title: Bioengineering of AAV2 capsid at specific serine, threonine, or lysine residues improves its transduction efficiency in vitro and in vivo.
abstract: We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20-90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T→alanine [A] or 9 K→arginine [R] single mutant or 2 double K→R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T→A and 7 K→R vectors were significantly higher (~63-90%) than the AAV2-WT vectors (~30-40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated ~8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.
authors: Department of Hematology, Christian Medical College, Vellore 632004, Tamil Nadu, India.
authors: Nishanth
authors: N
authors: Gabriel
authors: Sangeetha
authors: S
authors: Hareendran
authors: Dwaipayan
authors: D
authors: Sen
authors: Rupali A
authors: RA
authors: Gadkari
authors: Govindarajan
authors: G
authors: Sudha
authors: Ruchita
authors: R
authors: Selot
authors: Mansoor
authors: M
authors: Hussain
authors: Ramya
authors: R
authors: Dhaksnamoorthy
authors: Rekha
authors: R
authors: Samuel
authors: Narayanaswamy
authors: N
authors: Srinivasan
authors: Alok
authors: A
authors: Srivastava
authors: Giridhara R
authors: GR
authors: Jayandharan
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood / Q000276:immunology; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D017124:Conserved Sequence; D000229:Dependovirus / Q000145:classification / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D022781:Hepatocytes / Q000378:metabolism / Q000821:virology; D006801:Humans; D008239:Lysine; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016415:Sequence Alignment; D012694:Serine; D013912:Threonine; D014161:Transduction, Genetic; D054875:Ubiquitination; D019562:Viral Load
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D036022:Capsid Proteins; D047428:Protein Kinase Inhibitors; D013912:Threonine; D012694:Serine; D008239:Lysine
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