This suggests that in human lung, the short-term effect of glucocorticoids is to prevent AP-1 DNA binding within the nucleus via protein-protein interactions. In the long-term, glucocorticoids reduce the level of Fos and Jun available for AP-1 formation. This may be an important molecular mechanism of steroid action in asthma and other chronic inflammatory lung diseases.

Inclusion of the woodchuck hepatitis virus post-transcriptional response element (WPRE) in the 3' UTR of mRNA encoding zinc-finger or TALE nucleases results in up to a fifty-fold increase in nuclease expression and a several-fold increase in nuclease-modified chromosomes. Significantly, this increase is additive with the enhancement generated by transient hypothermic shock. The WPRE-mediated improvement is seen across several types of human and mouse primary and transformed cells and is translatable in vivo to the mouse liver.The woodchuck hepatitis virus posttranscriptional response element (WPRE) is thought to promote nuclear export of viral genomic mRNA (1). As a result, the WPRE is often included in viral gene transfer vectors where it can improve viral titer and subsequent transgene expression (2). The effect of the WPRE is idiosyncratic, improving expression of some transgenes in some cell types but occasionally having neutral or even negative effects (3,4,5,6,7,8,9).There are conflicting reports as to how the WPRE works. In some contexts its presence increases steady-state mRNA levels (10) while in others the WPRE seems to improve transcriptional termination, having no effect on nuclear export or mRNA half-life (11). As there is one claim of an improvement in cytoplasmic mRNA metabolism resulting in increased translation (12), we investigated whether inclusion of the WPRE in sitespecific nuclease mRNA would improve nuclease expression and genome modification.To determine whether inclusion of the WPRE in the 3' UTR of a nuclease transcript would improve non-homologous end joining (NHEJ) DNA repair, we transfected in vitro transcribed mRNAs encoding CCR5-specific zinc-finger nucleases (ZFNs) either containing the WPRE or not and assayed the CCR5 locus by high-throughput DNA sequencing. The presence of a WPRE increased the frequency of NHEJ by two-to ten-fold in CD34-positive hematopoietic stem and progenitor cells (HSPCs), CD4-positive T cells, and CD8-positive T cells (Figure 1a, b, c). An increase in mutagenic NHEJ was seen when the WPRE was included in CCR5 TALE nuclease mRNAs as well (Figure 1d). We quantitated ZFN protein levels in transfected K562 cells and found that the WPRE-containing mRNAs yielded a steady-state level of ZFN protein between four-and 67-fold higher depending on the dose transfected (Figure 2) with improvements in NHEJ similar to primary cells (data not shown).A brief hypothermic shock has also been shown to increase NHEJ activity. To test whether hypothermia and the WPRE effect increased NHEJ via the same mechanism, we individually transfected CD34-positive HSPCs with mRNA encoding three separate ZFN pairs either with or without the WPRE in their 3' UTRs. The transfected cells were divided in half, with one half incubated at 30° C for 18 hours and the other half maintained at 37° C. At AAVS1, IL2Rγ, and CCR5, the WPREmediated improvement in NHEJ was additive with the improvement gained from hypothermic shock (Figure 3). We conclude that hypothermic shock and the WPRE functi...