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

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.

Simultaneous presence of fhs and purT genes is disadvantageous for the fitness of Escherichia coli growth

In bacteria, alternate mechanisms are known to synthesize N-10-formyltetrahydrofolate (N10-formyl-THF) and formyl glycinamide ribotide (fGAR), which are important in purine biosynthesis. In one of the mechanisms, a direct transfer of one carbon unit from formate allows Fhs to convert tetrahydrofolate to N-10-formyl-THF, and PurT to convert glycinamide ribotide (GAR) to fGAR. Our bioinformatics analysis of fhs and purT genes (encoding Fhs and PurT) showed that in a majority of bacteria (similar to 94%), their presence was mutually exclusive. A large number of organisms possessing fhs lacked purT and vice versa. The phenomenon is so penetrating that even within a genus (Bacillus) if a species possessed fhs it lacked purT and vice versa. To investigate physiological importance of this phenomenon, we used Escherichia coli, which naturally lacks fhs (and possesses purT) as model. We generated strains, which possessed fhs and purT genes in singles or together. Deletion of purT from E. coli in the presence or absence of fhs did not confer a detectable growth disadvantage in pure cultures. However, growth competition assays revealed that the strains possessing either of the single genes outcompeted those possessing both the genes suggesting that mutual exclusion of purT and fhs in organisms confers fitness advantage in mixed cultures.