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

Abstract Abstract 2045 Recombinant adeno-associated virus vectors based on serotype (AAV)-8 have shown significant promise for liver directed gene therapy of hemophilia B. However, in a recent clinical trial, two patients who received highest dose (2×1012 vg/kg) of the self-complementary (sc)AAV8 vector developed capsid specific T cells that required glucocorticoid therapy to attenuate this response [Nathwani et al, New Eng J Med, 2011]. Thus, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less 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 degradation machinery, we modified specific serine/threonine kinase or ubiquitination targets on AAV8 capsid to improve its transduction efficiency. To test this, point mutations at specific serine (S), threonine (T) or lysine (K) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant capsids were evaluated for their liver transduction efficiency at a dose of 5 × 1010 vgs/ animal in C57BL/6 mice in vivo. Two of the AAV8-S>A mutants (S279A and S501A) and a K137R mutant vector, demonstrated significantly higher EGFP expression (3.6 to 12.5 fold) in the liver compared to animals that received WT-AAV8 vectors alone (Figure 1). The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response [interleukin (IL)-6, IL-12, tumor necrosis factor α, Kupffer cells (KC) and innate immune responsive toll like receptors (TLR)-9] with a concomitant 2-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies also revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (22 fold), lungs (9.7 fold) and muscle (8.4 fold) tissue when compared to WT-AAV8 vectors. Further on-going studies with the optimal mutant scAAV8 vector expressing human coagulation factor IX in murine models of hemophilia B, will demonstrate the feasibility of the use of these novel vectors for potential gene therapy of hemophilia B. Figure 1: Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Figure 1:. Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Disclosures: No relevant conflicts of interest to declare.