Gene therapy could offer improved treatment or a cure of cystic fibrosis. However current attempts at gene therapy for the disease, focusing on viral or conventional plasmid vectors to introduce the CFTR gene into target cells, have serious inherent limitations. Dr. Calos and colleagues propose a new approach that uses stable extrachromosomal replicating vectors to carry the CFTR gene. The applicant's laboratory has developed a unique class of DNA vectors that replicate autonomously and are retained for long periods of time in human and other mammalian cells. These vectors lack the immunogenicity and size limits of viruses and are retained in cells much longer than conventional plasmid DNA. Gene expression from the vectors is correspondingly prolonged. These vectors can be introduced efficiently into lung epithelial cells in vivo by aerosol or intratracheal instillation of novel lipid:DNA complexes. Stable autonomously replicating vectors will be first adapted to function in primary and differentiated cells by provision of a universal promoter to the EBNA-1 gene. In order to correct the mutant CFTR gene of cystic fibrosis, they will place a specially adapted CFTR expression cassette on the vectors. Gene expression and function will be assayed over a time course of at least two months in lung epithelial tissue culture cells. Vectors will be complexed with the novel lipid EDMPC and introduced into animals for pre-clinical testing. If warranted, clinical trial in patients will be arranged.
The Specific Aims of this proposal are thus to: 1) extend the utility of the extrachromosomal replicating vectors for application to primary cell cultures of airway epithelium, 2) to employ the extrachromosomal replicating vectors for expressing CFTR in airway epithelial cells, and 3) to employ the extrachromosomal vector for CFTR production in vivo in a rodent model system. This proposal pairs an effective new type of vector with a safe method for non-viral DNA delivery, creating a much needed new approach for gene therapy of cystic fibrosis.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Mckeon, Catherine T
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Stanford University
Schools of Medicine
United States
Zip Code
Sclimenti, Christopher R; Neviaser, Andrew S; Baba, Edward J et al. (2003) Epstein-Barr virus vectors provide prolonged robust factor IX expression in mice. Biotechnol Prog 19:144-51
Stoll, S M; Sclimenti, C R; Baba, E J et al. (2001) Epstein-Barr virus/human vector provides high-level, long-term expression of alpha1-antitrypsin in mice. Mol Ther 4:122-9
Groth, A C; Olivares, E C; Thyagarajan, B et al. (2000) A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci U S A 97:5995-6000
Sclimenti, C R; Baba, E J; Calos, M P (2000) An extrachromosomal tetracycline-regulatable system for mammalian cells. Nucleic Acids Res 28:E80
Thyagarajan, B; Guimaraes, M J; Groth, A C et al. (2000) Mammalian genomes contain active recombinase recognition sites. Gene 244:47-54
Phillips, J E; Calos, M P (1999) Effects of homology length and donor vector arrangement on the efficiency of double-strand break-mediated recombination in human cells. Somat Cell Mol Genet 25:91-100
Calos, M P (1998) Stability without a centromere. Proc Natl Acad Sci U S A 95:4084-5
Sclimenti, C R; Calos, M P (1998) Epstein-Barr virus vectors for gene expression and transfer. Curr Opin Biotechnol 9:476-9