Identification and elimination of replication-competent adeno-associated virus (AAV) that can arise by nonhomologous recombination during AAV vector production

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.