title: Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing.
abstract: Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research.
authors.affiliation: 1] Department of Molecular and Medical Genetics, Oregon Health and Science University School of Medicine, Portland, Oregon 97239, USA [2].
authors.forename: Kei
authors.initials: K
authors.lastname: Adachi
authors.affiliation: 1] Takara Bio Inc., Otsu, Shiga 520-2134, Japan [2].
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Enoki
authors.affiliation: 1] Department of Molecular and Medical Genetics, Oregon Health and Science University School of Medicine, Portland, Oregon 97239, USA [2] Takara Bio Inc., Otsu, Shiga 520-2134, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kawano
authors.affiliation: Department of Molecular and Medical Genetics, Oregon Health and Science University School of Medicine, Portland, Oregon 97239, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Veraz
authors.affiliation: 1] Department of Molecular and Medical Genetics, Oregon Health and Science University School of Medicine, Portland, Oregon 97239, USA [2].
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry* / Q000502:physiology; D002460:Cell Line; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics; D000229:Dependovirus / Q000737:chemistry* / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000737:chemistry* / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype
chemical_list: D004279:DNA, Viral; D018398:Homeodomain Proteins; C064658:RAG-1 protein