Oral Presentation 14th Lorne Infection and Immunity 2024

Potent, HIV-specific latency-reversal through CRISPR activation delivered by lipid nanoparticles (#21)

Paula M Cevaal 1 , Stan Kan 1 , Abdalla Ali 1 , Abigail Tan 1 , Marvin Holz 2 , Denzil L Furtado 3 , Damian Purcell 2 , Angus Johnston 4 , Colin Pouton 4 , Thomas Payne 4 , Frank Caruso 3 , Jori Symons 1 5 , Michael Roche 1 , Sharon R Lewin 1 6 7
  1. Department of Infectious Diseases, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
  2. Department of Microbiology and Immunology, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
  3. Department of Chemical Engineering, The University of Melbourne, Melbourne, VIC, Australia
  4. Monash Institute of Pharmaceutical Sciences, Melbourne, VIC, Australia
  5. Department of Medical Microbiology, University Medical Center, Utrecht, The Netherlands
  6. Victorian Infectious Diseases Service, Royal Melbourne Hostpital at The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
  7. Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, VIC, Australia

Introduction: Activation of HIV transcription through LTR-targeted CRISPRactivation (CRISPRa) provides a promising strategy of reversing HIV latency without affecting host-cell transcription. However, the advancement of this novel latency-reversing agent is hampered by the lack of a delivery vehicle for the CRISPRa machinery to resting CD4+ T cells. We hypothesized that targeted 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 CRISPRa system (CRISPRa-LNP) were formulated through microfluidic mixing using two different lipid mixes. T cell-targeting antibodies were captured onto the LNPs following controlled-orientation conjugation of anti-IgG1 nanobodies to the LNP surface.  Transfection efficiency and associated toxicity were assessed in non-stimulated or αCD3/αCD28 pre-stimulated primary CD4+ T cells or PBMCs from HIV-negative donors. Reactivation of HIV transcription was assessed in J-Lat LTR-Tat-IRES-GFP reporter cells.

Results: Transfection efficiency of pre-stimulated CD4+ T cells using standard LNPs 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 HIV 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%. Functionalization of the modified LNPs with T cell-targeting antibodies enhanced the T cell transfection efficiency in the presence of bystander cells by over 20-fold.

Conclusions: We developed a novel LNP formulation capable of delivering nucleic acid-based therapeutics to resting CD4+ T cells. Antibody-functionalization of the modified LNP surface further enhances the specificity towards T cells with great potency. The three-component dCas9-SAM CRISPRa 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.