HYBRID PLASMID-AAV VECTORS FOR CFTR GENE THERAPY

描述（直接取自应用程序） 通过基因治疗长期治疗囊性纤维化（CF）将 需要转基因稳定地保持在转导的细胞内 人口 此外，载体不应表达病毒抗原， 将引发免疫应答，导致转导的 细胞 目前最有前途的病毒载体系统之一是 开发用于治疗CF是基于重组腺相关病毒， 病毒（rAAV）。 AAV基因组含有末端重复（TR）序列， 每个末端促进基因组整合到宿主中 染色体 重组AAV载体提供了两个优于其他病毒载体的优点。 矢量系统：它们可以在集成状态下保持 转导细胞及其后代的寿命;并且，载体可以是 构建成使得没有病毒基因保持被表达并引发免疫应答。 免疫反应 然而，保留病毒rep基因的rAAV载体是不稳定的。 特异性整合到人类19号染色体上的位点。 这代表 具有这种性质的载体的重要安全特征。 缺点 rAAV载体对于包装是严格的大小限制，并且难以在载体中表达。 生长合适纯度的高滴度病毒储备液。 我们创造了一种新 DNA载体，双D，其利用修饰的AAV TR促进整合 但可以作为质粒在细菌中繁殖。 这种混合载体因此 解决了转基因编码能力和大规模生产的问题， 纯化载体的量。 我们建议制定方法， 将该载体有效递送到培养的细胞中，包括气道 上皮细胞，使用脂质体介导的转染。 这项工作还将 需要在BAN中瞬时共递送AAV Rep蛋白， 载体的整合将被导向染色体19位点。 最后，我们将利用双D载体将CFTR基因递送到 细胞，并确定其表达的幅度和持续时间。

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.