Persistence of high-capacity adenoviral vectors as replication-defective monomeric genomes in vitro and in murine liver

• Published in: Human gene therapy Vol. 20; no. 8; p. 883
• Main Authors: Jager, Lorenz, Ehrhardt, Anja
• Format: Journal Article
• Language: English
• Published: United States 01.08.2009
• Subjects: Adenoviridae - genetics; Adenoviridae - physiology; Animals; Cell Cycle; Cell Line; DNA Restriction Enzymes - metabolism; DNA, Concatenated - genetics; Electrophoresis, Gel, Pulsed-Field; Genetic Vectors - genetics; Genome, Viral - genetics; Humans; Liver - virology; Mice; Mice, Inbred C57BL; Polymerase Chain Reaction; Virus Replication - physiology
• ISSN: 1557-7422
• DOI: 10.1089/hum.2009.020

Liver-based gene therapy approaches demonstrated that high-capacity adenoviral vectors (HC-AdVs) can persist life-long in mice and for 2 years or longer in rats, dogs, and nonhuman primates. However, the molecular status of episomal HC-AdV DNA molecules and the mechanism of vector genome maintenance have not been analyzed. HC-AdV lacks all viral coding sequences including early gene region 4 (E4), which prevents concatemerization in wild-type adenovirus. Therefore, we addressed whether concatemerization or circularization of HC-AdV DNA occurs in transduced cells. We employed pulsed-field gel electrophoresis and a sensitive concatemer/circle-specific polymerase chain reaction (PCR). To test for replication as a potential mechanism for maintenance, we developed a methylase/restriction endonuclease-based system using methylation-marked HC-AdV. We found that unlike DeltaE4 mutant virus, only monomers of HC-AdV genomes were observable in vitro. Using our methylase/restriction endonuclease-based system, no replication of HC-AdV was sensed in various cell lines. However, concatemer formation of HC-AdV could be induced after coinfection with an E4-deleted helper virus, indicating that linkage of genomes may be supported by replication. To examine HC-AdV DNA molecules in vivo, C57BL/6 mice were injected and vector DNA in liver was analyzed. In concordance with our in vitro results, exclusively linear monomers were detected. To sense the replication status of HC-AdV genomes, we established a sensitive real-time PCR. Our results indicated that the input transduced DNA genomes were the persistent molecules in murine liver. In summary, we demonstrated that HC-AdV genomes persist predominantly as replication-defective monomeric genomes.