Eastman S J, Tousignant J D, Lukason M J, Chu Q, Cheng S H, Scheule R K
Genzyme Corporation, Framingham, MA 01701-9322, USA.
Hum Gene Ther. 1998 Jan 1;9(1):43-52. doi: 10.1089/hum.1998.9.1-43.
Previously, we have described the optimization of the aerosol delivery of a nonviral gene therapy vector to the lungs of rodents (Eastman et al., 1997b). Although aerosolizing cationic lipid:pDNA complexes into a whole-body exposure chamber resulted in high levels of reporter gene expression in the lungs of BALB/c mice, the conditions employed were not optimal for the delivery of lipid:pDNA complexes to the lungs of human patients. That is, the consumption rate of the material in the nebulizer, and thus the delivery time, were very slow and the aerosol was delivered in a continuous flow. Here we describe in vitro experiments used to develop a cationic lipid:pDNA aerosol with characteristics more suitable for delivery to the lungs of humans, as a necessary prerequisite for conducting a clinical study with human cystic fibrosis patients. Using cascade impactors and all-glass impingers, we have screened several commercially available nebulizers for their ability to deliver intact, respirable, active lipid:pDNA complexes in the shortest time possible, and have identified the Pari LC Jet Plus nebulizer as the optimal nebulizer that meets these criteria. Using this nebulizer in an intermittent mode to mimic breath actuation, consumption rates of approximately 0.6 ml/min of the cationic lipid:pDNA complexes (6 mM cationic lipid:8 mM pDNA) were obtained. The plasmid DNA remained intact and the complexes were shown to maintain activity throughout the nebulization run. Based on measurements of the nebulized dose and the mass median aerodynamic diameter, we calculate a delivered dose of approximately 22 micromol (7.2 mg) of pDNA for each 8 ml of cationic lipid:pDNA complex aerosolized to the lungs of a human patient. This dose should be sufficient to test the clinical efficacy of cationic lipid-mediated gene delivery for the treatment of cystic fibrosis.
此前，我们已经描述了将一种非病毒基因治疗载体以气溶胶形式递送至啮齿动物肺部的优化方法（伊斯特曼等人，1997b）。虽然将阳离子脂质：质粒DNA复合物雾化到全身暴露舱中可使BALB/c小鼠肺部的报告基因表达水平很高，但所采用的条件对于将脂质：质粒DNA复合物递送至人类患者肺部并非最佳。也就是说，雾化器中材料的消耗速率以及因此的递送时间非常缓慢，且气溶胶是以连续流的形式递送的。在此，我们描述了用于开发一种具有更适合递送至人类肺部特征的阳离子脂质：质粒DNA气溶胶的体外实验，这是对人类囊性纤维化患者进行临床研究的必要前提。使用级联冲击器和全玻璃冲击器，我们筛选了几种市售雾化器，以评估它们在尽可能短的时间内递送完整、可吸入、有活性的脂质：质粒DNA复合物的能力，并确定了帕里LC Jet Plus雾化器是符合这些标准的最佳雾化器。以间歇模式使用该雾化器模拟呼吸驱动，阳离子脂质：质粒DNA复合物（6 mM阳离子脂质：8 mM质粒DNA）的消耗速率约为0.6 ml/分钟。质粒DNA保持完整，且复合物在整个雾化过程中均显示保持活性。基于雾化剂量和质量中值空气动力学直径的测量，我们计算出，对于雾化至人类患者肺部的每8 ml阳离子脂质：质粒DNA复合雾气溶胶，递送的质粒DNA剂量约为22微摩尔（7.2毫克）。该剂量应足以测试阳离子脂质介导的基因递送治疗囊性纤维化的临床疗效。
