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Revision as of 16:38, 10 September 2016
Introduction
The CFTR gene encodes an ATP-binding cassette (ABC) transporter that functions as a low conductance Cl(-)-selective channel gated by cycles of ATP binding and hydrolysis at its nucleotide-binding domains (NBDs) and regulated tightly by an intrinsically disordered protein segment distinguished by multiple consensus phosphorylation sites termed the regulatory domain
Cystic fibrosis (CF) is the most common lethal genetic disorder in Caucasian populations. It affects mostly the lungs, and also the pancreas, liver and kidneys. The CF disorder is a result of mutations in a gene coding for an ATP-gated ion channel called cystic fibrosis trans membrane conductance regulator - CFTR. Nearly two thousand cystic fibrosis causing mutations in the CFTR gene have been described. The most common mutation is ΔF508, which results from a deletion of three nucleotides, and a consequent loss of the amino acid phenylalanine. A CFTRΔF508 protein is unable to leave the endoplasmic reticulum (ER) and translocate to the plasma membrane.
There is yet no cure for cystic fibrosis, and today CF patients suffering lung infections are treated with antibiotics. Excessive mucus blocking the lungs is removed by mucus thinning drugs and physical exercises. Previous research has demonstrated that the CRISPR technology has the potential to provide a successful treatment for CF. However; this potential has not yet been realized due to the lack of an efficient delivery vehicle which can specifically target, in vivo, the epithelium of the airways and lungs. Therefore, we wish to suggest a new way of targeting the CRISPR components in order to fix the ΔF508 mutation, as described below.
Project Description
The B subunit of the toxin produced by the pathogenic bacterium Vibrio cholera can specifically bind the GM1-like ganglioside receptor on the membrane of epithelial cells – including the asialo-GM1 variant that line the lungs. This binding leads to internalization of the toxin by endocytosis. We, therefore, intend to use this B subunit as a vehicle to specifically deliver the CRISPR/Cas9 expression plasmid into the epithelium of the lungs of CF patients. The CRISPR/Cas9 plasmid will be attached to the B subunit by a cleavable linker and the conjugated molecules will be applied by aerosol spray to the nose. A special “repair template”, designed to replace the ΔF508 mutation by the correct sequence (via homology directed repair - HDR) will also be delivered into the cells by this B subunit in a similar way. Both DNA molecules will be delivered as naked DNA.
