324. Successful Repeated Delivery of Helper-Dependent Adenoviral Vector Toachieve Efficient Long-Term Human CFTR Expression in Mouse Airwaythrough Transient Immunosuppression

Abstract

Cystic Fibrosis (CF) is a common life-threatening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel. As a chronic, lifelong disease, CF should be best treated with a continue level of CFTR expression. Pulmonary gene therapy may ultimately cure the CF lung disease. Thus, efficient long-term expression of the delivered CFTR gene in targeted cell types is essential to achieve this goal either by repeated application or with a long-duration expression system. Even though novel approaches to target airway progenitor/stem cells with gene editing may be more attractive, the efficiency for targeting specific progenitor cells will still be a challenge in airway delivery. Repeated administration of viral vectors may be required to increase the percentage of cells to be targeted and to boost long-term therapeutic effects. However, immune responses to viral proteins are the greatest barrier to repeated vector administration. Here, we demonstrated that repeated transduction of human CFTR gene in mouse airway can be successfully achieved with helper-dependent adenoviral (HD-Ad) vector through transient immunosuppression.We showed previously that cyclophosphamide significantly enhanced human CFTR expression in mice received HD-Ad vector readministration and sustained expression through inhibition of host immune reactions. In this study, we examined cyclophosphamide effects on expression of the human CFTR in multiple rounds of HD-Ad vector delivery to mouse airways. Four group mice were nasally delivered with an HD-Ad-K18-CFTR vector at 1.5×1010 vector particles per mouse at day 0, day 70 and day 120. Two groups were treated with cyclophosphamide 6 hours before and 4 day and 8 days after vector delivery each time (except the last round of vector delivery for mice sacrificed at day 123). The other two groups without treatment were used as controls. One treated group and one control group were sacrificed at day 123, and the others at day 153 for detecting transgene expression and immune reaction. We found that cyclophosphamide has significantly improved the expression of CFTR (4.1 times higher) compared to the control group as determined by real-time qPCR 3 days after last delivery. And the CFTR expression level was 3.1 times higher 30 days after last vector transduction. Cyclophosphamide also significantly reduced the levels of antiadenoviral and neutralizing antibodies in both BALF and serum as well as prevented leukocyte infiltration in lung tissues. This data suggested that efficient repeated transduction of human CFTR gene with HD-Ad vector can be achieved by transient immunopuression in mouse lung.