BACKGROUND: Activation of HIV transcription through LTR-targeted CRISPR activation (CRISPRa) provides a promising strategy of potently reversing HIV latency without affecting host-cell transcription. However, the advancement of this novel latency-reversing agent is hampered by the lack of an efficient delivery vehicle for the CRISPRa machinery to resting CD4+ T cells. We hypothesized that mRNA-lipid nanoparticles (LNPs) could be used to advance CRISPRa as a next-generation latency-reversing agent.
METHODS: Fluorescently labelled standard and modified LNPs encapsulating reporter mCherry mRNA (mCherry-LNP) or co-encapsulating the dCas9-SAM system – dCas9-VP64 mRNA, MS2-p65-HSF1 mRNA and either LTR-targeting or non-targeting control guideRNA (CRISPRa-LNP) – were formulated through microfluidic mixing. RNA concentration and encapsulation efficiency were determined using a RiboGreen assay. Transfection efficiency of mCherry-LNPs and viability were assessed in Jurkat cells, non-stimulated or 72h aCD3/aCD28 pre-stimulated primary CD4+ T cells from HIV-negative donors using flow cytometry. Reactivation of LTR-mediated transcription through CRISPRa-LNPs was assessed in J-Lat LTR-Tat-IRES-GFP reporter cells using flow cytometry.
RESULTS: LNPs were formulated reproducibly at sub-100 nm size and exhibited RNA encapsulation efficiencies of >90%. Standard mCherry-LNPs exhibited high transfection efficiency within 24h in Jurkat cells (99%) at <5% toxicity. Transfection efficiency of pre-stimulated CD4+ T cells was moderate (mean±SEM mCherry+ cells 29±5%) but toxic (43±5% viability) after 72h. In non-stimulated cells, few cells were transfected (2±0.3%) with lower toxicity (68±8% viability), which coincided with a 20-fold reduction in LNP association. In contrast, transfection of non-stimulated CD4+ T cells with modified LNPs resulted in a striking 92±2% efficiency at minimal toxicity (88±3% viability) within 72h. Similarly, treatment with modified but not standard CRISPRa-LNPs induced potent LTR-mediated transcription with all five targeting guideRNAs, reaching up to 76±13% GFP+ J-Lat cells compared with 0.89±0.1% using non-targeting guideRNA, both at viabilities >90%.
CONCLUSIONS: We developed a novel LNP formulation capable of delivering nucleic acid-based therapeutics to resting CD4+ T cells. The three-component dCas9-SAM CRISPR activation system can be co-encapsulated into one LNP and can induce strong latency reversal in a cell line model for HIV latency. These results provide compelling justification for the further assessment of CRISPRa-LNP as a ‘shock and kill’ strategy.

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