13. Persistence of AAV Capsid in Transduced Cells

Abstract

Adeno-associated virus (AAV) vector mediated liver-directed gene transfer has been used to effect long-term cure of hemophilia B in the canine model. Based on these data, a clinical trial was performed on subjects with severe hemophilia B. Vector was introduced through the hepatic artery and at the highest dose tested (2 |[times]| 1012 vg/kg) resulted in therapeutic levels of F.IX (|[sim]|10%) that persisted at this level for 4 weeks and then declined over the ensuing six weeks to baseline levels (<1%). As F.IX levels fell, liver transaminases rose and then returned to normal. Detailed study of a subsequent patient infused with AAV-F.IX documented a transient T cell response to AAV capsid but not to F.IX on IFN-|[gamma]| ELISPOT. We hypothesized that the transduced cells were destroyed by an immune response to AAV capsid and that since this antigen is not long-lived, transient immunosuppression may allow persistence of expression. We therefore initiated studies to determine the duration of AAV-2 capsid persistence in transduced cells. The AAV capsid is composed of three proteins, VP1, 2, and 3. The B1 antibody has been shown to recognize all three capsid proteins in the nonassembled form. We transduced HeLa cells with AAV2 vectors at a MOI of 100,000 at 37 |[deg]|C for 2 hrs. In the nuclear extract, capsid proteins could be detected by B1 antibody on Western blot after 0, 6, 16, and 24 hrs post transduction. Capsid proteins were no longer detectable after 48 hrs post transduction. In the cytoplasmic extract, no signal was detected by Western blot with B1 antibody at any time point except time 0 after transduction. The result indicates that vectors enter the nucleus or are tightly bound to the nuclear envelope at an early stage. A similar phenomenon was also observed using immunocytochemistry staining with the B1 antibody. In the nucleus, capsid proteins could be detected up to 16 hrs and disappeared to undetectable levels after 24 hrs post transduction.