Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

Two shortcomings of nonviral gene therapy are a lack of tissue-specific targeting of vectors and low levels of gene transfer. Our laboratory has begun to address these limitations by designing plasmids that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing expression in a controlled manner. We have shown that a 176 bp portion of the smooth muscle gamma-actin (SMGA) promoter can mediate plasmid nuclear import specifically in smooth muscle cells (SMCs). Here, we demonstrate that the binding sites for serum response factor (SRF) and NKX3-1/3-2 within this DNA nuclear targeting sequence (DTS) are required for plasmid nuclear import. Knockdown of these factors with siRNA abrogates plasmid nuclear import, indicating that they are necessary cofactors. In addition, coinjection of recombinant SRF and Nkx3.2 with the vector in TC7 epithelial cells rescues import. Finally, we show that the SRF nuclear localization sequence (NLS) is required for vector nuclear import. We propose that SRF and NKX3-1/3-2 bind the SMGA DTS in the cytoplasm, thus coating the plasmid with NLSs that mediate translocation across the nuclear pore complex. This discovery could aid in the development of more efficient nonviral vectors for gene transfer to SMCs.