PECAM-1 directed re-targeting of exogenous mRNA providing two orders of magnitude enhancement of vascular delivery and expression in lungs independent of apolipoprotein E-mediated uptake

Abstract

Systemic administration of lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) leads predominantly to hepatic uptake and expression. Here, we conjugated nucleoside-modified mRNA-LNPs with antibodies (Abs) specific to vascular cell adhesion molecule, PECAM-1. Systemic (intravenous) administration of Ab/LNP-mRNAs resulted in profound inhibition of hepatic uptake concomitantly with ~200-fold and 25-fold elevation of mRNA delivery and protein expression in the lungs compared to non-targeted counterparts. Unlike hepatic delivery of LNP-mRNA, Ab/LNP-mRNA is independent of apolipoprotein E. Vascular re-targeting of mRNA represents a promising, powerful, and unique approach for novel experimental and clinical interventions in organs of interest other than liver.

Keywords: Apolipoprotein E; Endothelial targeting; Inflammation; Vascular targeting; mRNA delivery.

Fig. 1.

Physicochemical characterization of targeted mRNA containing lipid nanoparticles. (A) Schematic illustration of the use of antibodies against endothelial cell surface markers for development of lung-targeted LNPs. Amino groups on antibodies were functionalized with heterobifunctional crosslinker (SATA) for introduction of thiol moieties on antibody surface followed by maleimide-thiol conjugation to maleimide-bearing LNP-mRNAs. (B) The average (n = 3) intensity size distribution curves for the unconjugated LNP-mRNA (gray trace) and antibody-conjugated LNP-mRNAs (black and red traces). (C) Particle size (z-average) and surface charge of particles measured using dynamic light scattering (DLS) and laser doppler velocimetry (LDV), respectively (n =3). Images taken by transmission electron microscopy of (D) unconjugated LNP-mRNA, and (E) antibody-conjugated LNP-mRNA, scale bar: 100 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

Binding and functional activity of targeted particles in vitro. (A) In vitro binding of targeted LNP-mRNAs to PECAM-1 positive and negative REN cells after 1 h incubation of ¹²⁵I-labeled anti-PECAM-1/LNP-mRNA with cells at RT (*P < 0.05). (B) mRNA encoded protein expression of anti-PECAM-1/LNP-mRNA in REN-PECAM-1 positive cells compared to control IgG/LNP-mRNA (#P < 0.05). The inset shows the luciferase activity for unconjugated LNP-mRNA. (C) In vitro eGFP expression of control IgG and anti-PECAM-1 conjugated eGFP-mRNA-LNPs in REN-PECAM-1 positive cells, 6 μg mRNA per well.

Fig. 3.

Targeting of LNP-mRNA to PECAM-1 in vivo. (A) Biodistribution of ¹²⁵I-labeled anti-PECAM mAb- and control IgG-LNP-mRNAs in mice at 30 min. Tissue uptake is indicated as mean ± SEM (n =3). (*P < 0.05 and **P < 0.001). (B) Immunospecificity index, calculated as the ratio of %ID/g of selected organs in mice treated with targeted (anti-PECAM-1) vs. non-targeted (control IgG)-LNP-mRNAs, normalized to blood levels. In vivo kinetics of LNP-binding as quantitative measurement of the percentage of PECAM-1-targeted (C), Control IgG- (D) and unconjugated (E) mRNA-loaded LNPs evaluated by radioactivity analysis in selected organs, after intravenous injection of nanoparticles.

Fig. 4.

Flow cytometric analysis of cell populations receiving PECAM-1 targeted LNPs in lung tissue. Staining was performed against CD31 for endothelial cells, CD45 for leukocytes, and F4/80 for monocytes/macrophages. (A) Pie chart representative of total cell recovery from lung. (B) Percent of sub-cell populations positive for LNPs.

Fig. 5.

Cell toxicity/inflammatory profile of LNP-mRNA. Effect of anti-PECAM mAb-, control IgG-, and unconjugated LNP-mRNAs on REN cell viability measured by colorimetric MTS assay upon 4.5 h (A1), 24 h (A2), and 48 h (A3) incubation with LNPs. %Viability is indicated as mean ± SEM (n = 3). (B) Western blot showing HUVEC cell lysates (10μg total protein/lane) stained for human VCAM-1 and actin. An increase in VCAM-1 protein expression was induced by LPS, but not by LNP-mRNA treatment. Pro-inflammatory cytokines IL-6 in plasma (C) and MIP-2 in liver homogenate (D) upon treatment with LNP-mRNA (8 μg/mouse) were compared to the untreated samples. LPS (2 mg/kg) was used as positive control here.

Fig. 6.

Organ distribution of firefly luciferase mRNA expression 4.5 h after intravenous administration of unconjugated, anti-PECAM-1 mAb- and control IgG/LNP-mRNAs demonstrated as (A) firefly luciferase activity and (B) luminescence imaging. (A) Quantitative expression as LU/mg protein values compared between non-targeted and targeted LNP. Data presented as mean ± SEM (n = 3), (*P < 0.05). (B) A representative sample set of mouse organs, which were analyzed 5 min after the administration of D-luciferin. (C) Transfection-specificity index, calculated as the ratio of luciferase activity in selected organs of mice treated with targeted (anti-PECAM-1) vs. non-targeted (control IgG)-LNP-mRNAs. (D) Lung to liver ratio, calculated as the ratio of transfection efficiency of lung to that of liver for each formulation. (E) Dose-response relationship of Luc mRNA containing anti-PECAM-1-LNPs. Mice received LNPs at doses of 1, 2, 4, and 8 μg mRNA per mouse via intravenous administration. Selected organs were harvested at 4.5 h post-treatment and firefly luciferase activity was measured in tissue extracts.

Fig. 7.

In vivo kinetics of firefly luciferase expression following LNP-mRNA administration. Quantitative measurement of firefly luciferase activity in (A) liver and (B) lung upon intravenous injection of unconjugated- and anti-PECAM-1/LNP-mRNA; mRNA dose: 8 μg/mouse.

Fig. 8.

Firefly luciferase mRNA expression in apoE knockout mice. Unconjugated, control IgG, and anti PECAM-1 Luc mRNA-LNPs were intravenously injected into mice. Mice were sacrificed 4.5 h after injection and firefly luciferase activity in livers and lungs of wild type mice was compared to apoE knockout mice. Data presented as mean ± SEM (n =3); (*P < 0.05).