Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Aug 1;26(8):2034-2046.
doi: 10.1016/j.ymthe.2018.05.014. Epub 2018 Jun 15.

Lipid Nanoparticle-Delivered Chemically Modified mRNA Restores Chloride Secretion in Cystic Fibrosis

Ema Robinson  1 , Kelvin D MacDonald  2 , Kai Slaughter  1 , Madison McKinney  1 , Siddharth Patel  1 , Conroy Sun  3 , Gaurav Sahay  4
Affiliations

Lipid Nanoparticle-Delivered Chemically Modified mRNA Restores Chloride Secretion in Cystic Fibrosis

Ema Robinson et al. Mol Ther. .

Abstract

The promise of gene therapy for the treatment of cystic fibrosis has yet to be fully clinically realized despite years of effort toward correcting the underlying genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR). mRNA therapy via nanoparticle delivery represents a powerful technology for the transfer of genetic material to cells with large, widespread populations, such as airway epithelia. We deployed a clinically relevant lipid-based nanoparticle (LNP) for packaging and delivery of large chemically modified CFTR mRNA (cmCFTR) to patient-derived bronchial epithelial cells, resulting in an increase in membrane-localized CFTR and rescue of its primary function as a chloride channel. Furthermore, nasal application of LNP-cmCFTR restored CFTR-mediated chloride secretion to conductive airway epithelia in CFTR knockout mice for at least 14 days. On day 3 post-transfection, CFTR activity peaked, recovering up to 55% of the net chloride efflux characteristic of healthy mice. This magnitude of response is superior to liposomal CFTR DNA delivery and is comparable with outcomes observed in the currently approved drug ivacaftor. LNP-cmRNA-based systems represent a powerful platform technology for correction of cystic fibrosis and other monogenic disorders.

Keywords: cystic fibrosis; gene therapy; ion transport; mRNA therapeutics; nanoparticles; nasal potential difference.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1
Figure 1
LNP Particle Characterization, Uptake, and Efficacy Particle size distribution of LNP-cmCFTR was measured through (A) nanoparticle tracking analysis and (B) dynamic light scattering. (C) CF and CF-WT bronchial epithelial cells were exposed to varying concentrations of LNP-cmRNA. Luciferase expression was normalized to cell viability (relative light units [RLU]/relative fluorescent units [RFU], gray bars), and Cy5 uptake was normalized to cell count (RFU, gray line) (n = 3, mean ± SD). (D) Intranasal lung instillation of LNP-cmFLuc (0.6 mg cmRNA/kg) was conducted in BALB/c mice (n = 4). At 12 hr, organs were harvested, and luciferase intensity was measured using IVIS.
Figure 2
Figure 2
Exposure to LNP-cmCFTR Enriches Membrane-Integrated CFTR In Vitro (A) CFTR-WT is core-glycosylated (CG) in the endoplasmic reticulum, after which it moves through the trans-Golgi network to become complex-glycosylated (CxG) and reaches the plasma membrane, where it acts as a chloride channel. CFTR-F508del fails to achieve complex glycosylation and rapidly undergoes degradation. (B) Western blot detection of CFTR in untreated and treated CF cell lysates (lanes 3 and 4). Lanes 1 and 2 contained complex- and core-glycosylated CFTR migration standards. β-Actin was used as a loading control. (C and D) Immunocytochemistry detection of CFTR was performed on treated (CF + LNP− cmCFTR) and untreated cells (CF-WT or CF) grown either under (C) standard growth conditions or (D) at the air-liquid interface until polarization. White arrows highlight the CFTR-enriched plasma membrane. Red, CFTR; blue, nucleus; green, actin.
Figure 3
Figure 3
In Vitro Translation of LNP-Delivered cmCFTR mRNA Yields Functional CFTR (A) Schematic diagram of the MQAE assay, illustrating that increasing intracellular fluorescence is proportional to chloride efflux, a quantifiable byproduct of CFTR function. (B) CF-WT and CF cells were grown in chamber slides and with or without treatment with LNP-cmCFTR. The MQAE assay was performed to measure chloride efflux over 240 s (expressed as fluorescence divided by fluorescence at 0 s, Ft/F0). CF-WT cells served as a positive control for chloride efflux, and CF-WT cells treated with CFTRInh-172, a CFTR-specific inhibitor, served as a negative control. (C) Ft/F0 values obtained for each condition at 30 s and 90 s. n = 25 cells/condition; mean ± SEM; ***p < 0.005; NSD, no significant difference by unpaired t test.
Figure 4
Figure 4
LNP-cmCFTR Delivery to the Nasal Epithelium of CFKO Mice Recovers Chloride Efflux (A) Schematic diagram illustrating the correlation between NPD traces and ion transport. (B) RT-PCR analysis of CFKO mouse nostrils 24 hr after treatment with LNP-cmCFTR. Negative control, untreated mice; positive control, lysed LNP-cmCFTR; loading control, GAPDH. (C) Representative NPD traces for a single mouse preceding and following LNP-cmCFTR exposure. (D–F) NPD was recorded in a cohort of CFKO mice (pretreatment NPD) prior to exposure to LNP-cmCFTR or sham exposure. Additional NPDs were recorded on days 1, 3, 7, and 14 post-transfection. NPD response at each time point is shown in normal control mice (n = 11), sham-treated mice (days 1, 7, and 14: n = 1; day 3: n = 4), and LNP-cmCFTR CFKO mice (n = 5) for (D) baseline NPDs, (E) ENaC response, and (F) CFTR response. Mean ± SEM; ***p < 0.005, **p < 0.01, *p < 0.05; paired t test. Symbols: arrow, activation; line with bar, inhibition; dashed arrow, no effect.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cystic Fibrosis Foundation (2018) About Cystic Fibrosis. https://www.cff.org/What-is-CF/About-Cystic-Fibrosis/.
    1. Riordan J.R., Rommens J.M., Kerem B., Alon N., Rozmahel R., Grzelczak Z., Zielenski J., Lok S., Plavsic N., Chou J.L. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989;245:1066–1073. - PubMed
    1. Welsh M.J. Abnormal regulation of ion channels in cystic fibrosis epithelia. FASEB J. 1990;4:2718–2725. - PubMed
    1. Chu C.S., Trapnell B.C., Curristin S., Cutting G.R., Crystal R.G. Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA. Nat. Genet. 1993;3:151–156. - PubMed
    1. Welsh M.J., Anderson M.P., Rich D.P., Berger H.A., Denning G.M., Ostedgaard L.S., Sheppard D.N., Cheng S.H., Gregory R.J., Smith A.E. Cystic fibrosis transmembrane conductance regulator: a chloride channel with novel regulation. Neuron. 1992;8:821–829. - PubMed

Publication types

Substances