Figure 3.

Streamlined vexosome purification using sucrose gradients. Vexosomes are banded by sucrose density ultracentrifugation. The resuspended microvesicle pellet from AAV1-Fluc producer cells or iodixanol purified AAV1-Fluc from cell lysates were loaded onto a sucrose gradient. After centrifugation sucrose gradient fractions were analyzed for (a) adeno-associated virus (AAV) genomes or (b) Fluc activity after transduction of 293T cells with an aliquot of each fraction. The unique peak in fraction 7 of media purified AAV is indicated by a black arrow. This likely represents the major vexosome fraction. To calculate total g.c., the number of g.c. in each fraction (titer in g.c./ml × sample volume) were added together to give the total contained in the entire gradient. The % total for each fraction was calculated by the following equation: %Total g.c. = total g.c. in fraction n/gradient total g.c. × 100. (c) Antibody neutralization assay of AAV1-Fluc vexosomes. 10⁸ g.c. of standard purified AAV1-Fluc or AAV1-Fluc vexosomes were incubated with a control anti-AAV2 antibody or serial dilutions of a neutralizing anti-AAV1 antibody. After 1-hour incubation, the mixture was added to cells and a transduction assay performed on U87 cells. Cells were harvested at 48 hours for a luciferase assay.