Efficient mRNA delivery to resting T cells to reverse HIV latency

Summary

LNP X enables delivery of mRNA to resting T cellsWe first assessed whether an LNP similar to an existing, FDA-approved LNP formulation (patisiran) was able to transfect non-stimulated primary CD4+ T cells by delivering a reporter mCherry mRNA. Our patisiran-like LNP (hereafter referred to as patisiran LNP) is formulated with DMG-PEG2000 instead of PEG2000-C-DMG. At the highest dose tested of 500 ng per 105 cells, mCherry expression was only detected in 2.1 ± 0.4% (mean ± SEM) of live cells (Fig. 1a and Fig. S1a) after 72 h incubation. Pre-stimulation of the CD4+ T cells with anti-CD3/anti-CD28 led to substantially higher transfection efficiencies of up to 51 ± 5.1% compared to non-stimulated T cells (Fig. 1a), though the toxicity associated with patisiran LNP treatment was greater in pre-stimulated compared to non-stimulated CD4+ T cells at higher LNP doses (Fig. S1b). These data show that successful mRNA delivery to primary CD4+ T cells using patisiran LNPs is dependent on the activation state of the T cell.Fig. 1: LNP X formulation potently transfects primary CD4+ T cells in the absence of pre-stimulation.a CD4+ T cells from HIV-negative donors were rested or pre-stimulated with anti-CD3/anti-CD28, then treated with LNPs (similar to the patisiran lipid composition) encapsulating mCherry mRNA for 72 h at the indicated doses. Highest dose of mCherry-LNP X corresponds to 2.5 µg/mL. Transfection efficiency was determined by measuring mCherry expression using flow cytometry. Mean ± SEM, n = 6 donors. b Schematic representation of patisiran-like LNP formulation versus novel LNP formulation X. Mol %, relative molar percentage of indicated lipid in lipid mixture. Created in BioRender: Cevaal, P. (2025) https://BioRender.com/ypaicaz. c Size distribution of patisiran LNP (yellow) versus LNP X (green) encapsulating mCherry mRNA. Lines represent individual LNP batches. d Relative mCherry expression levels in Jurkat T cells treated for 24 h with indicated doses of patisiran LN...