rAAV vector production and purification
The recombinant AAV2/8-CAG-GFP vector was produced in adherent HEK293
cells by double-plasmid transfection. The 6.6 kbp vector plasmid
pAAV-CAG-GFP-SV40pA-ISceI harbors the 3179 bp recombinant AAV genome
which is composed of the cytomegalovirus enhancer fused to the CMV early
enhancer/chicken beta-actin (CAG) promoter, followed by the enhanced
green fluorescent protein (eGFP) reporter gene and a simian virus 40
(SV40) polyadenylation signal. The rAAV genome is flanked by AAV2
inverted terminal repeats (ITR) from the plasmid pSub201 (Samulski et
al., 1987). The co-transfected helper plasmid pDP8 (Grimm et al., 1998)
contains the helper genes E2a, E4 and VA RNA of Adenovirus 5 and allows
the expression of AAV2 Rep proteins under the mouse mammary tumor virus
(MMTV) LTR promoter and a shortened p5 promoter and of the viral
proteins VP of AAV serotype 8 from the natural p40 promoter. The AAV
vector was produced and purified by ultracentrifugation on double cesium
chloride gradient as described in Ayuso et al. (Ayuso et al., 2010). The
titer in vector genomes (vg) was determined by real-time PCR targeting
the AAV2 ITRs and following the conditions described by D’Costa et al.
(D’Costa et al., 2016).