Efficient mRNA delivery to resting T cells to reverse HIV latency

Abstract

A major hurdle to curing HIV is the persistence of integrated proviruses in resting CD4⁺ T cells that remain in a transcriptionally silent, latent state. One strategy to eradicate latent HIV is to activate viral transcription, followed by elimination of infected cells through virus-mediated cytotoxicity or immune-mediated clearance. We hypothesised that mRNA-lipid nanoparticle (LNP) technology would provide an opportunity to deliver mRNA encoding proteins able to reverse HIV latency in resting CD4⁺ T cells. Here we develop an LNP formulation (LNP X) with unprecedented potency to deliver mRNA to hard-to-transfect resting CD4⁺ T cells in the absence of cellular toxicity or activation. Encapsulating an mRNA encoding the HIV Tat protein, an activator of HIV transcription, LNP X enhances HIV transcription in ex vivo CD4⁺ T cells from people living with HIV. LNP X further enables the delivery of clustered regularly interspaced short palindromic repeats (CRISPR) activation machinery to modulate both viral and host gene transcription. These findings offer potential for the development of a range of nucleic acid-based T cell therapeutics.

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 LNP (yellow) or LNP X (green) encapsulating mCherry mRNA. MFI, median fluorescent intensity. Mean ± SEM, n = 2. e CD4⁺ T cells from HIV-negative donors were rested or pre-stimulated with anti-CD3/anti-CD28, then treated with LNP X 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 using flow cytometry. Mean ± SEM, n = 6 donors.

Fig. 2. Superior potency of LNP X is not explained by differences in endosomal escape.

a The SNAP_switch assay simultaneously tracks the degree of nanoparticle association, endosomal escape and mRNA expression of LNPs co-encapsulating an AF488 oligo, Cy5-SNAP_switch oligo and mRNA encoding mScarlet. Figure adapted from Liu et al. and created in BioRender: Cevaal, P. (2025) https://BioRender.com/3ajxnam. b–g SNAP_switch-reporter Jurkat T cells were incubated with LNPs containing SM-102, DSPC, DMG-PEG2000 and either Cholesterol (Chol) or ß-sitosterol (Sito; LNP X) for 4 h. LNPs encapsulated a reporter mScarlet mRNA, AF488-tagged oligo and a Cy5-SNAP_switch oligo or constitutively active Cy5-oligo. Fluorescence was determined using flow cytometry. b Protein expression of the mScarlet reporter mRNA. c LNP association as determined by the fluorescence of the AF488-tagged oligo. d mScarlet protein expression (as in b) relative to LNP association (as in c). e Amount of cytosolic delivery of nucleic acid cargo as quantified by the Cy5-SNAP_switch oligo fluorescence. f Efficiency of endosomal escape based on the Cy5-SNAP_switch oligo fluorescence (as in e) normalised to a constitutively fluorescent Cy5-tagged oligo. g mScarlet protein expression (as in b) relative to the amount of cytosolic cargo delivery (as in e). b–g Bars represent aggregate mean of n = 3 independent experiments, each symbol representing the average of triplicate technical replicates. Significance was determined using a one-tailed paired ratio t test in (b–e,g) to allow comparisons of MFI values between experiments, or one-tailed unpaired t test in (f), ns non-significant.

Fig. 3. mRNA encoding HIV Tat exon 1 delivered by LNP X is a potent activator of HIV transcription.

a Schematic overview of the mechanism of action of HIV Tat on promoting transcription elongation. TAR; trans-activation response element. Created in BioRender: Cevaal, P. (2025) https://BioRender.com/cz40qwv. b J-Lat 10.6 cells were treated for 24 h with indicated doses of LNP X encapsulating codon-optimised mRNA expressing the 72 amino acids of the first coding exon of HIV Tat (Tat-LNP X) or mCherry (mCherry-LNP X) as control. Reactivation of HIV LTR-mediated transcription was determined after 24 h by measuring GFP expression. Highest dose of Tat-LNP X corresponds to 1 µg/mL. Treatment with PMA/ionomycin was included as a positive control. Mean ± SEM, n = 3 independent experiments. c–i CD4⁺ T cells from people living with HIV on suppressive ART were treated with 200 ng Tat-LNP X or mCherry-LNP X per 10⁵ cells (4 µg/mL) or PMA/PHA as a positive control. After 48 h (squares) or 72 h (circles), expression of HIV transcripts TAR (c) LongLTR (d), Pol (e), PolyA (f) and Tat-Rev (g) representing transcription initiation, proximal elongation, distal elongation, completion and splicing, respectively, was determined using digital RT-PCR. Data were normalised to RNA input, then presented as fold-change induction compared to the corresponding non-treated (NT) control. Horizontal dashed line (c–g) represents no change relative to untreated cells. h Cellular toxicity was determined using flow cytometry. Where datapoints are missing in (h), cell input was insufficient to perform an accurate measurement. i After 72 h, the number of copies of HIV RNA per mL of supernatant was quantified using RT-PCR and normalised to the non-treated control. c–i Short horizontal line represents the median of n = 7–8 donors. Significance in (c–g and i) was determined using a one-tailed Wilcoxon signed-rank test, ns non-significant. After 120 h, the number of intact (j), 5’ defective (k) or 3’ defective (l) HIV DNA copies were determined using digital PCR. Copy numbers were normalised to cell input. Datapoints represent individual donors (n = 7). Note different donors were used in (j–l and c–i). Significance was determined using a two-tailed Wilcoxon signed-rank test, ns non-significant.

Fig. 4. LNP X co-encapsulating CRISPR activation machinery can be used to induce expression of endogenous genes in T cells.

a Schematic overview of the dCas9-synergistic activation mediator (SAM) CRISPR activation system, consisting of a catalytically inactive (dead, d)Cas9 fused to transcriptional activator domain VP64, a gRNA and a further transcriptional activation MS2-p65-HSF1 fusion protein that is recruited to the Cas9-gRNA complex via MS2-binding to stem-loop structures in the gRNA scaffold. Created in BioRender: Cevaal, P. (2025) https://BioRender.com/p8j24y6. b–d LNP formulation X was used to encapsulate the CRISPR activation machinery (CRISPRa-LNP X) including a gRNA targeting the endogenous gene encoding CD25 (CD25 CRISPRa-LNP X) or a scrambled control gRNA (scr CRISPRa-LNP X). b Jurkat T cells were treated for 24 h with CRISPRa-LNP X, control mCherry-LNP X or PMA/ionomycin as positive control. Induction of CD25 expression was measured by surface stain using flow cytometry. CD4⁺ T cells from HIV-negative donors were treated with indicated doses of CRISPRa-LNP X per 10⁵ cells or mCherry-LNP X as a control for 72 h (c) or 6 days (d), after which CD25 expression was determined using flow cytometry. Highest dose of CD25 CRISPRa-LNP X corresponds to 4 µg/mL. Treatment with PMA/ionomycin was included as a positive control. Dotted line represented average baseline CD25 expression in the absence of treatment. Mean ± SEM, n = 4 independent experiments (b) or n = 4–6 donors (c, d). Significance was determined using a two-tailed Student’s t-test.

Fig. 5. CRISPRa-LNP X activates HIV transcription in CD4⁺ T cells from people living with HIV.

a Schematic overview of the CRISPRa system targeting the HIV LTR. Created in BioRender: Cevaal, P. (2025) https://BioRender.com/p8j24y6. b J-Lat 10.6 cells were treated for 24 h with indicated doses of CRISPRa-LNP X containing one of four HIV LTR-targeting gRNAs (L, O, B, C) or scrambled control gRNA. Reactivation of HIV LTR-mediated transcription was determined after 24 h by measuring GFP expression. Highest dose of CRISPRa-LNP X corresponds to 2.5 µg/mL. Treatment with PMA/ionomycin was included as a positive control. Mean ± SEM, n = 3 independent experiments. c–i CD4⁺ T cells from people living with HIV on suppressive ART were treated with 200 ng CRISPRa-LNP X containing gRNAs L and O (L + O CRISPRa-LNP X) or a scrambled gRNA control (scr CRISPRa-LNP X) per 10⁵ cells (4 µg/mL) or PMA/PHA as a positive control. After 48 h (squares) or 72 h (circles), expression of HIV transcripts TAR (c) LongLTR (d), Pol (e), PolyA (f) and Tat-Rev (g) representing transcription initiation, proximal elongation, distal elongation, completion and splicing, respectively, was determined using digital RT-PCR. Data were normalised to RNA input, then presented as fold-change induction compared to the corresponding non-treated (NT) control. Horizontal dashed line (c–g) represents no change relative to untreated cells. h Concurrently, cellular toxicity was determined using flow cytometry. Where datapoints are missing in (h), cell input was insufficient to perform an accurate measurement. i After 72 h, the number of copies of HIV RNA per mL of supernatant was quantified using RT-PCR and normalised to the non-treated control. c–i Short horizontal line represents the median of n = 7–8 donors. Significance in (c–g) and (i) was determined using a one-tailed Wilcoxon signed-rank test, ns non-significant.