13. Persistence of AAV Capsid in Transduced Cells

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