title: Vector design Tour de Force: integrating combinatorial and rational approaches to derive novel adeno-associated virus variants.
abstract: Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of "virtual family shuffling" to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 10(5) complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8.
authors.affiliation: Department of Pediatrics, Division of Cell & Molecular Therapy, University of Florida, Gainesville, Florida, USA.
authors.forename: Damien
authors.initials: D
authors.lastname: Marsic
authors.affiliation: 1] Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA [2] Current address: 3500 Windmeadows Blvd, Apt 68, Gainesville, Florida, USA.
authors.forename: Lakshmanan
authors.initials: L
authors.lastname: Govindasamy
authors.affiliation: Department of Pediatrics, Division of Cell & Molecular Therapy, University of Florida, Gainesville, Florida, USA.
authors.forename: Seth
authors.initials: S
authors.lastname: Currlin
authors.affiliation: Department of Pediatrics, Division of Cell & Molecular Therapy, University of Florida, Gainesville, Florida, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Markusic
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA.
authors.forename: Yu-Shan
authors.initials: YS
authors.lastname: Tseng
authors.affiliation: Department of Pediatrics, Division of Cell & Molecular Therapy, University of Florida, Gainesville, Florida, USA.
authors.forename: Roland W
authors.initials: RW
authors.lastname: Herzog
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, USA.
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
authors.affiliation: Department of Pediatrics, Division of Cell & Molecular Therapy, University of Florida, Gainesville, Florida, USA.
authors.forename: Sergei
authors.initials: S
authors.lastname: Zolotukhin
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D036022:Capsid Proteins / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis*; D000229:Dependovirus / Q000235:genetics*; D015723:Gene Library; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage*; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008099:Liver / Q000821:virology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009928:Organ Specificity; D017422:Sequence Analysis, DNA; D014161:Transduction, Genetic
chemical_list: D000596:Amino Acids; D036022:Capsid Proteins; D004279:DNA, Viral