drivers for developing virus integration detection methods are the fields of disease therapy and disease etiology. In gene therapy and immunotherapy studies, a major consideration is the non-integration 1 or at least the safe integration of a vector’s payload into the host genome 2 3 4 . In disease etiology, prominent examples of integrations into the host genome are the retroviruses human T-lymphotropic virus (HTLV) in adult T-cell leukemia 5 and human immunodeficiency virus (HIV) in acquired immune deficiency syndrome (AIDS).…”