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

Gabriel, Nishanth and Hareendran, Sangeetha and Sen, Dwaipayan and Gadkari, Rupali A and Sudha, Govindarajan and Selot, Ruchita and Hussain, Mansoor and Dhaksnamoorthy, Ramya and Samuel, Rekha and Srinivasan, Narayanaswamy and Srivastava, Alok and Jayandharan, Giridhara R (2013) Bioengineering of AAV2 Capsid at Specific Serine, Threonine, or Lysine Residues Improves Its Transduction Efficiency in Vitro and in Vivo. In: HUMAN GENE THERAPY METHODS, 24 (2). 80-U117.

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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 (similar to 63-90%) than the AAV2-WT vectors (similar to 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 similar to 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.

Item Type:	Journal Article
Publication:	HUMAN GENE THERAPY METHODS
Publisher:	MARY ANN LIEBERT, INC
Additional Information:	Copy right for this article belongs to the MARY ANN LIEBERT, INC, 140 HUGUENOT STREET, 3RD FL, NEW ROCHELLE, NY 10801 USA
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	14 Aug 2015 09:38
Last Modified:	14 Aug 2015 09:38
URI:	http://eprints.iisc.ac.in/id/eprint/52116

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