HYBRID PLASMID-AAV VECTORS FOR CFTR GENE THERAPY

描述(直接从应用程序中获取) 通过基因疗法长期治疗囊性纤维化将 要求转基因在转导细胞内保持稳定 人口。此外，载体不应表达病毒抗原 会引起免疫反应，导致被转导的 细胞。目前最有希望的病毒载体系统之一是 开发用于治疗CF的是基于重组腺相关的 病毒(RAAV)。甲型肝炎病毒基因组包含末端重复序列(Tr) 每一端都促进了基因组与宿主的整合 染色体。与其他病毒相比，重组AAV载体有两个优点 向量系统：它们可以在集成状态下维护 被转导细胞及其后代的寿命；并且，载体可以是 被构造成不再有病毒基因被表达，并引发 免疫反应。然而，保留病毒rep基因的rAAV载体是 专门整合到人类19号染色体上的一个位置。这代表了 具有此属性的向量中的一个重要安全功能。不足之处 RAAV载体对于包装是一个严格的大小限制，并且很难 培养具有合适纯度的高滴度病毒。我们创造了一个新的 DNA载体，双D，利用修饰的AAVTRs促进整合 但可以在细菌中以质粒的形式繁殖。因此，该混合载体 解决了转基因编码能力和大批量生产的问题 纯化载体的数量。我们建议开发方法来实现 将该载体高效地输送到培养细胞中，包括呼吸道 上皮细胞，采用脂质体介导的基因转染法。这项工作还将 需要在BANS中瞬时联合传递AAV Rep蛋白，从而 载体的整合将被定向到19号染色体上。 最后，我们将利用双D载体将CFTR基因转移到 并确定其表达的大小和持续时间。

DESCRIPTION (Taken directly from the application) Long term treatment of cystic fibrosis (CF) through gene therapy will require that the transgene be stably maintained within the transduced cell population. Further, the vector should not express viral antigens which will elicit an immune response leading to the destruction of the transduced cells. One of the most promising viral vector systems currently being developed for the treatment of CF is based on recombinant adeno-associated virus (rAAV). The AAV genome contains terminal repeat (TR) sequences at each end which promote the integration of the genome into the host chromosome. Recombinant AAV vectors offer two advantages over other viral vector systems: they can be maintained in the integrated state for the lifetime of the transduced cell and its progeny; and, the vectors can be constructed such that no viral genes remain to be expressed and to elicit an immune response. However, rAAV vectors which retain the viral rep gene are specifically integrated into a site on human chromosome 19. This represents an important safety feature in vectors with this property. Disadvantages of rAAV vectors are a stringent size limitation for packaging and difficulty in growing high titer virus stocks of suitable purity. We have created a new DNA vector, double D, which utilizes modified AAV TRs to promote integration but can be propagated as a plasmid in bacteria. This hybrid vector thus solves the problems of transgene coding capacity and production of large quantities of purified vector. We propose to develop methods for the efficient delivery of this vector into cultured cells, including airway epithelial cells, using liposome mediated transfection. This work will also entail the transient co-delivery of the AAV Rep protein in bans such that integration of the vector will be directed to the chromosome 19 site. Finally, we will utilize the double D vector to deliver the CFTR gene into cells and determine the magnitude and duration of its Expression.