Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis

Abstract

Safe and efficacious systemic delivery of messenger RNA (mRNA) to specific organs and cells in vivo remains the major challenge in the development of mRNA-based therapeutics. Targeting of systemically administered lipid nanoparticles (LNPs) coformulated with mRNA has largely been confined to the liver and spleen. Using a library screening approach, we identified that N-series LNPs (containing an amide bond in the tail) are capable of selectively delivering mRNA to the mouse lung, in contrast to our previous discovery that O-series LNPs (containing an ester bond in the tail) that tend to deliver mRNA to the liver. We analyzed the protein corona on the liver- and lung-targeted LNPs using liquid chromatography-mass spectrometry and identified a group of unique plasma proteins specifically absorbed onto the surface that may contribute to the targetability of these LNPs. Different pulmonary cell types can also be targeted by simply tuning the headgroup structure of N-series LNPs. Importantly, we demonstrate here the success of LNP-based RNA therapy in a preclinical model of lymphangioleiomyomatosis (LAM), a destructive lung disease caused by loss-of-function mutations in the Tsc2 gene. Our lung-targeting LNP exhibited highly efficient delivery of the mouse tuberous sclerosis complex 2 (Tsc2) mRNA for the restoration of TSC2 tumor suppressor in tumor and achieved remarkable therapeutic effect in reducing tumor burden. This research establishes mRNA LNPs as a promising therapeutic intervention for the treatment of LAM.

Keywords: lipid nanoparticles; lung-targeted delivery; lymphangioleiomyomatosis; mRNA; tuberous sclerosis complex.

Fig. 1.

Synthesis and in vivo screening of N-series LNPs. (A) Synthetic route and representative chemical structure of lipidoids. Representative whole-body bioluminescence images of mice (B) and in vivo mRNA delivery efficacy (C) of N-series LNPs measured by the IVIS imaging system. Mice were injected with either of the Luc mRNA–loaded N-series LNPs at a single dose of 0.5 mg/kg. Images were taken at 6 h postinjection (n = 3). Data are presented as mean ± SD; the error bar around each data point is the SEM.

Fig. 2.

Different pulmonary cell types can be targeted by tuning the head structure of N-series LNPs. (A) Schematic illustration of the delivery of Cre mRNA to the lung to activate tdTomato expression via Cre-mediated genetic deletion of the stop cassette in tdTomato transgenic Ai14 mice. (B, C) Chemical structure, representative ex vivo image of tdTomato fluorescence in edited Ai14 mouse organs captured by using the IVIS imaging system, representative immunofluorescence images of lung tissue taken by confocal microscopy, and quantification of the percentage of tdTomato+ cells within defined cell types of the lungs by flow cytometry of 306-N16B (B) and 113-N16B (C) LNPs. Mice were i.v. injected with Cre mRNA–loaded LNPs at a single dose of 0.75 mg mRNA equiv./kg. Endothelial cells were stained by FITC (fluorescein isothiocyanate)-CD31 antibody, epithelial cells were stained by CD326-PE-Cy7 antibody, and macrophages were stained by F4/80-eFluor 660 antibody (Scale bar, 20 μm).

Fig. 3.

Proteomics study of protein coronas formed on LNPs. (A) Schematic illustration of different organ targetability of O- and N-series LNPs. (B) Interaction of LNPs with proteins in the blood vessel. Quantification of percentage of total proteins of the top three protein components in the protein corona of the 306-O12B LNP (C) and the 306-N16B LNP (D) is shown (n = 3). The top 20 most abundant corona proteins were categorized based on their calculated molecular weight (E), isoelectric point (F), and biological function (G).

Fig. 4.

The 306-N16B LNP allows systemic codelivery of Cas9 mRNA and sgRNA in vivo. (A) Schematic illustration of codelivery of Cas9 mRNA and sgLoxP to activate the tdTomato expression in Ai14 mice. (B) Representative ex vivo image of organs collected from Cas9 mRNA and sgLoxP coloaded 306-N16B LNP–treated transgenic Ai14 mice. Mice were injected via tail vein at a dosage of 1.67 mg/kg of total RNA. (C) Representative microscopy images of the lung and liver dissected from Cas9 mRNA and sgLoxP coencapsulated 306-N16B LNP–treated Ai14 mice (Scale bar, 100 μm).

Fig. 5.

hLNP enables specific delivery of mRNA to the TTJ tumor cells in vivo. (A) Schematic illustration of the preparation of hybrid LNP. hLNP was formulated at a molar ratio of 25:25:38.5:10:1.5 of 306-N16B:306-O12B:cholesterol:DOPC:DMG-PEG2000. (B) Representative images of IHC analysis of EGFP in mouse LAM lungs. Syngeneic C57BL/6J mice were tail vein injected with TSC2-deficient TTJ cells to form tumor nodules in the lungs. Mice were tail vein injected with an hLNP loaded with EGFP mRNA. Lungs were collected 6 h postinjection. (C) Multiplex immunofluorescence staining shows colocalization of pS6 (a marker of tumor cell) and EGFP. (Left) Staining of pS6; (Right) staining of EGFP.

Fig. 6.

Therapeutic effect of TSC2 mRNA–loaded hLNP in antitumor growth. (A) Treatment design. Syngeneic C57BL/6J mice were tail vein injected with 2 × 10⁶ TSC2-deficient TTJ cells to form tumor nodules in the lung. On day 24 after tumor cell inoculation, mice were randomly assigned to three groups: untreated control, empty LNP treatment, and LNP-mTsc2 (0.75 mg/kg of mRNA per injection) treatment. Mice were treated every other day for a total of five times (n = 5). (B) Fraction of tumor nodules per lung in the three treatment groups. (C) Representative images of IHC assessment of ki67, cleaved caspase 3, macrophages, and CD3 in empty hLNP and TSC2 mRNA–loaded hLNP. (D) Quantitative analysis of C. Ten images were analyzed for each group. T cell: CD3 IHC; apoptosis: cleaved caspase 3 IHC; proliferation: Ki67 IHC; macrophage: F4/80 IHC. Data are shown in mean ± SD; the error bar around each data point is the SEM. Student’s t test was used to calculate the statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.