Abstract
816. RNAi Applications for Respiratory Epithelia: Delivery and Efficacy
Molecular therapy, Vol.11(S1), pp.S317-S317
05/01/2005
DOI: 10.1016/j.ymthe.2005.07.359
Abstract
The airway epithelium is a common site of disease pathogenesis, including viral infections and primary diseases associated with inflammation. The delivery of RNAi to respiratory epithelia offers an approach to inhibit replication of RNA viruses and modify host responses to infection. To this end, we are using respiratory syncytial virus (RSV) as a model disease target. RSV is a major cause of respiratory disease in infants and immunocompromised adults. There is no approved vaccine for RSV, and medical treatment is supportive. The primary site of RSV replication is in the respiratory epithelium. As a proof of principle for RNAi delivery to the conducting airways of mice, we used gel-formulated adenoviral vectors to deliver short hairpin RNAs (shRNAs) against GFP or an irrelevant control to respiratory epithelia of GFP-transgenic mice. Vector transduced cells were visualized by expression of a reporter gene (dsRed or beta-galactosidase) to localize cells with putative RNAi activity. This approach achieved efficient transduction of the conducting airway epithelium. The efficiency of RNAi against GFP is measured by reduction of GFP in transduced cells. In a complementary approach, we are evaluating uptake of Cy3 labeled siRNA oligos in polarized human airway epithelial (HAE) cell culture and in mouse models. To inhibit RSV expression in vitro and in vivo, shRNAs were designed against the viral nucleocapsid (N) and large polymerase (L) genes of the RSV A2 strain and cloned into an expression vector behind a mU6 promoter. A misdirected control shRNA was produced by the same method to control for off-target effects of shRNA. Short hairpin expression vectors were screened for RNAi activity against renilla luciferase-viral gene fusions in co-transfection experiments, and functional constructs were identified. Subsequently, antiviral activity of effective shRNAs was measured in an in vitro infection model by reduction of viral RNA (Real Time-PCR) and viral protein (western blot). We will present our progress in these studies.
Details
- Title: Subtitle
- 816. RNAi Applications for Respiratory Epithelia: Delivery and Efficacy
- Creators
- Anthony J FischerChristine L Wohlford-lenanePatrick L SinnHong Peng JiaSteven M VargaBeverly L DavidsonPaul B Mccray
- Resource Type
- Abstract
- Publication Details
- Molecular therapy, Vol.11(S1), pp.S317-S317
- Publisher
- Elsevier Limited
- DOI
- 10.1016/j.ymthe.2005.07.359
- ISSN
- 1525-0016
- eISSN
- 1525-0024
- Language
- English
- Date published
- 05/01/2005
- Academic Unit
- Pathology; Microbiology and Immunology; Internal Medicine; Stead Family Department of Pediatrics; Graduate College Admin and Gen; Pulmonary Medicine
- Record Identifier
- 9984297627502771
Metrics
3 Record Views