98. Bortezomib Enhances AAV Vector Mediated Transduction But Inhibits the Secretion of Transgene Product

Abstract

Adeno-associated virus (rAAV) mediated gene therapy has shown promise for the treatment of alpha1 antitrypsin deficiency (AATD). Sustained alpha 1 antitrypsin (AAT) expression has been achieved in clinical trails, but the serum AAT concentrations do not reach therapeutic levels. Enhancing transduction efficiency is critical to developing a successful treatment. Since rAAV transduction may be limited by the proteasome, which degrade AAV capsids, we tested the effect of proteasome inhibitors on rAAV mediated AAT production. We treated HeLa and 293 cells with proteasome inhibitors including bortezomib and infected HeLa cells with rAAV2-GFP, rAAV2-AAT or AAV1-AAT and we Infected 293 cells with rAAV2-AAT. We measured AAT in the medium and the lysates of cells. Consistent with previous reports, bortezomib significantly enhanced AAV2 mediated GFP transduction in Hela cells. In rAAV-AAT infected cells, bortezomib treatment resulted in 9.7-13.1 fold increase in of intracellular AAT, while only modest (1.16-1.6 fold) increases of secreted AAT compared to that in cells without bortezomib. Similarly, intramuscular injection of rAAV1-AAT to C57BL6 mice treated with bortezomib also resulted in a small (~1 fold) increases in serum AAT compared with no treatment. These results indicate that bortezomib inhibited AAT protein secretion. To test the role unfolded protein response (UPR) in AAT accumulation, we measured UPR-related genes and showed that bortezomib treatment increased the mRNA levels of the protein chaperone and UPR sensor GRP78 (BiP) indicating an activation of ER stress pathways. Together, our results indicate that bortezomib can enhance rAAV transduction, but it can also inhibit the secretion of transgene product, which will limit the application of this drug for enhancing the efficiency of certain gene therapy, such as alpha 1 antitrypsin gene therapy.