Towards human translation of lentiviral airway gene delivery for cystic fibrosis: A one-month CFTR and reporter gene study in marmosets

2021 ◽  
Author(s):  
Nigel R Farrow ◽  
Patricia Cmielewski ◽  
Juliette Delhove ◽  
Nathan Rout-Pitt ◽  
lewis Vaughan ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Delivery ◽  
Reporter Gene ◽  
Gene Study

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

Biomedicines ◽  
2020 ◽  
Vol 8 (9) ◽  
pp. 364
Author(s):  
A. Katharina Kolonko ◽  
Janes Efing ◽  
Yadira González-Espinosa ◽  
Nadine Bangel-Ruland ◽  
Willy van Driessche ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Delivery ◽  
Cell Line ◽  
Single Gene ◽  
Ussing Chamber ◽  
Hereditary Disease ◽  
Cellular Level ◽  
Hydrodynamic Diameter ◽  
Target Cells ◽  
Gene Coding

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.

Non–invasive liposome–mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice

1993 ◽  
Vol 5 (2) ◽  
pp. 135-142 ◽  
Author(s):  
E. W. F. W. Alton ◽  
P. G. Middleton ◽  
N. J. Caplen ◽  
S. N. Smith ◽  
D. M. Steel ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Delivery ◽  
Ion Transport ◽  
Mutant Mice ◽  
Non Invasive ◽  
Transport Defect

scholarly journals Gene delivery systems—gene therapy vectors for cystic fibrosis

2004 ◽  
Vol 3 ◽  
pp. 203-212 ◽  
Author(s):  
Daniel Klink ◽  
Dirk Schindelhauer ◽  
Andreas Laner ◽  
Torry Tucker ◽  
Zsuzsanna Bebok ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Therapy ◽  
Gene Delivery ◽  
Delivery Systems ◽  
Gene Therapy Vectors

Selenium Nanoparticles in Folate-Targeted delivery of the pCMV-Luc DNA Reporter Gene

2020 ◽  
Vol 16 ◽  
Author(s):  
Fiona C Maiyo ◽  
Londiwe S Mbatha ◽  
Moganavelli Singh
Keyword(s):  
Gene Delivery ◽  
Reporter Gene ◽  
Targeted Delivery ◽  
Transgene Expression ◽  
Folate Receptor ◽  
Selenium Nanoparticles ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Breast Adenocarcinoma ◽  
Mcf 7

Background: Selenium, an essential micronutrient, has been studied for decades for its anticancer properties. Selenium nanoparticles (SeNPs) have now emerged as an interesting alternative for drug and gene delivery. Aims: We aimed to demonstrate in proof of principle, the potential use of SeNPs in targeted pCMV-Luc DNA (pDNA) delivery in vitro. Objectives: To chemically synthesize, characterize and evaluate the transgene expression of functionalized SeNPs in five human cell lines. Methods: SeNPs were synthesized via chemical reduction, coated with chitosan (Ch) and a targeting moiety folic acid (FA). All nanoparticles were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), UV-vis and Fourier transform infra-red (FTIR) spectroscopy. Nanoparticle:pDNA interactions were assessed using the electrophoretic mobility shift, dye displacement and nuclease protection assays. The MTT and Luciferase reporter gene assays were used to determine cytotoxicity and transgene expression respectively, in the human colorectal adenocarcinoma (HT-29 and Caco-2), breast adenocarcinoma (MCF-7), oral epidermoid/cervical carcinoma contaminant (KB) and the embryonic kidney (HEK293) cells. Results: Homogenous nanoparticles of 60-70 nm were able to successfully bind, compact and protect the pDNA from enzyme digestion. Low cytotoxicity was observed in all cells, except for the MCF-7 cells, which could be attributed to apoptosis and necrosis. Luciferase gene expression was highest for the targeted nanocomplexes in the folate-receptor rich KB cell line, confirming nanocomplex uptake through folate receptor-mediated endocytosis. Conclusion: This study opens a new avenue for synergistic treatment of cancer, combining selenium’s bioactivity and its carrier potential for therapeutic gene delivery.

scholarly journals Modifier gene study of meconium ileus in cystic fibrosis: statistical considerations and gene mapping results

2009 ◽  
Vol 126 (6) ◽  
pp. 763-778 ◽  
Author(s):  
Ruslan Dorfman ◽  
Weili Li ◽  
Lei Sun ◽  
Fan Lin ◽  
Yongqian Wang ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Mapping ◽  
Modifier Gene ◽  
Meconium Ileus ◽  
Gene Study

scholarly journals Melittin Enables Efficient Vesicular Escape and Enhanced Nuclear Access of Nonviral Gene Delivery Vectors

2001 ◽  
Vol 276 (50) ◽  
pp. 47550-47555 ◽  
Author(s):  
Manfred Ogris ◽  
Robert C. Carlisle ◽  
Thierry Bettinger ◽  
Leonard W. Seymour
Keyword(s):  
Gene Expression ◽  
Gene Delivery ◽  
Cell Lines ◽  
Reporter Gene ◽  
Umbilical Vein ◽  
Reporter Gene Expression ◽  
Nonviral Gene Delivery ◽  
Nonviral Gene Transfer ◽  
Gene Delivery Vectors ◽  
C Terminus

Entry of exogenously applied DNA into the cytoplasm and subsequent transport into the nucleus are major cellular barriers for nonviral gene delivery vectors. To overcome these barriers, we have covalently attached the cationic peptide melittin to poly(ethylenimine) (PEI). This conjugate condensed DNA into small, discrete particles (<100 nm in diameter), and the membrane lytic activity of melittin enabled efficient release of the DNA into the cytoplasm, as monitored by fluorescence microscopy and flow cytometry. Compared with PEI, the transfection activity was strongly increased within a broad range of cell lines and types tested, including different tumor cell lines but also primary hepatocytes and human umbilical vein endothelial cells. The early onset of gene expression (within 4 h, reaching maximal values after 12 h) and the high reporter gene expression achieved in slowly dividing or confluent cells suggested a further role of melittin after releasing the DNA into the cytoplasm. Intracytoplasmic microinjection of melittin-containing PEI·DNA complexes into fibroblasts produced 40% cellular frequency of reporter gene expression that was inhibitable by co-injection of wheat germ agglutinin, whereas simple PEI·DNA complexes showed only 10%. These data suggest that melittin enables release of nonviral gene transfer particles into the cytoplasm and also enhances their transport into the nucleus, possibly via the cationic cluster KRKR near the C terminus of the peptide.

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