Repression of HIV-1 reactivation mediated by CRISPR/dCas9-KRAB in lymphoid and myeloid cell models

Abstract

Background Despite antiretroviral treatment efficacy, it does not lead to the complete eradication of HIV infection. Consequently, reactivation of the virus from latently infected cell reservoirs is a major challenge toward cure efforts. Two strategies targeting viral latency are currently under investigation: the “shock and kill” and the “block and lock.” The “Block and Lock” methodology aims to control HIV-1 latency reactivation, promoting a functional cure. We utilized the CRISPR/dCas9-KRAB platform, which was initially developed to suppress cellular genes transcription, to block drug-induced HIV-1 reactivation in latently infected T cells and myeloid cells. Results We identified a set of five sgRNAs targeting the HIV-1 proviral genome (LTR1-LTR5), having the lowest nominated off-target activity, and transduced them into the latently infected lymphoid (J-Lat 10.6) and myeloid (U1) cell lines. One of the sgRNAs (LTR5), which binds specifically in the HIV-1 LTR NFκB binding site, was able to promote robust repression of HIV-1 reactivation in latently infected T cells stimulated with Phorbol 12-Myristate 13-Acetate (PMA) and Ingenol B (IngB), both potent protein kinase C (PKC) stimulators. Reactivation with HDAC inhibitors, such as SAHA and Panobinostat, showed the same strong inhibition of reactivation. Additionally, we observed a hundred times reduction of HIV-1 RNA expression levels in the latently infected myeloid cell line, U1 induced with IngB. Conclusion Taken together, our results show that the KRAB fused CRISPR/dCas9 system can robustly prevent the HIV-1 latency reactivation process, mediated by PMA or IngB and SAHA or Panobinostat, both in myeloid and lymphoid HIV-1 latently infected cells. In addition, we demonstrated that KRAB repressor protein is crucial to reactivation resistance phenotype, and we have identified some useful hotspots sequences in HIV-1 LTR for the design sgRNAs.

Targeting Viral Transcription for HIV Cure Strategies

“…These positive results prompted a clinical trial, which is currently ongoing (NCT05144386). Several studies have evaluated the applicability of catalytically inactive Cas9 to reactivate or silence HIV and SIV transcription [114,[121][122][123][124][125][126][127][128], but all these studies are still in the pre-clinical stage.…”

CRISPR-Cas System: The Current and Emerging Translational Landscape

“…Another approach is to promote a permanent latent state by inhibiting HIV-1 transcription factors. In line with the second approach, da Costa et al used sgRNAs targeting the HIV-1 proviral genome (LTR1-LTR5) and CRISPR/dCas9-KRAB to block HIV-1 reactivation in latently infected T cells and myeloid cells treated with latency-reversing agents such as PKC agonists and HDAC inhibitors [ 208 ]. They found that targeting the LTR enhancer region provided the best repression, and one of their CRISPR constructs inhibited latency reactivation by 160×.…”