Abstract
Retroviral vectors are silenced by epigenetic mechanisms in embryonic stem (ES) cells that cannot be fully prevented and whose kinetics are poorly understood. The HSC1 self-inactivating retroviral vector is silenced in mouse ES cells rapidly after integration or over prolonged culture, even when favourable integration sites for transgene expression are selected by cell sorting or drug selection. To escape this silencing, we tested the D4Z4 insulator. We show that a 3'D4Z4 fragment directs retroviral expression in ES cells with persistent but variable expression for up to 5 months. Combining an internal 3'D4Z4 with HS4 insulators in the LTRs shows that these elements cooperate, and defines the first retroviral vector that fully escapes long-term silencing. Epigenetic analyses demonstrate that 3'D4Z4 and HS4 insulators together maintain hypomethylated DNA on retroviral transgenes and reduce H3K9me3 repressive marks to the same level as the expressed endogenous Nanog locus. In order to decipher the role of 3'D4Z4 activity at specific integration sites, we used FLP recombinase to induce deletion of 3'D4Z4 from ES cell clones that contain a single provirus. We observe that after 3'D4Z4 deletion retroviral silencing is established at many but not all integration sites. This finding shows that 3'D4Z4 does not target retrovirus integration into favourable epigenomic domains. Instead, epigenetic analyses demonstrate that 3'D4Z4 is a barrier element that blocks the spread of heterochromatin marks including DNA methylation and repressive histone modifications such as H3K9 methylation. In addition, our deletion system reveals three distinct kinetic classes of silencing (rapid, gradual or not silenced), and our epigenetic analyses suggest that multiple pathways participate in silencing at different integration sites. We conclude that vectors with both the 3'D4Z4 barrier and HS4 insulator elements block rapid and gradual kinetic classes of silencing, and may have unprecedented utility for gene transfer applications that require long-term gene expression in pluripotent stem cells. This vector is well suited for manipulation of iPS cells for genetic rescue in disease modeling studies and has potential for suicide vector applications to increase the safety of iPS or ES cell therapies.