GENE THERAPY FOR CYSTIC FIBROSIS USING AAV VECTORS

AAV载体对囊性纤维化的基因治疗。囊性纤维化（CF）影响3,200例新生儿中的1例，导致肺部疾病和过早死亡，中位年龄为32岁。基因治疗可以通过替换有缺陷的蛋白，囊性纤维化跨膜调节蛋白（CFTR）来治愈这种疾病。该应用的长期目标是利用源自非致病性人细小病毒腺相关病毒（AAV）的病毒载体，开发肺靶向基因治疗CF。在几种动物模型中，AAV载体已显示出促进基因转移和长期表达的作用，特别是AAV载体可以以大于或等于5%的速率在小鼠肺中转导气道和肺泡上皮细胞并持续8个月以上。然而，涉及AAV载体介导的CFTR基因向人类转移的临床试验尚未显示CFTR表达水平或临床疗效。这种表达缺失可能是由于CFTR cDNA的大尺寸导致难以制作表达CFTR的AAV载体，以及难以测量基因治疗可能导致的CF疾病严重程度的微小变化。为了克服这些困难，并提供更直接的测试AAV载体在人类中的效用，本提案的具体目标包括在健康受试者和CF患者中进行临床试验，以解决AAV载体的安全性，有效性和免疫反应，这些载体编码易于检测的组织化学标记基因，人胎盘碱性磷酸酶（hpAp）。我们发现，与常用的AAV血清型2载体相比，来自AAV血清型6的载体在小鼠气道中的转导率更高，尽管AAV血清型2在人类中的安全性数据更广泛。在这里，我们提出比较hpAP的表达，广泛的安全性数据可用于人类AAV2。本研究拟比较AAV2和AAV6在健康受试者中传递的hpAP的表达。随后将在CF患者中测试具有更好安全性和基因表达谱的载体血清型。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CFTR cDNA。这些方法旨在最有效地测试和开发用于治疗CF的AAV载体。

Gene Therapy for Cystic Fibrosis using AAV Vectors. 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 non-pathogenic human parvovirus, adeno-associated virus (AAV). AAV vectors have been shown to promote gene transfer and long-term expression in several animal models, in particular, AAV vectors can transduce airway and alveolar epithelial cells in the lungs of mice at rates of greater than or equal to 5% 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 or 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 the 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 healthy subjects and CF patients which 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 (hpAp). We have found that vectors derived from AAV serotype 6 show 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 extensive safety data in humans is available for AAV2. Here we propose to compare expression of hpAP delivered by both AAV2 and AAV6 in healthy subjects. The vector serotype with the better safety and gene expression profile will be tested subsequently in patients with CF. 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.