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

• Published in: PloS one Vol. 11; no. 6; p. e0158294
• Main Authors: Bialek, Julia K, Dunay, Gábor A, Voges, Maike, Schäfer, Carola, Spohn, Michael, Stucka, Rolf, Hauber, Joachim, Lange, Ulrike C
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 24.06.2016; Public Library of Science (PLoS)
• Subjects: Base Sequence; Binding Sites; Biology and Life Sciences; Caspases; Cell culture; CRISPR; CRISPR-Cas Systems; Deoxyribonucleic acid; DNA; Drug therapy; Gene Editing; Gene Targeting; Genetic aspects; Genome editing; Genomes; HIV; HIV Infections - virology; HIV Long Terminal Repeat; HIV-1 - physiology; Human immunodeficiency virus; Human immunodeficiency virus 1; Humans; Infections; Jurkat Cells; Kinases; Latency; Latent infection; Lentivirus; Long terminal repeat; Lymphocytes; Medicine and Health Sciences; Modulation; Nucleotide sequence; Physiological aspects; Protein Binding; Proteins; Proviruses - genetics; Research and Analysis Methods; Reservoirs; RNA, Guide, CRISPR-Cas Systems; Target recognition; Transcription (Genetics); Transcription activation; Transcriptional Activation; Viral infections; Virology; Virus Latency - genetics; Virus Replication; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0158294

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.