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Cystic fibrosis (CF) is the most common lethal recessive genetic disease in the Caucasian population. It is caused mutations in the CF transmembrane conductance regulator (CFTR) gene which lead to the production of a partially or completely non-functional protein. Lack of CFTR in epithelial cell membranes results in abnormal ion transport and dehydration of mucosal surfaces. In the respiratory tract this leads severe lung disease, the leading cause of morbidity and mortality in the CF population. Since CFTR was first cloned in 1989, a major goal of CF research has been to develop effective gene therapy for CF lung disease. Respiratory syncytial virus (RSV) naturally infects the ciliated cells that line the human respiratory tract, cells that express CFTR and play a major role in determining the hydration of the airway mucosal surface. In addition, although an immune response is mounted against RSV, this does not prevent subsequent infections suggesting that an RSV-based vector might be effectively re-administered a requirement since the apical ciliated cells of the respiratory tract are terminally differentiated and have a defined life span. To test whether the large, 4.5 kb, CFTR gene could be expressed by a recombinant RSV and whether infectious virus could be used to deliver CFTR to ciliated airway epithelium derived from CF patients, we have inserted the CFTR gene into four sites in the recombinant, green fluorescent protein-expressing (rg)RSV genome to generate virus expressing four different amounts of the CFTR protein. Two of these rgRSV-CFTR viruses were capable of expressing CFTR with little effect on viral replication. rgRSV-delivered CFTR was functional in primary CF airway epithelial cultures. Infection with these viruses resulted in chloride channel function similar to that seen in non-CF cultures and correction of additional characteristic defects seen in CF airway epithelia. Unfortunately, most ciliated airway cells infected with RSV die within 5 days. To create an improved RSV-based vector we generated an RSV “replicon” by removing the three glycoprotein genes from a full-length RSV cDNA. The replicon is able to replicate in cells without killing them or producing infectious virus, allowing the isolation and expansion of replicon-containing cells. Providing the viral glycoproteins in trans enables the production of “one-step virus” (OSV) which is capable of delivering a replicon to fresh cells where it produces its encoded proteins, but again cannot spread. We inserted the CFTR gene into the RSV replicon genome in four positions. All of these replicons were capable of producing the CFTR protein. When CFTR-expressing replicon OSV were used to infect primary human airway epithelial cultures, replicon containing cells remained within the cultures for 12-20 days, indicating that replicon containing cells have a survival advantage over those infected with complete virus. Our results indicate that RSV is capable of expressing CFTR, supplying enough functional CFTR to primary CF airway cells to correct their physiologic defects and with modification can remain in primary airway cells for a prolonged period of time. Collectively, our data support further investigation of RSV as a potential vector for CF gene therapy. |
