Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 28

1.3 Production
of AAV Vectors

The most commonly used method to produce AAV vectors is
based on a double or triple plasmid transfection of HEK_293 cells
with rAAV plasmid and plasmids encoding the rep and cap genes
and/or adenoviral helper functions [ 48 , 49 ]. Although such meth-
ods generate high vector titers suitable for small animals experi-
mentation, alternative strategies have been developed to achieve
large-scale productions for clinical trials including the generation
of producer cell lines [ 50 ], the use of adenovirus-AAV hybrids
[ 51 ] and herpes simplex virus [ 52 ] and infection of insect cells with
baculovirus vectors [ 53 , 54 ]. Cells are typically harvested 2-3 days
posttransfection and lysed by several freeze-thaw cycles to release
the recombinant virions (i.e., plasmid-based transfection).
Following clarification, the crude lysate is subjected to various
purification steps. Commonly used purification methods produc-
ing high-quality vector preparations include cesium chloride or
iodixanol-gradient ultracentrifugation [ 55 ], ion exchange [ 56 ,
57 ], or affinity chromatography (e.g., heparin affinity column for
AAV2) [ 58 ]. Recombinant vectors are finally formulated in a buff-
ered physiological solution suitable for long-term storage and in
vivo studies. Titration and quality controls of rAAV vectors are
subsequently performed as part of the testing for viral batch release.
Recombinant AAVs titering methods include the determination of
physical particles, infectious particles, and transducing particles,
whereas quality controls are aimed to detect any impurities within
the final vector preparation such as host cell-derived proteins, cell
culture related reagents, endotoxins, and contaminating viruses.

2 Materials

2.1 Plasmids
Characterization

Escherichia coli electrocompetent SURE (Agilent Technologies)
and DH5α cells (Life Technologies).
Shaking incubators at 30 °C and 37 °C (Fisher Scientific).
Endotoxin-free plasmid midi and maxi kits (Qiagen, Nucleobond).
DNA UV spectrophotometer (Fisher Scientific).
SmaI (New Englands Biolabs, cat. no. R0141S).

Low-passage HEK-293T cells (ATCC, CRL-11268).
High-glucose Dulbecco's modified Eagle's medium (DMEM)
with glutamax and sodium pyruvate (Life Technologies, cat.
no. 31966-021) supplemented with 10 % heat-inactivated
fetal bovine serum (HI-FBS Life Technologies, cat. no. 26140-
079) and 1× penicillin-streptomycin (Life Technologies, cat.
no. 15070-063).
DMEM with glutamax and sodium pyruvate supplemented with
2 % HI-FBS, 1× penicillin-streptomycin and 25 mM HEPES
(Life technologies, cat. no. 15630-056).

2.2 rAAV Vector
Production