Chu Q, St George J A, Lukason M, Cheng S H, Scheule R K, Eastman S J
Genzyme, Framingham, MA 01701, USA.
Hum Gene Ther. 2001 Mar 20;12(5):455-67. doi: 10.1089/104303401300042348.
Administration of recombinant adenoviral (AdV) vectors to animals can lead to inflammatory and immune responses. For therapeutic indications in which repeated treatment is necessary, such as cystic fibrosis (CF), these responses can limit the therapeutic usefulness of the vector. In principle, the utility of the vector can be improved by increasing its therapeutic index, that is, by either increasing its efficacy or decreasing its toxicity. A strategy that would enhance the efficacy of an adenoviral approach would allow the use of fewer virus particles to achieve a given level of transgene expression, and thereby also reduce unwanted effects such as immune responses. Following up on our observation that treating polarized normal human bronchial epithelial cells with calcium (Ca(2+))-free medium or the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) significantly enhanced the subsequent transfection of these cells with cationic lipid:pDNA complexes, we have now asked whether such a treatment protocol might also improve the ability of AdV to infect these cells. Treating polarized airway epithelial cells with EGTA led to a dramatic increase in AdV-mediated transduction, as demonstrated by an approximately 50-fold increase in transgene expression. This strategy was also tested in vivo and resulted in substantial increases (up to 50-fold) in the ability of AdV vectors to infect mouse tracheal epithelium. Transfection of mouse trachea with an AdV aerosol was also significantly increased by pretreatment with EGTA. The enhancing effects of EGTA could not be duplicated with hypo- or hyperosmotic treatments. Light microscopy of mouse trachea that had been EGTA treated and then infected with AdV demonstrated an EGTA-mediated AdV infection of airway epithelial cells. The apparent enhanced potency of AdV for airway cells resulting from this strategy provides a significant increase in the therapeutic index of this gene delivery vector, and may increase the likelihood that it can be used for clinical indications requiring chronic administration of the vector.
向动物体内注射重组腺病毒（AdV）载体可引发炎症和免疫反应。对于诸如囊性纤维化（CF）这类需要重复治疗的疾病，这些反应会限制载体的治疗效用。原则上，可通过提高其治疗指数来提升载体的效用，也就是要么增强其疗效，要么降低其毒性。一种能够增强腺病毒疗法疗效的策略，将允许使用更少的病毒颗粒来实现给定水平的转基因表达，从而也能减少诸如免疫反应等不良影响。基于我们的观察结果，即使用无钙（Ca(2+)）培养基或钙螯合剂乙二醇双（β-氨基乙基醚）-N,N,N',N'-四乙酸（EGTA）处理极化的正常人支气管上皮细胞，能显著增强这些细胞随后被阳离子脂质:pDNA复合物转染的能力，我们现在探究这样的处理方案是否也能提高AdV感染这些细胞的能力。用EGTA处理极化的气道上皮细胞会使AdV介导的转导显著增加，转基因表达增加了约50倍就证明了这一点。该策略也在体内进行了测试，结果是AdV载体感染小鼠气管上皮的能力大幅提高（高达50倍）。用EGTA预处理也显著提高了用AdV气溶胶转染小鼠气管的效率。用低渗或高渗处理无法重现EGTA的增强效果。对经EGTA处理然后感染AdV的小鼠气管进行光学显微镜检查，显示气道上皮细胞存在EGTA介导的AdV感染。这种策略使AdV对气道细胞的效力明显增强，显著提高了这种基因递送载体的治疗指数，并可能增加其用于需要长期给药载体的临床适应症的可能性。
