PACKAGING CELL LINES FOR ADENO ASSOCIATED VIRUS VECTORS

腺相关病毒是一种复制缺陷的细小病毒， 具有许多吸引人的特性，作为潜在的基因转移载体 人类基因疗法。高效率生产重组AAV的难点 滴度是分子事件研究的主要障碍。 参与AAV感染，以及特定部位的整合，以及 在安全考虑AAV作为一种 用于人类的基因转移载体。目前的生产方法 重组AAV(RAAV)是指将重组AAV与质粒DNA共转染细胞 编码甲型肝炎病毒复制和囊膜蛋白，以及 编码AAV末端两侧的目的基因的单独质粒 重复结构。在存在腺病毒(Ad)感染的情况下供应 辅助功能用于复制，编码目的基因的rAAV是 包装好了。申请人已经创建了带有AAV代表和CAP的细胞系 基因稳定整合。拟议研究的具体目标是 尝试提高包装细胞系产生的rAAV效价 通过几种不同的策略：1)上调Rep/Cap蛋白 通过将一个额外的Rep/帽导入细胞来表达 表达盒，其拷贝数将通过 额外的选择压力；2)消除转基因， 将rAAV载体整合到非同源的REP/CAP细胞系中 质粒，以致腺病毒单独感染会导致rAAV 生产；3)或者，编码所有 将创建腺病毒辅助功能，并将其与rAAV一起转染 载体；没有活体腺病毒会污染产生的rAAV，并 避免了重组腺病毒在纯化过程中的损失。

The adeno-associated virus (AAV) is a replication-defective parvovirus that has many attractive properties as a potential gene transfer vector for human gene therapy. The difficulty in producing recombinant AAV in high titer is a major obstacle to the investigation of the molecular events involved in AAV infection, and site-specific integration, as well as to the animal trials that are necessary prior to safe consideration of AAV as a gene transfer vector for humans. The current method of producing recombinant AAV (rAAV) involves co-transfection of cells with plasmid DNA encoding the replication and encapsidation proteins of AAV, along =with a separate plasmid encoding the gene of interest flanked by the AAV terminal repeat structures. In the presence of adenovirus (Ad) infection to supply helper functions for replication, rAAV encoding the gene of interest is packaged. The applicant has created cell lines with the AAV rep and cap genes stably integrated. The specific aims of the proposed research are to attempt to increase the titers of rAAV produced by the packaging cell line via several separate strategies: 1) Upregulation of Rep/Cap protein expression by transfecting into the cell lie an additional rep/cap expression cassette, the copy number of which will be amplified via additional selection pressures; 2) Elimination of transfection, integrating the rAAV vector into the rep/cap cell line on a non-homologous plasmid, such that adenovirus infection alone will result in rAAV production; 3) Alternatively, a transfectable element coding all of the adenovirus helper functions will be created and transfected with the rAAV vector; no live adenovirus will contaminate the rAAV produced, and obligate losses of rAAV during Ad purification will be avoided.