Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs

Abstract

Nucleoside-modified messenger RNA (mRNA)-lipid nanoparticles (LNPs) are the basis for the first two EUA (Emergency Use Authorization) COVID-19 vaccines. The use of nucleoside-modified mRNA as a pharmacological agent opens immense opportunities for therapeutic, prophylactic and diagnostic molecular interventions. In particular, mRNA-based drugs may specifically modulate immune cells, such as T lymphocytes, for immunotherapy of oncologic, infectious and other conditions. The key challenge, however, is that T cells are notoriously resistant to transfection by exogenous mRNA. Here, we report that conjugating CD4 antibody to LNPs enables specific targeting and mRNA interventions to CD4+ cells, including T cells. After systemic injection in mice, CD4-targeted radiolabeled mRNA-LNPs accumulated in spleen, providing ∼30-fold higher signal of reporter mRNA in T cells isolated from spleen as compared with non-targeted mRNA-LNPs. Intravenous injection of CD4-targeted LNPs loaded with Cre recombinase-encoding mRNA provided specific dose-dependent loxP-mediated genetic recombination, resulting in reporter gene expression in about 60% and 40% of CD4+ T cells in spleen and lymph nodes, respectively. T cell phenotyping showed uniform transfection of T cell subpopulations, with no variability in uptake of CD4-targeted mRNA-LNPs in naive, central memory, and effector cells. The specific and efficient targeting and transfection of mRNA to T cells established in this study provides a platform technology for immunotherapy of devastating conditions and HIV cure.

Keywords: LNP; T cell; genetic recombination; lipid nanoparticle; mRNA; targeted mRNA delivery.

Graphical abstract

Figure 1

Binding and functional activity of CD4-targeted particles in vitro (A) Specific in vitro binding of anti-human CD4/¹²⁵I-labeled mRNA-LNPs to human CD4⁺ T cells after 1 h incubation at room temperature (RT). (B) Binding of anti-CD4/mRNA-LNPs and control IgG/mRNA-LNPs to human CD4⁺ T cells, with increasing mRNA-LNP doses, and their corresponding mean fluorescence intensity (MFI). (C) Luc activity measured in human CD4⁺ T cells treated with anti-human CD4/mRNA-LNPs or control IgG/mRNA-LNPs.

Figure 2

Cre mRNA-mediated genetic recombination in vitro (A) Cre mRNA-induced genetic recombination and consequent reporter gene expression presented as % of ZsGreen1⁺ cells among CD3⁺CD8⁻ cells. Splenocytes were harvested from Ai6 mice and incubated with Cre mRNA-LNPs at doses of 1, 3, 6, or 9 μg per 2 million cells. %ZsGreen1⁺ cells upon anti-CD4/mRNA-LNP administration was compared to control IgG/mRNA-LNP and unconjugated mRNA-LNP administration (mean with SEM is shown; ∗∗∗∗p < 0.0001, two-way ANOVA with Bonferroni correction). (B) Gating strategy to identify ZsGreen1⁺ cells among CD3⁺CD8⁻ cells.

Figure 3

Targeting of mRNA-LNP to CD4⁺ cells in vivo (A) Biodistribution of ¹²⁵I-labeled anti-CD4/ and control IgG/poly(C) RNA-LNPs in mice at 0.5 h. Tissue uptake is indicated as mean ± SEM (∗∗∗∗p < 0.0001). (B) Localization ratio, calculated as the ratio of %ID/g of a given organ to that in the blood of mice treated with either ¹²⁵I-labeled anti CD4/ or control IgG/mRNA-LNP at 30 min post-injection. Mean ± SEM is shown. In vivo mRNA-LNP binding as quantitative measurement of the percentage of radiolabeled anti-CD4/mRNA-LNPs in selected organs (C) and localization ratios in spleens (D), after intravenous injection of mRNA-LNPs. Group size is 3 animals. Statistical analysis was performed by two-way ANOVA with Bonferroni correction (∗∗∗∗p < 0.0001).

Figure 4

Biodistribution of targeted mRNA-LNP expression in vivo Mice were i.v. injected with 8 μg of mRNA-LNPs. Organ distribution of Luc mRNA expression 5 h after administration of anti-CD4/ and control IgG/Luc mRNA-LNP was evaluated by (A) measuring Luc activity in lysed tissues and by (B and C) luminescence imaging. (A) Quantitative expression of Luc as light unit (LU)/mg protein. A representative sample set of dissected mouse organs (B) and whole carcasses after organ removal (showing luminescing lymph nodes) (C) were analyzed 5 min after the administration of D-luciferin. (D) Quantitative expression of Luc as LU/mg protein values in CD3⁺ cell preparation obtained from the spleens of mice injected with the mRNA-LNPs. (A and D) Error bars indicate SEM. Group size is 3 animals. Statistical analysis was performed by two-way ANOVA with Bonferroni correction, (∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).

Figure 5

Cre-mediated genetic recombination upon in vivo administration of CD4-targeted Cre mRNA-LNPs (A) Schematic diagram depicting targeted delivery of anti-CD4/mRNA-LNPs for selective genetic recombination in CD4⁺ T cells, and the principle of the Ai6 reporter allele: Cre-mediated excision of a loxP-flanked STOP cassette allows robust expression of ZsGreen1, a fluorescent protein. Ai6 mice received Cre mRNA-LNPs at doses of 3, 10, and 30 μg via i.v. administration. Spleens and lymph nodes were harvested at 24 h post treatment and % of ZsGreen1⁺ cells in the CD3⁺CD8⁻ cell population were determined in splenic (B) and lymph node (C) single-cell suspensions using flow cytometry. Changes in the number of ZsGreen1-expressing CD4⁺ T cells in spleens (D) and lymph nodes (E) over time were monitored after i.v. injection of 10 μg of mRNA-LNPs. Group size is 8 or 9 (B and C) or 6 (D and E) animals in a total of three independent experiments. Each symbol represents one animal, and horizontal lines show the mean with SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni correction. %ZsGreen1⁺ cells after injection of different doses of anti-CD4/mRNA-LNPs (∗p < 0.05, ∗∗∗∗p < 0.0001) and unconjugated mRNA-LNP (^####p < 0.0001) were compared.

Figure 6

In vivo uptake of Cre mRNA-LNP by different T cell subtypes Spleens were harvested at 24 h post-treatment with 10 μg of Cre mRNA-LNPs, and % of ZsGreen1⁺ cells in CD4⁺ T cell subpopulations (A) and versus CD25 marker (B) were determined using flow cytometry. Naive CD4⁺ T cells are considered as CD44⁻CD62L⁻, central memory T cells as CD44⁺CD62L⁺, and effector memory T cells as CD44⁺CD62L⁻. Group size is 3–11 animals. Each symbol represents one animal, and horizontal lines show the mean with SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni correction comparing T cell subtypes (∗∗p < 0.01, ∗∗∗∗p < 0.0001). (C) Gating strategy to identify ZsGreen1⁺ cells among different CD4⁺ T cell subtypes.

Figure 7

mRNA-LNP targeting efficiency using multiple administrations Ai6 mice received 10 μg (0.4 mg/kg) of anti-CD4/, control IgG/, or unconjugated Cre mRNA-LNPs via i.v. administration as daily injections for 3 or 5 days. Spleens and lymph nodes were harvested after three or five sequential injections, and the % of ZsGreen1⁺ cells in the CD3⁺CD8⁻ cell population was determined in splenic (A) and lymph node (B) single-cell suspensions using flow cytometry. Group size is 9 animals. Each symbol represents one animal, and horizontal lines show the mean. Error bars indicate SEM. Statistical analysis was performed by two-way ANOVA with Bonferroni correction. %ZsGreen1⁺ cells after different number of injections of anti-CD4/mRNA-LNP (∗∗p < 0.01, ∗∗∗∗p < 0.0001) were compared with unconjugated mRNA-LNP.