Xiao X, Li J, Samulski R J
Gene Therapy Center, Division of Pharmaceutics, University of North Carolina at Chapel Hill, 27599, USA.
J Virol. 1998 Mar;72(3):2224-32. doi: 10.1128/JVI.72.3.2224-2232.1998.
Recently, efficient and long-term in vivo gene transfer by recombinant adeno-associated virus type 2 (rAAV) vectors has been demonstrated in a variety of tissues. Further improvement in vector titer and purity will expedite this in vivo exploration and provide preclinical information required for use in human gene therapy. In an effort to obtain higher titers, we constructed a novel AAV helper plasmid which utilizes translational control of AAV Rep genes (J. Li et al., J. Virol. 71:5236-5243, 1997). To address the issue of purity, in this study we report the first rAAV production method which is completely free of adenovirus (Ad) helper virus. The new production system uses a plasmid construct which contains a mini-Ad genome capable of propagating rAAV in the presence of AAV Rep and Cap genes. This construct is missing some of the early and most of the late Ad genes and is incapable of producing infectious Ad. Transfection of 293 cells with the new mini-Ad helper and AAV packaging plasmids results in high-titer rAAV vectors with yields greater than 1,000 transducing units, or 10(5) viral particles per cell. When rAAV vectors were produced by using this production scheme and compared to traditional heat-inactivated rAAV preparations in vitro and in vivo, we observed transduction equivalent to or better than normal levels. The complete removal of infectious Ad from AAV production should facilitate a better understanding of immune response to AAV vectors in vivo, eliminate the need for developing replication-competent Ad assays, and provide a more defined reagent for clinical use.
最近，重组2型腺相关病毒（rAAV）载体在多种组织中实现了高效且长期的体内基因转移。进一步提高载体滴度和纯度将加速这种体内研究，并提供人类基因治疗所需的临床前信息。为了获得更高的滴度，我们构建了一种新型的AAV辅助质粒，该质粒利用AAV Rep基因的翻译控制（J. Li等人，《病毒学杂志》71:5236 - 5243，1997）。为了解决纯度问题，在本研究中我们报告了第一种完全不含腺病毒（Ad）辅助病毒的rAAV生产方法。新的生产系统使用一种质粒构建体，该构建体包含一个小型Ad基因组，能够在AAV Rep和Cap基因存在的情况下繁殖rAAV。这个构建体缺失了一些早期和大部分晚期Ad基因，无法产生有感染性的Ad。用新的小型Ad辅助质粒和AAV包装质粒转染293细胞可产生高滴度的rAAV载体，产量大于1000个转导单位，即每个细胞产生10⁵个病毒颗粒。当使用这种生产方案生产rAAV载体并在体外和体内与传统的热灭活rAAV制剂进行比较时，我们观察到转导效果等同于或优于正常水平。从AAV生产中完全去除有感染性的Ad应有助于更好地理解体内对AAV载体的免疫反应，消除开发具有复制能力的Ad检测方法的必要性，并为临床应用提供更明确的试剂。
