Bioengineering of AAV2 Capsid at Specific Serine, Threonine, or Lysine Residues Improves Its Transduction Efficiencyin Vitroandin 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.

K137R Mutation on Adeno-Associated Viral Capsids Had Minimal Effect on Enhancing Gene DeliveryIn Vivo

“…Targeted modifications of those amino acids were thought to circumvent the hostcellular serine/threonine kinase phosphorylation and ubiquitination of the viral capsids. Indeed, delivering the mutated AAV2 vector in vivo by tail vein injection resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression in hepatocytes (up to 14-fold) (Gabriel et al, 2013). This strategy was also applied to the other serotypes by the same group such as AAV1, AAV5 (Sen et al, 2013a), and AAV8 (Sen et al, 2013b), and similar increases of gene transduction efficiency (up to 16-fold) were observed.…”

“…In an effort to search for simple and efficient method to improve transduction efficiency of AAV vector, Gabriel et al (2013) hypothesized that the mutation of surface-exposed amino acids other than tyrosine on AAV capsid may facilitate AAV intracellular trafficking. Specifically, eight serine (S), seven threonine (T), and nine lysine (K) residues on AAV2 capsid were mutated to alanine (A) and arginine (R) individually or in combination.…”

“…Mutation of serine/ threonine to alanine or lysine (K) to arginine (R) on AAV capsid shares the same principle as tyrosine mutation. The rationale is to alter the enzymatic (kinase/ubiquitination ligase) targets on AAV capsid to circumvent capsid ubiquitination and increase the transduction efficiency of these vectors (Gabriel et al, 2013). This strategy was originally attempted on AAV2 capsid, and the mutant AAV2 vector resulted in the increase of transgene expression up to 14-fold (Gabriel et al, 2013).…”

“…The rationale is to alter the enzymatic (kinase/ubiquitination ligase) targets on AAV capsid to circumvent capsid ubiquitination and increase the transduction efficiency of these vectors (Gabriel et al, 2013). This strategy was originally attempted on AAV2 capsid, and the mutant AAV2 vector resulted in the increase of transgene expression up to 14-fold (Gabriel et al, 2013). Later, AAV1, AAV5 (Sen et al, 2013a), and AAV8 (Sen et al, 2013b) were mutated, making it appear that most of the mutant vectors mediated much higher transgene expression than the original capsid.…”

Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

“…These data suggest that amino acids other than tyrosines may be recognized on the AAV8 capsid by the host-cellular kinase machinery. Our recent studies in AAV2 vectors have implicated serine (S) and threonine (T) or lysine (K) amino acids as potential cellular kinase or ubiquitination targets, which, when substituted with compatible amino acids, improved AAV2 transduction in vitro and in vivo (Gabriel et al, 2013). Based on these data, the present study was designed to test the efficacy of AAV8 vectors, which are modified at critical S/T/K residues on the vector capsid.…”