In Vivo Nasal Potential Difference: Techniques and Protocols for Assessing Efficacy of Gene Transfer in Cystic Fibrosis

Abstract

Cystic fibrosis (CF) is a monogenetic disease that is associated with chronic airways disease and early death. The pulmonary disease reflects mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and associated abnormal epithelial ion transport, including defective cAMP-mediated (CFTR) Cl¯ secretion and an accelerated rate of basal Na⁺ transport. With the development of vectors for gene therapy, the airway epithelium of CF patients has been targeted for studies of gene transfer. The biological efficacy of gene transfer of the normal CFTR cDNA into CF respiratory epithelia can be assessed by in vivo measurements of the transepithelial potential difference (PD), a parameter of ion transport that reflects the expression and function of CFTR. This paper describes techniques that can be used to discriminate in vivo between the ion transport phenotype of normal subjects and patients with cystic fibrosis. Protocols are outlined to allow assessment of individual components of the electrolyte transport phenotype, i.e., the magnitude of the basal and cAMP-mediated (CFTR) Cl¯ secretion, and the rate of Na⁺ transport. The physiologic basis of the protocols and important technical features of these measurements are defined. If performed properly, the in vivo nasal PD technique clearly discriminates between normal subjects and cystic fibrosis patients, and can yield estimates of the biological efficacy of gene transfer to achieve correction of the electrolyte transport defects in CF patients. Cystic fibrosis (CF) is a recessive genetic disease that reflects mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Chronic pulmonary disease reflects abnormal ion transport by airway epithelium, which is the target for gene transfer using viral and liposomal gene transfer vectors. Protocols involving measurement of potential difference (PD) in vivo have been developed to discriminate between the ion transport phenotype for normal subjects and CF patients. These protocols allow assessment of the basal and cAMP-mediated (CFTR) Cl¯ secretion, and the rate of Na⁺ transport. Taken together, these methodologies allow in vivo estimates of the biological efficacy of gene transfer of the normal CFTR cDNA to respiratory epithelium of CF patients.