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Production of helper-dependent adenovirus vector relies on helper virus structure and complementing
Heshan Sam Zhou1, Tiejun Zhao, X Mei Rao
1Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA. hzhou@bcm.tmc.edu
Background:
The helper-dependent (HD) adenoviral (Ad) vector relies on a helper virus to provide viral proteins for vector amplification. HD-Ad vectors can significantly increase therapeutic gene expression and improve safety. However, the yield of an HD-Ad vector is generally lower than that of an E1-deleted first-generation vector, likely due to the alterations in viral E3 or packaging regions of a helper virus that attenuate its replication and complementing for an HD-Ad vector.
Methods:
To study this question and improve HD-Ad vector production, we have generated four different helper viruses with a wild-type or deleted E3 region, and with a relocated loxP. We have also constructed a first-generation vector with a wild-type E3 region and without the loxP site. We compared the replication of these viruses in Cre-positive and -negative cells and studied their complementing for HD-Ad vector production.
Results:
Viruses with deleted E3 formed smaller plaques and produced lower titer compared with viruses containing the E3 region. The site where a loxP is inserted can also affect virus replication. Higher yield of HD-Ad vector was obtained when a helper virus with wild-type E3 was used. We also showed that deletion of the packaging signal in a helper virus through loxP/Cre interaction decreased the viral DNA complementing ability.
Conclusions:
Although the E3 region is not essential for adenovirus replication in vivo, deletion of this region attenuates virus replication. Production of HD-Ad vector can be further improved by modifications in helper virus structure.