Comparative analysis of the effects of packaging signal, transgene orientation, promoters, polyadenylation signals, and E3 region on growth properties of first-generation adenoviruses.
First-generation adenovectors have been developed for gene therapy and vaccine applications. The construction of these adenovectors has entailed the use of numerous types of expression cassettes. It has long been known that first-generation adenovectors can be rescued more easily and to higher titers with some transgenes than with others. This study has systematically shown that there can be marked differences in growth properties of recombinant adenovectors attributable to the use of promoters, the orientation of the transgene within the E1A/E1B-deleted region, and the inclusion of the E3 region. In addition, we had demonstrated the benefit of extending the packaging signal region to include elements V, VI, and VII. The effects of the complete packaging region were studied by plasmid competition studies between original and modified adenovectors. Similar competition studies between E3(+) and E3(-) adenovectors were performed and showed that the E3(+) vector had a growth advantage over its E3(-) counterpart. By making various changes, we have enhanced the growth capacity of our recombinant adenovector by more than 3-fold under serum-free and cell suspension growth conditions. Along with this enhanced growth, our adenovectors have maintained their genetic stability after 21 successive passages in cell culture. This increased robustness will be critical when adapting first-generation recombinant adenovectors to commercial production.
Youil
Rima
R
Merck & Company, West Point, PA 19486, USA. rima_youil@merck.com
Toner
Timothy J
TJ
Su
Qin
Q
Casimiro
Danilo
D
Shiver
John W
JW
Chen
Ling
L
Bett
Andrew J
AJ
Rogers
Bethany M
BM
Burden
Eric C
EC
Tang
Aimin
A
Chen
Michelle
M
Emini
Emilio A
EA
Kaslow
David C
DC
Aunins
John G
JG
Altaras
Nedim E
NE
D000256:Adenoviridae / Q000235:genetics* / Q000254:growth & development; D017500:Adenovirus E1A Proteins / Q000096:biosynthesis; D017503:Adenovirus E3 Proteins / Q000235:genetics; D000818:Animals; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral; D015750:Genes, gag; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral; D017874:Immediate-Early Proteins / Q000235:genetics; D008565:Membrane Proteins; D051379:Mice; D010957:Plasmids; D011401:Promoter Regions, Genetic; D039104:RNA 3' Polyadenylation Signals; D019076:Transgenes; D019065:Virus Assembly
D017500:Adenovirus E1A Proteins; D017503:Adenovirus E3 Proteins; D036022:Capsid Proteins; C084301:E1A protein 243R, Human adenovirus type 5; D017874:Immediate-Early Proteins; C484408:Laptm5 protein, mouse; D008565:Membrane Proteins; C018315:hexon capsid protein, Adenovirus