Production of helper-dependent adenovirus vector relies on helper virus structure and complementing.
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.
Zhou
Heshan Sam
HS
Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA. hzhou@bcm.tmc.edu
Zhao
Tiejun
T
Rao
X Mei
XM
Beaudet
Arthur L
AL
D000256:Adenoviridae / Q000235:genetics* / Q000254:growth & development; D017503:Adenovirus E3 Proteins / Q000235:genetics; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics* / Q000254:growth & development; D019426:Integrases / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism
D017503:Adenovirus E3 Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases