Nanoparticles versus Dendritic Cells as Vehicles to Deliver mRNA Encoding Multiple Epitopes for Immunotherapy

Abstract

The efficacy of antigen-specific immunotherapy relies heavily on efficient antigen delivery to antigen-presenting cells and engagement of as many disease-relevant T cells as possible in various lymphoid tissues, which are challenging to achieve. Here, we compared two approaches to deliver mRNA encoding multiple epitopes targeting both CD4⁺ and CD8⁺ T cells: a lipid-based nanoparticle platform to target endogenous antigen-presenting cells in vivo versus ex vivo mRNA-electroporated dendritic cells. After intraperitoneal injection, the nanoparticle platform facilitated efficient entry of mRNA into various endogenous antigen-presenting cells, including lymph node stromal cells, and elicited robust T cell responses within a wider network of lymphoid tissues compared with dendritic cells. Following intravenous injection, mRNA-electroporated dendritic cells and the nanoparticle platform localized primarily in lung and spleen, respectively. When administered locally via an intradermal route, both platforms resulted in mRNA expression at the injection site and in robust T cell responses in draining lymph nodes. This study indicates that multiple epitopes, customizable for specific patient populations and encoded by mRNA, can be targeted to different lymphoid tissues based on delivery vehicle and route, and constitute the groundwork for future studies using mRNA to reprogram exogenous or endogenous APCs for immunotherapy.

Keywords: autoimmune diabetes; beta cell antigens; dendritic cell; mRNA vaccine; nanomedicine; nanoparticle; neoepitopes; precision medicine; stromal cell.

Graphical abstract

Figure 1

mRNA Construct and Biophysical Characterization of mRNA-NPs (A) mRNA construct encoding major epitopes and mimotopes related to four different β cell antigens (insulin, ChgA, GAD65, and IGRP). It uses an endosome-targeting signal (ETS) to direct CD4 epitopes for processing and loading onto MHC class II. Some of the epitopes highlighted in color are recognized by T cell clones indicated underneath and used in this study. (B) EMSA assay showing the binding efficiency of mRNA and jetMESSENGER at different ratios for antigen (Ag), mCherry (MC), and Luc mRNAs (2 μg). (C and D) Biophysical characterization of mRNA-NPs containing Luc-mRNA by Zetasizer showing particle size distribution (C) and zeta potential (D). Each color represents a replicate.

Figure 2

mRNA-NP Transfection Efficiency in Various Cell Types (A) In vitro transfection efficiency of mRNA-NPs in BM-DCs and stromal cell lines (LECs, FRCs, and DAPg7) after 48 h of culture using mCherry-encoding mRNA. The transfection efficiency of BM-DCs was significantly lower than any of the stromal cell lines (p ≤ 0.01). (B–E) In vivo transfection efficiency of mRNA-NPs in different cell types using reporter mRNA in PLNs 48 h after i.p. injection. Data shown in panels B and C are from a study using GFP mRNA and no tissue digestion, while those depicted in panels D and E are from a study using mCherry mRNA and collagenase digestion to retrieve all APC populations. Control mice were untreated mice whose untransfected tissues/cells were used to determine fluorescence background for each cell type. The data are shown as mean of three biological replicates ± SEM (B and D) or as representative plots (C and E). Significant differences in uptake and expression of mRNA indicated for several cell types are relative to LNECs (ECs) for the treated group (D).

Figure 3

mRNA-NPs and mRNA-DCs Differ in Their In Vivo Biodistribution after i.p. Injection (A and B) Luc expression 8 h after i.p. injection of Luc mRNA-NPs (A) or Luc mRNA-DCs (B) in live mice (top) and in excised lymphoid tissues (middle), and relative expression in each lymphoid tissue (bottom) at doses of 20 μg mRNA-NPs/mouse or 6 μg Luc-expressing mRNA/10⁶ electroporated DCs. (C and D) Quantified Luc signal from Luc mRNA-NPs (C) or mRNA-DCs (D) at both 4- and 8-h time points displayed as total photons per second (based on region of interest). Mean ± SD from n = 3 mice.

Figure 4

Localization of mRNA-NPs and mRNA-DCs after i.v. Injection (A and B) Luc expression 4 h after i.v. injection of Luc-expressing mRNA-NPs (A) or mRNA-DCs (B) in live mice (top) and in excised lymphoid tissues 8 h after injection (bottom). (C and D) Quantification of Luc expression (total photons/s) from Luc-expressing mRNA-NPs (C) or mRNA-DCs (D) at 4- and 8-h time points (based on region of interest). Mean ± SD from n = 3 mice.

Figure 5

Localized Luc Expression from mRNA-NPs and mRNA-DCs after i.d. Injection (A and B) Luc expression 4 h after i.d. injection of Luc-expressing mRNA-NPs at dose of 5 μg mRNA/mouse (A) or mRNA-DCs at dose of 2 μg mRNA/10⁶ DCs/mouse (B) in live animal (top) and in excised lymphoid tissues (middle), and relative Luc expression in draining lymph nodes and injection site (bottom). (C and D) Quantification of Luc expression from Luc mRNA-NPs (C) or mRNA-DCs (D) at the site of injection (based on region of interest). Mean ± SD from n = 3 mice.

Figure 6

mRNA-NPs Induce Antigen-Specific CD4⁺ T Cell Responses in a Wider Network of Lymphoid Tissues Compared with mRNA-DCs after i.p. Administration (A–F) Responses of transferred BDC2.5 CD4⁺ T cells to mRNA-NPs at a dose of 10 μg mRNA/mouse (A–C) and mRNA-DCs at a dose of 2 μg/1 × 10⁶ electroporated DCs/mouse (D–F) were analyzed in cervical (CLNs), inguinal (ILNs), pancreatic (PLNs), mesenteric lymph nodes (MLNs) and spleen. The results are depicted as representative dot plots (A and D), proliferation (percentage divided) (B and E), and CD25 upregulation (C and F). EGFP mRNA was used as control for both mRNA-NP and mRNA-DCs modalities. The bar graphs (B, C, E, and F) show the mean ± SEM from three biological replicates, and the significant differences indicated for several lymphoid tissues are relative to PLNs in the antigen-treated group.

Figure 7

mRNA-NPs Target a Broader Network of Lymphoid Tissues Than mRNA-DCs for Antigen Presentation (A–F) Endogenous antigen-specific CD4⁺ T cell responses to mRNA-NPs (A–C) and mRNA-DCs (D–F) in various lymphoid tissues. Mice were injected i.p. with mRNA-NPs (5 μg mRNA/mouse) or 1 × 10⁶ mRNA-DCs (1 μg mRNA/mouse), whereby mRNAs express multiple epitopes (antigen) or mCherry (control), and lymphoid tissues were analyzed 3 days later. The percentage of CD44^hi among p79-reactive CD4⁺ T cells indicating their engagement with antigen is shown in red on the dot plots (A and D). The bar graphs (B, C, E and F) show the mean ± SD from three biological replicates, and the significant differences indicated for several lymphoid tissues are relative to PLNs in the antigen-treated group.

Figure 8

Induction of Antigen-Specific T Cell Responses in Local Draining Lymphoid Tissues after i.d. Injection (A–F) Responses of transferred BDC2.5 CD4⁺ T cells to mRNA-NPs at a dose of 5 μg mRNA/mouse (A–C) or mRNA-DCs at a dose of 0.6 μg/1 × 10⁶ electroporated DCs/mouse (D–F) were analyzed 3 days after i.d. injection. The bar graphs (B, C, E, and F) show the mean ± SD from three to six biological replicates, and the significant differences indicated for several lymphoid tissues are relative to ILNs in the antigen-treated group.