Development of approaches to restore the cystic fibrosis transmembrane conductance regulator to treat cystic fibrosis

Abstract

Cystic fibrosis (CF) is a devastating monogenetic disorder affecting about 1 in 2,500 live births amongst the Caucasian population. The disorder affects secretory tissues including the lungs, liver, pancreas, intestine, kidneys and reproductive organs causing their clogging with thick mucus. Up to 80% of deaths result from lung-related disorders due to mucus buildup in the airways causing respiratory failure and chronic chest infections. The condition drastically reduces life expectancy and is currently the number one life-threatening disorder affecting children in New Zealand. CF is the result of autosomal recessive mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene encodes a cyclic-AMP regulated channel for chloride ion transport. The mutation perturbs the normal transport of chloride and sodium ions across the epithelial cell layer, which results in the formation of a detrimental thick mucus layer. Currently there is no cure for this disorder, and therapy is based on pharmacological intervention to provide symptomatic relief and slowing of disease progression. The ideal treatment would be to restore the normal gene and/or protein function to essentially provide a cure for the condition. The CFTR gene was cloned in 1989 and its cloning has paved the way for gene therapy. Modification of the genetic content of cells or tissues that contain the aberrant CFTR gene, or the introduction of the normal gene into the relevant tissues of a CF affected individual by use of a vector would alleviate and cure the disorder. Xentry (LCLRPVG) is a novel cell-penetrating peptide (CPP) encompassing residues 16 to 22 of the X-protein of the hepatitis B virus. The Xentry peptide is efficiently taken up into cells through energy-dependent clathrin-mediated endocytosis by a process involving heparan sulfate moieties expressed on cell-surface syndecan 4. Xentry has demonstrated ability to successfully deliver a wide variety of different types of cargo into cells. Thus, Xentry fused to a KALA peptide was seen to successfully deliver anti-B-raf siRNA to melanoma cells in culture that are B-raf dependent for survival, leading to tumor cell apoptosis. Xentry labeled with Cy7 and TAMRA and delivered intravenously into mice demonstrated uptake into the epithelial tissues of most major organs including lungs, intestine, pancreas, kidneys. There was heightened accumulation of Xentry within epithelia overlying the bronchiole airways, stomach and intestinal tract. The Xentry peptide was not sequestered or diluted by uptake into non-targeted blood cells when administered systemically. The above characteristics of Xentry make it a potential vector for application in gene therapy for the treatment of disorders that manifest in epithelial tissue such as cystic fibrosis. The aim of this project was to investigate the potential of the novel CPP Xentry to serve as a gene therapy vector for the treatment of single gene disorders like CF. The ability of a Xentry peptide to deliver nucleic acid cargoes into the CF airway epithelial cell line CuFi-1 in the form of peptide nucleic acids (PNAs), and corrective short single-stranded DNAs to potentially correct the mutant CFTR gene missing the delta508 codon was assessed. In addition, a Xentry peptide was examined for its ability to deliver eGFP mRNA into the CuFi-1 cell line as a proof of principle of its ability to deliver CFTR mRNA, as a potential cure for CF. The Xentry peptide fused to a nuclear localization sequence (NLS) from the SV40 virus and to a protamine peptide, which allowed for the binding and condensation of nucleic acid cargoes, demonstrated only low levels of transfection of eGFP mRNA due to endosomal entrapment. PEGylation of the Xentry-NLS-protamine peptide together with inclusion of the toll-like receptor (TLR) small molecule inhibitor E6446 resulted in efficient transfection of CuFi-1 cells with eGFP mRNA. This result suggests that Xentry has the potential to successfully deliver CFTR mRNA into cells to cure CF patients of disease. The above studies have provided important insights into the potential of the novel CPP Xentry, and how it might be modified and applied for the treatment of single gene disorders such as CF.