Title

Potent, HIV-specific latency reversal through CRISPR activation delivered by lipid nanoparticles exhibiting a high efficiency of transfecting resting CD4⁺ T cells with minimal toxicity

Presenter

Paula M. Cevaal

Authors

P.M. Cevaal¹, S. Kan², A. Tan², M. Holz¹, D.L. Furtado³, D. Purcell¹, A. Johnston⁴, C. Pouton⁴, T. Payne⁴, W. Zhao², F. Caruso³, J. Symons^2,5, M. Roche², S.R. Lewin^2,6,7

Institutions

¹The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Department of Microbiology and Immunology, Melbourne, Australia, ²The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Department of Infectious Diseases, Melbourne, Australia, ³The University of Melbourne, Department of Chemical Engineering, Melbourne, Australia, ⁴Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Australia, ⁵University Medical Center, Translational Virology, Department of Medical Microbiology, Utrecht, Netherlands, ⁶Royal Melbourne Hospital at The Peter Doherty Institute for Infection and Immunity, Victorian Infectious Diseases Service, Melbourne, Australia, ⁷Alfred Hospital and Monash University, Department of Infectious Diseases, Melbourne, Australia

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.

Download the e-Poster (Movie)

Download the e-Poster (PDF)

Go to Session