title: Engineering and selection of shuffled AAV genomes: a new strategy for producing targeted biological nanoparticles.
abstract: We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.
authors.affiliation: Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7352, USA.
authors.forename: Wuping
authors.initials: W
authors.lastname: Li
authors.forename: Aravind
authors.initials: A
authors.lastname: Asokan
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Wu
authors.forename: Terry
authors.initials: T
authors.lastname: Van Dyke
authors.forename: Nina
authors.initials: N
authors.lastname: DiPrimio
authors.forename: Jarrod S
authors.initials: JS
authors.lastname: Johnson
authors.forename: Lakshmanan
authors.initials: L
authors.lastname: Govindaswamy
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
authors.forename: Stefan
authors.initials: S
authors.lastname: Leichtle
authors.forename: D Eugene
authors.initials: DE
authors.lastname: Redmond
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: McCown
authors.forename: Kimberly B
authors.initials: KB
authors.lastname: Petermann
authors.forename: Norman E
authors.initials: NE
authors.lastname: Sharpless
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Samulski
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001921:Brain / Q000378:metabolism; D002213:Capsid / Q000276:immunology; D006224:Cricetinae; D032621:DNA Shuffling; D000229:Dependovirus / Q000235:genetics* / Q000648:ultrastructure; D019020:Directed Molecular Evolution; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification*; D016679:Genome, Viral / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000378:metabolism; D008545:Melanoma; D051379:Mice; D008807:Mice, Inbred BALB C; D018482:Muscle, Skeletal / Q000378:metabolism; D053758:Nanoparticles; D011323:Primates; D014161:Transduction, Genetic; D053586:Virus Internalization
chemical_list: D000906:Antibodies