Long Terminal Repeats of Gammaretroviruses Retain Stable Expression After Integration Retargeting or Knock-In into the Restrictive Chromatin of Lamina-Associated Domains

Abstract

Retroviruses integrate their genomes into the genomes of infected host cells and form a genetic platform for stable gene expression. Epigenetic silencing can, however, hamper the expression of integrated provirus. As gammaretroviruses ({gamma}RVs) preferentially integrate into sites of active promoters and enhancers, the high expression activity of {gamma}RVs can be attributed to the integration preference. Long terminal repeats (LTRs) of some {gamma}RVs were shown to act as potent promoters for gene expression. Here, we investigate the capacity of different {gamma}RV LTRs to drive stable expression inside a non-preferred epigenomic environment using diverse retroviral vectors and CRISPR-Cas9-directed vector knock-in. We demonstrate that different {gamma}RV LTRs are either rapidly silenced or long-term active with active proviral population prevailing under normal and retargeted integration. In addition, we show that lamina-associated domains (LADs) can be targeted by CRISPR-Cas9 for vector insertion leading to {gamma}RV LTR-driven long-term stable gene expression. Alternatively to established {gamma}RV systems, the LTRs of feline leukemia virus and koala retrovirus are capable of driving stable, albeit intensity-diverse, transgene expression in LADs. Altogether, we show that despite the occurrence of rapid silencing events, the majority of {gamma}RV LTRs can drive stable expression after retrovirus integration or CRISPR-Cas9-directed knock-in outside of the preferred chromatin landscape.