title: Optimization of the capsid of recombinant adeno-associated virus 2 (AAV2) vectors: the final threshold?
abstract: The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ∼24-fold over the WT AAV2 vector, and ∼2-3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.
authors.affiliation: Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, United States of America. aslanidi@peds.ufl.edu
authors.forename: George V
authors.initials: GV
authors.lastname: Aslanidi
authors.forename: Angela E
authors.initials: AE
authors.lastname: Rivers
authors.forename: Luis
authors.initials: L
authors.lastname: Ortiz
authors.forename: Liujiang
authors.initials: L
authors.lastname: Song
authors.forename: Chen
authors.initials: C
authors.lastname: Ling
authors.forename: Lakshmanan
authors.initials: L
authors.lastname: Govindasamy
authors.forename: Kim
authors.initials: K
authors.lastname: Van Vliet
authors.forename: Mengqun
authors.initials: M
authors.lastname: Tan
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
authors.forename: Arun
authors.initials: A
authors.lastname: Srivastava
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism / Q000821:virology; D006801:Humans; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D013912:Threonine; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D036022:Capsid Proteins; D013912:Threonine