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

Abstract

Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T/Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S/A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (*9-to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h.FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h.FIX:Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.Introduction R ecombinant adeno-associated virus (AAV) vectors have gained significant attention as a gene therapy vector due to their lack of pathogenicity and their ability to infect different tissues (Flotte and Carter, 1995;Choi et al., 2005;Wu et al., 2006;Mueller and Flotte, 2008). So far, 12 serotypes of AAV (AAV1 to AAV12) vectors have been studied extensively as gene therapy vectors (Schultz and Chamberlain, 2008). These AAV serotypes share a common genome, but their unique capsid structure helps them recognize different cell-surface receptors (Grimm and Kay, 2003). AAV serotype 2 is the most extensively studied but has demonstrated only limited precli...

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

“…A previous study indicated that the K137R-mutated AAV8 vector resulted in a 10-fold increase of hepatic gene transfer compared with the wild-type AAV8 (Sen et al, 2013b); however, our current data showed only moderate increase of hepatic gene transfer. These conflicted results prompted us to repeat the similar experiments in a different lab using a different reporter gene.…”

“…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 particular, the single-amino acid 137 K-R mutation on AAV1 (AAV1-K137R) and AAV8 (AAV8-K137R) resulted in consistently higher hepatocyte transgene expression in vivo with a 5-10-fold increase compared with the original capsids (Sen et al, 2013a,b).…”

“…Finally, total DNA was extracted from the liver, and quantitative PCR was performed to determine AAV vector DNA amounts. The higher AAV vector DNA copy numbers were expected in the mutant groups, since the K137R mutation on AAV capsid was supposed to decrease capsid ubiquitination and improve intracellular trafficking of AAV DNA (Sen et al, 2013b). Indeed, the vector DNA of hepatocytes was doubled in the AAV8-K137R group (6.17 -2.7 copies/cell) as compared with the AAV8 group (3.12 -1.11 copies/cell) (Fig.…”

“…enhancement for other serotypes, we created AAV7-K137R and AAV9-K137R, in addition to the AAV8-K137R packaging plasmid (see Materials and Methods for details) (Sen et al, 2013b). The mutations were independently confirmed by DNA sequencing.…”

“…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. In particular, the AAV8-K137R mutant resulted in a significantly higher vector copy numbers in the liver (22-fold), lungs (9.7-fold), and muscle (8.4-fold) tissues when compared with the original AAV8 vectors (Sen et al, 2013b).…”