title: Native molecular state of adeno-associated viral vectors revealed by single-molecule sequencing.
abstract: The single-stranded genome of adeno-associated viral (AAV) vectors is one of the key factors leading to slow-rising but long-term transgene expression kinetics. Previous molecular studies have established what is now considered a textbook molecular model of AAV genomes with two copies of inverted tandem repeats at either end. In this study, we profiled hundreds of thousands of individual molecules of AAV vector DNA directly isolated from capsids, using single-molecule sequencing (SMS), which avoids any intermediary steps such as plasmid cloning. The sequence profile at 3' ends of both the regular and oversized vector did show the presence of an inverted terminal repeat (ITR), which provided direct confirmation that AAV vector packaging initiates from its 3' end. Furthermore, the vector 5'-terminus profile showed inconsistent termination for oversized vectors. Such incomplete vectors would not be expected to undergo canonical synthesis of the second strand of their genomic DNA and thus could function only via annealing of complementary strands of DNA. Furthermore, low levels of contaminating plasmid DNA were also detected. SMS may become a valuable tool during the development phase of vectors that are candidates for clinical use and for facilitating/accelerating studies on vector biology.
authors.affiliation: Helicos BioSciences , Cambridge, MA 02139, USA.
authors.forename: Philipp
authors.initials: P
authors.lastname: Kapranov
authors.forename: Lingxia
authors.initials: L
authors.lastname: Chen
authors.forename: Debra
authors.initials: D
authors.lastname: Dederich
authors.forename: Biao
authors.initials: B
authors.lastname: Dong
authors.forename: Jie
authors.initials: J
authors.lastname: He
authors.forename: Kathleen E
authors.initials: KE
authors.lastname: Steinmann
authors.forename: Andrea R
authors.initials: AR
authors.lastname: Moore
authors.forename: John F
authors.initials: JF
authors.lastname: Thompson
authors.forename: Patrice M
authors.initials: PM
authors.lastname: Milos
authors.forename: Weidong
authors.initials: W
authors.lastname: Xiao
mesh_terms: D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016679:Genome, Viral; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods; D020079:Terminal Repeat Sequences / Q000235:genetics*; D014162:Transfection; D019065:Virus Assembly
chemical_list: D004279:DNA, Viral