The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Mol Ther. 2010 Jan;18(1):87-92. doi: 10.1038/mt.2009.258. Epub 2009 Nov 10.
Application of recombinant adeno-associated virus (rAAV) in gene therapy has been limited by its packaging capacity. Recent studies suggested that rAAV could achieve persistent transgene expression beyond 4.7-kb packaging limit. To clarify the mechanism leading to transgene expression from oversized rAAV vector, we constructed a series of rAAV vectors with genomes ranging from 2.9 to 7.2 kb. A plasmid replication origin and an ampicillin-resistant marker were included in the vector to facilitate the recovery of circularized, post-transduction AAV genome. Southern dot-blot analysis and silver staining confirmed that rAAVs could be produced at varying vector size. However, the vector yields decreased approximately tenfold for oversized vectors as compared to regular ones. Alkaline Southern blot hybridization suggested that the packaged genomes for oversized vectors were truncated. In the cells transduced by the above vectors, circularized rAAV monomers could be rescued at 24 hours after infection. Few recovered AAV genomes were >5 kb regardless of the initial vector size. In mice receiving the above vectors, larger circularized rAAV genomes could be recovered for oversized vectors at day 21 after vector administration. Our studies suggested that the partially packaged rAAV sequences may complement each other to restore full expression cassette.
腺相关病毒（rAAV）在基因治疗中的应用受到其包装能力的限制。最近的研究表明，rAAV 可以实现超过 4.7kb 包装限制的持续转基因表达。为了阐明导致超大 rAAV 载体中转基因表达的机制，我们构建了一系列基因组大小从 2.9 到 7.2kb 的 rAAV 载体。载体中包含一个质粒复制起点和氨苄青霉素抗性标记，以方便回收经转导后的环状 AAV 基因组。Southern 点印迹分析和银染证实 rAAV 可以在不同的载体大小下产生。然而，与常规载体相比，超大载体的载体产量下降了约十倍。碱性 Southern 印迹杂交表明，超大载体包装的基因组发生了截断。在上述载体转导的细胞中，感染后 24 小时即可回收环状 rAAV 单体。无论初始载体大小如何，回收的 AAV 基因组中很少有超过 5kb 的。在接受上述载体的小鼠中，载体给药后第 21 天，超大载体可回收更大的环状 rAAV 基因组。我们的研究表明，部分包装的 rAAV 序列可能相互补充，以恢复完整的表达盒。
