Oliveira, C. A., Sumner-Jones, S. G., Pringle, I. A., Hyde, S. C. & Gill, D. R.
Molecular Therapy, 20, Abstract 875Back Download
Gene therapy for chronic lung diseases will require vectors capable of persistent transgene expression. This has been achieved for several non-viral vectors by judicious promoter selection and removal of CG dinucleotides from the plasmid DNA. Plasmid pG4-hCEFI-soLux directs persistent, high-level transgene expression for at least 6 months in the mouse lung after aerosol delivery, utilising the hCEFI promoter, which is a hybrid of CpG-free versions of the human CMV enhancer (hC) and human Elongation Factor 1a (EF) promoter sequences [Hyde et al, 2008, Nat Biotech 26(5):549]. However, replacement of the 302bp human CMV enhancer with the equivalent murine sequence (mC) in plasmid pG4- mCEFI-soLux resulted in only transient expression lasting for a few days. To further study the cellular and molecular mechanisms involved, we investigated the potential use of two cell culture models to mimic the transgene silencing observed in vivo: the A549 lung cell line (ATCC) and human air-liquid interface (ALI) primary respiratory epithelial ex vivo cultures (Epithelix, Sarl, Switzerland). A549 cells were transiently transfected with both plasmids (5E4 cells, 3μg plasmid DNA, 0.1M polyethylenimine (PEI)) and luciferase activity in cell lysate was measured at t=6h, 12h, 24h, 48h and 72h (n=3). Although luciferase activity differed at 12h (p<0.01), 24h (p<0.001) and 48h (p<0.001) (2-way ANOVA with Bonferroni posttest), neither plasmid directed persistent transgene expression. ALI cultures were transiently transfected with both plasmids (5E5 cells per insert; 10μg plasmid DNA; 25μl lipofectamine) for 6h, after which the transfection mix was removed. Addition of 100ul of D-Luciferin at 30mg/ml was added prior to measurement of luciferase activity using the IVIS100 (Caliper Life Sciences) bioluminescent imaging system. Imaging was repeated at 12h and 24h after transfection and daily thereafter for 7 days. Average radiance in initial studies was very low with both plasmids at all time-points. To increase transgene expression, ALIs were treated with EGTA to disrupt tight junctions transiently, via pre-treatment with 10mM EGTA for 30 minutes, or co-treatment with 10mM EGTA present in the transfection mix. EGTA pre-treatment did not lead to increased transgene expression compared with non-treated controls, but co- treatment with EGTA resulted in a 63-fold increase in luciferase activity at 12h (p<0.001, 2-way ANOVA with Bonferroni posttest) over pre-treated inserts. Using the EGTA co-treatment transfection conditions, a time-course experiment was performed to determine transgene expression in ALI cultures when transfected with pG4-hCEFI-soLux or pG4-mCEFI-soLux (n=7 ALIs per plasmid). Luciferase activity directed by the two plasmids was different at 24h (p<0.001), 48h (p<0.001) and 72h (p<0.05) (2-way ANOVA with Bonferroni posttest). However, in each case, peak expression rapidly declined within a few days. Together these data demonstrate that non-viral transgene expression observed in mouse lungs following aerosol delivery is not recapitulated in A549 lung cells or in human ALI ex vivo cultures. Currently the reasons for this are unknown, but may be due to species and cell type differences.