Characterization of the function of Adenovirus L4 gene products and their impact on AAV vector production.

Efficient manufacturing of recombinant adenovirus-associated virus (AAV) vectors is critical to the successful development of genomic medicines. We attempted to optimize AAV vector production in a producer cell line platform. In this system, helper functions required for AAV replication and production are provided via infection with a replication-competent wild-type Adenovirus. To evaluate strategies for the reduction of replication and packaging of adenovirus and to understand the interplay of recombinant AAV and the helper virus during AAV vector production, wild-type adenovirus was compared to a mutant (Ad5ts149) containing a temperature-sensitive mutation in the DNA polymerase gene. Infection of a producer cell line with Ad5ts149 at the restrictive temperature reduced recombinant AAV titer and altered the pattern of AAV protein expression. Further investigation revealed that the adenoviral late L4-22K/33K gene products regulated both AAV rep/cap gene transcription and splicing of the rep/cap transcripts. Furthermore, the L4-33K gene products were found to impact AAV production in both the producer cell line and transient transfection platforms. Optimization of Adenovirus L4-22K/33K expression to facilitate efficient expression and splicing of AAV rep/cap transcripts therefore represents a unique opportunity to optimize AAV vector production.