GENE-TRANSFER INTO THE AIRWAY EPITHELIUM OF ANIMALS BY TARGETING THE POLYMERIC IMMUNOGLOBULIN RECEPTOR
GENE-TRANSFER INTO THE AIRWAY EPITHELIUM OF ANIMALS BY TARGETING THE POLYMERIC IMMUNOGLOBULIN RECEPTOR
复制标题
DOI：
10.1172/jci117690
复制
发表时间：
1995-02-01
影响因子：
15.9
通讯作者：
DAVIS, PB
中科院分区：
文献类型：
--
作者：
FERKOL, T;PERALES, JC;DAVIS, PB
通过利用受体介导的多聚免疫球蛋白受体的内吞作用，可以高效地将感兴趣的基因特异性靶向完整动物的呼吸道上皮细胞。一种DNA载体，由针对大鼠分泌成分的多克隆抗体的Fab部分共价连接到聚-L-赖氨酸，被用来引入含有不同的报告基因的质粒到气道上皮细胞在体内。我们在肝脏和肺的蛋白质提取物中观察到显著水平的荧光素酶活性，分别达到每毫克蛋白质提取物13，795 +/-4，431和346，954 +/-199，120积分光单位（ILU）的最大值。在不表达受体的脾或心脏中未检测到荧光素酶活性。使用由与真正载体结合的不相关质粒（pCMV lacZ）或基于不相关Fab片段与载体结合的表达质粒（pGEMluc）组成的复合物进行转染，导致所有检查的组织中荧光素酶活性的背景水平。因此，只有含有携带多聚免疫球蛋白受体的细胞的组织被转染，并且转染不能归因于不相关的载体-DNA复合物的非特异性摄取。在转染动物的肺中也检测到来自荧光素酶基因的特异性mRNA。为了确定肺中的哪些细胞通过该方法转染，制备了含有表达质粒的DNA复合物，所述表达质粒具有编码细菌β-半乳糖苷酶或人白介素2受体的基因。这些基因的表达定位于气道和粘膜下腺体的表面上皮，而不是细支气管和肺泡。受体介导的内吞作用可用于将功能基因导入大鼠呼吸道上皮，并可能成为一种有用的肺靶向基因治疗技术。
Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795+/-4,431 and 346,954+/-199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lung are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.