Development of novel E1-complementary cells for adenoviral production free of replication-competent adenovirus
| Autor: | Qun Li, Dominic Brignetti, Lucy Tao, Deborah Farson, Derek Ko, Thomas Harding, De-Chao Yu, Denice Mittelstaedt, Yuanhao Li, Karrie Segawa |
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| Rok vydání: | 2005 |
| Předmět: |
viruses
Biology Virus Replication Viral vector Adenoviridae Cell Line Cell Line Tumor Drug Discovery Genetics Humans Adenovirus E1B Proteins Molecular Biology Cytopathic effect Virus quantification Pharmacology Adenovirus genome HEK 293 cells Gene Transfer Techniques Virology Molecular biology Oncolytic virus Oncolytic Viruses Cell culture Molecular Medicine Adenovirus E1A Proteins Homologous recombination |
| Zdroj: | Molecular therapy : the journal of the American Society of Gene Therapy. 14(2) |
| ISSN: | 1525-0016 |
| Popis: | Top of pageAbstract Historically adenoviruses, especially E1-deficient adenoviral vectors, are produced from E1-complementary cells such as human embryonic kidney 293 cells or human embryonic retinoblast PerC6 cells. Each of these cell lines contain a fragment of the adenovirus genome that includes both the E1A and E1B coding regions, and have been shown to generate low levels of replication competent adenovirus (RCA) or other types of recombinants due to homologous recombination with cellular DNA. We have developed a novel E1A/1B complementing cell line that is suitable for the large-scale production of adenovirus that does not generate RCA by separately inserting the coding sequences for E1A and E1B into different regions of the host cell genome. Initially, retroviral vectors derived from Moloney Murine Leukemia Virus (MMLV) were separately constructed encoding the coding sequences for adenovirus E1A or E1B. Retroviral supernatants were then prepared from these vectors and used to co-infect A549 cells, a human lung cancer cell line. E1-complementary clones were selected by a functional assay using an E1A-deficient adenovirus and two cell clones, clone 51 and clone 139 were chosen based upon viral yield. Western blot analysis indicated that clone 51 and 139 express E1A, E1B 19kD and E1B 55kD proteins. PCR and Southern blot were then used to demonstrate that the E1A and E1B genes are inserted into separate regions of the A549 genome. Production of an E1-deleted adenovirus vector on the 2 clones indicated that viral yields are comparable to 293 or PerC6 cells. In comparison, production of E1-modified oncolytic adenoviruses was found to be superior for clones 51 and 139 than viral yields derived from 293 or PerC6 cells. The E1-complementation was stable over 10 weeks of continuous culture. To conduct industrial scale adenoviral production for clinical application, clones 51 and 139 were adapted to serum-free suspension culture and shown to produce adenovirus at levels comparable to 293 suspension cells. Most importantly, the adenoviral products from these cells were demonstrated to be free of RCA. In a blind passage study, an RCA-free Ad-GMCSF virus was serially amplified for 20 passages on the new E1-clones, 293 and PerC6 cells. The adenovirus from the last passage was then amplified in na|[iuml]|ve A549 cells and assayed for RCA or other recombinants that cause cytopathic effect (CPE). The result indicates that both 293 and PerC6 cells produce recombinants that form CPE in A549 cells, whereas the new E1-clones do not generate detectable recombinants by plaque assay or a PCR based recombination assay. These new E1-complimenting cell clones should be of considerable use in the large-scale production of RCA-free adenovirus vectors for clinical application. I own stock in Cell Genesys. |
| Databáze: | OpenAIRE |
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