AAV Virus Production
AAV2 recombinant genomes encoding wild-type human α-synuclein, or GFP,
under the transcriptional control of the PGK1 (phosphoglycerate kinase)
promoter were pseudotyped in serotype 6 capsids as previously described
(Berger et al., 2015). In brief, viral particles were produced by
co-transfection of HEK-293T cells with an adenovirus helper plasmid
(pXX6-80), an AAV packaging plasmid carrying the rep2 and cap6 genes,
and a plasmid encoding a recombinant AAV2 genome containing the
transgene expression cassette. Seventy-two hours after transfection,
viral particles were purified and concentrated from cell lysates and
supernatants by ultracentrifugation on an iodixanol density step
gradient, followed by dialysis against PBSMK buffer (0.5 mM
MgCl2 and 1.25 mM KCl in PBS). The concentration of
vector stocks was estimated by real-time PCR and expressed as vector
genomes per µL of concentrated stocks (vg/µL). On the day of surgery,
the vectors were diluted in PBS with 0.01% Pluronic F-68 to the
appropriate titer.