Abstract
Cystic fibrosis (CF), a lethal hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene coding for an epithelial chloride channel, is characterized by an imbalanced homeostasis of ion and water transports in secretory epithelia. As the disease is single-gene based, transcript therapy using therapeutic mRNA is a promising concept of treatment in order to correct many aspects of the fatal pathology on a cellular level. Hence, we developed chitosan nanocapsules surface-loaded with wtCFTR-mRNA to restore CFTR function. Furthermore, we loaded the nanocapsules with capsaicin, aiming to enhance the overall efficiency of transcript therapy by reducing sodium hyperabsorption by the epithelial sodium channel (ENaC). Dynamic light scattering with non-invasive back scattering (DLS-NIBS) revealed nanocapsules with an average hydrodynamic diameter of ~200 nm and a Zeta potential of ~+60 mV. The results of DLS-NIBS measurements were confirmed by asymmetric flow field-flow fractionation (AF4) with multidetection, while transmission electron microscopy (TEM) images confirmed the spherical morphology and size range. After stability measurements showed that the nanocapsules were highly stable in cell culture transfection medium, and cytotoxicity was ruled out, transfection experiments were performed with the CF cell line CFBE41o-. Finally, transepithelial measurements with a new state-of-the-art Ussing chamber confirmed successfully restored CFTR function in transfected cells. This study demonstrates that CS nanocapsules as a natural and non-toxic delivery system for mRNA to target cells could effectively replace risky vectors for gene delivery. The nanocapsules are not only suitable as a transcript therapy for treatment of CF, but open aspiring possibilities for safe gene delivery in general.
Highlights
Messenger RNA is a single-stranded nucleic acid, which transfers genetic information to the ribosome, where protein synthesis takes place
Nanocapsules, either naked or co-loaded with wtCFTR-Messenger RNA (mRNA) at P/N charge ratio 75, were determined with DLS-NIBS measurements and from their electrophoretic mobility. This specific P/N charge ratio was chosen based on the transfection procedure as at P/N 75 the amount of wtCFTR-mRNA, as well as capsaicin are ideal for transfection of CFBE41o- cells
MRNA, as well as capsaicin are ideal for transfection of CFBE41o- cells
Summary
Messenger RNA (mRNA) is a single-stranded nucleic acid, which transfers genetic information to the ribosome, where protein synthesis takes place. The use of mRNA for therapeutic purposes, such as the so-called transcript therapy, has been tested since the early 1990s and has undergone tremendous developments since [1]. MRNA displays many advantages for gene delivery compared to DNA. MRNA is smaller than DNA, providing easier transport into the cell; it . Biomedicines 2020, 8, 364 exhibits reduced immunogenicity and is efficient at any point of the cell cycle [2]. Three clinical trials are testing the efficiency of mRNA-based therapies targeting different proteins, including the cystic fibrosis transmembrane conductance regulator (CFTR) via lipid nanoparticle inhalation for the treatment of cystic fibrosis (CF) [3]
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