On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles

Abstract

Lipid nanoparticles (LNPs) are a clinically mature technology for the delivery of genetic medicines but have limited therapeutic applications due to liver accumulation. Recently, our laboratory developed selective organ targeting (SORT) nanoparticles that expand the therapeutic applications of genetic medicines by enabling delivery of messenger RNA (mRNA) and gene editing systems to non-liver tissues. SORT nanoparticles include a supplemental SORT molecule whose chemical structure determines the LNP's tissue-specific activity. To understand how SORT nanoparticles surpass the delivery barrier of liver hepatocyte accumulation, we studied the mechanistic factors which define their organ-targeting properties. We discovered that the chemical nature of the added SORT molecule controlled biodistribution, global/apparent pK_a, and serum protein interactions of SORT nanoparticles. Additionally, we provide evidence for an endogenous targeting mechanism whereby organ targeting occurs via 1) desorption of poly(ethylene glycol) lipids from the LNP surface, 2) binding of distinct proteins to the nanoparticle surface because of recognition of exposed SORT molecules, and 3) subsequent interactions between surface-bound proteins and cognate receptors highly expressed in specific tissues. These findings establish a crucial link between the molecular composition of SORT nanoparticles and their unique and precise organ-targeting properties and suggest that the recruitment of specific proteins to a nanoparticle's surface can enable drug delivery beyond the liver.

Keywords: endogenous targeting; gene editing; lipid nanoparticles; mRNA delivery.

Fig. 1.

SORT nanoparticles for tissue-specific mRNA delivery have unique biodistribution and ionization behavior. (A) By adding a fifth, supplemental SORT molecule to a conventional, four-component LNP (mDLNP: 23.8 mol % 5A2-SC8, 23.8 mol % DOPE, 47.6 mol % cholesterol, and 4.8 mol % C14-PEG2K), the tissue-specific activity of delivered mRNA changes based on the chemical structure of the included SORT molecule. An ionizable cationic lipid (DODAP) enhances liver-specific mRNA translation (liver SORT: 19 mol % 5A2-SC8, 19 mol % DOPE, 38 mol % cholesterol, 4 mol % C14-PEG2K, and 20 mol % DODAP), an anionic lipid (18PA) results in spleen-specific mRNA translation (spleen SORT: 16.7 mol % 5A2-SC8, 16.7 mol % DOPE, 33.3 mol % cholesterol, 3.3 mol % C14-PEG2K, and 30 mol % 18PA), and a cationic quaternary ammonium lipid (DOTAP) results in lung-specific mRNA translation (lung SORT: 11.9 mol % 5A2-SC8, 11.9 mol % DOPE, 23.8 mol % cholesterol, 2.4 mol % C14-PEG2K, and 50 mol % DOTAP). (B) Ex vivo fluorescence of Cy5-labeled mRNA in major organs extracted from C57BL/6 mice IV injected with SORT LNPs that incorporate increasing percentages of different SORT molecules (0.5 mg/kg mRNA/body weight, 6 h). (C) Relative average Cy5 fluorescence measured in the liver, lung, and spleen as a function of SORT molecule percent inclusion (0.5 mg/kg mRNA/body weight, n = 2). SORT molecules promote mRNA biodistribution to target organs. Data are shown as mean ± SEM. (D) Representative TNS assay curves for determining the apparent pK_a of SORT LNPs incorporating increasing percentages of ionizable cationic, anionic, or permanently cationic lipid SORT molecules. Apparent pK_a was defined as the point at which 50% of TNS fluorescence was achieved. (E) LNPs were assigned a tissue specificity index based on the tissues in which functional luciferase mRNA was detected. Intermediate tissue specificity indexes represent LNPs in which mRNA activity was detected in multiple organs; these LNPs are plotted in the region of the organ for which higher activity was measured. For the 67 LNPs tested with the TNS assay, LNP apparent pK_a was correlated with the specificity of luciferase mRNA tissue delivery.

Fig. 2.

Multiple steps are involved in the mechanism of SORT LNP tissue targeting, including formation of unique protein coronas. (A) A proposed three-step endogenous targeting mechanism for tissue-specific mRNA delivery by SORT LNPs in which 1) PEG lipid desorption 2) enables distinct plasma proteins to bind SORT LNPs, 3) resulting in cellular internalization in the target tissues by receptor-mediated uptake. (B) Ex vivo bioluminescence of major organs excised from C57BL/6 mice IV injected with liver, spleen, and lung SORT LNPs incorporating either sheddable PEG lipids (C14-PEG2K) or less sheddable PEG lipids (C18-PEG2K) (0.1 mg FLuc mRNA/kg body weight, 6 h). Total luminescence produced by each organ is reduced when less sheddable PEG lipid is used, suggesting that PEG lipid desorption is a key process for efficacious mRNA delivery by SORT LNPs. (C) Quantification of total luminescence produced by functional protein translated from FLuc mRNA in target organs of C57BL/6 mice IV injected with liver, spleen, and lung SORT LNPs incorporating either C14- or C18-PEG2K (0.1 mg FLuc mRNA/kg body weight, 6 h). (D) ELISA quantification of serum hEPO in C57BL/6 mice treated with liver, spleen, or lung SORT LNPs encapsulating hEPO mRNA (0.1 mg hEPO mRNA/kg body weight, 6 h). Using a less-sheddable PEG reduces SORT LNP potency. (E) SDS–PAGE of the plasma proteins adsorbed to the surface of mDLNP, liver SORT, spleen SORT, and lung SORT LNPs. LNPs with different organ-targeting properties bind distinct plasma proteins. (F) The average abundance of proteins with distinct biological functions in the protein coronas of mDLNP and liver, spleen, and lung SORT LNPs. The choice of SORT molecule leads to large-scale differences in the functional ensemble of plasma proteins which bind the LNP. (G) Isoelectric point distribution for the most enriched proteins which constitute 80% of the protein corona of the LNPs. A SORT molecule’s headgroup structure influences the pI distribution of the protein corona. (H) The top five most abundant plasma proteins that bind different SORT LNPs (n = 3). The chemical structure of SORT molecule affects the number one plasma protein that is most highly enriched on the surface of SORT LNPs. Data are shown as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student’s t test (*P < 0.05).

Fig. 3.

Distinct plasma proteins regulate SORT LNP uptake and efficacy in vitro. (A) SORT LNPs were preincubated with either ApoE, β2-GPI, or Vtn prior to treating relevant cell lines to measure cellular uptake (Cy5 mRNA tracking) or functional mRNA delivery (bioluminescence). (B) Representative images of cellular uptake of uncoated and coated SORT LNPs taken up by relevant cell types. Incubating a SORT LNP with the protein most avidly binds increases mRNA uptake in cell lines expressing the cognate receptor (250 ng mRNA per well, 1.5 h). (Scale bar: 50 µm.) (C) Quantification of Cy5 mRNA fluorescence in cells treated with uncoated or coated SORT LNPs (250 ng mRNA per well, 1.5 h, n = 10). Statistical significance was determined using an unpaired two-tailed Student’s t test (****P < 0.0001, *P < 0.05). (D) Activity of functional luciferase protein translated from mRNA delivered by uncoated or protein-coated SORT LNPs in relevant cell lines (25 ng mRNA, 24 h, n = 4). Statistical significance determined using one-way ANOVA with Brown–Forsythe test (****P < 0.0001, ***P < 0.001, *P < 0.05). Individual proteins exclusively bind to specific SORT LNPs and enhance mRNA delivery only to cell lines expressing the cognate receptor. Data are shown as mean ± SEM.

Fig. 4.

Extrahepatic mRNA delivery occurs via an ApoE-independent mechanism. (A) Ex vivo bioluminescence produced by functional protein translated from FLuc mRNA in major organs excised from WT C57BL/6 mice IV injected with mDLNP or liver, spleen, or lung SORT LNPs (0.1 mg/kg FLuc mRNA, 6 h). The role of ApoE on SORT LNP efficacy varies based on the chemical structure of the included SORT molecule. (B) Ex vivo bioluminescence produced by functional protein translated from FLuc mRNA in major organs excised from ApoE^−/− mice IV injected with mDLNP or liver, spleen, or lung SORT LNPs (0.1 mg/kg FLuc mRNA, 6 h). (C) Quantification of total bioluminescence produced by target organs excised from WT and ApoE^−/− mice treated with mDLNP or liver, spleen, or lung SORT LNPs (0.1 mg/kg FLuc mRNA, 6 h, n = 3). Data are shown as mean ± SEM. Statistical significance was determined using an unpaired two-tailed Student’s t test (**P < 0.01, *P < 0.05, ns, P > 0.05). Elimination of ApoE from the serum using genetic knockout results in a marked reduction of hepatic mRNA delivery by mDLNP and liver SORT LNPs. In contrast, spleen SORT LNPs have enhanced spleen targeting when ApoE is depleted from the serum, while the efficacy of lung SORT LNPs is unaffected by ApoE elimination.