Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems

Abstract

CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5’ long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

CRISPR/Cas9-Based Engineering of the Epigenome

“…These strategies can be a useful alternative to transgene overexpression or antisense-mediated gene repression. In fact, several studies have already demonstrated its applicability in the validation of gene functionality (Gilbert et al, 2014; Chavez et al, 2016; Radzisheuskaya et al, 2016), interrogation of promoter and enhancer regions (Kearns et al, 2015; Zalatan et al, 2015; Himeda et al, 2016; Klann et al, 2017), induction of direct cell fate conversion (Chakraborty et al, 2014; Balboa et al, 2015; Black et al, 2016), reprogramming to pluripotency (Balboa et al, 2015; Xiong et al, 2016), genome-scale transcriptional modifications (Gilbert et al, 2014; Konermann et al, 2015) and proof of concept therapeutic approaches against various diseases (Bialek et al, 2016; Braun et al, 2016; Garcia-Bloj et al, 2016). …”

Novel AIDS therapies based on gene editing

“…A novel means to activate HIV-1 is to use catalytically-deficient Cas9-synergistic activation mediator (dCas9-SAM) technology as a method to selectively and potently reactivate latent viral reservoirs. It has been possible to screen and identify gRNAs within the HIV-1 LTR that induce robust reactivation of provirus [79,80], which induced cellular suicide via toxic buildup of viral proteins suggesting that this might serve as a novel HIV-latency-reversing therapeutic tool for eliminating HIV-1 latent reservoirs.…”

CRISPR Editing Technology in Biological and Biomedical Investigation

“…However, since extended expression of CRISPR/Cas9 in the cells is necessary with this tactic, off-target effects may become a problem as explained above. A related approach has been to use a catalytically-deficient Cas9-synergistic activation mediator (dCas9-SAM) as a means to activate the HIV-1 provirus similar to the “shock and kill” approach only with a novel and more specific viral latency-reversing tool (Zhang et al, 2015a; Bialek et al, 2016). …”

“…In addition, catalytically inactive Cas9 (dCas9) can be fused to other proteins and used together with gRNA to exert sequence-specific targeted effects such as gene activation or gene silencing. For example, dCas9-SAM can be used as a means to activate HIV-1 provirus as discussed above (Zhang et al, 2015a; Bialek et al, 2016). dCas9 derivatives can also be used to execute target-specific epigenetic changes such as DNA methylation and histone acetylation (Cano-Rodriguez and Rots, 2016).…”