Common structure of rare replication-deficient E1-positive particles in adenoviral vector batches.
The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 x 10(12) vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad.
Murakami
Pete
P
Process Development Department, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA.
Havenga
Menzo
M
Fawaz
Farah
F
Vogels
Ronald
R
Marzio
Giuseppe
G
Pungor
Erno
E
Files
Jim
J
Do
Linh
L
Goudsmit
Jaap
J
McCaman
Michael
M
D017498:Adenovirus E1 Proteins / Q000378:metabolism; D000260:Adenoviruses, Human / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral; D004279:DNA, Viral / Q000235:genetics; D005822:Genetic Vectors; D016679:Genome, Viral; D006367:HeLa Cells; D006378:Helper Viruses; D006801:Humans; D011995:Recombination, Genetic; D019076:Transgenes; D014771:Virion / Q000235:genetics* / Q000378:metabolism; D019065:Virus Assembly; D014779:Virus Replication
D017498:Adenovirus E1 Proteins; D004279:DNA, Viral