title: High-efficiency transduction and correction of murine hemophilia B using AAV2 vectors devoid of multiple surface-exposed tyrosines.
abstract: Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity. A triple-mutant (Y444+500+730F) vector consistently had the highest level of in vivo gene transfer to murine hepatocytes, approximately threefold more efficient than the best single-mutants, and ~30-80-fold higher compared with the wild-type (WT) AAV2 capsids. Improvement of gene transfer was similar for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors, indicating that these effects are independent of viral second-strand DNA synthesis. Furthermore, Y730F and triple-mutant vectors provided a long-term therapeutic and tolerogenic expression of human factor IX (hF.IX) in hemophilia B (HB) mice after administration of a vector dose that only results in subtherapeutic and transient expression with WT AAV2 encapsidated vectors. In summary, introduction of multiple tyrosine-mutations into the AAV2 capsid results in vectors that yield at least 30-fold improvement of transgene expression, thereby lowering the required therapeutic dose and potentially vector-related immunogenicity. Such vectors should be attractive for treatment of hemophilia and other genetic diseases.
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32611, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Markusic
authors.forename: Roland W
authors.initials: RW
authors.lastname: Herzog
authors.forename: George V
authors.initials: GV
authors.lastname: Aslanidi
authors.forename: Brad E
authors.initials: BE
authors.lastname: Hoffman
authors.forename: Baozheng
authors.initials: B
authors.lastname: Li
authors.forename: Mengxin
authors.initials: M
authors.lastname: Li
authors.forename: Giridhara R
authors.initials: GR
authors.lastname: Jayandharan
authors.forename: Chen
authors.initials: C
authors.lastname: Ling
authors.forename: Irene
authors.initials: I
authors.lastname: Zolotukhin
authors.forename: Wenqin
authors.initials: W
authors.lastname: Ma
authors.forename: Sergei
authors.initials: S
authors.lastname: Zolotukhin
authors.forename: Arun
authors.initials: A
authors.lastname: Srivastava
authors.forename: Li
authors.initials: L
authors.lastname: Zhong
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D002836:Hemophilia B / Q000235:genetics* / Q000628:therapy*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014161:Transduction, Genetic; D014443:Tyrosine / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D014443:Tyrosine