693. Potent and Targeted Activation of Latent HIV-1 Using Multiplexed Guide RNAs and the CRISPR/dCas9 Activator Complex

Cells latently infected with HIV provide a reservoir that promotes new viral infection indefinitely, thus making it exceedingly difficult for antiretroviral therapy (ART) to completely ablate the infection. A powerful strategy to eradicate latent HIV reservoirs has been to reactivate proviral gene expression using histone deacetylase (HDAC) inhibitors followed by ART: an approach called “shock and kill”. However, this approach suffers from lack of efficacy and specificity. Gene specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising a nuclease deficient mutant of Cas9 (dCas9) fused to a C-terminal VP64 or VP160 acidic transactivation domain. We initially identified 18 sites for targeting of small guide RNAs (sgRNAs) in the U3 region, upstream of the canonical HIV transcriptional start site TSS (-450 to 0 bp). Activating sgRNA candidates were identified and verified by transient transfection in TZM-Bl cells and also in HEK293 cells with Subtype B and C LTR-driven luciferase reporters. Several candidate sgRNA individually induced a 10-fold increase in gene expression, with a “hotspot” region between -200 and -250 bp of the TSS. However, when combined in multiplex fashion, with four conserved gRNAs expressed off four independent Pol III promoters in a single vector, a synergistic enhancement of HIV transcription of ~30 fold was observed. Given the complexity and multifaceted mechanisms of latency, current cell line models are unable to completely recapitulate the conditions of endogenous HIV reservoirs. As an initial proof-of-concept that the CRISPR/Cas9 system can be used to purge the latent HIV proviral genome, we used a CEM T cell-based reporter system, comprising a single copy of HIV, that encodes mCherry-IRES-Tat from the full-length HIV LTR (LChIT). The LChIT system enables a Tat-mediated positive feedback loop that drives expression of Tat and mCherry allowing for exquisite sensitivity to alterations in viral gene expression, either directly or indirectly. CEM-LChIT clone 3.2 expressed mCherry in 50% of the population (a bimodal state). In this model, nucleofected sgRNAs and dCas9-VP64 led to a 200-fold activation of gene expression; a result higher than treatment controls using a combination of HDAC inhibitor trichostatin A (TSA) and TNFa. In conclusion, the potential for RNA-guided gene activation systems to provide maximum efficiency and specificity in a targeted reactivation strategy augurs well for the future of the “shock and kill” approach as an impending cure for HIV.