Miller A M, Dean D A
Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
Gene Ther. 2008 Aug;15(15):1107-15. doi: 10.1038/gt.2008.83. Epub 2008 May 22.
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.
非病毒基因治疗的两个缺点是载体缺乏组织特异性靶向性以及基因转移水平较低。我们实验室已开始通过设计在细胞不分裂的情况下能够进入特定细胞类型细胞核的质粒来解决这些局限性，从而以可控方式增强基因表达。我们已经表明，平滑肌γ-肌动蛋白（SMGA）启动子的176 bp部分可以特异性地介导质粒在平滑肌细胞（SMC）中导入细胞核。在此，我们证明该DNA核靶向序列（DTS）中血清反应因子（SRF）和NKX3-1/3-2的结合位点是质粒核导入所必需的。用小干扰RNA（siRNA）敲低这些因子可消除质粒核导入，表明它们是必需的辅助因子。此外，在TC7上皮细胞中将重组SRF和Nkx3.2与载体共注射可挽救导入过程。最后，我们表明SRF核定位序列（NLS）是载体核导入所必需的。我们提出，SRF和NKX3-1/3-2在细胞质中与SMGA DTS结合，从而用介导跨核孔复合体转运的NLS覆盖质粒。这一发现可能有助于开发更有效的用于向SMC进行基因转移的非病毒载体。
