Conrad C K, Allen S S, Afione S A, Reynolds T C, Beck S E, Fee-Maki M, Barrazza-Ortiz X, Adams R, Askin F B, Carter B J, Guggino W B, Flotte T R
Eudowood Division of Pediatric Respiratory Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Gene Ther. 1996 Aug;3(8):658-68.
Gene therapy for cystic fibrosis (CF) would ideally be accomplished with a vector capable of long-term expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in the absence of a host inflammatory response. Recombinant adeno-associated virus (AAV)-CFTR vectors possess these characteristics in rabbits. Because the utility of AAV vectors as gene transfer agents has only been recognized recently, AAV vector-mediated transduction has never been modeled in a primate host, which is an important step before its use in humans. In order to test the safety and biological activity of AAV-CFTR, single doses of AAV-CFTR vector were administered by fiberoptic bronchoscopy to the posterior basal segment of the right lower lobe (RLL) of the lungs of 10 rhesus macaques with four matched vehicle-treated controls. Animals were followed for 10, 21, 90 or 180 days following vector instillation. Vector DNA transfer occurred in bronchial epithelial cells in the RLL of each animal that received vector as assessed by in situ DNA PCR. Vector mRNA was detectable for 180 days after administration as detected by RT-PCR and by RNase protection assay. Safety of vector administration was determined by measurements of pulmonary mechanics, arterial blood gas analysis, chest radiographs, and bronchoalveolar lavage (BAL) fluid analysis including cell count and quantification of inflammatory cytokines. Gross and microscopic pathologic examination were also performed. There was no evidence of inflammation or other toxicity, although vector DNA was found in extrapulmonary organs of some animals. These results indicate that transduction of the primate airway epithelium with the AAV-CFTR mediates long-term CFTR cDNA transfer and is relatively safe.
理想情况下，用于囊性纤维化（CF）的基因治疗应通过一种载体来实现，该载体能够在不引发宿主炎症反应的情况下长期表达囊性纤维化跨膜传导调节因子（CFTR）。重组腺相关病毒（AAV）-CFTR载体在兔体内具备这些特性。由于AAV载体作为基因转移剂的效用直到最近才被认识到，AAV载体介导的转导从未在灵长类宿主中进行过模拟，而这是其用于人类之前的重要一步。为了测试AAV-CFTR的安全性和生物学活性，通过纤维支气管镜将单剂量的AAV-CFTR载体给予10只恒河猴肺右下叶（RLL）的后基底段，并设置4只匹配的接受载体对照的动物。在载体滴注后对动物进行10、21、90或180天的随访。通过原位DNA PCR评估，接受载体的每只动物RLL的支气管上皮细胞中均发生了载体DNA转移。通过RT-PCR和核糖核酸酶保护试验检测，给药后180天可检测到载体mRNA。通过测量肺力学、动脉血气分析、胸部X光片以及支气管肺泡灌洗（BAL）液分析（包括细胞计数和炎症细胞因子定量）来确定载体给药的安全性。还进行了大体和显微镜病理检查。尽管在一些动物的肺外器官中发现了载体DNA，但没有炎症或其他毒性的证据。这些结果表明，用AAV-CFTR转导灵长类气道上皮可介导长期的CFTR cDNA转移且相对安全。
