515. Sequence Requirements for Alveolar Epithelial Cell-Specific Plasmid Nuclear Import

Abstract

Top of pageAbstract Non-viral DNA gene delivery has many advantages over viral vectors, including potential for repeated administration and minimal immunological response. However, to date, the gene transfer efficiency remains low with this type of vector. This low efficiency is due to several barriers non-viral vectors must overcome prior to transgene expression. Perhaps the most important barrier in non-viral gene transfer is the nuclear envelope. In non-dividing cells, theplasmid DNA has no access to the nucleus where the transcriptional machinery resides, and thus transgenes are not expressed. However, inclusion of specific sequences termed DNA targeting sequences (DTS) on plasmids will mediate nuclear import via the nuclear pore complex and enhance non-viral DNA gene transfer. These sequences, when included on a plasmid, will bind newly-synthesized transcription factors in the cytoplasm and the plasmid DNA will translocate into the nucleus via the nuclear localization signals on the bound transcription factors. Additionally, we can use cell-specific DTSs to mediate nuclear import only in specific cell types, thus limiting transgene expression to the cell type of choice. Using this novel strategy, we can improve both safety and efficiency of non-viral gene therapy by targeting the nucleus of only specific cell types. We have demonstrated previously that the 365 bp fragment of the human SP-C promoter (|[minus]|318 to +47) will mediate nuclear import of plasmid DNA in alveolar epithelial cells but in no other cell types tested to date. Within this 365 nucleotide fragment, there exist several known binding sites for transcription factors. Several truncations of the SP-C promoter have been constructed, as well as mutations that will abrogate transcription factor binding to the SP-C promoter DNA. Using microinjection and in situ hybridization strategy in both A549 and MLE-12 cell lines, we have determined which sequences within the SP-C promoter are required for nuclear import these alveolar epithelial cell lines. These data gives us a further understanding as to the mechanism of DTS function, knowledge that will allow us in the future to predict or create new DTSs for targeted plasmid DNA transfer to many other cell types.