Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences
Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences
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DOI：
10.1038/gt.2008.83
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发表时间：
2008-08-01
期刊：
影响因子：
5.1
通讯作者：
Dean, D. A.
中科院分区：
文献类型：
--
作者：
Miller, A. M.;Dean, D. A.
非病毒基因治疗的两个缺点是缺乏载体的组织特异性靶向和低水平的基因转移。我们的实验室已经开始通过设计在没有细胞分裂的情况下进入特定细胞类型的细胞核的质粒来解决这些限制，从而以受控的方式增强表达。我们已经证明，平滑肌γ-肌动蛋白（SMGA）启动子的176 bp部分可以介导质粒核输入特异性平滑肌细胞（SMC）。在这里，我们证明了血清反应因子（SRF）和NKX 3 -1/3-2的结合位点在这个DNA核靶向序列（NKX 3）是质粒核输入所需的。用siRNA敲低这些因子消除质粒核输入，表明它们是必需的辅因子。此外，将重组SRF和Nkx3.2与载体共注射到TC 7上皮细胞中拯救了输入。最后，我们表明，SRF核定位序列（NLS）是必要的载体核进口。我们提出SRF和NKX 3 -1/3-2结合细胞质中的SMGA酶，从而用介导跨核孔复合物易位的NLS包被质粒。这一发现有助于开发更有效的非病毒载体，用于将基因转移到SMC。
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.