Niedzinski E J, Chen Y-J, Olson D C, Parker E A, Park H, Udove J A, Scollay R, McMahon B M, Bennett M J
Genteric, Inc., Alameda, CA 94502, USA.
Gene Ther. 2003 Dec;10(26):2133-8. doi: 10.1038/sj.gt.3302125.
Gene transfer to the major salivary glands is an attractive method for the systemic delivery of therapeutic proteins. To date, nonviral gene transfer to these glands has resulted in inadequate systemic protein concentrations. We believe that identification of the barriers responsible for this inefficient transfection will enable the development of enhanced nonviral gene transfer in salivary glands and other tissues. One potential barrier is the degradation of plasmid DNA by endonucleases. To test this hypothesis, we coadministered two endonuclease inhibitors ((zinc and aurintricarboxylic acid (ATA)) with plasmid DNA, containing the secreted alkaline phosphatase gene (SEAP), to the submandibular glands of rats. The effect of zinc and ATA on SEAP expression, tissue accumulation of plasmid DNA, and plasmid DNA stability was then characterized. We observed that mixtures containing zinc/DNA, ATA/DNA, and zinc/ATA/DNA significantly enhanced both systemic transgene expression and the amount of plasmid DNA associated with treated tissues. The relative endonuclease inhibitory activity of zinc, ATA, and zinc/ATA correlated with the observed effects on transfection efficacy. The use of zinc/ATA enhanced the efficacy of salivary gland transfection by at least 1000-fold versus DNA alone. Importantly, this improved performance resulted in robust systemic secretion of an exogenous protein (SEAP), thus demonstrating the potential this nonviral gene transfer technology has as a method to treat systemic protein deficiencies.
基因转移至主要唾液腺是一种用于全身递送治疗性蛋白质的有吸引力的方法。迄今为止，向这些腺体进行的非病毒基因转移导致全身蛋白质浓度不足。我们认为，确定造成这种低效转染的障碍将有助于开发增强的唾液腺及其他组织中的非病毒基因转移方法。一个潜在的障碍是核酸内切酶对质粒DNA的降解。为了验证这一假设，我们将两种核酸内切酶抑制剂（锌和金精三羧酸（ATA））与含有分泌性碱性磷酸酶基因（SEAP）的质粒DNA共同给予大鼠的颌下腺。然后对锌和ATA对SEAP表达、质粒DNA的组织蓄积以及质粒DNA稳定性的影响进行了表征。我们观察到，含有锌/DNA、ATA/DNA和锌/ATA/DNA的混合物显著增强了全身转基因表达以及与处理组织相关的质粒DNA量。锌、ATA和锌/ATA的相对核酸内切酶抑制活性与观察到的对转染效率的影响相关。与单独使用DNA相比，使用锌/ATA可使唾液腺转染效率提高至少1000倍。重要的是，这种性能的改善导致了外源蛋白（SEAP）的强劲全身分泌，从而证明了这种非病毒基因转移技术作为一种治疗全身蛋白质缺乏症方法的潜力。
