Gene transfer vector development to treat lung disease: The use of a dual-function Lentiviral vector containing ENaC RNAi and the CFTR gene to treat Cystic Fibrosis lung disaease

Cystic Fibrosis (CF) is a degenerative disorder that is often associated with chronic lung disease. CF is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel, which lead to defective chloride and sodium ion movement across epithelia. Subsequent dehydration of the airway surface liquid (ASL) on airway epithelia, is associated with poor mucociliary clearance and chronic lung infection. The monogenic nature of CF, along with the accessibility of the lung, makes the disease amenable to gene replacement therapy. Gene therapy clinical trials have focused on replacing the mutated CFTR with a functional copy, which has led to improved chloride transport, but has shown no significant effects on sodium transport. An alternative strategy for CF gene therapy therefore, could be to reduce the expression of the epithelial sodium channel (ENaC) in the lung, using RNA interference (RNAi), combined with CFTR delivery. Developing a dual-function gene transfer vector could potentially restore chloride and sodium levels in the ASL and help alleviate CF lung disease. The aim of this thesis was to develop a recombinant lentivirus delivery system capable of simultaneously delivering CFTR expression and knocking down ENaC expression in the airways. A modular HIV vector genome plasmid was developed to allow simple insertion of various promoter elements, transgenes and knockdown sequences, for subsequent virus production. Insertion of the CFTR transgene and a short-hairpin RNA (shRNA) sequence targeting the ENaC alpha subunit (ENaCα) resulted in significant knockdown of human ENaCα and simultaneous expression of CFTR in A549 (human lung carcinoma) cell culture. Replacement of the ENaCα shRNA with an shRNA targeting the transcription factor BACH1 resulted in target gene knockdown and concomitant HMOX1 up-regulation, confirming specific knockdown effects, and demonstrating that the dual-function rLV vector could mediate target gene knockdown irrespective of the target. Attempts were made to knock down BACH1 in primary cultures of human bronchial epithelial cells grown at the air-liquid interface (ALI), but improved transduction efficiencies from the apical surface will be required to generate successful knockdown in this experimental model. These studies provide proof-of-principle for the utility of this versatile dual-function prototype virus. The dual function vector not only has the potential for treatment of CF lung disease, but could be readily altered to target other lung diseases where combinations of prolonged target gene knockdown and gene expression/up-regulation could collectively provide an appropriate therapy. In conclusion, the focus on the rational design of gene transfer vectors for specific therapeutic effects will aid the development and translation of gene therapy approaches to human studies.

Keywords: Air-liquid interface
Gene therapy
Cystic fibrosis
Lentivirus
ENaC
Epithelial sodium channel
CFTR
RNA interference
shRNAmir
shRNA
Advisor: Gill, D
Hyde, S