Optimization of CFTR-mRNA transfection in human nasal epithelial cells

Fernández, Elena Fernández; Bangel-Ruland, Nadine; Tomczak, Katja; Weber, Wolf-Michael

doi:10.1186/s41231-016-0006-0

Cited by 4 publications

(5 citation statements)

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“…Chemical modification, using various techniques, has showed great improvements in the mRNA-based approach, especially in cancer therapy and vaccination for infectious diseases [148]. In vitro studies on differentiated human nasal epithelial primary cells revealed that the minimal dose of wt-CFTR-mRNA (0.6 µg/cm 2 ) delivered by lipofectamine 2000 unregulated the functional electrophysiology of the CFTR channel for over 72 h [149]. The efficacy of this approach was also demonstrated in vivo by Robinson et al (2018), where the CFTR mRNA restored the Clions efflux by 55% in CFTR knockout mice compared to healthy mice [150].…”

Section: Targeting Cftrmentioning

confidence: 99%

Cystic Fibrosis: Overview of the Current Development Trends and Innovative Therapeutic Strategies

et al. 2020

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Cystic Fibrosis (CF), an autosomal recessive genetic disease, is caused by a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This mutation reduces the release of chloride ions (Cl−) in epithelial tissues, and hyperactivates the epithelial sodium channels (ENaC) which aid in the absorption of sodium ions (Na+). Consequently, the mucus becomes dehydrated and thickened, making it a suitable medium for microbial growth. CF causes several chronic lung complications like thickened mucus, bacterial infection and inflammation, progressive loss of lung function, and ultimately, death. Until recently, the standard of clinical care in CF treatment had focused on preventing and treating the disease complications. In this review, we have summarized the current knowledge on CF pathogenesis and provided an outlook on the current therapeutic approaches relevant to CF (i.e., CFTR modulators and ENaC inhibitors). The enormous potential in targeting bacterial biofilms using antibiofilm peptides, and the innovative therapeutic strategies in using the CRISPR/Cas approach as a gene-editing tool to repair the CFTR mutation have been reviewed. Finally, we have discussed the wide range of drug delivery systems available, particularly non-viral vectors, and the optimal properties of nanocarriers which are essential for successful drug delivery to the lungs.

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Section: Targeting Cftrmentioning

confidence: 99%

Cystic Fibrosis: Overview of the Current Development Trends and Innovative Therapeutic Strategies

et al. 2020

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“…Generally, the average size diameter of chitosan-based complexes is determined by DLS. Complexes formed using a series of different chitosans range from few nanometers to less than a micron [ 81 , 93 , 101 , 113 ].…”

Section: Characterization Techniques Of the Biophysical Propertiesmentioning

confidence: 99%

“…Recently, a proof-of-concept mRNA-based functional restoration of impaired CFTR function have been demonstrated, not only in an immortalized human bronchial CF cell line, but also in primary human nasal epithelial (HNE) cells [ 113 , 249 ]. Transfection of the CFBE41o- cells with CFTR-mRNA restored cAMP induced CFTR currents similar to the values seen in control cells (16HBE14o-) and an almost two-fold increase in the cAMP-stimulated CFTR current after transfection using primary cultured HNE cells.…”

Section: Rare Diseasesmentioning

confidence: 99%

“…Transfection of the CFBE41o- cells with CFTR-mRNA restored cAMP induced CFTR currents similar to the values seen in control cells (16HBE14o-) and an almost two-fold increase in the cAMP-stimulated CFTR current after transfection using primary cultured HNE cells. The authors demonstrated that optimized CFTR-mRNA can be reduced to a minimal dose of 0.6 μg/cm 2 in primary HNE cells, and that this dose can be persistent for a period longer than 24 h after transfection procedures [ 113 ]. These experiments successfully established a new strategy for the delivery of CFTR-mRNA directly to airway epithelial cells.…”

Section: Rare Diseasesmentioning

confidence: 99%

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Chitosan in Non-Viral Gene Delivery: Role of Structure, Characterization Methods, and Insights in Cancer and Rare Diseases Therapies

2018

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Non-viral gene delivery vectors have lagged far behind viral ones in the current pipeline of clinical trials of gene therapy nanomedicines. Even when non-viral nanovectors pose less safety risks than do viruses, their efficacy is much lower. Since the early studies to deliver pDNA, chitosan has been regarded as a highly attractive biopolymer to deliver nucleic acids intracellularly and induce a transgenic response resulting in either upregulation of protein expression (for pDNA, mRNA) or its downregulation (for siRNA or microRNA). This is explained as the consequence of a multi-step process involving condensation of nucleic acids, protection against degradation, stabilization in physiological conditions, cellular internalization, release from the endolysosome (“proton sponge” effect), unpacking and enabling the trafficking of pDNA to the nucleus or the siRNA to the RNA interference silencing complex (RISC). Given the multiple steps and complexity involved in the gene transfection process, there is a dearth of understanding of the role of chitosan’s structural features (Mw and degree of acetylation, DA%) on each step that dictates the net transfection efficiency and its kinetics. The use of fully characterized chitosan samples along with the utilization of complementary biophysical and biological techniques is key to bridging this gap of knowledge and identifying the optimal chitosans for delivering a specific gene. Other aspects such as cell type and administration route are also at play. At the same time, the role of chitosan structural features on the morphology, size and surface composition of synthetic virus-like particles has barely been addressed. The ongoing revolution brought about by the recent discovery of CRISPR-Cas9 technology will undoubtedly be a game changer in this field in the short term. In the field of rare diseases, gene therapy is perhaps where the greatest potential lies and we anticipate that chitosans will be key players in the translation of research to the clinic.

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“…In our group, we have been working with wtCFTR-mRNA to improve chloride secretion of epithelial cells constantly optimizing delivery methods [10][11][12]. The most promising delivery systems of nucleic acids to epithelial cells proved to be self-assembled polyelectrolyte chitosan (CS)-based nanocomplexes due to their low cytotoxicity and high transfection efficiency [12][13][14].…”

Section: Introductionmentioning

confidence: 99%

Capsaicin-Loaded Chitosan Nanocapsules for wtCFTR-mRNA Delivery to a Cystic Fibrosis Cell Line

et al. 2020

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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.

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Optimization of CFTR-mRNA transfection in human nasal epithelial cells

Cited by 4 publications

References 53 publications

Cystic Fibrosis: Overview of the Current Development Trends and Innovative Therapeutic Strategies

Cystic Fibrosis: Overview of the Current Development Trends and Innovative Therapeutic Strategies

Chitosan in Non-Viral Gene Delivery: Role of Structure, Characterization Methods, and Insights in Cancer and Rare Diseases Therapies

Capsaicin-Loaded Chitosan Nanocapsules for wtCFTR-mRNA Delivery to a Cystic Fibrosis Cell Line

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