Gene Therapy for Cystic Fibrosis Using AAV Vectors

囊性纤维化(CF)影响每3,200名新生儿中的1名，并导致衰弱的肺部疾病和过早死亡，平均年龄为32岁。基因治疗可能通过替换有缺陷的蛋白-囊性纤维化跨膜调节因子(CFTR)来治愈这种疾病。这一应用的长期目标是开发肺靶向基因疗法 使用来自非致病性人类细小病毒-腺相关病毒(AAV)的病毒载体治疗CF。AAV载体能以20%的速率转导小鼠肺内的呼吸道和肺泡上皮细胞，并可持续8个月以上。然而，涉及AAV载体介导的CFTR基因转移到人类的临床试验尚未显示出有用的CFTR表达水平的临床疗效。这种缺乏表达可能是由于由于CFTRcDNA的大小而难以制造表达CFTR的AAV载体，以及难以测量可能导致的CF疾病严重程度的微小变化 基因疗法。为了绕过这些困难，并对AAV载体在人类中的实用性提供更直接的测试，这项建议的具体目标包括在CF患者中进行临床试验，以解决编码易于检测的组织化学标记基因--人胎盘碱性磷酸酶(AP)的AAV载体对鼻腔和支气管给药的安全性、有效性和免疫反应。我们发现，与常用的AAV 2型载体相比，来自AAV6型的载体在小鼠呼吸道中的转导率有很大提高，尽管AAV2在人类中有更广泛的安全性数据。在这里，我们建议比较AAV2和AAV6在CF患者中传递的HPAP的表达。 另一个具体目标是开发有效的AAV载体，用于转移和高效表达CFTRc DNA。这些方法旨在最有效地测试和开发用于治疗CF的AAV载体。

Cystic fibrosis (CF) affects 1 in 3,200 births and leads to debilitating lung disease and premature death at a median age of 32 years. Gene therapy may provide a cure for this disease by replacement of the defective protein, the cystic fibrosis transmembrane regulator (CFTR). The long-range objective of this application is the development of lung-targeted gene therapy for treatment of CF using viral vectors derived from a nonpathogenic human parvovirus, adeno-associated virus (AAV). AAV vectors can transduce airway and alveolar epithelial cells in the lungs of mice at rates of >20% and lasting for over 8 months. However, clinical trials involving AAV vector-mediated transfer of the CFTR gene to humans have yet to show useful levels of CFTR expression of clinical efficacy. This lack of expression is likely due to the difficulty of making AAV vectors that express CFTR because of the large size of CFTR cDNA, and the difficulty of measuring small changes in CF disease severity that might result from gene therapy. To circumvent these difficulties and to provide a more direct test of the utility of AAV vectors in humans, the specific aims of this proposal include clinical trials in CF patients that will address the safety, efficacy and immune responses to nasal and bronchial administration of AAV vectors that encode an easily detected histochemical marker gene, human placental alkaline phosphatase (AP). We have found that vectors derived from AAV serotype 6 show much improved transduction rates in mouse airway compared to commonly used AAV serotype 2 vectors, although more extensive safety data in humans is available for AAV2. Here we propose to compare expression of hpAP delivered by both AAV2 and AAV6 in CF subjects. Another specific aim will address the development of effective AAV vectors for transfer and efficient expression of the CFTR cDNA. These approaches are designed to most efficiently test and develop AAV vectors for treatment of CF.