Persistence of high-capacity adenoviral vectors as replication-defective monomeric genomes in vitro and in murine liver.
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.
Department of Virology, Max von Pettenkofer-Institute, Ludwig-Maximilian-University of Munich, Munich 80336, Germany.
Lorenz
L
Jager
Anja
A
Ehrhardt
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology*; D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D004262:DNA Restriction Enzymes / Q000378:metabolism; D044708:DNA, Concatenated / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D005822:Genetic Vectors / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000821:virology*; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D014779:Virus Replication / Q000502:physiology*
D044708:DNA, Concatenated; D004262:DNA Restriction Enzymes