Identification and elimination of replication-competent adeno-associated virus (AAV) that can arise by nonhomologous recombination during AAV vector production
Identification and elimination of replication-competent adeno-associated virus (AAV) that can arise by nonhomologous recombination during AAV vector production
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DOI：
10.1128/jvi.71.9.6816-6822.1997
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发表时间：
1997-09-01
影响因子：
5.4
通讯作者：
Miller, AD
中科院分区：
文献类型：
--
作者：
Allen, JM;Debelak, DJ;Miller, AD
腺相关病毒（AAV）载体制剂通常被不同量的复制型AAV（rcAAV）污染，这可能影响这些载体在培养细胞和动物中的行为。构建不含显著同源物且缺乏野生型AAV p5启动子的包装质粒/载体质粒系统以消除野生型的产生。通过重组的AAV。尽管如此，在通过大规模共转染这些质粒产生的载体中检测到rcAAV。序列分析表明，rcAAV是通过非同源重组产生的。构建了一种新的AAV包装质粒，该包装质粒携带两种不同的表达盒rep和cap，表达方向相反，用该包装质粒制备的AAV载体不含rcAAV。
Adeno-associated virus (AAV) vector preparations are often contaminated with variable amounts of replication-competent AAV (rcAAV), which may influence the behavior of these vectors both in cultured cells and in animals. A packaging plasmid/vector plasmid system containing no significant homolog and lacking the wild-type AAV p5 promoter was constructed to eliminate the production of wild-type. AAV by recombination. Still, rcAAV was detected in vector produced by cotransfection of these plasmids at large scale. Sequence analysis revealed that nonhomologous recombination was responsible for the generation of these novel rcAAVs, A new AAV packaging plasmid carrying separate rep and cap expression cassettes in opposite transcriptional orientations was constructed, AAV vector preparations produced by using this packaging construct did not contain rcAAV.