Gottfried L, Lin X, Barravecchia M, Dean D A
Department of Pediatrics, School of Medicine and Dentistry, University of Rochester, Rochester, NY, USA.
Gene Ther. 2016 Oct;23(10):734-742. doi: 10.1038/gt.2016.52. Epub 2016 Jul 1.
The ability to restrict gene delivery and expression to particular cell types is of paramount importance for many types of gene therapy, especially in the lung. The alveolar epithelial type I (ATI) cell, in particular, is an attractive cell type to target, as it comprises 95% of the internal surface area of the lung. We demonstrate, through microinjection of fluorescently labeled plasmids, that a DNA sequence within the rat T1α promoter was able to mediate ATI cell-specific plasmid DNA nuclear import due to the binding of ATI-enriched transcription factors. Promoter deletion analysis and site-directed mutagenesis of specific transcription-factor-binding sites within the +101 to -200 bp region of the T1α promoter identified HNF3 and TTF-1 as critical transcription factors for import. To test for nuclear import in vivo, plasmids expressing GFP from the CMV promoter were delivered into the lungs of mice by electroporation and evaluated immunohistochemically 48 h later. Plasmids carrying the 1.3 kbp T1α sequence resulted in GFP expression almost exclusively in ATI cells. This represents a new and highly efficient way to target a specific lung epithelial cell type both in vitro and in vivo based on the restriction of DNA nuclear import.
将基因传递和表达限制在特定细胞类型的能力对于许多类型的基因治疗至关重要，尤其是在肺部。特别是肺泡I型上皮（ATI）细胞，是一个有吸引力的靶向细胞类型，因为它占肺内表面积的95%。我们通过显微注射荧光标记的质粒证明，大鼠T1α启动子内的一个DNA序列能够介导ATI细胞特异性的质粒DNA核输入，这是由于富含ATI的转录因子的结合。对T1α启动子+101至-200 bp区域内特定转录因子结合位点的启动子缺失分析和定点诱变确定HNF3和TTF-1是核输入的关键转录因子。为了在体内测试核输入，将从CMV启动子表达GFP的质粒通过电穿孔导入小鼠肺部，并在48小时后进行免疫组织化学评估。携带1.3 kbp T1α序列的质粒几乎只在ATI细胞中导致GFP表达。基于DNA核输入的限制，这代表了一种在体外和体内靶向特定肺上皮细胞类型的新的高效方法。
