Harvest of produced viral vectors
Viral particles were harvested from the cell culture broth as previously
described [47, 56]. Briefly, the cells were harvested and lysed by
adding a 10X Lysis Buffer (20 mM MgCl2, 1% Triton X-100
in 500 mM Tris-buffered solution). Benzonase was added to a final
concentration of 5 U/mL to digest host-cell DNA and unpacked virus DNA.
After 1 hour of incubation at 37°C with agitation, MgSO4was added to a final concentration of 37.5 mM to prevent AAV aggregation
and binding to cellular components. After 30 minutes of incubation at
37°C with agitation, the lysate was clarified via centrifugation at
10,000 × g for 15 minutes. The clarified viral lysate was stored in a
-80°C freezer.