Disease relevance of CFTR

• Genomic DNA sequence of Rhesus (M. mulatta) cystic fibrosis (CFTR) gene [1].
• Here we describe a novel approach (aerosolization) to deliver a replication-deficient adenovirus carrying the CFTR gene (Ad.CFTR) to the airways [2].
• Hypotheses that link mutations in CFTR and defects in ion transport to CF lung disease predict that alterations in airway surface liquid (ASL) isotonic volume, or ion composition, are critically important [3].

High impact information on CFTR

• The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been proposed to be an epithelial cell receptor for Pseudomonas aeruginosa involved in bacterial internalization and clearance from the lung [4].
• Treatment of primate fetuses at 110 days gestation with an adenovirus expressing the cystic fibrosis transmembrane conductance regulator (cftr) gene resulted in accelerated differentiation of the lung [5].
• The technique of concurrent flow spirometry was used to determine the deposited dose of Ad2/CFTR-2, which ranged from 3 to 8 x 10(10) I.U. Transgene DNA was detected by the polymerase chain reaction (PCR) in lung tissue from all treated animals, and human CFTR mRNA was detected on days 3, 7, and 21 post-exposure [6].
• These results indicate that transduction of the primate airway epithelium with the AAV-CFTR mediates long-term CFTR cDNA transfer and is relatively safe [7].
• No CFTR-specific proliferation of peripheral blood lymphocytes was detected [8].

Biological context of CFTR

• Cystic fibrosis is a common human genetic disease caused by mutations in CFTR, a gene that codes for a chloride channel that is regulated by phosphorylation and cytosolic nucleotides [1].
• Expression of CFTR mRNA was demonstrated on Rhesus monkey tracheobronchial tissue by reverse transcription polymerase chain reaction (RT-PCR) analysis [9].
• Technology evaluation: AAV-CFTR vector, targeted genetics [10].
• To attenuate Ad-CFTR further, a mutation was introduced in the cis-acting regulatory sequences that control the encapsidation of the viral genome [11].
• We show that Ad5 can mobilize the defective Ad-CFTR genome in vitro and in cotton rats [11].

Anatomical context of CFTR

• The Rhesus monkey has been used as a model for evaluating the possibility of introducing the CFTR gene into the airway epithelium in vivo [9].
• By immunofluorescent light microscopy, CFTR was localized on the apical plasma membrane of ciliated cells as we previously described for human tracheobronchial surface epithelium [9].
• However, the extent of the complementation in vivo by wild-type virus is limited because no additional spreading or shedding of Ad-CFTR to trachea, lungs, and stools is elicited [11].

Analytical, diagnostic and therapeutic context of CFTR

• As part of a program to discover natural animal models for human genetic diseases, we have determined the genomic sequence of CFTR in the Rhesus monkey, Macaca mulatta [1].
• A sensitive, real-time, RNA-specific PCR method for the detection of recombinant AAV-CFTR vector expression [12].
• Finally, we demonstrate the utility of this new RS-PCR method in detecting recombinant AAV-CFTR expression, including, an in vitro transduction assay and methods to support both preclinical and clinical trials [12].
• In order to test the safety and biological activity of AAV-CFTR, single doses of AAV-CFTR vector were administered by fiberoptic bronchoscopy to the posterior basal segment of the right lower lobe (RLL) of the lungs of 10 rhesus macaques with four matched vehicle-treated controls [7].
• Treatment of cystic fibrosis by gene therapy will require the development of vectors capable of efficient and safe transfer of a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia [11].

References