575. CFTR Reporter Construct for Optimization of Airway Epithelium Differentiation

DNA VECTOROLOGY & GENE TARGETING II 575. CFTR Reporter Construct for Optimization of Airway Epithelium Differentiation

Myra E. Gordon,1 Alaleh Esmaeili-Shandiz,1 Shingo Suzuki,1 Roy G. Sargent,1 Dieter C. Gruenert.1,2,3 1 Otolaryngology- Head & Neck Surgery, UCSF, San Francisco, CA; 2Eli & Edythe Broad Institute for Regenerative Medicine and Stem Cell Research, UCSF, San Francisco, CA; 3Institute for Human Genetics, UCSF, San Francisco, CA. Cystic fibrosis (CF) is the most common genetic disorder in the Caucasian population with over 1,000 new cases diagnosed each year in the US alone. While >1800 mutations may lead to CF, a 3 base deletion that leads to the loss of a phenylalanine at codon 508 in the CF transmembrane conductance regulator (CFTR) gene (delF508) is found in 70% of all CF chromosomes. The CFTR protein is a cAMP dependent Cl channel found in the apical membrane of epithelial cells that regulates the Cl gradient at the cell surface. CFTR mutations prevent transport of Cl across the membrane causing thick mucus to build up in the airways and significant airway epithelial pathology. While lung transplantation has been a standard of care for CF patients with terminal disease, the recent development of pluripotent stem cell systems has opened the door to other potential therapeutic paradigms. The development of CF patient-specific induced pluripotent stem (iPS) cells in which the pathological mutation has been corrected has the potential of supplanting conventional allogenic transplantation with the transplantation of autologous cells/tissues. For these cells/tissues to have therapeutic value it will be necessary to generate precursor cells that have the potential of differentiating into cells that will express CFTR. While the CFTR gene has been well characterized, little is known about the regulation of human CFTR throughout development of the airway epithelium. The studies described will evaluate CFTR expression in an in vitro developmental system using iPS cells that are directed to differentiate along an endodermal lineage-specific pathway. A reporter construct consisting of a pcDNA3 backbone with an EGFP under the regulation of a human CFTR promoter region will be used to monitor the dynamics of CFTR expression throughout differentiation of the primitive pluripotent state to the more defined airway epithelial progenitors and their terminal daughter cells. The stably integrated construct will be used to define the expression of the CFTR gene through the expression of EGFP. The expression of EGFP provides a mechanism for the enrichment of CFTR expressing cell populations throughout differentiation. Thus far, the studies indicate that cell-type specific CFTR expression exists regardless of the promoter orientation. Studies are now underway to evaluate the expression of CFTR in the directed endodermal differentiation of CF and non-CF iPS cells. These studies have been supported by Pennsylvania Cystic Fibrosis, Inc (PACFI), Cystic Fibrosis Research, Inc (CFRI) and a Graduate Student Research Training Grant from the California Institute for Regenerative Medicine (CIRM).

576. Non-Viral Gene Delivery to Oral Squamous Cell Carcinoma and Cervical Carcinoma Cells and Suicide Gene Therapy

Nejat Düzgünes,1 Aruna Singh,1 Senait Gebremedhin,1 Krystyna Konopka.1 1 Department of Biomedical Sciences, University of the Pacific School of Dentistry, San Francisco, CA.

Objectives: Suicide gene therapy of oral squamous cell carcinoma (OSCC) may be a viable approach to the treatment of this cancer. However, human OSCC cells are relatively resistant to efficient transfection by non-viral vectors. To identify an optimal vector for gene delivery, we compared the transfection activities and efficiencies of Glycofect, Metafectene, Metafectene Pro, Metafectene Easy and Fugene HD, using the OSCC cell line, HSC-3, and the cervical Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

carcinoma cell line, HeLa. Methods: The cells were seeded at a density of 1.5 x105 in 1 ml of appropriate media in 48-well culture plates one day before transfection, and used at approximately 80% confluence. Complexes were prepared at the optimal ratio of reagent:DNA with the plasmid pCMV.Luc (VR-1216), added in serum-free DMEM, and incubated with the cells for 4 h at 37 °C. Serum-containing medium was added, and the cells were incubated for 48 h. Transfection activity was assayed using the Luciferase Assay System (Promega). To evaluate transfection efficiency, pCMV.lacZ was used in a similar manner, and X-gal was added as a substrate for -galactosidase. For gene therapy experiments, HSV-tk was delivered to the cells, and the cells were incubated with 20 ug/ml ganciclovir for 3, 6 and 9 days. Cell viability was assessed by the Alamar blue assay. Results: Metafectene Easy and Fugene HD mediated the highest transfection activity and efficiency in both cell lines. The activity was higher in HeLa cells than in HSC-3 cells. With suicide gene therapy, HeLa cell viability was 22±3% of controls by 9 days with Fugene HD and 26±3% with Metafectene Easy. With HSC-3 cells, cell viability was 42±25% with Fugene HD, and 58±28% with Metafectene Easy. The reduction in viability was statistically significant in both cases (p0.005; average of 3 independent experiments), although there was considerable variability between experiments with HSC-3 cells. Conclusions: Metafectene Easy and Fugene HD may be useful in gene therapy of OSCC and cervical carcinoma. The reasons for the differential susceptibility of these cells to transfection by non-viral vectors are not known. Supported by funds from the University of the Pacific, Arthur A. Dugoni School of Dentistry.

577. Orphan Product Development for DNA Therapeutics Gladice Wallraven,1 Phillip B. Maples,1 John Nemunaitis.1 1 Gradalis, Inc, Dallas.

The FDA marks its 30th year in establishing the Orphan Drug Act (ODA). This is a program to encourage pharmaceutical and biotech companies to research options for individuals with rare diseases. A rare disease is defined as “any disease or condition that affects less than 200,000 persons in the United States.” Many DNA therapeutics target rare diseases. Due to the cost of drug development, companies usually do not investigate rare diseases since the high costs of drug development are less likely to be recovered due to the smaller populations affected. Receiving orphan product designation, is an important step in the development of a DNA therapeutic. The designation qualifies the product for financial incentives in the form of grants and reduction in the clinical trial taxes and market exclusivity. Furthermore, the FDA fast track approval system allows companies to move a product faster through to commercialization. Gradalis has been granted orphan status for further development of FANG vaccine in 2 disease designations (IIIc ovarian cancer and advanced melanoma). Components of the orphan product request include: 1. A statement of request identifying the rare disease or condition; 2. The name and address of the sponsor; 3. A description of the rare disease or condition, the proposed indication for use, and the reasons why such therapy is needed; 4. A description of the drug and a discussion of the scientific rationale for the use of the drug for the rare disease or condition; 5. A summary of the regulatory status and marketing history of the drug in the United States and in foreign countries; 6. Documentation, with appended authoritative references, to demonstrate that the disease or condition for which the drug is intended affects fewer than 200,000 people in the United States; 7. A statement as to whether the sponsor submitting the request is the real party in interest of the development and the intended or actual production and sales of the product. The processes for obtaining orphan product designation (of three products 08-2671, 11-3360, and 11-3566) through product approval for DNA based experimental therapeutics will be presented. S221