ADENO-ASSOCIATED VIRUS VECTORS FOR CYSTIC FIBROSIS GENE THERAPY

AAV载体已经显示出用于囊性纤维化（CF）基因治疗的前景， 组织培养，小动物，灵长类动物和CF患者，特别是 关于安全性和持久性。然而，基因转移在 CF患者的气道效率低于预期， 体外研究和动物数据。存在多个可能的 对此的解释。首先，有可能的是， 人的下呼吸道缺乏AAV受体。在 例腺病毒（Ad）载体，载体嗜性为人鼻腔 上皮细胞似乎明显低于观察到的 实验动物的支气管上皮。如果一个类似的 在AAV的情况下存在差异，然后病毒的改变 可能需要衣壳来增强进入人支气管上皮细胞 细胞其次，AAV颗粒暴露于游离中性粒细胞 弹性蛋白酶、细胞外白细胞DNA、细菌胞外酶或超- CF支气管分泌物中存在的硫酸化蛋白多糖可能会导致 这是CF患者肺中体内转导的特别障碍。 第三，AAV的小包装容量导致使用 最小的内源性AAV启动子元件，用于在 AAV载体，其对于表达可能是次优的。以来 较大启动子与全长CFTR的组合将产生 超过AAV的5-kb包装限度的载体盒， 部分截断版本的CFTR将需要审查。 这些问题将在以下具体目标中解决：1。到 评估AAV载体对正常人支气管的向性 体内上皮。一种新的报告系统，基于荧光 支气管镜检测的绿色荧光蛋白（GFP），将 用于评估正常人类志愿者的基因转移。2. 确定是否存在AAV转导的特异性屏障 与正常人相比， 个体3.为了改变AAV载体的向性以靶向特异性表达， 支气管上皮细胞基底外侧膜上的受体。在 与托马斯Ferkol博士合作，我们将尝试增强AAV 通过偶联载体与 针对细胞受体的抗体，如表皮受体， 生长因子受体（EGFR）和多聚免疫球蛋白受体 （pIgR）。为了优化来自AAV载体系统的CFTR的表达， 特别是在使用更有活性的启动子来驱动 CFTR小基因的表达。我们预计，完成这些 目的将消除临床发展的剩余障碍， 用于囊性纤维化的有用的基于AAV的基因疗法。

AAV vectors have shown promise for cystic fibrosis (CF) gene therapy in tissue culture, small animals, primates and CF patients, particularly with regard to safety and persistence. However, gene transfer in the airways for CF patients has been less efficient than was predicted from the in vitro studies and the animal data. There are a number of possible explanations for this. First, it is possible that the luminal surface of the lower airways of humans is deficient in receptors for AAV. In the case of adenovirus (Ad) vectors, vector tropism for the human nasal epithelium appears to be substantially lower than that observed for the bronchial epithelium of experimental animals. If an analogous discrepancy is present in the case of AAV, then alteration of the viral capsid may be required to enhance entry into human bronchial epithelial cells. Secondly, the exposure of AAV particles to free neutrophil elastase, extracellular leukocyte DNA, bacterial exoenzymes, or hyper- sulfated proteoglycans present in CF bronchial secretions may pose a particular obstacle to in vivo transduction in the lungs of CF patients. Thirdly, the small packaging capacity of AAV has led to the use of minimal endogenous AAV promotor elements for expression of CFTR within AAV vectors, which may be suboptimal for expression. Since the combination of larger promoter with full-length CFTR would produce vector cassettes which exceed the 5-kb packaging limit of AAV, the use of partially-truncated versions of the CFTR will need to be examined. These issues will be addressed in the following specific aims: 1. To assess the tropism of AAV vectors for the normal human bronchial epithelium in vivo. A new reporter system, based on fluorescent bronchoscopic detection of the Green Fluorescent Protein (GFP), will be used to assess gene transfer efficiently in normal human volunteers. 2. To determine whether there are specific barriers to AAV transduction which are greater in individuals with CF as compared with normal individuals. 3. To alter the tropism of AAV vectors to target specific receptors on the basolateral membrane in bronchial epithelial cells. In collaboration with Dr. Thomas Ferkol we will attempt to enhance AAV uptake and bypass mucosal barriers by coupling of vectors with antibodies directed against cellular receptors, such as the epidermal growth factor receptor (EGFR) and the polymeric immunoglobulin receptor (pIgR). To optimize the expression of CFTR from the AAV vector system, particularly in the context of using more active promoters to drive expression of CFTR minigenes. We anticipate that the completion of these aims will remove the remain obstacles to the development of a clinically useful AAV-based gene therapy for cystic fibrosis.