title: Optimization of the helper-dependent adenovirus system for production and potency in vivo.
abstract: Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences provide for safe and highly efficient gene transfer with long-lasting transgene expression. High titer stocks of HD vectors can be generated by using the cre-recombinase system. However, we have encountered difficulties with this system, including rearranged HD vectors and variable efficiency of HD vector rescue. These problems represent a major hindrance, particularly with regard to large-scale production. To overcome these limitations, we have modified the system in two ways: We constructed a new helper virus with a modified packaging signal and enhanced growth characteristics. We also redesigned the vector backbones by including noncoding adenovirus sequences adjacent to the right inverted terminal repeat and by incorporated a number of different segments of noncoding DNA of human origin as "stuffer." Comparison of these vectors showed that the nature of the stuffer sequence affects replication of the HD vector. Optimization of the system resulted in a more robust and consistent production of HD vectors with low helper contamination and high in vivo potency.
authors.affiliation: Department of Virus and Cell Biology, Merck Research Laboratories, West Point, PA 19486, USA. volker_sandig@merck.com
authors.forename: V
authors.initials: V
authors.lastname: Sandig
authors.forename: R
authors.initials: R
authors.lastname: Youil
authors.forename: A J
authors.initials: AJ
authors.lastname: Bett
authors.forename: L L
authors.initials: LL
authors.lastname: Franlin
authors.forename: M
authors.initials: M
authors.lastname: Oshima
authors.forename: D
authors.initials: D
authors.lastname: Maione
authors.forename: F
authors.initials: F
authors.lastname: Wang
authors.forename: M L
authors.initials: ML
authors.lastname: Metzker
authors.forename: R
authors.initials: R
authors.lastname: Savino
authors.forename: C T
authors.initials: CT
authors.lastname: Caskey
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D016384:Consensus Sequence; D003587:Cytomegalovirus / Q000235:genetics; D004274:DNA, Recombinant / Q000737:chemistry / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics / Q000502:physiology*; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D017930:Genes, Reporter; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006378:Helper Viruses / Q000502:physiology*; D006801:Humans; D007121:Immunocompetence; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins; D011995:Recombination, Genetic; D012449:Safety; D014162:Transfection; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins; D004921:Erythropoietin; D008156:Luciferases