4.7 Article

Potent and Targeted Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex

Journal

MOLECULAR THERAPY
Volume 24, Issue 3, Pages 488-498

Publisher

CELL PRESS
DOI: 10.1038/mt.2015.202

Keywords

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Funding

  1. Strategic Health Innovation Partnerships (SHIP) Unit of South African Medical Research Council (SAMRC)
  2. South African Department of Science and Technology (DST)
  3. NIAID [PO1 AI099783-01, R01 AI111139-01, R01 DK104681-01]
  4. Australian Research Council [FT1300100572]

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HIV-1 provirus integration results in a persistent latently infected reservoir that is recalcitrant to combined antiret-roviral therapy (cART) with lifelong treatment being the only option. The shock and kill strategy aims to eradicate latent HIV by reactivating proviral gene expression in the context of cART treatment. Gene-specific transcriptional activation can be achieved using the RNA guided CRISPR-Cas9 system comprising single guide RNAs (sgRNAs) with a nuclease-deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64). We engineered this system to target 23 sites within the long terminal repeat promoter of HIV-1 and identified a hotspot for activation within the viral enhancer sequence. Activating sgRNAs transcriptionally modulated the latent proviral genome across multiple different in vitro latency cell models including T cells comprising a clonally integrated mCherry-IRES-Tat (LChlT) latency system. We detected consistent and effective activation of latent virus mediated by activator sgRNAs, whereas latency reversal agents produced variable activation responses. Transcriptomic analysis revealed dCas9-VP64/sgRNAs to be highly specific, while the well-characterized chemical activator TNF alpha induced widespread gene dysregulation. CRISPR-mediated gene activation represents a novel system which provides enhanced efficiency and specificity in a targeted latency reactivation strategy and represents a promising approach to a functional cure of HIV/AIDS.

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