Kobayashi N, Rosenthal E R, Yoshimura K, Crystal R G
Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Nucleic Acids Res. 1994 Oct 25;22(21):4470-6. doi: 10.1093/nar/22.21.4470.
The expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be down-regulated by inflammatory stimuli such as phorbol myristate acetate (PMA). Since the respiratory manifestations of cystic fibrosis (CF) are characterized by intense chronic airway inflammation very early in life, successful gene therapy for CF will require that expression of the transferred normal CFTR gene be resistant to down-regulation by inflammatory mediators. To evaluate the concept that a viral promoter--human CFTR cDNA unit would be resistant to this form of down-regulation, a retrovirus promoter (5' long terminal repeat of the Moloney murine leukemia virus)--human CFTR cDNA unit was transferred to T84 human colon carcinoma cell line using a retrovirus vector. Exposure of the retrovirus-modified T84 cells to PMA resulted in down-regulation of the endogenous CFTR mRNA transcripts (6.5 kb), but did not affect the level of exogenous CFTR transcripts (8.0 kb). Importantly, in parallel with the persistence of the exogenous CFTR transcripts, the modified cells still maintained cAMP-regulated CI- secretion in the presence of PMA. These in vitro data suggest that a constitutive viral promoter--CFTR cDNA unit should be resistant to modulation by inflammatory stimuli, a likely requirement for successful gene therapy for CF.
囊性纤维化跨膜传导调节因子（CFTR）基因的表达可被诸如佛波醇肉豆蔻酸酯乙酸酯（PMA）等炎性刺激下调。由于囊性纤维化（CF）的呼吸道表现特征为在生命早期就存在强烈的慢性气道炎症，因此成功的CF基因治疗将要求所转移的正常CFTR基因的表达对炎性介质的下调具有抗性。为了评估病毒启动子-人CFTR cDNA单位对这种下调形式具有抗性的概念，使用逆转录病毒载体将逆转录病毒启动子（莫洛尼鼠白血病病毒的5'长末端重复序列）-人CFTR cDNA单位转移至T84人结肠癌细胞系。将经逆转录病毒修饰的T84细胞暴露于PMA导致内源性CFTR mRNA转录本（6.5 kb）下调，但不影响外源性CFTR转录本（8.0 kb）的水平。重要的是，与外源性CFTR转录本的持续存在同时，修饰后的细胞在存在PMA的情况下仍维持cAMP调节的Cl-分泌。这些体外数据表明，组成型病毒启动子-CFTR cDNA单位应能抵抗炎性刺激的调节，这可能是CF成功基因治疗的一个必要条件。
