Repeat Dosing of Adeno-Associated Viral Vectors

在动物和第一批人类研究中，我们的团队表明AAV2-CFTR可以应用于鼻上皮、上颌窦和人肺右下叶，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持续表达。最近的一项多机构合作研究显示，在雾化3次1x10[13]DNase抗性颗粒tgAAVCF后，第30天的FEV1和第14-45天观察到的诱导痰IL-8水平有显著改善。因此，AAV载体具有很大的治疗潜力。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。这笔赠款将使用一种动物组合 以及人体研究，以解决这样一种假设，即通过支气管镜微喷雾器将新的更强大的启动子高效地输送到呼吸道将是安全的，并导致重组基因表达水平的提高。有三个总体问题将得到解决。1.使用基于AAV5病毒的伪型载体是否会导致重组载体的表达增加？这一特定目的的实验将专门针对AAV5-CFTR递送到哺乳动物的呼吸道是否引起免疫或炎症反应，如果是的话，针对载体的哪个方面进行免疫反应。2.包含一个更强大的启动子是否会增加重组载体中CFTR的表达？因此，我们将探索潜在的风险 以及使用改变的cftr结构和新的鸡β-肌动蛋白启动子来增强cftr表达的好处。预计在非人类灵长类动物模型中对这些问题的回答将提供必要的临床前数据，以启动在CF患者中雾化AAV5-CFTR载体的研究。3.雾化吸入新型高滴度AAV5-CFTR载体给轻度肺病患者是否能导致均匀的基因转移和CFTR表达？还将特别关注新的伪型载体AAV5-CFTR在人类中是否会导致重组载体的表达增加。对这一问题的回答有望带来一种实用的重组AAV5-CFTR递送系统，该系统具有潜在的新启动子来促进CFTR的表达。

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.