title: Characterization of empty capsids from a conditionally replicating adenovirus for gene therapy.
abstract: As virus vectors for gene therapy approach the goal of successful clinical treatment, it is increasingly necessary for the product to be fully characterized. Empty capsids are perhaps the main extraneous component of recombinant adenovirus (rAd) products that are purified by column chromatography. Two diverse rAd products, one a replication-defective rAd and the other a conditionally replicating rAd, show different protein compositions of their empty capsids. The empty capsid type from the replication-defective rAd carrying the gene for p53 was previously determined to have approximately 1400 copies per particle of pVIII, the precursor to the hexon-associated protein VIII (Vellekamp et al., Hum. Gene Ther. 2001;12:1923-1936). Quantification of this protein is a useful measure of the amount of empty capsids in preparations of this vector. Here we purify and characterize empty capsids from the conditionally replicating rAd. This empty capsid type lacks any appreciable amount of pVIII but contains pVI and multiple forms of the L1 52/55K protein, mostly as disulfidelinked oligomers. Empty capsid from conditionally replicating rAd present new challenges in terms of its quantification, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis densitometry analysis suggests that the amount of this empty capsid in a preparation, like that of rAd p53 empty capsid, declines with increased time of infection. This empty capsid demonstrates heterogeneity by anion-exchange chromatography, electron microscopy, and CsCl density gradient centrifugation.
authors.affiliation: Canji, Inc., San Diego, CA 92121, USA. suganto.sutjipto@canji.com
authors.forename: Suganto
authors.initials: S
authors.lastname: Sutjipto
authors.forename: Sundari
authors.initials: S
authors.lastname: Ravindran
authors.forename: Douglas
authors.initials: D
authors.lastname: Cornell
authors.forename: Yan-Hui
authors.initials: YH
authors.lastname: Liu
authors.forename: Mark
authors.initials: M
authors.lastname: Horn
authors.forename: Thomas
authors.initials: T
authors.lastname: Schluep
authors.forename: Beth
authors.initials: B
authors.lastname: Hutchins
authors.forename: Gary
authors.initials: G
authors.lastname: Vellekamp
mesh_terms: D000256:Adenoviridae / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002460:Cell Line; D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D018014:Gene Transfer Techniques; D016158:Genes, p53 / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D011817:Rabbits; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
chemical_list: D010446:Peptide Fragments; D014764:Viral Proteins