Repeat Dosing of Adeno-Associated Viral Vectors

在动物和第一个人类研究中，我们的小组表明，AAV 2-CFTR可以应用于鼻上皮、上颌窦和人肺的右下叶，而没有任何不良影响。这些研究和文献中发表的多项动物研究表明，从AAV载体表达的基因导致在各种组织中的持续表达。最近的一项多机构合作研究显示，在3次雾化剂量的1 × 10[13] DNA酶抗性颗粒tgAAVCF后，在第30天观察到FEV 1的统计学显著改善，在第14-45天观察到诱导痰IL-8水平。因此，AAV载体具有作为治疗剂的巨大潜力。尽管有这些有希望的研究，但在AAV载体成为有用的治疗剂之前，必须克服几个障碍。这笔赠款将使用动物的组合 和人类研究，以解决这样的假设，即开发具有通过支气管镜微喷雾器有效递送至气道的新的更强大的启动子的新血清型将是安全的，并导致重组基因表达水平的增加。将讨论三个总体问题。1.基于AAV 5病毒的假型载体给药是否会导致重组载体的表达增加？该特定目的的实验将具体针对解决是否将AAV 5-CFTR递送至哺乳动物气道引发免疫或炎症应答，以及如果是，则针对载体的哪个方面是免疫应答。2.包含更强的启动子是否会增加重组载体的CFTR表达？因此，我们将探讨潜在的风险 以及使用改变的CFTR构建体和新的鸡β-肌动蛋白启动子来增强CFTR表达的益处。预期在非人灵长类动物模型中对这些问题的回答将提供在患有CF的患者中启动雾化AAV 5-CFTR载体的研究所需的临床前数据。3.向患有轻度肺病的CF患者施用新的更高滴度AAV 5-CFTR载体的气溶胶递送是否导致均匀的基因转移和CFTR表达？还将特别关注新的假型化载体AAV 5-CFTR在人中是否导致重组载体的表达增加。预期对该问题的回答将导致用于重组AAV 5-CFTR的实用递送系统，其具有潜在的新启动子以促进CFTR表达。

In animal and in the first human studies, our group showed that AAV2-CFTR could be applied to the nasal epithelium, maxillary sinus, and right lower lobe of the human lung without any adverse affects. These studies and multiple animal studies published in the literature showed that genes expressed from AAV vectors result in persistent expression in a variety of tissues. A recent multi institutional collaborative study showed statistically significant improvement in FEV1 at Day 30 and induced sputum IL-8 levels observed between Days 14-45 following 3 aerosolized doses of 1 x 10[13] DNAse resistant particles tgAAVCF. Thus, AAV vectors have great potential as therapeutic agents. Despite these promising studies several hurdles must be overcome before AAV vectors will be useful therapeutic agents. This grant will use a combination of animal and human studies to address the hypothesis that development of a new serotype with new more powerful promoters delivered to the airways efficiently via a bronchoscopic Microsprayer will be safe and result in increased levels of recombinant gene expression. There are three overall questions that will be addressed. 1. Will Dosing with a pseudotyped vector based on the AAV5 virus lead to increased expression from the recombinant vector? Experiments in this specific aim will be directed specifically toward addressing, if delivery of AAV5-CFTR to the mammalian airway elicits an immunologic or inflammatory response and if so to what aspect of the vector is the immunologic response directed. 2. Will inclusion of a more powerful promoter augment CFTR expression from recombinant vectors? Thus, we will explore the potential risks and benefits of using an altered CFTR construct and a new chicken beta-actin promoter to enhance expression of CFTR. It is anticipated that answers to these questions in a non-human primate model will provide the preclinical data necessary to launch a study of aerosolized AAV5-CFTR vector in patients with CF. 3. Does aerosol delivery of new higher titer AAV5-CFTR vectors administered to CF patients with Mild Lung Disease lead to uniform gene transfer and CFTR expression? Particular attention will also be given to whether the new pseudotyped vector AAV5-CFTR in humans leads to increased expression of the recombinant vector. It is expected that answers to this question will lead to a practical delivery system for recombinant AAV5-CFTR with potential new promoters to boost CFTR expression.