Although non-viral gene therapy has great potential for use in the lung, several problems including inefficient gene transfer and expression and the relative lack of cell-specific targeting have limited its applications. The two approaches that have been used to target genes to desired cell types are through specific ligand-receptor interactions at the cell surface and through cell-specific promoters to limit transcription to the desired cell type. We have developed a novel third approach to target genes to specific cells based on selective nuclear import of plasmids in non-dividing cells. Using a microinjection and in situ hybridization approach, I tested several potential DNA sequences for their ability to mediate plasmid nuclear import in alveolar type II epithelial (ATII) cells. Of these, only a sequence within the human surfactant protein C (SP-C) promoter was able to mediate nuclear localization of plasmid DNA specifically in ATII cells but not in other cell types. I have mapped the minimal import sequence to the proximal 318 nucleotides of the promoter, and demonstrate that binding sites for NFI, TTF-1, and GATA-6 are required for import activity. Depletion of any of these three transcription factors from ATII cell lines using siRNA abolishes nuclear import activity of the SP-C promoter, further suggesting that the proteins are necessary for DNA nuclear import. However, they are not sufficient for ATII cell-specific import, since expression of all three in non-ATII cells does not allow SP-C DNA nuclear import. Following delivery of DNA to the lungs of mice using electroporation, I demonstrate that the SP-C promoter sequence will enhance delivery and gene expression specifically in ATII cells in mouse lung. I hypothesize that ATII-specific transcription factors bind this sequence in the cytoplasm and shuttle the plasmid into the nucleus via their nuclear localization signals. This mechanism represents a novel activity for the SP-C promoter. This ATII cell-specific nuclear import of DNA may prove to be a safe and effective method for restricted and enhanced gene expression in ATII cells

Last modified

• 08/16/2018

Creator

• James Vincent DeGiulio

DOI

• https://doi.org/10.21985/N2F42P

Subject

• Integrated Graduate Program in the Life Sciences

Keyword

• nuclear import
• SP-C
• gene therapy
• plasmid
• non-viral

Date created

• 2007-09-24

Resource type

• Dissertation

Rights statement

• In Copyright