Bioengineering of AAV2 Capsid at Specific Serine, Threonine, or Lysine Residues Improves Its Transduction Efficiency in Vitro and in Vivo

摘要: We hypothesized that the AAV2 vector is targeted for destruction in cytoplasm by host cellular kinase/ubiquitination/proteasomal machinery and modification of their targets on capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors serine/threonine kinases (protein kinase A, protein C, casein II) showed an increase (20-90%) AAV2-mediated gene expression. The three-dimensional structure was then analyzed to predict sites ubiquitination phosphorylation. Three phosphodegrons, which are phosphorylation recognized as degradation signals ubiquitin ligases, were identified. Mutation comprising eight serine (S) or seven threonine (T) nine lysine (K) residues selected around phosphodegrons basis solvent accessibility, overlap receptor binding regions, interaction interfaces proteins, evolutionary conservation across AAV serotypes. AAV2-EGFP vectors wild-type (WT) mutant capsids (15 S/T -> alanine A] 9 K arginine R] single 2 double R mutants) evaluated vitro. efficiencies 11 A 7 significantly higher (similar 63-90%) than AAV2-WT 30-40%). Further, hepatic transfer these vivo resulted copy numbers (up 4.9-fold) transgene expression 14-fold) observed from vector. One vectors, S489A, generated similar 8-fold fewer antibodies could be cross-neutralized AAV2-WT. This study thus demonstrates feasibility use novel therapy.