Background
Persistence of HIV-1 in latently infected CD4+T and myeloid cells necessitates the existence of lifelong ART. Latency development has been clinically associated with an exclusive increase in CCR5 receptor expression in the latently infected population. We have demonstrated that CXCR4 receptor-targeted LNPs can efficiently eliminate latent proviral DNA from human CD4+T cells. Hence, we propose that dual targeting of CCR5 and CXCR4 receptors through CRISPR-Cas9 encapsulated LNPs can be a potential strategy to excise proviral DNA from both the anatomical and cellular HIV-1 reservoirs, aiming to achieve a functional cure.
Methods
CCR5 receptor-targeted lipid nanoparticles (M5-LNPs) encapsulating CRISPR-Cas9 and gRNA were formulated using microfluidics; untargeted C-LNPs served as controls. The RiboGreen assay determined the mRNA encapsulation efficiency. LNP size and zeta potential were analyzed by dynamic light scattering. Luciferase and CellTiter-BlueTM assays were conducted to analyze the LNPs protein translation efficacy and toxicity profile in human macrophages. M5-LNP and C-LNP were tested for their HIV-1 DNA excision efficiency in latently infected human monocyte-derived macrophages (MDMs), and T lymphocytic and monocytic cell lines. The in vivo study was conducted to analyze the tissue-selective mRNA translation.
Results
LNPs exhibited >98% mRNA encapsulation efficiency and particle sizes <100 nm. They were non-toxic up to a dose of 16 µg mRNA per 10⁶ MDMs. M5-LNPs showed dose-dependent, enhanced protein expression than C-LNPs. Notably, M5-LNPs enabled spleen and lymph node tissue-selective mRNA translation. In HIV-1-infected MDMs and CD4⁺ T cells, M5-LNPs achieved ~98% viral DNA excision efficiency
Conclusions
M5-LNPs demonstrated superior HIV-1 proviral DNA excision efficiency in both monocytic and lymphocytic cellular compartments. In vivo mRNA translation resulted in protein expression localized to the spleen and lymph nodes, which harbor latently infected cells. Ultimately, the dual-targeted LNPs will enable concurrent CRISPR delivery to CD4+ T cells and MDMs, strengthening the viral elimination strategy.
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